Stimulation Of Guanylyl Cyclase Research Articles

Increased Cyclic Guanosine Monophosphate Synthesis and Calcium Entry Blockade Account for the Relaxant Activity of the Nitric Oxide–Independent Soluble Guanylyl Cyclase Stimulator BAY 41-2272 in the Rabbit Penile Urethra

To study the direct relaxant activity of 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) in the rabbit penile urethra and to investigate its modulatory effect on nitric oxide (NO)-mediated responses. Urothelium-intact (U+) and denuded (U-) rings were mounted in 10-mL organ baths for isometric force recording. Intracellular cyclic guanosine monophosphate (cGMP) levels were quantified with specific kits. BAY 41-2272 (0.0001 to 10 micromol/L) caused relaxation of urethral rings contracted with phenylephrine (10 micromol/L), with higher potency (P <0.01) in U+ (pEC(50) 7.77 +/- 0.09) compared with U- (pEC(50) 6.84 +/- 0.19) preparations. The NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (100 micromol/L) or the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ) (10 micromol/L) had no effect on BAY 41-2272 responses in U+ or U- rings. The phosphodiesterase-5 inhibitor vardenafil (0.1 micromol/L) potentiated the relaxant effects of BAY 41-2272 in both U+ (10-fold) and U- (sevenfold) tissues. Ca(2+)-induced contractions in K(+) depolarized rings were significantly attenuated by BAY 41-2272 (1 micromol/L) in an ODQ-insensitive manner. BAY 41-2272 (0.03-0.3 micromol/L) increased the amplitude and duration of electrical field stimulation-induced relaxations (1 to 32 Hz), as well as those evoked by the NO donor glyceryl trinitrate (0.0001 to 10 micromol/L). BAY 41-2272 induced ODQ-resistant increases in cGMP levels above baseline (approximately twofold) in both U+ and U- rings. BAY 41-2272 relaxes penile urethra in a synergic fashion with NO. Targeting soluble guanylate cyclase with BAY 41-2272 may represent a new therapy in the management of voiding disturbances associated with impaired NO-cGMP signaling.

DETANO and Nitrated Lipids Increase Chloride Secretion across Lung Airway Cells

We investigated the cellular mechanisms by which nitric oxide (NO) increases chloride (Cl-) secretion across lung epithelial cells in vitro and in vivo. Addition of (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1, 2-diolate (DETANONOate [DETANO];1-1,000 microM) into apical compartments of Ussing chambers containing Calu-3 cells increased short-circuit currents (I(sc)) from 5.2 +/- 0.8 to 15.0 +/- 2.1 microA/cm(2) (X +/- 1 SE; n = 7; P < 0.001). NO generated from two nitrated lipids (nitrolinoleic and nitrooleic acids; 1-10 microM) also increased I(sc) by about 100%. Similar effects were noted across basolaterally, but not apically, permeabilized Calu-3 cells. None of these NO donors increased I(sc) in Calu-3 cells pretreated with 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase). Scavenging of NO either prevented or reversed the increase of I(sc). These data indicate that NO stimulation of soluble guanylyl cyclase was sufficient and necessary for the increase of I(sc) via stimulation of the apical cystic fibrosis transmembrane regulator (CFTR). Both Calu-3 and alveolar type II (ATII) cells contained CFTR, as demonstrated by in vitro phosphorylation of immunoprecipitated CFTR by protein kinase (PK) A. PKGII (but not PKGI) phosphorylated CFTR immuniprecipitated from Calu-3 cells. Corresponding values in ATII cells were below the threshold of detection. Furthermore, DETANO, 8-Br-cGMP, or 8-(4-chlorophenylthio)-cGMP (up to 2 mM each) did not increase Cl- secretion across amiloride-treated ATII cells in vitro. Measurements of nasal potential differences in anesthetized mice showed that perfusion of the nares with DETANO activated glybenclamide-sensitive Cl- secretion. These findings suggest that small concentrations of NO donors may prove beneficial in stimulating Cl- secretion across airway cells without promoting alveolar edema.

POD-05.11: The soluble guanylyl cyclase stimulator BAY 41-2272 reverses endothelial and nerve dysfunction in corpus cavernosum from spontaneously diabetic ( db/db -/-) mice

POD-05.11: The soluble guanylyl cyclase stimulator BAY 41-2272 reverses endothelial and nerve dysfunction in corpus cavernosum from spontaneously diabetic ( db/db -/-) mice

Necessity of inositol (1,4,5)-trisphosphate receptor 1 and μ-calpain in NO-induced osteoclast motility

In skeletal remodeling, osteoclasts degrade bone, detach and move to new locations. Mechanical stretch and estrogen regulate osteoclast motility via nitric oxide (NO). We have found previously that NO stimulates guanylyl cyclase, activating the cGMP-dependent protein kinase 1 (PKG1), reversibly terminating osteoclast matrix degradation and attachment, and initiating motility. The PKG1 substrate vasodilator-stimulated protein (VASP), a membrane-attachment-related protein found in complexes with the integrin alphavbeta3 in adherent osteoclasts, was also required for motility. Here, we studied downstream mechanisms by which the NO-dependent pathway mediates osteoclast relocation. We found that NO-stimulated motility is dependent on activation of the Ca(2+)-activated proteinase mu-calpain. RNA interference (RNAi) showed that NO-dependent activation of mu-calpain also requires PKG1 and VASP. Inhibition of Src kinases, which are involved in the regulation of adhesion complexes, also abolished NO-stimulated calpain activity. Pharmacological inhibition and RNAi showed that calpain activation in this process is mediated by the inositol (1,4,5)-trisphosphate receptor 1 [Ins(1,4,5)P(3)R1] Ca(2+) channel. We conclude that NO-induced motility in osteoclasts requires regulated Ca(2+) release, which activates mu-calpain. This occurs via the Ins(1,4,5)P(3)R1.

S-nitrosylation of soluble guanylyl cyclase: a novel mechanism of nitrate tolerance?

Organic nitrates, such as nitroglycerin (GTN) have been used since the 19th century for the treatment of angina pectoris, ischemia and hypertension. Like S-nitrosothiols, organic nitrates are NO-generating compounds that are vasoactive via NO stimulation of the soluble guanylyl cyclase (sGC). The therapeutic effectiveness of GTN is severely limited by the development of nitrate tolerance, which is the loss of NO-dependent vasodilation after exposure to GTN. More than a century after its first description, the mechanism of nitrate tolerance remains a mystery. Recently, much interest has focused on another NO-dependent signal, S-nitrosylation, which could modulate vascular physiology and pathophysiology.

Effect of new sildenafil analogues in the rabbit isolated aorta

Background Nitric oxide (NO) diffuses into vascular smooth muscle cells to cause stimulation of guanylyl cyclase to elevate cGMP. The accumulated cGMP is degraded by phosphodiesterase-5 (PDE5) in cavernosal and vascular smooth muscle (Boollel et al., 1996). PDE5 inhibitors, such as sildenafil (SILD), block the degradation of cGMP, and lead vasorelaxation through increases in cGMP levels. The major side-effect of PDE5 inhibitors is associated with vasodilatation, producing a significant hypotension in individuals who take organic nitrates.

Effects of 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272) on Smooth Muscle Tone, Soluble Guanylyl Cyclase Activity, and NADPH Oxidase Activity/Expression in Corpus Cavernosum from Wild-Type, Neuronal, and Endothelial Nitric-Oxide Synthase Null Mice

