Inactivation Of Acetylcholinesterase Research Articles

Adenosine A1 receptor agonist N6-cyclopentyladenosine affects the inactivation of acetylcholinesterase in blood and brain by sarin.

The objective of the present study was to develop a kinetics of pharmacodynamics model to properly describe and investigate the in vivo interaction between the selective adenosine A(1) agonist N(6)-cyclopentyladenosine (CPA), acetylcholinesterase (AChE) in blood and brain, and the AChE-inhibitor sarin (isopropylmethylphosphonofluoridate). The direct interaction of CPA (2 microM) on the inhibition of AChE by sarin was studied in vitro in heparinized rat blood and in 10% (w/v) brain homogenate. CPA did not directly influence the sarin-mediated inactivation of AChE in either system. In sarin-poisoned (144 microg/kg s.c.) rats not treated with CPA, AChE was completely inactivated in blood and brain within 7 min. CPA (2 mg/kg i.m.) treatment, 1 min after sarin administration, caused a small delay in the inhibition of AChE in blood. Treatment with CPA, 2 min before sarin, protected the neuronal AChE partially from being inhibited, but not the enzyme localized in blood. With a dose-response-time model the proportion of the dose of sarin reaching the site of action was estimated to be 48 +/- 12 or 13 +/- 3% after CPA post- or pretreatment, respectively. A correlation between the residual AChE activity in the brain and the incidence of cholinergic symptoms could be established with logistic regression analysis: lower inhibition of AChE in the brain precluded the onset of critical symptoms. In conclusion, CPA affects the concentration of sarin reaching the site of action, which contributes to the protection previously observed in sarin-poisoned rats.

Stereochemical constraints on the substrate specificity of phosphotriesterase.

A series of achiral, chiral, and racemic mixtures of paraoxon analogues containing various combinations of methyl, ethyl, isopropyl, or phenyl substituents were synthesized as probes of the stereochemical constraints within the active site of phosphotriesterase. The kinetic constants for these paraoxon analogues with the enzyme varied significantly with the size of substituents surrounding the phosphorus center. These results indicate that binding and catalysis depend significantly on the relative size and orientation of the two subsites that must accommodate the coordination of the alkyl or aryl substituents within the enzyme active site. Individual enantiomers of paraoxon analogues were also synthesized and the stereochemical specificity for phosphotriesterase determined. In general, the kinetic constants, kcat and kcat/Km, for the (-)-enantiomers of these phosphotriesters were 1-2 orders of magnitude greater than the (+)-enantiomers. In every case, the preferred isomer is of the SP-configuration. For example, the kcat/Km for SP-(-)-ethyl phenyl p-nitrophenyl phosphate is 1.8 x 10(8) M-1 s-1 but is only 1.8 x 10(6) M-1 s-1 for the RP-(+)-isomer. These results suggest that one enantiomer is positioned for hydrolysis more favorably than the other enantiomer. The inactivation of acetylcholinesterase with the same series of organophosphate nerve agents was also measured. The stereoisomer that more rapidly inactivates human acetylcholinesterase is hydrolyzed more slowly than its enantiomer by the phosphotriesterase.

The importance of electrodiagnostic studies in acute organophosphate poisoning

Six patients with acute organophosphate pesticide poisoning in whom electrodiagnostic studies influenced or supported specific decisions in management are described. One patient was admitted to hospital with a diagnosis of acute alcoholic intoxication. Electrodiagnostic studies revealed single stimulus induced repetitive responses and decrement–increment responses at 30 and 50 Hz repetitive nerve stimulation, findings that are indicative of a depolarization block due to inactivation of acetylcholinesterase at the motor end-plate. The patient was subsequently treated as a case of acute organophosphate poisoning. The administration of edrophonium (0.1 mg) to another patient with normal neuromuscular transmission studies unmasked the latent electrophysiological abnormalities. Three instances are described in which electrodiagnostic studies were useful in predicting whether pralidoxime administration was likely to be useful and for how long was pralidoxime therapy to be continued. Phrenic nerve conduction study in one patient with impending respiratory failure revealed an unstimulable phrenic nerve. The potential role of phrenic nerve conduction studies and neuromuscular transmission studies in influencing decisions like intubation and mechanical ventilation is discussed.

Liposome-catalyzed unfolding of acetylcholinesterase from Bungarus fasciatus.

The kinetics of thermal inactivation of acetylcholinesterase from the venom of the snake, Bungarus fasciatus, were studied at 45-54 degrees C. An Arrhenius plot reveals an activation energy of 113 kcal/mol. The thermally denatured enzyme displays the spectroscopic characteristics of a partially unfolded 'molten globule' state. The rate of thermal denaturation is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, the energy barrier for the transition being lowered from 113 to 52 kcal/mol. In contrast to our findings for partially unfolded Torpedo californica acetylcholinesterase [Shin et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2848-2852], the thermally denatured snake enzyme does not remain bound to the liposomes but is released after unfolding and subsequently aggregates. The liposomes thus serve as catalysts for unfolding of the snake enzyme, and its rate of unfolding in the presence of liposomes can be described by the Michaelis-Menten equation (Km = 8 x 10(-7) M). The phospholipid vesicles display a catalytic turnover number of kcat approximately 4 min-1, assuming 15 binding sites per vesicle for the snake acetylcholinesterase.

Increased paraoxon detection with solvents using acetylcholinesterase inactivation measured with a choline oxidase biosensor

Inactivation by paraoxon of free and immobilized acetylcholinesterase, in the presence of various amounts of different organic solvents, and with choline oxidase immobilized on an oxygen electrode was studied. With acetylcholinesterase in solution it was found that 5% cyclohexane enhances paraoxon detection when compared to the detection without solvent. In these conditions, as low as 10 −9 m paraoxon can be detected. Free acetylcholinesterase was more sensitive to paraoxon than immobilized acetylcholinesterase, depending on the nature and concentration of solvent (500-fold in the presence of 5% cyclohexane). The stability of the biosensor in continuous use and stored at room temperature was at least 3–4 weeks with triton X100 and bovine serum albumin addition. The sensitivity of paraoxon detection with free acetylcholinesterase in the presence of 5% cyclohexane corresponds to 0·2 ppb paraoxon, 10-fold below the legal limit admitted in the European Economic Community. The same approach could be applied for detection of low amounts of different organophosphorus or carbamate pesticides.

Acetylcholine release from frontal cortex in the waking rat measured by microdialysis without acetylcholinesterase inhibitors: effects of diisopropylfluorophosphate

Acetylcholine (ACh) release was measured in frontal cortex of awake quietly resting rats by microdialysis without using cholinesterase blockers in the perfusate. Resting release was 16.61 ± 2.05 fmol/h (±.S.E.M., n = 18). Injection of sublethal doses of the acetylcholinesterase blocker, diisopropylfluorophosphate produced dose-dependent increases in ACh release, reaching 79.9 fmol/h with a dose of 0.7-times the LD 50 Although this irreversible inactivation of acetylcholinesterase increased ACh recovery to more than 700% of control values, levels of ACh in the perfusate never reached those seen in physostigmine-treated animals. The relationship between the amount of acetylcholinesterase inactivation and the quantity of ACh in the perfusate suggests that the extracellular ACh concentrations are controlled by simple enzyme kinetics. Within 2 h after enzyme inactivation, extracellular choline levels fell significantly, suggesting that ACh degradation by acetylcholinesterase plays an important role in regulating the amount of choline in the extracellular space.

Ecotoxicology of chlorpyrifos.

Chlorpyrifos is a broad-spectrum organophosphorothioate insecticide with a principal mechanism of toxicity by inactivation of acetylcholinesterase at nerve junctions. Unlike certain organochlorine pesticides, chlorpyrifos is relatively nonpersistent (Racke 1993), and its principal degradation products are less toxic than the parent chemical. Species sensitivity varies considerably across kingdom and phyla. In general, aquatic and terrestrial microorganisms and plants are tolerant to chlorpyrifos exposure. Aquatic invertebrates, particularly crustaceans and insect larvae, are sensitive to exposure: LC50s are generally less than 1 microgram/L, and no-observed-effect concentrations may be below 0.1 microgram/L in laboratory studies. Fish appear to be less sensitive, with LC50s generally between 1 and 100 micrograms/L and no-observed effect concentrations of approximately 0.5 microgram/L. In general, saltwater and freshwater organisms exhibit similar sensitivity to chlorpyrifos, considering the extreme phylogenetic and species differences in toxicity. Chlorpyrifos effects in aquatic ecosystems are complex because of the diversity of species assemblages and trophic interactions. In general, functional endpoints (e.g., community metabolism) are less sensitive than structural parameters of ecosystems (e.g., loss of sensitive species). Ecosystem recovery is dependent on the interaction of a variety of factors including treatment timing and application dose, rate of dissipation, species assemblages, trophic structure, and the reproductive capacity and growth rate of susceptible and tolerant populations. Terrestrial species are relatively tolerant of chlorpyrifos exposure, although contact toxicity to sensitive terrestrial invertebrates may occur at concentrations of 0.1 microgram/insect. Amphibians, birds, and mammals show similar sensitivity to orally administered chlorpyrifos, with LD50s ranging from 8 to > 400 mg/kg body weight. Long-term chronic feeding studies in birds and mammals have shown no observed effect concentrations to be greater than 1 mg/kg food. In general, field studies have shown limited or no acute toxicity to amphibians, reptiles, birds, or mammals.

Biochemical characterization of sheep platelet acetylcholinesterase after detergent solubilization

The biochemical characterization of detergent-solubilized acetylcholinesterase (AChE) from subcellular particles of sheep platelets and the effects of different effectors on AChE activity from solubilized platelet crude membranes have been undertaken and studied. Solubilization of AChE with detergent increased the thermal stability of the enzyme from all particulate fractions. Solubilized AChE from the mitochondria-granule fraction was the most thermostable at 55°C. The K m values against acetylthiocholine chloride and the Arrhenius plot obtained were very similar for the AChE from all the solubilized fractions. There were no differences in the ability of solubilized AChE from different subcellular fractions to bind concanavalin A (Con A). In solubilized platelet crude membranes, benzyl alcohol was a potent AChE inhibitor at a concentration of 10 −2 M, whereas ethanol was not. Mg 2+ cations and, to a lesser extent, Ca 2+ and Mn 2+ cations, activated AChE at concentrations higher than 1 mM. Serine hydrolase inhibitors and cholinesterase-specific inhibitors were very effective in the inactivation of AChE, whereas EDTA and EGTA had no effect. Of all the monosaccharides tested, only N-acetylneuraminic acid exerted an inhibitory effect on AChE activity. Immobilized-lectin binding studies demonstrated the interaction of solubilized crude membrane-bound AChE with Con A, lentil lectin and wheat germ agglutinin. Taken together, these data suggest the presence of a unique form of the membrane-bound AChE which has at least a-mannose and N-acetylglucosamine residues in the glycan chain.

Effect of hydroxamic acids from cereals on aphid cholinesterases

2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), a hydroxamic acid involved in the resistance of wheat to the aphid Rhopalosiphum padi, and known to react with serine and cysteine residues, inactivated acylcholinesterases (acylcholine hydrolases, EC 3.1.1.7) from homogenates of whole bodies of adult R. padi. The enzyme preparation was shown to consist mainly of acetylcholinesterase (AChE) containing an essential cysteine residue outside the substrate binding site. AChE from R. padi, and also ChE from other insects and from human erythrocytes which do not possess such a cysteine residue, were inhibited (I 50 in the mM range) by DIMBOA. 4-Hydroxy-7-methoxy-1,4-benzoxazin-3-one, a compound known to react with cysteine residues but not with serine residues, did not inactivate AChE from R. padi under similar conditions. The results suggest that inactivation of AChE from R. padi by DIMBOA does not occur through reaction with its characteristic cysteine residue.

IN vitro protection of acetylcholinesterase and butyrylcholinesterase by tetrahydroaminoacridine : Comparison with physostigmine

The protective action of 1,2,3,4-tetrahydro-9-aminoacridine (THA) against the long-lasting inactivation of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) brought about by diisopropylnuorophosphate (DFP) and physostigmine, as well as by neostigmine in the case of AChE only, was evaluated by a dilution technique using Electrophorus electricus AChE and horse serum BuChE as target enzymes. In parallel experiments, the ability of physostigmine itself to protect these enzymes from DFP was evaluated and compared with that of THA. THA pretreatment was seen to prevent in a dose-dependent manner the inhibition of both AChE and BuChE. However, it was appreciably more potent towards AChE than towards BuChE. THA mean ec 50 values for protecting AChE against 10, 40 and 100 μM DFP were 0.04, 0.16 and 0.45 μM, respectively; against 1 μM physostigmine the value was 1.8 μM and against 1.2 μm neostigmine it was 3.0 μM. The THA mean ec 50 value for protecting BuChE against 3 μM physostigmine was 0.55 μM and the values for protecting against 3, 10 and 40 μM DFP were 1.5, 3 and >10 μM, respectively. The protective action of THA was time independent: recovery of the maximal enzymic activity was immediate upon dilution. Unlike THA, the protective action of physostigmine developed progressively after dilution and was maximal within 3–4 (AChE) or 6–8 hr (BuChE). Under our experimental conditions, 0.3 μM physostigmine protected approximately 70% of AChE from 40 μM DFP and 5 μM physostigmine protected 9 and 47% of BuChE from 40 and 3 μM DFP, respectively. The results of this work suggest that THA exerts its protective action by shielding the active site of AChE and BuChE from the attack of the inactivating agents on account of its higher enzymic affinity, whereas the protective action of physostigmine against DFP takes advantage also of the carbamylation of the enzyme. These results are in line with the hypothesis that protection of AChE is the primary mechanism responsible for the antidotal action of THA against organophosphorus poisoning.

Measurement of Bimolecular Rate Constantskiof the Cholinesterase Inactivation Reaction by 55 Insecticides and of the Influence of Various Pyridiniumoximes onki

Abstract The cholinesterase (ChE) inhibition test is a biochemical method for screening insecticides. The possibilities for the detection of different cholinesterase inhibiting substances were investigated. The inactivation abilities against cholinesterases from 4 different biological origins were measured. General rules for detection and discrimination of insecticides were derived from the bimolecular rate constants k i of 55 relevant organophosphorus compounds, carbamates and carbamate metabolites. The thionophosphates were converted into the more powerful inhibiting oxones by oxidation with N-bromosuccinimide. The inactivation values k i differ in some cases up to 6 orders of magnitude for different insecticides and 3 orders of magnitude for different enzymes. The inactivation of acetylcholinesterase (AChE) by organophosphorous compounds can be suppressed by reactivation with obidoxime. On the other hand, an acceleration of inactivation of butyrylcholinesterase (BChE) by carbofuran and certain other ca...

Inhibition of acetylcholinesterase and butyrylcholinesterase by the organophosphorus insecticide methylparathion in the central nervous system of the golden hamster (Mesocricetus auratus)

The toxic effects of the organophosphorus pesticide methylparathion are primarily caused by the inhibition of acetylcholinesterase activity in the central nervous system, whereas the relationship between butyrylcholinesterase and poisoning symptoms is unclear. The presumed different effects of methylparathion on acetylcholinesterase in various regions of brain and spinal cord suggest differences in the distribution of molecular enzyme forms. In the present work, the in vitro and in vivo effects of methylparathion on acetylcholinesterase and butyrylcholinesterase were studied in whole brain homogenates of golden hamsters with biochemical methods. Furthermore, acetylcholinesterase activity was determined in regions of the nervous system by quantitative histochemistry (microdensitometry). Biochemically, very low IC50 values of the hydrophilic and lipophilic fractions of both enzymes were measured. Analysis of the time course of enzyme inhibition revealed maximum inhibition 45 min after methylparathion application. Using microdensitometry different degrees of acetylcholinesterase inhibition were found in various areas of the brain. The highest inactivation was observed in the Substantia nigra and in thalamic nuclei; in several regions of the cerebellum, the inhibition rate was comparatively lower. In conclusion, methylparathion acts as an potent inhibitor of acetylcholinesterase and butyrylcholinesterase in the hamster nervous system. The region-specific different inactivation of acetylcholinesterase might be caused by the existence of multiple forms of the enzyme in various brain regions.

Muscarinic effects on the hydroxy- and methoxyindole pathway in the rat pineal gland.

[3H]Quinuclidinyl benzylate ([3H]QNB)-binding sites, showing similar properties to cholinergic muscarinic receptors in other tissues, were disclosed in the rat pineal gland. Functionality of these receptors was demonstrated, as in-vitro muscarinic activation by pilocarpine increased the pineal metabolic production of the hydroxyindole derivatives 5-hydroxytryptophan and serotonin, with a slight effect on melatonin biosynthesis. Electric-field stimulation of pineal slices caused similar metabolic effects. These effects were inhibited by muscarinic blockade with atropine and enhanced by neostigmine inactivation of acetylcholinesterase. These results suggest that acetylcholine is the neurotransmitter involved. Cholinergic activity may, therefore, regulate indole metabolism in the pineal gland.

