Release of ATP from cholinergic synaptic vesicles during freeze-thaw cycling

Cholinergic synaptic vesicles were isolated from Torpedo californica and subjected to repeated freeze/thraw (F/T) cycles. Both vesicular (trapped) and released (free) ATP were measured after each cycle. It was found that a constant percentage of vesicular content was released during each F/T cycle. In solutions low in Ca2+ and Mg2+ and high in sucrose, 25% of the ATP is released by each F/T cycle. When synaptic vesicles are resuspended in Tornedo Ringer's (which contains less sucrose, higher Ca2+ and urea), 35-40% of the trapped ATP is released during each F/T cycle. These data contradict the hypothesis that F/T does not disrupt synaptic vesicle membranes (although F/T does disrupt cell membranes and synaptosomes). This nonrupture hypothesis was assumed by C. Solsona, C. Saltó, and A. Ymbern (1991, Biochim. Biophys. Acta 1095, 57-62) to calculate cytosolic ATP as 40% of total ATP. Our results indicate that cytosolic ATP is 10-15%. These results may also explain some of the discrepancies in reported values for cytosolic acetylcholine (ACh). Values of 35-50% were obtained by previous workers using the nonrupture hypothesis, and values of 8-22% in experiments that did not depend on the nonrupture hypothesis. Our results refute the nonrupture hypothesis and thus support a lower value for cytosolic ACh.