Activation Of Soluble Guanylyl Cyclase Research Articles

New concepts for the treatment of male lower urinary tract symptoms.

New insights into how lower urinary tract symptoms (LUTS) develop in the presence (or absence) of benign prostatic hyperplasia (BPH) led to a change of terminology from BPH to LUTS, and to the revelation that several other (not only prostatic) molecular structures are of value in treating LUTS. This review aims to summarize what new we know about 'why the prostate grows large', and what kind of therapeutic implications that bears, and second to present several new compounds, which show promising results in preclinical testings in regards to LUTS. Apart from the old concept of mainly hormone-dependent prostate growth, new concepts on BPH cause include genetic reasons, correlations with metabolic disorders, as well as an inflammatory origin. New pharmacological compound classes, which might be of value in treating LUTS, are transient receptor potential V member 1 antagonists, fatty acid amide hydrolase inhibitors, soluble guanylyl cyclase stimulators and activators, Rho-kinase inhibitors, and purinergic receptor antagonists. New concepts on BPH cause show interesting approaches, but are still in early stages. They carry, however, the highest prospect to bring up causal treatments. Some new compounds related to LUTS are on the verge to switch 'from bench to bedside'.

Cytokine-induced S-nitrosylation of soluble guanylyl cyclase and expression of phosphodiesterase 1A contribute to dysfunction of longitudinal smooth muscle relaxation.

The effect of proinflammatory cytokines on the expression and activity of soluble guanylyl cyclase (sGC) and cGMP-phosphodiesterases (PDEs) was determined in intestinal longitudinal smooth muscle. In control muscle cells, cGMP levels are regulated via activation of sGC and PDE5; the activity of the latter is regulated via feedback phosphorylation by cGMP-dependent protein kinase. In muscle cells isolated from muscle strips cultured with interleukin-1β (IL-1β) or tumor necrosis factor α (TNF-α) or obtained from the colon of TNBS (2,4,6-trinitrobenzene sulfonic acid)-treated mice, expression of inducible nitric oxide synthase (iNOS) was induced and sGC was S-nitrosylated, resulting in attenuation of nitric oxide (NO)-induced sGC activity and cGMP formation. The effect of cytokines on sGC S-nitrosylation and activity was blocked by the iNOS inhibitor 1400W [N-([3-(aminomethyl)phenyl]methyl)ethanimidamide dihydrochloride]. The effect of cytokines on cGMP levels measured in the absence of IBMX (3-isobutyl-1-methylxanthine), however, was partly reversed by 1400W or PDE1 inhibitor vinpocetine and completely reversed by a combination of 1400W and vinpocetine. Expression of PDE1A was induced and was accompanied by an increase in PDE1A activity in muscle cells isolated from muscle strips cultured with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice; the effect of cytokines on PDE1 expression and activity was blocked by MG132 (benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate), an inhibitor of nuclear factor κB activity. NO-induced muscle relaxation was inhibited in longitudinal muscle cells isolated from muscle strips cultured with IL-1β or TNF-α or obtained from the colon of TNBS-treated mice, and this inhibition was completely reversed by the combination of both 1400W and vinpocetine. Inhibition of smooth muscle relaxation during inflammation reflects the combined effects of decreased sGC activity via S-nitrosylation and increased cGMP hydrolysis via PDE1 expression.

PDE and sGC hand in hand to see the light.

In PNAS, Sikka et al. (1) suggest that melanopsin, a non–image-forming opsin (opsin4, Opn4), may play a physiological role in the regulation of vascular tone by mediating photorelaxation. The authors conclude that the response to light is due to hyperpolarization of the vascular smooth muscle cells resulting from the activation of soluble guanylyl cyclase (sGC), but does not involve protein kinase G (PKG). This study not only provides an intriguing molecular explanation for a phenomenon (photorelaxation) that has puzzled vascular biologists for more than half a century (2), but also forces us to rethink the fate of the products generated by sGC.

Reversal of severe angioproliferative pulmonary arterial hypertension and right ventricular hypertrophy by combined phosphodiesterase-5 and endothelin receptor inhibition.

BackgroundPatients with pulmonary arterial hypertension (PAH) are treated with vasodilators, including endothelin receptor antagonists (ERAs), phosphodiesterase-5 (PDE-5) inhibitors, soluble guanylyl cyclase activators, and prostacyclin. Despite recent advances in pharmacotherapy for individuals with PAH, morbidity and mortality rates in this patient population remain unacceptably high. Here, we tested the hypothesis that combination therapy with two PAH drugs that target distinct biochemical pathways will provide superior efficacy relative to monotherapy in the rat SU5416 plus hypoxia (SU-Hx) model of severe angioproliferative PAH, which closely mimics the human condition.MethodsMale Sprague Dawley rats were injected with a single dose of SU5416, which is a VEGF receptor antagonist, and exposed to hypobaric hypoxia for three weeks. Rats were subsequently housed at Denver altitude and treated daily with the PDE-5 inhibitor, tadalafil (TAD), the type A endothelin receptor (ETA) antagonist, ambrisentan (AMB), or a combination of TAD and AMB for four additional weeks.ResultsMonotherapy with TAD or AMB led to modest reductions in pulmonary arterial pressure (PAP) and right ventricular (RV) hypertrophy. In contrast, echocardiography and invasive hemodynamic measurements revealed that combined TAD/AMB nearly completely reversed pulmonary hemodynamic impairment, RV hypertrophy, and RV functional deficit in SU-Hx rats. Efficacy of TAD/AMB was associated with dramatic reductions in pulmonary vascular remodeling, including suppression of endothelial cell plexiform lesions, which are common in human PAH.ConclusionsCombined therapy with two vasodilators that are approved for the treatment of human PAH provides unprecedented efficacy in the rat SU-Hx preclinical model of severe, angioproliferative PAH.

Prolonged therapy with the soluble guanylyl cyclase activator BAY 60-2770 restores the erectile function in obese mice.

Cardiovascular and endocrine-metabolic diseases associated with increased oxidative stress such as obesity lead to erectile dysfunction (ED). Activators of soluble guanylyl cyclase (sGC) such as BAY 60-2770 reactivate the heme-oxidized sGC in vascular diseases. This study aimed to evaluate the effects of 2-week oral intake with BAY 60-2270 on a murine model of obesity-associated ED. C57BL/6 male mice were fed for 12 weeks with standard chow or high-fat diet. Lean and obese mice were treated with BAY 60-2770 (1 mg/kg/day, 2 weeks). Measurements of intracavernosal pressure (ICP), along with acetylcholine (10(-9) to 10(-5) M) and electrical field stimulation (EFS; 4-10 Hz)-induced corpus cavernosum relaxations in vitro, were obtained. Levels of cyclic guanosine monophosphate (cGMP), reactive oxygen species (ROS), and sGC protein expressions in cavernosal tissues were measured. Cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly lower in obese compared with lean mice (P < 0.05). Two-week therapy with BAY 60-2770 fully reversed the decreased ICP in obese group. Acetylcholine-induced cavernosal relaxations were 45% lower (P < 0.001) in obese mice, which were fully restored by BAY 60-2770 treatment. Likewise, the EFS-induced relaxations in obese mice were restored by BAY 60-2770. Basal cGMP content in erectile tissue was 68% lower (P < 0.05) in obese mice, an effect normalized by BAY 60-2770. Levels of ROS were 52% higher (P < 0.05) whereas protein expression of α1 sGC subunit was reduced in cavernosal tissue of obese mice, both of which were normalized by BAY 60-2770. In lean group, BAY 60-2770 did not significantly affect any functional, biochemical, or molecular parameter analyzed. Two-week therapy with BAY 60-2770 restores the erectile function in obese mice that is associated with reduced ROS levels, up-regulation of α1 sGC subunit, and increased cGMP levels in the erectile tissue.

P201 - Identification of new inhibitors of soluble guanylyl cyclase activity

P201 - Identification of new inhibitors of soluble guanylyl cyclase activity

Hyperoxia inhibits nitric oxide treatment effects in alveolar epithelial cells via effects on L-type amino acid transporter-1.

