Ultrafast and Slow Cholinergic Transmission. Different Involvement of Acetylcholinesterase Molecular Forms.

Acetylcholine (ACh), an ubiquitous mediator substance broadly expressed in nature, acts as neurotransmitter in cholinergic synapses, generating specific communications with different time-courses. (1) Ultrafast transmission. Vertebrate neuromuscular junctions (NMJs) and nerve-electroplaque junctions (NEJs) are the fastest cholinergic synapses; able to transmit brief impulses (1-4 ms) at high frequencies. The collagen-tailed A12 acetylcholinesterase is concentrated in the synaptic cleft of NMJs and NEJs, were it curtails the postsynaptic response by ultrafast ACh hydrolysis. Here, additional processes contribute to make transmission so rapid. (2) Rapid transmission. At peripheral and central cholinergic neuro-neuronal synapses, transmission involves an initial, relatively rapid (10-50 ms) nicotinic response, followed by various muscarinic or nicotinic effects. Acetylcholinesterase (AChE) being not concentrated within these synapses, it does not curtail the initial rapid response. In contrast, the late responses are controlled by a globular form of AChE (mainly G4-AChE), which is membrane-bound and/or secreted. (3) SlowAChsignalling. In non-neuronal systems, in muscarinic domains, and in most regions of the central nervous system (CNS), many ACh-releasing structures (cells, axon terminals, varicosities, boutons) do not form true synaptic contacts, most muscarinic and also part of nicotinic receptors are extra-synaptic, often situated relatively far from ACh releasing spots. A12-AChE being virtually absent in CNS, G4-AChE is the most abundant form, whose function appears to modulate the "volume" transmission, keeping ACh concentration within limits in time and space.

Presynaptic K(+) channels, vesicular Ca(2+)/H (+) antiport--synaptotagmin, and acetylcholinesterase, three mechanisms cutting short the cholinergic signal at neuromuscular and nerve-electroplaque junctions.

In neuromuscular and nerve-electroplaque junctions, nerve impulses can be transmitted at high frequencies. This implies that transmission of individual impulses must be very brief. We describe three mechanisms which curtail the time course of individual impulses at these synapses: (1) opening of presynaptic K(+) channels (delayed rectifier) efficiently curtails the presynaptic action potential. Inhibition of K(+) channel by aminopyridines transforms the normally brief postsynaptic potential (2-3 ms) to a long-lasting "giant" potential (exceeding half a second); (2) a low-affinity Ca(2+)/H(+) antiport ensures rapid Ca(2+) sequestration into synaptic vesicles, curtailing the calcium signal and thereby the duration of transmitter release. Indeed vesicular Ca(2+)/H(+) antiport inhibition by bafilomycin or Sr(2+) prolongs the duration of the postsynaptic potential. We recently showed that synaptotagmin-1 is required for this antiport activity; thus the vesicular Ca(2+)/H(+) antiport might be synaptotagmin itself, or regulated by it; and (3) it is recalled that, in these junctions, acetylcholinesterase is highly concentrated in the synaptic cleft and that anticholinesterases lengthen the endplate time course. Therefore, at three different steps of synaptic transmission, an efficient mechanism curtails the local synaptic signal. When one of these three mechanisms is inhibited, the duration of individual impulses is prolonged, but the synapse loses its faculty to fire at high frequencies.

Synaptotagmin 1 is required for vesicular Ca²âº/H⁺-antiport activity.

A low-affinity Ca²âº/H⁺-antiport was described in the membrane of mammalian brain synaptic vesicles. Electrophysiological studies showed that this antiport contributes to the extreme brevity of excitation-release coupling in rapid synapses. Synaptotagmin-1, a vesicular protein interacting with membranes upon low-affinity Ca²âº-binding, plays a major role in excitation-release coupling, by synchronizing calcium entry with fast neurotransmitter release. Here, we report that synaptotagmin-1 is necessary for expression of the vesicular Ca²âº/H⁺-antiport. We measured Ca²âº/H⁺-antiport activity in vesicles and granules of pheochromocytoma PC12 cells by three methods: (i) Ca²âº-induced dissipation of the vesicular H⁺-gradient; (ii) bafilomycin-sensitive calcium accumulation and (iii) pH-jump-induced calcium accumulation. The results were congruent and highly significant: Ca²âº/H⁺-antiport activity is detectable only in acidic organelles expressing functional synaptotagmin-1. In contrast, synaptotagmin-1-deficient cells--and cells where transgenically encoded synaptotagmin-1 was acutely photo-inactivated--were devoid of any Ca²âº/H⁺-antiport activity. Therefore, in addition to its previously described functions, synaptotagmin-1 is involved in a rapid vesicular Ca²âº sequestration through a Ca²âº/H⁺ antiport.

