A double-labeled preparation for simultaneous measurement of [3H]-noradrenaline and [14C]-glutamic acid exocytosis from streptolysin-O (SLO)-perforated synaptosomes.
We have developed a novel method to examine [3H]-noradrenaline and [14C]-glutamate release from the same sample of streptolysin-O (SLO) perforated rat cortical synaptosomes. Ca2+ -dependent [3H]-noradrenaline and [14C]-glutamate release was examined at different temperatures and was found to be greater at 30 degrees C than at 25 degrees C. Ca2+ -dependent release of [3H]-noradrenaline is more ATP dependent than Ca2+ -dependent release of [14C]-glutamate. No significant reuptake of either neurotransmitter by the perforated synaptosomes was detected, indicating all the synaptosomes were indeed perforated. Incubations with 1 mM ouabain, a specific Na+,K+ -ATPase inhibitor, slightly increased Ca2+ -dependent release of both neurotransmitters. [3H]-noradrenaline is released from large dense-core vesicles and [14C]-glutamate is released from small clear synaptic vesicles, so one can directly compare and contrast neurotransmitter release mechanisms between large dense-core vesicles and small clear synaptic vesicles using this preparation.
McFadden
S C
SC
Department of Chemistry, Texas Christian University, Forth Worth, TX 76129, USA. s.c.mcfadden@student.tcu.edu
Bobich
J A
JA
Zheng
Q
Q
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins; D001831:Body Temperature / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology; D002250:Carbon Radioisotopes / Q000493:pharmacokinetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism* / Q000493:pharmacokinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism* / Q000493:pharmacokinetics; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D012588:Scintillation Counting / Q000379:methods; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects / Q000378:metabolism; D013301:Streptolysins / Q000494:pharmacology; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology*; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium / Q000493:pharmacokinetics
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D013301:Streptolysins; C025964:streptolysin O; D014316:Tritium; D018698:Glutamic Acid; D010042:Ouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D009638:Norepinephrine