CAMP ACTIVATES AN ATP-CONDUCTIVE PATHWAY IN CULTURED SHARK RECTAL GLAND-CELLS

The molecular mechanisms associated with ATP transport and release int o the extracellular milieu are largely unknown. To assess the presence of endogenous ATP-conductive pathway(s) in shark rectal gland (SRG) c ells, patch-clamp techniques were applied to primary cultures of SRG c ells. Whole cell currents were obtained with either intracellular tris (hydroxymethyl)-aminomethane (Tris) or Mg2+ salts of ATP (200 mM nomin al ATP) and 280 mM NaCl bathing solution. Basal currents showed a siza ble ATP permeability for outward movement of MgATP. Adenosine 3',5'-cy clic monophosphate (cAMP) stimulation significantly increased the whol e cell conductance (with either intracellular Tris-ATP or MgATP). Symm etrical whole cell ATP currents were also observed after cAMP activati on, thus consistent with ATP as the main charge carrier. The cAMP-indu cible ATP currents were insensitive to the Cl- channel blockers 4,4'-d iisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylat e, and anthracene-9-carboxylic acid but were readily blocked by nifedi pine (400 mu M) and glibenclamide (400 mu M). The nature of the electr odiffusional ATP movement was further assessed by single-channel analy sis of either MgATP or Tris-ATP currents ill excised inside-out patche s, both spontaneous and after activation with protein kinase A. Single -channel ATP currents were inhibited by either nifedipine or glibencla mide. Thus SRG cells express endogenous ATP-permeable pathways both be fore and after cAMP stimulation. Electrodiffusional ATP movement by SR G cells may play a significant role in the transport and delivery of c ellular ATP to the extracellular milieu, which may help coordinate the dynamics of the epithelial secretory response in this cell model.