Cl-]i-dependent Phosphorylation of the Na-K-Cl Cotransport Protein of Dog Tracheal Epithelial Cells

Mark Haas,Donna Mcbrayer,Christian Lytle

doi:10.1074/jbc.270.48.28955

Mark Haas, Donna Mcbrayer + Show 1 more

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https://doi.org/10.1074/jbc.270.48.28955

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Abstract

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.

Highlights

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
The cellular signal for this secondary cotransport activation in dog tracheal cells is likely to be a decrease in intracellular [ClϪ] ([ClϪ]i) and/or in cell volume; both reduced [ClϪ]i and cell shrinkage can produce a level of cotransport activation similar to that seen with apical UTP [9]
The molecular mass of the major immunoprecipitated protein is in good agreement with that of a single peak of specific labeling identified by photolabeling of dog tracheal epithelial cells with the photosensitive bumetanide analogue, [3H]BSTBA (Fig. 2)

Summary

Introduction

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. In the shark rectal gland it was shown that increasing degrees of hypertonic cell shrinkage produce increasing and approximately proportional stimulation of cotransport protein phosphorylation and [3H]benzmetanide binding [14], the latter having previously been shown to correlate closely with Na-K-Cl cotransport activity [16, 20]. In these secretory epithelia it appears that Na-K-Cl cotransport activation involves phosphorylation of the cotransport protein itself. A preliminary report has been presented in abstract form [28]

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