Non-quantal Acetylcholine Release Research Articles

Prevention of diisopropylphosphorofluoridate-induced myopathy by botulinum toxin type A blockage of quantal release of acetylcholine

Botulinum toxin type A (BTx), which blocks quantal and partially reduces spontaneous nonquantal acetylcholine (ACh) release at neuromuscular junctions, was tested for its possible attenuating effect on diisopropylphosphorofluoridate (DFP)-induced muscle lesions. The extent of muscle lesion in extensor digitorum longus and soleus muscle of DFP injected rats with and without BTx pretreatment was evaluated using light and electron microscopic procedures. In parallel experiments, acetylcholinesterase (AChE) activity was measured and the functional state of muscles in experimental groups was determined by electrophysiological methods. The results show that pretreatment with BTx almost completely protects the muscles from DFP-induced spontaneous activity and lesions in spite of critically inhibited synaptic AChE. These results are consistent with the conclusion that the effect is not mediated by direct action of organophosphate on muscle, but by the accumulation of ACh resulting in muscle hyperactivity. Therefore, it is concluded that in conditions of acutely inhibited synaptic AChE, the quantal release of ACh is essential for lesion induction, whereas the spontaneous nonquantal ACh release, which is only partially affected in BTx-blocked nerve endings, seems not to be involved.

The dependence of non-quantal acetylcholine release on the choline-uptake system in the mouse diaphragm

The time course of local end-plate hyperpolarization after d-tubocurarine application measured by an intracellular microelectrode was followed in vitro in anticholinesterase-treated mouse diaphragm pinned to the bottom of the perfusion chamber. The d-tubocurarine-induced hyperpolarization, which served as an indicator of non-quantal acetylcholine release, started to decline from 6 mV after 1 h and was negligible after 3 h in continuously perfused preparations. This decline was slowed down by 10 mumol l-1 choline and almost completely prevented by long-term nerve stimulation with a frequency of 3 Hz. The rapid decrease of the d-tubocurarine-induced hyperpolarization was observed within 10-15 min after the application of 1 mumol l-1 hemicholinium-3 and substitution of lithium for sodium. Both these procedures inhibit the fast choline uptake into nerve terminals. Our data suggest that the amount of available acetylcholine for non-quantal release is proportional to the rate of its synthesis and to the number of available carriers in the nerve terminals. Some of our observations might also be explained by postulating that the choline-uptake system as such is responsible for the non-quantal release.

Kinetic differences in the effect of calcium on quantal and non-quantal acetylcholine release at the murine diaphragm

The effects of Ca 2+ withdrawal on non-quantal, evoked quantal and spontaneous quantal release of acetylcholine (ACh) from the motor nerve terminals were studied with standard intracellular recording techniques. Anticholinesterase-treated mouse diaphragms were used. In a Ca 2+-free solution all forms of ACh release decreased, but with different kinetics. As expected, evoked quantal release declined to zero within a few minutes. Spontaneous quantal release, i.e. the frequency of the miniature end-plate potentials (MEPPs), decreased to 15% of the control within 20 min after calcium withdrawal. The slowest decay was that of non-quantal release which declined very slowly and reached zero after 45–50 min. Following Ca 2+ re-admission, both quantal types of ACh release were rapidly restored (evoked in 10 min, spontaneous in 20 min). However, recovery of non-quantal release did not occur until after 50 to 60 min.

Non-quantal acetylcholine release at mouse neuromuscular junction: Effects of elevated quantal release and aconitine

The rate of non-quantal acetylcholine (ACh) release was estimated at the mouse neuromuscular junction by observing the effect of (+)-tubocurarine on endplate membrane potential or current in preparations pretreated with an irreversible anti-acetylcholinesterase (anti-AChE). Voltage clamping was an effective method for measuring nonquantal release. Non-quantal release was markedly inhibited by 10 μM aconitine. Non-quantal release was not significantly increased by 10 μM dihyroouabain (DHO). (It has been reported that ouabain increases the leak.) Non-quantal release was roughly doubled following exposure to hypertonic solution or to elevated K +-solution. This is in accord with the hypothesis that the leak is by way of ACh transporters incorporated into the terminal membrane following exocytosis, but other interpretations remain to be tested.

The effects of nerve terminal activity on non‐quantal release of acetylcholine at the mouse neuromuscular junction.

1. Local endplate depolarization induced by anticholinesterase application to mouse nerve-diaphragm preparations was taken as a measure of non-quantal release of acetylcholine. 2. Non-quantal acetylcholine release occurred within 20-60 s after anticholinesterase application, either spontaneously or evoked by nerve stimulation. Non-quantal release declined with time and disappeared after 3-5 min. 3. The amplitude of stimulation-evoked non-quantal release increased with the frequency of stimulation and was maximal at frequencies above 50 Hz. Two stimuli were sufficient to evoke the maximal effect. 4. Micromolar concentrations of atropine, pirenzepine and vesamicol reduced the amplitude and shortened the duration of non-quantal release. Oxotremorine (10(-8) M) enhanced the amplitude and ouabain (10(-4) M) prolonged the duration of non-quantal release. 5. Our results support the idea that the non-quantal release is due to the vesicular acetylcholine transport system which becomes transiently a part of the nerve terminal during exocytotic release of quantal acetylcholine.

Direct measurement of ACh release from exposed frog nerve terminals: constraints on interpretation of non-quantal release.

1. Acetylcholine (ACh) release from enzymatically exposed frog motor nerve terminals has been measured directly with closely apposed outside-out clamped patches of Xenopus myocyte membrane, rich in ACh receptor channels. When placed close to the synaptic surface of the terminal, such a membrane patch detects both nerve-evoked patch currents (EPCs) and spontaneous quantal 'miniature' patch currents (MPCs), from a few micrometres length of the terminal, in response to ACh release from the nearest three to five active zones. 2. Chemical measurements of ACh efflux from whole preparations revealed a spontaneous release rate of 4.1 pmol (2 h)-1, and no significant difference in resting efflux between enzyme-treated and control preparations. The ratio of enzyme-treated to contralateral control muscle efflux averaged 1.17, indicating that enzyme treatment did not affect spontaneous ACh release. Vesamicol (1.7 microM), which blocks the ACh transporter in synaptic vesicles, decreased the spontaneous release of ACh to 67% of control. 3. In the absence of nerve stimulation, the frequency of single-channel openings recorded by outside-out patch probes adjacent to nerve terminals was very low (1-2 min-1), and little different at a distance of hundreds of micrometres, suggesting that if ACh was continually leaking from the terminal in a non-quantal fashion, the amount being released near active zone regions on the terminal was below the limit of detection with the patches. 4. Direct measurements of the sensitivity of the patches, coupled with calculated ACh flux rates, lead to the conclusion that the amount of ACh released non-quantally from the synaptic surface of the frog nerve terminal is less than one-tenth the amount expected if all non-quantal release is from this region of the terminal membrane. 5. Following a series of single nerve shocks or a 50 Hz train of nerve stimuli, the frequency of asynchronous single-channel openings increased for several seconds. This transient increase in channel openings was not sensitive to movement of the patch electrode a significant distance (4 microns) away from the active sites, or to manipulations previously reported to block non-quantal transmitter leakage, including addition of 10 mM-Ca2+ or 1.7 microM-vesamicol to the bath. These channel openings appear to be due to an accumulation of ACh which originated from many evoked quanta, and not the effect of locally increased non-quantal ACh release due to nerve stimulation. 6. We conclude that transmitter leakage at adult frog terminals is either localized to a source other than the synaptic surface of the nerve terminal, or released in a widespread and diffuse fashion from many sources, which may include the nerve terminal.

Effect of Mg 2+ on non-quantal acetylcholine release at the mouse neuromuscular junction

The effects of extracellular concentrations of Mg 2+ on the non-quantal release of acetylcholine (ACh) from nerve terminals was studied by extra- and intracellular electrophysiological methods. Anticholinesterase-treated mouse diaphragms were used in vitro. In the presence of Ca 2+, the non-quantal release was maximal in the absence of Mg 2+ and was inhibited by 3 mmol/l Mg 2+. The inhibitory effect of Mg 2+ was antagonized by ouabain and was absent in Ca 2+-free (EGTA) solutions. The non-quantal release of ACh was found to be more sensitive to inhibition by Mg 2+ than the quantal one which was measured as the amplitude of miniature endplate currents.

Constraints on the interpretation of nonquantal acetylcholine release from frog neuromuscular junctions.

