Different Kinds of Acetylcholine Release From The Motor Nerve

Different Kinds of Acetylcholine Release From The Motor Nerve

DIFFERENT KINDS OF ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE By S. Thesleff Department of Pharmacology University of Lund 5-22362 Lund, Sweden Stu...

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DIFFERENT KINDS OF ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE By S. Thesleff Department of Pharmacology

University of Lund

5-22362 Lund, Sweden

Studies of transmitter release at the neuromuscular junction have revealed the presence of several distinct types of acetylcholine (ACh) secretion. The purpose of this presentation is to describe these processes and to discuss underlying release mechanisms and the possible physiological significance of the various kinds of transmitter action. It is not intended as a review coveringall aspects of the field. Excellent recent reviews already exist for most parts of the subject and reference will be made to them for further details. It is instead a rather personal presentation and interpretation of a complex area of research in which I have, to a small extent, been involved. Transmitter release from the motor nerve may be divided into those involving intermittent, quantal, or nonquantal release of ACh and those characterized by a continuous leakage of ACh. Figure 1 illustrates the three forms of ACh release (1-111) which will be considered. Intermittent secretion of ACh involves either a Ca2+-sensitive(I) or a Ca2+-insensitive(11) 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-risingminiature end-plate potentials (Thesleff and Molgb, 1983).Molecular leakage of ACh is a continuous process originating not only from the presynaptic nerve (111) but also from the postsynaptic muscle cell.

I. Intermittent Secretions of ACh

A. CALCIUM-SENSITIVE QUANTAL RELEASE OF ACh (I) This is the mechanism responsible for neuromuscular transmission, i.e., the chemical transfer of a nerve impulse to the muscle fiber (Fig. 1, I). INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 28

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Copyright 6 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Ca*+

FIG. 1. The diagram illustrates the three kinds of ACh secretion from the motor nerve (1-111) that will be considered in this presentation. I is a quantal, Ca2+-sensitive secretion of ACh, presumably originating from synaptic vesicles (SV) which discharge their content of ACh at active zones. I 1 is an intermittent, calcium-insensitive, but possibly cyclic AMP-sensitive, discharge of ACh from areas outside of the active zones, and 111 depicts a continuous, ATPase-dependent efflux of ACh from the terminal. The figure also illustrates the ionic mechanism responsible for nerve impulse evoked transmitter release, i.e., the presence of Na+ and K' channels in the axon and only Ca2+ and K + channels in the terminal. For further details, see text.

As elegantly shown 33 years ago, intracellular electrical recording at the synapse or end-plate region of a muscle fiber reveals the presence of spontaneous, small, intermittent electric potential changes of 0.5- 1 mV amplitude, rising rapidly within 1 msec and declining exponentially with a total duration of about 2 msec (Fig. 2B). These potentials are similar, but much smaller, than nerve impulse evoked end-plate potentials (epps) and were therefore called miniature end-plate potentials or mepps (Fatt and Katz, 1952; del Castillo and Katz, 1954). Subsequently Katz and coworkers in a series of classical papers that were also excellently reviewed (Katz, 1966, 1969)demonstrated the correspondence between mepps and the quantal components constituting the evoked epp. Furthermore, del Castillo and Katz (1955) postulated that each unit package of ACh, which when released produces a mepp, is preformed within a synaptic vesicle in the nerve terminal. The synaptic vesicle was supposed to accumulate ACh actively from its site of synthesis in the axoplasm. According to this socalled vesicular hypothesis for quantal ACh release, Ca2+inside the nerve

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terminal membrane is essential for the process which allows a transient fusion of terminal and synaptic vesicle membranes and thereby the release of a quantal package of ACh. Furthermore, it was suggested that this process was rapidly reversible so that a vesicle, having discharged its content, would quickly detach itself from the terminal membrane and start to reaccumulate the transmitter from the axoplasm (Katz, 1969, p. 15). Katz and Miledi (1965) demonstrated the Ca2+ dependency of this type of quantal ACh release. As amply supported by subsequent studies (see reviews by Llinas and Heuser, 1977; Silinsky, 1985) it is evident that the described spontaneous intermittent quantal secretion of ACh giving rise to the mepp is a Ca2+-sensitiveform of transmitter secretion. DepoIarization of the terminal part of the motor axon by the action potential increases the Ca2 conductance of the nerve terminal, allowing Ca2 ions to diffuse along their electrochemical gradient into the terminal and thereby activating ACh discharge. In the squid giant synapse, a linear proportionality is observed between Ca2+entry and the amount of transmitter released by this process (Llinh et al., 1981). Since the epp is the sum of a number of synchronous mepps evoked by the influx of Ca2+ during the nerve terminal action potential, one may call the process an electrically controlled Ca*+-sensitiveform of quantal ACh release. The vesicular hypothesis for ACh release has been critically examined in a number of electrophysiological,morphological, and biochemical studies of various types of cholinergic synapses, e.g., brain synaptosomes. electric organs of fish, and motor nerves from different species. Some of the results provide direct support for the hypothesis, such as the finding that synaptic vesicles actively accumulate and contain ACh (Whittaker et al., 1964; Israel et al., 1968; Whittaker et al., 1972) in amounts corresponding to that giving rise to a spontaneous mepp or a maximum of about 10,000 molecules of ACh (Kufller and Yoshikami, 1975; Fletcher and Forrester, 1975). Morphological studies have revealed depletion of synaptic vesicles followingstimulation of transmitter release to exhaustion and also evidence of vesicle fusion with axolemma (Peper et al., 1974: Heuser, 1977; Ceccarelli et al., 1979a,b; Heuser et al., 1974, 1979; PCcot-Dechavassine, 1982). By the use of extracellular marker molecules such as horseradish peroxidase, it has been possible to visualize what apparently constitutes the retrieval of vesicle membrane from the axolemma, i.e., vesicle reformation by an endocytic process (Heuser and Reese, 1973; Heuser, 1976; Ceccarelli and Hurlbut, 1980; Meldolesi and Ceccarelli, 1981). The membrane potential of the nerve terminal fails to influence the size of each quantum of ACh released (del Castillo and Katz, 1954), which fits with vesicular discharge, but argues against the possibility that quanta of ACh +

