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 (NH4+/NH3). Here, we examined the effects of NH4+/NH3 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 NH4+/NH3 on glutamate transporter activity by measuring D-asparate-induced pH(i) and [Na+](i) transients. In 20-34 days in vitro (DIV) astrocytes, NH4+/NH3 decreased steady-state pH(i) by 0.19 pH units and increased [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, pH(i) and [Na+](i) were minimally affected by NH4+/NH3, and D-asparate-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 all increased expression of NKCC1 in 20-34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH4+/NH3-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 NH4+/NH3 to 30% in 20-34 DIV astrocytes. Our results Suggest that NH4+/NH3 decreases steady-state pH(i) and increases steady-state [Na+](i), ill astrocytes by all age-dependent activation of NKCC. These NH4+/NH3-evoked changes ill the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, Contribute to elevated extracellular glutamate levels observed during HE. (C) 2008 Wiley-Liss, Inc.