Mechanism of aminopyridine-induced release of [3H]dopamine from rat brain synaptosomes.

1. Aminopyridines (APs) induced the release of [3H]dopamine (3H-DA) from rat synaptosomal preparations. 2. 4-AP and 3,4-DAP were of equal efficacy in inducing release of 3H-DA; 3-AP, 2-AP and 2,6-AP were less active; pyridine and pyridine-4-carboxylamide were inactive. 3. Cd2+ was more effective in inhibiting 4-AP-induced release of 3H-DA (IC50 approximately 4 microM) than Co2+ and Ni2+ (IC50s approximately 500 microM). 4. While 4-AP increased the 45Ca2+ content of whole synaptosomal preparations, no effect of 4-AP on 45Ca2+ content was observed in lysed synaptosomal preparations. 5. 4-AP-induced 45Ca2+ uptake was inhibited by Cd2+, Ni2+ and Co2+ in concentration ranges similar to those inhibiting 3H-DA release.     

Ca(2+)-dependent inhibition of Ca(2+)-independent contraction in uterine smooth muscle.

Uterine smooth muscle of the rat shows Ca(2+)-independent contraction in response to oxytocin in Ca(2+)-free medium. Micromolar Ca2+ inhibits this contraction. We now tested whether Ca2+ itself is the cause of this inhibition. The ratio of fura-2 fluorescence, the indicator of the intracellular level of Ca2+, was increased in parallel with the degree of inhibition by Ca2+. When inhibition was elicited by Ca2+, EGTA released the inhibition. Comparison of the dose-response curve for oxytocin in Ca(2+)-free solution and that in the medium with 1 microM Ca2+ showed that the inhibition by Ca2+ is non-competitive. EGTA chelation of the intracellular Ca2+ by loading of EGTA as its acetoxymethylester resulted in diminution of inhibition by Ca2+. EGTA suppressed the Ca(2+)-induced contraction but did not affect Ca(2+)-independent contraction. It is concluded that the inhibition is induced by intracellular Ca2+ itself.     

Influence of stimulation on Ca(2+) recruitment triggering [3H]acetylcholine release from the rat motor-nerve endings

The influence of rat phrenic nerve stimulation frequency (5-50 Hz) and of pulse duration (0.04-1 ms) on Ca(2+) mobilization triggering [3H]acetylcholine release was investigated. The P-type voltage-dependent Ca(2+) channel (VDCC) blocker, omega-agatoxin IVA (100 nM), decreased [3H]acetylcholine release evoked by pulses of 0. 04-ms duration delivered at 5 Hz frequency. When the stimulus pulse duration was increased to 1 ms (5 Hz frequency) or the stimulation frequency to 50 Hz (0.04-ms duration), inhibition of [3H]acetylcholine release became evident after blockade of L-type VDCC, with nifedipine (1 microM), and/or depletion of thapsigargin-sensitive internal stores. The inhibitory effect of thapsigargin (2 microM) was still observed in Ca(2+)-free medium. Neither omega-conotoxin GVIA (1 microM) nor omega-conotoxin MVIIC (150 nM) modified neurotransmitter release. The results suggest that, depending on the stimulus paradigm, both internal (thapsigargin-sensitive) and external (either P- or L-type channels) Ca(2+) pools can be mobilized to promote acetylcholine release from motor nerve terminals.     

Effects of mercuric chloride on [3H]dopamine release from rat brain striatal synaptosomes

Electrophysiological studies employing amphibian neuromuscular preparations have shown that mercuric chloride (HgCl2) in vitro increases both spontaneous and evoked neurotransmitter release. The present study examines the effect of HgCl2 on the release of [3H]dopamine from synaptosomes prepared from mammalian brain tissue. Mercuric chloride (3-10 microM) produces a concentration-dependent increase in spontaneous [3H]dopamine release from "purified" rat striatal synaptosomes, in both the presence and absence of extra-synaptosomal calcium. The effects of HgCl2 on transmitter release from amphibian neuromuscular junction preparations resemble those produced by the Na+, K+-ATPase inhibitor ouabain. Experiments were performed to determine whether the HgCl2 effects on mammalian synaptosomal dopamine release are a consequence of Na+, K+-ATPase inhibition. Na+, K+-ATPase activity in lysed synaptosomal membranes is inhibited by HgCl2 (IC50 = 160 nM). However, mercuric chloride in the presence of 1 mM ouabain still increased [3H]dopamine release. The specific inhibitor of Na+-dependent, high-affinity dopamine transport, RMI81,182 inhibited ouabain-induced [3H]dopamine release whereas it had no effect on HgCl2-induced [3H]dopamine release. These data suggest that augmentation of spontaneous [3H]dopamine release by HgCl2 probably is not mediated by an inhibition of Na+, K+-ATPase and HgCl2 does not act directly on the dopamine transporter.     

