Regulation of 5-hydroxytryptamine-induced signal transduction in canine cultured aorta smooth muscle cells by phorbol ester.
Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of ASMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min almost abolished the 5-HT-induced IPs formation and Ca2+ mobilization. This inhibition was reduced after long-term incubating the cells with PMA. Prior treatment of ASMCs with staurosporine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the 5-HT-induced IP formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of ASMCs with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta isozymes from the cytosol to the membrane was seen after 5-min, 30-min, 2-h, and 4-h treatment. However, 24-h treatment caused a partial down-regulation of these PKC isozymes. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca2+ mobilization in ASMCs.
Yang
C M
CM
Department of Pharmacology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan.
Chiu
C T
CT
Fan
L W
LW
Tsao
H L
HL
Wang
C C
CC
D000818:Animals; D001011:Aorta / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012701:Serotonin / Q000378:metabolism* / Q000494:pharmacology; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D007527:Isoenzymes; D012701:Serotonin; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium