Rat Cerebral Cortex Synaptosomes Research Articles

Mistletoe lectin I binding sites on the synaptosomes of the rat cerebral cortex

By means of combined lectinological and immunological methods were demonstrated mistletoe lectin I binding sites on rat cerebral cortex synaptosomes. The mistletoe lectin I binds specifically D-galactose. Galactosyl residues were established on the junctional and nonjunctional synaptosomal membrane, on the synaptic vesicles, mitochondria and on myelin contamination. The relative number of mistletoe lectin I receptors per unit area of synaptosomal membrane was calculated.

Differential localization of GABA-dependent and GABA-independent benzodiazepine binding sites within synapses of rat cerebral cortex

Fractions containing non-junctional membranes (fraction 1) could be separated from those enriched in junctional complexes (fraction 2) by sucrose gradient centrifugation of osmotically disrupted rat cerebral cortex synaptosomes. [ 3H]flunitrazepam binding sites were found to be homogeneous characterized by K d = 0.65 nM, B max = 13 pM for fraction 1; and K d = 2 nM, B max = 45 pM for fraction 2. GABA-dependent benzodiazepine receptor sites were found to be localized to the junctional complex fraction, while non-junctional membranes showed GABA-independent receptor and acceptor binding sites.

Benzodiazepine inhibition of adenosine uptake is not prevented by benzodiazepine antagonists

Uptake of [ 3H]adenosine into rat cerebral cortex synaptosomes was studied. Hexobendine (10 −5 M) and the benzodiazepine agonists diazepam (10 −5 M) and flurazepam (10 −4 M) significantly inhibited this uptake, but only if the compounds were pre-incubated for 10 min in the case of the benzodiazepines. The benzodiazepine antagonists Ro15-1788 (10 −5 M) and CGS 8216 (10 −5 M) failed to reverse the action of benzodiazepine agonists or hexobendine on [ 3H]adenosine uptake. The results add weight to the view that inhibition of adenosine uptake processes by benzodiazepines do not contribute to their behavioural effects.

Calcium/phospholipid regulates phosphorylation of a Mr "87k" substrate protein in brain synaptosomes.

Depolarization-induced calcium influx into rat cerebral cortex synaptosomes increased the phosphorylation of several synaptosomal proteins as examined by 32Pi incorporation. A phosphopeptide mapping technique involving NaDodSO4/polyacrylamide gels has been used to show that phosphorylation of a Mr 87,000 substrate protein is stimulated by depolarization-induced calcium influx. Phosphorylation of this Mr 87,000 substrate occurred in synaptosomal cytosol and was markedly stimulated by calcium/phosphatidylserine. Calmodulin inhibited this phosphorylation reaction. This substrate for calcium/phospholipid-dependent protein kinase is enriched in and appears to be specific to neurons.

The effect of sodium on depolarization-induced calcium uptake and acetylcholine release by synaptosomes

The influence of external sodium concentration on potassium (depolarizing agent)-stimulated calcium uptake and Ca +-dependent acetylcholine release by rat cerebral cortex synaptosomes has been studied. It was found that increased sodium concentration decreases both the Ca 2+ uptake and the acetylcholine release, whereas a low external sodium concentration is stimulatory.

The effects of dopamine agonists and antagonists on Na +, K +-ATPase and Mg 2+-atpase activities of synaptosomes

The effects of dopamine agonists and antagonists on the Na +,K +-ATPase and Mg 2+-ATPase activities of rat cerebral cortex synaptosomes have been determined. Dopamine, ADTN, apomorphine and S-584, but not piribedil, stimulated the activities of the enzymes. The stimulatory effect of dopamine was not antagonised by dopamine antagonists and apparently the catechol group is responsible for the enzyme stimulation.

Prostaglandins D 2, and E 2 are not regulators of amino acid release from rat cortical synaptosomes

Rat cerebral cortex synaptosomes synthesise prostaglandins, and analysis by gas chromatography-mass spectrometry revealed that of the prostaglandins quantified PGD 2 (10.9 ng mg protein) was produced in highest concentration. Treatment with high potassium or veratrine caused release of putative amino acid transmitters, but not of prostaglandins, and prostaglandins D 2 and E 2 were unable to stimulate release of amino acids. The release of putative amino acid transmitters, evoked by high potassium levels, was not inhibited by these prostaglandins. Prostacyclin receptors could not be identified on synaptosomes by radioligand binding techniques.

“On” and “Off” Responses of K+‐Induced Synaptosomal Proline Release: Involvement of the Sodium Pump

The K+-induced release of the putative neurotransmitter proline in rat cerebral cortex synaptosomes was studied by measuring [5-3H]proline released upon exposure to high-K+ media. In contrast to previous experiments, K+ was not substituted for Na+, which was held constant. Under the resulting conditions of increased osmolarity, two peaks of proline release were obtained, one upon exposure to increased [K+], another upon restoring normal [K+]. The dependence of the latter peak on ionic composition of the medium, and its sensitivity to ouabain are discussed.

Effects of adrenergic agonists and antagonists and of the catechol nucleus on the Na +, K +-ATPase and Mg 2+-ATPase activities of synaptosomes

The effects of adrenergic agonists, antagonists and other drugs on the Na +, K +-ATPase and Mg 2+-ATPase activities of rat cerebral cortex synaptosomes have been determined. Noradrenaline, isoprenaline, dopamine, 1-DOPA and catechol stimulated the activities of the enzymes. The stimulatory effects were not prevented by low concentrations of adrenergic antagonists and apparently depend upon the presence in the molecules of a catechol moiety.

Studies on the role of Na+, K+ and Cl- ion permeabilities in K+-induced release of 3H-noradrenaline from rat brain slices and synaptosomes and in its presynaptic alpha-adrenergic modulation.

Possible ionic mechanisms by which activation of presynaptic α-adrenoceptors might inhibit the K+-induced release of 3H-noradrenaline from superfused rat cerebral cortex slices or synaptosomes were investigated. (1) 4-Aminopyridine (4-AP), a blocker of K+-permeability, enhanced the release of 3H-noradrenaline from cortex slices induced by 13 mM K+ in a concentration-dependent manner; 1 mM 4-AP caused an enhancement by 400%. By itself, 4-AP also induced a Ca2+-dependent release of 3H-noradrenaline; the release caused by 1 mM 4-AP was of a similar magnitude as that caused by 13 mM K+. (2) Tetrodotoxin (TTX), a blocker of Na+-permeability, concentration-dependently inhibited the release of 3H-noradrenaline induced by 13 mM K+; 0.3 μM TTX caused an inhibition by 90%. (3) The enhancing effect of 4-AP and the inhibitory effect of TTX on K+-induced 3H-noradrenaline release were inversely related to the K+-concentration used to stimulate release. (4) The release of 3H-noradrenaline from synaptosomes induced by 6–10 mM K+ was also enhanced by 4-AP and inhibited by TTX. (5) In Cl−-free medium the baseline tritium efflux was increased to about 3 times that found in normal medium. The release of 3H-noradrenaline induced by 13 mM K+ was enhanced by about 40% in Cl−-free medium. (6) Both the inhibitory effect of the α-receptor agonist oxymetazoline and the enhancing effect of the antagonist phentolamine on K+-induced 3H-noradrenaline release were decreased by 4-AP (1 mM). The effect of oxymetazoline was fully restored, despite the presence of 4-AP, when the Ca2+-concentration in the medium was lowered from 1.2 to 0.1 mM. Under these low Ca2+ conditions the effect of phentolamine was partially restored. (7) No change in presynaptic modulation of K+-induced 3H-noradrenaline release by oxymetazoline and phentolamine was observed when TTX (0.3 μM) was present in the medium. Moreover, 3H-noradrenaline release induced by increasing Na+-permeability with veratrine was effectively modulated. (8) Effective modulation of K+-induced 3H-noradrenaline release by oxymetazoline and phentolamine was also observed in Cl−-free medium.

Endogenous phosphorylation in vitro: Differential effects of brain state (anesthesia, post-mortem) on electrophoretically separated brain proteins

In vitro phosphorylation of rat cerebral cortex synaptosomes was measured in animals that had been acutely treated with sodium pentobarbital. [ 32P]Labelled phosphoproteins were separated by SDS-slab gel electrophoresis, and the autoradiographs were analyzed by densitometry. We report here that Band F of our previous reports can be separated into two components, F 1 and F 2, using an improved gel system. This separation is particularly relevant in this report since these components appear to be differentially sensitive to the manipulations used. Specifically, we found that while F 1 phosphorylation was markedly diminished by deep barbiturate anesthesia, F 2 was relatively stable. While phosphorylation of F 2 was also stable 24 h post-mortem, Band F 1 phosphorylation was no longer detectable. Finally, while osmotic shock treatment of synaptosomes reduced phosphorylation of F 2 somewhat, it eliminated the in vitro phosphorylation of Band F 1. We found that under light barbiturate anesthesia, just at the time when the animals lost the righting reflex, the in vitro phosphorylation of Bands D (M R 78,000–80,000 daltons), F 1 (M R 47,000–49,000) and F 2 (M R 40,000–45,000) increased relative to unanesthetized controls. The in vitro labelling of Bands D and F 1 was depressed in tissue prepared from animals that were deeply comatose. These effects of pentobarbital were more pronounced when animals were sacrificed by liquid nitrogen immersion, rather than by decapitation. Cyclic AMP-dependent phosphorylation of Band D exhibited remarkable stability 24 h post-mortem (7 days in one case), even when brain tissue was left at room temperature (21–23 °C). Phosphorylation of Band F 1, however, was not detectable in post-mortem tissue. The results of these studies indicate that phosphorylation of Band F 1 is: (1) sensitive to pentobarbital, and (2) unstable post-mortem. Previous findings from our laboratory suggest that Bad F 1 is: (3) increased in phosphorylation in liquid nitrogen P 2 preparations, and may be (4) cAMP-independent, (5) rapidly turning over its phosphate in vivo, and (6) altered by a training experience. Other evidence suggests that: (7) Band F 1 phosphorylation may be Ca 2+-dependent and that: (8) its phosphorylation is sensitive to osmotic lysis of synaptosomes. These results suggest an important and perhaps unique role for Band F 1 in neuronal function.

Effet de la noradrénaline sur sa propre libération dans des synaptosomes de cortex de rat

Norepinephrine (NE) uptake velocity in rat cerebral cortex synaptosomes does not depend on internal NE contents: continuous exchange occurs. NE enhances spontaneous release and inhibits release elicited by KCl. Phenylephrine an a agonist, produces the same effect. Phentolamine, an a antagonist, did not modified the spontaneous release but enhanced release elicited by KCl 25 mM.

BINDING OF H3-IMIPRAMINE, H3-DIMETACRINE AND S35-CHLORPROMAZINE TO SYNAPTOSOMES

Binding of H3-imipramine, H3-dimetacrine and S35-chlorpromazine to synaptosomes of rat cerebral cortex was studied using a centrifugation method, and kinetic analysis of the experimental data. Three psychotropic drugs were shown to be rapidly bound to synaptosomes at 2 degrees C, representing a typical binding mode with two classes of binding components, i.e., saturable and non-saturable binding. A double reciprocal plot of the saturable binding component of these drugs revealed that H3-dimetacrine and S35-chlorpromazine represented a single binding mode, whereas H3-imipramine showed a multiple one. When the synaptosomes were treated by freezing and thawing 15 times, a high affinity binding component of H3-imipramine was not observed, while the other two drugs showed a single binding mode as well as those of the undisrupted synaptosomes. To investigate the specificity of this multiple binding mode, comparative binding studies of H3-imipramine were carried out using myelin fragments of rat cerebral cortex. In the myelin fragments preparation, two typical classes of binding mode as shown in the synaptosomes were also recognized. However, a double reciprocal plot of the saturable binding component showed only a straight line, i.e., single binding mode. These findings suggest that imipramine has multiple binding sites to synaptosomes and a high affinity binding component is affected by freezing and thawing procedure.

Efflux of 5-hydroxytryptamine from synaptosomes of rat cerebral cortex.

The efflux of 3H-5-HT from a crude synaptosome preparation of the cerebral cortex of reserpinized rats was examined. The synaptosomes were loaded with 3H-5-HT by pre-incubation of the homogenate in presence of pargyline and desipramine in order to inhibit deamination of 5-HT and uptake into noradrenergic neurons. The synaptosomes were collected by centrifugation, washed and resuspended in 0.25 M sucrose. No spontaneous efflux of 5-HT was detectable at 0 degrees C but marked efflux was observed at 27 degrees C and 37 degrees C. 4-Chloroamphetamine, low external Na+ concentration, ouabain and the depolarizing agent veratridine markedly accelerated the initial (5 min) efflux. Inhibitors of the neuronal 5-HT uptake, e.g. chlorimipramine, H 102/09 and A 23189, antagonized the 5-HT efflux evoked by these means, whereas desipramine, which is a poor inhibitor of the 5-HT uptake, had only slight effect on the 5-HT efflux. It is suggested that 5-HT can be actively transported out from the synaptosomes by the reversed 5-HT uptake mechanism.

Soluble and membrane-bound S-100 protein in rat cerebral cortex synaptosomes during postnatal development

Soluble and membrane-bound S-100 protein in rat cerebral cortex synaptosomes during postnatal development

Uptake of acetylcholine and choline into rat brain cortical slices and synaptosomes as related to 32Pi incorporation into their phospholipids.

Abstract—The role of ACh‐stimulated 32Pi incorporation into the phospholipids of rat cerebral cortex slices and isolated nerve endings (synaptosomes) has been studied. ACh stimulation is not connected with any carrier‐mediated uptake of ACh. Such uptake may occur in slices in the presence of the anticholinesterase Sarin but barely in the presence of eserine. Regardless of the nature of the anticholinesterase used, rat cerebral cortex synaptosomes that respire and show high and low affinity choline uptake do not accumulate ACh against a concentration gradient. At exogenous ACh concentrations of 10–5m and above, some ACh enters the synaptosomes by diffusion and significantly stimulates 32Pi incorporation into phosphatidic acid. It is discussed whether, in isolated nerve endings, an increase in cytoplasmic ACh concentration due to diffusion may induce vesicle turnover to keep a balance between ‘free’ and bound ACh or if a presynaptic ACh receptor is responsible for the observed changes in phosphatidic acid. The distribution of accumulated radioactivity derived from exogenous choline and ACh respectively between ACh, choline, phosphorylcholine and betaine has been studied in slices and isolated nerve endings.