Extracellular ATP activates both Ca²⁺- and cAMP-dependent Cl⁻ conductances in rat epididymal cells

Abstract

Activation of Ca²⁺ and cAMP-dependent Cl⁻ conductances by extracellular ATP was studied using the whole-cell patch clamp technique. Immediately after addition of extracellular ATP (10 μm), activation of wholecell Cl⁻ current exhibiting delayed inactivation and activation kinetics at hyperpolarizing and depolarizing voltages, respectively, was observed. After prolonged activation, the kinetic characteristics of the ATP-induced Cl⁻ current became time- and voltage-independent. When applied to the later phase of the ATP-activated whole-cell current, the disulfonic acid stilbene DIDS (200 μm) could only inhibit 64% of the current while diphenylamine-dicarboxylic acid (DPC, 1 mm) completely inhibited it. Inclusion of a peptide inhibitor for protein kinase A (PKI, 10 nm) in the pipette solution blocked ATP-induced time- and voltage-independent current activation but did not affect the delayed activating and inactivating current activation but did not affect the delayed activating and inactivating current which could be totally blocked by DIDS. Anion selectivity sequence was determined in the presence of either PKI or DIDS and found to be significantly different. Increased pipette EGTA (10 mm) or treatment of the cells with trifluoperazine (40 μm), an inhibitor of calmodulin, suppressed both types of ATP-induced Cl⁻ currents. No current activation by ATP was observed when cells were dialyzed with the IP₃ receptor blocker, heparin (10 ng/ml). These results suggest that extracellular ATP activates IP₃-linked Ca²⁺-dependent regulatory pathway, which in turn activates cAMP-dependent pathway, leading to activation of both Ca²⁺ and cAMP-dependent Cl⁻ conductances in epididymal cells.