Abstract 9710: Hydrogen Sulfide Increases Nitric Oxide Production by Calcium-Dependent Activation of Endothelial Nitric Oxide Synthase

Abstract

Purpose: Hydrogen sulfide (H 2 S), which has been regarded as a toxic gas, was recently discovered to be synthesized in mammalian tissues by several different enzymes, such as cystathionine gamma-lyase (CSE), cystathionine beta-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST). Many studies have shown that H 2 S has various physiological functions, including vasodilator and antihypertensive effects in cardiovascular system. Therefore, H 2 S is considered as one of endothelium-derived relaxing factors (EDRFs). However, intracellular mechanisms of H 2 S-induced vasodilation and its interactions between other EDRFs, such as nitric oxide (NO), still remain unclear. In the present study, we investigated whether H 2 S directly regulates endothelial NO synthase (eNOS) activity and NO production in endothelial cells. Methods: We treated bovine aortic endothelial cells (BAEC) with a hydrogen sulfide donor (NaHS) and determined NO production from BAEC into the culture medium by fluorometric measurement with 2,3-diaminonaphthalene. We determined the changes of intracellular calcium concentration by using Fura 2-AM, and analyzed the activity of eNOS by western blotting. Results: NaHS dose-dependently (12.5 - 200 µM) increased NO production from BAEC with 2.6-fold increase (n=8, p<0.01) by 200 µM. This NaHS-induced NO production was abolished by an intracellular calcium-chelator (BAPTA-AM), but was not affected by the inhibitor of phosphatidylinositol 3-kinase (wortmannin). This effect was partially blocked by a ryanodine receptor inhibitor (dantrolene) and an inositol 1,4,5-trisphosphate receptor inhibitor (xestosponginC). NaHS induced relatively slow increases in the intracellular calcium by 11.3 ± 1.1 nM after 15 minutes (n=4, p<0.01). NaHS induced phosphorylation of eNOS at the activating phosphoserine residue 1179. This effect was completely blocked by BAPTA-AM, but wasn't affected by wortmannin. Conclusions: H 2 S directly acts on endothelial cells to induce eNOS activation and NO production by releasing calcium from the intracellular store in endoplasmic reticulum, which may explain one of mechanisms of its vasodilator function as an EDRF.