Quantal acetylcholine release induced by mediatophore transfection

Mediatophore is a protein of approximately 200 kDa able to translocate acetylcholine in response to calcium. It was purified from the presynaptic plasma membranes of the electric organ nerve terminals. Mediatophore is a homooligomer of a 16-kDa subunit, homologous to the proteolipid of V-ATPase. Cells of the N18TG-2 neuronal line are not able to produce quantal acetylcholine release. We show here that transfection of N18TG-2 cells with a plasmid encoding the mediatophore subunit restored calcium-dependent release. The essential feature of such a release was its quantal nature, similar to what is observed in situ in cholinergic synapses from which mediatophore was purified.     

L-3,4-dihydroxyphenylalanine increases the quantal size of exocytotic dopamine release in vitro

The catecholamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) is used to augment striatal dopamine (DA), although its mechanism of altering neurotransmission is not well understood. We observed the effects of L-DOPA on catecholamine release in ventral midbrain neuron and PC12 pheochromocytoma cell line cultures. In ventral midbrain neuron cultures exposed to 40 mM potassium-containing media, L-DOPA (100 microM for 1 h) increased DA release by > 10-fold. The elevated extracellular DA levels were not significantly blocked by the DA/norepinephrine transport inhibitor nomifensine, demonstrating that reverse transport through catecholamine-uptake carriers plays little role in this release. In PC12 cells, where DA release from individual secretory vesicles can be observed, L-DOPA (50 microM for 1 h) elevated DA release in high-potassium media by 370%. Amperometric measurements demonstrated that L-DOPA (50 microM for 40-70 min) did not raise the frequency of vesicular exocytosis but increased the average size of quantal release to at least 250% of control levels. Together, these findings suggest that L-DOPA can increase stimulation-dependent transmitter release from DA cells by augmenting cytosolic neurotransmitter, leading to increased quantal size.     

Convulsant-anticonvulsant interactions on seizure activity and cortical acetylcholine release

The effects of leptazol and bicuculline on the efflux of endogenous acetylcholine (ACh) from the surface of the cerebral cortex have been related to EEG activity in urethane-anaesthetised rats. During seizure activity there was a calcium dependent increase in ACh efflux which was related to increase EEG activity and clonic muscle movements. ACh release and EEG activity were reduced during convulsive activity by trimethadione but not phenytoin. Phenobarbitone reduced convulsive EEG activity but left ACh release relatively unaffected. Blood pressure changes induced by convulsant and anticonvulsant drugs were not consistently related to EEG activity or ACh release. It is suggested that ACh efflux from the cerebral cortex is closely related to the activity of neurones within the cortex where it is released from nerve endings. Comparison of EEG changes induced by anticonvulsants and urethane during control and convulsant activity showed that only trimethadione produces anticonvulsant activity unaccompanied by general CNS depression.     

Nerve terminal currents induced by autoreception of acetylcholine release

The activation of autoreceptors is known to be important in the modulation of presynaptic transmitter secretion in peripheral and central neurons. Using whole-cell recordings made from the free growth cone of myocyte-contact motoneurons of Xenopus cell cultures, we have observed spontaneous nerve terminal currents (NTCs). These spontaneous NTCs are blocked by d-tubocurarine (d-TC) and alpha-bungarotoxin (alpha-BuTx), indicating that endogenously released acetylcholine (ACh) can produce substantial membrane depolarization in the nerve terminals. Local application of NMDA to the growth cone increased the frequency of spontaneous NTCs. When the electrical stimulations were applied at the soma to initiate evoked-release of ACh, evoked ACh-induced potentials were recorded in the nerve terminals, which were inhibited by d-TC and hexamethonium but not by atropine. Replacement of normal Ringer's solution with high-Mg2+, low-Ca2+ solution also reversibly inhibited evoked ACh-induced potentials. The possible regulatory role of presynaptic nicotinic autoreceptors on the synaptic transmission was also examined. When the innervated myocyte was whole-cell voltage-clamped to record synaptic currents, application of hexamethonium inhibited the amplitude of evoked synaptic currents at a higher degree than that of iontophoretic ACh-induced currents. Furthermore, hexamethonium markedly reduced the frequency of spontaneous synaptic currents at high-activity synapses. Pretreatment of neurons with alpha-BuTx also inhibited the evoked synaptic currents in manipulated synapses. These results suggest that ACh released spontaneously or by electrical stimulation may act on the presynaptic nicotinic autoreceptors of the same nerve terminals to produce membrane potential change and to regulate synaptic transmission.     