We aimed to characterize the relaxation induced by the soluble guanylyl cyclase (sGC) stimulator 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and its pharmacological interactions with nitric oxide (NO) in the corpus cavernosum (CC) from wild-type (WT), endothelial nitric-oxide synthase (eNOS)(-/-), and neuronal (n)NOS(-/-) mice. The effect of BAY 41-2272 on superoxide formation and NADPH oxidase expression was also investigated. Tissues were mounted in myographs for isometric force recording. Enzyme immunoassay kits were used for cGMP determination. sGC activity was determined in the supernatant fractions of the cavernosal samples by the conversion of GTP to cGMP. Superoxide formation and expression of NADPH oxidase subunits were studied using the reduction of ferricytochrome c and Western blot analysis, respectively. BAY 41-2272 (0.01-10 microM) relaxed CC with pEC(50) values of 6.36 +/- 0.07 (WT), 6.27 +/- 0.06 (nNOS(-/-)), and 5.88 +/- 0.07 (eNOS(-/-)). The relaxations were accompanied by increases in cGMP levels. N(omega)-Nitro-L-arginine methyl ester inhibited BAY 41-2272-evoked responses in CC from WT and nNOS(-/-), but not eNOS(-/-).1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one reduced and sildenafil potentiated the relaxations induced by BAY 41-2272 in all groups. BAY 41-2272 enhanced NO (endogenous and exogenous)-induced relaxations in a concentration-dependent manner. Expression and activity of sGC was similar among the different groups. Superoxide formation was reduced by BAY 41-2272 (0.1-1 microM). The compound also inhibited p22(phox) and gp91(phox) expression induced by 9,11-dideoxy-11 alpha,9 alpha-epoxymethanoprostaglandin F(2 alpha (U46619). Our results demonstrated that sGC activation in the penis by BAY 41-2272 directly or via enhancement of NO effects may provide a novel treatment for erectile dysfunction, particularly in the event of an increased intrapenile oxidative stress.

L‐Citrulline inhibits [3H]acetylcholine release from rat motor nerve terminals by increasing adenosine outflow and activation of A1 receptors

Nitric oxide (NO) production and depression of neuromuscular transmission are closely related, but little is known about the role of L-citrulline, a co-product of NO biosynthesis, on neurotransmitter release. Muscle tension recordings and outflow experiments were performed on rat phrenic nerve-hemidiaphragm preparations stimulated electrically. L-citrulline concentration-dependently inhibited evoked [(3)H]ACh release from motor nerve terminals and depressed nerve-evoked muscle contractions. The NO synthase (NOS) substrate, L-arginine, and the NO donor, 3-morpholinosydnonimine chloride (SIN-1), also inhibited [(3)H]ACh release with a potency order of SIN-1>L-arginine>L-citrulline. Co-application of L-citrulline and SIN-1 caused additive effects. NOS inactivation with N(omega)-nitro-L-arginine prevented L-arginine inhibition, but not that of L-citrulline. The NO scavenger, haemoglobin, abolished inhibition of [(3)H]ACh release caused by SIN-1, but not that caused by L-arginine. Inactivation of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) fully blocked SIN-1 inhibition, but only partially attenuated the effects of L-arginine. Reduction of extracellular adenosine accumulation with adenosine deaminase or with the nucleoside transport inhibitor, S-(p-nitrobenzyl)-6-thioinosine, attenuated the effects of L-arginine and L-citrulline, while not affecting inhibition by SIN-1. Similar results were obtained with the selective adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine. L-citrulline increased the resting extracellular concentration of adenosine, without changing that of the adenine nucleotides. NOS catalyses the formation of two neuronally active products, NO and L-citrulline. While, NO may directly reduce transmitter release through stimulation of soluble guanylyl cyclase, the inhibitory action of L-citrulline may be indirect through increasing adenosine outflow and subsequently activating inhibitory A(1) receptors.

Guanylate Cyclase Activators Influence Reactivity of Human Mesenteric Superior Arteries Retrieved and Preserved in the Same Conditions as Transplanted Kidneys

