CONTROL OF H+-HCO3- PLASMA-MEMBRANE TRANSPORTERS BY UREA HYPEROSMOLALITY IN RAT MEDULLARY THICK ASCENDING LIMB

Hyperosmolality inhibits bicarbonate absorption by the rat medullary thick ascending limb (MTAL) by unknown mechanisms. Intracellular pH (pH(i)) was monitored with use of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in rat MTAL tubule suspensions to specify the H+-HCO3- membrane transporters affected by hyperosmolality. Measurements were made after greater than or equal to 15-min incubation of the cells in media rendered hypertonic by urea to avoid any change in cell volume. Na+-H+ antiport activity, estimated from the Na+-induced initial rate of pH(i) recovery of Na+-depleted acidified cells in the presence of 0.1 mM furosemide to inhibit Na+-K+-2Cl(-) cotransport, was inhibited by 300 mM urea and 10(-8) M arginine vasopressin (AVP) in an additive manner. Na+-H+ antiport inhibition by urea hyperosmolality was maximal at 300 mM urea with a half-maximal inhibitory concentration of 75 mM and was due to a 28% decrease in maximum velocity (V-max) with no effect on the Michaelis constant for sodium. Urea hyperosmolality (300 mM) did not affect steady-state intracellular calcium concentration ([Ca2+](i)), assessed with use of fura 2 fluorescence, and still inhibited Na+-H+ antiport in MTAL cells loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to minimize any transient change in [Ca2+](i) during the preincubation in urea medium. Furthermore, 300 mM urea did not stimulate basal or AVP-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Plasma membrane H+-adenosinetriphosphatase (ATPase) activity and HCO, transport, assessed by appropriate experimental protocols, were unaltered by 300 mM urea. We conclude that urea hyperosmolality directly inhibits Na+-H+ antiport, but not H+-ATPase and K+-HCO3-, cotransport, of rat MTAL cells by affecting the V-max of the antiporter independently of change in cell volume, cytosolic calcium, or cAMP.