Osmotic stimulation of Na(+)-K(+)-Cl- cotransport in squid giant axon is [Cl-]i dependent

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Abstract
The effects of increasing extracellular osmolality on unidirectional Cl- fluxes through the Na(+)-K(+)-Cl- cotransporter were studied in internally dialyzed squid giant axons. Hyperosmotic seawater stimulated bumetanide-sensitive Cl-influx at 150 mM intracellular Cl- concentration ([Cl-]i), whereas Cl- efflux was unaffected under comparable ionic conditions. Stimulation of bumetanide-sensitive Cl- influx was proportional to the increase in extracellular osmolality. Bumetanide-sensitive Cl- influx began to increase after a latency of approximately 20 min after a stepwise increase of extracellular osmolality and continued to increase for at least 70 min. The increased bumetanide-sensitive Cl- influx measured after 65 min of exposure to hyperosmotic external fluid was a function of the intracellular Cl- concentration; stimulation by hyperosmotic external fluids was observed at physiological [Cl-]i levels (greater than 100 mM) but not at lower [Cl-]i levels. Under both normo- and hyperosmotic conditions, intracellular Cl- inhibited Na(+)-K(+)-Cl- cotransport influx in a concentration-dependent manner. However, in hyperosmotic seawater, the dose dependence of inhibition by intracellular Cl- was shifted to higher [Cl-]i values. Therefore, we conclude that hyperosmotic extracellular fluids stimulate influx via the Na(+)-K(+)-Cl- cotransport by resetting the relation between [Cl-]i and transport activity.