Characterization of L-glutamate uptake into and release from astrocytes and neurons cultured from different brain regions

Abstract

The uptake of L-glutamate was studied in astrocytes cultured from different brain areas of newborn rats as well as in two different cultures of neurons obtained from mouse brain. Both astrocytes and neurons exhibited high-affinity glutamate uptake with K_m values ranging from 34 [μM to 82 μM. V_max values for astrocytes cultured from the different brain regions were: prefrontal cortex: 13.9; occipital cortex: 11.4; neostriatum: 27.3 and cerebellum: 5.8 nmol · min⁻¹ · mg⁻¹ cell protein. For cerebellar granule cells and cerebral cortical neurons the V_max values were found to be 10.2 and 5.9 nmol · min⁻¹ · mg⁻¹ cell protein, respectively. The effect on L-glutamate uptake in astrocytes cultured from prefrontal cortex and in cultured cerebellar granule cells of a series of compounds structurally related to glutamate was studied, and detailed kinetic analyses of the inhibitory patterns of three potent inhibitors were performed. L-aspartate and L-aspartate-β-hydroxamate were found to be competitive inhibitors of L-glutamate uptake in both cell types with K_i values for astrocytes of 60 μM and 91 [μM, respectively, and for granule cells of 48 μM and 72 μM, respectively. D-aspartate was found to be a mixed-type noncompetitive inhibitor of L-glutamate uptake in astrocytes (K;: 106 μM), but in granule cells this compound showed simple competitive inhibition with a K_i of 49 μM. Sodium dependency of L-glutamate uptake in both cell types was studied at a series of Lglutamate and Na⁺ concentrations. It was found that the uptake of glutamate in astrocytes is coupled with one Na⁺ ion in contrast to two Na⁺ ions in granule cells. The K_m value for sodium was found to be 15 mM in both cell types. It was shown that release of exogenously supplied [³H] -L-glutamate from cerebel lar granule cells could be stimulated in a Ca²⁺-dependent manner by high concentrations (55 mM) of K⁺. In contrast to this no K⁺-induced release of glutamate could be demonstrated in cultured astrocytes.