Inhibitory effect of phorbol ester on carbachol-induced signal transduction in cultured canine tracheal smooth muscle cells |
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Authors: | Chuen-Mao Yang PhD Ming-Che Hsu Richard Ong Jen-Tsung Hsieh Hui-Liang Tsao Yi-Chin Chen Shue-Fen Luo |
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Institution: | (1) Cellular and Molecular Pharmacology Laboratory, Department of Pharmacology, Chang Gung College of Medicine and Technology, Tao-Yuan, Taiwan, Republic of China;(2) Department of Internal Medicine, Chang Gung College of Medicine and Technology, Tao-Yuan, Taiwan, Republic of China |
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Abstract: | Regulation of the increases in inositol 1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration (Ca2+]i) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP3 formation and caused an initial transient peak of Ca2+]i followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 µM) for 30 min blocked the carbachol-induced IP3 formation and Ca2+ mobilization. Following preincubation, carbachol-induced Ca2+ mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP3 formation and increase in Ca2+]i were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 µM), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4-phorbol 12,13-didecanoate at 1 µM, did not inhibit these responses to carbachol. The Kd and Bmax of the muscarinic receptor for 3H]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 Ca2+]i increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity. |
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Keywords: | Phorbol ester Carbachol Protein kinase C Inositol phosphates Calcium ion Muscarinic receptor Tracheal smooth muscle cell |
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