sGC Protein Levels Research Articles

Botulinum toxin type A suppresses arterial vasoconstriction by regulating calcium sensitization and the endothelium-dependent endothelial nitric oxide synthase/soluble guanylyl cyclase/cyclic guanosine monophosphate pathway: An in vitro study.

Botulinum toxin type A (BTX-A) is a potent neurotoxin that causes relaxation of striated muscle by inhibiting the release of acetylcholine at the neuromuscular junction. Some studies have suggested that BTX-A treatment for Raynaud syndrome is safe and effective with few adverse reactions. However, the underlying mechanism remains unclear. In the present study, we used both arterial rings isolated from rabbits and human microvascular endothelial cells (HMEC-1) to evaluate the mechanism underlying the effects of BTX-A on arterial vasoconstriction induced by 5-hydroxytryptamine. The roles of calcium sensitization and the endothelial nitric oxide synthase (eNOS)/soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway were investigated. BTX-A caused a concentration-dependent decrease in the contraction of endothelium-intact arteries and significantly reduced calcium sensitization in the arteries. Inhibitors of eNOS (L-NAME) and sGC (methylene blue) both significantly abolished the vasodilatory action of BTX-A. Furthermore, BTX-A increased eNOS activity and the cGMP level in dose- and time-dependent manners and increased eNOS and sGC protein levels in a time-dependent manner in HMEC-1. Taken together, these findings indicate that BTX-A suppresses arterial vasoconstriction by regulating smooth muscle calcium sensitization and the eNOS/sGC/cGMP pathway. Impact statement Raynaud syndrome (RS), usually caused by cold or mental stress, may lead to cyanosis and reactive hyperemia accompanied by pain or paresthesia. Although a variety of drugs have been used to alleviate the symptoms, the effects have not been satisfactory, so there is an urgent need to explore new alternative treatments. The present study investigated the mechanism underlying the effects of botulinum toxin type A (BTX-A) on arterial vasoconstriction, which may provide new approaches for RS. We found that BTX-A suppresses arterial vasoconstriction by regulating smooth muscle calcium sensitization and the endothelial nitric oxide synthase (eNOS)/soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway. These findings clarify the underlying mechanism of the effects of BTX-A on arterial vasoconstriction and provide new theoretical support for the use of BTX-A on RS. It may also help us to develop new medicines which regulate calcium sensitization and the eNOS/sGC/cGMP pathway against RS.

Downregulation of Guanylate Cyclase Enzyme in Human Asthma model to Investigate NO‐sGc‐ cGMP as a Therapeutic Pathway in Asthma

Asthma is characterized by airway smooth muscle hypersensitivity. Common treatments include β‐adrenergic agonist therapy, such as budesonide, to induce airway smooth muscle relaxation via increased cAMP. However, 70% of patients develop resistance to such therapies. Nitric oxide is a locally produced smooth muscle relaxant that operates via stimulation of soluble guanylate cyclase (sGc) to increase cGMP production. In this study, the NO‐sGc‐cGMP pathway was investigated as an alternate pharmacologic pathway for asthma therapy. Previous studies have found that asthma patients are insensitive to NO signaling and have used sGc drugs to directly stimulate the enzyme. The objective of this study was to examine the expression of the two sGc subunits, α and β, within smooth muscle cells isolated from asthma patients in order to understand how NO‐sGC‐cGMP signaling may be disrupted. Further, we examined how budesonide treatment altered sGc expression. RNA sequencing data showed increased mRNA for both sGc subunits in asthmatic patients and that budesonide downregulated the gene expression of both subunits. We hypothesized that this increased gene expression was in part due to a lack of cGMP signaling due to decreased protein levels that is further impaired with steroids such as budesonide. To examine sGc, we measured the protein levels of both the α and β subunits of sGC by western blot in cultured smooth muscle cells. Fatal asthma and non‐asthma donor derived primary airway smooth muscle cells were cultured with and 24 hours of budesonide treatment. Statistical analysis of western blotting was performed by ANOVA. Densitometry of sGC western blots indicated a 2‐fold downregulation of the α (p<0.0001) and 1.5 fold downregulation of the β (p<0.05) subunits in asthmatic compared to normal patients. No significant changes were observed with normal or asthmatic patients with budesonide treatment (p>0.05), indicating no effect of steroids on the NO‐sGc‐cGMP pathway. A downregulation of sGc protein levels in asthmatic patients would lower cGMP production, which could explain the lack of NO responsiveness in these patients and would contribute to increased airway constriction in fatal asthma. These results indicate the NO‐sGc‐cGMP pathway as a potential new therapeutic pathway in asthma, and future work will examine cGMP levels.Support or Funding InformationSupported by R25ES020721 and ASPET SURF Intern ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nitric oxide signalling pathway in Duchenne muscular dystrophy mice: up-regulation of L-arginine transporters

