Synaptic Membrane Antigens in Developing Rat Brain Cerebral Cortex and Cerebellum

Abstract: The contents of five synaptic membrane antigens (56K, 58K, 62K, 63K, and 64K) were determined in rat cerebral cortex and cerebellum at eight developmental time points: E9, E14, P < 1, P5, P14, P28, P60, and P180 (E, embryonic; P, postnatal). In cerebral cortex, the five antigens showed five different developmental patterns with respect both to specific content (i.e., quantity per unit of membrane) and total content (i.e., quantity per cortex). The 56K, 58K, and 62K polypeptides were first detected at E14, increased slightly to P5, then increased rapidly from P5 to P28 by 14-, 11-, and 18-fold, respectively. From P28 to PI80, the patterns of these antigens showed very large differences. The 63K and 64K antigens were first detected at P14 and P28, respectively. The specific content of 63K antigen continued to increase steadily throughout adult life; in contrast, the specific content of the 64K antigen did not change appreciably. In cerebellum only three antigens (56K, 58K, and 62K) were detected. These three antigens showed different developmental patterns. The 56K polypeptide was first detected at E14; its specific content increased very rapidly to a maximum at P < 1; it then decreased, first slowly, and then more rapidly, disappearing at P60. The 58K polypeptide also was detectable at E14 and increased very rapidly to a maximum at P < 1. It then decreased markedly to P5, followed by an increase, returning almost to its maximum level at P14. It then slowly decreased disappearing at P180. The 62K antigen was first detected at P14 and then it slowly decreased with disappearance at P60. The patterns with respect to total contents per cerebellum were similar for the three antigens, with a maximum at P28. We conclude that the highest increase in the contents of these antigens roughly corresponds to the period of maximal synaptogenesis (P9 to P28) in both regions. Differences among developmental patterns probably reflect changing molecular machinery required for development and functional differentiation of synapses in different brain regions. The fine structure of these patterns suggests that the quantitative measurement of synaptic membrane antigens will be useful for delineating complex processes occurring during synaptogenesis.     

Subcellular Localization of Bioactive Cholecystokinin-Like Peptides in the Rat Cerebral Cortex

Abstract: The subcellular distribution of cholecystokinin (CCK), especially its exact localization within the synaptosome, was studied in the rat cerebral cortex. Highest CCK-like bioactivity was measured in the synaptic membrane fractions, paralleling the distribution of (Na⁺+ K⁺)-dependent ATPase. In the synaptic vesicles, which were characterized by high acetylcholine content and by the absence of (Na⁺+ K⁺)-ATPase, only minimal quantities of CCK were detected.     

Effect of In Vivo Administration of Naloxone on ATP-ase's Enzyme Systems of Synaptic Plasma Membranes from Rat Cerebral Cortex

Naloxone is a specific competitive antagonist of morphine, acting on opiate receptors, located on neuronal membranes. The effects of in vivo administration of naloxone on energy-consuming non-mitochondrial ATP-ases were studied in two different types of synaptic plasma membranes from rat cerebral cortex, known to contain a high density of opiate receptors. The enzyme activities of Na⁺, K⁺-ATP-ase, Ca²⁺, Mg²⁺-ATP-ase and Mg²⁺-ATP-ase and of acetylcholinesterase (AChE) were evaluated on synaptic plasma membranes obtained from control and treated animals with effective dose of naloxone (12g · kg^–1 i.m. 30 minutes). In control (vehicle-treated) animals specific enzyme activities assayed on these two types of synaptic plasma membranes are different, being higher on synaptic plasma membranes of II type than of I type, because the first fraction is more enriched in synaptic plasma membranes. The acute treatment with naloxone produced a significant decrease in Ca²⁺,Mg²⁺-ATP-ase activity and an increase in AChE activity, only in synaptic plasma membranes of II type. The decrease of Ca²⁺,Mg²⁺-ATP-ase enzymatic activity and the increased AChE activity are related to the interference of the drug on Ca²⁺ homeostasis in synaptosoplasm, that leads to the activation of calcium-dependent processes, i.e. the extrusion of neurotransmitter. These findings give further evidence that pharmacodynamic characteristics of naloxone are also related to increase [Ca²⁺] _i , interfering with enzyme systems (Ca²⁺,Mg²⁺-ATP-ase) and that this drug increases acetylcholine catabolism in synaptic plasma membranes of cerebral cortex.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).     

O -·2增强谷氨酸与其受体的结合力及EBSELEN的保护作用

用放射配体测定受体法研究了黄嘌呤(X)/黄嘌呤氧化酶(XO)体系产生的超氧阴离子自由基(O -·2)对［3H］DL-谷氨酸与大鼠大脑皮层突触膜谷氨酸受体结合的影响,结果表明O -·2明显增强谷氨酸与其受体的结合力,此作用能被2-苯基-1,2-苯并异硒唑-3(2H)酮(EBSELEN)(1 μmol/L)所抑制.     

