Identification of residues crucially involved in soluble guanylate cyclase activation

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.     

Metabolites of orally active NO-independent pyrazolopyridine stimulators of soluble guanylate cyclase

The pyrazolopyridine stimulators of soluble guanylate cyclase BAY 41-2272 and 41-8543 were oxidised in rats and dogs at their 5-pyrimidinyl-cyclopropyl and -morpholino residue. These metabolites activate the soluble guanylate cyclase, induce vasoelaxation and thereby may contribute to the in vivo activity of BAY 41-2272 and BAY 41-8543.     

The Soluble Guanylyl Cyclase Activator Bay 58-2667 Selectively Limits Cardiomyocyte Hypertrophy

Background

Although evidence now suggests cGMP is a negative regulator of cardiac hypertrophy, the direct consequences of the soluble guanylyl cyclase (sGC) activator BAY 58-2667 on cardiac remodeling, independent of changes in hemodynamic load, has not been investigated. In the present study, we tested the hypothesis that the NO^•-independent sGC activator BAY 58-2667 inhibits cardiomyocyte hypertrophy in vitro. Concomitant impact of BAY 58-2667 on cardiac fibroblast proliferation, and insights into potential mechanisms of action, were also sought. Results were compared to the sGC stimulator BAY 41-2272.

Methods

Neonatal rat cardiomyocytes were incubated with endothelin-1 (ET₁, 60nmol/L) in the presence and absence of BAY 41-2272 and BAY 58-2667 (0.01–0.3 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. The impact of both sGC ligands on basal and stimulated cardiac fibroblast proliferation in vitro was also determined.

Results

We now demonstrate that BAY 58-2667 (0.01–0.3 µmol/L) elicited concentration-dependent antihypertrophic actions, inhibiting ET₁-mediated increases in cardiomyocyte 2D area and de novo protein synthesis, as well as suppressing ET₁-induced cardiomyocyte superoxide generation. This was accompanied by potent increases in cardiomyocyte cGMP accumulation and activity of its downstream signal, vasodilator-stimulated phosphoprotein (VASP), without elevating cardiomyocyte cAMP. In contrast, submicromolar concentrations of BAY 58-2667 had no effect on basal or stimulated cardiac fibroblast proliferation. Indeed, only at concentrations ≥10 µmol/L was inhibition of cardiac fibrosis seen in vitro. The effects of BAY 58-2667 in both cell types were mimicked by BAY 41-2272.

Conclusions

Our results demonstrate that BAY 58-2667 elicits protective, cardiomyocyte-selective effects in vitro. These actions are associated with sGC activation and are evident in the absence of confounding hemodynamic factors, at low (submicromolar) concentrations. Thus this distinctive sGC ligand may potentially represent an alternative therapeutic approach for limiting myocardial hypertrophy.     

Pulmonary vascular effects of nitric oxide-cGMP augmentation in a model of chronic pulmonary hypertension in fetal and neonatal sheep

Persistent pulmonary hypertension of the newborn (PPHN) is partly due to impaired nitric oxide (NO)-cGMP signaling. BAY 41-2272 is a novel direct activator of soluble guanylate cyclase, but whether this drug may be an effective therapy for PPHN is unknown. We hypothesized that BAY 41-2272 would cause pulmonary vasodilation in a model of severe PPHN. To test this hypothesis, we compared the hemodynamic response of BAY 41-2272 to acetylcholine, an endothelium-dependent vasodilator, and sildenafil, a selective inhibitor of PDE5 in chronically instrumented fetal lambs at 1 and 5 days after partial ligation of the ductus arteriosus. After 9 days, we delivered the animals by cesarean section to measure their hemodynamic responses to inhaled NO (iNO), sildenafil, and BAY 41-2272 alone or combined with iNO. BAY 41-2272 caused marked pulmonary vasodilation, as characterized by a twofold increase in blood flow and a nearly 60% fall in PVR at day 1. Effectiveness of BAY 41-2272-induced pulmonary vasodilation increased during the development of pulmonary hypertension. Despite a similar effect at day 1, the pulmonary vasodilator response to BAY 41-2272 was greater than sildenafil at day 5. At birth, BAY 41-2272 dramatically reduced PVR and augmented the pulmonary vasodilation induced by iNO. We concluded that BAY 41-2272 causes potent pulmonary vasodilation in fetal and neonatal sheep with severe pulmonary hypertension. We speculate that BAY 41-2272 may provide a novel treatment for severe PPHN, especially in newborns with partial response to iNO therapy.     

