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. © 2008 Wiley‐Liss, Inc.