Inhibition Of Phosphodiesterase 5A Research Articles

Abstract 8905: Protein Kinase G Positively Regulates Proteasome Proteolytic Function in the Heart

The Ubiquitin Proteasome System (UPS) plays a pivotal role in the intracellular protein homeostasis, thereby regulating a variety of cellular processes including protein quality control (PQC). Inadequate PQC and proteasome functional insufficiency are associated with and may play an important role in adverse cardiac remodeling and cardiac failure (CHF). This is best reflected by the increased presence of misfolded proteins and protein aggregates in failing human hearts. While the regulatory mechanisms of these processes remain poorly understood they carry significant clinical implications. Reduced protein kinase G (PKG) activity is well documented in CHF. Increasing the activity PKG, during various cardiovascular diseases improves patient prognosis. We hypothesize that PKG plays a role in regulating UPS function. Using both genetic and/or pharmacological manipulation of PKG activity in cultured cardiomyocytes and intact mice and taking advantage of a previously validated surrogate misfolded protein (GFPu or GFPdgn) as well as a bona fide misfolded protein (R120G-CryAB) that is known to cause proteinopathy in human and mice, our studies have revealed a significant role for PKG in UPS regulation. In cultured cardiomyocytes, PKG inhibition was achieved by PKG-specific siRNA transfection or administration of PKG pharmacological inhibitor while PKG activity was induced by adenoviral delivery of a constitutively active PKG or by the treatment of phosphodiesterase 5A inhibitor Sildenafil. PKG activation significantly stimulated UPS activity in cultured cardiomyocytes as indicated by a reduced protein level of GFPu and increased proteasome activities; while PKG inhibition reduced UPS proteolytic activity. Furthermore PKG activation resulted in enhanced removal of R120G-CryAB. These findings from cell culture studies were further corroborated by in vivo findings from manipulating the muscarinic (M) receptor in GFPdgn transgenic mice. The M receptor signals through PKG. Activating the M receptor deceased, while antagonizing the M receptor increased, the steady protein level of GFPdgn in the heart. Hence, we conclude that PKG positively regulates the UPS thereby supporting cardiomyocyte PQC.

Sildenafil enhances systolic adaptation, but does not prevent diastolic dysfunction, in the pressure‐loaded right ventricle

Right ventricular (RV) failure due to pressure or volume overload is a major risk factor for early mortality in congenital heart disease and pulmonary hypertension, but currently treatments are lacking. We aimed to demonstrate that the phosphodiesterase 5A inhibitor sildenafil can prevent adverse remodelling and improve function in chronic abnormal RV overload, independent from effects on the pulmonary vasculature. In rat models of either pressure or volume overload, we performed pressure-volume studies to measure haemodynamic effects and voluntary exercise testing as clinical outcome after 4 weeks of sildenafil (or vehicle) administration. In the pressure-loaded right ventricle, sildenafil enhanced contractility [end-systolic elastance (mmHg/mL) 247 ± 68 vs.155 ± 71, sildenafil vs. vehicle, P < 0.05], prevented RV dilatation [end-diastolic volume (μL) 733 ± 50 vs. 874 ± 39, P < 0.05], reduced wall stress [peak wall stress (mmHg) 323 ± 46 vs. 492 ± 62, P < 0.05], and partially preserved exercise tolerance [running distance (%) -33 ± 15 vs. -62 ± 12, P < 0.05]. Protein kinase A was not activated by sildenafil and thus did not mediate the observed effects. In contrast, protein kinase G-1 was activated by sildenafil, but hypertrophy was not inhibited. Importantly, sildenafil did not prevent diastolic dysfunction, whereas RV fibrosis appeared to be increased in sildenafil-treated rats. In the volume-loaded right ventricle, sildenafil treatment did not show any beneficial effects. We demonstrate sildenafil to have beneficial, afterload-independent effects on the pressure-loaded right ventricle, but not on the volume-loaded right ventricle. These results indicate that sildenafil may offer a specific treatment for the pressure-loaded right ventricle, although persistent diastolic dysfunction and RV fibrosis could be of concern.