+

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could be released from the cytoplasm along its electrochemical gradient (gated release of cytoplasmic ACh). Furthermore, no outward current which could correspond to ACh has been observed in mouse motor terminals. T h e expected ACh current would be 1.5 X lo-’‘ Nquantum for a 1-msec release period. This gives a current of about 1 nA in the presence of K +-channel blockers and is therefore larger than the currents carried by other ions (A. Mallart, personal communication). The shrinking or swelling of the nerve terminal should at least momentarily alter the ACh concentration in the cytoplasm and therefore also change the efflux through a gated channel. However, mepp amplitudes are not appreciably affected by massive alterations of the osmotic pressure of the extracellular solution that supports vesicular discharge of ACh (Van der Kloot, 1978). On the other hand, results have been published that at least superficially, are not readily reconciled with the vesicular hypothesis. For instance, it has repeatedly been demonstrated that newly synthesized ACh is released preferentially by nerve stimulation (Dunant et al., 1972; review by Israel et al., 1979). Since ACh is synthesized in the axoplasm and only subsequently accumulated in vesicles, this observation has been taken to indicate that stimulation preferentially releases ACh from the cytoplasm and that vesicular ACh is not primarily involved in the release process. Furthermore, it has been shown that neither the vesicular content of ACh nor the mean number of vesicles was modified by nerve stimulation at physiological frequencies (Dunant et al., 1972, 1974; Lynch, 1982). In contrast, the cytoplasmic-free ACh was depleted during such stimulation and renewed with precursor. Similarly, experiments using an inhibitor of precursor uptake (hemicholinium) or a “false”precursor have shown that the transmitter released by nerve stimulation is not stored in independent quanta but is continuously mixed with the cytoplasmic pool of transmitter (Elmqvist and Quastel, 1965; Large and Rang, 1978; Collier et al., 1979). Reports have also appeared questioning the validity of synaptic vesicle recycling on the ground that the labeling by extracellular horseradish peroxidase is too low to be consistent with synaptic vesicles undergoing continuous exo- and endocytosis along the presynaptic plasma membrane (Meshul and Pappas, 1984). On the basis of such conflicting evidence, there are proponents and opponents to the vesicular hypothesis for transmitter release. Their views have been excellently presented and summarized in several recent reviews (proponents: Zimmermann, 1979a; Meldolesi and Ceccarelli, 1981; Whittaker, 1984; opponents: Israel et al., 1979; Tauc, 1982). Opponents consider that the nerve terminal membrane contains a hypothetical structure, possibly located at “active”zones, that bind ACh to saturation and which, upon activation by Ca2+,releases ACh in a none-

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lectrogenic manner into the extracellular fluid. Isreal et al. (1979) have coined the term “operator” and Tauc (1982) the term “vesigate”for such a release mechanism. I believe that the contradiction between the vesicular and the cytoplasm-gate hypothesis for transmitter release, as discusssed by Zimmermann (1979b),Israel et al. (1979),and Whittaker (1984),could be overcome by certain assumptions. For instance, a portion of the synaptic vesicles have properties different from the rest, i.e., a functional heterogeneity exists among synaptic vesicles and certain vesicles are preferentially involved in transmitter release and these vesicles go through repeated cycles of exo- and endocytosis during transmitter release. Heterogeneity among cholinergic synaptic vesicles has been observed morphologically and biochemically (Zimmermann and Whittaker, 1977; Zimmermann, 1979a; Whittaker, 1984; and Agoston et al., 1985). Physiologically a functional heterogeneity is observed among quantal sizes (McLachlan, 1975;Doherty et al., 1984),and stimulation makes apparent metabolic and morphological heterogeneity of cholinergic synaptic vesicles (Zimmermann, 1979b). A motor nerve terminal contains 500-1000 “active”zones, and at each zone, a double row of 20-30 vesicles each are present (Couteaux and PCcot-Dechavassine, 1974). If vesicles in that position required the property to repeatedly discharge ACh, that population would constitute less than 10% of the total vesicular population in a terminal. Hence, changes in its content of transmitter would only marginally affect the total amount of vesicular ACh and thereby explain the failure to detect depletion during stimulation. The existence of a small population of synaptic vesicles, primarily involved in transmitter release and reaccumulation of ACh from the cytoplasm, would explain a preferential release of newly synthesized cytoplasmic transmitter and a lack of correspondence between the cytoplasmic and the total vesicular content of transmitter during stimulation. The idea that only a small portion of the total number of synaptic vesicles (operator vesicles; according to Isreal et al., 1979), presumably only those attached to the active zones of a nerve terminal under physiological conditions, participated in transmitter release as “shuttle gates” between cytoplasm and synaptic cleft is quite attractive. It would explain most, if not all, differences between the advocates of vesicluar and nonvesicular quantal transmitter release mechanisms (Israel and Dunant, 1979; Zimmermann, 1979b). The model would require that the vesicles discharge their content through a pore or a channel opening into the synaptic cleft. Upon discharge, the vesicles would quickly detach themselves and reaccumulate ACh from the cytoplasm. This would be the process of release during physiological stimulation,while during stimulation to transmitter exhaus-

