Title: In Vivo Regulation of Acetylcholinesterase in Slow and Fast Muscle of Rat Dettbarn WD, Groswald DE, Gupta RC, Misulis KE, Patterson GT Ref: In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology, (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC:71, 1991 : PubMed
Title: Comparison of cholinergic and neuromuscular toxicity following acute exposure to sarin and VX in rat Gupta RC, Patterson GT, Dettbarn WD Ref: Fundamental & Applied Toxicology, 16:449, 1991 : PubMed
Male Sprague-Dawley rats injected with a sublethal sc dosage of 110 micrograms/kg of sarin (isopropyl methylphosphonofluoridate), or 12 micrograms/kg of VX (S-(2-diisopropylaminoethyl) O-ethyl methylphosphonothioate), developed severe toxic signs within 5-15 min after sarin and 20-50 min after VX lasting for 5 to 7 hr. Myonecrotic lesions were seen in soleus and diaphragm muscles within 1 hr. A maximum number of lesions had developed after 24 hr, and lesions were also present in extensor digitorum longus (EDL) at this time. Regeneration of muscle fibers was slow since lesions were still evident past 7 days of treatment. Within 1 hr following VX, AChE activity was reduced to 8, 12, and 17% of control activity in soleus, diaphragm, and EDL, respectively, whereas with sarin the enzyme activity was reduced to 23, 48, and 82% of control. A still greater inhibition was seen 24 hr after sarin when AChE activity was reduced to 19, 13, and 43% in these muscles. In skeletal muscles the different molecular forms of AChE, such as 16 S, 12 S, 10 S, and 4 S vary in location and functional importance with the 16 S form highly concentrated at the neuromuscular junction. All forms in a given muscle were equally sensitive to the inhibitors. In EDL, sarin was the least effective in reducing AChE or its molecular forms. In the brain structures (cortex, brain stem, striatum, and hippocampus), AChE activity was reduced to 1-6% of control by sarin and VX with the exception that following VX striatal AChE was reduced to only 41% of control activity. AChE activity in the brain cortex following either of the agents was maximally affected (1%). A slow but significant recovery of brain AChE was evident after 24 hr and more so after Day 7. Butyrylcholinesterase (BCHE) activity was less sensitive to inhibition by both inhibitors compared to AChE activity and showed a rapid recovery. Based on the equitoxic doses (toxic signs of similar magnitude), VX was found to be 10 times more toxic than sarin. The mechanisms of this disparity may be due to differences in rate of uptake, circulation, susceptibility to hydrolysis, and reactivity with nonspecific binding sites.
Title: Biochemical and histochemical alterations following acute soman intoxication in the rat Gupta RC, Patterson GT, Dettbarn WD Ref: Toxicol Appl Pharmacol, 87:393, 1987 : PubMed
Rats injected with a nonlethal acute dose (100 micrograms/kg, sc) of soman (pinacolyl methylphosphonofluoridate) exhibited signs of anticholinesterase toxicity beginning at 5-15 min with increasing severity and lasting for 4-6 hr. Generalized tremors and seizure activity indicated comparatively greater involvement of the central cholinergic system than peripheral neuromuscular effects. During peak toxicity, all the brain regions tested showed more than 95% inhibition of acetylcholinesterase (AChE) activity. The cortex area was maximally affected (99% inhibition). Among skeletal muscles, soleus AChE was most severely affected (94%) and extensor digitorum longus (EDL) the least (72%). Inhibition of EDL AChE occurred at a much slower rate than in brain and other muscles. Significant recovery of AChE activity was seen by 48-72 hr after soman treatment in both brain and skeletal muscles. By Day 7, recovery was virtually complete in skeletal muscles but not in brain, although significant recovery had occurred by this time. Muscle fiber necrosis developed within 6 hr in the soleus and diaphragm, while no necrotic fibers were found in the EDL. The 16 S AChE molecular form showed the fastest recovery of the AChE isozymes in all three muscles. Full recovery was seen after 7 days in soleus and was increased to greater than control activity in diaphragm and EDL. The inhibition pattern of butyrylcholinesterase (BCHE) activity was similar to that described for AChE activity, but the recovery was comparatively faster. Carboxylesterase activity in plasma was decreased to less than 10% of control within 1 hr and recovered to 53% of control within 24 hr. No significant inhibition was seen in hepatic carboxylesterase activity. It can be concluded that soman-induced acute toxicity is directly related to the rate and degree of AChE inhibition. A significant amount of soman binds to non-AChE enzymes with serine sites such as BCHE and carboxylesterases.
Title: Acute tabun toxicity; biochemical and histochemical consequences in brain and skeletal muscles of rat Gupta RC, Patterson GT, Dettbarn WD Ref: Toxicology, 46:329, 1987 : PubMed
Male Sprague-Dawley rats injected s.c. with an acute non-lethal dose (200 micrograms/kg) of ethyl N,N-dimethylphosphoramidocyanidate (tabun) showed onset of hypercholinergic activity within 10-15 min. The maximal severity of toxicity signs was evident within 0.5-1 h and persisted for 6 h. Except for mild tremors no overt toxicity signs were evident after 24 h. Within 1 h a dramatic decline of acetylcholinesterase (AChE) activity occurred in all the brain structures (less than 3%) and skeletal muscles (less than 10% in soleus and hemi-diaphragm; and 32% in extensor digitorum longus (EDL)). No significant recovery was seen up to 48-72 h. Within 7 days rats became free of toxicity signs and AChE activity had recovered to about 40% in brain structures (except cortex, 14%) and 65-70% in skeletal muscles. Within 1 h the 16 S molecular form of AChE located at the neuromuscular junction was most severely inhibited in soleus, followed by hemi-diaphragm and least in the EDL, and had fully recovered in all the muscles when examined after day 7. Muscle fiber necrosis developed within 1-3 h in soleus and hemi-diaphragm and after a delay of 24 h in EDL. The highest number of necrotic lesions in all muscles was seen at 72 h with the hemi-diaphragm maximally affected and EDL the least. To determine detoxification of tabun by non-specific binding, the activity of butyrylcholinesterase (BCHE) and carboxylesterase (CarbE) was measured. The inhibition and recovery pattern of BCHE activity was quite similar to that of AChE, except that the rate of recovery was more rapid. Within 1 h the remaining activity of CarbE was 10% in plasma, about 30% in brain structures, and 79% in liver; recovery was complete within 7 days. The inhibition of BCHE and CarbE can serve as a protective mechanism against tabun toxicity by reducing the amount available for AChE inhibition. The prolonged AChE inhibition in muscle and brain may indicate storage of tabun and delayed release from non-enzymic sites. Since tabun is a cyanophosphorus compound, the toxic effects from the released cyanide (CN) could be another reason for the delayed recovery after tabun.
Title: Serum regulation of acetylcholinesterase in cultured myotubes Patterson GT, Wilson BW Ref: Experimental Neurology, 91:308, 1986 : PubMed
A large (20S) collagen-tailed form of acetylcholinesterase associated with the neuromuscular junction appears in cultures of chick embryo muscle cells when horse serum is withdrawn from the medium. In this report, 10-day-old cultures were incubated 2 days in serum-free medium or in medium containing either horse, bovine, fetal calf, chicken, heat-treated horse or chicken serum, low (less than 100K) or high (less than 100K) molecular weight fractions of horse serum, or fibronectin. Total acetylcholinesterase activity and activity of the 20S form increased in medium without serum, with fetal calf serum and with the low-molecular-weight fraction of horse serum. The largest increase occurred with fibronectin. The results suggest that a factor(s) greater than 100K in adult sera inhibits total acetylcholinesterase production and formation of the 20S form of the enzyme.
Title: Mechanisms involved in the development of tolerance to DFP toxicity Gupta RC, Patterson GT, Dettbarn WD Ref: Fundamental & Applied Toxicology, 5:S17, 1985 : PubMed
Rats treated daily with diisopropylfluorophosphate (DFP) (0.5 mg/kg, sc), an inhibitor of acetylcholinesterase (AChE) activity, exhibited the symptoms of cholinergic hyperactivity between Days 3 and 5 similar to those observed 15 min after a single acute dosage (1.5 mg/kg, sc). A significant (p less than 0.05) decrease in the activities of both AChE and cholinesterase (BuChE) (greater than 80%) occurred in muscles and in brain regions and of aliesterases in liver (greater than 92%) at this time. Further administration of DFP (0.5 mg/kg, for 7-14 days) led to behavioral tolerance, where symptoms of toxicity disappeared such as muscle fasciculations, tremors, and muscle necrosis. The activity of aliesterases in liver and AChE in muscles significantly (p less than 0.01) recovered, while no such recovery was seen in brain AChE. DFP toxicity was potentiated in rats that were pretreated with BuChE inhibitors, such as iso-OMPA (3 mg/kg, sc) or mipafox (0.05 mg/kg, sc), 30 min prior to DFP (0.5 mg/kg, sc). The severity of cholinergic hyperactivity and inhibition of aliesterase in liver, AChE and BuChE activity in brain and muscles was greater when compared to the effects of DFP alone. Both iso-OMPA and mipafox completely abolished the tolerance development to DFP, since no animal survived more than 5 days of combined treatment. The observed adaptation to DFP toxicity appears to be due to recovery of aliesterase, BuChE, and AChE activity as well as decreased nicotinic binding sites at the neuromuscular junction, as previously reported.
Title: Unifying mechanisms in AChE regulation and organophosphate induced neuropathy of the chicken Wilson BW, Ishikawa Y, Chow E, McNamee MG, Randall WR, Patterson GT, Entrikin RK, Nieberg PS Ref: In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases, (Brzin M, Barnard EA, Sket D, Eds) De Gruyter:493, 1984 : PubMed
High molecular forms of acetylcholinesterase (AChE) have been difficult to obtain in primary cultures of muscle from mammals and birds. This research used pectoral muscle cultures from Japanese quail and chick embryos grown in medium containing embryo extract and horse serum to study the growth and extraction conditions necessary for study of high molecular weight 20S AChE forms found in these birds. The results confirmed the presence of a 20S AChE form in quail muscle cultures and showed that its extraction from the cells was considerably improved by using a Mg-cholate extraction buffer rather than the more commonly used NaCl-Triton X buffer. A striking finding was that removal of serum from the medium for 1-2 days caused the preferential increase in a 20S form in the quail and the resolution of one from background activity in chick muscle cultures. Removal of embryo extract had no effect on levels of the AChE forms. The results suggest that serum factors inhibit the formation of the high molecular weight, motor end plate associated form of AChE in aneural cultures of avian muscle, and that effects of factors such as neural extracts and nerves on AChE forms in cultured muscle should be examined using a defined basal media.
Title: Phenytoin, methysergide, and penicillamine in hereditary muscular dystrophy of the chicken Entrikin RK, Patterson GT, Wilson BW Ref: Experimental Neurology, 72:82, 1981 : PubMed
Title: Major physiologic and histochemical characteristics of inherited dystrophy of the chicken. Wilson BW, Randall WR, Patterson GT, Entrikin RK Ref: Annals of the New York Academy of Sciences, 317:224, 1979 : PubMed
Chicks affected with hereditary muscular dystrophy were injected twice daily with 20 milligrams of diphenylhydantoin per kilogram of body weight on days 1 to 40 after hatching. The righting ability of dystrophic chicks treated with diphenylhydantoin was improved compared to that of untreated dystrophic chicks, and acetylcholinesterase activity was reduced to normal levels in the posterior latissimus dorsi muscles.
Title: Distribution of acetylcholinesterase activity in normal, dystrophic, and denervated muscles of the chicken Patterson GT, Wilson BW Ref: Experimental Neurology, 52:250, 1976 : PubMed
Title: Distribution of extrajunctional acetylcholinesterase in muscle of normal and dystrophic chickens Patterson GT, Wilson BW Ref: Experimental Neurology, 50:214, 1976 : PubMed
Fifty human muscle biopsies were examined for histochemical localization of acetylcholinesterase (AChE) activity. Six normal muscle samples had AChE at the myoneural junctions and around the periphery of many fibers. The AChE within the sarcoplasm itself was found in only a few atrophied fibers. However, 21 of 44 biopsies of abnormal muscles had sarcoplasmic AChE in more than 10% of their fibers. Such cases included Duchenne, limb-girdle and facio-scapulo-humeral dystrophy, neurogenic and spinal muscle atrophy, spinal cord injury, peripheral nerve injury, Schwartz-Jampel syndrome and myasthenic syndrome. Sarcoplasmic AChE is found in embryo muscle and usually declines after birth. It appears after denervation in the chicken but not the rat and remains in muscles of chickens with an inherited muscular dystrophy. The results of the human muscle study support the idea that in the human, as in the chicken, interruption of a neurally-mediated regulation of AChE results in the reappearance of high AChE activity in the sarcoplasm of the muscle fibers.
Fifty-nine biopsies of human muscle, 53 of them abnormal, 6 normal, were studied for the histochemical localization of acetylcholinesterase (AChE) using frozen sections and light microscopy. In addition to AChE which was found at the myoneural and myotendon junction, specific staining was found around the periphery of many fibers from normal and abnormal muscles. Moreover, AChE activity was found to be high in the sarcoplasm of more than 10% of the fibers from 28 biopsies of abnormal muscle including cases of hemiplegia, spinal cord injury, denervation and neuropathy, infantile spinal muscle atrophy, Duchenne, limb-girdle and facioscapulohumeral dystrophies, Schwartz-Jampel syndrome and a myasthenic syndrome. Of the muscles from experimental animals examined, only the Rhesus monkey exhibited AChE around the periphery of the fibers, and only the dystrophic chicken and not the dystrophic mouse or hamster, showed extensive sarcoplasmic AChE. Histograms of muscle fiber diameters indicated that AChE in the sarcoplasm was associated with fibers of all sizes, depending on the nature of the disorder examined. Fibers containing AChE were smaller than unstained fibers in dystrophic chicken muscle. The results suggest that in the human, sarcoplasmic AChE is reversibly repressed during muscle maturation and that its mode of regulation by motor neurons is similar to that found in the chicken.
