Rat Brain Acetylcholinesterase Research Articles

Rat Brain Acetylcholinesterase Visualized with [ 11C]Physostigmine

Physostigmine, a powerful cholinesterase inhibitor, has recently been labelled with 11C in view of its potential application for in vivo imaging of cerebral acetylcholinesterase (AChE) using positron emission tomography. Here we carried out autoradiography of the rat brain using [ 11C]physostigmine in order to characterize the cerebral targets of this ligand. Autoradiograms were obtained using phosphor storage plates which, compared to autoradiographic films, greatly improved the quality of 11 C images. Following autoradiography, brain sections were stained for AChE activity, allowing a direct comparison of autoradiographic and histoenzymatic localizations. The distributions of 11C label and of AChE activity were found to be essentially super-imposable, both after in vivo injection of and after in vitro incubation with [ 11C]physostigmine. Densitometric analysis showed that radioactivity and enzymatic activity distributions were regionally correlated. The fixation of [ 11C]physostigmine to cerebral tissue was abolished after incubation of the rat brain sections with BW 284C51, a specific AChE inhibitor, but not after incubation with iso-OMPA, a specific inhibitor of butyrylcholinesterase. Unilateral excitotoxic lesions of the striatum that eliminated local AChE expression concomitantly reduced the binding of the ligand in the lesioned area. These results indicate that autoradiographic images of the rat brain obtained with [ 11C]physostigmine reflect AChE distribution, thus supporting the use of this radioligand to trace cerebral AChE activity in humans with positron emission tomography.

Binding of 125I-fasciculin to rat brain acetylcholinesterase: P. Marchot, A. Khélif, Y.-H. Ji, P. Mansuelle and P. E. Bougis (CNRS URA 1455 ‘Ingenierie des Protéines’, Faculté de Médecine Secteur Nord, Bd Pierre Dramard, F-13916 Marseilla Cedex 20, France)

Binding of 125I-fasciculin to rat brain acetylcholinesterase: P. Marchot, A. Khélif, Y.-H. Ji, P. Mansuelle and P. E. Bougis (CNRS URA 1455 ‘Ingenierie des Protéines’, Faculté de Médecine Secteur Nord, Bd Pierre Dramard, F-13916 Marseilla Cedex 20, France)

Interaction of acetylcholinesterase with the enantiomers of malaoxon and isomalathion.

The biomolecular reaction constants (ki), dissociation constants (Kd), and phosphorylation constants (kp) were determined for the enantiomers of malaoxon against rat brain acetylcholinesterase, and for the stereoisomers of isomalathion against rat brain acetylcholinesterase and electric eel acetylcholinesterase. (R)-Malaoxon was an 8.6-fold more potent anti-cholinesterase than (S)-malaoxon. Isomalathion stereoisomers with the R configuration at carbon were 3-13-fold stronger inhibitors than those with the S configuration. The isomalathion stereoisomers with the R configuration at phosphorus were 4.3-8.8-fold stronger inhibitors of rat brain acetylcholinesterase, yet 3.4-5.8-fold weaker inhibitors of electric eel acetylcholinesterase, than the isomalathion stereoisomers with the S configuration at phosphorus. The rat brain acetylcholinesterase spontaneous (k0 = approximately 13.0 x 10(-3) min-1) and oxime-mediated (koxime) = 51.0 x 10(-3) min-1) reactivation rate constants following inhibition by isomalathion stereoisomers with the R configuration at phosphorus were comparable to spontaneous (11.3 x 10(-3) min-1) and oxime-mediated (50.2 x 10(-3) min-1) reactivation rates obtained for (S)-isoparathion methyl. These data support a common phosphorylation mechanism, namely, the displacement of the thiosuccinyl moiety from isomalathion stereoisomers with the R configuration at phosphorus, and displacement of the p-nitrophenoxy ligand from (S)-isoparathion methyl to form the same O,S-dimethyl phosphorothiolated enzyme. Rat brain acetylcholinesterase inhibited by the isomalathion stereoisomers with the S configuration at phosphorus were refractory to reactivation, suggesting an alternate mechanism of inhibition, i.e., the loss of the methylthio ligand. Several mechanisms are proposed to account for the subsequent nonreactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of 125I-fasciculin to rat brain acetylcholinesterase. The complex still binds diisopropyl fluorophosphate

