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

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 (NH4+/NH3). Here, we examined the effects of NH4+/NH3 on steady‐state intracellular pH (pHi) and sodium concentration ([Na+]i) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH4+/NH3 on glutamate transporter activity by measuring D‐aspartate‐induced pHi and [Na+]i transients. In 20–34 days in vitro (DIV) astrocytes, NH4+/NH3 decreased steady‐state pHi by 0.19 pH units and increased [Na+]i by 21 mM. D‐Aspartate‐induced pHi and [Na+]i transients were reduced by 80–90% in the presence of NH4+/NH3, indicating a dramatic reduction of glutamate uptake activity. In 9–16 DIV astrocytes, in contrast, pHi and [Na+]i were minimally affected by NH4+/NH3, and D‐aspartate‐induced pHi 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 NH4+/NH3‐evoked changes in steady‐state pHi and [Na+]i and attenuated the reduction of D‐aspartate‐induced pHi and [Na+]i transients by NH4+/NH3 to 30% in 20–34 DIV astrocytes. Our results suggest that NH4+/NH3 decreases steady‐state pHi and increases steady‐state [Na+]i in astrocytes by an age‐dependent activation of NKCC. These NH4+/NH3‐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. © 2008 Wiley‐Liss, Inc.