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.
Sulphur-containing excitatory amino acid transmitter candidates (500 microM) 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 CoCl2 (1 mM) in place of CaCl2. The stimulated release of D-[3H]aspartate was markedly attenuated by 200 microM 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 microM 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 constrast, agents which act as competitive inhibitors of the plasma membrane carrier in granule cells, e.g. beta-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.
Dunlop
J
J
Department of Biochemistry and Microbiology, University of St. Andrews, Fife, Scotland, U.K.
Grieve
A
A
Damgaard
I
I
Schousboe
A
A
Griffiths
R
R
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005971:Glutamates / Q000502:physiology; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D051379:Mice; D008815:Mice, Inbred Strains; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000494:pharmacology*; D011810:Quinoxalines / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D017470:Receptors, Glutamate / Q000187:drug effects / Q000502:physiology*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium
D002352:Carrier Proteins; D005971:Glutamates; D018377:Neurotransmitter Agents; D011810:Quinoxalines; D017470:Receptors, Glutamate; D006710:Homocysteine; D014316:Tritium; D001224:Aspartic Acid; C010982:homocysteinesulfinic acid; C056723:FG 9041; C011119:S-sulphocysteine; D003545:Cysteine; D002118:Calcium; C013461:cysteine sulfinic acid