Different regulatory pathways involved in ATP-stimulated chloride secretion in rat epididymal epithelium

The regulatory pathways involved in the ATP-stimulated Cl- secretion across rat epididymal epithelium were investigated by the short-circuit current (ISC) technique. Biphasic characteristic was observed in the ISC responded to ATP (0.01-10 microM). Inhibitor of P1 receptor, 8-phenyltheophylline (up to 100 microM), did not have any effect on both phases of the ATP-stimulated ISC. The order of potency for stimulation of the two phases in ISC was ATP > ADP >> AMP, adenosine, consistent with the presence of P2-purinoceptors. Cl- channel blocker, disulfonic acid stilbene (DIDS, 300 microM), only inhibited the first peak of the ATP-stimulated ISC while diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced both, indicating the involvement of different conductance pathways. DIDS was found to have an inhibitory effect on Ca(2+)-activated ISC (induced by ionomycin, 10 microM) but not cAMP-activated ISC (induced by forskolin, 1 microM) which could only be blocked by DPC. Both peaks of the ATP-activated ISC could be significantly inhibited by pretreatment with a Ca(2+)-chelating agent, BAPTA-AM (50 microM). An increase in cellular cAMP content upon stimulation of ATP was measured by radioimmunoassay. No significant increase in cAMP production was observed in cells stimulated with adenosine. The ATP-induced cAMP increase was prevented by pretreatment with BAPTA-AM (100 microM) indicating that cAMP production upon ATP stimulation was secondary to an increase in intracellular Ca2+ concentration. These results indicate that the ATP-stimulated Cl- secretion could be mediated by Ca2+ and cAMP-dependent regulatory pathways giving rise to the biphasic nature of the ATP-induced ISC.