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tion, a process involving the fusion and incorporation of synaptic vesicles with the axolemma would occur. The latter would involve the 90%or so “reserve”vesicles not readily available for release. In line with this view, Meldolesi and Ceccarelli (1981) propose that vesicular transmitter release and vesicle recycling is maintained by two different processes (Ceccarelli and Hurlbut, 1980).The first mechanism, as outlined above, would account for a fast recycling occurring at physiological frequency of stimulation, whereas a second, different mechanism would account for vesicles becoming completely incorporated into the axolemma and recycled as coated vesicles. The second mechanism might predominantly operate at higher frequencies of stimulation and during chemical stimulation by a Ca2+ionophore or black widow spider venom. A brief attachment of a synaptic vesicle to the axolemma and a discharge of ACh through a narrow membrane pore or channel, possibly by a cation exchange mechanism as suggested by Uvnas and Aborg (1984), is also attractive, because it offers an explanation for the release of ACh quanta of variable, particularly small, size, giving rise to small-amplitude mepps or sub-mepps (Uvnas and Aborg, 1984).It might therefore provide an explanation for the observation that various experimental procedures, which conceivably could affect the life-span of a secretory membrane pore, increase the number of sub-mepps present at the neuromuscular junction (see a review by Trernblay et al., 1983). Such a mechanism seems more plausible as an explanation for sub-mepps than the idea that one sub-mepp corresponds to the release of a single synaptic vesicle, whereas a mepp is caused by the simultaneous release of several vesicles (Wernig and Stirner, 1977). The mechanisms by which synaptic vesicles are attracted to the axolemma and made to discharge their content are so far unknown. It should, however, be mentioned that there is strong evidence that synaptic vesicles move along the surface of a specific set of presynaptic microtubules that direct the vesicles to dense bars at the presynaptic membrane. These dense bars constitute the active zone at which synaptic-vesicle discharge occurs. Freeze-fracture studies of motor end-plate active zones during or immediately after transmitter release reveal linear arrays of synaptopores perforating the presynaptic membrane (Dreyer et al., 1973; Heuser et al., 1974; Heuser, 1976; Akert and Tokunaga, 1980; Gray, 1983). Calcium ions, when entering the terminal along voltage-dependent channels, possibly exclusively located at active zones (Pumplin and Reese, 1978; Pumplin et al., 19Sl), may promote a vesicle movement, discharge, and detachment through a number of selective mechanisms, such as by activating actomyosin filaments, membrane phospholipase AP, adenylate cyclase, or cdlcium-calmodu~in and CAMP-dependent protein kinases.

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Screening of negative membrane charges by Ca" might also facilitate membrane apposition. The concerted action of many such events may explain how transmitter release is regulated (Greengard, 1978; Moskowitz and Puzskin, 1983; Cooper and Meyer, 19 4). However, as reviewed by Kelly et al. (1979), our knowledge about the iachemical steps involved in the release process is so far incomplete and marred by conflicting results caused by methodoIogica1 and experimental difficulties. In nerve impulse-evoked transmitter release not only intracellular Ca2+ but also the depolarization of the terminal directly controls the number of quanta released (Dudel, 1983; Dudel et ad., 1983). It seems that depolarization triggers a mechanism which acts as a gain control in the Ca2+-messengersystem. An example of such a mechanism would be inositol trisphosphate (Berridge and Irvine, 1984), but see also Rasmussen and Barrett (1984) and Kostyuk (1984) for other possible mechanisms. An interesting, but iittle-debated issue, is why a nerve impulse activates transmitter discharge only from a fraction of available active zones in the terminal. As previously mentioned, a frog motor-nerve terminal may contain about 1000 active zones. The quantum content of a normal epp is much smaller than that, about 200, and therefore only a part of the active zones is activated by a given impulse. Bennett and Lavidis (1982), Tremblay et al. (1984); and Dalonzo and Grinnell (1985) observed that the probability of quanta1 secretion from an active zone declined along the length of the terminal branch. Brigant and Mallart (1982) have shown that mammalian nerve terminals are practically devoid of Na+ channels while they are rich in K + and Ca2+ channels. Therefore, the terminal part of the axon cannot conduct an action potential and is depolarized passively by electrotonic spread. This initiates inward Ca2+and outward K + currents (Fig. 1). Normally the outward K + current overwhelms the inward Ca2+current and the terminal is repolarized and the Ca2+channels closed. Furthermore, the Ca"-dependent K + current is activated by Ca2+and therefore develops with a short delay after the start of the Ca2+ current (Mallart, 1984). Such ionic mechanisms are eminently suited as regulators of highfrequency phasic transmitter release but might also prevent the depolarization by an action potential from reaching the furthermost part of the nerve terminal network. These mechanisms provide an explanation of why drugs which block the outward K + current (see below) have such a dramatic potentiating effect on impulse-evoked ACh release. Another possibility is that heterogeneity exists in the probability of active zones to discharge transmitter. For instance, a proximodistal gradient might exist in the size of active zones as observed by Davey and Bennett (1982). A better knowledge of the role of aformentioned mech-

ti

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anisms in the regulation of transmitter discharge at individual active zones would undoubtedly be of great value for the understanding of phenomena such as activity-dependent facilitation and depression of transmitter release, as well as the mode of action of a number of drugs and toxins affecting the release process. 1. Effect of'Drugs and Toxins

Drugs or procedures which alter the free Ca2+concentration in the nerve terminal also alter the quantal release of ACh from the nerve, be it spontaneous, causing mepps, or impulse evoked, giving rise to epps. Table I enumerates a number of drugs and procedures which increase the level of free Ca2+ in the nerve cytoplasm, either by enhancing the influx of this ion from the extracellular medium or by releasing Ca2+from storage or binding sites within the teminal. This action greatly accelerates the frequency of the quantal discharge of ACh, giving rise to mepps. Several drugs selectively enhance nerve impulse-evoked Ca2+-semitive, quantal transmitter release without affecting spontaneous release. Such drugs are the aminopyridines (4-aminopyridine and SPdiaminopyridine) and tetraethylammonium, which all act by blocking the K + channel and thereby K currents in excitable tissues. These drugs enhance Ca2+influx into nerve terminals by prolonging the duration of depolarization due to the action potential by a blockade of the voltage-activated K + channels. For recent reviews of the pharmacology of these drugs see Thesleff (1980), Bowman and Savage (1981), and Glover (1982). Drugs which interfere with Ca2+entry into the terminal such as Mg2+ and the aminoglycoside antibiotics reduce evoked Ca2+-sensitive quantal +

TABLE I SUMMARY OF T H E EFFECTS OF VARIOUS PROCEDURES O N THE FREQUENCY OF CALCIUM-DEPENDENT FASTA N D CALCIUM-INDEPENDENT SLOWmepps AT NORMAL A N D BoTx POISONED ENDPLATES~ Agent or procedure

Normal muscle, fast mepps only

BoTx-poisoned, slow mepps only

Ca2+,8 mM K + , 20 mM K + , 0 mM Ouabain, 0.2 mM Ethanol, 0.5 M Mn2+, 10 mM Hypertonicity, 2~ Hypotonicity, 0.5X

Increase 4~ Increase 2 0 ~ Increase 15X Increase 5OX Increase IOX Increase 8~ Increase 8OX Decrease 0.3X

No change Increase 2X No change No change No change No change Decrease 0.3X Increase 3X

"From Thesleff el al. (1983). 'Approximate change times control.

