Regulation of NO Synthesis in Endothelial Cells

Localization The endothelial nitric oxide (NO) synthase (eNOS) is a constitutively expressed 135-kD protein predominantly associated with the particulate subcellular fraction, suggesting that the native enzyme is a membrane-bound protein [1]. Membrane association is achieved by attachment of myristic acid to the amino terminal end of the enzyme and as such is consistent with reports that eNOS cDNA contains a consensus sequence for co-translational modification of the enzyme by N-terminal myristoylation. Preventin of myristic acid inorporation in site-directed mutagenesis experiments converse the membrane-associated eNOS to a cytosolic form but has little effect on enzyme activity. Myristoylation alone, however, provides barely enough energy to anchor a protein to a lipid bilayer, therefore other factors are important in determining the association of the enzyme with the plasma membrane, e.g., additonal hydrophobic interactions owing to reversible palmitoylation of a nearby cysteine residue. A recent detailed analysis of the membrane association of eNOS has demonstrated that this enzyme is localized to specific structures in the plasmalemmal membrane identified as caveolae and that this membrane compartmentalization requires both myristoylation and palmitoylation. In cases of such a dual protein acylation the combined hydrophobic interactions of the two covalently attached lipid moieties anchor the protein firmly to the plasma membrane and attachment could be regulated by palmitate turnover. Although eNOS is reported to be reversibly depalmitoylated in response to certain stimuli, the role of actively regulated eNOS palmitoylation in determining its cellular localization remains controversial. The association of eNOS with a subcompartment of the plasma membrane in which several key signal-transducing complexes are concentrated (e.g., G proteins, src-family tyrosine kinases) is likely to have profound repercussions on enzyme activity as well as on its sensitivity to activation by ignal transduction cascades other than those resulting in an increase in intracellular Ca 2+ ([Ca]i). Indeed, the association of eNOS with the caveolar scaffolding and marker protein caveolin-1 attenuates enzyme activity [2]. Moreover, the variety of additional signal molecules detected within the caveolae render these structures ideal sites for cross-talk between receptor-dependent and independent signal-transduction pathways.