Inhibitory Effect Of Sildenafil Research Articles

Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ‑mediated downregulation of TRPC.

Persistent pulmonary hypertension of the newborn (PPHN) is a common pulmonary vascular disease during the neonatal period, and it is associated with a high clinical mortality rate and a poor prognosis. At present, the treatment of PPHN is based mainly on inhaled nitric oxide (iNO), high-frequency ventilation, and pulmonary vasodilators. Sildenafil has gradually begun to be used in recent years for the treatment of PPHN and has exhibited some success; however, its detailed mechanism of action requires further elucidation. An animal model of neonatal pulmonary hypertension (neonatal rats, 48 h after birth, 10% O2, 14 days) as well as a cell model [human pulmonary artery smooth muscle cells (PASMCs), 4% O2, 60 h] were established. The effects of sildenafil on pulmonary hypertension in neonatal rats were evaluated by hematoxylin and eosin staining, immunofluorescence analysis, western blotting and PCR, and the changes in peroxisome proliferator-activated receptor γ (PPARγ), transient receptor potential canonical (TRPC)1, TRPC6 and Ki67 expression levels were detected under hypoxic conditions. The results revealed that sildenafil reversed the increases in the right ventricular mean pressure and right ventricular hypertrophy index induced by hypoxia, and attenuated pulmonary arterial remodeling as well as PASMC proliferation. The inhibitory effects of sildenafil on TRPC expression and PASMC proliferation were attenuated by GW9662 and PPARγ small interfering RNA. In conclusion, sildenafil protects against hypoxia-induced pulmonary hypertension and right ventricular hypertrophy in neonatal rats by upregulating PPARγ expression and downregulating TRPC1 and TRPC6 expression.

Sildenafil drug as a safe anticorrosion for 6063 aluminum alloy in acidic and alkaline solutions: Theoretical and experimental studies

The corrosion inhibition of aluminum alloy surface (6063AA) by Sildenafil (SIL) in 1 M for both HCl and NaOH solutions was investigated using weight loss, electrochemical techniques and quantum calculations. The inhibitory effect of SIL was explained by the formation of the complex adsorbed on the 6063AA surface. The adsorption obeyed Langmuir isotherm. The adsorption of the SIL is a mix of physical and chemical adsorption, where the free energy of adsorption was found to equal −39.12 and −34.6 KJ/mol, in HCl and NaOH, respectively. Experimental outcomes showed that SIL significantly inhibits the dissolution of 6063Al in both acidic and alkaline media. SIL drug acts as a mixed inhibitor in both two aggressive solutions. The correlation of the inhibition performance of SIL on 6063Al with the molecular structure was adopted using Dmol3 (Materials Studio v7. 0) based on some descriptors as EHOMO and ELUMO. Fukui indices were computed to determine the active sites of the inhibitor molecules. The equilibrium adsorption behavior of SIL on Al (111) surface was examined using molecular dynamics simulation. The quantum chemical descriptors were found suitable in explaining the inhibitive action of SIL.

Inhibitory effects of sildenafil and tadalafil on inflammation, oxidative stress and nitrosative stress in animal model of bronchial asthma.

