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In this paper, we, for the first time, describe the interaction between the butyrylcholinesterase enzyme and echothiophate, a popular model compound and an analogue of the chemical warfare agents VX and VR, at the atomistic level. Competition between the two echothiophate conformations in the active site was found using molecular modeling techniques. The first one is close to the mode of binding of the substrates of choline series (butyrylcholine and butyrylthiocholine) and is inhibitory, since it is unable to react with the enzyme. The second one is characterized by a significantly worse estimated binding affinity and is reactive. Thus, echothiophate combines the features of two types of inhibitors: competitive and suicidal. This observation will help clarify the kinetic reaction scheme in order to accurately assess the kinetic constants, which is especially important when designing new butyrylcholinesterase variants capable of full-cycle hydrolysis of organophosphorus compounds.

Enzyme-catalyzed hydrolysis of echothiophate, a P&ndash, S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with Km = 0.20 &plusmn, 0.03 mM and kcat = 5.4 &plusmn, 1.6 min&minus, 1. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (Km = 2.6 &plusmn, 0.2 mM, kcat = 53400 min&minus, 1). With a kcat/Km = (2.6 &plusmn, 1.6) &times, 107 M&minus, 1min&minus, 1, GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P&ndash, S bonded OPs by thiol-free OP hydrolases.

Gene delivery using an adenoviral system has been effective in introducing therapeutic proteins in vitro and in vivo. This study tested the feasibility of using adenovirus to deliver clinically relevant amounts of butyrylcholinesterase (BChE), a proven bioscavenger of nerve agents. The adenovirus construct expressed full-length mouse BChE. Mice were injected with a single dose of adenovirus (1.5 × 10(10) infectious units) in the tail vein; plasma was collected through day 11 and assayed for BChE activity. Maximum activity, representing a 300- to 3400-fold increase over baseline, was found on day 4. Expression levels returned to baseline by day 10. Nondenaturing gel electrophoresis showed the recombinant BChE was a dimer that could be converted to tetramers by addition of polyproline. The toxic compounds chosen for protection studies were positively charged organophosphorus agents, echothiophate, and O-ethyl-S-2-N,N-diisopropylaminoethyl methylphosphonothiolate (VX). Mice containing elevated blood levels of BChE (300- to 3,000-fold over the control mice) were challenged with incremental doses of echothiophate or VX. Mice showed no signs of toxicity and were protected from up to 30× LD(50) dose of echothiophate and 5× LD(50) dose of VX. A good correlation was observed between tolerated echothiophate dose and plasma BChE levels at time of challenge. The absolute increases in levels of circulating BChE and the sustained nature of the response resulted in a very high enzyme concentration, deemed critical in acute toxicity (5× LD(50) or more) scenarios. These results suggest that gene-delivered BChE is a prophylactic and affords protection equivalent to that of a multimilligram injection of the same.

Butyrylcholinesterase (EC 3.1.1.8 BChE) is present in all human and mouse tissues, and is more abundant than acetylcholinesterase (EC 3.1.1.7 AChE) in all tissues except brain. People who have no BChE activity due to a genetic variation are healthy. This has led to the hypothesis that BChE has no physiological function. We tested this hypothesis by challenging BChE and AChE knockout mice, as well as wild-type mice, with the AChE specific inhibitors, (−)-huperzine A and donepezil, and with serine hydrolase inhibitors, echothiophate and chlorpyrifos oxon. (−)-Huperzine A and donepezil caused mortality and significant toxicity in the BChE−/− animals. The BChE heterozygote (BCHE+/−) mice with approximately one-half the BChE activity of the BChE wild type (BChE+/+) exhibited intermediate toxic symptoms, and survived a longer period. The BChE+/+ animals displayed comparatively minor toxic symptoms and recovered by 24 h post-dosing. Plasma AChE activity was inhibited to the same extent in BChE−/−, +/−, and +/+ mice, whereas BChE activity was not inhibited. This indicated that the protective effect of BChE was not due to scavenging (−)-huperzine A. AChE−/− mice were unaffected by (−)-huperzine A and donepezil, demonstrating the specificity of these inhibitors for AChE. AChE−/− mice treated with chlorpyrifos oxon lost all BChE activity, had severe cholinergic symptoms and died of convulsions. This showed that BChE activity was essential for survival of AChE−/− mice. In conclusion, we propose that the protective effect of BChE is explained by hydrolysis of excess acetylcholine in physiologically relevant regions such as diaphragm, cardiac muscle, and brain. Thus, BChE has a function in neurotransmission. People with BChE deficiency are expected to be intolerant of standard doses of the anti-Alzheimer's drugs, (−)-huperzine A and donepezil.

Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.

Organophosphorus poisons (OP) bind covalently to the active-site serine of cholinesterases. The inhibited enzyme can usually be reactivated with powerful nucleophiles such as oximes. However, the covalently bound OP can undergo a suicide reaction (termed aging) yielding nonreactivatable enzyme. In human butyrylcholinesterase (hBChE), aging involves the residues His438 and Glu197 that are proximal to the active-site serine (Ser198). The mechanism of aging is known in detail for the nerve gases soman, sarin, and tabun as well as the pesticide metabolite isomalathion. Aging of soman- and sarin-inhibited acetylcholinesterase occurs by C-O bond cleavage, whereas that of tabun- and isomalathion-inhibited acetylcholinesterase occurs by P-N and P-S bond cleavage, respectively. In this work, the crystal structures of hBChE inhibited by the ophthalmic reagents echothiophate (nonaged and aged) and diisopropylfluorophosphate (aged) were solved and refined to 2.1, 2.25, and 2.2 A resolution, respectively. No appreciable shift in the position of the catalytic triad histidine was observed between the aged and nonaged conjugates of hBChE. This absence of shift contrasts with the aged and nonaged crystal structures of Torpedo californica acetylcholinesterase inhibited by the nerve agent VX. The nonaged hBChE structure shows one water molecule interacting with Glu197 and the catalytic triad histidine (His438). Interestingly, this water molecule is ideally positioned to promote aging by two mechanisms: breaking either a C-O bond or a P-O bond. Pesticides and certain stereoisomers of nerve agents are expected to undergo aging by breaking the P-O bond.

