Abstract 126: Nox5 Regulation of Vascular Contraction Involves Oxidation of Endoplasmic Reticulum Calcium Channels and Calreticulin

Abstract

The biological function of calcium-sensitive superoxide-generating Nox5 is unclear, but it may play a role in regulating contraction, as we previously demonstrated. Here we explored molecular mechanisms whereby Nox5 controls contraction. Human arteries and mice expressing human NOX5 in smooth muscle cells (Nox5+SM22+) were studied. In arteries from hypertensive subjects, Nox5 expression, assessed by immunoblotting, was increased (50%, p<0.05 vs control). In human VSMCs, AngII-induced ROS generation (1 fold) and activation of myosin light chain (MLC) (2.5 fold) were exaggerated in VSMCs from hypertensive subjects (p<0.05 vs control); an effect that was attenuated by Nox5 siRNA. In arteries from Nox5+/SM22+ mice, contraction to U46619 was increased in (5.8±0.3 mN vs WT: 4.2±0.2 mN, p<0.05). These hypercontractile responses were inhibited by NAC (ROS scavenger), calmidazolium (calmodulin inhibitor), dantrolene (ryanodine receptor Ca 2+ channel inhibitors) and CDN1163 (SERCA channel activator), but not by a Nox1/Nox4 inhibitor (GKT137831). ONOO - levels were increased in vessels from Nox5+/SM22+ mice (5.8±0.9 vs WT 3.4±0.1 AU/mg, p<0.05). Inactivation of MYPT1 (181±1.8AU vs 164±1.9AU WT) and activation of MLC (207±10.3AU vs 155±2.7AU WT) were increased in VSMCs from Nox5+SM22+ (p<0.05). To assess the oxidative proteome in VSMCs, we immunoprecipitated reversibly oxidized proteins and observed oxidation of Nox5, decreased oxidation of MYPT1 and increased oxidation of SERCA2b in Nox5+/SM22+ mice . Proteome analysis of human VSMCs identified the ER Ca 2+ sensor, calreticulin, as a potential Nox5 binding protein. Calreticulin reversible oxidation was increased in VSMCs from Nox5+SM22+ mice and hypertensive subjects. Our study unravels crosstalk between oxidative stress and Ca 2+ in the vasculature, where Nox5 regulation of contraction involves ROS, Ca 2+ and endoplasmic reticulum localized Ca 2+ channels/proteins. We identify novel mechanisms whereby Nox5 influences pro-contractile signalling through processes involving oxidation of the ER Ca 2+ sensor, calreticulin, and ER Ca 2+ channels.