Regulation of acetylcholinesterase biogenesis by muscle activity

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Richard L. Rotundo - Committee Chair


The predominant forms of acetylcholinesterase (AChE) synthesized by quail skeletal muscle cultures are monomers/dimers, tetramers and asymmetric molecules. The relative abundance of oligomeric forms is strongly dependent upon the activity state of the cells. I examined several stages of AChE biogenesis to determine which ones were regulated by muscle activity. Inhibiting spontaneous contraction with tetrodotoxin (TTX) reduced AChE activity by approximately 30% of the levels found in actively contracting cells. This decrease was due primarily to the loss of 20S asymmetric (collagen-tailed) AChE. AChE down-regulation was not mediated through changes in the rates of synthesis or degradation of isotopically-labeled AChE polypeptides chains or acquisition of AChE catalytic activity; however, a larger percentage of catalytically active dimers and tetramers were secreted from TTX-treated cultures compared to controls.To study the mechanism of regulation, cultures incubated in the presence of TTX (sodium channel antagonist) or veratridine (sodium channel agonist) were compared. The abundance of asymmetric AChE was 10-fold greater in veratridine-treated cultures than in TTX-treated cultures, suggesting that sodium channel activation initiates a cascade of events that induces expression of asymmetric AChE. A23187, a calcium ionophore did not stimulate asymmetric AChE appearance in the presence of TTX; however, cobalt, an inorganic calcium channel blocker, reduced the abundance of all AChE forms in the presence or absence of veratridine. Results obtained with phorbol esters, potent activators of protein kinase C, and with neomycin, an inhibitor of phosphatidylinositol bisphosphate (PIP$\sb2$) hydrolysis, were consistent with a PKC-dependent expression of asymmetric AChE.Transcriptional regulation was examined by Northern blot analysis. Major AChE transcripts were 5-5.3 and 6.2 kb in length while minor transcripts were 4.8, 9 and 11 kb in length. The relative abundance of AChE transcripts increased in cultures treated with TTX or phorbol esters, but was unchanged by incubation with veratridine. These results suggest that the activity-dependent expression of asymmetric AChE in quail skeletal muscle cultures is regulated at the transcriptional as well as post-translational level. Calcium appears to be required for synthesis of all AChE forms, but is not sufficient to stimulate asymmetric AChE synthesis in TTX-treated cultures; whereas phorbol esterstimulated PKC activity is associated with the appearance of asymmetric AChE.


Biology, Neuroscience

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