Endothelial Nitric Oxide Synthase Targeting to Caveolae

Abstract

The endothelial isoform of nitric oxide synthase (eNOS) modulates cardiac myocyte function and is expressed in the particulate subcellular fraction. We have previously shown that eNOS is targeted to plasmalemmal caveolae in endothelial cells. Caveolae, specialized domains of the plasma membrane, may serve to sequester signaling proteins; a family of transmembrane proteins, the caveolins, form a key structural component of these microdomains. Caveolae in cardiac tissues contain the muscle-specific isoform caveolin-3, and caveolae in endothelial cells contain the widely expressed isoform caveolin-1, which shares limited sequence identity with caveolin-3. Our immunohistochemical analyses of rat cardiac muscle used isoform-specific caveolin antibodies to reveal prominent caveolin-3 staining in myocyte sarcolemmal membranes and at intercalated discs, whereas caveolin-1 staining was prominent in the vascular endothelium. Caveolin or eNOS antibodies were utilized to immunoprecipitate cardiac myocyte or cultured aortic endothelial cell lysates, which then were analyzed in immunoblots. In endothelial cells, we found that eNOS is quantitatively immunoprecipitated by antibodies to caveolin-1. In cardiac myocyte lysates, nearly all the eNOS is immunoprecipitated instead by antibodies to caveolin-3 and, conversely, eNOS antiserum immunoprecipitated primarily caveolin-3. These studies establish expression of eNOS in cardiac myocyte caveolae and document tissue-specific and quantitative associations of eNOS with caveolin. These findings may have important implications for the regulation of eNOS by caveolin isoforms and by other signaling proteins targeted to caveolae. The endothelial isoform of nitric oxide synthase (eNOS) modulates cardiac myocyte function and is expressed in the particulate subcellular fraction. We have previously shown that eNOS is targeted to plasmalemmal caveolae in endothelial cells. Caveolae, specialized domains of the plasma membrane, may serve to sequester signaling proteins; a family of transmembrane proteins, the caveolins, form a key structural component of these microdomains. Caveolae in cardiac tissues contain the muscle-specific isoform caveolin-3, and caveolae in endothelial cells contain the widely expressed isoform caveolin-1, which shares limited sequence identity with caveolin-3. Our immunohistochemical analyses of rat cardiac muscle used isoform-specific caveolin antibodies to reveal prominent caveolin-3 staining in myocyte sarcolemmal membranes and at intercalated discs, whereas caveolin-1 staining was prominent in the vascular endothelium. Caveolin or eNOS antibodies were utilized to immunoprecipitate cardiac myocyte or cultured aortic endothelial cell lysates, which then were analyzed in immunoblots. In endothelial cells, we found that eNOS is quantitatively immunoprecipitated by antibodies to caveolin-1. In cardiac myocyte lysates, nearly all the eNOS is immunoprecipitated instead by antibodies to caveolin-3 and, conversely, eNOS antiserum immunoprecipitated primarily caveolin-3. These studies establish expression of eNOS in cardiac myocyte caveolae and document tissue-specific and quantitative associations of eNOS with caveolin. These findings may have important implications for the regulation of eNOS by caveolin isoforms and by other signaling proteins targeted to caveolae.