cyclic-gmp and zaprinast

Alzheimer's disease (AD), characterized by a progressive impairment of memory and cognition, is a major health problem in both developing and developed countries. Currently, no drugs can reverse the progression of AD. Phosphodiesterase 5 (PDE5) is a critical component of the cyclic guanosine monophosphate/protein kinase G (cGMP/PKG) signaling pathway in neurons, the inhibition of which has produced neuroprotective effects, and PDE5 inhibitors have recently been thought to be potential therapeutic agents for AD. In this paper, we summarized the outstanding progress that has been made in PDE5 inhibitors as anti-AD agents with encouraging results in animal studies, clinical trials and the investigations on the underlying mechanisms. The novel PDE5 inhibitors reported recently in the treatment of AD were also reviewed and discussed.

Topics: Alzheimer Disease; Animals; Cognition; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Flavonoids; Humans; Neurons; Neuroprotective Agents; Phosphodiesterase 5 Inhibitors; Purinones; Pyrimidines; Signal Transduction; Sildenafil Citrate; Sulfonamides; Tadalafil; Vardenafil Dihydrochloride

In humans cone photoreceptors are responsible for high-resolution colour vision. A variety of retinal diseases can compromise cone viability, and, at present, no satisfactory treatment options are available. Here, we present data towards establishing a reliable, high-throughput assay system that will facilitate the search for cone neuroprotective compounds using the murine-photoreceptor cell line 661 W. To further characterize 661 W cells, a retinal marker study was performed, followed by the induction of cell death using paradigms over-activating cGMP-dependent protein kinase G (PKG). We found that 661 W cells may be used to mimic specific aspects of cone degeneration and may thus be valuable for future compound screening studies.

Topics: Animals; Biomarkers; Cell Line, Tumor; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 6; Drug Evaluation, Preclinical; Enzyme Activation; Eye Proteins; High-Throughput Screening Assays; Mice; Mice, Knockout; Neuroprotective Agents; Organ Specificity; Phosphodiesterase Inhibitors; Purinones; Retinal Cone Photoreceptor Cells

Renal fibrosis is an important factor for end-stage renal failure. However, only few therapeutic options for its treatment are established. Zaprinast, a phosphodiesterase 5 inhibitor, and serelaxin, the recombinant form of the naturally occurring hormone relaxin, are differently acting modulators of cyclic guanosine monophosphate (cGMP) signaling. Both agents enhance cGMP availability in kidney tissue. These substances alone or in combination might interfere with the development of kidney fibrosis. Therefore, we compared the effects of combination therapy with the effects of monotherapy on renal fibrosis. Renal fibrosis was induced by unilateral ureteral obstruction (UUO) for 7 days in wild-type (WT) and cGKI knockout (KO) mice. Renal antifibrotic effects were assessed after 7 days. In WT, zaprinast and the combination of zaprinast and serelaxin significantly reduced renal interstitial fibrosis assessed by α-SMA, fibronectin, collagen1A1, and gelatinases (MMP2 and MMP9). Intriguingly in cGKI-KO, mRNA and protein expression of fibronectin and collagen1A1 were reduced by zaprinast, in contrast to serelaxin. Gelatinases are not regulated by zaprinast. Although both substances showed similar antifibrotic properties in WT, they distinguished in their effect mechanisms. In contrast to serelaxin which acts both on Smad2 and Erk1, zaprinast did not significantly diminish Erk1/2 phosphorylation. Interestingly, the combination of serelaxin/zaprinast achieved no additive antifibrotic effects compared to the monotherapy. Due to antifibrotic effects of zaprinast in cGKI-KO, we hypothesize that additional cGKI-independent mechanisms are supposed for antifibrotic signaling of zaprinast.

Topics: Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Drug Therapy, Combination; Fibrosis; Kidney; Kidney Diseases; Mice; Mice, Knockout; Phosphodiesterase 5 Inhibitors; Purinones; Recombinant Proteins; Relaxin; Signal Transduction; Ureteral Obstruction

Topics: Adipocytes; Animals; Blotting, Western; Bradykinin; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dual-Specificity Phosphatases; Glucose; Guanylate Cyclase; Immunoprecipitation; Insulin Resistance; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinase Phosphatases; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphodiesterase Inhibitors; Proto-Oncogene Proteins c-akt; Purinones; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; RNA, Messenger; Signal Transduction

Microneme secretion is essential for motility, invasion, and egress in apicomplexan parasites. Although previous studies indicate that Ca(2+) and cGMP control microneme secretion, little is known about how these pathways are naturally activated. Here we have developed genetically encoded indicators for Ca(2+) and microneme secretion to better define the signaling pathways that regulate these processes in Toxoplasma gondii We found that microneme secretion was triggered in vitro by exposure to a single host protein, serum albumin. The natural agonist serum albumin induced microneme secretion in a protein kinase G-dependent manner that correlated with increased cGMP levels. Surprisingly, serum albumin acted independently of elevated Ca(2+) and yet it was augmented by artificial agonists that raise Ca(2+), such as ethanol. Furthermore, although ethanol elevated intracellular Ca(2+), it alone was unable to trigger secretion without the presence of serum or serum albumin. This dichotomy was recapitulated by zaprinast, a phosphodiesterase inhibitor that elevated cGMP and separately increased Ca(2+) in a protein kinase G-independent manner leading to microneme secretion. Taken together, these findings reveal that microneme secretion is centrally controlled by protein kinase G and that this pathway is further augmented by elevation of intracellular Ca(2.)

Topics: Calcium Signaling; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Humans; Protozoan Proteins; Purinones; Serum Albumin; Toxoplasma

In this work, we report the effect of post-training intraperitoneal administration of zaprinast on rat memory retention in the Morris water maze task that revealed a significant memory impairment at the intermediate dose of 10mg/kg. Zaprinast is capable of inhibiting both striatal and hippocampal PDE activity but to a different extent which is probably due to the different PDE isoforms expressed in these areas. To assess the possible involvement of cyclic nucleotides in rat memory impairment, we compared the effects obtained 30 min after the zaprinast injection with respect to 24h after injection by measuring both cyclic nucleotide levels and PDE activity. As expected, 30 min after the zaprinast administration, we observed an increase of cyclic nucleotides, which returned to a basal level within 24h, with the exception of the hippocampal cGMP which was significantly decreased at the dose of 10mg/kg of zaprinast. This increase in the hippocampal region is the result of a cGMP-specific PDE5 induction, confirmed by sildenafil inhibition, in agreement with literature data that demonstrate transcriptional regulation of PDE5 by cAMP/cGMP intracellular levels. Our results highlight the possible rebound effect of PDE inhibitors.

Topics: Analysis of Variance; Animals; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Dose-Response Relationship, Drug; Escape Reaction; Hippocampus; In Vitro Techniques; Male; Maze Learning; Memory Disorders; Motor Activity; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Wistar; Reaction Time; Time Factors

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, the causative agents of severe malaria and toxoplasmosis, respectively, undergo several critical developmental transitions during their lifecycle. Most important for human pathogenesis is the asexual cycle, in which parasites undergo rounds of host cell invasion, replication, and egress (exit), destroying host cell tissue in the process. Previous work has identified important roles for Protein Kinase G (PKG) and Protein Kinase A (PKA) in parasite egress and invasion, yet little is understood about the regulation of cyclic nucleotides, cGMP and cAMP, that activate these enzymes. To address this, we have focused upon the development of inhibitors of 3',5'-cyclic nucleotide phosphodiesterases (PDEs) to block the breakdown of cyclic nucleotides. This was done by repurposing human PDE inhibitors noting various similarities of the human and apicomplexan PDE binding sites. The most potent inhibitors blocked the in vitro proliferation of P. falciparum and T. gondii more potently than the benchmark compound zaprinast. 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO) was found to be a potent inhibitor of recombinant P. falciparum PfPDEα and activated PKG-dependent egress of T. gondii and P. falciparum, likely by promoting the exocytosis of micronemes, an activity that was reversed by a specific Protein Kinase G inhibitor. BIPPO also promotes cAMP-dependent phosphorylation of a P. falciparum ligand critical for host cell invasion, suggesting that the compound inhibits single or multiple PDE isoforms that regulate both cGMP and cAMP levels. BIPPO is therefore a useful tool for the dissection of signal transduction pathways in apicomplexan parasites.

Topics: Antiprotozoal Agents; Chemistry Techniques, Synthetic; Cyclic AMP; Cyclic GMP; Drug Evaluation, Preclinical; Female; Humans; Phosphodiesterase Inhibitors; Phosphorylation; Plasmodium falciparum; Purinones; Pyrazoles; Pyrimidinones; Sequence Alignment; Sequence Homology, Amino Acid; Structural Homology, Protein; Toxoplasma

Nitric oxide (NO) modulates the dopamine uptake and release processes and appears to be implicated in dopamine-related pathologies, such as schizophrenia. However, it is unclear whether there is excess or deficient NO synthesis in schizophrenia pathophysiology. Analyses of the intracellular pathways downstream of NO system activation have identified the cyclic nucleotide cyclic guanosine monophosphate (cGMP) as a possible target for drug development. Defects in the sensorimotor gating of the neural mechanism underlying the integration and processing of sensory information have been detected across species through prepulse inhibition (PPI).. The aim of this study was to investigate the effects of NO/cGMP increase on sensorimotor gating modulation during dopamine hyperfunction.. Mice were treated with NO donors and subjected to the PPI test. Treatment with the NO donor sodium nitroprusside was preceded by pretreatment with a soluble guanylate cyclase (sGC) inhibitor. Additionally, the mice were treated with NO donors and phosphodiesterases inhibitors prior to amphetamine treatment.. Pretreatment with the NO donors enhanced the PPI response and attenuated the amphetamine-disruptive effects on the PPI. The sGC inhibitor did not modify the sodium nitroprusside effects. Additionally, the cGMP increase induced by a specific phosphodiesterase inhibitor did not modify the amphetamine-disruptive effect.. This study provides the first demonstration that an increase in NO can improve the PPI response and block the amphetamine-disruptive effects on the PPI response. Our data are consistent with recent clinical results. However, these effects do not appear to be related to an increase in cGMP levels, and further investigation is thus required.

Topics: Acoustic Stimulation; Amphetamine; Animals; Cyclic AMP; Cyclic GMP; Dopamine Agents; Dose-Response Relationship, Drug; Guanylate Cyclase; Inhibition, Psychological; Male; Mice; Neuropsychological Tests; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Reflex, Startle; Sensory Gating

Intracellular cyclic nucleotides are eliminated by phosphodiesterases (PDEs) and by ATP Binding cassette transporters such as ABCC4 and ABCC5. PDE5 and ABCC5 have similar affinity for cGMP whereas ABCC5 has much higher affinity for cGMP compared with cAMP. Since the substrate (cGMP) is identical for these two eliminatory processes it is conceivable that various PDE inhibitors also modulate ABCC5-transport. Cyclic GMP is also transported by ABBC4 but the affinity is much lower with a Km 50-100 times higher than for that of ABBCC5. The present study aimed to determine Ki-values for specific or relative specific PDE5 inhibitors (vardenafil, tadalafil, zaprinast and dipyridamole) and the non-specific PDE inhibitors (IBMX, caffeine and theophylline) for ABCC5 and ABCC4 transport. The transport of [(3)H]-cGMP (2 µM) was concentration-dependently inhibited with the following Ki-values: vardenafil (0.62 µM), tadalafil (14.1 µM), zaprinast (0.68 µM) and dipyridamole (1.2 µM), IBMX (10 µM), caffeine (48 µM) and theophylline (69 µM). The Ki-values for the inhibition of the [(3)H]-cAMP (2 µM) transport were: vardenafil (3.4 µM), tadalafil (194 µM), zaprinast (2.8 µM), dipyridamole (5.5 µM), IBMX (16 µM), caffeine (41 µM) and theophylline (85 µM). The specificity for ABCC5 we defined as ratio between Ki-values for inhibition of [(3)H]-cGMP and [(3)H]-cAMP transport. Tadalafil showed the highest specificity (Ki-ratio: 0.073) and caffeine the lowest (Ki-ratio: 1.2).

Topics: ATP-Binding Cassette Transporters; Caffeine; Carbolines; Cyclic AMP; Cyclic GMP; Dipyridamole; Humans; Imidazoles; Kinetics; Multidrug Resistance-Associated Proteins; Phosphodiesterase 5 Inhibitors; Phosphodiesterase Inhibitors; Piperazines; Purinones; Sulfones; Tadalafil; Theophylline; Triazines; Vardenafil Dihydrochloride

Inherited retinal degenerations affecting both rod and cone photoreceptors constitute one of the causes of incurable blindness in the developed world. Cyclic guanosine monophosphate (cGMP) is crucial in the phototransduction and, mutations in genes related to its metabolism are responsible for different retinal dystrophies. cGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4-5% of the retinitis pigmentosa, a rare form of retinal degeneration. The aim of this study was to evaluate whether pharmacological PDE6 inhibition induced retinal degeneration in cone-enriched cultures of porcine retina similar to that found in murine models. PDE6 inhibition was induced in cone-enriched retinal explants from pigs by Zaprinast. PDE6 inhibition induced cGMP accumulation and triggered retinal degeneration, as determined by TUNEL assay. Western blot analysis and immunostaining indicated that degeneration was accompanied by caspase-3, calpain-2 activation and poly (ADP-ribose) accumulation. Oxidative stress markers, total antioxidant capacity, thiobarbituric acid reactive substances (TBARS) and nitric oxide measurements revealed the presence of oxidative damage. Elevated TNF-alpha and IL-6, as determined by enzyme immunoassay, were also found in cone-enriched retinal explants treated with Zaprinast. Our study suggests that this ex vivo model of retinal degeneration in porcine retina could be an alternative model for therapeutic research into the mechanisms of photoreceptor death in cone-related diseases, thus replacing or reducing animal experiments.

Topics: Animals; Apoptosis; Calpain; Caspase 3; Cyclic GMP; In Situ Nick-End Labeling; Organ Culture Techniques; Oxidative Stress; Phosphodiesterase Inhibitors; Purinones; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Swine; Swine, Miniature

The edema formation in nephrotic syndrome (NS) is associated with a blunted response to atrial natriuretic peptide (ANP). The natriuretic effects of ANP have been related to renal dopamine D1-receptors (D1R). We examined the interaction between ANP and renal D1R in rats with puromycin aminonucleoside-induced NS (PAN-NS). Urinary sodium, cyclic guanosine monophosphate (cGMP) excretion, and D1R protein expression and localization in renal tubules were evaluated in PAN-NS and control rats before and during volume expansion (VE). The effects of zaprinast (phosphodiesterase type 5 inhibitor), alone or in combination with Sch-23390 (D1R antagonist), were examined in both groups. The increased natriuresis and urinary cGMP excretion evoked by acute VE were blunted in PAN-NS despite increased levels of circulating ANP. This was accompanied in PAN-NS by a marked decrease of D1R expression in the renal tubules. Infusion of zaprinast in PAN-NS resulted in increased urinary excretion of cGMP and sodium to similar levels of control rats and increased expression of D1R in the plasma membrane of renal tubular cells. Combined administration of Sch-23390 and zaprinast prevented natriuresis and increased cGMP excretion induced by zaprinast alone. We conclude that D1R may play a major role in the ANP resistance observed in PAN-NS.

Topics: Animals; Atrial Natriuretic Factor; Benzazepines; Cyclic GMP; Glomerular Filtration Rate; Homeostasis; Kidney; Male; Natriuresis; Nephrotic Syndrome; Purinones; Puromycin Aminonucleoside; Rats; Receptors, Dopamine D1; Sodium

The C-type natriuretic peptide (CNP) exerts its action via stimulation of the cyclic GMP (cGMP) signalling pathway, which includes the activation of cGMP-dependent protein kinases. The pathway can also be activated by inhibitors of phosphodiesterases (PDE) that hydrolyse cGMP. The present report shows that activation of the cGMP pathway by CNP, by bromo-cGMP, a cell-permeant cGMP analogue, or by the PDE inhibitor zaprinast dose dependently reduces intravenous cocaine self-administration by rats. The effect was found when the compounds were injected in situ into the prefrontal cortex, but not when they were injected into the nucleus accumbens. A decrease in the number of cocaine infusions performed by rats was obtained under the fixed ratio-1 schedule of reinforcement as well as under a progressive ratio schedule, which evaluates the motivation of the animals for the drug. Decrease in cocaine self-administration was accompanied with reduced expression of the epigenetic markers methyl-CpG-binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2) in dopaminergic projection areas. An increase in the acetylation level of histone H3, but not of histone H4, was also noticed. Since MeCP2 and HDAC2 are known to modulate dynamic functions in the adult brain, such as synaptic plasticity, our results showing that activation of the cGMP signal transduction pathway decreased both cocaine intake and expression of the epigenetic markers strongly suggest that the MeCP2/HDAC2 complex is involved in the analysis of the reinforcing properties of cocaine in the prefrontal cortex.

Topics: Analysis of Variance; Animals; Brain; Cocaine; Conditioning, Operant; Cyclic GMP; Dopamine Uptake Inhibitors; Gene Expression Regulation; Histone Deacetylase 2; Male; Methyl-CpG-Binding Protein 2; Natriuretic Peptide, C-Type; Phosphodiesterase Inhibitors; Protein Serine-Threonine Kinases; Purinones; Rats; Rats, Wistar; Reinforcement Schedule; Self Administration

For most neurodegenerative diseases the precise duration of an individual cell's death is unknown, which is an obstacle when counteractive measures are being considered. To address this, we used the rd1 mouse model for retinal neurodegeneration, characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cyclic guanosine-mono-phosphate (cGMP) levels. Using cellular data on cGMP accumulation, cell death, and survival, we created mathematical models to simulate the temporal development of the degeneration. We validated model predictions using organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast. Together, photoreceptor data and modeling for the first time delineated three major cell death phases in a complex neuronal tissue: (1) initiation, taking up to 36 h, (2) execution, lasting another 40 h, and finally (3) clearance, lasting about 7 h. Surprisingly, photoreceptor neurodegeneration was noticeably slower than necrosis or apoptosis, suggesting a different mechanism of death for these neurons.

Topics: Animals; Apoptosis; Cells, Cultured; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Mice; Models, Biological; Mutation; Neurons; Phosphodiesterase Inhibitors; Photoreceptor Cells, Vertebrate; Purinones; Retina; Retinitis Pigmentosa

Vessel tension can be modulated by phosphoinositide 3-kinase (PI3K) acting on l-type calcium channel, rho kinase and phosphodiesterase (PDE) type 3 in smooth muscle cells. Inhibition of PI3K could increase the relaxation of porcine coronary arteries to nitroglycerin independent of this pathway, and the aim of the present study was therefore to determine the underlying mechanisms.. Isolated porcine coronary arteries were dissected from the heart and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The response of these vessels was studied by using the organ chamber technique; the content of cyclic guanosine monophosphate (cGMP) was determined by using enzyme-linked immunosorbent assay kit; and PI3K and Akt activity were determined by measuring the phosphorylation level of their downstream signaling molecule on Western blot. Inhibition of PI3K with 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) potentiated the relaxation of porcine coronary arteries to nitroglycerin and nitric oxide (NO), but not to 8-bromo-guanosine 3'5'-cyclic monophosphate, isoproterenol or (R)-(+)-trans-4-(1-Aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y27632). Increased relaxation induced by LY294002 was eliminated by Akt1/2 kinase inhibitor (Akt-I: 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo(4,5-g)quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one trifluoroacetate salt hydrate) or zaprinast, but was not affected by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, nifedipine or milrinone. Inhibition of Akt caused similar effects as LY294002. Incubation with LY294002 or Akt-I decreased the activity of PI3K and Akt but augmented the elevation of cGMP caused by NO. Enhanced cGMP elevation induced by LY294002 or Akt-I was also eliminated by zaprinast.. PI3K-Akt signaling may affect vascular tone through a stimulatory effect on PDE type 5.

Topics: Animals; Chromones; Coronary Vessels; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Inhibitors; Milrinone; Models, Animal; Morpholines; Nifedipine; Nitroglycerin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Purinones; Signal Transduction; Swine; Vasodilation; Vasodilator Agents

The airways of patients with cystic fibrosis (CF) exhibit decreased nitric oxide (NO) concentrations, which might affect airway function. The aim of this study was to determine the effects of NO on ion transport in human airway epithelia. Primary cultures of non-CF and CF bronchial and bronchiolar epithelial cells were exposed to the NO donor sodium nitroprusside (SNP), and bioelectric variables were measured in Ussing chambers. Amiloride was added to inhibit the Na(+) channel ENaC, and forskolin and ATP were added successively to stimulate cAMP- and Ca(2+)-dependent Cl(-) secretions, respectively. The involvement of cGMP was assessed by measuring the intracellular cGMP concentration in bronchial cells exposed to SNP and the ion transports in cultures exposed to 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (ODQ), or to 8Z, a cocktail of 8-bromo-cGMP and zaprinast (phosphodiesterase 5 inhibitor). SNP decreased the baseline short-circuit current (I(sc)) and the changes in I(sc) induced by amiloride, forskolin, and ATP in non-CF bronchial and bronchiolar cultures. The mechanism of this inhibition was studied in bronchial cells. SNP increased the intracellular cGMP concentration ([cGMP](i)). The inhibitory effect of SNP was abolished by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO scavenger (PTIO) and ODQ and was partly mimicked by increasing [cGMP](i). In CF cultures, SNP did not significantly modify ion transport; in CF bronchial cells, 8Z had no effect; however, SNP increased the [cGMP](i). In conclusion, exogenous NO may reduce transepithelial Na(+) absorption and Cl(-) secretion in human non-CF airway epithelia through a cGMP-dependent pathway. In CF airways, the NO/cGMP pathway appears to exert no effect on transepithelial ion transport.

Topics: Adenosine Triphosphate; Adult; Aged; Amiloride; Bronchi; Chloride Channels; Colforsin; Cyclic GMP; Cyclic N-Oxides; Cystic Fibrosis; Epithelial Sodium Channel Blockers; Epithelial Sodium Channels; Free Radical Scavengers; Guanylate Cyclase; Humans; Imidazoles; Middle Aged; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxadiazoles; Purinones; Quinoxalines; Young Adult

The aim of the present study was to evaluate the role of the nitric oxide/cyclic guanosine monophosphate pathway in corticostriatal long-term depression induction in a model of levodopa-induced dyskinesia in experimental parkinsonism. Moreover, we have also analysed the possibility of targeting striatal phosphodiesterases to reduce levodopa-induced dyskinesia. To study synaptic plasticity in sham-operated rats and in 6-hydroxydopamine lesioned animals chronically treated with therapeutic doses of levodopa, recordings from striatal spiny neurons were taken using either intracellular recordings with sharp electrodes or whole-cell patch clamp techniques. Behavioural analysis of levodopa-induced abnormal involuntary movements was performed before and after the treatment with two different inhibitors of phosphodiesterases, zaprinast and UK-343664. Levodopa-induced dyskinesia was associated with the loss of long-term depression expression at glutamatergic striatal synapses onto spiny neurons. Both zaprinast and UK-343664 were able to rescue the induction of this form of synaptic plasticity via a mechanism requiring the modulation of intracellular cyclic guanosine monophosphate levels. This effect on synaptic plasticity was paralleled by a significant reduction of abnormal movements following intrastriatal injection of phosphodiesterase inhibitors. Our findings suggest that drugs selectively targeting phosphodiesterases can ameliorate levodopa-induced dyskinesia, possibly by restoring physiological synaptic plasticity in the striatum. Future studies exploring the possible therapeutic effects of phosphodiesterase inhibitors in non-human primate models of Parkinson's disease and the involvement of striatal synaptic plasticity in these effects remain necessary to validate this hypothesis.

Topics: Animals; Corpus Striatum; Cyclic GMP; Dyskinesia, Drug-Induced; Levodopa; Long-Term Synaptic Depression; Male; Microinjections; Neurons; Oxidopamine; Parkinsonian Disorders; Phosphodiesterase Inhibitors; Piperazines; Purinones; Pyrimidinones; Rats; Rats, Wistar

It has recently been shown that systemic endothelial dysfunction is associated with airflow limitation in COPD. We conducted this ex vivo study to assess whether endothelial dysfunction of pulmonary arteries of former smokers was associated with modifications of airway functions.. Pharmacological experiments were conducted on arterial and bronchial rings obtained from lung specimen of 20 patients: 13 smokers without COPD and 7 smokers with mild to moderate COPD (GOLD class I or II). The impairment of acetylcholine-mediated vasodilation (constriction) of preconstricted arterial rings defined endothelial dysfunction. Resting tone (initial and after a contraction test) and cGMP-mediated dilation of bronchial rings in response to zaprinast were evaluated.. Initial airway resting tone was correlated with airflow limitation (FEV(1) % predicted: Rho = -0.49; p = 0.032). The acetylcholine response of arterial rings was correlated with zaprinast-induced bronchodilation (Rho = 0.54, p = 0.019). Patients with endothelial dysfunction (n = 5), as compared with those displaying no dysfunction (n = 15), were characterized by an increased resting tone (after contraction test), an impaired response to zaprinast but a similar degree of airflow limitation (FEV(1)).. Endothelial dysfunction of pulmonary arteries is associated with increased resting tone and impaired cGMP-mediated dilation of airways in former smokers, suggesting common underlying mechanisms of pulmonary arterial and bronchial dysfunctions.

Topics: Acetylcholine; Aged; Bronchi; Bronchodilator Agents; Cyclic GMP; Endothelium, Vascular; Female; Forced Expiratory Volume; Humans; In Vitro Techniques; Male; Middle Aged; Pulmonary Artery; Pulmonary Disease, Chronic Obstructive; Purinones; Smoking; Vasodilation

Within erythrocytes (RBCs), cAMP levels are regulated by phosphodiesterases (PDEs). Increases in cAMP and ATP release associated with activation of β-adrenergic receptors (βARs) and prostacyclin receptors (IPRs) are regulated by PDEs 2, 4 and PDE 3, respectively. Here we establish the presence of cytosolic PDEs in RBCs and determine a role for PDE5 in regulating levels of cGMP.. Purified cytosolic proteins were obtained from isolated human RBCs and western analysis was performed using antibodies against PDEs 3A, 4 and 5. Rabbit RBCs were incubated with dbcGMP, a cGMP analog, to determine the effect of cGMP on cAMP levels. To determine if cGMP affects receptor-mediated increases in cAMP, rabbit RBCs were incubated with dbcGMP prior to addition of isoproterenol (ISO), a βAR receptor agonist. To demonstrate that endogenous cGMP produces the same effect, rabbit and human RBCs were incubated with SpNONOate (SpNO), a nitric oxide donor, and YC1, a direct activator of soluble guanylyl cyclase (sGC), in the absence and presence of a selective PDE5 inhibitor, zaprinast (ZAP).. Western analysis identified PDEs 3A, 4D and 5A. dbcGMP produced a concentration dependent increase in cAMP and ISO-induced increases in cAMP were potentiated by dbcGMP. In addition, incubation with YC1 and SpNO in the presence of ZAP potentiated βAR-induced increases in cAMP.. PDEs 2, 3A and 5 are present in the cytosol of human RBCs. PDE5 activity in RBCs regulates cGMP levels. Increases in intracellular cGMP augment cAMP levels. These studies suggest a novel role for PDE5 in erythrocytes.

Topics: Animals; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 4; Cyclic Nucleotide Phosphodiesterases, Type 5; Cytosol; Erythrocytes; Humans; Isoenzymes; Isoproterenol; Male; Phosphodiesterase Inhibitors; Purinones; Rabbits; Spermine; Vinca Alkaloids

The similarity between Plasmodium falciparum phosphodiesterase enzymes (PfPDEs) and their human counterparts have been examined and human PDE9A was found to be a suitable template for the construction of homology models for each of the four PfPDE isoforms. In contrast, the architecture of the active sites of each model was most similar to human PDE1. Molecular docking was able to model cyclic guanosine monophosphate (cGMP) substrate binding in each case but a docking mode supporting cyclic adenosine monophosphate (cAMP) binding could not be found. Anticipating the potential of PfPDE inhibitors as anti-malarial drugs, a range of reported PDE inhibitors including zaprinast and sildenafil were docked into the model of PfPDEα. The results were consistent with their reported biological activities, and the potential of PDE1/9 inhibitor analogues was also supported by docking.

Topics: Antimalarials; Catalytic Domain; Computer Simulation; Cyclic GMP; Drug Design; Humans; Models, Molecular; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Plasmodium falciparum; Purines; Purinones; Sequence Alignment; Sequence Homology, Amino Acid; Sildenafil Citrate; Sulfones

Dendroaspis natriuretic peptide (DNP), a newly-described natriuretic peptide, relaxes gastrointestinal smooth muscle. L-type calcium channel currents play an important role in regulating smooth muscle contraction. The effect of DNP on L-type calcium channel currents in gastrointestinal tract is still unclear. This study was designed to investigate the effect of DNP on barium current (I(Ba)) through the L-type calcium channel in gastric antral myocytes of guinea pigs and cGMP-pathway mechanism. The whole-cell patch-clamp technique was used to record L-type calcium channel currents. The content of cGMP in guinea pig gastric antral smooth muscle and perfusion solution was measured using radioimmunoassay. DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in perfusion medium. DNP concentration-dependently inhibited I(Ba) in freshly isolated guinea pig gastric antral circular smooth muscle cells (SMCs) of guinea pigs. DNP-induced inhibition of I(Ba) was partially blocked by LY83583, an inhibitor of guanylate cyclase. KT5823, a cGMP-dependent protein kinase (PKG) inhibitor, almost completely blocked DNP-induced inhibition of I(Ba). However, DNP-induced inhibition of I(Ba) was potentiated by zaprinast, an inhibitor of cGMP-sensitive phosphodiesterase. Taken together, DNP inhibits L-type calcium channel currents via pGC-cGMP-PKG-dependent signal pathway in gastric antral myocytes of guinea pigs.

Topics: Aminoquinolines; Animals; Calcium Channels, L-Type; Carbazoles; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Elapid Venoms; Electrophysiology; Enzyme Inhibitors; Guanylate Cyclase; Guinea Pigs; Intercellular Signaling Peptides and Proteins; Myocytes, Smooth Muscle; Peptides; Purinones; Pyloric Antrum; Radioimmunoassay

Mice with targeted deletion of neuronal nitric oxide (NO) synthase (nNOS⁻(/)⁻) display inability to increase plasma renin concentration (PRC) in response to sodium restriction. nNOS has a distinct expression at the macula densa (MD), and in the present study, it was tested whether nNOS supports renin release by cyclic guanosine monophosphate (cGMP)-mediated inhibition of cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase 3 (PDE3) in juxtaglomerular (JG) cells.. The experiments were performed in conscious nNOS⁻(/)⁻ and wild types after 10 days on a low-sodium diet by acute treatment with the PDE3-inhibitor milrinone, the PDE5 inhibitor zaprinast, or vehicle, using a crossover study protocol. PRC was measured with the antibody-trapping technique and blood pressure with telemetry. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were estimated by measurements of inulin- and para-amino hippuric acid (PAH) clearances, respectively.. The basal PRC was reduced in nNOS⁻(/)⁻ compared to the wild types. Administration of milrinone caused a more pronounced PRC increase in nNOS⁻(/)⁻, resulting in normalized renin levels, whereas PDE5 inhibition did not affect PRC in any genotype. The blood pressure was similar in both genotypes, and milrinone did not affect blood pressure compared to vehicle. GFR and RPF were similar at baseline and were reduced by milrinone.. The present study provides in vivo evidence supporting the view that NO, selectively derived from nNOS, mediates renin release during sodium restriction by inhibiting PDE3, which would increase renin release by elevating cAMP levels in the JG cells.

Topics: Animals; Blood Pressure; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 5; Female; Juxtaglomerular Apparatus; Kidney Cortex; Male; Mice; Mice, Inbred Strains; Mice, Knockout; Milrinone; Nitric Oxide Synthase Type I; Phosphodiesterase 3 Inhibitors; Phosphodiesterase 5 Inhibitors; Purinones; Renal Circulation; Renin; Sodium Chloride, Dietary

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness in humans. Previously, excessive activation of enzymes belonging to the poly-ADP-ribose polymerase (PARP) group was shown to be involved in photoreceptor degeneration in the human homologous rd1 mouse model for RP. Since there are at least 16 different PARP isoforms, we investigated the exact relevance of the predominant isoform - PARP1 - for photoreceptor cell death using PARP1 knock-out (KO) mice. In vivo and ex vivo morphological analysis using optic coherence tomography (OCT) and conventional histology revealed no major alterations of retinal phenotype when compared to wild-type (wt). Likewise, retinal function as assessed by electroretinography (ERG) was normal in PARP1 KO animals. We then used retinal explant cultures derived from wt, rd1, and PARP1 KO animals to test their susceptibility to chemically induced photoreceptor degeneration. Since photoreceptor degeneration in the rd1 retina is triggered by a loss-of-function in phosphodiesterase-6 (PDE6), we used selective PDE6 inhibition to emulate the rd1 situation on non-rd1 genotypes. While wt retina subjected to PDE6 inhibition showed massive photoreceptor degeneration comparable to rd1 retina, in the PARP1 KO situation, cell death was robustly reduced. Together, these findings demonstrate that PARP1 activity is in principle dispensable for normal retinal function, but is of major importance for photoreceptor degeneration under pathological conditions. Moreover, our results suggest that PARP dependent cell death or PARthanatos may play a major role in retinal degeneration and highlight the possibility to use specific PARP inhibitors for the treatment of RP.

Topics: Animals; Apoptosis; Blotting, Western; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Electroretinography; Female; Humans; In Situ Nick-End Labeling; Male; Mice; Mice, 129 Strain; Mice, Inbred C3H; Mice, Knockout; Phosphodiesterase Inhibitors; Photoreceptor Cells, Vertebrate; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Purinones; Retina; Retinal Degeneration; Retinitis Pigmentosa; Tomography, Optical Coherence

Nitric oxide (NO) is an inhibitory signalling molecule in the gastrointestinal (GI) tract that is released from neurons and from leucocytes during inflammation. NO stimulates soluble guanylate cyclase (sGC), elevates cyclic guanosine 3',5'-monophospate (cGMP), and subsequently activates cGMP-dependent protein kinase (PKG). Targets for NO in the guinea pig caecum were investigated by characterizing the cellular distribution of sGC, cGMP and PKG. Immunoreactivity for both isoforms of sGC, sGCalpha1 and sGCbeta1, was observed in the interstitial cells of Cajal (ICC) and enteric neurons in the tunica muscularis. Double labelling with anti-Kit and anti-sGC antibodies showed sGCalpha1 and sGCbeta1-like immunoreactivity (LI) in almost all intramuscular (IM) and myenteric ICC. Neuronal processes with neuronal NO synthase were closely apposed to ICC expressing sGC-LI. Cells with sGC-LI possessed ultrastructural features of ICC-IM: caveolae, close association with nerve bundles and contacts with smooth muscle cells (SMC). Sodium nitroprusside, added with the phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine and zaprinast), enhanced cGMP-LI in almost all ICC and in some enteric neurons. Nerve stimulation also increased cGMP-LI in ICC and enteric neurons. In contrast, no resolvable increase in cGMP-LI was observed in any cells when the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was present. ICC and SMC also expressed PKG type I-LI. These data show that ICC express the downstream signalling molecules necessary to transduce nitrergic signals and activate inhibitory pathways and thus are primary targets for NO released from neurons and other cells in the GI tract.

Topics: Animals; Cecum; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Activation; Female; Guanylate Cyclase; Guinea Pigs; Humans; Isoenzymes; Mice; Mice, Inbred BALB C; Nitric Oxide; Nitric Oxide Synthase Type I; Nitroprusside; Phosphodiesterase Inhibitors; Purinones; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Stem Cell Factor

Guanosine-specific cyclic nucleotide signaling is suggested to serve protective actions in the vasculature; however, the influence of selective pharmacologic modulation of cyclic guanosine monophosphate- synthesizing soluble guanylate cyclase or cyclic guanosine monophosphate-degrading phosphodiesterase on vessel remodeling has not been thoroughly examined. In this study, rat carotid artery balloon injury was performed and the growth-modulating effects of the soluble guanylate cyclase stimulator YC-1 or the cyclic guanosine monophosphate-dependent phosphodiesterase-V inhibitor zaprinast were examined. YC-1 or zaprinast elevated vessel cyclic guanosine monophosphate content, reduced medial wall and neointimal cell proliferation, stimulated medial and neointimal cellular apoptosis, and markedly attenuated neointimal remodeling in comparable fashion. Interestingly, soluble guanylate cyclase inhibition by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one failed to noticeably alter neointimal growth, and concomitant zaprinast with YC-1 did not modify any parameter compared to individual treatments. These results provide novel in vivo evidence that YC-1 and zaprinast inhibit injury-induced vascular remodeling through antimitogenic and proapoptotic actions and may offer promising therapeutic approaches against vasoproliferative disorders.

Topics: Animals; Apoptosis; Carotid Artery Injuries; Cell Proliferation; Cyclic GMP; Disease Models, Animal; Enzyme Activators; Guanylate Cyclase; Indazoles; Male; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Sprague-Dawley

Endothelial nitric oxide synthase (NOS3) regulates the functional response to beta-adrenergic (beta-AR) stimulation via modulation of the L-type Ca(2+) current (I(Ca)). However, the NOS3 signaling pathway modulating I(Ca) is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of I(Ca). Myocytes were isolated from NOS3 knockout (NOS3(-/-)) and wildtype (WT) mice. We measured I(Ca) (whole-cell voltage-clamp), and simultaneously measured Ca(2+) transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased beta-AR stimulated (isoproterenol, ISO)-I(Ca), and Ca(2+) transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I(Ca), but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3(-/-) myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated I(Ca), or Ca(2+) transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I(Ca), and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I(Ca); YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca(2+) transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I(Ca) and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating beta-AR stimulated I(Ca), while limiting effects on cardiac contraction.

Topics: Animals; Calcium Channels, L-Type; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Activation; Guanylate Cyclase; Indazoles; Ion Channel Gating; Isoproterenol; Mice; Models, Biological; Myocardial Contraction; Myocytes, Cardiac; Nitric Oxide Synthase Type III; Phosphodiesterase 5 Inhibitors; Purinones; Receptors, Adrenergic, beta; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase

Relaxing action of sodium nitroprusside (SNP) was significantly reduced in the stomach fundus of mice lacking the kinin B(1) receptor (B(1)(-/-)). Increased basal cGMP accumulation was correlated with attenuated SNP induced dose-dependent relaxation in B(1)(-/-) when compared with wild type (WT) control mice. These responses to SNP were completely blocked by the guanylate cyclase inhibitor ODQ (10 microM). It was also found that Ca(2+)-dependent, constitutive nitric oxide synthase (cNOS) activity was unchanged but the Ca(2+)-independent inducible NOS (iNOS) activity was greater in B(1)(-/-) mice than in WT animals. Zaprinast (100 microM), a specific phosphodiesterase inhibitor, increased the nitrergic relaxations and the accumulation of the basal as well as the SNP-stimulated cGMP in WT but not in B(1)(-/-) stomach fundus. From these findings it is concluded that the inhibited phosphodiesterase activity and high level of cGMP reduced the resting muscle tone, impairing the relaxant responses of the stomach in B(1)(-/-) mice. In addition, it can be suggested that functional B(2) receptor might be involved in the NO compensatory mechanism associated with the deficiency of kinin B(1) receptor in the gastric tissue of the transgenic mice.

Topics: Animals; Cyclic GMP; Gastric Mucosa; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Smooth; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Phosphodiesterase Inhibitors; Purinones; Receptor, Bradykinin B1; Stomach

Nitric oxide (NO), a neurotransmitter in the lower urinary tract, stimulates soluble guanylyl cyclase (sGC) and in turn cGMP-dependent protein kinase G (PKG) to modulate a number of downstream targets. NO donors reduce bladder hyperactivity in some pathological models but do not affect normal bladder activity in the adult rat. In this study, the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP; 100 microM) decreased the amplitude and frequency of spontaneous and carbachol-enhanced contractions in neonatal rat bladder strips, which are intrinsically hyperactive. This effect was blocked by inhibition of sGC and mimicked by application of a membrane-permeable cGMP analog (8-bromo-cGMP, 100 microM). Inhibition of PKG prevented or reversed the inhibitory effects of 8-bromo-cGMP. A portion of the SNAP-mediated inhibition was also dependent upon PKG; however, a short-lasting, sGC-dependent inhibitory effect of SNAP was still present after PKG inhibition. Inhibition of NO synthase with L-NAME (100 microM) did not change the amplitude or frequency of contractions. However, inhibition of endogenous phosphodiesterase (PDE)-5 with zaprinast (25 microM) reduced the amplitude and frequency of phasic contractions and increased the magnitude of inhibition produced by maximal concentrations of SNAP, suggesting that endogenous PDEs are constitutively active and regulate cGMP production. These results suggest that the NO-cGMP-PKG pathway may be involved in inhibitory control of the neonatal rat bladder.

Topics: Animals; Animals, Newborn; Carbachol; Cholinergic Agonists; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Dose-Response Relationship, Drug; Female; Guanylate Cyclase; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Neural Inhibition; NG-Nitroarginine Methyl Ester; Nitrergic Neurons; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Phosphodiesterase Inhibitors; Protein Kinase Inhibitors; Purinones; Rats; Rats, Sprague-Dawley; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Time Factors; Urinary Bladder

A role for guanosine 3',5'-cyclic monophosphate (cGMP) and the protein kinase G (PKG) pathway in synaptic long-term depression (LTD) in the hippocampal CA1 region has been proposed, based on observations in vitro, where, for example, increases of [cGMP] result in short-term depression (STD) coupled with a reduction in presynaptic glutamate release. To date, no evidence exists to support that LTD in the intact, freely behaving animal involves these mechanisms. We examined the effect of increases of [cGMP] on basal transmission and electrically-induced STD at hippocampal CA1 synapses in vivo. We found that elevating [cGMP] dose-dependently caused a chemically-induced STD which occluded electrically-induced STD. Repeated administration of Zaprinast, an inhibitor of cGMP-degrading phosphodiesterase, resulted in persistent LTD (>24 h). Paired-pulse analysis supported a presynaptic mechanism of action. Application of an inhibitor of soluble guanylate cyclase prevented LTD induced by low-frequency stimulation (LFS), and impaired LFS-STD elicited in the presence of Zaprinast. These data suggest the involvement of cGMP in LTD in the CA1 region of freely behaving adult rats.

Topics: Animals; Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Hippocampus; Long-Term Synaptic Depression; Male; Movement; Phosphoric Diester Hydrolases; Presynaptic Terminals; Purinones; Rats; Rats, Wistar; Synapses; Synaptic Transmission; Up-Regulation

3',5'-Cyclic guanosine monophosphate (cGMP), a well-known intracellular second messenger, is released to the intestinal lumen by the tapeworm, Hymenolepis diminuta. Enzyme-linked immunosorbent assay analysis of tapeworm conditioned media shows that cGMP is released at a constant rate. Multidrug resistant (MDR) proteins are efflux transporters for cyclic nucleotides. Two MDR inhibitors, niflumic acid and zaprinast, inhibit cGMP secretion by tapeworms and change the cGMP localization within the tapeworm tegument, as assessed by immunochemistry. cGMP, normally present throughout the tapeworm tegumental cytoplasm, is absent from the outer cytoplasmic band upon treatment with inhibitors. Inhibition of cGMP secretion by colchicine indicates that cGMP secretion is cytoskeleton dependent. Binding studies of [3H]cGMP to ileal segments of intestine demonstrate 2 saturable, reversible, and high-affinity binding sites. These studies demonstrate that cGMP is secreted from the cestode via a cytoskeleton-dependent mechanism and MDR efflux transporters. In addition, cGMP reaching the intestinal lumen can bind to the mucosa via receptors for cGMP. These data, combined with earlier observations of cGMP altering intestinal motility and slowing lumenal transit, indicate that tapeworms alter the physiology of the host digestive process via the secretion and binding of extracellular cGMP to lumenal receptors in the host intestine.

Topics: Animals; Colchicine; Cyclic GMP; Cyclooxygenase Inhibitors; Hymenolepis diminuta; Ileum; Immunohistochemistry; Male; Niflumic Acid; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Sprague-Dawley; Tubulin Modulators

The 3'5'-cyclic GMP (cGMP) pathway is known to influence ovarian functions, including steroidogenesis, ovulation, and granulosa cell proliferation. We show here that cGMP-phosphodiesterase (PDE) activity increased in a gonadotropin-dependent manner more than 3-fold in the cumulus-oocyte complex (COC) after 24 h in vitro maturation (IVM) and up to 5-fold after 48 h. Further characterization of this increase demonstrated that the activity was located primarily in cumulus cells, and was sensitive to sildenafil and zaprinast, two inhibitors specific to both type 5 and 6 PDEs. RT-PCR experiments showed that the mRNAs for cGMP-degrading PDEs 5A and 6C are present in the COC before and after 30 h IVM. Western blotting confirmed the presence of PDE 5A in the COC. Western blotting of PDE 6C revealed a significant up-regulation in the COC during IVM. Isolation and analysis of detergent-resistant membranes suggested that PDE 6C protein, along with half of the total sildenafil-sensitive cGMP-degradation activity, is associated with detergent-resistant membrane in the COC after 30 h IVM. Treatment of porcine COC with sildenafil during IVM caused a significant decrease in gonadotropin-stimulated progesterone secretion. Together, these results constitute the first report exploring the contribution of cGMP-PDE activity in mammalian COC, supporting a functional clustering of the enzyme, and providing the first evidence of its role in steroidogenesis.

Topics: Animals; Cells, Cultured; Cumulus Cells; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 2; Female; Gene Expression Regulation, Enzymologic; Gonadal Steroid Hormones; Oocytes; Oogenesis; Phosphodiesterase Inhibitors; Piperazines; Progesterone; Purines; Purinones; Sildenafil Citrate; Sulfones; Swine; Up-Regulation

Nitric oxide (NO) is a biologically active molecule which has been reported to protect the heart against ischemia and reperfusion injury in different species. This study aimed to test the hypothesis that nitric oxide may induce the expression of heat shock protein 72 (HSP72) which may protect the heart against ischemia.. Rabbits were given intravenous saline or S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, or Zaprinast, an inhibitor of cyclic guanosine monophosphate (GMP)-phosphodiesterase, which may increase myocardial cyclic GMP content. Twenty-four hours later, the rabbits were either sampled to measure HSP72, or induced with a 30-minute coronary occlusion followed by a 120-minute reperfusion, and then the infarct size was measured. Meanwhile, chelerythrine (CHE, an inhibitor of protein kinase C) was given intravenously 5 minutes before SNAP injection and the effect on HSP72 expression and infarct size was determined.. Twenty-four hours after pretreatment, immunoblotting showed HSP72 expression increased in the SNAP group compared with control groups, and this was blocked by CHE. Myocardial infarct size in the SNAP group was smaller than that of the control group ((32.4 +/- 5.8)% vs (51.1 +/- 4.7)%, P < 0.05). Pretreated with CHE abolished the infarct size-limiting effect of SNAP ((46.0 +/- 5.1)%). Pretreatment with Zaprinast neither induced HSP72 expression nor reduced infarct size ((55.4 +/- 5.4)%).. NO induced HSP72 expression and a delayed protection to the heart via the activities of protein kinase C by a cyclic GMP-independent pathway.

Topics: Animals; Benzophenanthridines; Cyclic GMP; Hemodynamics; HSP72 Heat-Shock Proteins; Male; Myocardial Infarction; Myocardial Ischemia; Nitric Oxide; Nitric Oxide Donors; Phosphodiesterase Inhibitors; Protein Kinase C; Purinones; Rabbits; S-Nitroso-N-Acetylpenicillamine

Dysregulation of dopamine receptors is thought to underlie levodopa-induced dyskinesias in experimental models of Parkinson's disease. It is unknown whether an imbalance of the second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), is involved in the alterations of levodopa/dopamine signal transduction. We examined cAMP and cGMP signalling in the interconnected cortico-striatal-pallidal loop at the peak of levodopa-induced dyskinesias in rats with 6-hydroxydopamine lesions in the substantia nigra. In addition, we examined the role of phosphodiesterase (PDE) and the rate of cAMP and cGMP degradation on the severity of levodopa-induced dyskinesias in animals pretreated with PDE inhibitor, zaprinast. Unilateral lesion of substantia nigra led to an increase in cAMP but a decrease in cGMP levels in the ipsilateral basal ganglia. After chronic levodopa treatment, cAMP and cGMP were differentially regulated in eukinetic animals: the cAMP level increased in the cortex and striatum but decreased in the globus pallidus of both hemispheres, whereas the cGMP decreased below baseline levels in the contralateral cortico-striatal-pallidal regions. In dyskinetic animals chronic levodopa treatment led to an absolute decrease in cAMP and cGMP levels in cortico-striatal-pallidal regions of both hemispheres. Pretreatment with zaprinast reduced the severity of levodopa-induced dyskinesias, and partly prevented the decrease in cyclic nucleotides compared with pretreatment with saline-levodopa. In conclusion, using a rat model of hemiparkinsonism, we observed a significant reduction in the levels of cyclic nucleotides in both hemispheres at the peak of levodopa-induced dyskinesias. We propose that such a decrease in cyclic nucleotides may partly result from increased catabolism through PDE overactivity.

Topics: Animals; Animals, Newborn; Brain; Cerebral Cortex; Cyclic AMP; Cyclic GMP; Dopamine; Dopamine Agents; Down-Regulation; Dyskinesia, Drug-Induced; Globus Pallidus; Levodopa; Male; Neostriatum; Oxidopamine; Parkinsonian Disorders; Phosphodiesterase Inhibitors; Phosphorylation; Purinones; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Substantia Nigra; Sympatholytics; Synaptic Transmission

The role of cGMP in the myometrium of pregnant women is not completely known. We have previously shown in guinea pig, monkey and man that NO-induced relaxation of oxytocin-induced contractions is independent of cGMP accumulation. To approach an understanding of the role of cGMP in myometrium, we have developed smooth muscle cell cultures from pregnant women undergoing caesarian section at term. Cells, grown in standard media containing progesterone, express smooth muscle cell markers and are used within five doublings. Cells stimulated with NO donors increase their cGMP levels nearly 100 fold (basal = approximately 9 pmol/mg protein). In the presence of oxytocin (OT; 1 microM), cGMP accumulation in the presence of NO (100 microM) is significantly blunted (25 fold). Cyclic GMP-degradation is inhibited by the presence of the phosphodiesterase inhibitor zaprinast; suggesting that the ability of OT to attenuate cGMP accumulation is unlikely to be due to degradation. We propose that the elevation of intracellular calcium following the addition of OT suppress the activity of a calcium-sensitive guanylyl cyclase. The diminution of cGMP synthetic potential in myometrial cells from pregnant women is consistent with the absence of a role for cGMP in the NO-induced relaxation of uterine muscle.

Topics: Cell Culture Techniques; Cyclic GMP; Female; Humans; Myocytes, Smooth Muscle; Myometrium; Nitric Oxide; Oxytocin; Phosphodiesterase Inhibitors; Pregnancy; Purinones

We tested the hypothesis that the negative functional effects of natriuretic peptides would be blunted in thyroxine (T4)-induced hypertrophic cardiac myocytes. We also studied the causes of these changes. Ventricular myocytes were obtained from control (n=8) and T4 (0.5 mg/kg/16 days) treated rabbit hearts (n=7). Cell shortening parameters were studied with a video edge detector. We also determined particulate (pGC) and soluble (sGC) guanylyl cyclase activity and cyclic GMP levels. Myocyte function was examined at baseline and after brain natriuretic peptide (BNP 10(-7,-6) M) or C-type natriuretic peptide (CNP 10(-7,-6) M) or zaprinast (cyclic GMP phosphodiesterase inhibitor 10(-6)M) followed by BNP or CNP. Baseline function was similar in control and T4 myocytes. BNP (5.7 +/- 0.2 to 4.3 +/- 0.1%) and CNP (5.7 +/- 0.4 to 4.2 +/- 0.2%) significantly reduced percent shortening in control myocytes. These reductions were not observed with T4 (BNP, 5.7 +/- 0.6 to 5.6 +/- 0.6; CNP, 5.6 +/- 0.4 to 5.5 +/- 0.5). BNP and CNP responded similarly after zaprinast. Baseline cyclic GMP was similar in control and T4, but BNP only increased cyclic GMP in controls. The activity of pGC was similar at baseline in control and T4, but the stimulated activity was significantly lower in T4 myocytes. Both basal and stimulated sGC activity were similar in control and hypertrophic myocytes. These results demonstrated that the ability of natriuretic peptides to reduce ventricular myocyte function was blunted in T4 hypertrophic myocytes. This blunted response was related to the reduced ability of natriuretic peptides to increase cyclic GMP levels due to a reduced stimulated particulate guanylyl cyclase activity.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cell Size; Cyclic GMP; Guanylate Cyclase; In Vitro Techniques; Myocytes, Cardiac; Natriuretic Peptide, Brain; Natriuretic Peptide, C-Type; Natriuretic Peptides; Phosphodiesterase Inhibitors; Purinones; Rabbits; Thyroxine

Celecoxib carries a smaller cardiovascular risk for myocardial infarction and hypertension than other cyclooxygenase-2 (COX-2)-selective non-steroidal anti-inflammatory drugs NSAIDs ("coxibs") and may ameliorate endothelial dysfunction. We aimed to determine which mechanism possibly accounts for the beneficial effect by investigating its vascular action in different in vitro preparations in comparison with other coxibs and reference phosphodiesterase-5 (PDE5) inhibitors.. To uncover potential effects on coronary flow, the effects of celecoxib in comparison with other NSAIDs and the PDE5 inhibitors, sildenafil and zaprinast, were investigated in guinea-pig Langendorff heart. This was supported by studies for vasorelaxation, interaction with the NO/cGMP pathway, and measurement of cyclic nucleotide amounts released from rat aortic rings, and inhibition of human PDE5 as well as PDE4 activity.. Bolus injections of sildenafil, celecoxib, and zaprinast (at 100 nmol) into the Langendorff heart increased coronary flow by approximately 100, 65, and 25%, respectively, while rofecoxib, lumiracoxib, parecoxib, and diclofenac, except valdecoxib (>100 nmol), failed to increase coronary flow up to 300 nmol. In rat aorta, sildenafil, celecoxib and zaprinast caused endothelium-dependent relaxation with -log[EC(50)]M values of 8.90, 6.66 and 5.56, respectively; their rank order of potency corresponds to their coronary dilatory effect. Celecoxib-induced relaxation of aorta was attenuated by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) and by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 10(-5) M). In aortic rings, celecoxib (3x10(-5) M) caused a fivefold increase in the cGMP level and potentiated that induced by sodium nitroprusside (5x10(-7) M). Celecoxib and valdecoxib inhibited human PDE5A1 with an IC(50) of 1.6x10(-5) and 1x10(-4) M, respectively, whereas other coxibs were without inhibitory effect.. Celecoxib caused coronary vasodilatation in guinea-pig hearts and relaxation of rat aorta and had a potentiating effect on the NO/cGMP signaling pathway in rat aorta through specific blockade of PDE5. These unexpected findings clearly support the notion that celecoxib possesses an as yet undisclosed molecule-specific property that possibly compensates a decrease of prostacyclin-dependent cAMP generation by concomitantly increasing cGMP levels resulting from inhibition of PDE5.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Aorta; Celecoxib; Coronary Vessels; Cyclic GMP; Endothelium, Vascular; Guanylate Cyclase; Guinea Pigs; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Oxadiazoles; Perfusion; Phosphodiesterase 5 Inhibitors; Piperazines; Purines; Purinones; Pyrazoles; Quinoxalines; Rats; Regional Blood Flow; Signal Transduction; Sildenafil Citrate; Sulfonamides; Sulfones; Vasodilator Agents

Hyaluronan must be exported from its site of synthesis, the inner side of plasma membrane, to the extracellular matrix. Here, we identified the multidrug-associated protein MRP5 as the principle hyaluronan exporter from fibroblasts. The expression of the MRP5 (ABC-C5) transporter was silenced in fibroblasts using RNA interference, and a dose-dependent inhibition of hyaluronan export was observed. Hyaluronan oligosaccharides introduced into the cytosol competed with the export of endogenously labeled hyaluronan and the MRP5 substrate fluorescein. Because cGMP is a physiological substrate of MRP5, the intracellular concentrations of cGMP were modulated by the drugs 3-isobutyl-1-methylxanthin, propentofyllin, L-NAME, zaprinast, and bromo-cGMP, and the effects on hyaluronan export were analyzed. Increasing the cGMP levels inhibited hyaluronan export and decreasing it afforded higher concentrations of zaprinast to inhibit the export. Thus, cGMP may be a physiological regulator of hyaluronan export at the level of the export MRP5.

Topics: 1-Methyl-3-isobutylxanthine; Biological Transport; Cell Membrane; Cyclic GMP; Cytosol; Fibroblasts; Humans; Hyaluronic Acid; Models, Biological; Multidrug Resistance-Associated Proteins; NG-Nitroarginine Methyl Ester; Purinones; RNA Interference; Xanthines

The lysosomal destabilization that precedes mitochondrial apoptotic changes is an important step in cell death, particularly in oxidative cell death. This study describes the novel pharmacological effects of zaprinast, a cGMP-elevating phosphodiesterase inhibitor, on the inhibition of oxidative cell death in astrocyte cultures. H2O2-induced oxidative cytotoxicity was measured grossly by monitoring lactate dehydrogenase (LDH) release, and was found to be associated with lysosomal acridine orange relocation, lysosomal cathepsin D release into cytosol, and reduced mitochondrial potentials. Moreover, zaprinast (100 microM) inhibited all of these cytotoxic phenomena. In addition, H2O2-induced LDH release was not inhibited by 8-pCPT-cGMP, and the inhibition of this release by zaprinast was unaffected by Rp-8-pCPT-cGMP, a protein kinase G inhibitor. Zaprinast was found to inhibit sphingosine-induced lysosomal acridine orange relocation and the induced decrease in mitochondrial potential, but zaprinast had no effect on rotenone-induced mitochondrial collapse, which was not associated with lysosomal destabilization. However, zaprinast did not inhibit the cellular increase of reactive oxygen species induced by H2O2, which suggests that its protective mechanism differs from that of desferrioxamine, which does inhibit such cellular increase of oxygen free radicals. We suggest that the novel protective effect of zaprinast on H2O2-induced oxidative cell death is primarily associated with its inhibition of lysosomal destabilization.

Topics: Animals; Animals, Newborn; Astrocytes; Cathepsin D; Cell Death; Cells, Cultured; Cerebral Cortex; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytoprotection; Cytosol; Deferoxamine; Dose-Response Relationship, Drug; Hydrogen Peroxide; Intracellular Membranes; L-Lactate Dehydrogenase; Lysosomes; Membrane Potential, Mitochondrial; Mitochondria; Oxidative Stress; Phosphodiesterase Inhibitors; Protective Agents; Purinones; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Rotenone; Sphingosine; Thionucleotides; Uncoupling Agents

Previous behavioural studies which have administered phosphodiesterase type-5 (PDE5) inhibitors have consistently demonstrated improved retention. However, when young chicks were trained on a strongly reinforced passive avoidance task 100microM zaprinast caused two periods of transient retention loss. This is opposed to past findings and may suggest an effect on retrieval. It is hypothesised that the level of reinforcement is central to this phenomenon. The molecular corollary of this may be the need to maintain cGMP homeostasis such that strong reinforcement+zaprinast may impair retention through the production of excessive levels of cGMP. This was demonstrated by two challenge studies whereby increasing concentrations of 8-Br-cGMP were administered in the presence of the guanylate cyclase inhibitor ODQ (100microM; ic) resulting in an inverted "U-shaped" retention curve. These findings suggest a more complex role for PDE5 and cGMP in memory processing than previously described and question the role of PDE5 inhibitors as nootropes under all circumstances.

Topics: Analysis of Variance; Animals; Animals, Newborn; Avoidance Learning; Behavior, Animal; Chickens; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Discrimination, Psychological; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Memory Disorders; Phosphodiesterase 5 Inhibitors; Purinones; Quinoxalines; Reinforcement, Psychology; Retention, Psychology; Time Factors

Nitric oxide-independent agonists of soluble guanylate cyclase (sGC) have been developed.. We tested whether inhalation of novel dry-powder microparticle formulations containing sGC stimulators (BAY 41-2272, BAY 41-8543) or an sGC activator (BAY 58-2667) would produce selective pulmonary vasodilation in lambs with acute pulmonary hypertension. We also evaluated the combined administration of BAY 41-8543 microparticles and inhaled nitric oxide (iNO). Finally, we examined whether inhaling BAY 58-2667 microparticles would produce pulmonary vasodilation when the response to iNO is impaired.. In awake, spontaneously breathing lambs instrumented with vascular catheters and a tracheostomy tube, U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mm Hg.. Inhalation of microparticles composed of either BAY 41-2272, BAY 41-8543, or BAY 58-2667 and excipients (dipalmitoylphosphatidylcholine, albumin, lactose) produced dose-dependent pulmonary vasodilation and increased transpulmonary cGMP release without significant effect on mean arterial pressure. Inhalation of microparticles containing BAY 41-8543 or BAY 58-2667 increased systemic arterial oxygenation. The magnitude and duration of pulmonary vasodilation induced by iNO were augmented after inhaling BAY 41-8543 microparticles. Intravenous administration of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which oxidizes the prosthetic heme group of sGC, markedly reduced the pulmonary vasodilator effect of iNO. In contrast, pulmonary vasodilation and transpulmonary cGMP release induced by inhaling BAY 58-2667 microparticles were greatly enhanced after treatment with ODQ.. Inhalation of microparticles containing agonists of sGC may provide an effective novel treatment for patients with pulmonary hypertension, particularly when responsiveness to iNO is impaired by oxidation of sGC.

Topics: Administration, Inhalation; Aerosols; Animals; Benzoates; Cyclic GMP; Dose-Response Relationship, Drug; Drug Combinations; Guanylate Cyclase; Injections, Intravenous; Lung; Morpholines; Nitric Oxide; Oxadiazoles; Particle Size; Phosphodiesterase Inhibitors; Powders; Pulmonary Circulation; Purinones; Pyrazoles; Pyridines; Pyrimidines; Quinoxalines; Receptors, Cytoplasmic and Nuclear; Sheep; Soluble Guanylyl Cyclase; Vasodilation

cGMP was first discovered in urine, demonstrating that kidney cells extrude this cyclic nucleotide. Drosophila Malpighian tubules provide a model renal system in which a homologue of mammalian PDE (phosphodiesterase) 6 is expressed. In humans, this cG-PDE (cGMP-specific PDE) is specifically expressed in the retinal system, where it controls visual signal transduction. In order to gain insight into the functional role of DmPDE6 (Drosophila PDE6-like enzyme) in epithelial function, we generated transgenic animals with targeted expression of DmPDE6 to tubule Type I (principal) cells. This revealed localization of DmPDE6 primarily at the apical membranes. As expected, overexpression of DmPDE6 resulted in elevated cG-PDE activity and decreased tubule cGMP content. However, such targeted overexpression of DmPDE6 creates a novel phenotype that manifests itself in inhibition of the active transport and efflux of cGMP by tubules. This effect is specific to DmPDE6 action, as no effect on cGMP transport is observed in tubules from a bovine PDE5 transgenic line which display reduced rates of fluid secretion, an effect not seen in DmPDE6 transgenic animals. Specific ablation of DmPDE6 in tubule principal cells, via expression of a targeted DmPDE6 RNAi (RNA interference) transgene, conferred increased active transport of cGMP, confirming a direct role for DmPDE6 in regulating cGMP transport in tubule principal cells. Pharmacological inhibition of DmPDE6 in wild-type tubules using the cG-PDE inhibitor, zaprinast, similarly results in stimulated cGMP transport. We provide the first demonstration of a novel role for a cG-PDE in modulating cGMP transport and efflux.

Topics: Animals; Biological Transport, Active; Cell Polarity; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Drosophila melanogaster; Gene Expression Regulation, Enzymologic; Malpighian Tubules; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones

This study tested the hypothesis that the positive inotropic effect of beta-adrenoceptor stimulation would be inhibited by increases in cyclic GMP in control cardiomyocytes and that this response would be modified in hypertrophic cardiomyocytes. Cell functional data as well as GMP and cyclic AMP data were collected from 7 control and 7 1K1C (one-kidney-one-clip) renal hypertensive hypertrophic rabbits. Using isolated control and IKIC ventricular myocytes, data were obtained at baseline and after treatment with the beta-adrenoceptor agonist isoproterenol (10(-8, -6) mol/l) or the cyclic GMP-phosphodiesterase inhibitor zaprinast (10(-5) mol/l) followed by isoproterenol (10(-8, -6) mol/l). We found that in control rabbits, isoproterenol (10(-6) mol/l) increased percent shortening (4.8 +/- 0.2 to 6.4 +/- 0.3%) and cyclic AMP (2.3 +/- 0.3 to 5.0 +/- 0.7 pmol/10(5) cells). Zaprinast 10(-5) mol/l increased cyclic GMP (150 +/- 20 to 209 +/- 14 fmol/10(5) cells) and decreased percent shortening (6.2 +/- 0.4 to 5.2 +/- 0.3). Zaprinast 10(-5) mol/l prevented the functional response to isoproterenol in control (5.2 +/- 0.3 to 4.7 +/- 0.3), without changing cyclic AMP levels. In 1K1C rabbits, isoproterenol (10(-6) mol/l) increased cyclic AMP (4.9 +/- 0.8 to 7.6 +/- 1.4 pmol/10(5) cells) without changing function. Zaprinast 10(-5) mol/l increased cyclic GMP (182 +/- 23 to 233 +/- 24 fmol/10(5) cells) and decreased percent shortening (6.6 +/- 0.9 to 4.7 +/- 0.5), but did not alter the lack of effect of isoproterenol in 1K1C. In control cardiomyocytes, cyclic GMP blunted the isoproterenol contraction response without changing cyclic AMP levels, but isoproterenol's functional effect was not seen in 1K1C cardiomyocytes.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adrenergic beta-Agonists; Animals; Cardiomegaly; Cell Enlargement; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Hypertension, Renal; In Vitro Techniques; Isoproterenol; Myocardial Contraction; Myocytes, Cardiac; Purinones; Rabbits

The interaction between renin, nitric oxide (NO), and its second messenger cGMP is controversial. cAMP is the stimulatory second messenger for renin but is degraded by phosphodiesterases (PDEs). We previously reported that increasing endogenous cGMP in rats by inhibiting its breakdown by PDE-5 stimulated renin secretion rate (RSR). This could be reversed by selective inhibition of neuronal nitric oxide synthase (nNOS). PDE-3 metabolizes cAMP, but this can be inhibited by cGMP, suggesting that renal cGMP could stimulate RSR by diminishing PDE-3 degradation of cAMP. Rats were anesthetized with Inactin before determination of blood pressure (BP), renal blood flow (RBF), and sampling of renal venous and arterial blood to determine RSR. In 13 rats, basal BP was 104 +/- 2 mmHg, RBF was 6.1 ml x min(-1) x g kidney wt(-1) and RSR was 2.9 +/- 1.4 ng ANG I x h(-1) x min(-1). Inhibiting PDE-5 with 20 mg/kg body wt i.p. Zaprinast did not change hemodynamic parameters but increased RSR fivefold to 12.2 +/- 4.9 ng ANG I x h(-1) x min(-1) (P < 0.05). Renal venous cAMP was increased by Zaprinast from 93.8 +/- 27.9 to 149.2 +/- 36.0 pM x min(-1) x g kidney wt(-1) (P < 0.05). When another 10 rats were treated with the PDE-3 inhibitor Milrinone (0.4 microg/min over 30 min, which did not affect hemodynamics), RSR was elevated to 10.4 +/- 4.4 ng ANG I x h(-1) x min(-1). Milrinone also increased renal venous cAMP from 212 +/- 29 to 304 +/- 29 pM x min(-1) x g kidney wt(-1) (P < 0.025). Administration of Zaprinast to rats pretreated with Milrinone (n = 10) did not further increase in RSR (7.5 +/- 3.3 ng ANG I x h(-1) x min(-1)). These results are consistent with endogenous renal cGMP inhibiting PDE-3, which diminishes renal metabolism of cAMP. The resulting increase in cAMP serves as an endogenous stimulus for renin secretion. This suggests a pathway by which NO can indirectly stimulate RSR through its second messenger cGMP.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 5; Furosemide; Kinetics; Male; Milrinone; Nitric Oxide; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Rats; Rats, Sprague-Dawley; Renin; Second Messenger Systems

Vasomotion refers to periodic oscillations in vascular tone that ensure the intermittent supply of blood to adjacent microvascular units. Previous evidence from vessels outside the eye suggests that cyclic guanosine-monophosphate (cGMP) is involved in the regulation of vasomotion, but it is unknown whether this compound has an effect on vasomotion in retinal vessels.. Retinal arterioles from porcine eyes were studied in a wire myograph. After initiation of vasomotion, the vessels were stimulated with increasing concentrations of the cGMP agonist 8-Br-cGMP (n = 6), the phosphodiesterase inhibitor zaprinast (n = 6) and the cGMP synthesis inhibitor L-NAME (n = 6). High concentrations of L-NAME blocked vasomotion, and control experiments (n = 20) using 8-Br-cGMP, S-nitroso-N-acetylpenicillamine (SNAP), adenosine and pinacidil were carried out to elucidate whether this effect was related to changes in the general tone of the vessel. Additionally, the relationship between oscillations in vascular tone and intracellular calcium concentration was studied.. Induction of cGMP agonistic activity with either 8-Br-cGMP or zaprinast lowered the vasomotion frequency significantly, whereas L-NAME-induced inhibition of cGMP increased this frequency. Neither of the agents affected the amplitude of the oscillations. The control experiments indicated that the effect of cGMP on vasomotion frequency was independent of the accompanying increase in tone. The oscillations in tone during vasomotion were accompanied by similar oscillations in intracellular calcium concentration.. Cyclic GMP lowers the frequency without affecting the amplitude of vasomotion in isolated porcine retinal arterioles.

Topics: Animals; Arterioles; Calcium; Cyclic GMP; Enzyme Inhibitors; Muscle, Smooth, Vascular; Myography; NG-Nitroarginine Methyl Ester; Nitric Oxide; Phosphodiesterase Inhibitors; Purinones; Retinal Artery; Swine; Vasodilation; Vasomotor System

Human platelets contain the cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) 2, 3 and 5. The cGMP-PDE5 inhibitors Sildenafil and Zaprinast have been demonstrated to potentiate the anti-platelet aggregatory effect of NO donors like sodium nitroprusside (SNP) in vitro but the mechanisms of Sildenafil's action on the secretory function of human platelets have not been analysed in detail. In the present paper, we show (1) that both compounds potentiate the SNP-induced increase in cGMP in human platelets concentration-dependently. (2) However, whereas Sildenafil plus SNP treatment only partially inhibits thrombin-induced release of serotonin, the less selective Zaprinast plus SNP cause a complete inhibition. (3) The inhibition mediated by Sildenafil plus SNP is limited to low compound concentrations at which cAMP levels are increased, probably due to cGMP-mediated inhibition of PDE3. (4) High concentrations of Sildenafil (plus SNP) neither affect cAMP levels, likely due to the activation of PDE2, nor inhibits the release of serotonin. Thus, increases in both cyclic nucleotides seem to control platelet function. (5) Accordingly, treatment with increasing concentrations of Sildenafil plus SNP and a selective PDE2 inhibitor, which by its own has no effect, induced a concentration-dependent increase in cAMP and complete inhibition of platelet activation. In summary, our data indicate that Sildenafil inhibits secretory function of human platelets at least in part due to the cGMP-mediated effects on intracellular cAMP and that entire inhibition of serotonin release from thrombin-activated platelets is controlled by both cyclic nucleotides.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Blood Platelets; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 5; Humans; In Vitro Techniques; Nitric Oxide Donors; Nitroprusside; Phosphodiesterase I; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Platelet Activation; Platelet-Derived Growth Factor; Purines; Purinones; Pyridazines; Serotonin; Sildenafil Citrate; Sulfones

Non-steroidal anti-inflammatory drugs (NSAIDs) are used to treat the condition of rheumatoid arthritis, where levels of prostaglandin E2 (PGE2) and granulocyte macrophage-colony stimulating factor (GM-CSF) are elevated in the synovial fluid. NO-NSAIDs are a new class of cyclooxygenase (COX)-inhibitors developed by coupling a nitric oxide (NO)-donating moiety to conventional NSAIDs. We show that, in cytokine-treated synoviocytes (from non-rheumatic patients), NO-naproxen and NO-flurbiprofen like their parent compounds concentration-dependently reduce the levels of PGE2 (an index of COX-2 activity), with a corresponding rise in the release of GM-CSF. Unlike acetylsalicylic acid (ASA), NO-ASA reduces the levels of PGE2, without increasing GM-CSF release, although cell viability is reduced at the highest concentration (1 mM). The effects of NSAIDs and NO-NSAIDs on GM-CSF release were attributable to the PGE2 mediated cyclic (c) AMP pathway because PGE2 reversed the effects of COX blockade. Second, phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-201724 (both of which elevate cAMP levels) decreased GM-CSF release, in the presence of PGE2. Finally, neither sodium nitroprusside nor zaprinast (both of which elevate cGMP levels) affected GM-CSF or PGE2 release. Our findings demonstrate that GM-CSF is regulated by NSAIDs and NO-NSAIDs via inhibition of COX and appears to be mediated via the cAMP pathway. NO-ASA is the exception, because it does not increase GM-CSF release, although at millimolar concentrations cell viability is reduced.

Topics: 1-Methyl-3-isobutylxanthine; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Blotting, Western; Cells, Cultured; Cyclic AMP; Cyclic GMP; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Flurbiprofen; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Indomethacin; Membrane Proteins; Naproxen; Nitric Oxide Donors; Nitroprusside; Phosphodiesterase Inhibitors; Prostaglandin-Endoperoxide Synthases; Purinones; Synovial Membrane; Time Factors

Intellectual function is impaired in patients with hyperammonemia and hepatic encephalopathy. Chronic hyperammonemia with or without liver failure impairs the glutamate-nitric oxide-cGMP pathway function in brain in vivo and reduces extracellular cGMP in brain as well as the ability of rats to learn a Y maze conditional discrimination task. We hypothesized that the decrease in extracellular cGMP may be responsible for the impairment in learning ability and intellectual function and that pharmacological modulation of the levels of cGMP may restore learning ability. The aim of this work was to try to reverse the impairment in learning ability of hyperammonemic rats by pharmacologically increasing extracellular cGMP in brain. We assessed whether learning ability may be restored by increasing extracellular cGMP in brain by continuous intracerebral administration of: (1) zaprinast, an inhibitor of the phosphodiesterase that degrades cGMP or (2) cGMP. We carried out tests of conditional discrimination learning in a Y maze with control and hyperammonemic rats treated or not with zaprinast or cGMP. Learning ability was reduced in hyperammonemic rats, which needed more trials than control rats to learn the task. Continuous intracerebral administration of zaprinast or cGMP restored the ability of hyperammonemic rats to learn this task. Pharmacological modulation of extracellular cGMP levels in brain may be a useful therapeutic approach to improve learning and memory performance in individuals in whom cognitive abilities are impaired by different reasons, for example in patients with liver disease who present hyperammonemia and decreased intellectual function.

Topics: Animals; Brain; Cyclic GMP; Disease Models, Animal; Extracellular Fluid; Hepatic Encephalopathy; Hyperammonemia; Learning Disabilities; Male; Maze Learning; Memory Disorders; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Wistar; Recovery of Function; Treatment Outcome; Up-Regulation

Sustained increases in intracellular cGMP concentrations ([cGMP]i) inhibit cell growth and induce apoptosis. We now report that a cGMP-specific phosphodiesterase, PDE5, plays a dominant role in regulating [cGMP]i transitions that inhibit cell growth and control susceptibility to apoptosis in pulmonary endothelium. Atrial natriuretic peptide (ANP) activates guanylyl cyclase A/B and induces a rapid [cGMP]i rise 2-5 min after its application, in both pulmonary arterial endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMVECs). However, increased [cGMP]i in PAECs is transient and decays within 10 min due to cytosolic PDE5 hydrolytic activity. Increased [cGMP]i in PMVECs is sustained for >3 h due to the absence of PDE5. Indeed, at any ANP concentration, the sustained (30 min) [cGMP]i rise is greater in PMVECs than in PAECs, unless PAECs are also treated with the PDE5 inhibitor zaprinast. Using RT-PCR, Western blot analysis, immunoprecipitation, and DEAE chromatography, we resolved the expression and activity of PDE 5A1/A2 only in PAECs. Similarly, PDE5 expression was restricted to extra-alveolar endothelium in vivo. ANP induced growth inhibition and apoptosis in PMVECs, but similar effects were not seen in PAECs unless ANP treatment was combined with zaprinast. ANP blocked the VEGF-induced proliferation and migration in PMVECs. Collectively, these data suggest that PDE5-regulated [cGMP]i controls endothelial cell growth and apoptosis, representing a mechanism of heterogeneity between two endothelial phenotypes.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Apoptosis; Atrial Natriuretic Factor; Cell Movement; Cell Proliferation; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Endothelium, Vascular; Enzyme Activation; Guanylate Cyclase; Immunoprecipitation; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Pulmonary Alveoli; Pulmonary Artery; Purinones; Rats; Receptors, Atrial Natriuretic Factor; Vascular Endothelial Growth Factor A

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in the guinea pig gall bladder were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), and zaprinast (type 5), inhibited CCh-induced contractions in a concentration-dependent manner. The rank order of potency for the gall bladder was Ro20-1724 > vinpocetine > EHNA > milrinone > zaprinast, which was different from that of the trachea, taenia coli, and aorta. In the presence of CCh (0.3 muM), vinpocetine, milrinone, and Ro20-1724 each increased cAMP content, but not cGMP. By contrast, zaprinast increased cGMP content, but not cAMP, and EHNA increased both cAMP and cGMP contents. These results suggest that vinpocetine-, milrinone-, and Ro20-1724-induced relaxation was correlated with cAMP, zaprinast-induced relaxation was correlated with cGMP, and that EHNA-induced relaxation was correlated with cAMP and cGMP in the guinea pig gall bladder. In conclusion, the effect of PDE inhibitors in the guinea pig gall bladder was different from those in smooth muscles, such as the trachea, taenia coli, and aorta.

Topics: 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Adenine; Animals; Carbachol; Cyclic AMP; Cyclic GMP; Gallbladder; Guinea Pigs; Male; Milrinone; Muscle Contraction; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Purinones; Vinca Alkaloids

Zaprinast is a phosphodiesterase inhibitor that is active in various models of pain when administered locally. In addition, the antinociception of zaprinast is involved in the nitric oxide (NO)-cGMP pathway. However, the effect of zaprinast administered spinally has not been examined. Therefore, this study examined the effect of zaprinast on the formalin-induced nociception at the spinal level. Next, the role of the NO-cGMP-potassium channel pathway on the effect of zaprinast was further clarified. Catheters were inserted into the intrathecal space of male Sprague-Dawley (SD) rats. Pain was induced by applying 50 microl of a 5% formalin solution to the hindpaw. The change in the zaprinast-induced effect was examined after an intrathecal pretreatment with a NO synthase inhibitor (l-NMMA), a guanylyl cyclase inhibitor (ODQ) or a potassium channel blocker (glibenclamide). Zaprinast produced an antinociceptive effect during phase 1 and phase 2 in the formalin test. Intrathecal l-NMMA, ODQ and glibenclamide did not reverse the antinociception of zaprinast in either phase of the formalin test. These results suggest that zaprinast is effective against both acute pain and the facilitated pain state at the spinal level. However, the NO-sensitive cGMP-potassium channel pathway is not contributable to the antinociceptive mechanism of zaprinast in the spinal cord.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Analgesics; Animals; Cyclic GMP; Formaldehyde; Glyburide; Injections, Spinal; Male; Nitric Oxide; omega-N-Methylarginine; Oxadiazoles; Pain; Pain Measurement; Phosphodiesterase Inhibitors; Potassium Channels; Purinones; Quinoxalines; Rats; Rats, Sprague-Dawley; Signal Transduction

Guanosine 3', 5'-cyclic monophosphate (cGMP) acts as a relaxant second messenger in the cerebral vessels. cGMP-specific phosphodiesterase type 5 (PDE5) inhibitor increases intracellular cGMP levels. This study investigated the effect of the PDE5 inhibitor on the ischemic brain.. Regional cerebral blood flow (rCBF), cGMP concentration, and infarction volume were measured in the rat middle cerebral artery occlusion model. Ten minutes after ischemia, the animals received an intravenous (i.v.) infusion of vehicle (phosphate-buffered saline), PDE5 inhibitor, zaprinast (10 mg/kg), or nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP, 100 microg/kg). rCBF was measured continuously by laser-Doppler flowmetry in the ischemic penumbra of the ischemic and contralateral sides under continuous blood pressure monitoring. cGMP concentrations were determined using the enzyme immunoassay and infarct volumes were estimated by 2,3,5-triphenyltetrazolium chloride staining.. The administration of zaprinast significantly increased rCBF in the ischemic brain compared with the pre-drug control value despite the decreased mean blood pressure, whereas it did not affect rCBF in the contralateral side. The cGMP concentration was significantly higher in the ischemic cortex compared with the contralateral side. SNAP infusion increased the cGMP concentration in the bilateral cortices to a similar extent. The volume of cerebral infarction was significantly decreased by zaprinast administration.. The PDE5 inhibitor zaprinast may selectively increase CBF in the ischemic brain via increased cGMP levels, thus providing a new strategy against acute cerebral infarction.

Topics: Analysis of Variance; Animals; Blood Circulation Time; Blood Pressure; Brain Ischemia; Cerebrovascular Circulation; Cyclic GMP; Disease Models, Animal; Functional Laterality; Immunoenzyme Techniques; Infarction, Middle Cerebral Artery; Laser-Doppler Flowmetry; Male; Nitric Oxide Donors; Penicillamine; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Wistar; Regional Blood Flow; Tetrazolium Salts; Time Factors

Experimental studies have indicated the importance of cAMP and cGMP in modulation of peripheral sensory neurons leading to hyperalgesic response. The concentration of both depends upon the activity of phosphodiesterase, which is responsible for their degradation. The aim of the present study was to evaluate the effect of the PDE-5 inhibitor sildenafil on central or peripheral administration in formalin-induced hyperalgesia in rats. Sildenafil dose-dependently and significantly attenuated both the early and late phase of formalin-induced hyperalgesia on central administration. However, sildenafil on peripheral administration inhibited only the late phase of formalin-induced hyperalgesia in rats. The anti-nociceptive effect of sildenafil was blocked by L-NAME, a non-selective NOS inhibitor, and methylene blue (MB), a guanylate cyclase inhibitor, but sildenafil itself had little or no effect on the first phase of the formalin test in rats. The results from the present study indicates that sildenafil, besides peripheral actions, has a central anti-nociceptive effect, which may be due to activation of the NO-cGMP pathway, as this effect was blocked by L-NAME and MB. PDE-5 inhibitors could be considered as a new class of anti-nociceptive agents for future drug development.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Analgesics; Animals; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Female; Injections, Intraventricular; Injections, Spinal; Male; Motor Activity; NG-Nitroarginine Methyl Ester; Nitric Oxide; Phosphodiesterase Inhibitors; Piperazines; Purines; Purinones; Rats; Rats, Wistar; Sildenafil Citrate; Sulfones

Many infants with congenital diaphragmatic hernias (CDHs) experience persistent pulmonary hypertension that is refractory to treatment with inhaled nitric oxide (NO). We have examined the responses of isolated pulmonary arterioles from prenatal and postnatal rats with and without nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether)-induced CDH to a variety of activators of the NO-cyclic guanosine monophosphate (cGMP) pathway.. Right-sided CDH was induced in fetal rats by feeding nitrofen to pregnant rats on day 12 of gestation. Control rats were fed olive oil (vehicle). Third-generation pulmonary arterioles were isolated from the right lung of prenatal rats at term and from newborn rats within 8 hours after birth. Responses to increasing concentrations of sodium nitroprusside (SNP), atrial natriuretic peptide, or 8-bromo-cGMP were measured in pulmonary arterioles from control rats and from rats with nitrofen-induced CDH. Postnatal responses to 8-bromo-cGMP were also recorded in the presence of zaprinast, a type V phosphodiesterase inhibitor.. Pulmonary arterioles from prenatal rats did not dilate in response to SNP, atrial natriuretic peptide, or 8-bromo-cGMP. Vasodilatory responses of postnatal pulmonary arterioles from control rats to SNP and 8-bromo-cGMP were significantly greater than for arterioles from rats with CDH. Zaprinast pretreatment resulted in similar responses for postnatal CDH and control arterioles to 8-bromo-cGMP.. Postnatal pulmonary arterioles from CDH rats exhibit altered nitrovasodilator responsiveness, which may be due to rapid degradation of cGMP.

Topics: Animals; Arterioles; Atrial Natriuretic Factor; Cyclic GMP; Disease Models, Animal; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Hypertension, Pulmonary; Lung; Nitric Oxide; Nitroprusside; Pesticides; Phenyl Ethers; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Sprague-Dawley; Vasodilation; Vasodilator Agents

This study investigates the effect of protein kinase G (PKG) activation upon proliferation of human cultured prostatic stromal cells. The PKG II activator (8-pCPT-cGMP; IC50 of 113+/-42 nM) and the phosphodiesterase inhibitor, zaprinast (up to 50 microM), but not the PKG I isoform activators (APT-cGMP and PET-cGMP), reduced foetal calf serum-stimulated proliferation. The effect of 8-pCPT-cGMP (30 microM) was blocked by Rp-8-Br-cGMPS (5 microM) and Rp-8-pCPT-cGMP (5 microM), but not Rp-cAMPS (5 microM). 8-pCPT-cGMP (30 microM) and zaprinast (50 microM), but not PET-cGMP (30 microM), caused a significant increase in atypical nuclei and an increase in annexin-V staining. These data indicate that activation of PKG II induces apoptosis of human cultured prostatic stromal cells.

Topics: Aged; Apoptosis; Azides; Cell Division; Cell Nucleus; Cells, Cultured; Cyclic AMP; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Humans; Isoenzymes; Male; Phosphoric Diester Hydrolases; Prostate; Prostatic Hyperplasia; Purinones; Serum; Stromal Cells; Thionucleotides

Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex, and may contribute to the pathophysiology of stroke and migraine. Previous studies demonstrated that nitric oxide (NO) formation promotes the repolarisation phase of CSD, and this effect may be cyclic GMP (cGMP)-mediated. Here, we have examined how phosphodiesterase (PDE) inhibition, either alone or superimposed on NO synthase (NOS) inhibition, alters CSD and the associated changes in extracellular cGMP. Microdialysis probes incorporating an electrode were implanted into the frontoparietal cortex of anaesthetised rats for quantitative recording of CSD, pharmacological manipulations, and dialysate sampling for cGMP measurements. CSD was induced by cathodal electrical stimulation in the region under study by microdialysis. Extracellular cGMP increased, but only slightly, during CSD. Perfusion of either zaprinast or sildenafil through the microdialysis probe, at concentrations that inhibited both PDE5 and PDE9 (and possibly other PDE), increased significantly extracellular cGMP. Unexpectedly, these levels remained high when NOS was subsequently inhibited with N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME, 1mM). The most interesting pharmacological effect on CSD was obtained with sildenafil. This drug altered neither CSD nor the subsequent characteristic effect of NOS inhibition, i.e. a marked widening of CSD. The fact that NOS inhibition still widened CSD in the presence of the high extracellular levels of cGMP associated with PDE inhibition, suggests that NO may promote CSD recovery, independently of cGMP formation.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cortical Spreading Depression; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Electric Stimulation; Extracellular Space; Male; Microdialysis; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Phosphodiesterase Inhibitors; Piperazines; Purines; Purinones; Rats; Rats, Sprague-Dawley; Sildenafil Citrate; Sulfones

To evaluate the association between hypertension, male erectile function, Rho-kinase, and cyclic GMP pathways, we monitored neurogenic erectile response in spontaneously hypertensive (SHR) vs normotensive rats. We also evaluated SHR erectile function before and after intracavernosal injection of either the specific Rho-kinase inhibitor Y-27632 or a combination of Y-27632 and the PDE5 inhibitor zaprinast to prevent cGMP degradation. SHR had lower resting baseline corpus cavernosum pressure and a higher threshold for development of tumescence than normotensive rats. In SHR, Y-27632 administration reversed hypertension-related changes in male erectile function; Rho-kinase antagonism and PDE5 inhibition in combination had a synergistic effect in improving the neurogenic erectile response. Our data indicate that hypertension is associated with impairment in the SHR neurogenic erectile response that may involve a derangement in hemodynamic mechanisms in penile erectile tissue. Rho-kinase inhibition alone or combined with PDE5 inhibition may be of value in treating hypertension-related ED.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Amides; Animals; Antihypertensive Agents; Blood Pressure; Blotting, Western; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Drug Synergism; Electric Stimulation; Hypertension; Intracellular Signaling Peptides and Proteins; Male; Penile Erection; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Protein Serine-Threonine Kinases; Purinones; Pyridines; Rats; Rats, Inbred SHR; Rats, Inbred WKY; rho-Associated Kinases; rhoA GTP-Binding Protein; Up-Regulation

Adenosine is an important regulator of neuronal excitability. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to suppress excitation. This action can be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by exposure to zaprinast. To explore the mechanism of this phenomenon further, we examined the effect of zaprinast on adenosine release itself in cultured rat forebrain neurons. Zaprinast significantly stimulated extracellular adenosine accumulation. The effect of zaprinast on adenosine appeared to be mediated by increasing intracellular cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA): (i) zaprinast stimulated intracellular cAMP accumulation; (ii) a cAMP antagonist (Rp-8-Br-cAMP) significantly reduced the zaprinast effect on adenosine; (iii) an inhibitor of phosphodiesterase (PDE)1 (vinpocetine) and an activator of adenylate cyclase (forskolin) mimicked the effect of zaprinast on adenosine. We also found that zaprinast had no effect on adenosine in astrocyte cultures, and tetrodotoxin completely blocked zaprinast-evoked adenosine accumulation in neuronal cultures, suggesting that neuronal activity was likely to be involved. Consistent with a dependence on neuronal activity, NMDA receptor antagonists (MK-801 and D-APV) and removal of extracellular glutamate by glutamate-pyruvate transaminase blocked the effect of zaprinast. In addition, zaprinast was shown to stimulate glutamate release. Thus, our data suggest that zaprinast-evoked adenosine accumulation is likely to be mediated by stimulation of glutamate release by a cAMP- and PKA-dependent mechanism, most likely by inhibition of PDE1 in neurons. Furthermore, regulation of cAMP, either by inhibiting cAMP-PDE activity or by stimulating adenylate cyclase activity, may play an important role in modulating neuronal excitability. These data suggest the existence of a homeostatic negative feedback loop in which increases in neuronal activity are damped by release of adenosine following activation of glutamate receptors.

Topics: Adenosine; Animals; Animals, Newborn; Astrocytes; Cells, Cultured; Cyclic AMP; Cyclic GMP; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Enzyme Activation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Extracellular Space; Female; Glutamic Acid; Intracellular Space; Male; Membrane Potentials; Models, Neurological; N-Methylaspartate; Neurons; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Pregnancy; Prosencephalon; Purinones; Rats; Rats, Sprague-Dawley; Thionucleotides

Nitric oxide (NO) has been implicated in the anxiolytic-like behavioral effects of nitrous oxide (N2O). This study was conducted to determine whether NO activates a soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway in the behavioral response to N2O in the light-dark exploration test. In mice pretreated with an sGC inhibitor, the increased light-compartment activity normally induced by N2O was significantly attenuated. Pretreatment with a cGMP phosphodiesterase inhibitor antagonized the anxiogenic effect of 15% N2O and enhanced the anxiolytic effect of 25% N2O, implying that cGMP reduces anxiety. These preliminary findings suggest that a signaling pathway involving NO and cGMP may mediate the behavioral effects of N2O.

Topics: Animals; Anxiety; Behavior, Animal; Cyclic GMP; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Exploratory Behavior; Male; Mice; Nitrous Oxide; Oxadiazoles; Periodicity; Phosphodiesterase Inhibitors; Purinones; Quinoxalines

In bovine tracheal smooth muscle (TSM) strips, muscarinic antagonists (atropine, 4-DAMP, AFDX-116 and methoctramine) were able to increase simultaneously and a similar fashion the intracellular levels of cyclic nucleotides, with a cAMP/cGMP ratio higher than 2.0. These original pharmacological responses were time-and dose-dependent, exhibiting maximal values at 15 min, with a pEC(50) of 7.4 +/- 0.2 for atropine and 4-DAMP. These effects on cAMP and cGMP levels were similar to the ones obtained with isobutyl-methylxantine (IBMX, 10 microM), a non-selective cyclic nucleotide phosphodiesterase (PDE) inhibitor, suggesting the involvement of PDEs in these muscarinic antagonist responses. Neither, rolipram (10 microM), a specific PDEIV inhibitor, nor zaprinast (10 microM), a PDEV inhibitor, exhibited this "atropine-like" responses. Instead, atropine enhanced the increments of cAMP levels induced by rolipram and cGMP levels by zaprinast. However, vinpocetine (20 microM), a non-calmodulin dependent PDEIC inhibitor was able to mimic these muscarinic antagonist responses in intact smooth muscle strips. In addition, in cell free systems, muscarinic antagonists inhibited the membrane-bound PDEIC activity whereas soluble (cytosol) PDEIC activity was not affected by these muscarinic drugs. These results indicate that muscarinic antagonists acting possibly as inverse agonists on M(2)/M(3)mAChRs anchored to sarcolemma membranes can initiate a new signal transducing cascade leading to the PDEIC inhibition, which produced a simultaneous rise in both cAMP and cGMP intracellular levels in tracheal smooth muscle.

Topics: Animals; Atropine; Cattle; Cell-Free System; Cyclic AMP; Cyclic GMP; Muscarinic Antagonists; Muscle, Smooth; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Rolipram; Trachea; Vinca Alkaloids

After birth and during the first days of extrauterine life, pulmonary arterial pressure is progressively reduced to reach the adult values. We hypothesized that changes in PDE5 activity might be involved in the pulmonary postnatal maturation of the nitric oxide (NO)/cGMP pathway. The PDE5 inhibitor sildenafil produced vasorelaxant responses in isolated pulmonary arteries. These effects were similar in newborn (3-18 h) and 2-wk-old piglets, unchanged by endothelium removal, and markedly inhibited by the soluble guanylyl cyclase inhibitor ODQ. The peak of the transient vasorelaxant response to NO gas increased with postnatal age but was unaffected by PDE inhibition. However, the duration of the response to NO was significantly increased. The vasorelaxant response to sodium nitroprusside was potentiated by sildenafil in both age groups. The PDE5 inhibitors dipyridamole and zaprinast, produced qualitatively similar effects but with lower potency. Both total and PDE5-dependent cGMP hydrolytic activity and PDE5 protein expression increased with postnatal age. All these results suggest that PDE5 is a key regulator of NO-induced vasodilation in the postnatal pulmonary arteries. PDE5 inhibition is able to produce pulmonary vasodilation even in the absence of a functional endothelium and potentiates the vasorelaxant response to exogenous NO and nitroprusside. However, PDE5 is not responsible for the maturational increase of NO bioactivity during the first days of extrauterine life.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Animals, Newborn; Blotting, Western; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Dipyridamole; In Vitro Techniques; Male; Nitric Oxide; Nitroprusside; Phosphodiesterase Inhibitors; Piperazines; Pulmonary Artery; Purines; Purinones; Sildenafil Citrate; Sulfones; Swine; Vasodilation; Vasodilator Agents

Effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in guinea pig taenia coli were investigated. Forskolin and sodium nitroprusside inhibited carbachol (CCh)-induced contraction in a concentration-dependent manner. Various selective PDE inhibitors, vinpocetine (type 1), erythro -9-(2-hydroxy-3-nonyl)adenine (EHNA, type 2), milrinone (type 3), Ro20-1724(type 4) and zaprinast (type 5) inhibited CCh-induced contraction in a concentration-dependent manner, but the inhibition of milrinone was noticeably smaller than that of the other PDE inhibitors. The rank order of potency was zaprinast > vinpocetine > EHNA > Ro20-1724 > milrinone. In the presence of CCh (0.3 microM), vinpocetine and Ro20-1724 both increased cAMP content, but not cGMP. By contrast, EHNA and zaprinast both increased cGMP content, but not cAMP. Pretreatment with ODQ (30 microM), a soluble guanylyl cyclase inhibitor, decreased the inhibition of CCh-induced contraction by EHNA or zaprinast. Pretreatment with SQ22536 (100 microM), an adenylyl cyclase inhibitor, decreased the inhibition of CCh-induced contraction by vinpocetine or Ro20-1724. In conclusion, it was indicated that vinpocetine- or Ro20-1724-induced relaxation was correlated with cAMP but EHNA- or zaprinast- induced relaxation was correlated with cGMP.

Topics: Adenine; Animals; Carbachol; Colforsin; Colon; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Guinea Pigs; Male; Milrinone; Muscle Contraction; Nitroprusside; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Purinones; Vinca Alkaloids

Adenosine appears to be an endogenous somnogen. The lateral dorsal tegmental/pedunculopontine nucleus (LDT/PPT) located in the mesopontine tegmentum is important in the regulation of arousal. Neurons in this nucleus are strongly hyperpolarized by adenosine and express neuronal nitric oxide synthase. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to inhibit the field excitatory postsynaptic potential. This action could be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by zaprinast. In the present study we tested the effect of zaprinast on extracellular adenosine accumulation in pontine slices containing the LDT. Zaprinast at 10 microM evoked an increase in extracellular adenosine concentration. This effect was blocked by impermeant inhibitors of 5'-nucleotidase, indicating that the extracellular adenosine was derived from extracellular AMP. However, inhibitors of cAMP degradation had little or no effect on zaprinast-evoked adenosine accumulation, suggesting that extracellular cAMP was not the source. Removal of extracellular calcium inhibited the effect of zaprinast. These results demonstrate that a pathway exists by which zaprinast stimulates extracellular adenosine accumulation, and the presence of this pathway in the pontine slice suggests the possibility that it may be relevant for the regulation of behavioral state.

Topics: Adenosine; Animals; Cyclic AMP; Cyclic GMP; In Vitro Techniques; Phosphodiesterase Inhibitors; Pons; Purinones; Rats; Rats, Sprague-Dawley

We have shown that intracellular cGMP levels increase during retinoic acid- and mycophenolic acid-induced neuroblastoma differentiation and that a 6 days treatment with 1 mM dbcGMP lead LAN5 cell to elaborate a network of neuritic processes suggesting an involvement of cGMP in neuroblastoma differentiation. We have also investigated the effects of some specific inhibitors of phosphodiesterases (PDE1, PDE3, PDE4 and PDE5) on human neuroblastoma (LAN5 and SHEP) growth and differentiation. After six days of incubation in the presence of each specific inhibitor at 10 x IC50 levels a cytostatic and differentiating effect was only observed with the PDE5 inhibitors Zaprinast and MY-5445. The cytostatic effect of these compounds increased increasing their concentrations far above their IC50 levels for PDE5, suggesting that these compounds could act by interfering with other molecular events than direct cGMP-PDE inhibition. No appreciable effect was observed using Dipyridamole, another specific PDE5 inhibitor.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Cyclic GMP; Dipyridamole; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Inhibitory Concentration 50; Neuroblastoma; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Phthalazines; Purinones; Time Factors

The mechanisms underlying the hydrogen peroxide-induced relaxation of the norepinephrine-contraction were studied by measuring isometric force, myosin light chain (MLC(20)) phosphorylation and cyclic GMP in endothelium-denuded muscle from the guinea-pig aorta. Norepinephrine (5.2+/-1.3 microM) produced a phasic, followed by a tonic contraction. Hydrogen peroxide (10 and 100 microM), glyceryl trinitrate (30 and 300 nM) and 8-bromo cyclic GMP (30 and 100 microM) did not change the basal tone, but reduced the norepinephrine-induced contraction. Phosphorylation of MLC(20) (percentage of phosphorylated to total MLC(20)) was increased 1 min (5.9+/-1.0% vs. 35.9+/-4.9%) and, to a lesser extent, 20 min (3.7+/-1.7% vs. 13.9+/-1.6%) after the addition of norepinephrine. Hydrogen peroxide (100 microM) did not modify basal MLC(20) phosphorylation, but reduced the increase in MLC(20) phosphorylation induced by 1-min exposure to norepinephrine (20.9+/-4.1%). Its effect was abolished by catalase. When the tissue was incubated for 20 min with norepinephrine in the presence of hydrogen peroxide, norepinephrine-induced MLC(20) phosphorylation was not changed (13.6+/-1.5%), as compared to that in the absence of hydrogen peroxide. Hydrogen peroxide relaxed norepinephrine-stimulated aortas in a concentration-dependent fashion with EC(50) values of 5.9+/-0.2 microM. The relaxation was inhibited by soluble guanylate cyclase inhibitors and increased by an inhibitor of cyclic GMP-selective phosphodiesterase. In aorta precontracted with norepinephrine, hydrogen peroxide (100 microM) relaxed the tissue by 89+/-11% and almost doubled tissue concentrations of cyclic GMP, whereas sodium nitroprusside (1 microM) relaxed the tissue by 100% and increased cyclic GMP concentrations 30-fold. It is suggested that the inhibitory effects of hydrogen peroxide on the norepinephrine-induced phasic and sustained contractions are explained by a decrease in MLC(20) phosphorylation and by an alteration in MLC(20) phosphorylation-independent mechanisms, respectively. The effects of hydrogen peroxide were in part mediated by cyclic GMP.

Topics: Animals; Aorta, Thoracic; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Guinea Pigs; Hydrogen Peroxide; In Vitro Techniques; Male; Methylene Blue; Myosin Light Chains; Nitroglycerin; Norepinephrine; Oxadiazoles; Phosphorylation; Purinones; Quinoxalines; Rats; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

We previously reported that several nitric oxide (NO) donors, guanylate cyclase activators, and cyclic GMP lower intraocular pressure (IOP) in rabbits.. This study evaluated a novel method for studying cGMP production in the iris-ciliary body after the administration of different NO donors and guanylate cyclase activators. Tissue samples of porcine iris-ciliary body were incubated for 30 or 60 minutes with the test compounds and with or without the phosphodiesterase inhibitor zaprinast. The concentration of cGMP in the iris-ciliary body as an indicator of soluble guanylate cyclase activation was measured by radioimmunoassay.. The tested NO donors - SNOG, NONOate, NOR-3, and SNAP - were shown to release NO in incubation medium, and clearly increase cGMP concentration in the iris-ciliary body. Cyclic GMP production was 2-5 times higher with nitrosocaptopril and about 10 times higher with SNP than in the unstimulated control tissue incubation. Captopril, the reference for nitrosocaptopril, did not induce cGMP production in the porcine iris-ciliary body. ODQ, a guanylate cyclase inhibitor, shut down the production of cGMP after the administration of nitrosocaptopril and SNP. The guanylate cyclase activators YC-1 and atriopeptin III increased cGMP dose-dependently.. In this novel tissue incubation method, several NO donors and guanylate cyclase activators increased cGMP production in the porcine iris-ciliary body. This method can be used to screen new molecules in terms of cGMP production, since the ciliary body is important in lowering intraocular pressure.

Topics: Animals; Captopril; Ciliary Body; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Intraocular Pressure; Iris; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitrites; Nitro Compounds; Nitrogen Oxides; Oxadiazoles; Penicillamine; Phosphodiesterase Inhibitors; Purinones; Quinoxalines; Rabbits; S-Nitrosothiols; Spermine; Swine; Time Factors

(1) Cyclic GMP (cGMP) has been shown to be an important modulator of cardiac contractile function. A major component of cGMP regulation of contractility is cGMP-mediated inhibition of the cardiac calcium current (I(Ca)). An under-appreciated aspect of cyclic nucleotide signalling is hydrolysis of the cyclic nucleotide (i.e., breakdown by phosphodiesterases (PDEs)). The role of cGMP hydrolysis in regulating I(Ca) has not been studied. Thus the purpose of this study was to investigate if inhibition of cGMP hydrolysis can modulate I(Ca) in isolated guinea-pig ventricular myocytes. (2) Zaprinast, a selective inhibitor of cGMP-specific PDE (PDE5), caused a significant increase in cGMP levels in myocytes, but was without affect on basal or beta-adrenergic stimulated cAMP levels (consistent with its actions as a specific inhibitor of PDE5). (3) Zaprinast inhibited I(Ca) that was pre-stimulated with cAMP elevating agents (isoproterenol, a beta-adrenergic agonist; or forskolin, a direct activator of adenylate cyclase). The effect of zaprinast was greatly reduced by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). (4) Zaprinast also significantly inhibited basal I(Ca) when perforated-patch or whole-cell recording with physiological pipette calcium concentration (10(-7) M) was used. However, this effect was not observed when using standard calcium-free whole-cell recording conditions. (5) These results indicate that inhibition of cGMP hydrolysis can decrease both basal and cAMP-stimulated I(Ca). Thus, cGMP hydrolysis may likely be an important step for physiological modulation of I(Ca). This regulation may also be important in disease states in which cGMP production is increased and PDE5 expression is altered, such as heart failure.

Topics: Animals; Calcium Channel Blockers; Calcium Channels, L-Type; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Guinea Pigs; Heart Ventricles; Hydrolysis; Male; Membrane Potentials; Myocytes, Cardiac; Purinones

This study was designed to compare the haemodynamic, electrophysiological and pharmacodynamic effects of three selective inhibitors of the different isoenzyme forms of phosphodiesterase (PDE) on ischaemia-induced dysrhythmias in the anaesthetized rat. The drugs used were pimobendan, a selective PDE III inhibitor, rolipram, a selective PDE IV inhibitor, and zaprinast, a selective PDE V inhibitor.. The coronary artery was occluded 15 min after commencing drug administration, and myocardial ischaemia was maintained for 30 min during which the heart rate and mean arterial pressure were recorded. cAMP and cGMP were determined by radioimmunoassay.. Pretreatment with rolipram decreased the duration of ventricular tachycardia without any change in the incidences of dysrhythmias or the mortality rate. This drug did not modify ventricular content of adenosine 3',5'-cyclic monophosphate (cAMP) or guanosine 3',5'-cyclic monophosphate (cGMP). Pimobendan (1 mg kg(-1) + 0.1 mg kg(-1) min) decreased the duration of ventricular tachycardia. This dose of pimobendan and zaprinast (1 mg kg(-1) + 0.1 mg kg(-1) min(-1)) increased the incidence rate of ventricular fibrillation following coronary artery ligation and the mortality rate. Moreover, both drugs increased cGMP in the ventricle.. The results demonstrated that pimobendan and zaprinast increased the incidence of dysrhythmias and the mortality rate, which was accompanied by an increase in the ventricular content of cGMP. Rolipram decreased the duration of ventricular tachycardia without a change in the cyclic nucleotide content or in the mortality rate.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Anesthesia; Animals; Arrhythmias, Cardiac; Blood Pressure; Coronary Vessels; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 4; Cyclic Nucleotide Phosphodiesterases, Type 5; Heart Rate; Heart Ventricles; Ligation; Male; Myocardial Ischemia; Myocardium; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Pyridazines; Rats; Rats, Sprague-Dawley; Rolipram

Cyclic GMP plays an important role in maintaining homeostasis in the gastric mucosa. NSAIDs damage the mucosa by mechanisms that may be mediated by alterations in the intragastric concentration of cyclic GMP. To test this hypothesis we studied the effects of the oral administration of acetylsalicylic acid (100, 300, and 500 mg/kg), piroxicam (5, 10, and 20 mg/kg) and sodium diclofenac (10, 25, 50, and 100 mg/kg), and of their interaction with zaprinast (5 mg/kg) and IBMX (10 mg/kg), on intragastric concentrations of cyclic GMP and the gastric erosive index in rats. All determinations were done 3 hr after the NSAID was given. All NSAIDs induced dose-dependent decreases in mucosal concentrations of cyclic GMP, which correlated inversely with the surface area showing mucosal injury. In contrast, cyclic GMP concentrations remained normal, and no intragastric damage was seen in rats given zaprinast (cyclic GMP-specific phosphodiesterase inhibitor) or IBMX (non-specific phosphodiesterase inhibitor) or in combination with NSAIDs. These findings are in line with the hypothesis that cyclic GMP is involved in the biochemical mechanisms of NSAID-induced gastric injury.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cyclic GMP; Disease Models, Animal; Drug Interactions; Gastric Mucosa; Gastritis; Male; Phosphodiesterase Inhibitors; Primary Prevention; Purinones; Random Allocation; Rats; Rats, Wistar; Sensitivity and Specificity

Previously, four splice variants of human cGMP-specific phosphodiesterase (PDE) 9A (PDEs 9A1, 9A2, 9A3 and 9A4) have been identified. In this study, we have cloned a cDNA representing a new human PDE9A variant (PDE9A5). PDE9A5 encodes a protein of 492 amino acids, smaller than PDEs 9A1 and 9A2 but larger than PDEs 9A3 and 9A4. The exon structure of PDE9A5 is different from those of PDEs 9A1, 9A2, 9A3 and 9A4 in that, of the 20 exons of PDE9A gene, it lacks exons 2 and 5. PDE9A5 has been characterized in comparison with PDE9A1, the longest PDE9A variant. PDEs 9A5 and 9A1 have similar enzymatic properties. They both have a high affinity for cGMP with similar Km values (0.39 and 0.25 microM, respectively), although they have slightly different Vmax values (2.55 and 0.96 micromol/min/mg, respectively). They exhibit very similar divalent metal ion dependency and inhibitor sensitivity. Real-time quantitative PCR analysis shows that PDEs 9A5 and 9A1 exhibit differential tissue distribution. They are highly expressed in immune tissues (spleen, lymph node and thymus) and are more abundant in T cells than in B cells, neutrophils and monocytes. When transiently expressed in HEK293 cells, PDEs 9A5 and 9A1 proteins exhibit differential subcellular localization. PDE9A5 localizes exclusively in the cytoplasm, whereas PDE9A1 localizes in the nucleus only. The nuclear localization of PDE9A1 is dependent on a unique pat7 motif. By Western blot analysis, native PDE9A1 is detectable in the nucleus but not in the cytoplasm of T cells. Thus, to our knowledge, PDE9A1 is the only PDE isoform found to localize exclusively in the nucleus. We speculate that the physiological role of the PDE9A diversity may be imparting cGMP-metabolizing ability to specific cellular compartments in appropriate tissues.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Alternative Splicing; Amino Acid Sequence; Base Sequence; Blotting, Western; Cell Line; Cloning, Molecular; Cyclic GMP; DNA, Complementary; Gene Expression Regulation, Enzymologic; Humans; Imidazoles; Isoenzymes; Male; Molecular Sequence Data; Phosphodiesterase Inhibitors; Piperazines; Purines; Purinones; Recombinant Proteins; RNA, Messenger; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Sildenafil Citrate; Sulfones

The effects of the type V phosphodiesterase (PDE V) inhibitors, MBCQ, zaprinast and dipyridamole, on the relationship between relaxation and cyclic nucleotide content were investigated in rat ileal smooth muscle. Each of MBCQ (0.01-10 microM), zaprinast (0.1-100 microM) and dipyridamole (0.1-100 microM) inhibited carbachol (CCh; 10 microM)-induced contractions in a concentration-dependent manner. When compared with the concentrations of these agents producing 50% relaxation (IC50) of CCh-induced contraction, MBCQ was 14-20 fold more potent than the other agents. The inhibitory potency of these agents against high K+ (65 mM KCl)-induced contractions were similar to that for CCh. MBCQ (1, 10 microM) did not significantly increase the cGMP content above control levels in the presence of CCh (10 microM). Both Zaprinast (1-100 microM) and dipyridamole (1-100 microM) increased the cGMP content of smooth muscle preparations in a concentration-dependent manner. There was a positive correlation between the inhibition of the CCh-induced contraction and the increase in cGMP content elicited by zaprinast and dipyridamole (zaprinast; r=0.72, P<0.05, dipyridamole; r=0.92, P<0.05). However, MBCQ at a concentration which induced a medium-sized relaxation did not significantly increase the cGMP content. Neither MBCQ, zaprinast nor dipyridamole significantly increased the cAMP content of the preparations above control. In summary, it is suggested that the inhibition of CCh-induced contractions by zaprinast and dipyridamole involves increases in cGMP content via inhibition of PDE V. However the inhibition of CCh-induced contraction by MBCQ may not involve cyclic nucleotides in rat ileal smooth muscle.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Carbachol; Cholinergic Agonists; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Dipyridamole; Ileum; Male; Muscle Relaxation; Muscle, Smooth; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Potassium; Purinones; Quinazolines; Rats; Rats, Wistar

The effects of ibudilast, an inhibitor of phosphodiesterases (PDEs), on tension, levels of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) were investigated in bovine tracheal smooth muscle. We especially examined the combined effect of ibudilast with the cGMP-elevating agents on these parameters. Ibudilast was equipotent to attenuate the precontractions induced by both 0.3 microM methacholine and 40 mM K(+). By contrast, the relaxant effects of sodium nitroprusside and salbutamol on 40 mM K(+)-contracted preparations were smaller than those on 0.3 microM methacholine-contracted ones. Neither N(omega)-nitro-L-arginine (100 microM), an inhibitor of nitric oxide synthase, nor ODQ (1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 5 microM), an inhibitor of soluble guanylyl cyclase, affected the ibudilast-induced relaxation. The relaxations induced by ibudilast and diltiazem on 40 mM K(+)-contracted preparations were significantly attenuated when extracellular CaCl(2) was increased from 2.54 mM to 10 mM. Ibudilast (10 microM), which caused only minor effect by itself, significantly ( P<0.05) shifted the concentration-response curves for the relaxant responses to sodium nitroprusside (SNP), atrial natriuretic peptide (ANP) and salbutamol to the left. On the other hand, ibudilast did not change the relaxant responses to diltiazem. Unlike ibudilast, diltiazem (3 microM) failed to affect the SNP- and salbutamol-induced relaxations. Ibudilast significantly ( P<0.05) increased basal levels of cGMP and cAMP. Furthermore, ibudilast enhanced SNP (0.3 microM)- and ANP (0.3 microM)-induced cGMP accumulation and salbutamol (10 nM)-induced cAMP accumulation. Zaprinast (10 microM), a type 5 PDE inhibitor, enhanced both relaxation and cGMP accumulation induced by SNP and ANP without changing salbutamol-induced responses. These findings suggest that blockade of voltage-gated Ca(2+) channels is involved in the relaxing action of ibudilast in bovine tracheal smooth muscle. However, ibudilast potentiates relaxation responses to ANP and SNP by inhibition of PDE 5, not by blockade of Ca(2+) channels. The enhancement of cGMP-mediated response may contribute to the therapeutic effects of ibudilast.

Topics: Albuterol; Analysis of Variance; Animals; Atrial Natriuretic Factor; Bronchodilator Agents; Cattle; Cyclic GMP; Dose-Response Relationship, Drug; In Vitro Techniques; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Nitric Oxide Donors; Nitroprusside; Phosphodiesterase Inhibitors; Purinones; Pyridines; Trachea

Developmental changes (from 2 to 26 weeks) in phosphodiesterase (PDE) activity in the rat submandibular gland were investigated. Major activities for both cAMP- and cGMP-PDE were present in the 100000 x g supernatant fractions (70-90% of total activities), but not in the pellet fractions, during development. The effects of stimulators (Ca(2+)/calmodulin and cGMP) and inhibitors (cGMP, cilostamide, rolipram and zaprinast) were investigated in the supernatant fractions. During development, PDE4 (cAMP-specific PDE) was a major PDE, indicating that the majority of cAMP is hydrolysed by PDE4. In the young rat, PDE1 hydrolysed cGMP three-fold more than the control, and PDE2 (cGMP-stimulated PDE) was present, indicating that the concentration of intracellular cGMP may be enhanced, and cGMP may function in the growth pathway in the submandibular gland. Chromatograms eluted on a Mono Q HR5/5 ion-exchange column supported the results of the inhibition studies: PDE1, PDE2, PDE3, PDE4 and PDE5 were present in the young submandibular gland, and PDE1, PDE3, PDE4 and PDE5 in the adult gland. Expression of PDE5 was detected by inhibition studies, reverse transcriptase-polymerase chain reaction and Western blotting in the submandibular gland.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Blotting, Western; Calcium; Calmodulin; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 1; Cyclic Nucleotide Phosphodiesterases, Type 2; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 4; Cyclic Nucleotide Phosphodiesterases, Type 5; Male; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Quinolones; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Rolipram; Submandibular Gland

In view of the scarce information about the analgesic mechanism of kappa-opioid receptor agonists, the objective of the present study was to determine whether nitric oxide (NO) is involved in the peripheral antinociception of bremazocine, a kappa-opioid receptor agonist. Three drugs all interfering with the L-arginine/NO/cyclic GMP pathway were tested using the rat paw model of carrageenan-induced (250 microg) hyperalgesia: (a) N(G)-nitro-L-arginine (a nonselective NO-synthase inhibitor), (b) methylene blue (a guanylate cyclase inhibitor), and (c) zaprinast (a cyclic GMP phosphodiesterase inhibitor). Intraplantar administration of bremazocine (20, 40 and 50 microg) caused a dose-dependent peripheral antihyperalgesia against carrageenan-induced hyperalgesia. The possibility of the higher dose of bremazocine (50 microg) having central or systemic effect was excluded since administration of the drug into the left paw did not elicit antinociception in the contralateral paw. However, when the dose of bremazocine was increased to 100 microg, a significant increase in the nociceptive threshold was observed, as measured in the hyperalgesic contralateral paw. Peripheral antihyperalgesia induced by bremazocine (50 microg) was significantly reduced in a dose-dependent manner when N(G)-nitro-L-arginine (6, 9, 12 and 25 microg) or methylene blue (250, 375 and 500 microg) was injected before. Previous treatment with 50 microg of zaprinast (which had no effect when administered alone) potentiated the antihyperalgesic effect of bremazocine (20 microg). Our data suggest that bremazocine elicits peripheral antinociception by activation of the L-arginine/NO/cyclic GMP pathway and that nitric oxide is an intermediary in this mechanism, forming cyclic GMP.

Topics: Analgesics; Animals; Arginine; Benzomorphans; Cyclic GMP; Guanylate Cyclase; Hyperalgesia; Male; Methylene Blue; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Pain; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Wistar; Receptors, Opioid, kappa

Isolated rat retina was preloaded with [(14)C]glutamate and subsequently superfused to follow release of glutamate (Glu). After 20 min of superfusion in the dark, exposure of the [(14)C]Glu preloaded rat retina to a single train of white light pulses reduced Glu efflux significantly in the absence as well as in the presence of low (4 microM) and high (0.5 mM) concentrations of the Glu uptake inhibitor trans-L-pyrrolidine-2,4-dicarboxylate (t-PDC). The dark-light response was the highest in the presence of 4 microM t-PDC by establishing a plateau at 75% +/- 7% of the tonic Glu release in the dark (100%). Displaying transient to saturating responses with increasing relative luminance, time series of four trains of white light pulses arrived at a plateau of 85% +/- 10%. The cyclic guanosine monophosphate (cGMP) phosphodiesterase inhibitor Zaprinast (200 microM) antagonized the effect of the light series, leading to a plateau of 115% +/- 9%. Exposure of the retina to the guanylyl cyclase inhibitor LY83583 (30 and 100 microM) showed fast, transient responses characterized by peaks at 90% +/- 1% and 80% +/- 3%, respectively.

Topics: Aminoquinolines; Animals; Carbon Radioisotopes; Cyclic GMP; Dark Adaptation; Dicarboxylic Acids; Enzyme Inhibitors; Glutamic Acid; Male; Neurons; Neurotransmitter Uptake Inhibitors; Organ Culture Techniques; Phosphodiesterase Inhibitors; Photic Stimulation; Purinones; Pyrrolidines; Rats; Rats, Long-Evans; Retina; Synaptic Transmission; Vision, Ocular

Proinflammatory cytokines produced by activated glial cells may in turn augment the immune/inflammatory reactions of glial cells through autocrine and paracrine routes. The NO/cGMP signaling represents one of the reactions of activated glial cells. We investigated whether the production of proinflammatory cytokines by glial cells is affected by NO-dependent downstream cGMP signaling. In primary cultures of mixed astrocytes and microglial cells, zaprinast (0.1 mM), an inhibitor of cGMP-selective phosphodiesterases, enhanced the basal and LPS (1.0 microg/ml)-induced secretion of TNF-alpha and IL-1beta. Zaprinast also enhanced NO production induced by LPS or IFN-gamma (100 U/ml), and in microglial cell cultures, but not in astrocyte cultures, zaprinast enhanced the basal and the IFN-gamma-induced production of the cytokines, TNF-alpha and IL-1beta, and of NO. This upregulation by zaprinast was partially inhibited by KT5823 (1.0 microM), an inhibitor of protein kinase G. The LPS-induced production of TNF-alpha, IL-1beta, and NO was inhibited by ODQ (50 microM), an inhibitor of soluble guanylyl cyclase, and by KT5823. Immunohistochemical analysis of mixed glial cell cultures showed that LPS/IFN-gamma-induced iNOS expression and the enhanced expression of iNOS by zaprinast were restricted to microglial cells. Zaprinast enhanced the IFN-gamma (200 U/ml)-induced expression of MHC Class II molecules in astrocytes and microglial cells in mixed cultures, but did not enhance this IFN-gamma-induced expression in pure astrocytes, which lacked paracrine TNF-alpha from microglial cells. Summarizing, zaprinast, which is associated with cGMP/protein kinase G signaling, may augment central immune/inflammatory reactions, possibly via the increased production of TNF-alpha and IL-1beta by activated microglial cells.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Astrocytes; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Cyclic GMP; Histocompatibility Antigens Class II; Interferon-gamma; Interleukin-1; Microglia; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Purinones; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

The species-specific sound production of acoustically communicating grasshoppers can be stimulated by pressure injection of both nicotinic and muscarinic agonists into the central body complex and a small neuropil situated posterior and dorsal to it. To determine the role of muscarinic acetylcholine receptors (mAChRs) in the control of acoustic communication behavior and to identify the second-messenger pathways affected by mAChR-activation, muscarinic agonists and membrane-permeable drugs known to interfere with specific mechanisms of intracellular signaling pathways were pressure injected to identical sites in male grasshopper brains. Repeated injections of small volumes of muscarine elicited stridulation of increasing duration associated with decreased latencies. This suggested an accumulation of excitation over time that is consistent with the suggested role of mAChRs in controlling courtship behavior: to provide increasing arousal leading to higher intensity of stridulation and finally initiating a mating attempt. At sites in the brain where muscarine stimulation was effective, stridulation could be evoked by forskolin, an activator of adenylate cyclase (AC); 8-Br-cAMP-activating protein kinase A (PKA); and 3-isobuty-1-methylxanthine, leading to the accumulation of endogenously generated cAMP through inhibition of phosphodiesterases. This suggested that mAChRs mediate excitation by stimulating the AC/cAMP/PKA pathway. In addition, muscarine-stimulated stridulation was inhibited by 2'-5'-dideoxyadenonsine and SQ 22536, two inhibitors of AC; H-89 and Rp-cAMPS, two inhibitors of PKA; and by U-73122 and neomycin, two agents that inhibit phospholipase C (PLC) by independent mechanisms. Because the inhibition of AC, PKA, or PLC by various individually applied substances entirely suppressed muscarine-evoked stridulation in a number of experiments, activation of both pathways, AC/cAMP/PKA and PLC/IP(3)/diacylglycerine, appeared to be necessary to mediate the excitatory effects of mAChRs. With these studies on an intact "behaving" grasshopper preparation, we present physiological relevance for mAChR-evoked excitation mediated by sequential activation of the AC- and PLC-initiated signaling pathways that has been reported in earlier in vitro studies.

Topics: Acetylcholine; Adenine; Adenylyl Cyclases; Animal Communication; Animals; Brain; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Dideoxyadenosine; Diglycerides; Enzyme Inhibitors; Estrenes; Grasshoppers; Inositol 1,4,5-Trisphosphate; Isoquinolines; Muscarine; Muscarinic Agonists; Phosphodiesterase Inhibitors; Purinones; Pyrrolidinones; Receptors, Muscarinic; Second Messenger Systems; Sphingosine; Sulfonamides; Thapsigargin; Thionucleotides; Type C Phospholipases

L-arginine-nitric oxide (NO) pathway participates in the physiology and in many pathological processes in the eye, such as glaucoma. The aim of the present study was to compare the ocular hypotensive effect of different NO-donors, and to get more information on the role of cyclic guanosine 3',5'-monophosphate (cGMP) in this process. The test compounds were administered topically or intravitreally in the eye of a normotensive rabbit. Intraocular pressure (IOP) was measured with a pneumatonometer after topical anesthesia. The metabolites of NO (nitrite, nitrate, NOx) and cGMP were assayed from the aqueous humor and plasma. NO-synthase (NOS) protein expression was assayed in the ciliary body by Western blotting. The maximal lowering of IOP was achieved as follows: atriopeptin III (concentration 78 (microM, decrease in IOP 50%), atriopeptin II (84 (microM 37%). 8-Br-cGMP (90 mM, 37%), zaprinast + 8-Br-cGMP (1 mM + 90 mM, 34%), L-arginine (1 mM, 29%), SNP (40 mM, 28%), nitrosocaptopril (100 mM, 28%), S-nitrosothiol (SNOG) (10 mM, 27%), YC-1 (10 (microM, 25%), zaprinast + SNP (1 mM + 40 mM, 22%), spermine NONOate (100 mM, 20%) [corrected]. The decrease in IOP lasted for 2-5 hr, except with atriopeptin II and III, when IOP values were first normalized in 6 hr and 2 days, respectively. In conclusion, the results of the present study indicate that by increasing the activity of L-arginine/NO/cGMP-pathway it is possible to lower IOP in rabbits equally to the currently used antiglaucomatous drugs.

Topics: Animals; Arginine; Cyclic GMP; Female; Indazoles; Intraocular Pressure; Male; Nitric Oxide Donors; Nitric Oxide Synthase; Purinones; Rabbits

The endogenous substance lysophosphatidic acid (LPA) has been found to generate proliferation of cultured smooth muscle cells (SMC). Therefore, the effect of LPA on human benign prostate hyperplasia (BPH) could be of interest.. The proliferative effect of LPA on cultured human prostatic SMC from specimens obtained at trans-urethral resection of the prostate (TURP) because of BPH, was analyzed by [3H]-thymidine and [35S]-methionine incorporation. In addition, LPA stimulated BPH SMC were treated with papaverin, forskolin, sildenafil or zaprinast, well known to increase the intracellular level of cAMP or cGMP.. LPA produced a dose-dependent increase in BPH SMC, both regarding DNA- and protein-synthesis with EC50 values of 3 and 10 microM, respectively. Furthermore, both papaverin, a general phosphodiesterase inhibitor regarding cAMP hydrolyzes, and forskolin, an adenylyl cyclase stimulating agent, inhibited the LPA-stimulated DNA replication in a dose dependent manner with IC50 = 2.5, and 0.35 microM, respectively. cGMP increasing agents, such as the NO-donors SIN-1 and SNAP, produced a weak anti-proliferative response. However, both phosphodiesterase 5 inhibitors sildenafil (Viagra) and zaprinast efficiently blocked DNA replication. In addition, when the protein synthesis was examined, we found that the LPA response was significantly inhibited by forskolin and papaverin.. The major conclusion of this investigation is that the endogenous serum component LPA, is able to promote human BPH SMC growth. In addition, our study indicates that cyclic nucleotides can inhibit this effect. Future clinical studies will be needed to determine if different specific phosphodiesterase inhibitors per se or in combination could represent a new therapeutic possibility for the treatment of BPH.

Topics: Cell Division; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic GMP; DNA; Dose-Response Relationship, Drug; Growth Inhibitors; Humans; Lysophospholipids; Male; Muscle, Smooth; Papaverine; Phosphodiesterase Inhibitors; Piperazines; Prostatic Hyperplasia; Protein Biosynthesis; Proteins; Purines; Purinones; Sildenafil Citrate; Stimulation, Chemical; Sulfones

Regulation of vasoactive intestinal peptide (VIP) release by nitric oxide (NO) was investigated in the hamster jejunum. Electrical field stimulation and applied NO (3-100 microM) evoked biphasic hyperpolarizations consisting of an initial transient hyperpolarizing component followed by a second more slowly developing component (late component). The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (200 microM) abolished the biphasic inhibitory junction potential evoked by electrical field stimulation. The NO scavenger oxyhemoglobin (50 microM) and the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 10 microM) abolished both components of the inhibitory junction potentials and the NO-induced hyperpolarizations. VIP(6-28) (1 microM), which abolished VIP (3 microM)-induced hyperpolarizations, also inhibited the late components of the inhibitory junction potentials and the NO-induced hyperpolarizations. ODQ inhibited VIP release and cAMP production by electrical field stimulation and NO application. N(6)-2,0-Dibutyryladenosine 3',5'-cyclic monophosphate (0.1-3 mM) caused a membrane hyperpolarization. These results suggest that NO may stimulate VIP release from enteric nerves in the hamster jejunum. In addition, we propose that NO and NO-stimulated VIP contribute to the early and late components of the inhibitory junction potentials, respectively, in the circular smooth muscle cells of the hamster jejunum.

Topics: Animals; Cricetinae; Cyclic AMP; Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Guanylate Cyclase; Jejunum; Male; Membrane Potentials; Mesocricetus; Muscle, Smooth; Myenteric Plexus; Neural Inhibition; NG-Nitroarginine Methyl Ester; Nitrergic Neurons; Nitric Oxide; Oxadiazoles; Oxyhemoglobins; Peptide Fragments; Phosphodiesterase Inhibitors; Purinones; Rolipram; Synaptic Transmission; Vasoactive Intestinal Peptide

1. The effects of L-NAME and zaprinast were investigated (i.v.) on reflex-evoked changes in bladder and urethral pressures in urethane-anaesthetized female rats. 2. L-NAME attenuated reflex-evoked urethral relaxations (65+/-10%), while zaprinast potentiated these responses (68+/-24%). L-NAME and zaprinast also increased baseline urethral pressure and urethral striated muscle (EUS-EMG) activity. These drugs had little effect on the bladder. 3. Following pre-treatment with alpha-bungarotoxin (i.v.) to block urethral striated muscle, L-NAME and zaprinast failed to increase baseline urethral pressure. Further zaprinast failed to alter the size of reflex-evoked urethral relaxations. 4. Intra-urethral zaprinast caused a significant increase while sodium nitroprusside (SNP) and isoprenaline caused decreases in urethral pressure (+14+/-3%, -25+/-5%, -29+/-7%, respectively). These changes were associated with increases in EUS-EMG activity. After chlorisondamine (i.v.), zaprinast caused a significant fall in urethral pressure, while the decrease in urethral pressure caused by SNP and isoprenaline was potentiated. No changes in EUS-EMG activity occurred. 5. These results indicate that a nitrergic pathway mediates reflex-evoked urethral smooth muscle relaxations. The data also indicates that there is a background release of NO, which reduces sphincter skeletal muscle activity. Further, the ability of zaprinast to potentiate nitrergic evoked urethral relaxations involves an increase in striated muscle tone. This appears to be an indirect result of smooth muscle relaxation and is mediated, at least in part, by a chlorisondamine-sensitive mechanism.

Topics: Animals; Cyclic GMP; Electromyography; Enzyme Inhibitors; Female; Muscle Relaxation; Muscle, Skeletal; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Sprague-Dawley; Reflex; Urethra; Urinary Bladder

The effects of selective and non-selective 3',5'-cyclic nucleotide phosphodiesterase (PDE) inhibitors on cGMP and cAMP accumulation were studied in rat hippocampal slices incubated in vitro. The following PDE inhibitors were used: vinpocetine and calmidazolium (PDE1 selective), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, PDE2 selective), SK&F 95654 (PDE3 selective), rolipram (PDE4 selective), SK&F 96231 (PDE5 selective), the mixed type inhibitors zaprinast and dipyridamole, and the non-selective inhibitors 3-isobutyl-1-metylxanthine (IBMX) and caffeine. cGMP levels were increased in the presence of different concentrations of IBMX, EHNA, dipyridamole, vinpocetine and rolipram. cGMP immunocytochemistry showed that incubation with different inhibitors in the presence and/or absence of sodium nitroprusside resulted in pronounced differences in the extent and regional localization of the cGMP response and indicate that PDE activity in the hippocampus is high and diverse in nature. The results suggest an interaction between cGMP and cAMP signalling pathways in astrocytes of the rat hippocampus.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Caffeine; Colforsin; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 1; Guanylate Cyclase; Hippocampus; Image Processing, Computer-Assisted; Immunohistochemistry; In Vitro Techniques; Male; NG-Nitroarginine Methyl Ester; Nitroprusside; Norepinephrine; Phosphodiesterase Inhibitors; Purinones; Radioimmunoassay; Rats; Signal Transduction

The role of vascular endothelial growth factor (VEGF), a potent endothelium-specific angiogenic factor, in the regulation of angiotensin-converting enzyme (ACE) in cultured human umbilical vein endothelial cells (HUVECs) was studied. VEGF (0.07-1.2 x 10(-6) mmol/l) caused a dose-dependent increase in ACE measured in intact endothelial cells and increased the expression of ACE mRNA. The stimulatory effect of VEGF was inhibited by pretreatment of endothelial cells with the tyrosine kinase inhibitor herbimycin (4.35 x 10(-5) mmol/l). The stimulatory effect of VEGF was potentiated by the selective cGMP phosphodiesterase inhibitor zaprinast (0.1 mmol/l). The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l) suppressed the stimulatory effect of VEGF. The nonselective cyclooxygenase (COX) inhibitor indomethacin (5 microM) and the selective COX-2 inhibitor NS-398 (5 microM) potentiated the stimulatory effect of VEGF, whereas the selective COX-1 inhibitor resveratrol (5 microM) was without effect. ACE induction by VEGF was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (2.5 x 10(-3) mmol/l) and by downregulating PKC with phorbol 12-myristate 13-acetate. In summary, VEGF induced ACE in cultured HUVECs. Intracellular events such as tyrosine kinase activation, PKC activation, and increase of cGMP were probably involved in ACE induction by VEGF. Nitric oxide may partially contribute to ACE induction by VEGF. The powerful capacity of VEGF to increase ACE in endothelial cells shown here suggests a synergistic relation between VEGF and the renin-angiotensin system in vascular biology and pathophysiology.

Topics: Benzoquinones; Cell Division; Cells, Cultured; Cyclic GMP; Cyclooxygenase Inhibitors; Endothelial Growth Factors; Endothelium, Vascular; Enzyme Inhibitors; Epoprostenol; Gene Expression Regulation, Enzymologic; Humans; Indoles; Indomethacin; Lactams, Macrocyclic; Lymphokines; Maleimides; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitrobenzenes; Peptidyl-Dipeptidase A; Phosphodiesterase Inhibitors; Protein Kinase C; Protein-Tyrosine Kinases; Purinones; Quinones; Rifabutin; RNA, Messenger; Signal Transduction; Sulfonamides; Umbilical Veins; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Peripheral autonomic neurones release nitric oxide (NO) upon nerve activation. However, the regulation of neuronal NO formation is poorly understood. We used the cyclic guanosine 3',5'-monophosphate (cGMP) analogue 8-Br-cGMP, the soluble guanylyl cyclase (sGC) stimulator YC-1, the phosphodiesterase inhibitor zaprinast and the sGC inhibitor ODQ to study whether the sGC/cGMP pathway is involved in regulation of neuronal NO release in nerve plexus-containing smooth muscle preparations from guinea pig colon. Electrical stimulation of the preparation evoked release of NO/NO(-)(2). In the presence of 8-Br-cGMP, YC-1 and zaprinast (all at 10(-4) M) the NO/NO(-)(2)-release increased to 152 +/- 16% (P < 0.05), 164 +/- 37% (P < 0.05) and 290 +/- 67% (P < 0.05) of controls, respectively. Conversely, ODQ (10(-5) M) decreased the evoked release of NO/NO(-)(2) to 49 +/- 7% (P < 0.05) of controls. Our data suggest that the sGC/cGMP pathway modulates NO release. Thus it is likely that NO exerts a positive feedback on its own release from peripheral autonomic neurones.

Topics: Animals; Arginine; Colon; Cyclic GMP; Dimethyl Sulfoxide; Electrophysiology; Enzyme Inhibitors; Guanylate Cyclase; Guinea Pigs; Immunohistochemistry; Male; Muscle, Smooth; Neurons; Nitric Oxide; Oxadiazoles; Phosphodiesterase Inhibitors; Purinones; Quinoxalines

Current gene therapy models are limited by inadequate vector delivery. Increases in microvascular permeability have been shown to improve adenovirus-mediated gene transfer to ex vivo and in vivo models. We explored the intracellular mechanism underlying the permeabilizing effects of vascular endothelial growth factor (VEGF). Using an ex vivo model of coronary perfusion in rabbits, we found a dose-response relationship between VEGF and the efficiency of adenoviral gene transfer. Inhibitors of nitric oxide synthase and guanylate cyclase prevented the VEGF effect, and analogues of nitric oxide and cGMP mimicked the effect. Co-administration of phosphodiesterase-5 inhibitors and VEGF caused a synergistic increase in gene delivery. These results can be readily applied to existing models to further optimize vector delivery for gene therapy.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenoviruses, Human; Animals; beta-Galactosidase; Cyclic GMP; Drug Synergism; Endothelial Growth Factors; Gene Transfer Techniques; Genes, Reporter; Genetic Vectors; Guanylate Cyclase; Heart; Humans; Isoquinolines; Lymphokines; Myocardium; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroglycerin; omega-N-Methylarginine; Phosphodiesterase Inhibitors; Piperazines; Purines; Purinones; Pyridines; Rabbits; Sildenafil Citrate; Sulfones; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Liver disease resulting from common bile duct ligation (CBDL) causes abnormal sodium metabolism that is manifested by resistance to the natriuretic action of atrial natriuretic peptide (ANP). This resistance is corrected both in vitro and in vivo by zaprinast, a selective inhibitor of a guanosine cyclic-3'-5'-monophosphate (cGMP)-specific phosphodiesterase (PDE5). Several other PDEs with affinity for cGMP are expressed in kidney and could also be involved in this response.. We measured cGMP hydrolysis in inner medullary collecting duct (IMCD) cell homogenates from kidneys of sham-operated and CBDL rats and quantitated the amount of PDE5 protein by Western blotting and immunoprecipitation studies. We also characterized ANP responsiveness in vivo of kidneys of anesthetized sham and CBDL rats by measuring sodium excretion before and after volume expansion (VE).. Kinetic analysis of PDE5 activity in homogenates of IMCD cells isolated from kidneys of sham-operated rats indicated a Vmax of 85.3 +/- 1.7 versus 157 +/- 2.9 pmol/mg/min from CBDL rats (P < 0.01), without a difference in Km. Enzyme activity was inhibited competitively by 1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazol[3,4d]-pyrimidin-4-(5H)-one (DMPPO), a potent and specific inhibitor of PDE5, with an apparent Ki of 4.5 +/- 0.7 and 4.9 +/- 0.7 nmol/L and an IC50 of 6.1 +/- 0.8 and 8.7 +/- 0.7 nmol/L in sham and CBDL rats, respectively (P = NS). DMPPO exhibited very poor inhibitory activity against the calcium-calmodulin-dependent PDE1 in IMCD homogenates from sham rats (Ki 1.3 +/- 0.1 micromol/L and IC50 1.9 +/- 0.2 micromol/L). Western analysis using an antiserum made against bovine lung PDE5 revealed a twofold increase in PDE5 protein in cytosolic extracts from IMCD of CBDL rat kidneys compared with sham-operated controls, and immunoprecipitation studies indicated that the increase in PDE5 protein accounted for the observed increase in cGMP hydrolysis. DMPPO (10 nmol/L) normalized the blunted ANP-dependent cGMP accumulation by IMCD cells from CBDL rats in vitro. Intrarenal infusion of DMPPO (0.5 nmol/min) in CBDL rats corrected both the impaired natriuretic response to VE and the blunted VE-related increase in urinary cGMP excretion from the infused, but not the contralateral kidney.. These results demonstrate that renal resistance to ANP in CBDL rats is accompanied by heightened activity of PDE5, which is due largely to an increase in PDE5 protein. Other PDEs could contribute only a minor part to the enhanced cGMP hydrolysis observed in kidneys of CBDL rats. This PDE5-dependent ANP resistance may represent an important contributor to the sodium retention of liver disease.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Allopurinol; Animals; Atrial Natriuretic Factor; Common Bile Duct; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Drug Resistance; Hydrolysis; Kidney; Ligation; Male; Natriuresis; Phosphodiesterase Inhibitors; Plasma Substitutes; Purinones; Rats; Rats, Sprague-Dawley; Sodium Chloride

We studied the role of cGMP in nitric oxide (NO)-induced changes in lung liquid production (J(v)) in chronically instrumented fetal sheep. Forty-five studies were done in which J(v) was measured by a tracer dilution technique. Left pulmonary arterial flow (Q(lpa)) was measured by a Doppler flow probe. There were two series of experiments. In the first, we gave 8-bromo-cGMP, a cGMP analog, by either the pulmonary vascular or intraluminal route; in the second, we used agents to inhibit or enhance endogenous cGMP activity. When infused directly into the pulmonary circulation, 8-bromo-cGMP significantly increased Q(lpa) but had no effect on J(v). Conversely, when instilled into the lung liquid, 8-bromo-cGMP had no effect on Q(lpa) but significantly reduced J(v). Inhibition of guanylate cyclase activity with methylene blue totally blocked, whereas phosphodiesterase inhibition with Zaprinast significantly enhanced, the effect of instilled NO on J(v). Thus the reduction in lung liquid caused by NO appears to be mediated by cGMP, perhaps through a direct effect on the pulmonary epithelium.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Blood Gas Analysis; Blood Pressure; Cyclic GMP; Drug Administration Routes; Enzyme Inhibitors; Fetus; Guanylate Cyclase; Lung; Methylene Blue; Nitric Oxide; Pulmonary Circulation; Purinones; Sheep; Vascular Resistance

N-acetyl-L-cysteine, a thiol compound, has been shown to potentiate the inhibition of platelet aggregation exerted by organic nitrates and to increase the anti-aggregating effect of L-arginine, which promotes endogenous synthesis of nitric oxide (NO) acting as substrate of platelet constitutive nitric oxide synthase (NOS). It is not known whether this thiol can exert direct effects on platelet aggregability.. 14 healthy male volunteers provided platelet samples to investigate whether N-acetyl-L-cysteine directly influences platelet function and intraplatelet levels of 3',5' cyclic guanosine monophosphate (cGMP), which represents the second messenger involved in NO-induced antiaggregation. Some experiments were repeated in the presence of NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), of nitric oxide-sensitive guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), of the selective cGMP phosphodiesterase inhibitor zaprinast and of calcium ionophores (A23187, ionomycin).. N-acetyl-L-cysteine at 3000-6000 micromol L-1 decreases the responses of human platelets both in platelet-rich plasma (aggregation induced by adenosine 5-diphosphate) and in whole blood (aggregation induced by collagen). The anti-aggregating effect was prevented by preincubation with L-NMMA and guanylyl cyclase inhibitor ODQ. In resting platelets, N-acetyl-L-cysteine increased the levels of cGMP starting from a concentration of 3000 micromol L-1. Permeabilized platelets exhibited an increased sensitivity to the anti-aggregating effect of N-acetyl-L-cysteine. Also, cGMP phosphodiesterase inhibition or the increase in calcium availability, enhanced N-acetyl-L-cysteine effects on platelets.. N-acetyl-L-cysteine exerts direct anti-aggregating effects through an increased bioavailability of platelet nitric oxide.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Acetylcysteine; Adenosine Diphosphate; Adult; Calcimycin; Cell Membrane Permeability; Collagen; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Humans; Ionomycin; Male; omega-N-Methylarginine; Oxadiazoles; Platelet Aggregation; Platelet Aggregation Inhibitors; Purinones; Quinoxalines

To clarify the contribution of intracellular Ca(2+) concentration ([Ca(2+)](i))-dependent and -independent signaling mechanisms in arteriolar smooth muscle (aSM) to modulation of arteriolar myogenic tone by nitric oxide (NO), released in response to increases in intraluminal flow from the endothelium, changes in aSM [Ca(2+)](i) and diameter of isolated rat gracilis muscle arterioles (pretreated with indomethacin) were studied by fluorescent videomicroscopy. At an intraluminal pressure of 80 mmHg, [Ca(2+)](i) significantly increased and myogenic tone developed in response to elevations of extracellular Ca(2+) concentration. The Ca(2+) channel inhibitor nimodipine substantially decreased [Ca(2+)](i) and completely inhibited myogenic tone. Dilations to intraluminal flow (that were inhibited by N(omega)-nitro-L-arginine methyl ester) or dilations to the NO donor S-nitroso-N-acetyl-DL-penicillamine (that were inhibited by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) were not accompanied by substantial decreases in aSM [Ca(2+)](i). 8-Bromoguanosine cGMP and the cGMP-specific phosphodiesterase inhibitor zaprinast significantly dilated arterioles yet elicited only minimal decreases in [Ca(2+)](i). Thus flow-induced endothelial release of NO elicits relaxation of arteriolar smooth muscle by a cGMP-dependent decrease of the Ca(2+) sensitivity of the contractile apparatus without substantial changes in the pressure-induced level of [Ca(2+)](i).

Topics: Animals; Arterioles; Calcium; Cyclic GMP; In Vitro Techniques; Muscle, Skeletal; Muscle, Smooth, Vascular; Nitric Oxide; Penicillamine; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Wistar; Regional Blood Flow; S-Nitroso-N-Acetylpenicillamine; Vasodilator Agents; Vasomotor System

The aim was to investigate the presence and activity of cGMP hydrolysing phosphodiesterases in guinea pig basilar arteries and the effect of selective and non-selective phosphodiesterase inhibitors on cerebral artery dilatation involving the nitric oxide (NO)-guanosine cyclic 3'5-monophosphate (cGMP) pathway. Immunoreactivity to phosphodiesterases 1A, 1B and 5, but not phosphodiesterase 1C was found in fractions of homogenised cerebral arteries eluted by high-pressure liquid chromatography (HPLC). Both the phosphodiesterase 1 inhibitor 8-methoxymethyl-1-methyl-3-(2methylpropyl)-xanthine (8-MM-IBMX) and the phosphodiesterase 5 inhibitors zaprinast and dipyridamole induced dilatation of cerebral arteries. The dilatory response to 8-MM-IBMX was reduced by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 microM) and endothelial removal and restored by sodium nitroprusside (0.1 microM) pretreatment, indicating a close relation to the nitric oxide-cGMP pathway. The responses to zaprinast and dipyridamole, however, were not only moderately affected, but also restored by sodium nitroprusside (0.1 microM) pretreatment. At high concentrations, the dilatory effects of zaprinast and dipyridamole were partly caused by cGMP-independent mechanisms. Targeting the phosphodiesterases present in cerebral arteries, with selective inhibitors or activators of phosphodiesterase, may be a possible new way of treating cerebrovascular disease.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Basilar Artery; Cerebral Arteries; Cyclic AMP; Cyclic GMP; Dipyridamole; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; Guinea Pigs; In Vitro Techniques; Isoenzymes; Male; Milrinone; Nitroprusside; Oxadiazoles; Pentoxifylline; Phosphodiesterase Inhibitors; Purinones; Quinoxalines; Vasodilation; Vasodilator Agents

We examined the effect of 3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine (ibudilast), which has been clinically used for bronchial asthma and cerebrovascular disorders, on cell viability induced in a model of reperfusion injury. Ibudilast at 10 - 100 microM significantly attenuated the H(2)O(2)-induced decrease in cell viability. Ibudilast inhibited the H(2)O(2)-induced cytochrome c release, caspase-3 activation, DNA ladder formation and nuclear condensation, suggesting its anti-apoptotic effect. Phosphodiesterase inhibitors such as theophylline, pentoxyfylline, vinpocetine, dipyridamole and zaprinast, which increased the guanosine-3',5'-cyclic monophosphate (cyclic GMP) level, and dibutyryl cyclic GMP attenuated the H(2)O(2)-induced injury in astrocytes. Ibudilast increased the cyclic GMP level in astrocytes. The cyclic GMP-dependent protein kinase inhibitor KT5823 blocked the protective effects of ibudilast and dipyridamole on the H(2)O(2)-induced decrease in cell viability, while the cyclic AMP-dependent protein kinase inhibitor KT5720, the cyclic AMP antagonist Rp-cyclic AMPS, the mitogen-activated protein/extracellular signal-regulated kinase inhibitor PD98059 and the leukotriene D(4) antagonist LY 171883 did not. KT5823 also blocked the effect of ibudilast on the H(2)O(2)-induced cytochrome c release and caspase-3-like protease activation. These findings suggest that ibudilast prevents the H(2)O(2)-induced delayed apoptosis of astrocytes via a cyclic GMP, but not cyclic AMP, signalling pathway.

Topics: Alkaloids; Animals; Animals, Newborn; Apoptosis; Astrocytes; Carbazoles; Cell Survival; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytochrome c Group; Dipyridamole; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydrogen Peroxide; Indoles; Mitochondria; Pentoxifylline; Peptide Hydrolases; Phosphodiesterase Inhibitors; Purinones; Pyridines; Rats; Rats, Wistar; Reperfusion Injury; Signal Transduction; Theophylline; Vinca Alkaloids

Recent studies implicate increased cGMP synthesis as a postreceptor contributor to reduced cardiac sympathetic responsiveness. Here we provide the first evidence that modulation of this interaction by cGMP-specific phosphodiesterase PDE5A is also diminished in failing hearts, providing a novel mechanism for blunted beta-adrenergic signaling in this disorder. In normal conscious dogs chronically instrumented for left ventricular pressure-dimension analysis, PDE5A inhibition by EMD82639 had modest basal effects but markedly blunted dobutamine-enhanced systolic and diastolic function. In failing hearts (tachypacing model), however, EMD82639 had negligible effects on either basal or dobutamine-stimulated function. Whole myocardium from failing hearts had 50% lower PDE5A protein expression and 30% less total and EMD92639-inhibitable cGMP-PDE activity. Although corresponding myocyte protein and enzyme activity was similar among groups, the proportion of EMD82639-inhibitable activity was significantly lower in failure cells. Immunohistochemistry confirmed PDE5A expression in both the vasculature and myocytes of normal and failing hearts, but there was loss of z-band localization in failing myocytes that suggested altered intracellular localization. Thus, PDE5A regulation of cGMP in the heart can potently modulate beta-adrenergic stimulation, and alterations in enzyme localization and reduced synthesis may blunt this pathway in cardiac failure, contributing to dampening of the beta-adrenergic response.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenylyl Cyclases; Adrenergic beta-Agonists; Animals; Blood Pressure; Cardiac Output, Low; Colforsin; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Dobutamine; Dogs; Female; Heart; Hemodynamics; Immunohistochemistry; Male; Myocardial Contraction; Myocardium; Phosphodiesterase Inhibitors; Piperazines; Purines; Purinones; Pyrazoles; Receptors, Adrenergic, beta; Signal Transduction; Sildenafil Citrate; Sulfones

The interaction between nitric oxide (NO) and renin is controversial. cAMP is a stimulating messenger for renin, which is degraded by phosphodiesterase (PDE)-3. PDE-3 is inhibited by cGMP, whereas PDE-5 degrades cGMP. We hypothesized that if endogenous cGMP was increased by inhibiting PDE-5, it could inhibit PDE-3, increasing endogenous cAMP, and thereby stimulate renin. We used the selective PDE-5 inhibitor zaprinast at 20 mg/kg body wt ip, which we determined would not change blood pressure (BP) or renal blood flow (RBF). In thiobutabarbital (Inactin)-anesthetized rats, renin secretion rate (RSR) was determined before and 75 min after administration of zaprinast or vehicle. Zaprinast increased cGMP excretion from 12.75 +/- 1.57 to 18.67 +/- 1.87 pmol/min (P < 0.003), whereas vehicle had no effect. Zaprinast increased RSR sixfold (from 2.95 +/- 1.74 to 17.62 +/- 5.46 ng ANG I. h(-1) x min(-1), P < 0.024), while vehicle had no effect (from 4.08 +/- 2.02 to 3.87 +/- 1.53 ng ANG I x h(-1) x min(-1)). There were no changes in BP or RBF. We then tested whether the increase in cGMP could be partially due to the activity of the neuronal isoform of NO synthase (nNOS). Pretreatment with the nNOS inhibitor 7-nitroindazole (7-NI; 50 mg/kg body wt) did not change BP or RBF but attenuated the renin-stimulating effect of zaprinast by 40% compared with vehicle. In 7-NI-treated animals, zaprinast-stimulated cGMP excretion was attenuated by 48%, from 9.17 +/- 1.85 to 13.60 +/- 2.15 pmol/min, compared with an increase from 10.94 +/- 1.90 to 26.38 +/- 3.61 pmol/min with zaprinast without 7-NI (P < 0.04). This suggests that changes in endogenous cGMP production at levels not associated with renal hemodynamic changes are involved in a renin-stimulatory pathway. One source of this cGMP may be nNOS generation of NO in the kidney.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Blood Pressure; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Inhibitors; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Phosphoric Diester Hydrolases; Purinones; Rats; Rats, Sprague-Dawley; Renal Circulation; Renin

The vital dye methylene blue (MB) has been shown to be teratogenic when injected into the amnion in the second trimester. On the other hand, the teratogenic potential of transplacental exposure to MB has not been determined.. MB was administered subcutaneously to ICR (CD-1) mice at 0, 35, 50, 60, or 70 mg/kg on gestation day 8 (plug day = day 0). Teratological assessments were carried out at term gestation, on gestation day 18. Since MB inhibits soluble guanylate cyclase enzyme activity, zaprinast (ZPN), a selective cGMP-phosphodiesterase type V inhibitor, was administered to prevent developmental disorders initiated by MB at 50 mg/kg.. There was a dose-dependent increment of embryolethality. MB treatment also produced axial skeleton and neural tube defects. Coadministration of ZPN (20 mg/kg per three times) abolished completely MB-induced neural tube defects and reduced by one-half the incidence of fetuses exhibiting axial skeletal defects. ZPN did not provide protection against the embryocidal effects of MB.. This study showed that transplacental exposure to MB is teratogenic in the mouse. Coadministration of ZPN prevented partly MB-induced teratogenesis, which supports the hypothesis that imbalance of cGMP pathway accounts, in part, for the teratogenicity of MB.

Topics: Animals; Bone and Bones; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Maternal Exposure; Methylene Blue; Mice; Mice, Inbred ICR; Neural Tube Defects; Phosphodiesterase Inhibitors; Placenta; Purinones; Teratogens; Time Factors

A possible defect of guanosine 3'-5'-cyclic monophosphate (cGMP) content in the renal tissue caused by an increased activity of cGMP phosphodiesterase (PDE) has, so far, not been evaluated in the pathogenesis of renal resistance to endogenous natriuretic peptides (ENP) in cirrhosis with ascites. To test this hypothesis the activity of cGMP-PDE and the concentration of cGMP were evaluated in vitro in the renal tissue of 10 control rats and 10 cirrhotic rats with ascites before and after the intravenous (IV) administration of Zaprinast (Sigma, St. Louis, MO), a specific cGMP-PDE inhibitor (30 microgram/kg/min). Moreover, the effects of the intravenous administration of Zaprinast (15 microgram/kg/min and 30 microgram/kg/min) on renal plasma flow (RPF), glomerular filtration rate (GFR), and urinary sodium excretion (U(Na)V) were evaluated in 10 conscious control rats and 10 conscious cirrhotic rats with ascites. The effects of Zaprinast on plasma renin activity (PRA) was also evaluated in 10 control rats and in 10 cirrhotic rats with ascites. Finally, the effect of Zaprinast on RPF, GFR, and U(Na)V were evaluated in 10 cirrhotic rats after the IV administration of the ENP-receptor antagonist, HS-142-1. The renal content of cGMP was reduced in cirrhotic rats because of increased activity of cGMP-PDE. Zaprinast inhibited cGMP-PDE activity and increased the renal content of cGMP in these animals. The inhibition of cGMP-PDE was associated with an increase in RPF, GFR, and U(Na)V and a reduction in PRA. HS-142-1 prevented any renal effect of Zaprinast in cirrhotic rats. In conclusion, an increased activity of the cGMP-PDE in renal tissue contributes to the renal resistance to ENP in cirrhosis with ascites.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Ascites; Cyclic GMP; Hemodynamics; Kidney; Liver Cirrhosis, Experimental; Male; Phosphodiesterase Inhibitors; Polysaccharides; Purinones; Rats; Rats, Wistar; Renin-Angiotensin System

Calcitonin gene-related peptide (CGRP) is believed to play an important role in maintaining low pulmonary vascular resistance (PVR) and in modulating pulmonary vascular responses to chronic hypoxia; however, the effects of adenovirally mediated gene transfer of CGRP on the response to hypoxia are unknown.. In the present study, an adenoviral vector encoding prepro-CGRP (AdRSVCGRP) was used to examine the effects of in vivo gene transfer of CGRP on increases in PVR, right ventricular mass (RVM), and pulmonary vascular remodeling that occur in chronic hypoxia in the mouse. Intratracheal administration of AdRSVCGRP, followed by 16 days of chronic hypoxia (FIO(2) 0.10), increased lung CGRP and cAMP levels. The increase in pulmonary arterial pressure (PAP), PVR, RVM, and pulmonary vascular remodeling in response to chronic hypoxia was attenuated in animals overexpressing prepro-CGRP, whereas systemic pressure was not altered while in chronically hypoxic mice, angiotensin II and endothelin-1-induced increases in PAP were reduced, whereas decreases in PAP in response to CGRP and adrenomedullin were not changed and decreases in PAP in response to a cAMP phosphodiesterase inhibitor were enhanced by AdRSVCGRP.. In vivo CGRP lung gene transfer attenuates the increase in PVR and RVM, pulmonary vascular remodeling, and pressor responses in chronically hypoxic mice, suggesting that CGRP gene transfer alone and with a cAMP phosphodiesterase inhibitor may be useful for the treatment of pulmonary hypertensive disorders.

Topics: Adenoviridae; Adrenomedullin; Animals; beta-Galactosidase; Calcitonin Gene-Related Peptide; Cyclic AMP; Cyclic GMP; Endothelin-1; Genes, Reporter; Genetic Therapy; Genetic Vectors; Hemodynamics; Humans; Hypertension, Pulmonary; Hypoxia; Lung; Mice; NG-Nitroarginine Methyl Ester; Peptides; Phosphodiesterase Inhibitors; Potassium Channels; Protein Precursors; Purinones; Recombinant Fusion Proteins; Rolipram; Second Messenger Systems; Transfection; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

To determine the effect of combining inhaled nitric oxide (NO) with an inhibitor of guanosine 3',5'-cyclic monophosphate-specific phosphodiesterase on total and segmental lung resistances.. A controlled laboratory study in isolated blood-perfused lungs prepared from lambs.. Animal research facility affiliated with a university teaching hospital.. Five newborn lambs at <48 hrs of life.. Isolated blood-perfused lungs were prepared and treated with indomethacin (40 microg/mL) to inhibit prostaglandin synthesis. After a baseline period of normoxia (28% oxygen), pulmonary hypertension was induced with the thromboxane mimetic U46619 (0.1-0.4 microg/kg/min). During pulmonary hypertension, lungs were studied with inhaled NO only, with infusion of zaprinast only (0.25 mg/kg bolus and 0.05 mg/kg/min infusion), and with a combination of the two. For each study condition, the total pressure decrease across the lung was measured, and the inflow-outflow occlusion technique was used to partition the total pressure gradient measured at constant flow (100 mL/kg/min) into gradients across relatively noncompliant large arteries and veins and more compliant small arteries and veins.. U46619 infusion produced significant pulmonary vasoconstriction. The combination of inhaled NO and zaprinast decreased the total pressure decrease across the lung significantly more than NO alone. This effect was primarily attributable to a significantly greater decrease in gradient across the small artery segment after inhaled NO and zaprinast compared with NO alone.. Guanosine 3',5'-cyclic monophosphate phosphodiesterase inhibition with zaprinast enhances the effect of inhaled NO, particularly in conditions in which small arteries represent the site of resistance. Phosphodiesterase inhibition may be a promising adjunct to inhaled NO for the treatment of persistent pulmonary hypertension.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Administration, Inhalation; Animals; Animals, Newborn; Cyclic GMP; Cyclooxygenase Inhibitors; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Synergism; Drug Therapy, Combination; Hypertension, Pulmonary; In Vitro Techniques; Indomethacin; Nitric Oxide; Phosphodiesterase Inhibitors; Purinones; Time Factors; Vascular Resistance; Vasodilator Agents

The aim of this study was to assess the role of nitric oxide (NO) and endothelin (ET)-1 in the pathophysiology of persistent pulmonary hypertension of the newborn in fetal lambs with a surgically created congenital diaphragmatic hernia (CDH). The pulmonary vascular response to various agonists and antagonists was assessed in vivo between 128 and 132 days gestation. Age-matched fetal lambs served as control animals. Control and CDH lambs had similar pulmonary vasodilator responses to acetylcholine, sodium nitroprusside, zaprinast, and dipyridamole. The ET(A)-receptor antagonist BQ-123 caused a significantly greater pulmonary vasodilatation in CDH than in control animals. The ET(B)-receptor agonist sarafotoxin 6c induced a biphasic response, with a sustained pulmonary vasoconstriction after a transient pulmonary vasodilatation that was not seen in CDH animals. We conclude that the NO signaling pathway in vivo is intact in experimental CDH. In contrast, ET(A)-receptor blockade and ET(B)-receptor stimulation significantly differed in CDH animals compared with control animals. Imbalance of ET-1-receptor activation favoring pulmonary vasoconstriction rather than altered NO-mediated pulmonary vasodilatation is likely to account for persistent pulmonary hypertension of the newborn in fetal lambs with a surgically created CDH.

Topics: Acetylcholine; Animals; Antihypertensive Agents; Cyclic GMP; Dipyridamole; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Female; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Hypertension, Pulmonary; Nitric Oxide; Nitroprusside; Peptides, Cyclic; Phosphodiesterase Inhibitors; Pregnancy; Pulmonary Circulation; Purinones; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Sheep; Vasoconstrictor Agents; Vasodilator Agents; Viper Venoms

Phosphodiesterase type 5 (PDE5) hydrolyzes cyclic guanosine monophosphate in the lung, thereby modulating nitric oxide (NO)/cyclic guanosine monophosphate-mediated pulmonary vasodilation. Inhibitors of PDE5 have been proposed for the treatment of pulmonary hypertension. In this study, we examined the pulmonary and systemic vasodilator properties of sildenafil, a novel selective PDE5 inhibitor, which has been approved for the treatment of erectile dysfunction.. In an awake lamb model of acute pulmonary hypertension induced by an intravenous infusion of the thromboxane analog U46619, we measured the effects of 12.5, 25, and 50 mg sildenafil administered via a nasogastric tube on pulmonary and systemic hemodynamics (n = 5). We also compared the effects of sildenafil (n = 7) and zaprinast (n = 5), a second PDE5 inhibitor, on the pulmonary vasodilator effects of 2.5, 10, and 40 parts per million inhaled NO. Finally, we examined the effect of infusing intravenous l-NAME (an inhibitor of endogenous NO production) on pulmonary vasodilation induced by 50 mg sildenafil (n = 6).. Cumulative doses of sildenafil (12.5, 25, and 50 mg) decreased the pulmonary artery pressure 21%, 28%, and 42%, respectively, and the pulmonary vascular resistance 19%, 23%, and 45%, respectively. Systemic arterial pressure decreased 12% only after the maximum cumulative sildenafil dose. Neither sildenafil nor zaprinast augmented the ability of inhaled NO to dilate the pulmonary vasculature. Zaprinast, but not sildenafil, markedly prolonged the duration of pulmonary vasodilation after NO inhalation was discontinued. Infusion of l-NAME abolished sildenafil-induced pulmonary vasodilation.. Sildenafil is a selective pulmonary vasodilator in an ovine model of acute pulmonary hypertension. Sildenafil induces pulmonary vasodilation via a NO-dependent mechanism. In contrast to zaprinast, sildenafil did not prolong the pulmonary vasodilator action of inhaled NO.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 3',5'-Cyclic-GMP Phosphodiesterases; Acute Disease; Animals; Blood Pressure; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Inhibitors; Hypertension, Pulmonary; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Pulmonary Artery; Pulmonary Circulation; Purines; Purinones; Sheep; Sildenafil Citrate; Sulfones; Vascular Resistance; Vasoconstrictor Agents; Vasodilator Agents; Wakefulness

Phosphodiesterases (PDE) are key enzymes regulating intracellular cyclic nucleotide turnover and, thus, smooth muscle tension. Recent reports have indicated the presence of PDE isoenzymes 1, 2, 4, and 5 in cytosolic supernatants prepared from human ureteral smooth muscle homogenates and the ability of second-generation inhibitors of PDE 3, 4, and 5 to relax KCl-induced tension of human ureteral muscle in vitro. The aim of the present study was to evaluate the functional effects of recently developed, third-generation isoenzyme-selective PDE inhibitors, the nitric oxide (NO)-donating agents sodium nitroprusside (SNP) and dihydropyridine (DHP), which is also described as an antagonist of L-type calcium channels, and the adenylyl cyclase-stimulating drug forskolin on tissue tension and cyclic nucleotide levels of human ureteral smooth muscle segments in vitro. Relaxant responses of human ureteral smooth muscle were investigated in vitro using the organ bath technique. Cyclic nucleotides cAMP and cGMP were determined by specific radioimmunoassay following time and dose-dependent incubation of the ureteral tissue with the drugs. The most pronounced relaxing effects on KCl-induced tension of ureteral smooth muscle were exerted by nitrovasodilator SNP, PDE4 inhibitor rolipram, and PDE5 inhibitors E 4021 and morpholinosulfonyl-pyrazolopyrimidine (MSPP). Relaxing potency of the drugs was paralleled by their ability to elevate intracellular levels of cGMP and cAMP, respectively. Our data suggest the possibility of using selective inhibitors of PDE isoenzymes 4 and 5 in the treatment of ureteral stones and ureteral colic.

Topics: Colforsin; Cyclic AMP; Cyclic GMP; Humans; In Vitro Techniques; Isoquinolines; Muscle, Smooth; Nitroprusside; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperidines; Platelet Aggregation Inhibitors; Potassium Chloride; Purinones; Quinazolines; Rolipram; Tetrahydroisoquinolines; Ureter; Vasodilator Agents

We tested the hypothesis that lowering the level of cyclic GMP would have positive functional effects on isolated rabbit ventricular myocytes regardless of the basal cyclic GMP level. Cell shortening data were collected with a video detector; O(2) consumption data were obtained with a Clark electrode; intracellular cyclic GMP levels were obtained by radioimmunoassay. Data were obtained: (1) at baseline; (2) after the addition of 1H-[1,2,4]oxadiazolo[4, 3-alpha]quinoxaline-1-one (ODQ) 10(-6) and 10(-4) mol/l, a selective soluble guanylyl cyclase inhibitor, and (3) after zaprinast 10(-6) mol/l, a cyclic GMP phosphodiesterase inhibitor, followed by ODQ 10(-6) and 10(-4) mol/l. We found that ODQ 10(-4) mol/l significantly decreased the cyclic GMP level from 493 +/- 75 to 301 +/- 78 (fmol/100,000 myocytes) and increased percent shortening (Pcs, %; 4.9 +/- 0.3 vs. 5.8 +/- 0.6) and maximum rate of shortening (Rs, microm/s; 58.7 +/- 5.7 vs. 73.6 +/- 4.9). Zaprinast significantly increased the cyclic GMP level from 419 +/- 140 to 599 +/- 241 and decreased Pcs (6.2 +/- 0.5 vs. 4.4 +/- 0.4) and Rs (65.5 +/- 5.3 vs. 49.6 +/- 4.3). After zaprinast, ODQ 10(-4) mol/l decreased the cyclic GMP level to 439 +/- 139 and increased percent shortening and rate of shortening by a similar percentage compared to the non-zaprinast treated myocytes. We conclude that in rabbit ventricular myocytes, a reduction in the level of myocyte cyclic GMP increases myocyte function independent of the initial cyclic GMP level.

Topics: Animals; Cyclic GMP; Guanylate Cyclase; Heart; In Vitro Techniques; Myocardium; Oxadiazoles; Oxygen Consumption; Purinones; Quinoxalines; Rabbits

Previously, we reported an abrupt reduction in chick chorioallantoic membrane (CAM) microvascular permeability to macromolecules between days 4.5 and 5.0 of the 21-day gestation. Further, exogenous activation of the cAMP pathway at day 4.5 served to restrict normal macromolecular extravasation. Here, we evaluated the influence of the cGMP pathway on macromolecular efflux at day 5.0. Zaprinast (10(-4) M), a selective inhibitor of the cGMP-specific phosphodiesterase (PDE V), acutely increased basal levels of FITC-dextran 40 extravasation. Further, the cGMP analogue, 8 br-cGMP (10(-4) and 10(-3) M) and the soluble guanylate cyclase activator, sodium nitroprusside (SNP, 10(-5) and 10(-4) M) increased tracer extravasation in a dose-dependent fashion. The cGMP-mediated increase was not associated with gap formation along the junctional clefts, however, vesiculo-vacuolar structures were characteristic of CAM endothelial ultrastructure. KT 5823 (10(-5) M), the highly selective protein kinase G (PKG) inhibitor, also served to increase basal tracer extravasation. The nonselective PDE inhibitor, IBMX (10(-4) M) had no effect alone, but reduced the permeability effects of both 8 br-cGMP and SNP. Rolipram (10(-4) M), a selective PDE IV inhibitor, on the other hand, potentiated the effect of 8 br-cGMP. These results serve to suggest that cAMP degradation, rather than PKG activation, is a principal component of the cGMP-mediated increase in CAM endothelial permeability in vivo.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Allantois; Animals; Capillary Permeability; Chick Embryo; Chorion; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; Microcirculation; Microscopy, Electron; Neovascularization, Physiologic; Nitroprusside; Phosphodiesterase Inhibitors; Purinones; Signal Transduction

In order to determine the mechanism by which nitric oxide (NO) inhibits prolactin release, we investigated the participation of cGMP-dependent cAMP-phosphodiesterases (PDEs) and protein kinase G (PKG) in this effect of NO.. Anterior pituitary glands of male rats were incubated with inhibitors of PDE and PKG with or without sodium nitroprusside (NP). Prolactin release, and cAMP and cGMP concentrations were determined by RIA.. The inhibitory effect of NP (0.5 mmol/l) on prolactin release and cAMP concentration was blocked by EHNA (10(-4)mol/l) and HL-725 (10(-4)mol/l), inhibitors of cGMP-stimulated cAMP-PDE (PDE2). 8-Br-cGMP (10(-4) and 10(-3)mol/l), which mimics cGMP as a mediator of NP effects on prolactin release, also decreased cAMP concentration. Zaprinast (10(-4)mol/l), a selective inhibitor of specific cGMP-PDE (PDE5), potentiated the NP effect on cAMP concentration. Rp-8-[(4-chlorophenyl)thio]-cGMP triethylamine (Rp-8-cGMP, 10(-7)-10(-6)mol/l), an inhibitor of PKG, reversed the effect of NP on prolactin release. The present study suggests that several mechanisms are involved in the inhibitory effect of NO on prolactin release. The activation of PDE2 by cGMP may mediate the inhibitory effect of NO on cAMP concentration and therefore on prolactin release. NO-activated PKG may also be participating in the inhibitory effect of NO on prolactin release.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic AMP; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Male; Nitric Oxide; Nitroprusside; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Pituitary Gland, Anterior; Prolactin; Protein Kinases; Purinones; Rats; Rats, Wistar

Nitric oxide (NO) upregulates ciliary beat frequency (CBF). The present study evaluates mechanisms of the NO-cyclic guanosine monophosphate (cGMP) pathway regulation of CBF. Rat tracheal explants were loaded with 4,5-diaminofluorescein diacetate for the demonstration of NO production by ciliated epithelial cells after L-arginine (L-Arg) stimulation. CBF was measured using phase contrast microscopy and videotape analysis. The roles of NO, soluble guanylate cyclase (sGC), cGMP-dependent protein kinase (PK) G, and phosphodiesterase (PDE) V in regulation of CBF were evaluated. NO synthase (NOS) was activated with L-Arg or inhibited with N(G)-monomethyl-L-Arg. sGC was stimulated with NO donors 1-hydroxy-2-oxo-3- (N-ethyl-2-aminoethyl)-3-ethyl-1-triazene and S-nitroso-L-glutathione or mimicked by 8-bromo-guanosine 3', 5'-cyclic monophosphate (8-Br-cGMP) and inhibited with 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one. The effects of the PKG inhibition with KT5823 and PDE V inhibition with Zaprinast were also examined. The studies demonstrate that ciliated epithelial cells produce NO, which is correlated with CBF stimulation. L-Arg dose- and time-dependently increases CBF, and NO donors, 8-Br-cGMP, and Zaprinast also enhance CBF. Inhibitors of NOS, sGC, and PKG can block the stimulant effect of L-Arg on CBF. Thus, NO is a regulator of CBF acting via sGC and PKG. The NO-cGMP signaling pathway regulates CBF in an autocrine manner in cultured rat ciliated airway epithelium.

