The time course of miniature endplate currents and its modification by receptor blockade and ethanol

Miniature endplate currents (MEPCs) recorded from mouse diaphragms with a point voltage clamp, without inhibition of acetylcholinesterase (AChE) and in the absence of any drug, showed in their decay phase consistent deviations from an exponential time course, consisting of (a) "curvature," a progressive increase of decay rate during most of the decay phase, followed by (b) "late" tails. Both phenomena persisted when MEPCs (and channel lifetime) were prolonged by ethanol. Curvature was increased by muscle fiber depolarization and decreased by hyperpolarization. Receptor blockade by (+)-tubocurarine, alpha-bungarotoxin, hexamethonium, or myasthenic IgG accelerated the decay of the main part of MEPCs and eliminated curvature; the time constant of MEPCs became close to the channel time constant. We conclude that curvature arises from repeated action of ACh with cooperativity in ACh-receptor interaction; the voltage sensitivity of curvature follows from the voltage sensitivity of channel closing. Ethanol, in addition to its effect to prolong channel lifetime, enhances the tendency of ACh to act more than once to open channels before being lost to the system. Analysis of the rising phase of the MEPC, in terms of driving functions, also indicated that ethanol promotes channel opening by ACh; this action can account for a substantial increase of MEPC height by ethanol when MEPCs are made small by receptor blockade. Driving functions were also voltage sensitive, in a manner indicating acceleration of channel opening, but reduction of channel conductance, with hyperpolarization. Poisoning or inhibition of AChE prolonged MEPCs without altering the duration of ionic channels. Since ethanol caused further prolongation of MEPCs after poisoning of AChE, with little change in MEPC height, we conclude that the extension of mean channel lifetime by ethanol is accompanied by a similar extension of ACh binding to receptors. After poisoning of AChE, MEPCs became very variable in time course and the decay rate (tau-1) was correlated with MEPC height with a slope of log tau vs. log height of 0.77 for MEPCs of greater than 60% mean size. This slope is larger than expected from cooperativity in ACh-receptor interaction. Correlation of tau and height of MEPCs also exists when AChE is intact; the slope of log tau vs. log height was 0.12 with or without prolongation of MEPCs by ethanol.     

Atypical endplate miniature potentials in the frog neuromuscular junction after modification of the intercellular matrix and osmotic exposures]

In frog cutaneous-pectoris muscles the frequency of slowly rising atypical miniature endplate potentials (MEPPs) was significantly enhanced after collagenase (0.1%) treatment. Treatment with trypsin, hyaluronidase, hyper- and hypoosmotic solutions caused no changes in slowly rising MEPP (frequency in muscle fibers with intact acetylcholinesterase (AChE). Inhibition of AChE caused appearance of giant MEPPs. Acceleration of acetylcholine diffusion from synaptic cleft after treatment with hyaluronidase decreased giant MEPP frequency demonstrating their dependence upon nonhydrolyzed acetylcholine in synaptic cleft. The relation between slowly rising MEPPs and activity of synaptic Schwann cells in discussed.     

Down-regulation of quantal size at frog neuromuscular junctions: possible roles for elevated intracellular calcium and for protein kinase C

Previous work showed that quantal size can be at least doubled at the frog neuromuscular junction by pretreatment with hormones or hypertonic solutions, primarily by the release of more acetylcholine (ACh) per quantum. Once increased, quantal size slowly declined over hours. Quantal size was measured from miniature end-plate potentials (MEPPs) or currents (MEPCs). In the present experiments, preparations in which quantal size had been increased were exposed to 17-25 mM [K+], quantal size decreased within minutes. Release of comparable numbers of quanta by nerve stimulation did not decrease size. K(+)-solutions did not decrease size if Ca2+ was omitted or replaced with Sr2+. The phosphokinase C (PKC) activators phorbol 12,13-diacetate (PDA) and 1-oleoyl-2-acetyl-rac-glycerol (OAG) also decreased quantal size within minutes when applied in a hypertonic solution that increased the rate of spontaneous release. Phorbol 12,13-dideconate, which does not activate PKC, did not decrease quantal size. The size decrease triggered by K(+)-solutions or PKC activators was blocked by 100 microM 1-(5-isoquinolinyl-sulfonyl)-2-methyl-piperazine (H7), a protein kinase inhibitor. Apparently, increasing [K+] elevated intracellular [Ca2+], which activates PKC, and which leads to the down-regulation of quantal size. During the period in which size is decreasing, there appears to be large and normal subpopulations of MEPP sizes, with normal gradually replacing large. This suggests that large quanta are formed by adding additional ACh to preformed quanta shortly before they are available for release.     

