Phosphodiesterase-5 Activity Research Articles

Saw Palmetto Extract Enhances Erectile Responses by Inhibition of Phosphodiesterase 5 Activity and Increase in Inducible Nitric Oxide Synthase Messenger Ribonucleic Acid Expression in Rat and Rabbit Corpus Cavernosum

To evaluate whether saw palmetto extract (SPE) relaxes corpus cavernosum and explore the underlying mechanisms. Forty Sprague-Dawley rats and 30 New Zealand rabbits were randomly allocated into 3 SPE-treated groups (low-, middle-, and high-dose) and 1 saline-treated control group. SPE was administered intragastrically for 7 consecutive days. Another 23 rats treated with sildenafil were used to appraise the erectile response to electrical stimulation of nerves in the corpus cavernosum. The erectile functions of rats and rabbits were evaluated 24 hours after the last SPE administration or 15 minutes after intragastric sildenafil. Outcome measures included corpus cavernosum electrical activity recording, phosphodiesterase 5 (PDE5) activity detected by the colorimetric quantitative method, and messenger ribonucleic acid (mRNA) expression level for PDE5 and inducible nitric oxide synthase (iNOS) determined using real-time polymerase chain reaction. In the SPE-treated animals, the relaxant response to electrical stimulation of nerves in the corpus cavernosum, reflected by the amplitude of the electrical activity within the cavernosum, was significantly and dose-dependently augmented. Similar effects were observed in the sildenafil-treated rats. PDE5 activity in rat and rabbit corpus cavernosum tissues was significantly and dose-dependently inhibited in SPE-treated animals, whereas the iNOS mRNA level increased compared with the saline group. PDE5 mRNA, however, was only significantly enhanced in the rats treated with the middle dose of SPE. The results suggest that SPE may have potential application value for the prevention or treatment of erectile dysfunction through an increase in iNOS mRNA expression and inhibition of PDE5 activity in corpus cavernosum smooth muscles.

NO‐induced increases in cGMP and inhibition of NKCC2 activity are blunted in thick ascending limbs from angiotensin II‐hypertensive rats

Thick ascending limbs (THALs) absorb 25% of the filtered NaCl mainly via the Na/K/2Cl cotransporter NKCC2. Transport is enhanced in angiotensin II‐induced hypertension (AIH). NO inhibits NKCC2 activity via cGMP. Phosphodiesterase 5 (PDE5), which degrades cGMP, is increased by angiotensin II in smooth muscle cells. We hypothesized that inhibition of NKCC2 by NO is blunted in AIH due to increased PDE5 activity. Male rats were infused with vehicle (control) or 200 ng/kg/min angiotensin II for 5 days. THAL NKCC2 activity and cGMP levels were measured by fluorescence microscopy and radioimmunoassay respectively. 100 μM Spermine NONOate, a NO donor, reduced NKCC2 activity by 35% from 1.7 ± 0.3 to 1.1 ± 0.2 AU/s (p<0.01) in THALs from control but not AIH. NO increased cGMP in control THALs by 2.2 ± 0.4 fmol/μg protein but only by 1.2 ± 0.3 fmol/μg protein in THALs from AIH (p<0.04). Inhibiting PDE5 with 25 nM vardenafil (Vard) restored NO's ability to reduce NKCC2 activity in AIH (Vard: 1.2 ± 0.2; Vard + NO: 0.4 ± 0.1 AU/s, p<0.02). Vard also restored the ability of NO to increase cGMP in AIH (control: 2.1 ± 0.7; AIH: 2.2 ± 0.3 fmol/μg protein). 500 μM db‐cGMP reduced NKCC2 activity to the same extent in THALs from control and AIH suggesting the signaling cascade downstream of cGMP was not altered. We conclude that PDE5 blunts NO‐induced increases in cGMP and inhibition of NKCC2 activity in THALs from angiotensin II‐hypertensive rats.Sources of funding: NIH HL 028982 to J.L.G. and AHA 11PRE7510005 to V.D.R.

