Mechanisms of H+/HCO3- transport in the medullary thick ascending limb of rat kidney.

The rat MTAL secretes protons into the tubular fluid and thus absorbs bicarbonate at substantial rates. Yet the cellular mechanisms of H+/HCO3- transport in the rat MTAL remain largely unsettled. We have performed intracellular pH recovery studies with use of the fluorescent probe BCECF in suspensions of rat MTAL fragments. Luminal H+ secretion occurs by two mechanisms (each responsible for 50% of the normal pHi recovery rate): (1) an electroneutral Na+/H+ antiporter that has an Na-Km of about 11 mM and is inhibited by amiloride (Ki = 2.8 x 10(-5) M); (2) a primary H+ pump that is inhibited by 10(-4) M NEM and 10(-4) M omeprazole, but not by 10(-4) M vanadate or removal of external K. These results suggest the presence of a vacuolar H(+)-ATPase rather than a H(+)-K(+)-ATPase. Basolateral HCO3 exit occurs predominantly by a Cl(-)- and Na(+)-independent electroneutral K+/HCO3- symporter, that has an HCO3-Km of about 17 mM, and is partially inhibited by 10(-4) M DIDS. Basolateral HCO3- efflux was not accompanied by variations of membrane potential monitored with the Em-sensitive fluorescent probe DIS-C3-5, and was not affected by maneuvers that depolarize the cells. It was strongly inhibited by cellular K depletion and dependent on transmembrane K gradient. We conclude that the rat MTAL should secrete protons through both Na+/H+ antiporter and H(+)-ATPase, and that basolateral HCO3- exit should occur through an electroneutral K+/HCO3- symporter.