Pathways of NH3/NH 4 + permeation across Xenopus laevis oocyte cell membrane

Abstract

While acid loading with extracellular NH4Cl solutions usually first alkalinizes the cells through NH3 influx, and acidifies only when NH4Cl is removed, Xenopus oocytes became immediately acidic upon NH4Cl addition and the cells did not acidify further upon its removal. Since NH4Cl solutions also collapsed the membrane potential (Vm) and resistance (Rm), we conclude that primarily NH4+ entered the cells where it liberated H+, with NH3 being trapped in intracellular lipid stores. To identify the NH4+ permeation pathway we have used K+ channel blockers (Ba2+, Cs+, tetraethylammonium, quinidine), various cation transport inhibitors (ouabain, bumetanide, amiloride) and other inhibitors, some of which block non-selective cation channels (La3+, diphenylamine-2-carboxylate, and p-chloromercuribenzoate). However, only the latter substances partially prevented the collapse of Vm and Rm. This suggests, that NH4+ passes through non-selective cation channels. In accordance with the voltage dependence and/or stretch activation of such channels NH4+ fluxes appeared to be asymmetric. NH4+ influx, which depolarized and swelled the cells, was large and acidified rapidly, while the efflux, which repolarized and shrank the cells, was slow and alkalinized only slowly.

Aquaporin 4 as a NH3 Channel

“…The water permeability of the native oocyte membrane was unaffected by the choice of osmolyte and pH o , indicating that the observed changes in water permeability originated in the expressed AQPs and suggests NH 3 permeation through both AQP4 and AQP8. Exposure of uninjected oocytes to NH 4 Cl persistently caused an intracellular acidification, as previously observed (22,30,(51)(52)(53)(54), which is assigned to NH 4 ϩ entry through still unidentified pathways, presumably cation-selective ion channels (53,55). Although a small fraction of the AQP4-microinjected oocytes displayed a similar acidification, the majority of the tested AQP4-expressing oocytes displayed either a lesser acidification or a robust alkalization upon exposure to ammonia with no obvious correlation to initial pH i or days in culture.…”

Ammonia excretion in aquatic and terrestrial crabs

“…An excretion mechanism based predominately on NH 3 diffusion is not likely, however, because membrane permeability of NH 3 is much lower than that of CO 2 (Knepper et al, 1989). Indeed, some plasma membranes of animal epithelia are relatively impermeable to NH 3 as shown for frog oocytes (Burckhardt and Frömter, 1992), the renal proximal straight tubules (Garvin et al, 1987) and colonic crypt cells (Singh et al, 1995). Accordingly, other authors have obtained experimental evidence for at least partial excretion of ammonia in its ionic form (NH 4 + ) in Callinectes sapidus (Pressley et al, 1981) and Carcinus maenas (Lucu, 1989;Siebers et al, 1995).…”

Intracellular pH regulation in unstimulatedCalliphorasalivary glands is Na+ dependent and requires V-ATPase activity

“…This suggests that NH 4 + enters the cell. Such an acidifying effect of NH 4 Cl has also been observed in the retinal glial cells of A. mellifera (Marcaggi et al, 1999;Marcaggi and Coles, 2000), the Malphighian tubules of Drosophila hydei (Bertram and Wessing, 1994), the epithelial cells of the cockroach salivary glands (Hille and Walz, 2007), the colon epithelium in rats (Ramirez et al, 1999) and Xenopus oocytes (Burckhardt and Frömter, 1992 discussed as possible mechanisms (Betram and Wessing, 1994;Burckhardt and Frömter, 1992;Hille and Walz, 2007;Marcaggi et al, 1999;Marcaggi and Coles, 2000;Ramirez et al, 1999). In C. vicina salivary glands, the NH 4 Cl pulse under Na + -free conditions caused a transient alkalinization in some experiments (Fig.2A), which was not seen in the presence of EIPA (Fig.3A).…”