Novel Mechanism of Endothelial Nitric Oxide Synthase Activation Mediated by Caveolae Internalization in Endothelial Cells

Abstract

Abstract-Caveolin-1, the caveolae scaffolding protein, binds to and negatively regulates eNOS activity. As caveolin-1 also regulates caveolae-mediated endocytosis after activation of the 60-kDa albumin-binding glycoprotein gp60 in endothelial cells, we addressed the possibility that endothelial NO synthase (eNOS)-dependent NO production was functionally coupled to caveolae internalization. We observed that gp60-induced activation of endocytosis increased NO production within 2 minutes and up to 20 minutes. NOS inhibitor N G -nitro-L-arginine (L-NNA) prevented the NO production. To determine the role of caveolae internalization in the mechanism of NO production, we expressed dominant-negative dynamin-2 mutant (K44A) or treated cells with methyl-␤-cyclodextrin. Both interventions inhibited caveolae-mediated endocytosis and NO generation induced by gp60. We determined the role of signaling via Src kinase in the observed coupling of endocytosis to eNOS activation. Src activation induced the phosphorylation of caveolin-1, Akt and eNOS, and promoted dissociation of eNOS from caveolin-1. Inhibitors of Src kinase and Akt also prevented NO production. In isolated perfused mouse lungs, gp60 activation induced NO-dependent vasodilation, whereas the response was attenuated in eNOS Ϫ/Ϫ or caveolin-1 Ϫ/Ϫ lungs. Together, these results demonstrate a critical role of caveolae-mediated endocytosis in regulating eNOS activation in endothelial cells and thereby the NO-dependent vasomotor tone. Key Words: caveolin-1 Ⅲ endocytosis Ⅲ vasomotor tone Ⅲ albumin Ⅲ transcytosis E ndothelial NO synthase (eNOS) is modified by N-myristoylation and palmitoylation, which targets the enzyme to caveolae, 1 the plasma membrane cholesterol-rich microdomains. 2,3 Multiple mechanisms are involved in regulating NO production following eNOS activation. eNOS activity is regulated by Ca 2ϩ -calmodulin, phosphorylation activated by kinases such as Src and interactions with caveolin-1, dynamin, and heat shock protein 90 (HSP90). 4 The effects of phosphorylation are complex. Phosphorylation of Ser116 and Thr497 negatively regulates eNOS activity, whereas phosphorylation at Ser635 and Ser1179 has the opposite effect. 5-7 Src kinase activated eNOS by inducing phosphorylation at Tyr83. 8 Phosphorylation at Ser617 functions by affecting the phosphorylation of the above residues. 9 Insulin, estrogen, and shear stress were shown to induce phosphorylationdependent activation of eNOS at Ser1179 independent of increased intracellular [Ca 2ϩ ] 10 -12 eNOS in caveolae is held inactive by its association with caveolin-1, 13 but eNOS activity can be increased by Ca 2ϩ /calmodulin 3 and binding to HSP90 and dynamin-2. 14,15 HSP90 facilitates the phosphorylation of eNOS by forming a ternary complex with eNOS and Akt. 16 Dynamin-2 regulates eNOS activity through the binding of its proline-rich domain to the FAD domain of eNOS, promoting electron transfer between the bound flavins of the reductase domain and increasing NO production. 15 We have shown that activation of t...

Phosphorylation of Endothelial Nitric-oxide Synthase Regulates Superoxide Generation from the Enzyme

“…As such, our in vitro data are consistent with the hypothesis that Thr-495 phosphorylation negatively regulates eNOS activity (13,20,22,44,52,55,56). Previous reports have indicated that phosphorylation of eNOS at Ser-1177 leads to a dissociation of eNOS and caveolin; however, this dissociation was dependent upon the induction of caveolin-dependent endocytosis (57,58). We conclude that phosphorylation of heNOS at Thr-495 plays a role in the regulation of eNOS activity indirectly through the alteration of CaM binding and the association with caveolin.…”

Regulation of Pulmonary Vasoconstriction by Agonists and Caveolae

“…As the relationship between caveolin-1 and eNOS is dynamic and a prerequisite to homeostasis, it is also plausible that MβCD may reversibly disrupt eNOS localization by altering the membrane organization. These results also support a recent new perspective on the interrelationship between caveolae and eNOS [23] that endothelial dysfunction observed in hypertension is associated with eNOS dysfunction. A tight eNOS-caveolin-1 association attenuates the ability of caveolin to inhibit signaling moieties and enhances cell proliferation [24].…”

Role of Gi/o-Src kinase-PI3K/Akt pathway and caveolin-1 in β2-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

“…Interestingly, we have shown that Src kinase and Akt inhibitors exert additive inhibitory effects on the β 2 -adrenergic relaxant response. Furthermore, Src kinase is known to exert a striking role in eNOS-dependent signaling related to its ability to directly phosphorylate caveolin-1 at Tyr 14 [23] and Src kinase-dependent phosphorylation of caveolin-1 has recently been proposed to promote caveolin-1 dissociation from eNOS, leading to eNOS activation and caveolaemediated endocytosis [31,32]. In this context, we focused on the potential role of caveolin-1 in the additional mechanism responsible for β 2 -AR-dependent eNOS activation.…”

“…Our results are consistent with -and further supportthis model, as it is the first time, to our knowledge, that the effect of both pharmacological and genetic disruptions was investigated in parallel on a NO-dependent vasorelaxation in a given artery. This point is important because vasorelaxant responses studied in arteries isolated from Cav-1 KO mice have been shown to be decreased, increased or not modified, depending on the vasorelaxant agent or the vascular bed studied [32,[34][35][36]. Regarding the β 2 -AR-mediated vasorelaxation, Neidhold et al reported that it was not modified in the saphenous artery from Cav-1 KO mouse [37].…”