Cyclic adenosine monophosphate response in primary and subcultured bladder epithelial cells: Inhibition by 12- O-tetradecanoylphorbol-13-acetate

Abstract

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 μmol/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 μmol/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 μmol/L staurosporine and 25 μmol/L sphingosine, protein kinase C inhibitors; 35 μmol/L cycloheximide, a protein synthesis inhibitor; 3.0 μmol/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 μmol/L 4α-TPA (a stereoisomer of TPA), or 1.0 μmol/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. This TPA effect appears to involve protein kinase C and is specific for TPA. Thus, cross-talk between two major intracellular signaling pathways (cAMP and phosphatidyl inosital [PI] cycle) appears to exist in urothelial cells.