Synaptic vesicles control the time course of neurotransmitter secretion via a Ca²+/H+ antiport.

We investigated the physiological role of the vesicular Ca2+/H+ antiport in rapid synaptic transmission using the Torpedo electric organ (a modified neuromuscular system). By inhibiting V-type H+-transporting ATPase (V-ATPase), bafilomycin A1 dissipates the H+ gradient of synaptic vesicles, thereby abolishing the Ca2+/H+ antiport driving force. In electrophysiology experiments, bafilomycin A1 significantly prolonged the duration of the evoked electroplaque potential. A biochemical assay for acetylcholine (ACh) release showed that the effect of bafilomycin A1 was presynaptic. Indeed, bafilomycin A1 increased the amount of radio-labelled ACh released in response to paired-pulse stimulation. Bafilomycin A1 also enhanced Ca2+-dependent ACh release from isolated nerve terminals (synaptosomes). The bafilomycin-induced electroplaque potential lengthening did not arise from cholinesterase inhibition, since eserine (which also prolonged the electroplaque potential) strongly decreased evoked ACh release. Bafilomycin A1 augmented the amount of calcium accumulating in nerve terminals following a short tetanic stimulation and delayed subsequent calcium extrusion. By reducing stimulation-dependent calcium accumulation in synaptic vesicles, bafilomycin A1 diminished the corresponding depletion of vesicular ACh, as tested using both intact tissue and isolated synaptic vesicles. Strontium ions inhibit the vesicular Ca2+/H+ antiport, while activating transmitter release at concentrations one order of magnitude higher than Ca2+ does. In the presence of Sr2+ the time course of the electroplaque potential was also prolonged but, unlike bafilomycin A1, Sr2+ enhanced facilitation in paired-pulse experiments. It is therefore proposed that the vesicular Ca2+/H+ antiport function is to shorten 'phasic' transmitter release, allowing the synapse to transmit briefer impulses and so to work at higher frequencies.

Ultra-fast versus sustained cholinergic transmission: a variety of different mechanisms.

Although synaptic transmission was assumed to use the same mechanisms in the case of different synapses of the central and peripheral nervous system, recent research revealed a great variety of different processes. Time might be a crucial factor to be considered in this diversity. It is recalled that the speed of a chemical reaction is inversely related to affinity. "Time is gained at the expense of sensitivity" as noticed by Bernard Katz (1989). Therefore, synaptic transmission will occur at a high speed only if it is supported by low affinity reactions. In the present work, we compare two examples of ultra-rapid transmission (the Torpedo nerve electroplaque synapse and the rat hippocampus mossy fiber/CA3 synapses), with a cholinergic process operating with high affinity but at a low speed: the release of glutamate elicited by nicotine from mossy fibers of the rat hippocampus.

Nicotine-induced and depolarisation-induced glutamate release from hippocampus mossy fibre synaptosomes: two distinct mechanisms.

Hippocampus mossy fibre terminals activate CA3 pyramidal neurons via two distinct mechanisms, both quantal and glutamatergic: (i) rapid excitatory transmission in response to afferent action potentials and (ii) delayed and prolonged release following nicotinic receptor activation. These processes were analysed here using rat hippocampus mossy fibres synaptosomes. The relationships between synaptosome depolarisation and glutamate release were established in response to high-KCl and gramicidin challenges. Half-maximal release corresponded to a 52 mV depolarisation step. KCl-induced release was accompanied by transient dissipation of the proton gradient across synaptic vesicle membrane. Nicotine elicited a substantial glutamate release from mossy fibre synaptosomes (EC(50) 3.14 microM; V(max) 12.01 +/- 2.1 nmol glutamate/mg protein; Hill's coefficient 0.99). However, nicotine-induced glutamate release was not accompanied by any change in the membrane potential or in the vesicular proton gradient. The effects of acetylcholine (200 microM) were similar to those of nicotine (25 microM). Nicotinic alpha7 receptors were evidenced by immuno-cytochemistry on the mossy fibre synaptosome plasma membrane. Therefore, the same terminals can release glutamate in response to two distinct stimuli: (i) rapid neurotransmission involving depolarisation-induced activation of voltage-gated Ca(2+) channels and (ii) a slower nicotinic activation which does not involve depolarisation or dissipation of the vesicular proton gradient.

