Amino acid uptake, content, and metabolism by neuronal and glial enriched cellular fractions from mouse cerebellum

Abstract

A series of biochemical determinations was performed on five cellular fractions obtained from the cerebellum of 8- to 14-day-old mice. Cerebellar tissue was dissociated by mild trypsinization and mechanical agitation. The dissociated cellular material was separated into five fractions using a series of continuous density gradients formed with Percoll. Three of the fractions were comprised primarily of cell bodies. One of these was dominated by cells having the size and morphological appearance of granule cells, and based on several criteria the other two were were enriched in astrocyte cell bodies. Morphological analysis indicated that the remaining two fractions were enriched, respectively, in nerve terminals and large nucleated cell bodies. The uptake of 12 amino acids and 4 other metabolites by these cellular fractions was examined, and Km and Vmax values were determined for 10 of the compounds studied. High affinity transport carriers (Km approximately 1 to 20 microM) for most of the compounds studied were evident in neuronal and astrocyte-enriched fractions; however, for glutamate and gamma-aminobutyric acid (GABA) additional carriers with higher substrate affinities (Km approximately 0.1 to 0.3 microM) were evident in the astrocyte-enriched fraction. The fraction enriched in granule cell bodies was distinguished by an exceptionally low uptake of GABA and citrate, and a comparatively low uptake of beta-alanine, taurine, alpha-ketoglutarate, and glutamate. An analysis of the content of nine amino acids in the five fractions revealed that only glutamate, aspartate, and GABA were concentrated in the fraction enriched in nerve terminals. GABA was concentrated also in the fraction enriched in large cell bodies and was present at a low concentration in the fraction enriched in granule cell bodies. The other amino acids measured were distributed nearly evenly among the five fractions. Several differences in metabolic activity among the five fractions were observed. Radiolabel from several precursors was incorporated into GABA preferentially in the fractions enriched in large cell bodies and nerve terminals. In contrast, the accumulation of label in glutamine occurred preferentially in the fractions enriched in astrocytes and granule cell bodies. Labeling of alanine from [14C]pyruvate and of proline from [14C]ornithine was most prominent in the fractions enriched in astrocytes and granule cell bodies.