Cerebral Cortical Synaptosomes Research Articles

Effects of ovariectomy and estradiol benzoate on high affinity choline uptake, ACh synthesis, and release from rat cerebral cortical synaptosomes

Several presynaptic processes were studied in cerebral cortical synaptosomes prepared from intact adult female rats or from ovariectomized animals that received 3 subcutaneous injections of either estradiol benzoate (10 μg/kg) or vehicle. Injections were given 1/day, every other day, and animals were sacrificed 1 h after the last injection. High affinity choline uptake and coupled acetylcholine (ACh) synthesis were reduced by ovariectomy, and restored to control levels by the estradiol benzoate injections. In contrast, low affinity choline uptake and depolarization-induced [ 3H]ACh release were unaffected by either ovariectomy or estradiol benzoate injections. These results suggest that changes in estradiol levels may alter the high affinity transport process regulating ACh synthesis in this tissue.

Effect of 5-(3-[4-(4-fluorophenyl)-1-piperazinyl]-propoxy)indan (BP-528) on benzodiazepine receptor bindings and gamma-aminobutyric acid release.

The action of a new type of anti-anxiety compound, 5-(3-[4-(4-fluorophenyl)-1-piperazinyl]-propoxy)indan (BP-528), was tested on benzodiazepine receptor bindings and on [3H]-GABA release. BP-528 did not alter [3H]-diazepam binding to rat cerebral cortical and hippocampal membranes either in the presence or absence of GABA; and the binding of [3H]-propyl-beta-carboline-3-carboxylate at low concentration (0.04 nM), which labels only the type I benzodiazepine receptor, was not changed by BP-528. BP-528 did not interact with the GABA-benzodiazepine receptor complex, which is related to the anti-anxiety activity of benzodiazepines. This compound affected neither GABA binding nor GABA uptake. Ten micromolar BP-528 depressed high K+-induced [3H]-GABA release from preloaded rat hippocampal slices. However, the same concentration of BP-528 also inhibited high K+-induced calcium uptake by rat cerebral cortical synaptosomes.

Effect of 5-{3-[4-(4-Fluorophenyl)-1-Piperazinyl]-Propoxy}indan (BP-528) on Benzodiazepine Receptor Bindings and γ-Aminobutyric Acid Release

The action of a new type of anti-anxiety compound, 5-{3-[4-(4-fluorophenyl)-1-piperazinyl]-propoxy}indan (BP-528), was tested on benzodiazepine receptor bindings and on [3H]-GABA release. BP-528 did not alter [3H]-diazepam binding to rat cerebral cortical and hippocampal membranes either in the presence or absence of GABA; and the binding of [3H]-propyl-β-carboline-3-carboxylate at low concentration (0.04 nM). which labels only the type I benzodiazepine receptor, was not changed by BP-528. BP-528 did not interact with the GABA-benzodiazepine receptor complex, which is related to the antianxiety activity of benzodiazepines. This compound affected neither GABA binding nor GABA uptake. Ten micromolar BP-528 depressed high K+-induced [3H]-GABA release from preloaded rat hippocampal slices. However, the same concentration of BP-528 also inhibited high K+-induced calcium uptake by rat cerebral cortical synaptosomes.

Antibodies directed against ubiquitin inhibit high affinity [3H]choline uptake in rat cerebral cortical synaptosomes.

Sodium-dependent [3H]choline uptake and coupled [3H]acetylcholine synthesis were inhibited in rat cerebral cortical synaptosomes in a dose- (1-10 micrograms/ml) and time-dependent manner by affinity-purified antibodies directed against ubiquitin (anti-Ub). Neither sodium-independent [3H]choline uptake nor [3H]acetylcholine release was affected by up to 10 micrograms/ml anti-Ub, indicating that the cholinergic terminals were not depolarized by the anti-Ub. Binding of anti-Ub to synaptosomes, as measured with 125I-protein A, was saturable and occurred over the same concentration range (1-10 micrograms/ml) at which uptake inhibition was observed. Although preimmune IgG bound to the synaptosome preparation to a greater extent and was apparently not readily saturable, this fortuitous binding was without effect on high affinity choline uptake and conversion to acetylcholine. The results suggest the presence of a ubiquitin-protein conjugate on the synaptosomal surface and a functional relationship between this protein conjugate and the sodium-dependent choline transport system.

