Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P-S Bonded Organophosphorus as Monitored by Spectrofluorimetry.

Enzyme-catalyzed hydrolysis of echothiophate, a P-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 ± 0.03 mM and kcat = 5.4 ± 1.6 min-1. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (Km = 2.6 ± 0.2 mM; kcat = 53400 min-1). With a kcat/Km = (2.6 ± 1.6) × 107 M-1min-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-S bonded OPs by thiol-free OP hydrolases.

Engineering Dynamic Surface Peptide Networks on ButyrylcholinesteraseG117H for Enhanced Organophosphosphorus Anticholinesterase Catalysis.

The single residue mutation of butyrylcholinesterase (BChEG117H) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in KM and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.

Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad.

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 µs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.

Intraocular pressure control with echothiophate iodide in children's eyes with glaucoma after cataract extraction.

PURPOSE: To report the intraocular pressure (IOP)-lowering ability and side-effect profile of echothiophate iodide (EI) in the control of glaucoma in aphakic and pseudophakic eyes of children. METHODS: The medical records of all aphakic and pseudophakic children treated with EI for IOP lowering after developing glaucoma were retrospectively reviewed. RESULTS: A total of 32 eyes of 21 children were included. Mean age at cataract removal was 3.9 months (range, 5 days-2.7 years). Mean age of glaucoma diagnosis was 3.2 years (range, 40 days-12 years). Mean duration from cataract removal and diagnosis of glaucoma was 2.9 years (range, 16 days-12 years). EI reduced IOP in 31 of 32 eyes. Mean baseline IOP (29.1 ± 5.3 mm Hg) dropped to 19.6 ± 6.7 mm Hg. Six eyes had IOP spikes that could not be controlled with other medications when commercial unavailability led to discontinuation of EI. Average duration of use was 3.5 years. Mean final IOP on an average of 2.2 medications was 16.9 ± 5.1 mm Hg 7.9 years following initial glaucoma diagnosis. Four eyes required surgery for uncontrolled IOP. Side-effects included transient redness (3/32 eyes), not necessitating discontinuation of EI. CONCLUSIONS: EI lowered IOP to within an acceptable range with no significant adverse events. In several patients it was the only pharmacologic agent that was successful.

α7-Containing and non-α7-containing nicotinic receptors respond differently to spillover of acetylcholine.

We explored whether nicotinic acetylcholine receptors (nAChRs) might participate in paracrine transmission by asking if they respond to spillover of ACh at a model synapse in the chick ciliary ganglion, where ACh activates diffusely distributed α7- and α3-containing nAChRs (α7-nAChRs and α3*-nAChRs). Elevating quantal content lengthened EPSC decay time and prolonged both the fast (α7-nAChR-mediated) and slow (α3*-nAChR-mediated) components of decay, even in the presence of acetylcholinesterase. Increasing quantal content also prolonged decay times of pharmacologically isolated α7-nAChR- and α3*-nAChR-EPSCs. The effect upon EPSC decay time of changing quantal content was 5-10 times more pronounced for α3*-nAChR- than α7-nAChR-mediated currents and operated over a considerably longer time window: ≈ 20 vs ≈ 2 ms. Control experiments rule out a presynaptic source for the effect. We suggest that α3*-nAChR currents are prolonged at higher quantal content because of ACh spillover and postsynaptic potentiation (Hartzell et al., 1975), while α7-nAChR currents are prolonged probably for other reasons, e.g., increased occupancy of long channel open states. α3*-nAChRs report more spillover when α7-nAChRs are competitively blocked than under native conditions; this could be explained if α7-nAChRs buffer ACh and regulate its availability to activate α3*-nAChRs. Our results suggest that non-α7-nAChRs such as α3*-nAChRs may be suitable for paracrine nicotinic signaling but that α7-nAChRs may not be suitable. Our results further suggest that α7-nAChRs may buffer ACh and regulate its bioavailability.

X-ray crystallographic snapshots of reaction intermediates in the G117H mutant of human butyrylcholinesterase, a nerve agent target engineered into a catalytic bioscavenger.

