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 intr acellular Ca2+ concentration ([Ca2+](i)) by protein kinase C (PKC) was inve stigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an i nitial 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 lon g-term incubating the cells with PMA. Prior treatment of ASMCs with stauros porine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenu ate 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 wit h antibodies against different PKC isozymes. The results revealed that trea tment of ASMCs with PMA for various times, translocation of PKC-alpha, beta I, beta II, delta, epsilon, theta, and zeta isozymes from the cytosol to t he 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 c onclusion, these results demonstrate that translocation of PKC-alpha, beta I, beta II, delta, epsilon, theta, and zeta induced by PMA caused an attenu ation of 5-HT induced IPs accumulation and Ca2+ mobilization in ASMCs. CELL SIGNAL 11;8:581-589, 1999. (C) 1999 Elsevier Science Inc.