Effects of different secretagogues and intracellular messengers on the muscarinic modulation of [3H]acetylcholine release.

We have investigated whether muscarinic receptors modulate the release of [3H]ACh elicited by secretagogues that act by different mechanisms in rat cerebral cortical synaptosomes. Oxotremorine (10 microM) reduced the calcium-dependent [3H]ACh release induced by mild K+-depolarization (10 and 15 mM K+), but not that by higher K+ concentrations. The ACh-release induced by A23187 (0.2-5 micrograms/ml), liposomes laden with 113 mM CaCl2, or 4-aminopyridine (1-10 mM) was not modulated by oxotremorine. Ouabain (100 microM)-induced release of [3H]ACh was reduced by oxotremorine in normal but not calcium-free KR, indicating that extracellular calcium-uptake but not Na+, K+-ATPase activity may be necessary for release-modulation. With respect to possible second messenger systems, dibutyrylcyclic AMP (0.1-2 mM), dibutyrylcyclic GMP (0.1-2 mM), forskolin (100 microM), and phorbol ester (0.3-3 micrograms/ml) were without effect on release or release-modulation. These results are consistent with an involvement of K+-channels and voltage-sensitive calcium-channels in the muscarinic release-inhibition process. They argue against an involvement of Na+, K+-ATPase, adenylate cyclase, guanylate cyclase, and phosphatidylinositol turnover in the release-modulation process.

Opposite effects of pyracetam and proline on release of3H-D-aspartic acid from rat cerebral cortical synaptosomes

Opposite effects of pyracetam and proline on release of3H-D-aspartic acid from rat cerebral cortical synaptosomes

Ca2+-independent and Ca2+ -dependent γ-aminobutyrate release occur from distinct compartments within guinea-pig cerebral cortical synaptosomes

Ca2+-independent and Ca2+ -dependent γ-aminobutyrate release occur from distinct compartments within guinea-pig cerebral cortical synaptosomes

Action of ascorbic acid on binding of3H-GABA and3H-glutamic acid to cerebral cortical synaptosomes in rats

Action of ascorbic acid on binding of3H-GABA and3H-glutamic acid to cerebral cortical synaptosomes in rats

Synaptosomes possess an exocytotic pool of glutamate.

There is increasing evidence that L-glutamate is a major excitatory neurotransmitter in the central nervous system. Immunocytochemical studies indicate that glutamate within nerve terminals may be concentrated in vesicles and glutamate-accumulating vesicles have recently been isolated. Exocytotic release of glutamate from synaptosomes (isolated nerve terminals) has not been convincingly demonstrated, however, and remains highly controversial. In order to study the kinetics of release of endogenous L-glutamate from guinea pig cerebral cortical synaptosomes we have devised a continuous enzymatic assay. This has enabled us to identify a pool, equivalent to 15-20% of the total synaptosomal glutamate, which is capable of rapid Ca2+-dependent exocytotic release.

Beta-nerve growth factor in developing mouse cerebral cortical synaptosomes: measurement by competitive radioimmunoassay and bioassay.

The existence of beta-nerve growth factor (beta NGF) in mouse cerebral cortical synaptosomal extracts was assessed using a newly developed radioimmunoassay and by bioassay. Displacement curves of labeled beta NGF in the radioimmunoassay by synaptosomal extracts were parallel to the beta NGF standard curves. In contrast, both mitochondrial and nonsynaptosomal extracts produced displacement curves in the beta NGF radioimmunoassay which were nonparallel to standard beta NGF. Intraventricular injections of purified beta NGF in amounts 5 and 100 ng/mouse (six to 120 times that of whole brain beta NGF concentrations) did not alter the endogenous synaptosomal beta NGF concentrations. Synaptosomal extracts contained beta NGF in relatively higher concentrations than the extracts of whole brain or cerebral cortex. Synaptosomal beta NGF levels undergo a developmental change. The beta NGF concentrations were highest in synaptosomal extracts of 12-day-old mouse cerebral cortex (the youngest age studied), and levels decreased with increasing age. Synaptosomal extracts of 12-day-old mouse cerebral cortex produced neurite outgrowth in the pheochromocytoma cell line 12 bioassay system and this response was completely inhibited by beta NGF antibodies. In the pheochromocytoma cell line 12 cell bioassay, synaptosomal extracts also caused cell-cell aggregation which was unaffected by beta NGF antibodies. These results provide evidence for the existence of beta NGF in relatively high concentration in nerve terminals of mouse cerebral cortex.

Parkinsonism-inducing neurotoxin, N-methyl-4-phenyl-1,2,3,6 -tetrahydropyridine: uptake of the metabolite N-methyl-4-phenylpyridine by dopamine neurons explains selective toxicity.

N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces neuropathological and clinical abnormalities in humans, monkeys, and mice that closely resemble idiopathic parkinsonism. N-Methyl-4-phenylpyridine (MPP+), a metabolite of MPTP formed by monoamine oxidase B, is accumulated into striatal and cerebral cortical synaptosomes by the dopamine and norepinephrine uptake systems, respectively, whereas MPTP itself is not accumulated. The potencies of drugs in inhibiting [3H]MPP+ or [3H]dopamine uptake into striatal synaptosomes are very similar, as are potencies in inhibiting [3H]MPP+ or [3H]norepinephrine uptake into cortical synaptosomes. The Km values for [3H]MPP+ uptake are 170 and 65 nM and the Vmax values are 2 and 0.1 nmol/g of tissue per min in rat striatum and cortex, respectively, similar to values for [3H]dopamine uptake, Autoradiography of accumulated [3H]MPP+ in slices of rat brain shows high densities in the caudate-putamen and nucleus accumbens. Furthermore, blockade of dopamine uptake by mazindol prevents MPTP-induced damage to nigrostriatal dopamine neurons, indicating that MPP+ concentration into dopamine neurons explains their selective destruction by MPTP.

Estradiol and progesterone potentiate adenosine's depressant action on rat cerebral cortical neurons

1. 1. The effects of 17β-estradiol and progesterone on the uptake of adenosine by rat cerebral cortical synaptosomes have been correlated with the ability of these gonadal steroids to potentiate the depressant actions of adenosine on the spontaneous firing of rat cerebral cortical neurons. 2. 2. 17β-estradiol hemisuccinate competitively inhibited adenosine uptake with a K i of 0.5 μM (Lineweaver-Burk plot) or 0.78 μM (Dixon plot). The K i for progesterone was 0.34 μM (Lineweaver-Burk plot) or 0.36 μM (Dixon plot). 3. 3. When applied by iontophoresis, both steroids potentiated the depressant effects of adenosine on the firing of rat cerebral cortical neurons. 4. 4. Potentiation of the effect of endogenously released adenosine would account for the central depressant actions of these steroids.

Potentiation of the action of adenosine on cerebral cortical neurones by the tricyclic antidepressants.

The effects of four tricyclic antidepressants, nortriptyline, iprindole, chlorimipramine and desipramine on adenosine-evoked depressions of the firings of rat cerebral cortical neurones has been studied. When applied iontophoretically, all four substances enhanced the depressant actions of iontophoretically applied adenosine but did not affect the depressant actions of the uptake-resistant analogue, adenosine 5'-N-ethylcarboxamide (NECA). Nortriptyline and iprindole administered intravenously (1 mg kg-1) enhanced the depressant actions of iontophoretically applied adenosine. When applied by larger iontophoretic currents, all four antidepressants inhibited the firing of cerebral cortical neurones. Chlorimipramine- and desimipramine-elicited depressions were antagonized by intravenously administered caffeine, an adenosine antagonist. Earlier studies showed the tricyclic antidepressants inhibit the uptake of adenosine by rat brain cerebral cortical synaptosomes. The present results demonstrate that four antidepressants are able to potentiate the action of adenosine and that this occurs when these compounds are given in behaviourally meaningful doses. The specificity of the potentiation is demonstrated by the failure of these compounds to potentiate the depressant actions of an uptake-resistant analogue of adenosine, NECA. Antagonism of the inhibitory effects of the antidepressants on neuronal firings by caffeine, indicates that these compounds can enhance the extracellular levels of endogenously released adenosine sufficiently to depress cell firing.

Effects of N-substituted phenyltetrahydropyridines on cerebral high-affinity synaptosomal uptake of dopamine and other monoamines in several mammalian species

Effects of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (N-methyl-PTP) and its N-propyl congener (N-propyl-PTP) on the high-affinity uptake of tritiated dopamine (DA), norepinephrine, and serotonin by striatal or cerebral cortical synaptosomes were evaluated in several species (rat, guinea pig, rabbit, calf, and man). Both compounds inhibited uptake of 0.1 ωM labeled amines at IC 50s of 5–10 ωM. Effects of N-methyl-PTP were competitive, reversible, somewhat more potent, and more selective for serotonin than were actions of N-propyl-PTP. Similar effects were found in all species. Neither agent inhibited binding of 3H-labeled spiperone or ADTN to DA receptor sites. 3H-N-methyl-PTP did not appear to be taken up selectively into DA neurons. N-methyl-PTP was highly toxic to the rat in doses that did not alter the metabolism of DA or serotonin in brain. These results, overall, do not provide strong support for the hypothesis that reported neurotoxic actions of N-methyl-PTP are mediated by neuron-specific local transport and intracellular accumulation, or account for species differences in the actions of this toxin, but do suggest interactions with brain monoamine neurons. The actions of the neurotoxic effects of N-methyl-PTP remain unclear.

Further studies on the inhibition of adenosine uptake into rat brain synaptosomes by adenosine derivatives and methylxanthines

1. 1. Various adenosine derivatives, methylxanthines and other compounds were tested for their abilities to inhibit the rapid uptake of adenosine by rat cerebral cortical synaptosomes. 2. 2. Several pharmacologically potent derivatives of adenosine were weak inhibitors of uptake with IC 20 values in excess of 10 −5 M. Derivatives in this category were adenosine-5′-N-ethylcarboxamide, adenosine-5′-cyclopropylcarboxamide, N 6-cyclohexyladenosine, l-N 6-phenylisopropyladenosine, 1-methylisoguanosine, 2-phenylaminoadenosine and 5-iodotubercidin. 3. 3. Several methylxanthines were very weak inhibitors of adenosine uptake. These included pentoxifylline, n-hexyltheophylline, n-butyltheobromine, and isoamyltheobromine. 4. 4. HL 725, a pyrimido-isoquinoline with potent phosphodiesterase inhibitory activity, inhibited adenosine uptake with an IC 20 of 2.0 × 10 −6 M. 5. 5. PK 11195, a putative ligand for the peripheral benzodiazepine binding site did not alter uptake at a concentration of 10 −4 M.

Interactions between adenosine and nifedipine in the rat cerebral cortex

Nifedipine inhibits the uptake of [ 3H]adenosine into rat cerebral cortical synaptosomes with an IC 50 value of 1.1 μM. When applied by iontophoresis onto rat cerebral cortical neurons it potentiated the depressant effects of adenosine on spontaneous firing. Some of the calcium-antagonist actions of nifedipine may be mediated by adenosine.

Inhibition of adenosine uptake into rat brain synaptosomes by prostaglandins, benzodiazepines and other centrally active compounds

1. 1. A number of compounds have been tested for their abilities to inhibit the rapid uptake of adenosine by rat cerebral cortical synaptosomes. 2. 2. Prostaglandings PGI 2, PGA 2, and PGE 1 and PGE 2 were potent inhibitors of adenosine uptake with IC 20 values in the 10 −7 M–10 −6 M range. PGA 1, PGD 2 and PGF 2α also inhibited uptake but were less active. 3. 3. The benzodiazepine antagonist Ro 15–1788 inhibited adenosine uptake and failed to antagonize the effects of diazepam. Another antagonist, ethyl-β-carboline-3-carboxylate, was a weak inhibitor of adenosine uptake. Ro 5–4864, the so-called peripheral benzodiazepine ligand, inhibited adenosine uptake. 4. 4. Hydroxyzine and tracazolate, two anxiolytic agents, inhibited uptake as did flunarizine, a coronary vasodilator. 5. 5. Two calmodulin antagonists, W7 and R 24571, were effective inhibitors of adenosine uptake. Their IC 50 values were comparable to those at which they have been demonstrated to inhibit calmodulin-mediated reactions in other systems. These observations suggest that adenosine uptake may be a calmodulin-regulated process.

Alpha-latrotoxin of black widow spider venom depolarizes the plasma membrane, induces massive calcium influx, and stimulates transmitter release in guinea pig brain synaptosomes.

The effect of alpha-latrotoxin from black widow spider venom upon guinea pig cerebral cortical synaptosomes is described. Plasma membrane potential (delta psi p), in situ mitochondrial membrane potential (delta psi m), Ca2+ transport, gamma-amino[3H]butyrate release, [3H]noradrenaline release, and synaptosomal ATP were monitored under parallel conditions. Potentials were determined both isotopically and with a tetraphenylphosphonium-selective electrode. alpha-Latrotoxin depolarizes delta psi p selectively, both in the presence and absence of Ca2+. A slight toxin-induced depolarization of delta psi m is a consequence of a massive Ca2+ uptake across the plasma membrane. Depolarization of delta psi p is insensitive to tetrodotoxin, and Ca2+ entry is only partially inhibited by verapamil. Release of [3H]noradrenaline and gamma-amino[3H]butyrate is markedly stimulated by the toxin in the presence of Ca2+, and this effect is only slightly reduced in Ca2+-free conditions.

Morphine enhances the release of 3H-purines from rat brain cerebral cortical prisms

In vitro experiments have shown that 3H-purines can be released from 3H-adenosine preloaded rat brain cortical prisms by a KCl-evoked depolarization. The KCL-evoked release of 3H-purines is dependent on the concentration of KCl present in the superfusate. At concentrations of 10 −7⇔ld10 −5M morphine did not influence the basal release of 3H-purines from the prisms, although it enhanced the KCl-evoked release of 3H-purines. The enhancement of KCl-evoked 3H-purine release by morphine was concentration-dependent and was antagonized by naloxone, suggesting the involvement of opiate receptors. Uptake studies with rat brain cerebral cortical synaptosomes show that morphine is a very weak inhibitor of adenosine uptake. Comparisons with dipyridamole, a potent inhibitor of adenosine uptake, suggest that this low level of inhibition of the uptake did not contribute significantly to the release of 3H-purine by morphine seen in our experiments. It is therefore suggested that morphine enhances KCl-evoke 3H-purine release by an interaction with opiate receptors and that the resultant increase in extracellular purine (adenosine) levels may account for some of the actions of morphine.

Competitive inhibition of the uptake of adenosine into rat brain synaptosomes by prostaglandins

Summary Prostaglandins E1, E2 and F2α inhibit the rapid uptake of adenosine into rat cerebral cortical synaptosomes. Prostaglandin E1 (PGE1), the most potent member of the group, inhibits uptake competitively with a Ki value of 95 μM. These results suggest adenosine may be involved in some of the central actions of the prostaglandins.