OPs (organophosphylates) exert their acute toxicity through inhibition of acetylcholinesterase, by phosphylation of the catalytic serine residue. Engineering of human butyrylcholinesterase, by substitution of a histidine residue for the glycine residue at position 117, led to the creation of OP hydrolase activity. However, the lack of structural information and poor understanding of the hydrolytic mechanism of the G117H mutant has hampered further improvements in the catalytic activity. We have solved the crystallographic structure of the G117H mutant with a variety of ligands in its active site. A sulfate anion bound to the active site suggested the positioning for an OP prior to phosphylation. A fluoride anion was found in the active site when NaF was added to the crystallization buffer. In the fluoride complex, the imidazole ring from the His117 residue was substantially shifted, adopting a relaxed conformation probably close to that of the unliganded mutant enzyme. Additional X-ray structures were obtained from the transient covalent adducts formed upon reaction of the G117H mutant with the OPs echothiophate and VX [ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl](methyl)phosphinate]. The position of the His117 residue shifted in response to the introduction of these adducts, overlaying the phosphylserine residue. These structural data suggest that the dephosphylation mechanism involves either a substantial conformational change of the His117 residue or an adjacent nucleophilic substitution by water.

Lowering of IOP by echothiophate iodide in pseudophakic eyes with glaucoma.

PURPOSE: We retrospectively investigated the intraocular pressure (IOP)-lowering effects of echothiophate iodide (EI) as adjunctive treatment for pseudophakic glaucoma patients who were receiving maximal medical therapy (MMT), including the newer class of medications, i.e., prostaglandin analogs, alpha-2 agonists, and topical carbonic anhydrase inhibitors. METHODS: The medical records of all pseudophakic glaucoma patients (24 eyes) under MMT who received supplementary EI 0.125% between January 2002 and December 2003 were reviewed. IOP data and number of medications before, during and after EI treatment were collected. RESULTS: Adding EI to MMT further reduced IOP in 23 of 24 eyes. Three eyes (12.5%) showed some lowering of IOP, but not enough to be considered controlled (IOP above the target pressure). The mean baseline IOP of 30.4 +/- 8.2 mmHg (median 29 mmHg) dropped at final follow-up (11.2 +/- 3.9 months) to 16.6 +/- 4.2 mmHg (median 17 mmHg, p < 0.0001) in all eyes that had showed effective pressure reduction upon the addition of EI. Their IOP rose to 27.7 +/- 8.0 mmHg (median 28 mmHg, p < 0.001) when EI was discontinued because of commercial non-availability. IOP reduction was > or =20% in 18 (75%) eyes and > or =30% (a mean decrease of 16.7 +/- 8.3mmHg) in 15 eyes (63%). Four eyes (16.6%) required a trabeculectomy despite EI supplement. Five eyes were re-challenged with EI when a small amount was released for sale: their IOP of 26.6 +/- 7.1 mmHg after the first EI discontinuation had dropped to 16.4 +/- 4.3 mmHg (p < 0.0001) and rose to 29.6 +/- 7.1 mmHg when EI was again discontinued. The recorded EI-associated side effects were increased miosis in all eyes and headache (8/24 patients), neither of which were reasons for discontinuation of the drug in any patient. CONCLUSION: EI substantially decreased the IOPs in pseudophakic glaucoma eyes receiving maximal medical therapy, including the newer class of medications. This drug may be the last resort for post-cataract advanced glaucoma patients and may obviate the need for filtering surgery among the very elderly.

Autonomic drugs and the accommodative system in rhesus monkeys.

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 min 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 min after instillation. Epinephrine caused a 2.76 +/- 0.38 mm (p < 0.01) increase in pupil diameter with no change in resting refraction 60 min after instillation. Timolol maleate resulted in no significant change in either pupil diameter or resting refraction 60 min 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.

Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry.

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.

Kinetic analysis of butyrylcholinesterase-catalyzed hydrolysis of acetanilides.

