Translocation of protein kinase C isoforms is involved in propofol-induced endothelial nitric oxide synthase activation.

Abstract

Previous studies have indicated that protein kinase C (PKC) may enhance endothelial nitric oxide synthase (eNOS) activation, although the detailed mechanism(s) remains unclear. In this study, we investigated the roles of PKC isoforms in regulating propofol-induced eNOS activation in human umbilical vein endothelial cells (HUVECs). We applied western blot (WB) analysis to investigate the effects of propofol on Ser(1177) phosphorylation-dependent eNOS activation in HUVECs. Nitrite (NO(2)(-)) accumulation was measured using the Griess assay. The phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway was examined by WB assay. Propofol-induced translocation of individual PKC isoforms in subcellular fractions in HUVECs was analysed using WB assay. In HUVECs, protocol treatment (1-100 microM) for 10 min induced a concentration-dependent increase in phosphorylation of eNOS at Ser(1177). The NO production was also increased accordingly. PKC inhibitors, bisindolylmaleimide I (0.1-1 microM), and staurosporine (20 and 100 nM), effectively blocked propofol-induced eNOS activation and NO production. Further analyses in fractionated endothelial lysate showed that short-term propofol treatment (50 microM) led to translocation of PKC-alpha, PKC-delta, PKC-zeta, PKC-eta, and PKC-epsilon from cytosolic to membrane fractions, which could also be inhibited by both PKC inhibitors. These data revealed that the differential redistribution of these isozymes is indispensable for propofol-induced eNOS activation. In addition, Akt was not phosphorylated in response to propofol at Ser(473) or Thr(308). Propofol induces the Ser(1177) phosphorylation-dependent eNOS activation through the drug-stimulated translocation of PKC isoforms to distinct intracellular sites in HUVECs, which is independent of PI3K/Akt-independent pathway.