The Na⁺2Cl^–K⁺ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage

Abstract 

Effects of cAMP on Cl^– secretion, intracellular Cl^– activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (I _sc) showed that cAMP stimulates Na⁺Cl^– secretion in a biphasic manner. The first and rapid phase corresponds to Cl^– exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl^– conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1–2 min) activation of the Na⁺2Cl^–K⁺ cotransporter. During the first phase cytosolic Cl^– activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of I _sc activation. When cytosolic Cl^– activity was increased by exposing the RGT to a high K⁺ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na⁺2Cl^–K⁺ cotransport is transient cell shrinkage.