Hypertonicity in Fused Madin-Darby Canine Kidney Cells: Transient Rise in NaHCO3 Followed by Sustained KCl Accumulation

We investigated mechanisms of regulatory volume increase in fused Madin-Darby canine kidney (MDCK) cells, a cell line originally derived from renal collecting duct. The intracellular ion concentrations as well as the concentration of the volume marker tetramethylammonium+ were measured by means of ion-selective microelectrodes. Application of hypertonic Ringer bicarbonate solution (+150 mmol/l mannitol) resulted in cell shrinkage to 84 +/- 2% of the initial cell volume (shrinkage expected for an ideal osmometer = 66%), indicating a significant regulatory volume increase. During the first 90 s of the hypertonic stress, a transient increase in intracellular Na+ and HCO3- concentrations was observed. It was followed by a sustained increase in intracellular K+ and Cl- concentrations. Ouabain (0.1 mmol/l) as well as amiloride (1 mmol/l) reduced K+ accumulation significantly, whereas the H+/K(+)-ATPase inhibitor SCH 28080 had no effect. Hypertonic stress hyperpolarized the cell membrane potential by 19 +/- 2 mV, owing to the decrease of the ratio of Cl- conductance to K+ conductance of the cell membrane. We conclude: (a) acute hypertonic stress activates Na+/H+ exchange in MDCK cells; (b) transient alteration of intracellular Na+ and pH stimulates Na+/K(+)-ATPase and Cl-/HCO3- exchange, exchange, both leading to the sustained intracellular accumulation of KCl; (c) a high intracellular KCl concentration is maintained by the partial reversion of the Cl-/K+ conductance ratio of the plasma membrane.