The aims of this study were to determine hyperoxia effects on S-nitrosothiol (SNO) accumulation and L-type amino acid transporter 1 (LAT1) expression/function in alveolar epithelium and to determine whether hyperoxia impairs exogenous nitric oxide (NO) treatment effects in alveolar epithelium through effects on LAT1 expression and/or function. SNO accumulation in vitro and in vivo after NO treatment was dependent on the LAT1 system transport. Hyperoxia (60% or 90%) impaired NO effects on SNO accumulation and soluble guanylyl cyclase activation in proportion to the magnitude of hyperoxia and the duration of exposure, up to 12 h, in type I-like (R3/1) and type II-like (L2) rat and human (A549) alveolar epithelial cells. LAT function, determined by sodium-independent (3)H-leucine uptake, was impaired in a parallel manner. Hyperoxia impaired LAT1 expression in alveolar epithelial cells, determined by immunoblots and immunofluorescence, and in newborn rats exposed to 60% O2 for 4 days, determined by immunohistochemistry. Despite significant preclinical evidence, inhaled NO has shown disappointing limitations in clinical applications. Our studies suggest an important explanation: oxidative stress, a common feature of diseases in which therapeutic NO would be considered, impairs LAT1 expression and function, blocking a major route for inhaled NO (iNO) action, that is, the uptake of S-nitrosocysteine via LAT1. SNO uptake after NO treatment is dependent on LAT1. Hyperoxia impairs SNO uptake and NO effects during NO exposure and impairs LAT system function and LAT1 expression. Effects on SNO formation and transport must be considered for rational optimization of NO-based therapeutics.

Involvement of inducible nitric oxide synthase and dimethyl arginine dimethylaminohydrolase in Nω-nitro-L-arginine methyl ester (L-NAME)-induced hypertension.

Chronic administration of Nω-nitro-L-arginine methyl ester (L-NAME) in rats is a chemical method to study the induction and progression of nitric oxide (NO) deficiency-induced endothelial dysfunction. Male Wistar rats received L-NAME (50 mg/kg/day in drinking water) or no drug for 6 weeks. Mean arterial pressure (MAP) was measured on Day 43 by carotid artery cannulation. Plasma interleukin 1β (IL-1β) level was measured by enzyme-linked immunosorbent assay. Aorta and carotid artery were isolated for determination of basal nitrite, cGMP production, soluble guanylylcyclase (sGC) activity, phosphodiesterase-5 (PDE5) activity, and dimethylarginine dimethylaminohydrolase (DDAH) activity. mRNA expression studies were done by real time-polymerase chain reaction. L-NAME induced an increase in MAP and plasma IL-1β. The treatment had varied effect on endothelial nitric oxide synthase (eNOS), sGC, and PDE5 but showed an increase in inducible NOS (iNOS) mRNA expression and plasma asymmetric dimethyl arginine levels. Basal nitrite, cGMP levels, sGC activity, and DDAH activity were significantly decreased in the tissues. Brief incubation of tissues in vitro with 1400 W, a specific iNOS blocker, partially reversed sGC activity, and cGMP levels. The results of this study showed that L-NAME-mediated inhibition of eNOS is only partially responsible for the vascular pathology observed in this model. Secondary effects that include an increase in iNOS and a decrease in DDAH activity are likely to be the causative factors for the progression of vascular dysfunction.

The soluble guanylyl cyclase activator BAY 60-2770 potently relaxes the pulmonary artery on congenital diaphragmatic hernia rabbit model.

Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension which is often difficult to manage, and a significant cause of morbidity and mortality. In this study, we have used a rabbit model of CDH to evaluate the effects of BAY 60-2770 on the in vitro reactivity of left pulmonary artery. CDH was performed in New Zealand rabbit fetuses (n=10 per group) and compared to controls. Measurements of body, total and left lung weights (BW, TLW, LLW) were done. Pulmonary artery rings were pre-contracted with phenylephrine (10μM), after which cumulative concentration-response curves to glyceryl trinitrate (GTN; NO donor), tadalafil (PDE5 inhibitor) and BAY 60-2770 (sGC activator) were obtained as well as the levels of NO (NO3/NO2). LLW, TLW and LBR were decreased in CDH (p<0.05). In left pulmonary artery, the potency (pEC50) for GTN was markedly lower in CDH (8.25±0.02 versus 9.27±0.03; p<0.01). In contrast, the potency for BAY 60-2770 was markedly greater in CDH (11.7±0.03 versus 10.5±0.06; p<0.01). The NO2/NO3 levels were 62% higher in CDH (p<0.05). BAY 60-2770 exhibits a greater potency to relax the pulmonary artery in CDH, indicating a potential use for pulmonary hypertension in this disease.

