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. Methods and 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.