Topics: Alkaloids; Animals; Arginine; Carbazoles; Cells, Cultured; Cilia; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; Glutathione; Indoles; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; omega-N-Methylarginine; Oxadiazoles; Purinones; Quinoxalines; Rats; S-Nitrosoglutathione; Signal Transduction; Trachea

Increased vascular smooth muscle cyclic guanine monophosphate (cGMP) results in vascular relaxation. The vascular effects of stimulating cGMP production with 10(-8)-10(-4) M nitroprusside (NP) and inhibiting cGMP hydrolysis with 10(-8)-10(-4) M zaprinast (ZAP), a selective type V inhibitor of cGMP phosphodiesterase (PDE), were assessed in isolated guinea pig hearts and aortic rings. Coronary flow (CF) IC50 values for NP and ZAP, respectively, were 0.8+/-0.1 x 10(-6) M and 3.6+/-0.1 x 10(-6) M; for coronary sinus pO2 IC50 values were 0.7+/-0.1 x 10(-6) M and 3.7+/-0.1 x 10(-6) M. CF increased by 13+/-2% with 10(-6) NP, and by 12+/-2% with 10(-5) M ZAP; percentage O2 extraction (%O2E) decreased by 17+/-3% with NP and 28+/-4% with ZAP. Together, 10(-6) M NP + 10(-5) M ZAP augmented the increased in CF to 23+/-3% of control, and the decrease in percentage O2 extraction (%O2E) to 40+/-4% of control. Other cardiac effects of NP and ZAP were minimal. In norepinephrine preconstricted aortic rings, the IC50 for relaxation was elicited at 0.4+/-0.1 x 10(-6) M NP and 6.1+/-0.1 x 10(-6) M ZAP. NP given with ZAP gave a logarithmic relation so that IC50 [NP] = -(57 log10 [ZAP]) + 416; R2 = 0.95. NP, 3 x 10(-7) M; ZAP, 3 x 10(-6) M; and NP + ZAP combined increased aortic tissue cGMP by eight-, nine-, and 15-fold, respectively. Inhibiting cGMP hydrolysis may be an effective approach to augment vasorelaxation elicited by cGMP synthesis in the heart.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Antihypertensive Agents; Aorta; Coronary Circulation; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Drug Interactions; Electrophysiology; Guinea Pigs; Heart; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Myocardial Contraction; Myocardium; Nitroprusside; Oxygen Consumption; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Vasodilator Agents

Disruption of endothelial barrier properties with development of noncardiogenic pulmonary edema is a major threat in lung ischemia-reperfusion (I/R) injury that occurs under conditions of lung transplantation. Inhaled nitric oxide (NO) reduced vascular leakage in lung I/R models, but the efficacy of this agent may be limited. We coadministered NO and zaprinast, a cGMP-specific phosphodiesterase inhibitor, to further augment the NO-cGMP axis. Isolated, buffer-perfused rabbit lungs were exposed to 4.5 h of warm ischemia. Reperfusion provoked a transient elevation in pulmonary arterial pressure and a negligible rise in microvascular pressure followed by a massive increase in the capillary filtration coefficient and severe lung edema formation. Inhalation of 10 parts/million of NO or intravascular application of 100 microM zaprinast on reperfusion both reduced pressor response and moderately attenuated vascular leakage. Combined administration of both agents induced no additional vasodilation at constant microvascular pressures, but additively protected against capillary leakage paralleled by a severalfold increase in perfusate cGMP levels. In conclusion, combining low-dose NO inhalation and phosphodiesterase inhibition may be suitable for the maintenance of graft function in lung transplantation by amplifying the beneficial effect of the NO-cGMP axis and avoiding toxic effects of high NO doses.

Topics: Animals; Blood Pressure; Capillary Permeability; Compliance; Cyclic GMP; Female; In Vitro Techniques; Ischemia; Male; Nitric Oxide; Organ Size; Phosphodiesterase Inhibitors; Pulmonary Artery; Pulmonary Circulation; Purinones; Rabbits; Reperfusion Injury

Sildenafil (0.1 - 30 microM), a cyclic GMP phosphodiesterase 5 (PDE 5) inhibitor, induced inhibition of electrically evoked contractions of ring segments of human vas deferens from 34 vasectomies. Zaprinast (0.1 - 100 microM), another PDE 5 inhibitor, and the nitric oxide (NO) donor sodium nitroprusside (SNP) (0.1 - 100 microM) had no effect on neurogenic contractions. The inhibition induced by sildenafil was not modified by the inhibitor of guanylate cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) (1 - 30 microM) but it was abolished by the K(+) channel blockers tetraethylammonium (TEA, 1 mM), iberiotoxin (0.1 microM) and charybdotoxin (0.1 microM). Sildenafil, zaprinast and SNP did not affect the contractions induced by noradrenaline. SNP (10 microM) caused elevation of cyclic GMP levels that was potentiated by sildenafil (10 microM) and zaprinast (100 microM). ODQ (10 microM) inhibited the increase in cyclic GMP. Sildenafil inhibits adrenergic neurotransmission in human vas deferens. The inhibition is not related to accumulation of cyclic GMP but is probably due to activation of prejunctional large-conductance Ca(2+)-activated K(+) channels.

Topics: Adult; Cyclic GMP; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Male; Middle Aged; Muscle Contraction; Nitric Oxide; Nitroprusside; Norepinephrine; Phosphodiesterase Inhibitors; Piperazines; Potassium Channels; Purines; Purinones; Sildenafil Citrate; Sulfones; Synaptic Transmission; Vas Deferens

High-frequency stimulation (HFS) of corticostriatal glutamatergic fibers induces long-term depression (LTD) of excitatory synaptic potentials recorded from striatal spiny neurons. This form of LTD can be mimicked by zaprinast, a selective inhibitor of cGMP phosphodiesterases (PDEs). Biochemical analysis shows that most of the striatal cGMP PDE activity is calmodulin-dependent and inhibited by zaprinast. The zaprinast-induced LTD occludes further depression by tetanic stimulation and vice versa. Both forms of synaptic plasticity are blocked by intracellular 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, indicating that an increased cGMP production in the spiny neuron is a key step. Accordingly, intracellular cGMP, activating protein kinase G (PKG), also induces LTD. Nitric oxide synthase (NOS) inhibitors N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) and 7-nitroindazole monosodium salt (7-NINA) block LTD induced by either HFS or zaprinast, but not that induced by cGMP. LTD is also induced by the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and hydroxylamine. SNAP-induced LTD occludes further depression by HFS or zaprinast, and it is blocked by intracellular ODQ but not by L-NAME. Intracellular application of PKG inhibitors blocks LTD induced by HFS, zaprinast, and SNAP. Electron microscopy immunocytochemistry shows the presence of NOS-positive terminals of striatal interneurons forming synaptic contacts with dendrites of spiny neurons. These findings represent the first demonstration that the NO/cGMP pathway exerts a feed-forward control on the corticostriatal synaptic plasticity.

Topics: Animals; Cerebral Cortex; Corpus Striatum; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dendrites; Immunohistochemistry; Neuronal Plasticity; Neurons; Nitric Oxide; Phosphodiesterase Inhibitors; Protein Kinases; Purinones; Rats; Rats, Wistar; Synaptic Transmission; Tetany

The effect of nitric oxide (NO) on the release of bombesin-like immunoreactivity (BLI) was examined in synaptosomes of rat small intestine. The NO donor S-nitroso-N-acetylpenicillamine (SNAP; 10(-7) to 10(-4) M) significantly stimulated BLI release. In the presence of the NO scavenger oxyhemoglobin (10(-3) M) or the guanylate cyclase inhibitor ODQ (10(-5) M), SNAP-induced BLI release was antagonized. In addition, SNAP increased the synaptosomal cGMP content and elevation of cGMP levels by zaprinast (3 x 10(-5) M), an inhibitor of the cGMP-specific phosphodiesterase (PDE) type 5, and increased basal and SNAP-induced BLI release. NO-induced BLI release was blocked by Rp-adenosine 3',5'-cyclic monophosphorothioate (3 x 10(-5) M and 10(-4) M), an inhibitor of the cAMP-dependent protein kinase A, whereas KT-5823 (3 x 10(-6) M) and Rp-8-(4-chlorophenylthio)-cGMP (5 x 10(-5) M), inhibitors of the cGMP-dependent protein kinase G, had no effect. Because cGMP inhibits the cAMP-specific PDE3, thereby increasing cAMP levels, the role of PDE3 was investigated. Trequinsin (10(-8) M), a specific blocker of PDE3, stimulated basal BLI release but had no additive effect on NO-induced release, suggesting a similar mechanism of action. These data demonstrate that because of a cross-activation of cAMP-dependent protein kinase A by endogenous cGMP BLI can be released by NO from enteric synaptosomes.

Topics: Animals; Arginine; Bombesin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Intestine, Small; Male; Nitric Oxide; Nitric Oxide Donors; Nitroarginine; Oxadiazoles; Phosphodiesterase Inhibitors; Protein Kinase Inhibitors; Purinones; Quinoxalines; Rats; Rats, Wistar; Synaptosomes

The effects of transfer of the endothelial nitric oxide synthase (eNOS) gene to the lung were studied in mice. After intratracheal administration of AdCMVbetagal, expression of the beta-galactosidase reporter gene was detected in pulmonary airway cells, in alveolar cells, and in small pulmonary arteries. Gene expression with AdCMVbetagal peaked 1 day after administration and decayed over a 7- to 14-day period, whereas gene expression after AdRSVbetagal transfection peaked on day 5 and was sustained over a 21- to 28-day period. One day after administration of AdCMVeNOS, eNOS protein levels were increased, and there was a small reduction in mean pulmonary arterial pressure and pulmonary vascular resistance. The pressure-flow relationship in the pulmonary vascular bed was shifted to the right in animals transfected with eNOS, and pulmonary vasodepressor responses to bradykinin and the type V cGMP-selective phosphodiesterase inhibitor zaprinast were enhanced, whereas systemic responses were not altered. Pulmonary vasopressor responses to endothelin-1 (ET-1), angiotensin II, and ventilatory hypoxia were reduced significantly in animals transfected with the eNOS gene, whereas pressor responses to norepinephrine and U46619 were not changed. Systemic pressor responses to ET-1 and angiotensin II were similar in eNOS-transfected mice and in control mice. Intratracheal administration of AdRSVeNOS attenuated the increase in pulmonary arterial pressure in mice exposed to the fibrogenic anticancer agent bleomycin. These data suggest that transfer of the eNOS gene in vivo can selectively reduce pulmonary vascular resistance and pulmonary pressor responses to ET-1, angiotensin II, and hypoxia; enhance pulmonary depressor responses; and attenuate pulmonary hypertension induced by bleomycin. Moreover, these data suggest that in vivo gene transfer may be a useful therapeutic intervention for the treatment of pulmonary hypertensive disorders.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenoviridae; Angiotensin II; Animals; Antimetabolites, Antineoplastic; beta-Galactosidase; Bleomycin; Blood Flow Velocity; Blood Pressure; Bradykinin; Cyclic GMP; Endothelin-1; Gene Transfer Techniques; Genes, Reporter; Hypertension, Pulmonary; Hypoxia; Mice; Mice, Inbred Strains; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Norepinephrine; Phosphodiesterase Inhibitors; Pulmonary Alveoli; Pulmonary Artery; Pulmonary Circulation; Pulmonary Wedge Pressure; Purinones; Sympathomimetics; Vasoconstrictor Agents

1. The cellular mechanisms by which endogenous nitric oxide (NO) modulates spontaneous motility were investigated in rat isolated proximal colon. The mechanical activity was detected as changes in intraluminal pressure. 2. Apamin (1-100 nM) produced a concentration-dependent increase in the amplitude of the spontaneous pressure waves. The maximal contractile effect was of the same degree as that produced by Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM) and the joint application of apamin plus L-NAME had no additive effects. Apamin (0.1 microM) reduced the inhibitory effects (i.e. reduction in the amplitude of the pressure waves) induced by sodium nitroprusside (SNP) (1 nM - 10 microM) or 8-Br-cyclic GMP (1-100 microM). 3. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (0.1-5 microM), inhibitor of NO-stimulated guanylate cyclase, produced a concentration-dependent increase of the spontaneous contractions. ODQ (1 microM) in the presence of apamin (0.1 microM) did not produce any further increase in the contraction amplitude, whereas after L-NAME (100 microM) it decreased the spontaneous contractions. ODQ (1 microM) reduced the SNP inhibitory effects. 4. Zaprinast (1-50 microM), inhibitor of cyclic GMP phosphodiesterase, produced a concentration-dependent decrease of the spontaneous contractions. The effects of zaprinast were significantly reduced in the presence of apamin (0.1 microM) or L-NAME (100 microM). 5. These results suggest that small conductance Ca2+-dependent K+ channels and cyclic GMP are involved in the modulation of the spontaneous contractile activity in rat proximal colon. Cyclic GMP production system and opening of apamin-sensitive K+ channels appear to work sequentially in transducing an endogenous NO signal.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Colon; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Oxadiazoles; Phosphodiesterase Inhibitors; Potassium Channels; Purinones; Quinoxalines; Rats; Rats, Wistar

The role of 3',5'-cyclic guanosine monophosphate (cGMP) in the activation of mitogen-activated protein kinases (MAPKs) was investigated in rat pinealocytes. Treatment with dibutyryl cGMP (DBcGMP) dose-dependently increased the phosphorylation of both p44 and p42 isoforms of MAPK. This effect of DBcGMP was abolished by PD98059 (a MAPK kinase inhibitor), H7 (a nonspecific protein kinase inhibitor), and KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor]. Elevation of cellular cGMP content by treatment with norepinephrine, zaprinast (a cGMP phosphodiesterase inhibitor), or nitroprusside was effective in activating MAPK. Natriuretic peptides that were effective in elevating cGMP levels in this tissue were also effective in activating MAPK. Our results indicate that, in this neuroendocrine tissue, the cGMP/PKG signaling pathway is an important mechanism used by hormones and neurotransmitters in activating MAPK.

Topics: Alkaloids; Animals; Atrial Natriuretic Factor; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cells, Cultured; Cyclic GMP; Dibutyryl Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavonoids; Indoles; Isoquinolines; Male; Naphthalenes; Natriuretic Peptide, C-Type; Neurosecretory Systems; Nitroprusside; Phosphodiesterase Inhibitors; Phosphorylation; Pineal Gland; Protein Kinase C; Purinones; Rats; Rats, Sprague-Dawley; Sulfhydryl Reagents; Sulfonamides; Thionucleotides

We tested the hypothesis that in isolated cardiac myocytes, the negative metabolic and functional effects of cyclic guanosine monophosphate (GMP) are mediated by cyclic GMP protein kinase activity, and that these effects are altered in renal hypertensive (one-kidney, one-clip, 1K1C) cardiac hypertrophic rabbits. By using isolated cardiac myocytes from control and 1K1C rabbits, oxygen consumption (Mvo2; O2 nl/ min/10(5) cells), cyclic GMP (fmol/10(5) cells), and cell shortening (percentage) data were collected (a) at baseline; (b) with cyclic GMP protein kinase inhibitors KT5823 (10(-6) M) or Rp8-pCPT-cGMP (5 x 10(-6) M); (c) with the cyclic GMP phosphodiesterase inhibitor zaprinast (10(-6), 10(-4) M); and (d) with zaprinast (10(-6), 10(-4) M) and protein kinase inhibitors. Basal levels of cyclic GMP were similar in control versus 1K1C myocytes (62 +/- 10 vs. 66 +/- 17 pmol/10(5) myocytes). Zaprinast produced a dose-dependent increase in cyclic GMP in both control and 1K1C myocytes. The addition of KT5823 did not significantly affect cyclic GMP levels. Zaprinast significantly and dose dependently decreased Mvo2, and KT5823 partially restored it in control and 1K1C. Zaprinast also significantly decreased percentage shortening, and KT5823 partially restored it in control. Similar results were obtained with Rp-8pCPT-cGMP, although neither inhibitor was effective without zaprinast. The hypertrophied myocytes demonstrated comparable responses to all agents. These data suggest that the cyclic GMP protein kinase activity was not significant under basal conditions; however, the importance of cyclic GMP protein kinase in control and 1K1C myocytes was significant under conditions of increased intracellular cyclic GMP.

Topics: Alkaloids; Animals; Carbazoles; Cardiomegaly; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Indoles; Myocardium; Oxygen Consumption; Purinones; Rabbits

The management of sick newborn infants who have sustained a hypoxic insult is a common clinical problem but relatively little is known about the recovery process. The aim of this study was to investigate this process in newborn piglets.. Thirty five newborn piglets were exposed to chronic hypobaric hypoxia for three days, either from birth, three or 14 days of age, and were allowed to recover for one, three, or six days. Control animals of relevant age were also studied. The heart weight ratio and pulmonary arterial muscularity were measured. Endothelial dependent and independent relaxation of the isolated intrapulmonary conduit arteries was determined in classical organ chamber studies, together with measurement of basal and stimulated cGMP accumulation.. After six days of recovery the hypoxia induced right ventricular hypertrophy and pulmonary arterial medial hypertrophy had decreased in all animals but values were still abnormal in the two younger age groups. Relaxation was still impaired during the first three days of recovery in all groups, had normalised by six days in the two youngest groups, but relaxation (both endothelium dependent and independent) remained impaired in older animals. In these older animals basal nitric oxide (NO) production and basal and stimulated cGMP accumulation was normal.. The recovery of the smooth muscle cells lags behind that of the endothelial cells. A normal stimulated increase in cGMP with reduced relaxation suggests an altered threshold for cGMP effected relaxation. These findings help to explain why some hypoxic infants require protracted NO therapy.

Topics: Acetylcholine; Animals; Animals, Newborn; Calcimycin; Cyclic GMP; Enzyme Inhibitors; Hypertension, Pulmonary; Hypertrophy; Hypertrophy, Right Ventricular; Hypoxia; Nitric Oxide; omega-N-Methylarginine; Phosphodiesterase Inhibitors; Pulmonary Artery; Purinones; Swine; Tunica Intima; Vasoconstriction; Vasodilator Agents

Nitric oxide (NO), a mediator involved in penile erection, is synthesized by the nitric oxide synthase (NOS) family of enzymes. It has been shown that NOS activity decreases with age. To determine whether adenoviral-mediated overexpression of endothelial NOS (eNOS) could enhance erectile responses, we administered a recombinant adenovirus containing the eNOS gene (AdCMVeNOS) into the corpora cavernosum of the aged rat. Adenoviral expression of the beta-galactosidase reporter gene was observed in cavernosal tissue 1 day after intracavernosal administration of AdCMVbetagal; 1 day after administration of AdCMVeNOS, transgene expression was confirmed by immunoblot staining of eNOS protein, and cGMP levels were increased. The increase in cavernosal pressure in response to cavernosal nerve stimulation was enhanced in animals transfected with eNOS, and erectile responses to acetylcholine and zaprinast were enhanced at a time when the erectile response to the NO donor sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate was not altered. These results suggest that in vivo gene transfer of eNOS, alone or in combination with a type V phosphodiesterase inhibitor, may constitute a new therapeutic intervention for the treatment of erectile dysfunction.

Topics: Acetylcholine; Adenoviridae; Aging; Animals; Cyclic GMP; Electric Stimulation; Erectile Dysfunction; Gene Transfer Techniques; Genetic Therapy; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Penis; Purinones; Rats; Rats, Sprague-Dawley

Nitric oxide (NO), which is known to inhibit systemic vascular smooth muscle cell proliferation, is used in the management of neonatal pulmonary hypertension. Our objectives were to determine: (1) if endogenous NO production by neonatal porcine pulmonary artery smooth muscle cells (PASMCs) varied with oxygen tension in vitro, and (2) the effect of exogenous NO and inducible NO synthase (iNOS) stimulators and inhibitors on PASMC proliferation and apoptosis. PASMCs were exposed to different conditions (varying PO(2), NO donors and scavengers, iNOS stimulators and inhibitors) and proliferation, apoptosis, and cyclic guanosine 5(')-monophosphate (cGMP) assessed. PASMCs proliferated best between 5 and 10% O(2) but cGMP levels were similar at all oxygen levels. NO donors (S-nitroso-N-acetyl-penicillamine, NOC-12, NOC-18) inhibited PASMC proliferation in a dose-dependent manner with associated cGMP increases, while NO scavengers (carboxy-PTIO), iNOS stimulators (interleukin-1beta, lipopolysaccharide), and iNOS inhibitors (aminoethylisothiourea) did not affect proliferation or cGMP. No changes in apoptosis were found at the concentrations of NO donors or iNOS stimulators used. These results suggest that while exogenous NO inhibits PASMC proliferation, endogenous NO may not regulate proliferation during changes in oxygen tension or cytokine levels. Endothelial derived and inhaled NO may attenuate smooth muscle hyperplasia and vascular remodeling. Inducible NOS in porcine PASMCs appears resistant to stimulation with interleukin-1beta or lipopolysaccharide. The mechanisms underlying hypoxia-mediated changes in PASMC proliferation require investigation.

Topics: Animals; Animals, Newborn; Apoptosis; Cell Division; Cells, Cultured; Cyclic GMP; DNA Damage; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Developmental; Immunohistochemistry; Interleukin-1; Lipopolysaccharides; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroso Compounds; Oxygen; Penicillamine; Phosphodiesterase Inhibitors; Pulmonary Artery; Purinones; Swine

Phosphodiesterases (PDEs) are an important component of the signal transduction pathway during the erectile response. To determine the PDE isoforms in the corpora cavernosa in the cat and to establish the functional presence of PDE 4 in human cavernosal tissue, the erectile response to intracavernosal phosphodiesterase (PDE) inhibitors alone and the combination of PDE inhibitors and prostaglandin E1 (PGE1) was evaluated in the anesthetized cat. The in vitro formation of cAMP and cGMP in human cavernosal smooth muscle cells (HCSMCs) treated with PGE1 and rolipram in primary culture was also measured.. In pentobarbital-anesthetized cats, increases in intracavernosal pressure, penile length, and duration of erectile response were determined after intracavernosal injections of (i) the type 3 cAMP-specific, cGMP-inhibitable PDE inhibitor, milrinone, (ii) the type 4 cAMP-specific PDE inhibitor, rolipram, (iii) the type 5 cGMP-specific PDE inhibitor, zaprinast, and (iv) the combination of rolipram and PGE1. Systemic arterial pressure was concurrently assessed in these experiments. All responses to PDE inhibitors were compared with a control triple-drug combination comprised of papaverine (1.65 mg.), PGE1 (0.5 microg.), and phentolamine (25 microg.). HCSMCs were incubated with PGE1 (3 microM) and rolipram (10 microM) individually or in combination up to 2 hours at 37C. The intracellular cAMP and cGMP was extracted by cold absolute ethanol and measured (pmol./10(6) cells) by a commercially available EIA kit.. Milrinone (3 to 100 microg.), rolipram (3 to 100 microg.), and zaprinast (3 to 100 microg.) induced dose-dependent increases in intracavernosal pressure and penile length (p <0.05) when administered intracavernosally. The maximum increase in cavernosal pressure in response to zaprinast was associated with no significant change in systemic arterial pressure. When rolipram was combined with PGE1 (0.1 microg.), the increases in intracavernosal pressure and the duration of erectile response were significantly higher (p <0.05) and longer (p <0.05) than those observed when rolipram alone was injected intracavernosally. PGE1 (3 microM) and rolipram (10 microM) produced significant increases (p <0.05) in the accumulation of intracellular cAMP levels in HCSMCs in primary culture above those of the baseline values while intracellular levels of cGMP did not change.. PDE inhibitors administered intracavernosally caused dose-dependent increases in cavernosal pressure in the cat. When a specific cAMP PDE inhibitor was combined with PGE1, the erectile response was enhanced and intracellular levels of cAMP were increased in HCSMCs in primary culture. These data suggest further exploration of the combination of various PDE inhibitors and PGE1 in the pharmacologic treatment of erectile dysfunction and provide functional evidence for the presence of PDE 4 isoenzyme in human penile cavernosal cells.

Topics: Alprostadil; Animals; Cats; Cells, Cultured; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Male; Milrinone; Penile Erection; Penis; Phosphodiesterase Inhibitors; Purinones; Rolipram

Involvement of phosphodiesterase isoenzymes (PDEs) in guanosine-3',5'-cyclic monophosphate (cGMP) hydrolysis was analyzed in aortic smooth muscle cells. Four families of PDEs were separated from pig aorta: PDE1 (calcium-calmodulin-activated), PDE3 (cGMP-inhibited), PDE4 (adenosine 3',5'-cyclic monophosphate [cAMP]-specific), and PDE5 (cGMP-specific). Within this PDE complement, PDE1 and PDE5 mostly contributed to the hydrolysis of cGMP both in the presence and absence of calcium-calmodulin. The role of these isoenzymes in cGMP degradation was analyzed in primary cultures of porcine aortic smooth muscle cells after stimulation with sodium nitroprusside (SNP) or atrial natriuretic factor (ANF). Pretreatment with 10 microM zaprinast, a concentration that selectively inhibits PDE5, did not potentiate the SNP- or ANF-induced rise of cGMP, questioning the widespread opinion that only PDE5 accounts for cGMP hydrolysis in this tissue. Further evidence came from experiments assessing the effect of zaprinast or 3-isobutyl-1-methylxanthine at concentrations inhibiting both type 1 and type 5 isoenzymes, in which this potentiation was clearly seen. Contribution of cGMP egression to the control of intracellular cGMP levels after SNP or ANF stimulation was also investigated. Shortly after guanylate cyclase activation, extracellular cGMP levels surpassed intracellular levels. However, comparison of the amounts of cGMP extruded to the extracellular medium with those degraded by PDEs leads to the conclusion that efflux is of relatively minor importance in regulating intracellular cGMP levels. In cells made tolerant to SNP, selective PDE5 inhibition synergistically increased intra- and extracellular cGMP amounts after SNP stimulation. These results indicate a previously undescribed greater relevance of PDE5 after tolerance development in aortic smooth muscle cells.

Topics: Animals; Aorta; Atrial Natriuretic Factor; Biological Transport; Cells, Cultured; Cyclic GMP; Drug Interactions; Isoenzymes; Muscle, Smooth, Vascular; Nitroprusside; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Swine; Vasodilator Agents

We tested the hypothesis that the negative functional effects of cyclic GMP (cGMP) would be greater after increasing cyclic AMP (cAMP), because of the action of cGMP-affected cAMP phosphodiesterases in cardiac myocytes and that this effect would be altered in left ventricular hypertrophy (LVH) produced by aortic valve plication. Myocyte shortening data were collected using a video edge detector, and O2 consumption was measured by O2 electrodes during stimulation (5 ms, 1 Hz, in 2 mM Ca2+) from control (n = 7) and LVH (n = 7) dog ventricular myocytes. cAMP and cGMP were determined by a competitive binding assay. cAMP was increased by forskolin and milrinone (10(-6) M). cGMP was increased with zaprinast and decreased by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxilin-1-one (ODQ) both at 10(-6) and 10(-4) M, with and without forskolin or forskolin + milrinone. Zaprinast significantly decreased percent shortening in control (9 +/- 1 to 7 +/- 1%) and LVH (10 +/- 1 to 7 +/- 1%) myocytes. It increased cGMP in control (36 +/- 5 to 52 +/- 7 fmol/10(5) myocytes) and from the significantly higher baseline value in LVH (71 +/- 12 to 104 +/- 18 fmol/10(5) myocytes). ODQ increased myocyte function and decreased cGMP levels in control and LVH myocytes. Forskolin + milrinone increased cAMP levels in control (6 +/- 1 to 15 +/- 2 pmol/10(5) myocytes) and LVH (8 +/- 1 to 18 +/- 2 pmol/10(5) myocytes) myocytes, as did forskolin alone. They also significantly increased percent shortening. There were significant negative functional effects of zaprinast after forskolin + milrinone in control (15 +/- 2 to 9 +/- 1%), which were greater than zaprinast alone, and LVH (12 +/- 1 to 9 +/- 1%). This was associated with an increase in cGMP and a reduction in the increased cAMP induced by forskolin or milrinone. ODQ did not further increase function after forskolin or milrinone in control myocytes, despite lowering cGMP. However, it prevented the forskolin and milrinone induced increase in cAMP. In hypertrophy, ODQ lowered cGMP and increased function after forskolin. ODQ did not affect cAMP after forskolin and milrinone in LVH. Thus, the level of cGMP was inversely correlated with myocyte function. When cAMP levels were elevated, cGMP was still inversely correlated with myocyte function. This was, in part, related to alterations in cAMP. The interaction between cGMP and cAMP was altered in LVH myocytes.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Cardiomegaly; Colforsin; Cyclic AMP; Cyclic GMP; Dogs; Heart; Milrinone; Oxadiazoles; Oxygen Consumption; Purinones; Quinoxalines

1. The sensitivity of the soluble guanylate cyclase (sGC)-cyclic guanosine-3',5'-monophosphate (cyclic GMP) system to nitric oxide (NO) was investigated in mouse aorta from wild type (WT) and NO synthase (NOS) knockout (KO) animals. 2. The NO donor, spermine-NONOate (SPER-NO) was more potent in aortas from eNOS KO mice compared to WT (pEC50 7.30+/-0.06 and 6.56+/-0.04, respectively; n=6; P<0.05). In contrast, the non-NO based sGC activator, YC-1 was equipotent in vessels from eNOS WT and KO mice. The sensitivity of aortas from nNOS and iNOS KO animals to SPER-NO was unchanged. Forskolin (an adenylate cyclase activator), was equipotent in vessels from eNOS WT and KO animals. 3. The cyclic GMP analogue, 8-Br-cGMP was equipotent in eNOS WT and KO mice (pEC50 4. 38+/-0.04 and 4.40+/-0.05, respectively; n=5; P>0.05). Zaprinast (10-5 M) a phosphodiesterase type V (PDE V) inhibitor, had no effect on the response to SPER-NO in vessels from eNOS WT or KO mice. 4. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 3x10-4 M) increased the potency of SPER-NO in aortas from WT mice (pEC50 6. 64+/-0.02 and 7.37+/-0.02 in the absence and presence of L-NAME, respectively; n=4; P<0.05). 5. In summary, there is increased sensitivity of vessels from eNOS KO animals to NO. Cyclic AMP-mediated dilatation is unchanged, consistent with a specific up-regulation of sGC - cyclic GMP signalling. The functional activity of cyclic GMP-dependent protein kinase (G-kinase) and PDE V was also unchanged, suggesting that sGC is the site of up-regulation. These alterations in the sensitivity of the sGC - cyclic GMP pathway might represent a mechanism for the dynamic regulation of NO bioactivity.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; 8-Bromo Cyclic Adenosine Monophosphate; Animals; Aorta, Thoracic; Colforsin; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Guanylate Cyclase; Homeostasis; Indazoles; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrogen Oxides; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Platelet Aggregation Inhibitors; Purinones; Spermine

Airway smooth muscle (ASM) hypertrophy and hyperplasia are important determinants of bronchial responsiveness in asthma, and agents that interfere with these processes may prevent airway remodeling. We tested the hypothesis that activators of soluble and particulate guanylyl cyclases would inhibit human ASM cell (HASMC) proliferation. We report that the nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP; 10(-6) to 10(-4) M) and sodium nitroprusside (10(-5) to 10(-3) M) and human atrial natriuretic peptide [ANP-(1-28); 10(-8) to 10(-6) M], which activate soluble and particulate guanylyl cyclases, respectively, inhibited serum- and thrombin-induced proliferation of cultured HASMCs. The antimitogenic effect of SNAP was reversed by hemoglobin (10(-5) M), an NO scavenger, suggesting that NO donation was involved. The antiproliferative effects of SNAP and ANP-(1-28) were potentiated by the cGMP-specific phosphodiesterase zaprinast and mimicked by 8-bromo-cGMP (10(-6) to 10(-3) M), suggesting that cGMP-dependent mechanisms were involved. However, first, ANP-(1-28) produced a smaller antiproliferative effect than SNAP in contrast to their abilities to elevate cGMP, and second, rat ANP-(104-126), which binds selectively to ANP clearance receptors without elevating cGMP, had a small antiproliferative effect, suggesting that cGMP-independent mechanisms were also involved. These results provide evidence for a novel antiproliferative effect of NO and ANP in HASMCs mediated through cGMP-dependent and cGMP-independent mechanisms.

Topics: Asthma; Atrial Natriuretic Factor; Blood Proteins; Cell Division; Cells, Cultured; Coloring Agents; Cyclic GMP; Diuretics; Hemoglobins; Hemostatics; Humans; Hyperplasia; Lung; Mitogens; Muscle, Smooth; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Penicillamine; Peptide Fragments; Phosphodiesterase Inhibitors; Purinones; Tetrazolium Salts; Thiazoles; Thrombin; Vasodilator Agents

We studied the modes of activation of the salt-secreting rectal gland of the spiny dogfish, Squalus acanthias, by the native cardiac peptide CNP. The stimulatory action of CNP in isolated perfused glands is inhibited by 10 mM procaine, presumably by blocking release of vasoactive intestinal peptide (VIP) from nerves. Procaine reduces the slope of the dose-response curve of human CNP and that of shark CNP (each P < 0.0001). CNP increases short-circuit current in cultured rectal gland cells from 4.8 +/- 1.6 to 27.0 +/- 7.8 microA/cm2. It also stimulates the secretion of chloride in isolated perfused glands in the presence of 10 mM procaine from 72 +/- 31 to 652 +/- 173 microeq. h(-1). g(-1). These results suggest that CNP has a direct cellular action not mediated by the neural release of VIP. The residual stimulation of perfused glands in the presence of procaine was almost completely inhibited by staurosporine [10 nM; an inhibitor of protein kinase C (PKC)] from 652 +/- 173 to 237 +/- 61 microeq. h(-1). g(-1). Although CNP stimulates guanylyl cyclase in shark rectal gland, chloride secretion of perfused glands was not elicited by 8-bromoadenosine-cGMP (8-BrcGMP) alone nor by the activator of PKC phorbol ester. The combination of PKC activation and 8-BrcGMP infusion, however, stimulated chloride secretion in perfused glands from 94 +/- 30 to 506 +/- 61 microeq. h(-1). g(-1), a level comparable to that observed in glands blocked with procaine. Several parallel pathways appear to be synergistic in activating chloride secretion stimulated by CNP in the rectal gland.

Topics: Animals; Cells, Cultured; Chlorides; Cyclic GMP; Dogfish; Dose-Response Relationship, Drug; Humans; Kinetics; Membrane Potentials; Natriuretic Peptide, C-Type; Perfusion; Procaine; Purinones; Salt Gland; Swine; Tetradecanoylphorbol Acetate

The present study was performed to determine whether measurement of penile diameter in an in vivo rat model is useful for pharmacologic and physiologic investigations on penile erection. Penile erection induced by electrical stimulation of the cavernous nerve was monitored by measuring the penile diameter sonomicrometrically with a pair of 10-MHz piezoelectric crystals glued to the opposite surfaces of the adventitia of the penile erectile chamber in anesthetized rats. Using this method, we examined the effects of a nitric oxide (NO) synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), and a well-known phosphodiesterase 5 (PDE5) inhibitor, zaprinast, on the maximal developed penile diameter (D-max) and the time from the maximum response to 50% recovery (T50%) of the maximum response as an index of the duration of penile erection. An intravenous injection of L-NAME at a dose of 10 mg/kg significantly inhibited D-max produced by cavernous electrical stimulation at 5 to 50 V, without affecting T50%. Sequential intravenous infusions of 10, 30, 100, and 300 microg/kg/min of zaprinast at 30-min intervals did not show any effect on D-max, heart rate, and systolic arterial pressure, although doses of 100 and 300 microg/kg/min significantly prolonged T50% and the maximum dose decreased diastolic arterial pressure. Moreover, zaprinast produced a more prominent increase in cyclic guanosine monophosphate (cGMP) levels than cyclic adenosine monophosphate levels in the plasma taken at the end of the maximum dose infusion. Measurement of murine penile diameter with a sonomicrometrical device, indicating that a NO-cGMP-PDE5 pathway plays a pivotal role in the penile diameter increase and its maintenance, would be useful for pharmacologic and physiologic investigations on penile erection.

Topics: Animals; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Hemodynamics; Male; NG-Nitroarginine Methyl Ester; Penile Erection; Penis; Purinones; Rats; Rats, Sprague-Dawley; Sonication; Time Factors

Nitric oxide activates guanylate cyclase to form cGMP, comprising a signalling system that is believed to be a distinct mechanism for increasing glucose transport and metabolism in skeletal muscle. The effects of a selective cGMP phosphodiesterase inhibitor, zaprinast, on basal glucose utilization was investigated in incubated rat soleus muscle preparations isolated from both insulin-sensitive (lean Zucker; Fa/?) and insulin-resistant (obese Zucker; fa/fa) rats. Zaprinast at 27 microM significantly increased cGMP levels in incubated soleus muscle isolated from lean, but not obese, Zucker rats. Muscles were incubated with 14C-labelled glucose and various concentrations of zaprinast (3, 27 and 243 microM). Zaprinast (at 27 and 243 microM) significantly increased rates of net and 14C-labelled lactate release and of glycogen synthesis in lean Zucker rat soleus muscle; glucose oxidation was also increased by 27 microM zaprinast. In addition, regardless of concentration, the phosphodiesterase inhibitor failed to increase any aspect of 14C-labelled glucose utilization in soleus muscles isolated from obese Zucker rats. The maximal activity of nitric oxide synthase (NOS) was significantly decreased in insulin-resistant obese Zucker muscles. Thus the lack of effect of zaprinast in insulin-resistant skeletal muscle is consistent with decreased NOS activity. To test whether there is a defect in insulin-resistant skeletal muscle for endogenous activation of guanylate cyclase, soleus muscles were isolated from both insulin-sensitive and insulin-resistant Zucker rats and incubated with various concentrations of the NO donor sodium nitroprusside (SNP; 0.1, 1, 5 and 15 mM). SNP significantly increased rates of net and 14C-labelled lactate release, as well as glucose oxidation in muscles isolated from both insulin-sensitive and insulin-resistant rats. A decreased response to SNP was observed in the dose-dependent generation of cGMP within isolated soleus muscles from insulin-resistant rats. A possible link between impaired NO/cGMP signalling and abnormal glucose utilization by skeletal muscle is discussed.

Topics: Animals; Cell Fractionation; Cyclic GMP; Female; Glucose; Glycogen; Insulin; Insulin Resistance; Lactic Acid; Muscle, Skeletal; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Obesity; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Zucker; Signal Transduction

Increased nitric oxide (NO) production plays a critical role in the mammalian pulmonary vascular adaptation to extrauterine life. NO activates soluble guanylate cyclase, increasing intracellular cGMP concentrations, thereby inducing relaxation of vascular smooth muscle. cGMP is inactivated by cyclic nucleotide phosphodiesterases (PDEs). One PDE isozyme, PDE5, specifically hydrolyzes cGMP, is abundant in lung tissues, and modifies the pulmonary vasodilatory response to exogenous NO. To investigate the regulation of PDE5 gene expression during pulmonary development, PDE5 mRNA levels, as well as cGMP-metabolizing PDE enzyme activity, were measured in the lungs of perinatal and adult rats. RNA blot hybridization revealed that PDE5 mRNA was detectable in fetal lung tissue as early as 18.5 d of the 22-d term gestation and reached maximal levels in neonatal lungs. mRNA levels in adult rat lungs were 3-4-fold less than the levels measured in lungs of 1- and 8-d-old rats. Pulmonary cGMP hydrolytic activity in 1-d-old animals was 30-fold greater than the cGMP hydrolytic activity of adult rat lungs. Zaprinast, a specific PDE5 antagonist, inhibited 52 and 56% of cGMP hydrolytic activity in lungs of 1- and 8-d-old rats, respectively, but only 18% of the activity in adult lungs. In situ hybridization revealed that PDE5 mRNA transcripts were present in the vascular smooth muscle cells of neonatal and adult lungs. PDE5 mRNA was also detected in the alveolar walls of neonatal rat lungs. These results demonstrate that the gene encoding PDE5 is abundantly expressed in the lungs of perinatal rats, and is available to participate in the mammalian pulmonary vascular transition to extrauterine life. Extravascular PDE5 gene expression in neonatal lungs suggests a potentially important nonvascular role for this enzyme during pulmonary development.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Gene Expression Regulation, Enzymologic; Hydrolysis; In Situ Hybridization; Lung; Phosphodiesterase Inhibitors; Purinones; Rats; RNA, Messenger

Zaprinast, an inhibitor of guanosine-3',5'-cyclic monophosphate (cGMP)-selective phosphodiesterase, augments smooth muscle relaxation induced by endothelium-dependent vasodilators (including inhaled nitric oxide [NO]). The present study was designed to examine the effects of inhaled nebulized zaprinast, alone, and combined with inhaled NO.. Eight awake lambs with U46619-induced pulmonary hypertension sequentially breathed two concentrations of NO (5 and 20 ppm), followed by inhalation of aerosols generated from solutions containing four concentrations of zaprinast (10, 20, 30, and 50 mg/ml). The delivered doses of nebulized zaprinast at each concentration (mean +/- SD) were 0.23 +/- 0.06, 0.49 +/- 0.14, 0.71 +/- 0.24, and 1.20 +/- 0.98 mg x kg(-1) x min(-1), respectively. Each lamb also breathed NO (5 and 20 ppm) and zaprinast (0.23 +/- 0.06 mg x kg[-1] x min[-1]) in combination after a 2-h recovery period.. Inhaled NO selectively dilated the pulmonary vasculature. Inhaled zaprinast selectively dilated the pulmonary circulation and potentiated and prolonged the pulmonary vasodilating effects of inhaled NO. The net transpulmonary release of cGMP was increased by inhalation of NO, zaprinast, or both. The duration of the vasodilation induced by zaprinast inhalation was greater than that induced by NO inhalation.. Aerosolization of a cGMP-selective phosphodiesterase inhibitor alone or combined with NO may be a useful noninvasive therapeutic method to treat acute or chronic pulmonary hypertension.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Administration, Inhalation; Aerosols; Animals; Cyclic GMP; Dose-Response Relationship, Drug; Drug Combinations; Hemodynamics; Hypertension, Pulmonary; Lung; Nitric Oxide; Phosphodiesterase Inhibitors; Pulmonary Circulation; Purinones; Sheep; Vasoconstrictor Agents; Vasodilation

The known mammalian 3':5'-cyclic nucleotide phosphodiesterases (PDEs) contain a conserved region located toward the carboxyl terminus, which constitutes a catalytic domain. To identify amino acids that are important for catalysis, we introduced substitutions at 23 conserved residues within the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE; PDE5). Wild-type and mutant proteins were compared with respect to Km for cGMP, kcat, and IC50 for zaprinast. The most dramatic decrease in kcat was seen with H643A and D754A mutants with the decrease in free energy of binding (DeltaDeltaGT) being about 4.5 kcal/mol for each, which is within the range predicted for loss of a hydrogen bond involving a charged residue. His643 and Asp754 are conserved in all known PDEs and are strong candidates to be directly involved in catalysis. Substitutions of His603, His607, His647, Glu672, and Asp714 also produced marked changes in kcat, and these residues are likely to be important for efficient catalysis. The Y602A and E775A mutants exhibited the most dramatic increases in Km for cGMP, with calculated DeltaDeltaGT of 2.9 and 2.8 kcal/mol, respectively, that these two residues are important for cGMP binding in the catalytic site. Zaprinast is a potent competitive inhibitor of cGB-PDE, but the key residues for its binding differ significantly from those that bind cGMP.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Amino Acid Sequence; Binding Sites; Catalysis; Conserved Sequence; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphodiesterase Inhibitors; Purinones; Recombinant Proteins; Substrate Specificity; Thermodynamics

The involvement of the cGMP-protein kinase G (PKG) signaling pathway in the induction of long-term depression (LTD) and long-term potentiation (LTP) was investigated in the medial perforant path of the dentate gyrus in vitro. Low-frequency stimulation (LFS)-induced LTD of field EPSPs was inhibited by bath perfusion of the selective soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3, -a]quinoxalin-1-one (ODQ). LFS-induced LTD of EPSPs and whole-cell patch-clamped EPSCs was also blocked by bath perfusion and postsynaptic intracellular injection, respectively, of the selective PKG inhibitor KT5823. Elevation of intracellular cGMP by perfusion of the cGMP phosphodiesterase inhibitor zaprinast resulted in induction of LTD of field EPSPs and EPSCs. Occlusion experiments showed mutual inhibition between LFS-induced LTD and zaprinast-induced LTD. The zaprinast-induced LTD of field EPSPs was inhibited by perfusion of ODQ and KT5823. In addition, zaprinast-induced LTD of EPSCs was inhibited by postsynaptic application of KT5823. Glutamate receptor stimulation, especially that of metabotropic glutamate receptors (mGluRs), was required for zaprinast-induced LTD, because cessation of test stimulation or perfusion with the mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) inhibited zaprinast-induced LTD. No inhibitory effect of ODQ or KT5823 on the induction of LTP of EPSPs or EPSCs was found. These data indicate that the cGMP-guanyly cyclase-PKG signaling pathway in the dentate gyrus is essential for induction of LTD, although not of LTP, in the dentate gyrus.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Alkaloids; Animals; Benzoates; Carbazoles; Cyclic GMP; Dentate Gyrus; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glycine; GTP-Binding Proteins; Guanylate Cyclase; Indoles; Long-Term Potentiation; Neurons; Oxadiazoles; Phosphodiesterase Inhibitors; Protein Kinases; Purinones; Quinoxalines; Rats; Receptors, Metabotropic Glutamate; Signal Transduction

Sildenafil, an inhibitor of cGMP-specific phosphodiesterase 5 (PDE5), is currently undergoing evaluation as an oral therapy for penile erectile dysfunction. The aims of this study were to investigate the mechanism of action of sildenafil on the neurogenic relaxation of human corpus cavernosum (HCC) in vitro and to determine the activity of sildenafil against a full range of PDE isozymes.. Strips of HCC tissue were precontracted with phenylephrine. Relaxation responses resulting from electrical field stimulation (EFS) were then determined in the presence and absence of sildenafil. The effects of sildenafil on PDE1 to 5 prepared from human tissues and PDE6 from bovine retina were determined by measuring the conversion of [3H]-cGMP or [3H]-cAMP to their respective [3H]-5'-mononucleotides.. Sildenafil (0.001 to 1 microM) enhanced the EFS-induced, nitric oxide (NO) dependent, relaxation of HCC in a concentration-dependent manner to a maximum of 3 times the pretreatment level at 1 microM sildenafil. Compared with zaprinast, an early PDE5 inhibitor, sildenafil was approximately 240-fold more potent, inhibiting PDE5 from HCC with a geometric mean IC50 of 3.5 nM. For sildenafil, IC50 values for inhibition of PDE1 to 4 were 80 to more than 8500 times greater than that for PDE5 and the IC50 for PDE6 (33 nM) was approximately 9-fold greater.. The data support the proposal that enhancement of penile erection by sildenafil in patients with erectile dysfunction involves potentiation of the NO-stimulated cGMP signal mediating relaxation of cavernosal smooth muscle during sexual stimulation. Sildenafil is a potent inhibitor of PDE5 from HCC, with high selectivity for PDE5 relative to other PDE isozymes.

Topics: Animals; Cattle; Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Guanylate Cyclase; Humans; In Vitro Techniques; Isoenzymes; Male; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Penile Erection; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Purines; Purinones; Sildenafil Citrate; Sulfones

In human corpus cavernosum, release of nitric oxide from the non-adrenergic, non-cholinergic nerves and/or the endothelium activates guanylyl cyclase and increases intracellular cGMP levels. The increase in intracellular cGMP modulates intracellular calcium and in turn regulates smooth muscle contractility and erectile function. Phosphodiesterases play an important physiological role by regulating the intracellular levels of cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of phosphodiesterase (PDE) type 5 (E.C. 3.1.4.35 3',5'-cyclic GMP phosphodiesterase) by a novel, high affinity, selective PDE type 5 inhibitor, sildenafil, in soluble extracts of human corpus cavernosum smooth muscle cells. Sildenafil inhibited PDE type 5 cGMP-hydrolytic activity, in the crude extract (Ki=4-6 nM) and in partially purified preparations (Ki=2 nM) in a competitive manner, as determined by Dixon plots. Sildenafil (Ki=2-4 nM) was a more effective PDE type 5 inhibitor than zaprinast (Ki=250 nM). Stimulation of intracellular cGMP synthesis by the nitric oxide donor, sodium nitroprusside, resulted in less than a 5% increase in cGMP levels in the absence of sildenafil and a 35% increase in cGMP levels in the presence of sildenafil, in intact cells at physiological temperatures. These results are in accord with the clinical observations that sildenafil, taken orally, promotes penile erection through increased intracellular cGMP in response to sexual stimulation, potentiating smooth muscle relaxation.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Cell Fractionation; Cells, Cultured; Cyclic GMP; Cytosol; Enzyme Inhibitors; Humans; Hydrolysis; Kinetics; Male; Muscle, Smooth; Nitroprusside; Penis; Piperazines; Purines; Purinones; Sildenafil Citrate; Sulfones

Pulmonary hypertension plays a significant role in the pathophysiology of congenital diaphragmatic hernia (CDH). Although there has been an intensive research effort directed at mediators that may cause pulmonary vasoconstriction, no single agent has been identified. The authors hypothesize that there may be an alteration in the cGMP-nitric oxide (NO) pathway of vasodilatation contributing to the pulmonary hypertension observed in CDH. The purpose of these studies is to begin to elucidate vasoactive properties of pulmonary vessels with particular attention to the cGMP-NO pathway of vasodilatation in fetal lambs with CDH.. Fourth-generation pulmonary arteries and pulmonary veins were dissected from both right and left lungs of eight, 139-day gestational fetuses with surgically created CDH. Vessels were studied with standard isolated tissue bath techniques. Experiments examined basal release of NO in endothelium-intact PVs and PAs of both right and left lungs by measuring the contractile force of vessels constricted with norepinephrine (NE) in the presence and absence of the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NA). Concentration-response curves to the vasodilating agents zaprinast and A23187 were also obtained in vessels contracted by NE.. Left and right pulmonary artery responses to NE are enhanced over those of historic controls. Pretreatment of left pulmonary arteries with L-NA enhances the vasoconstrictor response to NE, whereas right PAs show no increased response. Relaxation responses to A23187 and zaprinast, in both left and right pulmonary arteries were not different from control lambs. Relaxation responses of both left and right pulmonary veins to A23187 and zaprinast are blunted compared with controls. This blunting is significantly more in left pulmonary veins than right. Further, right but not left pulmonary veins display enhanced vasoconstrictive response to NE after L-NA pretreatment.. The NO-cGMP pathway of vasodilatation is abnormal in the near term, fetal lamb with CDH. These abnormalities were most apparent in pulmonary veins and may reflect abnormal NOS activity or content between left and right lungs of the fetal lamb with CDH. Pulmonary arteries from CDH lambs have basal and stimulated NO release equal to that of historic controls but appear to be hypersensitive to exogenous vasoconstrictors.