Cyclic neucleotides are involved in many cellular functions including smooth muscle relaxation, inflammation, and signal transduction. Sildenafil and tadalafil are phosphodiesterase-5 (PDE-5) inhibitors which prevent the degradation of cyclic neucleotide i.e. guanosine 3',5' cyclic monophosphate (cGMP) and increase the levels of cGMP. In this study sildenafil and tadalafil were evaluated for their anti-inflammatory, anti-oxidative and anti-nitrosative stress potential in animal model of bronchial asthma. Wistar rats were sensitized with 10 mg intraperitoneal (ip) ovalbumin adsorbed to 10 μg of aluminum hydroxide on day 0. Animals were given sildenafil (1 and 3 mg/kg ip) and tadalafil (1 and 3 mg/kg ip) from day 1 to day 14. Also, on day 14 animals were challenged with ovalbumin (1 mg ip). After 24 h, samples were collected to analyze interleukin-4 (IL-4) and tumour necrosis factor-α (TNF-α), in serum and bronchoalveolar lavage fluid (BALF). The oxidative stress markers malondialdehyde (MDA), reduced glutathione (GSH) and nitric oxide metabolites (NOx) were also measured in serum. Pre-treatment with sildenafil (1 and 3 mg/kg ip) and tadalafil (1 and 3 mg/kg ip) significantly reduced the levels of pro-inflammatory cytokines IL-4 and TNF-α in rat serum and BALF. In addition, pre-treatment with both the drugs decreased the levels of MDA and NOx and increased the levels of GSH in serum. Sildenafil and tadalafil decreased pro-inflammatory cytokines in serum and BALF. Both drugs inhibit oxidative and nitrosative stress in animal model of bronchial asthma and could have a therapeutic potential in bronchial asthma.

Sildenafil protects neuronal cells from mitochondrial toxicity induced by β-amyloid peptide via ATP-sensitive K+ channels

To understand the molecular mechanisms underlying the beneficial effects of sildenafil in animal models of neurological disorders, we investigated the effects of sildenafil on the mitochondrial toxicity induced by β-amyloid (Aβ) peptide. Treatment of HT-22 hippocampal neuronal cells with Aβ25∼35 results in increased mitochondrial Ca2+ load, which is subsequently suppressed by sildenafil as well as by diazoxide, a selective opener of the ATP-sensitive K+ channels (KATP). However, the suppressive effects of sildenafil and diazoxide are significantly attenuated by 5-hydroxydecanoic acid (5-HD), a KATP inhibitor. The increased mitochondrial Ca2+ overload is accompanied by decrease in the intracellular ATP concentration, increase in intracellular ROS generation, occurrence of mitochondrial permeability transition, and activation of caspase-9 and cell death. Exposure to sildenafil inhibited the mitochondria-associated changes and cell death induced by Aβ. However, the inhibitory effects of sildenafil are abolished or weakened in the presence of 5-HD, suggesting that opening of the mitochondrial KATP is required for sildenafil to exert these effects. Taken together, these results indicate that at the mitochondrial levels, sildenafil plays a protective role towards neuronal cell in an environment rich in Aβ, and exerts its effects via the mitochondrial KATP channels-dependent mechanisms.

Phosphodiesterase Inhibitors Sildenafil and Vardenafil Reduce Zebrafish Rod Photoreceptor Outer Segment Shedding.

PurposeThe vertebrate rod photoreceptor undergoes daily growth and shedding to renew the rod outer segment (ROS), a modified cilium that contains the phototransduction machinery. It has been demonstrated that ROS shedding is regulated by the light–dark cycle; however, we do not yet have a satisfactory understanding of the molecular mechanisms that underlie this regulation. Given that phototransduction relies on the hydrolysis of cGMP via phosphodiesterase 6 (PDE6), we examined ROS growth and shedding in zebrafish treated with cGMP-specific PDE inhibitors.MethodsWe used transgenic zebrafish that express an inducible, transmembrane-bound mCherry protein, which forms a stripe in the ROS following a heat shock pulse and serves as a marker of ROS renewal. Zebrafish were reared in constant darkness or treated with PDE inhibitors following heat shock. Measurements of growth and shedding were analyzed in confocal z-stacks collected from treated retinas.ResultsAs in dark-reared zebrafish, shedding was reduced in larvae and adults treated with the PDE5/6 inhibitors sildenafil and vardenafil but not with the PDE5 inhibitor tadalafil. In addition, vardenafil noticeably affected rod inner segment morphology. The inhibitory effect of sildenafil on shedding was reversible with drug removal. Finally, cones were more sensitive than rods to the toxic effects of sildenafil and vardenafil.ConclusionsWe show that pharmacologic inhibition of PDE6 mimics the inhibition of shedding by prolonged constant darkness. The data show that the influence of the light–dark cycle on ROS renewal is regulated, in part, by initiating the shedding process through activation of the phototransduction machinery.

