Mechanisms controlling choline transport and acetylcholine synthesis in motor nerve terminals during electrical stimulation

Electrical stimulation of the chick ciliary nerve leads to a frequency-dependent increase in the Na+-dependent high affinity uptake of [3H]choline (SDHACU) and its conversion to acetylcholine (ACh) in the nerve terminals innervating the iris muscle. The forces that drive this choline (Ch) uptake across the presynaptic membrane were evaluated. Depolarization with increased [K+] out or veratridine decreases Ch accumulation. In addition to the electrical driving force, energy is provided by the Na+ gradient. Inhibition of the Na,K-ATPase decreased the Ch taken up. Thus, changes in the rate of Ch transport are dependent on the electrochemical gradients for both Ch and Na+. Ch uptake and ACh synthesis were increased after a conditioning preincubation with high [K+] out or veratridine. As is the case for electrical stimulation, this acceleration of Ch uptake and ACh synthesis was strongly dependent on the presence of Ca++ in the incubation medium. Na+ influx through a TTX-sensitive channel also contributed to this acceleration. Inasmuch as membrane depolarization reduces the initial velocity of Ch uptake and ACh synthesis, their increases during electrical stimulation therefore cannot be the direct effect of the depolarization phase of the action potential. Instead they are the result of the ionic fluxes accompanying the presynaptic spike. It is concluded that stimulation of Ch uptake and ACh synthesis by nerve activity depends first, on the ACh release elicited by Ca++ influx after depolarization and second, on the activation of the Na,K-ATPase due to Na+ entry. Furthermore, it is suggested that the release of ACh after stimulation drives translocation of cytoplasmic ACh into a protected compartment (probably vesicular). This recompartmentation of intraterminal ACh stimulates ACh synthesis by mass action, allowing further accumulation of Ch.     

Choline and acetylcholine metabolism in rat neostriatal slices

Choline (Ch) uptake and release and acetylcholine (ACh) synthesis and release have been studied by gas chromatography mass spectrometry (GCMS) in slices of rat neostriatum in vitro to assess the effects of depolarization by 25 mM K+ and the influence of elevated concentrations of Ch in the incubation medium. During the first 60 min after preparation, 25 mM K+ increased ACh release by 182% and reduced ACh levels by 40%. The rate of ACh synthesis was unchanged. After a 1-h equilibration period, the rate of ACh synthesis was considerably less (2.41 nmol mg-1 h-1, compared to 9.78 nmol mg-1 h-1). Exposure to 25 mM K+ during the second hour increased the rate to 6.47 nmol mg-1 h-1. During the first 10 min of exposure to 25 mM K+, ACh synthesis was reduced, regardless of incubation. Increasing concentrations of external [2H4]Ch apparently favored initial rates of net ACh synthesis, since the rank order of initial net ACh synthesis rates is the same as the rank order of external [2H4] Ch concentration under both normal and depolarized conditions. However, the only significant effect of external [2H4]Ch on ACh metabolism was that it increased ACh release during the initial 10 min, when the preparation was depolarized with K+. The efflux of endogenous [2H0]Ch was increased initially (10 min) and slowed over a 60-min period by 25 mM K+, and increased when [2H4]Ch in the medium was increased. Changes in ACh synthesis and release were dependent upon the time exposure of slices to high K+, and the results suggest that Ch favors initial rates of ACh synthesis, but that Ch influences ACh release primarily under conditions of stress (i.e., depolarization).     

Effects of tetrodotoxin and anaesthetics on brain metabolism and transport during anoxia

1. Tetrodotoxin, at concentrations at which it abolishes generation of action potentials in the nervous system, enhances by about 300% the rate of anaerobic glycolysis of brain-cortex slices from adult rats, or from adult and infant guinea pigs. This occurs to a greater extent in Ca(2+)-deficient incubation media than in Ca(2+)-rich media. Tetrodotoxin has no accelerative effect on cerebral aerobic glycolysis. 2. Tetrodotoxin does not affect the rate of anaerobic glycolysis of 2-day-old rat brain-cortex slices, nor that of adult rat kidney medulla, nor that of an extract of an acetone-dried powder of brain. 3. Tetrodotoxin does not affect the rate of penetration of glucose into brain slices. 4. Its effect is not apparent if it is added 10min or later after the onset of anoxia. 5. Its effect diminishes as the concentration of K(+) in the incubation medium is increased while that of Na(+) is decreased. 6. Its salient effect, at the onset of anoxia, is to diminish influx of Na(+) into, and efflux of K(+) from, the brain slices. 7. Substances that promote cerebral influx of Na(+), e.g. protoveratrine, sodium l-glutamate, diminish the accelerative action of tetrodotoxin. 8. It is concluded that tetrodotoxin exerts its effect on anaerobic glycolysis by suppressing, at the onset of anoxia, the generation of action potentials and thereby the accompanying influx of Na(+) and efflux of K(+). It is suggested that glycolytic stimulation occurs because a rate-limiting step, e.g. operation of pyruvate kinase, is stimulated by K(+) and depressed by Na(+). 9. Local anaesthetics behave in a manner similar to that of tetrodotoxin in enhancing cerebral anaerobic glycolysis. 10. Sodium Amytal has a marked effect at relatively high concentration. 11. Tetrodotoxin diminishes efflux of amino acids, particularly glutamate and aspartate, at the onset of anoxia.     

Release of Acetylcholine from Tissue Slices of the Rat Nucleus Basalis Magnocellularis

We investigated the release of acetylcholine (ACh) from tissue slices obtained from the nucleus basalis magnocellularis (nbM) of the rat brain. Potassium (35 mM) depolarization produced a 10- to 12-fold increase in the release of endogenous ACh above spontaneous release. Potassium-evoked ACh release was Ca2+ dependent. Injection of the excitotoxin quinolinic acid into the nbM produced a 72.8 +/- 13.0% decrease in spontaneous ACh release and a 60.4 +/- 8.2% decrease in potassium-evoked release. A fourfold increase in ACh release was observed following perfusion of the tissue with 1 mM 3,4-diaminopyridine (3,4-DAP) whereas 10 mM 3,4-DAP caused a sevenfold increase. The increase in ACh release caused by 3,4-DAP was inhibited by tetrodotoxin. Tissue slices accumulated [3H]choline by high-affinity choline uptake and this could be inhibited by hemicholinium-3. These results indicate that ACh can be released from tissue slices of the nbM by a calcium-dependent process and that a part of this release appears to be from the cholinergic neurons of the nbM.     

Inhibition of Glutamate Release by Arachidonic Acid in Rat Cerebrocortical Synaptosomes

The action of arachidonic acid on glutamate release in rat cerebrocortical synaptosomes was investigated. The Ca(2+)-dependent release of glutamate evoked by 4-aminopyridine (4-AP) was inhibited by arachidonic acid (0.5-10 microM), but the KCl-evoked release was not modified. The Ca(2+)-independent release of glutamate was insensitive to low concentrations of arachidonic acid, but higher concentrations of this free fatty acid (30 microM) induced a slow efflux of cytoplasmic glutamate. The decrease in the Ca(2+)-dependent release of glutamate by arachidonic acid was consistent with a reduction in both the depolarization and the subsequent rise in the cytoplasmic free Ca2+ concentration induced by 4-AP in the nerve terminal. The inhibitory action by arachidonic acid observed in glutamate release was reversed in the presence of the K(+)-channel blocker tetraethylammonium.     

Inhibition of choline efflux results in enhanced acetylcholine synthesis and release in the guinea-pig corticocerebral synaptosomes.

Synthesis and release of [3H]acetylcholine ([3H]ACh) were measured in synaptosomes from the guinea pig cerebral cortex after preloading with [3H]choline ([3H]Ch). We demonstrate here that inhibition of choline (Ch) efflux results in an increase in acetylcholine (ACh) synthesis and release. Our findings are as follows: (1) inhibition of [3H]Ch efflux by hemicholinium-3 (HC-3) (100 microM), increased the levels of both the released (116% of control) and the residing (115% of control) [3H]ACh. (2) The muscarinic agonist, McN-A-343 (100 microM), which was previously shown to inhibit Ch efflux, also increased the released (121% of control) and the residing (109% of control) [3H]ACh. (3) Omission of Na+ ions (which are required for Ch transport) from the incubation medium had similar effects to those observed with McN-A-343 and HC-3. These results suggest inverse relationships between Ch efflux on one hand, and ACh synthesis and release on the other hand. (4) Depolarization with 50 mM K+, or with the K+ channel blocker, 4-aminopyridine (100 microM), also increased the total level of [3H]ACh (113 and 107% of nondepolarized synaptosomes, respectively). However, whereas conditions that inhibit Ch transport such as HC-3, McN-A-343 and "no sodium" increased both the residing and the released [3H]ACh depolarization with high K+ or 4-aminopyridine reduced the residing (79 and 87% of control, respectively) and increased only the released [3H]ACh (182 and 148% of control, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of synthesis and release of radioactive acetylcholine in brain slices from the rat. Effects of neurotropic drugs

1. Studies of the synthesis and release of radioactive acetylcholine in rat brain-cortex slices incubated in Locke-bicarbonate-[U-(14)C]glucose media, containing paraoxon as cholinesterase inhibitor, revealed the following phenomena: (a) dependence of K(+)-or protoveratrine-stimulated acetylcholine synthesis and release on the presence of Na(+) and Ca(2+) in the incubation medium, (b) enhanced release of radioactive acetylcholine by substances that promote depolarization at the nerve cell membrane (e.g. high K(+), ouabain, protoveratrine, sodium l-glutamate, high concentration of acetylcholine), (c) failure of acetylcholine synthesis to keep pace with acetylcholine release under certain conditions (e.g. the presence of ouabain or lack of Na(+)). 2. Stimulation by K(+) of radioactive acetylcholine synthesis was directly proportional to the external concentration of Na(+), but some synthesis and release of radioactive acetylcholine occurred in the absence of Na(+) as well as in the absence of Ca(2+). 3. The Na(+) dependence of K(+)-stimulated acetylcholine synthesis was partly due to suppression of choline transport, as addition of small concentrations of choline partly neutralized the effect of Na(+) lack, and partly due to the suppression of the activity of the Na(+) pump. 4. Protoveratrine caused a greatly increased release of radioactive acetylcholine without stimulating total radioactive acetylcholine synthesis. Protoveratrine was ineffective in the absence of Ca(2+) from the incubation medium. It completely blocked K(+) stimulation of acetylcholine synthesis and release. 5. Tetrodotoxin abolished the effects of protoveratrine on acetylcholine release. It had blocking effects (partial or complete) on the action of high K(+), sodium l-glutamate and lack of Ca(2+) on acetylcholine synthesis and release. 6. Unlabelled exogenous acetylcholine did not diminish the content of labelled tissue acetylcholine, derived from labelled glucose, suggesting that no exchange with vesicular acetylcholine took place. In the presence of 4mm-KCl it caused some increase in the release of labelled acetylcholine. 7. The barbiturates (Amytal, pentothal), whilst having no significant effects on labelled acetylcholine synthesis in unstimulated brain except at high concentration (1mm), diminished or abolished (at 0.25 or 0.5mm) the enhanced release of acetylcholine, due to high K(+) or lack of Ca(2+). The fall in tissue content of acetylcholine, due to lack of Ca(2+), was diminished or abolished by pentothal (0.25 or 0.5mm) or Amytal (0.25mm).     

Depolarization-evoked release of gamma-hydroxybutyrate from rat brain slices

The release of gamma-hydroxybutyrate from preloaded rat brain striatal slices was investigated. K+-induced depolarization caused an efflux of gamma-hydroxybutyrate of about 50 fmol min-1 mg-1 (wet weight), but in a Ca2+-free medium containing Mg2+, the evoked release was reduced by 50-60%. The release was higher when 100 microM veratridine was used as a depolarizing agent. The efflux of gamma-hydroxybutyrate is related to veratridine and K+ concentration, and is strongly inhibited by 10 microM tetrodotoxin. The Ca2+ channel blocker verapamil induces a large decrease in the efflux of gamma-hydroxybutyrate after both K+- and veratridine-induced depolarization. These results are in favour of a possible transmitter function for gamma-hydroxybutyrate in rat striatum.     