Topics: Animals; Calcimycin; Cyclic GMP; Disease Models, Animal; Dose-Response Relationship, Drug; Fetus; Hernia, Diaphragmatic; Ionophores; Nitric Oxide; Phosphodiesterase Inhibitors; Pulmonary Artery; Pulmonary Veins; Purinones; Sheep; Vasoconstriction; Vasodilation

This study describes the phosphodiesterase inhibitory potency and cardiovascular actions of WIN 65579 (1-cyclopentyl-3-ethyl-6-(3-ethoxy-4-pyrridyl)-1H-pyrazolo[3,4-d]p yrimidin-4-one), a potent, new cGMP phosphodiesterase 5 inhibitor. WIN 65579 is a competitive inhibitor of phosphodiesterase 5, with IC50 values of 2-3 nM for phosphodiesterase 5 from human or canine vascular sources. WIN 65579 has low affinity for phosphodiesterases 1, 2 and 3 (IC50 > 3-10 microM), and is somewhat selective for phosphodiesterase 4 (IC50 approximately 100 nM). WIN 65579 is an endothelial-dependent relaxant of rat aortic smooth muscle (EC50 = 60 nM) and lowers mean arterial blood pressure in conscious spontaneous hypertensive rats following intravenous or oral dosing. WIN 65579 also increases plasma cGMP levels, and reinstates vascular responsiveness to nitroglycerin in conscious rats that are nitroglycerin-tolerant. These data show that WIN 65579 is one of the more potent phosphodiesterase 5 inhibitors, and that WIN 65579 possesses cardiovascular activities consistent with vascular phosphodiesterase 5 inhibition in vivo.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aorta; Blood Pressure; Cyclic GMP; Heart Rate; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Phosphodiesterase Inhibitors; Purinones; Pyrimidines; Rats; Rats, Inbred SHR

The influence of the atrial natriuretic factor (ANF) on heart-cell communication was investigated in cell pairs isolated from the ventricle of cardiomyopathic hamsters (BIO TO-2; 11 months old), and the results were compared with controls (F1B) of same age. The results indicated that ANF (10(-8) M) added to the bath caused a decline in junctional conductance (gj) of 48 +/- 2% (n = 15) within 90 s. The effect of ANF was suppressed by HS-142-1, a specific antagonist of guanylyl cyclase ANF receptor. Moreover, the decline in gj elicited by ANF was related to the synthesis of cyclic guanosine monophosphate (cGMP). Indeed, dibutyryl-cGMP (10(-4) M) decreased gj by 80 +/- 3.5% (n = 15) within 90 s, and zaprinast, a selective inhibitor of cGMP phosphodiesterase, enhanced the effect of ANF on gj. The possible relationship between ischemia, ANF release, and impairment of cell coupling is discussed.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Atrial Natriuretic Factor; Bucladesine; Cardiomyopathies; Cell Communication; Cricetinae; Cyclic GMP; Electrophysiology; Gap Junctions; Heart Failure; Heart Ventricles; Male; Mesocricetus; Phosphodiesterase Inhibitors; Purinones; Receptors, Atrial Natriuretic Factor

Guanosine 3',5'-cyclic monophosphate (cGMP) is an important second messenger in many biological systems including vascular smooth muscle where it mediates relaxation. Cellular levels of cGMP are regulated primarily by three enzymes; nitric oxide (NO) synthase, soluble guanylate cyclase, and cGMP-phosphodiesterase. Basal cGMP levels of isolated endothelium intact porcine pulmonary vein are five fold higher than in pulmonary artery. The objective of this study was to investigate possible reasons for this difference. Therefore, we compared NO synthase activity of pulmonary vein with artery and used pharmacologic approaches to compare soluble guanylate cyclase and phosphodiesterase activities in these vessels. NO synthase activities of pulmonary vein and artery were measured by monitoring the conversion of exogenous L-[14C]arginine to L-[14C]citrulline and by quantifying NO formation from endogenous L-arginine. Rates (pM/min per mg protein) of basal L-citrulline and NO formation from endothelium intact pulmonary vein (29.0 +/- 4.8 and 44 +/- 7.1, respectively) were significantly higher than from artery (8.3 +/- 2.2 and 17.1 +/- 3.3). Western blot analysis indicated higher constitutive NO synthase protein in the vein than in artery. N-nitro-L-arginine (0-100 microM), a potent inhibitor of NO synthase, induced contractions of the pulmonary vein which were significantly higher than those of the artery. N-nitro-L-arginine (5 and 20 microM) in the presence of phosphodiesterase inhibitors, decreased basal cGMP levels of endothelium intact blood vessels. In endothelium denuded pulmonary vein and artery, basal cGMP levels were not different from each other, but increased significantly following stimulation of soluble guanylate cyclase with exogenous NO. In the presence of both non-specific and specific cGMP phosphodiesterase inhibitors, exogenous NO-induced cGMP levels of endothelium denuded tissues were not significantly different from each other. However, in the absence of the phosphodiesterase inhibitors, exogenous NO-induced cGMP was significantly less in the artery than in the vein. These results suggest that (I) the intact porcine pulmonary vein contains higher levels of NO synthase activity than pulmonary artery, and that (II) the soluble guanylate cyclase activities in pulmonary vein and artery are equally responsive to NO, and finally (III) pulmonary artery expresses greater phosphodiesterase activity than vein. Higher NO synthase and lower phosphodiesterase

Topics: 1-Methyl-3-isobutylxanthine; Animals; Arginine; Blotting, Western; Carbon Radioisotopes; Citrulline; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Female; Guanylate Cyclase; Male; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Nitroarginine; Pulmonary Veins; Purinones; Swine

To determine whether phosphodiesterase (PDE) is involved in the degradation of cGMP in human erythrocytes, we studied the cell cGMP content in the presence of different PDE inhibitors: zaprinast and dipyridamole, specific inhibitors of cGMP-binding, cGMP-specific PDE (cG-BPDE); vinpocetine, a specific inhibitor of Ca2+, calmodulin-dependent phosphodiesterase (CaM-PDE); an unspecific inhibitor, 3-isobutyl-1-methylxanthine (IBMX). IBMX, zaprinast, and dipyridamole at 30 microM did not affect the intracellular cGMP content. However, vinpocetine at this concentration increased the cGMP content by 102 +/- 14% (p < 0.05). The effect of vinpocetine was dose-dependent, reached the maximal level after 1 min of incubation and flattened at the same level. Ca2+ (10 microM) in the presence of the Ca(2+)-ionophore, A23187 (5 microM), decreased the cGMP content (-23% +/- 4; p < 0.05), which can be explained by the CaM-PDE activation. The Ca(2+)-induced decrease in cGMP was completely inhibited by the CaM antagonist, W-7 (100 microM). These data suggest that erythrocytes contain Ca2+, CaM-PDE.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; Calcium; Calmodulin; Cyclic GMP; Erythrocytes; Humans; Hydrolysis; Phosphodiesterase Inhibitors; Purinones; Sulfonamides; Vinca Alkaloids

We tested the hypothesis that preventing cyclic GMP degradation with zaprinast, (a selective cyclic GMP-phosphodiesterase inhibitor) would produce a blunted reduction in myocardial O2 consumption in renal hypertension (One Kidney-One Clip, 1K1C)-induced cardiac hypertrophy. Four groups of anesthetized open-chest New Zealand white rabbits (n = 26) were utilized. Either vehicle or zaprinast (3 x 10(-3) M) was applied topically to the left ventricular surface of control or 1K1C rabbits. Coronary blood flow (radioactive microspheres) and O2 extraction (microspectrophotometry) were used to determine O2 consumption. Myocardial cyclic GMP levels were determined by radioimmunoassay. The 1K1C rabbits had a greater heart weight-to-body weight ratio (2.94 +/- 0.08 g/kg) than controls (2.58 +/- 0.17). Systolic blood pressure was higher in 1K1C (102 +/- 9 mm Hg) than in controls (86 +/- 3). Zaprinast significantly and similarly increased cyclic GMP in both control (3.90 +/- 0.47 to 4.66 +/- 0.89 pmol/g) subepicardium (EPI) and (5.08 +/- 0.69 to 7.06 +/- 1.36) subendocardium (ENDO) and 1K1C hearts (5.53 +/- 0.61 to 7.48 +/- 1.51 EPI and 6.48 +/- 0.42 to 8.88 +/- 1.08 ENDO). Myocardial O2 consumption (ml O2/min/ 100 g) was significantly lower in controls treated with zaprinast (EPI: 8.8 +/- 0.1; ENDO: 9.5 +/- 1.9) than in controls treated with vehicle (EPI: 13.6 +/- 1.3; ENDO: 16.2 +/- 2.9). This effect was diminished in 1K1C rabbits treated with zaprinast (EPI: 10.3 +/- 2.4; ENDO: 11.2 +/- 2.6) compared with the vehicle-treated 1K1C group (EPI: 13.3 +/- 1.2; ENDO: 14.5 +/- 2.4). There was a similar increase in myocardial cyclic GMP after treatment with zaprinast, but a greater depression of myocardial O2 consumption in control animals than in 1K1C after treatment with zaprinast. This suggested that the reduction in myocardial O2 consumption, related to increases in cyclic GMP caused by cyclic GMP-phosphodiesterase blockade, was less in 1K1C cardiac hypertrophy.

Topics: Analysis of Variance; Animals; Cardiomegaly; Coronary Circulation; Cyclic GMP; Hemodynamics; Hypertension, Renovascular; Microspheres; Myocardium; Oxygen Consumption; Phosphodiesterase Inhibitors; Purinones; Rabbits

Phosphodiesterases play an important physiological role by regulating the intracellular levels of cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of phosphodiesterase (PDE) type 5 (EC 3.1.4.35, 3',5'-cyclic GMP phosphodiesterase) by a novel, high-affinity, selective PDE type 5 inhibitor, sildenafil, in intact cells and in soluble extracts of human clitoral corpus cavernosum smooth muscle cells. Sildenafil inhibited cGMP hydrolysis in the crude extract (Ki = 7.2 +/- 2.7) and in partially purified preparations (Ki = 9 nM) in a competitive manner, as determined by Dixon plots. Sildenafil was a more effective PDE type 5 inhibitor than zaprinast (Ki = 400.0 +/- 76.4 nM, crude extracts; 250 nM, partially purified). Stimulation of intracellular cGMP synthesis by the nitric oxide donor sodium nitroprusside resulted in a 3.3- and 2.9-fold increase in cGMP concentration in the presence of sildenafil or zaprinast, respectively, compared to sodium nitroprusside treatment alone in intact cells at physiological temperatures. These observations suggest that human clitoral corpus cavernosum smooth muscle tone may be regulated by the synthesis and release of nitric oxide and that this pathway is dependent on PDE type 5 activity.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Cells, Cultured; Clitoris; Colforsin; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Female; Humans; Hydrolysis; Kinetics; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Purines; Purinones; Sildenafil Citrate; Sulfones

1. Vascular endothelium plays a pivotal role in the control of vascular tone through the release of vasoactive factors such as EDRF (NO). 2. The aim of this study was to investigate whether the addition of exogenous L-citrulline, the byproduct of the NO-synthesis, could relax vascular smooth muscle. 3. L-citrulline relaxed both endothelium-denuded and endothelium-intact rabbit aortic rings precontracted with noradrenaline 10(-6) M (maximum relaxations induced by L-citrulline 10(-8) M were 74.1+/-5.2% vs 51.3+/-2.8% in endothelium-denuded and endothelium-intact arteries, respectively). 4. This relaxant effect was enhanced by zaprinast (a phosphodiesterase type 5 inhibitor) and inhibited by HS-142-1 (a particulate guanylate cyclase inhibitor) and by apamin (a K(Ca)-channel blocker). 5. L-citrulline (10(-13)-10(-8) M) increased cGMP levels in aortic rings (maximum value with L-citrulline 10(-8) M was 0.165+/-0.010 pmol cGMP mg(-1) of tissue vs 0.038+/-0.009 pmol mg(-1) of tissue in basal). 6. L-citrulline as well as NO were released from endothelial cells in culture stimulated with ACh. The values were 6.50+/-0.50 microM vs 2.30+/-0.20 microM (stimulated with ACh and basal respectively) for L-citrulline and 4.22+/-0.10 microM vs 0.87+/-0.26 microM (stimulated with ACh and basal respectively) for NO. 7. These results suggest that L-citrulline could be released together with NO from endothelium and may have actions complementary to those of NO in the control of vascular smooth muscle relaxation.

Topics: Animals; Aorta; Cells, Cultured; Citrulline; Cyclic GMP; Dexamethasone; Endothelium, Vascular; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oxadiazoles; Polysaccharides; Potassium Channels; Purinones; Quinoxalines; Rabbits

The transition from fetal to newborn life is marked by a reduction in pulmonary vascular tone mediated by the intracellular second messengers, cGMP and cAMP. We have compared the rates of phosphodiesterase (PDE)-catalyzed hydrolysis of cGMP and cAMP in intrapulmonary vessels of fetal (146 +/- 2 days gestation) and newborn (3-7-day-old) lambs, each n = 6. Lung vessels of second to sixth generations were dissected and cytosol was prepared by differential centrifugation. PDE activity in cytosol was determined by radiometric assay of the hydrolysis of exogenous nucleotides at 30 degrees C for 10 min. Rates of hydrolysis (pmol/min/mg protein) of cGMP were 225 +/- 38 in fetal arteries and different from 151 +/- 7 in veins. In newborn vessels, the rates were 155 +/- 49 and 63 +/- 13 in arteries and veins, respectively. Rates of cAMP hydrolysis by the fetus were 80 +/- 11 in arteries and 45 +/- 16 veins. In newborn lambs the rates were 69 +/- 10 in arteries and different from 18 +/- 4 in veins. Inhibition of PDE activity by zaprinast, a cGMP-specific PDE inhibitor, and rolipram, a cAMP-specific PDE inhibitor, was more in veins of fetal and newborn lambs. Our data show that rates of hydrolysis of the cyclic nucleotides were faster in fetal vessels than in the newborn. We speculate that this would result in a greater accumulation of the cyclic nucleotides in newborn vessels, particularly the veins, and therefore endow the veins with less vascular tone.

Topics: Animals; Animals, Newborn; Cyclic AMP; Cyclic GMP; Cytosol; Female; Hydrolysis; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Pregnancy; Pulmonary Artery; Pulmonary Veins; Purinones; Pyrrolidinones; Rolipram; Sheep

The phototransduction mechanism of the extra-ocular photoreceptor cells Ip-2 and Ip-1 in the mollusc Onchidium ganglion was examined. Previous work showed that the depolarizing receptor potential of another extra-ocular photoreceptor cell, A-P-1 is produced by a decrease of the light-sensitive K+ conductance activated by a second messenger, cGMP and is inactivated by the hydrolysis of cGMP. Here, a hyperpolarizing receptor potential of Ip-2 or Ip-1 was associated with an increase in membrane conductance. When Ip-2 or Ip-1 was voltage-clamped near the resting membrane potential, light induced an outward photocurrent corresponding to the above hyperpolarization. The spectral sensitivity had a peak at 510 nm. The shift of reversal potentials of the photocurrent depended on the Nernst equation of K(+)-selective conductance. The photocurrent was blocked by 4-AP and L-DIL, which are effective blockers of the A-P-1 light-sensitive K+ conductance. These results suggested that the hyperpolarization is mediated by increasing a similar light-sensitive K+ conductance to that of A-P-1. The injection of cGMP or Ca2+ into a cell produced a K+ current that mimicked the photocurrent. 4-AP and L-DIL both abolished the cGMP-activated K+ current, while TEA suppressed only the Ca(2+)-activated K+ current. These results indicated that cGMP is also a second messenger that regulates the light-sensitive K+ conductance. The photocurrent was blocked by LY-83583, a guanylate cyclase (GC) inhibitor, but was unaltered by zaprinast, a phosphodiesterase (PDE) inhibitor. Together, the present results suggest that increasing the internal cGMP in Ip-2 or Ip-1 cells light-activates GC rather than inhibits PDE, thereby leading to an increase of the light-sensitive K+ conductance and the hyperpolarization.

Topics: 1-Methyl-3-isobutylxanthine; 4-Aminopyridine; 8-Bromo Cyclic Adenosine Monophosphate; Abdomen; Aminoquinolines; Animals; Cyclic GMP; Electric Conductivity; Electrophysiology; Enzyme Inhibitors; Membrane Potentials; Mollusca; Phosphodiesterase Inhibitors; Photic Stimulation; Photoreceptor Cells, Invertebrate; Potassium; Purinones; Second Messenger Systems; Tetraethylammonium; Vision, Ocular

Recent studies suggest that increased lipid peroxidation and lipid peroxidation products, such as 4-hydroxynonenal (HNE), contribute to neuronal loss in conditions associated with oxidative stress. The focus of the present study was to determine possible neuroprotective effects of elevated cyclic nucleotide levels against lipid peroxidation and HNE-mediated neural toxicity. Application of 8-bromo derivative analogs of cAMP or cGMP resulted in attenuation of HNE-induced increases in mitochondrial calcium, reactive oxygen species, and neuron loss. Similar results were obtained when neural cells were pretreated with the phosphodiesterase inhibitors zaprinast or isobutylmethylxanthanine (IBMX). These data are consistent with a possible neuroprotective role for elevated cyclic nucleotide levels in disorders associated with increases in lipid peroxidation and HNE.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Aldehydes; Animals; Calcium; Cell Death; Cell Survival; Cells, Cultured; Cyclic AMP; Cyclic GMP; Cysteine Proteinase Inhibitors; Lipid Peroxidation; Mitochondria; Neurons; Neurotoxins; PC12 Cells; Phosphodiesterase Inhibitors; Purinones; Rats; Reactive Oxygen Species

To examine the role of atrial natriuretic peptide (ANP) and cyclic GMP in the regulation of angiotensin converting enzyme (ACE) in cultured human endothelial cells.. Cultured endothelial cells from human umbilical veins (HUVEC) were treated with ANP (0.3-30 nM), 8-Br-cGMP (1-100 microM), Rp-8-Br-PET-cGMPS (1 microM), or the phosphodiesterase inhibitors, zaprinast (10-100 microM), dipyridamole (1-10 microM), or isobutyl methyl xanthine (IBMX, 0.1-0.5 mM). ACE amounts were measured by inhibitor binding assay and cellular cGMP levels by radioimmunoassay.. ANP caused a dose dependent increase in ACE measured in intact endothelial cell culture. The stimulatory effect of ANP was blocked by Rp-8-Br-PET-cGMPS, a protein kinase G inhibitor. The cyclic GMP analog, 8-Br-cGMP and the cyclic GMP specific phosphodiesterase inhibitor, zaprinast, both increased ACE. Increase of ACE was also caused by nonspecific phosphodiesterase inhibitors, dipyridamole and IBMX. Intracellular cGMP levels were shown to increase by ANP, and phosphodiesterase inhibitors.. These data suggest that cGMP is an intracellular mediator regulating ACE and that ANP induced increase of ACE is mediated via a cGMP dependent mechanism.

Topics: 1-Methyl-3-isobutylxanthine; Atrial Natriuretic Factor; Cells, Cultured; Cyclic GMP; Dipyridamole; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Humans; Peptidyl-Dipeptidase A; Phosphodiesterase Inhibitors; Protein Kinase Inhibitors; Purinones; Stimulation, Chemical; Thionucleotides; Umbilical Veins; Vasodilator Agents

1. The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca2+]i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10(-1) M), NO (10(-10) - 10(-6) M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca2+]i and cation influx as indicated by fura-2 fluorimetry. 2. Zaprinast (10(-4) M), increased NO-stimulated levels of cyclic GMP by 3-20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca2+]i reached after administration of NO, the initial decreases in [Ca2+]i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast. 3. The guanylyl cyclase inhibitor, H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (ODQ, 10(-5) M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp-8-Br-cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10(-10) - 10(-8) M, but the decrease in [Ca2+]i or cation influx caused by higher concentrations of NO (10(-7) - 10(-6) M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10(-7) - 10(-5) M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO. 4. Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca2+]i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute significantly to the decrease in [Ca2+]i and smooth muscle relaxation to NO.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; Angiotensin II; Animals; Aorta; Calcium; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Manganese; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Oxadiazoles; Phosphodiesterase Inhibitors; Protein Kinase C; Purinones; Quinoxalines; Rabbits; Thionucleotides; Vasoconstrictor Agents; Vasodilator Agents

1. We examined whether zaprinast, a putative cGMP-specific phosphodiesterase inhibitor, affects neural control of renal function in pentobarbital-anaesthetized dogs. 2. Renal nerve stimulation (1 Hz, 1 ms duration) reduced urine flow rate, urinary Na+ excretion (UNaV) and fractional excretion of Na+ (FENa) with little change in either renal blood flow (RBF) or glomerular filtration rate (GFR). 3. Intrarenal arterial infusion of zaprinast (10 and 100 micrograms/kg per min) increased basal urine flow rate, UNaV and FENa but not RBF or GFR. Zaprinast infusion (100 micrograms/kg per min) also increased renal venous plasma cGMP concentration and urinary cGMP excretion. 4. Renal nerve stimulation-induced reductions in UNaV and FENa were attenuated during zaprinast infusion, whereas the reduction in urine flow rate was resistant to zaprinast. 5. Renal nerve stimulation increased the renal venous plasma noradrenaline concentration and renal noradrenaline efflux, which remained unaffected during infusion of zaprinast (100 micrograms/kg per min). 6. The results of the present study suggest that zaprinast induces natriuresis and counteracts adrenergically induced antinatriuresis by acting on renal tubular sites in the dog kidney in vivo.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Blood Pressure; Cyclic GMP; Dogs; Electric Stimulation; Female; Glomerular Filtration Rate; Kidney; Male; Natriuresis; Norepinephrine; Phosphodiesterase Inhibitors; Purinones; Renal Circulation

In the coronary circulation, endothelin-1 (ET-1) evokes spasms which are difficult to treat when the endothelial integrity is compromised. This study compares several classes of relaxing agents on already established contractions to ET-1 in an in vitro model using ring segments of the porcine left descending coronary artery (pLAD). All segments were precontracted with 10 nmol/L ET-1. The calcium channel blocker isradipine was 300 times more potent than verapamil, but was only a partial relaxant; the maximal relaxation obtained was 52 +/- 2% (n = 6). Atrial natriuretic peptide (ANP) was an equally potent relaxant of the ET-1 contraction; however, it too was an incomplete relaxant, maximal relaxation being < 60%. A 50% relaxation of the ET-1 contraction was obtained with 0.28 +/- 0.24 mumol/L ANP, n = 4 (IC50). Comparison of cyclic nucleotide analogues revealed a 30 times higher potency for 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP)(IC50 44 +/- 11 mumol/L, n = 6) than for 8-bromo-cyclic adenosine monophosphate (8-Bi-cAMP) (IC50 1600 mumol/L, n = 6). The cyclic nucleotide phosphodiesterase (PDE) inhibitor milrinone, a PDE 3-inhibitor with an IC50 2.4 +/- 1.8 mumol/L, (n = 6) was 10 times more potent than rolipram (PDE 4-inhibitor), zaprinast (PDE 5-inhibitor) and vinpocentine (PDE 1-inhibitor). Withdrawal of these analogues and inhibitors from segments continuously exposed to 10 nmol/l ET-1 revealed that vinpocentine and 8-Br-cGMP were irreversible relaxants, in contrast to milrinone and 8-Br-cAMP. In conclusion, this study has demonstrated that cGMP-enhancing agents, such as the naturally occurring ANP, the calcium channel blocker isradipine, and the synthetic inhibitor of PDE 3, were the most effective relaxants of ET-1 evoked contractions in pLAD in vitro.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Atrial Natriuretic Factor; Caffeine; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Colforsin; Coronary Vessels; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Endothelin-1; In Vitro Techniques; Isradipine; Milrinone; Muscle, Smooth, Vascular; Papaverine; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Pyrrolidinones; Rolipram; Swine; Vasoactive Intestinal Peptide; Vasoconstriction; Vasodilation; Verapamil; Vinca Alkaloids

Rolipram was previously reported to elevate plasma cyclic adenosine 3',5'-monophosphate (cAMP) and inhibit serum tumor necrosis factor-alpha (TNF-alpha) production in mice. CP-80,633, a new cyclic nucleotide phosphodiesterase (PDE4) inhibitor, has been shown to augment intracellular cAMP levels and to inhibit TNFalpha release from human monocytes in vitro. This study was undertaken to determine the effect of p.o. CP-80,633 on plasma cAMP levels and lipopolysaccharide-induced TNFalpha production in mice with and without adrenal glands. CP-80,633 dose-dependently (3-32 mg/kg p.o.) elevated plasma cAMP levels and decreased systemic TNFalpha production in response to i.p. injection of lipopolysaccharide. Elevated plasma cAMP levels can be detected for up to 4 hr. CP-80,633 (10 mg/kg p.o.) caused a 6-fold increase in the plasma cAMP level, a 2-fold increase in the plasma epinephrine level and a greater than 95% reduction in TNFalpha production. Unlike CP-80,633, neither vinpocetine, dipyridamole, SKB-94,120 nor zaprinast, at 100 mg/kg p.o., modified the cAMP response, which suggests that this response is mediated by inhibition of PDE4. Adrenalectomy reduced the cAMP response and completely blocked the epinephrine response; however, the levels of plasma cAMP in the CP-80,633-treated mice (10 mg/kg p.o.) remained elevated (vehicle: 47.3 +/- 6.8 vs. CP-80,633: 98.4 +/- 10.3 pmol/ml, n = 7, P < .05). This effect is mimicked by treatment of control mice with propranolol, which demonstrates that beta adrenoreceptors contribute to the cAMP response. Removal of adrenal glands significantly increased the LPS-induced elevation of serum TNFalpha. The ability of CP-80,633 to block the TNFalpha response was only slightly affected by adrenalectomy (ED50 = 1.2 mg/kg in controls vs. 3.9 mg/kg in adrenalectomized mice). Taken together, these results show that CP-80,633, when given p.o. to mice, is capable of elevating plasma cAMP and inhibiting TNFalpha production and that adrenal catecholamines contribute significantly to the effect of CP-80,633 on the cAMP response but only slightly to its effect on the systemic TNFalpha response.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adrenalectomy; Animals; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 4; Dipyridamole; Epinephrine; Humans; Kinetics; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Monocytes; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piroxicam; Propranolol; Purinones; Pyrimidinones; Thromboxane B2; Time Factors; Tumor Necrosis Factor-alpha; Vinca Alkaloids

The mechanism by which changes in cyclic GMP (cGMP) regulate glutamate release was investigated in rat cerebrocortical nerve terminals. The elevation of cGMP levels by inhibition of cGMP-phosphodiesterase with 2-o-propoxy-phenyl-8-azapurin-6-one (zaprinast) reduced the Ca(2+)-dependent glutamate release evoked by depolarization with 30 mM KCl or 1 mM 4-aminopyridine. The nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine also enhanced cGMP and reduced glutamate release. In addition, the membrane-permeable analogs 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) and N,2'-o-dibutyrylguanosine (dbcGMP) at 10 microM also mimic glutamate release inhibition. The reduction in glutamate release was observed with no modifications in the ATP/ADP ratio, and was reversed in the presence of the protein kinases inhibitor [N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide, HCl] (H-8). Interestingly, higher concentrations of dbcGMP (1 mM) abolished the inhibition observed with low concentrations although no facilitation was observed. This finding seems to indicate the existence of a dual role for cGMP in the control of glutamate exocytosis.

Topics: Adenosine Triphosphate; Animals; Calcium; Cerebral Cortex; Cyclic GMP; Enzyme Activation; Exocytosis; Glutamic Acid; Guanylate Cyclase; Male; Nerve Endings; Nitric Oxide; Phosphodiesterase Inhibitors; Protein Kinases; Purinones; Rats; Rats, Wistar; Signal Transduction; Synaptosomes

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are important second messengers in mediating relaxation of various smooth-muscle cells. This second-messenger pathway also appears to be essential for cavernous smooth-muscle relaxation on the basis of the assumption it would be of theoretical and clinical interest to determine the functional relevance of various phosphodiesterase (PDE) isoenzymes in human cavernous smooth-muscle This study was concentrated on characterizing PDE isoenzymes that exist in cavernous smooth muscle and evaluating the effect of selective PDE inhibitors on relaxation that is needed for the initiation of erection. Separation of PDE isoenzymes was performed using anion-exchange chromatography [diethylaminoethanol (DEAE)-Sepharose column], and a modification of the PDE-assay method proposed by Thompson and Lakey was used. The relaxation effect of PDE inhibitors was evaluated in an organ-bath study. Three different PDE isoenzymes have been shown in human cavernous smooth-muscle homogenate: cGMP-inhibited PDE (PDE III), cAMP-specific PDE (PDE IV), and cGMP-specific PDE (PDE V). All PDE inhibitors tested showed a relaxation effect on isolated human cavernous smooth-muscle, albeit with differing potency. Quazinone (a selective PDE III inhibitor) had potency at least equal to that of papaverine (a non-selective PDE inhibitor) and had a superior effect as compared with Rolipram (a selective PDE IV inhibitor) and zaprinast (a selective PDE V inhibitor). The present study provides the rationale and opens the possibility of using selective PDE inhibitors in the treatment of patients with erectile dysfunction.

Topics: Animals; Cyclic AMP; Cyclic GMP; Humans; In Vitro Techniques; Isoenzymes; Male; Muscle, Smooth; Papaverine; Penile Erection; Penis; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Pyrrolidinones; Quinazolines; Rolipram

High concentrations of nitric oxide (NO) inhibit bone resorption by mature osteoclasts. We examined the effects of low NO concentrations on osteoclast formation in mouse bone marrow cultures. The NO releasers sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine inhibited the formation of multinucleated cells expressing tartrate-resistant acid phosphatase (a marker for osteoclasts) when administered during the last 3 days of 6-day cultures (differentiation stage) but not during the first 3 days (proliferation stage). SNP (1 microM) completely inhibited pit formation on dentine wafers when added to cultures during osteoclast formation, but 100 microM SNP was required to inhibit pitting by mature osteoclasts. Conversely, the NO synthase inhibitors aminoguanidine and nitro-L-arginine methyl ester both increased osteoclast formation. Inhibition of osteoclast formation by NO likely was guanosine 3',5'-cyclic monophosphate (cGMP) dependent, as SNP increased cGMP in marrow cultures, and 1 mM 8-bromo-cGMP or dibutyryl-cGMP reduced osteoclast formation when administered during the differentiation stage. The cGMP-specific type V phosphodiesterase inhibitor, zaprinast (M & B 22948) also inhibited osteoclast formation (half-maximal inhibitory constant, 100 microM) only when added during the differentiation stage. We conclude that the differentiation stage of osteoclast formation is inhibited by increases in cGMP levels elicited by NO.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Bone Marrow Cells; Bone Resorption; Bucladesine; Calcitriol; Cells, Cultured; Cyclic GMP; Dentin; Dibutyryl Cyclic GMP; Guanidines; In Vitro Techniques; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Osteoclasts; Penicillamine; Purinones; S-Nitroso-N-Acetylpenicillamine; Whales

Cyclic GMP phosphodiesterase (PDE) is the effector enzyme in the visual transduction cascade of vertebrate photoreceptor cells. In the dark, the activity of the enzyme catalytic alpha and beta subunits (Palphabeta) is inhibited by two gamma subunits (Pgamma). Previous results have established that approximately 5-7 C-terminal residues of Pgamma comprise the inhibitory domain. To study the interaction between the Pgamma C-terminal region and Palphabeta, the Pgamma mutant (Cys68 --> Ser, and the last 4 C-terminal residues replaced with cysteine, Pgamma-1-83Cys) was labeled with the fluorescent probe 3-(bromoacetyl)-7-diethylaminocoumarin (BC) at the cysteine residue (Pgamma-1-83BC). Pgamma-1-83BC was a more potent inhibitor of PDE activity than the unlabeled mutant, suggesting that the fluorescent probe in part substitutes for the Pgamma C terminus in PDE inhibition. HoloPDE (Palphabetagamma2) had no effect on the Pgamma-1-83BC fluorescence, but the addition of Palphabeta to Pgamma-1-83BC resulted in an approximately 8-fold maximal fluorescence increase. A Kd for the Pgamma-1-83BC-Palphabeta interaction was 4.0 +/- 0.5 nM. Zaprinast, a specific competitive inhibitor of PDE, effectively displaced the Pgamma-1-83BC C terminus from its binding site on Palphabeta (IC50 = 0.9 microM). cGMP and its analogs, 8-Br-cGMP and 2'-butyryl-cGMP, also competed with the Pgamma-1-83BC C terminus for binding to Palphabeta. Our results provide new insight into the mechanism of PDE inhibition by showing that Pgamma blocks the binding of cGMP to the PDE catalytic site.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Binding Sites; Cattle; Cell Membrane; Coumarins; Cyclic GMP; Cysteine; Fluorescent Dyes; Kinetics; Macromolecular Substances; Phosphodiesterase Inhibitors; Point Mutation; Purinones; Rod Cell Outer Segment; Serine; Vertebrates

The mechanism by which proadrenomedullin NH2-terminal 20 peptide (PAMP) decreases vascular resistance was investigated in the hindlimb vascular bed in the cat. Injections of PAMP, a shortened form of the peptide PAMP-(12-20), and adrenomedullin (ADM) into the hindlimb perfusion circuit elicit dose-related decreases in perfusion pressure. The order of potency was ADM > PAMP > PAMP-(12-20), and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) had no effect on vasodilator responses to PAMP or ADM. Vasodilator responses to PAMP were increased in duration by the adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase inhibitor Rolipram, whereas inhibitors of nitric oxide synthase and guanosine 3',5'-cyclic monophosphate phosphodiesterase had no effect. Vasodilator responses to PAMP were not altered by treatment with alpha-receptor or adrenergic nerve terminal blocking agents and were similar in innervated and denervated hindlimb preparations. Responses to PAMP were similar when vasoconstrictor tone was increased by stimulation of the sympathetic nerves or infusion of phenylephrine and were not altered by the passage of time. These data suggest that PAMP dilates the hindlimb vascular bed by a direct cAMP-dependent mechanism and that inhibition of norepinephrine release plays little if any role in mediating responses to the peptide in the regional vascular bed of the cat.

Topics: Adrenomedullin; Animals; Antihypertensive Agents; Blood Pressure; Calcitonin Gene-Related Peptide; Calcitonin Gene-Related Peptide Receptor Antagonists; Cats; Cyclic AMP; Cyclic GMP; Female; Hindlimb; Male; NG-Nitroarginine Methyl Ester; Norepinephrine; Peptide Fragments; Phentolamine; Phosphodiesterase Inhibitors; Protein Precursors; Proteins; Purinones; Pyrrolidinones; Receptors, Adrenergic, alpha; Regional Blood Flow; Rolipram; Vascular Resistance; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Early heart failure is characterized by elevated plasma atrial natriuretic peptide (ANP) levels, but little is known about the direct effects of ANP on cardiac myocytes. In neonatal rat cardiac myocytes, ANP induced apoptosis in a dose-dependent and cell type-specific manner. Maximum effects occurred at 1 microM ANP, with a 4-5-fold increase in apoptotic cells, reaching a maximum apoptotic index of 19%. In contrast, the maximum apoptotic index of ANP-treated non-myocytes was 1.1 +/- 0.2%, equivalent to control cultures. ANP treatment also sharply reduced levels of Mcl-1 mRNA, a Bcl-2 homologue, coincident with the increase in the incidence of apoptosis. ANP induction of apoptosis was receptor-dependent and mediated by cyclic GMP: the effect was mimicked by 8-bromo-cGMP, a membrane-permeable analog, and by sodium nitroprusside, an activator of soluble guanylyl cyclase, and was potentiated by a cGMP-specific phosphodiesterase inhibitor, zaprinast. Interestingly, norepinephrine, a myocyte growth factor, inhibited ANP-induced apoptosis via activation of the beta-adrenergic receptor and elevation of cyclic AMP. These results show that ANP is a specific effector of cardiac myocyte apoptosis in culture via receptor-mediated elevation of cGMP. Furthermore, at least in this model, ANP and norepinephrine may have opposing roles in the modulation of cardiac myocyte growth and survival.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adrenergic alpha-Antagonists; Animals; Animals, Newborn; Apoptosis; Atrial Natriuretic Factor; Cell Nucleus; Cells, Cultured; Cyclic GMP; Desmin; DNA Fragmentation; Dose-Response Relationship, Drug; Heart; Kinetics; Myeloid Cell Leukemia Sequence 1 Protein; Myocardium; Neoplasm Proteins; Nitroprusside; Norepinephrine; Phosphodiesterase Inhibitors; Polymerase Chain Reaction; Prazosin; Propranolol; Proto-Oncogene Proteins c-bcl-2; Purinones; Rats; Receptors, Atrial Natriuretic Factor

Neonatal pulmonary hypertension is associated with increased pulmonary vascular reactivity. We studied the responses of isolated porcine intrapulmonary arteries after exposure of piglets to chronic hypobaric hypoxia (CHH) from 0 to 2.5, 3 to 6, or 14 to 17 days of age. CHH inhibited the postnatal development of endothelium-dependent vasorelaxation to acetylcholine (ACh) and the calcium ionophore A-23187. Basal accumulation of guanosine 3', 5'-cyclic monophosphate (cGMP) was unaffected, but cGMP response to ACh was inhibited. Endothelium-independent relaxation to nitric oxide (NO) and zaprinast (a phosphodiesterase inhibitor) was also inhibited, but cGMP accumulation in response to these agonists was normal. The ability of sodium nitroprusside (SNP) to cause vasorelaxation and increase cGMP accumulation was unaffected. Contractile responses to potassium chloride and prostaglandin F2 alpha (PGF2 alpha) were similar to normal after exposure from birth and 3 days and were decreased in the older group, but the ability of NG-monomethyl-L-arginine acetate to increase PGF2 alpha-induced contractions decreased. Thus exposure of newborn piglets to CHH causes 1) no increase in contractile responses and 2) impairment of endothelium-dependent and -independent relaxation by impairing signal transduction mechanisms involved in the release of NO and the effectiveness of cGMP.

Topics: Acetylcholine; Animals; Animals, Newborn; Atmospheric Pressure; Calcimycin; Chronic Disease; Cyclic GMP; Dinoprost; Endothelium, Vascular; Heart; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Nitric Oxide; omega-N-Methylarginine; Potassium Chloride; Pulmonary Artery; Purinones; Swine; Vasodilation; Vasomotor System

The mechanisms underlying smooth muscle relaxations of cerebral arteries in response to nitric oxide (NO) and cyclic GMP (cGMP) are still not completely understood. The present study was designed to determine the role of potassium channels in the relaxations to NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), as well as 8-bromo-3',5' -cGMP (a synthetic analogue of cGMP) and zaprinast (a selective cGMP phosphodiesterase inhibitor).. Rings of canine middle cerebral asteries without endothelium were suspended in Krebs-Ringer bicarbonate solution for isometric tension recording. The levels of cGMP were measured by radioimmunoassay. Relaxations to NO donors 8-bromo-cGMP and zaprinast were studied in the presence and in the absence of K+ channel blockers charybdotoxin (large-conductance Ca(2+)-activated K+ channels), glyburide (ATP-sensitive K+ channels), 4-aminopyridine (delayed rectifier K+ channels), and BaCl2 (multiple types of K+ channels).. Concentration-dependent relaxations caused by NO donors (SIN-1 and SNP) were significantly reduced in arteries treated with BaCl2 (3 x 10(-4) mol/L) or charybdotoxin (3 x 10(-8) mol/L). Relaxations to 8-bromo-cGMP were not affected by the same concentrations of BaCl2 and charybdotoxin; however, they were reduced by higher concentrations of BaCl2 (3 x 10(-3) mol/L) or charybdotoxin (10(-7) mol/L). Zaprinast-induced relaxations were significantly reduced by BaCl2 (3 x 10(-4) mol/L) or charybdotoxin (3 x 10(-8) mol/L). Glyburide (10(-5) mol/L) and 4-aminopyridine (10(-3) mol/L) did not alter the relaxations to SIN-1 or SNP. The production of cGMP stimulated by SIN-1 in the vascular smooth muscle was not affected by BaCl2 (3 x 10(-3) mol/L) or charybdotoxin (10(-7) mol/L).. These results indicate that in canine middle cerebral arteries, a significant portion of relaxations to NO liberated from nitrovasodilators is mediated by large-conductance Ca(2+)-activated K+ channels. Other K+ channels, sensitive to BaCl2, may also be involved in the mechanism of relaxations induced by NO.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; 4-Aminopyridine; Animals; Barium Compounds; Cerebral Arteries; Charybdotoxin; Chlorides; Cyclic GMP; Diltiazem; Dogs; Glyburide; In Vitro Techniques; Molsidomine; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Potassium Channels; Purinones; Vasodilation; Vasodilator Agents

We report the regulation of cGMP accumulation induced by the heat-stable enterotoxin, STh, in the T84 human colonic cell line. STh binding to its receptor, guanylyl cyclase C (GCC), leads to elevated intracellular levels of cGMP. Prolonged exposure of T84 cells to STh induced refractoriness to further cGMP accumulation, without significant receptor internalization, but with reduced STh-induced cGMP synthesis by the receptor. Significantly, increased degradation of cGMP by a cGMP-specific phosphodiesterase was observed in desensitized cells. This is the first report on the desensitization of GCC, as well as the role of the Type V phosphodiesterase in inducing cellular refractoriness.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Bacterial Toxins; Cell Line; Cell Membrane; Cyclic GMP; Enterotoxins; Escherichia coli Proteins; Guanylate Cyclase; Humans; Kinetics; Milrinone; Phenothiazines; Purinones; Pyridones

Perfusate levels of nitric oxide (NO)-containing compounds and guanosine 3',5'-cyclic monophosphate (cGMP) are increased in hypoxia-induced hypertensive rat lungs. To test if increased cGMP was due to NO stimulation of soluble guanylate cyclase (sGC), we examined effects of inhibition of NO synthase with N omega-nitro-L-arginine (L-NNA) on perfusate accumulation of cGMP in physiological salt solution (PSS)-perfused hypertensive lungs isolated from rats exposed for 3-4 wk to hypobaric hypoxia. Because 200 microM L-NNA did not reduce cGMP, we next examined inhibitors of other pathways of stimulation of either sGC or particulate GC (pGC). Neither 5 microM Zn-protophorphyrin, an inhibitor of CO production by heme oxygenase, nor 10 mM aminotriazole, an inhibitor of H2O2 metabolism by catalase, reduced perfusate cGMP. However, an antiserum to atrial natriuretic peptide (ANP; 100 microliters antiserum/30 ml PSS), to inhibit ANP activation of pGC, completely prevented accumulation of the nucleotide. ANP antiserum was also more effective than L-NNA in reducing lung tissue cGMP. In contrast, L-NNA but not ANP antiserum increased resting vascular tone. These results suggested that whereas ANP determined perfusate and tissue levels of cGMP, NO regulated vascular tone. To test if perfusate cGMP reflected ANP stimulation of pGC in endothelial rather than smooth muscle cells, we examined effects of 10 microM Zaprinast, an inhibitor of cGMP hydrolysis in smooth muscle but not endothelial cells, and found no increase of cGMP in hypertensive lungs. ANP levels were not elevated in hypertensive lungs, and it is unclear by what mechanism the ANP-stimulated activity of pGC is increased in hypertensive pulmonary vascular endothelial cells.

Topics: Altitude; Amitrole; Animals; Atrial Natriuretic Factor; Catalase; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Heme Oxygenase (Decyclizing); Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Immune Sera; Kinetics; Lung; Male; Nitroarginine; Protoporphyrins; Purinones; Rats; Rats, Sprague-Dawley; Reference Values

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility in guinea pig ileal longitudinal smooth muscle were investigated. 1) 3-Isobutyl-1-methyl xanthine (IBMX) or zaprinast markedly inhibited the high K(+)- or carbachol (CCh)-induced contraction and increased cGMP content of the muscle strip in a concentration-dependent manner. However, these agents only slightly increased the cAMP content. Milrinone or Ro20-1724 also slightly inhibited the high K(+)- or CCh-induced contraction and increased the cAMP content, but did not increase cGMP. 2) In a fura2-loaded muscle, IBMX or zaprinast inhibited both contractions and the increase in intracellular Ca2+ ([Ca2+]i) level induced by high K+ or CCh, although the inhibitory effect on the [Ca2+]i level was smaller than that on muscle tension. 3) In alpha-toxin-permeabilized muscles, cGMP, IBMX or zaprinast significantly inhibited the Ca(2+)-induced contraction. These results suggest that IBMX and zaprinast inhibit muscle contraction in the ileal longitudinal smooth muscles mainly through an increase in cGMP and the inhibitory mechanism of IBMX or zaprinast is involved in the decreases in the [Ca2+]i level and sensitivity of contractile elements to Ca2+.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Calcium; Carbachol; Cell Membrane Permeability; Cyclic AMP; Cyclic GMP; Detergents; Guinea Pigs; Ileum; Male; Muscle Contraction; Muscle, Smooth; Nitroprusside; Octoxynol; Phosphodiesterase Inhibitors; Potassium; Purinones; Type C Phospholipases; Vasodilator Agents

The stimulation of NMDA receptor increased [3H]GABA release from preloaded cultured retina cells. This effect appears to be mediated by NO production, since addition of L-NA reduces NMDA-evoked [3H]GABA release. Spermine/NO complex, an NO donor, mimics the effect produced by NMDA. The addition of zaprinast, a phosphodiesterase inhibitor, as well as 8-Br-cGMP enhances the NMDA-evoked [3H]GABA release. These results agree with the existence in chick retina cells of NO/cGMP pathways and support a role for NO in NMDA-evoked events. The activation of this receptor complex through maturative stages of the retina together with the NO-mediated increase in GABA release may account for NMDA differentiative effect in culturing retina cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Cell Differentiation; Cells, Cultured; Chick Embryo; Cyclic GMP; Dizocilpine Maleate; gamma-Aminobutyric Acid; Isoquinolines; N-Methylaspartate; Nitric Oxide; Nitroarginine; Nitrogen Oxides; Purinones; Receptors, N-Methyl-D-Aspartate; Retina; Spermine; Tritium

1. The effect of systemic administration of the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME) on the antihypertensive effects of the angiotensin AT1 receptor antagonist, GR138950, the angiotensin-converting enzyme (ACE) inhibitor, enalapril, or hydralazine has been evaluated in unrestrained, conscious renal artery ligated hypertensive (RALH) rats. The effect of the phosphodiesterase type V inhibitor, zaprinast on the antihypertensive effect of GR138950 in RALH rats was also examined. The effect of GR138950 on blood pressure, and plasma and urine cyclic GMP levels was compared to that of zaprinast in conscious RALH rats. 2. GR138950, enalapril or hydralazine caused marked reductions in blood pressure associated with immediate tachycardia in conscious RALH rats. L-NAME pretreatment attenuated the antihypertensive effects of GR138950 or enalapril but not that of hydralazine in conscious RALH rats. The initial tachycardia caused by GR138950 or enalapril but not hydralazine was attenuated by L-NAME pretreatment. L-NAME alone caused a transient (20 min) pressor response and a prolonged (6 h) bradycardia in conscious RALH rats. 3. Pretreatment with indomethacin did not affect the cardiovascular effect of GR138950 in conscious RALH rats. Indomethacin alone did not significantly change basal blood pressure or heart rate in RALH rats. 4. Zaprinast pretreatment did not affect the antihypertensive effect of GR138950 in conscious RALH rats but potentiated the depressor response to sodium nitroprusside. Zaprinast alone caused a small reduction in basal blood pressure but did not change basal heart rate in RALH rats. 5. The antihypertensive effect of GR138950 was not associated with an increase in plasma or urine cyclic GMP levels in conscious RALH rats, whereas zaprinast caused a small fall in blood pressure associated with increases in plasma and urine cyclic GMP. 6. The ability of L-NAME to inhibit the antihypertensive action of GR138950 or enalapril suggests that these agents release nitric oxide (NO) and/or enhance the cardiovascular effects of NO as part of their mechanism of action. However, the inability of zaprinast to potentiate the antihypertensive effects of GR138950 and the finding that GR138950 did not increase urine and plasma cyclic GMP levels are not consistent with this view. Attenuation of the response to GR138950 or enalapril, but not hydralazine, suggests a selective interaction between L-NAME and inhibitors of the ren

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antihypertensive Agents; Benzofurans; Blood Pressure; Cyclic GMP; Drug Interactions; Enalapril; Enzyme Inhibitors; Hydralazine; Hypertension; Indomethacin; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Phosphodiesterase Inhibitors; Purinones; Rats; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Angiotensin

The effects of 7-nitroindazole, a putative selective inhibitor of neuronal nitric oxide (NO) synthase and zaprinast, a cGMP-selective phosphodiesterase inhibitor, were evaluated on recognition memory of rats in the object recognition test. This test is based on the differential exploration of a new and a familiar object. Two doses of 7-nitroindazole (10 and 30 mg/kg) and zaprinast (3 and 10 mg/kg) were used. The substances were administered i.p. immediately after the exposure to two identical objects, i.e., at the start of the delay interval. After a delay interval of 1 h, control rats spent more time exploring the new object which demonstrates that they recognized the familiar one. Both doses of 7-nitroindazole impaired the discrimination between the two objects after the 1 h interval. After a 4 h interval, control rats did not discriminate between the objects. The highest dose of zaprinast facilitated object recognition after the 4 h interval. In addition, this dose of zaprinast (10 mg/kg) reversed the recognition memory deficit induced by 7-nitroindazole (10 mg/kg) at the 1 h interval. The highest dose of 7-nitroindazole slightly increased mean arterial blood pressure 1 h after its administration. 4 h after administration of zaprinast (10 mg/kg), mean arterial blood pressure was also slightly increased, but not after 1 h after zaprinast administration. However, these effects on blood pressure do not explain the differential effects on object recognition memory. These results therefore suggest that NO-cGMP signal transduction is involved in object recognition memory independently of its cardiovascular role. Finally, since 7-nitroindazole affected mean arterial blood pressure it can not be regarded as a selective inhibitor of neuronal NO synthase.

Topics: Animals; Blood Pressure; Cyclic GMP; Drug Interactions; Exploratory Behavior; Indazoles; Male; Memory; Nitric Oxide; Nitric Oxide Synthase; Phosphodiesterase Inhibitors; Purinones; Rats; Signal Transduction; Visual Perception

While it is known that nitric oxide (NO) is an important modulator of tone in the hypertensive pulmonary circulation, the roles of cyclic 3'-5'-guanosine monophosphate (cGMP) and cGMP-phosphodiesterase (PDE) are uncertain. We found that isolated lung perfusate levels of cGMP were over ninefold elevated in hypertensive vs. normotensive control rats. 98-100% of lung cGMP hydrolytic activity was cGMP-specific PDE5, with no significant decrease in PDE activity in hypertensive lungs, suggesting that the elevation in cGMP was due to accelerated production rather than reduced degradation. In pulmonary hypertensive rat lungs, in vitro, cGMP-PDE inhibition by E4021[1-(6-chloro-4-(3,4-methylbenzyl) amino-quinazolin-2-yl)piperdine-4-carboxylate], increased perfusate cGMP threefold, reduced hypoxic vasoconstriction by 58 +/- 2%, and reduced baseline pulmonary artery pressure by 37 +/- 5%. In conscious, pulmonary hypertensive rats, intravenous administration of E4021 reduced hypoxic vasoconstriction by 68 +/- 8%, pulmonary artery pressure by 12.6 +/- 3.7% and total pulmonary resistance by 13.1 +/- 6.4%, with no significant effect on cardiac output, systemic pressure, and resistance. Comparison of E4021 to inhaled nitric oxide demonstrated that cGMP-PDE inhibition was as selective and as effective as inhaled NO.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calcium Channel Blockers; Cyclic GMP; Diltiazem; Hemodynamics; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Lung; Male; Nitric Oxide; Perfusion; Phosphodiesterase Inhibitors; Piperidines; Pulmonary Circulation; Purinones; Quinazolines; Rats; Rats, Sprague-Dawley; Vasodilation

The effects of the nonspecific cyclic nucleotide inhibitors 1-methyl-3-isobutylxanthine (IBMX) and dipyridamole, and the cGMP-specific phosphodiesterase inhibitor Zaprinast were studied on parallel fiber-Purkinje cell synaptic responses in rat cerebellar slices. Bath application of all three compounds, at concentrations shown to inhibit cGMP breakdown, led to stable and robust long-term depression of PF responses. Injections of dipyridamole directly into the Purkinje cell dendrites were similarly effective as bath applications, confirming a postsynaptic site of action. Inhibitors of both protein kinase G and C and also the metabotropic glutamate receptor antagonist MCPG completely prevented the induction of LTD by dipyridamole and Zaprinast. The extent of phosphodiesterase-induced synaptic depression was dependent on the frequency of parallel fiber stimulation, and this form of LTD both occluded and was occluded by LTD induced by pairing parallel and climbing fiber inputs. The degree of LTD induced by IBMX was dose-dependent, and also required PKC and PKG activity, but was preceded by a large, transient potentiation of parallel fiber responses occurring by a postsynaptic mechanism independent of cGMP. These data not only confirm that cGMP is capable of inducing cerebellar LTD when paired with parallel fiber stimulation but indicate that cGMP is an endogenous intermediate in this form of synaptic plasticity.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Cerebellum; Cyclic GMP; Dipyridamole; Long-Term Potentiation; Male; Phosphodiesterase Inhibitors; Purinones; Purkinje Cells; Rats; Rats, Wistar; Synaptic Transmission

Cardiac endothelium releases a number of factors that may modulate performance of underlying cardiac muscle. Nitric oxide (NO), which accounts for the biological activity of the vascular endothelium-derived relaxing factor and relaxes vascular smooth muscle by elevating intracellular cGMP, may be involved in this cardiac modulation.. We examined the myocardial contractile effects of the NO-releasing nitrovasodilators sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1), and S-nitroso-N-acetyl-penicillamine (SNAP); of a cGMP analogue, 8-bromo-cGMP; and of the cGMP-phosphodiesterase inhibitor zaprinast in isolated cat papillary muscle. Modulation of these effects by endocardial endothelium (EE) and by cholinergic and adrenergic stimulation was also investigated. Concentration-response curves with addition of NO-releasing nitrovasodilators (SNP, SIN-1, SNAP) and 8-bromo-cGMP resulted in a biphasic inotropic response. Although administration of low concentrations induced a positive inotropic effect, higher concentrations induced a negative inotropic effect. Both NO-induced positive and negative inotropic effects were attenuated by methylene blue, suggesting a role for cGMP. The response to high concentrations of 8-bromo-cGMP was shifted to the right in muscles with damaged EE, whereas cholinergic stimulation shifted the curve leftward. Zaprinast caused a monophasic concentration-dependent positive inotropic effect; damaging the EE shifted the terminal portion of the curve upward. Concomitant cholinergic or adrenergic stimulation modified the response to zaprinast into a negative inotropic response.. NO and cGMP induced a concentration-dependent biphasic contractile response. The myocardial contractile effects of NO and cGMP were modulated by the status of EE and by concomitant cholinergic or adrenergic stimulation.

Topics: Animals; Cats; Cyclic GMP; Endothelium, Vascular; In Vitro Techniques; Molsidomine; Myocardial Contraction; Nitric Oxide; Nitroprusside; Penicillamine; Purinones; S-Nitroso-N-Acetylpenicillamine

Experimental nephrotic syndrome is characterized by abnormal sodium metabolism, reflected in a blunted natriuretic response both to volume expansion and to infused atrial natriuretic peptide (ANP). The studies presented here examined the relationships among plasma ANP concentration and urinary sodium (VNaV) and cyclic GMP excretion (UcGMPV) in vivo, and the responsiveness of isolated glomeruil and inner medullary collecting duct (IMCD) cells to ANP and urodilatin (renal natriuretic peptide; RNP) in vitro in rats with Heymann nephritis, an immunologically mediated model of nephrotic syndrome. Nine to 14 days after Ip injection of anti-Fx1A antiserum, rats were proteinuric and had a blunted natriuretic response to intravenous infusion of isotonic saline (2% body weight, given over 5 min). Thirty min after the onset of the infusion, plasma ANP concentration was increased to the same extent in both normal and nephritic rats, compared with their respective hydropenic controls. Despite this increase, UcGMPV was significantly less in nephritic rats after the saline infusion. Accumulation of cGMP by isolated glomeruil and IMCD cells from nephritic rats after incubation with ANP and RNP was also significantly reduced, compared with normal rats. This difference was not related to differences in either density or affinity of renal ANP receptors, but was abolished when accumulation of cGMP was measured in the presence of 10(-3) M isobutylmethylxanthine or Zaprinast, two different inhibitors of cyclic nucleotide phosphodiesterases (PDE). Infusion of Zaprinast into one renal artery in nephritic rats normalized both the natriuretic response to volume expansion and the increase in UcGMPV from the infused, but not the contralateral, kidney. Furthermore, cGMP-PDE activity was increased in IMCD cell homogenates from nephritic compared with normal rats (388 +/- 32 versus 198 +/- 93 pmol/min per mg protein, P < 0.03). These results indicate that blunted volume expansion natriuresis accompanied by cellular resistance to ANP in vitro occurs in an immunologic model of renal injury. The resistance is not related to an alteration in ANP release or binding to its renal receptors, but is suppressed by PDE inhibitors and is associated with increased renal cGMP. PDE activity, thus suggesting that enhanced cGMP-PDE activity may account for resistance to the natriuretic actions of ANP observed in vivo. This defect may represent the intrinsic sodium transport abnormality linked to sodium

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Atrial Natriuretic Factor; Cyclic GMP; Diuretics; Glomerular Filtration Rate; Glomerulonephritis; In Vitro Techniques; Kidney Glomerulus; Kidney Tubules, Collecting; Male; Natriuresis; Peptide Fragments; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Sprague-Dawley; Receptors, Atrial Natriuretic Factor

In this study, we investigated the effects of a 3-min insulin incubation both at physiological and at supraphysiological concentrations on platelet aggregation and intraplatelet cyclic guanosine monophosphate (cGMP) levels both in the absence and in the presence of phosphodiesterase inhibition. We observed that insulin at concentration in the range 0.25-2 nmol/L decreases platelet response to adenosine 5-diphosphate (ADP), being Effective Dose 50 (ED50) for ADP with 2 nmol/L insulin 164 +/- 15% of the basal value, p = 0.005; furthermore, insulin increases intraplatelet content of cGMP (from basal 7.3 +/-0.6 pmol/10(9) plts to 14.6 +/- 1.2 pmol/10(9) plts with 2 nmol/L insulin, p=0.0001) and does not affect the platelet cGMP increase induced by nitrates. On the contrary, at very elevated concentrations (25-200 nmol/L) insulin increases platelet aggregation to ADP (ADP ED50 with 200 nmol/L insulin being 81 +/- 4% of the basal value, p = 0.01), decreases intraplatelet content of cGMP (from basal 7.2 +/- 0.1 pmol/10(9) plts to 5.7 +/- 0.2 pmol/10(9) plts with 200 nmol/L insulin, p = 0.01) and attenuates the platelet cGMP increase induced by nitrates. When cGMP catabolism is inhibited by theophylline or the selective cGMP phosphodiesterase inhibitor zaprinast, insulin shows anti-aggregating effects also at highly supraphysiological concentration (25-200 nmol/L). These results indicate that insulin, depending on the concentrations employed, shows opposite effects on platelet function, and they provide information about the mechanisms involved: actually, insulin is able to increase both cGMP synthesis, through guanylate cyclase activation, and cGMP catabolism, through phosphodiesterase activation. At physiological or slightly supraphysiological concentrations the first phenomenon is prevailing, so that cGMP intraplatelet values increase and insulin shows antiaggregating properties, whereas, at supraphysiological concentrations, insulin reduces cGMP levels through a prevailing phosphodiesterase activation, as supported by the fact that, when cGMP catabolism is prevented, insulin shows anti-aggregating properties also at the highest concentrations used.