In the frog nerve-muscle preparation, there is evidence for nonquantal release of acetylcholine (ACh) at a level 100 times that attributable to spontaneous quantal events (miniature endplate potentials). It is widely assumed that nonquantal release occurs near the sites of quantal release (active zones) in the nerve terminal, close to the postsynaptic muscle membrane. This high level of nonquantal ACh release has led to the suggestion that it may serve a trophic function at the nerve-muscle junction. However, the precise origin and mechanism of nonquantal release have not been determined. We have used outside-out patches of ACh receptor-rich membrane as a sensitive technique for the direct measurement of ACh release from highly localized regions of enzymatically treated nerve terminals and have found little detectable ACh "leakage" from active-zone regions. If all of the nonquantal ACh release were localized to the under surface of the nerve terminal, we estimate that we would have detected more than 10 times the low level detected with our patch probe. Furthermore, although quantal release was easily measurable, vesicular exocytosis (hypothesized to insert ACh transport proteins into the plasma membrane, thereby producing the leak) did not increase single-channel activity in the patch probe above that attributable to quantal release. We conclude that, at rest, the active-zone region of nerve terminals is not a major source of nonquantal ACh release and that vesicular exocytosis does not noticeably increase the level of nonquantal release from the nerve terminal. Thus, with biochemical measurements that indicate that spontaneous ACh release is relatively unchanged by prior denervation, these results question the assumed source and mechanism of nonquantal release and the suggestion that leakage of ACh from active-zone regions plays a trophic role in nerve-muscle interaction.

A study on early post-denervation changes of non-quantal and quantal acetylcholine release in the rat diaphragm.

The d-tubocurarine (dTC) induced hyperpolarization of antiesterase-treated muscles at the endplate zone, miniature endplate potentials (mepps), resting membrane potentials (RMPs) and the input resistances of single muscle fibres (Rin) were measured in rat diaphragm at various times after denervation. The dTC-induced hyperpolarization decreased in two phases: 2 h after denervation it decreased transiently to 25%, after 4 h it had partially recovered to 60% and from 6 h it progressively decreased up to 12 h after which time it changed to depolarization. The initial fall and recovery were also present in muscles from sham-operated animals. The frequency of mepps decreased by 25% and the amplitude diminished by 10% within the first 2-4 h. After 10 h the frequency had decreased by 35% and the amplitude by 65%. After 12 h no mepps were present. The RMP was not significantly changed during the first 16 h after denervation. From 16 to 24 h the membrane became depolarized at a rate of about 1 mV/h. The input resistance of a single muscle fibre was constant for 12 h after denervation and from 12 to 24 h it increased by 25%. It is concluded that the early decrease in the dTC-induced hyperpolarization is probably due to the desensitization of acetylcholine (ACh) receptors caused by stress-activated non-quantal ACh release. The later decrease of dTC-hyperpolarization reflects a fall in the non-quantal ACh release. The depolarization of the resting membrane after denervation is related to the decrease in passive membrane permeability which is a secondary consequence of transmission failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature dependence of spontaneous quantal and nonquantal transmitter release from motor nerve endings in the mouse

Spontaneous quantal and nonquantal acetylcholine release were investigated at an ambient temperature range of 10–35°C in a preparation of white mouse hemidiaphragm. Quantal transmitter release was assessed by the frequency of miniature endplate potentials and nonquantal secretion by the level of H-effect. Spontaneous quantal release rose exponentially in step with increasing temperature. Two relative maxima, one of 20°C and the other of 35°C were noted in the temperature dependence of nonquantal transmitter release. Nonquantal release of acetylcholine did not take place at a temperature of 10°C. The effective energy of activation of quantal release was calculated as 57.0 kJ/mole over the temperature range investigated; that of the nonquantal release process at intervals of 15–20°C and 25–35°C measured 45.5 and 38.2 kJ/mole respectively. It is suggested that an active transport system process rather than simple diffusion of acetylcholine molecules is responsible for nonquantal release of this neurotransmitter.