Organophosphorus toxicants (OP) include chemical nerve agents and pesticides. The goal of this work was to find out whether an animal could be made resistant to OP toxicity by genetic engineering. The human butyrylcholinesterase (BChE) mutant G117H was chosen for study because it has the unusual ability to hydrolyze OP as well as acetylcholine, and it is resistant to inhibition by OP. Human G117H BChE, under the control of the ROSA26 promoter, was expressed in all tissues of transgenic mice. A stable transgenic mouse line expressed 0.5 microg/ml of human G117H BChE in plasma as well as 2 microg/ml of wild-type mouse BChE. Intestine, kidneys, stomach, lungs, heart, spleen, liver, brain, and muscle expressed 0.6-0.15 microg/g of G117H BChE. Transgenic mice were normal in behavior and fertility. The LD50 dose of echothiophate for wild-type mice was 0.1 mg/kg sc. This dose caused severe cholinergic signs of toxicity and lethality in wild-type mice, but caused no deaths and only mild toxicity in transgenic animals. The mechanism of protection was investigated by measuring acetylcholinesterase (AChE) and BChE activity. It was found that AChE and endogenous BChE were inhibited to the same extent in echothiophate-treated wild type and transgenic mice. This led to the hypothesis that protection against echothiophate toxicity was not explained by hydrolysis of echothiophate. In conclusion, the transgenic G117H BChE mouse demonstrates the factors required to achieve protection from OP toxicity in a vertebrate animal.

In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BuChE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10-fold decreased IC50 values for DFP, iso-OMPA, and echothiophate. These findings add Tyr440 and Phe329 to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BuChE, and foreshadow the design of recombinant BuChEs with tailored scavenging properties.

Previous studies in this and other laboratories have suggested an important role for central cholinergic neurons in the expression of morphine withdrawal symptoms. This study was designed to determine whether the symptoms of withdrawal could be mitigated by normalization of the effect of morphine on cholinergic neurons. Since this effect is generally inhibitory, we used centrally acting cholinergic agonists to augment central cholinergic tone during chronic morphine infusion. Rats were made dependent following the intra-arterial (i.a.) infusion of increasing concentrations (35–100 mg kg −1 day −1 ) of morphine over 5 days. I.a. injection of 0.5 mg/kg of naloxone precipitated a profound withdrawal response that included a dramatic increase in mean arterial pressure (MAP) which was maintained over the 60-min observation period, a short duration increase in heart rate (HR), and characteristic opiate withdrawal symptoms. In separate groups of rats, non-toxic doses (50 and 250 μg/kg) of the acetylcholinesterase (AChE) inhibitor, diisopropylflurophosphate (DFP) were administered as single daily injections concomitant with the morphine infusion. DFP treated rats, exhibited significantly reduced expression of the naloxone-evoked pressor response. The apparent anti-withdrawal effect of DFP was not reproduced by the selective peripherally acting AChE inhibitor, echothiophate, although both compounds effectively reduced the expression of certain other withdrawal symptoms. The centrally acting muscarinic cholinergic receptor agonist, arecoline, resulted in an even more impressive suppression of withdrawal symptoms. While not all symptoms associated with morphine withdrawal are mediated via central cholinergic pathways, these results suggest that physical dependence on morphine can be suppressed to a significant degree by the augmentation of central cholinergic activity during morphine administration.

The G117H mutant of human butyrylcholinesterase (EC 3.1.1.8) was expressed in Chinese hamster ovary cells. Substitution of Gly 117 with His to make the G117H mutant endowed butyrylcholinesterase with the ability to catalyze the hydrolysis of organophosphate esters. G117H was still able to hydrolyze butyrylthiocholine, benzoylcholine, and o-nitrophenyl butyrate, but in addition it had acquired the ability to hydrolyze the antiglaucoma drug echothiophate and the pesticide paraoxon. Wild-type butyrylcholinesterase was irreversibly inhibited by echothiophate and paraoxon, but G117H regained 100% activity within 2-3 min following reaction with these compounds. On a polyacrylamide gel, the same bands that stained for activity with butyrylthiocholine also stained for activity with echothiophate. G117H is the only enzyme known that hydrolyzes echothiophate. Diethoxyphosphorylated G117H aged with a half-time of 5.5 h, a rate 600 times slower than the rate of hydrolysis. Echothiophate and paraoxon were hydrolyzed with the same kcat of 0.75 min-1. This calculates to a rate acceleration of 100,000-fold for hydrolysis of echothiophate and paraoxon by the G117H mutant compared to the nonenzymatic rate.

The aim of this study was to characterize the molecular forms of acetylcholinesterase (AChE) associated with the synaptic basal lamina at the neuromuscular junction. The observations were made on the neuromuscular junctions of cutaneous pectoris muscles of frog, Rana pipiens, which are similar to junctions of most other vertebrates including mammals, but are especially convenient for experimentation. By measuring relative AChE activity in junctional and extrajunctional regions of muscles after selective inactivation of extracellular AChE with echothiophate, or of intracellular AChE with DFP and 2-PAM, we found that > 66% of the total AChE activity in the muscle was junction-specific, and that > 50% of the junction-specific AChE was on the cell surface. More than 80% of the cell surface AChE was solubilized in high ionic strength detergent-free buffer, indicating that most, if not all, was a component of the synaptic basal lamina. Sedimentation analysis of that fraction indicated that while asymmetric forms (A12, A8) were abundant, globular forms sedimenting at 4-6 S (G1 and G2), composed > 50% of the AChE. It was also found that when muscles were damaged in various ways that caused degeneration of axons and muscle fibers but left intact the basal lamina sheaths, the small globular forms persisted at the synaptic site for weeks after phagocytosis of cellular components; under certain damage conditions, the proportion of globular to asymmetric forms in the vacated basal lamina sheaths was as in normal junctions. While the asymmetric forms required high ionic strength for solubilization, the extracellular globular AChE could be extracted from the junctional regions of normal and damaged muscles by isotonic buffer. Some of the globular AChE appeared to be amphiphilic when examined in detergents, suggesting that it may form hydrophobic interactions, but most was non-amphiphilic consistent with the possibility that it forms weak electrostatic interactions. We conclude that the major form of AChE in frog synaptic basal lamina is globular and that its mode of association with the basal lamina differs from that of the asymmetric forms.