Iodination of fasciculin 3 (FAS3) from Dendroaspis viridis venom provided us with a fully active specific probe of fasciculin binding sites on rat brain acetylcholinesterase (AChE). Binding and inhibition are concomitant, as association and inhibition rate constants k1 and ki are identical. The 125I-FAS3.AChE complex dissociates very slowly (t 1/2 = 48 h) and is characterized by a dissociation constant, Kd, of 0.4 pM. All the specific binding of 125I-FAS3 to AChE is prevented by FAS3 as from D. angusticeps venom (Kd = 0.4, 14, and 25 pM, respectively). It is also prevented by propidium iodide, BW284C51, and d-tubocurarine, which bind to peripheral anionic sites of AChE, by Ca2+ and Mg2+, known to enhance AChE activity through an allosteric phenomenon and by acetylthiocholine concentrations which lead to excess substrate inhibition of the enzyme. Diisopropyl fluorphosphate and paroxon, which inhibit AChE by phosphorylating the catalytic serine, have no effect on either the binding rate or the number of binding sites of 125I-FAS3. O-Ethyl-S2-diisopropylaminoethyl methylphosphonothionate, however, which binds irreversibly to the AChE catalytic site but reversibly to a peripheral site, induces a 130% increase in the binding rate of 125I-FAS3, without changing the total number of 125I-FAS3 binding sites. Our results demonstrate that fasciculins bind on a peripheral site of AChE, distinct from the catalytic site and, at least partly, common with the sites on which some cationic inhibitors and the substrate in excess bind. Since phosphorylation of the catalytic serine (esteratic subsite) by [1,3-3H]diisopropyl fluorophosphate can still occur on the FAS3.AChE complex, the structural modification induced by fasciculins may affect the anionic subsite of AChE catalytic site.

Kinetics of the postinhibitory reactions of acetylcholinesterase poisoned by chiral isomalathion: a surprising nonreactivation induced by the RP stereoisomers.

Inhibitory (ki), spontaneous (k0), and oxime-mediated reactivation (k(oxime)) reaction kinetics for the four stereoisomers of isomalathion (SPRC,SPSC,RPRC, and RPSC) were determined against rat brain acetylcholinesterase (AChE). (SPRC)-Isomalathion was the most potent anticholinesterase agent and RPSC-isomalathion the least potent with racemic material approximately midway in activity. Following inhibition of rat brain AChE by (SPRC)- or (SPSC)-isomalathion, k0 and k(oxime) values were obtained that were comparable to (SP)-isoparathion methyl, indicating that the same mechanism of inhibition was shared, namely, formation of an O,S-dimethyl phosphorothiolated enzyme. Conversely, no appreciable reactivation occurred with or without oxime following inhibition of rat brain AChE by (RPSC)- or (RPRC)-isomalathion. This observation was not consistent with (RP)-isoparathion methyl, and a switch in inhibition mechanism to the loss of the thiomethyl moiety is suggested. The nonreactivation of rat brain AChE following inhibition by the (RP)-isomalathion stereoisomers is postulated to result from a mechanism involving either a beta-elimination of diethyl fumarate or displacement of the thiosuccinate moiety from the phosphate moiety.

Rat brain acetylcholinesterase response to monocrotophos and abate.

All the reagents used in the present study were obtained from SIGMA and used without further purification. Technical grade of MCP (purity > 95%) was a gift from NOCIL, Bombay and abate (purity > 95.5%) from VOLHRO, Hyderabad. Male Wistar strain rats weighing about 120 + 5 gms used in the present experiment were obtained from Centre for Cellular & Molecular Biology, Hyderabad. They were housed in polypropylene cages (shoe box cage with stainless steel grill tops 29x22x13.5 cms.) maintained at 22 + 2 ~ C with 55+10% relative humidity at 12 hours dark and 12 hours light cycles. The animals had free access to food (supplied by Lipton India Ltd., Bangalore) and sterile drinking water. The rats were divided into two groups and orally administered LD50 doses of 20 mg/kg MCP and 8,600 mg/kg abate respectively. Send reprint request to J V Rao at the above address