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transmitter release (Elmqvist and Josefsson, 1962; Vital Brazil and PradoFranceschi, 1969; Molgo et al., 1979; Fiekers, 1983; review by Pittinger and Adamson, 1972), as expected from this mode of action. Of particular interest are the neurotoxins of Clostridium botulinum and Clostridium tetani which block Ca2+-sensitivequantal transmitter release from cholinergic nerve terminals. The block presumably results from an interference with the role of Ca2+ as a trigger of transmitter discharge, see reviews by Simpson (198l), Sellin (19Sl), and Mellanby (1984). Apparently, the toxins do not affect Ca2+entry into the nerve terminal (Gundersen et al., 1982; Dreyer et al., 1983) but reduce the efficacy of Ca2+ to release transmitter (Cull-Candy et al., 1976a). Molgo and Thesleff (1984) have suggested that botulinal toxins (BoTx), upon entering the nerve terminal by an endocytic mechanism, catalyze intracellular processes involved in the disposal of Ca2+from the cytoplasm. I n normal conditions, Ca2+ turnover in the nerve terminal is balanced so that the Ca2+ which enters the terminal reaches active zones in a sufficient amount to cause synaptic-vesicle discharge. An enhanced disposal of a Ca2+ would lower the concentration of this ion to a level in which the amount of Ca2+that entered the terminal would be insufficient to trigger this type of transmitter release. Mellanby (1984) has a somewhat similar suggestion. She proposes that the toxins inactivate a Ca2+-dependentenzyme within the nerve terminal which is involved in the release of transmitter, possibly by phosphorylating critical membrane proteins either on vesicles or on the presynaptic membrane. In that connection, it is interesting that Simpson (1984) has shown botulinum toxin type C2 to possess ADP-ribosylating activity.

2. Physiological Role The physiological importance of Ca2+-sensitive,quantal ACh release is to act as a chemical mechanism for propagating the phasic information contained in nerve impulses across the synaptic cleft. This type of ACh release mechanism is ideally suited to respond to high-frequency stimulation because of an efficient mechanism for phasic Ca2+ entry, which proportionately triggers transmitter release, and a subsequent rapid Ca2+ inactivation, which assures that each nerve impulse is faithfully transmitted. This mechanism is responsible for low- and high-frequency impulse propagation across the synaptic cleft and thereby for the neurogenic control of muscle tone and activity. Since the pattern of muscle activity has long-term consequences for the chemical and physiological properties of a muscle (Lomo and Westgaard, 1975; review by Lomo, 1976, one may consider that this type of transmitter release mechansim also is a part of the trophic influence exerted on muscle by the nerve.

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B. CALCIUM-INSENSITIVE SECRETION OF ACh (11)

This type of ACh release is not involved in impulse propagation across the synaptic cleft, but represents a spontaneous intermittent form of ACh secretion which is particularly prominent when synaptic impulse transmission is blocked and during synaptic development (Fig. 1, 11). i t is characterized by spontaneous mepps with highly variable times-to-peak and amplitudes (see review by Thesleff and Molgo, 1983). Typically, such mepps have a prolonged time-to-peak, the mean exceeding more than twice that of Ca2+-sensitive quantal mepps or nerve impulse-evoked epps. In many instances, times-to-peak as long as 10 msec are recorded (Kim et al., 1984). The amplitude of such mepps is also highly variable, with generally much larger amplitudes than the Ca"-sensitive quantal mepp. Amplitudes as large as 15 mV are not uncommon and may therefore be of sufficient size to trigger an action potential in the muscle cell. Examples of Ca2+-insensitive and Ca2'-sensitive mepps are shown in Fig. 2A and B, respectively. Figure 2C illustrates Ca*+-sensitive multiquantal ePPs.

n

B

L FIG. 2. Examples of Ca2+-insensitive, intermittent secretion of ACh giving rise to slow-rising, large amplitude mepps in A. The record was obtained from a muscle poisoned by botulinum toxin. Record B illustrates intermittent, Cay+-sensitivequantal release of ACh giving rise to fast-rising, uniform amplitude mepps in a normal, untreated muscle. Record C is from the same fiber as A showing evoked epps. Record D is a recording from a normal, untreated muscle showing that slow-rising, giant mepps may also exist under normal conditions. Record E illustrates the effect of 4-aminoquinoline on a normal muscle. Note that the drug has induced a population of large-amplitude, slow-rising mepps of a type similar to that in A. Voltage calibrations are 1 mV for all tracings except C, for which it is 2 mV. Time calibrations are 2 msec for all tracings except B, D, and E for which it is 4 msec. Temperature, 30°C.

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Calcium-insensitive transmitter release is neither activated nor influenced by nerve impulses and resultant transmembrane Ca2+fluxes in the nerve terminal. Consequently, this type of transmitter release does not participate in impulse transmission. Spontaneous mepps resulting from the Ca2+-insensitivetype of transmitter release are uninfluenced in frequency by procedures which affect intra- or extracellular Ca2+concentrations of the nerve terminal (see Table I). Hypertonic media, which increase the frequency of Ca2+-sensitivemepps, decrease the frequency of Ca2 -insensitive mepps, while hypotonic solutions exert an opposite effect on both types of release (Table I). Furthermore, the temperature dependence of the two release processes is quite different; Ca*+sensitive, quantal ACh-release frequency is enhanced by temperatures with a Qlo of 2-3, while the Ca2+-insensitive,intermittent release rate is enhanced with a Qlo of about 12 and is virtually blocked at temperatures below 15°C (Thesleff et al., 1983). The first one to observe the Ca'+-independent type of intermittent ACh release was Liley, who in 195'7described unusually large mepps (giant mepps) occurring at a low rate in normal rat neuromuscuIar junctions. These potentials varied in frequency among fibers, but occasionally constituted up to 20% of all mepps recorded. Their frequency was unaltered by nerve stimulation, by nerve terminal depolarization,and by changes in the extracellular Ca2+ or Mg2+ concentration. They also differed, as reported by Liley, from nerve stimuIus-evokedquantal epps (Fig. 2C). Subsequently, Jansen and van Essen (1976) pointed out that the giant potentials had a slow and protracted shape which did not correspond to a simple summation of normal mepps, so that they could not be considered quantal in nature, as estimated from Poisson analysis of epp failure. According to Heinonen el al. (1982), ColmCus et al. (1982) and Kim et al. (1984)giant, slow-rising mepps constitute,on the average, 4% of all mepps recorded at rat neuromuscular junctions at 30°C with great variability in their frequency between fibers. Slow-rising, large-amplitude mepps of the Ca2 -insensitive type are present also at early stages of regenerating neuromuscularjunctions (Bennett et d.,19'73). These mepps constitute a majority of all mepps in this condition and are reduced in number when the synapse matures and normal fast-rising Ca2+-sensitive quantal mepps appear (Colm6us et al., 1982). Slow mepps are also present at neuromuscular junctions of dystrophic mice, strain 1291ReJ (Carbonetto, 1977), and in mice with a hereditary end-plate disease (Weinstein, 1980).They appear in skeletal muscle of chickens curarized during early development (Ding et al., 1983). Growth cones of embryonic cholinergic neurons in culture intermittently secrete large amounts of ACh by an apparently Ca'+-insensitive mechanism (Hume et al., 1983; Young and Poo, 1983). +

