Abstract P279: Regulation Of H 2 S Production By Cystathionine Gamma-lyase In Endothelial Cells.

Abstract

Hydrogen sulfide (H 2 S) is the most recently described endothelium-derived vasodilator. In the endothelium, H 2 S is predominantly produced by cystathionine γ-lyase (CSE). We and others have previously shown CSE inhibition diminishes acetylcholine (ACh)-induced dilation in aortic ring segments. However, the mechanism of CSE regulation is not well-defined. The goal of this study was to identify endogenous regulators of CSE in endothelial cells. Previous reports suggest that [Ca 2+ ] and phosphorylation regulate CSE activity. We previously reported that there was an increase in H 2 S production in rat aortic endothelial cells loaded with an H 2 S fluorescence indicator, sulfide fluor-7 acetoxymethylester (SF7-AM, 10 μM) after exposure to either ACh (10 μM) or an H 2 S donor (NaHS, 10 μM). Pretreatment with the CSE inhibitor, β-cyanoalanine (BCA, 100 μM) prevented the ACh-induced but not the NaHS-induced increases in SF 7 flourescence. Our current study tested the hypothesis that increasing intracellular calcium stimulates CSE-dependent H 2 S production. Cultured rat aortic endothelial cells were loaded with SF 7 and a nuclear dye (Nuclear-ID Red DNA stain, to correct for cell number) and fluorescence was measured at baseline and in response to a calcium ionophore (ionomycin, 100 nM). Ionomycin increased H 2 S production (Control: 0.8293, +ionomycin: 1.0365, p = 0.0001, n=3 of 7-15 wells per replicate). Pretreatment with BCA significantly decreased ionomycin-induced H 2 S production (+ionomycin: 1.0365, +BCA: 0.6600, p < 0.0001, n=3 of 7-15 wells per replicate). Preliminary ongoing studies in pressurized mesenteric arteries suggest a tendency of CSE inhibition to reduce ACh-induced dilation (Control: 77.06%, +BCA: 42.04%, p=0.08, n=3-4 arteries per group). However, a larger sample size is needed. Taken together, these studies suggest that ACh elicits vasodilation through a Ca 2+ -dependent activation of CSE.