Inhibitory Effect of Phorbol Ester on Carbachol-Induced Signal Transduction in Cultured Canine Tracheal Smooth Muscle Cells.

Regulation of the increases in inositol 1,4,5-trisphosphate (IP(3)) production and intracellular Ca(2+) concentration (Ca(2+)) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP(3) formation and caused an initial transient peak of Ca(2+) followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 &mgr;M) for 30 min blocked the carbachol-induced IP(3) formation and Ca(2+) mobilization. Following preincubation, carbachol-induced Ca(2+) mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP(3) formation and increase in Ca(2+) were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 &mgr;M), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate at 1 &mgr;M, did not inhibit these responses to carbachol. The K(d) and B(max) of the muscarinic receptor for [(3)H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the Ca(2+) increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity. Copyright 1995 S. Karger AG, Basel