Differential control of Ca2+-dependent [3H]noradrenaline release from rat brain slices through presynaptic opiate receptors and alpha-adrenoceptors

The inhibitory actions of the Ca2+ antagonist Cd2+, morphine and noradrenaline (exogenously added + endogenously released) on electrically evoked release of [3H]noradrenaline from superfused rat neocortical slices were strongly reduced when release was enhanced by 4-aminopyridine. In the presence of 4-aminopyridine the release inhibiting effects of these drugs were restored by lowering the extracellular Ca2+ concentration. When release was enhanced by prolonging the pulse duration, only the release inhibiting effect of noradrenaline was reduced but the effects of Cd2+ and morphine were unchanged. Irrespective of the pulse duration, blockade of presynaptic alpha-adrenoceptors with phentolamine did not affect the release inhibiting effects of Cd2+ and morphine. The inhibitory effects of morphine and noradrenaline remained unchanged in Cl--free medium. Furthermore, these drugs strongly reduced the [3H]noradrenaline release induced by 20 mM K+ in the presence of tetrodotoxin. The results suggest that activation of presynaptic opiate-receptors inhibits Ca2+ entry through voltage-sensitive Ca2+ channels, whereas presynaptic alpha-adrenoceptors affect a step in the secretory process subsequent to Ca2+ influx. Moreover, the involvement of (direct) changes in Na+, K+ or Cl- permeability appears unlikely for both receptor systems.     

Effect of morphine in vivo on uptake of [3H]choline and release of [3H]acetylcholine from rat striatal synaptosomes

The effect of morphine on the rat striatal cholinergic system was investigated in vitro by measuring the rates of [³H]choline uptake and [³H]acetylcholine release in striatal synaptosomes after in vivo injections of morphine sulfate. Morphine caused a 50 per cent increase in the V_max of [³H]choline uptake. Although a concomitant increase was also measured in the amount of [³H] acetylcholine released, it could be explained by the previous increase in uptake. It is suggested that morphine had an overall stimulatory effect on the striatal cholinergic system which may be a transynaptic phenomenon rather than a direct effect on the cholinergic cell.     

The effects of depolarizing agents and neuropeptides on dopamine release from striatal synaptosomes

Synaptosomes (isolated nerve endings) from rat corpus striatum responded to several depolarizing agents by releasing dopamine. Among these agents were KCl, glutamic acid, ouabain, and veratrine. Substance P hexapeptide (SP6) also caused dopamine release, but the magnitude of this effect was small and variable. A number of other neuropeptides (cholecystokinin 1–8, des-Tyr-γ-endorphin, Leu⁵-β-endorphin and substance P) did not alter dopamine release. SP6-induced dopamine release may result from substance P receptor-induced depolarization; however, the lack of robustness of the response in this preparation makes it unsuitable for studying agonists and antagonists of substance P.     

Nicotine-evoked [3H]5-hydroxytryptamine release from rat striatal synaptosomes

The aim of this study was to characterize the pharmacology of presynaptic nicotinic cholinoceptors (nAChRs) that modulate release of 5-hydroxytryptamine (5-HT) from superfused rat brain synaptosomes preloaded with [3H]5-HT. Nicotine increased 5-HT release from striatal synaptosomes (maximally by 15-30%) but not from cerebral cortex or hippocampal synaptosomes. Release of striatal 5-HT was increased in a concentration-dependent manner by nicotine, epibatidine, cytisine, and ACh (with added esterase inhibitor and muscarinic antagonist). Respective EC50 values were: 0.5, 0.003, 0.1 and 0.7 microM. The maximal effect of each agonist was virtually completely blocked by a high concentration of the insurmountable nicotinic antagonist mecamylamine; at a higher concentration of epibatidine (3 microM), a mecamylamine-insensitive effect was revealed. Nicotine, ACh and epibatidine appeared equally efficacious, whereas cytisine was of lower efficacy (60-70% of ACh). Release evoked by a half-maximal concentration of nicotine was inhibited by the nicotinic antagonists dihydro-beta-erythroidine (IC50 0.04 microM) and methyllycaconitine (IC50 0.06 microM). Nicotine-evoked 5-HT release was not reduced by tetrodotoxin given in a concentration that blocked veratridine-evoked release. These findings provide functional evidence for a direct action of nicotine on 5-HT neurons in the brain. The presynaptic nAChRs that modulate striatal 5-HT release appear to possess a novel pharmacological profile.     