Relation between subsynaptic receptor blockade and response to quantal transmitter at the mouse neuromuscular junction

When a quantum of transmitter is released into a synaptic cleft, the magnitude of the subsynaptic response depends upon how much transmitter becomes bound to receptors. Theoretical considerations lead to the conclusion that if receptor density is normally high enough that most of the quantal transmitter is captured, subsynaptic quantal responses may be insensitive to receptor blockade. The effectiveness of receptor blockers in depressing the subsynaptic response should be diminished by interference with processes that normally dispose of transmitter, but increased if receptor density is reduced. In conformity with equations derived from a simple mathematical model, the apparent potency of (+)- tubocurarine (dTC) to depress the peak height of miniature end-plate currents (MEPCs) in mouse diaphragm was substantially reduced by poisoning of acetylcholinesterase (AChE) and increased by partial blockade of receptors by immunoglobulin G from patients with myasthenia gravis or alpha-bungarotoxin. We calculated from the data that normally capture of quantal acetylcholine (ACh) by receptors is approximately 75% of what it would be if there were no loss of ACh by hydrolysis or diffusion of ACh form the synaptic cleft. This fraction is increased to approximately 90% by poisoning of AChE. Conversely, it normally requires blockade of approximately 80% of receptors-and after AChE poisoning, approximately 90% of receptors-to reduce ACh capture (and MEPC height) by 50%. The apparent potency of dTC to alter MEPC time- course (after AChE poisoning) and to depress responses to superperfused carbachol was much greater than its apparent potency to depress MEPC height, but corresponded closely with the potency of dTC to block receptors as calculated from the action of dTC on MEPC height. These results indicate that the amplitude of the response to nerve-applied acetylcholine does not give a direct measure of receptor blockade; it is, in general, to be expected that an alteration of subsynaptic receptor density may not be equally manifest in responses to exogenous and endogenous neurotransmitter.     

Quantal transmitter release at somatic motor-nerve terminals: stochastic analysis of the subunit hypothesis.

Here we analyze the problem of determining whether experimentally measured spontaneous miniature end-plate currents (MEPCs) indicate that quanta are composed of subunits. The properties of MEPCs at end plates with or without secondary clefts at the neuromuscular junction are investigated, using both stochastic and deterministic models of the action of a quantum of transmitter. It is shown that as the amount of transmitter in a quantum is increased above about 4000 acetylcholine (ACh) molecules there is a linear increase in the size of the MEPC. It is possible to then use amplitude-frequency histograms of such MEPCs to detect a subunit structure, as there is little potentiation effect above 4000 ACh molecules. Autocorrelation and power spectral analyses of such histograms establish that their subunit structure can be detected if the coefficient of variation of the subunit size is less than about 0.12 or, if electrical noise is added, about 0.1. Positive gradients relate the rise time and half-decay times of MEPCs to their amplitude, even in the absence of potentiating effects; these gradients are shallower at motor nerve terminals that possess secondary clefts. The effect of asynchronous release of subunits is also investigated. The criteria determined by this analysis for identifying a subunit composition in the quantum are applied to an amplitude-frequency histogram of MEPCs recorded from a small group of active zones at a visualized amphibian motor-nerve terminal. This did not provide evidence for a subunit structure.     

Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.     