The effects of nerve section on the non-quantal release of ACh from the motor nerve terminal

The spontaneous release of acetylcholine (ACh) from motor nerve terminals is now thought to occur by two mechanisms: (a) quantal release, giving rise to miniature endplate potentials; and (b) non-quantal release. In this study we have examined the effect of nerve section on spontaneous non-quantal ACh release, and have compared the time-course of cessation of non-quantal and quantal ACh release. Non-quantal ACh release, measured by an electrophysiological technique, declined 4 h after nerve section to approximately 50% of the control value. At 8–10 h it briefly rose again, then gradually declined to undetectable levels. Spontaneous quantal release (frequency of miniature endplate potentials) in the same muscle fibers remained close to control levels for 8 h after nerve section, and also increased prior to failure. Decline of non-quantal ACh release appears to be the earliest change in the nerve terminal following nerve transection; it may therefore be relevant in understanding the effects of denervation on the consequent changes in muscle properties.

Abolishment of non-quantal release of acetylcholine from the mouse phrenic nerve endings by toosendanin.

The hyperpolarizing effect (H-effect) of d-tubocurarine on the end-plate of the isolated diaphragm pretreated with an anticholinesterase was irreversibly abolished by toosendanin (1 X 10(-5) g/ml), indicating the blockade of spontaneous non-quantal release of acetylcholine (ACh). The H-effect was also inhibited, but temporarily, when toosendanin (a dose of 0.6 LD50) was subcutaneously injected into the mouse and the diaphragm was isolated 40-120 min after injection. During such an inhibitory period, however, spontaneous release of ACh remained facilitated. It is concluded that the effect of toosendanin on non-quantal release of ACh was different from its effect on quantal release not only at the direction but also at the time course.

Different Kinds of Acetylcholine Release From The Motor Nerve

Publisher Summary Studies of transmitter release at the neuromuscular junction have revealed the presence of several distinct types of acetylcholine (ACh) secretion. This chapter describes these processes and discusses the underlying release mechanisms and the possible physiological significance of the various kinds of transmitter action. Transmitter release from the motor nerve may be divided into those involving intermittent, quantal, or nonquantal release of ACh as well as those characterized by a continuous leakage of ACh. Intermittent secretion of ACh involves either a Ca 2+ –sensitive(I) or a Ca 2+ – insensitive (II) type of transmitter release process. The former characterizes phasic, nerve impulse evoked or spontaneous quantal ACh release and the latter the spontaneous intermittent, nonquantal secretion of ACh giving rise postsynaptically to so-called giant and slow-rising miniature end-plate potentials. Molecular leakage of ACh is a continuous process originating not only from the presynaptic nerve (III) but also from the postsynaptic muscle cell.

Has nonquantal acetylcholine secretion from motor nerve endings a role in neurotrophic control of resting membrane potential in rat muscle fibers?

The resting membrane potential of fibers of the rat diaphragm was measured by a microelectrode technique 3 h after division of the phrenic nerve and incubation in culture medium for 5 days after denervation. The membrane potential was recorded in synaptic regions of fibers close to (2–3 mm) and distant from (9–11 mm) the site of nerve division. The membrane potential of the synaptic region of the close fibers 3 h after denervation became smaller, whereas that of the synaptic region of distant fibers did not change relative to the control. Placing the muscle 3 h after denervation into medium with carbamylcholine (1·10−8 M), cGMP (1·10−4 M), or dibutyryl-cGMP (1·10−6 M) led to hyperpolarization of the synaptic region of the close fibers but did not change the resting potential in the synaptic region of the distant fibers, and abolished differences between them. Five days after division of the nerve, incubation of the muscle in a solution with the above-mentioned substances did not affect the resting membrane potential. Nonquantal release of acetylcholine from motor nerve endings, assessed by the amplitude of hyperpolarization of the postsynaptic membrane, induced by application of curarine against the background of acetylcholine esterase inhibition, 3 h after denervation was identical in the synaptic region of the close and distant fibers and did not differ from the control. It is postulated that the postdenervation fall of membrane potential of rat muscle fibers is not due to disturbance of nonquantal secretion of acetylcholine from motor nerve endings.

Is an acetylcholine transport system responsible for nonquantal release of acetylcholine at the rodent myoneural junction?

Experiments were performed to investigate the effects on the spontaneous, nonquantal release of acetylcholine (AcCho) from motor nerve terminals of substances known to inhibit the AcCho transport system present in cholinergic synaptic vesicles. In mouse diaphragms, the hyperpolarization normally produced by d-tubocurarine in the endplate area of muscle fibers that had been treated by an anticholinesterase was partly or completely blocked by 2-(4-phenylpiperidino)cyclohexanol (AH5183, 0.1-1 microM), quinacrine (0.1 microM), and tetraphenylborate (1 microM). Since the sensitivity of the subsynaptic area to AcCho was not changed, the block of the hyperpolarizing action of d-tubocurarine indicated in inhibition of the spontaneous, nonquantal release of AcCho. This was confirmed in experiments on rat diaphragm using direct radioenzymatic measurement of the AcCho released into the incubation medium. The release of AcCho from the innervated diaphragm was decreased by about 50% in the presence of AH5183 (0.01-1 microM) and by 42% in the presence of quinacrine (0.1-1 microM). The AcCho released was presumably neural, since the release of AcCho from 4-day denervated diaphragms was not diminished by either AH5183 or quinacrine. The results indicate that the spontaneous release of AcCho from the motor nerve terminals is highly sensitive to low concentrations of specific inhibitors and is probably mediated by a carrier. It is proposed that spontaneous release is due to the incorporation into the membrane of the nerve terminal during exocytosis of the vesicular transport system responsible for moving AcCho into the vesicle.

Non-quantal release of acetylcholine at a developing neuromuscular synapse in culture

Local, pulsed application of d-tubocurarine at neuromuscular synapses in embryonic Xenopus nerve-muscle culture resulted in a transient hyperpolarization of muscle membrane potential. Miniature endplate potentials (MEPPs) were abolished during the hyperpolarization and recovered after the return of resting membrane potential. The magnitude of hyperpolarization was independent of the frequency of MEPPs before curarization, and it had an average peak value of 4.3 mV in medium containing physiological levels of Ca2+. Prolonged application of curare or alpha-bungarotoxin led to sustained hyperpolarizations up to 8 mV in magnitude. Denervation produced by mechanically removing the neurite from the muscle cell also produced similar hyperpolarization, and curarization after denervation was without significant hyperpolarizing effect. Increasing the extracellular Ca2+ concentration to about 8 mM abolished the curare-induced hyperpolarization response, in sharp contrast to its effect in elevating the frequency of MEPPs. Taken together, our results indicate that innervated embryonic muscle cells were maintained at a depolarized state relative to that of uninnervated muscle cells by a steady, spontaneous release of acetylcholine (ACh) from the innervating neurite. The cellular mechanism underlying this mode of ACh release appears to be different from that of the quantal ACh release responsible for MEPPs.

Passive transfer of Lambert-Eaton myasthenic syndrome in mice: decreased rates of resting and evoked release of acetylcholine from skeletal muscle.

Mice were injected for 1-2 months daily with 10 mg immunoglobulin G (IgG) from four patients with Lambert-Eaton myasthenic syndrome (LEMS); control mice were injected with pooled human IgG from normal donors. Gastrocnemius muscles were homogenised for the assay of acetylcholine (ACh), choline acetyltransferase (ChAT), and cholinesterase (ChE). The ACh, ChAT, and ChE contents of gastrocnemius muscles from "LEMS mice" were about the same as the control values, which were 180 pmol, 40 nmol X h-1 (37 degrees C), and 15 mumol X h-1 (37 degrees C), respectively. Hemidiaphragms were treated with an irreversible ChE inhibitor (Soman) and incubated at 20 degrees C for estimation of ACh release. Resting ACh release from experimental muscles was reduced by about 25% (P2 less than 0.05) and the release evoked by 3 s-1 nervous stimulation by 50% (P2 less than 0.05). On the other hand, 50 mM KCl-induced transmitter release was not abnormal in LEMS mice. The findings indicate that IgG antibody from patients with LEMS may bind to nerve terminal determinants that are involved in quantal and nonquantal ACh release.

