cyclic-gmp and trequinsin

To study the roles of nitric oxide (NO) in growth of nerve fibers, (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamine (NOR3), an NO-donor, was applied to cultured dorsal root ganglion (DRG) neurites from a micropipette. Ejection of a small volume of 1 mM NOR3 solution (not more than 1 pl/s) from a micropipette to terminal branches of neurites caused enlargement of the neurites, and often, elongation of their growth cones. This neurite enlargement was blocked by inhibitors for soluble guanylate cyclase. The neurite enlargement did not occur when protein kinase A (PKA) was inhibited. To prove that NOR3 activated PKA, we introduced a fluorescence peptide probe, ARII that reduces its fluorescence by activated PKA, to monitor PKA activity in DRG neurites. ARII fluorescence was reduced by NOR3, which was not observed when PKA was inhibited by its specific inhibitors. These indicated that PKA was indeed activated by NO. To examine whether the PKA activation is due to inhibition of phosphodiesterase III (PDE III) by cyclic GMP, we applied PDE III-specific inhibitors and found that the inhibitions activated PKA. Since PKA regulates various neuronal functions, our finding that NO activates PKA is important to understand roles of NO in nerve fibers.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Enzyme Activation; Fluorescent Dyes; Ganglia, Spinal; Growth Cones; Guanylate Cyclase; Isoquinolines; Milrinone; Neurites; Nitric Oxide; Nitric Oxide Donors; Nitro Compounds; Peptides; Phosphodiesterase Inhibitors; Platelet Aggregation Inhibitors; Rats; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Tetrahydroisoquinolines

We tested the hypothesis that cGMP stimulates renin release through inhibition of the cAMP-specific phosphodiesterase 3 (PDE3) in isolated rat juxtaglomerular (JG) cells. In addition, we assessed the involvement of PDE4 in JG-cell function. JG cells expressed PDE3A and PDE3B, and the PDE3 inhibitor trequinsin increased cellular cAMP content, enhanced forskolin-induced cAMP formation, and stimulated renin release from incubated and superfused JG cells. Trequinsin-mediated stimulation of renin release was inhibited by the permeable protein kinase A antagonist Rp-8-CPT-cAMPS. PDE4C was also expressed, and the PDE4 inhibitor rolipram enhanced cellular cAMP content. Dialysis of single JG cells with cAMP in whole-cell patch-clamp experiments led to concentration-dependent, biphasic changes in cell membrane capacitance (C(m)) with a marked increase in C(m) at 1 micromol/L, no net change at 10 micromol/L, and a decrease at 100 micromol/L cAMP. cGMP also had a dual effect on C(m) at 10-fold higher concentration compared with cAMP. Trequinsin, milrinone, and rolipram mimicked the effect of cAMP on C(m). Trequinsin, cAMP, and cGMP enhanced outward current 2- to 3-fold at positive membrane potentials. The effects of cAMP, cGMP, and trequinsin on C(m) and cell currents were abolished by inhibition of protein kinase A with Rp-cAMPs. We conclude that degradation of cAMP by PDE3 and PDE4 contributes to regulation of renin release from JG cells. Our data provide evidence at the cellular level that stimulation of renin release by cGMP involves inhibition of PDE3 resulting in enhanced cAMP formation and activation of the cAMP sensitive protein kinase.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 4; Isoproterenol; Isoquinolines; Juxtaglomerular Apparatus; Membrane Potentials; Mice; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Renin; Tetrahydroisoquinolines; Thionucleotides

Cellular export of cyclic nucleotides has been observed in various tissues and may represent an elimination pathway for these signaling molecules, in addition to degradation by phosphodiesterases. In the present study we provide evidence that this export is mediated by the multidrug resistance protein isoform MRP5 (gene symbol ABCC5). The transport function of MRP5 was studied in V79 hamster lung fibroblasts transfected with a human MRP5 cDNA. An MRP5-specific antibody detected an overexpression of the glycoprotein of 185 +/- 15 kDa in membranes from MRP5-transfected cells and a low basal expression of hamster Mrp5 in control membranes. ATP-dependent transport of 3',5'-cyclic GMP at a substrate concentration of 1 micrometer was 4-fold higher in membrane vesicles from MRP5-transfected cells than in control membranes. This transport was saturable with a K(m) value of 2.1 micrometer. MRP5-mediated transport was also detected for 3',5'-cyclic AMP at a lower affinity, with a K(m) value of 379 micrometer. A potent inhibition of MRP5-mediated transport was observed by several compounds, known as phosphodiesterase modulators, including trequinsin, with a K(i) of 240 nm, and sildenafil, with a K(i) value of 267 nm. Thus, cyclic nucleotides are physiological substrates for MRP5; moreover, MRP5 may represent a novel pharmacological target for the enhancement of tissue levels of cGMP.

Topics: Adenosine Triphosphate; ATP-Binding Cassette Transporters; Biological Transport; Cloning, Molecular; Cyclic AMP; Cyclic GMP; Glucuronates; Glutathione; Humans; Isoquinolines; Multidrug Resistance-Associated Proteins; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Piperazines; Purines; Recombinant Proteins; Sildenafil Citrate; Substrate Specificity; Sulfones; Tetrahydroisoquinolines

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

Natural killer (NK) cells are lymphocytes that are capable of destroying tumor cells and virally infected cells (cytolysis) without prior sensitization. When cyclic AMP (cAMP) is elevated artificially in NK cells, it is a potent inhibitor of their cytolytic function. Recently, we have shown that when NK cells are exposed to a range of lysis-sensitive (LS) tumor target cells, there is an increase in intracellular cAMP levels in the NK cells over a 60-min period. There is no increase in NK-cell cAMP in response to lysis-resistant (LR) tumor target cells. We determined that this cAMP elevation is due, in part, to an LS target-induced activation of adenylyl cyclase (AC), and that the AC-activation component appears to require a protein tyrosine kinase (PTK) activity. In the present study, we demonstrated that an LS target-induced inhibition of phosphodiesterase (PDE) is also contributing to the overall elevation of cAMP. Direct measurement of PDE activity showed an inhibition in lymphocytes that were exposed to LS targets but not in those exposed to LR targets. The inhibition of PDE activity was maximal by 30 min. Lymphocytes were exposed to targets and then lysed, so that PDE activity could be measured. Addition of class-selective inhibitors of PDE (at levels sufficient to completely block that class of PDE) to the lysate focused the measurement of PDE activity on those classes of PDE that were unaffected by the selective inhibitor. Using the PDE IV selective inhibitor rolipram and the PDE III selective inhibitors trequinsin and milrinone, we showed that a PDE III is being inhibited in lymphocytes by exposure to LS targets. As PDE III is known to be inhibited by elevated cyclic GMP (cGMP) levels, increased cGMP in NK cells following exposure to LS targets was a possible mechanism by which a PDE III in NK cells might be inhibited. However, when we measured cGMP levels in control and LS target-stimulated lymphocytes, we saw no change.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 4; Humans; Isoquinolines; K562 Cells; Killer Cells, Natural; Milrinone; Phosphodiesterase Inhibitors; Platelet Aggregation Inhibitors; Rolipram; Tetrahydroisoquinolines

Nitric oxide (NO) donors increase heart rate (HR) through a guanylyl cyclase-dependent stimulation of the pacemaker current I(f), without affecting basal I(Ca-L). The activity of I(f)is known to be enhanced by cyclic nucleotides and by an increase in cytosolic Ca(2+). We examined the role of cGMP-dependent signaling pathways and intracellular Ca(2+)stores in mediating the positive chronotropic effect of NO donors. In isolated guinea pig atria, the increase in HR in response to 1-100 micromol/l 3-morpholino-sydnonimine (SIN-1; with superoxide dismutase, n=6) or diethylamine-NO (DEA-NO, n=8) was significantly attenuated by blockers of the cGMP-inhibited phosphodiesterase (PDE3; trequinsin, milrinone or Ro-13-6438, n=22). In addition, the rate response to DEA-NO or sodium nitroprusside (SNP) was significantly reduced following inhibition of PKA (KT5720 or H-89, n=15) but not PKG (KT5728 or Rp-8-pCPT-cGMPs, n=16). Suppression of sarcoplasmic (SR) Ca(2+)release by pretreatment of isolated atria with ryanodine or cyclopiazonic acid (2 micromol/l and 60 micromol/l, n=16) significantly reduced the chronotropic response to 1-100 micromol/l SIN-1 or DEA-NO. Moreover, in isolated guinea pig sinoatrial node cells 5 micromol/l SNP significantly increased diastolic and peak Ca(2+)fluorescence (+13+/-1% and +28+/-1%, n=6, P<0.05). Our findings are consistent with a functionally significant role of cAMP/PKA signaling (via cGMP inhibition of PDE3) and SR Ca(2+)in mediating the positive chronotropic effect of NO donors.

