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 transepithel ial HCO3- absorption in the rat medullary thick ascending limb (MTAL). Regulation of the apical exchanger by intracellular pH (pH(i)) and hy perosmolality was studied in the isolated, perfused MTAL by measuremen t of pH(i) using the fluorescent probe 2',7'bis-(carboxyethyl)-5,6-car boxyfluorescein. Under isosmotic conditions (290 mosmol/kg H2O), the N a+/H+ exchange rate increased sigmoidally over the pH(i) range 7.8 to 6.5 (Hill coefficient = 2.1), consistent with cooperative activation o f the exchanger by internal H+. The exchanger had a high apparent affi nity for intracellular H+ (apparent pK = 7.36), which resulted in the exchanger being maximally active at resting pH(i) and insensitive to c hanges in pH(i) over the physiologic py. range (6.5-7.2). Hyperosmolal ity (590 mosmol/kg H2O) inhibited Na+/H+ exchange by at least 35% at a ll 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 excha nger. These functional properties of apical membrane Na+/H+ exchange c an account for our previous observations that hyperosmolality inhibite d net HCO3- absorption and that the rate of HCO3- absorption did not c orrelate with pH(i). Apical membrane Na+/H+ exchange in the MTAL diffe rs functionally from Na+/H+ exchange in other cell types in which exch anger activity is stimulated rather than inhibited by hyperosmolality.