Abstract 62: Sildenafil Inhibits Neointimal Hyperplasia After Angioplasty, and Reduces Platelet Aggregation

Background: Restenosis and stent thrombosis after stent implantation are one of the devating issues in interventional cardiology. Sildenafil has shown its effect in reducing cardiac hypertrophy as well as improving erectile dysfunction through protein kinase G (PKG) activation. Some studies have demonstrated that PKG occupies a central switching role in modulating vascular smooth muscle cell (VSMC) phenotype in response to vascular injury. In this study, we investigated the effects of PKG activation by sidenafil on platelet aggregation and neointimal hyperplasia. Methods and Results: In terms of restenosis after vascular injury, sildenafil significantly reduced neointimal hyperplasia in rat carotid arteries compared to control group. This effect of sildenafil was accompanied by the reduction of viability, cell cycle progression, and migration of VSMCs. This was also confirmed in the injured arteries in vivo. Further studies showed that the increased PKG activity by sildenafil inhibited PDGF-stimulated phenotype change of VSMCs from a contractile to a synthetic form. Conversely, the use of PKG inhibitor or gene transfer of dominant-negative PKG reversed the effects of sildenafil, resulting in the increased viability of VSMCs and neointimal formation. In addition, the mice treated with sidenafil showed the facilitated re-endothelialization, compared to control group. Furthermore, we confirmed the effect of sidenafil through PKG activation using cGK-KO mice. Interestingly, sildenafil significantly inhibited platelet aggregation induced by ADP or thrombin. This effect was reversed by PKG inhibitor, suggesting that sildenafil inhibits platelet aggregation via PKG pathway. Furthermore, assays for VASP phosphorylation and P-selectin activation showed the same inhibitory effect of sildenafil on platelet activation. Conclusions: This study showed that sildenafil inhibits not only platelet aggregation, but also neointimal hyperplasia through the PKG pathway. These findings suggest that sildenafil could be a promising candidate drug of drug-eluting stents for the prevention of restenosis without other complications such as stent thrombosis.

Inhibition of phosphodiesterase-5 suppresses calcineurin/NFAT- mediated TRPC6 expression in pulmonary artery smooth muscle cells

The up-regulation of transient receptor potential channel 6 (TRPC6) has been found to contribute to the proliferation of pulmonary artery smooth muscle cells (PASMCs), and inhibition of phosphodiesterase-5 (PDE5) has been shown to suppress TRPC6 expression in PASMCs. However, the molecular mechanisms underlying the up-regulation of TRPC6 expression and PDE5 modulation of TRPC6 expression in PASMCs remain largely unclear. The aim of this study is to address these issues. Endothelin-1 (ET-1) dose and time-dependently up-regulated TRPC6 expression in primary cultured rat PASMCs, and this was accompanied with the activation of calcineurin and subsequent translocation of NFATc4 to the nucleus. Further study indicated that inhibition of calcineurin by cyclosporine A or knockdown of NFATc4 using small interfering RNA suppressed ET-1-induced TRPC6 up-regulation. In addition, luciferase reporter assay showed that NFATc4 directly regulated the expression of TRPC6 in PASMCs. Inhibition of PDE5 by sildenafil suppressed ET-1-induced activation of calcineurin/NFATc4 signaling pathway and consequent TRPC6 up-regulation in PASMCs, while these inhibitory effects of sildenafil were abolished by PKG inhibitor Rp-8Br-cGMPs. Taken together, our study indicates that ET-1 stimulates TRPC6 expression by activation of calcineurin/NFATc4 signaling pathway, and inhibition of PDE5 suppresses calcineurin/NFATc4- mediated TRPC6 expression in PASMCs in a cGMP-PKG-dependent manner.

