Brain Synaptosomal Fractions Research Articles

Similarities of physico-chemical and antigenic properties of neurocupreins and extremely acidic copper proteins from chromaffin granules

Extremely acidic copper-containing proteins, neurocupreins, were isolated from brains of various mammals (bovine, rabbit, pig and sheep). Neurocupreins from all these sources were found to have similar physico-chemical and antigenic properties. Using the immunological approach, it was shown that neurocuprein is located only in brain cytosol and synaptosomal fractions. Extremely acidic copper-containing proteins were also isolated from soluble and membranous fractions of chromaffin granules from bovine adrenal medulla. The soluble form of the protein from the granules has practically the same physico-chemical and antigenic properties as neurocupreins. The copper protein isolated from membranes of granules has slightly higher molecular weight and somewhat different amino acid composition, although their EPR spectra are identical. However, both copper proteins from chromaffin granules are immunoprecipitated with antibodies to neurocuprein. It is suggested the the membranous form differs from the soluble one in possessing a peptide which prolongs the protein chain without changes in its antigenic properties.

Immunological characterization of sn-1,2-diacylglycerol and sn-2-monoacylglycerol kinase from pig brain.

Rabbit antisera were raised against diacylglycerol kinase purified from pig brain cytosol. Upon immunoblot analysis, the antibody was specifically reactive with the kinase (Mr = 79,000-80,000). Pig brain cytosol, microsomal, and synaptosomal fractions all contained the immunoreactive Mr = 80,000 polypeptide, thus showing that the same enzyme is present in the soluble as well as membrane fractions of the brain. The antibody could precipitate only 60% of the kinase activity present in the crude cytosol. Further, the antibody exhibited very little or no cross-reactivity toward liver cytosolic enzymes obtained from different animals including pigs. Immunostaining of brain tissues demonstrated that neurons, in particular, their nuclei, were positively stained, whereas glial cells were not stained. It is likely that there exists a tissue-and/or cell-dependent immunological multiplicity of diacylglycerol kinase. The enzyme activities phosphorylating sn-1 and sn-2 monoacylglycerols were co-precipitated by the antibody, indicating their identity with diacylglycerol kinase. The enzyme activity toward sn-1 monoolein was much lower than that obtained with sn-2 monoolein. Enzymic as well as chemical analyses of acyl isomers of the reaction products showed that even tested with pure (greater than 95%) sn-1 monoolein, about 70% of the formed lysophosphatidate was of the sn-2 acyl type. The results show that diacylglycerol kinase phosphorylates almost exclusively the sn-2 acyl type of monoacyl-glycerol.

Effects of calcium antagonists on KCl-evoked calcium uptake by rat cortical synaptosomes

1. 1. A series of calicum antagonists were used to study their blocking effect on high potassium-induced calcium uptake into rat cortical synaptosomes; these antagonists were classified into five groups: (1) dihydropyridine group (i.e. nifedipine and nitrendipine), (2) benzothiazepine group (i.e. diltiazem), (3) phenylalkylamine group (i.e. verapamil and D600), (4) phenothiazine group (i.e. trifluoperazine) and (5) diphenylpiperazine group (i.e. flunarizine and cinnarizine). 2. 2. Voltage-dependent 45Ca 2+-uptake into this fraction was measured after 20 see KCl-induced depolarization. The ID 30 values of the above-mentioned antagonists affecting 45Ca 2+-uptake were calculated to be nitrendipine (80 μM), nifedipine (100 μM), verapamil (50 μM), D600 (15 μM), diltiazem (70 μM), trifluoperazine (7 μM), cinnarizine (1.2 μM) and flunarizine (0.7 μM). 3. 3. Our results reveal that in rat brain synaptosomal fractions, calcium influx via the voltage-gated calcium channel appears to be more sensitive to diphenylpiperazine and phenothiazine groups; whereas, phenylalkylamine, benzothiazepine and dihydropyridine groups were relatively insensitive. 4. 4. This contrasts with the well known data obtained from vascular smooth muscle, in which the dihydropyridine group is the most sensitive of all the groups studied. 5. 5. Our results suggest that calcium channels in neuronal tissue are most likely different from those in non-neuronal tissue.

