The effect of spontaneous electromechanical activity on the metabolism of acetylcholinesterase in cultured embryonic rat myotubes

Abstract

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.

Increases in muscle Ca2+ mediate changes in acetylcholinesterase and acetylcholine receptors caused by muscle contraction.

“…RESULTS A12 AcChoE Synthesis Is Enhanced by Calcium lonophore Treatment. As reported previously (32)(33)(34), rat myotubes start to contract spontaneously 4-7 days after plating and then synthesize three primary AcChoE forms, all present as significant percentages of the total: the globular forms, G1 and G4, and the collagen-tailed asymmetric form, A12. There were also variable amounts of another asymmetric form, A8.…”

“…Numerous investigators have shown that a variety of types of muscle fibers grown in neuron-free primary cell culture and permitted to undergo spontaneous contraction are able to synthesize several forms of AcChoE including the A12 (28)(29)(30)(31). However, if contraction of rat muscle fibers, for example, is blocked by growing the fibers in the presence of tetrodotoxin (TTX), both the total amount of AcChoE and the percentage of AcChoE assembled as the A12 form are decreased severalfold (32)(33)(34). In this paper, we present data that suggest an increase in muscle cell Ca2' in the absence of contraction is able to stimulate cultured muscle cells to synthesize greatly increased amounts of A12 AcChoE.…”

Local Control of Acetylcholinesterase Gene Expression in Multinucleated Skeletal Muscle Fibers: Individual Nuclei Respond to Signals from the Overlying Plasma Membrane

“…We also know that AChE is regulated at least in part by muscle activity, the spontaneous or nerve-evoked depolarization of the plasma membrane. Drugs that block membrane depolarization, such as the sodium channel antagonist tetrodotoxin (TTX), inhibit assembly of the collagen-tailed (A 12 ) AChE form that in turn decreases accumulation of AChE on the cell surface (Rieger et al, 1980;Brockman et al, 1984;Fernandez-Valle and Rotundo, 1989). Conversely, sodium channel agonists such as veratridine (Ver) dramatically increase A 12 AChE assembly (De La Porte et al, 1984).…”

“…Studies from several laboratories have shown that synthesis and assembly of AChE are regulated by the activity state of the muscle (Koenig and Vigny, 1978;Brockman et al, 1984;Rubin, 1985;Fernandez-Valle and Rotundo, 1989). For example, sodium channel antagonists such as TTX suppress A 12 AChE assembly and cell surface AChE accumulation (Rieger et al, 1980;Koenig et al, 1982;Brockman et al, 1984;Rubin, 1985;Fernandez-Valle and Rotundo, 1989), whereas sodium channel agonists such as Ver increase it (De La Porte et al, 1984;Rubin et al, 1985).…”

“…For example, sodium channel antagonists such as TTX suppress A 12 AChE assembly and cell surface AChE accumulation (Rieger et al, 1980;Koenig et al, 1982;Brockman et al, 1984;Rubin, 1985;Fernandez-Valle and Rotundo, 1989), whereas sodium channel agonists such as Ver increase it (De La Porte et al, 1984;Rubin et al, 1985). Two other sodium channel agonists, ScVn (Sigma) and brevetoxin (BvTx) (kindly provided by Dr. D. Baden), were tested in our experiments with the same results as Ver.…”