Diaphragm Acetylcholinesterase Research Articles

Post-VX exposure treatment of rats with engineered phosphotriesterases

The biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M−1 min−1) towards VX and a preferential hydrolysis of the more toxic P(−) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD50) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg−1 i.v.: PTE-2 dosed at 1.3 mg kg−1 i.v. (PTE-2.1) and 2.6 mg kg−1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg−1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.

A comparison of the reactivating and therapeutic efficacy of two novel bispyridinium oximes (K727, K733) with the oxime HI-6 and obidoxime in sarin-poisoned rats and mice

The ability of two novel bispyridinium oximes K727 and K733 and currently available oximes (HI-6, obidoxime) to reactivate sarin-inhibited acetylcholinesterase and to reduce acute toxicity of sarin was evaluated. To investigate the reactivating efficacy of the oximes, the rats were administered intramuscularly with atropine and oximes in equitoxic doses corresponding to 5% of their LD50 values at 1 min after the intramuscular administration of sarin at a dose of 24 µg/kg (LD50). The activity of acetylcholinesterase was measured at 60 min after sarin poisoning. The LD50 value of sarin in non-treated and treated mice was assessed using probit-logarithmical analysis of death occurring within 24 h after intramuscular administration of sarin at five different doses. In vivo determined percentage of reactivation of sarin-inhibited rat blood, diaphragm and brain acetylcholinesterase showed that the potency of both novel oximes K727 and K733 to reactivate sarin-inhibited acetylcholinesterase roughly corresponds to the reactivating efficacy of obidoxime. On the other hand, the oxime HI-6 was found to be the most efficient reactivator of sarin-inhibited acetylcholinesterase. While the oxime HI-6 was able to reduce the acute toxicity of sarin >3 times, both novel oximes and obidoxime decreased the acute toxicity of sarin <2 times. Based on the results, we can conclude that the reactivating and therapeutic efficacy of both novel oximes K727 and K733 is significantly lower compared to the oxime HI-6 and, therefore, they are not suitable for the replacement of the oxime HI-6 for the antidotal treatment of acute sarin poisoning.

The evaluation of the reactivating and therapeutic efficacy of three novel bispyridinium oximes (K454, K456, K458) in comparison with the oxime K203 and trimedoxime in tabun-poisoned rats and mice

The potency of three newly developed bispyridinium compounds (K454, K456, K458) to reactivate tabun-inhibited acetylcholinesterase and reduce tabun-induced lethal toxic effects was compared with the oxime K203 and trimedoxime using in vivo methods. The study determining percentage of reactivation of tabun-inhibited diaphragm and brain acetylcholinesterase in poisoned rats showed that the reactivating efficacy of all newly developed oximes is comparable with K203 but lower than the reactivating potency of trimedoxime in diaphragm. In the brain, their potency to reactivate tabun-inhibited acetylcholinesterase is lower compared with trimedoxime and the oxime K203. All three newly developed oximes were also found to be relatively effective in reducing lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy is consistent with the therapeutic potency of the oxime K203. On the other hand, their potency to reduce acute toxicity of tabun is significantly lower compared with trimedoxime. In conclusion, the reactivating and therapeutic potency of all three newly developed oximes does not prevail the effectiveness of the oxime K203 and trimedoxime and, therefore, they are not suitable for their replacement of commonly used oximes for the treatment of acute tabun poisoning.

Different sensitivities of rat skeletal muscles and brain to novel anti‐cholinesterase agents, alkylammonium derivatives of 6‐methyluracil (ADEMS)

