ACh release from osmotically shocked synaptosomes refilled with transmitter

It is still uncertain whether or not acetylcholine (ACh) released at the synapse is derived from vesicles. We have recently described a sensitive chemiluminescent technique which allows continuous measurement of ACh release from tissue slices or synaptosomes incubated in the reaction mixture 1,2 . From the luminescence obtained when synaptosomes are opened in the reaction mixture by a single freezing and thawing we estimated that 30% of the total ACh of Torpedo electric organ synaptosomes is cytoplasmic. The vesicular ACh compartment which resists freezing and thawing requires a detergent to be liberated. These estimations showed that the ACh compartment considered as cytoplasmic was readily released from stimulated synaptosomes 3 . If we are dealing with a real, soluble cytoplasmic ACh compartment, it should in principle be possible to recover, in controlled ionic conditions, a calcium-dependent ACh release from synaptosomes emptied by an osmotic shock, and refilled at the desired ACh concentration. We show here that this is the case. These results suggest the possibility of a non-vesicular release of ACh from the nerve terminal.