Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides.

In endothelia, NO is synthesized by endothelial NO synthase (eNOS), which is negatively regulated by caveolin-1 (Cay-1), the primary coat protein of caveolae. We show that delivery of Cay-I amino acids 82-101 (Cay) fused to an internalization sequence from Antennapedia (AP) blocks NO release in vitro and inflammation and tumor angiogenesis in vivo. To characterize the molecular mechanism by which the AP-Cav peptide and Cay-I mediate eNOS inhibition, we subdivided the Cay portion of AP-Cav into three domains (Cay-A, -B, and -C), synthesized five overlapping peptides (AP-Cav-A, -AB, -B, -BC, and -C), and tested their effects on eNOS- dependent activities. Peptides containing the Cay-B domain (amino acids 89-95) induced time- and dose-dependent inhibition of eNOS-dependent NO release in cultured endothelial cells, NO- dependent inflammation in the ear, and hydraulic conductivity in isolated venules. Alanine scanning of AP-Cav-B revealed that Thr-90 and -91 (T90,91) and Phe-92 (F92) are crucial for AP-Cav-B- and AP-Cav-mediated inhibition of eNOS. Mutation of F92 to A92 in the Cay-1 cDNA caused the loss of eNOS inhibitory activity compared with wild-type Cay-1. These data highlight the impor- tance of amino acids 89-95 and particularly F92 in mediating eNOS inhibition by AP-Cav and Cay-1. [ABSTRACT FROM AUTHOR]