Topics: Animals; Calcium; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guinea Pigs; Heart Atria; Heart Rate; Hydrazines; Indoles; Isoquinolines; Male; Milrinone; Models, Biological; Molsidomine; Nitric Oxide Donors; Nitrogen Oxides; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Platelet Aggregation Inhibitors; Pyrroles; Quinazolines; Ryanodine; Sarcoplasmic Reticulum; Signal Transduction; Spectrometry, Fluorescence; Sulfonamides; Superoxide Dismutase; Tetrahydroisoquinolines; Thionucleotides; Time Factors; Vasodilator Agents

Bovine aortic endothelial cells contain cyclic GMP-stimulated phosphodiesterase (PDE) regulating intracellular cyclic AMP and cyclic GMP levels. To investigate the roles of this PDE isoform for cyclic AMP hydrolysis in intact endothelial cells (EC), we used an adenine prelabeling method to determine cyclic AMP accumulation in response to agents that might produce effects that increase cyclic GMP levels. Atrial natriuretic peptide (ANP), which dramatically increased cyclic GMP accumulation, reduced cyclic AMP in cultured EC from bovine aorta with or without addition of L-isoproterenol (L-ISO), whereas sodium nitroprusside (SNP) and 8-bromo cyclic GMP had no effect. The reduction in cyclic AMP by ANP was dose dependent (> or = 1.0 nM) and rapid (significant reduction was induced in < or = 15 s) and was abolished when EC were preincubated with 3-isobutyl-1-methylxanthine (IBMX) and HL-725, both nonselective PDE inhibitors. ANP had no effects on adenylate cyclase activity, nor any direct effects on the activities of partially purified cyclic GMP-stimulated PDE isoform or cyclic AMP-specific isoform. However, cyclic AMP hydrolyzing activities of EC were enhanced when EC were pretreated with 0.1 microM ANP. ANP activates cyclic GMP-stimulated PDE and induces reduction of cyclic AMP accumulation in intact EC, which may modify cyclic GMP-dependent endothelial function involved in ANP.

Topics: 1-Methyl-3-isobutylxanthine; Adenine; Adenylyl Cyclases; Animals; Aorta; Atrial Natriuretic Factor; Cattle; Cells, Cultured; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Drug Interactions; Endothelium, Vascular; Isoenzymes; Isoproterenol; Isoquinolines; Nitroprusside; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Tetrahydroisoquinolines

We have created a series of deletion mutants of a human cardiac cAMP phosphodiesterase in order to define sequences necessary for function and to identify residues required for inhibition by cGMP and by the drugs milrinone and trequinsin. These truncated constructs were expressed in yeast cells, and their biochemical properties were analyzed. The mutations define an amino acid sequence that is essential for function. Among the active constructs, there was considerable variability in the level of expression and in the stability of the proteins, with the full-length and near full-length constructs being the least stable. There were, however, no significant changes in Km values among the active enzymes. Cation studies confirmed that Mn2+ is a more efficient cofactor than Mg2+ or Co2+. Interestingly, Mn2+ acts as a more efficient cofactor for cGMP inhibition as well. Although IC50 values for the drugs trequinsin and milrinone were not significantly altered by deletions, there was a decrease in cGMP IC50 values for the smaller constructs, indicating a role for amino acid residues outside the catalytic region in cGMP inhibition. We also demonstrate in vivo inhibition of this enzyme in yeast cells grown in the presence of pharmacological inhibitors, allowing for the selection of drug-resistant mutants. Finally, we have constructed and analyzed chimeric genes in which portions of this phosphodiesterase are replaced with homologous sequences from a closely related phosphodiesterase isozyme that is expressed in brain. Our results demonstrate that sequence variations between related isozymes account for more than just pharmacological distinctions and may reflect significant structural differences.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Amino Acid Sequence; Base Sequence; Cyclic GMP; DNA Mutational Analysis; DNA Primers; Humans; Isoquinolines; Kinetics; Milrinone; Molecular Sequence Data; Myocardium; Phosphodiesterase Inhibitors; Polymerase Chain Reaction; Pyridones; Recombinant Fusion Proteins; Recombinant Proteins; Sequence Deletion; Tetrahydroisoquinolines

