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

Abstract

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 [ u3H]inositol phosphates exhibiting EC 50 values in the range 60–200 μM 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 μM) 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 μM (a concentration near to its EC 50 for phosphoinositide hydrolysis), APV inhibited induced responses by 70%. Sulphur-containing amino acid-stimulated [ 3H]inositol phosphate formation was unaffected by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM). 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 Ca 2+ 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 ([Ca 2+ 1) were measured in the presence of 500 μM 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 [Ca 2+] i by 70–100%. HCA and SSC co-administered with the less efficacious but selective metabotropic glutamate receptor agonist, ( ± )-1-aminocyclopentane- trans-1,3-dicar☐ylic acid ( trans-ACPD) at maximally effective concentrations (1 mM) of each agonist stimulated [ 3H]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 [ 3H]inositol phosphate formation compared to that observed in the presence of either sulphur-containing amino acid alone. In cells pre-treated with 1 μg/ml pertussis toxin, HCA-stimulated [ 3H]inositol phosphate formation was not significantly affected, however CSA-, CA- and HCSA-stimulated [ 3H]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 [ 3H]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. The data are consistent with the sulphur-containing amino acids possibly having a physiological role as endogenous activators of both ionotropic and metabotropic excitatory amino acid receptors. The present findings may, however, be of more significance in suggesting possible mechanisms by which the sulphur-containing amino acids or disturbances in sulphur-containing amino acid metabolism are linked to the aetiology of a number of neuropathologies.