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Elevated Endothelin-1 Levels Impair Nitric Oxide Homeostasis Through a PKC-Dependent Pathway

2006; Lippincott Williams & Wilkins; Volume: 114; Issue: 1_supplement Linguagem: Inglês

10.1161/circulationaha.105.001503

1524-4539

Danny Ramzy, Vivek Rao, Laura C. Tumiati, Ning Xu, Rohit Sheshgiri, Santiago Miriuka, Diego Delgado, Heather J. Ross,

Renin-Angiotensin System Studies

Background— Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone and pathological states such as ischemia/reperfusion (I/R) injury, coronary vasospasm, and cardiac allograft vasculopathy. We assessed the effects of elevated ET-1 levels as seen after I/R to determine if ET-1 modulates nitric oxide (NO) production via the translocation of specific protein kinase C (PKC) isoforms. Methods and Results— Human saphenous vein endothelial cells (HSVECs) (n=8) were incubated with ET-1 or phosphate-buffered saline (PBS) for 24 hours. NO production was determined in the supernatant by measuring nitrate/nitrite levels. Protein expression of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), caveolin-1 and PKC were determined. Lastly, PKC translocation and activity were assessed after exposure to the drug of interest. HSVECs exposed to ET-1 displayed decreased NO production. PKC inhibition reduced NO production, whereas PKC activation increased production. NO production was maintained when HSVECs exposed to ET-1 were treated with the PKC agonist, PMA. eNOS protein expression was reduced after ET-1 treatment. PKC inhibition also downregulated eNOS protein expression, whereas PMA upregulated expression. ET-1 exposure led to a significant increase in PKCδ and PKCα translocation compared with control, whereas translocation of PKCλ was inhibited. ET-1 exposure significantly reduced overall PKC activity compared with control. Conclusions— Our study demonstrates that high levels of ET-1 impair endothelial NO production via an isoform-specific PKC-mediated inhibition of eNOS expression. ET-1 antagonism with bosentan stimulates translocation of PKCλ and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.