Viable pancreas fragments from five human donors were incubated in oxygenated buffered Eagle Medium. The preparation and incubation conditions were based on the method of Scheele and Palade. In Group 1 there was 1-h preincubation with echothiophate (10(-4) M); then, acetylcholine (10(-5) M) was added. After 2 h tissues were prepared for electron microscopy. Acinar injury with vacuole formation was apparent. Many of these changes were observed in fragments incubated only with acetylcholine (10(-5) M) (Group 2) and in incubates with echothiophate only (10(-4) M) (Group 3); only minor changes were seen in controls with Eagle's Medium (Group 4). Large vacuoles were significantly more numerous in Group 1 than in Control Group 4 (p < 0.05). Zymogen granules were depleted in Groups 1, 2, and 3. This depletion was significant in Group 1 when compared with Group 4 (p < 0.02). These results extend previous in vitro results that showed increased amylase release after echothiophate treatment in human pancreas and a left shift in response to acetylcholine.

Accommodation and pupil constriction result from parasympathetic stimulation from the Edinger-Westphal (EW) nucleus of the midbrain resulting in release of acetylcholine at the neuromuscular junctions of the ciliary muscle and iris. Cholinergic and adrenergic drugs can be applied topically to evaluate the effects on the pupil and accommodative system without input from the EW nucleus. This study is directed at characterizing how topical low dose echothiophate, an anti-cholinesterase inhibitor (i.e., an indirect cholinergic agonist), epinephrine, an adrenergic agonist, and timolol maleate, a beta adrenergic antagonist, affect pupil diameter, resting refraction and accommodative amplitude and dynamics in rhesus monkeys. The effects of 0.015% echothiophate, 2% epinephrine, 0.5% timolol maleate and saline on pupil diameter and resting refraction were measured in one eye each of four normal rhesus monkeys for 60-90 minutes following topical instillation. Pupil diameter was measured with infrared videography and refraction was measured with a Hartinger coincidence refractometer. Effects on static and dynamic EW stimulated accommodation were studied in three iridectomized monkeys (ages 5, 6 and 12 years) with permanent indwelling stimulating electrodes in the EW nucleus. Dynamic accommodative responses were measured with infrared photorefraction for increasing current amplitudes before and during the course of action of the pharmacological agents. Echothiophate caused a significant decrease in pupil diameter of 3.07 ± 0.65 mm (mean ± SEM, p < 0.01), and a myopic shift in resting refraction of 1.30 ± 0.39 D (p < 0.05) 90 minutes after instillation. Epinephrine caused a 2.76±0.38 mm (p < 0.01) increase in pupil diameter with no change in resting refraction 60 minutes after instillation. Timolol maleate resulted in no significant change in either pupil diameter or resting refraction 60 minutes after instillation. There was no significant change in maximum EW stimulated accommodative amplitude after any agent tested. The amplitude vs. peak velocity relationship for accommodation was significantly different after echothiophate and timolol maleate, and for disaccommodation after echothiophate, epinephrine and timolol maleate. In conclusion, when tested objectively in anesthetized monkeys, epinephrine and timolol maleate did not alter resting refraction or accommodative amplitude, but did have small, significant affects on accommodative dynamics. This suggests that there is an adrenergic component to the accommodative system. Low dose echothiophate had significant effects on pupil diameter and resting refraction, with only small effects on the dynamics of the accommodative response.

Human pancreas contains two cholinesterase isoenzymes: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). In the present study, binding potency of two organophosphates for human cholinesterases were compared by the Ellman method. Echothiophate was found to have much greater potency than iso-OMPA for both cholinesterases. Using Karnovsky histochemical stains on human pancreatic tissue, the same results were confirmed. Dose-response studies with acetylcholine were done on viable pancreas fragments from nine human donors, without pancreatic disease (group I). Cold-preservation time was less than 30 h. Pancreas was minced into fragments, after the technique of Scheele and Palade, placed in Eagle's medium, and gassed with O2. Amylase release was measured by the Phadebas Method and corrected for basal release. There was a dose-dependent response to acetylcholine at 1 and 2 h, with a shift in peak amylase release to the left, when fragments were preincubated in 10(-4) M echothiophate. This indicated a 100-fold increase in sensitivity to acetylcholine. In three patients with chronic pancreatitis (Group II), there were variable patterns of response of amylase release to acetylcholine, and higher basal outputs. In Group III, prolonged storage conditions of over 40 h were tested for 4 pancreas donor tissues. There was no response to acetylcholine. These studies show that for up to 30 h cold storage, fragments of pancreas from human organ donors respond to acetylcholine in dose-dependent manner. An organophosphate, echothiophate (10(-4) M) which inhibits both cholinesterases, increases pancreatic sensitivity to acetylcholine, and these results are similar to findings from canine pancreas fragments, which also showed increased sensitivity.

An optical sensor for anticholinesterases (AntiChEs) was constructed by immobilizing fluorescein isothiocyanate (FITC)-tagged eel electric organ acetylcholinesterase (AChE) on quartz fibers and monitoring enzyme activity. The pH-dependent fluorescent signal generated by FITC-AChE, present in the evanescent zone on the fiber surface, was quenched by the protons produced during acetylcholine (ACh) hydrolysis. Analysis of the fluorescence response showed Michaelis-Menten kinetics with a Kapp value of 420 microM for ACh hydrolysis. The reversible inhibitor edrophonium (0.1 mM) inhibited AChE and consequently reduced fluorescence quenching. The biosensor response immediately recovered upon its removal. The carbamate neostigmine (0.1 mM) also inhibited the biosensor response but recovery was much slower. In the presence of ACh, the organophosphate (OP) diisopropylfluorophosphate (DFP) at 0.1 mM did not interfere with the ACh-dependent fluorescent signal quenching, but preexposure of the biosensor to DFP in absence of ACh inhibited totally and irreversibly the biosensor response. However, the DFP-treated AChE biosensor recovered fully after a 10-min perfusion with pralidoxime (2-PAM). Echothiophate, a quaternary ammonium OP, inhibited the ACh-induced fluorescence quenching in the presence of ACh and the phosphorylated biosensor was reactivated with 2-PAM. These effects reflected the mechanism of action of the inhibitors with AChE and the inhibition constants obtained were comparable to those from colorimetric methods. The biosensor detected concentrations of the carbamate insecticides bendiocarb and methomyl and the OPs echothiophate and paraoxon in the nanomolar to micromolar range. Malathion, parathion, and dicrotophos were not detected even at millimolar concentrations; however, longer exposure or prior modification of these compounds (i.e., to malaoxon, paraoxon) may increase the biosensor detection limits. This AChE biosensor is fast, sensitive, reusable, and relatively easy to operate. Since the instrument is portable and can be self-contained, it shows potential adaptability to field use.

The effects of sustained vagotonia induced by 7 consecutive days of pretreatment with an anticholinesterase, echothiophate iodide (ETP, 20μg/kg/day, s.c.) on carbachol-induced responses and muscarinic receptor binding characteristics in lung, heart and ileum of guinea-pigs have been investigated. The study was aimed at elucidating the mechanisms involved in the ETP-induced airway subsensitivity to carbachol observed in our previous studies.ETP-pretreatment reduced cholinesterase activity by about 40-50% in lung, heart, ileum and serum. In the lung and heart, neither the sensitivity of isolated tissues to carbachol nor the binding of [3H] quinuclidinyl benzilate ([3H]QNB) were influenced by ETP-pretreatment. In contrast, the sensitivity of isolated trachea and ileum to carbachol was reduced by ETP-pretreatment, and this sensitivity change of the ileum was associated with a fall in muscarinic receptor density without a change in receptor affinity, as determined in [3H]QNB binding experiments. ETP-pretreatment also reduced the sensitivity of trachea and ileum to histamine, although [3H] pyrilamine binding in the ileum was not affected.These results suggest that sustained vagotonia induced by chronic ETP-pretreatment reduces the sensitivity of isolated ileum and possibly trachea to carbachol and histamine through a reduction in muscarinic receptor density and also some alteration of receptor coupling to intracellular mechanisms.

The transsynaptic regulation of acetylcholinesterase (AChE) was studied by recording the changes in enzymatic activity following denervation in two types of autonomic ganglia in the frog, Rana pipiens. Opposite effects on AChE were found in the parasympathetic cardiac ganglion and in the sympathetic lumbar ganglion; denervation produced a significant increase in AChE activity in cardiac ganglia but a significant decrease in lumbar ganglia. The relative effects of denervation on intracellular and total AChE were examined by selectively inhibiting extracellular AChE with echothiophate, a poorly lipid-soluble cholinesterase inhibitor. Denervation resulted in a significant increase in intracellular AChE in cholinergic cardiac ganglia but had no effect on intracellular AChE activity in adrenergic lumbar ganglia. Histochemical studies revealed little change in extracellular AChE staining upon denervation in the cardiac ganglion, whereas in the lumbar ganglia there was a loss of AChE-specific reaction product. These results raise the possibility that the transsynaptic control of AChE activity by innervation in the frog is influenced by the transmitter synthetic properties of the postsynaptic ganglion cells.

Studies were performed to determine whether endothelium-derived relaxing factor (EDRF) can inhibit platelet aggregation within the vascular lumen, and if so, whether the inhibition persists in the presence of red blood cells (RBCs). Canine femoral arteries mounted in an organ bath were perfused with physiologic saline solution to which acetylsalicylic acid was added to block prostacyclin formation. During contraction with phenylephrine, addition of acetylcholine to the perfusing solution to evoke EDRF release relaxed the vessel wall. Washed human platelets labeled with 14C-5-hydroxytryptamine were added to the perfusing solution, and activated by thrombin infused via a branch vessel. The perfusate was collected downstream and centrifuged; the fraction of 14C-5-hydroxytryptamine appearing in the supernatant reflected the degree of platelet activation. Stimulation of EDRF release with acetylcholine inhibited 14C-5-hydroxytryptamine release. Hemoglobin (Hb) (10(-5) mol/L) blocked vascular relaxation and platelet- inhibition. RBCs at a hematocrit of 10% (treated with echothiophate to block erythrocyte cholinesterase) did not prevent relaxation but reversed the platelet inhibition. Lower hematocrits did not completely block the inhibition. Thus, erythrocyte Hb may modulate the inhibition of intraluminal platelet aggregation by EDRF.

Sublethal doses of organophosphate anticholinesterases cause acute pancreatitis in dogs within 2 h. In vitro studies using canine pancreatic fragments have also demonstrated that the peak of amylase release in response to acetylcholine is shifted far to the left after incubation with the organophosphates echothiophate (10(-4) M) or tetraisopropyl pyrophosphoramide (iso-OMPA) (10(-3) M), indicating an increased sensitivity of response. The present in vitro study examined whether there was also an increased susceptibility to acinar cell damage at the electron microscopic level after acetylcholine or cholecystokinin. Minced pieces of whole fresh canine pancreas 2-3 mm in size were placed in buffered Eagle's solution and gassed with 100% O2. After pretreatment 1 h with echothiophate or iso-OMPA, they were then incubated with acetylcholine (10(-5) M). Other tissues preincubated with echothiophate were stimulated with cholecystokinin (10(-9) M). These are submaximal doses for untreated canine pancreatic fragments. After acetylcholine and echothiophate or acetylcholine and iso-OMPA, there was extensive acinar damage with the appearance of large vacuoles and lakes, and interstitial edema. There was evidence of intense supramaximal stimulation and lateral exocytosis. Similar destructive changes were seen after echothiophate and cholecystokinin. In control sections from tissues stimulated with acetylcholine (10(-5) M) or cholecystokinin (10(-9) M, there were lumenal exocytotic patterns typical of submaximal stimulation. Other controls, organophosphate alone and unstimulated basal conditions, showed only minor changes. It is concluded that the increased sensitivity to acetylcholine after organophosphate incubation correlates with an increased susceptibility to acinar ultrastructural damage from acetylcholine and cholecystokinin.

Some organophosphorus compounds are toxic because they inhibit acetylcholinesterase (AChE) by phosphylation of the active site serine, forming a stable conjugate: Ser-O-P(O)-(Y)-(XR) (where X can be O, N, or S and Y can be methyl, OR, or SR). The inhibited enzyme can undergo an aging process, during which the X-R moiety is dealkylated by breaking either the P-X or the X-R bond depending on the specific compound, leading to a nonreactivatable enzyme. Aging mechanisms have been studied primarily using AChE. However, some recent studies have indicated that organophosphate-inhibited butyrylcholinesterase (BChE) may age through an alternative pathway. Our work utilized matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to study the aging mechanism of human BChE inhibited by dichlorvos, echothiophate, diisopropylfluorophosphate (DFP), isomalathion, soman, sarin, cyclohexyl sarin, VX, and VR. Inhibited BChE was aged in the presence of H2O18 to allow incorporation of (18)O, if cleavage was at the P-X bond. Tryptic-peptide organophosphate conjugates were identified through peptide mass mapping. Our results showed no aging of VX- and VR-treated BChE at 25 degrees C, pH 7.0. However, BChE inhibited by dichlorvos, echothiophate, DFP, soman, sarin, and cyclohexyl sarin aged exclusively through O-C bond cleavage, i.e., the classical X-R scission pathway. In contrast, isomalathion aged through both X-R and P-X pathways; the main aged product resulted from P-S bond cleavage and a minor product resulted from O-C and/or S-C bond cleavage.

