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.

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.

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.

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.

Effects of organophosphates on cholinesterase activity and neurite regeneration in Aplysia.

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.

The effects of multiple low doses of organophosphates on target enzymes in brain and diaphragm in the mouse.

1. Multiple low doses of the direct acting organophosphates, ecothiopate, paraoxon and mipafox produced persistent and additive inhibition of diaphragm acetylcholinesterase. Paraoxon and mipafox had similar effects on brain acetylcholinesterase. There was greater recovery from inhibition between doses for paraoxon and ecothiopate than for mipafox. 2. Ecothiopate did not inhibit brain acetylcholinesterase but there was a small increase in activity. 3. Mipafox also had a cumulative inhibitory effect on brain neuropathy target esterase. 4. These results have particular implication for the use of multiple low doses of organophosphates occupationally by man.

Construction and characterization of secreted and chimeric transmembrane forms of Drosophila acetylcholinesterase: a large truncation of the C-terminal signal peptide does not eliminate glycoinositol phospholipid anchoring.

Despite advances in understanding the cell biology of glycoinositol phospholipid (GPI)-anchored proteins in cultured cells, the in vivo functions of GPI anchors have remained elusive. We have focused on Drosophila acetylcholinesterase (AChE) as a model GPI-anchored protein that can be manipulated in vivo with sophisticated genetic techniques. In Drosophila, AChE is found only as a GPI-anchored G2 form encoded by the Ace locus on the third chromosome. To pursue our goal of replacing wild-type GPI-anchored AChE with forms that have alternative anchor structures in transgenic files, we report the construction of two secreted forms of Drosophila AChE (SEC1 and SEC2) and a chimeric form (TM-AChE) anchored by the transmembrane and cytoplasmic domains of herpes simplex virus type 1 glycoprotein C. To confirm that the biochemical properties of these AChEs were unchanged from GPI-AChE except as predicted, we made stably transfected Drosophila Schneider Line 2(S2) cells expressing each of the four forms. TM-AChE, SEC1, and SEC2 had the same catalytic activity and quaternary structure as wild type. TM-AChE was expressed as an amphiphilic membrane-bound protein resistant to an enzyme that cleaves GPI-AChE (phosphatidylinositol-specific phospholipase C), and the same percentage of TM-AChE and GPI-AChE was on the cell surface according to immunofluorescence and pharmacological data. SEC1 and SEC2 were constructed by truncating the C-terminal signal peptide initially present in GPI-AChE: in SEC1 the last 25 residues of this 34-residue peptide were deleted while in SEC2 the last 29 were deleted. Both SEC1 and SEC2 were efficiently secreted and are very stable in culture medium; with one cloned SEC1-expressing line, AChE accumulated to as high as 100 mg/liter. Surprisingly, 5-10% of SEC1 was attached to a GPI anchor, but SEC2 showed no GPI anchoring. Since no differences in catalytic activity were observed among the four AChEs, and since the same percentage of GPI-AChE and TM-AChE were on the cell surface, we contend that in vivo experiments in which GPI-AChE is replaced can be interpreted solely on the basis of the altered anchoring domain.

Sustained inhibition of acetylcholinesterase activity does not disrupt early geniculocortical ingrowth to developing rat visual cortex.

Esterase activity of endogenous transiently expressed acetylcholinesterase was locally suppressed in visual cortex of infant rats for 2-5 days by the irreversible inhibitor phospholine iodide, delivered from Elvax implants. Tissue processed for anterograde movement of the carbocyanine dye DiI or anterograde transneuronal transport of wheat germ agglutinin-horseradish peroxidase revealed normal geniculocortical growth into layer IV of visual cortex. These results suggest that the catalytic activity of transiently expressed acetylcholinesterase may play little, if any, role in early development of thalamocortical systems.

Distribution of acetylcholinesterase activity in the deutocerebrum of the sphinx moth Manduca sexta.

We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the deutocerebrum of the brain of the sphinx moth Manduca sexta. To distinguish between extra- and intracellular pools of the enzyme, some brains were treated prior to histochemical staining with echothiophate, an irreversible AChE inhibitor which penetrates cell membranes very slowly and, therefore, inhibits only extracellular AChE. In the antennal nerve, fascicles of presumably mechanosensory fibers show echothiophate-insensitive AChE activity. They bypass the antennal lobe and project to the antennal mechanosensory and motor center of the deutocerebrum. In the antennal lobe, fibers in the coarse neuropil, cell bodies in the lateral cell group, and all glomeruli exhibit AChE activity. In most ordinary glomeruli, echothiophate-sensitive AChE activity is concentrated in the outer cap regions, corresponding to the terminal arborizations of olfactory afferents. A previously unrecognized glomerulus in the ventro-median antennal lobe shows uniform and more intense AChE-specific staining that the other glomeruli. No AChE activity appeared to be associated with male-specific pheromone-sensitive afferents in the macroglomerular complex. About 67 interneurons with somata in the lateral cell group of the antennal lobe show echothiophate-insensitive AChE activity. These neurons seem to be members of two types of antennal-lobe projection neurons with fibers passing through the outer-antennocerebral tract to the protocerebrum. AChE-stained arborizations of these neurons appear to invade all glomeruli, including three distinguishable subunits of the male-specific macroglomerular complex. In echothiophate-treated animals, the projections of one of these types of fiber form large terminals in the lateral horn of protocerebrum, which partly protrude into the adjacent glial cell layer. The results suggest that extracellularly accessible AChE is associated with ordinary olfactory receptor terminals but apparently not with pheromone-sensitive afferents. Intracellular AChE appears to be present in antennal mechanosensory fibers and in two types of olfactory projection neurons of the antennal lobe. The study provides further evidence for cholinergic neurotransmission of most antennal afferents. The AChE-containing interneurons might be cholinergic as well or use the enzyme for functions unrelated to hydrolysis of acetylcholine.