Exocytosis, mediatophore, and vesicular Ca2+/H+ antiport in rapid neurotransmission.

In rapid synapses, neurotransmitter quanta are emitted in less than 100 mus, often at a high frequency. Using fast cryofixation of synapses, we found a very brief (2-3 ms) change affecting intramembrane particles in presynaptic membrane. Vesicle openings also occurred but after a significant delay. The particle change is most probably linked to mediatophore, a proteolipid of 220 kDa. Mediatophore aggregates were demonstrated in active zones of the presynaptic membrane. Reconstituted in liposomes, Xenopus oocytes, and neuroblastoma cells, mediatophore releases acetylcholine in a Ca(2+)-dependent and quantal manner, mimicking physiological release. In restricted presynaptic "nanodomains," Ca(2+) concentration explosively reaches a high level and then vanishes with a time constant of 300-400 micros. Among the processes contributing to the fast phase of Ca(2+) buffering, a vesicular Ca(2+)/H(+) antiport plays a major role. Energized by the Vesicular-ATPase-dependent proton gradient, the antiport has a low affinity for Ca(2+). We inactivated the Ca(2+)/H(+) antiport using bafilomycin A1, which annihilates the proton gradient. As a result, the postsynaptic potential was increased in duration for about 3 ms, an effect caused by persistence of transmitter release. A similar change was obtained by replacing extracellular Ca(2+) by strontium, which inhibits the antiport. The antiport function, therefore, is to abbreviate the presynaptic Ca(2+) signal, making transmitter release briefer. This allows transmission to operate at high frequency. Following a brief period of stimulation, calcium transiently accumulates in synaptic vesicles where it is exchanged against transmitter. Calcium is subsequently cleared from the terminal, most probably by exocytosis.

Acetylcholine release and choline uptake by cuttlefish (Sepia officinalis) optic lobe synaptosomes.

Acetylcholine (ACh), which is synthesized from choline (Ch), is believed to hold a central place in signaling mechanisms within the central nervous system (CNS) of cuttlefish (Sepia officinalis) and other coleoid cephalopods. Although the main elements required for cholinergic function have been identified in cephalopods, the transmembrane translocation events promoting the release of ACh and the uptake of Ch remain largely unsolved. The ACh release and Ch uptake were quantitatively studied through the use of in vitro chemiluminescence and isotopic methods on a subcellular fraction enriched in synaptic nerve endings (synaptosomes) isolated from cuttlefish optic lobe. The ACh release evoked by K+ depolarization was found to be very high (0.04 pmol ACh.s(-1).mg(-1) protein). In response to stimulation by veratridine, a secretagogue (a substance that induces secretion) that targets voltage-gated Na+ channels, the release rate and the total amount of ACh released were significantly lower, by 10-fold, than the response induced by KCl. The high-affinity uptake of choline was also very high (31 pmol Ch.min(-1).mg(-1) protein). The observed ACh release and Ch uptake patterns are in good agreement with published data on preparations characterized by high levels of ACh metabolism, adding further evidence that ACh acts as a neurotransmitter in cuttlefish optic lobe.

Comparative effects of aluminum and ouabain on synaptosomal choline uptake, acetylcholine release and (Na+/K+)ATPase.

Closing the gap between adverse health effects of aluminum and its mechanisms of action still represents a huge challenge. Cholinergic dysfunction has been implicated in neuronal injury induced by aluminum. Previously reported data also indicate that in vivo and in vitro exposure to aluminum inhibits the mammalian (Na(+)/K(+))ATPase, an ubiquitous plasma membrane pump. This study was undertaken with the specific aim of determining whether in vitro exposure to AlCl(3) and ouabain, the foremost utilized selective inhibitor of (Na(+)/K(+))ATPase, induce similar functional modifications of cholinergic presynaptic nerve terminals, by comparing their effects on choline uptake, acetylcholine release and (Na(+)/K(+))ATPase activity, on subcellular fractions enriched in synaptic nerve endings isolated from rat brain, cuttlefish optic lobe and torpedo electric organ. Results obtained show that choline uptake by rat synaptosomes was inhibited by submillimolar AlCl(3), whereas the amount of choline taken up by synaptosomes isolated from cuttlefish and torpedo remained unchanged. Conversely, choline uptake was reduced by ouabain to a large extent in all synaptosomal preparations analyzed. In contrast to ouabain, which modified the K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions, AlCl(3) induced reduction of stimulated acetylcholine release was only observed when rat synaptosomes were challenged. Finally, it was observed that the aluminum effect on cuttlefish and torpedo synaptosomal (Na(+)/K(+))ATPase activity was slight when compared to its inhibitory action on mammalian (Na(+)/K(+))ATPase. In conclusion, inhibition of (Na(+)/K(+))ATPase by AlCl(3) and ouabain jeopardized the high-affinity (Na(+)-dependent, hemicholinium-3 sensitive) uptake of choline and the Ca(2+)-dependent, K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions. The effects of submillimolar AlCl(3) on choline uptake and acetylcholine release only resembled those of ouabain when rat synaptosomes were assayed. Therefore, important differences were found between the species regarding the cholinotoxic action of aluminum. The variability of (Na(+)/K(+))ATPase sensitivity to aluminum of cholinergic neurons might contribute to their differential susceptibility to this neurotoxic agent.