Phosphodiesterase‐5 activity exerts a coronary vasoconstrictor influence in awake swine that is partly mediated via an increase in endothelin production

Nitric oxide (NO)‐induced coronary vasodilation is mediated through production of cyclic guanosine monophosphate (cGMP), and through inhibition of endothelin (ET). We previously demonstrated that both phosphodiesterase 5 (PDE5) and ET each exert a vasoconstrictor influence on coronary resistance vessels. Here we investigated the integrated control by PDE5 and ET of coronary resistance vessel tone in exercising swine. ETA/ETB receptor blockade with tezosentan (3 mg/kg iv) and PED5 inhibition with EMD360527 (300 μg/kg/min iv) each produced coronary vasodilation at rest that waned with incremental levels of exercise. Interestingly, however, tezosentan failed to produce further coronary vasodilation in the presence of EMD360527. Similarly, tezosentan (10−9–3×10−5 M) and EMD360527 (10−9–3×10−5 M) each produced concentration‐dependent vasodilation in preconstricted isolated porcine coronary small arteries in vitro, while tezosentan again failed to produce additional vasodilation in the presence of EMD360527. Importantly, EMD360527 (3×10−6 M) significantly attenuated Big ET‐induced (10−7 M), but not ET‐induced (10−7 M), coronary small artery constriction. In conclusion, PDE5 activity exerts a vasoconstrictor influence on coronary resistance vessels that is partly mediated via an increase in ET production.

Repurposing phosphodiesterase-5 inhibitors as chemoadjuvants

Repurposing phosphodiesterase-5 inhibitors as chemoadjuvants

Avanafil, a Potent and Highly Selective Phosphodiesterase-5 Inhibitor for Erectile Dysfunction

We investigated the in vitro inhibitory effects of avanafil, a novel, potent inhibitor of phosphodiesterase-5, on 11 phosphodiesterases. We also studied its potentiation of penile tumescence in dogs. Phosphodiesterase assay was done with the 4 phosphodiesterase-5 inhibitors avanafil, sildenafil, vardenafil and tadalafil using 11 phosphodiesterase isozymes. In anesthetized dogs the pelvic nerve was repeatedly stimulated to evoke tumescence. Intracavernous pressure was measured after avanafil or sildenafil administration. Avanafil specifically inhibited phosphodiesterase-5 activity at a 50% inhibitory concentration of 5.2 nM. Avanafil showed higher selectivity (121-fold) against phosphodiesterase-6 than sildenafil and vardenafil (16 to 21-fold) and showed excellent selectivity (greater than 10,000-fold) against phosphodiesterase-1 compared with sildenafil (375-fold). Avanafil also had higher selectivity against phosphodiesterase-11 than tadalafil (greater than 19,000 vs 25-fold). Avanafil also showed excellent selectivity against all other phosphodiesterases. After intravenous administration in anesthetized dogs the 200% effective dose of avanafil and sildenafil on the penile tumescence was 37.5 and 34.6 μg/kg, respectively. After intraduodenal administration the 200% effective dose of avanafil and sildenafil on tumescence was 151.7 and 79.0 μg/kg at the peak time, respectively. Time to peak response with avanafil and sildenafil was 10 and 30 minutes, respectively, indicating a more rapid onset of avanafil. Avanafil has a favorable phosphodiesterase-5 selectivity profile compared to that of marketed phosphodiesterase-5 inhibitors. Avanafil shows excellent in vitro and in vivo potency, and fast onset of action for penile erection. Cumulative data suggest that avanafil has a promising pharmacological profile for erectile dysfunction.