The effect of acetylcholinesterase on outgrowth of dopaminergic neurons in organotypic slice culture of rat mid-brain.

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 origin of the effects of an anticholinesterase on the latencies of action potentials in mouse skeletal muscles.

1. Subcutaneous injection in mice of a single dose of an organophosphorous anticholinesterase, ecothiopate (0.5 mumol kg-1), produced increased variability in the latency (jitter) of indirectly-elicited action potentials in diaphragm muscles 5 days after treatment, but there was no effect on the variability of latencies of endplate potentials. This study was designed to elucidate the mechanism(s) of the increase in action potential jitter. 2. Action potentials evoked directly by electrical stimulation at one end of muscle fibres and recording near the other end had less jitter than indirectly-evoked action potentials and ecothiopate had no effect on directly-evoked action potentials. 3. In preparations with uncut fibres, pretreatment with ecothiopate reduced by about 20% both muscle fibre input resistance and the amplitude of spontaneous miniature endplate potentials. Ecothiopate had no effect on muscle fibre resting membrane potential or on the threshold potential for excitation. 4. In untreated preparations, indirectly-evoked action potentials recorded at the endplate had similar jitter to action potentials recorded at the tendon when latencies were measured at 10% of peak amplitude. However, when latencies were measured at peak, there was greater jitter of action potentials at the endplate. Ecothiopate increased jitter of action potentials recorded at the endplate at 10% of peak but did not significantly increase jitter of action potentials recorded at the endplate when measured at the peak. 5. In cut-fibre preparations, the first endplate potential of trains was significantly increased after ecothiopate but there was no effect of ecothiopate on the amplitude of plateau endplate potentials later in the train. Analysis of plateau endplate potentials showed that 5 days after administration, ecothiopateproduced an increase in the variance of endplate potential amplitudes and changes in the binomial parameters n and p.6. It was concluded that the increased jitter produced by ecothiopate is not a generalized effect on the plasma membrane and that none of the above observations could explain the increased jitter. The possibility is discussed that increased jitter is produced by variability in times to threshold of endplate potentials and/or by variability in the locus of generation of the action potential in the perijunctional area.

Effect of topically applied demecarium bromide and echothiophate iodide on intraocular pressure and pupil size in beagles with normotensive eyes and beagles with inherited glaucoma.

Topically applied demecarium bromide (0.125 and 0.25%) and echothiophate iodide (0.125 and 0.25%) solutions were evaluated in Beagles with normotensive eyes and Beagles with inherited glaucoma. In single-dose studies, the effects of intraocular pressure (IOP) and pupil size (PS) were measured in eyes before drug treatment and in drug- and nondrug-treated eyes. Both concentrations of the 2 drugs induced long-term miosis and decrease in IOP in normotensive eyes of Beagles and of eyes of Beagles with inherited glaucoma. Demecarium bromide (0.125 and 0.5%) decreased IOP for 49 and 55 hours, respectively. Echothiophate iodide (0.125 and 0.5%) reduced IOP for 25 and 53 hours, respectively. The miosis associated with both concentrations of the 2 drugs generally paralleled the decreases in IOP.

Nonopioid effect of morphine on electrically evoked acetylcholine release from Torpedo electromotor neurons.

The release of acetylcholine from Torpedo electric organ slices following their electrical stimulation was modulated by morphine, by the muscarinic antagonist atropine, and by the nicotinic antagonist tubocurarine. Addition of either atropine or tubocurarine in the presence of the acetylcholinesterase inhibitor phospholine iodide enhanced acetylcholine release. The effects of the two antagonists were additive, a result suggesting that the secreted acetylcholine regulates its own release by activating both muscarinic and nicotinic cholinergic receptors and that these receptors inhibit acetylcholine release by different mechanisms. The effects of opiates on acetylcholine release were examined under conditions in which the cholinergic modulation of release is blocked, i.e., in the presence of atropine and tubocurarine. These experiments revealed that electrically evoked release of acetylcholine is blocked by the opiate agonists morphine and levorphanol. However, the inhibitory effect of morphine on acetylcholine release was not reversed by the opioid antagonist naloxone. Furthermore, dextrorphan, the nonopioid stereoisomer of levorphanol, had the same inhibitory effect as its opioid counterpart. These findings suggest that the effects of opiates on electrically evoked release of acetylcholine are not mediated by opioid receptors. The possible mechanisms underlying these nonopioid effects of morphine and levorphanol are discussed.

The role of acetylcholinesterase in denervation supersensitivity in the frog cardiac ganglion.

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.