Interplay of endothelial and inducible nitric oxide synthases modulates the vascular response to ischaemia–reperfusion in the rabbit lung

Abstract

Lung ischaemia-reperfusion induces nitric oxide synthesis and reactive nitrogen species, decreasing nitric oxide bioavailability. We hypothesized that in the ventilated lung, this process begins during ischaemia and intensifies with reperfusion, contributing to ischaemia-reperfusion-induced pulmonary vasoconstriction. The aim was to determine whether ischaemia-reperfusion alters inducible and endothelial nitric oxide synthase expression/activity, reactive nitrogen species generation, and nitric oxide bioavailability, potentially affecting pulmonary perfusion. Ischaemia-reperfusion was induced for various times in anesthetized rabbits with ventilated lungs by reversibly occluding the right pulmonary artery and initiating reperfusion. Nitric oxide synthase activity/expression and phosphorylation, reactive nitrogen species generation and total nitrate/nitrite were determined in lung tissue. Inducible nitric oxide synthase expression and activity, and reactive nitrogen species formation coincided with increased pulmonary vascular resistance during reperfusion and increased with ischaemia duration, further increasing after 2-h reperfusion. Total nitrate/nitrite also increased with ischaemia but decreased after 2-h reperfusion. Pre-treatment with an inducible nitric oxide synthase inhibitor (1400W; Cayman Chemical Company, Ann Arbor, MI, USA) attenuated inducible nitric oxide synthase activity, reactive nitrogen species generation and pulmonary vascular resistance, but did not affect total nitrate/nitrite. Endothelial nitric oxide synthase expression was unchanged by ischaemia-reperfusion; however, its phosphorylation on serine 1177 and dephosphorylation on threonine 495 was uncoupled, suggesting decreased endothelial nitric oxide synthase activity. 1400W prevented uncoupling of endothelial nitric oxide synthase phosphorylation, maintaining its activity during reperfusion. Ischaemia-reperfusion up-regulates inducible nitric oxide synthesis and/activity, which coincides with reduced endothelial nitric oxide synthase activity as suggested by its uncoupling and may contribute to ischaemia-reperfusion-induced pulmonary vasoconstriction.