Endothelial [Ca2+]i and caveolin-1 antagonistically regulate eNOS activity and microvessel permeability in rat venules

In this study, we investigated the mechanisms by which caveolin-1 (CAV) inhibits increases in permeability induced by platelet activating factor (PAF) and elucidated the relationship between the endothelial intracellular Ca2+ concentration ([Ca2+](i)) and CAV in regulating endothelial nitric oxide synthase (eNOS) activity and microvessel permeability in intact microvessels. Experiments were conducted in individually perfused mesenteric venules in Sprague-Dawley rats. Permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca2+](i) and nitric oxide (NO) production were measured in fura-2- and DAF-2-loaded microvessels. Perfusion of the CAV scaffolding domain, AP-CAV, at 1 mu M for 30 min did not affect PAF-induced increases in endothelial [Ca2+](i) but significantly attenuated PAF-induced NO production from 143 +/- 2 to 110 +/- 3% of control fluorescence intensity (FI). The PAF-induced Lp increase was correlatively reduced from a mean peak value of 7.5 +/- 0.9 to 1.9 +/- 0.5 times that of the control. Increasing extracellular [Ca2+] that potentiated PAF-induced peak [Ca2+](i) from 500 to 1225 nM augmented NO production to 193 +/- 13% and further increased Lp to 17.3 +/- 1.6 times the control value. More importantly, enhanced Ca2+ influx restored the reduced NO production and Lp by AP-CAV with NO FI at 149% and Lp at 7.7 +/- 1.1 times the control value. Our results indicate that eNOS inhibition and reduced NO production contribute to the inhibitory action of AP-CAV on PAF-induced increases in permeability. CAV and endothelial [Ca2+](i) antagonistically regulate eNOS activity in intact microvessels, and the level of produced NO is the key determinant of the degree of permeability increases during inflammation.