DMD (Duchenne muscular dystrophy) is an incurable rapidly worsening neuromuscular degenerative disease caused by the absence of dystrophin. In skeletal muscle a lack of dystrophin disrupts the recruitment of neuronal NOS (nitric oxide synthase) to the sarcolemma thus affecting NO (nitric oxide) production. Utrophin is a dystrophin homologue, the expression of which is greatly up-regulated in the sarcolemma of dystrophin-negative fibres from mdx mice, a mouse model of DMD. Although cardiomyopathy is an important cause of death, little is known about the NO signalling pathway in the cardiac muscle of DMD patients. Thus we used cardiomyocytes and hearts from two month-old mdx and mdx:utrophin-/- (double knockout) mice (mdx:utr) to study key steps in NO signalling: L-arginine transporters, NOS and sGC (soluble guanylyl cyclase). nNOS did not co-localize with dystrophin or utrophin to the cardiomyocyte membrane. Despite this nNOS activity was markedly decreased in both mdx and mdx:utr mice, whereas nNOS expression was only decreased in mdx:utr mouse hearts, suggesting that utrophin up-regulation in cardiomyocytes maintains nNOS levels, but not function. sGC protein levels and activity remained at control levels. Unexpectedly, L-arginine transporter expression and function were significantly increased, suggesting a novel biochemical compensatory mechanism of the NO pathway and a potential entry site for therapeutics.

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 AimsNitric 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.MethodsHuman washed platelet aggregation and adhesion assays, as well as flow cytometry for αIIbβ3 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).ResultsBAY 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 αIIbβ3 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.ConclusionThe inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca2+ levels and αIIbβ3 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.

Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells

In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e.g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions.

Oxidative stress induces CHIP-mediated ubiquitination and roteasomal degradation of soluble guanylyl cyclase

Oxidative stress attenuates the NO-cGMP pathway, e.g. in the vascular system, through scavenging of free NO radicals by superoxide O2, by inactivation of soluble guanylyl cyclase (sGC) via oxidation of its central Fe2+ ion, and by down-regulation of sGC protein levels. While the former pathways are well established, the molecular mechanisms underlying the latter are still obscure. Using oxidative sGC inhibitor ODQ we demonstrate rapid down-regulation of sGC protein in mammalian cells. Coincubation with proteasomal inhibitor MG132 results in accumulation of ubiquitinated sGC whereas sGC activator BAY 58–2667 prevents ubiquitination. ODQ-induced down-regulation of sGC is mediated through selective ubiquitination of its b subunit, and BAY 58–2667 abrogates this effect. Ubiquitination of sGC-b is dramatically enhanced by E3 ligase CHIP. Our data indicate that oxidative stress promotes ubiquitination of sGC b subunit through E3 ligase CHIP, and that sGC activator 58–2667 reverts this effect, most likely through stabilization of the heme-free b subunit. Thus the deleterious effects of oxidative stress can be counter-balanced by an activator of a key enzyme of vascular homeostasis. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007

Soluble guanylyl cyclase expression is reduced in LPS-induced lung injury

Soluble guanylyl cyclase (sGC) is a cGMP-generating enzyme implicated in the control of smooth muscle tone that also regulates platelet aggregation. Moreover, sGC activation has been shown to reduce leukocyte adherence to the endothelium. Herein, we investigated the expression of sGC in a murine model of LPS-induced lung injury and evaluated the effects of sGC inhibition in the context of acute lung injury (ALI). Lung tissue sGC alpha1 and beta1 subunit protein levels were determined by Western blot and immunohistochemistry, and steady-state mRNA levels for the beta1 subunit were assessed by real-time PCR. LPS inhalation resulted in a decrease in beta1 mRNA levels, as well as a reduction in both sGC subunit protein levels. Decreased alpha1 and beta1 expression was observed in bronchial smooth muscle and epithelial cells. TNF-alpha was required for the LPS-triggered reduction in sGC protein levels, as no change in alpha1 and beta1 levels was observed in TNF-alpha knockout mice. To determine the effects of sGC blockade in LPS-induced lung injury, mice were exposed to 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-l-one (ODQ) prior to the LPS challenge. Such pretreatment led to a further increase in total cell number (mainly due to an increase in neutrophils) and protein concentration in the bronchoalveoalar lavage fluid; the effects of ODQ were reversed by a cell-permeable cGMP analog. We conclude that sGC expression is reduced in LPS-induced lung injury, while inhibition of the enzyme with ODQ worsens lung inflammation, suggesting that sGC exerts a protective role in ALI.

Maturational changes in NO sensitivity are due to increases in soluble gaunylyl cyclase in the developing rat lung

Nitric oxide (NO) is an important regulator of pulmonary vascular tone in the neonate. We have shown that maturational changes exist in pulmonary NO production during the first weeks of neonatal life. We tested the hypothesis that maturational changes in sensitivity to NO exist in the pulmonary vascular bed. NO has its vasodilatory effect via increased cGMP levels, which depend on the balance between soluble gaunylyl cyclase (sGC) and phosphodiesterase type 5 (PDE-5) activities. The vasoconstrictor responses to K+ and the subsequent vasodilator responses to 0.1 μM spermineNONOate, a NO donor, were studied in isolated, perfused lungs from 7 and 14 day old Sprague-Dawley rats in the presence of 300 μM Nù-nitro-L-arginine. Baseline total pulmonary vascular resistance (PVR) was not different between age groups. The addition of KCl to the perfusate caused a concentration-dependent increase in PVR. The addition of spermineNONOate resulted in greater vasodilation in 14 day lungs than in 7 day lungs. Lungs were harvested from a separate group of age matched animals for determination of cGMP levels, as well as immunoblotting for sGC and PDE-5. The lung levels of cGMP where greater at 14 days than at 7 days (14.4±2.3 pmol/mL vs. 5.6±0.4 pmol/mL; p≤0.003). Protein levels of sGC were greater at 14 days than at 7 days. There were no differences between 7 day and 14 day lung protein levels of PDE-5. Our results demonstrate that maturational differences exist in the pulmonary vascular responsiveness to NO, and that these maturational differences in NO sensitivity are due, at least in part, to developmental increases in sGC protein levels.