Imaging of quantal calcium release in the inositol 1,4,5-trisphosphate-sensitive organelles of permeabilized HSY cells

Twenty-nine cases of histiocytic neoplasms, some resembling juvenile xanthogranuloma (JXG) and others resembling reticulohistiocytoma (RH), were evaluated. Immunohistochemical stains were performed. In this series, seven cases were identified that expressed S-100 protein positive cells. The S-100 positive cells were predominantly large mononuclear and multinucleated histiocytes with eosinophilic cytoplasm, but also in some cases xanthomatous cells and Touton giant cells. These cells also expressed a positive reaction for vimentin, KP-1, and Factor XIIIa. There was no reactivity observed for monoclonal antibody 010(CD1a). A positive reaction for S-100 protein is conventionally accepted as a useful differentiating feature between histiocytosis X and non-X histiocytosis such as JXG and RH. The conflicting results of the immunohistochemical stains in the lesions we studied could be potential pitfalls in diagnosing histiocytic neoplasms.     

Galparan induces in vivo acetylcholine release in the frontal cortex

The chimeric peptide galparan (galanin(1-13)-mastoparan) induced the in vivo release of acetylcholine in the frontal cortex of rats when injected intracerebroventricularly, i.c.v. The ACh-releasing effects of galparan are reversible, dose-dependent, and not exerted at galanin receptors or at sites where mastoparan acts. Pertussis toxin pretreatment (i.c.v.) of the rats for 96 h prior to injection of galparan or of mastoparan completely prevented the ACh-releasing effects of both galparan and mastoparan. It appears that galparan acts at a novel site in the release of ACh in the cerebral cortex in vivo.     

Differential acetylcholine and GABA release from cultured chick retina cells

In the present work we investigated the mechanisms controlling the release of acetylcholine (ACh) and of gamma-aminobutyric acid (GABA) from cultures of amacrine-like neurons, containing a subpopulation of cells which are simultaneously GABAergic and cholinergic. We found that 81.2 +/- 2.8% of the cells present in the culture were stained immunocytochemically with an antibody against choline acetyltransferase, and 38.5 +/- 4.8% of the cells were stained with an antibody against GABA. Most of the cells containing GABA (87.0 +/- 2.9%) were cholinergic. The release of acetylcholine and GABA was mostly Ca2+-dependent, although a significant release of [3H]GABA occurred by reversal of its transporter. Potassium evoked the Ca2+-dependent release of [3H]GABA and [3H]acetylcholine, with EC50 of 31.0 +/- 1.0 mm and 21.6 +/- 1.1 mm, respectively. The Ca2+-dependent release of [3H]acetylcholine was significantly inhibited by 1 micrometer tetrodotoxin and by low (30 nm) omega-conotoxin GVIA (omega-CgTx GVIA) concentrations, or by high (300 nm) nitrendipine (Nit) concentrations. On the contrary, the release of [14C]GABA was reduced by 30 nm nitrendipine, or by 500 nm omega-CgTx GVIA, but not by this toxin at 30 nm. The release of either transmitters was unaffected by 200 nm omega-Agatoxin IVA (omega-Aga IVA), a toxin that blocks P/Q-type voltage-sensitive Ca2+ channels (VSCC). The results show that Ca2+-influx through omega-CgTx GVIA-sensitive N-type VSCC and through Nit-sensitive L-type VSCC induce the release of ACh and GABA. However, the significant differences observed regarding the Ca2+ channels involved in the release of each neurotransmitter suggest that in amacrine-like neurons containing simultaneously GABA and acetylcholine the two neurotransmitters may be released in distinct regions of the cells, endowed with different populations of VSCC.     