The Soluble Guanylate Cyclase Activator BAY 58-2667 Protects against Morbidity and Mortality in Endotoxic Shock by Recoupling Organ Systems

Sepsis and septic shock are associated with high mortality rates and the majority of sepsis patients die due to complications of multiple organ failure (MOF). The cyclic GMP (cGMP) producing enzyme soluble guanylate cyclase (sGC) is crucially involved in the regulation of (micro)vascular homeostasis, cardiac function and, consequently, organ function. However, it can become inactivated when exposed to reactive oxygen species (ROS). The resulting heme-free sGC can be reactivated by the heme- and nitric oxide (NO)-independent sGC activator BAY 58-2667 (Cinaciguat). We report that late (+8 h) post-treatment with BAY 58-2667 in a mouse model can protect against lethal endotoxic shock. Protection was associated with reduced hypothermia, circulating IL-6 levels, cardiomyocyte apoptosis, and mortality. In contrast to BAY 58-2667, the sGC stimulator BAY 41-2272 and the phosphodiesterase 5 inhibitor Sildenafil did not have any beneficial effect on survival, emphasizing the importance of the selectivity of BAY 58-2667 for diseased vessels and tissues. Hemodynamic parameters (blood pressure and heart rate) were decreased, and linear and nonlinear indices of blood pressure variability, reflective for (un)coupling of the communication between the autonomic nervous system and the heart, were improved after late protective treatment with BAY 58-2667. In conclusion, our results demonstrate the pivotal role of the NO/sGC axis in endotoxic shock. Stabilization of sGC function with BAY 58-2667 can prevent mortality when given in the correct treatment window, which probably depends on the dynamics of the heme-free sGC pool, in turn influenced by oxidative stress. We speculate that, considering the central role of sGC signaling in many pathways required for maintenance of (micro)circulatory homeostasis, BAY 58-2667 supports organ function by recoupling inter-organ communication pathways.     

Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice

High tidal volume (HV(T)) ventilation causes pulmonary endothelial barrier dysfunction. HV(T) ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV(T) lung injury, but the role of cGMP is unknown. In the current study, ventilation of isolated C57BL/6 mouse lungs increased perfusate cGMP as a function of V(T). Ventilation with 20 ml/kg V(T) for 80 min increased the filtration coefficient (K(f)), an index of vascular permeability. The increased cGMP and K(f) caused by HV(T) were attenuated by nitric oxide synthase (NOS) inhibition and in lungs from endothelial NOS knockout mice. Inhibition of soluble guanylyl cyclase (sGC) in wild-type lungs (10 muM ODQ) also blocked cGMP generation and inhibited the increase in K(f), suggesting an injurious role for sGC-derived cGMP. sGC inhibition also attenuated lung Evans blue dye albumin extravasation and wet-to-dry weight ratio in an anesthetized mouse model of HV(T) injury. Additional activation of sGC (1.5 muM BAY 41-2272) in isolated lungs at 40 min increased cGMP production and K(f) in lungs ventilated with 15 ml/kg V(T). HV(T) endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 muM IBMX) as well as an inhibitor (10 muM BAY 60-7550) specific for the cGMP-stimulated PDE2A. Concordantly, we found a V(T)-dependent increase in lung cAMP hydrolytic activity and PDE2A protein expression with a decrease in lung cAMP concentration that was blocked by BAY 60-7550. We conclude that HV(T)-induced endothelial barrier dysfunction resulted from a simultaneous increase in NO/sGC-derived cGMP and PDE2A expression causing decreased cAMP.     

Effects of BAY 41-2272, a soluble guanylate cyclase activator, on pulmonary vascular reactivity in the ovine fetus

Nitric oxide (NO)-cGMP signaling plays a critical role during the transition of the pulmonary circulation at birth. BAY 41-2272 is a novel NO-independent direct stimulator of soluble guanylate cyclase that causes vasodilation in systemic and local circulations. However, the hemodynamic effects of BAY 41-2272 have not been studied in the perinatal pulmonary circulation. We hypothesized that BAY 41-2272 causes potent and sustained fetal pulmonary vasodilation. We performed surgery on 14 fetal lambs (125-130 days gestation; term = 147 days) and placed catheters in the main pulmonary artery, aorta, and left atrium to measure pressures. An ultrasonic flow transducer was placed on the left pulmonary artery (LPA) to measure blood flow, and a catheter was placed in the LPA for drug infusion. Pulmonary vascular resistance (PVR) was calculated as pulmonary artery pressure minus left atrial pressure divided by LPA blood flow. BAY 41-2272 caused dose-related increases in pulmonary blood flow up to threefold above baseline and reduced PVR by 75% (P < 0.01). Prolonged infusion of BAY 41-2272 caused sustained pulmonary vasodilation throughout the 120-min infusion period. The pulmonary vasodilator effect of BAY 41-2272 was not attenuated by N(omega)-nitro-l-arginine, a NO synthase inhibitor. In addition, compared with sildenafil, a phosphodiesterase 5 inhibitor, the pulmonary vasodilator response to BAY 41-2272 was more prolonged. We conclude that BAY 41-2272 causes potent and sustained fetal pulmonary vasodilation independent of NO release. We speculate that BAY 41-2272 may have therapeutic potential for pulmonary hypertension associated with failure to circulatory adaptation at birth, especially in the setting of impaired NO production.     

Effects of hydrogen peroxide on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries

Abstract

The production of reactive oxygen species, including hydrogen peroxide (H₂O₂), is increased in diseased blood vessels. Although H₂O₂ leads to impairment of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP signaling pathway, it is not clear whether this reactive molecule affects the redox state of sGC, a key determinant of NO bioavailability. To clarify this issue, mechanical responses of endothelium-denuded rat external iliac arteries to BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator), nitroglycerin (NO donor), acidified NaNO₂ (exogenous NO) and 8-Br-cGMP (cGMP analog) were studied under exposure to H₂O₂. The relaxant response to BAY 41-2272 (pD₂: 6.79?±?0.10 and 6.62?±?0.17), BAY 60-2770 (pD₂: 9.57?±?0.06 and 9.34?±?0.15) or 8-Br-cGMP (pD₂: 5.19?±?0.06 and 5.24?±?0.08) was not apparently affected by exposure to H₂O₂. In addition, vascular cGMP production stimulated with BAY 41-2272 or BAY 60-2770 in the presence of H₂O₂ was identical to that in its absence. On the other hand, nitroglycerin-induced relaxation was markedly attenuated by exposing the arteries to H₂O₂ (pD₂: 8.73?±?0.05 and 8.30?±?0.05), which was normalized in the presence of catalase (pD₂: 8.59?±?0.05). Likewise, H₂O₂ exposure impaired the relaxant response to acidified NaNO₂ (pD₂: 6.52?±?0.17 and 6.09?±?0.16). These findings suggest that H₂O₂ interferes with the NO-mediated action, but the sGC redox equilibrium and the downstream target(s) of cGMP are unlikely to be affected in the vasculature.     

Characterization of two different five-coordinate soluble guanylate cyclase ferrous-nitrosyl complexes

Soluble guanylate cyclase (sGC), a hemoprotein, is the primary nitric oxide (NO) receptor in higher eukaryotes. The binding of NO to sGC leads to the formation of a five-coordinate ferrous-nitrosyl complex and a several hundred-fold increase in cGMP synthesis. NO activation of sGC is influenced by GTP and the allosteric activators YC-1 and BAY 41-2272. Electron paramagnetic resonance (EPR) spectroscopy shows that the spectrum of the sGC ferrous-nitrosyl complex shifts in the presence of YC-1, BAY 41-2272, or GTP in the presence of excess NO relative to the heme. These molecules shift the EPR signal from one characterized by g 1 = 2.083, g 2 = 2.036, and g 3 = 2.012 to a signal characterized by g 1 = 2.106, g 2 = 2.029, and g 3 = 2.010. The truncated heme domain constructs beta1(1-194) and beta2(1-217) were compared to the full-length enzyme. The EPR spectrum of the beta2(1-217)-NO complex is characterized by g 1 = 2.106, g 2 = 2.025, and g 3 = 2.010, indicating the protein is a good model for the sGC-NO complex in the presence of the activators, while the spectrum of the beta1(1-194)-NO complex resembles the EPR spectrum of sGC in the absence of the activators. Low-temperature resonance Raman spectra of the beta1(1-194)-NO and beta2(1-217)-NO complexes show that the Fe-NO stretching vibration of the beta2(1-217)-NO complex (535 cm (-1)) is significantly different from that of the beta1(1-194)-NO complex (527 cm (-1)). This shows that sGC can adopt different five-coordinate ferrous nitrosyl conformations and suggests that the Fe-NO conformation characterized by this unique EPR signal and Fe-NO stretching vibration represents a highly active sGC state.     

A real-time fluorescent assay of the purified nitric oxide receptor, guanylyl cyclase

Nitric oxide (NO) mediates intercellular signaling through activation of its receptor, soluble guanylyl cyclase (sGC), leading to elevation of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP) levels. Through this signal transduction pathway, NO regulates a diverse range of physiological effects, from vasodilatation and platelet disaggregation to synaptic plasticity. Measurement of sGC activity has traditionally been carried out using end-point assays of cGMP accumulation or by transfection of cells with “detector” proteins such as fluorescent proteins coupled to cGMP binding domains or cyclic nucleotide gated channels. Here we report a simpler approach: the use of a fluorescently labeled substrate analog, mant-GTP (2′-O-(N-methylanthraniloyl) guanosine 5′-triphosphate), which gives an increase in emission intensity after enzymatic cyclization to mant-cGMP. Activation of purified recombinant sGC by NO led to a rapid rise in fluorescence intensity within seconds, reaching a maximal 1.6- to 1.8-fold increase above basal levels. The sGC inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), eliminated the fluorescence increase due to NO, and the synergistic activator of sGC, BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), increased the rate at which the maximal fluorescence increase was attained. High-performance liquid chromatography (HPLC) confirmed the formation of mant-cGMP product. This real-time assay allows the progress of purified sGC activation to be quantified precisely and, with refinement, could be optimized for use in a cellular environment.     

The use of alpha-blockers for the treatment of nephrolithiasis

Medical expulsion therapy has been shown to be a useful adjunct to observation in the management of ureteral stones. Alpha-1-adrenergic receptor antagonists have been studied in this role. Alpha-1 receptors are located in the human ureter, especially the distal ureter. Alpha-blockers have been demonstrated to increase expulsion rates of distal ureteral stones, decrease time to expulsion, and decrease need for analgesia during stone passage. Alpha-blockers promote stone passage in patients receiving shock wave lithotripsy, and may be able to relieve ureteral stent-related symptoms. In the appropriate clinical scenario, the use of alpha-blockers is recommended in the conservative management of distal ureteral stones.     

BAY 41-2272 activates host defence against local and disseminated Candida albicans infections

In our previous study, we have found that 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a guanylate cyclase agonist, activates human monocytes and the THP-1 cell line to produce the superoxide anion, increasing in vitro microbicidal activity, suggesting that this drug can be used to modulate immune functioning in primary immunodeficiency patients. In the present work, we investigated the potential of the in vivo administration of BAY 41-2272 for the treatment of Candida albicans and Staphylococcus aureus infections introduced via intraperitoneal and subcutaneous inoculation. We found that intraperitoneal treatment with BAY 41-2272 markedly increased macrophage-dependent cell influx to the peritoneum in addition to macrophage functions, such as spreading, zymosan particle phagocytosis and nitric oxide and phorbol myristate acetate-stimulated hydrogen peroxide production. Treatment with BAY 41-2272 was highly effective in reducing the death rate due to intraperitoneal inoculation of C. albicans, but not S. aureus. However, we found that in vitro stimulation of peritoneal macrophages with BAY 41-2272 markedly increased microbicidal activities against both pathogens. Our results show that the prevention of death by the treatment of C. albicans-infected mice with BAY 41-2272 might occur primarily by the modulation of the host immune response through macrophage activation.     

BAY 41-2272 activates two isoforms of nitric oxide-sensitive guanylyl cyclase

Soluble guanylyl cyclase is an important target for endogenous nitric oxide and the guanylyl cyclase modulator, YC-1. Recently BAY 41-2272 was identified as a similar but more potent and more specific substance. While YC-1 also acts as non-specific phosphodiesterase inhibitor, BAY 41-2272 is devoid of an effect on phosphodiesterases. BAY 41-2272 has so far only been tested on the alpha(1)/beta(1) heterodimeric isoform of soluble guanylyl cyclase and its binding site has been mapped to a region in the alpha(1) subunit amino-terminal sequence. Although this region is poorly conserved in the alpha(2) subunit, we show in the current study that the alpha(2)/beta(1) heterodimeric enzyme isoform is activated by BAY 41-2272. Deletion analysis of the alpha(2) subunit and co-expression with the beta(1) subunit in the baculovirus/Sf9 system is consistent with the amino-terminal amino acids 104 to 401 of the alpha(2) subunit as binding site for BAY 41-2272.     

