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Pharmacology of the nitric oxide receptor, soluble guanylyl cyclase, in cerebellar cells.

ABSTRACT: The nitric oxide (NO) receptor, soluble guanylyl cyclase (sGC), is commonly manipulated pharmacologically in two ways. Inhibition of activity is achieved using 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ) which oxidizes the haem prosthetic group to which NO binds, while the compound 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1) is considered an 'allosteric' activator. Knowledge of how these agents function and interact in a normal cellular environment is limited. These issues were addressed using rat cerebellar cells. Inhibition by ODQ was not simply competitive with NO. The rate of onset was ODQ concentration-dependent and developed in two kinetic phases. Recovery from inhibition occurred with a half-time of approximately 5 min. YC-1 slowed the rate at which sGC deactivated on removal of NO by 45 fold, consistent with YC-1 increasing the potency of NO for sGC. YC-1 also enhanced the maximal response to NO by 2 fold. Furthermore, when added to cells in which sGC was 90% desensitized, YC-1 abruptly enhanced sGC activity to a degree that indicated partial reversal of desensitization. After pre-exposure to YC-1, sGC became resistant to inhibition by ODQ. In addition, YC-1 rapidly reversed inhibition by ODQ in cells and for purified sGC, suggesting that YC-1 either increases the NO affinity of the oxidized sGC haem or reverses haem oxidation. It is concluded that the actions of ODQ and YC-1 on sGC are broadly similar in cells and purified preparations. Additionally, YC-1 transiently reverses sGC desensitization in cells. It is hypothesized that YC-1 has multiple actions on sGC, and thereby both modifies the NO binding site and enhances agonist efficacy.

SUBMITTER: Bellamy TC

PROVIDER: S-EPMC1762114 | biostudies-other | 2002 May

REPOSITORIES: biostudies-other

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RNA splicing in regulation of nitric oxide receptor soluble guanylyl cyclase.

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Nitric oxide activation of guanylyl cyclase in cells revisited.

Project description:Nitric oxide (NO) elicits physiological effects in cells largely by activating guanylyl cyclase (GC)-coupled receptors, leading to cGMP accumulation. Like other receptor-coupled effector mechanisms, NO stimulation of GC activity was previously considered to be a graded, concentration-dependent response, with deactivation following swiftly once the agonist disappeared. Recently, a new and unconventional mechanism has been proposed from experiments on purified protein [Cary, S. P. L., Winger, J. A. & Marletta, M. A. (2005) Proc. Natl. Acad. Sci. USA 102, 13064-13069]. It was concluded that GC in vivo will display a dual regulation by NO: a long-lasting tonic activity (10-20% of maximum) due to persistent occupation by NO of the heme binding site and phasic activity due to engagement of another unidentified, lower affinity site. The hypothesis was first tested by monitoring GC activity in rat platelets maintained in vitro and exposed to calibrated NO transients. The kinetics was as expected for a single binding site for NO (EC(50) = 10 nM), with activation and deactivation of enzyme activity conforming to the predictions of a simple receptor model. No tonic GC activity attributable to long-term NO binding was detected after exposure to the full range of active NO concentrations (peaking at 2-500 nM). Comparable results were obtained by using neural cells isolated from the cerebellum. After exposure to high NO concentrations, persistent GC activity could be recorded, but this activity was caused artifactually by secondary NO sources being formed in the medium. The new scheme for regulation of GC activity by NO is of doubtful relevance to cells.

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Soluble guanylyl cyclase mediates noncanonical nitric oxide signaling by nitrosothiol transfer under oxidative stress

Project description:Soluble guanylyl cyclase (GC1) is an α/β heterodimer producing cGMP when stimulated by nitric oxide (NO). The NO-GC1-cGMP pathway is essential to cardiovascular homeostasis but is disrupted by oxidative stress, which induces GC1 desensitization to NO by S-nitrosation (SNO) of its cysteines (C). We discovered that under these conditions, GC1-α subunit increases cellular S-nitrosation via transfer of its nitrosothiols to other proteins (transnitrosation). One of the SNO-targets was the oxidized form of the oxido-reductase Thioredoxin1 (oTrx1), which is unilaterally transnitrosated by GC1. GC1-αC610 was a major SNO-donor to oTrx1-C73. Because oTrx1 itself drives transnitrosation, we sought and identified several SNO-proteins targeted by both GC1 and oTrx1. Among them, transnitrosation of RhoA by SNO-GC1 requires oTrx1 as a nitrosothiol relay, suggesting a SNO-GC1→oTrx1→RhoA cascade. We showed that RhoA pathway, which is antagonized by the canonical NO-cGMP signaling, was alternatively inhibited by GC1-α-dependent S-nitrosation under oxidative conditions. We propose that some SNO-GC1’ functions are adaptive responses triggered by oxidation of the canonical NO-cGMP pathway

2022-10-15 | PXD033105 | Pride

Role of Nitric Oxide, Nitric Oxide Synthase, Soluble Guanylyl Cyclase, and cGMP-Dependent Protein Kinase I in Mouse Stem Cell Cardiac Development.

Project description:Introduction and Aim. Nitric oxide (NO) can trigger cardiac differentiation of embryonic stem cells (ESCs), indicating a cardiogenic function of the NO synthetizing enzyme(s) (NOS). However, the involvement of the NO/NOS downstream effectors soluble guanylyl cyclase (sGC) and cGMP activated protein kinase I (PKG-I) is less defined. Therefore, we assess the involvement of the entire NO/NOS/sGC/PKG-I pathway during cardiac differentiation process. Methods. Mouse ESCs were differentiated toward cardiac lineages by hanging drop methodology for 21 days. NOS/sGC/PKG-I pathway was studied quantifying genes, proteins, enzymatic activities, and effects of inhibition during differentiation. Percentages of beating embryoid bodies (mEBs) were evaluated as an index of cardiogenesis. Results and Discussion. Genes and protein expression of enzymes were increased during differentiation with distinctive kinetics and proteins possessed their enzymatic functions. Exogenous administered NO accelerated whereas the blockade of PKG-I strongly slowed cardiogenesis. sGC inhibition was effective only at early stages and NOS blockade ineffective. Of NOS/sGC/PKG-I pathway, PKG-I seems to play the prominent role in cardiac maturation. Conclusion. We concluded that exogenous administered NO and other pharmacological strategies able to increase the activity of PKG-I provide new tools to investigate and promote differentiation of cardiogenic precursors.

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