In developing murine primary skeletal muscle cells spontaneous electrical activity is associated with [Ca²⁺]_i transients and twitches. We have already observed that such spontaneous [Ca²⁺]_i oscillations were modulated by α-bungarotoxin. Moreover, patch-clamp experiments showed spontaneous openings (i.e. in the absence of exogenous acetylcholine) of the acetylcholine receptor (AChR) channel in oscillating cells. Thus we suggested a role for AChR channel activity in the generation of the spontaneous [Ca²⁺]_i oscillation in developing murine primary skeletal muscle cells. To further understand this phenomenon, experiments have been performed on murine primary skeletal muscle cells (i28) isolated from hindlimb muscles of a mouse killed by cervical dislocation. Then, i28 cells were differentiated in vitro and loaded with the dye fura-2. The videoimaging technique was used to measure the [Ca²⁺]_i oscillations.

In the first series of experiments we investigated the effect of agents capable of enhancing the probability of the AChR channel openings. In particular, nicotine was applied at submaximal concentrations (50Ð200 nM). In i28 oscillating cells, the addition of the agonist invariably caused a sudden increase in the frequency of spontaneous [Ca²⁺]_i oscillations. In addition, nicotine was also able to trigger the oscillatory activity when administered during the silent periods between two consecutive bursts of spontaneous [Ca²⁺]_i oscillations (n = 20). We also considered the possibility that developing i28 cells could release acetylcholine, activating their own AChRs (Hamann et al. 1995). Thus the effect of acetylcholinesterase on spontaneous [Ca²⁺]_i oscillations was investigated. When the enzyme (200 units ml^-1) was administered, the oscillatory activity stopped in 45 % of the cells observed (n = 22). In contrast, the acetylcholinesterase inhibitor edrophonium (500 nM) caused an increase in the frequency of spontaneous [Ca²⁺]_i transients (6/8). Moreover, preliminary immunocytochemistry experiments demonstrated the presence of choline acetyltransferase immunoreactivity in developing i28 cells.

To conclude, our results confirmed the involvement of AChR channels in the generation of spontaneous [Ca²⁺]_i oscillations. Interestingly, the secretion of acetylcholine by differentiating i28 cells may sustain the activity of the AChR channels and consequently the [Ca²⁺]_i oscillations.

This work was supported by grants from MURST-Italy and European Union.

All procedures accord with current National guidelines.