NH4-H+ EXCHANGERS OF THICK ASCENDING LIMBS OF RAT-KIDNEY - EVIDENCE FROM ISOLATED MEMBRANES( AS A SUBSTRATE FOR APICAL AND BASOLATERAL NA+)

1. We have used highly purified right-side-out luminal and basolateral membrane vesicles (LMVs and BLMVs) isolated from rat medullary thick ascending limb (MTAL) to study directly the possible roles of the LMV and BLMV Na+-H+ exchangers in the transport of NH4+. 2. Extravesicular NH4+ ((NH4+)(o)) inhibited outward H+ gradient-stimulated Na-22(+) up take in both types of vesicles. This inhibition could not be accounted for by alteration of intravesicular pH (pH(i)). 3. Conversely, in bot h plasma membrane preparations, the imposition of outward NH4+ gradien ts stimulated Na-22(+) uptake at the acidic pH(i) (6.60) of MTAL cells , under conditions in which possible alterations in pH(i) were prevent ed. All NH4+ gradient-stimulated Na+ uptake was sensitive to 0.5 mM 5- (N,N-dimethyl)-amiloride. 4. The BLMV and LMV Na+-H+ exchangers had a similar apparent affinity for internal H+ (H-i(+)), with pK(-log of di ssociation constant) values of 6.58 and 6.52, respectively. 5. These f indings indicate that NH4+ interacts with the external and internal tr ansport sites of the LMV and BLMV Na+-H+ antiporters, and that both of these exchangers can mediate the exchange of internal NH4+ ((NH4+)(i) ) for external Na+ (Na-o(+)) at the prevailing pH(i) of MTAL cells. 6. We conclude that operation of the BLMV Na+-H+ exchanger on the NH4+-N a+ mode may represent an important pathway for mediating the final ste p of NH4+ absorption, whereas transport of NH4+ on the apical antiport er may provide negative feedback regulation of NH4+ absorption.