Introduction This study sought to investigate the mechanisms of relaxation induced by the (nitric oxide (NO)-independent soluble guanylyl cyclase (sGC) stimulators 3-[5′-hydroxymethyl-2′-furyl]-1-benzylindazole (YC-1) in human mesenteric arteries relaxed and precontracted with 1 μmol/L 5-hydroxytryptamine (serotonin). Material and methods Human mesenteric arteries obtained during kidney retrieval were preserved in the same conditions as transplanted kidneys. All experiments were performed after reperfusion with Krebs buffer in 37°C and 100% oxygen exposure. Results In endothelium-intact rings, YC-1 (0.001 to 30 mmol/L) caused concentration-dependent relaxation (pEC 50: 6.59 ± 0.12), which shifted to the right in endothelium-denuded rings. The sGC inhibitor 1 H- [1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ 10 mmol/L) partially attenuated the maximal responses to YC-1 (E max = 51.30% ± 3.70%; n = 6) and displaced its curve to the right in intact and denuded vessels. Both, the NO synthesis inhibitor N-nitro-L-arginine methyl ester (100 mmol/L) and the NO scavenger carboxy-2-[4-carboxyphenyl]-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (100 mmol/L) significantly reduced YC-1 relaxation. The sodium pump inhibitor ouabain (1μmol/L) produced a greater decrease in the vasodilator response of YC-1 (E max = 18.7% ± 4.55%; n = 9). ODQ (10 μmol/L) plus 1 μmol/L ouabain abolished the relaxant response of YC-1 (E max = 9.4% ± 2.94%, n = 9). Conclusions This study demonstrated that sodium pump stimulation by YC-1 as an additional mechanism of sGC activation independent of cGMP relaxed human mesenteric artery, including blockade of Ca 2+ influx. Furthermore, this study suggested an ability of NO to mediate relaxation of resistance-like arteries through the activation of soluble guanylate cyclase and K + channels.

Nicorandil Elevates Tissue cGMP Levels in a Nitric-Oxide-Independent Manner

The K+ channel opener nicorandil is a hybrid compound that contains nitrate in its structure. It has been reported that nicorandil can relax vascular tissue in vitro via a mechanism that involves activation of KATP channels and stimulation of soluble guanylyl cyclase. However, it is not known whether the increase of cGMP levels occurs through an elevation of nitric oxide (NO). The aim of the present study was to determine whether NO release was a direct effect of nicorandil. We reported here that nicorandil did not generate NO using ozone chemiluminescence detection methods in human or rat liver microsomes (P450-rich fractions) with addition of NADPH. However, nicorandil elevated cGMP levels in rat liver, aorta, and human coronary smooth muscle cells in vitro. The elevation was not inhibited by the NO trapping agent carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO). These results suggest that nicorandil elevates cGMP without NO generation.

PAK-in' Up cGMP for the Move

Although guanylyl cyclases have been implicated in cell migration, how they are activated to promote cell motility is unknown. In this issue, report direct communication between guanylyl cyclases and the Rac-p21-activated kinase (PAK) signaling pathway--which is essential for cell migration--to promote cell motility, through allosteric activation of guanylyl cyclases by autophosphorylated PAK.

Cyclic GMP Synthesis by Human Retinal Pigment Epithelial Cells Is Mainly Mediated via the Particulate Guanylyl Cyclase Pathway

Background/Aims: Cyclic 3′,5′-guanosine monophosphate (cGMP), a central molecule in the phototransduction cascade, is also involved in a number of other physiological processes in the retina, like stimulating the absorption of subretinal fluid by activating the retinal pigment epithelium (RPE) cell pump. The aim of this study was to quantify cGMP synthesis by RPE cells and to investigate the role of two separate enzymatic pathways (soluble versus particulate guanylyl cyclase) in its production. Methods: cGMP expression was evaluated by immunochemistry and radioimmunoassay following culture of the D407 RPE cell line in the presence of a nonselective phosphodiesterase inhibitor (IBMX), in combination with the particulate guanylyl cyclase stimulator atrial natriuretic peptide (ANP) or the soluble guanylyl cyclase stimulator sodium nitroprusside (SNP). Results: Stimulation of the particulate guanylyl cyclase in RPE cells with ANP resulted in high intra- and extracellular cGMP levels. Stimulation of the soluble guanylyl cyclase by SNP resulted in a slight elevation of cGMP levels compared to controls. Conclusions: These results show that cultured human RPE cells are capable of producing cGMP and that most cGMP is generated following stimulation of the particulate guanylyl cyclase pathway.

Induction of Heme Oxygenase

Carbon monoxide (CO), produced within the body as a product of the catabolism of heme by the enzyme heme oxygenase (HO), has been reported to be a local vasodilator. One of its possible pathways is to stimulate guanylyl cyclase to produce cGMP as a second messenger.1 The parallel to NO, another gaseous signaling molecule that also works through cGMP-mediated pathways (although more potent), is obvious. In fact, the vasodilator actions of these 2 may be integrally linked. Both are endothelium derived.2 NO stimulation of cGMP may facilitate CO-mediated dilation of resistance vessels through calcium-activated potassium channels.3 However, the HO–CO story has not received the same interest and attention as NO, nor is it as generally well understood by those who work outside the field. In the present issue, we are presented with a provocative study by one of the well-established laboratories within the field of HO–CO. Wang et al4 focus on HO in the genetic model of hypertension, the spontaneously hypertensive rat (SHR). They report that 3 weeks of administration of hemin, the oxidation product of heme, which is both a substrate for and an inducer of HO, produced a profound 80-mmHg decrease in blood pressure that was sustained completely for 9 months after ceasing treatment (a duration in a rat’s life, which roughly translates into 17.5 human years). Although this is a remarkable, hopeful, and possibly profound observation, the study probably raises more questions than it answers. First, it is important to point out that this sustained decrease in blood pressure and reversal of hypertension is an unprecedented result. The potential importance of this observation is that a single 3-week treatment regimen induced a protracted reduction in blood pressure, long after treatment had ceased. To put this in perspective, treatment of virtually all forms of …

Involvement of arginase in regulating myometrial contractions during gestation in the rat

This study was designed to investigate the role of arginase in regulating myometrial contractions during gestation in the rat. Arginase activity in the myometrium was significantly decreased during the 7th-21st day of gestation, with the lowest value on the 14th day. However, the enzyme activity became significantly higher at term gestation (22nd day) than that in the non-pregnant myometrium. Arginase I protein was undetectable in the non-pregnant myometrium, at 7th and 14th day of gestation and after delivery. A slight positive signal for arginase I was detectable at 21st day of gestation. However, the protein was clearly up-regulated at term gestation (22nd day), although arginase II protein was down-regulated during gestation, with the lowest value on the 14th day. Gestational changes in arginase activity negatively correlated with those in cyclic GMP production, whereas the changes positively correlated with those in endogenous nitric oxide synthase (NOS) inhibitors and endothelin-1 (ET-1) contents. Myometrial arginase activity was inhibited by N(G)-hydroxy-L-arginine as an intermediate of NO production from L-arginine in a concentration-dependent manner. Both basal and stimulated guanylyl cyclase activities were enhanced at mid- and reduced at term gestation and after delivery, thereby partly increasing cyclic GMP production at mid- and partly decreasing the nucleotide production at term gestation and after delivery. These results suggest that the decreased arginase activity at mid-gestation possibly results from the down-regulation of arginase II protein. Whereas, the enhanced overall arginase activity at term gestation seems to be because of the induced functional arginase I in concert with the attenuated arginase II expression. The enhanced arginase activity at term gestation would be implicated in increasing myometrial contractions mediated by the increased ET-1. The increased peptide production at term gestation is possibly because of the reduced cyclic GMP production resulting from enhanced arginase activity, accumulated endogenous NOS inhibitors and attenuated guanylyl cyclase activity.

P061. Effects of the soluble guanylyl cyclase stimulator (sGC) BAY 41-2272 on vascular tone and cyclic GMP levels in mesenteric artery from spontaneously hypertensive rats

P061. Effects of the soluble guanylyl cyclase stimulator (sGC) BAY 41-2272 on vascular tone and cyclic GMP levels in mesenteric artery from spontaneously hypertensive rats

Cyclooxygenase-2 and Nitric Oxide

Nitric oxide (NO) is a simple but pluripotent molecule that is mainly released from vascular endothelial cells where it is formed intracellularly by nitric oxide synthase from L-arginine in response to several stimuli, including shear stress or muscarinic receptor stimulation. NO stimulates guanylyl cyclase to form cyclic guanosine monophosphate, which results in relaxation and vasodilatation of vascular smooth muscle cells (VSMCs). In addition, NO prevents adhesion and aggregation of platelets, and it possesses anti-inflammatory, antiproliferative, and antimigratory effects on leukocytes, endothelial cells, and VSMCs, thus offering protection from atherosclerosis. Dysfunction of the vascular endothelium has been documented in most conditions that promote or are associated with atherosclerosis and is characterized by a reduced bioavailability of NO. The healthy endothelium prevents adhesion and migration of leukocytes, proliferation of VSMCs, and platelet adhesion and aggregation. Maintaining the balance of blood flow and thrombus formation is also a major task of the vascular endothelium. It has been shown that both NO and prostacyclin, a cyclooxygenase-derived relaxing factor, inhibit activation of platelets and regulate vasomotion. Reduced NO and prostacyclin levels can result in endothelial dysfunction, which is recognized as the first step in the atherogenic process. It is of note that chronic inflammation conditions, such as rheumatoid arthritis, are associated with endothelial dysfunction. The reduced NO bioavailability may therefore explain the increased risk for cardiovascular events in patients with chronic low-grade inflammation, such as rheumatoid arthritis and osteoarthritis. Thus, this article provides an overview of the impact of inflammation and anti-inflammatory treatment with cyclooxygenase inhibitors on endothelial function.

