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 intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition 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 concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 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, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.
Yang
C M
CM
Department of Pharmacology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan. chuenmao@mail.cgu.edu.tw
Tsai
Y J
YJ
Pan
S L
SL
Lin
C C
CC
Wu
W B
WB
Wang
C C
CC
Huang
S C
SC
Chiu
C T
CT
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001920:Bradykinin / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D010716:Phosphatidylinositols; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D019311:Staurosporine; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin