Hypoxia – induced upregulation of arginase II (Arg II) and argininosuccinate synthetase (ASS I) in human lung microvascular endothelial cells (HMVEC)

We studied the effect of hypoxia on expression of Arg II and ASS I which are able to modulate the availability of arginine, for endothelial nitric oxide (NO) synthase (eNOS). We found that exposure of HMVEC to 1% oxygen (hypoxia) for 24 hr upregulated Arg II and ASS I. Next we studied the role of extracellular and intracellular pools of arginine in NO production under hypoxia and how inhibition of Arg II and ASS I would affect it. HMVEC were able to produce NO in media with and without arginine, although in arginine‐free media NO levels were lower. Inhibition of Arg II which competes with eNOS for substrate led to an increase in NO production in HMVEC incubated in media with arginine; inhibition of Arg II had little or no effect on NO production in arginine‐free media. eNOS can use citrulline recycled to arginine and ASS I is the rate‐limiting enzyme in this reaction. Inhibition of ASS I blocked NO production in arginine‐free media and led to substantial inhibition of NO production in cells incubated with L‐arginine. We conclude that eNOS uses two pools of L‐arginine: extracellular and intracellular, to generate NO under hypoxic conditions. The intracellular pool is largely composed of recycled citrulline. This raises the possibility of using citrulline to supply eNOS with substrate. In this case arginase would not compete or interfere with NO production. This work was supported by Florida DOH (KK), VA and NIH (JMP).