Brief Hyperoxia Increases Mitochondrial Oxidation and Increases Phosphodiesterase 5 Activity in Fetal Pulmonary Artery Smooth Muscle Cells

Oxygen is a pulmonary vasodilator, but data suggest high O(2) concentrations impede that response. We previously reported 24 h of 100% O(2) increased phosphodiesterase 5 (PDE5) activity in fetal pulmonary artery smooth muscle cells (FPASMC) and in ventilated neonatal lambs. PDE5 degrades cyclic GMP (cGMP) and inhibits nitric oxide (NO)-mediated cGMP-dependent vasorelaxation. We sought to determine the mechanism by which hyperoxia initiates reactive oxygen species (ROS) production and regulates PDE5. Thirty minutes of hyperoxia increased mitochondrial ROS versus normoxia (30.3±1.7% vs. 21.1±2.8%), but had no effect on cytosolic ROS, measured by roGFP, a ratiometric protein thiol redox sensor. Hyperoxia increased PDE5 activity (220±39%) and decreased cGMP responsiveness to NO (37±17%). Mitochondrial catalase overexpression attenuated hyperoxia-induced mitochondrial roGFP oxidation, compared to FPASMC infected with empty adenoviral vector (50±3% of control) or mitochondrial superoxide dismutase. MitoTEMPO, mitochondrial catalase, and DT-3, a cGMP-dependent protein kinase I alpha inhibitor, decreased PDE5 activity (32±13%, 26±21%, and 63±10% of control, respectively), and restored cGMP responsiveness to NO (147±16%,172±29%, and 189±43% of control, respectively). C57Bl6 mice exposed to 90%-100% O(2) for 45 min±mechanical ventilation had increased PA PDE5 activity (206±39% and 235±75%, respectively). This is the first description that hyperoxia induces ROS in the mitochondrial matrix prior to the cytosol. Our results indicate that short hyperoxia exposures can produce significant changes in critical cellular signaling pathways. These results indicate that mitochondrial matrix oxidant signals generated during hyperoxia, specifically H(2)O(2), activate PDE5 in a cGMP-dependent protein kinase-dependent manner in pulmonary vascular smooth muscle cells.

Phosphodiesterase 5 Inhibition Attenuates Cerebral Vasospasm and Improves Functional Recovery After Experimental Subarachnoid Hemorrhage

Cerebral vasospasm is an independent predictor of poor outcome after subarachnoid hemorrhage (SAH). The nitric oxide-cyclic guanosine monophosphate (NO-cGMP) vasodilatory pathway is strongly implicated in its pathophysiology. Preliminary studies suggest that phosphodiesterase 5 (PDE5), an enzyme that degrades cGMP, may play a role because the PDE5 inhibitor sildenafil was found to reduce vasospasm after SAH. However, several questions that are critical when considering translational studies remain unanswered. To elucidate the mechanism of action of sildenafil against vasospasm and to assess whether sildenafil attenuates SAH-induced neuronal cell death, improves functional outcome after SAH, or causes significant physiological side effects when administered at therapeutically relevant doses. SAH was induced via endovascular perforation in male C57BL6 mice. Beginning 2 hours later, mice received sildenafil citrate (0.7, 2 or 5 mg/kg orally twice daily) or vehicle. Neurological outcome was assessed daily. Vasospasm was determined on post-SAH day 3. Brain PDE5 expression and activity, cGMP content, neuronal cell death, arterial blood pressure, and intracranial pressure were examined. We found that PDE5 activity (but not expression) is increased after SAH, leading to decreased cGMP levels. Sildenafil attenuates this increase in PDE5 activity and restores cGMP levels after SAH. Post-SAH initiation of sildenafil was found to decrease vasospasm and neuronal cell death and markedly improve neurological outcome without causing significant physiological side effects. Sildenafil, a US Food and Drug Administration-approved drug with a proven track record of safety in humans, is a promising new therapy for vasospasm and neurological deficits after SAH.

PDE5 inhibition counteracts β- adrenergic induction of cardiac hypertrophy

The b-adrenoreceptors play important roles in cardiovascular function regulation mediated by the sympathetic nervous system. It is known that sustained b-adrenergic stimulations promotes cardiac hypertrophy (Oleg et al., 2007). Recently an antihypertrophic role of sildenafil, that acts as a phosphodiesterase 5 (PDE5) inhibitor, has been demonstrated in mice where hypertrophy was mechanically induced (Takimoto et al., 2005). We report the results obtained on a cellular system of cardiac hypertrophy in vitro. By using three-dimensional cultures of mouse ventricular cardiomyocytes (Xiang et al., 2005) and isolated cardiomyocytes we show that: 1) these cells express levels of PDE5 comparable with the ones in normal heart, 2) treatment of the cultures with the b-adrenoreceptors agonist isoproterenol induces cell hypertrophy accompanied by an increment of the level of PDE5 expression and 3) sildenafil prevents the development of such hypertrophy through specific b-adrenoreceptors and signaling pathways 4) the inhibition of other members of PDE family might contribute to the prevention of hypertrophy following b-adrenergic stimulation. In summary, we present a test system that may contribute to clarify intracellular signaling pathways leading to cardiac hypertrophy and to identify molecular targets, like the ones involved in PDE5 activity, on which to steer the development of new drugs and to design new clinical therapies.

