Calcium antagonists ameliorate ischemia-induced endothelial cell permeability by inhibiting protein kinase C.
BACKGROUND
Dihydropyridines block calcium channels; however, they also influence endothelial cells, which do not express calcium channels. We tested the hypothesis that nifedipine can prevent ischemia-induced endothelial permeability increases by inhibiting protein kinase C (PKC) in cultured porcine endothelial cells.


RESULTS
Ischemia was induced by potassium cyanide/deoxyglucose, and permeability was measured by albumin flux. Ion channels were characterized by patch clamp. [Ca2+]i was measured by fura 2. PKC activity was measured by substrate phosphorylation after cell fractionation. PKC isoforms were assessed by Western blot and confocal microscopy. Nifedipine prevented the ischemia-induced increase in permeability in a dose-dependent manner. Ischemia increased [Ca2+]i, which was not affected by nifedipine. Instead, ischemia-induced PKC translocation was prevented by nifedipine. Phorbol ester also increased endothelial cell permeability, which was dose dependently inhibited by nifedipine. The effects of non-calcium-channel-binding dihydropyridine derivatives were similar. Analysis of the PKC isoforms showed that nifedipine prevented ischemia-induced translocation of PKC-alpha and PKC-zeta. Specific inhibition of PKC isoforms with antisense oligodeoxynucleotides demonstrated a major role for PKC-alpha.


CONCLUSIONS
Nifedipine exerts a direct effect on endothelial cell permeability that is independent of calcium channels. The inhibition of ischemia-induced permeability by nifedipine seems to be mediated primarily by PKC-alpha inhibition. Anti-ischemic effects of dihydropyridine calcium antagonists could be due in part to their effects on endothelial cell permeability.
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002199:Capillary Permeability / Q000187:drug effects*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D007511:Ischemia; D009543:Nifedipine / Q000494:pharmacology*; D011042:Poisons / Q000633:toxicity; D011190:Potassium Cyanide / Q000633:toxicity; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D013552:Swine