Axonal transport of antibodies to subcellular and protein fractions of rat brain

Experiments examined the feasibility of using the axonal transport of antibodies as a possible means to characterize nerve membrane composition and the fate of internalized macromolecules. Polyspecific antibodies were generated in rabbits against rat brain synaptosomal and microsomal subcellular fractions and against wheat germ agglutinin-binding proteins isolated by lectin affinity chromatography. Antisera were injected into the vitreal chamber of the eye and into the facial musculature of anesthetized rats to test, respectively, for anterograde transport in retinotectal neurons and for retrograde transport in facial motoneurons. Control injections of preimmune serum were made into the opposite side. After survival for 4-168 h, animals were perfused and the axonally transported rabbit immunoglobulins detected in frozen sections of the brainstem using a modified peroxidase-antiperoxidase immunocytochemical procedure. Antisera against all 3 classes of neuronal antigens contained antibodies that underwent retrograde axonal transport. No evidence of anterograde transport was seen. Neurons containing retrogradely transported immunoglobulins exhibited punctate as well as diffuse staining of the cytoplasm and proximal dendrites, exclusive of the nucleus. Following retrograde transport of antibodies to the synaptosomal fraction, staining of the neuropil around motoneurons was also observed, suggesting transcellular transport of these antibodies. Concentrations of injected antibodies as low as 1% of whole antiserum led to detectable retrograde transport. Increasing concentrations of antibodies above the amount in whole antiserum did not increase the intensity of staining in retrogradely labeled neurons, suggesting saturation. The findings support the view that antibodies to neural membranes are taken up and transported by binding to specific sites on nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic disturbances of synaptosomes isolated from ischemic gerbil brain.

The effects of cerebral ischemia, induced for 10 min by bilateral common carotid ligation in the Mongolian gerbil, on the brain and synaptosomal content of phospholipids and free fatty acids were measured. Moreover, the incorporation of arachidonic acid and oleoyl-CoA into phospholipids, as well as the respiration and the accumulation of 45Ca, norepinephrine, dopamine, choline, glutamate, and gamma-aminobutyrate in the ischemic brain synaptosomal fraction were studied. Analyses of lipids showed a drop in phospholipids content with concomitant increase of lysocompounds and free fatty acids in ischemic cerebral cortex. Disturbances in lipid metabolism including rapid phospholipids hydrolysis and changes in the incorporation of arachidonic acid into inositol and choline phosphoglycerides were also shown in the synaptosomal fraction of ischemic brain. The uptake of neurotransmitter substances, expressed as a percent of control value, was reduced 21% for norepinephrine, 40% for dopamine, 20% for choline, 24% for glutamate and 13% for gamma-aminobutyrate in ischemic synaptosomes. There was no significant effect of ischemia on synaptosomal respiration and 45Ca uptake in both control and high potassium media. The inhibition of neurotransmitter uptake in ischemic brain synaptosomes may be caused by the disturbance of fatty acid metabolism.

Circulating digitalis-like substance is increased in doca-salt hypertension

Blood pressure and digitalis-like substance were measured in the plasma of control, salt-treated, and DOCA-salt treated rats. Blood pressure in DOCA-salt treated rats was significantly higher than that of either control or salt-treated animals. Digitalis-like activity was measured by two methods, radioimmunoassay for digoxin, and a receptor binding assay employing a rat brain synaptosomal membrane fraction. Digoxin-like immunoreactivity in plasma was not detected in either control or salt-treated rats, but was detected in DOCA-salt treated rats. Receptor binding activity in salt-treated rats was slightly but significantly higher than that of control rats. In DOCA-salt treated rats, receptor binding activity was significantly higher than that of salt-treated rats. Partial purification of the digitalis-like substance in plasma was performed by gel filtration using Sephadex G-25. Two peaks containing digoxin-like immunoreactivity were observed. Receptor binding activity, as well as Na +-K + ATPase inhibitory activity, was detected only in the second peak, in which approximately 70% of the digoxin-like immunoreactivity was eluted. These results indicate that a circulating digitalis-like substance is increased in DOCA-salt hypertension.

