NH4+ as a substrate for apical and basolateral Na+‐H+ exchangers of thick ascending limbs of rat kidney: evidence from isolated membranes

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Abstract
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+. Extravesicular NH4+ ((NH4+)o) inhibited outward H+ gradient-stimulated 22Na+ uptake in both types of vesicles. This inhibition could not be accounted for by alteration of intravesicular pH (pHi). Conversely, in both plasma membrane preparations, the imposition of outward NH4+ gradients stimulated 22Na+ uptake at the acidic pHi (6.60) of MTAL cells, under conditions in which possible alterations in pHi were prevented. All NH4+ gradient-stimulated Na+ uptake was sensitive to 0.5 mM 5-(N,N-dimethyl)-amiloride. The BLMV and LMV Na+-H+ exchangers had a similar apparent affinity for internal H+ (H+i), with pK (-log of dissociation constant) values of 6.58 and 6.52, respectively. These findings indicate that NH4+ interacts with the external and internal transport 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 pHi of MTAL cells. We conclude that operation of the BLMV Na+-H+ exchanger on the NH4+-Na+ mode may represent an important pathway for mediating the final step of NH4+ absorption, whereas transport of NH4+ on the apical antiporter may provide negative feedback regulation of NH4+ absorption.