Desensitization of NO/cGMP Signaling in Smooth Muscle: Blood Vessels Versus Airways

The NO/cGMP signaling pathway plays a major role in the cardiovascular system, in which it is involved in the regulation of smooth muscle tone and inhibition of platelet aggregation. Under pathophysiological conditions such as endothelial dysfunction, coronary artery disease, and airway hyperreactivity, smooth muscle containing arteries and bronchi are of great pharmacological interest. In these tissues, NO mediates its effects by stimulating guanylyl cyclase (GC) to form cGMP; the subsequent increase in cGMP is counteracted by the cGMP-specific phosphodiesterase (PDE5), which hydrolyzes cGMP. In platelets, allosteric activation of PDE5 by cGMP paralleled by phosphorylation has been shown to govern the sensitivity of NO/cGMP signaling. Here, we demonstrate that the functional responsiveness to NO correlates with the relative abundance of GC and PDE5 in aortic and bronchial tissue, respectively. We show a sustained desensitization of the NO-induced relaxation of aortic and bronchial rings caused by a short-term exposure to NO. The NO treatment caused heterologous desensitization of atrial natriuretic peptide-induced relaxation, whereas relaxation by the cGMP analog 8-pCPT-cGMP was unperturbed. Impaired relaxation was shown to be paralleled by PDE5 phosphorylation; this indicates enhanced cGMP degradation as a mechanism of desensitization. In summary, our results demonstrate the physiological impact of PDE5 activation on the control of smooth muscle tone and provide an explanation for the apparent impairment of NO-induced vasorelaxation.

Vasorelaxing Effect of BAY 41-2272 in Rat Basilar Artery

Decreases in intrinsic NO cause cerebral vasospasms because of the dysregulation of cGMP formation by NO-mediated pathways. Because 5-cyclopropyl-2-{1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}pyrimidin-4-ylamine (BAY 41-2272) is a potent soluble guanylyl cyclase (sGC) stimulator in an NO-independent manner, this study aimed to investigate the mechanisms underlying the relaxant effects of BAY 41-2272 in the rat basilar artery. BAY 41-2272 (0.0001 to 1 μmol/L) induced relaxations in a concentration-dependent manner, with pEC 50 values of 8.13±0.03 and 7.63±0.05 in intact and denuded rings, respectively. The sGC inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ) markedly displaced the curve for BAY 41-2272 to the right in intact or denuded rings (&amp;10-fold). The NO synthesis inhibitor N G -nitro- l -arginine methyl ester caused a rightward shift in the curve for BAY 41-2272 (4-fold), whereas the phosphodiesterase type 5 inhibitor sildenafil enhanced BAY 41-2272–induced relaxations (3- to 4-fold). The Na + -K + -ATPase inhibitor ouabain caused 3-fold rightward shifts in the curves for BAY 41-2272. Ca 2+ -induced contractions in K + depolarized rings were significantly attenuated by BAY 41-2272 in an ODQ-insensitive manner. The NO donor glyceryl trinitrate and BAY 41-2272 caused rightward shifts in the contractile responses to serotonin. Their coincubation caused a synergistic inhibition of serotonin-induced contractions. BAY 41-2272 and glyceryl trinitrate increased cGMP levels (but not cAMP) by 10-fold and 4-fold above baseline, respectively, in an ODQ-sensitive manner. cGMP levels increased by 50-fold after coincubation. BAY 41-2272 potently relaxes the rat basilar artery in a synergistic fashion with NO. Targeting the sGC with selective activators, such as BAY 41-2272, may represent a new therapy to treat cerebrovascular disease.