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.     

Depolarization affects the binding properties of muscarinic acetylcholine receptors and their interaction with proteins of the exocytic apparatus.

Membrane depolarization is the signal that triggers release of neurotransmitter from nerve terminals. As a result of depolarization, voltage-dependent Ca(2+) channels open, level of intracellular Ca(2+) increases. and release of neurotransmitter commences. Previous study had shown that in rat brain synaptosomes, muscarinic acetylcholine (ACh) receptors (mAChRs) interact with soluble NSF attachment protein receptor proteins of the exocytic machinery in a voltage-dependent manner. It was suggested that this interaction might control the rapid, synchronous release of acetylcholine. The present study investigates the mechanism for such a voltage-dependent interaction. Here we show that depolarization shifts mAChRs, specifically the m2 receptor subtype, to a low affinity state toward its agonists. At resting potential, mAChRs are in a high affinity state (K(d) of approximately 20 nM) and they shift to a low affinity state (K(d) of tens of microM) upon membrane depolarization. In addition, interaction between m2 receptor subtype and the exocytic machinery increases with receptor occupancy. Both phenomena are independent of Ca(2+) influx. We propose that these results may explain control of ACh release from nerve terminals. At resting potential the exocytic machinery is clamped due to its interaction with the occupied mAChR and depolarization relieves this interaction. This, together with Ca(2+) influx, enables release of ACh to commence.     

Effects of streptozotocin-induced diabetes on acetylcholine metabolism in rat brain

The main objective of this study was to determine whether uncontrolled hyperglycemia, as a consequence of diabetes, altered the metabolism of acetylcholine (ACh) in rat brain. To accomplish this, rats received injections of streptozotocin (STZ, 60 mg/kg, i.v.) or vehicle, and were maintained for up to 7 weeks after the injections. Various indices of ACh metabolism were determined in striatum and hippocampus, two brain regions densely innervated by cholinergic neurons. STZ induced diabetes in 96% of the rats injected, as evidenced by glucose spillage into the urine within 48 hours. Serum glucose levels increased to 326% of control values by 1 week and remained at this level for the duration of the study. The steady-state concentrations of ACh and choline, determined in brain tissue from animals killed by head-focused microwave irradiation, did not differ between the control and STZ-injected groups. However, the synthesis and release of neurotransmitter by striatal slices, measured in vitro, decreased in a time-dependent manner. Although the basal release of ACh was unaltered at 1 week, neurotransmitter release decreased significantly by 21% at 5 weeks and by 26% at 7 weeks. The release of ACh evoked by incubation with 35 mM KCl was inhibited significantly by 20% at all time points studied. ACh synthesis by slices incubated under basal conditions decreased by 13% and 27% at 5- and 7-weeks, respectively, the latter significantly less than controls. Synthesis by striatal slices incubated with 35 mM KCl was inhibited by 17% at 7 weeks. Although the synthesis and release of ACh by hippocampal slices from diabetic animals tended to be less than controls, these alterations were not statistically significant. Investigations into the mechanism(s) mediating the deficit in ACh synthesis exhibited by striatal slices indicated that it did not involve alterations in precursor choline availability, nor could it be attributed to alterations in the activities of the synthetic or hydrolytic enzymes choline acetyltransferase or acetylcholinesterase; rather, the decreased turnover of ACh may be secondary to other STZ-induced, hyperglycemia-mediated neurochemical alterations.     

Ca2+-Surrogate Action of Pb2+ on Acetylcholine Release from Rat Brain Synaptosomes

The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 microM Pb2+ to intact synaptosomes in Ca2(+)-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2(+)-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1-50 nM (EC50 congruent to 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2-5 microM (EC50 congruent to 0.5 microM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.     

