Na-K-Cl cotransport in the shark rectal gland. II. Regulation in isolated tubules.
We examined the binding of [3H]benzmetanide, a potent inhibitor of Na-K-Cl cotransport, to secretory tubules isolated from dogfish shark rectal glands. Specific binding increased dramatically (from 3 to 40 pmol/mg protein) when the tubules were exposed to secretory stimuli [e.g., vasoactive intestinal peptide, adenosine, forskolin, and permeable adenosine 3',5'-cyclic monophosphate (cAMP) analogues]. Binding was also promoted by osmotically induced changes in cell volume; a 45% reduction in cell water content mimicked the effect of secretagogues on binding, whereas a 40% increase in cell water was only half as effective. Volume-responsive binding required extracellular sodium and chloride. The effect of cell shrinkage on binding was rapid and reversible (half-activation time = approximately 3 min, half-deactivation time = approximately 2 min). The binding sites evoked by secretagogues and by cell shrinkage had similar affinities for [3H]benzmetanide (Kd approximately 0.35 microM). Forskolin, a potent secretagogue, increased cell cAMP content 10-fold and respiration 7-fold, whereas hypertonicity affected neither parameter. The effects of cAMP-dependent stimuli and hypertonicity on binding were not additive. These results suggest the following. 1) Na-K-Cl cotransporters acquire the ability to bind [3H]benzmetanide with high affinity when activated. 2) Hormonal modulation of rectal gland secretion involves a coordinated regulation of basolateral Na-K-Cl cotransporters and apical Cl channels. 3) Separate signal transduction pathways, one sensitive to cAMP and another to cell volume, regulate the Na-K-Cl cotransporter.
Lytle
C
C
Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672.
Forbush
B
B
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004284:Dogfish / Q000378:metabolism*; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D066298:In Vitro Techniques; D007477:Ions; D008565:Membrane Proteins / Q000378:metabolism; D009712:Nucleotides, Cyclic / Q000378:metabolism; D010101:Oxygen Consumption; D012491:Salt Gland / Q000166:cytology / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters; D013424:Sulfanilamides / Q000378:metabolism
D002352:Carrier Proteins; D007477:Ions; D008565:Membrane Proteins; D009712:Nucleotides, Cyclic; D028021:Sodium-Potassium-Chloride Symporters; D013424:Sulfanilamides; C039438:benzmetanide; D000242:Cyclic AMP