The aryl-acylamidase (AAA) activity of butyrylcholinesterase (BuChE) has been known for a long time. However, the kinetic mechanism of aryl-acylamide hydrolysis by BuChE has not been investigated. Therefore, the catalytic properties of human BuChE and its peripheral site mutant (D70G) toward neutral and charged aryl-acylamides were determined. Three neutral (o-nitroacetanilide, m-nitroacetanilide, o-nitrophenyltrifluoroacetamide) and one positively charged (3-(acetamido) N,N,N-trimethylanilinium, ATMA) acetanilides were studied. Hydrolysis of ATMA by wild-type and D70G enzymes showed a long transient phase preceding the steady state. The induction phase was characterized by a hysteretic "burst". This reflects the existence of two enzyme states in slow equilibrium with different catalytic properties. Steady-state parameters for hydrolysis of the three acetanilides were compared to catalytic parameters for hydrolysis of esters giving the same acetyl intermediate. Wild-type BuChE showed substrate activation while D70G displayed a Michaelian behavior with ATMA as with positively charged esters. Owing to the low affinity of BuChE for amide substrates, the hydrolysis kinetics of neutral amides was first order. Acylation was the rate-determining step for hydrolysis of aryl-acetylamide substrates. Slow acylation of the enzyme, relative to that by esters may, in part, be due suboptimal fit of the aryl-acylamides in the active center of BuChE. The hypothesis that AAA and esterase active sites of BuChE are non-identical was tested with mutant BuChE. It was found that mutations on the catalytic serine, S198C and S198D, led to complete loss of both activities. The silent variant (FS117) had neither esterase nor AAA activity. Mutation in the peripheral site (D70G) had the same effect on esterase and AAA activities. Echothiophate inhibited both activities identically. It was concluded that the active sites for esterase and AAA activities are identical, i.e. S198. This excludes any other residue present in the gorge for being the catalytic nucleophile pole.

Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters.

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.

Function-specific blockage of M(1) and M(3) muscarinic acetylcholine receptors by VX and echothiophate.

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.

Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging.

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.

H-7 effect on outflow facility after trabecular obstruction following long-term echothiophate treatment in monkeys.

PURPOSE: To determine whether H-7 can enhance outflow facility after trabecular meshwork obstruction by extracellular material that accumulates after long-term treatment of monkeys with the cholinesterase inhibitor echothiophate iodide (ECHO). METHODS: Cynomolgus monkeys were treated topically with 150 microg ECHO in one (n = 4 eyes) or both (n = 8 eyes) eyes for up to 48 weeks. Accommodation response to topical pilocarpine was monitored periodically. Outflow facility response to H-7 was measured by two-level constant pressure perfusion on three or four different occasions after intraocular pressure was elevated for 12 to 18 weeks. RESULTS: Long-term treatment with ECHO decreased the accommodative response to pilocarpine and increased intraocular pressure, as has been reported. Baseline outflow facility was decreased by 46% +/- 7% (n = 12, P < 0.001). H-7 partially restored baseline outflow facility measured during subsequent perfusions while ECHO treatment was continued. Concurrent H-7 enhanced outflow facility by 73% +/- 18% (n = 12, P < 0.005) beyond the same-day baseline in ECHO-treated eyes. Cessation of ECHO treatment further restored baseline outflow facility, and the outflow facility response to H-7. CONCLUSIONS: H-7 can enhance OF in the presence of trabecular obstruction produced by long-term ECHO treatment. This suggests that H-7 may be useful in treating glaucoma, even in the presence of accumulated plaque material that has been described previously.

Resistance to organophosphorus agent toxicity in transgenic mice expressing the G117H mutant of human butyrylcholinesterase.

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.

Rapid activation of presynaptic nicotinic acetylcholine receptors by nerve-released transmitter.

Nicotine's ability to enhance neurotransmitter release has implicated presynaptic nicotinic acetylcholine receptors (nAChRs) in synaptic modulation, but there aze few examples where presynaptic nAChRs are known to be activated by nerve-released transmitter. We searched for endogenous activation of presynaptic nAChRs in the calyceal nerve terminals of the chick ciliary ganglion by imaging presynaptic calcium transients using dextran-coupled indicator dyes. The amplitude of Ca(+)signals recorded in individual nerve terminals was frequency dependent over 2-50 Hz. Calcium transients evoked by stimulation of the preganglionic nerve were significantly reduced (approximately 10-15%) by the nonspecific nAChR antagonist d-tubocurarine (d-TC; 100 microM) and the alpha7-specific antagonist methyllycaconitine (20-50 nM) but were not affected by 10 microM dihydro-beta-erythroidine, which should inhibit several non-alpha7 nAChRs. Feedback was rapid and did not require a stimulation-dependent build-up of transmitter, as d-TC and MLA reduced the amplitude of the first calcium transient in a 2-Hz train. Choline is an agonist at alpha7 nAChRs but is not the sole agonist in this system, as inhibition of acetylcholinesterase by echothiophate failed to reduce calcium transients. These results show that nerve-released acetylcholine (ACh) feeds back onto presynaptic alpha7 nAChRs to enhance calcium signals within the terminal. This feedback may help maintain the high rate of transmission at this cholinergic synapse.

