Quantification of eNOS mRNA in the canine cardiac vasculature by competitive PCR

The goal of the present study was to develop a competitive PCR assay to mea sure changes in the expression of endothelial nitric oxide synthase (eNOS) mRNA levels throughout the canine vascular tree. A partial sequence of cani ne eNOS cDNA (1.86 kb), inducible NOS (1.95 kb), and neuronal NOS (1.16 kb) was cultured from canine aortic endothelial cells, LPS-treated canine sple nic vein endothelial cells, and from canine left ventricle, respectively. C ompetitor eNOS cDNA (eNOS-C) was constructed via recombinant PCR. Thus, wit h the use of a standard curve competitive PCR with eNOS-C, the amount of eN OS mRNA in 500 ng of total RNA was greatest in the circumflex > right coron ary artery > left anterior descending coronary artery > aorta. The isolatio n of coronary microvessels from the left ventricle was associated with an e nrichment of endothelial cell markers such as eNOS, von Willebrand factor, and caveolin-1, an observation supported by the detection of up to 15-fold higher levels of eNOS mRNA in coronary microvessels relative to the larger arteries. The ability to quantify changes in eNOS mRNA. levels throughout t he canine vasculature should provide greater insight into the molecular mec hanisms of how this gene is regulated in physiological and pathophysiologic al states.