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 (NHCl)-Cl-4 pulse acidified controls as well as depolarized cells. In contrast, when Ba2+, Cs+ or tertiapin were present, an (NHCl)-Cl-4 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. Copyright (c) 2007 S. Karger AG, Basel.