Abstract 320: Activation of Protein Kinase G Enhances Proteasome-Mediated Degradation of Misfolded Proteins

The Ubiquitin Proteasome System (UPS) plays a pivotal role in the intracellular protein homeostasis, thereby regulating a variety of cellular processes including protein quality control (PQC). Inadequate PQC and proteasome functional insufficiency are associated with and may play an important role in adverse cardiac remodeling and cardiac failure (CHF). This is best reflected by the increased presence of misfolded proteins and protein aggregates in failing human hearts. While the regulatory mechanisms of these processes remain poorly understood they carry significant clinical implications. Reduced protein kinase G (PKG) activity is well documented in CHF. Increasing the activity PKG, during various cardiovascular diseases improves patient prognosis. We hypothesize that PKG plays a role in regulating UPS function. Using both genetic and/or pharmacological manipulation of PKG activity in cultured cardiomyocytes and intact mice and taking advantage of a previously validated surrogate misfolded protein (GFPu or GFPdgn) as well as a bona fide misfolded protein (R120G-CryAB) that is known to cause proteinopathy in human and mice, our studies have revealed a significant role for PKG in UPS regulation. In cultured cardiomyocytes, PKG inhibition was achieved by PKG-specific siRNA transfection or administration of PKG pharmacological inhibitor while PKG activity was induced by adenoviral delivery of a constitutively active PKG or by the treatment of phosphodiesterase 5A inhibitor Sildenafil. PKG activation significantly stimulated UPS activity in cultured cardiomyocytes as indicated by a reduced protein level of GFPu and increased proteasome activities; while PKG inhibition reduced UPS proteolytic activity. Furthermore PKG activation resulted in enhanced removal of R120G-CryAB. These findings from cell culture studies were further corroborated by in vivo findings from manipulating the muscarinic (M) receptor in GFPdgn transgenic mice. The M receptor signals through PKG. Activating the M receptor deceased, while antagonizing the M receptor increased, the steady protein level of GFPdgn in the heart. Hence, we conclude that PKG positively regulates the UPS thereby supporting cardiomyocyte PQC.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

A non-brain penetrant PDE5A inhibitor improves functional recovery after stroke in rats

Phosphodiesterase 5A (PDE5A) inhibitors improve functional recovery in experimental models of stroke in rats when treatment is delayed and without effect on infarct volume. PDE5A is expressed to only a very limited extent in forebrain tissues, raising the possibility that the locus of effect for the inhibitors is outside the brain. To start to address this question, we determined whether PDE5A inhibitors must have the ability to cross the blood brain barrier to improve recovery. After permanent middle cerebral artery occlusion in rats, PF-5 and UK-489,791, PDE5A inhibitors that do or do not pass the blood brain barrier, were administered starting 24 h after occlusion and continued for 1 week. Motor function was assessed at intervals to 28 days using body swing and limb placement measures. Both PF-5 and UK-489,791 produced improvement in motor scores over 28 days that were significantly greater than in vehicle treated animals. There was no difference in efficacy between the two PDE5A inhibitors. Brain penetrability appears not to be critical to the ability of a PDE5A inhibitor to improve functional recovery after experimental stroke in rats. This finding is discussed with regard to the cellular target(s) for PDE5A inhibitors mediating this effect.

