Characterization of the receptor-mediated sulphur amino acid-evoked release of [(3)H]d-aspartate from primary cultures of cerebellar granule cells.

A continuous superfusion system was used to study the release of [(3)H]d-aspartate from primary cultures of mouse cerebellar granule cells. A number of neuroactive acidic sulphur amino acids (viz. cysteine sulphinate, cysteate, homocysteine sulphinate, homocysteate and S-sulpho-cysteine) of endogenous origin were shown to evoke a dose-dependent, saturable and stereospecific release of [(3)H]d-aspartate. Substitution of Ca(2+) in the superfusion medium by Co(2+) (which blocks voltage-gated Ca(2+)-channels) resulted in a marked attenuation of evoked release following stimulation by l-enantiomers of all sulphur amino acids tested, thus demonstrating a "Ca(2+)-dependent" release of [(3)H]d-aspartate. Cysteine sulphinate-evoked release of endogenous glutamate was shown to be Ca(2+)-dependent whereas negligible release of endogenous aspartate was observed. All sulphur amino acids inhibited to varying degrees, the intracellular accumulation of the [(3)H]tetraphenylphosphonium cation, an observation consistent with either partial or complete depolarization of the plasma membrane. The sulphur amino acid-evoked release of [(3)H]d-aspartate from the granule cells was inhibited by a variety of excitatory amino acid receptor antagonists. At a concentration range of antagonist between 0.1-500 ?M, the sulphur amino acid-evoked release of [(3)H]d-aspartate was inhibited by: (i) the selective, competitive N- methyl- d -aspartate receptor antagonists; 3-(+/-)-2-carboxypiperazin-4-yl) propyl-2-phosphonic acid and d-2-amino-5-phosphonopentanoic acid; (ii) the competitive quisqualate/kainate receptor antagonist, 6,7-dinitroquinoxalinedione; and (iii) the broad spectrum excitatory amino acid receptor antagonist, kynurenate. Such observations demonstrate that excitatory sulphur amino acid-evoked release of [(3)H]d-aspartate from primary cultures of cerebellar granule cells occurs primarily by a receptor-mediated event in which both N- methyl- d -aspartate and quisqualate/kainate receptors are involved. The findings of this study provide additional evidence to support the transmitter candidacy of certain endogenous neuroactive sulphur amino acids and furthermore, suggest the mechanism by which their epileptogenic and neurotoxic actions may be explained.