Ethanol inhibits veratridine-stimulated sodium uptake in synaptosomes

The effects of ethanol and pentobarbital on voltage-sensitive sodium channels in whole brain (rat) synaptosomes were studied using isotopic flux measurements. Incubation of synaptosomes with ethanol or pentobarbital $\text{in}$$\text{vitro}$ inhibited veratridine-stimulated ²²Na⁺ uptake. The effect of ethanol is dose-dependent, occurs at sublethal, pharmacologically relevant concentrations and is fully reversible. These results suggest that ethanol and pentobarbital directly interfere with sodium channel function in nervous tissue. Alterations in sodium channel function may be a possible mechanism for the central nervous system (CNS) depressant action of ethanol and related compounds.     

Muscarinic receptor activation inhibits both release and synthesis of noradrenaline in rat hippocampal synaptosomes

Rat hippocampal synaptosomes were used to investigate the effects of muscarinic cholinergic drugs on the release of [³H]noradrenaline and the rate of noradrenaline synthesis. Oxotremorine and acetylcholine caused depression of K⁺-evoked release of [³H]noradrenaline; these effects were reversed by atropine and pirenzepine. Muscarinic agonists depressed 2-chloroadenosine- and isoprenaline-stimulated noradrenaline synthesis but had no effect on 8-Br-cyclic AMP-stimulated synthesis. Oxotremorine also depressed the K⁺-acceleration of noradrenaline synthesis. The action of pirenzepine suggests that the inhibition of release and synthesis are mediated by separate muscarinic receptor subtypes.     

Bombesin-like peptides in rat brain: Localization in synaptosomes and release from hypothalamic slices

The subcellular distribution of bombesin-like immunoreactivity in rat brain was investigated. The number of receptors was significantly greater in the synaptosomal than mitochondrial fraction and quantity of bombesin-like immunoreactivity was greatest in the synaptosomal fraction. Also, the release of bombesin-like peptides from rat hypothalamic slices was investigate K⁺ and veratridine stimulated release of immunoreactivity in a Ca⁺⁺-dependent manner.     

Effect of amiodarone on Na+-, K+-ATPase and Mg2+-ATPase activities in rat brain synaptosomes

Amiodarone hydrochloride is a diiodinated antiarrhythmic agent widely used in the treatment of cardiac disorders. With the increasing use of amiodarone, several untoward effects have been recognized and neuropathy following amiodarone therapy has recently been reported. The present studies were carried out to study the effect of amiodarone on rat brain synaptosomal ATPases in an effort to understand its mechanism of action. Na+, K+-ATPase and oligomycin sensitive Mg2+ ATPase activities were inhibited by amiodarone in a concentration dependent manner with IC50 values of 50 microM and 10 microM respectively. [3H]ouabain binding was also decreased in a concentration dependent manner with an IC50 value of 12 microM, and 50 microM amiodarone totally inhibited [3H]ouabain binding. Kinetics of [3H]ouabain binding studies revealed that amiodarone inhibition of [3H]ouabain binding is competitive. K+-activated p-nitrophenyl phosphatase activity showed a maximum inhibition of 32 per cent at 200 microM amiodarone. Synaptosomal ATPase activities did not show any change in rats treated with amiodarone (20 mg kg-1 day-1) for 6 weeks, when compared to controls. The treatment period may be short, since the reported neurological abnormalities in patients were observed during 3-5 years of treatment. The present results suggest that amiodarone induced neuropathy may be due to its interference with sodium dependent phosphorylation of Na+, K+-ATPase reaction, thereby affecting active ion transport phenomenon and oxidative phosphorylation resulting in low turnover of ATP in the nervous system.     

Uptake and release of kyotorphin in rat brain synaptosomes

Uptake and release of kyotorphin (TyrArg) in rat brain synaptosomes were studied. Synthetic kyotorphin was taken up into crude synaptosomes (P₂), in a temperature-dependent manner. The Km and Vmax of the uptake were 1.31 ± 0.12 × 10⁻⁴M and 5.9 ± 0.5 pmol/mg protein/min, respectively. Metabolic inhibitors such as dinitrophenol and iodoacetamide and ouabain which is known as an inhibitor of Na⁺ dependent uptake mechanism significantly inhibited the uptake. When the synaptosomes previously preloaded with synthetic kyotorphin at 10⁻⁴M were exposed to high K⁺ medium, kyotorphin was released in a Ca²⁺-dependent manner. These findings support the view that kyotorphin plays a role as neurotransmitter/neuroregulator.     

Phenylarsine oxide inhibits alpha-latrotoxin-stimulated [3H]GABA release from rat brain synaptosomes

Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of membrane-trafficking processes, including exocytosis of neurotransmitters. However, there are contradictory findings concerned ability of phenylarsine oxide (PAO), an inhibitor of phosphatidylinositol 4-kinase, to affect exocytotic release of different types of neurotransmitters. We bent our efforts to a detailed analysis of action of PAO on Ca(2+)-dependent and Ca(2+)-independent [3H]GABA release produced by exposure of rat brain synaptosomes to different concentrations of alpha-latrotoxin. We also compared PAO action on alpha-latrotoxin- and 4-aminopyridine (4-AP)-evoked [3H]GABA release. The experiments have shown that release of [3H]GABA evoked by the depolarization with 4-AP was decreased by 80% as a result of action of 3 microM PAO and the complete inhibition of release was observed with 10 microM PAO. When alpha-latrotoxin as a stimulant was applied, release of [3H]GABA was increased as toxin concentration used was elevated from 0.5 to 3.0 nM, however, concomitantly, the response of the toxin-induced [3H]GABA release to PAO became attenuated: 10 microM PAO led to almost complete inhibition of the effect of 0.5 nM alpha-latrotoxin and only partly decreased (by 40%) the response to 3.0 nM alpha-latrotoxin. To test whether the efficacy of PAO depended on the toxin-induced outflow of cytosolic [3H]GABA, synaptosomes with depleted cytosolic [3H]GABA pool were also exploited. Depletion was performed by means of heteroexchange of cytosolic [3H]GABA with nipecotic acid. The experiments have shown that treatment of loaded synaptosomes with nipecotic acid resulted in some increase of [3H]GABA release evoked by 0.5 nM alpha-latrotoxin, but in the two-fold decrease of the response to 3.0 nM alpha-latrotoxin. PAO essentially inhibited [3H]GABA release from depleted synaptosomes irrespective of alpha-latrotoxin concentration used. Therefore, the amount of [3H]GABA released from cytosolic pool determined, in considerable degree, the insensitivity of alpha-latrotoxin action to PAO. Thus, our data show that subnanomolar concentrations of alpha-latrotoxin may be used for stimulation of exocytotic release of [3H]GABA. Exposure of synaptosomes with nanomolar toxin concentrations leads not only to stimulation of exocytosis, but also to leakage of [3H]GABA from cytosolic pool. PAO potently inhibits exocytotic release of [3H]GABA and its inhibitory effectiveness is diminished as far as the outflow of [3H]GABA is elevated.     

Stereoselective effects of ketamine on dopamine,serotonin and noradrenaline release and uptake in rat brain slices

Ketamine (2-(2-chlorophenyl)-(1-methylamino)-cyclohexanone) is a rapid-acting dissociative general anaesthetic whose hallucinogenic properties have made it a popular drug of abuse. Ketamine comprises two optical isomers, with differing pharmacology. In the present study, the effects of (+)- and (-)-ketamine on stimulated efflux and reuptake of dopamine (DA), noradrenaline (NA) and serotonin (5-HT) were compared in isolated superfused slices of the rat caudatoputamen (CPu), ventral bed nucleus of the stria terminalis (BSTV) or dorsal raphe nucleus (DRN), respectively. Monoamine efflux was elicited by local electrical stimulation (20 pulses, 100 Hz trains) at tungsten microelectrodes and measured at adjacent carbon fibre microelectrodes using fast cyclic voltammetry (FCV). In CPu (+)-ketamine increased stimulated DA efflux and slowed DA reuptake in a concentration-dependent manner (25-200 microM). At 100 microM (+)-ketamine increased DA efflux by 109+/-20% (mean+/-S.E.M., n=13) of control values after 30 min (P<0.001 versus control) and prolonged uptake half-time (t(1/2)) by 76+/-38% (n=9, P<0.001) of control. In contrast (-)-ketamine (100 microM) had no effect on DA efflux or uptake. In DRN, both isomers (100 microM) increased stimulated 5-HT efflux. (-)-Ketamine had a larger effect (P<0.001), an 88+/-15% increase in 5-HT efflux (n=9) versus 46+/-10% (n=8) for the (+)-isomer. The isomers had similar effects on 5-HT uptake, increasing t(1/2) by approximately 200%. No evidence of stereospecificity was seen in BSTV: both isomers had small effects (+)- and (-)-ketamine (100 microM) increasing NA efflux by 43+/-10% (n=7, P<0.001) and 29+/-8% (n=7, P<0.001), respectively. The isomers also had identical effects on NA uptake, each increasing uptake t(1/2) by approximately 100%. In summary, our data show that the optical isomers of ketamine have strikingly different stereospecificity for the monoamine systems and one might predict, therefore, a different psychotomimetic potential.     

