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

Abstract

The regulatory pathways involved in the ATP-stimulated CI⁻ secretion across rat epididymal epithelium were investigated by the short-circuit current (I_SC) technique. Biphasic characteristic was observed in the I_SC responded to ATP (0.01-10 m̈M). Inhibitor of P₁ receptor, 8-phenyltheophylline (up to 100 m̈M), did not have any effect on both phases of the ATP-stimulated I_SC. The order of potency for stimulation of the two phases in I_SC was ATP>ADP> AMP, adenosine, consistent with the presence of P₂-purinoceptors. CI⁻ channel blocker, disulfonic acid stilbene (DIDS, 300 m̈M), only inhibited the first peak of the ATP-stimulated I_SC 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 Ca²⁺-activated I_SC (induced by ionomycin, 10 m̈M) but not cAMP-activated I_SC (induced by forskolin, 1 m̈M) which could only be blocked by DPC. Both peaks of the ATP-activated I_SC could be significantly inhibited by pretreatment with a Ca²⁺-chelating agent, BAPTA-AM (50 m̈M). 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 m̈M) indicating that cAMP production upon ATP stimulation was secondary to an increase in intracellular Ca²⁺ concentration. These results indicate that the ATP-stimulated CI⁻ secretion could be mediated by Ca²⁺ and cAMP-dependent regulatory pathways giving rise to the biphasic nature of the ATP-induced I_SC.