Sildenafil and B-Type Natriuretic Peptide Acutely Phosphorylate Titin and Improve Diastolic Distensibility In Vivo

In vitro studies suggest that phosphorylation of titin reduces myocyte/myofiber stiffness. Titin can be phosphorylated by cGMP-activated protein kinase. Intracellular cGMP production is stimulated by B-type natriuretic peptide (BNP) and degraded by phosphodiesterases, including phosphodiesterase-5A. We hypothesized that a phosphodiesterase-5A inhibitor (sildenafil) alone or in combination with BNP would increase left ventricular diastolic distensibility by phosphorylating titin. Eight elderly dogs with experimental hypertension and 4 young normal dogs underwent measurement of the end-diastolic pressure-volume relationship during caval occlusion at baseline, after sildenafil, and BNP infusion. To assess diastolic distensibility independently of load/extrinsic forces, the end-diastolic volume at a common end-diastolic pressure on the sequential end-diastolic pressure-volume relationships was measured (left ventricular capacitance). In a separate group of dogs (n=7 old hypertensive and 7 young normal), serial full-thickness left ventricular biopsies were harvested from the beating heart during identical infusions to measure myofilament protein phosphorylation. Plasma cGMP increased with sildenafil and further with BNP (7.31±2.37 to 26.9±10.3 to 70.3±8.1 pmol/mL; P<0.001). Left ventricular diastolic capacitance increased with sildenafil and further with BNP (51.4±16.9 to 53.7±16.8 to 60.0±19.4 mL; P<0.001). Changes were similar in old hypertensive and young normal dogs. There were no effects on phosphorylation of troponin I, troponin T, phospholamban, or myosin light chain-1 or -2. Titin phosphorylation increased with sildenafil and BNP, whereas titin-based cardiomyocyte stiffness decreased. Short-term cGMP-enhancing treatment with sildenafil and BNP improves left ventricular diastolic distensibility in vivo, in part by phosphorylating titin.

Pulmonary Hypertension and Right Ventricular Function in Advanced Heart Failure

Pulmonary hypertension (PH) and right ventricular (RV) dysfunction are frequently encountered in patients with advanced heart failure (HF). Both conditions aggravate prognosis and influence clinical decisions. Echocardiography is the screening tool of choice for pulmonary pressures and RV function, although invasive assessment of PH is necessary when advanced therapies are considered. Reversibility of PH in response to short-term pharmacologic treatment or even to long-term unloading after left ventricular assist device (LVAD) implantation is a favorable prognostic sign for both medically treated patients and heart transplant candidates. Although patients with severe PH secondary to HF have not derived benefit from pulmonary arterial hypertension therapies thus far, agents that modulate the cyclic guanosine monophosphate pathway, including phosphodiesterase 5A inhibitors, hold promise and are being actively investigated in advanced HF. Therapies that lead to reduction in left-sided pressures, including cardiac resynchronization and LVAD placement, also have a favorable effect on pulmonary pressures and RV function. However, no specific medical treatment for RV dysfunction exists to date, highlighting an important gap in the management of patients with advanced HF.

Phosphodiesterase-5A (PDE5A) is localized to the endothelial caveolae and modulates NOS3 activity

It has been well demonstrated that phosphodiesterase-5A (PDE5A) is expressed in smooth muscle cells and plays an important role in regulation of vascular tone. The role of endothelial PDE5A, however, has not been yet characterized. The present study was undertaken to determine the presence, localization, and potential physiologic significance of PDE5A within vascular endothelial cells. We demonstrate primary location of human, mouse, and bovine endothelial PDE5A at or near caveolae. We found that the spatial localization of PDE5A at the level of caveolin-rich lipid rafts allows for a feedback loop between endothelial PDE5A and nitric oxide synthase (NOS3). Treatment of human endothelium with PDE5A inhibitors resulted in a significant increase in NOS3 activity, whereas overexpression of PDE5A using an adenoviral vector, both in vivo and in cell culture, resulted in decreased NOS3 activity and endothelium-dependent vasodilation. The molecular mechanism responsible for these interactions is primarily regulated by cGMP-dependent second messenger. PDE5A overexpression also resulted in a significant decrease in protein kinase 1 (PKG1) activity. Overexpression of PKG1 rapidly activated NOS3, whereas silencing of the PKG1 gene with siRNA inhibited both NOS3 phosphorylation (S1179) and activity, indicating a novel role for PKG1 in direct regulation of NOS3. Our data collectively suggest another target for PDE5A inhibition in endothelial dysfunction and provide another physiologic significance for PDE5A in the modulation of endothelial-dependent flow-mediated vasodilation. Using both in vitro and in vivo models, as well as human data, we show that inhibition of endothelial PDE5A improves endothelial function.