Desensitization of the acetylcholine receptor of frog end‐plates measured in a Vaseline‐gap voltage clamp.

1. Desensitization of the nicotinic acetylcholine receptor of the frog end-plate was investigated in dissociated frog muscle fibres using the Vaseline-gap clamp method so that a wide range of well-defined agonist concentrations could be used without having to use alpha-bungarotoxin to reduce currents, and so that the intracellular medium could be controlled. 2. Acetylcholine (ACh) concentrations between 1 and 1000 microM were used, after inactivation of acetylcholinesterase. The intracellular calcium concentration was usually kept near zero by using 80 mM-K2EGTA as the intracellular solution. 3. When using the low intracellular calcium solution, desensitization proceeded as a biphasic process with estimates of fast and slow time constants of about 8 and 80 s at 4 degrees C and 20 microM-ACh (the rates increased with concentration). In contrast, only one (fast) component of desensitization was detected when the intracellular calcium concentration was allowed to increase during ACh application. 4. Despite rapid application of ACh the time to peak response was 0.2 s (with 400 microM-ACh) to 2 s (with 1 microM-ACh); this slow rise was shown to result from diffusion delays. Nevertheless the peak current with 200 microM-ACh corresponded to opening of most of the channels present, so there is probably not much desensitization in the millisecond time range. 5. Both fast and slow time constants for onset of desensitization showed only slight dependence on membrane potential when [Ca2+]i was buffered with 80 mM-K2EGTA. 6. Increasing the intracellular cyclic AMP concentration directly, or indirectly with forskolin and IBMX, had no effect on the time course of desensitization. 7. Intracellular application of submicromolar concentrations of phorbol-12,13-dibutyrate (PDBu) and phorbol-12-myristate-13-acetate (PMA) yielded a small but reproducible reduction of the peak response to ACh. The time course of desensitization was, however, not modified by these substances. 8. The implications of these observations for the mechanism of desensitization, and their relationship to single-channel observations, are discussed.

Rate-limiting phosphorus-oxygen fission in the self-stimulated inactivation of acetylcholinesterase by 4-nitrophenyl 2-propyl methylphosphonate

Rate-limiting phosphorus-oxygen fission in the self-stimulated inactivation of acetylcholinesterase by 4-nitrophenyl 2-propyl methylphosphonate

Effects of glycyl-L-glutamine in vitro on the molecular forms of acetylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.

Normal and preganglionically denervated cat superior cervical ganglia were sectioned and cultured for 24 or 48 hr, with or without preliminary inactivation of acetylcholinesterase, and in the presence or absence of 10(-5) M glycyl-L-glutamine. They were then homogenized, and the molecular forms of acetylcholinesterase were analyzed by sucrose gradient sedimentation. We observed an increased proportion of the globular monomeric G1 form, and to a lesser extent of the dimeric G2 and tetrameric membranous G4 forms, of acetylcholinesterase in the glycyl-L-glutamine-treated compared with the control cultures. There was only a small increase in the total acetylcholinesterase activity and no significant variation in the activity of the metabolic enzyme lactate dehydrogenase. It therefore seems likely that glycyl-L-glutamine, or the endogenous neurotrophic factor, maintains acetylcholinesterase in the preganglionically denervated ganglia in vivo by specifically increasing the biosynthesis of the monomeric G1 form, but not that of other proteins; these trophic factors do not seem to promote the polymerization of G1 into the more complex G2 and G4 forms.

Catalytic recruitment in the inactivation of acetylcholinesterase by Soman: temperature dependence of the solvent isotope effect

3,3-Dimethyl-2-butyl methylphosphonofluoridate (soman) recruits at least 60-70% of the catalytic power of acetylcholinesterase during phosphonylation of the active-site serine, as compared with the acetylation by acetylcholine. The solvent isotope effect is 1.34 +/- 0.11 and is temperature independent within experimental error between 5 and 45/sup 0/C. This observation is quite similar to what has been found for the reactions of aryl acetates and anilides. Average activation parameters for the same temperature range are ..delta..H* = 7.3 +/- 0.6 kcal/mol and ..delta..S* = -4 +/- 2 eu (H/sub 2/O) and ..delta..H* = 7.1 +/- 0.4 kcal/mol and ..delta..S* = -6 +/- 1 eu (D/sub 2/O). A rate-limiting process with 50% contribution of an induced-fit conformational change is supported by the data.

Inactivation of acetylcholinesterase with a bretylium tosylate photoaffinity probe

Azidobretylium tosylate (ABT), the p-azido analogue of bretylium tosylate, has been synthesized to serve as a photoaffinity probe for bretylium binding sites. Bretylium tosylate has antiarrhythmic action and also interacts with amiloride-sensitive sodium ion transport sites. Acetylcholinesterase was used as a model protein, and both bretylium and ABT are reversible inhibitors of this enzyme. The kinetic inhibition constants ( K i) were determined to be 40 μM for bretylium tosylate and 6 μM for ABT. The azido compound is photochemically labile and apparently irreversibly inactivates the enzyme. The rate was retarded by the addition of bretylium tosylate or 4-oxo- N, N, N-trimethylpentanaminium iodide (OTI). Sephadex G-25 chromatography further demonstrated the irreversible nature of the photoinactivation. Since ABT binds at or near the acetylcholinesterase active site, it may be a useful probe for the characterization of the enzyme active site.

Brain neuronal chromatin responses in acute soman intoxicated rats.

Male Sprague-Dawley rats (200 g) were injected subcutaneously with soman, a potent neuronal acetylcholinesterase (AChE) inhibitor, at doses of 0.5, 0.8 and 1.0 LD50 (1 LD50 = 135 micrograms/kg) before decapitation at 1 and 24 h post-exposure. Correlative data were obtained on the severity of brain AChE inactivation and physicochemical changes in nuclear chromatin of cerebrocortical (layer V) and striatal neurons using Feulgen-DNA (F-DNA) cytophotometry and ocular filar micrometry. Decreased lability of neurons to F-DNA acid hydrolysis (reduced F-DNA yield), nuclear shrinkage and chromatin aggregation (decreased chromophore area) were used as indices of suppression of genomic template activity; conversely, increases in F-DNA yield and chromophore area signify enhanced neuroexcitation. At 1 hr post-soman there was a dose-dependent inactivation of AChE with a moderate increase in chromatin activation, i.e., nuclear hypertrophy and chromatin dispersion. At 24 hr post-soman there was a partial restoration of AChE activity, notably in striatal neurons, with a suppression in chromatin template activity. These data indicate that actions of soman on neuronal functioning are time-dependent. The absence of any dose-related neuronal chromatin changes may signify existence of non-cholinergic mediated events.

Acetylcholine hydrolysis during neuromuscular transmission in the synaptic cleft of skeletal muscle of mouse and chick

Inhibition of acetylcholinesterase (AChE) by more than 80% by neostigmine or physostigmine resulted in a failure of tetanic contraction (100 Hz) in the isolated mouse nerve-diaphragm preparation. In the chick biventer cervicis muscle, however, the tetanic contraction was well maintained and even outlasted the period of nerve stimulation after inactivation of AChE. The concentration of (+)tubocurarine for 70% block of the indirect twitch response of the mouse diaphragm at 0.1 Hz was increased from 0.67 to 0.99 to 1.21–2.03 μM in the presence of neostigmine (0.15–1.5 μM) which inhibited AChE by 70% or more, while that to depress the tetanic contraction (50 Hz) was increased from 0.38 to 0.42 to 0.53–0.69 μM. In the chick muscle, physostigmine at 2.4 μM increased the concentration of (+)tubocurarine for 70% block of the twitch response from 1.68 to 4.14 μM, whereas that for block of the response to exogenous acetylcholine (ACh) was increased from 1.47 to 74.6 μM. On single stimulation, the relative peak concentrations of acetylcholine (ACh) at the postsynaptic receptor site of the mouse diaphragm and chick biventer cervicis were estimated to be increased by about 110 and 120% respectively, after complete inhibition of AChE. In the chick muscle, physostigmine increased the relative concentration of ACh by about 40-fold at the receptor site for exogenously applied ACh. It is concluded that the intrinsic ACh released from the nerve terminal is hydrolyzed by about 50% during the time of diffusion across the synaptic cleft whereas most of exogenous ACh is hydrolyzed before reaching the target.