Thrombospondin-1 and angiotensin II inhibit soluble guanylyl cyclase through an increase in intracellular calcium concentration

Nitric oxide (NO) regulates cardiovascular hemostasis by binding to soluble guanylyl cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca(2+)](i)), and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, we show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells and that inhibition requires an increase in [Ca(2+)](i). Using flow cytometry, we show that E3CaG1 binds directly to CD47 on the surface of Jurkat T cells. Using digital imaging microscopy on live cells, we further show that E3CaG1 binding results in a substantial increase in [Ca(2+)](i), up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca(2+)](i), also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca(2+) remains inhibited, while addition of kinase inhibitor staurosporine prevents inhibition, indicating inhibition is likely due to phosphorylation. Inhibition is through an increase in K(m) for GTP, which rises to 834 μM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca(2+)](i) in response to NO, inducing vasodilation, but also is inhibited by high [Ca(2+)](i), providing a fine balance between signals for vasodilation and vasoconstriction.     

The nitric oxide-cGMP signaling pathway plays a significant role in tolerance to the analgesic effect of morphine

Although the phenomenon of opioid tolerance has been widely investigated, neither opioid nor nonopioid mechanisms are completely understood. The aim of the present study was to investigate the role of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway in the development of morphine-induced analgesia tolerance. The study was carried out on male Wistar albino rats (weighing 180-210 g; n = 126). To develop morphine tolerance, animals were given morphine (50 mg/kg; s.c.) once daily for 3 days. After the last dose of morphine was injected on day 4, morphine tolerance was evaluated. The analgesic effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), BAY 41-2272, S-nitroso-N-acetylpenicillamine (SNAP), N(G)-nitro-L-arginine methyl ester (L-NAME), and morphine were considered at 15 or 30 min intervals (0, 15, 30, 60, 90, and 120 min) by tail-flick and hot-plate analgesia tests (n = 6 in each study group). The results showed that YC-1 and BAY 41-2272, a NO-independent activator of soluble guanylate cyclase (sGC), significantly increased the development and expression of morphine tolerance, and L-NAME, a NO synthase (NOS) inhibitor, significantly decreased the development of morphine tolerance. In conclusion, these data demonstrate that the nitric oxide-cGMP signal pathway plays a pivotal role in developing tolerance to the analgesic effect of morphine.     

cGMP-dependent and -independent angiogenesis-related properties of nitric oxide

Nitric oxide exerts a stimulatory role during postnatal angiogenesis. Although soluble guanylyl cyclase (sGC) mediates many of the effects of nitric oxide (NO) in the vascular system, the contribution of cGMP-dependent vs cGMP-independent pathways in NO-induced angiogenesis remains unclear. Herein, we determined the effects of a NO donor (sodium nitroprusside; SNP) and a NO-independent sGC activator (BAY 41-2272) in the growth and migration of rat aortic endothelial cells (RAEC). RAEC lack enzymatically active sGC as suggested by their inability to accumulate cGMP upon exposure to SNP. However, treatment of RAEC with SNP promoted a modest increase in their proliferation and migration that was dependent on extracellular signal regulated kinase1/2 activation. Moreover, when RAEC were exposed to vascular endothelial growth factor we observed an increase in migration that was inhibited by NO synthase, but not sGC, inhibition. Infection of cells with adenoviruses containing sGC greatly increased the efficacy of SNP as a mitogenic and migratory stimulus. We conclude that NO is capable of stimulating EC proliferation and mobility in the absence of sGC; however, increased intracellular levels of cGMP following sGC activation greatly amplify the angiogenic potential of NO.     

The G-protein regulator LGN modulates the activity of the NO receptor soluble guanylate cyclase

sGC (soluble guanylate cyclase) is the main mediator of NO signalling. Biochemical and physiological studies suggest that, besides NO, in vivo regulation of sGC involves direct interaction with other proteins. Using yeast two-hybrid screening, we identified that the multidomain LGN (Leu-Gly-Asn repeat-enriched protein) interacts with both α1 and β1 sGC subunits. LGN and sGC co-localized in the cell cytoplasm, and the LGN-sGC complex was co-immunoprecipitated from cells expressing both proteins and from native tissues. Their interaction requires the N-terminal tetratricopeptide repeats of LGN, but does not require the N-terminal portions of α1 or β1 sGC subunits. Overexpression of LGN decreases the activity of cellular sGC, whereas knockdown of LGN mRNA and protein correlated with increased sGC activity. Although purified LGN interacts directly with purified sGC, the inhibitory effect in vitro is observed only after supplementation of cell lysate to the reaction. Although resting sGC and sGC activated by the stimulator BAY41-2272 have very similar LGN-IC50 values to the NO-stimulated sGC, they have a much higher Hill coefficient, suggesting co-operative binding with respect to LGN in the low-activated state of sGC. AGS3 (activator of G-protein signalling 3), the closest LGN homologue, also inhibits sGC. The interaction of sGC with these scaffolding proteins may expand the cross-talk between NO/cGMP signalling and other cellular pathways and tailor sGC function to specific tissues or signals.     

Activation of Haem-Oxidized Soluble Guanylyl Cyclase with BAY 60-2770 in Human Platelets Lead to Overstimulation of the Cyclic GMP Signaling Pathway

Background and Aims

Nitric oxide-independent soluble guanylyl cyclase (sGC) activators reactivate the haem-oxidized enzyme in vascular diseases. This study was undertaken to investigate the anti-platelet mechanisms of the haem-independent sGC activator BAY 60-2770 in human washed platelets. The hypothesis that sGC oxidation potentiates the anti-platelet activities of BAY 60-2770 has been tested.

Methods

Human washed platelet aggregation and adhesion assays, as well as flow cytometry for α_IIbβ₃ integrin activation and Western blot for α1 and β1 sGC subunits were performed. Intracellular calcium levels were monitored in platelets loaded with a fluorogenic calcium-binding dye (FluoForte).

Results

BAY 60-2770 (0.001–10 µM) produced significant inhibition of collagen (2 µg/ml)- and thrombin (0.1 U/ml)-induced platelet aggregation that was markedly potentiated by the sGC inhibitor ODQ (10 µM). In fibrinogen-coated plates, BAY 60-2770 significantly inhibited platelet adhesion, an effect potentiated by ODQ. BAY 60-2770 increased the cGMP levels and reduced the intracellular Ca²⁺ levels, both of which were potentiated by ODQ. The cell-permeable cGMP analogue 8-Br-cGMP (100 µM) inhibited platelet aggregation and Ca²⁺ levels in an ODQ-insensitive manner. The cAMP levels remained unchanged by BAY 60-2770. Collagen- and thrombin-induced α_IIbβ₃ activation was markedly inhibited by BAY 60-2770 that was further inhibited by ODQ. The effects of sodium nitroprusside (3 µM) were all prevented by ODQ. Incubation with ODQ (10 µM) significantly reduced the protein levels of α1 and β1 sGC subunits, which were prevented by BAY 60-2770.

Conclusion

The inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca²⁺ levels and α_IIbβ₃ activation are all potentiated in haem-oxidizing conditions. BAY 60-2770 prevents ODQ-induced decrease in sGC protein levels. BAY 60-2770 could be of therapeutic interest in cardiovascular diseases associated with thrombotic complications.     

High-salt diets during pregnancy increases renal vascular reactivity due to altered soluble guanylyl cyclase-related pathways in rat offspring

Adverse prenatal factors such as overtake of salt or fat food are potential risks for cardiovascular diseases in offspring. This study tested the hypothesis that prenatal high-salt (HS) diets may influence renal vascular tone and attenuates signaling pathways related to soluble guanylyl cyclase (sGC) or/and large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in the offspring.Pregnant rats were fed either normal salt (NS) (1% NaCl) or HS (8% NaCl) diet for the whole gestation. Offspring were maintained on NS diets. Renal interlobar arteries in offspring were tested for vascular responses to phenylephrine (Phe), K⁺ channels and signal pathways related to sGC.Phe induced higher vessel tension in interlobar arteries of the HS offspring. Following pretreatment with BK_Ca channel inhibitor iberiotoxin, Phe-mediated vasoconstrictions were decreased in HS offspring compared to NS. Phe-mediated constrictions following pretreatment with NO synthase inhibitor N(G)-nitro-l-arginine methyl ester or sGC inhibitor 1H-1,2,4-oxadiazolo-4,3-quinoxalin-1-one in the HS offspring were less sensitive than NS. The whole-cell K⁺ currents and the component of BK_Ca channels were not changed in smooth muscle cells from interlobar arteries, whereas the K⁺ currents stimulated by sGC activator BAY41-2272 were reduced in the HS offspring. The protein expressions of sGC β1 and β2 in the interlobar arteries of HS offspring were reduced.The results showed that chronic overintake of salt during pregnancy could increase renal vascular tone in the offspring. The affected signal pathways included down-regulation of sGC function and expression.