Diet-Induced Muscle Insulin Resistance Is Associated With Extracellular Matrix Remodeling and Interaction With Integrin α2β1 in Mice

OBJECTIVEThe hypothesis that high-fat (HF) feeding causes skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J mice and that this remodeling contributes to diet-induced muscle insulin resistance (IR) through the collagen receptor integrin α2β1 was tested.RESEARCH DESIGN AND METHODSThe association between IR and ECM remodeling was studied in mice fed chow or HF diet. Specific genetic and pharmacological murine models were used to study effects of HF feeding on ECM in the absence of IR. The role of ECM-integrin interaction in IR was studied using hyperinsulinemic-euglycemic clamps on integrin α2β1-null (itga2−/−), integrin α1β1-null (itga1−/−), and wild-type littermate mice fed chow or HF. Integrin α2β1 and integrin α1β1 signaling pathways have opposing actions.RESULTSHF-fed mice had IR and increased muscle collagen (Col) III and ColIV protein; the former was associated with increased transcript, whereas the latter was associated with reduced matrix metalloproteinase 9 activity. Rescue of muscle IR by genetic muscle-specific mitochondria-targeted catalase overexpression or by the phosphodiesterase 5a inhibitor, sildenafil, reversed HF feeding effects on ECM remodeling and increased muscle vascularity. Collagen remained elevated in HF-fed itga2−/− mice. Nevertheless, muscle insulin action and vascularity were increased. Muscle IR in HF-fed itga1−/− mice was unchanged. Insulin sensitivity in chow-fed itga1−/− and itga2−/− mice was not different from wild-type littermates.CONCLUSIONSECM collagen expansion is tightly associated with muscle IR. Studies with itga2−/− mice provide mechanistic insight for this association by showing that the link between muscle IR and increased collagen can be uncoupled by the absence of collagen-integrin α2β1 interaction.

Myocardial Reperfusion Injury: Reactive Oxygen Species vs. NHE-1 Reactivation

Background/Aims: Flow restoration to ischemic myocardium reduces infarct size (IS), but it also promotes reperfusion injury. A burst of reactive oxygen species (ROS) and/or NHE-1 reactivation were proposed to explain this injury. Our study was aimed to shed light on this unresolved issue. Methods: Regional infarction (40 min-ischemia/2 hs-reperfusion) was induced in isolated and perfused rat hearts. Maximal doses of N-(2-mercaptopropionyl)-glycine (MPG 2mmol/L, ROS scavenger), cariporide (10µmol/L, NHE-1 inhibitor), or sildenafil (1µmol/L, phosphodiesterase5A inhibitor) were applied at reperfusion onset. Their effects on IS, myocardial concentration of thiobarbituric acid reactive substances (TBARS), ERK1/2, p90^RSK, and NHE-1 phosphorylation were analyzed. Results: All treatments decreased IS ∼ 50% vs. control. No further protection was obtained by combining cariporide or MPG with sildenafil. Myocardial TBARS increased after infarction and were decreased by MPG or cariporide, but unaffected by sildenafil. In line with the fact that ROS induce MAPK-mediated NHE-1 activation, myocardial infarction increased ERK1/2, p90^RSK, and NHE-1 phosphorylation. MPG and cariporide cancelled these effects. Sildenafil did not reduce the phosphorylated ERK1/2-p90^RSK levels but blunted NHE-1 phosphorylation suggesting a direct dephosphorylating action. Conclusions: 1) Reperfusion injury would result from ROS-triggered MAPK-mediated NHE-1 phosphorylation (and reactivation) during reperfusion; 2) sildenafil protects the myocardium by favouring NHE-1 dephosphorylation and bypassing ROS generation.

