Stimulation Of Soluble Guanylyl Cyclase Research Articles

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.

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.

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

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.

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 (&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.

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.

Stimulation of soluble guanylyl cyclase by BAY 41-2272 relaxes anococcygeus muscle: Interaction with nitric oxide

The compound BAY 41-2272 stimulates the soluble guanylyl cyclase in a nitric oxide (NO)-independent manner. We have investigated the potency and efficacy of BAY 41-2272 in the rat anococcygeus muscle, as well as the effects of BAY 41-2272 on NO-mediated anococcygeus relaxations. BAY 41-2272 (0.01–10 μM) potently relaxed precontracted anococcygeus muscle strips, with a pEC 50 value of 6.44 ± 0.03 and maximum response of 100 ± 2%. The soluble guanylyl cyclase inhibitor 1 H-[1,2,4]-oxidiazolo[4,3- a] quinoxalin-1-one (ODQ, 1 μM) and the NO inhibitor N ω-nitro- l-arginine methyl ester ( l-NAME, 100 μM) caused significant rightward shifts in the concentration–response curves to BAY 41-2272. The phosphodiesterase type-5 inhibitor tadalafil (0.1 μM) markedly enhanced the relaxations evoked by BAY 41-2272. In addition, BAY 41-2272 increased the duration of nitrergic relaxations by approximately 55%. The relaxations induced by glyceryl trinitrate were also significantly potentiated by BAY 41-2272. In conclusion, BAY 41-2272 interacts with endogenous and exogenous NO causing a potent relaxation of rat anococcygeus muscle.

Molecular Mechanisms Underlying Rat Mesenteric Artery Vasorelaxation Induced by the Nitric Oxide-Independent Soluble Guanylyl Cyclase Stimulators BAY 41-2272 [5-Cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine] and YC-1 [3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl Indazole

The aim of this study was to investigate the mechanisms of relaxation to the nitric oxide (NO)-independent soluble guanylyl cyclase (sGC) stimulators 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) in the rat mesenteric artery. In endothelium-intact rings, BAY 41-2272 (0.0001-1 microM) and YC-1 (0.001-30 microM) caused concentration-dependent relaxations (pEC(50) values of 8.21 +/- 0.05 and 6.75 +/- 0.06, respectively), which were shifted to the right by 6-fold in denuded rings. The sGC inhibitor H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ) (10 microM) partially attenuated the maximal responses to BAY 41-2272 and YC-1 and displaced their curves to the right by 9- to 10-fold in intact and 3-fold in denuded vessels. The NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (100 microM) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (100 microM) reduced BAY 41-2272 and YC-1 relaxations, whereas the phosphodiesterase type 5 inhibitor sildenafil (0.1 microM) potentiated these responses. The phosphatase inhibitor calyculin A (50 nM) reduced the relaxant responses, and high concentrations of BAY 41-2272 (1 micorM) and YC-1 (10 microM) inhibited Ca(2+)-induced contractions in K(+)-depolarized rings. BAY 41-2272 (0.1 microM) and YC-1 (1 microM) markedly elevated cGMP levels in an ODQ-sensitive manner. Coincubation of BAY 41-2272 or YC-1 with a NO donor resulted in a synergistic inhibition of phenylephrine-induced contractions paralleled by marked increases in cGMP levels. In conclusion, BAY 41-2272 and YC-1 relax the mesenteric artery through cGMP-dependent and -independent mechanisms, including blockade of Ca(2+) influx. The synergistic responses probably reflect the direct effects of NO and NO-independent sGC stimulators on the enzyme, thus representing a potential therapeutic effect by permitting reductions of nitrovasodilator dose.