The rat respiratory muscle diaphragm has markedly lower sensitivity than the locomotor muscle extensor digitorum longus (EDL) to the new acetylcholinesterase (AChE) inhibitors, alkylammonium derivatives of 6-methyluracil (ADEMS). This study evaluated several possible reasons for differing sensitivity between the diaphragm and limb muscles and between the muscles and the brain. Increased amplitude and prolonged decay time of miniature endplate currents were used to assess anti-cholinesterase activity in muscles. In hippocampal slices, induction of synchronous network activity was used to follow cholinesterase inhibition. The inhibitor sensitivities of purified AChE from the EDL and brain were also estimated. The intermuscular difference in sensitivity to ADEMS is partly explained caused by a higher level of mRNA and activity of 1,3-bis[5(diethyl-o-nitrobenzylammonium)pentyl]-6-methyluracildibromide (C-547)-resistant BuChE in the diaphragm. Moreover, diaphragm AChE was more than 20 times less sensitive to C-547 than that from the EDL. Sensitivity of the EDL to C-547 dramatically decreased after treadmill exercises that increased the amount of PRiMA AChE(G4), but not ColQ AChE(A12) molecular forms. The A12 form present in muscles appeared more sensitive to C-547. The main form of AChE in brain, PRiMA AChE(G4), was apparently less sensitive because brain cholinesterase activity was almost three orders of magnitude more resistant to C-547 than that of the EDL. Our findings suggest that ADEMS compounds could be used for the selective inhibition of AChEs and as potential therapeutic tools.

The influence of combinations of oximes on the reactivating and therapeutic efficacy of antidotal treatment of soman poisoning in rats and mice

The influence of the combination of oximes on the reactivating and therapeutic efficacy of antidotal treatment of acute soman poisoning was evaluated. The ability of two combinations of oximes (HI-6 + trimedoxime and HI-6 + K203) to reactivate soman-inhibited acetylcholinesterase and reduce acute toxicity of soman was compared with the reactivating and therapeutic efficacy of antidotal treatment involving single oxime (HI-6, trimedoxime, K203) using in vivo model. Studies determining percent of reactivation of soman-inhibited blood and diaphragm acetylcholinesterase in poisoned rats showed that the reactivating efficacy of both combinations of oximes is slightly greater than the reactivating efficacy of the most effective individual oxime, but the difference among them is not significant. Both combinations of oximes were found to be as effective in the reduction of acute lethal toxic effects in soman-poisoned mice as the antidotal treatment involving the most efficacious individual oxime. Thus, the efficacy of oximes is comparative in rats vs mice. A comparison of reactivating and therapeutic efficacy of individual oximes showed that the newly developed oxime K203 is approximately as effective as commonly used trimedoxime; nevertheless, their reactivating and therapeutic efficacy is markedly lower compared to the oxime HI-6. Based on the obtained data, one can conclude that the antidotal treatment involving chosen combinations of oximes does not significantly influence the potency of the most effective individual oxime (HI-6) to reactivate soman-inhibited rat acetylcholinesterase and to reduce acute toxicity of soman.

Comparative studies of two organophosphorus compounds in the mouse

A rodent model, the albino mouse, was used to investigate the in vitro and in vivo capacity of 2 organophosphate (OP) compounds, mipafox and ecothiopate, to inhibit enzymes considered to be involved in the mechanisms of OP toxicity. Mipafox and ecothiopate were chosen as model compounds because the former can produce a delayed neuropathy whereas the latter does not. Mipafox (110 μmol/kg s.c.) inhibited brain acetylcholinesterase (AChE), neuropathy target esterase (NTE) and phenylvalerate hydrolases by 58, 64 and 65%, while diaphragm AChE and phenylvalerate hydrolases were inhibited by 66 and 80%, respectively. In contrast, ecothiopate (0.5 μmol/kg) had no effect on brain NTE or on brain or diaphragm phenylvalerate hydrolases. At the same time, diaphragm AChE was inhibited by 60% while brain AChE activity had increased by 15% of control. Mipafox was a potent inhibitor of AChE and NTE in vitro. Although ecothiopate was a highly potent anti-ChE in vitro, it had no inhibitory effect on NTE.

