Abstract

Background: The effect of hypoxia on the formation of superoxide (O₂⁻), the expression of gp91^phox and endothelial NO synthase (eNOS) were studied in pig intact pulmonary artery (PA) segments and PA vascular smooth muscle cells (PAVSMCs) and PA endothelial cells (PAECs).

Methods: Segments and cells were incubated under hypoxic conditions for 2 hours (with or without enzyme inhibitors) and the formation of O₂⁻ measured spectrophotometrically. Protein expression was assessed using Western blotting and immunocytochemistry.

Results: Hypoxia promoted the formation of O₂⁻ in PA segments, PAVSMCs and PAECs, an effect inhibited by diphenylene iodonium and apocynin (NAD[P]H oxidase inhibitors). Hypoxia induced O₂⁻ formation was enhanced by inhibition of eNOS and augmented by endotoxin and cytokines and re-oxygenation. Hypoxia also promoted the expression of gp91^phox and eNOS. In intact PA segments hypoxia induced the expression of nitrotyrosine and eNOS in the endothelium.

Conclusions: The simultaneous upregulation of NAD[P]H oxidase and eNOS in response to hypoxia in the PA results in the simultaneous formation of O₂⁻, NO, and ONOO⁻. This may represent either a protective mechanism designed to counter the pro-oxidant effect of hypoxia or a novel pathological mechanism underlying the progression of acute respiratory distress syndrome (ARDS).