Targeting and translocation of endothelial nitric oxide synthase

Abstract

This review explores advances in our understanding of the intracellular regulation of the endothelial isoform of nitric oxide synthase (eNOS) in the context of its dynamically regulated subcellular targeting. Nitric oxide (NO) is a labile molecule, and may play important biological roles both within the cell in which it is synthesized and in its interactions with nearby cells and molecules. The localization of eNOS within the cell importantly influences the biological role and chemical fate of the NO produced by the enzyme. eNOS, a Ca 2+ / calmodulin-dependent enzyme, is subject to a complex pattern of intracellular regulation, including co-and post-translational modifications and interactions with other proteins and ligands. In endothelial cells and cardiac myocytes eNOS is localized in specialized plasmalemmal signal-transducing domains termed caveolae; acylation of the enzyme by the fatty acids myristate and palmitate is required for targeting of the protein to caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation. In resting cells, eNOS is tonically inhibited by its interactions with caveolin, the scaffolding protein in caveolae. However, following agonist activation, eNOS dissociates from caveolin, and nearly all the eNOS translocates to structures within the cell cytosol; following more protracted incubations with agonists, most of the cytosolic enzyme subsequently translocates back to the cell membrane. The agonist-induced internalization of eNOS is completely abrogated by chelation of intracellular Ca 2+ . These rapid receptor-mediated effects are seen not only for classic eNOS agonists such as bradykinin, but also for estradiol, indicating a novel non-genomic role for estrogen in eNOS activation. eNOS targeting to the membrane is labile, and is subject to receptorregulated Ca 2+ -dependent reversible translocation, providing another point for regulation of NO-dependent signaling in the vascular endothelium.

Docking of Endothelial Nitric Oxide Synthase (eNOS) to the Mitochondrial Outer Membrane

“…Cells that were only exposed to secondary antibodies served as sample blanks. These data further support the view that eNOS antigen resides on the surface of mitochondrial membranes.eNOS is unique among the NOS isoforms in its dual acylation by myristic and palmitic acids (25,26). N-terminal myristoylation is required for the subcellular targeting of eNOS to cell membranes (where it associates with caveolin-1) and the Golgi complex (27).…”

“…eNOS is unique among the NOS isoforms in its dual acylation by myristic and palmitic acids (25,26). N-terminal myristoylation is required for the subcellular targeting of eNOS to cell membranes (where it associates with caveolin-1) and the Golgi complex (27).…”

Reduced Endothelial NO-cGMP–Mediated Vascular Relaxation and Hypertension in IL-6–Infused Pregnant Rats

“…For example, IL-6 has been localized in the plasma membrane caveolae, 48 which may promote tighter association of eNOS with the inhibitory protein caveolin-1 and thereby prevent the initial activation of eNOS and its dissociation from the plasma membrane. 49 IL-6 may also affect the activity of mitogen-activated protein . Western blot analysis of eNOS using anti eNOS antibody (1:1000; Transduction Laboratory) as normalized to ␤-actin (A) and the basal and ACh-induced nitrite/nitrate production (B) in endothelium-intact vascular strips of control and IL-6 -infused pregnant and virgin rats.…”

TNF-α enhances contraction and inhibits endothelial NO-cGMP relaxation in systemic vessels of pregnant rats

“…Instead, the acute effects of TNF-␣ are more likely related to one of several potential posttranscriptional and/or posttranslational effects. For example, TNF-␣-treated endothelial cells could exhibit a tighter association of eNOS with inhibitory molecules such as caveolin-1 (44). Also, TNF-␣ may interfere with the myristoylation and palmitolyation of eNOS and its translocation to and association with plasmalemmal caveolae, a process required for its full activation (44,57).…”

“…For example, TNF-␣-treated endothelial cells could exhibit a tighter association of eNOS with inhibitory molecules such as caveolin-1 (44). Also, TNF-␣ may interfere with the myristoylation and palmitolyation of eNOS and its translocation to and association with plasmalemmal caveolae, a process required for its full activation (44,57). TNF-␣ may also change the activity of mitogenactivated protein kinase, a signaling pathway that induces eNOS phosphorylation and enzyme inhibition (8).…”