Coronary Hemodynamics in Endothelial NO Synthase Knockout Mice

Abstract

Abstract —For the specific analysis of endothelial NO synthase (eNOS) function in the coronary vasculature, we generated a mouse homozygous for a defective eNOS gene (eNOS−/−). Western blot as well as immunohistochemical staining revealed the absence of eNOS protein in eNOS−/− mice. Aortic endothelial cells derived from eNOS−/− mice displayed only background levels of NO x formation compared with wild-type (WT) cells (88 versus 1990 pmol NO x · h −1 /mg protein −1 ). eNOS−/− mice were hypertensive (mean arterial pressure, 135±15 versus 107±8 mm Hg in WT) without the development of cardiac hypertrophy. Coronary hemodynamics, analyzed in Langendorff-perfused hearts, showed no differences either in basal coronary flow or in maximal and repayment flow of reactive hyperemia. Acute NOS inhibition with N ω -nitro- l -arginine methyl ester (L-NAME) in WT hearts substantially reduced basal flow and reactive hyperemia. The coronary response to acetylcholine (ACh) (500 nmol/L) was biphasic: An initial vasoconstriction (flow, −35%) in WT hearts was followed by sustained vasodilation (+190%). L-NAME significantly reduced vasodilation in WT hearts (+125%) but did not alter the initial vasoconstriction. In eNOS−/− hearts, the initial vasoconstriction was augmented (−70%), whereas the ACh-induced vasodilation was not affected. Inhibition of cyclooxygenase with diclofenac converted the ACh-induced vasodilation into vasoconstriction (−49% decrease of basal flow). This effect was even more pronounced in eNOS−/− hearts (−71%). Our results demonstrate that (1) acute inhibition of eNOS reveals a role for NO in setting the basal coronary vascular tone as well as participation in reactive hyperemia and the response to ACh; (2) chronic inhibition of NO formation in eNOS−/− mutant mice induces no changes in basal coronary flow and reactive hyperemia, suggesting the activation of important compensatory mechanisms; and (3) prostaglandins are the main mediators of the ACh-induced vasodilation in both WT and eNOS−/− mice.