Suppression by cholinesterase inhibition of a Ca(2+)-independent efflux of [3H]acetylcholine from the neuromuscular junction of the isolated rat diaphragm.

Endplate preparations of the left rat hemidiaphragm were incubated with [3H]choline to label neuronal acetylcholine stores. Elevation of the concentration (13.5-135 mmol/l) of extracellular potassium chloride (KCl) stimulated the release of [3H]acetylcholine in a concentration-dependent manner. KCl (27 mmol/l) still caused a significant efflux of [3H]acetylcholine in a Ca(2+)-free medium. Inhibitors of cholinesterase (physostigmine, diisopropylfluorophosphate) suppressed by 80% this Ca(2+)-independent efflux of [3H]acetylcholine. Vesamicol (10 mumol/l), the blocker of the vesicular acetylcholine carrier, also suppressed the stimulated, Ca(2+)-independent efflux of [3H]acetylcholine. The inhibitory effect of physostigmine was not prevented by muscarine or nicotine receptor antagonists, but the inhibitory effect was lost when the stimulus strength was increased (81 mmol/l KCl). The present experiments showed cholinesterase inhibition to suppress a Ca(2+)-independent efflux of [3H]acetylcholine, probably by interference with a membrane-bound acetylcholine carrier.     

Ca(2+)-induced translocation of protein kinase C during Ca(2+)-dependent histamine release from beta-escin-permeabilized rat mast cells.

When rat mast cells were cultured for a short period in plastic dishes and adhering cells were permeabilized with beta-escin and exposed to Ca2+ at concentrations higher than 10(-7) mol/l, histamine release was induced dose-dependently. Protein kinase C (PKC) activity in the crude extracts obtained from adhering mast cells was induced in the presence of Ca2+, phospholipid and diacylglycerol. The apparent Km value of PKC for Ca2+ was 0.33 mumol/l, and this Ca2+ concentration was equivalent to that which can elicit half the maximum of the Ca(2+0-induced histamine release. After permeabilization, approximately 80% of the total PKC activity remained in the cytosolic fraction. In the resting state, 95% of the total PKC activity was detected in the soluble fraction, and the rest was detected in the membrane fraction. When permeabilized mast cells were incubated with Ca2+ at micromolar concentrations, which are effective in releasing histamine, the total PKC activity did not change. However, the translocation of PKC took place from the cytosolic fraction to the membrane fraction, corresponding to Ca2+ concentrations in the medium. When the crude PKC extract of mast cells was incubated with phospholipid vesicles and centrifuged, the PKC activity in the supernatant was diminished; the amount of PKC binding to the vesicles was dependent upon Ca2+ concentrations in the medium. Calphostin C, a potent PKC inhibitor, interacts with PKC in a noncompetitive manner, and it does not inhibit Ca(2+)-induced translocation of PKC. It can be concluded that PKC is translocated into the cell membrane along with an increase in intracellular Ca2+ concentrations and the subsequent activation of PKC may be crucial for the process leading to histamine release.     

Cyclothiazide unmasks AMPA-evoked stimulation of [3H]-L-glutamate release from rat hippocampal synaptosomes.

The effect of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) on Ca(2+)-sensitive, tetrodotoxin (TTX)-insensitive K(+)-stimulated [3H]-L-glutamate release from rat hippocampal synaptosomes was determined. AMPA in the presence, but not in the absence of cyclothiazide, a drug which blocks AMPA receptor desensitization, elicited a dose-dependent increase in K(+)-stimulated [3H]-L-glutamate release but had no effect on basal release. The AMPA/cyclothiazide stimulation was blocked by CNQX and by GYKI 52466, an antagonist at the cyclothiazide site. These results indicate that AMPA receptors are present on presynaptic terminals and suggest that they may play a role in the regulation of neurotransmitter release.     

Effect of 1-aminocyclopropanecarboxylic acid on N-methyl-D-aspartate-stimulated [3H]-noradrenaline release in rat hippocampal synaptosomes.

