sildenafil-citrate 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

Persistent pulmonary hypertension of the newborn (PPHN) is a complex syndrome with multiple causes, with an incidence of 0.43-6.8/1,000 live births and a mortality of 10-20%. Survivors have high morbidity in the forms of neurodevelopmental and audiological impairment, cognitive delays, hearing loss, and a high rate of rehospitalization. The optimal approach to the management of PPHN remains controversial. Inhaled nitric oxide (iNO) is currently regarded as the gold standard therapy, but with as many as 30% of cases failing to respond, has not proven to be the single magic bullet. Given the complex pathophysiology of the disease, any such magic bullet is unlikely. A number of recent studies have suggested a role for specific phosphodiesterase (PDE) inhibitors in the management of PPHN. Sildenafil, a specific PDE5 inhibitor, appears the most promising of such agents. We aim to review the current status and limitations of iNO and the potential of PDE inhibitors in the management of PPHN. The reasons why caution is warranted before specific PDE5 inhibitors like sildenafil are labelled as potential magic bullets for PPHN will be discussed. The need for randomized-controlled trials to determine the safety, efficacy, and long-term outcome following treatment with sildenafil in PPHN is emphasized.

Topics: Dipyridamole; Endothelium-Dependent Relaxing Factors; Humans; Infant, Newborn; Nitric Oxide; Pentoxifylline; Persistent Fetal Circulation Syndrome; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Purines; Purinones; Sildenafil Citrate; Sulfones

Phosphodiesterase 5 terminates the cellular actions of the second messenger molecule cyclic GMP; inhibitors of phosphodiesterase 5 will therefore increase and prolong the actions of endogenous substances that signal via the cyclic GMP pathway, including nitric oxide released as a neurotransmitter from nitrergic nerves. To date, the most widely used phosphodiesterase 5 inhibitors, zaprinast and sildenafil, have proved vital in the elucidation of the widespread role of cyclic GMP in nitrergic transmission and, specifically in the case of sildenafil, have provided a major breakthrough in the treatment of erectile dysfunction in men. Although still a matter of debate, early evidence indicates that sildenafil may also be of benefit in some forms of sexual dysfunction in women. The remarkable clinical success of sildenafil has prompted the search for further novel phosphodiesterase 5 inhibitors which might be used to enhance nitrergic function in other disease states.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic Nucleotide Phosphodiesterases, Type 5; Humans; Nitric Oxide; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Purines; Purinones; Sildenafil Citrate; Sulfones; Synaptic Transmission

We recently reported that administration of the non-selective cyclic GMP-phosphodiesterase (cGMP-PDE) inhibitor zaprinast to cortically cryoinjured rats results three days post-lesion in reduced neuronal cell death that was associated to decreased macrophage/microglial activation and oxidative stress and increased astrogliosis and angiogenesis. Similar effects have been observed in cryoinjured animals overexpressing metallothioneins I/II (MT-I/II), metal-binding cysteine-rich proteins that are up-regulated in response to injury. In this work we have examined the effect of administration of the selective PDE5 inhibitor sildenafil (10mg/kg, sc) 2h before and 24 and 48h after induction of cortical cryolesion in wild-type and MT-I/II-deficient mice. Our results show that in wild-type animals sildenafil induces similar changes in glial reactivity, angiogenesis and antioxidant and antiapoptotic effects in the cryolesioned cortex as those observed in rats with zaprinast, indicating that inhibition of PDE5 is responsible for the neuroprotective actions. However, these effects were not observed in mice deficient in MT-I/II. We further show that sildenafil significantly increases MT-I/II protein levels in homogenates of lesioned cortex and MT-I/II immunostaining in glial cells around the lesion. Taken together these results indicate that cGMP-mediated pathways regulate expression of MT-I/II and support the involvement of these proteins in the neuroprotective effects of sildenafil in focal brain lesion.

Topics: Animals; Apoptosis; Blotting, Western; Brain Injuries; Cerebral Cortex; Cold Temperature; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme-Linked Immunosorbent Assay; Gliosis; Immunohistochemistry; Macrophage Activation; Metallothionein; Mice; Mice, Knockout; Microglia; Neovascularization, Physiologic; Neuroprotective Agents; Oxidative Stress; Phosphodiesterase Inhibitors; Piperazines; Purines; Purinones; Sildenafil Citrate; Sulfones; Up-Regulation

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

Xanthine oxidase (XO) plays an important role in various forms of ischemic and vascular injuries, inflammatory diseases and chronic heart failure. The XO inhibitors allopurinol and oxypurinol held considerable promise in the treatment of these conditions both in experimental animals and in human clinical trials. More recently, an endothelium-based protective effect of sildenafil has been reported in preconditioning prior to ischemia/reperfusion in healthy human subjects. Based on the structural similarities between allopurinol and oxypurinol with sildenafil and with zaprinast the authors have investigated the potential effects of these latter compounds on the buttermilk XO and on non-tumourigenic (HMEC) and malignant (MCF7) human mammary epithelial cells. Both sildenafil and zaprinast induced a significant and consistent decrease of XO expression and activity in either cell line. In MCF7 cells only, this effect was associated with the abrogation of xanthine-induced cytotoxicity. Overall, the data suggest that the protective effect of sildenafil on epithelial cells is a consequence of the inhibition of the XO and of the resulting decrease of free oxygen radical production that may influence the expression of NADPH oxidase and PDE-5.

Topics: Cell Survival; Epithelial Cells; Humans; Piperazines; Purines; Purinones; Reactive Oxygen Species; Sildenafil Citrate; Structure-Activity Relationship; Sulfones; Tumor Cells, Cultured; Xanthine Oxidase

Cyanoacetamides 3 were prepared via reacting ethyl cyanoacetate with benzylamine. Yields and reaction times needed for reaction completion at room temperature, by microwaves (muomega) heating and under ultrasound (US) irradiations are compared. The formed cyanoacetamides were coupled with aromatic diazonium salts and the formed arylhydrazones were used as precursors to title triazoles and pyrazoles via reacting the former with hydroxylamine and chloroacetonitrile. Yields of products formed via conventional heating are compared with those of muomega and US irradiation.

Topics: Green Chemistry Technology; Hot Temperature; Microwaves; Nitriles; Piperazines; Purines; Purinones; Pyrazoles; Sildenafil Citrate; Sulfones; Triazoles; Ultrasonics

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

Inhalation of nitric oxide (NO) and inhibition of phosphodiesterase type 5 (PDE5) selectively dilate the pulmonary circulation in patients with acute lung injury (ALI) associated with pulmonary hypertension. PDE5 inhibitors administered at doses that decrease pulmonary artery pressures have been shown to worsen arterial oxygenation. We investigated the efficacy of doses of PDE5 inhibitors that do not reduce pulmonary artery pressure alone (subthreshold doses) to improve the response to inhaled NO in an animal model of ALI.. Adult Sprague-Dawley rats were pre-treated with 0.5 mg/kg Escherichia coli 0111:B4 endotoxin and 16 to 18 h later, their lungs were isolated perfused and ventilated. The thromboxane mimetic U46619 was used to induce pulmonary hypertension. After the determination of subthreshold doses of two different PDE5 inhibitors, either 50 microg zaprinast or 10 ng sildenafil was added to the perfusate and the decrease of pulmonary artery pressure measured in the presence and absence of inhaled NO.. In the presence of 4 or 10 ppm NO, zaprinast (-1.6 +/- 0.4 and -2.9 +/- 0.6 mmHg, respectively) and sildenafil (-1.9 +/- 0.4 and -2.4 + 0.3 mmHg, respectively) improved responsiveness to inhaled NO compared to lungs from rats treated with LPS only (0.7 +/- 0.1 and -1.0 +/- 0.1 mmHg, respectively; P<0.05). Neither zaprinast nor sildenafil prolonged the pulmonary vasodilatory response to inhaled NO.. Subthreshold doses of PDE5 inhibitors improved responsiveness to inhaled NO. Combining inhaled NO with subthreshold doses of PDE5 inhibitors may offer a therapeutic strategy with minimal side-effects in ALI associated with pulmonary hypertension.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Administration, Inhalation; Animals; Bronchodilator Agents; Cyclic Nucleotide Phosphodiesterases, Type 5; Dose-Response Relationship, Drug; Drug Synergism; Endotoxemia; In Vitro Techniques; Nitric Oxide; Phosphodiesterase Inhibitors; Piperazines; Pulmonary Circulation; Pulmonary Wedge Pressure; Purines; Purinones; Rats; Respiratory Distress Syndrome; Sildenafil Citrate; Sulfones; Vasodilation

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

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

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

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

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

Erectile dysfunction is common in men with chronic renal failure. Previously nitrergic and endothelium-dependent relaxation responses have been shown to be reduced in chronic renal failure rabbits. We have therefore investigated the efficacy of phosphodiesterase inhibitors on the corpora cavernosa obtained from uremic rabbits. Uremia was induced with 5/6 nephrectomy and 4 weeks later cavernosal tissue strips were isolated. The relaxant effect of phosphodiesterase 5 inhibitors, zaprinast (1-300 microM) and sildenafil (0.01-300 microM), phosphodiesterase 3 inhibitor amrinone (1-100 microM) and non-specific phosphodiesterase inhibitor papaverine (1-300 microM) were investigated on phenylephrine (10 microM)-induced tone. We found a shift in the dose-response curve of only phosphodiesterase 5 inhibitors. These results suggest that the decreased production or availability of endogenous nitric oxide in chronic renal failure animals leads to decreased efficacy of phosphodiesterase 5 inhibitors to induce relaxation.

Topics: Amrinone; Animals; Body Weight; Calcium; Creatinine; Dose-Response Relationship, Drug; Electric Stimulation; In Vitro Techniques; Isoenzymes; Kidney Failure, Chronic; Male; Muscle Relaxation; Nephrectomy; Papaverine; Parathyroid Hormone; Penis; Phosphates; Phosphodiesterase Inhibitors; Phospholipases; Piperazines; Purines; Purinones; Rabbits; Sildenafil Citrate; Sulfones; Testosterone

Here we report that the active component of Viagra, Sildenafil and the first metabolite, N-desmethyl-sildenafil (UK-103, 320) increased the amplitude of flash-evoked electroretinogram (ERG) of dark-adapted albino rat retina. Effects of Sildenafil and N-desmethyl-sildenafil were comparable to those of the known phosphodiesterase inhibitor, Zaprinast. The photoreceptor cell response was isolated by blocking the glial K(+) ion-buffering and the on-bipolar components of the ERG with the use of BaCl(2) (500 microM) and the specific type VI metabotropic glutamate receptor agonist, DL-2-amino-4-phosphonobutyric acid (25 microM), respectively. Zaprinast, Sildenafil and N-desmethyl-sildenafil (1 microM each) increased the amplitude of photoreceptor cell response either. Besides, Sildenafil was significantly more effective than N-desmethyl-sildenafil. These findings suggest an increased sensitivity of photoreceptor cells in the presence of Sildenafil and it is metabolite.

