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