Journal of Neuroscience, Vol 4, 131-140, Copyright © 1984 by Society for Neuroscience
The effect of spontaneous electromechanical activity on the metabolism of acetylcholinesterase in cultured embryonic rat myotubes
SK Brockman, LH Younkin and SG Younkin
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.
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