Transmitter packaging at frog neuromuscular junctions exposed to anticholinesterases; the role of second-stage...

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 acetylcholinesterase (AChE) makes the MEPCs larger than normal. Our unexpected finding was that with the AChE inhibitor present, adding 2 microM (-)-vesamicol decreases the size of the MEPCs by approximately 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: 1) elevating extracellular K+ to diminish the Na+ electrochemical gradient required for Ch uptake; 2) replacing extracellular Na+ with methylamine+; and 3) 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 microM 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.