Differential effects of extracellular ATP on chloride transport in cortical collecting duct cells

Extracellular ATP in the cortical collecting duct can inhibit epithelial sodium channels (ENaC) but also stimulate calcium-activated chloride channels (CACC). The relationship between ATP-mediated regulation of ENaC and CACC activity in cortical collecting duct cells has not been clearly defined. We used the mpkCCD _c14 cortical collecting duct cell line to determine effects of ATP on sodium (Na ⁺) and chloride (Cl ^-) transport with an Ussing chamber system. ATP, at a concentration of 10 ^-6 M or less, did not inhibit ENaC-mediated short-circuit current (I _sc) but instead stimulated a transient increase in I _sc. The macroscopic current-voltage relationship for ATP-inducible current demonstrated that the direction of this ATP response changes from positive to negative when transepithelial voltage (V _te) is clamped to less than -10 mV. We hypothesized that this negative V _te might be found under conditions of aldosterone stimulation. We next stimulated mpkCCD _c14 cells with aldosterone (10 ^-6 M) and then clamped the V _te to -50 mV, the Vte of aldosterone-stimulated cells under open-circuit conditions. ATP (10 ^-6 M) induced a transient increase in negative clamp current, which could be inhibited by flufenamic acid (CACC inhibitor) and BAPTA-AM (calcium chelator), suggesting that ATP stimulates Cl ^- absorption through CACC. Together, our findings suggest that the status of ENaC activity, by controlling V _te, may dictate the direction of ATP-stimulated Cl ^- transport. This interplay between aldosterone and purinergic signaling pathways may be relevant for regulating NaCl transport in cortical collecting duct cells under different states of extracellular fluid volume.