Antiproliferative Effects of Phosphodiesterase Type 5 Inhibition in Human Pulmonary Artery Cells

Phosphodiesterase Type 5 (PDE5) inhibition represents a novel strategy for the treatment of pulmonary hypertension. Our aim was to establish the distribution of PDE5 in the pulmonary vasculature and effects of PDE5 inhibition on pulmonary artery smooth muscle cells (PASMCs). PDE5 expression was examined by immunohistochemistry and Western blotting, in both normal and hypertensive lung tissues. DNA synthesis, proliferation, PDE activity, and apoptosis were measured in distal human PASMCs treated with soluble guanylyl cyclase activators (nitric oxide donors and BAY41-2272) and sildenafil. Cells containing PDE5 and alpha-smooth muscle actin occurred throughout the pulmonary vasculature, including obstructive intimal lesions. Three molecular forms of PDE5 were identified and protein expression was greater in hypertensive than control lung tissue. Most cyclic guanosine monophosphate hydrolysis (about 80%) in cultured cells was attributed to PDE5. Sildenafil induced a greater elevation of intracellular cyclic guanosine monophosphate levels compared with nitric oxide donors and BAY41-2272 (about 10-fold versus about 2-fold) and cotreatment had a synergistic effect, increasing cyclic nucleotide levels up to 50-fold. Dual stimulation of soluble guanylyl cyclase and inhibition of PDE5 activities also had significant downstream effects, increasing phosphorylation of vasodilator-stimulated phosphoprotein, reducing DNA synthesis and cell proliferation, and stimulating apoptosis, and these effects were mimicked by cyclic guanosine monophosphate analogs. Phosphodiesterase Type 5 is the main factor regulating cyclic guanosine monophosphate hydrolysis and downstream signaling in human PASMCs. The antiproliferative effects of this signaling pathway may be significant in the chronic treatment of pulmonary hypertension with PDE5 inhibitors such as sildenafil.

Localization and characterization of cGMP-immunoreactive structures in rat brain slices after NO-dependent and NO-independent stimulation of soluble guanylyl cyclase

Possible differences in the localization of the cGMP response were investigated in rat brain coronal slices after in vitro incubation and NO-dependent or NO-independent stimulation of soluble guanylyl cyclase (sGC). Dose-dependent stimulation of cGMP synthesis by the NO donors, sodium nitroprusside, S-nitrosoglutathione, 3-morpholinosydnonimine and diethylamino NONOate was studied in the somatoparietal cortex, the hippocampus and the thalamus. cGMP accumulation was evaluated using a radioimmunoassay and by measuring cGMP-immunofluorescence using image analysis. All four NO donors induced similar cGMP staining patterns in the somatoparietal cortex, the hippocampus and the thalamus. NO-mediated cGMP synthesis in the cortical areas colocalized predominantly with the acetylcholine transporter and occasionally with parvalbumin (GABAergic cells) or the neuronal glutamate transporter. Incubation of the slices in the combined presence of a NO donor and the NO-independent activators YC-1 or BAY 41-2272 strongly potentiated cGMP synthesis and induced abundant cGMP-immunoreactivity in cortical GABAergic and glutamatergic cells. These findings indicate that the mechanism of NO release from the NO donors used does not determine the location of the cGMP response. The results suggest that YC-1 and BAY 41-2272 trigger a NO-sensing mechanism in cells in which the sGC is otherwise not sensitive to NO.

Stimulation of soluble guanylyl cyclase inhibits mesangial cell proliferation and matrix accumulation in experimental glomerulonephritis

To date, no specific treatment is established in mesangial proliferative glomerulonephritis in humans. Specific stimulation of soluble guanylyl cyclase (sGC), an enzyme catalyzing the synthesis of cGMP from GTP, can be achieved by the novel pyrazolopyridine derivative BAY 41-2272. The effect of sGC stimulation via BAY 41-2272 on mesangial proliferation was assessed in vivo using a mesangial proliferative glomerulonephritis model in rats (anti-Thy1 model). Renal biopsies, as well as glomerular isolates, urine samples, and blood samples were compared in BAY 41-2272- and placebo-treated groups during anti-Thy1 nephritis. The sGC beta(1)-subunit is upregulated during anti-Thy1 nephritis and mainly confined to mesangial areas by immunohistochemistry. Specific therapeutic sGC stimulation during anti-Thy1 nephritis in vivo was achieved via BAY 41-2272 treatment as demonstrated by increased glomerular cGMP levels causing inhibition of mesangial proliferation, glomerular matrix accumulation, and proteinuria compared with placebo-treated animals. sGC is tightly regulated in glomeruli during experimental glomerulonephritis. Considering its beneficial antiproliferative, antifibrotic, and antiproteinuric effect in experimental glomerulonephritis, the therapeutic stimulation of sGC could become a promising future goal in mesangial proliferative glomerulonephritis in humans.

Production and role of extracellular guanosine cyclic 3', 5' monophosphate in sodium uptake in human proximal tubule cells.

The present study was designed to determine the capability of human renal proximal tubule (RPT) to generate and export guanosine cyclic 3', 5' monophosphate (cGMP) in response to direct stimulation of soluble guanylyl cyclase by nitric oxide (NO) donors. In addition, we investigated whether cGMP extrusion from human RPT cells is required for inhibition of cellular sodium uptake. RPT cells were cultured from fresh human kidneys (normotensive subjects, n=4, mean age 65+/-4.7 years, 3 men, 1 woman; hypertensive patients, n=6, mean age 64+/-6.1 years, 4 men, 2 women) after unilateral nephrectomy. The fluorescence dye Sodium Green was employed to determine cytoplasmic Na+ concentration. In the presence of the Na+/K+ ATPase inhibitor ouabain, fluorescence was monitored at the appropriate wavelength (excitation 485 nm, emission 535 nm). Nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-4) M), increased both intracellular and extracellular cGMP (from 1.26+/-0.21 to 88.7+/-12.6 pmol/mg protein and from 0.58+/-0.10 to 9.24+/-1.9 pmol/mL, respectively, P<0.01) and decreased cellular Na+ uptake by 37.4+/-6.8% (P<0.05) compared with control. The effects of SNAP on cGMP production were similar in normotensive and hypertensive subjects. The increases in intracellular and extracellular cGMP concentration because of SNAP were blocked completely by soluble guanylyl cyclase inhibitor ODQ (1-H-[1,2,4] oxadiazolo [4,2-alpha] quinoxalin-1-one). Probenecid, an organic anion transport inhibitor, augmented the SNAP (10(-6) M)-induced increase in intracellular cGMP accumulation (from 4.9+/-0.9 to 9.8+/-1.5 pmol/mg protein, P<0.05), abrogated the SNAP-induced increase in extracellular cGMP extrusion (from 1.07+/-0.4 to 0.37+/-0.1 pmol/L, P<0.05) and blocked the SNAP-induced reduction in cellular Na+ uptake. Neither intracellular nor extracellular cGMP were influenced by l-arginine, the metabolic precursor of NO, or N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase. After exogenous administration of cGMP (10(-5) M) or its membrane-permeable analogue 8-Br-cGMP (10(-5) M), only 8-Br-cGMP crossed the cell membrane to increase intracellular cGMP (from 1.36+/-0.19 to 289.7+/-29.4 pmol/mg protein, P<0.01). However, both cGMP and 8-Br-cGMP were effective in decreasing cellular Na+ uptake. In conclusion, human RPT cells contain soluble guanylyl cyclase and are able to generate and export cGMP in response to NO. Because human RPT cells do not themselves contain constitutive NO synthase, the NO-generating cGMP must be derived from sources outside the human RPT. The cGMP cellular export system is critical in the regulation of RPT cellular Na+ absorption in humans.

Antitumor antibiotic streptonigrin and its derivatives as inhibitors of nitric oxide-dependent activation of soluble guanylyl cyclase

The influence of streptonigrin on the activity of human platelet guanylyl cyclase was investigated. Streptonigrin (0.1–5 μM) had no effect on the basal activity of the enzyme, but inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet soluble guanylyl cyclase with an IC 50 value of 4.16 μM. Streptonigrin (10 μM) also inhibited (by 28%) the activation of the enzyme by the direct nitric oxide (NO) donor–spermine–NONO (100 μM), but had no influence on the stimulation of soluble guanylyl cyclase by protoporphyrin IX. The absence of a correlation between the inhibition of NO-stimulated guanylyl cyclase activity by streptonigrin ( I) and its derivatives (streptonigrone ( IV), streptonigrone-2′-imine ( V), amide of 1 and 2′-deoxy-2′-amino- d-glucose ( VI), amide of 1 and 2′-deoxy-2′-amino-2′- d-galactose ( VII), amide of 1 and 1- O-methyl-6-deoxy-6-amino- d-glucose ( VIII), diphenylmethyl ester of I ( IX), conjugate of I and daunorubicin ( X)), and the level of cytotoxic effects of these compounds excludes the involvement of guanylyl cyclase in the mechanism of antitumor action of streptonigrin. Inhibition of guanylyl cyclase activation by NO donors but not by protoporphyrin IX represents a new biochemical effect of streptonigrin, which should be taken into account in addition to its antitumor action.

The NO signaling pathway differentially regulates KCC3a and KCC3b mRNA expression

Nitric oxide (NO) donors and protein kinase G (PKG) acutely up-regulate K–Cl cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in vascular smooth muscle cells (VSMCs). Here, we report the presence, relative abundance, and regulation by sodium nitroprusside (SNP) of the novel KCC3a and KCC3b mRNAs, in primary cultures of rat VSMCs. KCC3a and KCC3b mRNAs were expressed in an approximate 3:1 ratio, as determined by semiquantitative RT-PCR analysis. SNP as well as YC-1 and 8-Br-cGMP, a NO-independent stimulator of soluble guanylyl cyclase (sGC) and PKG, respectively, increased KCC3a and KCC3b mRNA expression by 2.5-fold and 8.1-fold in a time-dependent manner, following a differential kinetics. Stimulation of the NO/sGC/PKG signaling pathway with either SNP, YC-1, or 8-Br-cGMP decreased the KCC3a/KCC3b ratio from 3.0 ± 0.4 to 0.9 ± 0.1. This is the first report on a differential regulation by the NO/sGC/PKG signaling pathway of a cotransporter and of KCC3a and KCC3b mRNA expression.

Relaxing effects induced by the soluble guanylyl cyclase stimulator BAY 41-2272 in human and rabbit corpus cavernosum

5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1 H-pyrazolo[3,4- b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent soluble guanylyl cyclase stimulator in a nitric oxide (NO)-independent manner. The relaxant effect of BAY 41-2272 was investigated in rabbit and human corpus cavernosum in vitro. BAY 41-2272 (0.01–10 μM) relaxed both rabbit (pEC 50=6.82±0.06) and human (pEC 50=6.12±0.10) precontracted cavernosal strips. The guanylyl cyclase inhibitor (ODQ, 10 μM) caused significant rightward shifts in the concentration–response curves for BAY 41-2272 in rabbit (4.7-fold) and human (2.3-fold) tissues. The NO synthesis inhibitor ( N-nitro- l-arginine methyl ester ( l-NAME), 100 μM) also produced similar rightward shifts, revealing that BAY 41-2272 acts synergistically with endogenous NO to elicit its relaxant effect. The results also indicate that ODQ is selective for the NO-stimulated enzyme, since relaxations evoked by BAY 41-2272 were only partly attenuated by ODQ. The present study shows that both BAY 41-2272 and sildenafil evoke relaxations independent of inhibition of haem in soluble guanylate cyclase. Moreover, there is no synergistic effect of the two compounds in corpus cavernosum.

