Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells

Regulation of the increase in inositol phosphate (IP) production and intrac ellular Ca2+ concentration ([Ca2+](i) by protein kinase C (PKC) was investi gated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 mu M) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibiti on was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The con centrations of PMA giving a half-maximal (pEC(50)) and maximal inhibition o f BK induced an increase in [Ca2+](i), were 7.8 +/- 0.3 M and 1 mu M, n = 8 , respectively. Prior treatment of VSMCs with staurosporine (1 mu M) a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activa tion of PKC. Paralleling the effect of PMA on the BK-induced IP formation a nd Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC i sozymes, The results revealed that treatment of the cells with PMA for vari ous times, translocation of PKC-alpha, beta I, beta II, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 m in, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial dow nregulation of these PKC isozymes in both fractions. Treatment of VSMCs wit h 1 mu M PMA for either 1 or 24 h did nor significantly change the K-D and B-max of the BK receptor for binding (control: K-D = 1-7 +/- 0.2 nM; B-max 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, th ese resuts demonstrate that translocation of PKC-alpha, beta I, beta II, de lta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced I Ps accumulation and Ca2+ mobilisation in VSMCs.