Calcium-independent activation of endothelial nitric oxide synthase by ceramide

被引:102
作者
Igarashi, J
Thatte, HS
Prabhakar, P
Golan, DE
Michel, T [1 ]
机构
[1] Brigham & Womens Hosp, Div Hematol, Dept Med, Boston, MA 02115 USA
[2] Brigham & Womens Hosp, Div Cardiol, Boston, MA 02115 USA
[3] W Roxbury Vet Affairs Med Ctr, Boston, MA 02132 USA
[4] Harvard Univ, Sch Med, Dept Biol Chem & Mol Pharmacol, Boston, MA 02115 USA
关键词
D O I
10.1073/pnas.96.22.12583
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The endothelial isoform of NO synthase (eNOS) is targeted to sphingolipid-enriched signal-transducing microdomains in the plasma membrane termed caveolae. Among the caveolae-targeted sphingolipids are the ceramides, a class of acylated sphingosine compounds that have been implicated in diverse cellular responses. We have explored the role of ceramide analogues in eNOS signaling in cultured bovine aortic endothelial cells (BAEC). Addition of the ceramide analogue N-acetylsphingosine (C-2-ceramide; 5 mu M) to intact BAEC leads to a significant increase in NO synthase activity (assayed by using the fluorescent indicator 4,5-diaminofluorescein) and translocation of eNOS from the endothelial cell membrane to intracellular sites (measured by using quantitative immunofluorescence techniques); the biologically inactive ceramide N-acetyldihydrosphingosine is entirely without effect. C-2-ceramide-induced eNOS activation and translocation are unaffected by the intracellular calcium chelator 1,2-bis-o-aminophenoxyethane-N,N,N',N'-tetraacetic acid (BAPTA), Using the calcium-specific fluorescent indicator fluo-3, we also found that C-2-ceramide activation of eNOS is unaccompanied by a drug-induced increase in intracellular calcium. These findings stand in sharp contrast to the mechanism by which bradykinin, estradiol, and other mediators acutely activate eNOS, in which a rapid, agonist-promoted increase in intracellular calcium is required. Finally, we show that treatment of BAEC with bradykinin causes a significant increase in cellular ceramide content; the response to bradykinin has an EC50 Of 3 nM and is blocked by the bradykinin B-2-receptor antagonist HOE140. Bradykinin-induced ceramide generation could represent a mechanism for longer-term regulation of eNOS activity. Our results suggest that ceramide functions independently of Ca2+-regulated pathways to promote activation and translocation of eNOS, and that this lipid mediator may represent a physiological regulator of eNOS in vascular endothelial cells.
引用
收藏
页码:12583 / 12588
页数:6
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