Effect of pyridostigmine pretreatment on cardiorespiratory function in tabun poisoning

1. The effect of pyridostigmine on cardiorespiratory function after oxime + atropine injection was investigated in tabun poisoned guinea-pigs and without tabun poisoning. 2. The trachea, a carotid artery and jugular vein were cannulated in female urethane-anaesthetised Pirbright-white guinea-pigs. After baseline measurements the animals received pyridostigmine (0.05 mumol kg-1) and 30 min later atropine (29.5 mumol kg-1) plus obidoxime, HI 6 or HLö 7 (30 or 100 mumol kg-1) or tabun (1.85 mumol kg-1 = 5 x LD50) followed by oxime + atropine treatment (all i.v.). Erythrocyte, brain and diaphragm acetylcholinesterase (AChE) activity were determined. Similar groups without pretreatment were included for comparison. 3. Pyridostigmine aggravated the oxime + atropine induced hypotension and prevented the increase in heart rate but not the respiratory stimulation. The pyridostigmine inhibited AChE recovered only in the 100 mumol kg-1 kg oxime groups at the end of the experiment. 4. In tabun poisoning, pyridostigmine reduced the oxime + atropine induced circulatory recovery and decreased the survival time and rate. It did not affect the therapeutic oxime + atropine effect on respiratory function. 5. These results suggest that pyridostigmine enhances oxime + atropine related circulatory depression which may be the reason for the reduced efficacy of oxime + atropine treatment in tabun poisoning. The possible mechanisms are discussed.

Paraoxonase protects against chlorpyrifos toxicity in mice

Paraoxonase can hydrolyze paraoxon (PO), chlorpyrifos-oxon (CPO) and other organophosphates. Previous studies have indicated that the levels of serum paraoxonase can influence the toxicity of PO and CPO. In the present study we have investigated whether exogenous paraoxonase administered to mice would offer protection toward the acute toxicity of a phosphorothioate, chlorpyrifos (CPS). Paraoxonase was purified from rabbit serum and injected i.v., or i.v. plus i.p., in mice. Inhibition of acetylcholinesterase (AChE) in brain, diaphragm, plasma and red blood cells was measured as an index of CPS (100 mg/kg) toxicity. Administration of paraoxonase 30 min before CPS increased plasma enzyme activity toward CPO by 35-fold, and protected against its toxicity; protection was still present at 24 h, when enzyme activity was still 20-fold over basal. When paraoxonase was given 30 min after CPS, a significant protection against CPS toxicity was still observed, while after 3 h the protective effect was decreased. To mimic conditions of severe acute poisoning, a higher dose of CPS (150 mg/kg) was also administered. Administration of paraoxonase 30 min after this exposure abolished cholinergic signs and significantly protected against AChE inhibition. These results indicate that exogenous paraoxonase offers significant protection against CPS toxicity when administered both before and after the organophosphate, suggesting that it may be considered as a potential additional treatment of organophosphate poisoning.

Cardiorespiratory function in nerve agent poisoned and oxime + atropine treated guinea-pigs: effect of pyridostigmine pretreatment.

The effect of pyridostigmine pretreatment on the efficacy of oxime + atropine treatment in sarin and VX poisoning was investigated in a guinea-pig model with on-line monitoring of respiratory and circulatory parameters. The carotid artery, jugular vein and trachea were cannulated in female urethane-anesthetized Pirbright-white guinea-pigs. After baseline measurements the animals received pyridostigmine (PYR, 0.05 mumol/kg, i.v.), 30 min later sarin (100 or 200 micrograms/kg = 5 or 10 x LD50, i.v.) or VX (45 or 90 micrograms/kg = 10 or 20 x LD50, i.v.), followed 2 min later by atropine (10 mg/kg, i.v.) plus HI 6 or HLö 7 (30 mumol/kg each, i.v.). Sixty-minute survival time and rate and respiratory and circulatory parameters were recorded. Diaphragm acetylcholinesterase (AChE) activity was determined spectrophotometrically. Identical groups without PYR were included for comparison. With regard to survival time and rate, PYR pretreatment slightly improved the efficacy of HI 6 plus atropine in sarin 5 x LD50 poisoned animals, reduced the efficacy of oxime + atropine treatment in the other sarin groups and had no effect in VX poisoning. Compared to non-pretreated oxime + atropine groups, PYR slightly improved respiratory function in sarin and in VX poisoning (only HI 6). PYR did not affect circulatory function in VX poisoning but reduced circulatory parameters in sarin poisoning to varying extent's. The oxime efficacy in reactivating diaphragm AChE decreased in the order sarin > VX without significant differences between pretreated and non-pretreated groups. The data suggest that pyridostigmine pretreatment does not enhance the efficacy of oxime + atropine in sarin or VX poisoning.

Effect of atropine and bispyridinium oximes on respiratory and circulatory function in guinea-pigs poisoned by sarin

During the past decade the oxime HI 6(l-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyimino)methyl] pyridinium dichloride) was shown to improve survival in nerve agent poisoning (in combination with atropine). Recent studies indicate, that HLö 7 (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2,4-bis[(hydroxyimino)methyl]pyridinium diiodide or dimethanesulfonate) is also an effective antidote in nerve agent poisoning but, with both oximes, data on restoration of respiration and circulation are scarce. The ability of HLö 7 or HI 6 with atropine to improve the respiratory and circulatory function in sarin-poisoned guinea-pigs was therefore investigated. Female Dunkin-Hartley guinea-pigs were anaesthetised with urethane (1.8 g/kg) and the arteria carotis, vena jugularis and trachea were cannulated. After baseline measurements the animals received 100 or 200 μg/kg sarin, and 2 min later the antidotes (all i.v.): 10 mg/kg atropine sulfate or a combination of atropine and HLö 7 or HI 6 (30 μmnol/kg, each). Respiratory and circulatory parameters were recorded for the whole experimental period of 60 min or until the death of the animal. Brain and diaphragm acetylcholinesterase (AChE) activity was determined in each animal after the experiment. Poisoning by sarin resulted in a rapid respiratory arrest within 5 min. Atropine treatment was only partially effective in improving respiration after 100 μg/kg sarin but was ineffective after 200 μg/kg sarin. Therapy of sarin-poisoned animals with atropine plus oxime further improved respiration to various extents, restored circulation and increased survival time, HLö 7 being more effective than HI 6. Diaphragm and brain AChE were reactivated by HLö 7 and, to a minor extent, by HI 6. The results of this investigation suggest, that at equimolar doses (30 μmol/kg) the new bispyridinium dioxime HLö 7 has a higher therapeutic efficacy in sarin-poisoned guinea-pigs when compared to HI 6 (both in combination with atropine).

Effect of Atropine, HLö 7 and HI 6 on Respiratory and Circulatory Function in Guinea-pigs Poisoned by O-ethyl S-[2-(diisopropylamino) ethyl] Methylphosponothioate (VX)

In a guinea-pig model with on-line respiratory and circulatory monitoring the therapeutic efficacy of atropine, HLö 7 and HI 6 in VX poisoning was compared. In female urethane-anaesthetized Pirbright-white guinea-pigs the a. carotis, v. jugularis and trachea were cannulated. After base line measurements the animals received VX (22.5, 45 or 90 micrograms/kg = 5, 10 or 20 x LD50) intravenously and 2 min. later the antidotes: HLö 7 or HI 6 (30 mumol/kg, each) or atropine 10 mg/kg or a combination of atropine and one of the oximes (all intravenously). Respiratory and circulatory parameters were recorded for 60 min. or until death of the animal. Erythrocyte, brain and diaphragm acetylcholinesterase (AChE) activity was determined after the experiment. VX poisoning caused a rapid respiratory arrest within 4-5 min. Atropine treatment was effective in improving the respiratory function after VX, 22.5 micrograms/kg, but had only a small effect after the higher VX doses. The treatment of VX (10 or 20 x LD50) poisoned animals with oxime plus atropine improved respiration to various extents, restored circulation and prolonged the survival time, HLö 7 being more effective than HI 6 after VX 90 micrograms/kg. Oximes alone were completely ineffective. Erythrocyte and diaphragm AChE was reactivated by HLö 7 and, less effectively, by HI 6, while brain AChE remained almost completely inhibited in all groups. The results of this investigation demonstrate a reasonable efficacy of atropine after lower VX doses and of HLö 7 and HI 6 (plus atropine) after high-dose VX poisoning, HLö 7 being slightly more effective than HI 6.

Electrophysiological and biochemical effects following single doses of organophosphates in the mouse.

Single doses of organophosphates (mipafox or ecothiopate) were given subcutaneously to mice. At intervals up to 77 days after dosing animals were killed and muscle action potentials and endplate potentials were recorded intracellularly in mouse phrenic-nerve/hemidiaphragm preparations. Activities of acetylcholinesterase and neuropathy target esterase in brain and acetylcholinesterase in diaphragm were also measured. Mipafox (0.11 mmol/kg), a neurotoxic organophosphate, produced an increase in prejunctional jitter (i.e. the variabilities of the latencies) of endplate potentials. This increase began 14-21 days after administration and lasted more than 23 days. No clinical signs of neuropathy were observed during this study. Mipafox also produced an increase in postjunctional (muscle action potential) jitter. Mipafox inhibited brain and diaphragm acetylcholinesterase and brain neuropathy target esterase. By comparison, a non-neurotoxic organophosphate, ecothiopate (0.5 mumol/kg), was a potent inhibitor of diaphragm acetylcholinesterase and produced a large increase in postjunctional jitter but ecothiopate did not inhibit brain neuropathy target esterase and had no effect on prejunctional jitter. Doses were chosen so that the inhibition of diaphragm acetylcholinesterase by each of the two organophosphates was similar. It is concluded that the neurotoxic organophosphate, mipafox, produced measurable changes in nerve function. These long-term changes may represent a new phenomenon, unrelated to the classical organophosphate induced delayed neuropathy. Alternatively, they may represent a neuropathic process which precedes or is below the threshold for clinical signs.

Chromodacryorrhea in Rats: Absence Following Soman Poisoning

Chromodacryorrhea is the secretion of so-called "bloody tears" from the harderian gland which nearly circumscribes the eye within the bony orbit. Direct-acting cholinergic agonists such as oxotremorine, carbachol, and pilocarpine caused chromodacryorrhea but nicotine did not. Atropine blocked chromodacryorrhea induced by systemic administration of direct-acting cholinergic agonists. Thus, chromodacryorrhea appears to be a muscarinic receptor-related event. Soman (pinacolyl methylphosphonofluoridate), a potent irreversible inhibitor of acetylcholinesterase which increases the synaptic concentration of the neurotransmitter acetylcholine, did not induce chromodacryorrhea in rats. Similarly, physostigmine, a tertiary, carbamate acetylcholinesterase inhibitor, did not induce chromodacryorrhea. In vivo soman-induced inhibition of harderian gland acetylcholinesterase was independent of the soman dose and the inhibition was significantly less than brain acetylcholinesterase. In vitro soman-induced inhibition of harderian gland acetylcholinesterase was not significantly different from that of diaphragm acetylcholinesterase. The lack of inhibition of acetylcholinesterase in the harderian gland does not appear to be due to a difference in sensitivity to inhibition by soman. The distribution of the various molecular forms of acetylcholinesterase between the diaphragm and harderian gland was different. There was a great deal more of the 4S form of acetylcholinesterase in the harderian gland than in the diaphragm. The lack of the following, inhibition of harderian gland acetylcholinesterase and elevation of the synaptic concentration of acetylcholine, could explain the absence of chromodacryorrhea following soman poisoning. The discrepancy between the significant soman induced inhibition of brain acetylcholinesterase and the lack of inhibition of harderian gland acetylcholinesterase allows one to speculate that there may be a very efficient scavenger of soman present in the rat harderian gland.

Biochemical Changes Associated with Muscle Fibre Necrosis after Experimental Organophosphate Poisoning

1. This study was initiated to ascertain the possibility of biochemically monitoring the rhabdomyonecrosis that occurs after organophosphate poisoning. The evolution of different parameters has been assessed in the rat 6, 16, 24 and 48 h following 0.67 x LD50 of soman. 2. Acetylcholinesterase (AChE) was inhibited to 60% of the control value in the diaphragm at 6 and 16 h and serum ChE levels inhibited to an average of 30% of the control value. At 24 h, total blood, brain and diaphragm AChE were inhibited by 40, 69 and 38%, respectively. 3. Rhabdomyonecrosis lesions occurred in the diaphragm after 24 h and were accompanied by a concurrent increase in urinary creatine excretion rate (300% of the control) and serum total creatine phosphokinase activity (280% of the control). Calcium-activated neutral protease and phosphorylase a activities were elevated in the muscle at the same time. 4. These biochemical markers will prove useful for investigating the possible relationships between the different neuromuscular syndromes occurring in the course of an OP poisoning and potential therapeutic or protective pharmacological measures.

Diaphragm acetylcholinesterase multimeric forms in mice in response to burn trauma.

This report continues our investigations concerning relative changes in acetylcholinesterase (AChE) activity in response to burn trauma. This experiment was performed in mouse diaphragm. AChE is fundamental to the operating efficiency of the neuromuscular junction. It is through such junctions that all skeletal muscle contraction occurs. This paper tests the hypothesis that burn trauma induces changes in diaphragm AChE with respect to total content, specific molecular forms, and relationships of the polymorphic forms to each other. After mice were exposed to increasing size of body surface area (20%, 30%, and 50%) diaphragms were excised at 3 weeks after burn injury. AChE activity was measured by differential extraction and velocity sedimentation on sucrose gradients. In the sham-treated control groups most of the AChE activity consisted entirely (92%) of the globular forms (S1 and S2). Asymmetric (S3 and S4) and nonextractable (H5) forms made up 2% and 6% of the AChE activity, respectively. In the burn groups all total globular activity was decreased by 1/6 to 1/3 of control groups. For these groups the asymmetric forms (S3 and S4) were increased ninefold to fourteenfold. Nonextractable forms (S5) among the groups were not different. Individual soluble globular forms (S1: 4S, 6.5S, 10S) were reduced by 50% for only the 20% and 30% BSA groups. Residual globular forms (S2) were increased threefold to ninefold. Some of the asymmetric forms (S3: 4S, 10S, 12S, and 16S) were increased fivefold to eightfold among the burn groups. Residual asymmetric (S4) and nonextractable (S5) forms were not different. This report shows that in response to burn trauma the AChE activity of the diaphragm and the interdependence of the polymorphic forms were changed at the chronic postburn period. These findings may aid in understanding the adaptive response of diaphragmatic preservation in burn trauma.

Pharmacological nature of soman-induced hypothermia in mice

The object of the study was to determine the pharmacological nature of pinacolyl methylphosphonofluoridate (soman)-induced hypothermia in mice. This was accomplished by examining the soman hypothermia dose response and the effect of various pharmacological antagonists in comparison to the hypothermia responses of muscarinic and nicotinic cholinergic agonists such as oxotremorine and nicotine and another anticholinesterase, physostigmine. Core temperature in mice was monitored by telemetry. In general, atropine antagonized oxotremorrine, physostigmine, and soman hypothermia but not nicotine hypothermia whereas mecamylamine antagonized nicotine hypothermia but not that produced by the other agonists. Soman hypothermia was not affected significantly by various pharmacological antagonists, suggesting that other neurotransmitters were not involved in the expression of soman hypothermia. Soman hypothermia appears to be due to muscarinic receptor stimulation and can be effectively antagonized, but not completely, by the use of atropine. Acetylcholinesterase oxime reactivators, such as HI-6 and toxogonin, were ineffective in antagonizing soman-induced hypothermia and reactivating hypothalamic acetylcholinesterase, whereas HI-6 was effective in reactivating soman-inhibited diaphragm acetylcholinesterase when administered up to 10 min after soman, indicating that aging of the soman-inhibited acetylcholinesterase had not occurred. Soman hypothermia appears to be primarily a muscarinic receptor-related event.

Isolation of a tripeptide (Ala-Gly-Ser) exhibiting weak acetylthiocholine hydrolyzing activity from a high-salt soluble form of monkey diaphragm acetylcholinesterase.