Serotonergic modulation of acetylcholine release from cortex of freely moving rats

The modulation of acetylcholine (ACh) release by 5-HT3 receptor activation was studied using in vivo microdialysis. Spontaneous and K+-stimulated ACh release were measured in frontoparietal cortex and hippocampus of freely moving rats. Two consecutive exposures to high K+ produced ACh release of similar magnitude. In the cortex, serotonin (5-HT) failed to alter spontaneous ACh release, but caused a concentration-dependent decrease of K+-evoked ACh release. Phenylbiguanide (PBG) and m-chlorophenylbiguanide, two selective 5-HT3 agonists, mimicked the 5-HT responses, but 8-hydroxy-2-(di-n-propylamino)tetralin, a selective 5-HT1A agonist, was without effect. However, PBG failed to modify K+-evoked ACh release from the hippocampus. Systemic and local administration of a highly selective 5-HT3 antagonist, tropisetron ((3-alpha-tropanyl)1H-indole-carboxylic acid ester) blocked the effect of both 5-HT and PBG. The inhibition of ACh release by PBG was sensitive to tetrodotoxin. These observations provide direct evidence that, in rat cortex, 5-HT modulates in-vivo release of ACh through activation of 5-HT3 receptors.     

Influence of selective opiate antagonists on striatal acetylcholine and dopamine release

Ty3 is a retroviruslike element found in Saccharomyces cerevisiae. It encodes GAG3 and GAG3-POL3 polyproteins which are processed into mature proteins found in the Ty3 viruslike particle. In this study, the region encoding a protease that is homologous to retroviral aspartyl proteases was identified and shown to be required for production of mature Ty3 proteins and transposition. The Ty3 protease has the Asp-Ser-Gly consensus sequence found in copia, Ty1, and Rous sarcoma virus proteases, rather than the Asp-Thr-Gly found in most retroviral proteases. The Asp-Ser-Gly consensus is flanked by residues similar to those which flank the active sites of cellular aspartyl proteases. Mutations were made in the Ty3 active-site sequence to examine the role of the protease in Ty3 particle maturation and to test the functional significance of the Ser active-site variant in the consensus sequence. Mutation of the active-site Asp blocked processing of Gag3 and Gag3-Pol3 and allowed identification of a GAG3-POL3 polyprotein. This protein was turned over rapidly in cells expressing the mutant Ty3. Changing the active-site Ser to Thr caused only a modest reduction in the levels of certain Ty3 proteins. Five putative cleavage sites of this protease in Ty3 GAG3 and GAG3-POL3 polyproteins were defined by amino-terminal sequence analysis. The existence of an additional protein(s) of unknown function, encoded downstream of the protease-coding region, was deduced from the positions of these amino termini and the sizes of known Ty3 proteins. Although Ty3 protease cleavage sites do not correspond exactly to known retroviral protease cleavage sites, there are similarities. Residues P3 through P2' in the regions encompassing each of the five sites are uncharged, and no P1 position is occupied by an amino acid with a branched beta carbon.     

Cyclosporin A affects functions concerning acetylcholine release of cholinergic Torpedo synaptosomes

The effect of cyclosporin A was investigated on Torpedo synaptosomes. Cyclosporin A inhibits KCl-evoked acetylcholine release (up to 50% at 1 mu M) and was inactive on acetylcholine release induced by a Ca2+ ionophore, A23187. Interestingly, when the synaptosomes were pretreated with cyclosporin A, this immunosuppressor did abolish the modulation of A23187-induced acetylcholine release produced by two other drugs, cetiedil (alpha-cyclohexyl-3-thienyl acetic acid 2-(hexahydro-1H-azepin-1-yl) ethyl ester, citrate salt) and MR16728 (N-(N'-hexamethylene imino)-propyl-phenyl-cyclohexyl-methyl acetamide, chlorhydrate), which were previously shown to be inhibitory and stimulatory, respectively. Moreover, cyclosporin A and MR16728 are competitive inhibitors of [3H]cetiedil binding to purified synaptosomal presynaptic membranes (dissociation constant of 181.9 nM). These results suggest that presynaptic proteins involved in acetylcholine release (directly or indirectly through cyclophilin) are potential targets of cyclosporin A in Torpedo synaptosomes.     

Can barium support the release of acetylcholine by nerve impulses?

Conventional electrophysiological techniques were used to study the effects of Ba on the release of acetylcholine (ACh) from frog motor nerve terminals. Equimolar substitution of Ba for Ca eliminated end-plate potentials (e.p.ps) without a corresponding decline in the amplitude of the nerve terminal action potential. Miniature end-plate potentials (m.e.p.ps) were readily detectable in Ba solutions despite a depolarized muscle membrane. Studies on the e.p.p. in curarized preparations bathed with different concentrations of Ca and Ba suggest that Ba may compete with Ca in the process by which depolarization of the nerve terminal leads to the release of ACh. Repetitive nerve stimulation at 1 Hz in Ba solutions caused 5-20 fold increases in m.e.p.p. frequencies (7 experiments). Stimulation of Ba-bathed preparations at 10 Hz elevated m.e.p.p. frequencies to very high levels that could not be measured accurately ('100/s). It is suggested that the asynchronous discharge of m.e.p.ps produced by repetitive nerve stimulation is the electrophysiological correlate of the evoked ACh outflow in Ba solutions detected previously by bioassay of ther perfusion fluid.     

Modulation of acetylcholine release via GABAA and GABAB receptors in rat striatum

Investigation of a human T-lymphotropic virus type II (HTLV-II) infection in a female Australian blood donor identified a human bite as the likely mode of transmission, confirmed by nucleotide sequencing of the proviral tax/rex from both donor and contact. We believe this to be the first report of the transmission of an HTLV by a human bite.     

Evoked acetylcholine release expressed in neuroblastoma cells by transfection of mediatophore cDNA

Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real-time recording with an embryonic Xenopus myocyte. Glioma C6-Bu-1 cells were found to be competent for Ca(2+)-dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG-2 cells. However, acetylcholine release could be restored when N18TG-2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine-releasing capacity. The transfected N18TG-2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed, discrete amplitude levels, like in naturally occurring synapses.     

Effect of transient cerebral ischaemia on acetylcholine release in the gerbil hippocampus

To clarify the relationship between presynaptic cholinergic dysfunction and postsynaptic cell death in the hippocampus, extracellular levels of acetylcholine (ACh) were assayed and CA1 pyramidal cells were histologically investigated in gerbils which had undergone 2, 5 and 10 min ischaemia. It was found that the KCl- and atropine-induced release of ACh, an index of the functioning cholinergic system at the presynaptic terminals, was significantly lower in the ischaemic groups than in control groups. The hippocampal CA1 pyramidal cell area of the 5 and 10 min ischaemic animals was also significantly decreased, but the 2 min ischaemia caused no cell damage. These findings indicate that the presynaptic terminals of the cholinergic neurone are vulnerable to ischaemic insult and that cholinergic dysfunction precedes postsynaptic CA1 pyramidal cell death in the hippocampus.     

The effect of a reduction in temperature on the quantal release of transmitter at the mouse neuromuscular junction

1. The effects of a reduction in temperature were examined on evoked and spontaneous release of transmitter quanta and on presynaptic negative signals, blocked by Cd2+, measured externally at neuromuscular junctions in mouse diaphragm muscles in low-Ca2+, high-Mg2+ Krebs-Ringer solutions. 2. The evoked release was enhanced with lowering of the temperature, whereas the extent of spontaneous release was reduced. Cooperativity of Ca2+ in the evoked release was slightly reduced by lowering the temperature. 3. The presynaptic negative signals increased in duration with lowering of the temperature. 4. These results support the hypothesis that the effect of a reduction in temperature reflects the improved efficacy of the calcium-mediated mechanism of transmitter release, manifested as a prolongation of the inflow of Ca2+. The process involved in the evoked release is probably attributable to an almost passive mechanism.     

