Ca 2+-dependent and Ca 2+- independent glutamate release, energy status and cytosolic free Ca 2+ concentration in isolated nerve terminals following metabolic inhibition: Possible relevance to hyoglycaemia and anoxia

Abstract

Hypoglycaemia and anoxia both cause massive release of glutamate from the brain in vitro, 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 Ca 2+ (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. Ca 2+-dependent glutamate release had a high energy requirement which could only he satisfied by aerobic glycolysis. Respiration using endogenous substrates, or anaerobic glycolysis following rotenone, caused a progressive inhibition of Ca 2+-dependent release, correlating with a decline in the total ATP ADP ratio and creatine phosphate. With rotenone, an increase in Ca 2+-independent glutamate release was observed, correlating with a decline in plasma membrane potential. Only a slight increase in free Ca 2+ was seen. Rotenone plus iodoacetate caused an almost immediate collapse of ATP ADP ratio and a parallel loss of Ca 2+-dependent glutamate release before free Ca 2+ had risen to a level sufficient for exocytosis. In contrast, Ca 2+-independent glutamate release increased. The Ca 2+-dependent release of l-glutamate had the characteristics of an exocytotic transmitter release mechanism, being energy-dependent and triggered by elevated cytoplasmic free Ca 2+ concentration. A distinct Ca 2+-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 Ca 2+-independent release of cytoplasmic glutamate may make the major contribution to the excitotoxic release of glutamate in hypoglycacmic and anoxic conditions.