A high-salt soluble form of acetylcholinesterase (AChE) was purified from monkey (Macaca radiata) whole diaphragm by a two step affinity chromatographic procedure using m-aminophenyl trimethylammonium-chloride hydrochloride-Sepharose and procainamide-Sepharose columns. The purified enzyme showed three major protein bands at 80 kDa, 78 kDa and 60 kDa on SDS-gel electrophoresis. [3H]Diisopropyl fluorophosphate ([3H]DFP) labeled enzyme also gave three radioactive peaks corresponding to these three bands. The purified enzyme pretreated with dithiothreitol and subjected to limited trypsin digestion gave a peptide fragment of molecular weight approximately 300 Da showing weak acetylthiocholine hydrolyzing activity as identified by Sephadex G-25 gel filtration. Sequence analysis showed that the active peptide fragment was a tripeptide with the sequence Ala-Gly-Ser. When the purified AChE was labeled with [3H]DFP, digested with trypsin and subjected to Sephadex G-25 chromatography, a radioactive peak that would correspond to the tripeptide fragment was seen. The kinetics, inhibition characteristics and binding characteristics to lectins of the active peptide fragment was compared with the parent enzyme. A synthetic peptide of sequence Ala-Gly-Ser was also found to exhibit acetylthiocholine hydrolyzing activity. The kinetics and inhibition characteristics of the synthetic peptide was similar to those of the peptide derived from the purified enzyme, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 29480 times less than that of the purified AChE.

Soman and sarin inhibition of molecular forms of acetylcholinesterase in mice: Time course of recovery and reactivation by the oxime HI-6

The in vivo sensitivity of the molecular forms of the enzyme acetylcholinesterase to inhibition by either soman or sarin, reactivation by HI-6 and the time course of recovery following inhibition by soman were investigated in mice. Administration of HI-6 (50 mg/kg, i.p.) immediately after soman (100 μg/kg, s.c.) or sarin (150 μg/kg, s.c.) resulted in an apparent selective reactivation of the 10S and 16S molecular forms of acetylcholinesterase and no reactivation of the 4S form of diaphragm acetylcholinesterase. The apparent selectivity of the reactivation of the molecular forms of the acetylcholinesterase was probably due to the fact that the 10S and 16S forms of acetylcholinesterase are located primarily extracellularly and the 4S form intracellularly. The HI-6 was restricted primarily to the extracellular compartment due to its quaternary, hydrophilic nature. If the administration of HI-6 was delayed until 60 min following soman (100 μg/kg, s.c.) injection, no reactivation of any of the molecular forms of acetylcholinesterase could be found in the diaphragm. The soman-inhibited acetylcholinesterase had probably aged and, thus, was not susceptible to reactivation by HI-6. The time course of recovery of the molecular forms in the diaphragm occurred rather quickly with the smaller 4S and 10S forms recovering to control levels faster than the larger 16S form. It took between 8 and 16 days for the 16S form to recover to normal. In the brain, hypothalamic acetylcholinesterase molecular forms such as the 4S recovered faster than the 10S form which had not recovered to control 16 days after soman administration; the 16S form of acetylcholinesterase was not detected in the brain.

DOXAPRAM AND THE NEUROMUSCULAR JUNCTION

We have studied the action of doxapram on neuromuscular transmission in the rat phrenic nerve-diaphragm preparation. Doxapram augmented neuromuscular transmission in a dose-related manner when a threshold concentration of 5 x 10(-5) mol litre-1 had been exceeded. The activity of the acetylcholinesterase in rat diaphragm has been examined also in the presence of doxapram. No inhibitory effect was seen in the concentration range which augmented neuromuscular transmission, thus excluding cholinesterase inhibition as the underlying mechanism. In contrast, in the presence of partial neuromuscular block, a dose-related depression of neuromuscular transmission with doxapram was revealed. This was greatest when the neuromuscular blocking agents possessed significant presynaptic activity (beta-bungarotoxin and tubocurarine). In this situation any facilitatory action of doxapram was severely reduced or abolished. In contrast, the facilitatory effects of doxapram were apparent in the presence of partial block produced by agents with less or no presynaptic activity (pancuronium and alpha-bungarotoxin). This study suggests that doxapram has a presynaptic facilitatory action at the neuromuscular junction. In the presence of partial neuromuscular block, an inhibitory action is revealed which may be post-junctional. The concentrations of doxapram at which these effects appear are approximately five times greater than those reached in plasma after a standard clinical dose.

Effect of denervation on the molecular forms of acetylcholinesterase in rat diaphragm.

Effect of denervation on the molecular forms of acetylcholinesterase in rat diaphragm.