Appearance of acetylcholinesterase molecular forms in noninnervated cultured primary chick muscle cells

Asymmetric forms of AChE have generally not been detected in cultured chick skeletal muscle cells in the absence of cocultured neurons. To explore further neurotrophic effects of adult peripheral nerve extracts (NE) on muscle in vitro, we reexamined the appearance of various molecular forms of AChE in cultured chick muscle cells in the presence of NE. The various molecular forms of AChE were distinguished by sucrose gradient sedimentation and radioenzymatic techniques. In the presence of NE, cells proliferated during the first 48 hr of culture, then fused and formed spontaneously contracting myotubes by 6-8 days in culture. Total AChE, 5.4 S, and 11.5 S molecular forms reached activity plateaus by 8 days in culture which persisted until cultures were terminated at day 20. Between 1 and 6 days in culture, 19.5 S AChE (A12) was not detected. The A12 form was first observed at 7 days reaching a maximum of 11.3% of the total AChE at 14 days and then gradually declined to a level of 1% at day 20. Since the A12 form declined in older cultures but comprised 25% in embryonic muscle tissue, we examined the possible requirement of neurons in culture to attain higher levels of A12 AChE. Spinal cord neurons were plated onto 6-day muscle cultures and AChE activities were measured between 8 and 20 days. The results showed that 19.5 S AChE activity in the presence of both spinal cord neurons and NE was no greater than that found in the presence of NE alone. To suppress spontaneous contraction, 0.6 microM tetrodotoxin (TTX) or 15 microM d-tubocurarine (dTC) were added to 5-day-old muscle cultures at a time when myotubes were differentiated but contractile activity had not begun. TTX had cytotoxic effects and inhibited further development of myotubes. In contrast, dTC had no deleterious effect on morphological development, eliminated contraction, but did not interfere with the appearance of any forms of AChE including the A12 form. These studies show that primary chick muscle cells are capable of producing the A12 form of AChE if cultured in NE-supplemented medium. In this culture system, production of the A12 form does not require activity or innervation.