Inhibitory effect of phorbol ester on bradykinin-induced phosphoinositide hydrolysis and calcium mobilization 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 bradykinin (BK) 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 BK-induced IP3 formation and Ca2+ mobilization. However, this inhibition was reduced after incubating the cells for 4 h with PMA. Inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate at 1 microM, did not inhibit these responses to BK. Prior treatment with staurosporine (1 microM), a PKC inhibitor, inhibited the effect of PMA on the BK-induced response, suggesting that the effect of PMA is mediated by the activation of PKC. In parallel experiments, a change of PKC activity was observed. PMA rapidly decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the cell membranes within 30 min. Thereafter the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Moreover, treatment with 1 microM PMA for 2 and 24 h did not significantly change the KD and Bmax of the BK receptor for [H]BK binding (control: KD = 2.3 +/- 0.3 nM, Bmax = 25.2 +/- 1.4 fmol/mg protein). These results suggest that activation of PKC inhibit IP3 accumulation and consequently attenuate [Ca2+]i increase or inhibit independently both responses. The PMA-induced inhibition of responses to BK was associated with an increase in membranous PKC activity.
Luo
S F
SF
Internal Medicine and Cellular and Molecular Pharmacology Laboratory, Chang Gung College of Medicine and Technology, Kwei-San, Tao-Yuan, Taiwan, Republic of China.
Tsao
H L
HL
Ong
R
R
Hsieh
J T
JT
Yang
C M
CM
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001920:Bradykinin / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004285:Dogs; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006868:Hydrolysis; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D007700:Kinetics; D008297:Male; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D018002:Receptors, Bradykinin / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014132:Trachea
D000470:Alkaloids; D004791:Enzyme Inhibitors; D010703:Phorbol Esters; D010716:Phosphatidylinositols; D018002:Receptors, Bradykinin; C008876:phorbol-12,13-didecanoate; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D002118:Calcium