Impairment of Ca(2+) mobilization in circular muscle cells of the inflamed colon

This study investigated whether inflammation modulates the mobilization of Ca(2+) in canine colonic circular muscle cells. The contractile response of single cells from the inflamed colon was significantly suppressed in response to ACh, KCl, and BAY K8644. Methoxyverapamil and reduction in extracellular Ca(2+) concentration dose-dependently blocked the response in both normal and inflamed cells. The increase in intracellular Ca(2+) concentration in response to ACh and KCl was significantly reduced in the inflamed cells. However, Ca(2+) efflux from the ryanodine- and inositol 1,4, 5-trisphosphate (IP(3))-sensitive stores, as well as the decrease of cell length in response to ryanodine and IP(3), were not affected. Heparin significantly blocked Ca(2+) efflux and contraction in response to ACh in both conditions. ACh-stimulated accumulation of IP(3) and the binding of [(3)H]ryanodine to its receptors were not altered by inflammation. Ruthenium red partially inhibited the response to ACh in normal and inflamed states. We conclude that the canine colonic circular muscle cells utilize Ca(2+) influx through L-type channels as well as Ca(2+) release from the ryanodine- and IP(3)-sensitive stores to contract. Inflammation impairs Ca(2+) influx through L-type channels, but it may not affect intracellular Ca(2+) release. The impairment of Ca(2+) influx may contribute to the suppression of circular muscle contractility in the inflamed state.     

Acetylcholine Releases Prostaglandins from Brain Slices Incubated In Vitro

A variety of neurotransmitters elicit a phosphoinositide response in the CNS; however, their effects on prostaglandin (PG) formation in the brain are not well characterized. In the present study, we investigated the effect of acetylcholine (ACh) on the synthesis of PGs E and F in slices from various regions of guinea pig brain incubated in glucose-fortified Krebs-Henseleit bicarbonate saline. Slices were prewashed in the presence of 1% albumin to reduce basal PG levels followed by incubation for 30 min at 37 degrees C in the presence or absence of ACh. Under these conditions, 5 mM ACh significantly increased the efflux of PGE and PGF from brain regions enriched in muscarinic cholinergic receptors, i.e., cerebral cortex, temporal cortex, corpus striatum, and hippocampus. Depolarization by 45 mM KCl also significantly enhanced PG synthesis, and the relative magnitude of the effect was similar to that of ACh. The stimulation of PG synthesis by ACh was inhibited by 20 microM atropine, whereas the K+-induced stimulation was not. The effects of potassium and ACh were additive at maximally effective ACh concentrations, an observation that suggests that ACh and K+ increase PG efflux through independent mechanisms. Norepinephrine, histamine, and serotonin, three other neurotransmitters that evoke a phosphoinositide response in the brain, were ineffective in stimulating PG release from brain cortex slices.     

Decrease of Acetylcholine Release from Cortical Slices in Aged Rats: Investigations into Its Reversal by Phosphatidylserine

The release of total acetylcholine (ACh) and [3H]ACh was investigated in electrically stimulated cortical slices prepared from 4- and 18-month-old male Wistar rats. The slices were prelabeled with [3H]choline ([3H]Ch) and perfused with Krebs solution containing physostigmine. Total ACh was measured and the nature of the tritium efflux identified by HPLC. The total tritium content in the slices at the end of the incubation period was half as great in the old as in young rats. A linear relationship was found between stimulation frequencies (2, 5, and 10 Hz) and fractional [3H]ACh release in both young and old rats. In the latter the release was significantly smaller. At 10 Hz stimulation frequency the ratio between the two 2-min stimulation periods, S2/S1, was higher in the 18-month-old rats than in the young rats. Specific activity of the evoked ACh release was significantly smaller in S2 than in S1 in 4-month-old rats only. These findings indicate that the young synthetize ACh from endogenous unlabeled Ch more than older rats. In 18-month-old rats both the evoked total ACh and [3H]ACh release, expressed as picograms per minute, showed an approximately 50% decrease in both S1 and S2 stimulation periods, with no significant difference in specific activity. Phosphatidylserine (PtdSer) administration (15 mg/kg, i.p. daily) for 1 week to 18-month-old rats prevented the reduction in total evoked ACh release but not the reduction in evoked [3H]ACh release. The specific activity of ACh release was therefore significantly smaller than that of the young and untreated old rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca(2+)-dependent desensitization of insulin secretion by strong potassium depolarization