Certain organophosphate (OP) cholinesterase inhibitors (ChEIs) are also known to bind to the muscarinic acetylcholine receptor (mAChR). The functional consequences of such binding were investigated here using the following OP compounds: VX, echothiophate, sarin, and soman. VX (charged at physiological pH) and echothiophate (formally charged) inhibited a specific signal transduction pathway in CHO cells expressing either the M(1) or M(3) mAChR. Hence, they blocked carbamylcholine (CCh)-induced cyclic adenosine monophosphate (cAMP) synthesis (muM) and had almost no effect on CCh-induced phosphoinositide (PI) hydrolysis. These substances were inactive on forskolin-induced cAMP inhibition signaling in CHO cells expressing M(2) mAChR. In binding studies, using [(3)H]-N-methyl scopolamine ([(3)H]NMS) as the competitor ligand, the ChEIs, VX and echothiophate exhibited binding to rat cortical mAChR with K(i) values in the muM range. The non-charged compounds, sarin and soman, were inert in modulating both cAMP metabolism and PI hydrolysis in CHO cells expressing M(1), M(2), and M(3) mAChRs, and no binding was observed in presence of [(3)H]NMS. These data suggest that VX and echothiophate act as function-specific blockers via a non-classical path of antagonistic activity, implying the involvement of allosteric/ectopic-binding site in M(1) and M(3) mAChRs. The functionally selective antagonistic behavior of echothiophate and VX makes them potential tools for dissecting the interactions of the mAChR with different G proteins.

The origins of human acetylcholinesterase (HuAChE) reactivity toward the lethal chemical warfare agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) and its stereoselectivity toward the P(S)-VX enantiomer (VX(S)) were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere O-ethyl S-(3-isopropyl-4-methylpentyl) methylphosphonothioate (nc-VX) as well as echothiophate and its noncharged analogue. Reactivity of wild-type HuAChE toward VX(S) was 115-fold higher than that toward VX(R), with bimolecular rate constants of 1.4 x 10(8) and 1.2 x 10(6) min(-1) M(-1). HuAChE was also 12500-fold more reactive toward VX(S) than toward nc-VX(S). Substitution of the cation binding subsite residue Trp86 with alanine resulted in a 3 order of magnitude decrease in HuAChE reactivity toward both VX enantiomers, while this replacement had an only marginal effect on the reactivity toward the enantiomers of nc-VX and the noncharged echothiophate. These results attest to the critical role played by Trp86 in accommodating the charged moieties of both VX enantiomers. A marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 with asparagine (D74N) practically abolished stereoselectivity toward VX(S) (130-fold decrease), while a substitution which retains the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers is mainly a result of a different interaction of the charged leaving group with Asp74.

Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1 mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5 microg/ml human G117H BChE, 2 microg/ml wild-type mouse BChE, and 0.06 microg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1 mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.

Recently, several bis-pyridiniumaldoximes linked by a variable-length alkylene chain were rationally designed in our laboratories as cholinesterase reactivators. Extensive in vitro tests of these oximes with acetylcholinesterase inhibited by two different organophosphate agents, echothiophate and diisopropylfluorophosphate, revealed one compound with particularly good reactivation kinetics and affinity for phosphorylated acetylcholinesterase (AChE). This compound, designated "ortho-7", with a heptylene chain bridging two aldoximes ortho to a pyridinium ring nitrogen, was chosen for detailed comparison with the classic reactivator pyridine-2-aldoxime methochloride (2-PAM). In vitro, ortho-7 reactivated AChE selectively, without restoring activity of the related enzyme butyrylcholinesterase (BChE). For in vivo studies, rats were injected with ortho-7 or 2-PAM before or after organophosphate exposure, and the activities of AChE and BChE were determined at multiple intervals in blood and solid tissues. Ortho-7 behaved nearly as well in the animal as in vitro, reactivating AChE to the same extent as 2-PAM in all peripheral tissues studied (serum, red blood cell, and diaphragm), but at doses up to 100-fold smaller. Like other oxime reactivators, ortho-7 did not reactivate brain AChE after systemic administration. Nonetheless, this agent could be useful in combination therapy for organophosphate exposure, and it may provide a platform for development of additional, even more effective reactivators.

In this study neurite outgrowth from cultured hippocampal neurones was increased by addition of acetylcholinesterase acting in a non-cholinergic manner. Only monomeric acetylcholinesterase, a form of acetylcholinesterase dominant in development, increased neurite outgrowth (3-10 U/ml); moreover this effect was not blocked by active site blockers (echothiophate and galanthamine) but was sensitive to the addition of peripheral site blockers (fasciculin and BW284c51). It appears therefore that acetylcholinesterase has alternative, non-cholinergic functions, one of which could be in development, via a peripheral site. The possibility of a causal relationship between neurite outgrowth and calcium influx was explored using a spectrum of acetylcholinesterase variants, inhibitors and calcium channel blockers. Acetylcholinesterase regulation of outgrowth was shown to depend on an influx of extracellular calcium specifically via the L-type voltage-gated calcium channel. In summary, we propose that, independent of its catalytic activity, a selective form of acetylcholinesterase has a role in the development of hippocampal neurones via a selective voltage-gated calcium channel.

In the cercarial and schistosomal stages of the life cycle of the trematode Schistosoma mansoni, acetylcholinesterase occurs as two principal molecular forms (both globular), present in approximately equal amounts, with sedimentation coefficients of 6.5 S and 8 S. The 6.5 S form is solubilized by bacterial phosphatidylinositol-specific phospholipase C from intact schistosomula. It is thus located on the outer surface of the schistosomal tegument and is most probably analogous to the glycosylphosphatidylinositol-anchored G2 form of acetylcholinesterase found in the electric organ of Torpedo, on the surface of mammalian erythrocytes, and elsewhere. Both forms are fully solubilized by the non-ionic detergent Triton X-100. Upon passing such a detergent extract over a heparin-Sepharose column, only the 8 S form was retained on the column. The bound acetylcholinesterase could be progressively eluted by increasing the salt concentration, with approx. 0.5-0.6 M NaCl being needed for complete elution. Selective inhibition experiments carried out on live parasites using the covalent acetylcholinesterase inhibitor echothiophate (phospholine), which does not penetrate the tegument, selectively inhibited the 6.5 S form, but not the 8 S form, suggesting an internal location for the latter. Monoclonal antibodies raised against S. mansoni acetylcholinesterase also distinguished between the two forms. Thus monoclonal antibody SA7 bound the 6.5 S form selectively, whereas SA57 recognized the 8 S form. The selective binding of the 8 S form to heparin suggests that, within the parasite, this form may be associated with the extracellular matrix of the musculature.

In Aplysia, a marine mollusc, acetylcholinesterase (AChE) is present in cholinergic and non-cholinergic neurons and in hemolymph. Aplysia hemolymph has a very high level of AChE which promotes neurite growth in primary cultures of dopaminergic neurons via a non-catalytic mechanism. In contrast, AChE is known to facilitate neurite growth in cholinoceptive neurons by hydrolyzing ACh which inhibits neurite growth. In order to test whether AChE's site-specific neurotrophic action varies with the neuronal phenotype, we investigated the effects of active-site inhibited hemolymph AChE on neurite growth of cholinergic neurons of Aplysia in primary culture. Organophosphates being long-acting active site inhibitors of AChE were chosen for this study. The effects of active site inhibited hemolymph AChE was tested on large cholinergic neurons, R2 (abdominal ganglion) and LPL1 (left pleural ganglion) as well as small cholinergic neurons (buccal ganglion) of Aplysia, maintained in culture. Partially purified hemolymph AChE was inhibited by either 10 microM of echothiophate or 5 microM of paraoxon. Neurons were maintained in (1) L15 (defined medium) alone; (2) L15 + echothiophate; (3) L-15 + paraoxon; (4) L-15 + hemolymph AChE; (5) L15 + hemolymph AChE + echothiophate; and (6) L-15 + hemolymph AChE + paraoxon. Addition of uninhibited hemolymph AChE significantly increased neurite growth of cultured neurons compared to L15 alone. In the presence of echothiophate-inhibited or praoxon-inhibited AChE, neurite growth was significantly reduced when compared to L15 + uninhibited AChE. While the presence of echothiophate by itself did not reduce survival or neurite growth when compared to L-15 alone, the presence of paraoxon by itself markedly reduced survival and neurite growth of cultured neurons. The results show that AChE's catalytic action contributes to enhance neurite growth in cholinergic neurons and the effects of paraoxon appears to differ from that of echothiophate on cholinergic neurons of Aplysia.

Studies with mutant AChEs where up to seven surface negative charges were neutralized demonstrated that electrostatic attraction does not contribute to the catalytic efficiency of the enzyme [1,2]. Yet, electrostatic steering due to the negatively charged residues in the gorge (D74 at its rim and E202 and E450 near its bottom) remained difficult to assess [3, 4]. We therefore extended the studies of these mutants with isosteric pairs of charged and noncharged substrates (ATC- acetylthiocholine; 3, 3-dimethylbutyl thioacetate-TB), charged and noncharged phosphate inhibitors (echothiophate, S-3,3-di-methylbutyl diethylthiophosphate) as well as with the transition state analog m-trimethylammonio trifluoroacetophenone (TFK). Replacements of D74 by negatively charged (D74E) positively charged (D74K) or neutral (D74N, D74G) residues resulted in small and uniform increase in values of Km for ATC except for D74K where the increase was much larger. On the other hand, the bimolecular rates of reactions with echothiophate and TFK were affected much more in cases of D74N and D74K than in those of D74E and E74G. All these mutations have almost no effect on catalytic activity toward the non-charged substrate, yet a small increase of phosphorylation rates by the noncharged OP inhibitor was observed for enzymes with noncharged residues at position 74 but not for D74K or D74E. These findings, together with the observation that ionic strength had small and equivalent effect on reactivities of all these enzymes, indicate that D74 probably does not enhance reactivity by steering charged ligands into the gorge but rather influences indirectly specific interactions of charged ligands with elements of the active center.

Using the 3-dimensional crystal structure of acetylcholinesterase (AChE) and a sophisticated docking program (1), we designed bis-oximes predicted to have optimal characteristics for reactivating AChE. Each compound contained two pyridine-aldoxime moieties linked by a methylene chain. The best orientation for the oxime substituent proved to be ortho to the ring nitrogen. When tested in vitro with human AChE irreversibly inhibited by the organophosphate drug, echothiophate, efficacy of reactivation depended on linker chain length in the bis-oxime. The length dependency was exactly as predicted if one oxime group interacted with the active site serine while the second interacted with AChE’s peripheral site. The most potent compound (7-methylene residues) caused nearly 100% reactivation at 10−6 M in 1 hr at 37°C. Full dose response curves showed that this compound was 100-fold more potent than the “standard” oxime (2-pralidoxime) in reactivating AChE exposed to either echothiophate or di-isopropylflurophosphate. A 100-fold enhancement in potency was also observed in vivo, when the compound was tested for ability to promote survival of rats challenged with echothiophate in doses up to 10-LD50. These results show that, as with AChE inhibitors, oximes gain affinity and efficacy from dual site interactions, which can be predicted by computational chemistry.

Asp-70 is the defining amino acid in the peripheral anionic site of human butyrylcholinesterase (BuChE), whereas acetylcholinesterase has several additional amino acids, the most important one being Trp-277 (Trp-279 in TorpedoAChE). We studied mutants D70G, D70K and A277W to evaluate the role of Asp-70 and Trp-277 in reactions with organophosphates. We found that Asp-70 was important for binding positively charged echothiophate, but not neutral paraoxon and iso-OMPA. Asp-70 was also important for binding of positively charged pralidoxime (2-PAM) and for activation of re-activation by excess 2-PAM. Excess 2-PAM had an effect similar to substrate activation, suggesting the binding of 2 mol of 2-PAM to wild-type but not to the D70G mutant. A surprising result was that Asp-70 was important for irreversible aging, the D70G mutant having a 3-and 8-fold lower rate of aging for paraoxon-inhibited and di-isopropyl fluorophosphate-inhibited BuChE. Mutants of Asp-70 had the same rate constants for phosphorylation and re-activation by 2-PAM as wild-type. The A277W mutant behaved like wild-type in all assays. Our results predict that people with the atypical (D70G) variant of BuChE will be more sensitive to the toxic effects of echothiophate, but will be equally sensitive to paraoxon and di-isopropyl fluorophosphate. People with the D70G mutation will be resistant to re-activation of their inhibited BuChE by 2-PAM, but this will be offset by the lower rate of irreversible aging of inhibited BuChE, allowing some regeneration by spontaneous hydrolysis.