The depolarization-induced release of cholecystokinin C-terminal octapeptide (CCK-8) from rat synaptosomes and brain slices

Studies on the subcellular distribution of immunoreactive cholecystokinin (CCK) in homogenates of rat cerebral cortex showed that approximately 95% was associated with particulate fractions, including presynaptic terminals (synaptosomes). Chromatography of extracts of tissue and medium from incubated synaptosomes revealed that this material was almost exclusively in the form of COOH-terminal octapeptide (CCK-8), very little CCK-33 being present. There was a wide range of CCK-8 concentrations in synaptosomes from different brain regions (cortex > striatum ? hypothalamus > brain stem). Cerebral cortex synaptosomes were incubated in vitro and showed a complex pattern of CCK-8 release with varying concentrations of tissue: amounts in the medium rose rapidly with increasing synaptosome concentrations, then fell to a plateau at higher tissue values. A mechanism for the rapid disposal of extracellular CCK-8 was associated with synaptosomal fractions. Depolarization-induced (high K⁺) release of CCK-8 was observed with cortex and corpus striatum synaptosomes. A rapid and reversible enhancement of CCK-8 release from cortex slices was observed in response to elevated K⁺. Veratrine also released CCK-8 from cortex slices, although this was not reversible. Stimulus-induced release of CCK-8 from synaptosomes and slices required extracellular Ca²⁺. The storage, release and degradation of CCK-8 by nerve-endings suggest a synaptic function for this peptide.     

Involvement of noradrenaline transporters in S-nitrosocysteine-stimulated noradrenaline release from rat brain slices: existence of functional Na(+)-independent transporter activity

Noradrenaline (NA) can be released by both exocytosis and by the membrane transporter responsible for transmitter uptake. Previously, we reported that S-nitrosocysteine (SNC), an S-nitrosothiol, stimulated [3H]NA release from the rat hippocampus. In this study, we investigated the involvement of the NA transport system in SNC-stimulated NA release from rat brain (cerebral cortex and hippocampus) slices. [3H]NA release by SNC in normal Na(+) (148 mM)-containing buffer from both slices was slightly, but significantly, inhibited by 1 microM desipramine, an NA transporter inhibitor. [3H]NA release in low Na(+) (under 14 mM)-containing buffer was inhibited by over 50% by desipramine. [3H]NA release by tyramine from both slices in normal and low Na(+) buffer was almost completely inhibited by desipramine. [3H]NA uptake into cerebral cortical slices was observed in low Na(+) buffer at 20-30% of normal Na(+) buffer levels. [3H]NA uptake in both normal and low Na(+) buffers was inhibited by desipramine and by SNC. Although [3H]NA uptake in normal Na(+) buffer was almost completely inhibited by 500 microM ouabain, the uptake in low Na(+) buffer was resistant to ouabain. These findings suggest the existence of a functional Na(+)-independent NA transport system and that SNC stimulates NA release at least partially via this system in brain slices.     

Action of neurotropic substances on gamma-aminobutyric acid release from brain synaptosomes during K+ depolarization

The effects of aminazine (0.25 mM), phthoracyzine (0.5 mM), trifluperidole (0.5 mM) and imipramine (0.5 mM) on GABA release from rat brain synaptosomes depolarized with K+ (50 mM) were investigated. Incubation of synaptosomes with aminazine led to a 2-fold and that with phthoracyzine, trifluperidole and imipramine to a 1.5-fold increase in GABA release from synaptosomes as compared with its basic level. The raising of K+ in the incubation medium to 50 mM brought about a 2-fold augmentation of GABA release. Exposure of synaptosomes to drugs and a higher K+ concentration at a time did not change GABA release as compared to its basic level. Introduction into the incubation medium of the Ga-ionophore A23187 together with 50 mM K+ and trifluperidole or with K+ and imipramine led to the same increase in GABA release from synaptosomes as that produced by the psychotropic drugs as regards native synaptosomes. It is assumed that the lack of the influence of the psychotropic drugs under study of GABA release from synaptosomes depolarized with K+ is caused by blockade of synaptic membrane conductibility for Ca2+.     

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.     

