[Cl-]i-dependent phosphorylation of the Na-K-Cl cotransport protein of dog tracheal epithelial cells.

Basolateral Na-K-Cl cotransport activity in primary cultures of dog tracheal epithelial cells is stimulated by beta-adrenergic agents, such as isoproterenol, and by apical UTP, which acts through an apical P2-purinergic receptor. While at least part of the stimulatory effect of isoproterenol appears to involve direct activation of the cotransporter via cAMP-dependent protein kinase, cotransport stimulation by apical UTP is entirely secondary to apical Cl- efflux and a resultant decrease in intracellular [Cl-] ([Cl-]i) and/or cell shrinkage (Haas, M., and McBrayer, D. G. (1994) Am. J. Physiol. 266, C1440-C1452). In the secretory epithelia of the shark rectal gland and avian salt gland, Na-K-Cl cotransport activation by both cAMP-dependent and cAMP-independent secretagogues has been shown to be accompanied by phosphorylation of the cotransport protein itself (Lytle, C., and Forbush, B., III (1992) J. Biol. Chem. 267, 25438-25443; Torchia, J., Lytle, C., Pon, D. J., Forbush, B., III, and Sen, A. K. (1992) J. Biol. Chem. 267, 25444-25450). In the present study, we immunoprecipitate the approximately 170-kDa Na-K-Cl cotransport protein of dog tracheal epithelial cells with a monoclonal antibody against the cotransporter of the intestinal cell line T84. Incubation of confluent primary cultures of tracheal cells with isoproterenol and apical UTP increases basolateral-to-apical 36Cl- flux 3.4- and 2.6-fold, respectively, and produces similar increases (3.2- and 2.8-fold, respectively) in 32P incorporation into the approximately 170-kDa cotransport protein. Decreasing [Cl-]i (without concomitant cell shrinkage) by incubating cultures with apical nystatin and reduced apical [Cl-] ([Cl-]alpha) likewise increases both cotransport activity and cotransport protein phosphorylation. These effects become more pronounced with greater reductions in [Cl-]alpha; after 20 min of incubation with nystatin and 32 mM [Cl-]alpha, cotransport activity and 32P incorporation into the cotransport protein are increased 2.8- and 2.7-fold, respectively, similar to increases seen with apical UTP. 2-3-fold increases in cotransporter activity and phosphorylation are also seen in nystatin-treated cells under hypertonic conditions (50 mM sucrose added apically and basolaterally). These findings suggest a close correlation between Na-K-Cl cotransport activity and phosphorylation of the approximately 170-kDa cotransport protein. The latter is phosphorylated in response to both reduced [Cl-]i and cell shrinkage, either or both of which are likely to be involved in secondary cotransport activation in response to apical UTP.