Ocular hypotensive effects of cholinergic and adrenergic drugs may be influenced by prostaglandins E2 in the human and rabbit eye.

PURPOSE: Increased PGE2 production by the iris and ciliary body regulate intraocular pressure (IOP) in vivo. Various cholinergic and adrenergic compounds are traditionally used as antiglaucoma drugs, and their effect on IOP reduction is antagonised by cyclooxygenase inhibitors, indicating a role for eicosanoids in their hypotensive activity. One of the most potent antiglaucoma drugs, PG2 alpha (Latanoprost), reduces IOP by increasing uveoscleral outflow and also increases PGE2 production by the iris and ciliary body in vivo. We investigated whether cholinergic and adrenergic antiglaucoma drugs induce the production of prostaglandin E2 (PGE2) in vitro by: 1) the iris-ciliary body (ICB) of rabbits and, 2) irises of glaucoma patients. METHODS: Pilocarpine 2%, epinephrine 1% and echothiophate iodide 0.125% were applied topically to both eyes of Albino rabbits. Control groups were treated with the corresponding vehicles, or untreated completely. Human iris specimens were obtained from nine untreated cataract eyes, and five eyes under antiglaucoma medication undergoing surgery. PGE2 were determined by a radioimmunoassay. RESULTS: PGE2 production by the ICB of treated rabbits in vitro was twice that of vehicle-treated or untreated rabbit eyes (p<0.001, for either group). In vitro PGE2 production by treated glaucoma patients' irises was three times higher (p<0.001) than in cataract control patients. CONCLUSIONS: The study found an increase in in vitro production of PGE2 by the irises of eyes treated with cholinergic and adrenergic antiglaucoma medications. This suggests a role for endogenous PG production in the hypotensive effect of both classes of drug.

DNA sequence of butyrylcholinesterase from the rat: expression of the protein and characterization of the properties of rat butyrylcholinesterase.

The rat is the model animal for toxicity studies. Butyrylcholinesterase (BChE), being sensitive to inhibition by some organophosphorus and carbamate pesticides, is a biomarker of toxic exposure. The goal of this work was to characterize the purified rat BChE enzyme. The cDNA sequence showed eight amino acid differences between the active site gorge of rat and human BChE, six clustered around the acyl binding pocket and two below the active site serine. A prominent difference in rat was the substitution of arginine for leucine at position 286 in the acyl pocket. Wild-type rat BChE, the mutant R286L, wild-type human BChE, and the mutant L286R were expressed in CHO cells and purified. Arg286 was found responsible for the resistance of rat BChE to inhibition by Triton X-100. Replacement of Arg286 with leucine caused the affinity for Triton X-100 to increase 20-fold, making it as sensitive as human BChE to inhibition by Triton X-100. Wild-type rat BChE had an 8- to 9-fold higher K(m) for the positively charged substrates butyrylthiocholine, acetylthiocholine, propionylthiocholine, benzoylcholine, and cocaine compared with wild-type human BChE. Wild-type rat BChE catalyzed turnover 2- to 7-fold more rapidly than human BChE, showing the highest turnover with propionylthiocholine (201,000 min(-1)). Human BChE does not reactivate spontaneously after inhibition by echothiophate, but rat BChE reactivates with a half-life of 4.3hr. Human serum contains 5mg/L of BChE and 0.01mg/L of AChE. Male rat serum contains 0.2mg/L of BChE and approximately 0.2mg/L of AChE.

Prevention of precipitated withdrawal symptoms by activating central cholinergic systems during a dependence-producing schedule of morphine in rats.

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 microg/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.