Transmitter packaging at frog neuromuscular functions exposed to
anticholinesterases; the role of second-stage acetylcholine loading.
Naves, Ligia A. and William Van der Kloot.
Department of Physiology and Biophysics, Health Sciences Center, SUNY,
Stony Brook, NY 11794-8661.
APStracts 3:0135N, 1996.
SUMMARY AND CONCLUSIONS
1. This investigation was undertaken to explore an unexpected effect of
vesamicol, an agent that inhibits active acetylcholine (ACh) uptake into
isolated synaptic vesicles. Previous studies at the neuromuscular junction
showed that vesamicol makes miniature end-plate currents (MEPCs) smaller only
after tens of thousands of quanta have been released. Inhibiting AChE makes
the MEPCs larger than normal. Our unexpected finding was that with the AChE
inhibitor present, adding 2 (M (-)-vesamicol decreases the size of the MEPCs
by about 30%. The decrease was apparent within 15-30 min, during which only a
few thousand quanta had been released. 2. Experimental tests showed that the
(-)-vesamicol treatment is unlikely to be acting postsynaptically. For
example, it did not slow the rise of MEPCs, which would occur if the endplate
receptors were blocked. 3. When AChE was inhibited, three treatments expected
to block active choline (Ch) uptake into the presynaptic terminals decreased
MEPC size. a). Elevating extracellular K+ to diminish the Na+ electrochemical
gradient required for Ch uptake. b). Replacing extracellular Na+ with
methylamine+. c). Adding hemicholinium-3 (HC-3), an inhibitor of the Ch
transporter. These treatments did not act by reactivating AChE, blocking the
endplate ACh receptor, or by enhancing the desensitization of the ACh
receptor. 4. Previous evidence suggests that synaptic vesicles are formed and
partially filled with ACh in the cytoplasm, and then receive additional ACh
when they attach to the active zones, a process that is called second-stage
loading. We conclude that the MEPCs are becoming smaller when second-stage
loading is blocked by (-)-vesamicol or when the supply of ACh in the cytoplasm
of the motor nerve terminal is depleted. 5. To follow the time course of
second-stage loading we used the false transmitter precursor monoethylcholine
(MECh). It enters the terminal and is transformed into acetylmonoethylcholine
(AMECh). When 200 (M MECh was placed in the extracellular solution and the
AChE was inhibited, MEPC size was significantly smaller after 10 min. MEPC
size increased once again over a period of time when MECh was removed from the
extracellular solution and replaced with Ch. 6. We conclude that at the
neuromuscular junction second-stage loading is responsible for loading a
significant fraction of the ACh into the quanta.
Received 25 January 1996; accepted in final form 6 June 1996.
APS Manuscript Number J49-6.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 4 July 96