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

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 PD_b is the result of the falling cellular Cl⁻ activity.