Smoking in Andrology: State of Art

Andrology (from Greek andros, man; and -λογία, -logia) is a medical specialty that deals with male health, particularly related to the problems of the male reproductive system and urological problems that are unique to men. However, the scope and mission of Andrology is expanded from being traditionally concerned with issues related to male infertility, erectile dysfunction and prostate diseases in the past to broader interests in wide aspects related to male reproductive health and male health in general. A matter of concern in science as well as experience is smoking as an andrologically related issue. Actually, despite worldwide anti- smoking campaigns, cigarette smoking is rather common with strong evidences that smoking behavior is related to social factors, particularly the influence of parents and peer groups. Taste and smell also influence the inclination to smoke where exciting sensory organs in the lips, mouth and throat provide sensations of touch, taste and irritation. Also, it has been suggested that high negative mood variability is a risk factor for future smoking escalation and that its mood-stabilizing effects may reinforce and maintain daily cigarette use among youths. Strong body of evidence has indicated the negative effect of cigarette smoking on male fertility affecting every system involved in the reproductive process. Different articles have demonstrated a negative impact of smoking on human semen parameters, correlated with cigarettes intensity and duration. Most articles argued that smokers demonstrate lower semen volume, sperm count, sperm motility, sperm viability and normal sperm morphology compared with non-smokers. In addition, they experience increased seminal leukocytes, oval sperm percentage, spermatozoa with cytoplasmic droplets, apoptotic markers, seminal lead and seminal cadmium. In addition, passive exposure to environmental tobacco smoke has been shown to result in measurable seminal nicotine and cotinine levels correlated with the degree of the reported exposure. Meanwhile, the combination of smoking and varicocele has been shown to be strongly related to the incidence of oligozoospermia having an incidence 10 times greater than non- smokers with varicocele and 5 times greater than smokers devoid of varicocele (1-3). On the other hand, many opinions were raised to deny that affection on male infertility where millions of smokers are fathers. However, although smokers as a group may not experience reduced fertility, males with marginal semen quality may benefit from quitting smoking. Also, smokers should quit smoking for the sense of responsibility for their future generation as tobacco smoke contains numerous mutagenic substances. Nevertheless, while it is clear that Erectile Dysfunction (ED) is multi-factorial, the direct and negative effects of smoking on erectile function are well documented where several epidemiological studies demonstrated that smoking or even chronic passive smoking not only increases the risk of ED, but also amplifies its risk associated with other risk factors as hypertension, diabetes, dyslipidaemia or aging. Cigarette smoking-induced ED in human or animal models has been associated with impaired arterial flow to the penis, acute vasospasm of penile arteries, decreased synthesis/availability of nitric oxide leading to endothelial damage with architectural and functional changes, oxidative damage to endothelial cells, increased production of cyclooxygenase dependent and independent vasoconstrictor eicosanoids, activated Rho-associated kinase activity and vascular stiffness. Another interesting aspect that should be considered is the association between smoking and decreased testosterone that plays a major role in men's sexual function, libido and cGMP formation, through nitric oxide synthase stimulation/catabolism through phosphodiesterase-5 activity. Although cessation of cigarette smoking can improve ED in a considerable proportion of smokers, age and severity of ED before cessation are inversely related to the chance of improvement. Prevention of smoking represents a very important approach for reducing the risk of ED.

