GLUTAMATE UP-REGULATES ALPHA-1 AND ALPHA-2 SUBUNITS OF THE SODIUM-PUMP IN ASTROCYTES OF MIXED TELENCEPHALIC CULTURES BUT NOT IN PURE ASTROCYTE CULTURES

Prior work employing an in vitro model of the cerebral cortex has shown that sodium pump activity is a critical determinant for neuronal survival of glutamate stimulation. We have hypothesized that up-regulation of total brain sodium pump activity will protect against potential excitotoxins. Increased sodium pump activity could theoretically occur by changes in the reaction rate (short-term) and/or by increased levels of sodium pump protein (long-term) and is potentially complex since the three catalytic (a) subunit isoforms of the sodium pump are distributed in a highly variable, cell-specific pattern in the brain. Short-term regulation (seconds to minutes) has been well studied: brain sodium pump exhibits a large dynamic range. In contrast, the possibility of long-term modulation of sodium pump activity has not been extensively explored. We used isoform specific antibodies and [H-3]ouabain binding to determine whether prolonged stimulation of sodium pump activity in rodent telencephalic cultures increased total sodium pump enzyme. Exposure of mixed neuronal-glial cultures to high levels of glutamate (10 mM) for 18 h, which is highly toxic to neurons, was associated with an approximate to 80% increase in alpha 1 and alpha 2 subunit expression by glia. Induction of alpha 2 subunit immunoreactivity was also associated with comparable changes in [H-3]ouabain binding, suggesting that the up-regulation corresponded to functional alpha 2 protein. Shorter (30 min) glutamate treatments, which also killed neurons, did not produce similar changes in sodium pump expression. In contrast to mixed cultures, pure astrocyte cultures had undetectable alpha 2 and alpha 3 and moderate levels of alpha 1 protein, as confirmed by low levels of [H-3]ouabain binding. Glutamate treatment using this protocol was associated with a decrease in alpha 1 sodium pump expression. We conclude that long-term regulation of the sodium pump can be demonstrated in glia which have developed in the presence of neurons. Both alpha 1 and alpha 2 isoforms of the sodium pump are involved in this response to glutamate.