Hydrogen peroxide down-regulates inositol 1,4,5-trisphosphate receptor content through proteasome activation

Abstract

Hydrogen peroxide (H 2O 2) is implicated in the regulation of signaling pathways leading to changes in vascular smooth muscle function. Contractile effects produced by H 2O 2 are due to the phosphorylation of myosin light chain kinase triggered by increases in intracellular calcium (Ca 2+) from intracellular stores or influx of extracellular Ca 2+. One mechanism for mobilizing such stores involves the phosphoinositide pathway. Inositol 1,4,5-trisphosphate (IP 3) mobilizes intracellular Ca 2+ by binding to a family of receptors (IP 3Rs) on the endoplasmic–sarcoplasmic reticulum that act as ligand-gated Ca 2+ channels. IP 3Rs can be rapidly ubiquitinated and degraded by the proteasome, causing a decrease in cellular IP 3R content. In this study we show that IP 3R 1 and IP 3R 3 are down-regulated when vascular smooth muscle cells (VSMC) are stimulated by H 2O 2, through an increase in proteasome activity. Moreover, we demonstrate that the decrease in IP 3R by H 2O 2 is accompanied by a reduction in calcium efflux induced by IP 3 in VSMC. Also, we observed that angiotensin II (ANGII) induces a decrease in IP 3R by activation of NADPH oxidase and that preincubation with H 2O 2 decreases ANGII-mediated calcium efflux and planar cell surface area in VSMC. The decreased IP 3 receptor content observed in cells was also found in aortic rings, which exhibited a decreased ANGII-dependent contraction after treatment with H 2O 2. Altogether, these results suggest that H 2O 2 mediates IP 3R down-regulation via proteasome activity.