Stimulation of NO production and of eNOS phosphorylation in the microcirculation in vivo

The role of nitric oxide (NO) in microvascular permeability is controversia l, in part because the regulation of its endothelial constitutive synthase, eNOS, has been studied in vitro but not in vivo. Our study was designed to detect the morphologic and functional presence of eNOS and to test whether eNOS could be phosphorylated by platelet-activating factor (PAF), an agent that induces hyperpermeability. Immunocytochemistry was applied using huma n anti-eNOS antibodies in the hamster cheek pouch (hcp). The hcp microvesse ls demonstrated positive reaction products in the endothelium. The function al presence of eNOS in hcp was investigated by topical application of 10(-7 ) M PAF to the hcp and by measuring NO production by chemiluminescence. The mean baseline value of NO release was 63.3 +/- 6.9 pmol/ml (mean +/- SE), Application of PAF led to an increase in mean NO release to 120.8 +/- 31.2 pmol/ml (P < 0.05). In another series of experiments, 10(-7) M PAF was appl ied topically to hcp preincubated with [P-32]orthophosphoric acid. Immunopr ecipitation and Western blots detected P-32-labeled bands that migrated wit h the mobility of positive eNOS indicating phosphorylated eNOS protein. The intensity of the radioactive bands was evaluated by computer;assisted imag e analysis. Comparison of the net band intensities yielded a mean PAF-treat ed/control ratio of 1.6 +/- 0.1. Our data demonstrate the morphologic and f unctional presence of eNOS in the microcirculation. The data also provide e vidence that the function of microvascular eNOS is subject to regulation by phosphorylation. (C) 2000 Academic Press.