Ammonium‐evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity

Abstract

The clearance of extracellular glutamate is mainly mediated by pH- and sodium-dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH(4) (+)/NH(3)). Here, we examined the effects of NH(4) (+)/NH(3) on steady-state intracellular pH (pH(i)) and sodium concentration ([Na(+)](i)) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH(4) (+)/NH(3) on glutamate transporter activity by measuring D-aspartate-induced pH(i) and [Na(+)](i) transients. In 20-34 days in vitro (DIV) astrocytes, NH(4) (+)/NH(3) decreased steady-state pH(i) by 0.19 pH units and increased [Na(+)](i) by 21 mM. D-Aspartate-induced pH(i) and [Na(+)](i) transients were reduced by 80-90% in the presence of NH(4) (+)/NH(3), indicating a dramatic reduction of glutamate uptake activity. In 9-16 DIV astrocytes, in contrast, pH(i) and [Na(+)](i) were minimally affected by NH(4) (+)/NH(3), and D-aspartate-induced pH(i) and [Na(+)](i) transients were reduced by only 30-40%. Next we determined the contribution of Na(+), K(+), Cl(-)-cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20-34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH(4) (+)/NH(3)-evoked changes in steady-state pH(i) and [Na(+)](i) and attenuated the reduction of D-aspartate-induced pH(i) and [Na(+)](i) transients by NH(4) (+)/NH(3) to 30% in 20-34 DIV astrocytes. Our results suggest that NH(4) (+)/NH(3) decreases steady-state pH(i) and increases steady-state [Na(+)](i) in astrocytes by an age-dependent activation of NKCC. These NH(4) (+)/NH(3)-evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE.

Intracellular ion homeostasis

“…The NH 4 + /NH 3 -evoked increases in intracellular sodium were reversible, with sodium concentrations returning to baseline following removal of NH 4 + /NH 3 . Inhibiting HCO 3 --dependent pH regulatory mechanisms using HCO 3 --free media increased the NH 4 + /NH 3 -evoked acidifications and attenuated the sodium increases, suggesting that NH 4 + /NH 3 activates sodium-dependent intracellular pH regulatory mechanisms [5]. However, the NH 4 + /NH 3 -evoked sodium increases were not simply a consequence of intracellular pH regulatory mechanisms.…”

“…Fig. 3; [5]). Due to the ability of ammonium to change intracellular pH, the 'ammonium pre-pulse' technique is commonly used to investigate mechanisms of intracellular pH regulation and buffering.…”

“…Fig. 3a; [5]). Upon removal of NH 4 + /NH 3 , transient acidifications were observed before pH returned to its original baseline value of around pH 7.25.…”

“…Investigations recently carried out in our laboratory focussed initially on the question of whether, in addition to intracellular pH, NH 4 + /NH 3 also influences intracellular sodium concentrations [5]. To this end, primary cultured rat hippocampal astrocytes were washed in saline solution containing 5 mM NH 4 + /NH 3 in exchange for sodium ions.…”

“…These ionic fluxes are achieved not only by the opening of ionotropic transmitter receptors and ion channels, but also largely mediated by the activity of various ion transporters. The activation of the sodiumdependent glutamate transporter EAAT1 (glutamate transporter excitatory amino acid carrier 1) and 2 results in significant changes in intracellular sodium concentrations and pH in astrocytes (see [5]; see box. 1).…”