Cl- currents activated by extracellular nucleotides in human bronchial cells

The perforated-patch technique was used to study the response of human bronchial cells to extracellular nucleotides. ATP or UTP (100 μM) elicited a complex response consisting of a large transient membrane current increase followed by a relatively small sustained level. These two phases were characterized by different current kinetics. Throughout the transient phase (2-3 min) the membrane current (I(p)) displayed slow activation and deactivation kinetics at depolarizing and hyperpolarizing potentials respectively. At steady-state (I(s)) the relaxation at hyperpolarizing potential disappeared whereas at positive membrane potentials the current became slightly deactivating. The I(s) amplitude was dependent on the extracellular Ca²⁺ concentration, being completely inhibited in Ca²⁺-free medium. Cell preincubation with the membrane-permeable chelating agent BAPTA/AM prevented completely the response to nucleotides, thus suggesting that both I(p) and I(s) were dependent on intracellular Ca²⁺. The presence of a hypertonic medium during nucleotide stimulation abolished I(s) leaving I(p) unchanged. On the contrary, niflumic acid, a blocker of Ca²⁺-activated Cl^- channels, prevented completely I(p) without reducing significantly I(s). 1,9-dideoxyforskolin fully inhibited I(s) but also reduced I(p). Replacement of extracellular Cl^- with aspartate demonstrated that the currents activated by nucleotides were Cl^- selective. I(p) resulted five times more Cl^- selective than I(s) with respect to aspartate. Taken together, our results indicate that ATP and UTP activate two types of Cl^- currents through a Ca²⁺-dependent mechanism.