TRANSMITTER PACKAGING AT FROG NEUROMUSCULAR-JUNCTIONS EXPOSED TO ANTICHOLINESTERASES - THE ROLE OF 2ND-STAGE ACETYLCHOLINE LOADING

1. This investigation was undertaken to explore an unexpected effect o f 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 (ME PCs) smaller only after tens of thousands of quanta have been released . Inhibiting acetylcholinesterase (AChE) makes the MEPCs larger than n ormal. Our unexpected finding was that with the AChE inhibitor present , adding 2 mu M (-)-vesamicol decreases the size of the MEPCs by simil ar to 30%. The decrease was apparent within 15-30 min, during which on ly a few thousand quanta had been released. 2. Experimental tests show ed that the (-)-vesamicol treatment is unlikely to be acting postsynap tically. For example, it did not slow the rise of MEPCs, which would o ccur if the endplate receptors were blocked. 3. When AChE was inhibite d, three treatments expected to block active choline (Ch) uptake into the presynaptic terminals decreased MEPC size: 1) elevating extracellu lar K+ to diminish the Na+ electrochemical gradient required for Ch up take; 2) replacing extracellular Na+ with methylamine(+); and 3) addin g hemicholinium-3 (HC-3), an inhibitor of the Ch transporter. These tr eatments did not act by reactivating AChE, blocking the endplate ACh r eceptor, or by enhancing the desensitization of the ACh receptor. 4. P revious evidence suggests that synaptic vesicles are formed and partia lly filled with ACh in the cytoplasm and then receive additional ACh w hen they attach to the active zones, a process that is called second-s tage loading. We conclude that the MEPCs are becoming smaller when sec ond-stage loading is blocked by (-)-vesamicol or when the supply of AC h in the cytoplasm of the motor nerve terminal is depleted. 5. To foll ow the time course of second-stage loading, we used the false transmit ter precursor monoethylcholine (MECh). It enters the terminal and is t ransformed into acetylmonoethylcholine (AMECh). When 200 mu 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 extracellu lar solution and replaced with Ch. 6. We conclude that at the neuromus cular junction second-stage loading is responsible for loading a signi ficant fraction of the ACh into the quanta.