Sulphur-containing excitatory amino acid-stimulated inositol phosphate formation in primary cultures of cerebellar granule cells is mediated predominantly by N-methyl-D-aspartate receptors.
The stimulatory effect of excitatory sulphur-containing amino acids on inositol phosphate formation was investigated in primary cultures of cerebellar granule cells. L-Cysteine sulphinate (CSA), L-cysteate (CA), L-homocysteine sulphinate (HCSA), L-homocysteate (HCA) and S-sulpho-L-cysteine (SSC) dose-dependently stimulated the formation of [3H]inositol phosphates exhibiting EC50 values in the range 60-200 microM and maximal effects of six- to 17-fold that of basal [3H]inositol phosphate levels. Endogenous L-glutamate spontaneously released into the extracellular medium or following exposure of cells to HCSA, HCA or SSC did not contribute significantly to formation of [3H]inositol phosphates, whereas 10% of the total [3H]inositol phosphates accumulated following exposure to CSA and CA was due to released L-glutamate. The selective N-methyl-D-aspartate receptor antagonist, D,L-2-amino-5-phosphonopentanoic acid (APV, 500 microM) attenuated by 20% (HCSA) to between 80 and 100% (CSA, CA, SSC, HCA) the formation of [3H]inositol phosphates induced by 1 mM sulphur-containing amino acids. When, however, HCSA was used at 100 microM (a concentration near to its EC50 for phosphoinositide hydrolysis), APV inhibited induced responses by 70%. Sulphur-containing amino acid-stimulated [3H]inositol phosphate formation was unaffected by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM). Inhibition of sulphur-containing amino acid-stimulated [3H]inositol phosphate formation by co-administration of APV and CNQX was similar to that obtained in the presence of APV alone. CSA-, CA-, SSC- and HCA-stimulated [3H]inositol phosphate formation was markedly reduced by removal of Ca2+ from the extracellular medium whereas that stimulated by HCSA was less affected. A similar inhibitory profile was observed when the levels of sulphur-containing amino acid-induced increases in intracellular free calcium ([Ca2+]i) were measured in the presence of 500 microM APV; 1 mM HCSA-induced responses being inhibited by only 30% whereas responses to the remaining sulphur-containing amino acid (also at 1 mM) were inhibited by > 45%. When the sulphur-containing amino acids were used at concentrations approximating their EC50 values for phosphoinositide hydrolysis, APV inhibited the induced increases in [Ca2+]i by 70-100%. HCA and SSC co-administered with the less efficacious but selective metabotropic glutamate receptor agonist, (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) at maximally effective concentrations (1 mM) of each agonist stimulated [3H]inositol phosphate formation in an additive manner.(ABSTRACT TRUNCATED AT 400 WORDS)
Gorman
A
A
Division of Cell and Molecular Biology, School of Biological and Medical Sciences, University of Saint Andrews, Fife, Scotland, U.K.
Griffiths
R
R
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D003515:Cycloleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D003544:Cysteic Acid / Q000494:pharmacology*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005971:Glutamates / Q000378:metabolism / Q000494:pharmacology; D006710:Homocysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018377:Neurotransmitter Agents; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D005971:Glutamates; D007295:Inositol Phosphates; D018377:Neurotransmitter Agents; D016194:Receptors, N-Methyl-D-Aspartate; D010566:Virulence Factors, Bordetella; D006710:Homocysteine; D003515:Cycloleucine; C007956:homocysteic acid; C036361:1-amino-1,3-dicarboxycyclopentane; C010982:homocysteinesulfinic acid; C011119:S-sulphocysteine; D003544:Cysteic Acid; D003545:Cysteine; D002118:Calcium; C013461:cysteine sulfinic acid