Effects of Sodium and Bicarbonate Ions on γ-Aminobutyric Acid Receptor Binding in Synaptic Membranes of Rat Brain

Crude synaptic membranes treated with Triton X-100 (TX) bound gamma-aminobutyric acid (GABA) to two classes of receptor site in Na+-free 10 mM-Tris-sulfate buffer (pH 7.4), but to only a single class of receptor site in 10 mM Tris-sulfate buffer (pH 7.4), containing 150 mM-NaCl. The high-affinity receptor site in TX membranes was specifically masked in the presence of Na+. However, TX membranes incubated in Krebs-Ringer bicarbonate solution (pH 7.4) bound GABA to two classes of receptor site despite the presence of Na+. It was found that addition of bicarbonate ions to the Na+-containing 10 mM-Tris-sulfate buffer (pH 7.4) could restore that high-affinity class of GABA receptors, rendering both classes detectable. This finding suggests that both Na+ and HCO-3 may have a regulatory function on GABA binding to the receptor.     

增强谷氨酸与其受体的结合力及EBSELEN的保护作用

用放射配体测定受体法研究了黄嘌呤 (X) /黄嘌呤氧化酶 (XO)体系产生的超氧阴离子自由基 (O·2 )对 [3H]DL 谷氨酸与大鼠大脑皮层突触膜谷氨酸受体结合的影响 ,结果表明O·2 明显增强谷氨酸与其受体的结合力 ,此作用能被 2 苯基 1 ,2 苯并异硒唑 3 ( 2H)酮 (EBSELEN) ( 1 μmol/L)所抑制     

Distribution of Six Synaptic Membrane Antigens in Subcellular Fractions of Rat Brain Cortex

Abstract: The subcellular distribution in rat brain cortex of six synaptic membrane antigens (56K, 58K, 62K, 63K, 64K, 66K) was studied by rocket immunoelectrophoresis, using antiserum to a highly purified synaptic plasma membrane fraction. Initial analysis of the insoluble portion of subcellular fractions showed that these antigens were also present in smooth microsomes, rough microsomes, and synaptic vesicles; that only traces were present in synaptic junctions; and that none was present in nuclei, mitochondria, and myelin. A trace amount of activity was also present in synaptic vesicle cytosol, but none in whole brain cytosol. Quantitative measurements of synaptic plasma membranes, smooth microsomes, and synaptic vesicles showed that all six antigens were present in synaptic plasma membranes and smooth microsomes, but that the 66K antigen was absent from synaptic vesicles. The 56K, 58K, 62K, 63K, and 64K antigens were present in highest concentration in synaptic plasma membranes, whereas the 66K antigen content was highest in smooth microsomes. Only the 58K, 62K, and 63K antigens were detectable in the membrane fraction of whole brain. Their enrichments in synaptic plasma membranes were 10.9, 5.4, and 5.9, respectively. We conclude that the 56K, 58K, 62K, 63K and 64K antigens are primary components of synaptic plasma membranes. The presence of synaptic plasma membrane antigens in smooth microsomes and synaptic vesicles probably represents material being actively transported, consistent with the hypothesis that proteins of synaptic plasma membranes and synaptic vesicles are transported via smooth endoplasmic reticulum.     

Calcium-Stimulated Adenosine Triphosphatases in Synaptic Membranes

We have investigated the properties of several ATPases present in synaptic membrane preparations from the cerebral cortex of rat. In addition to the intrinsic (Na+ + K+)-ATPase and a low level of contaminating Mg2+-ATPase of mitochondrial origin, both of which could be controlled by the addition of ouabain and azide, respectively, four activities were studied: (1) a Mg2+-ATPase; (2) a Mg2+-independent activity requiring Ca2+ ions at high concentrations; (3) a (Ca2+ + Mg2+)-ATPase with a high affinity for Ca2+, which were enhanced further (4) by the inclusion of calmodulin (33 nM for half-maximal activity). In the presence of 0.5 mM-EGTA in the buffer used, half saturation for these respective metal ions was observed at 0.9 mM for (1), 1.0 mM for (2), and approximately 0.3 mM for (3) and (4); the latter values correspond to concentrations of free Ca2+ of 0.38 and 0.18 microM for (3) and (4), respectively. The level of activities observed, all in nmol X min-1 X mg-1, under optimal conditions of 37 degrees C, was in a number of preparations (n in parenthesis): for (1) 446 +/- 19 (19); for (2) 362 +/- 18 (3) for (3) 87 +/- 13 (12); and for (4) 161 +/- 29 (12). The (Ca2+ + Mg2+)-ATPase, both in the presence and absence of calmodulin, could be inhibited specifically by a number of agents (approximate I0.5 in parentheses) which, at these concentrations, showed little or no potency against the other activities; among them were vanadate (less than or equal to 10 microM), La3+ (75 microM), trifluoperazine, and other phenothiazines (50 microM). These properties suggest that the (Ca2+ + Mg2+)-ATPase described may be responsible for calcium transport across one (or more) of the several membranes present in nerve endings and contained in the preparation used.     

In Vitro and In Vivo Binding of S-Adenosyl-L-Homocysteine to Membranes from Rat Cerebral Cortex

Membranes from rat cerebral cortex are able to bind S-adenosyl-L-homocysteine (SAH) with a KD of 5 . 10(-7) M and n of 170 pmol/g fresh tissue (i.e. 20 mg protein). The binding is enhanced by Mg2+ and Ca2+ but not K+ and Na+. gamma-Aminobutyric acid, diazepine, noradrenaline and alpha antagonists are without any effect; S-adenosyl-L-methionine, adenosine and adenosine triphosphate inhibit SAH binding. Linkage with an adenosine receptor has not been expressly demonstrated by our method. SAH binding proteins are more abundant in the crude synaptosomal pellet (P2). A similar fixation seems to occur on brain membranes after [3H]SAH administration to rat. The binding might be linked to a methylase activity or an adenosine receptor.     

Interference of S-Nitrosoglutathione with the Binding of Ligands to Ionotropic Glutamate Receptors in Pig Cerebral Cortical Synaptic Membranes

The interactions of S-nitrosoglutathione (GSNO) with the ionotropic glutamate receptors were studied on synaptic membranes isolated from the pig cerebral cortex. GSNO displaced the binding of [³H]glutamate, 3-[(R)-2-carboxypiperazin-4-yl][³H]propyl-1-phosphonate ([³H]CPP), a competitive N-methyl-D-aspartate (NMDA) antagonist, and [³H]kainate, with IC₅₀ values in the low micromolar range. It failed to displace (S)-5-fluoro-[³H]willardiine, a selective agonist of 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Reduced and oxidized glutathione were almost as effective as GSNO in glutamate and CPP binding. Of the three, GSNO was the most potent in kainate binding. They all stimulated [³H]dizocilpine binding in a concentration-dependent manner. This effect was additive to that of glycine and not mimicked by NO donors such as S-nitroso-N-acetylpenicillamine, 5-amino-3-morpholinyl-1,2,3-oxadiazolium chloride (SIN-1) and nitroglycerin. We assume that GSNO may act as an endogenous ligand at the NMDA and non-NMDA classes of glutamate receptors. In this manner it may facilitate NO transfer and target its delivery to specific sites in these receptors.     

Solubilisation of a Glutamate Binding Protein from Rat Brain

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.     

Neurone-Specific Enolase and Creatine Phosphokinase Are Protein Components of Rat Brain Synaptic Plasma Membranes

Abstract: Neuron-specific enolase and creatine phosphokinase were found, by 2-dimensional gel analysis, in rat brain synaptic plasma membranes (SPM). The identity of these enzymes was confirmed by comigration with purified rat brain NSE and CPK and by peptide analysis. The specific enzymatic activities of enolase and creatine phosphokinase, as well as of pyruvate kinase, also present on the membranes, were comparable to those in the homogenates when these three enzymes were fully activated. In the SPM all three enzymes, particularly enolase, were partially cryptic in that enzymatic activities were very low unless the membranes were treated with Triton X-100. They were resistant to both low-salt and high-salt extraction and to trypsin, except when Triton X-100 was present. These results suggest that the enzymes are tightly bound protein components of the membrane and that they may constitute an assembly capable of generating ATP.     

Calmodulin Stimulation of Ca2+-Dependent ATP Hydrolysis and ATP-Dependent Ca2+ Transport in Synaptic Membranes

We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+.     