A single class of neurotensin receptors with high affinity in neuroblastoma X glioma NG108-15 hybrid cells that mediate facilitation of synaptic transmission

Receptor binding of [3H]neurotensin was examined on membrane preparations derived from neuroblastoma X glioma NG108-15 hybrid cells. The specific binding was saturable and reversible, and a dissociation constant (Kd) was calculated to be about 0.24 nM from the rate constants. Scatchard analysis of neurotensin binding at equilibrium revealed a single class of binding sites with a Kd of 0.86 nM and a maximal binding capacity (Bmax) of 250 fmol/mg of protein (7700 receptor sites/cell). [D-Arg9]-Neurotensin had a high affinity (IC50 = 0.5 nM) for the neurotensin receptors, but [D-Phe11]-neurotensin had a lower affinity (IC50 = 280 nM), while angiotensin II and bradykinin had almost no affinity for [3H]neurotensin-binding sites. Under similar conditions [3H]neurotensin binding to mouse and rat brain synaptosomal fractions showed two binding sites with high (0.86 and 0.44 nM) and low (13 and 19 nM) affinities. We have examined several possible physiological consequences of neurotensin receptor binding. Neurotensin (10 microM) exhibited no influence on adenylate cyclase activity, 45Ca uptake, or 32Pi incorporation into phosphatidylinositol fractions of NG108-15 cells. Electrophysiological study of isolated NG108-15 cells revealed neurotensin-induced transient hyperpolarization followed by sustained depolarization with enhanced membrane excitability. Application of neurotensin to NG108-15 cells that had formed synapses with cultured striated muscle cells caused a considerable increase in frequency of miniature endplate potentials from the muscle cells. These data show that NG108-15 cells possess a single class of neurotensin receptors similar to a high affinity site of synaptosomal membranes from the murine brains.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional significance of the effects of neurotransmitters on the Na+,K+-ATPase system.

The aim of the present experiments was to study the effects of the neurotransmitters acetylcholine, noradrenaline, 5-hydroxytryptamine, and dopamine on the Na+,K+-ATPase of rat brain synaptosomal fractions. It is shown that dopamine at low concentrations specifically inhibits the Na+,K+-ATPase of synaptic membranes from the brain regions rich in dopaminergic endings, but has no effect on the synaptosomal Na+,K+-ATPase from the other parts of brain. Acetylcholine and noradrenaline have similar specific effects on Na+,K+-ATPase from cholinergic and adrenergic synaptosomes. The Na+,K+-ATPase of synaptic membranes from the different brain regions, characterised by different distributions of cholinergic, adrenergic, and 5-hydroxytryptaminergic endings, show different reactions with neurotransmitters. These data indicate a functional significance of the effects of the neurotransmitters on the synaptosomal Na+,K+-ATPase.

Critical assessment of noradrenaline uptake in synaptosomal preparations.

Synaptosomes and other subcellular organelles were prepared from rat brain using a vertical rotor. The preparation time was reduced by up to 60% compared to conventional techniques. Uptake of [3H]-(-)-noradrenaline into subcellular fractions was characterized. The characteristics of this uptake were dependent on the subcellular composition and anatomical origin. Various methods of correction for energy independent processes were compared, but only sodium ion removal from the medium selectively inhibited the energy dependent uptake mechanism. Kinetic analysis of data revealed that high and low affinity uptake systems were dependent on the fraction under analysis. Noradrenaline uptake was not exclusively localised in noradrenergic terminals. Selective inhibitors of the noradrenaline uptake process (tricyclic antidepressants) inhibited energy dependent uptake completely only in purified synaptosomes prepared from cortex. In whole brain synaptosomal fractions, noradrenaline was partially accumulated into dopaminergic neurones; this uptake process was not inhibited by tricyclic antidepressants.

Studies on the mechanisms of the epileptiform activity induced by U186661. I Gross alteration of the lipids of synaptosomes and myelin.

U18666A, an inhibitor of desmosterol reductase (a terminal enzyme in cholesterol synthesis), has been found to produce chronic epileptiform activity in laboratory animals. Since desmosterol might substitute for cholesterol in neuronal membranes without detriment, the present study was undertaken to examine the possibility that this drug-induced epilepsy was related to changes in other brain lipids. Chronic treatment of rat with U18666A, beginning at one day of age, resulted in pronounced decreases in the concentration of phospholipids and increases in gangliosides of brain microsomal, synaptosomal, and crude myelin fractions. Since total sterol levels were not changed, the ratio of sterols to phospholipids also increased. If drug treatment was stopped at 4 weeks of age, brain lipids of all subcellular fractions examined returned to normal levels by 8 weeks, and no epileptiform activity was detected. However, following 8 weeks of continuous treatment, epileptiform activity was present, and the changes in brain lipids were focused in the myelin fraction. Phospholipid levels and the sterol:phospholipid ratio of microsomes and synaptosomes, in contrast to myelin, were near normal; however, gangliosides were still clearly elevated in all fractions. A reported ability to induce epileptiform activity in rats by treatment with antiserum to brain gangliosides could indicate a special significance of the altered myelin and synaptic gangliosides to the U18666A-induced epilepsy. We suggest that some epileptiform conditions could be directly related to alterations in the lipid composition of critical neuronal structures.

Analysis of alpha-bungarotoxin binding in the goldfish central nervous system.

We report here the equilibrium, kinetic, and pharmacological analysis of alpha-125I-bungarotoxin (alpha-125I-Bgt) binding to a Triton X-100-solubilized goldfish brain synaptosomal fraction. In addition, a refined analysis of equilibrium binding to a particulate synaptosomal fraction is presented. Equilibrium binding from both particulate and soluble fractions revealed an apparent heterogeneity of binding sites. Kinetic analysis of the soluble receptor revealed linear association kinetics and nonlinear dissociation kinetics. The dissociation curve suggested the presence of at least two rate constants. Potential sources of the binding heterogeneity found in both the equilibrium binding and dissociation kinetics experiments are (1) multiple receptor species, (2) multiple ligand species, and (3) different, possibly interconvertible, states of a single receptor type. No evidence for the first two alternatives was found. Support for the third alternative was obtained by observing the effect of cholinergic ligands on alpha-125I-Bgt dissociation. Carbamylcholine and d-tubocurarine increased the apparent proportion of rapidly dissociating sites, suggesting that the two binding affinities can be interconverted and may arise from a single receptor type. Evidence concerning the identity of the alpha-Bgt binding protein as a nicotinic acetylcholine receptor is discussed.

Notexin preferentially inhibits the release of newly synthesized acetylcholine from rat brain synaptosomal fractions

An investigation was made of the effects of the snake venom neurotoxin, notexin, on acetylcholine turnover in rat brain P2 fractions using a gas chromatographic mass spectrometric assay for acetylcholine and choline. In contrast to earlier reports, we found a stimulation of the uptake and acetylation of labeled choline by toxin-treated P2 fractions. More significantly, notexin inhibited the release of this newly synthesized transmitter. These effects were found to be dependent on the dose of the toxin and the time of exposure of the P2 fraction to notexin. Longer exposure to notexin or experiments involving resuspension of notexin-treated P2 fractions appeared to result in considerable lysis of the transmitter-containing particles. Thus, notexin may alter acetylcholine compartmentation in the nerve ending and thereby affect acetylcholine synthesis.

Effect of inhibitors of GABA aminotransferase on the metabolism of GABA in brain tissue and synaptosomal fractions.

