SULFUR-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 excitatoryl 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 [H-3]inositol phosphates exhibiting EC(50) values in the range 60-200 mu M and maximal effects of six- to 17-fold that of basal [H-3]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 [H-3]inositol phosphates, whereas 10% of the total [H-3]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 mu M) 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 mu M (a concentration near to its EC(50) for phosphoinositide hydrolysis), APV inhibited induced responses by 70%. Sulphur-containing amino acid-stimulated [H-3]inositol phosphate formation was unaffected by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropronic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 mu M). Inhibition of sulphur-containing amino acid-stimulated [H-3]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 [H-3]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 mu M 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 EC(50) 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 [H-3]inositol phosphate formation in an additive manner. In the case of CSA, CA and HCSA, however, co-administration with trans-ACPD caused no increase in level of [H-3]inositol phosphate formation compared to that observed in the presence of either sulphur-containing amino acid alone. In cells pre-treated with 1 mu g/ml pertussis toxin, HCA-stimulated [H-3]inositol phosphate formation was not significantly affected, however CSA-, CA- and HCSA-stimulated [H-3]inositol phosphate formation was significantly but only partially (by 40%) inhibited. Taken together, these results indicate that in cerebellar granule cells sulphur-containing amino acid-stimulated [H-3]inositol phosphate formation is mediated predominantly by N-methyl-D-aspartate receptor activation. There is evidence however to support an additional role for CSA, CA and particularly HCSA as agonists of the metabotropic glutamate receptor.