sGC Stimulator Research Articles

Sodium nitrite causes relaxation of the isolated rat aorta: By stimulating both endothelial NO synthase and activating soluble guanylyl cyclase in vascular smooth muscle

Ingestion of dietary nitrites lowers arterial blood pressure in experimental animals and in humans. However, the exact mechanism underlying the hypotensive effect of nitrite remains unclear. The present study compared nitrite-induced responses in rings (with or without endothelium) of aortae of 18–20weeks old Wistar–Kyoto Rats (WKY) and spontaneously hypertensive (SHR) rats and investigated the underlying mechanism. Relaxations of aortae from WKY and SHR to increasing concentrations (1nM–100μM) of sodium nitrite (NaNO2) were determined during sustained contractions to phenylephrine, in the absence and presence of pharmacological agents. The nitrite-induced relaxations were concentration-dependent and larger in SHR than in WKY aortic rings. Inhibition of endothelial nitric oxide synthase (eNOS) and the absence of endothelium decreased nitrite-induced relaxations in both WKY and SHR aortae, indicating the role of endothelium-derived nitric oxide (NO) in the response. The involvement of eNOS was further confirmed by increases in phosphorylation of eNOS at ser1177 in HUVEC cells following treatment with sodium nitrite. The presence of NO scavengers decreased the relaxation to nitrite in both WKY and SHR preparations while inhibition of soluble guanylyl cyclase (sGC) abolished the response, indicating that besides producing NO, nitrite also induces relaxation by directly activating the enzyme. Thus, the present study demonstrates that the sensitivity to exogenous nitrite is increased in the aorta of the SHR compared to that of the WKY. The endothelium-dependent component of the relaxation to nitrite involves activation of eNOS with production of endothelium-derived NO, while the endothelium-independent component is due to stimulation of sGC.

Stimulation of soluble guanylyl cyclase protects against obesity by recruiting brown adipose tissue.

Obesity is characterized by a positive energy balance and expansion of white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) combusts energy to produce heat. Here we show that a small molecule stimulator (BAY 41-8543) of soluble guanylyl cyclase (sGC), which produces the second messenger cyclic GMP (cGMP), protects against diet-induced weight gain, induces weight loss in established obesity, and also improves the diabetic phenotype. Mechanistically, the haeme-dependent sGC stimulator BAY 41–8543 enhances lipid uptake into BAT and increases whole-body energy expenditure, whereas ablation of the haeme-containing β1-subunit of sGC severely impairs BAT function. Notably, the sGC stimulator enhances differentiation of human brown adipocytes as well as induces ‘browning' of primary white adipocytes. Taken together, our data suggest that sGC is a potential pharmacological target for the treatment of obesity and its comorbidities.

The soluble guanylyl cyclase stimulator riociguat reduces liver fibrosis and portal pressure in cirrhotic rats

Background: In liver cirrhosis nitric oxide (NO) signaling, including function of its receptor soluble guanylyl cyclase (sGC), is distorted. The sGC stimulator riociguat (RIO) is used as treatment for pulmonary hypertension and it has been shown that RIO acts antifibrotic. We thus aimed to investigate effects of RIO on experimental liver cirrhosis and portal hypertension.

Riociguat: a novel new drug for treatment of pulmonary hypertension.

Riociguat is the first approved medication from the novel class of soluble guanylate cyclase (sGC) stimulators and the only agent approved for treating both chronic thromboembolic hypertension (CTEPH) and pulmonary arterial hypertension (PAH). The novel mechanism of riociguat lies in its ability to restore the homeostatic and therapeutic effects of nitric oxide that are diminished as a result of phenotypic alterations associated with pulmonary hypertension (PH). Improvements in 6-minute walk distance (6MWD) in patients with PAH during the phase 3 PATENT-1 trial were comparable to other oral agents approved for the treatment of PAH. Improvements in 6MWD in patients with CTEPH during the phase 3 CHEST-1 trial were greater than those previously observed with other oral PAH-directed therapies. Hypotension is the dose-limiting adverse effect of riociguat and dose titration is performed gradually according to systolic blood pressure. Riociguat was tolerated at maximal doses by most patients during PATENT-1 and CHEST-1 and was well tolerated during long-term extension studies. Key factors to consider with riociguat are a patient's systolic blood pressure, drug interactions mediated by CYP1A1, CYP3A4, and P-glycoprotein, cost, and teratogenicity requiring enrollment in a Risk Evaluation and Mitigation Strategy program. Recently published guidelines recommend riociguat monotherapy as an option for treatment-naïve patients with World Health Organization Functional Class (WHO FC) II or III symptoms or as add-on therapy for patients with persistent WHO FC III or IV symptoms being treated with an ERA or inhaled prostanoid. Postmarketing experience and ongoing clinical investigations will further define the safety and role of riociguat in patients with PAH and other types of PH.

