AJP - Cell Physiology, Vol 264, Issue 1 C189-C200, Copyright © 1993 by American Physiological Society

ARTICLES

Dual mechanisms for Na-K-Cl cotransport regulation in airway epithelial cells

M. Haas, D. G. McBrayer and J. R. Yankaskas
Department of Pathology, University of Chicago, Illinois 60637.

To investigate cellular mechanisms involved in the regulation of basolateral Na-K-Cl cotransport in airway epithelia, we determined saturable basolateral [3H]bumetanide binding, a measure of functioning cotransporters, in primary cultures of canine tracheal and human nasal epithelial cells, including cells from patients with cystic fibrosis (CF). As we previously reported [M. Haas, L. G. Johnson, and R. C. Boucher. Am. J. Physiol. 259 (Cell Physiol. 28): C557-C569, 1990], isoproterenol and hypertonic cell shrinkage produce an equivalent stimulation of [3H]bumetanide binding to dog tracheal cells. We now find that apical ATP and UTP, which stimulate apical Cl channels and Cl secretion in normal and CF airway cells by an adenosine 3',5'-cyclic monophosphate (cAMP)-independent mechanism (S. J. Mason, A. M. Paradiso, and R. C. Boucher. Br. J. Pharmacol. 103: 1649-1656, 1991), increase basolateral [3H]bumetanide binding to dog tracheal cells to the same extent as do isoproterenol and hypertonic shrinkage. The stimulatory effects of ATP and UTP on binding are inhibited by apical addition of a Cl channel blocker, the indanyloxyacetic acid derivative IAA-94 (0.2 mM), or by raising basolateral K concentration ([K]b) from 3.3 to 40 mM, suggesting these effects are secondary to apical Cl efflux via channels. Apical IAA-94 and increased [K]b also inhibit stimulation of binding by isoproterenol by approximately 50%, suggesting that part (but not all) of the effect of the beta-agonist on basolateral cotransport is secondary to apical Cl efflux, with an additional component of direct stimulation of cotransport via cAMP. In support of this interpretation, we find that isoproterenol and a membrane-permeable cAMP analogue increase [3H]bumetanide binding to primary cultures of CF nasal epithelial cells, in which significant cAMP-mediated stimulation of apical Cl efflux does not occur. [3H]bumetanide binding to CF nasal cells is also stimulated by apical ATP, and levels of saturable [3H]bumetanide binding to CF cells are 1.3-1.5 times those in non-CF nasal cells under both basal and stimulated conditions. The results suggest that basolateral Na-K-Cl cotransport in airway cells may be upregulated in two distinct ways: 1) directly via a cAMP-dependent cascade, and 2) as a secondary response to apical Cl channel activation. Both of these mechanisms appear to be intact in CF.

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