Five inhibitors of the GABA degrading enzyme GABA-aminotransferase (GABA-T), viz., gabaculine, gamma-acetylenic GABA, gamma-vinyl GABA, ethanolamine O-sulphate, and aminooxyacetic acid, as well as GABA itself and the antiepileptic sodium valproate were administered to mice in doses equieffective to raise the electroconvulsive threshold by 30 V. The animals were killed at the time of maximal anticonvulsant effect of the respective drugs and GABA, GABA-T and glutamate decarboxylase (GAD) were determined in whole brain and synaptosomes, respectively. The synaptosomal fraction was prepared from brain by conventional ultracentrifugation procedures. All drugs studied brought about significant increases in both whole brain and synaptosomal GABA concentrations, and, except GABA itself, inhibited the activity of GABA-T. Furthermore, all drugs, except GABA and gamma-acetylenic GABA, activated GAD in the synaptosomal fraction. This was most pronounced with ethanolamine O-sulphate, which induced a twofold activation of this enzyme but exerted only a weak inhibitory effect on GABA-T. The results suggest that activation of GAD is an important factor in the mechanism by which several inhibitors of GABA-T and also valproate increase GABA concentrations in nerve terminals, at least in the relatively non-toxic doses as used in this study.

Degradation of cholecystokinin-like peptides by a crude rat brain synaptosomal fraction: A study by high pressure liquid chromatography

Degradation of CCK-8, CCK-4, and related peptides by a crude synaptosomal fraction of rat brain was investigated by monitoring the tryptophan fluorescence of reaction products after HPLC fractionation. At 20°C, the half disappearance time was 52 min for CCK-8, 35 min for unsulphated CCK-8, 20 min for unsulphated CCK-7, 6 min for Tyr(SO 3H)-Trp-Met-Asp-Phe-NH 2, and 3 min only for CCK-4. Caerulein was much more resistant than CCK-8,and Boc-CCK-4 and Aoc-CCK-4 remained stable for at least 3 h. The apparent K m for CCK-8 and CCK-4 was 40 μM and maximal activity on CCK-8 was observed at pH 7.0. Zn 2+ was strongly inhibitory. The protease inhibitors puromycin and bacitracin, the metal chelator 1,10-phenanthroline, and the sulphydryl blocking agents N-ethylmaleimide and p-chloromercuribenzoate greatly reduced the release of tryptophan from CCK-8. Puromycin inhibition of CCK-8 degradation provoked the accumulation of a CCK-7-like peptide, and that of CCK-4 degradation was of a competitive type ( K i = 2 μM). The CCK-8 degrading activity of brain synaptosomes was present in the cytosol as well as in synaptic membranes.

Alterations of acetylcholine and choline metabolism in mammalian preparations treated with beta-bungarotoxin.

We have studied the effects of beta-bungarotoxin on acetylcholine and choline metabolism in central and peripheral cholinergic preparations using a gas chromatographic-mass spectrometric assay for acetylcholine and choline. In contrast with previous reports, beta-Bungarotoxin did not inhibit the high-affinity uptake of labeled choline or the synthesis of acetylcholine in rat brain synaptosomal fractions. However, the toxin did cause a significant increase of medium choline when it was incubated with synaptosomal fractions. This increase of endogenous choline in the medium may account for the previously reported inhibition of choline uptake because of a dilution of the specific activity of the labeled choline in the medium. Several experiments are reported in which a further characterization was made of the effect of beta-bungarotoxin on medium choline. beta-Bungarotoxin was also shown to cause a large increase of acetylcholine release from rat brain minces and a depletion of the acetylcholine content of minces. A similar phenomenon was found in diaphragm preparations that were exposed continuously to beta-bungarotoxin. However, diaphragms that were treated for only 30 min with toxin showed the previously reported increase of acetylcholine content. beta-bungarotoxin did not have any measurable effect on acetylcholine turnover in smooth muscle preparations from guinea pig ileum. These results help to explain certain inconsistencies in the literature regarding the action of beta-bungarotoxin.

Influence of acetylcholine on rebinding of soluble acetylcholinesterase to a synaptosomal fraction from rat brain

Acetylcholinesterase (Acetylcholine acetylhydrolase E.C. 3.1.1.7.) released from a rat brain synaptosomal fraction was shown to rebind upon incubation in the presence of 3 mM acetylcholine. This action was shown to be reversible. Glutamate, aspartate and gamma-aminobutyrate antagonise this effect. Solubilization of both bulk protein and acetylcholinesterase by Lubrol WX and triton X 100 after acetylcholine incubation of synaptosomal fractions is much lower than in the non incubated preparation. Local production of protons due to the hydrolysis of acetylcholine by the enzyme could partially explain the reassociation of the enzyme. We suggest that the observed phenomenon may play some physiological role in the function of acetylcholinesterase.

Solubilization of γ-aminobutyric acid receptor from rat brain

Gamma-aminobutyric acid (GABA) binding sites were solubilized from rat brain synaptosomal fractions by extraction with a combination of sodium deoxycholate and potassium chloride. Specific 3H-GABA binding to the solubilized fraction was saturable with the apparent dissociation constant, Kd = 23.4 ± 0.2 nM. GABA agonists and an antagonist inhibited the binding. The relative potencies of these drugs in competing for 3H-GABA binding to the solubilized fraction are in good agreement with findings with the membrane fraction, suggesting that the binding sites in the solubilized fraction retain the characteristics of membrane-bound GABA receptor. The sedimentation coefficient value of 3H-GABA binding site was estimated to be 11.3S by sucrose density gradient centrifugation, and this value was identical with that of 3H-flunitrazepam binding site in the same solubilized fraction.

Binding of scorpion toxins to rat brain synaptosomal fraction. Effects of membrane potential, ions, and other neurotoxins

Binding of scorpion toxins to rat brain synaptosomal fraction. Effects of membrane potential, ions, and other neurotoxins

Solubilization of the benzodiazepine receptor from rat brain

A macromolecular moiety having high binding affinity for benzodiazepines was solubilized from a rat brain synaptosomal fraction by extraction with a combination of sodium deoxycholate and potassium chloride. This solubilized fraction exhibits pharmacological specificity for benzodiazepines. Specific 3H-flunitrazepam binding to the solubilized fraction was saturable with the apparent dissociation constant Kd = 1.8 ± 0.3 nM. The binding is sensitive to proteolytic enzymes. The binding is increased by α-aminobutyric acid and decreased by inosine and hypoxanthine. These results are similar to those obtained with intact membranes, suggesting that this moiety may be the benzodiazepine receptor. The molecular weight was estimated to be approximately 200, 000 by sucrose density gradient centrifugation.

Nicotinic cholinergic receptors in brain synaptosomes

In order to elucidate pharmacological characteristics of nicotinic receptors in the brain, competitive binding of nicotine analogues or cholinergic agents to brain particles was studied utilizing [ 3H]nicotine. The binding of [ 3H]nicotine to brain crude mitochondrial or synaptosomal fraction was progressively inhibited by the addition of increasing amounts of nicotine or nornicotine, but cotinine had little effect. Of the myelin, synaptosomal and mitochondrial subfractions of the crude mitochondrial fraction, [ 3H]nicotine binding was almost exclusively confined to synaptosomes. In this binding, the affinity constant was 1.97 × 10 9/M and the maximum binding capacity was 7.30 × 10 −8 M/g of protein. This binding was reduced by 50% in the presence of 4 × 10 −7 M D-tubocurarine and 10 −6 M carbamylcholine, while atropine was the least effective of the drugs tested. These findings suggest the possible existence of nicotinic cholinergic receptors in the synaptic region. A study of regional differences in nicotinic cholinergic receptors showed that higher specific bindings of nicotine to synaptosomes occured in the hypothalamus, hippocampus and thalamus. These findings suggest that the nicotinic cholinergic mechanism plays an important role in these regions of the central nervous system.