Ca2+-dependent and Ca2+-independent glutamate release, energy status and cytosolic free Ca2+ concentration in isolated nerve terminals following metabolic inhibition: possible relevance to hypoglycaemia and anoxia.

Hypoglycaemia and anoxia both cause massive release of glutamate from the brain in vivo, and the nature of this release was investigated using guinea-pig cerebral-cortical synaptosomes and iodoacetate and rotenone to simulate the energetic consequences of these conditions. Glutamate release (by continuous fluorimetry), cytoplasmic free Ca2+ (by fura-2), membrane potentials, ATP, ADP and creatine phosphate were determined in parallel, following the addition of iodoacetate or rotenone, alone or in combination. Ca2+-dependent glutamate release had a high energy requirement which could only be satisfied by aerobic glycolysis. Respiration using endogenous substrates, or anaerobic glycolysis following rotenone, caused a progressive inhibition of Ca2+-dependent release, correlating with a decline in the total ATP/ADP ratio and creatine phosphate. With rotenone, an increase in Ca2+-independent glutamate release was observed, correlating with a decline in plasma membrane potential. Only a slight increase in free Ca2+ was seen. Rotenone plus iodoacetate caused an almost immediate collapse of ATP/ADP ratio and a parallel loss of Ca2+-dependent glutamate release before free Ca2+ had risen to a level sufficient for exocytosis. In contrast, Ca2+-independent glutamate release increased. The Ca2+-dependent release of L-glutamate had the characteristics of an exocytotic transmitter release mechanism, being energy-dependent and triggered by elevated cytoplasmic free Ca2+ concentration. A distinct Ca2+-independent release of cytoplasmic glutamate occurred by reversal of the Na+-coupled uptake carrier, which was accelerated by a decline in the Na+ gradient. It is concluded that the Ca2+-independent release of cytoplasmic glutamate may make the major contribution to the excitotoxic release of glutamate in hypoglycaemic and anoxic conditions.