We have investigated the effect of electromechanical activity on the molecular forms of acetylcholinesterase (AChE) in cultured embryonic rat myotubes. Both globular and asymmetric forms of AChE are present on the 5th day of culture when myotubes are just beginning to fibrillate. Between days 5 and 8, the 4 S (G1), 10 S (G4), and 16 S (A12) forms increase dramatically, and appreciable 12.5 S (A8) AChE appears. When fibrillation is prevented by adding tetrodotoxin on day 4, the increases in the A12 and A8 forms are prevented, and the increases in the G4 and G1 forms are significantly impaired. At 8 days, fibrillating myotubes have 19 times more A12 AChE and over 4 times more G1 and G4 enzyme than do nonfibrillating myotubes. The effect of tetrodotoxin is reversible. When tetrodotoxin is removed at 7 days, fibrillation resumes promptly, and globular and asymmetric forms recover. Light microscopic examination of fibrillating and nonfibrillating myotubes showed that tetrodotoxin does not affect the gross morphological development of the myotubes. Titration of AChE-active sites with O-ethyl-S2-diisopropyl methyl-phosphonothionate demonstrated that the increase in AChE activity associated with fibrillation is due to an increase in the number of AChE molecules present and not to an increase in the rate at which individual AChE molecules turn over acetylcholine. To evaluate AChE metabolism in fibrillating and nonfibrillating myotubes, we examined the enzyme after inactivating it with paraoxon. Paraoxon readily penetrates cells and diethylphosphorylates a serine in the active site of AChE, thereby inactivating it. The diethylphosphorylated enzyme is stable, but it can be reactivated rapidly and quantitatively with pyridine-2-aldoxime methiodide (2-PAM). After inactivating AChE with paraoxon, we simultaneously evaluated synthesis (by following the newly synthesized active AChE) and turnover (by following the 2-PAM-reactivatable AChE). Our results show that globular and asymmetric forms of AChE are both synthesized more rapidly in fibrillating than in nonfibrillating myotubes.

Substances which have an innervation-like effect on the cholinesterase activity of organ-cultured rat extensor digitorum longus muscles are moved in nerve by axonal transport, are released from nerve by stimulation, and are present in innervated muscle but apparently absent from denervated muscle. Substances which increase the acetylcholine sensitivity of cultured muscles behave similarly.

SUMMARY1. It has been proposed that the influence of innervation on the cholinesterase activity (ChE) of skeletal muscle and on end-plate ChE in particular is mediated by trophic substances) moved by axonal transport and released from nerve. We have tested this hypothesis using rat extensor digitorum longus (e.d.l.) and diaphragm muscles denervated in vitro for several days and then maintained in organ culture to assay putative trophic substancess.2. The cholinesterase activity (ChE) of rat extensor digitorum longus (e.d.l.) muscles decreased dramatically after 5 days of denervation in vivo as previously reported. The ChE of rat e.d.l. muscles denervated in vivo for 3 days and then maintained in organ culture for 2 days was essentially identical to that of muscles denervated 5 days in vivo.3. The ChE of e.d.l. muscles denervated in vivo for 3 days and then maintained for 2 days in culture medium supplemented with sciatic nerve or innervated muscle extract was significantly higher than that of muscles denervated in vivo for 5 days or denervated in vivo for 3 days and then cultured for 2 days in culture medium alone. Supplementing the culture medium with brain or spinal cord extract also significantly increased the ChE of organ-cultured e.d.l. muscles.4. Supplementing the culture medium with liver or spleen extract or with the extract of muscle denervated for 3-7 days in vivo before extraction did not increase the ChE of organ-cultured e.d.l. muscles.5. The effect of muscle extract on the ChE of organ-cultured e.d.l. muscles was dose dependent and occurred gradually reaching a maximum after approximately 24 h of culture.6. Substance(s) which increased the ChE of organ-cultured e.d.l. muscles were found to accumulate in transacted sciatic nerve in the region just proximal to the site of transaction where substances moved by axonal transport are known to accumulate.7. Media conditioned with neurally stimulated e.d.l. or diaphragm muscles caused a substantial and highly significant increase in the ChE of e.d.l. or diaphragm muscles denervated in vivo and then maintained in organ culture. Media conditioned in the same way with unstimulated muscles did not increase the ChE of organ-cultured muscles.

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