+

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The Ca"4nsensitive type of transmitter release is particularly prominent in muscles totally or partly paralyzed by the use of botulinum neurotoxin t y p e A (Colmeus et al., 1982; Kim et al., 1984; Dolly et al., 1985).As previously mentioned, this neurotoxin blocks the Ca2+-sensitive, quantal release mechanism for ACh and thereby neuromuscular transmission. A few days after a neuromuscular block is produced slow-rising mepps of the Ca2+-insensitivetype increase in frequency so that, about 10- 14 days after poisoning, their occurrence reaches 0.3-1 Hz with large variations between fibers (see Fig. 2A). As the effects of the toxin subside and neuromuscular transmission is gradually restored, the frequency of these mepps is reduced, while the number of fast-rising Ca2 sensitive, quantal mepps increases. It is of interest that the occurrence of slowrising, Ca2+-insensitive mepps is more marked in muscles only partially paralyzed by botulinum toxin than in totally paralyzed ones (Kim et al., 1984). The difference between Ca2+-sensitive and Ca2+-insensitive ACh release is particularly evident in muscles poisoned with botulinum toxin since it is possible to observe both types of release simultaneously. At botulinum toxin type A poisoned junctions, it is possible to reintroduce the Ca2+-sensitivetype of quantal transmitter release by procedures which elevate the intracellular Ca2+ concentration. The administration of mitochondrial blocking agents such as dinitrophenol (Sellin et al., 1983) or of a Ca2 ionophore such as A23187 (Cull-Candy et al., 1976a)reintroduces fast-rising, Ca2+-sensitive quantal mepps which appear simultaneously with the slow-rising mepps. Similarly,it is possible to restore nerve impulseevoked quantal release of ACh by drugs such as the aminopyridines or tetraethylammonium (Lundh et al., 1977). Quanta1 epps evoked in such a manner are identical to fast-rising, Ca2+-sensitive mepps and without resemblance to the slow mepps representing Ca2+-insensitive release, which can be observed simultaneously at the same junction as in Fig. 2C (Sellin and Thesleff, 1981). +

+

1. Drugs Which Induce Calcium-Insensitive,

Intermittent Secretion of ACh

The calcium-insensitive secretion of ACh, as observed in BoTx-poisoned muscle, is stimulated in the presence of cyclic AMP or dibutyryl cyclic AMP and further enhanced in the presence of caffeine, a phosphodiesterase inhibitor. The effect of cyclic nucleotides is variable but generally characterized by an increase in the amplitude and frequency of the slow-rising mepps, the potentials frequently appearing as bursts of activity (Tabti et al., 1986). Thus, it seems possible that this type of spontaneous intermittent ACh secretion is somehow modulated by the intraterminal concentration of cyclic AMP.

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4-Aminoquinoline in a concentration of 100-250 p M induces, within minutes of its application to mammalian, but not to amphibian, skeletal muscle, the appearance of a population of mepps with a larger than normal amplitude and a prolonged time-to-peak (Fig. 2E). The slow and large mepps induced by 4-aminoquinoline have all the characteristics of a Ca2+-insensitive,intermittent, nonquantal secretion of ACh (Molgo and Thesleff, 1982; Thesleff and Molgo, 1983). They are unaffected in frequency by nerve stimulation, by nerve terminal depolarization, and by increases in intra- or extracellular Ca2+concentrations. Their frequency has a high positive temperature coefficient,Qlo of 12,and they are virtually absent at temperatures below 15°C (Thesleff et al., 1983). These effects of 4-aminoquinoline occur without observable changes in the number of fast-rising,Ca2+-sensitivequantal mepps or epps (Molgo and Thesleff, 1982). 4-Aminoquinoline exerts a similar effect on botulinum toxin-poisoned end plates in which it markedly enhances the frequency of slow mepps, but does not affect the number of fast mepps (Thesleff et at., 1983). At muscles with regenerating nerve terminals, the drug increases the frequency of slow mepps about three times without affecting the frequency of fast mepps (Molg6 et al., 1982). One the basis of those findings, Thesleff and Molgd (1983) have concluded that 4aminoquinoline,by a hitherto unknown mechanism, selectivelystimulates the Ca2+-insensitivetype of intermittent ACh secretion at mammalian neuromuscular junctions. Other drugs, ions, and procedures have been reported to induce the appearance of giant mepps. Thus, an acidic pH and hypertonic solutions, substitution of Na+ by Li+,and prolonged tetanic stimulation may induce giant mepps (PCcot-Dechavassine, 1970; Pkcot-Dechavassine and Couteaux, 1971, 1972; Benoit et al., 1973; Heuser, 1974). Drugs affecting microtubules such as vinblastine (Pkcot-Dechavassine, 1976) and phospholipase A2-containing elapid neurotoxins (e.g. taipoxin and notexin) (Cull-Candy et al., 1976b) have similar effects. In all these instances, the giant potentials have a fast rise time and the mepps are in some instances accompanied by the appearance of large or aggregated synaptic vesicles which has been interpreted to account for the discharge of larger than normal amounts of ACh. Thus, it seems possible that the giant mepps recorded in these conditions might reflect a modification of the Ca2+sensitive, quantal release system rather than a stimulation of the Ca2+insensitive type of intermittent ACh release.

2. Origin and Mechanism of Release The agent responsible for the slow type of mepps is presumably ACh, since tubocurarine blocks such potentials and cholinesterase inhibitors enhance and prolong their duration (Liley, 1957; Molg6 and Thesleff,

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1982). However, it cannot be excluded that other substances, possibly neuropeptides or ATP (Dowdall et al., 1974), are coreleased with ACh (Thesleff and Molg6, 1983). Presumably the ACh release originates from the nerve, since it is abolished by denervation, and only reappears following reinnervation of the end plate (Cull-Candy et al., 1976a; Molgo and Thesleff, 1982). Studies of the time course of the disappearance of fast and slow mepps, i.e., of Ca2+-sensitiveand -insensitive ACh release, respectively,followingdenervation by surgical sectioningof the motor nerve, showed that both kinds of secretion disappeared simultaneously. This is the opposite of that seen with rnepps which originate from Schwann cells in the amphibian. These potentials appear only after denervation with a few days delay following the cessation of neuromuscular transmission and the disappearance of fast mepps (Bevan et al., 1976). Kim et al. (1984) and Thesleff et al. (1983) have investigated the possibility that the Ca2+-insensitivetype of transmitter release might originate from nerve terminal sprouts, particularly since such sprouting is prominent in botulinum toxin-poisoned muscles in which this type of transmitter release dominates. However, when botulinum toxinpoisoned muscles were directly stimulated in viuo with a frequency pattern which inhibits nerve terminal sprouting no change was observed in the appearance and frequency of slow mepps. Neither did X-ray irradiation of poisoned muscles, a procedure which prevents sprouting, affect the frequency of such rnepps. Consequently there is, at present, no evidence that would indicate that the Ca"4nsensitive type of intermittent transmitter secretion might originate from structures other than the motor nerve terminal. Postsynaptic factors such as a heterogeneity among ACh receptor properties are unlikely as an explanation for the slow mepps, since normal, fast mepps and quanta1epps occur, or can be induced, concomitantly with the spontaneous appearance of slow mepps. Intra-end-plate differences in cholinesterase activity also seem to be excluded, since neostigmine only increases the amplitude and time course of slow mepps without affecting their frequency (Liley, 1957). Nerve terminals treated with 4-aminoquinoline have been examined for the presence of unusually large synaptic vesicles or subaxolemmal cisternae which might account for the release of the amounts of ACh responsible for the generation of slow, giant potentials. No ultrastructural alterations were, however, observed that could account for the release of such large amounts of ACh (PCcot-Dechavassine and Molg6, 1982).One must consider that the potentials might result from a protracted discharge of transmitter from a row or a cluster of normal-sized synaptic vesicles. Support for such a possibility is the observation that slow mepps sometimes have notches in their rising or falling phases indicating a composite nature. Sellin and Thesleff (1981) and Molg6 and Thesleff (1982) have

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discussed the possibility that the discharge of transmitter might occur from areas outside of the active zones, i.e., from sites more distant from the postsynaptic receptor which would tend to prolong the time-to-peak of the resulting mepps. Such a nonspecific release site in the axolemma might also explain the failure of Ca2+to influence the frequency of ACh discharges and why this type of ACh release dominates during development, i.e., before the presynaptic specializationof active zones. It cannot, however, be excluded that the discharge reflects a mechanism of protracted, gated release of ACh from the cytoplasmic pool of free transmitter. Calcium ions may be involved ip the discharge process but their presence seems less critical than with quantal release at active zones. Hence, I have used the terminology intermittent, Ca2+-' insensitive transmitter release for this type of ACh secretion.

3. Physiological Role Calcium-insensitive, intermittent secretion of ACh is particularly marked at junctions where neuromuscular transmission is blocked or impaired and during synapse development (botulinurn toxin poisoning, regenerating neuromuscular junctions, and growth cones). This suggests that the release process might constitute a chemical signal which assists the axon in finding and maintaining its proper synaptic connection. Intermittent release of large amount of ACh from the growth cone could be a chemical signal to responsive cells, which in turn could direct axonal growth by reply signals. Once an appropriate innervation was established, the release could be suppressed by (a) factor(s) coming from the target. In favor of the latter possibility, is the delay of several days in the onset of this type of secretion following paralysis by botulinum toxin and the gradual suppression of secretion, as neuromuscular transmission is reestablished (Kim et al., 1984). Presently, very little is known about the chemical signals which act as messengers between nerve growth cones and target cells. The described secretion of ACh might be such a messenger or one among a number of messengers in a complex system of signals operating between the two (see Schmitt, 1984).

11. Continuous ACh Leakage (111)

In addition to the intermittent quantal and nonquantal discharges of ACh previously described, there is biochemical (Mitchell and Silver, 1963; Fletcher and Forrester, 1975) and electrophysiological (Katz and Miledi, 1977; Vyskocil and IllCs, 1977) evidence that ACh may escape from nerve

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terminals by a continuous process (Fig. 1, 111). At frog and mammalian neuromuscularjunctions, a steady leakage builds u p an ACh concentration of the order of lo-* to lo-' M in the synaptic cleft (Katz and Miledi, 1977; Vyskocil et al., 1983). Since the leakage is continuous, the total amount of ACh secreted can be estimated to exceed the efflux due to spontaneous quanta1 discharge by two orders of magnitude, and therefore it accounts for the largest part of ACh released from resting muscle (Katz and Miledi, 1977; Vizi and Vyskocil, 1979). It should, however, be mentioned that not only the nerve but also the muscle synthesizes and releases ACh. The amount of ACh released under resting conditions from nonneural parts of normal rat diaphragms has been estimated to be 30-50% of that released from their neural parts (Dolezal and Tucek, 1983). Accordingly, denervation reduces resting release of ACh by 50-70% (Straughan, 1960;Mitchell and Silver, 1963; Card Linden et al., 1983). One way to detect spontaneous leakage of ACh from the nerve is to apply curare to the end-plate region of a muscle fiber. The resulting blockade of cholinergic postsynaptic receptors causes a local hyperpolarization amounting to 0.04-9 mV depending upon species and experimental conditions (Fig. 3). This hyperpolarization or H-response is enhanced in the presence of cholinesterase inhibitors indicating that its origin is a blockade of cholinergic receptors continuously stimulated by ACh. The ion selectivity of the receptor channel blocked by curare and giving rise to the H-response is similar to that of the channel giving the epp, i.e., presumably the channel of the nicotinic-cholinergic receptor (Vyskocil et al., 1983).The H-response is abolished by denervation in murine and rat muscle (Vyskocil et al., 1983; Dolezal et al., 1983) but not in frog muscle (Katz and Miledi, 1977), indicating that, at least in these mammals, nerve terminals are a main source of the ACh responsible for this effect. This also suggests that in frog muscle at least a part of the continuous leakage of ACh comes from the muscle or from the Schwann cell. T h e release of ACh which causes a persistent depolarization of the end plate is not affected by nerve stimulation (Vizi and Vyskocil, 1979; Katz and Miledi, 1981; Vyskocil et al., 1983). It is however, influenced by the Cay+ concentration of the medium. At physiological Ca2+ levels (2 mM), the release of ACh is maximal, while lowering or elevating the Ca2+ concentration reduces release (Vyskocil et al., 1983). Inhibition of the Na+*K+-activatedmembrane ATPase increases the continuous nonquantal leakage of ACh from nerve terminals, but activation of the enzyme has the opposite effect, (see Fig. 3A, C and D) (Paton et al., 1971; Vizi, 1973, 1977, 1978; Vizi and Vyskocil, 1979). Since Ca2+ ions have been shown to inhibit membrane ATPase (Skou, 1957; Somogyi, 1964), it has been suggested that Ca2+ entering the terminal during

ACETYLCHOLINE RELEASE FROM THE MOTOR NERVE a

b

TC -

TC

TC

TC

TC

75

4 1

-

TC

FIG. 3. Local curarization of the end-plate region of a mouse diaphragm causes, in the presence of cholinesterase inhibition by prostigmine 6 x M (a) or by pretreatment with soman (b), a hyperpolarizing response (H-response). Inhibition of Na+-K+M (B) or a K+-free solution (C) enhances the activated ATPase (A) by ouabain 2 X H-response, while activation of ATPase by readmission of K + (D) blocks the response. Horizontal bars indicate the time of tubocurarine (TC) diffusion from a pipette located in the end-plate area. From Vyskocil and 1116s (1978).

stimulation might affect the activity of the ATPase and thereby also control this type of ACh secretion (Vizi, 1978). However, as mentioned above, this has not been experimentally confirmed. Recently, Edward et al. (1985) suggested that nonquantal, continuous leakage of ACh from nerve terminals might be the result of synaptic

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vesicle membrane incorporation into the nerve terminal membrane. The synaptic vesicular membrane accumulates ACh synthesized in the cytoplasm by an active transport process which depends on a Ca2+-or a Mg2+ATPase system driving the uptake of ACh when linked to an internally acidic proton gradient (Anderson et al., 1982, 1983). If this ACh transport system maintains its orientation following the opening of the vesicle into the synaptic cleft or the incorporation of the vesicle membrane into the terminal membrane, it would move ACh from the axoplasm into the extracellular space and could thereby account for a continuous ATPasedependent secretion of ACh. Indeed, Edwards et al. (1985) observed that drugs which block this transport system (AH 5183 and quinacrine) also blocked the H-response and reduced the release of ACh. Similarly an alkaline pH (9.4) completely blocked the H-response, as might be expected if the ACh transport depended upon a pH gradient. Botulinum toxin type A which, as previously mentioned, blocks Ca2'sensitive, quantal discharge of ACh, also reduces, by about 40%, the continuous leakage of ACh from nerve terminals (Polak et al., 1981; Vyskocil et al., 1983; Dolezal et al., 1983). This effect occurs within minutes of toxin application to the nerve muscle preparation and therefore possibly reflects a mode of action different from its blocking effect on quantal transmitter discharge (Vyskocil el al., 1983). The results from several studies (Miledi et al., 1982a, b, 1983; Dolezal and Tucek, 1983) of ACh produced and released from muscle indicate that nonneural ACh is synthesized in the cytoplasm of the muscle, possibly by the enzyme, carnitine acetyltransferase (EC 2.3.1.7.) (Tucek, 1982), while the neural enzyme is choline acetyltransferase (EC 2.3.1.6.). The way the formed ACh leaves the muscle is not known. It might, however, be mentioned that endocytic and therefore presumably also exocytic activity is quite marked in muscle tissue (Thesleff et al., 1979; TPgerud and Libelius, 1985). Consequently, it seems possible that exocytic membrane vesicles might deliver ACh, located in the cytoplasm, to the extracellular fluid.

PHYSIOLOGICAL ROLE As already stated, the continuous secretion of ACh represents by far the largest portion of ACh released under resting conditions. Even if onethird to one-half of this leakage originates from the muscle cell, it is tempting to assume that the release from the nerve has a physiological function. In that context, it is interesting that Po0 (1984) and Sun and Po0 (1985) have demonstrated that growth cones of cholinergic neurons in

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culture have this mechanism of transmitter secretion. Furthermore Po0 et al. (1978, 1979), Po0 and Robinson (1977),and Lin-Liu et al. (1984) have demonstrated that a uniform electric field across the surface of an embryonic muscle cell results in the asymmetric accumulation of lectin and ACh-receptor proteins toward the cathodal pole of the cell. The accumulation of ACh receptors by the electric field causes the formation in the membrane of stable, localized receptor aggregates. The field strength required to induce a detectable receptor accumulation was between 1 and 1.5 Vlcm, corresponding to a voltage difference of 2-3 mV across the cell membrane. Thus, it is possible, as suggested by Po0 (1985), that the local continuous depolarization of the end-plate area in a muscle fiber by leakage of ACh generates an electric field of sufficient strength to cause the aggregation and immobilization of nicotinic-cholinergic receptors in the area of contact and that this mechanism might assist in the development of the postsynaptic receptor accumulation typical of an end plate. Ziskind-Conhaim el al. (1984) have demonstrated that ACh-receptor clusters at developing end plates arise from receptors that previously were uniformly distributed on the muscle surface. It might also be mentioned that the membrane proteins which constitute the Na' channel are present at a greater density at the end-plate region than in other parts of the muscle membrane (Thesleff et al., 1974; Betz et al., 1984). Other neurotrophic factors are, however, also likely to be involved (see a review by Fischbach et al., 1979). Botulinum toxin poisoning induces, in skeletal muscle, changes similar to those seen following denervation, i.e., chemical supersensitivity, fall of resting membrane potential, fibrillation potentials, and the development of tetrodotoxin-resistant action potentials (Thesleff, 1960;Josefsson and Thesleff, 1961; Mathers and Thesleff, 1978),but these changes are quantitatively less marked than those seen after surgical denervation. A subsequent blockade of cholinergic receptors by curare or a-bungarotoxin brings the denervation changes to the same level as those resulting from surgical denervation (Pestronk et al., 1976; Mathers and Thesleff, 1978; Drachman et al., 1982). On that basis these authors concluded that the motor nerve, even when transmission is paralyzed by botulinum toxin, exerts a trophic influence on the muscle and that this influence is mediated by ACh. In botulinum toxin-paralyzed muscles ACh is released intermittently in a Ca2+-insensitivemanner and also continuously a molecular leakage, as previously explained. It seems possible that these releases of ACh might have a trophic influence, not only on botulinum-poisoned muscles, but also on normal muscle (Thesleff and Sellin, 1980; Bray et al., 1982; Card Linden et al., 1983; McArdle, 1983). We have no knowledge of the functional role of nonneural ACh re-

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leased from the muscle. It has been suggested that it might participate in the control of the Na+ ,K+-ATPase of muscle fibers or play a role in the control of the metabolism of membrane phospholipids (for further details and references see Dolezal et aE., 1983; Dolezal and Tucek, 1983).

111. Comments

T h e aim of this presentation has been to show that a nerve may use the same chemical substance ACh as a transmitter of different kinds of messages. ACh is used not only as a transmitter of the nerve impulse across the synaptic cleft and thereby for motor control of the muscle, but presumably also as a messenger for other types of information. Release of ACh might serve as a signal which helps the axon to find its proper target, to establish the synapse, and to maintain its target cell (muscle) in an optimal-functional state (neurotrophism). ACh seems to be involved in all these functions, although the release mechanism for the substance may vary. A weakness of these speculations is that we do not understand the mechanisms by which ACh could exert such different actions on muscle. Are second messengers, such as Ca2+,calcium-mobilizing polyphosphoinositides, or cyclic nucleotides, involved in the transfer of information from the cholinergic membrane receptor to metabolic and catabolic regulating centers of the muscle cell? Perhaps the mechanisms are similar to those involved in the regulation of muscle metabolism by mechanical activity. Studies of acute denervation changes in skeletal muscle indicate that several mechanisms and factors participate and interact as neurotrophic influences (see reviews by Thesleff and Sellin, 1980; McArdle, 1983). The mechanisms of the different types of ACh release are also insufficiently understood. To what extent and how are synaptic vesicles involved, and is it possible that there are common steps in different release mechanisms? For instance could there be a relation between the described intermittent, Ca2+-insensitivesecretion of ACh and synaptic vesicles and possibly also with the spontaneous continuous efflux of ACh? Maybe it is the site of release, presynaptic active zones, or remaining axolemma, that determines the Ca2 sensitivity of the discharge process? Similarly, little is known about the biochemical steps which initiate ACh discharge. In fact, so little is known that it discourages speculations about the molecular mechanisms underlying different kinds of ACh secretion. Despite the above, it might be worthwhile to pursue certain ideas regarding possible mechanisms involved. Greengard and co-workers, ex+

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cellently reviewed by Nestler and Greengard (1984), consider phosphorylation of a specific synaptic vesicle protein (synapsin I) as a priming step in the vesicle fusion-ACh release process. Synapsin I phosphorylation is proposed to be regulated both by a calcium-calmodulin-dependent protein kinase and by a cyclic AMP-dependent kinase present in nerve terminals. Synapsin I is a protein closely associated with synaptic vesicles and, when phosphorylated, it is detached from the vesicle membrane. As hypothesized by Nestler and Greengard (1984), dephosphosynapsin I is the active form of the molecule and acts to hinder the association of neurotransmitter vesicles with the plasma membrane. Phosphorylation of synapsin I would therefore allow vesicle aggregation and fusion with the nerve terminal membrane. As previously explained (Fig. l), calcium-sensitive, intermittent secretion of ACh (I) is believed to originate from the active zones in nerve terminals while the calcium-insensitive type of intermittent secretion (11) might originate from areas outside of these specialized parts of the nerve terminal membrane. If voltage-dependent Ca2+channels are preferentially located at active zones, as believed, Ca'+ would primarily enter the terminal at such sites during stimulation and nerve terminal depolarization. Hence, synapsin I phosphorylation by a Ca2+-calmodulin-dependentkinase would be most prominent at those sites and thereby account for a preferential release of ACh from this part of the terminal. Changes in the level of cyclic AMP or another second messenger concentration in the terminal are presumably generalized and not located to specific areas. Therefore, protein kinases dependent on such messengers can be expected to phosphorylate synapsin I and thereby induce synaptic vesicle fusion and ACh release from all parts of the terminal. If accompanied by vesicle aggregation, giant, slow-risingmepps would result, as observed for the Ca2+-insensitive,but perhaps cyclic AMP-modulated, type of intermittent ACh release. Maybe a cyclic nucleotide-regulated secretion of ACh represents an embryonic, more primitive type of transmitter release which is followed by a Ca2+-regulated mechanism once active zones with functional calcium channels develop in the terminal. Skeletal muscle has been shown to contain a protein kinase inhibitor specific for cyclic AMP-dependent pro1971).The retrograde transfer of such an inhibitor tein kinase (Walsh et d., from muscle to nerve as a result of neuromuscular transmission could explain the variability of this type of release between fibers and muscles and why cyclic AMP-dependent ACh release, to a large extent, is inhibited in synapses with intact transmission and its gradual reappearance and disappearance once transmission is respectively blocked and reestablished.

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One might also speculate that regulation of synapsin I phosphorylation by the cyclic AMP-dependent protein kinase present in nerve terminals is involved in an embryonic presynaptic chemoreceptor-regulated control of transmitter release. At present all these suggestions, however, are speculations, and this presentation raises far more questions than it answers. Acknowledgments 1 am grateful to Dr. E. Heilbronn, Stockholm, J. Molgo, Paris, and F. Vyskocil, Prague, for their valuable comments on the manuscript. References

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