A double-labeled preparation for simultaneous measurement of [H-3]-noradrenaline and [C-14]-glutamic acid exocytosis from streptolysin-O (SLO)-perforated synaptosomes

We have developed a novel method to examine [H-3]-noradrenaline and [C-14]- glutamate release from the same sample of streptolysin-O (SLO) perforated r at cortical synaptosomes. Ca2+-dependent [H-3]-noradrenaline and [C-14]-glu tamate release was examined at different temperatures and was found to be g reater at 30 degreesC than at 25 degreesC. Ca2+-dependent release of [H-3]- noradrenaline is more ATP dependent than Ca2+-dependent release of [C-14]-g lutamate. No significant reuptake of either neurotransmitter by the perfora ted synaptosomes was detected, indicating all the synaptosomes were indeed perforated. Incubations with 1 mM ouabain, a specific Na+, K+-ATPase inhibi tor, slightly increased Ca2+-dependent release of both neurotransmitters. [ H-3]-noradrenaline is released from large dense-core vesicles and [C-14]-gl utamate is released from small clear synaptic vesicles, so one can directly compare and contrast neurotransmitter release mechanisms between large den se-core vesicles and small clear synaptic vesicles using this preparation. (C) 2001 Elsevier Science B.V. All rights reserved.