Neurotransmitter release at rapid synapses

The classical concept of the vesicular hypothesis for acetylcholine (ACh) release, one quantum resulting from exocytosis of one vesicle, is becoming more complicated than initially thought. 1) synaptic vesicles do contain ACh, but the cytoplasmic pool of ACh is the first to be used and renewed on stimulation. 2) The vesicles store not only ACh, but also ATP and Ca(2+) and they are critically involved in determining the local Ca(2+) microdomains which trigger and control release. 3) The number of exocytosis pits does increase in the membrane upon nerve stimulation, but in most cases exocytosis happens after the precise time of release, while it is a change affecting intramembrane particles which reflects more faithfully the release kinetics. 4) The SNARE proteins, which dock vesicles close to Ca(2+) channels, are essential for the excitation-release coupling, but quantal release persists when the SNAREs are inactivated or absent. 5) The quantum size is identical at the neuromuscular and nerve-electroplaque junctions, but the volume of a synaptic vesicle is eight times larger in electric organ; at this synapse there is enough ACh in a single vesicle to generate 15-25 large quanta, or 150-200 subquanta. These contradictions may be only apparent and can be resolved if one takes into account that an integral plasmalemmal protein can support the formation of ACh quanta. Such a protein has been isolated, characterised and called mediatophore. Mediatophore has been localised at the active zones of presynaptic nerve terminals. It is able to release ACh with the expected Ca(2+)-dependency and quantal character, as demonstrated using mediatophore-transfected cells and other reconstituted systems. Mediatophore is believed to work like a pore protein, the regulation of which is in turn likely to depend on the SNARE-vesicle docking apparatus.     

EFFECT OF DENERVATION AND OF AXOPLASMIC TRANSPORT BLOCKAGE ON THE IN VITRO RELEASE OF MUSCLE ENDPLATE ACETYLCHOLINESTERASE

Abstract— Cat geniohyoid muscle samples containing endplate regions, when incubated in vitro at 37°C in phosphate buffer (pH 73, release acetylcholinesterase (AChE; EC 3.1.1.7) to the bathing medium. By treating the muscle samples with collagenase (EC 3.4.4.19), it was confirmed that most of the AChE released came from the endplates. Enzyme liberation was studied 10 days after either local injection of 10mM-cokhicine into the hypoglossal nerve or following nerve transection. Results showed that the rate of release is increased by denervation, but is not affected by axoplasmic transport blockage. It is postulated that the cellular contact between nerve and muscle—altered by denervation but not by interruption of axoplasmic transport—is an essential factor in maintaining the localization of end-plate AChE within the synaptic cleft substance. This does not invalidate the possible participation of ACh and muscle activity in such enzyme localization.     

Acetylcholine compartments in mouse diaphragm. Comparison of the effects of black widow spider venom, electrical stimulation, and high concentrations of potassium

We have studied the effects of 25 mM potassium, electrical stimulation of the phrenic nerve, and crude black widow spider venom on the ultrastructure, electrophysiology, and acetylcholine (ACh) contents of mouse diaphragms. About 65% of the ACh in diaphragms is contained in a depletable store in the nerve terminals. The rest of the ACh is contained in a nondepletable store that may correspond to the store that remains in denervated muscles and includes, in addition, ACh in the intramuscular branches of the phrenic nerve. About 4% of the ACh released from the depletable store at rest is secreted as quanta and may come from the vesicles, while 96% is secreted in a nonquantized form and comes from an extravesicular pool. The size of the extravesicular pool is uncertain: it could be less than 10%, or as great as 50%, of the depletable store. K causes a highly (but perhaps not perfectly) selective increase in the rate of quantal secretion so that quanta account for about 50% of the total ACh released from K- treated diaphragms. K, or electrical stimulation of the phrenic nerve, depletes both the vesicular and extravesicular pools of ACh when hemicholinium no. 3 (HC-3) is present. However, most of the vesicles are retained under these conditions so that the diaphragms are able to increase slightly their rates of release of ACh when K is added. Venom depletes the terminals of their vesicles and abolishes the release of quanta of ACh. It depletes the vesicular pool of ACh (since it depletes the vesicles), but may only partially deplete the extravesicular pool (since it reduces resting release only 10--40%). The rate of release of ACh from the residual extravesicular pool does not increase when 25 mM K is added. Although we cannot exclude the possibility that stimulation may double the rate of release of ACh from the extravesicular pool, our results are compatible with the idea that the ACh released by stimulation comes mainly from the vesicles and that, when synthesis is inhibited by HC-3, ACh may be exchanged between the extravesicular pool and recycled vesicles.     

Quantal Mechanism of Transmitter Release during Progressive Depletion of the Presynaptic Stores at a Ganglionic Synapse : The action of hemicholinium-3 and thiamine deprivation

In the present experiments we interfered with the mechanism of acetylcholine (ACh) synthesis in the rat superior cervical ganglion by impairing the supply of either the choline group (hemicholinium no. 3 [HC-3]treatment) or the acetyl group (thiamine deprivation). Under both conditions stimulation causes in the ganglion a progressive decline in ACh output associated with a depletion of transmitter tissue content. ACh release from the terminals of a single preganglionic fiber was estimated from the quantum content value of the evoked excitatory postsynaptic potentials (EPSP's) recorded intracellularly in the ganglion neuron under test. The present observations indicate that Poisson statistics describe transmitter release at either low or high release levels. Furthermore, the progressive decline in the rate of ACh output occurring during repetitive stimulation is shown to correspond to a progressive decrease in the number of transmitter quanta released per impulse and not to any modification in the size of individual quanta. Some 8,000 transmitter quanta proved to represent the presynaptic transmitter store initially present in those terminals on a neuron that are activated by stimulation of a single preganglionic fiber. Speculations are considered about synaptic efficacy and nerve connections in rat autonomic ganglia. It is suggested that six preganglionic fibers represent the mean input to a ganglion neuron.     

Effects of LF-14, THA and physostigmine in rat hippocampus and cerebral cortex

The effects of physostigmine, tetrahydroaminoacridine (THA) and LF-14 [3,3-dimethyl-1(4- amino-3-pyridyl)urea], a 3,4-diaminopyridine derivative, were compared on inhibition of acetyl- cholinesterase (AChE) activity, and release of [³H]acetylcholine (ACh) from rat brain cortical and hippocampal slices. All three compounds caused a concentration dependent inhibition of AChE, with an order of potency physostigmine > THA > LF-14. The electrically stimulated release of ACh from hippocampal and cortical slices was decreased by 10⁻⁵M physostigmine, although the effect was significant only in cortex. THA (5 × 10⁵M) caused a slight, but not significant, decrease in ACh release from both tissues. In contrast, LF-14 (5 × 10⁻⁵ M) caused an approx. 3-fold enhancement of stimulated release. When AChE was inhibited by prior addition of physostigmine, THA caused only a slight enhancement of ACh release, whereas LF-14 greatly increased release. ACh release was also reduced by stimulation of presynaptic muscarinic receptors with oxotremorine. In this case, THA had no effect on ACh release, while LF-14 was able to reverse the inhibition. This study suggests that LF-14 acts to promote ACh release through blocking K⁺ channels, and has a less potent AChE inhibitory effect. It is possible that a compound like LF-14 could be useful in treating diseases of cholinergic dysfunction such as Alzheimer's disease, by both promoting the release of ACh and inhibiting its breakdown.     

Translocation of cytosolic acetylcholine into synaptic vesicles and demonstration of vesicular release

The rate of translocation of newly synthesized acetylcholine (ACh) from the presynaptic cytosol of Torpedo electric organ nerve terminals into synaptic vesicles and the extent to which ACh release from these neurons is mediated by a vesicular mechanism were investigated. For this purpose the compound 2(4-phenylpiperidino)cyclohexanol (AH5183), which inhibits the active transport of ACh into isolated cholinergic synaptic vesicles, was employed. Preincubation of purified Torpedo nerve terminals (synaptosomes) with AH5183 does not affect the intraterminal synthesis of [3H]ACh but results in a marked inhibition (85%) of its Ca2+-dependent K+-evoked release. By contrast, the evoked release of the endogenous nonlabeled ACh is not affected by this compound. When AH5183 is added during radiolabeling, it causes a progressively smaller inhibition of [3H]ACh release which is completely abolished if the drug is added after the preparation has been labeled. These findings suggest that most of the newly synthesized synaptosomal [3H]ACh (85%) is released by a vesicular mechanism and that some [3H]ACh (15%) may be released by a different process. The translocation of cytosolic [3H]ACh into the synaptic vesicles was monitored by determining the time course of the loss of susceptibility of [3H]ACh release to AH5183. It was found not to be coupled kinetically to [3H]ACh synthesis and to lag behind it. The nature of the intraterminal processes underlying this lag is discussed.     

Simultaneous Release of Acetylcholine and ATP from Stimulated Cholinergic Synaptosomes

Abstract: The release of acetylcholine (ACh) and ATP from pure cholinergic synaptosomes isolated from the electric organ of Torpedo was studied in the same perfused sample. A presynaptic ATP release was demonstrated either by depolarization with KCl or after the action of a venom extracted from the annelid Glycera convoluta (GV). The release of ATP exhibited similar kinetics to that of ACh release and was therefore probably closely related to the latter. The ACh/ATP ratio in perfusates after KCl depolarization was 45; this was much higher than the ACh/ATP ratio in cholinergic synaptic vesicles, which was 5. The ACh/ATP ratio released after the action of GV was also higher than that of synaptic vesicles. These differences are discussed. The stoichiometry of ACh and ATP release is not consistent with the view that the whole synaptic vesicle content is released by exocytosis after KCl depolarization, as is the case for chromatin cells in the adrenal medulla.     

Receptor-mediated presynaptic facilitation of quantal release of acetylcholine induced by pralidoxime inAplysia

1. Possible interactions of contrathion (pralidoxime sulfomethylate), a reactivator of phosphorylated acetylcholinesterase (AChE), with the regulation of cholinergic transmission were investigated on an identified synapse in the buccal ganglion of Aplysia californica. 2. Transmitter release was evoked either by a presynaptic action potential or, under voltage clamp, by a long depolarization of the presynaptic cell. At concentrations higher than 10(-5) M, bath-applied contrathion decreased the amplitude of miniature postsynaptic currents and increased their decay time. At the same time, the quantal release of ACh was transiently facilitated. The facilitatory effect of contrathion was prevented by tubocurarine but not by atropine. Because in this preparation, these drugs block, respectively, the presynaptic nicotinic-like and muscarinic-like receptors involved in positive and negative feedback of ACh release, we proposed that contrathion activates presynaptic nicotinic-like receptors. 3. Differential desensitization of the presynaptic receptors is proposed to explain the transience of the facilitatory action of contrathion on ACh release. 4. The complexity of the synaptic action of contrathion raises the possibility that its therapeutic effects in AChE poisonings are not limited to AChE reactivation.     

Microdialysis measures of functional increases in ACh release in the hippocampus with and without inclusion of acetylcholinesterase inhibitors in the perfusate

Because brain extracellular acetylcholine (ACh) levels are near detection limits in microdialysis samples, an acetylcholinesterase (AChE) inhibitor such as neostigmine is often added to microdialysis perfusates to increase ACh levels in the dialysate, a practice that raises concerns that the inhibitor might alter the results. Two experiments compared functional differences in ACh release with and without neostigmine. In the first experiment, 30-60% increases in extracellular ACh concentrations in the hippocampus were evident during food-rewarded T-maze training with 20-500 nm neostigmine in the perfusate but no increases were seen without neostigmine. In the second experiment, 78% increases in ACh release in the hippocampus were seen after injections of the GABA(A) receptor antagonist, bicuculline, into medial septum only if neostigmine (50 nm) was included in the perfusate. These findings suggest that, in the hippocampus, endogenous brain AChEs are very efficient at removing extracellular ACh, obscuring differences in ACh release in these experiments. Therefore, inclusion of AChE inhibitors in the microdialysis perfusate may be necessary under some conditions for observations of functional changes in release of ACh in the hippocampus.     

Catalytic activity of acetylcholinesterase immobilized on mesoporous molecular sieves

MCM-41 and FSM-16 were used for enzyme immobilization on account of their good physical and chemical properties. In this work, the catalytic activity of acetylcholinesterase (AChE) immobilized on these materials was investigated, using neostigmina as AChE inhibitor. The results show that AChE was adsorbed on MCM-41 and on FSM-16-TIPB. AChE immobilized on the latter material maintained 70% of its activity and the material did not hydrolyze ACh (as MCM-41) by itself. Therefore, FSM-16-TIPB was the best material, considering also that when neostigmine was applied to AChE immobilized on FSM-16-TIPB, the activity of AChE decreased as occurs in its free from. Hence, this model could be useful in the evaluation of different kinds of AChE inhibitors, allowing the recycling of enzymes and making possible several assays and thereby, lowering cost.     

Effects of quantal content,membrane potential,and cholinoceptor density on the time course of end-plate currents in the rat under during acetylcholinesterase inhibition

The time-course of multiquantal end-plate currents (EPCs) was compared with monoquantal synaptic responses, i.e., miniature end-plate currents (MEPCs), in voltage-clamped rat diaphragm muscle fibers. In the presence of active acetylcholinesterase (AChE), the time constant of the decay of EPCs, that were composed of 25–140 quanta, was 1.2 times greater than that of MEPCs. After inhibition of AChE with armine or proserine the decay of the EPC was longer than the decay of the MEPC by 10–100 times, and unlike the MEPC, in the majority of synapses it could be described by the sum of two (n=34) or three (n=9) exponentials: monoexponential EPCs were noted in only three cases. The nature and duration of the EPC decay depended on its quantal content. After a reduction in the quantal content a three-exponential EPC decay could be successively reduced to a two- and a mono-exponential decay. A ,slow, component of the EPC decay, unlike the MEPC decay, was extremely sensitive to changes in the membrane potential, and extracellular magnesium ion concentration. When the cholinoceptors were irreversibly blocked by -bungarotoxin the MEPC decay accelerated, and the monoexponential EPC decay initially slowed down before accelerating, but even during a profound blockade the open-times of the ion channels were not affected. It therefore appears that during the generation of multiquantal EPCs when AChE is inhibited, not only does the synchronicity of the ion channel opening change, but so do their kinetics, possibly because of ion channel blockade by endogenous acetylcholine.S. V. Kurashov Institute of Medicine, Russian Ministry of Public Health, Kazan. Translated from Neirofiziologiya, Vol. 24, No. 3, pp. 269–279, May–June, 1992.     

Acetylcholine Release from Isolated Synaptic Vesicles Related to Ionic Permeability Changes: Continuous Detection with a Chemiluminescent Method

The effect of ionic permeability changes on acetylcholine (ACh) release from isolated cholinergic synaptic vesicles of Torpedo was studied using a chemiluminescent method for continuous ACh detection. Vesicles rendered freely permeable to potassium by valinomycin lost most of their ACh content in K+ media, if the accompanying anion was permeant; it thus appeared that ACh leakage occurred as the result of internal osmotic changes. Upon addition of ionophores that catalyse monovalent cation/H+ exchange (gramicidin D or a mixture of valinomycin plus protonophore FCCP), a rapid but transient ACh release was observed. Surprisingly, nigericin which also catalyses K+/H+ exchange, had no effect on ACh release. The divalent cation ionophore A23187 promoted ACh release only when calcium (and not magnesium) was introduced into the external medium in a millimolar concentration range. As the simultaneous addition of the protonophore FCCP and A23187 decreased this calcium-dependent ACh leakage, a releasing effect of A23187 through Ca2+/H+ exchange is suspected. The present results emphasise the role of internal protons for ACh retention inside synaptic vesicles.     

Effect of local acetylcholinesterase inhibition on sweat rate in humans.

ACh is the neurotransmitter responsible for increasing sweat rate (SR) in humans. Because ACh is rapidly hydrolyzed by acetylcholinesterase (AChE), it is possible that AChE contributes to the modulation of SR. Thus the primary purpose of this project was to identify whether AChE around human sweat glands is capable of modulating SR during local application of various concentrations of ACh in vivo, as well as during a heat stress. In seven subjects, two microdialysis probes were placed in the intradermal space of the forearm. One probe was perfused with the AChE inhibitor neostigmine (10 microM); the adjacent membrane was perfused with the vehicle (Ringer solution). SR over both membranes was monitored via capacitance hygrometry during microdialysis administration of various concentrations of ACh (1 x 10(-7)-2 M) and during whole body heating. SR was significantly greater at the neostigmine-treated site than at the control site during administration of lower concentrations of ACh (1 x 10(-7)-1 x 10(-3) M, P < 0.05), but not during administration of higher concentrations of ACh (1 x 10(-2)-2 M, P > 0.05). Moreover, the core temperature threshold for the onset of sweating at the neostigmine-treated site was significantly reduced relative to that at the control site. However, no differences in SR were observed between sites after 35 min of whole body heating. These results suggest that AChE is capable of modulating SR when ACh concentrations are low to moderate (i.e., when sudomotor activity is low) but is less effective in governing SR after SR has increased substantially.