Hydrocortisone normalizes oxygenation and cGMP regulation in lambs with persistent pulmonary hypertension of the newborn

In the pulmonary vasculature, cGMP levels are regulated by soluble guanylate cyclase (sGC) and phosphodiesterase 5 (PDE5). We previously reported that lambs with persistent pulmonary hypertension of the newborn (PPHN) demonstrate increased reactive oxygen species (ROS) and altered sGC and PDE5 activity, with resultant decreased cGMP. The objective of this study was to evaluate the effects of hydrocortisone on pulmonary vascular function, ROS, and cGMP in the ovine ductal ligation model of PPHN. PPHN lambs were ventilated with 100% O(2) for 24 h. Six lambs received 5 mg/kg hydrocortisone every 8 h times three doses (PPHN-hiHC), five lambs received 3 mg/kg hydrocortisone followed by 1 mg·kg(-1)·dose(-1) times two doses (PPHN-loHC), and six lambs were ventilated with O(2) alone (PPHN). All groups were compared with healthy 1-day spontaneously breathing lambs (1DSB). O(2) ventilation of PPHN lambs decreased sGC activity, increased PDE5 activity, and increased ROS vs. 1DSB lambs. Both hydrocortisone doses significantly improved arterial-to-alveolar ratios relative to PPHN lambs, decreased PDE5 activity, and increased cGMP relative to PPHN lambs. High-dose hydrocortisone also increased sGC activity, decreased PDE5 expression, decreased ROS, and increased total vascular SOD activity vs. PPHN lambs. These data suggest that hydrocortisone treatment in clinically relevant doses improves oxygenation and decreases hyperoxia-induced changes in sGC and PDE5 activity, increasing cGMP levels. Hydrocortisone reduces ROS levels in part by increasing SOD activity in PPHN lambs ventilated with 100% O(2.) We speculate that hydrocortisone increases cGMP by direct effects on sGC and PDE5 expression and by attenuating abnormalities induced by oxidant stress.

Apocynin improves oxygenation and increases eNOS in persistent pulmonary hypertension of the newborn

NADPH oxidase is a major source of superoxide anions in the pulmonary arteries (PA). We previously reported that intratracheal SOD improves oxygenation and restores endothelial nitric oxide (NO) synthase (eNOS) function in lambs with persistent pulmonary hypertension of the newborn (PPHN). In this study, we determined the effects of the NADPH oxidase inhibitor apocynin on oxygenation, reactive oxygen species (ROS) levels, and NO signaling in PPHN lambs. PPHN was induced in lambs by antenatal ligation of the ductus arteriosus 9 days prior to delivery. Lambs were treated with vehicle or apocynin (3 mg/kg intratracheally) at birth and then ventilated with 100% O(2) for 24 h. A significant improvement in oxygenation was observed in apocynin-treated lambs after 24 h of ventilation. Contractility of isolated fifth-generation PA to norepinephrine was attenuated in apocynin-treated lambs. PA constrictions to NO synthase (NOS) inhibition with N-nitro-l-arginine were blunted in PPHN lambs; apocynin restored contractility to N-nitro-l-arginine, suggesting increased NOS activity. Intratracheal apocynin also enhanced PA relaxations to the eNOS activator A-23187 and to the NO donor S-nitrosyl-N-acetyl-penicillamine. Apocynin decreased the interaction between NADPH oxidase subunits p22(phox) and p47(phox) and decreased the expression of Nox2 and p22(phox) in ventilated PPHN lungs. These findings were associated with decreased superoxide and 3-nitrotyrosine levels in the PA of apocynin-treated PPHN lambs. eNOS protein expression, endothelial NO levels, and tetrahydrobiopterin-to-dihydrobiopterin ratios were significantly increased in PA from apocynin-treated lambs, although cGMP levels did not significantly increase and phosphodiesterase-5 activity did not significantly decrease. NADPH oxidase inhibition with apocynin may improve oxygenation, in part, by attenuating ROS-mediated vasoconstriction and by increasing NOS activity.