Effect of REM sleep deprivation on rat brain acetylcholinesterase

Acetylcholinesterase activity was compared in control, rapid eye movement sleep-deprived and recovered rat brain. The activity was estimated in the whole brain, cerebrum, brain stem and cerebellum. Flower pot technique was used for continuing deprivation for two, four and eight days. The results showed that the enzyme activity increased signficantly in the deprived rat brain and it returned to control/normal level on recovery. The enzyme activity increased first in the brain stem, while the activity in the cerebellum showed no significant change. Control experiments suggest that the increase was primarily caused by the deprivation. The finding fits well with existing knowledge and would possibly help in explaining earlier observations.

The effect of imipramine and lithium on “learned helplessness” and acetylcholinesterase in rat brain

The effect of short- and long-term treatment with imipramine and lithium on shock stress-induced escape failures in a shuttlebox (the “learned helplessness” model of depression) was investigated in rats. Acetylcholinesterase (AChE) activity was measured in the frontal cortex, hippocampus and striatum after the shuttlebox test. Imipramine was found to normalize escape behavior, whereas lithium further aggravated escape behavior. No correlation was found between escape behavior and AChE activity in the three brain areas investigated. However, a significant decrease in AChE activity in striatum was found in rats exposed either to shock stress and no drug treatment or to drug treatment and no shock stress. In rats exposed to the combination of shock stress and drug (imipramine or lithium), a slight or no decrease of AChE activity occured. Exposure to shock stress alone produced no changes in AChE activity in the hippocampus and frontal cortex. In conclusion, lithium did not have an antidepressant effect on “learned helplessness” and AChE activity was not correlated to escape behavior. However, both imipramine and lithium normalized the decreased level of AChE activity in striatum in rats exposed to shock stress.

Are soluble and membrane-bound rat brain acetylcholinesterase different?

Salt-soluble and detergent-soluble acetylcholinesterases (AChE) from adult rat brain were purified to homogeneity and studied with the aim to establish the differences existing between these two forms. It was found that the enzymatic activities of the purified salt-soluble AChE as well as the detergent-soluble AChE were dependent on the Triton X-100 concentration. Moreover, the interaction of salt-soluble AChE with liposomes suggests amphiphilic behaviour of this enzyme. Serum cholinesterase (ChE) did not bind to liposomes but its activity was also detergent-dependent. Detergent-soluble AChE remained in solution below critical micellar concentrations of Triton X-100. SDS polyacrylamide gel electrophoresis of purified, Biobeads-treated and iodinated detergent-soluble 11 S AChE showed, under non reducing conditions, bands of 69 kD, 130 kD and greater than 250 kD corresponding, respectively, to monomers, dimers and probably tetramers of the same polypeptide chain. Under reducing conditions, only a 69 kD band was detected. It is proposed that an amphiphilic environment stabilizes the salt-soluble forms of AChE in the brain in vivo and that detergent-soluble Biobeads-treated 11 S AChE possess hydrophobic domain(s) different from the 20 kD peptide already described.

In vivo and in vitro effects of diisopropyl fluorophosphate and paraoxon on individual molecular forms of rat brain acetylcholinesterase

Previous study in this laboratory showed that following a sc injection of an organophosphorus compound, diisopropyl fluorophosphate (DFP), into rats the inhibition of 10S molecular forms was considerably more pronounced than that of 4S forms of brain acetylcholinesterase (AChE). This could depend on different accessibility of the two forms or on their different intrinsic sensitivity to the antiChE compound. In the present study the effects of DFP and Paraoxon on 10S and 4S forms were evaluated in vivo, i.e., after systemic administration, and in vitro by adding the organophosphorus compounds to each of the two forms after extraction from brain of untreated rats, solubilization and separation. The in vivo preferential inhibition of 10S forms was confirmed. The 10S/4S ratios for control and DFP-treated rats were 9.05 and 5.01, respectively; these ratios were 8.46 and 3.33 for Paraoxon. On the other hand, in the in vitro experiments there were no significant differences between IC50 values for 10S and 4S forms both in the case of DFP (2.66 and 2.98 microM) and Paraoxon (32.4 and 42.4 nM, respectively). The overall data suggest that the preferential in vivo inhibition of 10S molecular forms with respect to 4S forms depends on their different accessibility probably due to different subcellular localization of the two forms and not on their different intrinsic sensitivity.

Multiple effects of tetrahydroaminoacridine on the cholinergic system: Biochemical and behavioural aspects

9-Amino-1,2,3,4-tetrahydroaminoacridine (THA) in combination with lecithin has been reported to improve the memory of Alzheimer's disease patients. We have examined some properties of THA in vitro and in vivo so as to define some of the mechanism(s) by which THA might produce its therapeutic effects. In vitro, THA was more potent at inhibiting human plasma cholinesterase (IC50 = 0.03 microM) than human erythrocyte acetylcholinesterase (IC50 = 0.3 microM) and rat brain acetylcholinesterase (IC50 = 0.32 microM). Radioligand binding studies indicated that THA binds reversibly and competitively to primary M1 and M2 human cortical muscarinic receptors with similar affinities. Moreover, THA showed similar affinity for temporal cortices muscarinic receptors from Alzheimer and non-Alzheimer (control) brains. In vivo, subcutaneous administration of THA (1-8 mg/kg body weight) to adult rats (6 months old) produced a dose dependent decrease in general activity compared to saline-treated rats. However, at a concentration of 0.5 mg/kg body weight, the general activity of the rats was increased compared to saline-treated rats. The cognitive function of the THA-treated adult rats (subcutaneously 2 mg/kg body weight) was not significantly improved compared to saline-treated rats. It is concluded that the mechanisms of action of THA on the cholinergic system involve reversible inhibition of cholinesterases and reversible and competitive interaction with muscarinic acetylcholine receptors. These effects might be of therapeutic value in the treatment of Alzheimer's disease.

Noncatalytic detoxication of six organophosphorus compounds by rat liver homogenates

The ability of rat liver aliesterases to noncatalytically detoxify the oxons of six phosphorothionate insecticides was studied; the insecticides were methyl parathion, parathion, chlorpyrifos-methyl, chlorpyrifos, leptophos, and EPN. All oxons were more potent inhibitors (n M range) of rat liver aliesterases than the target rat brain acetylcholinesterase, with the exception of methyl paraoxon. Rat liver homogenates (including EDTA to eliminate possible A-esterase contributions) increased apparent I 50s of the oxons to bovine brain acetylcholinesterase, indicating a detoxication of an appreciable amount of the oxon. Except for EPN-oxon, detoxication ability correlated with aliesterase sensitivity to inhibition. Liver homogenates from rats treated in vivo with the phosphorothionates had a reduced detoxication capability which correlated highly with residual aliesterase activity. With the exception of methyl parathion, animals treated for 90 min with high doses of the phosphorothionates displayed higher liver aliesterase inhibition than brain acetylcholinesterase inhibition. Thus, liver aliesterases represent a significant alternative phosphorylation site for organophosphates, and their efficacy for detoxication is a function of relative affinities of the oxon for the aliesterases and acetylcholinesterase.

Effect of fixatives on rat brain acetylcholinesterase activity

Biochemical measurement of acetylcholinesterase (AChE) activity on sonicates of samples of rat cortex, striatum and cerebellum which had been treated with either 4% formaldehyde (HCHO) or 4% HCHO/0.5% glutaraldehyde suggest that fixation of tissue causes a rather large loss (50–80%) in AChE activity which may vary considerably from region to region of brain. Attempts to protect the activity by treatment with diisopropyl fluorophosphate (DFP) before fixation and reactivation after fixation with pyridine-2-aldoxime methiodide (PAM) were unsuccessful. The results suggest care must be exercised in attempting to use AChE histochemistry on fixed tissue for quantitative measurements.

Activation and degradation of the phosphorothionate insecticides parathion and EPN by rat brain

Cytochrome P-450-dependent monooxygenases are known to activate phosphorothionate insecticides to their oxon (phosphate) analogs by oxidative desulfuration. These activations produced potent anticholinesterases, decreasing the I 50 values to rat brain acetylcholinesterase almost 1000-fold (from the 10 −5 M range to the 10 −8 M range). Since the usual cause of death in mammals from organophosphorus insecticide poisoning is respiratory failure resulting, in part, from a failure of the respiratory control center of the brain, we investigated the ability of rat brain to activate and subsequently degrade two phosphorothionate insecticides, parathion (diethyl 4-nitrophenyl phosphorothioate) and EPN (ethyl 4-nitrophenyl phenylphosphonothioate). Microsomes from specific regions (cerebral cortex, corpus striatum, cerebellum, and medulla/pons) of the brains of male and female rats and from liver were incubated with the phosphorothionate and an NADPH-generating system. Oxon production was quantified indirectly by the amount of inhibition resulting in an exogenous source of acetylcholinesterase added to the incubation mixture as an oxon trap. The microsomal activation specific activity was low for brain when compared to liver [0.23 to 0.44 and 5.1 to 12.0 nmol · min −1 · (g tissue) −1 respectively]. The mitochondrial fraction of the brain possessed an activation activity for parathion similar to that of microsomes [about 0.35 nmol · min −1 · (g tissue) −1 for each fraction], but mitochondrial activity was slightly greater than microsomal activity for EPN activation [0.53 to 0.58 and 0.23 to 0.47 nmole · min −1 · (g tissue) −1]. Whole homogenates were tested for their ability to degrade paraoxon and EPN-oxon (ethyl 4-nitrophenyl phenylphosphonate), quantitated by 4-nitrophenol production. Specific activity for oxon degradation in liver was greater than that in brain [31 to 74 and 1.1 to 10.7 nmole · min −1 · (g tissue) −1 respectively]. Overall, the brain and liver had about 1.5- to 12-fold higher specific activities for degradation than activation depending on the compound used. These findings demonstrate that the brain possesses both phosphorothionate activation and oxon degradation abilities, both of which may be significant during exposures to organophosphorus insecticides.

Tacrine slows the rate of ageing of sarin-inhibited acetylcholinesterase

Bovine erythrocyte acetylcholinesterase was inhibited by the organophosphate sarin, and the rate of ageing (the time-dependent decrease in the ability of an oxime to reactivate the enzyme) was studied. At pH 7.0 and 37°C, 10 −5 M or 10 −6 M tacrine (tetrahydroaminoacridine) decreased the rate of ageing in low ionic strength buffer. Tacrine at 10 −5 M also significantly decreased the rate of ageing in 150 mM NaCl. The results indirectly demonstrated that the inhibition of substrate hydrolysis by tacrine is reversible, and that tacrine does not prevent reactivation of sarin-inhibited acetylcholinesterase. Both these observations, which were also made for rat brain acetylcholinesterase, are in contrast with reports in the literature.

Effect of papaverine on acetylcholinesterase in rat brain

Papaverine administration produced significant increases in acetylcholinesterase activity in cerebral cortex and striatum of rat brain. Two related compounds, tetrahydropapaverine and tetrahydropapaveroline, also gave similar effects. However, their actions seem to be indirect because papaverine has noin vitro effect on the enzymatic activity.

Anticholinesterase effect and toxicity of bis(trichloromethyl) sulfone (N-1386 ® biocide) in rats

The oral LD50 for bis(trichloromethyl) sulfone (N-1386 ® Biocide) in male rats was 691 mg/kg. Deaths occurred 1–5 days after treatment and signs of toxicity suggestive of an anticholinesterase effect were noted. However, neither plasma cholinesterase nor brain acetylcholinesterase was inhibited 2, 4 or 24 hours after a single, oral dose of 500 mg/kg. Atropine (300 mg/kg, s.c.) or scopolamine (670 mg/kg, s.c.) pretreatments did not protect against the acute lethality of bis(trichloromethyl) sulfone although signs of toxicity were alleviated by both pretreatments. Bis(trichloromethyl) sulfone produced in vitro inhibition of rat plasma cholinesterase and brain acetylcholinesterase. The inhibition was competitive in brain. IC50's for these 2 enzymes were 8 uM in plasma and 25 uM in brain. In summary, bis(trichloromethyl) sulfone produced in vitro cholinesterase inhibition not demonstrated in vivo . Doses of anticholinergic compounds that ameliorated many toxic signs did not protect against lethality produced by bis(trichloromethyl) sulfone.

An inhibitory monoclonal antibody to human acetylcholinesterases

The monoclonal antibody AE-2 raised against acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from human erythrocytes is shown to inhibit the enzyme activity. The reaction of the antibody with a structural epitope is investigated further. The epitope resides on monomeric, dimeric and tetrameric species of the enzyme. The rate of phosphorylation of the enzyme by diisopropylfluorophosphate was not affected by the antibody. On the other hand, inhibitors directed towards the anionic site(s) competed with antibody binding, suggesting that one of these is the epitope. The titration with antibody is biphasic and yields about 80% inhibition even in the presence of a large excess of antibody. Inhibition is fully reversible upon dilution, in a time-dependent manner. AE-2 also inhibited human adult and fetal brain acetylcholinesterase (to the same extent). However bovine brain acetylcholinesterase was inhibited to a lesser extent and rat brain acetylcholinesterase did not interact with the antibody. Butyrylcholinesterase (EC 3.1.1.8) also showed no reactivity towards the antibody.

Generation of subunit-specific antibody probes for Torpedo acetylcholinesterase: cross-species reactivity and use in cell-free translations.

The assembly of the collagen tailed A12 form of acetylcholinesterase (AChE) is regulated by muscle contraction. To begin to study this regulation, we derived antibody probes for the three subunits (100 kd, catalytic, and collagen tail) of AChE purified from Torpedo californica electric tissue. These included a polyclonal antiserum that recognizes all 3 subunits and 19 monoclonal antibodies; 16 of the monoclonals recognized the catalytic subunit, 2 recognized the tail subunit, and 1 recognized the 100 kd subunit on Western blots. We used immunohistochemical procedures to show that several of the anticatalytic and one of the antitail monoclonals cross-reacted with frog muscle AChE and Western blotting to show that several of the anticatalytic monoclonals cross-react with rat brain AChE. These antibodies were then used to immunoprecipitate AChE precursors from a cell-free translation system. There were generally three primary translation products, corresponding to the three enzyme subunits. Therefore, each subunit is probably derived from a separate mRNA. Occasionally there were two translation products corresponding to the catalytic subunit alone. The catalytic subunit was glycosylated following addition of canine microsomal membranes to the translation mix. The mRNA coding for this subunit appeared to be present in the poly(A)- RNA pool.

Inactivation of rat brain acetylcholinesterase by pyridoxal 5′-phosphate

Effects of pyridoxal 5′-phosphate on the activity of crude and purified acetylcholinesterase from cerebral hemispheres of adult rat brain were examined. Acetylcholinesterase was completely inactivated by incubation with 0.5 mM pyridoxal 5′-phosphate. The enzyme activity remained unaltered in the presence of analogs of pyridoxal 5′-phosphate, pyridoxal, pyridoxamine and pyridoxamine 5′-phosphate. The inhibition of acetylcholinesterase activity by pyridoxal 5′-phosphate appeared to be of a noncompetitive nature, as determined by Lineweaver-Burk analysis. The inhibitory effect of pyridoxal 5′-phosphate on acetylcholinesterase appeared to be a general one, as the activity of the enzyme from the brains of immature chick and egg-laying hen, and from different tissues of the adult male rats, exhibited a similar pattern in the presence of the inhibitor. The inhibitory effects of pyridoxal 5′-phosphate could be reversed upon exhaustive dialysis of the pyridoxan 5′-phosphate-treated acetylcholinesterase preparations. We propose that the effects of pyridoxal 5′-phosphate are due to its interaction with acetylcholinesterase, and that it can be employed as a useful tool for studying biochemical aspects of this important brain enzyme.