Abbreviations used: AM, acetoxymethyl ester; BM, basal medium; [Ca2+]c, cytoplasmic free Ca2+ concentration; ωCmTx-MVIIC, ω-conotoxin-MVIIC; CN/2-dOG, 1 mM sodium cyanide and 10 mM 2-deoxyglucose; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FM1-43, N-(3-triethylammoniumpropyl)-4-(dibutylaminostyryl)pyridinium dibromide; MK-801, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-iminehydrogen maleate; NBQX, 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione; TnTx, tetanus toxin.
Abstract: The mechanism of glutamate release from cultured cerebellar granule neurones in response to a chemical model of ischaemia (10 mM 2-deoxyglucose plus 1 mM sodium cyanide) was investigated. In the first 2 min of ischaemia, release of preloaded d-[3H]aspartate could be extensively attenuated by tetanus toxin and bafilomycin A1 and was dependent on the activation of Ca2+ channels sensitive to the “Q” type Ca2+ channel antagonist, ω-conotoxin-MVIIC. During this period, ATP/ADP ratios fell rapidly. The extent of release in the first 2 min was comparable to that evoked by 2-min depolarization by 50 mM KCl. Free Ca2+ concentrations, determined in neurites and somata, did not increase until after 2 min. The neurite increase in cellular Ca2+ precedes that of the cell somata. Release of d-[3H]aspartate was partially inhibited by the NMDA receptor antagonist MK-801, which also delayed the increase in free Ca2+ concentration. Prolonging the period of ischaemia to 6 and 10 min produced no further increase in the apparently exocytotic component of release, but initiated an extensive nonexocytotic release of the amino acid. Studies with the synaptic vesicle membrane probe FM1-43 in which released amino acid was removed by superfusion indicated that Ca2+-dependent exocytosis was delayed in this system. It is concluded that chemical ischaemia initiates an initial exocytotic followed by nonexocytotic release and that the former is facilitated by NMDA receptor activation. These events occur in cells that are still able to exclude propidium iodide, indicating that cell death has not yet occurred.