Topics: Adenosine Diphosphate; Adult; Cyclic GMP; Dose-Response Relationship, Drug; Female; Humans; Insulin; Male; Nitroglycerin; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Platelet Aggregation; Platelet Aggregation Inhibitors; Purinones; Theophylline; Vasodilator Agents

To investigate early endothelial function associated with increased pulmonary blood flow, vascular shunts were placed between the ascending aorta and main pulmonary artery in 18 late-gestation fetal sheep. Four weeks after delivery, the lambs were instrumented to measure vascular pressures and blood flows, and blood was collected to measure plasma concentrations of guanosine 3',5'-cyclic monophosphate [cGMP, the second messenger to nitric oxide (NO)-mediated vasodilation] and L-arginine (the precursor for NO synthesis). The responses to the endothelium-dependent vasodilators acetylcholine (ACh, 1.0 microgram/kg) and ATP (0.1 mg.kg-1.min-1), the endothelium-independent vasodilators M & B-22948 (a cGMP-specific phosphodiesterase inhibitor, 2.5 mg/kg) and inhaled NO (40 ppm), and N omega-nitro-L-arginine (an inhibitor of NO synthase, 5 mg/kg) were then compared with responses in 12 age-matched controls. Vasodilator responses in control lambs were determined during pulmonary hypertension induced by U-46619 (a thromboxane A2 mimic). Shunted lambs displayed a selective impairment of endothelium-dependent pulmonary vasodilation, an augmented pulmonary vasoconstricting response to NO synthase inhibition, increased plasma cGMP concentrations, and decreased L-arginine concentrations. Taken together, these data suggest that lambs with pulmonary hypertension and increased pulmonary blood flow have early aberrations in endothelial function, as manifested by increased basal NO activity, that cannot be further increased by agonist-induced endothelium-dependent vasodilators.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Adenosine Triphosphate; Animals; Animals, Newborn; Arginine; Cyclic GMP; Endothelium, Vascular; Hemodynamics; Hypertension, Pulmonary; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Phosphodiesterase Inhibitors; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; Purinones; Sheep; Thromboxane A2; Vasoconstrictor Agents

1. Incubation of endothelial cells with platelets in the absence or the presence of PAF (10 nM) markedly increased platelet cyclic AMP levels, which were significantly decreased by indomethacin (3 microM). Co-incubation of endothelial cells and platelets with polymorphonuclear leukocytes (PMNs) did not change the platelet cyclic AMP levels. 2. Incubation of endothelial cells with platelets in the absence of PAF increased platelet cyclic GMP levels, which were increased 3.5 fold by PAF. These cyclic GMP levels were significantly decreased by NG-nitro-L-arginine (100 microM), and completely by methylene blue (10 microM). When endothelial cells and platelets were co-incubated with PMNs, the cyclic GMP level in the cell mixture was 42.5 and 65.3% lower than that in endothelial cells and platelets without and with PAF stimulation, respectively. 3. PAF induced platelet adhesion to endothelial cells only when PMNs were present. Methylene blue dose-dependently potentiated the PMN-dependent platelet adhesion induced by PAF, although it had no effect in the absence of PMNs. 4. Sodium nitroprusside and 8-bromo cyclic GMP but not dibutyryl cyclic AMP significantly, although partially, inhibited the platelet adhesion. Inhibition of cyclic GMP-specific phosphodiesterase by zaprinast slightly inhibited the PMN-induced platelet adhesion and potentiated the inhibitory effect of 8-bromo cyclic GMP, while these drugs markedly inhibited the adhesion of platelet aggregates induced by PMN sonicates. 5. These results suggest that the impairment by activated PMNs of EDRF-induced platelet cyclic GMP formation is involved in part in the mechanism of PMN-dependent platelet adhesion to endothelial cells induced by PAF in vitro. The precise mechanism still remains to be clarified.

Topics: Animals; Blood Platelets; Cyclic AMP; Cyclic GMP; Cyclooxygenase Inhibitors; Endothelium, Vascular; Humans; In Vitro Techniques; Indomethacin; Leukocytes; Methylene Blue; Neutrophils; Nitric Oxide; Nitroprusside; Phosphodiesterase Inhibitors; Platelet Activating Factor; Platelet Adhesiveness; Purinones; Rabbits

To examine the role of cyclic 3', 5'-guanosine monophosphate (cGMP) in airway responsiveness the effects of substances known to interfere with nitric oxide (NO) or cGMP were investigated on guinea pig airways. Using a perfused organ bath system, it was possible to apply the chemicals from either the serosal or the mucosal side independently. In addition, levels of intracellular cGMP were determined in tissues after various treatments. Sodium nitroprusside (a donor of NO), zaprinast (a specific inhibitor of cGMP phosphodiesterase) and 8-bromo-cGMP (8-Br-cGMP) caused a concentration-dependent relaxation of guinea pig trachea. These results indicate that cGMP is an important second messenger mediating tracheal relaxations. The above mentioned drugs caused a more profound relaxation when applied to the serosal side compared to the mucosal side, suggesting a barrier function of the epithelial layer. Incubation on the mucosal side of the tissues with 100 microM pyrogallol (a generator of superoxide that may inactivate NO) increased the contractile response to histamine at concentrations 0.3-3.2 microM (P < 0.05). Treatment of the preparations with 1 mM cystamine (an inactivator of guanylate cyclase) caused a 5-fold increase in the sensitivity to histamine (P < 0.05), indicating the involvement of the NO/cGMP pathway in the development of airway hyperresponsiveness. Incubation of the tissues with 100 microM histamine elevated the intracellular cGMP levels 10-fold; this effect was completely prevented by incubation of the tissues with methylene blue (a potent inactivator of guanylate cyclase). Mucosal incubation of the tracheal tubes with 10 microM methylene blue induced an 8-fold increase in sensitivity to histamine (P < 0.01) and the Emax was slightly increased. 25 min after instillation of 0.4 mumol methylene blue into the airways of anaesthetized guinea pigs, the lung resistance in response to histamine was elevated up to 395 +/- 82% (P < 0.001). The present study revealed that inactivation of NO or guanylate cyclase enhances the histamine-induced contractions of guinea pig tracheas. Therefore, it is suggested that the NO/cGMP pathway may be implicated in the pathogenesis of airway hyperresponsiveness and that drugs which enhance cGMP levels in airway smooth muscle may be of significance in the treatment of airway obstruction and enhanced reactivity.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Airway Resistance; Analysis of Variance; Animals; Coloring Agents; Cyclic GMP; Dose-Response Relationship, Drug; Epithelium; Guinea Pigs; Histamine; Male; Methylene Blue; Muscle Contraction; Muscle Relaxation; Nitric Oxide; Purinones; Trachea

The effect of inhibition of 3',5'-phosphodiesterase (PDE) activity on the cGMP accumulation was studied in control and nitric oxide (NO) stimulated hippocampal slices incubated in vitro using immunohistochemical visualisation of cGMP. Isobutylmethylxanthine (IBMX) was used as a non-selective PDE inhibitor and zaprinast was used as a selective inhibitor of cGMP-specific PDE activity. In the absence of PDE inhibitors cGMP-immunoreactivity (cGMP-IR) was found in blood vessel walls only. After incubation with the NO-donor sodium nitroprusside (SNP) cGMP-IR was found in a few isolated varicose fibres which were distributed throughout the slice. Incubation in the presence of either 1 mM IBMX or 10 microM zaprinast resulted in cGMP-IR in small numbers of varicose fibres distributed throughout the hippocampal slice. SNP in combination with IBMX resulted in cGMP-IR in small numbers multitude of varicose fibres throughout the slice; occasionally cell somata were observed. After incubation with SNP and zaprinast cGMP-IR was found in varicose fibres, although with a more restricted distribution and less numerous than in the presence of IBMX. In the latter combination, varicose fibres were observed predominantly in the CA2/CA3 region and in the stratum lacunosum molecular of the hippocampus, and cell somata were occasionally observed throughout the hippocampus. The differential distribution of cGMP-IR in the presence of different PDE inhibitors is consistent with the notion that there are regional differences in the localization of cGMP hydrolyzing enzymes in the hippocampus.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Cyclic GMP; Hippocampus; Immunohistochemistry; Nitric Oxide; Organ Culture Techniques; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Inbred Strains

1. While exposure of smooth muscle cells to sodium nitroprusside (SNP) leads to the development of tolerance to soluble guanylate cyclase (sGC) activation, the mechanisms responsible for this phenomenon in intact cells remain unclear. In the present study, possible mechanisms of tolerance were investigated in a cell culture model where sGC activity was estimated from the accumulation of cyclic GMP in response to 10 microM SNP over a 15 min period in the presence of a phosphodiesterase (PDE) inhibitor. 2. Pretreatment of rat aortic smooth muscle cells with 10-500 microM SNP led to a dose-dependent downregulation of cyclic GMP accumulation upon subsequent SNP stimulation. This effect was evident as early as 2 h following incubation with 10 microM SNP, reached a plateau at 4 h and was blocked by co-incubation with 30 microM oxyhaemoglobin. 3. Pretreatment of smooth muscle cells with the PDE inhibitor, zaprinast, resulted in downregulation of the SNP-induced cyclic GMP accumulation in a time- and concentration-dependent manner, that was first evident after 12 h. Moreover, while the zaprinast-induced downregulation of cyclic GMP accumulation was completely inhibited by the protein kinase A (PKA) inhibitor, H89, tolerance to SNP was partially reversed by H89. 4. beta 1 sGC steady state mRNA levels of S-nitroso N-acetylpenicillamine (SNAP)- or 8Br-cyclic GMP-pretreated cells were unchanged, as indicated by Northern blot analysis. However, Western blot analysis revealed that alpha 1 protein levels were decreased in zaprinast, but not in SNP, SNAP or 8Br-cyclic GMP pretreated cells. 5. While thiol depletion did not prevent the development of tolerance, pretreatment of cells with SNP in the presence of reducing agents partially or completely restored the ability of cells to respond to SNP. 6. We conclude that tolerance to SNP results from two distinct mechanisms: an early onset, NO-mediated event that is reversed by reducing agents and a more delayed, PKA-sensitive process that is mediated through increases in cyclic GMP and a decrease in sGC protein levels.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Antioxidants; Aorta; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Dose-Response Relationship, Drug; Drug Tolerance; Ethylmaleimide; Isoquinolines; Maleates; Muscle, Smooth, Vascular; Nitroprusside; Purinones; Rats; Rats, Wistar; Sulfhydryl Reagents; Sulfonamides; Vasodilator Agents

As previously described for endothelin-3, platelet exposure to cyclic GMP-elevating agents such as sodium nitroprusside and M&B-22948 (2-o-propoxyphenyl-8-azapurin-6-one), a cGMP phosphodiesterase inhibitor, lowered Ca2+ mobilization in response to thrombin. Interestingly, when cGMP phosphodiesterases were blocked, endothelin-3 produced a dose-dependent cGMP accumulation (P < 0.001). Since endothelin-3 has been proposed to decrease the activity of Ca2+ accumulating pumps, we examined whether this latter effect could be mediated by a rise in cGMP content. Cyclic GMP decreased in a dose-dependent manner the initial rate and plateau value of the ATP-dependent 45Ca2+ uptake in platelet membrane vesicles (P = 0.006 for each). Furthermore, combined treatment with endothelin-3 and M&B-22948 or a moderate concentration of Na(+)-nitroprusside further reduced the thrombin-evoked Ca2+ discharge (P = 0.004 and 0.01, respectively), suggesting that endothelin-3 pre-exposure had reduced the amount of mobilizable Ca2+. We propose that the depletion of platelet Ca2+ stores and the reduction of Ca2+ release evoked by endothelin-3 could be due, at least in part, to the elevation of cGMP content and to a decrease in Ca2+ accumulating pump activity.

Topics: Analysis of Variance; Biological Transport, Active; Blood Platelets; Calcium; Cyclic GMP; Dose-Response Relationship, Drug; Endothelin-3; Enzyme Inhibitors; Humans; Indicators and Reagents; Nitroprusside; Phosphodiesterase Inhibitors; Purinones; Thapsigargin; Thrombin

Hemodynamic tolerance to nitroglycerin was developed in spontaneously hypertensive rats following 2-3 days of pretreatment with 100 mg/kg of nitroglycerin administered s.c. 3 times/day. Tolerance was evaluated both in vivo, by administering ascending bolus doses of nitroglycerin of 1-300 micrograms/kg i.v., and ex vivo in isolated, denuded aortic vascular rings by exposure to ascending concentrations of nitroglycerin of 0.0003-100 microM. Tolerance was observed as a significant blunting of the hypotensive and vasorelaxant effect of nitroglycerin. Co-incubation of tolerant aortic rings and pretreatment of tolerant SHR with 10 microM and 0.1-10 mg/kg zaprinast, respectively, resulted in full restoration of the vasorelaxant and hypotensive effect of nitroglycerin. Zaprinast partially reversed hemodynamic tolerance at 0.01 mg/kg. Conversely, dipyridamole (10 microM) reversed tolerance ex vivo, but was ineffective in reversing tolerance in vivo at pretreatment doses of 30 and 60 mg/kg. Following a 100-micrograms/kg i.v. challenge dose of nitroglycerin, aortic cyclic guanosine monophosphate (cGMP) levels were lower in nitroglycerin tolerant SHR when compared to non-tolerant SHR. Pretreatment of tolerant SHR with 10 mg/kg zaprinast restored the increase in cGMP levels to nitroglycerin to that seen in non-tolerant SHR. Conversely, dipyridamole (30 mg/kg) pretreatment was not effective in restoring cGMP levels. These data therefore suggest that reversal of hemodynamic tolerance in vivo is related to restoration of changes in vascular cGMP levels. Zaprinast, a selective cGMP phosphodiesterase inhibitor, effectively reverses tolerance and dipyridamole, a rather non-selective inhibitor, does not.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Analysis of Variance; Animals; Aorta, Abdominal; Blood Pressure; Cyclic GMP; Dipyridamole; Dose-Response Relationship, Drug; Drug Tolerance; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Purinones; Rats; Rats, Inbred SHR; Vasodilator Agents

Blunted volume expansion (VE) natriuresis and renal resistance to atrial natriuretic peptide (ANP) characterize states of pathological sodium retention. This study examined rats 1 to 3 wk after common bile-duct ligation (CBDL), at which time they had hyperbilirubinemia and hypoalbuminemia. Sham-operated normal rats (Sham) showed an increased sodium excretion rate (UNaV) from 1.0 +/- 0.1 to 16.3 +/- 3.9 muEq/min in response to acute VE (iv saline, 2 mL/100 g body wt over 5 min), whereas CBDL rats had a blunted response that was apparent after 1 wk and became maximal at 2 and 3 wk (0.3 +/- 0.1 to 3.2 +/- 0.4 muEq/min at 3 wk, P < 0.01 versus Sham response). The peak urinary cGMP excretion rate (UcGMPV) was also blunted (37.9 +/- 3.6 versus 87.5 +/- 8.3 pmol/min, P < 0.01) despite an even greater increase in plasma ANP concentration (Sham, 9.6 +/- 0.4 pg/mL in hydropenia to 22.8 +/- 2.6 pg/mL after VE; CBDL, 15.3 +/- 2.3 to 41.8 +/- 6.8 pg/mL). ANP-dependent cGMP accumulation by isolated inner medullary collecting duct (IMCD) cells from both Sham and CBDL rat kidneys was dose-dependent; however, at higher concentrations of ANP (> 10(-8) M), accumulation by cells from CBDL rats was significantly blunted, indicating resistance to ANP. Binding of 125I-ANP to IMCD cells was not different in CBDL rats compared with Sham control rats. Renal denervation improved but did not completely reverse the blunted natriuresis, and ANP resistance persisted in IMCD cells from denervated kidneys of CBDL rats. Incubation of IMCD cells with the phosphodiesterase inhibitors isomethylbutylxanthine or Zaprinast (each at 10(-3) M) restored ANP responsiveness in both innervated and denervated kidneys from CBDL rats, and intrarenal infusion of Zaprinast (10 micrograms/min) corrected the blunted increase in UNaV and UcGMPV after VE in rats with CBDL. These results suggest that ANP resistance in a model of abnormal sodium metabolism devoid of intrinsic renal disease may be related to increased activity of phosphodiesterase in renal target cells for ANP as well as to heightened renal nerve activity.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Atrial Natriuretic Factor; Blood Volume; Common Bile Duct; Cyclic GMP; Drug Resistance; Kidney; Ligation; Liver Diseases; Male; Natriuresis; Nervous System; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Sprague-Dawley

Guanylate cyclase activity in human erythrocytes is investigated by evaluating the intracellular guanosine 3',5'-cyclic monophosphate (cGMP) content in the presence of various agents that exert specific effects on soluble or particulate guanylate cyclase. The increase in the intraerythrocyte cGMP content by the soluble guanylate cyclase activators nitroprusside and NaNO2 suggests the presence of this enzyme in human erythrocytes. The effects of four different atrial natriuretic peptide (ANP) fragments on the intraerythrocyte cGMP content is also studied. ANP II and ANP III increase the intraerythrocyte cGMP content, whereas ANP I and des-Ser5,des-Ser6-ANP III are ineffective. Thus our data show that human erythrocytes possess particulate guanylate cyclase together with the soluble enzyme. The ANP fragments ANP II and ANP III also activate the erythrocyte Na+/H+ exchange. Nitroprusside, M & B 22948 (an inhibitor of cGMP phosphodiesterase), and the cGMP analogues dibutyryl cGMP and 8-bromoguanosine 3',5'-cyclic monophosphate also increase the erythrocyte Na+/H+ exchange rate. The latter data also suggest that the erythrocyte Na+/H+ exchange is regulated by cGMP.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Triphosphate; Atrial Natriuretic Factor; Calcimycin; Cyclic GMP; Enzyme Activation; Erythrocytes; Guanylate Cyclase; Hemoglobins; Homeostasis; Humans; Kinetics; Manganese; Methemoglobin; Nitroprusside; Peptide Fragments; Phosphodiesterase Inhibitors; Purinones; Sodium Nitrite; Sodium-Hydrogen Exchangers

Administration of bacterial lipopolysaccharide (LPS) to rats stimulates synthesis of nitric oxide (NO), a free radical molecule that activates soluble guanylate cyclase, thereby increasing intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration and inducing systemic vasodilatation. To investigate the effect of endotoxemia on the pulmonary NO/cGMP signal transduction system, we measured the release of cGMP by isolated-perfused lungs of rats that received an intraperitoneal injection of LPS (1 mg/kg) or saline 2 days earlier. Over 90 min, 1.4 +/- 0.78 and 0.079 +/- 0.016 nmol cGMP accumulated in pulmonary perfusates of saline- and LPS-treated rats, respectively (P < 0.05). Despite addition to the perfusate of Zaprinast, superoxide dismutase, or A23187, markedly less cGMP was released from the lungs of rats exposed to LPS than from the lungs of control rats. In contrast, after ventilation with 100 parts per million NO gas, cGMP accumulating in the perfusate of the lungs of both groups of rats was markedly increased, and the quantity of cGMP released from the lungs of LPS-treated rats was similar to that released by control rat lungs (2.8 +/- 0.57 vs. 3.3 +/- 0.88 nmol, P = NS). With the use of immunoblot techniques, equal concentrations of constitutive endothelial NO synthase were detected in the lungs of rats treated with saline or LPS. These results demonstrate that the NO/cGMP signal transduction system is abnormal in the lungs of rats exposed to LPS, at least in part, at the level of endothelial NO synthase activation.

Topics: Animals; Blood Vessels; Body Water; Cyclic GMP; Endothelium, Vascular; In Vitro Techniques; Lipopolysaccharides; Lung; Nitric Oxide; Nitric Oxide Synthase; Perfusion; Phosphodiesterase Inhibitors; Pulmonary Circulation; Purinones; Rats; Rats, Sprague-Dawley; Respiration, Artificial; Superoxide Dismutase

Guanosine cyclic 3':5'-monophosphate (cGMP) plays a crucial role in regulating vascular smooth muscle contractile state. In rat aortic smooth muscle cells (RSMC) three isozymes of phosphodiesterase (PDE) may be involved in the degradation of cGMP, namely PDE I, PDE III, and PDE V. To study the effective contribution of PDE V to the control of intracellular cGMP levels, a specific and potent PDE V inhibitor 1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazolo[3, 4d]- pyrimidin-4-(5H)-one (DMPPO) was synthesized. DMPPO is a competitive inhibitor with respect to cGMP (Ki = 3 nM) and displayed high selectivity for PDE V as compared to other PDE isozymes. DMPPO strongly potentiated the cGMP response of atrial natriuretic peptide- or sodium nitroprusside-treated RSMC (EC50 = 0.5 microM). In addition, similar intracellular cGMP levels were obtained in the presence of a saturating concentration of DMPPO or 3-isobutyl-1-methylxanthine, a nonspecific PDE inhibitor, suggesting that cGMP is almost exclusively hydrolyzed by PDE V in RSMC. Stimulation of RSMC with atrial natriuretic factor resulted in accumulation of cGMP in the extracellular media. This egression was shown to be proportional to the intracellular level of cGMP and a first-order rate constant of 0.04 min-1 was determined for the egression process. DMPPO did not interfere with the efflux and allowed us to show that intracellular cGMP levels are mainly controlled by PDE V, rather than by egression in RSMC. DMPPO is, therefore, a useful tool for determining the role of PDE V in the control of cGMP levels in living cells and tissues.

Topics: Allopurinol; Animals; Atrial Natriuretic Factor; Cattle; Cyclic AMP; Cyclic GMP; Imidazoles; Isoenzymes; Muscle, Smooth, Vascular; Nitroprusside; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Quinoxalines; Rats; Sensitivity and Specificity; Stimulation, Chemical

The breakdown of the relaxation-inducing second messengers cAMP and cGMP is mediated by phosphodiesterases. Inhibitors of functionally present phosphodiesterases can be expected to induce relaxation by increasing the basic amount of cAMP and/or cGMP. In the cat gastric fundus, vinpocetine, which has some selectivity for phosphodiesterase type I, only induced contractions, but the inhibitors of type III [5-(4-acetimidophenyl)pyrazin-(1H)-one; SKF 94120], type IV (rolipram) and type V (zaprinast) phosphodiesterase all caused concentration-dependent relaxation, as did the non-specific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). The most potent relaxant agent was rolipram (EC50 9 +/- 5 x 10(-7) M and 3 +/- 1 x 10(-7) M in longitudinal and circular smooth muscle strips, respectively). These results suggest that type III, IV and V phosphodiesterases are functionally present in the cat gastric fundus and are involved in the regulation of tone. The possible influence of the phosphodiesterase inhibitors on non-adrenergic non-cholinergic (NANC) relaxation induced by nitric oxide (NO), vasoactive intestinal polypeptide (VIP) and train and sustained electrical field stimulation was then tested. Rolipram (3 x 10(-8) M), SKF 94120 (10(-5) M) and IBMX (10(-6) M) did not potentiate any of the relaxant stimuli studied. Zaprinast (10(-5) M), the cGMP specific type V phosphodiesterase inhibitor, caused a significant increase of the relaxation induced by exogenous NO and by train electrical field stimulation. These stimuli are thought to induce relaxation via an increase of intracellular cGMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Animals; Cats; Cyclic GMP; Dose-Response Relationship, Drug; Female; Gastric Fundus; In Vitro Techniques; Isoenzymes; Male; Muscle Relaxation; Nitric Oxide; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Pyrrolidinones; Rolipram; Vasoactive Intestinal Peptide

1. Possible contribution of K+ channel opening to the relaxation by glyceryl trinitrate (GTN) was examined using isolated rabbit aorta. 2. While glibenclamide and apamin failed to affect relaxation by GTN, both charybdotoxin (ChTx) and iberiotoxin (IbTx) effectively attenuated GTN-induced relaxation. 3. The increase in cGMP produced by GTN was not attenuated by ChTx and IbTx. 4. The inhibitory effect of ChTx on GTN-induced relaxation was not reduced in the presence of zaprinast, indicating that cGMP but not GMP was responsible for activation of the K+ channel. 5. Okadaic acid, a selective inhibitor of protein phosphatase 2A, had no effect on the relaxation by GTN. These results indicate that, though small in degree, activation of a ChTx-sensitive K+ channel (large conductance Ca(2+)-activated K+ channel) is involved in the GTN-induced relaxation in rabbit aorta.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aorta, Thoracic; Apamin; Charybdotoxin; Cyclic GMP; Endothelium, Vascular; Ethers, Cyclic; Glyburide; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Okadaic Acid; Peptides; Phosphoprotein Phosphatases; Potassium Channels; Protein Phosphatase 2; Purinones; Rabbits; Scorpion Venoms

Inhaled nitric oxide (NO) has been shown to selectively dilate the pulmonary vasculature. Zaprinast, an inhibitor of guanosine 3',5'-cyclic monophosphate-specific phosphodiesterase, augments smooth muscle relaxation induced by endothelium-dependent vasodilators. The present study was designed to determine whether intravenous administration of Zaprinast potentiates the vasodilating effects or prolongs the duration of action of intermittent NO inhalation. Eight awake lambs with U-46619-induced pulmonary hypertension breathed three concentrations of NO (5, 10, and 20 ppm) in a random order before and during an intravenous Zaprinast infusion (0.1 mg.kg-1.min-1). Inhaled NO decreased pulmonary arterial pressure (PAP) in a dose-dependent fashion, with mean PAP reduction at 5, 10, and 20 ppm NO inhalation of 6 +/- 1, 7 +/- 1, and 9 +/- 1 (SE) mmHg, respectively. Although the Zaprinast infusion did not change the magnitude of mean PAP reduction, it caused a statistically significant reduction of pulmonary vascular resistance and prolonged the duration of action of inhaled NO (half-times of vasodilator response to 5, 10, and 20 ppm NO inhalation: 1.9 +/- 0.1, 2.1 +/- 0.2, and 2.1 +/- 0.2 min, respectively; half-times of NO inhalation with Zaprinast: 9.7 +/- 1.7, 11.5 +/- 2.2, and 12.3 +/- 2.0, respectively). Plasma concentrations as well as the transpulmonary differences of guanosine 3',5'-cyclic monophosphate were increased by the Zaprinast infusion during NO inhalation. A stable level of pulmonary vasodilation was demonstrated in four additional lambs by combining intermittent NO breathing with an intravenous infusion of Zaprinast.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Administration, Inhalation; Animals; Cyclic GMP; Hemodynamics; Hypertension, Pulmonary; Infusions, Intravenous; Nitric Oxide; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Purinones; Sheep; Thromboxane A2; Vascular Resistance; Vasodilation

Nonadrenergic, noncholinergic relaxations were elicited by field stimulation (1-16 Hz, 1 msec, 8 V for 15 sec) of guinea pig trachea desensitized with capsaicin (3 microM), pretreated with atropine (1 microM), propranolol (1 microM), indomethacin (3 microM) and treated with alpha-chymotrypsin (2 U/ml) and contracted with 3 microM histamine. The effect of the phosphodiesterase (PDE) isozyme selective inhibitors siguazodan (PDE III-selective), rolipram (PDE IV-selective), denbufylline (PDE IV-selective) and zaprinast (PDE V-selective) was examined on the relaxant responses to field stimulation and on relaxations elicited by the nitric oxide donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1). The response to field stimulation in the presence of alpha-chymotrypsin (the putative nitric oxide component), at all the frequencies tested, was potentiated significantly by the PDE IV inhibitors rolipram (1 and 10 microM) and denbufylline (3 and 10 microM) as were responses to SIN-1. The PDE V inhibitor zaprinast (30 microM) potentiated relaxations elicited by field stimulation at 8 and 16 Hz and also potentiated responses to SIN-1. The PDE III inhibitor siguazodan (1 microM), however, was without effect on relaxant responses to field stimulation or to SIN-1. These results suggest that the nitric oxide component of the nonadrenergic, noncholinergic relaxant response is mediated primarily via cyclic AMP whose action is inactivated by a PDE IV isozyme and also by cyclic GMP which is inactivated by a PDE V isozyme.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Atropine; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 4; Electric Stimulation; Guanidines; Guinea Pigs; Indomethacin; Isoenzymes; Muscle Relaxation; Nitric Oxide; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Propranolol; Purinones; Pyridazines; Pyrrolidinones; Receptors, Adrenergic; Receptors, Cholinergic; Rolipram; Trachea; Vasoactive Intestinal Peptide; Xanthines

In the presence of 3-isobutyl-methylxanthine (IBMX), induction of cyclic 3',5'-guanosine monophosphate (GMP) production in human washed platelets (HWP) by nitric oxide donors (NOD) is followed by its accumulation in the surrounding medium in a time- and concentration-dependent manner. Thirty minutes incubation of HWP with 3-morpholino-sydonimine (SIN-1, 10 microM) at 37 degrees C resulted in a 4.6-fold increase of cyclic GMP in platelets, whereas in the extracellular medium the increase was 17.6-fold. Similar results were obtained when other NOD such as S-nitroso-N-acetylpenicyllamine (SNAP) and 3-(2-methoxy-5-chlorophenyl)oxatriazol-5-imine (GEA 3184) and the selective phosphodiesterase inhibitor, zaprinast (M&B 22948, 10 microM), were used. Probenecid (1-300 microM), an inhibitor of organic anion transport, or ouabain (1-300 microM), an inhibitor of Na+/K+ adenine triphosphate (ATP)-ase had no effect on cyclic GMP production or extrusion after stimulation with SIN-1. Significantly prostaglandin A1 (PGA1) and prostaglandin D2 (PGD2) inhibited the efflux of cyclic GMP from platelets induced by SNAP (10 microM) in a concentration-dependent fashion, with an IC50 of 63 +/- 16 and 143 +/- 17 microM, respectively. These studies suggest that the extrusion of cyclic GMP from human platelets after activation of soluble guanylate cyclase by NOD may contribute to the control of cyclic GMP levels in platelets with potential physiological and therapeutic consequences.

Topics: 1-Methyl-3-isobutylxanthine; Blood Platelets; Cells, Cultured; Culture Media; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Space; Guanylate Cyclase; Humans; Ion Transport; Molsidomine; Nitric Oxide; Ouabain; Penicillamine; Phosphodiesterase Inhibitors; Platelet Aggregation Inhibitors; Probenecid; Prostaglandin D2; Prostaglandins; Prostaglandins A; Purinones; S-Nitroso-N-Acetylpenicillamine; Sodium-Potassium-Exchanging ATPase; Time Factors; Triazoles

The effects of ethanol on cyclic GMP (cGMP) in washed human platelets were studied in the presence and absence of sodium nitroprusside (SNP), nitric oxide donor which stimulates guanylate cyclase. SNP stimulated cGMP accumulation in a dose-dependent fashion. After 1 min exposure to 100 microM SNP, the level of cGMP was approximately four-fold that in vehicle-treated platelets. Alcohol had no effect on basal cGMP, but inhibited SNP-induced cGMP accumulation at 17, 85 and 170 mM. In further experiments, platelets were incubated for 0, 0.5, 1 2 or 5 min with 10 microM SNP, with or without 100 microM zaprinast, a selective cGMP-phosphodiesterase (PDE) inhibitor and 85 mM ethanol. In the presence of zaprinast but not alcohol, cGMP levels rose continuously, to 10-fold the basal level at 5 min. Without zaprinast, cGMP levels were lower and reached a plateau by 2 min. Accumulation of cGMP was attenuated by alcohol 2 and 5 min after SNP addition, both in zaprinast-treated platelets and those without zaprinast. Thus, alcohol inhibits platelet cGMP accumulation stimulated by nitric oxide donor. Its mechanism probably does not involve a major effect on PDE, because the inhibition was observed in the presence or absence of zaprinast. We hypothesize that alcohol inhibits guanylate cyclase, contributing to its complex functional effects in platelets.

Topics: Blood Platelets; Cyclic GMP; Ethanol; Guanylate Cyclase; Humans; Nitroprusside; Phosphodiesterase Inhibitors; Platelet Activation; Purinones; Radioimmunoassay; Signal Transduction

Phosphodiesterases (PDE) are key enzymes regulating intracellular cyclic nucleotide metabolism and, thus, contraction and relaxation of the muscle. At present, five different families of isoenzymes of PDE exist that show a distinct species-specific and organ-specific distribution. The aim of the present study was to analyze the PDE isoenzymes present in the human ureter and to evaluate the functional effects of isoenzyme-specific inhibitors in this tissue. Normal ureteral tissue was obtained during radical nephrectomies, homogenized, centrifuged, and the supernatant fraction was separated using DEAE-Sephacel anion-exchange chromatography. PDE assay was then performed and the isoenzymes characterized on the basis of their kinetic characteristics and their sensitivity to allosteric modulators and inhibitors. In vitro, longitudinal ureteral strips as well as ureteral rings were precontracted, and different selective and nonselective PDE inhibitors were added incrementally. Three different PDE isoenzymes were identified: PDE I (Ca/calmodulin-stimulated), PDE II (cyclic guanosine monophosphate-stimulated), and PDE IV (cyclic adenosine monophosphate-specific). All PDE inhibitors relaxed the strips dose-dependently with an EC50 of 30 microM for papaverine, 40 microM for zaprinast, 25 microM for quazinone, and 0.1 microM for rolipram. The existence of three different PDE isoenzymes was shown in this study. The ureter-relaxing effect of the PDE IV inhibitor at low concentrations, combined with its low effect on the systemic circulatory parameters, may open a possibility of using selective PDE IV inhibitors in the treatment of ureteral colics or ureteral stones.

Topics: Chromatography, Ion Exchange; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Humans; Hydrolysis; In Vitro Techniques; Isoenzymes; Muscle Contraction; Muscle, Smooth; Papaverine; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Pyrrolidinones; Quinazolines; Rolipram; Ureter

1. After a period of myocardial ischaemia, reperfusion of the myocardium can elicit cardiac arrhythmias. Susceptibility to these arrhythmias declines with time, such that a preceding period of more than approximately 40 min ischaemia is associated with few reperfusion-induced arrhythmias. We have tested the hypothesis that this decline in susceptibility occurs, in part, because of protection by endogenous guanosine 3':5'-cyclic monophosphate (cyclic GMP). 2. Rat isolated hearts were subjected to 60 min left regional ischaemia followed by reperfusion (n = 10 per group). Methylene blue (20 microM), a soluble guanylate cyclase inhibitor, raised the incidence of reperfusion-induced ventricular fibrillation (VF) from 10% in control hearts to 80% (P < 0.05). This effect of methylene blue was abolished by co-perfusion with zaprinast (100 microM), a phosphodiesterase inhibitor which, in the rat heart, is cyclic GMP-specific (specific for the type-V phosphodiesterase isozyme). 3. Methylene blue reduced cyclic GMP levels in the ischaemic, non-ischaemic and reperfused myocardium (P < 0.05) to 50 +/- 10, 52 +/- 12 and 70 +/- 7 fmol mg-1 tissue wet weight, respectively from control values of 143 +/- 38, 147 +/- 43 and 156 +/- 15 fmol mg-1. Co-perfusion with zaprinast prevented this effect, and cyclic GMP levels were actually elevated (P < 0.05) to 366 +/- 102, 396 +/- 130 and 293 +/- 22 fmol mg-1 in ischaemic, non-ischaemic and reperfused myocardium, respectively. Zaprinast by itself also elevated cyclic GMP content. Cyclic AMP levels were not affected by zaprinast or methylene blue. 4. In conclusion, when endogenous cardiac cyclic GMP synthesis is reduced, susceptibility to reperfusion-induced VF after sustained ischaemia is substantially increased. The effect is prevented by inhibiting cyclic GMP degradation. Therefore cyclic GMP appears to be an endogenous intracellular cardioprotectant, and its actions may account for the low susceptibility to VF normally encountered in hearts reperfused after sustained ischaemia.

Topics: Animals; Cyclic GMP; Disease Susceptibility; Electrocardiography; Hemodynamics; In Vitro Techniques; Male; Methylene Blue; Myocardial Reperfusion Injury; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Wistar; Ventricular Fibrillation

Differentiation of murine erythroleukemia (MEL) cells induced by hexamethylene bisacetamide (HMBA) and DMSO was inhibited by several structurally unrelated nitric oxide (NO)-releasing agents and two membrane-permeable cGMP analogues. Since the effect of the NO-releasing agents was augmented by a cGMP phosphodiesterase inhibitor, at least some of their effect appeared to be mediated by activation of cytosolic guanylate cyclase. The drugs did not globally block differentiation since hemin-induced differentiation was undisturbed. In HMBA-treated cells, the NO-releasing agents and cGMP analogues reduced beta-globin and delta-aminolevulinate synthetase mRNA expression and inhibited the late down-regulation of c-myb mRNA that is required for HMBA-induced differentiation of MEL cells; the regulation of c-myc mRNA was not changed by the drugs. Nuclear run-off analyses showed that the drugs inhibited the HMBA-induced changes in beta-globin and c-myb transcription rates, and transient transfection of a reporter gene construct demonstrated that the drugs inhibited HMBA-inducible enhancer function of the alpha-globin control region, which contains binding sites for the erythroid transcription factors NF-E2 and GATA-1. The NO-releasing agents and cGMP analogues largely prevented HMBA-induced increases in DNA binding of NF-E2, whereas DNA binding of GATA-1 and SP-1 was not affected. The inhibition of erythroid gene expression by NO and cGMP analogues may be physiologically important under conditions of high NO production by endothelial cells and macrophages, i.e. during acute or chronic inflammation.

Topics: 5-Aminolevulinate Synthetase; Acetamides; Animals; Base Sequence; Cell Differentiation; Cell Line; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Cyclic GMP; Dimethyl Sulfoxide; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; GATA1 Transcription Factor; Gene Expression Regulation, Neoplastic; Globins; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Molsidomine; NF-E2 Transcription Factor; NF-E2 Transcription Factor, p45 Subunit; Nitric Oxide; Nuclear Proteins; Oligonucleotide Probes; Oncogenes; Phosphodiesterase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Purinones; Recombinant Proteins; RNA, Messenger; Thionucleotides; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The role of cGMP as a second messenger for renin secretion is contentious. This was investigated using a superfused collagenase-dispersed rat kidney cortex cell preparation devoid of indirect influences on renin secretion. Nitroprusside, atriopeptin II and 8-Br-cGMP all increased renin release but the dose-response relationships were biphasic. At low dose ranges there was a positive correlation between increasing drug concentration and renin secretion, but at high drug concentrations, a negative correlation was apparent. Methylene blue, a guanylate cyclase inhibitor, also suppressed baseline renin release at 10(-5) and 10(-6) M, but stimulated release at 10(-3) M. Using mid-range drug concentrations, the cGMP specific phosphodiesterase inhibitor MB22948 potentiated renin release in response to nitroprusside and 8-Br-cGMP. Inhibition of guanylate cyclase with either methylene blue or LY83583 attenuated renin release in response to nitroprusside, but, as expected, had no effect on 8-Br-cGMP induced release. We conclude that, under physiological conditions, cGMP is a stimulatory second messenger for renin release. This activity is mimicked at low dose ranges by 8-Br-cGMP, nitroprusside and atriopeptin II. In response to high doses of these drugs an unknown inhibitory pathway is activated and this opposes, in a dose-related manner, the stimulatory actions of cGMP for renin release.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Aminoquinolines; Animals; Atrial Natriuretic Factor; Cyclic GMP; Guanylate Cyclase; In Vitro Techniques; Kidney Cortex; Methylene Blue; Nitroprusside; Peptide Fragments; Purinones; Rats; Renin; Second Messenger Systems

Although the ability of somatostatin (ST) to relax cultured rat mesangial cells has recently been described, the intimate cellular mechanisms responsible for this effect have not been adequately clarified. The present experiments were designed to test the hypothesis that cyclic GMP (cGMP) could be involved in the genesis of this relaxation. ST increased cGMP synthesis by cultured rat mesangial cells, in basal conditions and in the presence of isobutylmethylxanthine or zaprinast. This effect was dose-dependent, with a threshold value of about 1 nM and a maximal response at ST concentrations between 0.1 and 1 microM. This increased cGMP synthesis was dependent on the stimulation by ST of a particulate guanylate cyclase, as the synthesis of cGMP by a particulate membrane fraction obtained from the cells increased in the presence of ST. When the cGMP-specific phosphodiesterase of mesangial cells was blocked with zaprinast, the ST-dependent relaxation, assessed both by morphological and biochemical criteria, significantly increased with respect to the experiments performed without zaprinast. These results support a role for cGMP in the ST-dependent relaxation of cultured rat mesangial cells. The increased cGMP synthesis appears to be the consequence of the activation of some form of particulate guanylate cyclase.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cell Division; Cells, Cultured; Cyclic GMP; Enzyme Activation; Glomerular Mesangium; Guanylate Cyclase; Purinones; Rats; Second Messenger Systems; Somatostatin; Vasodilation

1. We tested the hypothesis that increasing myocardial cyclic GMP levels would reduce myocardial O2 consumption and areas of low O2 supply/consumption balance, using zaprinast, a selective cyclic GMP-phosphodiesterase inhibitor. 2. The study was conducted in three groups (vehicle, 10(-3) and 3 x 10(-3) mol/L zaprinast) of anaesthetized open-chest New Zealand white rabbits (n = 24). Coronary blood flow (radioactive microspheres), arterial and venous O2 saturation (microspectrophotometry), O2 consumption, cyclic GMP content (competitive binding) and cyclic GMP-phosphodiesterase activity (conversion of 3H-cyclic GMP to 3H-GMP) were determined. 3. Agents were applied to a patch on the myocardial surface and did not cause significant haemodynamic changes, except for bradycardia in the vehicle and low dose group. 4. The total myocardial cyclic GMP-phosphodiesterase activity was 148 +/- 14 while the zaprinast (10 mumol/L) inhibitable activity averaged 63 +/- 8 pmol/mg protein per min. Cyclic GMP content was increased with increasing doses of zaprinast (vehicle, 4.308 +/- 0.349 pmol/g; low dose zaprinast, 4.803 +/- 0.279 and high dose zaprinast, 7.938 +/- 1.304 pmol/g). 5. Coronary blood flow was not different after treatment (198 +/- 11, 209 +/- 10 and 153 +/- 9 mL/min per 100 g for the vehicle, low and high dose zaprinast, respectively). 6. Under control conditions, 48% of the small veins had O2 saturations below 50%. With zaprinast, this value was reduced to 19% for the low and 24% for the high dose. 7. Average venous O2 saturation increased with zaprinast (49 +/- 2%, 61 +/- 3% and 59 +/- 1%).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Binding, Competitive; Blood Pressure; Coronary Circulation; Cyclic GMP; Dose-Response Relationship, Drug; Heart; Heart Rate; Microspheres; Myocardium; Oxygen; Oxygen Consumption; Purinones; Rabbits; Spectrophotometry, Ultraviolet

We investigated the presence and features of "low Km" 3'-5' cyclic nucleotide phosphodiesterase activity in the homogenates and extracts of rat superior cervical ganglion. The DEAE chromatographic elution profile of a Triton X-100 extract showed two peaks of cAMP phosphodiesterase activity eluted at 280 and 600 mM sodium acetate and two peaks of cGMP phosphodiesterase activity eluted at 300 and at 500 mM sodium acetate. The activity was poorly stimulated by calcium-calmodulin and neither stimulated or inhibited by cGMP. Both cGMP PDE peaks were inhibited by zaprinast, with IC50's of 1.4 microM and 0.28 microM: their Km values were 4.4 and 3.8 microM, respectively. These features, together with cGMP binding activity, indicate that both enzymes belong to the phosphodiesterase V family. The Km values of the first and second cAMP phosphodiesterase peaks were 1.7 and 3.8 microM. Although both peaks displayed a cAMP specific hydrolysis, only the second peak was inhibited by RO 20-1724, with an IC50 of 8 microM. Preganglionic denervation indicated that the bulk of phosphodiesterase activity is localized in ganglion cells. In order to investigate possible effects of aging on the ganglionic function, phosphodiesterase activity was assayed in the ganglia of young (3 months) and old (25 months) male Fisher rats. The chromatographic profiles and kinetic features revealed no significant differences between young and old rats.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Age Factors; Animals; Cyclic AMP; Cyclic GMP; Kinetics; Male; Purinones; Rats; Rats, Sprague-Dawley; Superior Cervical Ganglion

The ability of exogenous nitric oxide (NO) to modify synaptic transmission was investigated in area CA1 of the rat hippocampal slice. The NO donors S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (SNOG) depressed field excitatory postsynaptic potentials evoked by low frequency stimulation of the Schaffer collateral-commissural pathway. Upon washout of the NO donors, synaptic transmission rapidly returned to control levels. A similar reversible synaptic depression was produced by SNAP when tetanic stimulation (100 Hz; 1 s) was delivered in its presence. The effect of SNAP was not mimicked by its precursor or breakdown product and was blocked by haemoglobin, indicating that the effect involved NO. Roussin's black salt, a photolabile NO donor, also depressed transiently field excitatory postsynaptic potentials following photolysis. The depression was induced rapidly following a flash of UV light (20 s duration) focused onto the slice using a confocal microscope. The depressant effect of the NO donors on synaptic transmission was mimicked by zaprinast, a specific cGMP-phosphodiesterase inhibitor. Zaprinast depressed to a similar extent both the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and N-methyl-D-aspartate receptor-mediated components of excitatory postsynaptic currents without affecting passive membrane properties, indicating a presynaptic locus of action. SNAP, SNOG and zaprinast all elevated cGMP levels in rat hippocampal slices. Immunocytochemical staining revealed that the cGMP accumulation was mainly in a network of varicose fibres running throughout the CA1 region, consistent with a presynaptic site of action of NO. We conclude that NO, possibly through activation of guanylate cyclase, may be involved in transient presynaptic depression in the CA1 region of the hippocampus.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Electric Stimulation; Evoked Potentials; Female; Glutathione; Hippocampus; Immunochemistry; In Vitro Techniques; Nitric Oxide; Nitroso Compounds; Penicillamine; Photolysis; Purinones; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; S-Nitrosoglutathione; Synaptic Transmission

An increase in cyclic nucleotide monophosphate levels is suggested to play a prominent role in mediating smooth-muscle relaxation. Cyclic nucleotide phosphodiesterase (PDE) influences smooth-muscle tone by decreasing the level of cyclic nucleotides. At present, five different families of isoenzymes of PDE exist that show a distinct species- and organ-specific distribution. Our study was done to evaluate the existence of specific PDE isoenzymes and its functional role in human ureteral tissue. Normal ureteral tissue was homogenized and centrifuged and the supernatant fraction was separated using anioin-exchange diethylaminoethyl (DEAE)-Sephacel chromatography. A PDE assay was then performed and the peak fractions were added to different specific PDE activators and inhibitors. In vitro, longitudinal ureteral strips were precontracted and different selective and non-selective PDE inhibitors were added incremently. Three different PDE isoenzymes were characterized: PDE I (calmodulin-sensitive), PDE II (cGMP-stimulated), and PDE IV (cAMP-specific). All PDE inhibitors relaxed the strips dose-dependently, with the 50% effective concentrations (EC50) being 30 microM for papaverine, 40 microM for zaprinast, 25 microM for quazinone, and 0.1 microM for rolipram. The ureter-relaxing effect of the PDE IV inhibitor at low concentrations, combined with its low-level effect on the systemic circulatory parameters, may open the possibility of using selective PDE IV-inhibitors in the treatment of ureteral colics or for ureteral stone passage.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Calmodulin; Chromatography, DEAE-Cellulose; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Humans; Hydrolysis; In Vitro Techniques; Muscle Relaxation; Muscle Tonus; Muscle, Smooth; Papaverine; Phosphodiesterase Inhibitors; Purinones; Pyrrolidinones; Quinazolines; Rolipram; Ureter

Activation of the NO-cGMP pathway or adenosine receptors depresses reversibly synaptic transmission in the hippocampus. Here we demonstrate, using the selective A1 receptor antagonist DPCPX, a convergence in the mechanisms of action of the NO donor SNAP, the cGMP phosphodiesterase inhibitor zaprinast and adenosine.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Hippocampus; In Vitro Techniques; Neural Pathways; Nitric Oxide; Penicillamine; Purinergic P1 Receptor Antagonists; Purinones; Rats; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Synapses; Synaptic Transmission; Vasodilator Agents; Xanthines

According to their respective elution order, specificity for cAMP and cGMP, their sensitivity to calmodulin, and their modulation by cGMP and rolipram, four cyclic nucleotide phosphodiesterases (PDE) were separated from the cytosol: PDE I (calmodulin-sensitive), PDE II (stimulated by cGMP, PDE IV (cGMP specific-PDE and inhibited by rolipram) and PDE V (cGMP specific). PDE IV (Km = 1.4 microM) was competitively inhibited by rolipram (Ki = 1.2 microM) whereas PDE V (Km = 0.83 microM) was competitively inhibited by zaprinast in the mumolar range (Ki = 0.12 microM). Moreover the microsomal fraction contained three PDE isoforms: PDE II, PDE III (inhibited by cGMP or indolidan) and PDE IV. These results show that cAMP degradation in cytosolic and membrane fractions is modulated by cGMP and selectively inhibited by rolipram and, in addition, by indolidan in membrane fractions.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cattle; Cell Membrane; Cyclic GMP; Cytosol; Egtazic Acid; Epithelium; Indoles; Isoenzymes; Kinetics; Microsomes; Oxindoles; Phosphodiesterase Inhibitors; Purinones; Pyridazines; Pyrrolidinones; Rolipram; Substrate Specificity; Trachea

Platelets have been implicated in the pathophysiology of ischemia-reperfusion injury. In this study, antiplatelet effects of cyclic GMP (cGMP)- and cyclic AMP (cAMP)-mediated agents were evaluated in renal ischemia in pentobarbital-anesthetized rats. Renal ischemia was induced by unilateral occlusion of the left renal artery (40 min) followed by reperfusion (30 min) with the contralateral kidney serving as control. 111Indium-labeled platelets, drugs or vehicle were administered 30 min before induction of renal ischemia. Occlusion of the left renal artery for 20, 40 or 60 min resulted in a 100, 300 and 600% increase (over contralateral right kidney) in the platelet-associated 111indium activity in the ischemic kidney. In all subsequent studies the kidney was occluded for 40 min to test the antiplatelet activity of individual agents. 8-Br-cGMP (0.1 and 0.3 mg/kg/min i.v.), zaprinast (0.1 mg/kg/min i.v.) and sodium nitroprusside (0.003 and 0.01 mg/kg/min i.v.) significantly attenuated platelet accumulation in renal ischemia, whereas 8-Br-cAMP (0.3 mg/kg/min i.v.) or milrinone (0.1 mg/kg i.v. bolus, plus 0.01 mg/kg/min) did not. Minoxidil (0.01 and 0.03 mg/kg/min i.v.), a vasodilator which produced equihypotensive effects as the cGMP-mediated agents, and milrinone failed to prevent platelet accumulation. These results demonstrate that modulation of the platelet function by cGMP agents can be dissociated from their blood pressure lowering effects. cGMP is known to inhibit both platelet adhesion and aggregation, whereas cAMP is only active against aggregation. The present findings provide further evidence that cGMP-mediated drugs may afford effective antiplatelet action in an in vivo model of ischemia-reperfusion injury.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Blood Platelets; Cyclic AMP; Cyclic GMP; Ischemia; Kidney; Male; Nitroprusside; Platelet Aggregation Inhibitors; Purinones; Rats; Reperfusion

Fibroblast growth factor is present in blood vessels and is thought to play an important role in promoting vascular cell proliferation in vivo. In the current study, we show that three agents that activate the guanosine 3',5'-cyclic monophosphate (cGMP) system, including the nitric oxide-generating agents S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) as well as the stable cGMP analogue 8-bromo-cGMP, increased fibroblast growth factor-2 (FGF-2; basic fibroblast growth factor)-induced [3H]thymidine incorporation by severalfold in primary cultures of rat aortic smooth muscle cells. SNAP increased the efficacy, but not the potency, of FGF-2. The stimulatory effect of SNAP was selective for FGF-2-induced mitogenesis as shown by the lack of a significant effect on [3H]thymidine incorporation induced by several other growth factors. Consistent with thymidine incorporation experiments, SNAP amplified the increase of the cellular DNA content induced by FGF-2 as well as the proliferation of cells. A selective inhibitor of cGMP phosphodiesterases, zaprinast, potentiated the comitogenic effect of SNAP and its ability to increase cGMP levels, supporting the involvement of cGMP as second messenger. Consistent with previous results, and opposite to that found in primary and early subculture, SNAP decreased mitogen-induced [3H]thymidine incorporation in cells in later subculture. Because macrophage- and vascular smooth muscle-derived nitric oxide is likely to be present in relatively large concentrations after vascular injury, we speculate that endogenous nitric oxide may amplify the activity of FGF-2 in vivo.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aorta; Cell Division; Cells, Cultured; Cyclic GMP; DNA; Drug Synergism; Fibroblast Growth Factor 2; Growth Substances; Male; Mitogens; Muscle, Smooth, Vascular; Nitric Oxide; Penicillamine; Purinones; Rats; Rats, Sprague-Dawley; S-Nitroso-N-Acetylpenicillamine; Thymidine; Time Factors

To elucidate the sequence of events between the release of neurotransmitters and cavernous smooth muscle relaxation in erection, we studied the role of the cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) systems. In a well-established simian model, the effects of specific agonists and antagonists of the intracellular sequence for smooth muscle relaxation and potassium channel openers on the intracavernous pressure were examined. Sodium nitroprusside (10(-3) M), a nitric oxide releaser and thus a stimulant of the cGMP system, caused an increase in the intracavernous pressure from 82 to 115 cm H2O for 7 to 19 min and penile diameter from 24.8 +/- 2.28 to 43 +/- 4.87 mm. When nitroprusside was injected after methylene blue (10(-3) M), a specific antagonist of the enzyme guanylate cyclase, intracavernous pressure rise decreased significantly, but cromakalin, a potassium channel opener, provoked excellent increases after the block. A smaller dose of sodium nitroprusside (10(-4) M) caused an increase in intracavernous pressure from 35 to 85 cm H2O for 7 to 11.5 min. When nitroprusside was injected after zaprinast, a phosphodiesterase inhibitor, the increase in pressure ranged from 80 to 116 cm H2O for 15 to 30 min. Prostaglandin E1, an activator of the cAMP system, caused an increase in the intracavernous pressure of 20-80 cm H2O for 5 to 10 min, and an increase in penile diameter from 25 +/- 2.22 to 35 +/- 3.48 mm. The erectile response to PGE1, but not to cromakalin, was nearly abolished by ethylmaleimide, an adenylate cyclase blocker. The response to nitroprusside was significantly greater (P < 0.05) than to PGE1. Both systems, cAMP and cGMP, may be involved in cavernous smooth muscle relaxation, and cGMP is probably the predominant intracellular second messenger in penile erection in monkeys. Stimulants of the cGMP system, such as nitric oxide releasers, could represent a more physiological and effective approach in the treatment of erectile dysfunction.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Alprostadil; Animals; Benzopyrans; Cromakalim; Cyclic AMP; Cyclic GMP; Ethylmaleimide; Macaca nemestrina; Male; Methylene Blue; Nitroprusside; Penile Erection; Penis; Phosphodiesterase Inhibitors; Purinones; Pyrroles; Ultrasonography

There is growing evidence that nitric oxide serves as a neurotransmitter released from enteric inhibitory nerves in the gastrointestinal tract. The distribution of nitric oxide synthase suggests that nitric oxide may also be a neurotransmitter within enteric ganglia. Since many actions of nitric oxide are mediated by stimulation of soluble guanylate cyclase and a subsequent increase in 3',5'-cyclic guanosine monophosphate (cGMP) concentration, targets for nitric oxide in the canine proximal colon were investigated by immunohistochemical localization of cGMP. In the presence of phosphodiesterase inhibitors (M&B 22948, 100 microM and 3-isobutyl-1-methyl-xanthine, 1 mM), exogenous nitric oxide and electrical field stimulation caused an accumulation of cGMP-like immunoreactivity in several cell-types including colonic smooth muscle cells. cGMP-like immunoreactivity was also observed in a subpopulation of neurons in both myenteric and submucosal ganglia. Sequential labeling with the NADPH diaphorase technique showed that 94% of neurons that responded to exogenous nitric oxide with an increase in cGMP-like immunoreactivity were NADPH diaphorase negative. None of the myenteric neurons that responded to electrical field stimulation with an increase in cGMP-like immunoreactivity were NADPH diaphorase positive, and only one submucosal neuron with cGMP-like immunoreactivity was also NADPH diaphorase positive. The electrical field-stimulated increase in cGMP-like immunoreactivity was blocked by nitroarginine (100 microM). An increase in cGMP-like immunoreactivity also occurred in interstitial cells located at the submucosal surface of the circular muscle layer. These cells are interposed between nerve varicosities and smooth muscle cells and may partially mediate neuromuscular transmission. Sodium nitroprusside and nitric oxide also caused an accumulation of cGMP-like immunoreactivity in smooth muscle cells of intramural arterioles and venules. The results of this study further support the role of nitric oxide as a neurotransmitter in colonic muscles, and provide support for the hypothesis that interstitial cells are functionally innervated by enteric inhibitory neurons. The data also suggest that nitric oxide may serve as a neurotransmitter in enteric ganglia.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Colon; Cyclic GMP; Dogs; Electric Stimulation; Enteric Nervous System; Female; Ganglia, Autonomic; Male; Nitric Oxide; Nitroprusside; Purinones; Second Messenger Systems

Cyclic nucleotides mediate relaxation of gastrointestinal smooth muscle. The intracellular concentration of these second messengers is determined by a balance between their synthesis and metabolism. Because cyclic nucleotide phosphodiesterase enzymes (PDE) are the sole enzymes responsible for their degradation, it is essential to determine the role of the various PDE isozymes in regulating cyclic nucleotide content of gastrointestinal smooth muscle. To examine the role of different PDE isozymes in colonic smooth muscle motility, soluble PDE activity was measured in fractions obtained from homogenates of canine colon using DEAE sepharose chromatography. PDE activity was determined using [3H]cyclic AMP (cAMP) (1 microM) or [3H]cyclic GMP (cGMP) (1 microM) as a substrate. Results indicated that colonic smooth muscle contains at least two forms of PDE with a high affinity for cGMP. One form was stimulated by calmodulin (type I) and the other was inhibited by low concentrations of zaprinast (type V). In addition, colonic smooth muscle contains at least two isozymes that prefer cAMP as a substrate. One form was inhibited by SB 94120 and cGMP (type III) and the other by rolipram (type IV). An additional peak of PDE activity was identified. The hydrolysis of cAMP by this peak was greatly enhanced by the presence of cGMP, suggesting that this activity belonged to type II or cGMP-stimulated PDE. The functional role of these isozymes was evaluated by determining the ability of selective PDE inhibitors to antagonize a carbachol (0.3 microM)-induced contraction of isolated circular colonic muscle strips in the presence of forskolin (0.2 microM). Concentration-dependent decreases in contractile activity were observed with the following potency order: rolipram > Ro 20-1724 > isobutyl methylxanthine > SB 94120 > zaprinast. These results demonstrate that colonic smooth muscle contains several PDE isozymes and that selective inhibition of PDE isozymes can increase cyclic nucleotide content and antagonize contractile responses. Functionally, PDE IV appears to be very important in reducing contractile activity, suggesting that selective PDE IV inhibitors might be useful in the treatment of gut hypermotility disorders.

