Chloride activity in cells of isolated perfused cortical thick ascending limbs of rabbit kidney

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Abstract
Rabbit cortical thick ascending limb segments were perfused in vitro, and intracellular Cl− activity was estimated in three types of experiments using conventional and chloride selective microelectrodes. In series 1 Ringer like solutions were present on the two epithelial sides. In series 2 lumen Cl− was replaced by gluconate, and in series 3 furosemide, 10–20 · 10−6 mol · 1−1, was added to the lumen perfusate. It was found that under control conditions intracellular Cl− activity, as estimated from the difference of the reading of the conventional (n=53) and ion selective electrodes (n=118) was 26±1 mmol · 1−1. This value is approximately three times higher than expected for passive distribution of Cl−1. After removal of lumen Cl− (series 2) intracellular Cl− activity fell to 9 mmol · 1−1 which is only some 4 mmol · 1−1 above passive distribution. We argue that these 4 mmol · 1−1 reflect mainly the interference with the Cl− electrode by other anions, such as phosphate. The above estimates for intracellular Cl− activity, have to be diminished by these 4 mmol·1−1, and, thus, are close to 22 mmol·1−1. In series 3 a rapid and reversible fall in intracellular Cl− from 23 to 7 mmol·1−1 was observed. We conclude that the Cl− activity in cTAL cells is clearly above equilibrium under control conditions and that it falls rapidly to values close to equilibrium when Cl− reabsorption is blocked by either removing lumen Cl− or by blocking the Cl− entry via the Na+−2Cl−−K+-carrier with furosemide. The present data directly support our concept that the furosemide induced hyperpolarization of PDb is the result of the falling cellular Cl− activity.