Cyclic adenosine monophosphate response in primary and subcultured bladder epithelial cells: inhibition by 12-O-tetradecanoylphorbol-13-acetate.
Primary and first-passage dog urothelial cells (DUC and DUC-P1, respectively) exhibited an active catalytic subunit for the cyclic adenosine monophosphate (cAMP) second messenger system. Dramatic increases in cAMP levels were observed following the addition of forskolin, which elicited a time- and dose-response--dependent increase in cAMP levels. Increases in intracellular cAMP levels preceded media increases in cyclic nucleotide levels and were observed at the earliest time examined (5 minutes). The lowest effective concentration of forskolin was between 1 and 10 mumol/L. cAMP level increases as large as 20- to 100-fold were observed in cells and media. Preincubation of primary and subcultured cells with 0.1 mumol/L 12-O-tetradecanoylphorbol-13-acetate (TPA) for 60 minutes reduced the magnitude of the forskolin-induced increase in cAMP levels. To determine the mechanism by which TPA elicits its effect in primary cultures, the following test agents were used: 1.0 mumol/L staurosporine and 25 mumol/L sphingosine, protein kinase C inhibitors; 35 mumol/L cycloheximide, a protein synthesis inhibitor; 3.0 mumol/L indomethacin, an inhibitor of prostaglandin synthesis; and 0.5 mmol/L RO-20-1724, a cyclic nucleotide phosphodiesterase inhibitor. Staurosporine and sphingosine were the only agents that prevented the effect of TPA. The specificity of the TPA effect was evaluated with the following test agents: 0.2 nmol/L epidermal growth factor (EGF), 0.1 mumol/L 4 alpha-TPA (a stereoisomer of TPA), or 1.0 mumol/L A23187. In contrast to TPA, none of these agents reduced forskolin-mediated increases in cAMP. Results indicate forskolin cAMP responsiveness and regulation of this response by TPA in both primary and subcultured cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Thomas
D J
DJ
Veterans Administration Medical Center, St Louis, MO 63125.
Zenser
T V
TV
Davis
B B
BB
D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism / Q000502:physiology; D007213:Indomethacin / Q000494:pharmacology; D008954:Models, Biological; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D015290:Second Messenger Systems / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D013110:Sphingosine / Q000494:pharmacology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D001743:Urinary Bladder / Q000166:cytology* / Q000378:metabolism* / Q000502:physiology
D000470:Alkaloids; D005576:Colforsin; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; D000001:Calcimycin; D004815:Epidermal Growth Factor; D003513:Cycloheximide; D000242:Cyclic AMP; D011493:Protein Kinase C; D019311:Staurosporine; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D007213:Indomethacin