Regulation of soluble guanylyl cyclase redox state by hydrogen sulfide

Abstract

Soluble guanylate cyclase (sGC) is a receptor for nitric oxide (NO). Binding of NO to ferrous (Fe²⁺) heme increases its catalytic activity, leading to the production of cGMP from GTP. Hydrogen sulfide (H₂S) is a signaling molecule that exerts both direct and indirect anti-oxidant effects. In the present, study we aimed to determine whether H₂S could regulate sGC redox state and affect its responsiveness to NO-releasing agents and sGC activators. Using cultured rat aortic smooth muscle cells, we observed that treatment with H₂S augmented the response to the NO donor DEA/NO, while attenuating the response to the heme-independent activator BAY58-2667 that targets oxidized sGC. Similarly, overexpression of H₂S-synthesizing enzyme cystathionine-γ lyase reduced the ability of BAY58-2667 to promote cGMP accumulation. In experiments with phenylephrine-constricted mouse aortic rings, treatment with rotenone (a compound that increases ROS production), caused a rightward shift of the DEA/NO concentration-response curve, an effect partially restored by H₂S. When rings were pre-treated with H₂S, the concentration-response curve to BAY 58-2667 shifted to the right. Using purified recombinant human sGC, we observed that treatment with H₂S converted ferric to ferrous sGC enhancing NO-donor-stimulated sGC activity and reducing BAY 58-2667-triggered cGMP formation. The present study identified an additional mechanism of cross-talk between the NO and H₂S pathways at the level of redox regulation of sGC. Our results provide evidence that H₂S reduces sGC heme Fe, thus, facilitating NO-mediated cellular signaling events.

Introduction

Nitric oxide (NO) is a signaling molecule that affects diverse physiological and pathophysiological processes in practically all systems and organs [1], [2]. In the cardiovascular system, NO regulates vascular tone, inhibits platelet aggregation, promotes angiogenesis, modulates inflammatory responses and exerts cardioprotective effects [3]. Although NO is capable of reacting with a plethora of molecular targets, the response to low physiological levels of NO is predominantly mediated by the heterodimeric soluble guanylyl cyclase (sGC) [4]. sGC is a heme protein with high affinity and specificity towards NO [5], [6], [7] that converts GTP to the second messenger cGMP; sGC activity is increased several hundred fold upon binding of NO [6].

Maintenance of sGC heme moiety in the ferrous state is essential for the sGC NO sensing function [8]. Oxidation of sGC heme by specific agents [9] or oxidative stress induced by various pathological conditions [10] renders sGC insensitive to normal levels of NO and attenuates NO/cGMP signaling. Moreover, it has been demonstrated that sGC with oxidized heme has a propensity to lose the heme moiety [11], [12]. The pool of ferric or heme-free sGC is much more susceptible to degradation [12], [13]. About fifteen years ago, NO-independent sGC activators and stimulators were discovered [14], [15]; these agents enhance sGC activity in a heme-dependent or ⿿independent manner. sGC activators are unique for their ability to increase the catalytic activity of heme-free/oxidized sGC. sGC stimulators have already been granted approval for human use for certain types of pulmonary hypertension [16].

For many decades, H₂S was considered a toxic gas that penetrates cells by simple diffusion [17]. Following the discovery that mammalian cells are capable of producing H₂S, this molecule underwent a dramatic metamorphosis from a dangerous pollutant to a biologically relevant molecule, reminiscent of the transformation of NO [18], [19]. H₂S is now accepted as a signaling molecule with important roles in physiology and disease [20], [21], [22]. H₂S triggers many of the same responses as NO in the cardiovascular system; it reduces blood pressure, promotes angiogenesis and limits infarct size following ischemia/reperfusion injury [21], [23]. Although distinct effectors have been identified for H₂S and NO, both agents are capable of enhancing cGMP levels. Much like NO donors, exposure of cells or tissues to H₂S donors leads to intracellular cGMP accumulation [24], [25]. However, unlike NO which stimulates sGC, H₂S raises cGMP by preventing its breakdown [26], by enhancing endothelial NO synthase (eNOS) activity [25], [27] as well as by liberating NO from stable biological stores of NO [28]. The interdependence of NO and H₂S signaling is an area of intense investigation, with several groups aiming to unravel its importance for normal cell function, as well as various pathophysiological states.

Our previous studies demonstrated that H₂S does not directly affect the activity of ferrous sGC [25]. In the current study, we investigated whether H₂S affects the function of sGC carrying oxidized ferric heme. We report that H₂S reduces ferric sGC heme into a ferrous state, which is accompanied by the restoration of NO activation of purified and cellular sGC, and the recovery of NO-dependent vasodilation. Conversely, H₂S-dependent reduction of sGC heme diminishes the response to ferric-sGC by BAY58-2667; this later observation has significant implications for the pharmacology of sGC activators, especially in light of the translational efforts of this class of agents.