Topics: Animals; In Vitro Techniques; Male; Phosphodiesterase Inhibitors; Photoreceptor Cells, Vertebrate; Piperazines; Purines; Purinones; Rats; Rats, Wistar; Retina; Sildenafil Citrate; Sulfones

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 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

Zaprinast, dipyridamole and sildenafil were injected into the corpora cavernosa of cats to determine whether changes in the steady state level of cyclic guanosine monophosphate (cGMP) induced by inhibiting type 5 phosphodiesterase would cause an erectile response.. Increases in intracavernous pressure, penile length and erectile response duration were determined after intracavernous injection of the type 5 cGMP specific phosphodiesterase inhibitors zaprinast, dipyridamole and sildenafil as well as combined zaprinast and prostaglandin E1 (PGE1), and zaprinast and sodium nitroprusside. Systemic arterial pressure was concurrently assessed in these experiments. All responses to phosphodiesterase inhibitors were compared to a control triple drug combination of 1.65 mg papaverine, 0.5 microg PGE1 and 25 microg phentolamine.. Each selective type 5 phosphodiesterase inhibitor caused dose related increases in intracorporeal pressure and penile length. However, none of the compounds was as effective as the control drug combination of papaverine, phentolamine and PGE1. Combining zaprinast with sodium nitroprusside led to further increases in pressure and erectile response duration that more closely resembled the control drug response. Combining zaprinast with PGE1 led to a response that was indistinguishable from the control response.. The results of these feline studies establish that administering a type 5 phosphodiesterase inhibitor without concomitant administration of a nitric oxide donor or stimulation of the cavernous nerves may have a direct effect on the erectile response. These data also suggest that combining a selective type 5 phosphodiesterase inhibitor with PGE1 may be highly effective local therapy for erectile dysfunction and an acceptable alternative to other current forms of treatment.

Topics: Alprostadil; Animals; Cats; Dipyridamole; Dose-Response Relationship, Drug; Male; Nitroprusside; Penile Erection; Phosphodiesterase Inhibitors; Piperazines; Purines; Purinones; Sildenafil Citrate; Sulfones; Vasodilator Agents

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

Human, dog and rabbit corpus cavernosum type 5 phosphodiesterases (PDE5) were isolated and their characteristics were compared. The three enzymes showed Km values of 0.8, 2.1 and 2.3 uM, respectively. They exhibited similar pH-dependence with optimal pH being 7.5. They required Mg++ for activity and the activity was suppressed by high concentrations of Zn++ (0.1-1 mM). Sildenafil potently inhibited the three enzymes with IC50 values of 3.6, 1.7 and 3.0 nM, respectively. Dipyridamole and IBMX (3-isobutyl-1-methylxanthine) each also inhibited the three enzymes with similar, albeit lower, potencies (IC50 about 1.1 and 5.7 uM, respectively). However, zaprinast exhibited a significantly higher potency against the rabbit enzyme (IC50 53 nM) than against the human and dog PDE5s (IC5s 332 and 217 nM, respectively). Thus, the corpus cavernosum PDE5s are very similar among the various species with the only significant difference being their sensitivity to zaprinast. Human platelet PDE5 was also characterized by comparison with the corpus cavernosum enzyme. The platelet enzyme exhibited a Km, pH-, Mg++- and Zn++-dependence, and sensitivity to sildenafil and zaprinast very similar to those of the corpus cavernosum PDE5. However, compared with corpus cavernosum PDE5, the platelet enzyme exhibited higher sensitivity to dipyridamole and IBMX (IC50 0.46 and 1.8 uM, respectively). This study shows that despite similar kinetics and enzymatic properties, corpus cavernosum PDE5s from different species, and corpus cavernosum and platelet PDE5s, can have differential sensitivity to pharmacological inhibitors.