Histamine Acting on H2 Receptors Stimulates Phospholipid Methylation in Synaptic Membranes of Rat Brain

Histamine stimulated [3H]methyl group incorporation into phospholipids in crude synaptic membranes of rat whole brain (without cerebellum) in modified Krebs-Ringer solution containing the methyl donor S-adenosyl-[methyl-3H]methionine. The transient increase of [3H]methyl incorporation into lipids peaked within 45 s after addition of histamine (5 or 10 microM) and decreased the basal level in 60 s. Histamine-stimulated [3H]methyl incorporation was increased linearly in a protein concentration-dependent manner. The stimulation was temperature and histamine concentration dependent. TLC analysis of a chloroform/methanol extract indicated that radioactive phospholipids (phosphatidylcholine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidyl-N-monomethylethanolamine) accounted for 60-65% of the total radioactivity recovered. The synaptosomal fraction had the highest specific activity of all the subfractions of crude synaptic membranes (P2). Histamine-induced [3H]methyl incorporation was inhibited by addition of cimetidine (0.01-10 microM) or famotidine (0.01-1.0 microM) in a concentration-dependent manner but not by mepyramine (0.1-10 microM) or diphenhydramine (0.1-10 microM). The stimulation of [3H]methyl incorporation was also observed by addition of impromidine (0.01-10 microM) or dimaprit (1.0 microM-1.0 mM) in a concentration-dependent manner but not by 2-pyridylethylamine (1.0 microM-1.0 mM). These results indicate that phospholipid methylation is induced by histamine acting on H2 receptors in rat brain synaptosomes.     

Quinolinic Acid-induced Seizures Stimulate Glutamate Uptake into Synaptic Vesicles from Rat Brain: Effects Prevented by Guanine-based Purines

Glutamate uptake into synaptic vesicles is a vital step for glutamatergic neurotransmission. Quinolinic acid (QA) is an endogenous glutamate analog that may be involved in the etiology of epilepsy and is related to disturbances on glutamate release and uptake. Guanine-based purines (GBPs) guanosine 5′-monophosphate (GMP and guanosine) have been shown to exert anticonvulsant effects against QA-induced seizures. The aims of this study were to investigate the effects of in vivo administration of several convulsant agents on glutamate uptake into synaptic vesicles and investigate the role of MK-801, guanosine or GMP (anticonvulsants) on glutamate uptake into synaptic vesicles from rats presenting QA-induced seizures. Animals were treated with vehicle (saline 0.9%), QA 239.2 nmoles, kainate 30 mg/kg, picrotoxin 6 mg/kg, PTZ (pentylenetetrazole) 60 mg/kg, caffeine 150 mg/kg or MES (maximal transcorneal electroshock) 80 mA. All convulsant agents induced seizures in 80–100% of animals, but only QA stimulated glutamate uptake into synaptic vesicle. Guanosine or GMP prevented seizures induced by QA (up to 52% of protection), an effect similar to the NMDA antagonist MK-801 (60% of protection). Both GBPs and MK-801 prevented QA-induced glutamate uptake stimulation. This study provided additional evidence on the role of QA and GBPs on glutamatergic system in rat brain, and point to new perspectives on seizures treatment.     

Amphetamine Increases Extracellular Concentrations of Glutamate in the Prefrontal Cortex of the Awake Rat: A Microdialysis Study

Using microdialysis, the effect was investigated of intracerebral infusions of different doses of amphetamine (1.25, 2.5, 5, 10, and 20 g/l) on the extracellular concentrations of glutamate in the medial prefrontal cortex of the rat. Amphetamine produced a dose-related increase in extracellular concentrations of glutamate. At the highest dose, amphetamine increased extracellular glutamate by 445% of baseline as well as extracellular concentrations of taurine, and reduced extracellular concentrations of glutamine. Amphetamine did not modify other amino acids such as arginine. Increases in extracellular concentrations of glutamate and taurine were independent of calcium in the perfusion medium. This is the first study showing that amphetamine produces a calcium-independent increase in extracellular concentrations of glutamate and taurine in the medial prefrontal cortex of the rat.     

Characterization of γ-Aminobutyric Acid Binding Sites on Crude Synaptic Membranes

[3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X-100 [membranes (TX)] involved two classes of GABA binding sites (KD = 38.7 and 78.0 nM) in the absence of Na+, but the high-affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD = 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high-affinity class of GABA receptor.     

A Comparison of Some Metabolic Effects of N-Methylaspartate Stereoisomers,Glutamate and Depolarization: A Multinuclear MRS Study

Exposure of guinea pig brain slices to low concentrations (10 M) of NMDA caused decreases in PCr and ATP within 30 min, with a slower decrease in NAA and increase in lactate, both detectable after 1 h. Exposure to NMDA for over 1 h or at higher concentrations caused further increases in lactate and decreases in NAA, with no further change in PCr or ATP. The L-isomer, NMLA, and the racemic mixture, NMDLA, caused similar changes in lactate and NAA, but both produced greater decreases in the energy state than NMDA, similar to those caused by prolonged exposure to glutamate. MK-801 prevented the changes in the energy state caused by NMDA, but not those caused by NMLA or by glutamate. The results are compared to previous studies on depolarization and discussed in terms of the role of the NMDA sub-type of glutamate receptor in the excitotoxic hypothesis of neuronal degeneration.