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

Anne Blanchard, Dominique Eladari, Françoise Leviel, Michel Tsimaratos, Michel Paillard and René-Alexandre Podevin

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 ²²Na⁺ uptake in both types of vesicles. This inhibition could not be accounted for by alteration of intravesicular pH (pH_i).

3. Conversely, in both plasma membrane preparations, the imposition of outward NH₄⁺ gradients stimulated ²²Na⁺ uptake at the acidic pH_i (6·60) of MTAL cells, under conditions in which possible alterations in pH_i were prevented. 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 dissociation constant) values of 6·58 and 6·52, respectively.

5. 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 pH_i of MTAL cells.

6. 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.

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