Reciprocal Regulation of Endothelial Nitric-oxide Synthase by Ca2+-Calmodulin and Caveolin

Abstract

The endothelial nitric-oxide synthase (eNOS) is a key determinant of vascular homeostasis. Like all known nitric-oxide synthases, eNOS enzyme activity is dependent on Ca 2؉ -calmodulin. eNOS is dynamically targeted to specialized cell surface signal-transducing domains termed plasmalemmal caveolae and interacts with caveolin, an integral membrane protein that comprises a key structural component of caveolae. We have previously reported that the association between eNOS and caveolin is quantitative and tissue-specific (Feron, O., Belhassen, L., Kobzick, L., Smith, T. W., Kelly, R. A., and Michel, T. (1996) J. Biol. Chem. 271, 22810 -22814). We now report that in endothelial cells the interaction between eNOS and caveolin is importantly regulated by Ca 2؉ -calmodulin. Addition of calmodulin disrupts the heteromeric complex formed between eNOS and caveolin in a Ca 2؉ -dependent fashion. In addition, overexpression of caveolin markedly attenuates eNOS enzyme activity, but this inhibition is reversed by purified calmodulin. Caveolin overexpression does not affect the activity of the other NOS isoforms, suggesting eNOSspecific inhibition of NO synthase by caveolin. We propose a model of reciprocal regulation of eNOS in endothelial cells wherein the inhibitory eNOS-caveolin complex is disrupted by binding of Ca 2؉

Caveolin versus Calmodulin

“…Immunoprecipitation, SDS-PAGE, and Immunoblotting-Immunoprecipitation of eNOS and caveolin followed our previously reported protocols (8,11) as summarized below. BAEC were harvested, lysed and sonicated, then solubilized with a Ca 2ϩ -free CHAPS buffer containing 50 mM Tris-HCl (pH-7.4), 16 mM CHAPS, 1 mM dithiothreitol, 1 mM EGTA, 1 mM EDTA, 10 M tetrahydrobiopterin, 4 M FAD, 4 M FMN, 1 M L-arginine, and protease inhibitors.…”

“…Protein G-Sepharose was then added for 1 h, and the immunoprecipitated complexes were washed four times, eluted by boiling in Laemmli sample buffer, separated on 12% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Bio-Rad). Mouse monoclonal ␣-eNOS and ␣-caveolin-1 antibodies (Transduction Laboratories) were then used to detect eNOS and caveolin-1 as described previously (8,11). Quantitation of proteins detected by immunoblotting was performed using laser densitometry of x-ray films.…”

“…The key role of Ca 2ϩ -calmodulin in modulating eNOS-caveolin interactions led us to explore whether caveolin might also interact with calmodulin directly, although our previous studies had found no evidence for co-immunoprecipitation of caveolin with calmodulin in endothelial cell membranes (8). The specificity of the enzyme inhibitory effect of the caveolin scaffolding domain was explored in studies of calmodulin-dependent kinase activity in endothelial cell membranes in which we found that the caveolin scaffolding domain peptide inhibits eNOS activity but not calmodulin-dependent kinase activity in endothelial cell lysates (data not shown).…”

“…We have previously shown that eNOS and caveolin can be immunoprecipitated in a stable heteromeric complex (11) and that addition of Ca 2ϩ -calmodulin disrupts the association between eNOS and caveolin (8). To explore whether sequences corresponding to the caveolin scaffolding domain could be implicated in the calmodulin-regulated interactions between eNOS and caveolin, we designed synthetic oligopeptides based on the scaffolding domain of caveolin (5) and the calmodulin-binding domain of eNOS (9).…”

“…Recently, we reported the results of transient transfection experiments in COS-7 cells in which we showed that the coexpression of eNOS and caveolin leads to a marked inhibition of eNOS enzyme activity (8). The enzyme activities of iNOS and nNOS, which are not targeted to caveolae, are not affected by co-transfection with caveolin.…”