Topics: 1-Methyl-3-isobutylxanthine; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Blood Platelets; Chromatography; Cyclic Nucleotide Phosphodiesterases, Type 5; Dipyridamole; Dogs; Humans; Isoenzymes; Kinetics; Male; Muscle, Smooth; Penis; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Purines; Purinones; Rabbits; Sildenafil Citrate; Species Specificity; Sulfones

We have produced a polyclonal antibody that specifically recognizes cGMP-binding cGMP-specific phosphodiesterase (PDE5). The antibody was raised in rabbit using as immunogen a fusion protein, in which glutathione S-transferase was coupled to a 171 amino acid polypeptide of the N-terminal region of bovine PDE5. The antibody is able to immunoprecipitate PDE5 activity from mouse tissues and neuroblastoma extracts while it has no effect on all other PDE isoforms present in the extracts. PDE5 activity recovered in the immunoprecipitates retains its sensitivity to specific inhibitors such as zaprinast (IC(50)=0.6 microM) and sildenafil (IC(50)=3.5 nM). Bands of the expected molecular mass were revealed when solubilized immunoprecipitates were analysed in Western blots. The antibody selectively stained cerebellar Purkinje neurones, which are known to express high levels of PDE5 mRNA. Western blot analysis of mouse tissues revealed the highest expression signal in mouse lung, followed by heart and cerebellum, while a lower signal was evident in brain, kidney and a very low signal was present in the liver. In the hybrid neuroblastoma-glioma NG108-15 cells the antibody revealed a high PDE5 induction after dibutyryl-cAMP treatment.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Antibody Specificity; Blotting, Western; Brain; Cyclic Nucleotide Phosphodiesterases, Type 5; Immune Sera; Immunohistochemistry; Liver; Lung; Mice; Myocardium; Phosphodiesterase Inhibitors; Piperazines; Precipitin Tests; Purines; Purinones; Sildenafil Citrate; Sulfones; Tissue Extracts; Tumor Cells, Cultured

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

Phosphodiesterase type 5 (PDE5) inhibitors with improved PDE isozyme selectivity relative to sildenafil may result in agents for the treatment of male erectile dysfunction (MED) with a lower incidence of PDE-associated adverse effects. This paper describes the discovery of 14, a PDE5 inhibitor with improved potency and selectivity in vitro compared to sildenafil. This compound shows activity in a functional assay of erectile function comparable to that of sildenafil.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic Nucleotide Phosphodiesterases, Type 5; Erectile Dysfunction; Imidazoles; In Vitro Techniques; Male; Penis; Phosphodiesterase Inhibitors; Piperazines; Purines; Purinones; Quinazolines; Rabbits; Sildenafil Citrate; Structure-Activity Relationship; Sulfones

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

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

Sildenafil is currently used in the treatment of erectile dysfunction. However, assessment of direct effects of sildenafil on coronary arteries and on arteries used as coronary grafts is unknown. This study was designed to investigate the effects of sildenafil on contracted human coronary, internal mammary, and radial arteries obtained from multiorgan donors. The observations were extended to forearm veins. Zaprinast was included in this study for comparison.. Segments of left coronary, internal mammary, and radial arteries, and forearm veins were obtained from 16 multiorgan donors. Vascular rings were suspended in organ bath chambers and isometric tension was recorded. Then the effects of sildenafil, zaprinast, and sodium nitroprusside on precontracted vessels were studied.. Sildenafil (10(-8) - 3 x 10(-5) mol/L) caused concentration-dependent relaxation in the internal mammary arteries, radial arteries, and forearm veins. In the coronary arteries, sildenafil had a modest relaxant effect. In addition, sildenafil amplified the relaxation induced by sodium nitroprusside in all four vessels. Relaxation was unaffected by the inhibitor of nitric oxide synthase NG-monomethyl-L-arginine (10(-4) mol/L). Compared with zaprinast, sildenafil was eight to ten times more potent in terms of EC50 values.. The direct relaxant effects of sildenafil together with its synergistic interaction with nitric oxide donors should be considered in patients undergoing coronary bypass surgery, patients with low blood pressure, and patients receiving antihypertensive regimes.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Coronary Vessels; Dose-Response Relationship, Drug; Humans; Mammary Arteries; Muscle, Smooth, Vascular; Phosphodiesterase Inhibitors; Piperazines; Purines; Purinones; Radial Artery; Sildenafil Citrate; Sulfones; Vasodilation; Veins

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

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