Small alterations in cobinamide structure considerably influence sGC activation.

Specially designed B-ring-modified cobalamin derivatives were synthesized and tested as potential activators of soluble guanylyl cyclase (sGC). Herein, we disclose the influence of substituents at the c- and d-positions in hydrophilic and hydrophobic cobyrinic acid derivatives on their capacities to activate sGC. The presence of the amide group at c-/d-position in cobyrinic acid derivatives strongly influence the level of sGC activation. Removal of the d-position altogether has a profound effect for hydrophobic compounds. In contrast, little differences were observed in hydrophilic ones.

Towards structural insights into activation and modulation of the soluble guanylyl cyclase

Towards structural insights into activation and modulation of the soluble guanylyl cyclase

Future Pharmacotherapies for Male Lower Urinary Tract Symptoms

Lower urinary tract symptoms (LUTS) in men are a prevalent condition with complex and multifactorial etiology. Different types of symptoms overlap among them and treatment has evolved to a more holistic and individualized approach. Advances in the understanding of LUTS pathophysiology has led to the development of new drugs and compounds to treat male LUTS. Recently, new drugs, such as phosphodiesterase type 5 inhibitors and beta3 adrenergic agonists, have been approved for the treatment of LUTS and have just made it to clinical practice. New agents for intraprostatic injection, for instance PRX302 and NX-1207, are currently under evaluation in clinical trials with promising results. Rho-kinase inhibitors, transient receptor potential channel blockers and activators of soluble guanylyl cyclase are examples of compounds targeting different pathways involved in the pathophysiology of male LUTS, which have been tested in experimental studies. New advances in pharmacotherapy may also allow combination of drugs to achieve synergistic effect.

The nitric oxide-cGKII system relays death and survival signals during embryonic retinal development via AKT-induced CREB1 activation

During early neurogenesis, retinal neuronal cells display a conserved differentiation program in vertebrates. Previous studies established that nitric oxide (NO) and cGMP accumulation regulate essential events in retinal physiology. Here we used pharmacological and genetic loss-of-function to investigate the effects of NO and its downstream signaling pathway in the survival of developing avian retinal neurons in vitro and in vivo. Six-day-old (E6) chick retinal cells displayed increased calcium influx and produced higher amounts of NO when compared with E8 cells. L-arginine (substrate for NO biosynthesis) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP; a nitrosothiol NO donor) promoted extensive cell death in E6 retinas, whereas in E8 both substances decreased apoptosis. The effect of NO at both periods was mediated by soluble guanylyl cyclase (sGC) and cGMP-dependent kinase (cGK) activation. In addition, shRNA-mediated cGKII knockdown prevented NO-induced cell death (E6) and cell survival (E8). This, NO-induced cell death or cell survival was not correlated with an early inhibition of retinal cell proliferation. E6 cells also responded differentially from E8 neurons regarding cyclic AMP-responsive element-binding protein (CREB) activation in the retina in vivo. NO strongly decreased nuclear phospho-CREB staining in E6 but it robustly enhanced CREB phosphorylation in the nuclei of E8 neurons, an effect that was completely abrogated by cGKII shRNAs at both embryonic stages. The ability of NO in regulating CREB differentially during retinal development relied on the capacity of cGKII in decreasing (E6) or increasing (E8) nuclear AKT (V-Akt murine thymoma viral oncogene) activation. Accordingly, inhibiting AKT prevented both cGKII shRNA-mediated CREB upregulation in E6 and SNAP-induced CREB activation in E8. Furthermore, shRNA-mediated in vivo cGKII or in vitro CREB1 knockdown confirmed that NO/cGKII dualistically regulated the downstream CREB1 pathway and caspase activation in the chick retina to modulate neuronal viability. These data demonstrate that NO-mediated cGKII signaling may function to control the viability of neuronal cells during early retinal development via AKT/CREB1 activity.

M‐Atrial Natriuretic Peptide and Nitroglycerin in a Canine Model of Experimental Acute Hypertensive Heart Failure: Differential Actions of 2 cGMP Activating Therapeutics

BackgroundSystemic hypertension is a common characteristic in acute heart failure (HF). This increasingly recognized phenotype is commonly associated with renal dysfunction and there is an unmet need for renal enhancing therapies. In a canine model of HF and acute vasoconstrictive hypertension we characterized and compared the cardiorenal actions of M‐atrial natriuretic peptide (M‐ANP), a novel particulate guanylyl cyclase (pGC) activator, and nitroglycerin, a soluble guanylyl cyclase (sGC) activator.Methods and ResultsHF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II infusion. We characterized the cardiorenal and humoral actions prior to, during, and following intravenous M‐ANP (n=7), nitroglycerin (n=7), and vehicle (n=7) infusion. Mean arterial pressure (MAP) was reduced by M‐ANP (139±4 to 118±3 mm Hg, P<0.05) and nitroglycerin (137±3 to 116±4 mm Hg, P<0.05); similar findings were recorded for pulmonary wedge pressure (PCWP) with M‐ANP (12±2 to 6±2 mm Hg, P<0.05) and nitroglycerin (12±1 to 6±1 mm Hg, P<0.05). M‐ANP enhanced renal function with significant increases (P<0.05) in glomerular filtration rate (38±4 to 53±5 mL/min), renal blood flow (132±18 to 236±23 mL/min), and natriuresis (11±4 to 689±37 mEq/min) and also inhibited aldosterone activation (32±3 to 23±2 ng/dL, P<0.05), whereas nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation.ConclusionsOur results advance the differential cardiorenal actions of pGC (M‐ANP) and sGC (nitroglycerin) mediated cGMP activation. These distinct renal and aldosterone modulating actions make M‐ANP an attractive therapeutic for HF with concomitant hypertension, where renal protection is a key therapeutic goal.

Soluble Guanylyl Cyclase (sGC) Degradation and Impairment of Nitric Oxide-Mediated Responses in Urethra from Obese Mice: Reversal by the sGC Activator BAY 60-2770

Obesity has emerged as a major contributing risk factor for overactive bladder (OAB), but no study examined urethral smooth muscle (USM) dysfunction as a predisposing factor to obesity-induced OAB. This study investigated the USM relaxant machinery in obese mice and whether soluble guanylyl cyclase (sGC) activation with BAY 60-2770 [acid 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4-(trifluoromethyl) biphenyl-4-yl] methoxy} phenyl) ethyl] amino} methyl) benzoic] rescues the urethral reactivity through improvement of sGC-cGMP (cyclic guanosine monophosphate) signaling. Male C57BL/6 mice were fed for 12 weeks with a high-fat diet to induce obesity. Separate groups of animals were treated with BAY 60-2770 (1 mg/kg per day for 2 weeks). Functional assays and measurements of cGMP, reactive-oxygen species (ROS), and sGC protein expression in USM were determined. USM relaxations induced by NO (acidified sodium nitrite), NO donors (S-nitrosoglutathione and glyceryl trinitrate), and BAY 41-2272 [5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine] (sGC stimulator) were markedly reduced in obese compared with lean mice. In contrast, USM relaxations induced by BAY 60-2770 (sGC activator) were 43% greater in obese mice (P < 0.05), which was accompanied by increases in cGMP levels. Oxidation of sGC with ODQ [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one] (10 μM) potentiated BAY 60-2770-induced USM responses in the lean group. Long-term oral BAY 60-2770 administration fully prevented the impairment of USM relaxations in obese mice. Reactive-oxygen species (ROS) production was enhanced, but protein expression of β1 second guanylate cyclase subunit was reduced in USM from obese mice, both of which were restored by BAY 60-2770 treatment. In conclusion, impaired USM relaxation in obese mice is associated with ROS generation and down-regulation of sGC-cGMP signaling. Prevention of sGC degradation by BAY 60-2770 ameliorates the impairment of urethral relaxations in obese mice.

YC-1 enhances the anti-tumor activity of sorafenib through inhibition of signal transducer and activator of transcription 3 (STAT3) in hepatocellular carcinoma

BackgroundTraditional systemic chemotherapy does not provide survival benefits in patients with hepatocellular carcinoma (HCC). Molecular targeted therapy shows promise for HCC treatment, however, the duration of effectiveness for targeted therapies is finite and combination therapies offer the potential for improved effectiveness.MethodsSorafenib, a multikinase inhibitor, and YC-1, a soluble guanylyl cyclase (sGC) activator, were tested in HCC by proliferation assay, cell cycle analysis and western blot in vitro and orthotopic and ectopic HCC models in vivo.ResultsIn vitro, combination of sorafenib and YC-1 synergistically inhibited proliferation and colony formation of HepG2, BEL-7402 and HCCLM3 cells. The combination also induced S cell cycle arrest and apoptosis, as observed by activated PARP and caspase 8. Sorafenib and YC-1 respectively suppressed the expression of phosphorylated STAT3 (p-STAT3) (Y705) in a dose- and time-dependent manner. Combination of sorafenib and YC-1 significantly inhibited the expression of p-STAT3 (Y705) (S727), p-ERK1/2, cyclin D1 and survivin and SHP-1 activity compared with sorafenib or YC-1 used alone in all tested HCC cell lines. In vivo, sorafenib-YC-1 combination significantly suppressed the growth of HepG2 tumor xenografts with decreased cell proliferation and increased apoptosis observed by PCNA and PARP. Similar results were also confirmed in a HCCLM3 orthotopic model. There was a reduction in CD31-positive blood vessels and reduced VEGF expression, which suggested a combinational effect of sorafenib and YC-1 on angiogenesis. The reduced expression of p-STAT3, cyclin D1 and survivin was also observed with the combination of sorafenib and YC-1.ConclusionsOur data show that sorafenib-YC-1 combination is a novel potent therapeutic agent that can target the STAT3 signaling pathway to inhibit HCC tumor growth.

Soluble guanylyl cyclase activators increase the expression of tolerance to morphine analgesic effect.

It is aimed to investigate the effects of guanylyl cyclase activation and inhibition on acute morphine antinociception and the development of tolerance to its effect. Nitric oxide-soluble guanylyl cyclase signal transduction cascade suggested to play an important role in the development of tolerance to antinociceptive effects of morphine. Nociception was evaluated by tail flick and hot plate tests in male Wistar rats. The analgesic effects of intraperitoneal protoporphyrin IX (PPIX; an activator of soluble guanylyl cyclase), 3-morpholinosydnonimine hydrochloride (SIN-1; NO donor and activator of guanylyl cyclase), S-Nitroso-N-acetylpenicillamine (SNAP; an activator of guanylyl cyclase), 3,3-Bis (amino ethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18; NO donor activating guanylyl cyclase) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; an inhibitor of guanylyl cyclase) alone or in combination with subcutaneous morphine injection were evaluated. Their effects on morphine tolerance development were evaluated by giving these agents 20 minutes prior to twice daily morphine injection during tolerance development for 5 days. On day 6, the expression of morphine tolerance was determined. PPIX, SIN-1, SNAP and NOC-18 significantly increased expression of morphine tolerance while ODQ decreased. These data suggested that sGC activators have a significant role in tolerance to the analgesic effect of morphine (Tab. 1, Fig. 4, Ref. 29).

Modulation of Sarcolemmal ATP-Sensitive Potassium Channels by Atrial Natriuretic Peptide in Ventricular Cardiomyocytes

ATP-sensitive potassium (KATP) channels couple cell metabolic status to membrane excitability and are crucial for stress adaptation in the heart. Natriuretic peptides (NPs) produced by the heart, the vasculature and the kidney exert physiologically important actions such as natriuresis and inhibition of sympathetic tone and cardiomyocyte hypertrophy. Our recent study reveals that the gaseous messenger nitric oxide evokes cardiac KATP channel stimulation via activation of soluble guanylyl cyclase and subsequently a cGMP-dependent signaling pathway. Binding of NPs like atrial natriuretic peptide (ANP) to the type 1 NP receptor (NPR-A) also increases intracellular cGMP level, through activating guanylyl cyclase intrinsic to the receptor; however, how ANP modulates the function of cardiac KATP channels remains to be established. By performing cell-attached patch recordings in ventricular cardiomyocytes acutely isolated from adult rabbits, we found that the single-channel activity of sarcolemmal KATP (sarcKATP) channels induced by sodium azide through metabolic inhibition was potentiated by addition of ANP (100 nM) (n=16), whereas the potentiation by ANP was abolished when the NPR-A antagonist anantin (1 μM) was coapplied (n=5); these findings suggest that the stimulatory action of ANP is mediated by NPR-A activation, which may depend on generation of diffusible cGMP. Indeed, sarcKATP channel stimulation by ANP in intact ventricular myocytes was significantly abated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823 (1 μM) (n=5). Moreover, the ANP effect was prevented by selective suppression of extracellular signal-regulated protein kinase (ERK)1/2 (n=6) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) (n=6). Collectively, here we demonstrate that the polypeptide hormone ANP positively modulates ventricular KATP channel function via activation of NPR-A, PKG, ERK1/2 and CaMKII. By opening myocardial KATP channels, this signaling pathway may regulate cardiac excitability and contribute to cytoprotection against ischemia-reperfusion injury.

Mechanisms of antiplatelet activity of nifedipine

Nifedipine, an L-type calcium channel blocker, is widely used in the treatment of hypertension and coronary heart diseases, and also exhibits an antiplatelet activity. Activation of peroxisome proliferator-activated receptors (PPARs; α, β/δ, and γ) inhibits the platelet aggregation. Therefore, the purpose of this study was to evaluate the contribution of PPAR-mediated processes to the antiplatelet activity of nifedipine. We assessed human platelet aggregation by using an aggregometer and measured several platelet activating markers and related signaling pathways in platelets treated with nifedipine in the presence or absence of PPAR agonists. Nifedipine treatment (1, 5 μmol/l) dose-dependently increased the activity and intracellular expression of PPAR-β/-γ by inhibiting the release of PPAR-β/-γ from activated platelets. Nifedipine treatment also upregulated cyclic 3',5'-cyclic monophosphate (GMP)/protein kinase G (PKG) expression, and increased PI(3)K/Akt pathway, endothelial nitric oxide synthase, and soluble guanylyl cyclase activities. In the presence of a selective PPAR-β antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibitory effects of nifedipine on collagen-induced platelet aggregation, intracellular Ca mobilization, and protein kinase C (PKC-α) activation were abrogated. Similarly, PPAR-β-γ antagonists markedly attenuated nifedipine-mediated upregulation of nitric oxide/cyclic GMP/PKG cascade. In a mouse model of thrombosis, the administration of nifedipine substantially inhibited fluorescein sodium-induced vessel thrombus formation; however, the antithrombotic effect was considerably reduced in the presence of PPAR-β/-γ antagonists. This study is the first to show that the PPAR-β/-γ-dependent upregulation of PI(3)K/Akt/nitric oxide/cyclic GMP/PKG pathway and the inhibition of PKC-α activity and intracellular Ca(+) mobilization in platelets may be the mechanisms underlying the antiplatelet and antithrombotic activities of nifedipine.

Nitrosopersulfide (SSNO−) accounts for sustained NO bioactivity of S-nitrosothiols following reaction with sulfide

Sulfide salts are known to promote the release of nitric oxide (NO) from S-nitrosothiols and potentiate their vasorelaxant activity, but much of the cross-talk between hydrogen sulfide and NO is believed to occur via functional interactions of cell regulatory elements such as phosphodiesterases. Using RFL-6 cells as an NO reporter system we sought to investigate whether sulfide can also modulate nitrosothiol-mediated soluble guanylyl cyclase (sGC) activation following direct chemical interaction. We find a U-shaped dose response relationship where low sulfide concentrations attenuate sGC stimulation by S-nitrosopenicillamine (SNAP) and cyclic GMP levels are restored at equimolar ratios. Similar results are observed when intracellular sulfide levels are raised by pre-incubation with the sulfide donor, GYY4137. The outcome of direct sulfide/nitrosothiol interactions also critically depends on molar reactant ratios and is accompanied by oxygen consumption. With sulfide in excess, a ‘yellow compound’ accumulates that is indistinguishable from the product of solid-phase transnitrosation of either hydrosulfide or hydrodisulfide and assigned to be nitrosopersulfide (perthionitrite, SSNO−; λmax 412nm in aqueous buffers, pH 7.4; 448nm in DMF). Time-resolved chemiluminescence and UV–visible spectroscopy analyses suggest that its generation is preceded by formation of the short-lived NO-donor, thionitrite (SNO−). In contrast to the latter, SSNO− is rather stable at physiological pH and generates both NO and polysulfides on decomposition, resulting in sustained potentiation of SNAP-induced sGC stimulation. Thus, sulfide reacts with nitrosothiols to form multiple bioactive products; SSNO− rather than SNO− may account for some of the longer-lived effects of nitrosothiols and contribute to sulfide and NO signaling.