Vesicular calcium transport shapes rapid acetylcholine secretion.

Rapid secretion relies on the occurrence of spike-like Ca2+ transients in active zones (Llinás et al., 1992; Yazejian et al., 2000; Dunant and Bloc, 2003). Presynaptic Ca2+ nanodomains are to be restricted both in time and in space as to assure rapid onset and termination of transmitter release (Llinás et al., 1992; Pozzan et al., 1994; Yazejian et al., 2000; Dunant and Bloc, 2003). A very fast Ca2+-buffering mechanism should allow Ca2+ rise above approximately 100 microM for less than approximately 250 micros and then rapid reduction of Ca2+ to subthreshold levels of release (Llinás et al., 1992; Pozzan et al., 1994; Yazejian et al., 2000; Dunant and Bloc, 2003). Swift Ca2+ clearance by vesicular Ca2+/H+ antiport as a low-affinity, high-capacity extrusion mechanism was postulated in the past (Pozzan et al., 1994; Dunant and Bloc, 2003). We demonstrated pH gradient (DeltapH)-dependent Ca2+ uptake by mammalian brain synaptic vesicles (Gonçalves et al., 1998, 2000). Moreover, this antiport activity is effective at [Ca2+] ranging from approximately 100 to 800 microM (max. at approximately 500 microM) (Gonçalves et al., 1998, 2000). We now show that the time course of acetylcholine (ACh) secretion in Torpedo neuroelectrocytic synapse is modified by bafilomycin A1 (baf.), which compromises antiport activity. Along with this mechanism, synaptic vesicles also have a P-type Ca2+ ATPase, exhibiting half-maximal activation for 0.6 microM Ca2+ (Gonçalves et al., 2000). Here, we demonstrate the role of P-type Ca2+ ATPase in preventing desensitization of the release mechanism by inhibiting it with orthovanadate.

Acetylcholine release in rapid synapses: two fast partners--mediatophore and vesicular Ca2+/H+ antiport.

Rapid neurotransmission is like lightning: a spark of calcium in the nerve terminal, a spark of transmitter in the cleft, and the signal is over. But "time is gained at the expense of sensitivity" (Katz, 1988); transmission relies on low-affinity, high-speed reactions. These fast processes are modulated by regulating reactions that do not need to be so rapid.

Two SUR1-specific histidine residues mandatory for zinc-induced activation of the rat KATP channel.

Zinc at micromolar concentrations hyperpolarizes rat pancreatic beta-cells and brain nerve terminals by activating ATP-sensitive potassium channels (KATP). The molecular determinants of this effect were analyzed using insulinoma cell lines and cells transfected with either wild type or mutated KATP subunits. Zinc activated KATP in cells co-expressing rat Kir6.2 and SUR1 subunits, as in insulinoma cell lines. In contrast, zinc exerted an inhibitory action on SUR2A-containing cells. Therefore, SUR1 expression is required for the activating action of zinc, which also depended on extracellular pH and was blocked by diethyl pyrocarbonate, suggesting histidine involvement. The five SUR1-specific extracellular histidine residues were submitted to site-directed mutagenesis. Of them, two histidines (His-326 and His-332) were found to be critical for the activation of KATP by zinc, as confirmed by the double mutation H326A/H332A. In conclusion, zinc activates KATP by binding itself to extracellular His-326 and His-332 of the SUR1 subunit. Thereby zinc could exert a negative control on cell excitability and secretion process of pancreatic beta-and alpha-cells. In fact, we have recently shown that such a mechanism occurs in hippocampal mossy fibers, a brain region characterized, like the pancreas, by an important accumulation of zinc and a high density of SUR1-containing KATP.

