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

Hyperosmolality inhibits bicarbonate absorption by the rat medullary t hick ascending limb (MTAL) by unknown mechanisms. Intracellular pH (pH (i)) was monitored with use of 2',7'-bis(carboxyethyl)-5(6)-carboxyflu orescein in rat MTAL tubule suspensions to specify the H+-HCO3- membra ne transporters affected by hyperosmolality. Measurements were made af ter greater than or equal to 15-min incubation of the cells in media r endered hypertonic by urea to avoid any change in cell volume. Na+-Hantiport activity, estimated from the Na+-induced initial rate of pH(i ) recovery of Na+-depleted acidified cells in the presence of 0.1 mM f urosemide to inhibit Na+-K+-2Cl(-) cotransport, was inhibited by 300 m M 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 th e 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+ a ntiport in MTAL cells loaded with ,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid to minimize any transient change in [Ca2+](i) duri ng the preincubation in urea medium. Furthermore, 300 mM urea did not stimulate basal or AVP-induced adenosine 3',5'-cyclic monophosphate (c AMP) accumulation. Plasma membrane H+-adenosinetriphosphatase (ATPase) activity and HCO, transport, assessed by appropriate experimental pro tocols, were unaltered by 300 mM urea. We conclude that urea hyperosmo lality directly inhibits Na+-H+ antiport, but not H+-ATPase and K+-HCO 3-, cotransport, of rat MTAL cells by affecting the V-max of the antip orter independently of change in cell volume, cytosolic calcium, or cA MP.