Protease inhibitors reduce the loss of nerve terminals induced by activity and calcium in developing rat soleus muscles in vitro.

The end-plate of a mammalian skeletal muscle fibre is innervated by several axons at the time of birth but by only one axon in the adult. In the rat soleus muscle the transition from polyneuronal to single innervation occurs during the first 2-3 weeks after birth. While it is evident that the loss of the excess nerve terminals depends to some extent on neuromuscular activity, the mechanism involved is not known. In the present experiments neonatal rat soleus muscles were stimulated in vitro in the presence of a variety of combinations of calcium, the cholinesterase inhibitor edrophonium and the proteolytic enzyme inhibitors leupeptin, pepstatin and Ep-475. Electron microscopical examination revealed that stimulation alone had little effect on the morphology of the end-plate region but stimulation in the presence of raised levels of calcium caused severe disruption of the nerve terminals and a marked reduction in the number of intact nerve terminal profiles contacting each end-plate. Contraction measurements showed that, in spite of this, the muscles were not functionally denervated to any large extent. The addition of edrophonium potentiated the morphological alterations but caused no further reduction in the number of profiles. Conversely, the protease inhibitors wholly or partially (in the case of Ep-475) prevented the effects of stimulation and calcium on the nerve terminals. These results are consistent with the idea that neuromuscular activity induces the secretion of proteolytic enzymes into the end-plate region, where they digest the immature nerve terminals. The importance of calcium suggests that the calcium-dependent neutral protease may be involved, and is also consistent with a secretory mechanism. The possibility that the nerve terminals are digested by their own proteases is also discussed.