Zinc inhibits glutamate release via activation of pre-synaptic K channels and reduces ischaemic damage in rat hippocampus.

Zinc is concentrated in certain CNS excitatory tracts, especially in hippocampal mossy fibres where it has been suggested to modulate synaptic transmission and plasticity. Using rat mossy fibre synaptosomes depolarized by 4-aminopyridine, we show here that low zinc concentrations restore the membrane potential and reduce glutamate release. Both effects arose from activation of ATP-sensitive potassium channels (KATP), since they were mimicked by the KATP opener diazoxide and antagonized by the KATP blocker tolbutamide. Using recombinant channels expressed in COS-7 cells, we confirmed that micromolar zinc did activate KATP of the type found in hippocampus. We tested the hypothesis that this action of zinc could be beneficial during an ischaemic challenge by using organotypic hippocampal slice cultures. When zinc was applied at micromolar concentrations during a brief anoxic-hypoglycaemic episode, it significantly attenuated the ensuing neuronal death, whereas chelation of endogenous zinc markedly aggravated cell damage. Protective effect of zinc was mediated through KATP, as was shown by using the opener diazoxide and the blocker tolbutamide. Thus, by activating pre-synaptic KATP channels, zinc protects neurones from hyper-excitation, excessive transmitter release and exitotoxicity, and may thus act as an endogenous neuroprotector in conditions such as epilepsy or stroke.

Low- and high-affinity reactions in rapid neurotransmission.

Until 1950-1960, most physiologists were reluctant to accept chemical neurotransmission. They believed that chemical reactions were much too slow to account for the speed of synaptic processes. The first breakthrough was to discover the impressive velocity of acetylcholinesterase. Then, nicotinic receptors provided an example of complex ultrarapid reactions: fast activation at a low ligand affinity, then desensitization if the ligand is not rapidly removed. Here, we describe synaptic transmission as a chain of low-affinity rapid reactions, assisted by many slower regulatory processes. For starting quantal acetylcholine release, mediatophores are activated by high Ca2+ concentrations, but they desensitize at a higher affinity if Ca2+ remains present. Several mechanisms concur to the rapid removal of Ca2+ from the submembrane microdomains. Among them, the Ca2+/H+ antiport is a typical low-affinity, high-speed process that certainly contributes to the rapidity of neurotransmission.

Tumor necrosis factor-alpha and angiostatin are mediators of endothelial cytotoxicity in bronchoalveolar lavages of patients with acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is characterized by an extensive alveolar capillary leak, permitting contact between intra-alveolar factors and the endothelium. To investigate whether factors contained in the alveolar milieu induce cell death in human lung microvascular endothelial cells, we exposed these cells in vitro to bronchoalveolar lavage fluid (BALF) supernatants from control patients, patients at risk of developing ARDS, and patients with early- and late-phase ARDS. In contrast to BALF from control patients, a significant cytotoxicity was found in BALF from patients at risk of developing ARDS, with late-phase ARDS, and especially from patients with early-phase ARDS. Subsequently, we determined the levels of factors known to exert cytotoxicity in endothelial cells, i.e., tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, and angiostatin. BALF from patients at risk of developing ARDS, with early-phase ARDS, and with late-phase ARDS, contained increased levels of TNF-alpha and angiostatin, but not of TGF-beta1, as compared with BALF from control patients. Whereas inhibition of TGF-beta1 had no effect in this setting, neutralization of TNF-alpha or angiostatin inhibited the cytotoxic activity on endothelial cells of part of the early-phase ARDS BALF. These results indicate that TNF-alpha and angiostatin may contribute to ARDS-related endothelial injury.

An invertebrate defense molecule activates membrane conductance in mammalian cells by means of its lectin-like domain.

The invertebrate defense molecule Coelomic Cytolytic Factor-1 (CCF-1) and the mammalian cytokine Tumor Necrosis Factor (TNF) share a similar lectin-like domain that, upon interaction with specific sugars, causes lysis of African trypanosomes. In contrast to TNF, CCF-1 does not require an acidification of a lysosomal compartment for this activity. Moreover, we could demonstrate using the whole cell patch clamp technique that both TNF and CCF-1 activate amiloride-sensitive channels in mammalian cells, in a TNF receptor-independent way, but, unlike TNF, CCF-1 does not require acidic conditions for this activity. These data confirm the functional analogies of an invertebrate defense molecule and a mammalian cytokine, based on a similar lectin-like interaction.