NA-K-CL COTRANSPORT IN THE SHARK RECTAL GLAND .1. REGULATION IN THE INTACT PERFUSED GLAND

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 [H-3]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). [H-3]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 [H-3]benzmetanide was determined in the membrane fraction. Most of the membrane-associated [H-3]-benzmetanide appeared to be associated with the Na-K-Cl cotransporter as judged by the dissociation rates at 0-degrees-C and 20-degrees-C, by labeling with a photosensitive analogue, and by continued association of [H-3]benzmetanide with membrane protein on solubilization. With the use of [H-3]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 [H-3]-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 [H-3]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 [H-3]benzmetanide binding was completely inhibited when Ba was added to the perfusate to block K channel-mediated K exit across the basolateral membrane. This suggests that modulation of the cotransporter occurs subsequent to the activation of apical membrane Cl channels and loss of KCl. In glands not treated with VIP, increasing perfusate osmolarity to 1.4 times normal to induce cell shrinkage was found to stimulate [H-3]benzmetanide binding fivefold with no appreciable increase in salt secretion. Na-K-Cl cotransport may thus play a dual role in the shark rectal gland: participation in secretagogue-stimulated net salt secretion and in the regulation of epithelial cell volume in the face of large transcellular salt and water fluxes.