Rapid HPLC-UV method for quantification of l-citrulline in watermelon and its potential role on smooth muscle relaxation markers

A rapid and versatile HPLC-UV method for the separation and quantification of l-citrulline from watermelon was developed for the first time. Freeze dried juice and rind of watermelon were extracted with acidic methanol and analysed by HPLC using 0.03mM phosphoric acid as a mobile phase. The separation was performed using Zorbax SB-Aq, Synergi Hydro-RP and Gemini C-18 columns and l-citrulline was detected at 207nm. l-Citrulline was well separated using Gemini C-18 column and the identity of peak was confirmed by mass spectral analysis. These results were further confirmed by derivatisation method. The developed method was used for the quantification of l-citrulline in three varieties of watermelons such as Petite treat, Jamboree and Yellow crimson. Thus, the method is simple and involves direct analysis of aqueous extracts and can be used for rapid routine analysis of commercial samples. In addition, the role of watermelon extracts on smooth muscle relaxation markers was studied. Petite treat and Jamboree extracts induced production of nitric oxide (NO) significantly at 25ppm. This was also confirmed by measuring the change in the intracellular calcium. Additionally, extracts of petite treat rind and juice have shown inhibition of phosphodiesterase-5A (PDE-5A) in human uterine smooth muscle cells (UtSMC). The induction of NO and the inhibition of PDE-5A seems to suggest the potency of these extracts to induce relaxation of smooth muscle. It is possible that watermelon extracts may have the potential to induce PDE-5A mediated smooth muscle relaxation. However, further studies are critical before any conclusion of smooth muscle relaxation effect.

Phosphodiesterase inhibition with tadalafil provides longer and sustained protection of stem cells

We hypothesized that inhibition of the cGMP-specific enzyme phosphodiesterase 5A (PDE5A) promoted cGMP/protein kinase G (PKG) activity to condition stem cells for enhanced survival and proliferation. One-time tadalafil treatment (1 μM for 30 min) of mesenchymal stem cells ((Tada)MSCs) provided sustained protection of cells for 36 h. Higher cGMP activity with concomitantly increased PKG1 activity was observed in (Tada)MSCs, which peaked within 12 h after tadalafil treatment. Pretreatment with PKG1 blockers (1 μM KT-5823 or 20 nM K-252a) or transduction with adenoviral PKG1-short-hairpin RNA abolished tadalafil-induced cytoprotection of the cells. A higher proliferation rate was observed in (Tada)MSCs compared with nontreated MSCs ((Cont)MSCs). In a rat model of acute myocardial infarction, (Tada)MSCs transplanted 0 and 24 h after tadalafil treatment showed higher survival compared with (Cont)MSCs on day 2 and day 4 after engraftment. (Tada)MSCs transplanted 48 h after tadalafil treatment lost their protection on both day 2 and day 4 after engraftment, and their rate of survival was similar to (Cont)MSCs. Reduced terminal dUTP nick end-labeling positivity (P < 0.01 vs. (Cont)MSCs) and higher proliferation of (Tada)MSCs (P < 0.01 vs. (Cont)MSCs) was observed in the infarcted heart. Fluorescence immunostaining revealed neomyogenesis in both the infarct and peri-infarct areas. Blood vessel density was significantly increased in group 2 compared with group 1. Transthoracic echocardiographic heart function revealed significant preservation of the indexes of left ventricle contractility and attenuation of remodeling in (Tada)MSC-engrafted animal hearts (group 2) compared with (Cont)MSCs (group 1). PDE5A inhibition using long-acting tadalafil is an innovative approach to promote stem cell survival and proliferation in the infarcted heart.

Decreased Activity of the Na + /H + Exchanger by Phosphodiesterase 5A Inhibition Is Attributed to an Increase in Protein Phosphatase Activity

The beneficial effect of phosphodiesterase 5A inhibition in ischemia/reperfusion injury and cardiac hypertrophy is well established. Inhibition of the cardiac Na(+)/H(+) exchanger (NHE-1) exerts beneficial effects on these same conditions, and a possible link between these therapeutic strategies was suggested. Experiments were performed in isolated cat cardiomyocytes to gain insight into the intracellular pathway involved in the reduction of NHE-1 activity by phosphodiesterase 5A inhibition. NHE-1 activity was assessed by the rate of intracellular pH recovery from a sustained acidic load in the absence of bicarbonate. Phosphodiesterase 5A inhibition with sildenafil (1 μmol/L) did not affect basal intracellular pH; yet, it did decrease proton efflux (J(H); in millimoles per liter per minute) after the acidic load (proton efflux: 6.97±0.43 in control versus 3.31±0.58 with sildenafil; P<0.05). The blockade of both protein phosphatase 1 and 2A with 100 nmol/L of okadaic acid reverted the sildenafil effect (proton efflux: 6.77±0.82). In contrast, selective inhibition of protein phosphatase 2A (1 nmol/L of okadaic acid or 100 μmol/L of endothall) did not (3.86±1.0 and 2.61±1.2), suggesting that only protein phosphatase 1 was involved in sildenafil-induced NHE-1 inhibition. Moreover, sildenafil prevented the acidosis-induced increase in NHE-1 phosphorylation without affecting activation of the extracellular signal-regulated kinase 1/2-p90(RSK) pathway. Our results suggest that phosphodiesterase 5A inhibition decreases NHE-1 activity, during intracellular pH recovery after an acidic load, by a protein phosphatase 1-dependent reduction in NHE-1 phosphorylation.

Oral sildenafil increases skin hyperaemia induced by iontophoresis of sodium nitroprusside in healthy volunteers

Sildenafil, a specific inhibitor of phosphodiesterase 5A (PDE5A), is currently tested as a treatment for severe Raynaud's phenomenon. Here, we tested whether sildenafil, alone or combined with local sodium nitroprusside (SNP) delivered through skin iontophoresis, increased forearm cutaneous blood conductance in healthy volunteers, and to assess how well this combination was tolerated. Ten healthy volunteers were enrolled. Variations in cutaneous vascular conductance (CVC) following oral administration of 50 or 100 mg of sildenafil with or without SNP iontophoresis were expressed as a percentage of maximal CVC, and were monitored using laser Doppler imaging. SNP iontophoresis was performed on the ventral surface of the forearm, 1 h after application of lidocaine/prilocaine cream. Sildenafil at 100 mg, but not 50 mg, increased overall responses (area under the curve) (44%) and peak responses (29%) to SNP iontophoresis. Sildenafil at 100 mg, but not 50 mg, increased baseline CVC (75%). Incidence of headache was not changed when SNP iontophoresis was combined with sildenafil. One episode of symptomatic arterial hypotension occurred in a volunteer given 50 mg sildenafil, 30 min after the beginning of SNP iontophoresis. Oral sildenafil at 100 mg potentiated local skin hyperaemia induced by SNP iontophoresis, with no increased incidence of headaches. The combination of oral specific PDE5A inhibitor and nitrates administered through skin iontophoresis deserves further investigation in diseases such as severe Raynaud's phenomenon, with particular attention to the incidence of arterial hypotension.

Phosphodiesterase 5A Inhibition Decreases NHE-1 Activity Without Altering Steady State pHi: Role of Phosphatases

Background/Aims: This study aimed to identify the signaling pathway for the proposed link between phosphodiesterase-5A (PDE5A) inhibition and decreased cardiac Na⁺/H⁺ exchanger (NHE-1) activity. Methods: NHE-1 activity was assessed in rat isolated papillary muscles by the Na⁺-dependent initial pH_i recovery from a sustained acidosis (ammonium prepulse). ERK1/2, p90RSK and NHE-1 phosphorylation state during acidosis was determined. Results: PDE5A inhibition (1 µmol/L sildenafil, SIL) did not modify basal pH_i but significantly blunted pH_i recovery after sustained acidosis. Although preventing ERK1/2- p90RSK signaling pathway (10 µmol/L U0126) mimicked SIL effect, SIL did not blunt the acidosis-mediated increase in kinases activation. SIL+U0126 did not show additive effect on NHE-1 activity. Then, we hypothesized that SIL could be activating phophasatases (PP1 and/or PP2A) to directly dephosphorylate NHE-1 despite preserved ERK1/2-p90RSK activation. Non-specific phosphatases inhibition (1 µmol/L okadaic acid) canceled SIL effect on pH_i recovery from acidosis. Same result was observed by inhibiting PP2A either with a lower dose of okadaic acid (1 nmol/L) or, more specifically, with 100 µmol/L endothall. Consistently, NHE-1 phosphorylation at Ser703 increased after acidosis, SIL prevented this effect and PP2A inhibition (endothall) reverted SIL effect. Conclusion: We suggest that PDE5A inhibitors decrease NHE-1 phosphorylation and activity through a mechanism that involves PP2A activation.

Sildenafil increases the force of right atrial contractions in vitro via the NO-guanylyl cyclase pathway involving β-adrenoceptor linked mechanisms

Sildenafil, a drug used in the treatment of erectile dysfunction, is a phosphodiesterase 5A inhibitor that increases cyclic guanosine monophosphate (cGMP) levels. In addition to its vascular actions, sildenafil is also known to alter cardiac functions. This study was undertaken to elucidate the effect of sildenafil on cardiac contractility and the underlying mechanisms. The experiments were conducted on spontaneously-beating right atria isolated from adult rats. The effect of sildenafil on the isometric contractions in vitro was examined in the absence or presence of antagonists. Sildenafil (0.001–10μM) produced a concentration-dependent increase in the atrial force of contraction without altering the atrial rate, even up to 10μM. A concentration as low as 0.001μM produced a significant increase (16%) in force and the increase was about 50% at 10μM. Pretreatment with methylene blue (a guanylyl cyclase inhibitor) or N-ω-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) blocked the force changes induced by sildenafil. Sildenafil-induced increase in force of contraction was also blocked by propranolol (a β-adrenoceptor antagonist) and diltiazem (an L-type Ca2+ channel antagonist). The present results demonstrate that sildenafil increases the atrial force of contraction involving cGMP-β-adrenoceptor-Ca2+ channel-dependent mechanisms.

Letter by Cingolani et al Regarding Article, “Ventricular Phosphodiesterase-5 Expression Is Increased in Patients With Advanced Heart Failure and Contributes to Adverse Ventricular Remodeling After Myocardial Infarction in Mice”

To the Editor: Pokreisz et al1 recently published an interesting article in which they show increased cardiac left ventricular phosphodiesterase-5A (PDE5A) expression in patients with heart failure. They also generated a PDE5A transgenic mouse in which overexpression of this cGMP-selective phosphodiesterase worsened ventricular remodeling and function after myocardial infarction. Therefore, the obvious conclusion seems to be that inhibition of PDE5A might protect against postmyocardial infarction remodeling. Although the authors show cGMP to be related to these cardiac architectural changes, …

Response to Letter Regarding Article, “Ventricular Phosphodiesterase-5 Expression Is Increased in Patients With Advanced Heart Failure and Contributes to Adverse Ventricular Remodeling After Myocardial Infarction in Mice”

We appreciate the comments by Cingolani et al on a possible involvement of Na+/H+ exchanger (NHE-1) in signaling mechanisms downstream of cGMP that can mediate cardioprotection after myocardial infarction. These authors previously reported that NHE-1 expression and activity are increased after myocardial infarction in rats (as a result of myocardial acidosis) and that sustained …

Phosphodiesterase 5A Inhibitors Improve Functional Recovery after Stroke in Rats: Optimized Dosing Regimen with Implications for Mechanism

Phosphodiesterase 5A (PDE5A) inhibitors improve functional recovery after middle cerebral artery occlusion (MCA-o) in rats. We used the PDE5A inhibitor 3-(4-(2-hydroxyethyl)piperazin-1-yl)-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one hydrochloride (PF-5) to determine the timing, duration, and degree of inhibition that yields maximum efficacy. We also investigated the localization of PDE5A to determine the tissues and cells that would be targets for PDE5 inhibition and that may mediate efficacy. Nearly complete inhibition of PDE5A, starting 24 h after MCA-o and continued for 7 days, resulted in nearly complete recovery of sensorimotor function that was sustained for 3 months. Delaying administration until 72 h after MCA-o resulted in equivalent efficacy, whereas delaying treatment for 14 days was ineffective. Treatment for 7 days was equivalently efficacious to 28 or 84 days of treatment, whereas treatment for 1 day was less effective. In the normal forebrain, PDE5A immunoreactivity was prominent in smooth muscle of meningeal arteries and a few smaller blood vessels, with weak staining in a few widely scattered cortical neurons and glia. At 24 and 48 h after MCA-o, the number and intensity of blood vessel staining increased in the infarcted cortex and striatum. PDE5A immunoreactivity also was increased at 48 h in putative microglia in penumbra, whereas there was no change in staining of the scattered cortical neurons. Given the window for efficacy and the PDE5A distribution, we hypothesize that efficacy results from an effect on vasculature, and perhaps modulation of microglial function, both of which may facilitate recovery of neuronal function.

Transpulmonary B-Type Natriuretic Peptide Uptake and Cyclic Guanosine Monophosphate Release in Heart Failure and Pulmonary Hypertension: The Effects of Sildenafil

We sought to identify factors that discriminate heart failure (HF) patients with normal and elevated pulmonary vascular resistance (PVR) and to elucidate the role of cyclic guanosine monophosphate (cGMP)-dependent vasodilation. Mechanisms of PVR increase in patients with chronic HF are incompletely understood. Twenty-two HF patients with high pulmonary vascular resistance (H-PVR) (>200 dyn.s.cm(-5)) were compared with 24 matched low pulmonary vascular resistance (L-PVR) patients of similar age, sex, body size, HF severity, and volume status who were undergoing invasive hemodynamic study. Pulmonary arterial (PA) and venous blood samples from a wedged PA catheter were used to calculate transpulmonary B-type natriuretic peptide (BNP) uptake and cGMP release. The H-PVR patients were re-examined 1 h after a 40-mg oral dose of sildenafil. Although transpulmonary BNP uptake was similar (p = 0.2), cGMP release was diminished in the H-PVR patients (-1.9 vs. 27.8 nmol.min(-1); p = 0.005). Transpulmonary BNP uptake and cGMP release correlated in the L-PVR patients (R = 0.6, p = 0.004) but not in the H-PVR. The H-PVR patients also had lower PA compliance, systemic arterial compliance (by 47% and 20%, p < 0.001 and p < 0.03), and cardiac index. Sildenafil reduced PVR (-47%), systemic resistance (-24%) and heart rate (-8%), increased cardiac index (+24%), and PA compliance (+87%, all p < 0.001), with a parallel increase of cGMP release (from -5.6 to 16.5 nmol.min(-1), p = 0.047), without affecting BNP uptake or norepinephrine(PA). The PVR response was not dependent on PA wedge pressure or pulmonary hypertension reversibility with prostaglandin E(1). The H-PVR patients have stiffening of both pulmonary and systemic arteries, preserved transpulmonary BNP uptake, but diminished cGMP release, which is reversible by the administration of sildenafil. This study provides in vivo evidence that phosphodiesterase 5A inhibition restores sensitivity of pulmonary vasculature to endogenous cGMP-dependent vasodilators.