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

Rolipram [(R,S)-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidone] has been shown to inhibit selectively the cAMP phosphodiesterase (PDE) of vascular smooth muscle. In order to further explore the structural requirements for selective PDE inhibition, we synthesized a series of rolipram derivatives differently substituted either at the pyrrolidinone or at the aromatic ring. Among these compounds, rolipram was the most active compound. Semirigid analogues were prepared and used for an evaluation of the active conformation of rolipram. Structural comparison with two other potent and chemically different smooth muscle cAMP-PDE inhibitors, trequinsin and Ro 20-1724, allows us to propose a first topological model of the smooth muscle cAMP-PDE pharmacophore.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Cattle; Chemical Phenomena; Chemistry; Cyclic GMP; Hydrolysis; Imidazoles; In Vitro Techniques; Isoquinolines; Molecular Conformation; Muscle, Smooth, Vascular; Phosphodiesterase Inhibitors; Pyrrolidinones; Rolipram; Tetrahydroisoquinolines

Recent observations imply the involvement of an endothelium-derived relaxing factor (EDRF) in the vasodilation of isolated vascular preparations accompanied by an increase of cyclic guanosine 3',5'-monophosphate (cGMP). To investigate the changes of cGMP and cyclic adenosine 3',5'-monophosphate (cAMP) in endothelium-dependent relaxation of isolated rabbit thoracic aortic rings we used colforsin (forskolin, FOR) as an adenylate cyclase stimulator, trequinsin (TRE) as a phosphodiesterase inhibitor and isoprenaline (ISO) as a beta-adrenoceptor agonist. Norepinephrine (NE, 10(-8) mol/l) evoked a contractile response in intact rings of rabbit aorta. In these precontracted rings with endothelium, acetylcholine (ACh) induced a concentration-dependent relaxation at 10(-8)-10(-6) mol/l. FOR, TRE and ISO reduced NE-vasoconstrictor responses in a concentration-dependent manner with an IC50 of 4.1 x 10(-8) mol/l, 8.5 x 10(-7) mol/l and 4.0 x 10(-7) mol/l, respectively, in rabbit aortic rings with endothelium. These effects were associated with elevations (p less than 0.05) in cAMP and cGMP in vascular tissue. In segments with disrupted endothelium the IC50 for FOR and TRE were increased about 3.5- and 2.3-fold, without changes in cyclic nucleotides. All three compounds attenuated ACh-induced relaxations of aortic rings in a concentration-dependent manner. High concentrations of FOR (10(-7) mol/l) and TRE (10(-5)) which increased cAMP even reversed ACh-induced relaxations, comparable to ACh effects in de-endothelialized vascular tissue. It is suggested that FOR-, TRE- and ISO-induced relaxations of isolated aortic preparations, accompanied by increased cAMP, interact with EDRF-dependent relaxations.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Colforsin; Cyclic AMP; Cyclic GMP; Female; In Vitro Techniques; Isoproterenol; Isoquinolines; Male; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Platelet Aggregation Inhibitors; Rabbits; Tetrahydroisoquinolines

Low-density lipoproteins activate isolated human platelets. The mechanism of this activation is unknown, but may involve increased phosphoinositide turnover. We have examined the effect of low-density lipoproteins on intracellular calcium concentrations in platelets loaded with the photoprotein aequorin. The lipoproteins induced concentration-dependent increases in intracellular calcium, associated with shape change and aggregation. These responses could be partially inhibited by the removal of extracellular calcium and by pre-incubation with acetylsalicylic acid. They were also antagonised by agents which increase cellular concentrations of cyclic adenosine and guanosine monophosphates. It is not clear whether the platelet-lipoprotein interaction involves a 'classical' lipoprotein receptor.

Topics: Aequorin; Aspirin; Blood Platelets; Calcium; Cyclic GMP; Humans; Isoquinolines; Lipoproteins, LDL; Luminescence; Luminescent Proteins; Platelet Aggregation; Platelet Aggregation Inhibitors; Tetrahydroisoquinolines