Effects of nitric oxide on sGC redox states

Soluble guanylyl cyclase (sGC), the best-established nitric oxide receptor is a heterodimeric heme-protein synthesizing cGMP upon NO binding to its Fe(II)heme. cGMP in turn plays an important role in the regulation of vascular tone and platelet aggregation. The redox state of the sGC heme is fundamental for the activation by NO. A novel class of sGC activators, such as the recently described BAY 58-2667, are however NO-independent and target sGC in its Fe(III) and heme-free state. Given the therapeutic potential of such sGC activators, the mechanisms that modulate the sensitivity of sGC towards these agonists is of great interest. In this study we found, that treatment of primary porcine pulmonary artery endothelial cells with the NO donor DETA-NO (100 μmol/l) leads to a downregulation of DEA-NO stimulated and an up-regulation of BAY 58-2667 stimulated sGC activities (pmol/106 cells) [DEA-NO: 4.83 ± 0.11 vs. 3.02 ± 0.08***, BAY 58-2667: 0.77 ± 0.01 vs. 1.91 ± 0.04***; control vs. 2 h DETA-NO pre-treatment]. Furthermore, we observed a down-regulation of sGC (α1/β1) protein levels upon treatment with DETA-NO in a doseand time-dependent manner. Interestingly, the decrease in protein levels (maximal at t = 72 h: 56.7 ± 7% of control for sGC α1 and 44.4 ± 8% of control for sGC β1) occured subsequent to the apparent reduction in NO stimulatable sGC activity (maximal at t = 2 h). Downregulation of cellular cGMP responses has recently been attributed to upregulation of cGMP breakdown via enhanced PDE5 activity. Thus we tested the effects of the cGMP analogue 8-Br-cGMP (125/250/500 μmol/l), the mixed NO-independent sGC activator and PDE inhibitor YC-1 (10/50/100 μmol/l) and the selective phosphodiesterase-5 inhibitor zaprinast (50/100/200 μmol/l) on sGC protein levels. In contrast to DETA-NO, none of these modulators altered sGC protein expression arguing against a cGMP/PDE dependent mechanism. To determine if endogenously produced NO mimicked the effects of DETA-NO on sGC we utilised two models. First, porcine endothelial cells were pretreated with the endothelial NO synthase (eNOS) inhibitor L-NAME (100 μmol/l from 2 to 72 hours) and, second, aortas from eNOS deficient mice were investigated. L-NAME induced changes in endothelial NO and BAY 58-2667 stimulated sGC activity, that were reciprocal to those observed with DETA-NO treatment. While sGC expression and the response to DEA-NO were enhanced, the response to BAY 58-2667 was un-altered. Concurrent to these findings, in aortic homogenates from eNOS -/mice, an increased DEA-NO induced sGC response was observed as compared to wild type controls. In conclusion, our data suggests endogenous regulation of the sGC redox state via NO. The decrease in the cGMP response to DEA-NO and the increase to BAY 58-2667 induced cGMP production following DETA-NO pretreatment, indicates a change in the redox state of the sGC heme from a ferrous to ferric or even heme free. Furthermore, these changes in the redox state of sGC, seem to induce a protein degradation of sGC (α1/β1). Hence, desensitization of the NO/cGMP-pathway is heterogenous, occurring either as a result of PDE-5 upregulation or, as in pulmonary endothelial cells, via a PDE-5-independent mechanism involving a change in sGC redox state and protein stability. from 2nd International Conference of cGMP Generators, Effectors and Therapeutic Implications Potsdam, Germany, 10–12 June, 2005

Endostatin Down-Regulates Soluble Guanylate Cyclase (sGC) in Endothelial Cells In Vivo: Influence of Endostatin on Vascular Endothelial Growth Factor (VEGF) Signaling

Endostatin was suggested to be an antiangiogenic agent with the potential for clinical use in cancer therapy. Unfortunately, up to now no antiangiogenic effect was seen in clinical trials using this substance. The lack of response might be caused by an incomplete understanding of endostatin signaling. Endostatin is known to influence the vascular endothelial growth factor (VEGF) signaling pathway. It has been reported to bind to the VEGF receptor KDR directly and to decrease the phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 via the protein phosphatase 2A (PP2A). But so far no details of endostatin signaling with regard to NO downstream effectors have been revealed. In the present work the authors demonstrate that endostatin down-regulates the protein level of soluble guanylate cyclase (sGC) in endothelial cells of newly formed blood vessels in 5 day-old wounds (control: 62.5 +/- 33 vessels/mm2, endostatin: 9.2 +/- 3.2 vessels/mm2). This was confirmed in experiments with endothelial tubes of embryoid bodies and endothelial cells derived from embryonic stem cells (eESCs; control: 126 +/- 20, endostatin: 58 +/- 10). The decrease of sGC protein levels in response to endostatin was abolished after preincubation with the PP2A inhibitor okadaic acid. No alterations of sGC mRNA levels could be found under endostatin treatment in eESC. The authors conclude that endostatin affects VEGF signaling in endothelial cells by a post-transcriptional PP2A-dependent down-regulation of sGC protein levels.

Upregulation of lung soluble guanylate cyclase during chronic hypoxia is prevented by deletion of eNOS.

Hypoxia upregulates endothelial (e) nitric oxide synthase (NOS), but how eNOS affects soluble guanylate cyclase (sGC) protein expression in hypoxia-induced pulmonary hypertension is unknown. Wild-type (WT), eNOS-deficient [eNOS(-/-)], and inducible NOS (iNOS)-deficient [iNOS(-/-)] mice were used to investigate the effects of lack of NO from different NOS isoforms on sGC activity and protein expression and its relationship to the muscularization of the pulmonary vasculature. After 6 days of hypoxic exposure (10% O2), the ratios of the right ventricle to left ventricle + septum weight (RV/LV+S) and right ventricle weight to body weight, the lung sGC activity, and vascular muscularization were determined, and protein analysis for eNOS, iNOS, and sGC was performed. Results demonstrated that there were significant increases of RV/LV+S in all animals treated with hypoxia. In hypoxic WT and iNOS(-/-) mice, eNOS and sGC alpha1- and beta1-protein increased twofold; cGMP levels and the number of muscularized vessels also increased compared with hypoxic eNOS(-/-) mice. There was a twofold increase of iNOS protein in WT and eNOS(-/-) mice, and the basal iNOS protein concentration was higher in eNOS(-/-) mice than in WT mice. In contrast, the eNOS(-/-) mouse lung showed no eNOS protein expression, lower cGMP concentrations, and no change of sGC protein levels after hypoxic exposure compared with its normoxic controls (P > 0.34). These results suggest that eNOS, but not iNOS, is a major regulator of sGC activity and protein expression in the pulmonary vasculature.

Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus.

Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

Mechanisms of tolerance to sodium nitroprusside in rat cultured aortic smooth muscle cells.

1. While exposure of smooth muscle cells to sodium nitroprusside (SNP) leads to the development of tolerance to soluble guanylate cyclase (sGC) activation, the mechanisms responsible for this phenomenon in intact cells remain unclear. In the present study, possible mechanisms of tolerance were investigated in a cell culture model where sGC activity was estimated from the accumulation of cyclic GMP in response to 10 microM SNP over a 15 min period in the presence of a phosphodiesterase (PDE) inhibitor. 2. Pretreatment of rat aortic smooth muscle cells with 10-500 microM SNP led to a dose-dependent downregulation of cyclic GMP accumulation upon subsequent SNP stimulation. This effect was evident as early as 2 h following incubation with 10 microM SNP, reached a plateau at 4 h and was blocked by co-incubation with 30 microM oxyhaemoglobin. 3. Pretreatment of smooth muscle cells with the PDE inhibitor, zaprinast, resulted in downregulation of the SNP-induced cyclic GMP accumulation in a time- and concentration-dependent manner, that was first evident after 12 h. Moreover, while the zaprinast-induced downregulation of cyclic GMP accumulation was completely inhibited by the protein kinase A (PKA) inhibitor, H89, tolerance to SNP was partially reversed by H89. 4. beta 1 sGC steady state mRNA levels of S-nitroso N-acetylpenicillamine (SNAP)- or 8Br-cyclic GMP-pretreated cells were unchanged, as indicated by Northern blot analysis. However, Western blot analysis revealed that alpha 1 protein levels were decreased in zaprinast, but not in SNP, SNAP or 8Br-cyclic GMP pretreated cells. 5. While thiol depletion did not prevent the development of tolerance, pretreatment of cells with SNP in the presence of reducing agents partially or completely restored the ability of cells to respond to SNP. 6. We conclude that tolerance to SNP results from two distinct mechanisms: an early onset, NO-mediated event that is reversed by reducing agents and a more delayed, PKA-sensitive process that is mediated through increases in cyclic GMP and a decrease in sGC protein levels.