Na-K-Cl cotransport in the shark rectal gland. I. Regulation in the intact perfused gland

Abstract

To investigate regulation of the Na-K-Cl cotransport system in the rectal gland of the dogfish shark Squalus acanthias, we examined binding of the loop diuretic [3H]benzmetanide to the intact gland. Glands were perfused with a shark Ringer solution, either in a basal state or stimulated with vasoactive intestinal peptide (VIP). [3H]benzmetanide was added to the perfusion solution for the last 25 min of perfusion, after which the gland was homogenized and the amount of bound [3H]benzmetanide was determined in the membrane fraction. Most of the membrane-associated [3H]-benzmetanide appeared to be associated with the Na-K-Cl cotransporter as judged by the dissociation rates at 0 degree C and 20 degrees C, by labeling with a photosensitive analogue, and by continued association of [3H]benzmetanide with membrane protein on solubilization. With the use of [3H]4-benzoyl-5-sulfamoyl-3-(3- thenyloxy)benzoic acid, a photosensitive analogue of benzmetanide, a 200-kDa protein was selectively labeled on exposure to ultraviolet light. It was also possible to detect [3H]-benzmetanide binding during the perfusion period as an arterial-venous difference, thereby providing a time course of the binding process. In comparing two groups of five glands each, VIP stimulated NaCl secretion 20-fold and [3H]benzmetanide binding 16-fold, providing strong evidence that the Na-K-Cl cotransport system is activated as part of the process of stimulation of secretion. The VIP-stimulated increase in [3H]benzmetanide binding was completely inhibited when Ba was added to the perfusate to block K channel-mediated K exit across the basolateral membrane.(ABSTRACT TRUNCATED AT 250 WORDS)