Aplysia, a marine mollusc, has significant amounts of acetylcholinesterase in its hemolymph, reaching maximum levels in the adults with reproductive maturity [Srivatsan M., et al. (1992) J. comp. Physiol. 162, 29-37]. Since hemolymph of mature Aplysia is neurotrophic to Aplysia neurons in culture [Schacher S. and Proshanski E. (1983) J. Neurosci. 3, 2403-2413], we examined whether acetylcholinesterase is a hemolymph neurotrophic factor. Dopaminergic neurons from the pedal ganglia of young adult Aplysia were maintained in culture in defined medium or defined medium supplemented with hemolymph. After 24 h, neurons in defined medium supplemented with hemolymph were well attached to the substratum and exhibited multiple, long neurites. In contrast, neurons in defined medium alone attached poorly and exhibited one or two short neurites. When acetylcholinesterase was inhibited with a specific, membrane-impermeable inhibitor (1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide) which binds to its catalytic and peripheral anionic sites, the neurotrophic effect of hemolymph was significantly reduced. However, inhibition of the catalytic site alone with membrane impermeable echothiophate still resulted in enhanced neurite growth. An analogue of acetylcholine, carbachol, which is not hydrolysed by acetylcholinesterase, did not interfere with neurite growth when added to the supplemented medium. Acetylcholinesterase isolated from the hemolymph and highly purified human acetylcholinesterase also promoted neurite growth in Aplysia neurons. These results show that i) acetylcholinesterase circulating in the hemolymph promotes neurite growth of adult neurons in culture; ii) the growth promoting action of acetylcholinesterase is independent of its function of hydrolysing acetylcholine and iii) the peripheral anionic site of acetylcholinesterase appears to be involved in neurite regeneration.

Rat hippocampal minces were loaded with [acetyl 1- 14 C]acetylcholine ([ 14 C]ACh) in the presence of the “poorly penetrating” acetylcholinesterase (EC 3.1.1.7; AChE) inhibitor echothiophate and the effect of high K + depolarization determined on the subcellular storage and release of [ 14 C]ACh and its metabolites. Results indicated that high K + did not augment the release of [ 14 C]ACh. Rather, it increased the release of [ 14 ]Clacetate while simultaneously reducing the level of [ 14 C]ACh in the cytosolic (S 3 ) fraction. When the identical experiment was performed with paraoxon, a “penetrating” AChE inhibitor, high K + still did not increase the release of [ 14 C]ACh. However, paraoxon prevented the K + -induced loss of [ 14 C]ACh from the cytosolic fraction as well as the K + -induced gain of [ 14 C]acetate in the release medium. When minces were loaded with [ 14 C]ACh in the presence of echothiophate and subsequently subjected to high K + depolarization in the absence or presence of vesamicol (AH5183; (−)-trans-2-[4-phenylpiperidino] cyclohexanol), a drug which blocks the refilling of synaptic vesicles with ACh, the amount of endogenous ACh released was reduced approximately 50%. Conversely, the amount of [ 14 C]ACh released was not reduced at all. These results suggest that cytosolic ACh is not directly transported into synaptic vesicles for release when hippocampal nerve terminals are depolarized. Rather, its hydrolysis is accelerated in response to depolarization. A working hypothesis explaining the importance of the depolarization-induced breakdown of cytosolic ACh to central ACh metabolism is presented.

Intrathecal (IT) administration of acetyl cholinesterase inhibitors produces analgesia through a muscarinic action. The addition of IT cholinergic agonists to IT opioids or alpha 2-adrenergic agonists results in enhanced analgesic effects, but it is not clear whether these interactions are synergistic, additive, or less than additive. Dose-response curves for hot plate and tail immersion tests were established for IT neostigmine, physostigmine, and echothiophate in rats. Dose-response curves for hot plate testing were established for IT morphine and clonidine. The effect of maximally effective doses of each of the three cholinergic drugs on hot plate testing was plotted over time. Isobolographic analysis was performed for hot plate testing using neostigmine-morphine and neostigmine-clonidine combinations. The three cholinesterase inhibitors produced profound analgesia on hot plate testing but incomplete analgesia using the tail immersion test. Duration of analgesia on hot plate testing ranged from 45 min for physostigmine to more than 24 h for echothiophate. IT administration of combinations of neostigmine plus morphine and neostigmine plus clonidine both produced significantly more profound analgesia than the calculated additive effects and are, therefore, synergistic in their actions.

This study has investigated the possibility that acetylcholinesterase could play a non-classical role as an adhesion factor or growth factor in the development of dopaminergic neurons in organotypic slice culture of postnatal day 1 rats. When the culture medium was supplemented with acetylcholinesterase (3 U/ml), outgrowth of tyrosine hydroxylase-immunoreactive neurites was significantly enhanced. Addition of a specific inhibitor of acetylcholinesterase, BW284c51, caused a decrease in the number of tyrosine hydroxylase neurons and a reduction in the cell body size and extent of neurite outgrowth of remaining neurons. However, echothiophate which also inhibits AChE activity, did not produce these effects. Therefore acetylcholinesterase could act as a growth enhancing factor for dopaminergic neurons, and disruption of an as yet unidentified site on the acetylcholinesterase molecule by BW284c51 could decrease the survival and outgrowth of these neurons.

The severity of poisoning following acetylcholinesterase (AChE) inhibition correlates weakly with total AChE activity. This may be partly due to the existence of functional and non-functional pools of AChE. AChE consists of several molecular forms. The aim of the present study was to investigate which of these forms will correlate best with neuromuscular transmission (NMT) remaining after partial inhibition of this enzyme. Following sublethal intoxication of rats with the irreversible AChE inhibitor soman, diaphragms were isolated after 0.5 or 3 h. It appeared that at 3 h after soman poisoning the percentage of G1 increased, while those of G4 and A12 decreased. NMT was inhibited more strongly than in preparations obtained from the 0.5 h rats with the same level of AChE inhibition, but with a normal ratio of molecular forms. NMT correlated positively with G4 as well as with A12, but inversely with G1. In vitro inhibition with the charged inhibitors DEMP and echothiophate resulted in higher levels of total AChE, relatively less G1 and more G4 and A12 than after incubation with soman, but led to less NMT. Treatment of soman-intoxicated rats with the reactivating compound HI-6 resulted in preferential reactivation of A12, persisting low levels of G1 and concurrent recovery of NMT as compared with saline-treated soman controls with equal total AChE activity. Apparently, in rat diaphragm G4 and A12 are the functional AChE forms.

