Regulation of 5-Hydroxytryptamine-Induced Signal Transduction in Canine Cultured Aorta Smooth Muscle Cells by Phorbol Ester

Abstract

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca 2+ concentration ([Ca 2+] 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 [Ca 2+] i peak followed by a sustained elevation of [Ca 2+] 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 Ca 2+ 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 Ca 2+ 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-α, βI, βII, δ, ϵ, θ, and ζ 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-α, βI, βII, δ, ϵ, θ, and ζ induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca 2+ mobilization in ASMCs.