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Thick ascending limb of Henle's loop; Rabbit; Furosemide; Loop diuretics; Na−Cl2−K cotransport; Isolated perfused tubule
Abstract:
Previously we have shown that chloride reabsorption in the isolated perfused cortical thick ascending limb of Henle's loop of rabbit (cTAL) is dependent on the presence of sodium and potassium. The present study was performed to elucidate the dependence on chloride in quantitative terms. Ninety-four cTAL segments were perfused at high rates with solutions of varying chloride concentration. Chloride was substituted by sulfate, methylsulfate or nitrate. The open circuit transepithelial electrical potential difference (PDte, mV) and the specific transepithelial resistance (Rt, Ωcm2) were measured, and from both the equivalent short circuit current (Isc) was calculated. The correlation of Isc versus Cl− concentration (in 294 observations) revealed a saturation kinetics depending on the (Cl−)2 concentration. The apparent constants were K1/2 50 mmol·l−1 and Isc,max 198 μA cm−2. These results are compatible with the assumption of 2 Cl− interacting with the luminal cotransport system. Although this finding in conjunction to our previous observations already is highly suggestive for a non-charged carrier this question was pursued further by recording the membranePD across both cell membranes during luminal application of furosemide. The data (n=26) indicate that furosemide (10−5 mol·l−1, lumen) produces an immediate decline inPD te to values close to zero, and a simultaneous hyperpolarization of both cell membranes by 15±2 mV for the basolateral and by 8±2 mV for the lumen membrane. These data exclude the possibility that the lumen positive transepithelialPD is generated by a net negative current flow from the lumen into the cell. It is concluded that the cotransport across the luminal membrane of the cTAL segment is electroneutral and involves 1 Na+∶2 Cl−∶1K+. The hyperpolarization of both cell membranes observed immediately after application of furosemide is caused most likely by a rapid fall in intracellular chloride activity.