Phosphodiesterase-5 Inhibition Mimics Intermittent Reoxygenation and Improves Cardioprotection in the Hypoxic Myocardium

Although chronic hypoxia is a claimed myocardial risk factor reducing tolerance to ischemia/reperfusion (I/R), intermittent reoxygenation has beneficial effects and enhances heart tolerance to I/R. Aim of the study: To test the hypothesis that, by mimicking intermittent reoxygenation, selective inhibition of phosphodiesterase-5 activity improves ischemia tolerance during hypoxia. Adult male Sprague-Dawley rats were exposed to hypoxia for 15 days (10% O2) and treated with placebo, sildenafil (1.4 mg/kg/day, i. p.), intermittent reoxygenation (1 h/day exposure to room air) or both. Controls were normoxic hearts. To assess tolerance to I/R all hearts were subjected to 30-min regional ischemia by left anterior descending coronary artery ligation followed by 3 h-reperfusion. Whereas hypoxia depressed tolerance to I/R, both sildenafil and intermittent reoxygenation reduced the infarct size without exhibiting cumulative effects. The changes in myocardial cGMP, apoptosis (DNA fragmentation), caspase-3 activity (alternative marker for cardiomyocyte apoptosis), eNOS phosphorylation and Akt activity paralleled the changes in cardioprotection. However, the level of plasma nitrates and nitrites was higher in the sildenafil+intermittent reoxygenation than sildenafil and intermittent reoxygenation groups, whereas total eNOS and Akt proteins were unchanged throughout. Conclusions: Sildenafil administration has the potential to mimic the cardioprotective effects led by intermittent reoxygenation, thereby opening the possibility to treat patients unable to be reoxygenated through a pharmacological modulation of NO-dependent mechanisms.

Combined ligand based pharmacophore modeling, virtual screening methods to identify critical chemical features of novel potential inhibitors for phosphodiesterase-5

The central role of phosphodiesterase-5 (PDE5) is to hydrolyze the cyclic guanidine monophosphate which leads male erectile dysfunction. To inhibit the PDE5 activity, integrated computer-aided drug design technologies were utilized to generate pharmacophore modeling, database screening and docking methodologies. 3D pharmacophore models are generated using HypoGen to identify the critical chemical features of PDE5 inhibitors. Among the top ten generated hypotheses, the first hypothesis (Hypo1) was selected as best one. The best pharmacophore model, Hypo1, characterized by one hydrogen bond acceptor-lipid ( HBAL), one hydrophobic ( H), two ring aromatic ( RA) features, also have high cost difference (111.04), good correlation coefficient (0.94) and low root mean square deviation (1.53). Hypo1 model was cross validated by using Fischer's randomization method, test and decoy sets. The well validated Hypo1 was used as 3D query to screen NCI database. The screened molecules were sorted by applying rule of five, ADMET properties and molecular docking study to refine the retrieved hits. Finally, 3 molecules were showed good interaction with important amino acids in PDE5 active site. All above said validation analysis strongly suggested that Hypo1 will act as reliable and useful tool to identify new potential leads against PDE5 by screen the large databases and also it predicted 3 compounds in our present study which may act as potent inhibitor of PDE5 and these 3 compounds were chosen for further development.

Conformation Changes, N-terminal Involvement, and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.

Structural complexity of the testis and PKG I / StAR interaction regulate the Leydig cell adaptive response to repeated immobilization stress

The role of the structural complexity of the testis and the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway was analysed in adult male rats exposed to acute and repeated immobilization stress (IMO). In whole testis preparations, exposure to acute and repeated IMO caused an increase in NO production. In contrast, NO production was inhibited in interstitial cell preparations after exposure to all types of stress. In purified Leydig cell preparations, NO production was inhibited only after exposure to prolonged IMO. These findings indicate that biologically active compounds released from various testicular compartments exert both stimulatory and inhibitory effects on NO production. TaqMan Low Density Array of rat phosphodiesterases revealed a decrease in the expression of cGMP-specific phosphodiesterase 5 (PDE5) in Leydig cells of animals exposed to repeated IMO. In contrast, the expression of cGMP-dependent protein kinase type I (PKG I), total and phosphorylated steroidogenic acute regulatory protein (StAR), and PKG I/StAR immunoprecipitated complex was increased during repeated exposure to IMO. The increase in both total and phosphorylated StAR formation was effectively blocked by inhibition of PKG I in vitro. Thus, increased expressions of PKG I and StAR complex, accompanied by decreased PDE5 activity, suggest that the NO-cGMP signalling pathway and consequent activation of the StAR protein regulate the adaptive response of Leydig cells to repeated IMO stress.

Mitochondrial oxidant stress increases PDE5 activity in persistent pulmonary hypertension of the newborn

In the pulmonary vasculature, phosphodiesterase-5 (PDE5) degrades cGMP and inhibits nitric oxide-mediated, cGMP-dependent vasorelaxation. We previously reported that ventilation with 100% O2 increased PDE5 activity in pulmonary arteries (PAs) of pulmonary hypertension lambs (PPHN) more than in control lambs. In the present study, PA smooth muscle cells (PASMCs) from PPHN lambs had increased basal PDE5 activity, decreased cGMP-responsiveness to NO, and increased mitochondrial matrix oxidant stress compared to control PASMC. Hyperoxia (24 h) increased PDE5 activity and mitochondrial matrix oxidant stress above baseline to a similar degree in PPHN and control PASMC. Mitochondrially targeted catalase decreased PDE5 activity at baseline and after hyperoxia in PPHN PASMC. Similarly, catalase treatment of PPHN lambs ventilated with 100% O2 decreased PDE5 activity and increased cGMP in PA. We conclude that baseline PDE5 activity and oxidative stress is increased in PPHN PASMC, and scavenging H2O2 is sufficient to block oxidant-mediated increases in PDE5 activity in PPHN.

Damiana (Turnera diffusa Willd.) – a traditionally used aphrodisiac as modern PDE-5 inhibitor

Selective phosphodiesterase-5 (PDE-5) inhibitors like Sildenafil, Tardanafil or Vardenafil are commonly used for the treatment of Erectile Dysfunction (ED). PDE-5 inhibitors are generally not considered as aphrodisiacs because they do not have any direct effect on the libido. However, increased ability to attain an erection may be interpreted as increased sexual arousal by users of these drugs. An aphrodisiac is a substance that increases sexual desire. One link between PDE-5 inhibitors and aphrodisiacs might be the herbal drug Folia Damiana. The leaves of Damiana (Turnera aphrodisiaca) have a long history of traditional use as aphrodisiac in Mexico [1]. Nowadays Damiana products claimed to increase libido and sexual performance are widely marketed, whereas scientific data are scarce: Damiana extracts were shown to stimulate sexual behaviour in rats [2,3]. Our previous investigations showed for the first time, that Damiana leaf preparations influenced PDE-5 activity in vitro [4]. In the present study a crude 60% w/w ethanolic Damiana extract was investigated for its inhibitory activity on PDE-5 in vitro and was further optimised using different purification techniques. In conclusion, PDE-5 inhibition might be a possible mechanism of action of Damiana. An human study on the effects of FB9389 on mild Erectile Dysfunction in male subjects is currently in progress.

Increased renal phosphodiesterase-5 activity mediates the blunted natriuretic response to a nitric oxide donor in the pregnant rat

Pregnancy is characterized by plasma volume expansion and renal sodium retention with loss of natriuretic response to atrial natriuretic peptide due to increased medullary phosphodiesterase-5 (PDE5). Here, we determined whether natriuretic responses to nitric oxide (NO) are also blunted in pregnancy due to increased PDE5. Anesthetized 16-day pregnant and virgin rats were studied at baseline and during intrarenal infusion of the NO donor spermine NONOate (2.5 nmol/min), the PDE5 inhibitor sildenafil (SILD; 0.5 μg/min), or a combination. The right (noninfused) kidney served as a control. Intrarenal NONOate had no effect on mean arterial pressure (MAP); however, SILD reduced MAP in virgin rats, and the combination of NONOate+SILD reduced MAP in both virgin and pregnant rats. Neither NONOate nor SILD altered glomerular filtration rate. NONOate and SILD each stimulated sodium excretion (U(Na)V) and fractional excretion of sodium (FE(Na)) in virgin rats, but the combination did not result in an additional natriuretic response. However, NONOate infusion did not increase U(Na)V or FE(Na) in pregnant rats, but the natriuretic response to NONOate was restored with SILD, and SILD alone produced a natriuresis during pregnancy. Sodium nitroprusside (10(-4) mol/l)-stimulated cGMP accumulation from inner medullary collecting duct cells was blunted in cells from pregnant vs. virgin or postpartum rats and was restored by treatment with the PDE5 inhibitor DMPPO (10(-7) mol/l). Therefore, increased intrarenal PDE5 mediates the blunted natriuretic response to NO, and loss of responsiveness to the cGMP-dependent, natriuretic agents may contribute to volume expansion during pregnancy.

SOD and inhaled nitric oxide normalize phosphodiesterase 5 expression and activity in neonatal lambs with persistent pulmonary hypertension

Phosphodiesterase 5 (PDE5) and soluble guanylate cyclase (sGC) are key regulators of cGMP and pulmonary vascular tone. We sought to determine the impact of mechanical ventilation with O(2) with or without inhaled nitric oxide (iNO) or recombinant human Cu/Zn SOD (rhSOD) on sGC, PDE5, and cGMP in the ovine ductal ligation model of persistent pulmonary hypertension of the newborn (PPHN). PPHN lambs were ventilated with 100% O(2) for 24 h alone or combined with either inhalation of 20 parts per million (ppm) iNO continuously or a single intratracheal dose of rhSOD (5 mg/kg). Ventilated PPHN lambs were compared with PPHN fetuses, control fetuses, and 1-day-old spontaneously breathing lambs (1DSB). In the small pulmonary arteries of 1DSB lambs, sGC expression increased, PDE5 expression decreased, and cGMP concentrations increased relative to fetal levels. In PPHN lambs ventilated with 100% O(2), sGC activity increased to levels comparable with 1DSB levels. However, PDE5 expression and activity increased, and cGMP levels remained at fetal levels. Addition of either iNO or rhSOD decreased PDE5 expression and activity in PPHN lambs and increased cGMP levels to levels comparable with 1DSB lambs. These data suggest that ventilation of PPHN lambs with 100% O(2) impairs cGMP-mediated vasodilation in part due to increased PDE5 expression and activity. The addition of either iNO or rhSOD normalized PDE5 and cGMP levels. Thus therapies designed to decrease PDE5 and increase cGMP, such as iNO and rhSOD, may prove useful in the treatment of PPHN in newborn infants.

Mechanisms of Activity-dependent Plasticity in Cellular Nitric Oxide-cGMP Signaling

Cellular responsiveness to nitric oxide (NO) is shaped by past history of NO exposure. The mechanisms behind this plasticity were explored using rat platelets in vitro, specifically to determine the relative contributions made by desensitization of NO receptors, which couple to cGMP formation, and by phosphodiesterase-5 (PDE5), which is activated by cGMP and also hydrolyzes it. Repeated delivery of brief NO pulses (50 nm peak) at 1-min intervals resulted in a progressive loss of the associated cGMP responses, which was the combined consequence of receptor desensitization and PDE5 activation, with the former dominating. Delivery of pulses of differing amplitude showed that NO stimulated and desensitized receptors with similar potency (EC50 = 10–20 nm). PDE5 activation was highly sensitive to NO, with a single pulse peaking at 2 nm being sufficient to evoke a 50% loss of response to a subsequent near-maximal NO pulse. However, the activated state of the PDE subsided quickly after removal of NO, the half-time for recovery being 25 s. In contrast, receptor desensitization reverted much more slowly, the half-time being 16 min. Accordingly, with long (20-min) exposures, NO concentrations as low as 600 pm provoked significant desensitization. The results indicate that PDE5 activation and receptor desensitization subserve distinct short term and longer term roles as mediators of plasticity in NO-cGMP signaling. A kinetic model explicitly describing the complex interplay between NO concentration, cGMP synthesis, PDE5 activation, and the resulting cGMP accumulation successfully simulated the present and previous data.