Nonquantal acetylcholine release from motor nerve endings and denervation changes in rat muscle fiber membranes after axonal transport blockade

Microelectrode experiments on the rat diaphragm showed that application of colchicine, which disturbs axonal transport, to the motor nerve leads after 5 days to a decrease in resting potential and an increase in input resistance of the electrogenic membrane, disappearance of differences of input resistance between the postsynaptic and extrasynaptic membranes, the appearance of extrasynaptic sensitivity to acetylcholine, and the appearance of anode-break action potentials resistant to tetrodotoxin. Similar changes develop in the muscle membrane after division of the motor nerve. Application of colchicine to the nerve, unlike its division, does not cause cessation of contractile activity of the muscle or disturbance of quantal and reduction of nonquantal acetylcholine secretion in motor nerve endings, as reflected in the degree of hyperpolarization of the postsynaptic membrane (H effect) in response to the action of D-tubocurarine chloride on the muscle after inhibition of acetylcholinesterase. The results confirmed the view that neurotrophic control of the mammalian muscle fiber membrane is effected mainly by means of substances carried to the muscle by axonal transport. Synaptic acetylcholine, secreted from nerve endings in nonquantal form, does not play a leading role in neurotrophic control of the muscle membrane.

An analysis of the mechanisms underlying the non-quantal release of acetylcholine at the mouse neuromuscular junction

The hyperpolarization produced by the application of 10 −5 M d-tubocurarine to the end-plate of mouse diaphragm-phrenic nerve preparation pretreated with an irreversible anticholinesterase was studied. This hyperpolarization has been attributed to a non-quantal release of acetylcholine by the nerve terminals. The usual hyperpolarization effect of about 10 mV was mimicked by adding 10 −7 M acetylcholine to the bath which contained 15mM Ca 2+ to block the non-quantal release. The hyperpolarization effect was maximal (9.2 ± 0.8mV) in saline solution with the normal concentrations of Ca 2+ (2mM) and K + (5mM). Reducing [Ca 2+] in the bath decreased the hyperpolarization effect. In Ca-free solution with 4.10 −4 M ethleneglycoltetra-acetate the hyperpolarization effect was3.8 ± 0.6mV. An increase of [Ca 2+] also reduced the hyperpolarization effect and it was absent in 15mM of Ca 2+. When the external concentration of Ca 2+ was kept constant (2mM), both decreasing and increasing K + concentration from 5 mM diminished the hyperpolarization effect. However, the decrease at elevated K + concentrations was due to a reduction of membrane resistance and when the hyperpolarization effect was corrected for the change in size of the miniature endplate potential, it was unchanged in solutions with increased [K +] up to 13 mM. No traces of non-quantal release were observed during repetitive stimulation of the phrenic nerve in a medium containing 0.3 mM Ca 2+. The hyperpolarization effect was not found in denervated muscles that lacked signs of neuromuscular transmission. The hyperpolarization effect was blocked by botulinum toxin while some evoked release of ACh was present. The time required for the block by 1 μg/ml toxin was shorter than for 0.25 μg/ml. The application of α-bungarotoxin produced a hyperpolarization of the membrane in the junctional region. The addition of d-tubocurarine failed to produce the hyperpolarization effect. The hyperpolarization effect was also absent when muscles were incubated with the desensitization potentiating drug, SKF-525A. The hyperpolarization effect was unchanged by replacement of Na + with guanidine and was absent following the replacement of Na + with arginine. The data suggest the channel responsible for the hyperpolarization effect is the same as that giving the endplate potential. The results are in agreement with the hypothesis that the hyperpolarization effect reflects a sustained non-quantal release of acetylcholine from the nerve terminal into the synaptic cleft.

Decrease of the spontaneous non-quantal release of acetylcholine from the phrenic nerve in botulinum-poisoned rat diaphragm

Botulinum type A toxin (BoTx) has been found to diminish by 40% the spontaneous release of acetylcholine (ACh) from normal (acutely denervated) rat diaphragms incubated in the presence of 5 mM K+, while the release of ACh from chronically (4 days) denervated diaphragms was not affected during 2 h incubations. The toxin has been found to rapidly remove (within 10 min) the local depolarization of about 8 mV which developed in the end-plate zones of the diaphragms after the inhibition of cholinesterases; after the administration of BoTx, tubocurarine lost its ability to increase the resting membrane potential (H-response, Katz and Miledi 1977) in the end-plate area of anticholinesterase-treated muscles. It is concluded that BoTx inhibits the non-quantal release of ACh from the motor nerve fibres and that it probably acts directly on the nerve terminal surface membrane (without internalization). The H-response in the rat diaphragm reflects the non-quantal release of ACh from the nerve terminals and not from the muscle fibres.