Developmental differences in pulmonary nitric oxide (NO) production and endogenous nitric oxide synthase inhibitors in the neonatal rat

Abstract

We recently observed differences in exhaled NO production in isolated perfused lungs in 7 and 14 d/o rats, where 7 d lungs produced less than 14 d lungs. These differences were independent of NO synthase (NOS) protein levels. Therefore, mechanisms responsible for NOS activation and inhibition were evaluated. We tested the hypothesis that the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) plays a role in modulating NOS activity in the developing pulmonary vasculature. Whole lung homogenates and plasma were obtained from 7 and 14 d/o rats and snap frozen. The homogenates were then compared by immunoblot analysis for site specific eNOS phosphorylation and compared by immune‐mediated co‐precipitation experiments to assess eNOS associated HSP90 or Caveolin 1 (Cav 1) levels. Finally, we used HPLC to measure plasma ADMA and arginine (L‐Arg) levels. We found no differences in eNOS associated HSP90 or Cav 1 protein levels, nor were there differences in eNOS phosphorylation profiles. However, plasma ADMA levels were 2 fold greater in the 7 vs. 14 d/o rats and the plasma L‐Arg/ADMA ratio was shifted lower in the 7 vs. 14 d/o rats. The shift in the L‐Arg/ADMA ratio is significant because NOS uses LArg as its substrate and ADMA is a competitive inhibitor of L‐Arg binding. This ratio allows one to predict the degree of NOS inhibition. Based on previously published Ki’s for NOS, we expect that the decreased ratio observed could inhibit NOS by as much as 30–40%, which may result in impaired endothelial function in the early neonatal course.