Some compounds that inhibit acetylcholinesterase (AChE) activity compete directly with quinuclidinyl benzilate (QNB) binding, a muscarinic antagonist which binds to all subtypes equally, and with cis-methyldioxolane (CD), an agonist that binds with high affinity to the M2 subtype of muscarinic receptors. The relationship between inhibition of AChE activity and the capability to affect muscarinic receptors directly has not been systematically explored. The interaction of eight organophosphates with muscarinic receptors was compared to their ability to inhibit AChE activity in vitro in tissue homogenates from rat hippocampus and frontal cortex, two cholinergically enriched areas of the brain. Of the compounds tested only echothiophate competed for [3H]QNB binding and only at concentrations greater than 100 μM. The anticholinesterase compounds were also tested for their ability to compete with a muscarinic receptor agonist, [3H]CD, which binds with high affinity (approximate KD = 3.5 nM) to 10 and 3% of the muscarinic receptors in the frontal cortex and hippocampus, respectively. The anticholinesterase compounds inhibited high-affinity [3H]CD binding up to 80% and the effects were similar in both tissues. Echothiophate and DFP were potent inhibitors of [3H]CD binding, as were the active "oxon" forms of parathion, malathion, and disulfoton. The parent "thio" forms of these insecticides, however, were much less effective in competing for [3H]CD binding. A similar pattern of potency was observed for the inhibition of brain AChE activity. A strong correlation was found between the ability of a compound to inhibit AChE activity and the ability to compete with [3H]CD binding. These data suggest that the biological effects of cholinesterase-inhibiting compounds may be due to more than their ability to inhibit AChE.

Cholinesterases present homologies with some cell adhesion molecules; however, it is unclear whether and how they perform adhesive functions. Here, we provide the first direct evidence showing that neurite growth in vitro from various neuronal tissues of the chick embryo can be modified by some, but not all, anticholinesterase agents. By quantifying the neuritic G4 antigen in tectal cell cultures, the effect of anticholinesterases on neurite growth is directly compared with their cholinesterase inhibitory action. BW 284C51 and ethopropazine, inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively, strongly decrease neurite growth in a dose-dependent manner. However, echothiophate which inhibits both cholinesterases, does not change neuritic growth. These quantitative data are supplemented by morphological observations in retinal explant cultures grown on striped laminin carpets, viz., defasciculation of neurite bundles by BW 284C51 and Bambuterol occurs, indicating that these drugs disturb adhesive mechanisms. These data strongly suggest that a) cholinesterases can participate in regulating axonal growth, b) both AChE and BChE can perform such a nonsynaptic function, and c) this function is not the result of the enzyme activity per se, since at least one drug was found that inhibits all cholinesterase activities but not neurite growth. Thus, a secondary site on cholinesterase molecules must be responsible for adhesive functions.

The effect of eight different acetylcholinesterase inhibitors (AChEIs) on the activity of acetylcholinesterase (AChE) molecular forms was investigated. Aqueous-soluble and detergent-soluble AChE molecular forms were separated from rat brain homogenate by sucrose density sedimentation. The bulk of soluble AChE corresponds to globular tetrameric (G4), and monomeric (G1) forms. Heptylphysostigmine (HEP) and diisopropylfluorophosphate were more selective for the G1 than for the G4 form in aqueous-soluble extract. Neostigmine showed slightly more selectivity for the G1 form both in aqueous- and detergent-soluble extracts. Other drugs such as physostigmine, echothiophate, BW284C51, tetrahydroaminoacridine, and metrifonate inhibited both aqueous- and detergent-soluble AChE molecular forms with similar potency. Inhibition of aqueous-soluble AChE by HEP was highly competitive with Triton X-100 in a gradient, indicating that HEP may bind to a detergent-sensitive non-catalytic site of AChE. These results suggest a differential sensitivity among AChE molecular forms to inhibition by drugs through an allosteric mechanism. The application of these properties in developing AChEIs for treatment of Alzheimer disease is considered.

1. The sensitivity of normal and denervated cardiac ganglion cells to the cholinergic agonists acetylcholine and carbamylcholine (carbachol) were compared in the frog, Rana pipiens. Acetylcholine and carbachol bind to the same acetylcholine receptors, but, unlike acetylcholine, carbachol is resistant to hydrolysis by acetylcholinesterase. 2. Sensitivity was assessed by the peak depolarization elicited in response to a sustained pulse of ligand emitted from a pipette positioned 10 microns from the ganglion cell surface. This technique allows the sensitivity of the entire cell to be recorded with a single measurement. 3. The acetylcholine sensitivity of normal cardiac ganglion cells was increased by inhibiting extracellular acetylcholinesterase with echothiophate. 4. Denervation increased the sensitivity of cardiac ganglion cells to acetylcholine but not to carbachol. 5. Following the inhibition of extracellular acetylcholinesterase with echothiophate, sensitivity to acetylcholine was similar in normal and in denervated ganglion cells. 6. The increased sensitivity to acetylcholine of cardiac ganglion cells following denervation is caused by a reduction in the hydrolysis of the transmitter by acetylcholinesterase rather than by changes in the number and/or properties of acetylcholine receptors.

In 20 patients with open-angle glaucoma in aphakia or pseudophakia whose intraocular pressure had remained uncontrolled on their current medical therapy, the medication was changed from pilocarpine or carbachol to echothiophate iodide. In all, 12 patients (60%) showed a statistically significant improvement in pressure control, 7 (35%) showed no change, and 1 had higher pressure. One-third of the patients with improved intraocular-pressure control eventually required laser or incisional surgery after a mean of 23 months, whereas the remaining subjects were controlled for the duration of the follow-up, which averaged 26 months. Side effects encountered during echothiophate iodide treatment included ocular irritation, decreased vision, and one retinal detachment.