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(Circulation. 2006;114:I-319 – I-326.)
© 2006 American Heart Association, Inc.

Myocardial Protection and Vascular Biology

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

Danny Ramzy, MD; Vivek Rao, MD, PhD; Laura C. Tumiati, BSc; Ning Xu, MD, MSc; Rohit Sheshgiri, BSC; Santiago Miriuka, MD; Diego H. Delgado, MD; Heather J. Ross, MD, MSc

From the Heart Transplant Program (R.S.), Toronto General Hospital, University Health Network, Division of Cardiac Surgery (D.R., V.R., L.C.T., N.X.), University of Toronto, Division of Cardiology (S.M., D.H.D., H.J.R.), University of Toronto, Toronto, Ontario, Canada.

Correspondence to Vivek Rao, Alfredo and Teresa deGasperis Chair in Heart Failure Surgery, 4N-464, Toronto General Hospital, 200 Elizabeth St, Toronto, Ontario, M5G 2C4. E-mail vivek.rao{at}uhn.on.ca

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 PKC and PKC translocation compared with control, whereas translocation of PKC 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 PKC and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.

Key Words: endothelin-1 • endothelial dysfunction • nitric oxide • protein kinase C