Elevated endothelin-1 levels impair nitric oxide homeostasis through a PKC-dependent pathway.
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.


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 PKCdelta and PKCalpha translocation compared with control, whereas translocation of PKClambda 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 PKClambda and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.
Ramzy
Danny
D
Heart Transplant Program, University of Toronto, Toronto, Ontario, Canada.
Rao
Vivek
V
Tumiati
Laura C
LC
Xu
Ning
N
Sheshgiri
Rohit
R
Miriuka
Santiago
S
Delgado
Diego H
DH
Ross
Heather J
HJ
D000470:Alkaloids; D053119:Benzophenanthridines; D000077300:Bosentan; D051242:Caveolin 1 / Q000096:biosynthesis / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D019332:Endothelin-1 / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D006706:Homeostasis / Q000187:drug effects; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology*; D009281:Naphthalenes / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis / Q000235:genetics; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis* / Q000235:genetics; D010617:Phenanthridines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D051571:Protein Kinase C-alpha / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D012501:Saphenous Vein / Q000166:cytology; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000099293:Protein Kinase C-lambda
D000470:Alkaloids; D053119:Benzophenanthridines; D000077300:Bosentan; D051242:Caveolin 1; D019332:Endothelin-1; D007527:Isoenzymes; D009281:Naphthalenes; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D010617:Phenanthridines; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D013449:Sulfonamides; D013755:Tetradecanoylphorbol Acetate; D000099293:Protein Kinase C-lambda; C495540:CAV1 protein, human; C016299:chelerythrine; C058819:calphostin C