Inhibitory effect of sildenafil on pyloric sphincter from streptozotocin-diabetic rats: role of no-cGMP transduction pathway

Long-standing and poorly controlled diabetes may result disturbances in gastrointestinal function of which gastroparesis is one of the more common. Previous studies have shown that nitric oxide-cyclic guanosine monophosphate transduction pathway has a regulatory role in gastrointestinal motility. But, this drive is adversely affected by diabetes mellitus. Sildenafil, as a selective inhibitor of cyclic guanosine monophosphate-selective phosphodiesterase type 5, has also beneficial effect in diabetic patients with gastrointestinal symptoms. In this study, we aimed to investigate the effect of sildenafil and its underlying mechanisms on pylorus motility of diabetic rats. Acetylcholine (10−8–10−3 M) produced concentration-dependent contraction responses that is significantly attenuated by sildenafil (10−6 M) incubation. On the other hand, sildenafil elicited relaxant effect in rat pylorus precontracted by acetylcholine. The relaxant responses to sildenafil partially, but significantly, inhibited by nitric oxide synthase blocker N-nitro-L-arginine methyl ester (L-NAME, 10−4 M) or soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10−5 M). These results demonstrated that sildenafil continue to relax diabetic rat pylorus despite a nitric oxide-cyclic guanosine monophosphate pathway inhbition. Hence, other mechanisms (calcium dynamics, acetylcholine metabolism) may also contribute to its relaxant effect. The results also suggest the potential use of sildenafil for improving gastroparesis in diabetic patients.

Inhibitory effect of sildenafil on the human isolated seminal vesicle

To investigate the effects of sildenafil on noradrenaline- and potassium-induced contractions of isolated human seminal vesicles (SVs), as premature ejaculation is a relatively common male sexual dysfunction that currently lacks an adequate therapy, and recent in vitro tests showed that sildenafil induces relaxation of rodent isolated SVs, but it is not known whether it also inhibits isolated human SV. Isolated strips of human SVs were suspended in an organ bath and cumulative concentration-response curves to either noradrenaline or KCl were constructed in the presence and absence of sildenafil to evaluate its inhibitory actions. Sildenafil (25-100 microm) induced concentration-dependent inhibitory effects on the human SV contracted by either noradrenaline or KCl. These actions of sildenafil do not therefore appear to be mediated via a competitive antagonism of alpha-adrenoceptors and are consistent with its recognized ability to inhibit phosphodiesterase-5. The present results show an important inhibitory action of sildenafil in the isolated human SV, supporting the therapeutic indication of this drug for treating premature ejaculation.

Involvement of α-receptors and potassium channels in the mechanism of action of sildenafil citrate

Modulation of the adrenergic activity and interfering with channels such as potassium channels may affect relaxation and contraction of the corpus cavernosum. Sildenafil is a selective phosphodiesterase-5 inhibitor, proven effective in treating erectile dysfunction. In this study, the effect of sildenafil citrate on alpha-receptors modulation and potassium channels was tested. The direct relaxant effect of sildenafil citrate was studied by measuring changes in isometric tension in isolated strips of rabbit corpus cavernosum and rat aortic ring precontracted with phenylephrine or KCl compared to that of diazoxide in the presence and absence of tetraethylammonium. The inhibitory effect of sildenafil on electrical field stimulation-induced contraction of rabbit corpus cavernosum and rat anococcygeus muscle was also studied compared to that of phentolamine. Muscle relaxant effect of sildenafil (1 x 10(-9)-1 x 10(-6) M on phenylephrine-precontracted rabbit corpus cavernosum strips was not attenuated by N(G)-nitro-L-arginine (3 x 10(-5) M). Cumulative addition of sildenafil (1 x 10(-9)-1 x 10(-6) M) and phentolamine (1 x 10(-9)-1 x 10(-6) M) to the organ bath dose-dependently inhibited electrical field stimulation-induced contraction of rabbit corpus cavernosum and rat anococcygeus muscle, with almost similar EC(50) values. Sildenafil (1 x 10(-7) M) also inhibited phenylephrine-induced contraction of rat aortic rings by 39.83+/-3.01%. In addition, tetraethylammonium (1 x 10(-3) M) significantly attenuated the muscle relaxant effect of sildenafil (1 x 10(-9)-1 x 10(-6) M) on phenylephrine-precontracted strips of rabbit corpus cavernosum. Sildenafil citrate is capable of producing cavernosal smooth muscle relaxation by an additional mechanism that may involve alpha-receptors and potassium channel opening.

Inhibitory effects of sildenafil on gastric motility and gastric slow waves in dogs

The aim of this study was to investigate the effects of sildenafil on gastric motility and gastric slow waves in dogs. The study was performed in healthy dogs and composed of three experiments. The first experiment was designed to study the effects of sildenafil on gastric emptying and gastric slow waves. The second experiment was used to investigate the effects of sildenafil on gastric tone. The third experiment was used to study the effects of sildenafil on postprandial antral contractions. (i) Sildenafil did not alter gastric emptying of liquid. (ii) Sildenafil had no effects on dominant frequency and percentage of normal gastric slow waves. The dominant power of gastric slow waves was, however, significantly reduced with sildenafil (P < 0.02). (iii) Fasting gastric volume with sildenafil was significantly higher than that at baseline (P < 0.0005) or in the control session (P < 0.002). However, the postprandial gastric volume was not altered with sildenafil. (iv) Sildenafil inhibited gastric antral motility. The contraction index was 338.5 +/- 39.99 at baseline and 122.5 +/- 20.3 after the injection of sildenafil (P = 0.003). Sildenafil inhibits fundic tone and antral motility but does not seem to delay gastric emptying of liquid in dogs. The amplitude but not the frequency or regularity of the gastric slow wave is inhibited by sildenafil.

Phosphodiesterase-5 Isoforms: Differential Cyclic Guanyl Monophosphate Binding and Cyclic Guanyl Monophosphate Catalytic Activities, and Inhibitory Effects of Sildenafil and Vardenafil

We determined the differential cyclic guanyl monophosphate catalytic and cyclic guanyl monophosphate binding activity of phosphodiesterase-5 isoforms and the inhibitory effects of sildenafil (Pfizer, New York, New York) and vardenafil (Bayer Pharmaceutical Research, Wuppertal, Germany). Coding sequences of the human phosphodiesterase-5 isoforms A1, A2 and A3 were cloned into pBlueBac4.5/V5-His (Invitrogen, Carlsbad, California), which allows the tagging of histidines at the carboxyl terminal of the expressed protein. Isoforms were expressed with the Bac-N-Blue baculoviral system and purified with the ProBond system. Expression clones were identified by polymerase chain reaction using vector and phosphodiesterase-5 specific primers. Purified proteins were verified by Western blotting. Purified proteins were analyzed for cyclic guanyl monophosphate catalytic and cyclic guanyl monophosphate binding activity, and used to determine the differential potencies of the phosphodiesterase-5 selective inhibitors sildenafil and vardenafil. Cloning and expression of phosphodiesterase-5A1 to A3 isoforms in the baculoviral system resulted in the isolation of purified isoform proteins. Mean cyclic guanyl monophosphate catalytic activity (K(m)) +/- SD was 4.76 +/- 0.37, 4.52 +/- 0.09 and 11.39 +/- 0.22 microM for A1 to A3, respectively. Mean cyclic guanyl monophosphate binding activity (K(d)) was 3.24 +/- 0.47, 1.95 +/- 0.60 and 1.70 +/- 0.47 microM for A1 to A3, respectively. Mean IC(50) of sildenafil against phosphodiesterase-5A1 to A3 was 1.20 +/- 0.34, 2.83 +/- 0.56 and 2.28 +/- 0.38 nM, respectively. Mean IC(50) of vardenafil against phosphodiesterase-5A1 to A3 was 0.41 +/- 0.15, 0.23 +/- 0.08 and 0.45 +/- 0.06 nM, respectively. Phosphodiesterase-5A1 and A2 had similar K(m) values, while phosphodiesterase-5A3 had a much higher K(m) and, thus, lower cyclic guanyl monophosphate catalytic activity. Phosphodiesterase-5A2 and A3 had similar K(d) values, while phosphodiesterase-5A1 had higher K(d) and, thus, lower cyclic guanyl monophosphate binding activity. Vardenafil was more potent (3 to 12-fold) than sildenafil for inhibiting the catalytic activity of all 3 human phosphodiesterase-5 isoforms with phosphodiesterase-5A2 showing the highest differentiation (12-fold).

Inhibitory Effects of Sildenafil on Small Intestinal Motility and Myoelectrical Activity in Dogs

Previous studies have shown that sildenafil inhibits the esophageal motility in both humans and animals. The aim of this study was to investigate the effects of sildenafil on intestinal myoelectrical activity and motility. The study was composed of 2 experiments and performed in 7 healthy female dogs with a duodenal cannula 20 cm beyond pylorus (19-26 kg). The first experiment was designed to study the effects of sildenafil on intestinal myoelectrical activity and it included 2 sessions each consisting of 30-minute baseline, 15-minute posttreatment (placebo or 100 mg sildenafil) and 90 minutes after a liquid meal. Intestinal myoelectrical activity was recorded during the entire experiment period. The second experiment was aimed to investigate the effect of sildenafil on intestinal motility and was performed immediately after a solid meal. Intestinal motility was measured by a manometric catheter inserted into the small intestine via the duodenum cannula for 30 minutes at baseline and 60 minutes after sildenafil. Sildenafil significantly reduced the amplitude but had no effect on the frequency and regularity of the intestinal myoelectrical activity. Sildenafil significantly inhibited postprandial intestinal contractions. Although the frequency of the contractions was not altered, the mean area under the curve was significantly reduced during the first 30 minutes (P < .03) and second 30 minutes after sildenafil (P < .03); the power of intestinal contractile activities was also significantly reduced during the first 30 minutes (P < .0004) and second 30 minutes after sildenafil (P < .0003) in comparison with baseline. In conclusion, sildenafil inhibits the amplitude of both intestinal contractile activity and intestinal slow waves.

Inhibition of RhoA/Rho kinase pathway is involved in the beneficial effect of sildenafil on pulmonary hypertension.

Inhibition of the type 5 phosphodiesterase and inhibition of Rho kinase are both effective in reducing pulmonary hypertension (PH). Here we investigate whether Rho kinase inhibition is involved in the beneficial effect of the type 5 phosphodiesterase inhibitor sildenafil on PH. Chronic hypoxia-induced PH in rats is associated with an increase in RhoA activity in pulmonary artery that was maximal after 2 days (10.7+/-0.9-fold increase, n=6, P<0.001). The activity of Rho kinase assessed by measuring the level of myosin phosphatase target subunit 1 (MYPT1) phosphorylation was also increased (5.7+/-0.8-fold over control, n=8). Chronic fasudil (30 mg kg(-1) day(-1); 14 days) and sildenafil (25 mg kg(-1) day(-1); 14 days) treatments reduced PH and pulmonary cardiovascular remodelling, and inhibited the MYPT1 phosphorylation in pulmonary artery from hypoxic rats by 82.3+/-3% (n=4) and by 76.6+/-2% (n=4), respectively. The inhibitory effect of sildenafil (10 microM) on MYPT1 phosphorylation was demonstrated by the loss of actin stress fibres in vascular smooth muscle cells. However, in vitro kinase assays indicated that sildenafil had no direct inhibitory action on Rho kinase activity. Sildenafil treatment induced increased RhoA phosphorylation and association to its cytosolic inhibitory protein, guanine dissociation inhibitor (GDI) in pulmonary artery.We propose that sildenafil inhibits RhoA/Rho kinase-dependent functions in pulmonary artery through enhanced RhoA phosphorylation and cytosolic sequestration by GDI. The inhibition of intracellular events downstream of RhoA thus participates in the beneficial effect of sildenafil on PH.