Depolarization by a high K(+) concentration is a widely used experimental tool to stimulate insulin secretion. The effects occurring after the initial rise in secretion were investigated here. After the initial peak a fast decline occurred, which was followed by a slowly progressive decrease in secretion when a strong K(+) depolarization was used. At 40 mM KCl, but not at lower concentrations, the decrease continued when the glucose concentration was raised from 5 to 10 mM, suggesting an inhibitory effect of the K(+) depolarization. When tolbutamide was added instead of the glucose concentration being raised, a complete inhibition down to prestimulatory values was observed. Equimolar reduction of the NaCl concentration to preserve isoosmolarity enabled an increase in secretion in response to glucose. Unexpectedly, the same was true when the Na(+)-reduced media were made hyperosmolar by choline chloride or mannitol. The insulinotropic effect of tolbutamide was not rescued by the compensatory reduction of NaCl, suggesting a requirement for activated energy metabolism. These inhibitory effects could not be explained by a lack of depolarizing strength or by a diminished free cytosolic Ca(2+) concentration ([Ca(2+)](i)). Rather, the complexation of extracellular Ca(2+) concomitant with the K(+) depolarization markedly diminished [Ca(2+)](i) and attenuated the inhibitory action of 40 mM KCl. This suggests that a strong but not a moderate depolarization by K(+) induces a [Ca(2+)](i)-dependent, slowly progressive desensitization of the secretory machinery. In contrast, the decline immediately following the initial peak of secretion may result from the inactivation of voltage-dependent Ca(2+) channels.     

Role of the choline exchanger in Na(+)-independent Mg(2+) efflux from rat erythrocytes

Two types of Na(+)-independent Mg(2+) efflux exist in erythrocytes: (1) Mg(2+) efflux in sucrose medium and (2) Mg(2+) efflux in high Cl(-) media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na(+)-independent Mg(2+) efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K(+),Cl(-)- and Na(+),K(+),Cl(-)-symport, Na(+)/H(+)-, Na(+)/Mg(2+)-, Na(+)/Ca(2+)- and K(+)(Na(+))/H(+) antiport, Ca(2+)-activated K(+) channel and Mg(2+) leak flux. We suggest that, in choline Cl medium, Na(+)-independent Mg(2+) efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg(2+) efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg(2+) to the same degree. The K(d) value for inhibition of [(14)C]choline efflux and for inhibition of Mg(2+) efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg(2+) efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg(2+) efflux was reduced to the same degree by these inhibitors as was the [(14)C]choline efflux.     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of Endogenous Release of Glutamate and γ-Aminobutyric Acid from Hippocampus CA1 Slices After In Vivo Long-Term Potentiation

The effect of long-term potentiation (LTP) on endogenous amino acid release from rat hippocampus slices was studied. LTP was induced in vivo by application of a tetanus (200 Hz, 200 ms) to the Schaffer collateral fibers in unanesthetized rats. Endogenous release of glutamate and gamma-aminobutyric acid (GABA) was investigated 60 min after tetanization in CA1 subslices of potentiated and control rats. No significant effects of LTP were observed in basal and K(+)-induced Ca(2+)-independent release components of these amino acids. In contrast, K(+)-induced Ca(2+)-dependent release of both glutamate and GABA increased approximately 100% in slices from potentiated rats. No differences were observed in total content of glutamate and GABA between the subslices from control and LTP animals. These results suggest a persistent increase in the recruitment of the presynaptic vesicular pool of glutamate and GABA during LTP.     

CB1 receptors diminish both Ca(2+) influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals

We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals.