EXTRACELLULAR ATP ACTIVATES BOTH CA2-DEPENDENT AND CAMP-DEPENDENT CL-CONDUCTANCES IN RAT EPIDIDYMAL CELLS()

Activation of Ca2+ and cAMP-dependent Cl- conductances by extracellula r ATP was studied using the whole-cell patch clamp technique. Immediat ely after addition of extracellular ATP (10 mu M), activation of whole cell Cl- current exhibiting delayed inactivation and activation kineti cs at hyperpolarizing and depolarizing voltages, respectively, was obs erved. After prolonged activation, the kinetic characteristics of the ATP-induced Cl- current became time- and voltage-independent. When app lied to the later phase of the ATP-activated whole-cell current, the d isulfonic acid stilbene DIDS (200 mu M) could only inhibit 64% of the current while diphenylamine-dicarboxylic acid (DPC, 1 mM) completely i nhibited it. Inclusion of a peptide inhibitor for protein kinase A (PK I, 10 nM) in the pipette solution blocked ATP-induced time- and voltag e-independent current activation but did not affect the delayed activa ting and inactivating current activation but did not affect the delaye d activating and inactivating current which could be totally blocked b y DIDS. Anion selectivity sequence was determined in the presence of e ither PKI or DIDS and found to be significantly different. Increased p ipette EGTA (10 mM) or treatment of the cells with trifluoperazine (40 mu M), an inhibitor of calmodulin, suppressed both types of ATP-induc ed 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+-depe ndent regulatory pathway, which in turn activates cAMP-dependent pathw ay, leading to activation of both Ca2+ and cAMP-dependent Cl- conducta nces in epididymal cells.