Hypoxia Impairs the Citrulline‐Nitric Oxide Cycle in Aortic Endothelial Cells

Abstract

Hypoxia, a condition caused by a significant and prolonged decrease in oxygen, results in endothelial dysfunction, in part, due to the impairment of nitric oxide (NO) production. Endothelial NO is an “endogenous vasodilator” signaling vascular smooth muscle relaxation which lowers resistance to blood flow and consequently decreases blood pressure. Previous studies have shown that under hypoxic conditions, endothelial nitric oxide synthase (eNOS) expression is reduced in bovine aortic endothelial cells (BAEC). Our laboratory has shown that endothelial argininosuccinate synthase (AS) is essential for NO production, providing arginine, the substrate for eNOS. Thus, we investigated whether hypoxia induced a coordinate suppression of both eNOS and AS, resulting in impaired NO production. Initially, cobalt chloride or desferrioxamine treatment of BAEC was used to simulate hypoxic conditions. In each case, western blot analysis demonstrated a coordinate and dose-dependent decrease in AS and eNOS protein expression. Subsequently, the direct effect of hypoxia was investigated by culturing BAEC under 2% O2 conditions. In this case, quantitative real-time PCR demonstrated a significant reduction of AS and eNOS mRNAs, and western blot analysis showed a corresponding decrease in protein. In conclusion, hypoxia induces the coordinate suppression of AS and eNOS, thus compromising the citrulline-nitric oxide cycle in endothelial cells by reducing the availability of substrate, as well as eNOS activity, which results in impaired endothelial NO production.