DECREASED NITRIC-OXIDE SYNTHASE ACTIVITY CAUSES IMPAIRED ENDOTHELIUM-DEPENDENT RELAXATION IN THE POSTISCHEMIC HEART

Endothelial nitric-oxide synthase (eNOS) is an important regulator of endothelial function and vascular tone in biological tissues, While en dothelial dysfunction occurs following ischemia and has been attribute d to altered NO. formation, the biochemical basis for this dysfunction is unknown, Therefore, studies were performed to determine the effect s of myocardial ischemia and reperfusion on eNOS in isolated rat heart s subjected to periods of global ischemia or ischemia followed by repe rfusion. eNOS activity was assayed by L-[C-14]arginine to L-[C-14]citr ulline conversion and alterations in the amount and distribution of eN OS determined by Western blotting and immunohistochemistry. While acti vity was preserved after 30 min of ischemia with a value of 1.1 +/- 0. 1 pmol x min(-1) x mg of protein(-1), it decreased by 77% after 60 min and became nearly undetectable after 120 min. Reperfusion resulted in only a partial restoration of activity, The decline in activity with ischemia was due, in part, to a loss of eNOS protein, Hemodynamic stud ies showed that the onset of impaired vascular reactivity paralleled t he loss of functional eNOS, Subjecting isolated eNOS to conditions of acidosis, which occur during ischemia, followed by restoration of pH a s occurs on reperfusion, caused a combination of reversible and irreve rsible loss of activity similar 60 that seen in ischemic and reperfuse d hearts, Thus, Loss of endothelial function following ischemia is par alleled by a loss of eNOS activity due to a combination of pa-dependen t denaturation and proteolysis.