NA-K-CL COTRANSPORT IN NYSTATIN-TREATED TRACHEAL CELLS - REGULATION BY ISOPROTERENOL, APICAL UTP, AND [CL](I)

Chloride secretion in mammalian airway epithelia is stimulated by p-ad renergic agonists via an adenosine 3',5'-cyclic monophosphate (cAMP)-d ependent mechanism and by apical triphosphate nucleotides (ATP, UTP) v ia a cAMP-independent mechanism. Both types of secretagogues are known to stimulate apical Cl channels in airway cells; however, to maintain a stimulated rate of secretion, basolateral Cl influx via Na-K-Cl cot ransport must be upregulated in parallel with apical Cl efflux. To exa mine the regulation of basolateral cotransport activity and its relati onship to apical Cl efflux, we examined Cl transport in confluent prim ary cultures of dog tracheal epithelial cells treated with nystatin, a n antibiotic that increases the permeability of plasma membranes to sm all monovalent ions, including Cl. By applying nystatin to the apical membrane of these cultures, apical Cl permeability could be increased to the point where transepithelial Cl transport is limited by transpor t across the basolateral membrane, which reflects primarily the activi ty of the cotransporter. In cultures of tracheal cells not treated wit h nystatin, transepithelial (basolateral-to-apical) Cl-36 flux was inc reased two- to threefold by exposure to isoproterenol (5 mu M, basolat eral) or apical UTP (10 mu M). Apical application of nystatin (400 uni ts/ml) increased the basal level of transepithelial Cl-36 flux similar to 1.5-fold and eliminated UTP stimulation of this flux, although an approximately twofold stimulation by isoproterenol persisted. Nystatin treatment also abolished UTP stimulation of saturable, basolateral [H -3]bumetanide binding, a measure of functioning Na-K-Cl cotransporters in these cells; isoproterenol stimulation of binding was only mildly inhibited by nystatin treatment. Lowering intracellular CI concentrati on ([Cl](i)) by incubating cultures with apical media containing nysta tin and reduced [Cl] (NO3 replacement) increased both basolateral-to-a pical Cl-36 flux and [H-3]bumetanide binding in the absence of secreta gogues or cell shrinkage. The results support our previous suggestion, based entirely on [H-3]bumetanide binding [M. Haas, D. G. McBrayer, a nd J. R. Yankaskas. Am. J. Physiol. 264 (Cell. Physiol. 32): C189-C200 , 1993], that UTP stimulation of basolateral Na-K-Cl cotransport in ai rway epithelial cells is entirely secondary to, and requires, an incre ase in apical Cl efflux, and further suggest that a decrease in [Cl](i ) may be a signal for cotransport activation in response to UTP. In ad dition, a cAMP-dependent cascade initiated by isoproterenol appears to directly stimulate the cotransporter.