Topics: 1-Methyl-3-isobutylxanthine; 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Colforsin; Colon; Cyclic AMP; Cyclic GMP; Dogs; Isoenzymes; Muscle, Smooth; Phosphodiesterase Inhibitors; Purinones; Pyrrolidinones; Rolipram

We have investigated the role played by cyclic nucleotide phosphodiesterases (EC 3.1.4.17) in the control of T-lymphocyte response to mitogenic agents by their ability to influence the cellular level of cAMP. The importance of this messenger as a negative regulator in this cell type is well established. Multiple isoenzymes of phosphodiesterase were fractionated from the cytosol of rat thymic lymphocytes by high performance liquid chromatography on an anion exchange column. In addition to the type II, III, IV isoforms that we have already described [Valette et al., Biochem. Biophys. Res. Commun. 169:864-872 (1990)], a phosphodiesterase fraction sharing several of the characteristics of type V, cGMP-binding phosphodiesterase, was detected. Non-isoform-selective inhibitors of phosphodiesterase such as dipyridamole, papaverine, and methyl-isobutylxanthine were able to totally prevent the proliferative response of thymocytes to stimulation by the mitogenic lectin concanavalin A. In contrast, the selective inhibitor of type IV phosphodiesterases rolipram induced a rather moderate inhibition of proliferation, not exceeding 60%; and the selective inhibitors of type III and type V phosphodiesterases, milrinone and M&B 22,948, respectively, displayed only marginal inhibitory effects. The association of the type III and IV phosphodiesterase inhibitors produced synergistic inhibition of proliferation, which could then be almost totally suppressed. These inhibitory effects on cell multiplication were reflected at the level of the cell cAMP content; only rolipram was able to induce a significant (approximately 50%) increase in cAMP, and this increase was potentiated by the presence of milrinone, reaching almost 100%. The type V phosphodiesterase selective inhibitor M&B 22,948 displayed similar properties to those of milrinone, which suggests that it indirectly inhibited the type III, cGMP-inhibitable isoenzyme, by inducing a cGMP rise. This hypothesis was supported by evidence of a significant raising effect of M&B 22,948 on cGMP level, and by the ability of a cGMP-elevating agent, sodium nitroprusside, to mimic the synergistic effects of milrinone associated with rolipram. Furthermore, 8-bromo-cGMP, a potent activator of cGMP-dependent protein kinase, which showed only weak inhibitory effects on thymic type III phosphodiesterase, failed to alter the effects of rolipram on the cell proliferation. These results allow us to delineate a role for types III, IV, and V phosp

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Concanavalin A; Cyclic AMP; Cyclic GMP; Cytosol; In Vitro Techniques; Isoenzymes; Lymphocyte Activation; Male; Milrinone; Nitroprusside; Purinones; Pyridones; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Rolipram; T-Lymphocytes

A newly synthesized compound, 4-((3,4-(methylenedioxy)benzyl)amino)-6,7,8-trimethoxyquinazoline (6), had a potent (IC50 = 0.36 microM) inhibitory action on cyclic GMP phosphodiesterase (cGMP-PDE) isolated from porcine aorta; its inhibitory activities toward other PDE isozymes were at least 10-fold weaker. In addition, 6 relaxed porcine coronary arteries precontracted with PGF2 alpha (EC50 = 1.96 +/- 0.58 microM). At the concentration of 30 microM, 6 caused elevation of the intracellular cGMP level in porcine coronary arteries without any change in cAMP level. Various other 4-substituted 6,7,8-trimethoxyquinazolines were also synthesized and evaluated for cGMP-PDE inhibitory activity. From their structure-activity relationships, we concluded that the 4-((3,4-(methylenedioxy)benzyl)-amino) group is essential for potent inhibition of cGMP-PDE.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aorta; Chromones; Coronary Vessels; Cyclic GMP; Dinoprost; Isoenzymes; Molecular Structure; Muscle Relaxation; Purinones; Quinazolines; Structure-Activity Relationship; Swine

Alterations in either cyclic AMP (cAMP) or cyclic GMP (cGMP) may modulate the production of aqueous humor by the ciliary epithelium of the eye, thereby affecting intraocular pressure. We have found distinct profiles of phosphodiesterase (PDE) isozyme activity in cultured cells derived from bovine pigmented ciliary epithelium (PE) and cells derived from human nonpigmented ciliary epithelium (NPE), as well as corresponding differences in the effects of selective PDE inhibitors on the accumulation of cAMP and cGMP. In NPE cells, but not in PE cells, the major peak of PDE activity was stimulated by Ca++/calmodulin-stimulated (PDE I), and hydrolyzed both cAMP and cGMP. In contrast, PE cells contained a cGMP-specific PDE V not found in NPE cells. Rolipram, a selective inhibitor of PDE IV, was more potent and effective than the selective PDE III inhibitor CI-930 at potentiating intracellular cAMP accumulation in both cell types. Zaprinast, a selective inhibitor of PDE V, potentiated cGMP accumulation in PE but not in NPE cells. The results suggest that selective PDE inhibitors may modulate aqueous humor production by pigmented and nonpigmented ciliary epithelium, the two cell types may have different functional roles, and selective modulation of their functions may be possible. Furthermore, there may be distinct roles for intracellular calcium in regulating cGMP and cAMP in pigmented vs. nonpigmented ciliary epithelial cells.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calcium; Cattle; Cell Line, Transformed; Ciliary Body; Cyclic AMP; Cyclic GMP; Drug Synergism; Epithelial Cells; Epithelium; Humans; Isoenzymes; Pigment Epithelium of Eye; Purinones; Pyridazines; Pyrrolidinones; Rolipram

Following in vitro exposure of rat aortic rings to 550 microM nitroglycerin for 1 h, tolerance was demonstrated by a significant increase in EC50 values for nitroglycerin-induced relaxation. However, cross-tolerance to sodium nitroprusside was not observed. Co-incubation of aortic rings with the cGMP-phosphodiesterase (cGMP-PDE) inhibitor zaprinast (10 microM), during incubation with 550 microM nitroglycerin, did not prevent the development of tolerance. However, the addition of 0.30 or 10 microM zaprinast to tolerant aortic rings did restore responsiveness to nitroglycerin. The increase in cGMP in tolerant aortic rings in response to 300 nM nitroglycerin (2-4 fmol/micrograms) was significantly less than that observed for non-tolerant rings (6.6-12 fmol/micrograms), but cGMP levels were restored in tolerant rings by zaprinast (7-12 fmol/micrograms). These data suggest that inhibition of vascular cGMP-PDE activity does not prevent the development of tolerance in vitro, but does reverse the loss of vasorelaxant potency to nitroglycerin via restoration of intracellular cGMP levels.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aorta, Thoracic; Cyclic GMP; Drug Tolerance; Hydrolysis; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Nitroglycerin; Purinones; Rats; Rats, Sprague-Dawley

The purpose of this study was to determine if significant relationships exist between plasma and aortic cyclic GMP (cGMP) levels and pharmacodynamic effect after the i.v. administration of the cGMP-selective phosphodiesterase inhibitor zaprinast to conscious, spontaneously hypertensive rats. Zaprinast dose-dependently increased plasma and aortic cGMP levels at 10, 18 and 30 mg/kg and decreased mean arterial blood pressure (MAP) at 18 and 30 mg/kg. The concentrations of cGMP in the plasma and in the aorta were significantly correlated (r = 0.765, P < 0.0001). The changes in MAP were significantly correlated to aortic (r = -0.750, P < 0.0001) and plasma (r = -0.762, P < 0.0001) cGMP levels. We conclude that plasma cGMP may be an index of cGMP-selective phosphodiesterase inhibition in vivo.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Analysis of Variance; Animals; Aorta, Abdominal; Blood Pressure; Cyclic GMP; Dose-Response Relationship, Drug; Hypertension; Injections, Intravenous; Male; Purinones; Radioimmunoassay; Rats; Rats, Inbred SHR

We sought to determine whether the L-arginine-nitric oxide-guanosine 3',5'-cyclic monophosphate (cGMP) pathway, known to mediate neurostimulation-induced smooth muscle relaxation in penile tissue of rabbits and humans in vitro, is operative also in vivo. Adult male dogs (n = 9) were subjected to direct electrical stimulation of the pelvic nerves to induce penile tumescence. Intracavernous injection of the nitric oxide-releasing substance S-nitroso-N-acetylpenicillamine resulted in similar tumescence. Intracavernous injection of a specific inhibitor of nitric oxide synthesis, NG-nitro-L-arginine, blocked pelvic nerve-stimulated tumescence, and this was partially reversed by intracavernous injection of the nitric oxide precursor L-arginine. Furthermore, neurostimulated tumescence was inhibited by methylene blue, an inhibitor of cytosolic guanylate cyclase and enhanced by M&B 22948, a cGMP phosphodiesterase inhibitor. These in vivo findings support the hypothesis that cavernous smooth muscle relaxation and penile tumescence are mediated by nitric oxide and cGMP.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Arginine; Cyclic GMP; Dogs; Electric Stimulation; Male; Methylene Blue; Nervous System Physiological Phenomena; NG-Nitroarginine Methyl Ester; Nitric Oxide; Pelvis; Penile Erection; Purinones

Experiments were performed to test the hypothesis that enteric inhibitory neurotransmission in pyloric muscles is mediated by NO. Junction potentials were recorded with intracellular microelectrodes from cells near the myenteric and submucosal surfaces of the circular muscle layer. Inhibitory junction potentials (IJPs) were apamin sensitive and were reduced by arginine analogues [NG-nitro-L-arginine-methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA)]. The effects of arginine analogues were reversed by L-arginine. Inhibition of IJPs unmasked excitatory JPs (EJPs) in the myenteric region and increased excitability of cells in the submucosal region. IJPs were also reduced by oxyhemoglobin. As with arginine analogues, reduction in IJPs increased EJP amplitude. Combination of L-NAME and oxyhemoglobin completely blocked IJPs, suggesting that NO, or an NO-containing compound, mediated the enteric inhibitory nerve responses. Exogenous NO hyperpolarized membrane potential, and these responses were also reduced by apamin. The magnitude of the responses to a given dose of NO was similar in cells of the myenteric and submucosal regions, suggesting that relatively smaller IJPs in submucosal cells may be due to a lower density of enteric inhibitory innervation in the submucosal region. The effects of NO were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (cGMP) and M & B 22948, a specific cGMP phosphodiesterase inhibitor, suggesting that the hyperpolarization response to NO may be mediated by enhanced production of cGMP. IJPs were also prolonged by M & B 22948. IJPs and NO disrupted normal electrical rhythmicity in cells in the myenteric region. This may provide a basis for inhibitory effects of enteric inhibitory nerve stimulation on sphincter pressure in pyloric canal in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Arginine; Cyclic GMP; Dogs; Duodenum; Electric Stimulation; Female; Gastric Mucosa; In Vitro Techniques; Intercellular Junctions; Intestinal Mucosa; Male; Membrane Potentials; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide; omega-N-Methylarginine; Oxyhemoglobins; Phentolamine; Propranolol; Purinones; Pylorus; Stomach; Tetrodotoxin

Characterization of 'low Km' 3':5' cyclic nucleotide phosphodiesterase activities (PDE) expressed in mouse N18TG2 neuroblastoma cells is reported. At least 3 peaks of activity were isolated by DEAE chromatography, none of which was calcium-calmodulin stimulated and cGMP stimulated or inhibited. A first peak elutes at 200 mM sodium acetate; it specifically hydrolyzes cGMP with a Km of 4.7 microM and shows sensitivity to zaprinast [M&B 22948] (1.8 microM). A second peak eluting at 410 mM sodium acetate hydrolyzes both cyclic nucleotides. A third peak, specific for cAMP hydrolysis, elutes at 580 mM sodium acetate, has a Km of 3.2 microM and is sensitive to RO 20 1724 (7.6 microM) and rolipram (2 microM). Hydrodynamic analysis showed for the first peak a Stokes radius of 5.3 nm with a sedimentation coefficient of 8.1 S, a frictional ratio (f/fo) of 1.41 and a native molecular mass of 182 kDa. The same analysis for peak 3 showed a Stokes radius of 4.1 nm with a sedimentation coefficient of 3.2 S, a frictional ratio of 1.63 and a native molecular mass of 56 kDa. The biochemical features reported for the enzyme eluting in the first peak, and its cGMP-binding activity stimulated by inhibitors of phosphodiesterase activity, demonstrate that it belongs to the PDE V subfamily; on the other hand the cAMP specific enzyme eluting in the third peak can be assigned to the 'RO 20 1724 inhibited' form. The significance of these findings is discussed in relation to the functional characteristics of the N18TG2 cell line.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Blood Platelets; Centrifugation, Density Gradient; Chromatography, DEAE-Cellulose; Chromatography, Gel; Cyclic GMP; Cytosol; Electrophoresis, Polyacrylamide Gel; Kinetics; Lung; Mice; Molecular Weight; Neuroblastoma; Purinones; Rats; Tumor Cells, Cultured

Effects of glyceryl trinitrate (GTN) and nitric oxide (NO) on the cardiac functions and myocardial cyclic GMP (cGMP) contents were examined in comparison with those in the aorta and correlated with myoglobin (an inhibitor of soluble guanylate cyclase) contents using the preparations isolated from the reserpinized rabbit. GTN (10(-10)-10(-4) mol/l) produced a dose-dependent relaxation in the aorta. However, this compound exerted no effect on the rate of the spontaneous beat of the right atrium and the contraction of the papillary muscle. A transient and significant increase in cGMP was observed in the aorta with GTN (3 x 10(-6) mol/l). Although the increase was also observed in the right atrium, it was much smaller. No definite change was observed in papillary muscle. Increases in cGMP produced by NO (3 x 10(-6) mol/l) were larger and significant in all tissues; (AUCcGMP(GTN)/AUCcGMP(NO)) ratio was 30.1 for the aorta, 65.0 for the right atrium and 16.3% for the papillary muscle. Although higher concentrations of NO were necessary in the right atrium and papillary muscle to induce increases in cGMP, no differences were noted in the three tissues as regards the maximum accumulation of this substance. Furthermore, kinetic analysis of NO-induced increases in tissue cGMP indicated no marked difference in the production rate among the three tissues, while the rate of elimination of cGMP was lower in the aorta than in the atrium or the papillary muscle. The increases in cGMP observed in these three tissues were inversely related to the contents of myoglobin in respective tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aorta, Thoracic; Atrial Function, Right; Cyclic GMP; Kinetics; Male; Muscle, Smooth, Vascular; Myocardial Contraction; Myocardium; Myoglobin; Nitric Oxide; Nitroglycerin; Papillary Muscles; Purinones; Rabbits

The effects of the NO-donor 3-morpholinosydnonimin (SIN-1) on isometric tension, cyclic guanosine 3',5'-monophosphate (cyclic GMP) accumulation and neuronal release of 3H-noradrenaline were investigated in rabbit isolated corpus cavernosum (CC), and compared to the actions of sodium nitroprusside (SNP) and the cyclic GMP-specific phosphodiesterase inhibitor zaprinast. SIN-1, zaprinast and SNP concentration dependently relaxed rabbit CC preparations contracted by 1 microM. phenylephrine. All the drugs were highly effective, and the order of potency was SNP > zaprinast > SIN-1. SIN-1 had a biphasic effect on contractions evoked by electrical field stimulation of nerves: at low concentrations (1 and 10 microM.), SIN-1 inhibited the contractions, while at concentrations > or = 100 microM., the contractions were again increased. There were no changes in baseline tension. Electrically evoked contractions were inhibited by zaprinast in a concentration-dependent manner. Compared with controls, 1 mM. SIN-1 caused a significant (p < 0.05) increase in both the basal efflux and in the electrically induced release of 3H from CC preparations incubated with 3H-noradrenaline. SIN-1, zaprinast and SNP increased tissue levels of cyclic GMP. There was no positive correlation between cyclic GMP accumulation and the relaxant effects of the drugs. The effects of SIN-1 and SNP on the tissue content of cyclic GMP were not significantly affected by methylene blue, an inhibitor of soluble guanylate cyclase. It may be concluded that SIN-1, zaprinast and SNP are effective in relaxing isolated penile erectile tissue, and this effect is associated with an increase in the tissue content of cyclic GMP via pathways not sensitive to methylene blue. However, additional mechanisms beside stimulation of adrenergic neurotransmission and activation of guanylate cyclase in the smooth muscle cell seem to participate in the action of SIN-1 on rabbit penile erectile tissue.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Male; Molsidomine; Nitric Oxide; Nitroprusside; Penile Erection; Penis; Purinones; Rabbits; Vasodilator Agents

Five phosphodiesterase isozymes were separated from the supernatant of pig aortic smooth muscle homogenates, using DEAE-Toyopearl 650S chromatography in the presence of 0.1 mM Ca2+ followed by re-chromatography in the absence of Ca2+ and affinity chromatography on immobilized rolipram or cGMP. Type I (calmodulin-dependent family) preferentially hydrolysed cGMP and its activity was stimulated by calmodulin. Type II (cGMP-stimulated family), which had not yet been identified in aortic smooth muscle, hydrolysed both cGMP and cAMP. Its cAMP hydrolysis was stimulated by 10 microM cGMP. Type III (cGMP-inhibited family) and IV (cAMP-specific family) preferentially hydrolysed cAMP. The cAMP hydrolytic activity of Type III was inhibited by cGMP, but that of Type IV was not. Type V (cGMP-specific family) preferentially hydrolysed cGMP and its activity did not depend on calmodulin. The inhibition of all five phosphodiesterase isozymes by various phosphodiesterase inhibitors was investigated, and the potency and selectivity of each phosphodiesterase inhibitor discussed.

Topics: Animals; Aorta; Calmodulin; Chromatography, Affinity; Cyclic AMP; Cyclic GMP; Dipyridamole; Isoenzymes; Muscle, Smooth, Vascular; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Purinones; Swine; Vinca Alkaloids

Nitric oxide has been identified as an endothelium-derived relaxing factor in blood vessels. We tried to determine whether it is involved in the relaxation of the corpus cavernosum that allows penile erection. The relaxation of this smooth muscle is known to occur in response to stimulation by nonadrenergic, noncholinergic neurons.. We studied strips of corpus cavernosum tissue obtained from 21 men in whom penile prostheses were inserted because of impotence. The mounted smooth-muscle specimens were pretreated with guanethidine and atropine and submaximally contracted with phenylephrine. We then studied the smooth-muscle relaxant responses to stimulation by an electrical field and to nitric oxide.. Electrical-field stimulation caused a marked, transient, frequency-dependent relaxation of the corpus cavernosum that was inhibited in the presence of N-nitro-L-arginine and N-amino-L-arginine, which selectively inhibit the biosynthesis of nitric oxide from L-arginine. The addition of excess L-arginine, but not D-arginine, largely reversed these inhibitory effects. The specific liberation of nitric oxide (by S-nitroso-N-acetylpenicillamine) caused rapid, complete, and concentration-dependent relaxation of the corpus cavernosum. The relaxation caused by either electrical stimulation or nitric oxide was enhanced by a selective inhibitor of cyclic guanosine monophosphate (GMP) phosphodiesterase (M&B 22,948). Relaxation was inhibited by methylene blue, which inhibits cyclic GMP synthesis.. Our findings support the hypothesis that nitric oxide is involved in the nonadrenergic, noncholinergic neurotransmission that leads to the smooth-muscle relaxation in the corpus cavernosum that permits penile erection. Defects in this pathway may cause some forms of impotence.

Topics: Adult; Aged; Arginine; Cyclic GMP; Electric Stimulation; Erectile Dysfunction; Humans; Male; Methylene Blue; Middle Aged; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Nitroarginine; Organ Culture Techniques; Penile Erection; Penis; Purinones; Synaptic Transmission

Endothelium-dependent relaxations are inhibited during chronic vasospasm after subarachnoid hemorrhage in the canine basilar artery, although the luminal release of endothelium-derived relaxing factor (EDRF) is maintained. The present study investigated the mechanisms underlying the impaired vascular reactivity and in particular whether the loss of responsiveness of the smooth muscle to EDRF is due to an impaired production of cGMP. Bradykinin and nitric oxide evoked concentration-dependent relaxations in isolated canine basilar arteries with and without endothelium, respectively, which were reduced in the subarachnoid hemorrhage group. Relaxations evoked by M&B22,948 (an inhibitor of cGMP phosphodiesterases) were smaller, but those evoked by the lipophilic cGMP analogue 8-bromo-cGMP were potentiated slightly in the subarachnoid hemorrhage group. The resting levels of cGMP in rings with endothelium (reflecting the effect of spontaneous release of EDRF) and those evoked by bradykinin in rings with endothelium and by nitric oxide in rings without endothelium were diminished in the subarachnoid hemorrhage group. These data indicate that the altered endothelium-mediated relaxations of the smooth muscle after subarachnoid hemorrhage is due, at least in part, to an impaired activation of soluble guanylate cyclase leading to a reduced production of cGMP in the smooth muscle.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Basilar Artery; Bradykinin; Cyclic GMP; Dogs; Endothelium, Vascular; Female; Male; Nitric Oxide; Purinones; Radioimmunoassay; Subarachnoid Hemorrhage; Vasodilation

Microvascular functions have been shown to be sensitive to agents associated with changes in cyclic nucleotide levels. The central hypothesis of the current study was that one measure of capillary exchange capacity, hydraulic conductivity (Lp), would be elevated by agents shown to elevate cellular levels of cGMP. To evaluate the hypothesis, frog mesenteric capillary Lp was measured during luminal exposure to 1) atrial natriuretic peptide (ANP), 2) the truncated atriopeptins ANP-I and ANP-III, 3) the nitrovasodilator sodium nitroprusside (SNP), 4) the cGMP phosphodiesterase inhibitor M&B 22948, and 5) methylene blue dye, both alone and in combination with ANP or SNP. ANP (100 nM) elevated Lp by 2.3 +/- 0.2-fold from control levels (n = 15); 10 nM ANP induced a 2.1 +/- 0.3-fold change (n = 8), while 10 nM ANP-III elicited a 1.7 +/- 0.4-fold change (n = 8). In contrast, Lp did not change from basal levels during 10 nM ANP-I infusion (Lp ANP-I/Lp control = 1.2 +/- 0.2; n = 14). SNP (1 microM) induced a reversible, 2.6 +/- 0.5-fold increase in Lp (n = 30) that was inhibitable by methylene blue dye (Lp SNP + MetB/Lp control = 1.1 +/- 0.1; n = 8). Methylene blue did not mask the response to 100 nM ANP (Lp ANP + MetB/Lp control = 2.1 +/- 0.5; n = 7). M&B 22948 (30 microM) increased Lp by 2.8 +/- 0.6-fold (n = 9). These data constitute strong inference that agents demonstrated to elevate cGMP also mediate an increase in capillary Lp in in situ, perfused exchange vessels.

Topics: Animals; Atrial Natriuretic Factor; Capillaries; Cyclic GMP; Drug Synergism; Methylene Blue; Nitroprusside; Purinones; Rana pipiens; Vasodilator Agents

Nitric oxide (NO) caused a potent, marked, and transient relaxation of precontracted strips of corpus cavernosum isolated from humans and rabbits. The relaxation response elicited by NO was very similar to the relaxation evoked by electrical field stimulation via the nonadrenergic-noncholinergic pathway. Sodium nitroprusside, nitroglycerin, and S-nitroso-N-acetylpenicillamine, which are nitrovasodilators known to generate NO, also caused marked concentration-dependent relaxation of corpus cavernosum. Relaxant responses to NO were enhanced by the cyclic GMP phosphodiesterase inhibitor M&B 22,948 and inhibited by oxyhemoglobin. Similarly, relaxation of corpus cavernosum in response to electrical field stimulation or acetylcholine was enhanced by M&B 22,948 and inhibited by oxyhemoglobin. NO stimulated cyclic GMP formation in corpus cavernosum and a close positive correlation was found between the magnitudes of relaxation and cyclic GMP formation. The data suggest that NO-elicited activation of guanylate cyclase and cyclic GMP formation represents the signal transduction mechanism responsible for relaxation and nonadrenergic-noncholinergic-mediated penile erection. These observations indicate that NO is a potent relaxant of human and rabbit corpus cavernosum and support our hypothesis that endogenous NO is the principal mediator of penile erection caused by nonadrenergic-noncholinergic stimulation.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Electric Stimulation; Humans; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Nitric Oxide; Nitroglycerin; Nitroprusside; Penicillamine; Penis; Purinones; Rabbits; S-Nitroso-N-Acetylpenicillamine; Vasodilator Agents

We have demonstrated previously that in response to hypoxia, isolated rat pulmonary arteries show an initial endothelium-dependent relaxation followed by an endothelium-independent transient contraction. In the presence of increased extracellular Ca2+, both of these responses were enhanced in endothelium-intact arteries. Nitro-L-arginine, a blocker of the biosynthesis of endothelium-derived relaxing factor (EDRF), abolished the initial endothelium-dependent relaxation and Ca(2+)-induced enhancement of hypoxic contraction in endothelium-intact arteries but did not alter responses in endothelium-denuded vessels. Inhibition of prostaglandin formation with indomethacin had no effect on the hypoxia-elicited responses. Preincubation with LY 83583, an inhibitor of guanylate cyclase activation, abolished the initial hypoxia-elicited relaxation and subsequent contraction. M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor, decreased tone under O2 but not under N2, causing an apparent enhancement of the contraction to hypoxia. Thus the modulation of hypoxic responses by the endothelium is dependent on changes in EDRF production, and a decrease in smooth muscle cGMP not involving an EDRF mechanism appears to mediate the endothelium-independent hypoxic contraction observed in the isolated rat pulmonary artery.

Topics: Aminoquinolines; Animals; Arginine; Cyclic GMP; Hypoxia; In Vitro Techniques; Indomethacin; Male; Nitroarginine; Pulmonary Artery; Purinones; Rats; Rats, Inbred Strains

1. Nitric oxide (NO) may serve as a non-adrenergic, non-cholinergic (NANC) neurotransmitter released from enteric inhibitory nerves in the gastrointestinal tract. We tested whether guanosine 3':5'-cyclic monophosphate (cyclic GMP) may serve as a second messenger in transducing the NO signal into inhibitory junction potentials (i.j.ps) and relaxation in the canine proximal colon. 2. The membrane permeable analogue of cyclic GMP, 8-bromo cyclic GMP (8-Br-cyclic GMP) mimicked the effects of NO by hyperpolarizing cells near the myenteric border of the circular muscle layer and shortening slow waves in cells near the submucosal surface of the circular muscle layer. 8-Br-cGMP also inhibited spontaneous phasic contractions. 3. The specific cyclic GMP phosphodiesterase inhibitor, M&B 22948, hyperpolarized cells near the myenteric border and prolonged the duration of i.j.ps. M&B 22948 also inhibited phasic contractile activity. 4. Methylene blue failed to reduce significantly the amplitude and duration of i.j.ps and had variable effects on contractions. 5. Cyclic GMP levels were assayed in unstimulated muscles and in muscles exposed to exogenous NO and electrical field stimulation. Both stimuli hyperpolarized membrane potential, inhibited contractions, and elevated cyclic GMP levels. 6. Treatment of muscles with L-NG-nitroarginine methyl ester (L-NAME) increased spontaneous contractile activity and lowered cyclic GMP levels. The inhibitory effect of M&B 22948 on contractions was greatly reduced after muscles were treated with L-NAME. 7. These data support the concept that the effects of NANC nerve stimulation and NO (which may be one of the enteric inhibitory transmitters) may be mediated by cyclic GMP.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Arginine; Autonomic Nervous System; Colon; Cyclic GMP; Dogs; Electric Stimulation; Female; Gastrointestinal Motility; Male; Methylene Blue; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide; Purinones; Signal Transduction; Synaptic Transmission

1. The possible roles of the L-arginine-NO pathway and of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in regulating the prejunctional release of noradrenaline and neurogenic vasoconstriction were investigated in the perfused rat tail artery. 2. In the presence of N omega-nitro-L-arginine methyl ester (L-NAME; 30 microM), an inhibitor of NO formation, the vasoconstrictor responses to perivascular nerve stimulation (24 pulses at 0.4 Hz, 0.3 ms, 200 mA) and to exogenous noradrenaline (1 microM) were significantly enhanced, whereas the stimulation-evoked tritium overflow from [3H]-noradrenaline preloaded arteries was not modified. The vasoconstriction enhancing effect of L-NAME was prevented by L-arginine (1 mM) but not D-arginine (1 mM) and was abolished by removal of the endothelium. 3. The NO donor, 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1; 0.1-30 microM), and the cyclic GMP phosphodiesterase inhibitor, zaprinast (0.1-30 microM) both induced a concentration-dependent inhibition of the electrical field stimulation-induced vasoconstriction, while atrial natriuretic peptide (ANP; 100 nM) produced only a slight decrease of the vasoconstrictor response. Methylene blue (3 microM), a known inhibitor of soluble guanylate cyclase increased the electrical field stimulation-induced vasoconstriction. SIN-1 and methylene blue when administered simultaneously, antagonized each others effect. None of the compounds tested (SIN-1, zaprinast, ANP or methylene blue) had any significant effect on the stimulation-evoked [3H]-noradrenaline overflow. 4. 8-Bromo-cyclic GMP, a potent activator of cyclic GMP-dependent protein kinase, markedly and concentration-dependently (3-300 microM) increased [3H]-noradrenaline overflow but decreased field stimulation-induced vasoconstriction. Dibutyryl-cyclic GMP (100 JM), a weak activator of cyclic GMP-dependent protein kinase, affected neither the pre- nor the postjunctional response to electrical field stimulation.5. These data show that an NO-like substance of endothelial origin, derived from L-arginine, attenuates vasoconstriction in the rat tail artery, whether neurally-induced or evoked by exogenous noradrenaline.Since noradrenaline release was unaltered by compounds modifying NO production, this NO-like compound acted through a postjunctional mechanism. The lack of prejunctional effects of both soluble and membrane-associated guanylate cyclase activators, despite a large effect of 8-bromo-cyclic GMP,suggests that endogenou

Topics: Animals; Arginine; Arteries; Atrial Natriuretic Factor; Cyclic GMP; Electric Stimulation; Endothelium, Vascular; In Vitro Techniques; Male; Methylene Blue; Molsidomine; NG-Nitroarginine Methyl Ester; Nitric Oxide; Norepinephrine; Purinones; Rats; Tail; Vasoconstriction; Vasodilator Agents

8-(4-Chlorophenyl)thio-cyclic AMP (8-CPT-cAMP), extensively used as selective activator of cyclic AMP-dependent protein kinase, has been found to be a potent inhibitor of the cyclic GMP-specific phosphodiesterase (PDE VA). Indeed, 8-CPT-cAMP (IC50 = 0.9 microM) inhibited PDE VA with a potency identical to that of zaprinast. 8-CPT-cAMP was also metabolized by PDE VA at a rate half that of cyclic GMP. The cyclic GMP-inhibited phosphodiesterase (PDE III) (IC50 = 24 microM) and the cyclic AMP-specific phosphodiesterase (PDE IV) (IC50 = 25 microM) were also inhibited by 8-CPT-cAMP. In contrast, most of the other cAMP-derivative studies showed little inhibition of any phosphodiesterase isoenzyme. These observations provide further reasons why the mechanism of the physiological effects of 8-CPT-cAMP should be interpreted with caution.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Cyclic AMP; Cyclic GMP; Humans; Infant, Newborn; Isoenzymes; Kinetics; Mathematics; Purinones; Thionucleotides

1. Nonadrenergic, noncholinergic (NANC) nerves mediate vasodilatation in guinea-pig pulmonary artery (PA) by both endothelium-dependent and endothelium-independent mechanisms. The transmitter(s) involved in the endothelium-independent pathway have not yet been identified. We have therefore investigated the possibility that nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) may mediate this neural vasodilator response in guinea-pig branch PA rings denuded of endothelium. 2. Electric field stimulation (EFS, 50 V, 0.2 ms) induced a frequency-dependent (1-24 Hz), tetrodotoxin-sensitive relaxation of the U44069-precontracted PA rings in the presence of adrenergic and cholinergic blockade. 3. The NO synthase inhibitors NG-monomethyl L-arginine (L-NMMA, 100 microM) and NG-nitro L-arginine methyl ester (L-NAME, 30 microM), and the guanylyl cyclase inhibitor methylene blue (5 microM) inhibited the EFS (16 Hz)-induced relaxation by 53 +/- 5, 74 +/- 9 and 82 +/- 9% respectively (n = 5-7, P < 0.01, compared with control rings). 4. Excess concentrations of L-, but not D-arginine (300 microM) completely reversed the inhibitory effect of L-NMMA. 5. The EFS-elicited relaxation (4 Hz) was potentiated by 1 microM zaprinast, a type V phosphodiesterase inhibitor which inhibits guanosine 3':5'-cyclic monophosphate (cyclic GMP) degradation, but was unaffected by 0.1 microM zardaverine, a type III/IV phosphodiesterase inhibitor which inhibits cyclic AMP degradation. 6. EFS (50 V, 0.2 ms, 16 Hz) induced a 3 fold increase in tissue cyclic GMP content, an action which was inhibited by L-NMMA (100 microM). 7. Pyrogallol (100microM), a superoxide anion generator, also inhibited the EFS-induced relaxation by 53 +/- 9%, and this effect was prevented by superoxide dismutase.8. Chemical sympathetic denervation with 6-hydroxydopamine had no effect on the relaxant response to EFS in the endothelium-denuded PA rings.9. In endothelium-denuded branch PA rings at resting tone, L-NMMA (100 microM) significantly augmented the adrenergic contractile response, an effect which was completely reversed by L-arginine,but not by D-arginine. In the same groups of vessel rings, L-NMMA had no significant effect on the matched contractile response to exogenous noradrenaline.10. These results suggest that NO may be released from intramural nerve endings other than adrenergic nerves (probably NANC nerves), and this leads to vasodilatation via activation of guanylyl cyclase.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Arginine; Autonomic Nervous System; Cyclic GMP; Electric Stimulation; Guinea Pigs; In Vitro Techniques; Male; Methylene Blue; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Norepinephrine; omega-N-Methylarginine; Oxidopamine; Phosphodiesterase Inhibitors; Purinones; Pyrogallol; Sympathectomy, Chemical

We have previously demonstrated that the catalytic sub-unit of protein kinase A can catalyse a potent activation of partially purified Type V cyclic GMP-specific phosphodiesterase activity (Burns et al., 1992, Biochem. J. 283, 487-491). We now demonstrate that this phosphodiesterase most likely has a sub-unit mass of 90kDa, based upon 32P-cyclic GMP photo-affinity labelling, that activation of the phosphodiesterase does not require the prior binding of cyclic GMP to the phosphodiesterase, and that alkaline phosphatase can reverse the protein kinase A-dependent activation of phosphodiesterase activity. Zaprinast is a mixed inhibitor of non-activated cyclic GMP phosphodiesterase activity. However, inhibition of the protein kinase A-activated phosphodiesterase is competitive. These results suggest that protein kinase A can modulate the inhibitory effects of zaprinast via perturbations of a non-catalytic binding site.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Alkaline Phosphatase; Animals; Cyclic GMP; Enzyme Activation; Guinea Pigs; Isoenzymes; Kinetics; Lung; Macromolecular Substances; Protein Kinases; Purinones; Pyrazines; Pyrrolidinones; Rolipram

We investigated the effect of aging on atrial natriuretic peptide (ANP)-induced relaxation and cyclic GMP (cGMP) formation in the rat thoracic aorta. In the aorta from young rats (4 weeks old), removal of the endothelium, and treatment with the nitric oxide synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), the radical scavenger, hemoglobin (Hb), and the soluble guanylate cyclase inhibitor, methylene blue (MB), attenuated ANP-induced relaxation and considerably reduced ANP-stimulated cGMP formation. With increasing age of the rats, the ANP-induced relaxation and cGMP formation in endothelium-intact aorta decreased, and Hb, L-NAME and MB no longer inhibited the ANP-induced effects, irrespective of whether the endothelium was present or absent. In the arteries without endothelium, the age-associated reduction in ANP-induced relaxation was less than in arteries with endothelium. Aging also decreased the relaxation induced by the soluble guanylate cyclase activator, nitroprusside. Potentiation due to the cGMP-phosphodiesterase (cGMP-PDE) inhibitor, M&B 22948, of the ANP-induced relaxation was greater in aortas from old rats than in those from young rats, suggesting that the degradation of cGMP may be accelerated in old rats. These results suggest that the relaxant action of ANP on the thoracic aorta from young rats is in part modulated by endothelium-derived relaxing factor (EDRF/nitric oxide), which in turn activates soluble guanylate cyclase, thus elevating the cGMP level. Aging may decrease the ANP-induced relaxation and ANP-stimulated increase in cGMP level by decreasing the ability of endothelial cells to produce EDRF, by decreasing guanylate cyclase activity, and by enhancing cGMP-PDE activity.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Aging; Animals; Aorta, Thoracic; Arginine; Atrial Natriuretic Factor; Cyclic GMP; Endothelium, Vascular; Guanylate Cyclase; Hemoglobins; In Vitro Techniques; Male; Methylene Blue; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Purinones; Rats; Rats, Inbred Strains

Previously we showed that atrial natriuretic factor (ANF) decreases cardiac cell volume by inhibiting ion uptake by Na+/K+/2Cl- cotransport. Digital video microscopy was used to study the role of guanosine 3',5'-monophosphate (cGMP) in this process in rabbit ventricular myocytes. Each cell served as its own control, and relative cell volumes (volume(test)/volume(control)) were determined. Exposure to 10 microM 8-bromo-cGMP (8-Br-cGMP) reversibly decreased cell volume to 0.892 +/- 0.007; the ED50 was 0.77 +/- 0.33 microM. Activating guanylate cyclase with 100 microM sodium nitroprusside also decreased cell volume to 0.889 +/- 0.009. In contrast, 8-bromo-adenosine 3',5'-monophosphate (8-Br-AMP; 0.01-100 microM) neither altered cell volume directly nor modified the response to 8-Br-cGMP. The idea that cGMP decreases cell volume by inhibiting Na+/K+/2Cl- cotransport was tested by blocking the cotransporter with 10 microM bumetanide (BUM) and removing the transported ions. After BUM treatment, 10 microM 8-Br-cGMP failed to decrease cell volume. Replacement of Na+ with N-methyl-D-glucamine or Cl- with methanesulfonate also prevented 8-Br-cGMP from shrinking cells. The data suggest that 8-Br-cGMP, like ANF, decreases ventricular cell volume by inhibiting Na+/K+/2Cl-cotransport. Evidence that ANF modulates cell volume via cGMP was also obtained. Pretreatment with 10 microM 8-Br-cGMP prevented the effect of 1 microM ANF on cell volume, and ANF suppressed 8-Br-cGMP-induced cell shrinkage. Inhibiting guanylate cyclase with the quinolinedione LY83583 (10 microM) diminished ANF-induced cell shrinkage, and inhibiting cGMP-specific phosphodiesterase with M&B22948 (Zaprinast; 100 microM) amplified the volume decrease caused by a low dose of ANF (0.01 microM) approximately fivefold. In contrast, neither 100 microM 8-Br-cAMP nor 50 microM forskolin affected the response to ANF. The effects of ANF, LY83583, and M&B29948 on cGMP levels in isolated ventricular myocytes were confirmed by 125I-cGMP radioimmunoassay. These data argue that ANF shrinks cardiac cells by increasing intracellular cGMP, thereby inhibiting Na+/K+/2Cl- cotransport. Basal cGMP levels also appear to modulate cell volume.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Aminoquinolines; Animals; Atrial Natriuretic Factor; Chlorides; Cyclic GMP; Guanylate Cyclase; In Vitro Techniques; Myocardium; Potassium; Purinones; Rabbits; Second Messenger Systems; Sodium; SRS-A

The cyclic nucleotide phosphodiesterase (PDE) forms present in frog atrial fibers were isolated and characterized by their drug sensitivities. DEAE-sephacel chromatography of cytosolic PDE activity resolved three major PDE forms: peak A hydrolyzed both adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) and was activated by calcium-calmodulin (PDE I); peak B also hydrolyzed both cAMP and cGMP but was activated by 5 microM cGMP (PDE II); peak C specifically hydrolyzed cAMP (PDE IV). Rolipram specifically inhibited PDE IV (Ki = 1.1 microM), whereas dipyridamole potently inhibited both PDE II (Ki = 4.6 microM) and PDE IV (Ki = 0.8 microM). Atrial fiber PDE I was preferentially inhibited by zaprinast (Ki = 10 microM). 3-Isobutyl-1-methyl xanthine (IBMX) and theophylline inhibited nonspecifically all three different enzymes. The positive inotropic drug CI 930 only inhibited the different isolated atrial PDE forms at concentrations greater than 200 microM. However, under assay conditions for which PDE IV was specifically inhibited (presence of 100 microM rolipram), an IC50 of 17 microM for CI 930 was observed on the remaining 26% cAMP hydrolytic activity of peak C (which could represent a cGMP-inhibited PDE form: PDE III). The same PDE forms were also found in frog ventricle. The major difference between frog atrial fiber (and ventricular tissue) PDEs and mammalian cardiac PDEs is that the main cytosolic cAMP-specific hydrolytic activity in frog heart is due to PDE IV rather than PDE III. Rolipram, dipyridamole, and zaprinast might be useful tools to investigate the participation of cAMP in frog atrial contraction (unpublished observations).

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calcium Chloride; Calmodulin; Chromatography, Ion Exchange; Cyclic GMP; Cytosol; Dipyridamole; Heart Atria; Heart Ventricles; Kinetics; Myocardium; Papaverine; Purinones; Pyrrolidinones; Rana esculenta; Rana ridibunda; Rolipram; Substrate Specificity; Theophylline

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cell Membrane; Cyclic AMP; Cyclic GMP; Electric Conductivity; Ion Channels; Leeches; Purinones; Salivary Glands

Down-regulation of atrial natriuretic peptide (ANP) receptors was investigated using a cultured bovine pulmonary artery endothelial (CPAE) cell line. Endothelial cells have been shown to possess two subtypes of ANP receptors, a guanylate cyclase-coupled receptor (B-receptor) and a clearance receptor (C-receptor). The treatment with APIII, rat ANP (103-126), at concentrations of 10(-8) to 10(-6) M for 24 h, resulted in a significantly (p less than 0.01) greater decrease in maximum 125I-APIII binding to CPAE cells than the identical concentration of API, rat ANP (103-123). APIII at concentrations of 10(-8) to 10(-6) M stimulated cyclic GMP (cGMP) production 3.3-17.5-fold greater than similar concentrations of API. From these findings, we hypothesized that cGMP produced following ANP binding to the B-receptor participates in ANP receptor regulation. M&B 22948, a selective inhibitor of cGMP-specific phosphodiesterase, significantly (p less than 0.01) potentiated the effect of both API and APIII on 125I-APIII binding, while M&B 22948 itself had no significant effect on 125I-APIII binding. Treatment of the cells with 1 mM 8-bromo-cGMP also significantly (p less than 0.01) decreased 125I-APIII binding to the cells, and a potentiation of this effect was observed by M&B 22948. Scatchard analysis of binding data from 8-bromo-cGMP-treated cells showed a significant decrease in Bmax (1.79 +/- 0.15 to 1.20 +/- 0.07 fmol/mg protein, p less than 0.05) without a significant change in Kd. Affinity cross-linking of 125I-APIII to 8-bromo-cGMP-treated cells showed a decrease in the labeling of 60- and 70-kDa bands corresponding to the C-receptor. In addition, the APIII-stimulated cGMP response remained unchanged in the 8-bromo-cGMP-treated cells, indicating that the B-receptor was not down-regulated. We conclude that cGMP regulates ANP-binding sites on the endothelial cell and that the evidence indicates that the C-receptor may preferentially be down-regulated by cGMP in CPAE cells.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Atrial Natriuretic Factor; Autoradiography; Cattle; Cell Line; Cross-Linking Reagents; Cyclic GMP; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular; Purinones; Rats; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface

1. The effects of zaprinast (M&B 22948), a selective guanosine 3':5'-cyclic monophosphate (cyclic GMP) phosphodiesterase inhibitor, and sodium nitroprusside on cyclic GMP content, phosphoinositide hydrolysis and airway smooth muscle tone were examined in flurbiprofen pretreated bovine tracheal smooth muscle (BTSM). 2. Anion-exchange chromatography of the soluble fraction of BTSM homogenates resolved three peaks of Ca2+/calmodulin-independent phosphodiesterase (PDE) activity that corresponded to type Ia (cyclic GMP-specific, zaprinast-inhibitable), type II (cyclic GMP-stimulated) and type IV (Ro 20 1724-inhibitable) PDE isoenzymes. Zaprinast caused a selective inhibition of the type Ia PDE isoenzyme (IC50 0.94 microM) with respect to the type II and IV (IC50 s 93 microM and 197 microM respectively) isoenzymes. 3. Pretreatment of BTSM strips with zaprinast (10 microM) for 20 min affected neither the initial rate of force development, nor the resultant magnitude of contraction induced by methacholine (10 microM). In addition, zaprinast (10 microM; 20 min) did not affect the cumulative concentration-response relationship induced by methacholine. In contrast, sodium nitroprusside (300 microM) either alone, or in combination with zaprinast (10 microM), significantly attenuated tone induced by low, but not high concentrations of methacholine. This resulted in a non-parallel, rightwards shift of the methacholine concentration-response curves (nitroprusside: 4.0 fold; nitroprusside/zaprinast: 4.8 fold at the EC50 values), without a reduction in the maximum tone generated. 4. In BTSM slices, zaprinast (10 or 100 microM) did not influence basal or methacholine (10 microM)-stimulated cyclic GMP accumulation or inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation over a 60s incubation period, although it did significantly increase cyclic GMP content over longer (30 min) stimulation periods. 5. In [3H]-inositol prelabelled BTSM slices, stimulated in the presence of 5mM LiCl, methacholine (10 microM) caused a marked increase in total [3H]-inositol phosphate accumulation. This effect was not inhibited by zaprinast (10 microM), sodium nitroprusside (300 microM), or a combination of these drugs despite these agents markedly increasing tissue cyclic GMP content. 6. These findings demonstrate that despite zaprinast being a potent and selective inhibitor of the type Ia PDE isoenzyme in a cell-free system, this drug only increases cyclic GMP content in BTSM following

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cattle; Cyclic GMP; Flurbiprofen; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Isoenzymes; Methacholine Compounds; Muscle Contraction; Muscle Tonus; Muscle, Smooth; Nitroprusside; Purinones; Trachea

1. In the present study the effects of M&B 22,948, a guanosine 3':5'-cyclic monophosphate (cyclic GMP) selective phosphodiesterase inhibitor and of 8-bromo cyclic GMP were examined on the synthesis of dopamine from L-3,4-dihydroxyphenylalanine (L-DOPA) in rat cortical slices and in whole kidney homogenates. The deamination of newly-formed dopamine into 3,4-dihydroxyphenylacetic acid (DOPAC) was also studied. The assay of L-DOPA, dopamine, noradrenaline and DOPAC was performed by high performance liquid chromatography (h.p.l.c.) with electrochemical detection. 2. Incubation of renal slices and homogenates of whole kidney with exogenous L-DOPA (0.1-10.0 microM) resulted in a concentration-dependent formation of both dopamine and DOPAC. 3. The addition of M&B 22,948 (10 microM) to the incubation medium resulted in a marked reduction in the accumulation of both newly-formed dopamine and DOPAC in kidney slices; the inhibitory effect of M&B 22,948 on DOPAC formation was greater than that on dopamine. 8-Bromo cyclic GMP (250 microM) produced only a slight decrease in the tissue levels of newly-formed dopamine (5-13% reduction), but was found to decrease significantly (51-68% reduction) the formation of DOPAC in kidney slices. The addition of 8-bromo cyclic GMP plus M&B 22,948 to the incubation medium resulted in similar effects to those described for M&B 22,948 alone. 4. In kidney homogenates, in contrast to results observed in kidney slices, M&B 22,948 (10 microM) and 8-bromo cyclic GMP (250 microM) were found to affect neither the formation of dopamine nor its deamination to DOPAC. 5. In conclusion, the results presented here suggest that cyclic GMP may be involved in the regulation of dopamine synthesis, probably through the control of the entry of L-DOPA into the tubular epithelial cells.

Topics: Animals; Cyclic GMP; Dopamine; In Vitro Techniques; Kidney; Levodopa; Purinones; Rats; Rats, Inbred Strains

Four cyclic nucleotide phosphodiesterase activities (PDEs) could be resolved from rat mesenteric artery by DEAE-Sephacel chromatography: a calmodulin-activated fraction, a cyclic GMP-inhibited fraction, a cyclic AMP-specific rolipram-sensitive fraction and a cyclic GMP-specific fraction containing PDE I, III, IV and V. Cardiotonic drugs (CI 930 and LY 195115) selectively inhibited PDE III; rolipram and zaprinast selectively inhibited PDE IV and PDE V, respectively. These results show that the rat mesenteric artery contains the same PDEs as previously found in the aorta, and suggest that these PDEs may be implicated in the regulation of arterial contraction.

Topics: Animals; Calmodulin; Chromatography, DEAE-Cellulose; Cyclic AMP; Cyclic GMP; Female; Male; Mesenteric Arteries; Phosphoric Diester Hydrolases; Purinones; Pyrrolidinones; Rats; Rats, Inbred Strains; Rolipram

Electrical field stimulation (EFS) of phenylephrine-contracted bovine mesenteric arteries pretreated with guanethidine elicited a relaxation that amounted to roughly 40%. This relaxation was sensitive to tetrodotoxin pretreatment, suggesting a neurogenic origin. The EFS-induced relaxation was correlated to an increase in cGMP level, from 14.2 +/- 2.5 pmol/g wet wt in nonstimulated arteries to 31.6 +/- 3.4 pmol/g wet wt after 1 minute of EFS. cAMP values were not affected by EFS. Methylene blue (5 microM) and the compound LY 83583 (10 microM), inhibitors of soluble guanylate cyclase, inhibited the EFS-induced relaxation by 60% and 50%, respectively. Zaprinast (1 microM), a selective inhibitor of cGMP degradation, significantly (p = 0.005) potentiated the EFS-induced relaxation. The relaxation induced by EFS in bovine mesenteric arteries exhibits characteristics similar to the relaxations evoked by organic nitroesters and endothelium-dependent vasodilators, both of which are suggested to be mediated by cGMP and probably with nitric oxide as the common activator of the cGMP system. The possible involvement of nitric oxide as a mediator of EFS-induced relaxations was investigated with the use of known modulators of endogenous nitric oxide production. Preincubation of the arteries with 1 mM arginine or 1 mM N-alpha-benzoyl-L-arginine, both reported to potentiate endogenous nitric oxide production, or 5 mM L-canavanine, 0.25 mM NG-monomethyl-L-arginine, or 0.1 mM NG-nitro-L-arginine, alleged inhibitors of endogenous nitric oxide production, were without effect on the relaxation induced by EFS. However, pyrogallol, a generator of superoxide anions, was a potent inhibitor of relaxations induced by EFS in bovine mesenteric arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Arginine; Cattle; Cyclic GMP; Electric Stimulation; Endothelium, Vascular; In Vitro Techniques; Mesenteric Arteries; Nervous System Physiological Phenomena; Nitric Oxide; Purinones; Vasodilation

The biochemical differences among cGMP phosphodiesterases in platelets have not been thoroughly examined, primarily due to the lack of sufficient purified material. This report describes a simple method developed to isolate a specific bovine platelet cGMP phosphodiesterase. This enzyme is cytosolic in its native form and was purified to an apparent homogeneity by ion-exchange chromatography, affinity chromatography, and density gradient centrifugation. Cyclic GMP binds to a "pseudo-site" when the catalytic site is deprived of Mg++. The affinity for cGMP at alkaline pH in presence of EDTA and IBMX (Kd = 60 nM) suggests that the removal of Mg++ by EDTA converts the catalytic site to a binding site. A ligand affinity chromatography was designed to take advantage of these features. The core enzyme has a molecular weight 190,000 composed of 2 subunits (MW 95,000) and has a specific activity of 2.5 mumol/min/mg. Moreover, this enzyme was phosphorylated by cAMP- and cGMP-dependent protein kinases, suggesting that its activity could be indirectly regulated by cyclic nucleotides. Agents elevating cGMP and cAMP inhibit platelet activation by inhibiting protein kinase C and thrombin induced hydrolysis of phosphatidylinositol 4,5 diphosphate. The antiaggregating properties of some of these agents might therefore be attributed to the fact that they are inhibitors of phosphodiesterases.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Blood Platelets; Catalysis; Cattle; Chromatography, Affinity; Cyclic AMP; Cyclic GMP; Dipyridamole; Electrophoresis, Polyacrylamide Gel; Kinetics; Phosphorylation; Platelet Activation; Protein Kinases; Purinones; Substrate Specificity

To determine if N omega-nitro-L-arginine (NNA), an inhibitor of the synthesis and/or release of endothelium-derived relaxing factor (EDRF), alters the response to zaprinast, a selective inhibitor of cyclic GMP (cGMP) phosphodiesterase, zaprinast (3-30 mg/kg) or vehicle (1 ml/kg) was given to conscious, spontaneously hypertensive rats (SHR) in a cumulative i.v. dose-response manner 30 min after pretreatment with NNA (1 or 3 mg/kg) or saline (1 ml/kg). Mean arterial pressure (MAP) was measured 5 min after each dose of zaprinast. Five minutes after the last dose of zaprinast (30 mg/kg), the rats were anesthetized with pentobarbital (25 mg i.v.). A segment of the abdominal aorta was freeze-clamped in situ and removed for the determination of cGMP levels. NNA (3 mg/kg) decreased basal aortic cGMP levels by 54% and increased MAP by 37 +/- 2 mm Hg. Zaprinast (30 mg/kg) increased aortic cGMP by 187% and decreased MAP by 49 +/- 4 mm Hg. NNA (3 mg/kg) reduced the accumulation of cGMP in aortic tissue (from 4.1 +/- 0.4 to 1.3 +/- 0.1 fmol/microgram protein) and attenuated the depressor response (from -49 +/- 4 to -31 +/- 4 mm Hg) produced by zaprinast. These data are consistent with the hypothesis that NNA inhibits the tonic release of EDRF and that the depressor effects of zaprinast are due, at least in part, to the potentiation of the vasodilator effects of EDRF in vivo. Moreover, since the changes in MAP produced by NNA and zaprinast were significantly correlated with cGMP levels in aortic tissue, the concentration of cGMP in vascular tissue may be a determinant of blood pressure in SHR.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Analysis of Variance; Animals; Aorta, Abdominal; Arginine; Blood Pressure; Cyclic GMP; Hypotension; Male; Muscle, Smooth, Vascular; Nitroarginine; Purinones; Radioimmunoassay; Rats; Rats, Inbred SHR

Low concentrations of sodium nitroprusside (0.2 and 1 microM) relaxed carbachol-induced tone of the rat anococcygeus but did not affect the content of either cGMP or cAMP; higher concentrations (10,100 and 1000 microM) produced greater relaxation (greater than 60%) and a rise in cGMP but not cAMP. In the presence of the cGMP-phosphodiesterase inhibitor M&B 22948 (10 microM), 1 microM sodium nitroprusside produced greater relaxation and a selective increase in cGMP. Forskolin (0.5-250 microM) caused relaxation and a selective increase in cAMP; the concentration-response relationships of the two effects were similar. Non-adrenergic, non-cholinergic (NANC) field stimulation (10 Hz; 20 s trains) reduced tone by 52% but had no effect on cyclic nucleotide content; in the presence of 10 microM M&B 22948 or 1 microM sodium nitroprusside, NANC stimulation produced a greater degree of relaxation and increased cGMP but not cAMP content. The results show that NANC stimulation acts like sodium nitroprusside, causing a selective increase in cGMP, and this supports the proposal that NANC transmission in the rat anococcygeus involves an endogenous nitrate; the possibility that multiple pools of cGMP exist in the anococcygeus is discussed.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Autonomic Nervous System; Carbachol; Colforsin; Cyclic AMP; Cyclic GMP; Electric Stimulation; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Nitroprusside; Purinones; Rats; Rats, Inbred Strains

The role of cyclic GMP (cGMP) in mediating relaxation of canine trachealis produced by nitrovasodilators (NVDs), compounds that activate guanylate cyclase, was examined. Sodium nitroprusside (SNP) produced a concentration-dependent relaxation of the canine trachealis that was accompanied by a concentration-related increase in cGMP content. In time course studies, relaxation of isolated trachealis strips induced by 30 microM SNP was paralleled by an increase in cGMP that reached a maximum of 18-fold above basal levels within 2 min. Zaprinast, an inhibitor of the cGMP-specific phosphodiesterase, potentiated both SNP-induced relaxation and cGMP accumulation. A cell-permeable analog of cGMP, 8-bromo-cGMP, mimicked the relaxant effects of SNP. Also assessed were the effects of methylene blue, an agent that inhibits soluble guanylate cyclase activity, and hemoglobin, an agent that competitively binds NO-containing compounds. In these experiments, tissues were pretreated with the above agents for 10 min, contracted with 1 or 3 microM methacholine, and then relaxed by the cumulative addition of SNP or two other NVDs, S-nitroso-N-acetyl-penicillamine (SNAP) and glyceryl trinitrate (GTN). Tissues were flash-frozen after adding the final concentration of the various NVDs and assayed for cGMP. Methylene blue and hemoglobin suppressed both cGMP accumulation and relaxation in response to SNAP and GTN. in contrast, methylene blue and hemoglobin inhibited SNP-induced cGMP accumulation but, paradoxically, potentiated SNP-induced relaxation. The results of this study generally support a role for cGMP in NVD-induced relaxation of airway smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Dogs; Drug Synergism; Female; Guanylate Cyclase; Hemoglobins; Isoproterenol; Male; Methylene Blue; Muscle Relaxation; Nitroglycerin; Nitroprusside; Penicillamine; Purinones; S-Nitroso-N-Acetylpenicillamine; Time Factors; Trachea; Vasodilator Agents

Nitrovasodilators increase both cyclic GMP and cyclic AMP in isolated platelets (Maurice DH, Haslam RJ. Mol Pharmacol 1990;37:671-81). To determine whether this occurs in blood, platelet cyclic[3H]GMP and cyclic [3H]AMP were measured in prelabeled rabbit platelets resuspended in modified Tyrode's solution or citrated blood. In the former medium, increases in cyclic [3H]nucleotides in response to nitroprusside (NP) and 3-morpholinosydnonimine (SIN-1) were maximal by 1 min; in blood, maximal increases were observed only after 10 min and were much smaller. In blood, SIN-1 was more effective than the same concentration of NP. After 10 min, 100 microM SIN-1 increased platelet cyclic[3H )GMP by 475 +/- 58% and cyclic[3H]AMP by 29 +/- 7% (means +/- SEM, 18 experiments). Supraadditive increases in platelet cyclic [3H]AMP in blood were observed when SIN-1 was combined with prostaglandin E1 (PGE1). Thus, after 10 min, SIN-1 (100 microM), PGE1 (20 nM), and SIN-1 + PGE1 increased cyclic[3H]AMP by 25 +/- 7, 35 +/- 6, and 130 +/- 17%, respectively (four experiments). In the same experiments, release of platelet [14C]serotonin by platelet-activating factor (PAF) was inhibited by 22 +/- 5, 2 +/- 2, and 61 +/- 5%, respectively. Increases in platelet cyclic[3H]GMP with SIN-1 were unaffected by PGE1. These results suggest that although cyclic GMP may mediate the effects of SIN-1 alone on platelet function, cyclic AMP mediates the synergistic action of SIN-1 and PGE1. M&B 22,948 (a selective cyclic GMP phosphodiesterase inhibitor) enhanced the increases in platelet cyclic[3H]GMP and cyclic[3H]AMP caused by SIN-1 and also increased the associated inhibition of [14C]serotonin release. M&B 22,948 also augmented the synergistic increases in cyclic[3H]AMP and inhibition of platelet function caused by SIN-1 + PGE1. The results show that a selected nitrovasodilator (e.g., SIN-1), a prostaglandin and a cyclic GMP phosphodiesterase inhibitor can exert synergistic effects on platelets in blood. This may be relevant to the pharmacologic management of thromboembolic disease.

Topics: Alprostadil; Animals; Blood Platelets; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Drug Synergism; Epoprostenol; Male; Molsidomine; Nitric Oxide; Purinones; Rabbits; Serotonin; Vasodilator Agents

We examined the vasoinhibitory effect of (3E)-4-ethyl-2-hydroximino-5-nitro-3-hexamide FK409, a new vasodilator, on contractile responses in isolated rabbit arteries. FK409 (10(-8)-10(-5) M) inhibited contractile responses to norepinephrine (NE), histamine (His), and 5-hydroxytryptamine (5-HT) in rabbit aorta. The pattern of inhibition by FK409 was not competitive. The inhibitory effect of FK409 on the 5-HT response was much greater than that of nitroglycerin (NG). A high concentration of FK409 (10(-5) M) was necessary to inhibit the response to KCl (10-70 mM). The effect of combined treatment with FK409 (10(-5) M) and a subthreshold concentration of nifedipine (10(-9) M) on the KCl response was much greater than a single treatment with either agent. In addition, 3 x 10(-6) M D600, but not FK409 (10(-6) or 10(-5) M), inhibited the increase in the rate of 45Ca influx stimulated by a 40-mM KCl substituted solution. In a Ca2(+)-free medium containing EGTA and nifedipine, FK409 (10(-9)-10(-5) M) inhibited phasic responses to NE, His, and 5-HT, and subsequent sustained responses owing to addition of Ca2+. The response to caffeine in rabbit iliac arteries incubated in Ca2(+)-free medium was also inhibited by FK409 (10(-6) and 10(-5) M). In rabbit aorta precontracted with NE (10(-5) M) and partially inhibited by prior exposure to NG (10(-5) M), the relaxing effect of FK409 was slightly attenuated. Pretreatment of tissues with FK409 (10(-6) M) inhibited the relaxing action of NG much more than prior NG inhibited the relaxing action of FK409. Methylene blue (10(-5) M), but not hemoglobin (10(-6) M), inhibited the relaxing action of FK409, whereas M&B 22,948 (3 x 10(-4) M) potentiated it. FK409 caused a relaxation of precontracted aorta without endothelium that was inhibited by methylene blue. In rabbit aorta precontracted with NE, FK409 (10(-6) M) increased cyclic GMP but not cyclic AMP content. FK409 (10(-5) M) had no effect on the NE-mediated increase in tissue inositol monophosphate (IP). These results suggest that FK409 inhibits the responses attributed to both intracellular Ca2+ release and Ca2+ influx through receptor-operated channels. The inhibitory effect of FK409 on both the KCl contractile response and KCl-stimulated 45Ca influx appears to be different from that of nifedipine or D600. Furthermore, the inhibitory action of FK409 may be partially mediated by cyclic GMP.

Topics: Animals; Arteries; Caffeine; Calcium; Cyclic GMP; Hemoglobins; Histamine; In Vitro Techniques; Inositol Phosphates; Male; Methylene Blue; Nitro Compounds; Nitroglycerin; Norepinephrine; Purinones; Rabbits; Serotonin; Vasoconstriction; Vasodilator Agents

1 The present study has examined the relaxant effects of alpha-human atrial natriuretic peptide (alpha-hANP) and sodium nitroprusside on dog saphenous vein ring preparations; responses were tested on a background of tone corresponding to 30% and 80% of maximal contraction. Also tested on the lower background of tone were the responses to carbachol, M&B 22,948, forskolin and 3-isobutyl-1-methylxanthine (IBMX). The vascular responses to alpha-hANP and carbachol were also examined in dog splenic artery ring preparations. 2 On a background of tone corresponding to 30% of maximal contraction, both alpha-hANP and sodium nitroprusside were found to relax venous ring preparations with IC50 values of 6 x 10(-8) and 2 x 10(-7) M, respectively. When the background of tone was raised to 80% of maximal contraction, the relaxant effect of sodium nitroprusside was significantly reduced and that of alpha-hANP became almost completely abolished; the IC50 value for sodium nitroprusside in these experimental conditions was 2 x 10(-6) M. 3 The cGMP phosphodiesterase inhibitor M&B 22,948 was found to produce a concentration-dependent relaxation, with an IC50 value of 2 x 10(-6)M in saphenous vein rings with a background of tone corresponding to 30% of maximal contraction. By contrast, saphenous vein rings were found to be insensitive to carbachol. Forskolin and IBMX were also found to produce concentration-dependent relaxing responses of venous preparations, with IC50 values of 2 x 10(-7) and 7 x 10(-7) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Animals; Atrial Natriuretic Factor; Carbachol; Colforsin; Cyclic AMP; Cyclic GMP; Dogs; Female; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroprusside; Peptide Fragments; Purinones; Saphenous Vein; Splenic Artery; Vasodilation

The interactions between isoprenaline, sodium nitroprusside, and the isozyme-selective phosphodiesterase inhibitors OPC 3911 ("cAMP specific") and zaprinast ("cGMP specific") on platelet aggregation induced by adenosine diphosphate (ADP) and on levels of cAMP and cGMP were studied. Isoprenaline at 10(-6)M diminished aggregation by 28%, and this effect was enhanced by 10(-7)-10(-6)M OPC 3911. Neither 10(-6) M isoprenaline nor 10(-7)M OPC 3911 elevated cAMP, but in combination they caused a significant rise in cAMP (27% above the basal level), accompanying the synergistic functional inhibition, without affecting cGMP levels. Sodium nitroprusside at 10(-5) M diminished aggregation by 39%, elevated cGMP levels (81-110%), but also caused a statistically significant increase in cAMP (21-32%), and enhanced the effects of 10(-6)M isoprenaline on cAMP levels. Zaprinast at 10(-5) M caused a modest inhibition of aggregation by 20%, and a small increase in cGMP (20%), and it clearly enhanced the effects of 10(-5)M sodium nitroprusside on both cGMP and cAMP levels, but not on aggregation. The cAMP-increasing effect of sodium nitroprusside might be a consequence of a cGMP-mediated inhibition of the "low-Km cGMP-inhibited phosphodiesterase" that is also inhibited by OPC 3911. The effects of all of the drugs on ADP-induced aggregation seem to depend more on their effect on cAMP levels than on the levels on cGMP.

Topics: Cyclic AMP; Cyclic GMP; Drug Interactions; Humans; Isoproterenol; Nitroprusside; Phosphodiesterase Inhibitors; Platelet Aggregation; Platelet Aggregation Inhibitors; Purinones; Quinolones

The effects of the cyclic phosphodiesterase (PDE) inhibitors, rolipram, 3-isobutyl-1-methylxanthine (IBMX), amrinone and zaprinast on pancreatic exocrine secretion were investigated in anesthetized dogs. Rolipram (1-30 nmol), IBMX (44-440 nmol) or zaprinast (1-10 mumol) injected i.a. elicited a dose-dependent increase in the secretion of pancreatic juice, but amrinone (up to 53 mumol) did not. The bicarbonate concentration in pancreatic juice was increased and the protein concentration was decreased by rolipram and IBMX, but neither was affected by zaprinast. Rolipram elicited more than the respective additive secretory response when added together with secretin, although the stimulatory effects of CCK-8 with rolipram were additive. Rolipram and IBMX, but not zaprinast, increased cyclic AMP concentration but did not affect cyclic GMP concentration. These results suggest that rolipram, IBMX and zaprinast have direct secretory properties on pancreatic exocrine glands of the dog, which may be mediated through the increase of intracellular cyclic AMP concentration, by inhibiting PDE activity. Furthermore, the pancreatic PDE enzyme in the dog pancreas may be mainly a type IV.

Topics: 1-Methyl-3-isobutylxanthine; 2',3'-Cyclic-Nucleotide Phosphodiesterases; Amrinone; Animals; Bicarbonates; Cyclic AMP; Cyclic GMP; Dogs; Female; Male; Pancreas; Pancreatic Juice; Perfusion; Phosphodiesterase Inhibitors; Proteins; Purinones; Pyrrolidinones; Rolipram; Secretin; Sincalide

Increases in cyclic adenosine monophosphate and cyclic guanosine monophosphate content accompany relaxation of isolated strips of opossum and canine lower oesophageal sphincter muscle. The aim of this investigation was to characterise these responses in isolated muscle from the human lower oesophageal sphincter. Electrical stimulation of enteric neurons produced a frequency dependent relaxation of the human lower oesophageal sphincter that was sensitive to tetrodotoxin. Furthermore, as previously shown in the opossum and canine lower oesophageal sphincter, cyclic guanosine monophosphate content was significantly raised in muscle strips frozen during maximum electrical field stimulation whereas cyclic adenosine monophosphate content was unchanged. In addition, sodium nitroprusside (EC50 = 0.1 microM) produced a concentration dependent relaxation of human lower oesophageal sphincter, significantly increased cyclic guanosine monophosphate content, but did not alter cyclic adenosine monophosphate content. Zaprinast (M&B 22948) and SK&F 94120, selective inhibitors of cyclic guanosine monophosphate and cyclic adenosine monophosphate phosphodiesterases, respectively, both relaxed human lower oesophageal sphincter with a potency similar to that seen in the dog or opossum lower oesophageal sphincter. Finally, the 8-bromo analogues of both cyclic adenosine monophosphate (EC50 = 420 microM) and cyclic guanosine monophosphate (EC50 = 100 microM) relaxed the human lower oesophageal sphincter. These studies suggest that in the human, as well as the canine and opossum lower oesophageal sphincter, increases in cyclic nucleotide content are associated with relaxation and increases in cyclic guanosine monophosphate are associated with the relaxation induced by stimulation of enteric neurons.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Cyclic AMP; Cyclic GMP; Electric Stimulation; Esophagogastric Junction; Humans; In Vitro Techniques; Muscle Relaxation; Nitroprusside; Nucleotides, Cyclic; Purinones; Pyrazines; Tetrodotoxin

The present study was designed to investigate whether cyclic GMP or cyclic AMP modulates the release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) in cultured bovine aortic endothelial cells (BAE cells). BAE cell-conditioned medium was transferred onto rat fetal lung fibroblasts (RFL-6 cells) and the increase in cyclic GMP in these cells was used as a sensitive bioassay of EDRF/NO activity. BAE cells released a material that markedly enhanced cyclic GMP in RFL-6 cells. The synthesis of this substance could be stimulated with bradykinin (10 nM) or Ca++ ionophore A23187 (1 microM) and was completely prevented by treatment of the BAE cells with the EDRF/NO synthesis inhibitors NG-nitro-L-arginine (100 microM) or NG-methyl-L-arginine (1 mM). Addition of hemoglobin (10 microM) or incubation of the RFL-6 detector cells with methylene blue (10 microM) also abolished the cyclic GMP increase in the RFL-6 cells. The release of EDRF/NO by bradykinin and A23187 was accompanied by an approximately 2-fold increase in the cyclic GMP content in the producing BAE cells (in the presence of the cyclic GMP phosphodiesterase inhibitor M&B 22,948, 0.1 mM). Incubation of BAE cells with atrial natriuretic peptide (0.1 microM) or sodium nitroprusside (10 microM) enhanced cyclic GMP content of BAE cells 6.5-fold and 4.1-fold, respectively (in the presence of M&B 22,948, 0.1 mM). These increases in the cyclic GMP levels in BAE cells had no effect on basal or bradykinin- and A23187-stimulated release of EDRF/NO. Bradykinin (10 nM) and A23187 (1 microM) also stimulated prostacyclin production in BAE cells 2.4-fold and 5.6-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Aorta; Arginine; Calcimycin; Cattle; Cells, Cultured; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; Epoprostenol; Nitric Oxide; Nitroarginine; Purinones

1. Sodium nitroprusside (NP) caused both an inhibition of a noradrenaline (NA)-induced contraction and an elevation of cyclic guanosine 3',5'-monophosphate (cyclic GMP) in rat aorta. Both NP-induced responses were enhanced by the selective cyclic GMP phosphodiesterase inhibitor, M&B 22948 (2-o-propoxyphenyl-8-aza-purin-6-one, 30 microM). 2. The inhibition of a NA-induced contraction by NP was characterized by dissociating the intracellular Ca2+ release component from the extracellular Ca2+ influx component of the contraction. The transient contraction stimulated by NA in the absence of extracellular Ca2+ was inhibited by NP. Also, the slowly developed tension stimulated by NA in aortas depleted of stored Ca2+ and subsequently exposed to extracellular Ca2+ was inhibited by NP. Both components of contraction were equally sensitive to NP. 3. NA stimulated both unidirectional 45Ca2+ influx in the presence of extracellular Ca2+ and 45Ca2+ efflux into a 0 Ca2+ solution that contained 2 mM-ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid (EGTA). The increased 45Ca2+ efflux is thought to reflect release of stored Ca2+ followed by membrane transport. NP greater than 10 nM inhibited both 45Ca2+ influx and release components whereas NP at 1-3 nM enhanced NA-stimulated 45Ca2+ efflux and relaxed the maintained tension caused by NA in 0 Ca2+, 2 mM-EGTA. 4. NP also inhibited the Ca2(+)-dependent 42K+ and 36Cl- effluxes from rat aorta stimulated either by NA or by high potassium. NP inhibited the contractile and flux responses to NA more effectively than the responses to high potassium. 5. These data indicate that: (1) NP reduces cytosolic Ca2+ by the combined inhibitory effects on Ca2+ influx and intracellular Ca2+ release, and by the stimulation of a Ca2(+)-ATPase; and (2) the differential sensitivity of the NA and high-potassium responses to NP may reflect underlying differences in Ca2+ handling induced by receptor occupancy and depolarization.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Aorta, Thoracic; Calcium; Calcium-Transporting ATPases; Chlorides; Cyclic GMP; Egtazic Acid; Ferricyanides; In Vitro Techniques; Ion Channels; Male; Muscle Contraction; Muscle, Smooth, Vascular; Nitroprusside; Norepinephrine; Potassium; Purinones; Rats; Rats, Inbred Strains

Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aorta; Calcium; Cells, Cultured; Chromatography, Ion Exchange; Colforsin; Cyclic AMP; Cyclic GMP; Dipyridamole; Endothelium, Vascular; Isoquinolines; Kinetics; Purinones; Pyrazines; Pyrrolidinones; Rolipram; Swine; Tetrahydroisoquinolines

To investigate the relationship between AP, cyclic GMP, and sodium excretion, we studied the effect of a cyclic GMP phosphodiesterase inhibitor (M + B22948) on the natriuretic response to (a) an infusion of AP (103-126) and (b) acute volume expansion in rats. The phosphodiesterase inhibitor markedly potentiated the effect of low-dose AP infusions on urinary sodium and cyclic GMP excretion without potentiating the fall in blood pressure. Acute volume expansion (1% body wt) led to small but significant (P less than 0.01) rises in plasma AP and urinary cyclic GMP levels. Pretreatment with the phosphodiesterase inhibitor enhanced the natriuretic and cyclic GMP response to volume loading, an effect that was attenuated by administration of a monoclonal antibody directed against AP. These data indicate that cyclic GMP mediates the natriuretic activity of AP and AP and cyclic GMP play active roles in the natriuresis of acute volume expansion. Moreover, pharmacological manipulation of cyclic GMP levels may prove a useful therapeutic strategy for facilitating the natriuretic but not the hypotensive effects of AP.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Antibodies, Monoclonal; Atrial Natriuretic Factor; Blood Pressure; Cyclic GMP; Male; Natriuresis; Purinones; Rats; Rats, Inbred Strains

Neutrophils harvested from the peritoneal cavities of rats have been shown to release a factor that relaxes precontracted aorta and has a pharmacologic profile similar to that previously reported for endothelium-derived relaxing factor (EDRF). The present study was designed to determine if this neutrophil-derived relaxing factor (NDRF) relaxes rat aortic smooth muscle by affecting the intracellular cGMP levels. Aortic sheets (endothelium removed) were incubated in organ chambers in a physiological salt solution containing phenylephrine (1 x 10(-7) M) and superoxide dismutase (10 or 100 U/ml). Basal cGMP levels (10-15 pmoles/g tissue) were not affected by the incubation reagents. Neutrophils (3 x 10(6) to 1 x 10(8) cells/10 ml) increased cGMP, but not cAMP, levels in a cell number-dependent manner. Peak induction occurred at 5 min of incubation. Methylene blue (1 x 10(-5) M) inhibited and zaprinast (1 x 10(-5) M) potentiated the neutrophil-induced increases in cGMP. The data thus support the hypothesis that neutrophil-induced vascular smooth muscle relaxation is mediated through a factor, NDRF, which increases intracellular cGMP levels.

Topics: Animals; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; In Vitro Techniques; Male; Methylene Blue; Muscle, Smooth, Vascular; Myocardial Contraction; Neutrophils; Nitric Oxide; Peritoneal Cavity; Phenylephrine; Purinones; Rats; Rats, Inbred Lew; Superoxide Dismutase

The effect of pharmacological manipulation of atriopeptin (AP) activity on sodium excretion and blood pressure was examined in the rat aortovenocaval (A-V) fistula model of cardiac failure. Introduction of an A-V shunt led to a marked and sustained elevation of plasma AP immunoreactivity and urinary cGMP levels. Further elevation of plasma AP levels by infusion of exogenous peptide induced modest increases in urinary sodium and cGMP excretion and a decrease in blood pressure but these responses were significantly attenuated compared to sham-operated animals. In contrast, low-dose infusion of M + B 22948 (a cGMP phosphodiesterase inhibitor) or thiorphan [a neutral endopeptidase (membrane metallo-endopeptidase, EC 3.4.24.11) inhibitor] induced a natriuresis in A-V fistula rats, which exceeded that seen in control animals given these compounds and matched the peak natriuresis produced in sham-operated animals by high doses of AP. In the doses used, these compounds had little effect on blood pressure. The greater renal efficacy of M + B 22948 in A-V fistula rats is consistent with postreceptor facilitation of AP activity. The effect of thiorphan on sodium excretion was accompanied by a pronounced increase in urinary cGMP and AP immunoreactivity excretion (and was attenuated by anti-AP monoclonal antibody) but could not be explained solely in terms of an increase in circulating AP levels. It is proposed that thiorphan allows filtered AP to reach renal tubule sites that are normally inaccessible to the peptide and are thus protected from down-regulation by high circulating AP levels. The implication of these observations for patients in cardiac failure is the potential for using pharmacological agents to maximize the response to endogenous AP without compromising cardiac function.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Antibodies, Monoclonal; Atrial Natriuretic Factor; Blood Pressure; Cyclic GMP; Disease Models, Animal; Heart Failure; Male; Purinones; Rats; Rats, Inbred Strains; Reference Values; Sodium; Thiorphan

Previous studies in our laboratory showed that ANP inhibits increases in endothelial monolayer permeability to macromolecules induced by thrombin. In this present study, we investigated the second messenger system involved in the influence of ANP on monolayer permeability. In bovine aortic endothelial cells (BAEC), ANP (100 nM) caused increased cGMP levels which were measurable at 30 sec and maximal at 3 min. Addition of 8-bromo cGMP (1 mM) to BAEC monolayers mimicked the actions of ANP by inhibiting thrombin- mediated increases in permeability to [125I]-labeled bovine serum albumin. Inhibition of increases in permeability by lower concentrations of ANP was enhanced by the cGMP-selective phosphodiesterase inhibitor, M&B 22948 (100 microM). The use of ANP structural analogs which stimulate cGMP production (AP III or BNP) prevented thrombin-induced increases in monolayer permeability, whereas AP-I, which does not increase cGMP levels, was ineffective.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Aorta; Atrial Natriuretic Factor; Cattle; Cell Membrane Permeability; Cyclic GMP; Endothelium, Vascular; Kinetics; Purinones; Serum Albumin, Bovine; Structure-Activity Relationship; Thrombin

We examined, in isolated blood perfused rat lungs, the effect of the cell permeable 8-bromo derivative of cGMP on pulmonary vasoconstriction induced by either alveolar hypoxia or angiotensin II. 8-Bromo cGMP dose-dependently reduced both hypoxia-(IC50 = 2.2 X 10(-5) M) and angiotensin-II-induced pulmonary vasoconstriction (IC50 = 5.0 X 10(-5) M). This effect of 8-bromo cGMP on pulmonary vasoconstriction was not affected by cyclooxygenase blockade. M & B 22948 (0.1 mM), an inhibitor of cGMP-phosphodiesterase, reduced synergistically with 8-bromo cGMP the hypoxia or angiotensin-II-induced vasoconstriction. The cGMP-phosphodiesterase inhibitor M & B 22948, by itself, selectively reduced hypoxia-induced vasoconstriction, suggesting a modulating effect of endogenous cGMP during hypoxic vasoconstriction.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Angiotensin II; Animals; Cyclic GMP; Dose-Response Relationship, Drug; Hypoxia; Lung; Male; Meclofenamic Acid; Purinones; Rats; Vasoconstriction

1. The mechanism by which M&B 22,948, MY-5445, vinpocetine and 1-methyl-3-isobutyl-8-(methylamino)xanthine (MIMAX), which have been described as selective cyclic GMP phosphodiesterase (PDE) inhibitors, relax rat aorta was investigated. 2. Three cyclic nucleotide PDEs were identified in the soluble fraction of rat aorta; a Ca2+-insensitive form exhibiting substrate selectivity for cyclic GMP (cGMP PDE), a Ca2+/calmodulin-stimulated form which also preferentially hydrolyzed cyclic GMP (Ca2+ PDE), and a form demonstrating substrate selectivity for cyclic AMP (cAMP PDE). 3. M&B 22,948 and MIMAX inhibited cGMP PDE (Ki = 0.16 microM and 0.43 microM, respectively) and Ca2+ PDE (Ki = 9.9 microM and 0.55 microM, respectively), but exhibited weak activity against cAMP PDE (Ki = 249 microM and 42 microM, respectively). MY-5445 selectivity inhibited cGMP PDE (Ki = 1.3 microM) and vinpocetine selectively inhibited Ca2+ PDE (Ki = 14 microM). 4. M&B 22,948 and MIMAX induced dose-dependent increases in the accumulation of cyclic GMP, but not cyclic AMP, in rat aorta pieces. These effects were greatly reduced by endothelial denudation and by methylene blue (5 microM) which blocks the actions of endothelium-derived relaxant factor. MY-5445 and vinpocetine had no effect on rat aorta cyclic GMP or cyclic AMP accumulation. 5. All four compounds caused dose-related relaxation of 5-hydroxytryptamine (10 microM) contracted, endothelium-intact rat aorta, the effects of M&B 22,948 and MIMAX being greatly reduced by methylene blue (5 microM). Methylene blue also caused 10 fold and 100 fold rightward shifts in the dose-response curves of MY-5445 and vinpocetine, respectively. 6. The results are consistent with the smooth muscle relaxant actions of M&B 22,948 and MIMAX, but not vinpocetine and MY-5445, being mediated through a mechanism involving inhibition of cyclic GMP hydrolysis.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aorta, Thoracic; Cyclic AMP; Cyclic GMP; In Vitro Techniques; Kinetics; Male; Methylene Blue; Muscle Relaxants, Central; Nitric Oxide; Phthalazines; Purinones; Pyridazines; Rats; Rats, Inbred Strains; Theophylline; Vinca Alkaloids

Electrical field stimulation (EFS) of isolated strips of opossum lower esophageal sphincter (LES) produced a relaxation that was accompanied by an elevation of intracellular cyclic GMP content. In order to compare the time dependence of the EFS-induced relaxation with that of the elevation of cyclic GMP, the ability of EFS to produce relaxation and increase cyclic GMP was measured. The results of these experiments showed that cyclic GMP content increased before the onset of relaxation. Cumulative addition of atriopeptin II, an activator of particulate guanylate cyclase, produced a concentration-dependent relaxation of this tissue and increased cyclic GMP content. In other experiments, zaprinast, an inhibitor of a cyclic GMP selective-phosphodiesterase, produced a concentration-related relaxation of opossum LES and increased cyclic GMP content. However, pretreatment with zaprinast (3 microM) did not potentiate the EFS-induced relaxation or the increase in cyclic GMP content. At this concentration, however, zaprinast increased the basal content of cyclic GMP. Finally, 8-Br-cyclic GMP, a membrane-permeable analog of cyclic GMP, produced a concentration-dependent relaxation of isolated strips of opossum LES. In conclusion, these data extend the initial findings that an elevation in cyclic GMP content is associated with relaxation and suggest that cyclic GMP is a potential intracellular messenger of neurally- and drug-induced relaxation of opossum LES.

Topics: Animals; Cyclic GMP; Electric Stimulation; Esophagogastric Junction; Female; Male; Methylene Blue; Muscle Relaxation; Nitroprusside; Opossums; Purinones

Atrial natriuretic peptide (ANP) has been shown to increase plasma insulin levels in vivo and to act on various target cells as a potent stimulator of the cGMP system. It has, therefore, been investigated whether ANP has a direct insulinotropic effect mediated by specific binding sites and by affecting the cGMP system in isolated rat pancreatic islets. Unlabelled ANP inhibited 125I-ANP binding in a concentration-related manner (Kd1 and Kd2 = 0.02 and 11.2 nM, Bmax1 and Bmax2 = 0.0147 and 0.0328 pmoles per 1 mg protein). ANP was able to augment cGMP levels in islets, but was not able to enhance insulin secretion at various glucose concentrations. Since the role of cGMP for the glucose-mediated insulin release is controversial, in addition to ANP M&B 22,948 (a cGMP phosphodiesterase inhibitor) was investigated to evaluate the possible role of cGMP for insulin release more precisely. Like ANP M&B 22,948 increased cGMP levels but did not affect insulin release. The data indicate no direct insulinotropic effect of ANP, although ANP binding sites are present on rat pancreatic islets and question the claimed role of cGMP for insulin secretion in general. Therefore, the recently observed in vivo elevation of plasma insulin levels in response to ANP is rather an indirect than a direct effect.

Topics: Animals; Atrial Natriuretic Factor; Cyclic GMP; Female; Glucose; In Vitro Techniques; Insulin; Insulin Secretion; Iodine Radioisotopes; Islets of Langerhans; Male; Phosphodiesterase Inhibitors; Proteins; Purinones; Rats; Rats, Inbred Strains; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface

1. Production of endothelium-derived relaxing factor (EDRF) by primary cultures of pig aortic endothelial cells was assessed indirectly by measuring endothelial cyclic GMP content, and prostacyclin production was measured by radioimmunoassay of 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha). 2. The resting level of cyclic GMP fell significantly following removal of extracellular calcium (1 mM EGTA present), but elevations of cyclic GMP content induced by sodium azide (10 microM) or atriopeptin II (10 nM) were similar in the absence and presence of extracellular calcium. 3. Haemoglobin (10 microM) reduced the resting level of cyclic GMP in the presence, but not the absence of extracellular calcium. M&B 22,948 (100 microM), superoxide dismutase (30 u ml-1), bradykinin (0.1 microM), ATP (10 microM) and ionophore A23187 (0.1 microM) each induced an increase in endothelial cyclic GMP content that was reduced in the absence of extracellular calcium. 4. In cascade bioassay experiments using endothelial cells on microcarrier beads and perfused in columns, continuous infusion of bradykinin (0.1 microM) induced release of EDRF, assayed on rabbit aortic rings, that was maximal after 2 min and still detectable up to about 16 min. 5. In the presence of extracellular calcium, the time course of bradykinin (0.1 microM)-stimulated production of EDRF, assessed as endothelial cyclic GMP content was maximal within 1 min, declined thereafter, but was still significant after 30 min. Production of 6-keto PGF1 alpha, measured simultaneously rose rapidly but was complete within 3 min. 6. In the absence of extracellular calcium the resting endothelial content of cyclic GMP fell, but resting production of 6-keto PGF1 alpha was unaffected. 7. In the presence of TMB-8 (100 microM) resting endothelial content of cyclic GMP rose slightly, but production of 6-keto PGFg fell. The bradykinin (0.1 microM)-stimulated increase in cyclic GMP content was augmented, but the stimulation of 6-keto PGF1I production was blocked. Results from cascade bioassay experiments confirmed that TMB-8 (100 microM) did not inhibit bradykinin-induced production of EDRF. 8. The data suggest that resting production of EDRF but not prostacyclin is dependent upon the presence of extracellular calcium. Bradykinin-stimulated production of EDRF is sustained and requires the presence of extracellular calcium, but stimulated production of prostacyclin is transient and may result from discharge of an intracellular po

Topics: Adenosine Triphosphate; Animals; Atrial Natriuretic Factor; Azides; Biological Factors; Bradykinin; Calcimycin; Calcium; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Epoprostenol; Gallic Acid; Hemoglobins; Humans; Nitric Oxide; Purinones; Sodium Azide; Superoxide Dismutase; Swine

Few high affinity inhibitors of the photoreceptor phosphodiesterases have been identified. We show here that dipyridamole and M&B 22,948 (Zaprinast), potent inhibitors of the cGMP-binding, cGMP-specific phosphodiesterase (PDE), also inhibit trypsin- or transducin-activated bovine rod and cone photoreceptor phosphodiesterases at submicromolar concentrations. Dixon plots demonstrated that the inhibition of trypsin-activated rod PDE was competitive, with Ki values of 140 nM for M&B 22,948 and 380 nM for dipyridamole. Both of these drugs were much more potent than other PDE inhibitors, including isobutylmethylxanthine (IBMX). These results reinforce the suggestion that the photoreceptor and the cGMP-binding, cGMP-specific PDE are closely related. In addition, the high affinity and selectivity of these agents should make them useful for probing the regulation and function of PDE in the photoreceptor. At low substrate concentrations, the effects of these drugs on basal unactivated PDE activity were similar to those seen with trypsin- or transducin-activated PDE. At millimolar substrate concentrations, however, the effects of the drugs were biphasic; PDE activity was stimulated at drug concentrations from 1 to 10 microM and inhibited at higher concentrations. Stimulation was not observed with IBMX. This stimulation of activity apparently was not an allosteric effect caused by direct binding of the dipyridamole and M&B 22,948 to the high affinity noncatalytic cGMP binding sites on the PDEs; whereas no cooperativity of cGMP binding to this site has been demonstrated, the drugs actually stimulated the binding of low concentrations of cGMP to this site. In addition, whereas preincubation with cGMP and cGMP analogs blocked the stimulation exerted by the drugs, they did so only at much higher concentrations than those necessary for saturation of the high affinity noncatalytic cGMP site. Because the stimulation can only be seen at higher substrate levels than are thought to exist in the photoreceptor, only the inhibitory effects of the drugs are likely to be pharmacologically relevant. However, the stimulation exerted by these drugs may point to a hitherto unknown allosteric interaction between the catalytic and regulatory sites on the PDE or to a previously unrecognized regulatory site.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Binding Sites; Cattle; Cyclic GMP; Dipyridamole; Enzyme Activation; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Phosphodiesterase Inhibitors; Photoreceptor Cells; Purinones; Rats; Rod Cell Outer Segment; Trypsin

We examined the effects of an acute infusion of M&B 22,948 (2-o-propoxyphenyl-8-azapurin-6-one), a (cGMP)-selective phosphodiesterase inhibitor, on mean arterial pressure (MAP) and urinary sodium excretion in anesthetized rats. M&B 22,948 (at doses of 0.34-2.72 mg/kg/min for 30 min) lowered MAP in a dose-dependent manner, with a 60 mm Hg fall in pressure produced at the highest dose. Despite large decreases in MAP, a profound natriuresis was observed at all doses. Plasma concentrations of cGMP increased in parallel with the depressor action of M&B 22,948, whereas increases in the urinary excretion of cGMP temporally correlated with the natriuresis. The concentration of cyclic AMP in plasma increased transiently in rats treated with M&B 22,948 but the urinary excretion of cyclic AMP was not elevated in these animals. Because changes in cGMP correlated with the physiological effects of M&B 22,948, and the increase in cyclic AMP did not, it is likely that the depressor and natriuretic actions of M&B 22,948 are mediated by increases in cGMP. M&B 22,948 administered chronically at an oral dose of 200 mg/kg/day normalized MAP in spontaneously hypertensive rats; whereas MAP in vehicle-treated spontaneously hypertensive rats remained at hypertensive levels. cGMP-selective phosphodiesterase inhibitors (like M&B 22,948) could be more effective antihypertensive drugs than currently available vasodilators because, when administered acutely, M&B 22,948 simultaneously lowers blood pressure and promotes sodium excretion in the anesthetized rat.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Blood Pressure; Cyclic AMP; Cyclic GMP; Male; Natriuresis; Purinones; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Sodium

The novel neuropeptide, brain natriuretic peptide (BNP), causes concentration-dependent relaxations in rat isolated arterial rings. The pD2 value of BNP in rat thoracic aorta is 8.05 +/- 0.06, almost identical to the pD2 value of atrial natriuretic peptide (the 28 amino acid peptide, rat sequence, AP-28, 8.11 +/- 0.08), indicating that BNP and ANP have the same potency in relaxing thoracic aorta. In addition, BNP is equally potent at causing relaxation in abdominal aorta and mesenteric and renal arteries. However, BNP is less potent in causing vasorelaxation in the common iliac and femoral arteries and shows no relaxant effects in caudal arteries. This pharmacological profile of BNP in different rat arteries is very similar to that of ANP. Like ANP, BNP induces a vasorelaxation that is independent of endothelium and is associated with very sustained increases in cyclic GMP, but not cyclic AMP, levels in rat thoracic aorta. The BNP-induced cyclic GMP elevation, like the vasorelaxation, is also independent of endothelium and is not blocked by methylene blue (10 microM), a soluble guanylate cyclase inhibitor. Furthermore, BNP-induced cyclic GMP elevation is independent of extracellular calcium and potentiated by the cyclic GMP-phosphodiesterase inhibitor M & B 22948. Therefore, the pharmacological characteristics of BNP in rat blood vessels are very similar to those of ANP, suggesting that BNP and ANP may act through a common receptor and post-receptor mechanism to cause vasodilation.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aorta, Thoracic; Atrial Natriuretic Factor; Cyclic GMP; Endothelium, Vascular; In Vitro Techniques; Male; Natriuretic Peptide, Brain; Nerve Tissue Proteins; Purinones; Rats; Rats, Inbred Strains; Vasodilation

We have examined the interaction of zaprinast with mediators of guanylate cyclase on the relaxation of aortic smooth muscle. Zaprinast, a selective inhibitor of the low Km-cyclic GMP (cGMP) phosphodiesterase [low Km cGMP phosphodiesterase (PDE)], was equally effective in relaxing phenylephrine-contracted aortas from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) with an intact endothelium [EC50 = 7.6 (3.5-16.6) microM vs. 9.3 (4.1-21.3) microM, respectively]. In contrast, the vasorelaxant activity of zaprinast in intact and denuded phenylephrine-contracted guinea pig aortas, as well as denuded (SHR and WKY) aortas was minimal. Sodium nitroprusside and atriopeptin II were significantly (P less than .05) more potent as vasorelaxants in denuded SHR aortas when compared with denuded aortas from WKY. Pretreatment with zaprinast potentiated the vasorelaxant potency of sodium nitroprusside in both SHR and WKY aortas whereas atriopeptin II responses were potentiated only in WKY aortas. In studies with the low Km cGMP PDE, isolated via DEAE column chromatography, the apparent Km for cGMP and potency of zaprinast were approximately 2-fold greater (P less than .05) in WKY when compared with the same PDE isozyme isolated from SHR aortic preparations. However, the Vmax (picomoles per milligram per minute) for cGMP hydrolysis was greater in SHR than in WKY. In conclusion, these data show that, although there are no apparent differences in the influence of spontaneously released endothelium-derived relaxing factor from SHR and WKY aortas, reactivity differences to other agents known to stimulate guanylate cyclase activity exist between SHR and WKY denuded aortas.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Atrial Natriuretic Factor; Biological Factors; Cyclic GMP; Guanylate Cyclase; Guinea Pigs; In Vitro Techniques; Isoenzymes; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Purinones; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Vasoconstriction

Nitric oxide (NO) was compared with prostacyclin as an inhibitor of the activation of human platelets during isolation, washing and storage. The use of NO throughout the procedure prevented the activation of platelets. The morphology and behaviour of NO-washed platelets was similar to that of prostacyclin-washed platelets when stored at 4 degrees C for up to 24 h. Prolonged storage resulted in deterioration of the platelets which occurred earlier in the NO-washed than in the prostacyclin-washed platelets. The protective effect of NO was potentiated by the selective cGMP phosphodiesterase inhibitor M & B 22948, suggesting that it is mediated by the activation of guanylate cyclase.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Blood Platelets; Blood Preservation; Cell Separation; Cyclic AMP; Cyclic GMP; Drug Synergism; Epoprostenol; Humans; Nitric Oxide; Platelet Aggregation; Purinones; Thromboxane A2

Low doses of insulin (0.1-50 nM) when presented to intact ureteral segments increase cyclic AMP (cAMP) phosphodiesterase (PDE) activity in subsequently isolated supernatant and particulate fractions. The stimulation of cAMP PDE occurs within 5 to 10 min of the introduction of insulin. When cyclic GMP or high concentrations of cAMP (greater than 5 microM) are used as substrate, insulin does not increase PDE activity. Although the insulin-increased cAMP PDE exhibits the same sensitivity as control PDE from untreated preparations to isobutylmethyl xanthine, a nonspecific PDE inhibitor, and M & B 22,948, a relatively selective cyclic GMP PDE inhibitor, differences in the degree of inhibition of PDE activity are seen in the insulin-treated and untreated preparations with the low Km cAMP PDE inhibitors Ro20-1724, rolipram, amrinone and milrinone and with cyclic GMP. Pertussis toxin, which modifies GTP regulatory proteins of the adenylate cyclase enzyme and the photoreceptor PDE, blocks cAMP PDE activation by insulin.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Amrinone; Animals; Cyclic GMP; Enzyme Activation; Female; Guinea Pigs; Insulin; Kinetics; Milrinone; Purinones; Pyridones; Pyrrolidinones; Rolipram; Ureter

The same factors that regulate the activation of purified hepatic soluble guanylate cyclase by diverse agents possessing distinct requirements for enzyme activation were found to modulate cyclic GMP formation in intact viable hepatic cells. A comparison was made between activation of heme-deficient or heme-reconstituted guanylate cyclase and stimulation of cyclic GMP formation in mouse hepatic slices that were 95% viable and showed no active efflux of cyclic GMP. Heme-dependent activators of guanylate cyclase elicited a greater -fold increase in hepatic cyclic GMP levels in slices from phenobarbital-pretreated than control mice. Brilliant cresyl blue and KCN inhibited both enzyme activation and hepatic cyclic GMP accumulation caused by agents that generate nitric oxide. Hepatic slices from 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated mice, which are known to develop sharp increases in hepatic protoporphyrin IX/heme concentration ratios, showed elevated resting cyclic GMP levels whereas phenobarbital pretreatment produced decreased resting cyclic GMP levels compared to controls. Guanylate cyclase activation by azide required added catalase, and both enzyme activation and hepatic cyclic GMP formation were inhibited by aminotriazole. Enzyme activation by glyceryl trinitrate and NaNO2 required added thiols. Hepatic slices from acetaminophen-pretreated mice showed marked depletion of sulfhydryls and decreased cyclic GMP formation in response to these enzyme activators. Both effects were completely restored by treatment of thiol-depleted mice with N-acetylcysteine. These observations lend support to the general view that information gained from studies on the regulatory properties of purified soluble guanylate cyclase bears a close relationship to studies on regulatory mechanisms that modulate cyclic GMP formation in intact cells.

Topics: 1-Methyl-3-isobutylxanthine; Acetaminophen; Acetylcysteine; Animals; Cyclic AMP; Cyclic GMP; Guanylate Cyclase; Heme; Liver; Methylnitronitrosoguanidine; Mice; Nitroglycerin; Penicillamine; Phenobarbital; Phenylhydrazines; Potassium Cyanide; Probenecid; Protoporphyrins; Purinones; S-Nitroso-N-Acetylpenicillamine

The inhibition of platelet adhesion by nitric oxide (NO) and prostacyclin and their mechanism of action was studied. Platelet adhesion to collagen fibrils and endothelial cell matrix was inhibited completely by NO but only partially by prostacyclin. Adhesion of platelets to endothelial cell monolayers was inhibited by bradykinin. This effect of bradykinin was unaffected by aspirin, and was accounted for by the amounts of NO released by the endothelial cells. Inhibition of platelet adhesion by NO and prostacyclin was potentiated by selective inhibitors of cGMP phosphodiesterase, but not of cAMP phosphodiesterase, indicating that elevation of cGMP regulates platelet adhesion.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Aspirin; Blood Platelets; Collagen; Cyclic GMP; Endothelium, Vascular; Epoprostenol; Extracellular Matrix; Humans; In Vitro Techniques; Nitric Oxide; Phthalazines; Platelet Adhesiveness; Purinones; Superoxide Dismutase; Thrombin

The role of the endothelium in modulating cyclic nucleotide levels and intrinsic smooth muscle tone was studied in isolated rings of bovine intrapulmonary artery and vein. Cyclic 3',5'-guanosine monophosphate (cGMP) levels were threefold to fourfold higher in unrubbed artery and vein than in vessels that had been denuded of endothelium. Cyclic 3',5'-adenosine monophosphate (cAMP) levels were twofold higher in unrubbed than in endothelium-denuded artery, but no differences were observed in veins. Methylene blue, an inhibitor of guanylate cyclase, decreased cGMP but not cAMP levels, and this was accompanied by increases in smooth muscle tone. M&B 22,948, an inhibitor of cGMP-phosphodiesterase, increased cGMP but not cAMP levels, and this was accompanied by decreases in smooth muscle tone. Unrubbed vessels were more sensitive than endothelium-denuded vessels to the actions of both methylene blue and M&B 22,948, and this may be attributed to endothelium-dependent increases in cGMP turnover. Moreover, unrubbed vessels were more sensitive than endothelium-denuded vessels to contractile responses to phenylephrine and potassium, and these responses were potentiated by methylene blue and attenuated by M&B 22,948. Although indomethacin lowered cAMP levels in unrubbed artery, no changes in tone or contractile responsiveness were observed. A consistent observation was that the smaller branches of unrubbed but not endothelium-denuded intrapulmonary artery and vein had higher levels of cGMP but not cAMP, were sensitive to endothelium-dependent vasodilators, were more sensitive to methylene blue, and would not maintain a steady level of submaximal tone to phenylephrine when compared with larger branches from a common vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cattle; Cyclic AMP; Cyclic GMP; Endothelium; In Vitro Techniques; Indomethacin; Methylene Blue; Muscle Contraction; Muscle, Smooth, Vascular; Phenylephrine; Pulmonary Circulation; Purinones

The selective cyclic GMP phosphodiesterase inhibitor M&B 22948 and the less selective phosphodiesterase inhibitors papaverine and isobutylmethylxanthine (IBMX) each induced a component of relaxation of rat aortic rings that was endothelium-dependent. The most selective agent at inducing endothelium-dependent relaxation was M&B 22948, which caused little relaxation of endothelium-denuded rings at concentrations that produced almost complete relaxation of endothelium-containing rings. Although endothelium-dependent components of relaxation induced by papaverine and IBMX were clearly present, they were less well separated from the endothelium-independent components of relaxation. In the aorta of the rabbit, M&B 22948 and papaverine were less affective at inducing an endothelium-dependent component of relaxation than in the aorta of the rat, and IBMX produced no discernible endothelium-dependent component. The endothelium-dependent components of relaxation induced by M&B 22948, papaverine and IBMX on rat and rabbit aorta were probably dependent on endothelium-derived relaxing factor (EDRF), because they were associated with concomitant endothelium-dependent rises in cyclic GMP, and these components of relaxation as well as the rises in cyclic GMP were completely blocked by the EDRF-blocking agent hemoglobin. The action of hemoglobin was entirely specific, as none of the endothelium-independent components of relaxation induced by any of the phosphodiesterase inhibitors was affected by this hemoprotein. It is likely that the phosphodiesterase inhibitors induce their endothelium-dependent components of relaxation by inhibiting the hydrolysis of cyclic GMP formed in response to EDRF released spontaneously from endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Animals; Aorta; Cyclic GMP; Drug Synergism; Endothelium; Hemoglobins; In Vitro Techniques; Isoproterenol; Male; Nitric Oxide; Nitroglycerin; Papaverine; Phosphodiesterase Inhibitors; Purinones; Rabbits; Rats; Rats, Inbred Strains; Vasodilation; Vasodilator Agents

Cyclic nucleotide phosphodiesterase (PDE) activity from the 105,000 g supernatant of human, bovine and rat aorta smooth muscle cells was resolved by DEAE-trisacryl chromatography into three major forms showing similar properties in each species. In addition to the two PDE forms previously characterized in vascular tissues (a cAMP-PDE and a calmodulin-dependent PDE), a cGMP-PDE, insensitive to calmodulin, was isolated and characterized in the aorta of the three species. Each isolated PDE form was differently inhibited by various chemical compounds, and these compounds produced effects on cyclic nucleotide levels in isolated rat aorta which could be expected from their inhibitory effect on isolated PDE forms. At concentrations non-selectively inhibiting the three isolated PDE forms (including the calmodulin-dependent one), IBMX (3-isobutyl-1-methylxanthine) and trequinsin markedly and dose-dependently increased both cAMP and cGMP aorta levels (up to 7-fold, in presence of 500 microM IBMX). By contrast selective inhibitors of cGMP-PDE or cAMP-PDE could only induce a moderate elevation (by 1.5-3-fold) in cGMP or cAMP levels, respectively. In the case of M&B 22,948, a highly specific and potent inhibitor of cGMP-PDE, a concentration-dependent increase in tissue cGMP levels was produced by concentrations (in the microM range) active in inhibiting the isolated enzyme. In the case of selective cAMP-PDE inhibitors (rolipram and Ro 20-1724), however, a significant increase in aorta cAMP content was induced only in the presence of drug concentrations which were much higher (200 and 500 microM, respectively) than those inhibiting the isolated enzyme (IC50:5 and 18 microM, respectively). Inhibitors of both cGMP-PDE and cAMP-PDE (dipyridamole, cilostamide and its derivative AAL 05) produced the same moderate effects as did the combination of a selective cGMP-PDE inhibitor and a selective cAMP-PDE inhibitor on the levels of both cGMP and cAMP. These results show that the three forms of PDE isolated from aortic smooth muscle retain properties that they exhibit in the tissue and which are similar in the three species examined, including man. They suggest that each form participates in a specific manner to the regulation of cAMP and cGMP concentrations in aorta smooth muscle cells.

Topics: 1-Methyl-3-isobutylxanthine; 2',3'-Cyclic-Nucleotide Phosphodiesterases; 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aorta; Cattle; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 1; Humans; Kinetics; Muscle, Smooth, Vascular; Purinones; Rats; Species Specificity; Substrate Specificity

Atrial natriuretic factor (ANF) has been shown to bind to specific receptors on vascular smooth muscle cells (VSMC) and to cause an increase in intracellular cyclic GMP (cGMP) content. We have recently demonstrated that a prominent Na+,K+,Cl- cotransport system is present in VSMC and that a permeable cGMP analog (8-bromo-cGMP) stimulates activity of the cotransporter. We have also shown that the ANF peptide, rat atriopeptin III, stimulates Na+,K+,Cl- cotransport and elevates intracellular cGMP levels in VSMC. In the present study, we tested the hypothesis that ANF stimulation of Na+,K+,Cl- cotransport occurs via an increase in cGMP levels. When the quinolinedione, 6-anilo-5,8-quinolinedione (LY83583) (10 microM), was used to block formation of cGMP in VSMC from primary cultures of rat thoracic aorta, it was found that both basal and rat atriopeptin III (100 nM)-stimulated Na+,K+,Cl- cotransport were significantly inhibited. The effect of LY83583 was dose-dependent and the half-maximal inhibitory concentration was 0.5 microM. LY83583 also inhibited cotransport in the presence of a maximal concentration of 8-bromo-cGMP. However, this inhibition was not seen in cells also treated with 2-O-propoxyphenyl-8-azapurin-6-one (M&B 22,948), an inhibitor of cGMP phosphodiesterase. M&B 22,948 alone also increased levels of cotransport. Since inhibition of cGMP formation blocks ANF-stimulated Na+,K+,Cl- cotransport and inhibition of cGMP breakdown enhances Na+, K+, Cl- cotransport, we conclude that ANF stimulation of Na+,K+,Cl- cotransport in VSMC is mediated via increase in intracellular cGMP levels.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; 8-Bromo Cyclic Adenosine Monophosphate; Aminoquinolines; Animals; Aorta, Thoracic; Atrial Natriuretic Factor; Carrier Proteins; Cyclic GMP; Muscle, Smooth, Vascular; Purinones; Rats; Sodium-Potassium-Chloride Symporters

We examined the effects of haemoglobin (which inhibits the vascular responses to stimulation of soluble guanylate cyclase) and of M&B 22,948 (which selectively inhibits cyclic GMP phosphodiesterase) on the relaxation induced in rabbit aorta by the atrial natriuretic peptide, atriopeptin II (which stimulates particulate guanylate cyclase). Pretreatment with M&B 22,948 (100 microM) produced a 2.3 fold potentiation of atriopeptin II-induced relaxation of endothelium-denuded rings of rabbit aorta. Pretreatment with haemoglobin (10 microM) had no effect on the relaxation or the 10.9 fold increase in cyclic GMP content induced by atriopeptin II in endothelium-denuded rings of rabbit aorta. The potentiation by M&B 22,948 suggests a causal role for cyclic GMP in mediating atriopeptin II-induced vasodilatation of rabbit aorta. The inability of haemoglobin to block the atriopeptin II-induced rise in cyclic GMP suggests that it does not block stimulation of particulate guanylate cyclase. Thus, it is unlikely that a ferrous haem-containing receptor site is involved in the activation of the particulate form of guanylate cyclase as it is with soluble guanylate cyclase.

Topics: Animals; Aorta; Atrial Natriuretic Factor; Cyclic GMP; Drug Synergism; Endothelium; Hemoglobins; In Vitro Techniques; Male; Purinones; Rabbits; Vasodilation

The mechanism of action of endothelium-derived relaxant factor (EDRF) was studied using aortic strip preparations of the rabbit and a bioassay system of a rabbit coronary artery perfused in series with an intact aorta. Methylene blue (an inhibitor of guanylate cyclase) inhibited, and 2-O-propoxyphenyl-8-azapurine-6-one (MB22948, an inhibitor of cGMP phosphodiesterase) potentiated the vascular effects of EDRF whether these were due to its basal or to stimulated release. Infusion of these agents at different sites in the bioassay indicated that they act pharmacologically at the smooth muscle level and not on release of EDRF or by chemical interaction with EDRF. The data are consistent with the hypothesis that EDRF-induced relaxation is mediated by elevation of smooth muscle cGMP levels.

Topics: Animals; Aorta, Thoracic; Blood Vessels; Cyclic GMP; Endothelium; In Vitro Techniques; Methylene Blue; Purinones; Rabbits; Vasodilation

1 Cyclic adenosine 3',5'-monophosphate (cyclic AMP)- and cyclic guanosine 3',5'-monophosphate (cyclic GMP)-phosphodiesterase activities from rat lung were selectively inhibited by ZK 62711 and M & B 22948, respectively. Theophylline and papaverine inhibited both activities. 2 Rat lung strips contracted by carbachol were relaxed by 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 26711, EC25 = 7 x 10(-8)M) and 2-O-propoxyphenyl-8-azapurin-6-one (M & B 22948, EC25 = 5 x 10(-7)M) indicating relaxant properties of both cyclic AMP and cyclic GMP. 3 The antigen-induced histamine release from human basophils was inhibited by ZK 62711 (IC25 = 8 x 10(-7)M), whereas M & B 22948 had no effect. On the contrary, the release from rat mast cells was inhibited by M & B 22948 (IC25 = 10(-6)M), while ZK 62711 had no effect. 4 These data show an inhibitory effect of cyclic AMP on histamine release to be involved with basophils, whereas cyclic GMP is predominantly involved with mast cells. Is is suggested that the antianaphylactic properties of cyclic nucleotide phosphodiesterase inhibitors are mainly linked to the increase of cyclic GMP.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Aza Compounds; Cromolyn Sodium; Cyclic AMP; Cyclic GMP; Cyclopentanes; Histamine Release; Humans; In Vitro Techniques; Lung; Male; Papaverine; Purinones; Pyrrolidinones; Rats; Rolipram; Theophylline

The effect of different phosphodiesterase (PDE) inhibitors on the antigen or 48/80 induced histamine release from isolated Hooded Lister rat mast cells was tested. The unselective PDE inhibitors theophylline (2.5 mM) and IBMX (0.2 mM) and the selective cyclic GMP PDE inhibitor M & B 22948 (0.1 mM) inhibited the antigen induced histamine release by 50% while 48/80 induced release was inhibited by about 25%. The cyclic AMP selective PDE inhibitors ICI 63197 (0.5 mM) or Ro 20-1724 (0.2 mM) had no effect on 48/80 induced histamine release but tended to enhance antigen induced release. There was no correlation between the measured levels of cyclic AMP and the effect on histamine release by the investigated PDE inhibitors. Cyclic AMP or cyclic GMP up to 10(-3) M did not affect the anaphylactic histamine release. Dibutyryl-cAMP and dibutyryl-cGMP (10(-4) M) both inhibited the release about 20% but this effect could be explained by the effect of butyric acid as sodium butyrate (2 X 10(-4) M) also inhibited the release by 20%. The presence results suggest that cyclic nucleotides are not important regulators of histamine release from isolated mast cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Aza Compounds; Cyclic AMP; Cyclic GMP; Histamine Release; Male; Mast Cells; Purinones; Rats; Rats, Inbred Strains; Theophylline