Endothelial nitric oxide synthase protein distribution and nitric oxide production in endothelial cells along the coronary vascular tree.

Abstract

OBJECTIVE\nFreshly isolated endothelial cells from both conduit arteries and microvasculature were used to test the hypothesis that eNOS protein content and nitric oxide production in coronary endothelial cells increases with vessel radius.\n\n\nMETHODS\nPorcine hearts were obtained from a local abattoir. Large and small arteries as well as arterioles were dissected free of myocardium and homogenized as whole vessels. Additionally, endothelial cells were isolated from both conduit arteries and left ventricular myocardium by tissue digestion with collagenase, followed by endothelial cell isolation using biotinylated-anti-CD31 and streptavidin-coated paramagnetic beads. Purity of isolated endothelial cells was confirmed by immunofluorescence and immunoblot.\n\n\nRESULTS\nIn whole vessel lysate, immunoblot analysis revealed that protein content for eNOS was greater in arterioles compared to small and large arteries. Nitric oxide metabolites (nitrite plus nitrate; NOx) levels measured from whole vessel lysate decreased as vessel size increased, with both arterioles and small arteries displaying significantly greater NOx content than conduit. Consistent with our hypothesis, both eNOS protein level and NOx were significantly greater in endothelial cells isolated from conduit arteries compared with those from coronary microvasculature. Furthermore, confocal microscopy revealed that eNOS protein was present in all conduit and microvascular endothelial cells, although eNOS staining was less intense in microvascular cells than those of conduit artery.\n\n\nCONCLUSIONS\nThese findings demonstrate increased eNOS protein and NOx content in endothelial cells of conduit arteries compared with the microcirculation and underscore the importance of comparing endothelial-specific molecules in freshly isolated endothelial cells, rather than whole lysate of different sized vessels.