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 measure changes in the expression of endothelial nitric oxide synthase (eNOS) mRNA levels throughout the canine vascular tree. A partial sequence of canine 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 splenic vein endothelial cells, and from canine left ventricle, respectively. Competitor eNOS cDNA (eNOS-C) was constructed via recombinant PCR. Thus, with the use of a standard curve competitive PCR with eNOS-C, the amount of eNOS mRNA in 500 ng of total RNA was greatest in the circumflex > right coronary artery > left anterior descending coronary artery > aorta. The isolation of coronary microvessels from the left ventricle was associated with an enrichment 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 the canine vasculature should provide greater insight into the molecular mechanisms of how this gene is regulated in physiological and pathophysiological states.