Abstract 227: Activity of Soluble Guanylyl Cyclase, the Nitric Oxide Receptor, is Modulated by Cysteines Redox
The enzyme soluble guanylyl cyclase (sGC) increases cellular level of the second messenger cGMP upon stimulation by nitric oxide (NO), whose role is exemplified in the cardiovascular system where it governs smooth muscle cell proliferation, vascular relaxation and platelet aggregation. Various pathologies like diabetes, hypertension and hypercholesterolemia are associated with enhanced oxidative stress with increased formation of reactive oxygen (ROS) and nitrogen species (RNS). We recently showed that cysteines beta1C243 and beta1C122 are S-nitrosylated in sGC leading to its desensitization. We have now identified by Mass Spectrometry, two additional cysteines (beta1C571 and alpha1C516) as potentially modified. Our hypothesis is that loss in vascular reactivity in oxidative pathologies is mediated, in part, by desensitization of sGC via thiols oxidation. We studied the effect of oxidative stress on sGC in primary vascular smooth muscle cells (VSMC) by directly exposing them to hydrogen peroxide (H2O2) in the absence or presence of a catalase inhibitor Amino-Triazole (AT). H2O2 induced inhibition of NO-stimulated sGC activity in VSMC and in COS 7 cells transiently transfected with sGC. Desensitization of sGC was further increased in VSMC treated with both H2O2 and AT. Next, we conducted mutational analysis of the 4 cysteines to assess their involvement in redox sensing. Replacement of C571 and C122 eliminated the oxidative-dependent desensitization observed in the wild-type. In addition, peroxynitrite, a strong oxidant, inhibited the NO-stimulated sGC activity in a purified system without oxidation of the heme. In conclusion, our results suggest that sGC activity is directly modulated by the redox state of the cells. Moreover, the identified 4 cysteines can be modified by both ROS and RNS suggesting a complex modulation. Ultimately, our goal is to determine whether loss of vascular reactivity in oxidative pathophysiologies is in part mediated by desensitization of sGC and what are the involved cysteines.
This work was supported by NIH GM067640, HL089771.