Preparing nurses for the inevitable: the New Madrid earthquake

Although this article discusses earthquake preparedness for the New Madrid seismic zone, registered nurses in any location will be on the front line as patient caregivers and managers in the event of a damaging earthquake. Two self-instructional modules were developed to educate registered nurses about earthquake preparedness. Statistical analyses of pretest and posttest scores from nurses who completed the modules and from nurses who participated in a control group reveal that the modules are effective educational tools. This information will make them more effective as nurses during and after an earthquake and emphasizes the need for their involvement in disaster mitigation and planning.     

M2 muscarinic autoreceptors modulate acetylcholine release in the medial pontine reticular formation

Muscarinic autoreceptors regulate acetylcholine (ACh) release in several brain regions, including the medial pontine reticular formation (mPRF). This study tested the hypothesis that the muscarinic cholinergic receptor mediating mPRF ACh release is the pharmacologically defined M2 subtype. In vivo microdialysis was used to deliver muscarinic cholinergic receptor (MAChR) antagonists to the feline mPRF while simultaneously measuring endogenously released ACh. The lowest concentration of each antagonist that caused a significant increase in mPRF ACh release was determined and defined as the minimum ACh-releasing concentration. Data obtained from 41 mPRF dialysis sites in 10 animals showed that the order of potency (followed by the minimum ACh-releasing concentration) was scopolamine (1 nM) > AF-DX 116 (3 nM) > pirenzepine (300 nM). Comparison of these minimum ACh-releasing concentrations to the known affinities of the antagonists for the five mAChR subtypes is consistent with the conclusion that the autoreceptor regulating mPRF ACh release is the M2 subtype. Considerable evidence supports a role for cholinergic neurotransmission and postsynaptic M2 receptors in the mPRF in regulating levels of arousal. The present data suggest that presynaptic M2 receptors contribute to the regulation of arousal states by modulating mPRF ACh release.     

Zinc blocks acetylcholine release but not vesicle fusion at the Torpedo nerve-electroplate junction

The combined effects of Zn2+ treatment and nerve stimulation were studied on cholinergic synapses of the Torpedo marmorata electric organ. Incubation of small pieces of electric tissue in 250 microM ZnCl2 for 2 h irreversibly blocked synaptic transmission by inhibiting the release of acetylcholine. This treatment, however, did not cause any significant fine structural alteration in the nerve-electroplate junctions. Preparations treated with Zn2+ were submitted to electrical stimulation. In spite of the fact that no transmitter was released, stimulation resulted in the accumulation of calcium in the tissue, and in marked ultrastructural changes. The density of synaptic vesicles was significantly reduced and many of the remaining vesicles were found in close proximity to the presynaptic membrane. Images of vesicles fused with the plasmalemma were abundant, indicating that numerous vesicles were caught in different phases of exocytosis or endocytosis. Freeze-fracture replicas made from quick-frozen or chemically fixed material showed a high number of vesicle openings (pits) in the presynaptic plasmalemma. No recovery occurred even after a prolonged period of rest, indicating that retrieval was impaired by zinc treatment. In conclusion, the present experimental paradigm created an unusual situation where fusion of synaptic vesicles to the plasma membrane could be activated independently from the release of transmitter.     

Role of dopaminergic neuronal system in dizocilpine-induced acetylcholine release in the rat brain

The effects of dopaminergic receptor antagonists on dizocilpine-induced increase in extracellular acetylcholine (ACh) levels in the rat parietal cortex were examined in freely-moving rats, using an in vivo brain microdialysis method. Dizocilpine (0.5 mg/kg) significantly increased extracellular ACh levels in the rat parietal cortex and hippocampus, but not in the striatum. Pretreatment with alpha-methyl-p-tyrosine methyl ester (alpha MpT) delayed the onset but prolonged the duration of the dizocilpine-induced increases in extracellular ACh levels. The dopamine D2 receptor antagonist, haloperidol, showed dual effects similarly to alpha MpT, while the dopamine D1 receptor antagonist, SCH23390, prolonged, but did not delay, the onset of the dizocilpine-induced increases in ACh levels. These results suggest that the dopaminergic system is involved in the dizocilpine-induced increase in the extracellular ACh level in the parietal cortex in two ways, through both dopamine D1 and D2 receptors.