Investigation of the Ba2+-Sensitive NH4+ Transport Pathways in the Apical Cell Membrane of Primary Cultured Rabbit MTAL Cells

Abstract

Background: Several apical ammonium (NH₄⁺/NH₃) transport pathways have been described in medullary thick ascending limb (MTAL) cells. The exact nature and importance of some of these pathways remain controversial. Methods: Ammonium transport in primary cultured rabbit MTAL cells was investigated by measuring intracellular pH (pH_i). Results: To create physiological conditions, experiments were performed in the symmetrical presence of NH₄Cl, which acidified the cells to pH_i 6.89. When blockers of apical NH₄⁺ transport were used, the cells alkalinized due to a decreased NH₄⁺ loading. The following values (pH units) were observed: bumetanide, +0.05; verapamil, +0.04; Ba²⁺ and Cs⁺, +0.19; tertiapin, +0.09. Tetraethylammonium had no effect. Depolarizing the cells by increasing the K⁺ concentration alkalinized the cells by 0.16 pH units. Because NH₄⁺ might enter through nonspecific channels, ammonium pulse experiments were performed: an NH₄Cl pulse acidified controls as well as depolarized cells. In contrast, when Ba²⁺, Cs⁺ or tertiapin were present, an NH₄Cl pulse alkalinized the cells. The pharmacological profile of this apical NH₄⁺ transport pathway correlates with the renal outer medullary K⁺ (ROMK) channel. Indirect immunofluorescence showed the presence of the ROMK protein. Conclusion: In these MTAL cells the Ba²⁺-sensitive component of NH₄⁺ transport is predominant and consists of permeation of NH₄⁺ through an apical ROMK-related channel.