Apical membrane Na⁺/H⁺ exchange in rat medullary thick ascending limb: pH_i-dependence and inhibition by hyperosmolality

Apical membrane Na⁺/H⁺ exchange mediates virtually all of transepithelial HCO^-₃ absorption in the rat medullary thick ascending limb (MTAL). Regulation of the apical exchanger by intracellular pH (pH_i) and hyperosmolality was studied in the isolated, perfused MTAL by measurement of pH_i using the fluorescent probe 2′,7′-bis-(carboxyethyl)-5,6-carboxyfluorescein. Under isosmotic conditions (290 mosmol/kg H₂O), the Na⁺/H⁺ exchange rate increased sigmoidally over the pH_i range 7.8 to 6.5 (Hill coefficient = 2.1), consistent with cooperative activation of the exchanger by internal H⁺. The exchanger had a high apparent affinity for intracellular H⁺ (apparent pK = 7.36), which resulted in the exchanger being maximally active at resting pH_i and insensitive to changes in pH_i over the physiologic pH_i range (6.5-7.2). Hyperosmolality (590 mosmol/kg H₂O) inhibited Na⁺/H⁺ exchange by at least 35% at all pH_i values studied and induced pH_i dependence of the exchanger between 6.5 and 7.2. The inhibition by hyperosmolality appeared to be the result of an acid shift of the pH_i dependence curve of the exchanger. These functional properties of apical membrane Na⁺/H⁺ exchange can account for our previous observations that hyperosmolality inhibited net HCO^-₃ absorption and that the rate of HCO^-₃ absorption did not correlate with pH_i. Apical membrane Na⁺/H⁺ exchange in the MTAL differs functionally from Na⁺/H⁺ exchange in other cell types in which exchanger activity is stimulated rather than inhibited by hyperosmolality.