Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization induced by endothelin-1 in cultured canine tracheal smooth muscle cells.
Regulation of the increase in inositol 1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by endothelin-1 (ET-1) led to IP3 formation and caused an initial transient peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min blocked the ET-1-induced IP3 formation and Ca2+ mobilization. However, this inhibition was reduced after incubating the cells for 8 h with PMA. Following preincubation, ET-1-induced Ca2+ mobilization recovered with time and reached the same extent of control cells within 48 h. The concentrations of PMA that gave half-maximal inhibition (-logEC50) of ET-1-induced IP3 formation and increase in [Ca2+]i were 8.6 and 8.4 M, respectively. Prior treatment of TSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate ET-1-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 ET-1-induced IP3 formation and Ca2+ mobilization, a change of PKC activity was observed in TSMCs. PMA rapidly 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 the phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit independently both responses. The PMA-induced inhibition of responses to ET-1 was associated with an increase in membranous PKC activity.
Yang
C M
CM
Department of Pharmacology, Chang Gung College of Medicine and Technology, Kwei-San, Tao-Yuan, Taiwan, Republic of China.
Ong
R
R
Chen
Y C
YC
Hsieh
J T
JT
Tsao
H L
HL
Tsai
C T
CT
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004075:Diglycerides / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004347:Drug Interactions; D016232:Endothelins / Q000494:pharmacology*; D006868:Hydrolysis; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D009119:Muscle Contraction / Q000502:physiology; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014132:Trachea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D000470:Alkaloids; D004075:Diglycerides; D016232:Endothelins; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium