Inhibition of mesangial iNOS by reduced extracellular pH is associated with uncoupling of NADPH oxidation

Abstract

Background. Since chronic renal failure is associated with metabolic acidosis and down-regulation of intrarenal nitric oxide (NO) synthesis, I tested the hypothesis that acidosis may impair the intrarenal NO synthesis. The effects of alterations in extracellular pH were examined on inducible NO synthesis in murine mesangial cells (MMC) in culture. Methods. NO synthesis was induced in MMC by bacterial lipopolysaccharide and tumor necrosis factor-α and assayed by an NO analyzer that measured nitrites and nitrates (NOx). The activity of inducible NO synthase (iNOS) enzyme was assayed by conversion of [3H]-arginine to [3H]-citrulline. Experimental groups included cells cultured with a pH of 7.3 (normal), or 7.0 (low) or 7.6 (high), and the assigned pH values were maintained by HEPES and Tris. Results. NOx was decreased in MMC exposed to the reduced pH compared to other groups. [3H]-citrulline assay showed an 80% reduction in iNOS activity in stimulated MMC exposed to a reduced pH versus control pH (P < 0.01). iNOS mRNA and protein expression were similar in control and low pH cells. The iNOS inhibition was not reversed by supplementation of MMC with either L-arginine or tetrahydrobiopterin, a major co-factor for NOS enzyme. MMC re-incubated in control pH after being exposed to the low pH demonstrated re-inducibility of NOS activity. Furthermore, MMC exposed to low pH were associated with a higher NADP+/+[H]-citrulline ratio (3.2) compared to standard pH (1.7), indicating an increase in NADP+/+[H]-citrulline stoichiometries and uncoupling of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation. In contrast, macrophages exposed to the reduced pH did not demonstrate uncoupling of NADPH oxidation. Conclusion. Acidosis impairs iNOS activity in MMC by a post-translational mechanism that involves uncoupling of NADPH oxidation.