Regulation of Na-K-Cl cotransport in cultured canine airway epithelia: a [3H]bumetanide binding study

Abstract

We examined [3H]bumetanide binding to membranes isolated from canine tracheal and bronchial epithelia and to confluent primary cultures of these cells. Crude plasma membranes from trachea and bronchus bind [3H]bumetanide in a saturable manner; tracheal membranes have a higher affinity but lower maximal binding (K1/2 approximately equal to 0.7 microM; Bmax approximately equal to 2.5 pmol/mg protein) than do bronchial membranes (K1/2 approximately equal to 3.5 microM; B(max) approximately equal to 7.5 pmol/mg). In both cases, saturable binding is reduced by greater than 65% when either Na, K, or Cl is removed from the medium. In primary cultures, saturable [3H]bumetanide binding (inhibited by a 30-fold excess of unlabeled bumetanide) occurs when [3H]bumetanide (1.0 microM) is added to the solution bathing the basolateral side of tracheal (1.20 +/- 0.10 pmol bound/mg total cell protein) and bronchial (1.79 +/- 0.52 pmol/mg) cultures; minimal binding is seen with apical [3H]bumetanide. Isoproterenol (10(-5) M; basolateral exposure) produces approximately 100% increase in saturable basolateral [3H]bumetanide binding to tracheal cultures and approximately 30% increase in bronchial cultures. Similar augmentation of binding is seen when apical Cl is reduced from 134 to 4 mM and when both apical and basolateral media are made hypertonic by addition of 100 mM sucrose. Under these latter two conditions, isoproterenol produces little or no additional increase in binding. Our results indicate that the increase in basolateral Cl influx via Na-K-Cl cotransport that must occur during beta-adrenergic stimulation of net salt secretion in canine airway epithelia is related to an actual increase in the number of functioning cotransporters in the basolateral membrane and is not simply due to a change in ion gradients. The increase in cotransport sites, however, may be secondary to initial stimulation of apical Cl channels, with resultant cell shrinkage.