Hyperosmolality inhibits bicarbonate absorption by the rat medullary thick ascending limb (MTAL) by unknown mechanisms. Intracellular pH (pHi) 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 > or = 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 pHi 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 (Vmax) 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 HCO3- transport, assessed by appropriate experimental protocols, were unaltered by 300 mM urea.(ABSTRACT TRUNCATED AT 250 WORDS)