NO-dependent and NO-independent stimulation of soluble guanylyl cyclase in the rat frontal cortex: localization and characterization.

NO-dependent and NO-independent stimulation of soluble guanylyl cyclase in the rat frontal cortex: localization and characterization.

Particulate and soluble guanylyl cyclase stimulation in the treatment of heart failure

Particulate and soluble guanylyl cyclase stimulation in the treatment of heart failure

Mechanisms of nitric oxide independent activation of soluble guanylyl cyclase

The heterodimeric heme-protein soluble guanylyl cyclase (sGC) is the only proven receptor for nitric oxide (NO). Recently, two different types of NO-independent soluble guanylyl cyclase stimulators have been discovered. The heme-dependent stimulator 2-[1-[2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-5(4-morpholinyl)-4,6-pyrimidinediamine (BAY 41-8543) stimulates the enzyme in a synergistic fashion when combined with NO, requires the presence of the heme group and can be blocked by the soluble guanylyl cyclase inhibitor 1H-(1,2,4)-Oxadiazole-(4,3-a)-quinoxalin-1-one (ODQ). The heme-independent activator 4-[((4-carboxybutyl)[2-[(4-phenethylbenzol) oxy]phenethyl]amino)methyl[benzoic]acid (BAY 58-2667) activates soluble guanylyl cyclase even in the presence of ODQ or rendered heme-deficient. In the present study, BAY 41-8543, BAY 58-2667 and NO strongly increased V(max). Combination of BAY 58-2667 and NO increased V(max) in an additive manner, whereas the synergistic effect of BAY 41-8543 and NO on enzyme activation was reflected in an overadditive increase of V(max). ODQ potentiated V(max) of BAY 58-2667-stimulated soluble guanylyl cyclase. BAY 41-8543 prolonged the half-life of the nitrosyl-heme complex of NO-activated enzyme, an effect that was not observed with BAY 58-2667. These results show the different activation patterns of both compounds and demonstrate their value as tools to investigate the mechanisms that underlie soluble guanylyl cyclase activation.

Differential relaxing responses to particulate or soluble guanylyl cyclase activation on endothelial cells: a mechanism dependent on PKG-I alpha activation by NO/cGMP.

cGMP is generated in endothelial cells after stimulation of soluble guanylyl cyclase (sGC) by nitric oxide (NO) or of particulate guanylyl cyclase (pGC) by natriuretic peptides (NP). We examined whether localized increases in cytosolic cGMP have distinct regulatory roles on the contraction induced by H2O2 treatment in human umbilical vein endothelial cells. cGMP concentrations and temporal dynamics were different upon NO stimulation of sGC or C-type NP (CNP) activation of pGC and did not correlate with their relaxing effects measured as planar cell surface area after H2O2 challenge. cGMP production due to sGC stimulation was always smaller and more brief than that induced by pGC stimulation with CNP, which was greater and remained elevated longer. The NO effects on cell relaxation were cGMP dependent because they were blocked by sGC inhibition with 1H-(1,2,4)Oxadiazolo(4,3-a)quinoxaline-1-one and mimicked by 8-Br-cGMP. An antagonist of the cGMP-dependent protein kinase type-I (PKG-I) also inhibited the NO-induced effects. The cell contraction induced by H2O2 produces myosin light chain (MLC) phosphorylation and NO prevented it completely, whereas CNP only produced a partial inhibition. Transfection with a dominant negative form of PKG type-I alpha completely reversed the NO-induced effects on MLC phosphorylation, whereas it only partially inhibited the effects due to CNP. Taken together, these results demonstrate that the NO/sGC/cGMP pathway induces endothelial cell relaxation in a more efficient manner than does CNP/pGC/cGMP pathway, an effect that might be related to a selective stimulation of PKG-1 alpha by NO-derived cGMP. Consequently, stimulated PKG-I alpha may phosphorylate important protein targets that are necessary to inhibit the endothelial contractile machinery activated by oxidative stress.