PnTx3-6 a spider neurotoxin inhibits K+-evoked increase in [Ca2+](i) and Ca2+-dependent glutamate release in synaptosomes

The present experiments investigated the effect of a neurotoxin purified from the venom of the spider Phoneutria nigriventer. This toxic component, P. nigriventer toxin 3-6 (PnTx3-6), abolished Ca(2+)-dependent glutamate release with an IC(50) of 74.4nM but did not alter Ca(2+)-independent secretion of glutamate when brain cortical synaptosomes were depolarized by KCl (33mM). This effect was most likely due to interference with the entry of calcium through voltage activated calcium channels (VACC), reducing the increase in the intrasynaptosomal free calcium induced by membrane depolarization with an IC(50) of 9.5nM. We compared the alterations induced by PnTx3-6 with the actions of toxins known to block calcium channels coupled to exocytosis. Our results indicate that PnTx3-6 inhibition of glutamate release and intrasynaptosomal calcium involves P/Q type calcium channels and this toxin can be a valuable tool in the investigation of calcium channels.     

Identification of a third isoform of Na+, K+-ATPase activity in rat brain synaptosomes

³H-ouabain binding and ouabain-inhibitable ⁸⁶Rb⁺ (K⁺) uptake were investigated as a means to identify a third isoform of Na⁺, K⁺-ATPase in crude synaptosome preparations. The specific binding of low concentrations (10 nM and 1 uM) of ³H-ouabain, in crude synaptosome preparations, was markedly inhibited by K⁺ (0.5–5 mM). Accordingly, ⁸⁶Rb⁺ (K⁺) uptake, in the presence of 5 mM K⁺ was not sensitive to inhibition by low concentrations (10⁻¹¹–10⁻⁷ M) of ouabain. Higher concentrations (10⁻⁶–10^−2.6 M) of ouabain resulted in a biphasic inhibition of K⁺ uptake, which distinguished the activities of the presumed alpha 2 and alpha 1 isozymes of Na⁺, K⁺-ATPase. Reduction of K⁺ (1.25 mM and 0.5 mM) in the incubation, resulted in the observation of a third component of ouabain- sensitive K⁺ uptake. This Na⁺, K⁺-ATPase activity, which was defined, pharmacologically, as very sensitive (VS) to ouabain, exhibited IC₅₀s of 3.6 nM and 92 nM at 1.25 mM K⁺ and 0.5 mM K⁺, respectively. Inhibition of ouabain binding and VS-dependent K⁺ uptake, at a high, physiological cocentration (5 mM) of K⁺, suggests that VS may be an inactive isoform of brain Na⁺, K⁺-ATPase under resting conditions.     

Osmotic regulation of furosemide-sensitive K+ (86Rb+) transport in rat brain synaptosomes]

In the rat brain synaptosomes the furosemide-sensitive component of 86Rb+ uptake constituted 30.8% of the total uptake in the medium containing 132 mM Na+. A decrease in the medium tonicity from 310 to 230 mOsm increased the rate of 86Rb+ uptake from 2.38 +/- 0.58 to 7.12 +/- 0.52 nMoles/mg of protein/min.     

Clostridium neurotoxins influence serotonin uptake and release differently in rat brain synaptosomes

Clostridium neurotoxins produce inhibition of both basal and K(+)-evoked serotonin release in rat brain synaptosomes. To produce these effects, tetanus toxin (TeTx), as well as botulinum neurotoxin type A (BoNT/A), added to brain synaptosomes, must be incubated at 37 degrees C over a long interval (hours). This serotonin exocytosis inhibition was abolished with previous treatment with specific Zn2(+)-metalloprotease inhibitors. Nevertheless, a short incubation time produces different behavior of the indicated neurotoxins: TeTx significantly blocks the sodium-dependent, high-affinity serotonin uptake, whereas a small increase of this uptake was found with BoNT/A. Both Zn2(+)-metalloprotease active fragments, light chains of TeTx and BoNT/A, are unable to reproduce the block of the serotonin uptake, whereas the C-terminal portion of the TeTx heavy chain (Hc-TeTx), which binds specifically to the target tissue, inhibited the serotonin uptake in a dose-dependent manner. The IC50 of Hc-TeTx ranges from 0.62 to 2.08 nM. Binding of [3H]imipramine and [3H]serotonin did not change after toxin treatments, which indicates that these clostridium neurotoxins do not act on the serotonin high-affinity site at the serotonin transporter or at other serotonin high-affinity sites. These results could indicate that TeTx and Hc-TeTx bind to different targets than BoNT/A in the plasma membrane.     