Role of the nitric oxide (NO)/cGMP signaling in the relaxations of mouse corpus cavernosum (CC) induced by the soluble guanylyl cyclase stimulator (sGC) BAY 41‐2272.

OBJECTIVE To characterize the in vitro effects of the NO-independent sGC stimulator BAY 41-2272 on CC from wild-type (WT), nNOS −/− and eNOS −/− mice. METHODS CC strips were mounted in 4-ml myographs and isometric force was recorded. cGMP was measured using EIA kits. RESULTS BAY 41-2272 (0.01–10 μM) relaxed CC with pEC50 values of 6.34±0.04, 6.36±0.06 and 5.70±0.07 in WT, nNOS μ/μ and eNOS μ/μ mice, respectively. L-NAME (100 μM) rightward shifted the curves to BAY 41-2272 by 3-fold in CC from WT and nNOS μ/μ, but not eNOS μ/μ. ODQ (10 μM) reduced BAY 41-2272-evoked relaxations as evidenced by rightward shifts of 4-fold. Sildenafil (0.1 μM) potentiated the relaxations induced by BAY 41-2272 in all groups. BAY 41-2272 (0.1 μM) enhanced SNP-induced relaxations of CC from WT (CTL: 6.58±0.05; BAY: 7.06±0.01), nNOS μ/μ (CTL: 6.47±0.06; BAY: 6.84±0.03) and eNOS μ/μ (CTL: 6.74±0.04; BAY: 7.16±0.03). BAY 41-2272 potentiated acetylcholine (ACh, 0.01–1 μM)- and electrical field stimulation (EFS, 1–16 Hz)-induced relaxations. BAY 41-2272 (1 μM) caused a 30-fold increase in cGMP concentration in WT and nNOS −/− samples versus an 18-fold increase in eNOS −/− CC. Co-incubation with SNP (1 μM) resulted in a synergistic increase in cGMP levels in an ODQ-sensitive manner. CONCLUSION sGC activation in the penis by BAY 41-2272 directly or via enhancement of NO effects may provide a novel treatment for erectile dysfunction. Support: HL74167, AHA.

Effects of the soluble guanylyl cyclase stimulator (sGC) BAY 41‐2272 on vascular tone and cyclic GMP levels in spontaneously hypertensive rats.

OBJECTIVE This study aimed to investigate the vasomotor activity of BAY 41-2272 and its effects on cGMP levels in intact (E+) and denuded (E-) rings of mesenteric artery from normotensive (WKY) and hypertensive (SHR) rats. METHODS Rings were mounted in myographs and data were recorded in a PowerLab system. Cyclic GMP was measured using EIA kits. RESULTS In E+ rings, BAY 41-2272 (0.0001-1 μM) caused concentration-dependent relaxations with pEC50 values of 8.57 ± 0.06 (WKY) and 8.28 ± 0.09 (SHR). In E- rings, the curves for BAY 41–2272 were shifted to the right (WKY, 6-fold; SHR, 4-fold) in comparison to E+ preparations. The sGC inhibitor ODQ (10 μM) caused significant inhibition of BAY 41–2272-induced relaxations in WKY (E+, 6.98 ± 0.18; E-, 6.31 ± 0.27) and SHR (E+, 7.89 ± 0.12; E-, 7.16 ± 0.25). In E+ and E- rings, BAY 41–2272-induced relaxations were reduced by approximately 50% in all concentrations used. Relaxations to acetylcholine (ACh, 0.001–10 μM) in SHR rings were markedly potentiated by BAY 41–2272 (0.01–0.1 μM). BAY 41–2272 (0.1 μM) increased cGMP levels by 30- and 15-fold WKY and SHR rings, respectively, in an ODQ-sensitive manner. Levels of cGMP increased by 300- (WKY) and 35-fold (SHR) when BAY 41–2272 was co-incubated with SNP (1 μM). CONCLUSION These findings ascertained the functional and biochemical basis that BAY 41–2272 acts synergistically with NO in vessels from normotensive and hypertensive animals. Financial Support: HL-74167.