Investigation of the Ba2+-sensitive NH4+ transport pathways in the apical cell membrane of primary cultured rabbit MTAL cells.

BACKGROUND

Several apical ammonium (NH(4)(+)/NH(3)) 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 NH(4)Cl, which acidified the cells to pH(i) 6.89. When blockers of apical NH(4)(+) transport were used, the cells alkalinized due to a decreased NH(4)(+) loading. The following values (pH units) were observed: bumetanide, +0.05; verapamil, +0.04; Ba(2+) 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 NH(4)(+) might enter through nonspecific channels, ammonium pulse experiments were performed: an NH(4)Cl pulse acidified controls as well as depolarized cells. In contrast, when Ba(2+), Cs(+) or tertiapin were present, an NH(4)Cl pulse alkalinized the cells. The pharmacological profile of this apical NH(4)(+) 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 Ba(2+)-sensitive component of NH(4)(+) transport is predominant and consists of permeation of NH(4)(+) through an apical ROMK-related channel.