Bromoacetylcholine and acetylcholinesterase introduced via liposomes into motor nerve endings block increases in quantal size

Abstract

We incorporated bromoacetylcholine (an inhibitor of choline acetyltransferase), acetylcholinesterase, or both into liposomes made of phosphatidylcholine. Frog sartorius muscles were exposed to these liposomes for 30-60 min. The liposome treatment itself did not decrease the size of the quanta compared to untreated controls. Then the preparations were exposed for 10-20 min to a hypertonic solution, which increases the rate of spontaneous quantal release and elicits an increase in the amount of acetylcholine released per quantum. Following the hypertonic treatment the quanta were significantly smaller in the liposome-treated preparations. Most of the difference occurred because in the preparations not exposed to the liposomes quantal size increased following the hypertonic treatment. This increase is thought to be due to the incorporation of more acetylcholine into each quantum. Our conclusions are that the treatments decreased the acetylcholine concentration in the cytoplasm, and that the increase in size occurs because additional acetylcholine is added to the vesicles containing the quanta from the cytoplasm.

Calmodulin increases transmitter release by mobilizing quanta at the frog motor nerve terminal

“…The increase in m was associated primarily with an increase in n , the number of functional transmitter release sites (to 130% of control; filled circles in Figure 1B), as there were no significant changes in p , the mean probability of release (filled circles in Figure 1C) or var s p , the spatial variance in p (filled circles in Figure 1D). The effects were reversed after 40 min of wash. For a molecule the size of CaM, the final concentration attained in the nerve terminal was estimated to be about 10 −2 of the concentration contained in the liposomes (Brailoiu & van der kloot, 1996; Brailoiu et al ., 1999). Heat‐inactivated CaM (1000 units ml −1 ) had no significant effects on any of the quantal release parameters (open circles in Figure 1A–D).…”

“…There was no effect on var s p (filled circles in Figure 3D). The effects of antiCaM were not reversed, even after 1 h of wash. Again, it was estimated that the concentration attained in the cytoplasm for large molecules such as antibodies was about 10 −2 of the concentration encapsulated in the liposomes (Brailoiu & van der kloot, 1996; Brailoiu et al ., 1999). Liposomes containing heat‐inactivated CaM antibodies (50 μl ml −1 ) had no consistent effect on any of the quantal release parameters (open circles in Figure 3A–D).…”

“…Encapsulation of agents in liposomes is an accepted procedure for delivering small and large molecules (Brailoiu & van der kloot, 1996; Brailoiu et al ., 1999) into the motor nerve terminal (de paiva & Dolly, 1990; Silinsky et al ., 1995). Multilamellar liposomes were prepared with 60 mg ml −1 egg phosphatidylcholine (Sigma, type X‐E), as previously described (Brailoiu et al ., 2001).…”

TLC Characterization of Liposomes Containing Angiotensinogen, Angiotensine I, Angiotensine II and Saralazin

“…These data are also in accordance with the results of Ahl et al (1994) which demonstrated that, in the presence of ethanol, liposomes obtained from phosphatidylcholine did not induce interdigitation. Therefore, since in physiological studies we used liposomes prepared from egg phosphatidylcholine type X-E (Sigma; also containing other lipids) (Brailoiu et al, 1995a;Brailoiu and Van der Kloot, 1996) we cannot exclude the possibility that impurities could induce interdigitation.…”

“…An alternative method for intracellular delivery of drugs for physio-pharmacological experiments is the use of liposomes (Brailoiu et al, 1995a;Brailoiu and Van der Kloot, 1996). However, there are some disadvantages in using this method.…”