Sulphur-containing excitatory amino acid-evoked Ca 2+-independent release of d-[ 3H]aspartate from cultured cerebellar granule cells: The role of glutamate receptor activation coupled to reversal of the acidic amino acid plasma membrane carrier

Abstract

Sulphur-containing excitatory amino acid transmitter candidates (500 μM) stimulated the Ca 2+independent efflux of exogenously-supplied d-[ 3H]aspartate from primary cultures of cerebellar granule cells superfused continuously with HEPES-buffered saline containing CoCl 2 (1 mM) in place of CaC 2. The stimulated release of d-[ 3H]aspartate was markedly attenuated by 200 μM 6,7-dinitroquinoxalinedione, a concentration at which the antagonist inhibits both non- N-methyl- d-aspartate and N-methyl- d-aspartate ionotropic excitatory amino acid receptors. The Ca 2+-independent component of evoked release was also markedly attenuated and, in some cases, abolished by removing NaCl from the superfusion medium. Furthermore, when 700 μM dihydrokainate (demonstrated herein as a mixed/non-competitive inhibitor of the high-affinity dicarboxylic amino acid transporter in cultured granule cells) was included in the superfusion medium, stimulated efflux of d-[ 3H]aspartate was reduced by between 15–78% of the control response; the extent of inhibition varying with the agonist employed. In contrast, agents which act as competitive inhibitors of the plasma membrane carrier in granule cells, e.g. β-methylene- d, l-aspartate. potentiated the release of d-[ 3H]aspartate in a synergistic manner. Taken together, these findings are consistent with a mechanism for the Ca 2+-independent release of d-[ 3H]aspartate that is mediated predominantly by activation of excitatory amino acid receptors resulting in a reversal of the high-affinity dicarboxylic amino acid transport system. Although the physiological relevance of such non-vesicular release from the cytosol remains obscure and is still a matter of some debate, this mode of release may be of pathological significance.