Histochemical demonstration of transmitter release from noradrenaline,dopamine and 5-hydroxytryptamine nerve terminals in field stimulated rat brain slices

Summary The effect of electrical field stimulation on noradrenaline (NA), dopamine (DA) and 5-hydroxytryptamine (5-HT) nerve terminals in rat brain slicesin vitro was investigated. Slices prepared from the cerebral cortex or the neostriatum were incubated in physiologic buffer for 30 min and then superfused by buffer and stimulated by an electrical field (biphasic pulses, 10 Hz, 12 mA, 2 ms) for various time periods. The effect of the stimulation was studied at the cellular level with the histochemical fluorescence technique of Falck and Hillarp. The transmitter overflow into the superfusing buffer caused by the stimulation was studied with isotope technique. Cerebral Cortex NA Nerve Terminals. Stimulation caused release of NA from cortical NA nerve terminals. Already after 2 min stimulation a slight decrease of the fluorescence intensity of the nerve terminals could be found. Stimulation for 15 to 30 min greatly reduced the fluorescence intensity. In slices preincubated with³H-NA the stimulation-induced overflow of tritium during 2 min stimulation was about 15% (i.e. 15% of the tissue tritium content was overflowing into the superfusing buffer in response to stimulation for 2 min). During prolonged stimulation there was a considerable decline of the tritium efflux. Cerebral Cortex 5-HT Nerve Terminals. The 5-HT-analogue 6-hydroxytryptamine (6-HT) which is readily taken up into 5-HT nerve terminals was used to permit good visualization of these nerve terminals. Uptake of 6-HT into cortical NA nerve terminals was prevented by preincubation with 6-hydroxydopamine (6-OH-DA) or protriptyline. Stimulation for 15 to 30 min reduced the fluorescence intensity of the 5-HT nerve terminals. In slices preincubated with³H-5-HT the stimulation-induced overflow of tritium during 2 min stimulation was about 5%. The tritium efflux slowly decreased during continuous stimulation. Neostriatal DA Nerve Terminals. In slices frozen directly after preparation an intense diffuse fluorescence could be seen. After incubation in drug-free buffer at 37° C the fluorescence was localized in the varicosities of the DA nerve terminals. The central parts of the slices almost completely lacked specific fluorescence, while the outer zones were brightly fluorescent. No clear reduction of the fluorescence intensity of the DA nerve terminals in the outer zone could be observed after stimulation for 30 min. In slices preincubated with³H-DA the stimulation-induced overflow of tritium during 2 min stimulation was about 2%. The tritium efflux slowly decreased during continuous stimulation.It is suggested that the differences in release between the various nerve terminal systems foundin vitro reflect differences in transmitter release occurringin vivo. The comparably high release of NA per impulse from the cortical NA nerve terminals implicate that the discharge rate of these neuronsin vivo is very low.This investigation has been supported by grants from the Swedish Medical Research Council (B72-14X-2330-05A) and Magnus Bergwall's Foundation.The author is greatly indebted to Mrs. Annika Hamberger for her skillful technical assistance. For generous supplies of drugs thanks are due to Hässle, Göteborg, Sweden, through Dr. H. Corrodi (6-HT, 6-OH-DA and H44/68).     

Effect of leu-enkephalin and the delta-sleep-inducing peptide, M (DSIP), on endogenous noradrenaline release by brain synaptosomes in the rat

Fluorometry was employed to measure the noradrenaline (NA) content in rat brain synaptosomes depending on the duration of incubation, depolarization effects (40 mM KCl or 1.5 mM ouabain), composition of the synaptosomal fraction and concentration of the peptides. The 10-minute incubation in a potassium medium of a suspension of light synaptosomes was used as an optimal test-system for studying the peptide action. Leu-enkephalin inhibited the depolarization-induced NA release. The effect was abolished by naloxone. The delta-sleep-inducing peptide (DSIP) did not influence the neurotransmitter release at concentrations of 10(-8)-10(-5) M. A mixture of amino acids imitating the amino acid composition of the DSIP influenced spontaneous release of NA. This effect is discussed in connection with the physiological action of the peptide on its intraventricular injection.     

Charybdotoxin blocks both Ca-activated K channels and Ca-independent voltage-gated K channels in rat brain synaptosomes

Charybdotoxin (ChTX), a 4.3 kDa polypeptide toxin from the venom of the scorpion Leiurus quinquestriatus, blocks both a Ca-activated K channel (IC₅₀ ≈ 15 nM) and a Ca-independent voltage-gated K channel (IC₅₀ ≈ 40 nM) in rat brain synaptosomes. These results indicate that in this preparation ChTX is not specific for the Ca-activated K channel and suggest that there may be structural homology among the toxin-binding sites on various types of K channels.