Pamela S. Nieberg

Production and release of acetylcholinesterase by cultured chick embryo muscle.

Abstract The acetylcholinesterase (AChE) activity of cultures from 11-day-old chick embryo muscle cells was studied for up to 4 weeks in vitro. AChE activity was found in mononucleated cells and multinucleated myotubes. The activity increased greatly after fusion. Maximum AChE levels were reached after 7–10 days of incubation and tended to decline thereafter. Multiple forms of AChE found in embryo muscle in situ were present in cultures before and after fusion. Selective inhibitors and substrates were used to show that AChE was released by the cells into their medium. Within a 2-day period the AChE that accumulated in the medium averaged over 6 times that remaining in the cells. Release of AChE from the cells was inhibited by cycloheximide, and AChE levels in cells and medium were much reduced when differentiation was inhibited by bromodeoxyuridine. Little AChE was present in subcultures of fibroblasts from muscle cultures. Acetyl-β-methylcholine and, to a lesser degree, choline itself, prevented the decrease in AChE levels of 2- to 3-week-old muscle cultures.

Tissue acetylcholinesterase in plasma of chick embryos and dystrophic chickens

Abstract The plasma cholinesterases of chickens (line 304) with inherited muscular dystrophy and 2 normal lines were examined from 12 days of incubation to 14 weeks after hatching. Acetylcholinesterase (AChE) and non-specific cholinesterase (BChE) activities were distinguished by spectrophotometric, electrophoretic and titrimetric analyses using acetylcholine, butyrylcholine, and their thioester analogs acetylthiocholine and butyrylthiocholine as substrates and 284C51 and iso-OMPA as selective inhibitors. Plasma from normal and dystrophic embryos and from dystrophic chicks had high AChE activity. For example, by 12 weeks of age, 37% of plasma acetylthiocholine hydrolysis was inhibited by the anti-AChE agent 284C51. The results support the view that embryo plasma contains AChE and BChE forms. After hatching the AChE forms decrease greatly in normal plasma and a shift in BChE forms occurs. Later, the AChE forms return in dystrophic line plasma. AChE activity in plasma was circumstantially associated with multiple molecular forms of AChE in the sarcoplasm of embryo and dystrophic muscles and it is likely that these muscles are sources of the plasma AChE activity. The results also confirmed that acetyl-β-methylcholine, unlike the situation which exists in mammals, is hydrolyzed by both AChE and BChE forms in the chicken, and cannot be used to distinguish these cholinesterases.

Myogenic defect in muscular dystrophy of the chicken

Abstract Inherited muscular dystrophy of the chicken is thought to arise from abnormal development of trophic regulation of skeletal muscles by their innervating nerves. To determine whether expression of muscular dystrophy in the chicken is a property of the nerves or of the muscles, wing limb buds were transplanted between normal and dystrophic chick embryos at 3 1 2 days of incubation (stage 19–20). Muscles of donor limbs innervated by nerves of the hosts were compared to contralateral unoperated host limb muscles in chicks from 6 to 25 weeks after hatching. Expression of normal or dystrophic phenotype was determined by examination of five different properties which are altered in dystrophic chick muscle: electromyographic evidence of myotonia; fiber diameter; acetylcholinesterase activity, localization, and isozymes; lactic dehydrogenase activity; and succinic dehydrogenase activity. Genetically normal muscle innervated by nerves of normal or dystrophic hosts was phenotypically normal while genetically dystrophic muscle innervated by normal nerves was phenotypically dystrophic. The results suggest that inherited muscular dystrophy of the chicken arises from a defect of muscle rather than from a lesion in the nerves themselves.

Ultrastructural localization of acetylcholinesterase in cultured cells. I. Embryo muscle

Several techniques were employed to examine the localization of acetylcholinesterase (EC 3.1.1.7, AChE) in cultured chick embryonic skeletal muscle. Glutaraldehyde produced the best cellular preservation but less enzyme activity was lost when the cells were fixed in paraformaldehyde. Two staining methods were examined: in one (Karnovsky MJ, Roots L: J Histochem Cytochem 12:219, 1964) potassium ferricyanide was added with the primary reactants, and in the other (Tsuji S: Histochemistry 42:99, 1974) the potassium ferricyanide was added at the end of the staining procedure. Localizations of AChE were similar with both stains; activity was present in the nuclear envelope, the perinuclear sarcoplasm, the sarcoplasmic reticulum, subsurface vesicles and bound outside the cells. /owever, a granular artifact was found with the method of Karnovsky and Roots that did not appear with the method of Tsuji. The localization of AChE are consistent with kinetic data that AchE binds, moves and is released from cultured muscle fibers.

Growth and Metabolism of Chick Embryo Muscle Cultures

Malathion and malaoxon inhibited growth (cell number, protein, DNA) of chick embryo pectoral muscle cultures. Low-protein phosphate-buffered medium with pesticide was more toxic than high-protein, carbon dioxide-buffered medium. Growth was also inhibited by parathion and paraoxon. Malathion, but not malaoxon, inhibited the respiration of cells, tissue homogenates, and mitochondrial fractions. Oxygen consumption of intact cells was inhibited by malathion under conditions in which it was toxic to growth. The results suggested oxidation of α-ketoglutarate as one site of malathson action. Acetylcholinesterase (AChE) activity was studied by spectrophotometry, cytochemistry, and gel electrophoresis. Muscle cultures and embryo muscle tissue had similar AChE isozymes. Cells grown with malaoxon had less AChE activity than those grown with malathion. Levels of AChE activity did not correlate with toxicity of the organophosphorus agents.

Ultrastructural localization of acetylcholinesterase in cultured cells. II. Cycloheximide-treated embryo muscle.

The acetylcholinesterase activity (AChE) of cultured chick embryo muscle fibers that remains after the cells have been treated with the protein synthesis inhibitor cycloheximide was examined with cytochemical stains and the electron microscope. AChE activity that decreased rapidly after addition of the inhibitor was associated with enzyme within the cells, and AChE activity that was relatively insensitive to the inhibitor was associated with AChE outside of the cells. The results support the view that there are at least two fractions of AChE in developing muscle fibers, one intracellular and labile, the other extracelullar and stable.

Ultrastructural localization of acetylcholinesterase in cultured cells. III. DFP treated embryo muscle.

Brief treatment with 10(-4)M diisopropylfluorophosphate (DEP) irreversibly inactivates acetylcholinesterase (E.C.3.1.1.7; acetylcholine hydrolase) (AChE) activity in 10 day old chick embryonic muscle cultures. Electron microscopic cytochemistry was employed to follow the distribution of new AChE during recovery from DEP treatment. In normal 10 day cultures of embryo pectoralis muscles AChE is localized in the nuclear envelope, perinuclear sarcoplasm, sarcotubular system, subsurface vesicles and bound outside the cells. Immediately after DFP treatment AChE activity is absent in large myotubes. Within 15 min, activity is randomly present in small amounts in the sarcotubular system and nuclear envelope. There is a dramatic increase in activitv in the nuclear envelope during the 1st hr of recovery, and connections between the nuclear envelope and sarcotubular system are often seen. The next few hr of recovery show increased AChE activity. By 4 hr activity approaches that of controls. Six to 8 hr after treatment, AChE activity can be detected spectrophotometrically in the medium and can be seen bound outside the cells with the electron microscope. The spatial and temporal patterns of AChE activity demonstrate that the recovery of AChE and its mobilization and release from DFP-treated cells are not governed solely by the levels attained by the enzyme in the cultured embryo muscle.

Recovery of acetylcholinesterase forms in quail muscle cultures after intoxication with diisopropylfluorophosphate

Studies of recovery of acetylcholinesterase (AChE, EC 3.1.1.7) after inhibition by organophosphates (OPs) have been hampered by the low number of in vitro systems with large collagen-tailed forms of AChE characteristic of motor end plates. Pectoral muscle cultures from Japanese quail with high levels of a large 20S form of AChE were used to study recovery of cells from acute treatment with diisopropylfluorophosphate (DFP), an irreversible AChE inhibitor. Low molecular weight AChE forms were synthesized more rapidly than the large 20S form following a 15-min treatment with 10(-4)M DFP. Most of the activities of the small forms, but only part of the activity of the 20S form, disappeared within 48 hr after cycloheximide was added to block protein synthesis. To the contrary, virtually all the activity of the 20S AChE that was newly synthesized after DFP treatment was lost within 24 hr after cycloheximide treatment. The results were generally consistent with the idea that the 20S AChE form is assembled inside the cell near to its surface and then is released to bind to its outside.

Ryanodine induces maturation of embryonic acetylcholinesterase forms in cultured quail myotubes

[3H]Ryanodine is shown to specifically bind to cultured myotubes from 10 day quail embryo pectoralis. The binding of [3H]ryanodine increases in a time-dependent manner reaching 38 +/- 3 fmol/mg protein at 4 h. A level of theophylline (THEO; 5mM) that induces propagated wave-like contractures, doubles the capacity of the myotubes to bind [3H]ryanodine (78 +/- 7 fmol/mg protein at 4 h). Polycationic ruthenium red (100 microM) only partially inhibits (56%) [3H]ryanodine-binding, whereas the membrane permeable channel antagonist [2,6-dichloro-4-dimethyl-amino-phenyl]-isopropylamine (20 microM) inhibits occupancy > 80%. Ryanodine (10 microM) interferes with THEO-induced contractures. Pretreatment with micromolar ryanodine for 48 h, followed by washout for 48 h, causes a persistent decrease in [3H]ryanodine-binding sites. Persistent [3H]ryanodine receptor blockade coincides with a dramatic shift in AChE forms found in the myotubes. A transition from the embryonic 4S and 7S globular forms to the 20S collagen-tailed (adult) form is evident within 12 hr exposure to ryanodine and progresses after removal of the alkaloid from the culture medium, mimicking the transition that normally occurs during myocyte maturation in vivo. These results suggest that SR Ca++ movements and excitation-contraction coupling may, at least in part, contribute to AChE maturation.

ACTIONS OF PYRIDOSTIGMINE AND ORGANOPHOSPHATE AGENTS ON CHICK CELLS, MICE, AND CHICKENS*

ABSTRACT Gulf War veterans were given pyridostigmine bromide (PB) tablets to enhance the therapeutic effect of antidotes to nerve agents in the event of exposure. The goal of this research is to examine whether combined exposure to PB and sarin (agent GB) is more neurotoxic to sensitive surrogate animals, mice and chickens, than if given separately. Scoping trials were performed to establish appropriate dose-response ranges for sarin and control chemicals. IC50 values were determined in chickens and mice for in vitro inhibition of acetylcholinesterase (AChE) and neuropathy target esterase (NTE). The results indicated PB neither inhibits NTE nor does it spare sarins inhibition of AChE. Chick embryo nerve cells in vitro showed more inhibition of AChE activity and no faster recovery when PB treatment was followed by DFP treatment than the other way around. Experiments on chickens also indicated that PB treatment did not inhibit NTE and that it crossed the blood brain barrier inhibiting brain AChE although to a lesser extent than it inhibited blood cholinesterases. Other experiments determined multiple dose levels in chickens for sarin and DFP that inhibited >80% of NTE, considered a threshold for triggering organophosphate-induced delayed neuropathy.