1. The effect of 1-aminocyclopropanecarboxylic acid (ACPC), a partial agonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor complex that exhibits neuroprotective, anxiolytic and antidepressant-like actions, was investigated in a functional assay for presynaptic NMDA receptors. 2. NMDA (100 microM) produced a 36% increase of tritium efflux above basal efflux in rat hippocampal synaptosomes preincubated with [3H]-noradrenaline ([3H]-NA), reflecting a release of tritiated noradrenaline. This effect was prevented by 10 microM 7-chlorokynurenic acid, an antagonist of the glycine site of the NMDA receptor. 3. Glycine enhanced the effect of NMDA with Emax and EC50 values of 84 +/- 11% and 1.82 +/- 0.04 microM, respectively. ACPC potentiated the effect of NMDA on tritium overflow with a lower EC50 (43 +/- 6 nM) and a lower maximal effect (Emax = 40 +/- 9%) than glycine. Furthermore, ACPC (0.1 microM) shifted the EC50 of glycine from 1.82 microM to > or = 3 mM. 4. These results show that ACPC can reduce the potentiation by glycine of NMDA-evoked [3H]-NA release and hence, may act as an antagonist at the glycine site of presynaptic hippocampal NMDA receptors when the concentration of glycine is high.     

Binding of potassium [3H]embelate to rat brain synaptosomes.

[3H]Embelate (potassium salt of 2,5-dihydroxy-3-undecyl-1,4- benzoquinone), a new analgesic compound, showed a specific and saturable binding in rat brain synaptosomes which was not influenced by naloxone and morphine. The binding characteristics were correlated with its non-narcotic central analgesic action.     

Cannabinoid receptor agonists inhibit Ca(2+) influx to synaptosomes from rat brain

We examined the effects of cannabinoid receptor agonists on (45)Ca(2+) uptake in rat brain synaptosomes. A cannabinoid receptor agonist, (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-merpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone (WIN 55212-2) dose-dependently inhibited (45)Ca(2+) uptake in rat synaptosomes. Only an endogenous cannabinoid receptor agonist, anandamide, dose-dependently inhibited (45)Ca(2+) uptake in rat synaptosomes, but not an endogenous cannabinoid receptor agonist, palmitoylethanolamide. Only a cannabinoid CB1 antagonist, [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride] (SR 141716A), reversed the inhibitory effect of these WIN 55212-2 and anandamide on (45)Ca(2+) uptake in rat synaptosomes, but not a cannabinoid CB2 receptor antagonist, [N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide] (SR 144528). The inhibitory effects of WIN 55212-2 and anandamide on (45)Ca(2+) uptake in rat synaptosomes were reversed by the pretreatment of a voltage-sensitive A-type K(+) channel blocker, dendrotoxin, but no other type of K(+) channel blockers, i.e. iberiotoxin, charybdotoxin or glibenclamide. These findings suggest that cannabinoid receptors inhibit Ca(2+) influx into rat brain nerves via the activation of CB1 receptors and the opening of voltage-sensitive A-type K(+) channels.     

Effect of cocaine and 5-HT3 receptor antagonists on 5-HT-induced [3H]dopamine release from rat striatal synaptosomes.

The effect of serotonin (5-HT) on the release of tritium from striatal synaptosomes previously loaded with [3H]dopamine ([3H]DA) was studied. 5-HT stimulated both the spontaneous and Ca(2+)-evoked efflux of tritium in a concentration-dependent manner. This effect was not mimicked by the non-selective 5-HT agonist, d-lysergic acid diethylamide. Further, the stimulatory effects of 5 muM 5-HT were unaffected by the selective 5-HT3 receptor antagonists, MDL-72222 and GR-38032F. On the other hand, cocaine and the selective DA uptake inhibitor, nomifensine completely antagonized the effect of 5 muM 5-HT on spontaneous tritium efflux with IC50 values of 0.2 and 0.09 muM, respectively. The effect of 5-HT on Ca(2+)-evoked tritium efflux was also blocked by these DA uptake inhibitors, albeit at somewhat higher concentrations. These data support the hypothesis that 5-HT induces the release of DA from striatal nerve terminals via a mechanism involving the transport of 5-HT into the dopaminergic terminal, rather than by activating 5-HT3 receptors as has been proposed to account for the effect of 5-HT observed in striatal slices.     

Modulation of Ca(2+)-dependent and Ca(2+)-independent miniature endplate potentials by phorbol ester and adenosine in frog

Phorbol esters and adenosine modulate transmitter release from frog motor nerves through actions at separate sites downstream of calcium entry. However, it is not known whether these agents have calcium-independent sites of action. We therefore characterised calcium independent miniature endplate potentials (mepps) generated in response to 4-aminoquinaldine (4-AQ(A)) and then compared the modulation of these mepps by phorbol esters and adenosine with that of normal calcium dependent mepps. Application of 30 microM 4-AQ(A) resulted in the appearance of a population of mepps with amplitudes greater than twice the total population mode (mepp(>2M)). In the presence of 4-AQ(A), K(+) depolarisation or hypertonicity increased the numbers of normal amplitude mepps (mepp(N)) but had no effect on the frequency of mepp(>2M) events, suggesting that mepp(>2M) are not dependent on calcium. Treatment with the botulinum toxin (Botx) fractions C, D, or E (which selectively cleave syntaxin, synaptobrevin and SNAP-25, respectively) produced equivalent reductions in both normal and 4-AQ(A) induced mepps, suggesting that both mepp populations have equal dependence on the intact SNARE proteins.Phorbol dibutyrate (PDBu, 100 nM) increased the frequencies of both populations of mepps recorded in the presence of 4-AQ(A). Adenosine (25 microM) selectively reduced the numbers of mepp(N) with no effect on the frequency of mepp(>2M) events. These results suggest that mepp(>2M) events released in response to 4-AQ(A) are dependent on intact forms of syntaxin, synaptobrevin and SNAP-25, but unlike mepp(N) are independent of a functional calcium sensor. The selective action of adenosine, to reduce the numbers of normal amplitude mepps without effecting the frequency of mepp(>2M) events, suggests that adenosine normally inhibits transmitter release through a mechanism that is dependent on the presence of a functional calcium sensor.     

Influence of mercury on uptake of [3H]dopamine and [3H]norepinephrine by rat brain synaptosomes

Mercuric compounds have been shown to alter several membrane-bound enzymes and associated receptor activities. The present studies were initiated to investigate the in vitro effects of mercuric chloride (HgCl2) and methylmercury chloride (CH3HgCl) on the uptake of [3H]dopamine (3HDA), [3H]norepinephrine (3HNE), and Na+, K+-ATPase in rat brain synaptosomes. Brain synaptosomes were prepared by the ficoll-sucrose gradient method from normal, adult male Sprague-Dawley rats, weighing approx. 200 g. The effect of mercury on Na+, K+-ATPase was determined by using a coupled enzymatic method. Uptake of DA and NE by brain synaptosomes was determined by filtration in the presence and absence of 0-30 microM HgCl2 and 0-100 microM CH3HgCl. A parallel inhibition in the synaptosomal uptake of 3HDA and 3HNE, and the activity of the synaptosomal membrane Na+, K+-ATPase, was observed in both mercuric chloride and methylmercury treatments. The mercury compounds also significantly inhibited the mitochondrial ATPase (Mg2+-oligomycin-sensitive ATPase). The inhibitory influences of the toxins were concentration-dependent. The results suggest that the mercury compound mediated decrease in DA and NE uptake in brain synaptosomes may be related to the inhibition of Na+, K+-ATPase by the same toxins.     

Effect of psychotropic substances on the uptake of [H3]-gamma-aminobutyric acid by rat brain synaptosomes]

The in vitro effects of some neuroleptics and antidepressants on the accumulation of [3H]/-GABA by the synaprosomes of the rat brain cortex were studied. Chloropromazine, trifluoperazine, fluphenazine, perphenazine, thioproperazine, haloperidol, trifluperidol, droperidol, imipramine, haloanison and phthoracyzine were found (in order of a decreasing activity) to inhibit the [3H]/-GABA uptake of synaptosomes. Neuroleptics, except for a new drug carbidine, proved to be more potent inhibitors than antidepressants are. The tranquilized diazepan failed to have any effect on the [3H]/-GABA uptake. The rats synaptosomes treated with chlorpromazine and imipramine were found to display a decreased ability to accumulate [3H]/-GABA. The suppressive effect of psychotropic agents on the [3H]/-GABA uptake by synaptosomes is suggested to be due, at least partly, to their known inhibitory influence on the Na+, K+-dependent ATPase.