Effect of soluble guanylyl cyclase activator and stimulator therapy on nitroglycerin-induced nitrate tolerance in rats

Chronic nitroglycerin (GTN) anti-ischemic therapy induces side effects such as nitrate tolerance and endothelial dysfunction. Both phenomena could be based on a desensitization/oxidation of the soluble guanylyl cyclase (sGC). Therefore, the present study aims at investigating the effects of the therapy with the sGC activator BAY 60-2770 and the sGC stimulator BAY 41-8543 on side effects induced by chronic nitroglycerin treatment. Male Wistar rats were treated with nitroglycerin (100mg/kg/d for 3.5days, s.c. in ethanol) and BAY 60-2770 (0.5 or 2.5mg/kg/d) or BAY 41-8543 (1 and 5mg/kg/d) for 6days. Therapy with BAY 60-2770 but not with BAY 41-8543 improved nitroglycerin-triggered endothelial dysfunction and nitrate tolerance, corrected the decrease in aortic nitric oxide levels, improved the cGMP dependent activation of protein kinase I in aortic tissue and reduced vascular, cardiac and whole blood oxidative stress (fluorescence and chemiluminescence assays; 3-nitrotyrosine staining). In contrast to BAY 41-8543, the vasodilator potency of BAY 60-2770 was not impaired in isolated aortic ring segments from nitrate tolerant rats. sGC activator therapy improves partially the adverse effects of nitroglycerin therapy whereas sGC stimulation has only minor beneficial effects pointing to a nitroglycerin-dependent sGC oxidation/inactivation mechanism contributing to nitrate tolerance.

SGC Stimulation Results in Potent Anti‐hypertensive Effects as a Stand‐alone or Combination Therapy in the Rat

Soluble guanylate cyclase (sGC) stimulators have the potential to treat a variety of cardiovascular diseases via the nitric oxide (NO)‐sGC‐cyclic guanosine monophosphate (cGMP) pathway. Ironwood Pharmaceuticals has synthesized a number of novel sGC stimulators with the objective of creating a once‐a‐day oral anti‐hypertensive.A screening paradigm that evaluated sGC enzyme stimulation, pharmacokinetics (PK), and pharmacodynamics (PD) identified three novel sGC stimulators that induce sustained cardiovascular effects after oral dosing. Potency against sGC was evaluated using an in vitro cell based assay that measured cGMP production. Compounds were subsequently evaluated in PK and blood pressure PD in vivo rat models. All compounds were potent stimulators of sGC in vitro (EC50 = 40‐220nM), had sustained compound exposure in vivo (Thalf 7‐12 hours) after oral dosing, and significantly decreased mean arterial blood pressure (MABP) in vivo (蠅 10mmHg) after oral dosing.The potential for sGC stimulators to work in combination with reference anti‐hypertensive therapies was assessed in our in vivo PD assay. Doses of Losartan, Atenolol, Amlodipine or each of our sGC stimulators that induced a decrease in MABP (蠅 10mmHg) when dosed alone were chosen. Combining sGC stimulators with anti‐hypertensives resulted in an additional 5‐50% decrease in MABP over either therapy given alone. These data indicate that IWP sGC stimulators possess potent anti‐hypertensive effects in the rat and that their efficacy increases when dosed in combination with standard anti‐hypertensive agents.This research was funded by Ironwood Pharmaceuticals.

IWP‐427, an sGC Stimulator, Has Cardioprotective Effects in the Monocrotaline Model of Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) results in increased resistance of blood return to the pulmonary vasculature and concomitant hypertrophy of the right ventricle of the heart (RHH). Stimulation of the NO/sGC/cGMP pathway has been shown to reduce RHH in preclinical models of PAH and improves clinical outcomes in patients with PAH. We therefore hypothesized that prophylactic treatment with an sGC stimulator would prevent RHH in a preclinical model of PAH. IWP‐427 is a potent (HEK EC50=167nM), orally bioavailable sGC stimulator. In this study, a single subcutaneous injection of monocrotaline (MCT; 60mg/kg) induced significant RHH in male rats compared to naïve controls after 4 weeks as measured by the ratio of right heart weight to the left ventricle and septum (0.33±0.02 vs 0.22±0.01; p<0.001; n=9‐12). Prophylactic treatment with IWP‐427 prevented MCT‐induced RHH as compared to vehicle treated animals (0.21±0.03 vs 0.33±0.02; p<0.01; n=9‐12). Additionally, plasma levels of NT‐proBNP, a marker of right ventricular damage, were significantly lower in animals treated with IWP‐427 compared to vehicle (10913±1024 vs 15961±1993 ECL units; p<0.05; n=8). IWP‐427 is therefore a cardioprotective sGC stimulator in a monocrotaline model of PAH.

Differential synergy with nitric oxide across a panel of soluble guanylate cyclase stimulators

Soluble guanylate cyclase (sGC) is the endogenous receptor for nitric oxide (NO). Upon binding of NO, sGC catalyzes the synthesis of cGMP which is involved in numerous downstream processes, including vasorelaxation, fibrosis, and inflammation. Dysregulation of this pathway has been implicated in multiple pathologies. sGC stimulators are a class of molecules which are capable of increasing the activity of the enzyme. While these stimulators are capable of activating the enzyme in the absence of NO, their effect is greatly amplified in the presence of NO. However, it is unclear if all stimulators synergize with NO equally. To examine differences in NO synergy across stimulators, we assessed a panel of compounds for their ability to stimulate cGMP synthesis with or without NO in a human whole cell assay. All compounds selected for this analysis were potent in vitro under high NO conditions (with EC50 values ranging from 10 to 200nM) and were effective at lowering blood pressure in rodents at doses between 1 and 10 mg/kg. We found that all compounds tested had similar EC50 values (~5µM) when examined in the absence of NO. However, in the presence of high NO the compounds exhibited a wide range of potency shifts relative to the NO‐free state (16‐320 fold). Emax values for the compounds were also differentially affected by NO, with some compounds reaching the same Emax regardless of the presence of NO, and others exhibiting a 4‐6 fold increase in Emax upon addition of NO. These data suggest that synergy between NO and sGC stimulators is complex and varies considerably between compounds. We hypothesize that differences between compounds in NO synergy may be harnessed to maximize their therapeutic value in a variety of disease states and patient populations.

SGC Stimulator Efficacy Beyond Blood Pressure in Dahl‐Salt Sensitive Model of Hypertension

Soluble guanylate cyclase (sGC) is an important intracellular receptor that can be activated by sGC stimulators to produce cyclic guanosine monophosphate (cGMP)protein kinase G (PKG) mediated effects. We have identified a novel series of sGC stimulators that decreased blood pressure (BP) in rats in a dose‐dependent manner at oral doses between 1‐10 mg/kg. These compounds possess a rat pharmacokinetic profile consistent with predicted once daily administration in humans. In this study we evaluated IWP‐121 in the Dahl Salt‐Sensitive (DSS) model of hypertension. Male DSS rats received 2 weeks of high‐salt diet prior to high salt plus compound for 6 additional weeks. Compounds were tested at doses of 1, 3, and 10 mg/kg/day eq in the chow (n=8/group) using Losartan as a positive control at a dose of 30 mg/kg/day eq in the water and compared to a high‐salt (hs) control group who received high salt diet for 8 weeks. IWP‐121 dose dependently decreased mean BP at week 8 with a maximal effect of 49 mmHg at the 10mg/kg dose. In comparison, losartan reduced mean BP by 27 mmHg. Both IWP‐121 (at all doses tested) and losartan had statistically significant effects on heart hypertrophy compared with the high‐salt (HS) group (3.5± 0.1 vs 4.5 ±0.3 g/Kg; p<0.001). IWP‐121 was efficacious at decreasing microalbuminaria in the urine (an indicator of kidney end organ protection) and serum biomarkers known to be involved in inflammatory and fibrotic processes (TIMP‐1, OPN and MCP‐1) in a dose‐dependent manner. In summary, IW‐121 demonstrated a potent anti‐hypertensive effect, additional effects in end organ protection, and effects on biomarkers consistent with anti‐fibrotic/anti‐inflammatory activity.

Aminolevulinic Acid Treatment of Pulmonary Arteries Attenuates Endothelin‐1 and Angiotensin II Elicited Increases in Mitochondrial, but not Extra‐Mitochondrial Superoxide

We recently reported that treatment of mice with the heme biosynthesis precursor δ‐aminolevulinic acid (ALA) attenuates the development of hypoxia‐induced pulmonary hypertension. In addition, we previously reported that 24‐hour organoid culture of bovine pulmonary arteries (BPAs) with ALA caused an increase in soluble guanylate cyclase (sGC) activity by generating protoporphyrin IX, a stimulator of sGC and cGMP‐mediated activation of PKG. This study examines how heme biosynthesis modulation with ALA potentially functions to prevent the effects of 24‐hour organoid culture of BPAs with the hypoxia and pulmonary hypertension mediators endothelin‐1 (ET‐1) and angiotensin II (AngII), with a focus on detecting subcellular changes in superoxide production. Organoid culture of BPAs with 10 nM ET‐1 or 0.1 µM AngII for 24‐hours caused increases in mitochondrial (MitoSox/HPLC) and extra‐mitochondrial/cytosolic (DHE/HPLC) superoxide, but ALA only attenuated the increases in mitochondrial superoxide. Since ALA also attenuates an ET‐1 elicited remodeling regulatory mechanism controlled by src kinase phosphorylation of STAT3 and its associated lowering of miR204 in organoid cultured BPA and the chronic hypoxia‐elicited depletion of miR204 in mouse lungs, the prevention of increased mitochondrial, but not extra‐mitochondrial superoxide generation could be a key factor in how ALA attenuates processes in pulmonary arteries that contribute to hypertension development.

Reduced Relaxation Responses to Heme‐dependent and –independent Soluble Guanylyl Cyclase Activation in Mesenteric Arteries of a Rat Model of Type 2 Diabetes

Endothelial dysfunction is a main feature of type 2 diabetes. Impairment in vascular smooth muscle cell function may also contribute to diabetes‐associated microvascular and macrovascular complications. We hypothesized that mesenteric resistance arteries (MA) from non‐obese rats with type 2 diabetes (Goto‐Kakizaki, GK) would have reduced responses to soluble guanylyl cyclase (sGC) activation as compared to controls (Wistar). Endothelium‐dependent and –independent relaxation was assessed in isolated arterial segments mounted in a wire myograph. GK MA had reduced responses to acetylcholine (ACh; ‐logEC50: 7.92 ± 0.08 vs. 7.65 ± 0.06, p<0.05) and sodium nitroprusside (SNP; ‐logEC50: 8.43 ± 0.08 vs. 7.84 ± 0.06, p<0.05), but there were no group differences in 8‐Br‐cGMP‐induced relaxation and PKG‐1 and phosphorylated VASPSer239 expression (p>0.05). GK MA had attenuated responses to BAY41‐2272 (heme‐dependent sGC stimulator; ‐logEC50: 7.55 ± 0.07 vs. 6.88 ± 0.08, p<0.05) and BAY58‐2667 (heme‐independent sGC stimulator; ‐logEC50: 10.82 ± 0.07 vs. 10.27 ± 0.08, p<0.05) compared to control values but pharmacological inhibition of sGC abolished these differences. A reduction in sGC activity may account for impaired vascular smooth muscle responsiveness to dilatory stimuli in type 2 diabetes.

Different influences of extracellular and intracellular superoxide on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries.

Superoxide production is increased in diseased blood vessels, which is considered to lead to impairment of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway. To investigate the respective influence of extracellular and intracellular superoxide on vascular function through the NO/sGC/cGMP pathway, mechanical responses of rat external iliac arteries without endothelium were studied under exposure to a superoxide-generating agent, pyrogallol, or menadione. Exposure to pyrogallol impaired the relaxation induced by acidified NaNO2 (exogenous NO) but not that by nitroglycerin (organic nitrate), BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator), or 8-Br-cGMP (cGMP analog). Superoxide dismutase (SOD) and tempol restored the impaired relaxation by acidified NaNO2. Superoxide production in the bathing solution, but not in artery segments, was significantly increased by exposure to pyrogallol, which was abolished in the presence of SOD or tempol. However, exposure to menadione impaired the relaxant response to acidified NaNO2, nitroglycerin, or BAY 41-2272, whereas it augmented that to BAY 60-2770. Also, this exposure had no effect on the 8-Br-cGMP-induced vasorelxation. Superoxide production in artery segments was dramatically enhanced by exposure to menadione, whereas that in the bathing solution was not affected. This increase in vascular superoxide production was normalized by tempol but not by SOD. These findings suggest that extracellular superoxide reacts with NO only outside the cell, whereas intracellular superoxide not only scavenges NO inside the cell but also shifts the sGC redox equilibrium.

Effects of Peroxynitrite on Relaxation through the NO/sGC/cGMP Pathway in Isolated Rat Iliac Arteries

Background/Aims: The present study investigated the mechanism by which peroxynitrite impairs vascular function through the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway. Methods: Mechanical responses of rat external iliac arteries without endothelium were studied under exposure to peroxynitrite. cGMP concentrations were determined by enzyme immunoassay. Results: Relaxation induced by BAY 41-2272 (sGC stimulator) was impaired under exposure to peroxynitrite, whereas that by BAY 60-2770 (sGC activator) was enhanced. These responses were correlated with tissue levels of cGMP. Effects of peroxynitrite on the relaxant responses to BAY compounds were also observed in the presence of superoxide dismutase (SOD) or tempol, both of which scavenge a certain kind of reactive molecules other than peroxynitrite. As is the case with the relaxant response to BAY 41-2272, acidified NaNO₂- and nitroglycerin-induced relaxations were markedly attenuated by exposing the arteries to peroxynitrite, which was not abolished by preincubation with SOD or tempol. On the other hand, peroxynitrite exposure had no effect on the 8-Br-cGMP-induced vasorelxation. Conclusion: These findings suggest that peroxynitrite interferes with the NO/sGC/cGMP pathway by altering the redox state of sGC. It is likely that peroxynitrite can shift the sGC redox equilibrium to the NO-insensitive state in the vasculature.

Soluble guanylate cyclase is required for systemic vasodilation but not positive inotropy induced by nitroxyl in the mouse.

Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO·), confers unique physiological effects including vasorelaxation and enhanced cardiac contractility. These features have spawned current pharmaceutical development of HNO donors as heart failure therapeutics. HNO interacts with selective redox sensitive cysteines to effect signaling but is also proposed to activate soluble guanylate cyclase (sGC) in vitro to induce vasodilation and potentially enhance contractility. Here, we tested whether sGC stimulation is required for these HNO effects in vivo and if HNO also modifies a redox-sensitive cysteine (C42) in protein kinase G-1α to control vasorelaxation. Intact mice and isolated arteries lacking the sGC-β subunit (sGCKO, results in full sGC deficiency) or expressing solely a redox-dead C42S mutant protein kinase G-1α were exposed to the pure HNO donor, CXL-1020. CXL-1020 induced dose-dependent systemic vasodilation while increasing contractility in controls; however, vasodilator effects were absent in sGCKO mice whereas contractility response remained. The CXL-1020 dose reversing 50% of preconstricted force in aortic rings was ≈400-fold greater in sGCKO than controls. Cyclic-GMP and cAMP levels were unaltered in myocardium exposed to CXL-1020, despite its inotropic-vasodilator activity. In protein kinase G-1α(C42S) mice, CXL-1020 induced identical vasorelaxation in vivo and in isolated aortic and mesenteric vessels as in littermate controls. In both groups, dilation was near fully blocked by pharmacologically inhibiting sGC. Thus, sGC and cGMP-dependent signaling are necessary and sufficient for HNO-induced vasodilation in vivo but are not required for positive inotropic action. Redox modulation of protein kinase G-1α is not a mechanism for HNO-mediated vasodilation.

Soluble guanylate cyclase: a new therapeutic target for pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension.

Nitric oxide (NO) activates soluble guanylate cyclase (sGC) by binding its prosthetic heme group, thereby catalyzing cyclic guanosine monophosphate (cGMP) synthesis. cGMP causes vasodilation and may inhibit smooth muscle cell proliferation and platelet aggregation. The NO-sGC-cGMP pathway is disordered in pulmonary arterial hypertension (PAH), a syndrome in which pulmonary vascular obstruction, inflammation, thrombosis, and constriction ultimately lead to death from right heart failure. Expression of sGC is increased in PAH but its function is reduced by decreased NO bioavailability, sGC oxidation and the related loss of sGC's heme group. Two classes of sGC modulators offer promise in PAH. sGC stimulators (e.g., riociguat) require heme-containing sGC to catalyze cGMP production, whereas sGC activators (e.g., cinaciguat) activate heme-free sGC. Riociguat is approved for PAH and yields functional and hemodynamic benefits similar to other therapies. Its main serious adverse effect is dose-dependent hypotension. Riociguat is also approved for inoperable chronic thromboembolic pulmonary hypertension.

S39 The Role Of Soluble Guanylate Cyclase Stimulator Bay 41-2272 On Remodelling Processes Relevant To The Pathogenesis Of Pulmonary Arterial Hypertension

Introduction and objectives Pulmonary arterial hypertension (PAH) is characterised by remodelling of small, muscular pre-capillary blood vessels. The subsequent rise in pulmonary vascular resistance leads to right ventricular failure and death. The aetiology of the remodelling process is largely unknown although defects in the bone morphogenetic protein receptor II (BMPR II) pathway are likely to be involved. Most of the therapies used thus far are aimed at pulmonary vasodilation. However it is unclear how much of the benefit seen with these medications is related to reverse remodelling. Riociguat is a “first in class” drug that stimulates soluble guanylate cyclase, with a consequent increase in cyclic GMP (and vasodilation). Riociguat has recently been shown to improve haemodynamics and exercise capacity in patients with idiopathic PAH and chronic thromboembolic PH (PATENT and CHEST). Here we sought to determine the effects of Bay 41–2272, the tool compound for riociguat, on remodelling processes in pulmonary vascular cells. Methods We used primary human endothelial (HPAECs) and smooth muscle cells (HPASMCs) as our target cells. Proliferation was measured using the CyQUANT proliferation kit after cells were treated with various concentration of Bay 41–2272 (kind gifted by Bayer Pharmaceuticals Ltd) for 72 h in the presence of 15% serum. Apoptosis was measured using Cell death ELISA kit and DAPI staining after cells were treated with various concentration of Bay 41–2272 for 24 and 48 h in the absence of serum. Results Bay41–2272 treatment increased HPASMC apoptosis after 24 and 48 h (Figure 1) by Cell death ELISA; this was further confirmed by DNA condensation assay (DAPI staining). Bay 41–2272 treatment also increased HPAEC apoptosis at 24 h. It was not clear at 48 h treatment whether HPAEC cell death was significant in the absence of serum. Bay 41–2272 treatment reduced HPASMC proliferation (Figure 2), but had no effect on HPAECs. Conclusions Our preliminary indicate that Bay 41–2272 increases apoptosis and inhibits proliferation, at least in HPASMCs, in vitro . Further studies are needed to fully characterise these effects on remodelling processes and to compare sGC stimulators, such as Bay 41–2272 to Type V phosphodiesterases.

P158 - IWP-051, a novel, orally available small molecule soluble guanylate cyclase (sGC) stimulator with once-daily dosing potential for the treatment of cardiovascular diseases

P158 - IWP-051, a novel, orally available small molecule soluble guanylate cyclase (sGC) stimulator with once-daily dosing potential for the treatment of cardiovascular diseases

P154 - Evaluation of methodologies to assess intracellular cGMP levels induced by soluble guanylate cyclase (sGC) stimulators in whole cells

P154 - Evaluation of methodologies to assess intracellular cGMP levels induced by soluble guanylate cyclase (sGC) stimulators in whole cells

Reduction of obstruction related bladder overactivity by the guanylyl cyclase modulators BAY 41-2272 and BAY 60-2770 alone or in combination with a phosphodiesterase type 5 inhibitor.

To assess the urodynamic effects of soluble guanylyl cyclase (sGC) stimulator, BAY 41-2272, and activator, BAY 60-2770, (which both are able to induce cGMP synthesis even in the absence of nitric oxide (NO)) alone or in combination with a phosphodiesterase type 5 (PDE5) inhibitor, vardenafil, in a model of partial urethral obstruction (PUO) induced bladder overactivity (BO). Fifty-six male Sprague-Dawley rats were used, 31 of them underwent PUO. Fourteen rats were used for Western blots to assess PDE5 and sGC expression. For drug evaluation cystometry without anesthesia was performed three days following bladder catheterization. Obstructed rats showed higher micturition frequency and bladder pressures than non-obstructed animals (Intermicturition Interval, IMI, 2.28 ± 0.55 vs. 3.60 ± 0.60 min (± standard deviation, SD); maximum micturition pressure, MMP, 70.1 ± 8.0 vs. 48.8 ± 7.2 cmH2O; both P < 0.05). In obstructed rats vardenafil, BAY 41-2272, and BAY 60-2770 increased IMI (2.77 ± 1.12, 2.62 ± 0.52, and 3.22 ± 1.04 min; all P < 0.05) and decreased MMP (54.4 ± 2.8, 61.5 ± 11.3, and 51.2 ± 6.3 cmH2O; all P < 0.05). When vardenafil was given following BAY 41-2272 or BAY 60-2770 no further urodynamic effects were observed. PDE5 as well as sGC protein expression was reduced in obstructed bladder tissue. Targeting sGC via stimulators or activators, which increase the levels of cGMP independent of endogenous NO, is as effective as vardenafil to reduce urodynamic signs of BO. Targeting the NO/cGMP pathway via compounds acting on sGC might become a new approach to treat BO.

Deficiency of endothelial nitric oxide signaling pathway exacerbates peritoneal fibrosis in mice

Long-term peritoneal dialysis (PD) causes peritoneal dysfunction and structural alterations, eventually leading to peritoneal fibrosis. The endothelial nitric oxide synthase (eNOS)-NO signaling pathway contributes to the progression of organ fibrosis. However, it remains unknown whether NO signaling is involved in the process of peritoneal fibrosis. We evaluated the role of the eNOS-NO signaling pathway in the development of peritoneal fibrosis and whether stimulation of soluble guanylate cyclase (sGC), a downstream effector of NO, could attenuate peritoneal fibrosis. We used wild-type (WT) and eNOS-deficient mice (eNOSKO). The mice underwent mechanical peritoneal stripping-induced peritoneal fibrosis at day 0. At 3, 7, 14, and 28 days after peritoneal stripping, the mice were killed. In some eNOSKO mice, the sGC stimulator Bay 41-2272 was administered by intraperitoneal injection. In WT mice, granulomatous tissue formation was observed in the submesothelial area at days 3 and 7. After day 7, the peritoneal membrane thickness gradually decreased and peritoneal tissue was repaired with leaving only slight fibrosis at day 28. However, eNOSKO mice demonstrated more progression of peritoneal fibrosis than WT mice at 28 days after peritoneal stripping. Expression of vimentin in the thickened peritoneum was prolonged after day 7 in eNOSKO mice. Treatment with Bay 41-2272 significantly attenuated peritoneal vimentin expression and fibrosis in the eNOSKO mice. Disruption of the eNOS-NO signaling pathway exacerbates peritoneal fibrosis by delaying wound healing. sGC stimulation may be a useful therapy for prevention of peritoneal fibrosis.