Synaptosomal Membrane Fragments Research Articles

Anticonvulsant activity of 4-urea-5,7-dichlorokynurenic acid derivatives that are antagonists at the NMDA-associated glycine binding site.

Twelve 4-urea-5,7-dichlorokynurenic acid derivatives were synthesized by reacting the 4-tosylimino derivative of 5,7-dichlorokynurenate methyl ester first with triphosgene and then with a secondary amine. Compounds were screened in mice for anticonvulsant activity using maximal electroshock (MES), subcutaneous pentylenetetrazole (Met), and threshold tonic extension (TTE) tests. A rotorod test was used to determine neurotoxicity. Seven of the derivatives had anticonvulsant activity in TTE testing at 100 mg/kg. One compound, 2-methyl carboxylate-5,7-dichloro-4-([¿diphenylamino¿-carbonyl]amino)-quino line, had an ED50 value of 134 mg/kg (95% conf. int.: low-78.5, high-205.7; slope 1.9, SE = 0.44) in TTE testing. Two derivatives had MES activity. Only one compound, an N,N-diethylamino derivative, was neurotoxic in the rotorod test. Compounds were screened at a 10-microM concentration for activity in displacing 5,7-dichlorokynurenic acid from synaptosomal membrane fragments. Since 9 of the 12 compounds synthesized and tested have demonstrated anticonvulsant activity, this class of chemicals offers promise for the production of useful therapeutic agents.

Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes

Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes. Toxicon 29, 973–988 1991.—Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus, was iodinated and used to demonstrate high affinity, specific binding to guineapig ( Cavia porcellus) brain synaptosomes and synaptosomal membrane fragments. 125I-crotoxin binding to the membrane fragments displays two binding plateaus, ( K d1 = 4 nM and K d2 = 87 nM, B max1 = 2 and B max2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau ( K d = 2 nM and B max = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125I-crotoxin binding to synaptosomal membrane fragments gave a K d-value of 3 nM. The K d value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, β-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A 2, dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A 2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins.

Ca2+ Sensitivity of Ca2+‐Dependent Protein Kinase Activities Toward Intrinsic Proteins in Synaptosomal Membrane Fragments from Rat Cerebral Tissue

The Ca2+ and calmodulin sensitivity of endogenous protein kinase activity in synaptosomal membrane fragments from rat brain was studied in medium containing Ca2+ plus EGTA using a modified computer programme to calculate free Ca2+ concentrations that took into account the effect of all competing cations and chelators. The Ca2+-dependent phosphorylation of 10 major polypeptide acceptors with Mr values ranging from 50 to 360 kilodaltons required calmodulin in reactions that were all equally sensitive to Ca2+; half-maximal phosphorylation required a free Ca2+ concentration of 45 nM and maximal phosphorylation approximately 110 nM. The significance of these values in relation to published data on the intracellular concentration of free Ca2+ in the nervous system is discussed. One acceptor of 45 kilodaltons was phosphorylated in a Ca2+-dependent reaction that did not require calmodulin. This polypeptide appeared to correspond to the B-50 protein, an established substrate of the lipid-dependent protein kinase C. Further study of this phosphorylating system showed that the reaction was only independent of calmodulin at saturating concentrations of Ca2+; at subsaturating concentrations (in the range 50-130 nM), a small but significant stimulation of the enzyme by calmodulin was demonstrated. The possible significance of this finding is discussed.

Stimulation and inhibition by magnesium ions of intrinsic protein phosphorylating systems in synaptosomal membrane fragments from rat brain.

Synaptosomal membrane fragments from rat brain were incubated with [gamma-32P]ATP in the presence of cyclic AMP or Ca2+ plus calmodulin and a range of Mg2+ concentrations. Incorporation of 32P into membrane polypeptides was examined by electrophoresis and radioautography. Cyclic AMP-stimulated reactions were stimulated by low concentrations and inhibited to varying degrees by high concentrations of Mg2+ in the range 1-50 mM. In general the Ca2+ plus calmodulin-stimulated reactions were maximally active in the range 30-50 mM Mg2+, but the Ca2+ plus calmodulin dependent phosphorylation of Protein I was progressively inhibited by concentrations of Mg2+ above 5 mM. These results emphasize the importance of establishing optimum Mg2+ concentrations in the study of specific membrane protein phosphorylating systems.

Intrinsic protein phosphorylation in synaptosomal plasma membrane fragments: a comparison of cerebral cortex tissue from several species, including human biopsy specimens.

Intrinsic protein phosphorylation was studied in synaptosomal membrane fragments made from cerebral cortex tissue taken from the following species: human (biopsy specimens), ox, rat, rabbit, guinea pig and mouse. Membrane fragments from all species exhibited a qualitatively similar range of protein acceptors phosphorylated by cyclic AMP-dependent protein kinase activity; contrary to a previous report, no evidence for cyclic GMP-dependent protein kinase activity was found in the human material. With the exception of membrane fragments prepared from ox brain, all the preparations exhibited the same range of Ca2+-dependent protein kinase activity. Ox brain obtained from a slaughterhouse yielded membranes containing no Ca2+-dependent protein kinase activity, but this may have been due to unavoidable postmortem losses.

Ca2+-sensitivity of Ca24 -plus-calmodulin-dependent protein kinase activity towards intrinsic proteins in synaptosomal membrane fragments from rat cerebral cortex

Ca2+-sensitivity of Ca24 -plus-calmodulin-dependent protein kinase activity towards intrinsic proteins in synaptosomal membrane fragments from rat cerebral cortex

Resolution of two biochemically and pharmacologically distinct benzodiazepine receptors

Brain-specific binding sites have been isolated on synaptosomal membrane fragments which recognize pharmacologically active benzodiazepine (BDZ's) and triazolopyridazines (TPZ's). While early evidence indicated the existence of a single homogeneous class of BDZ binding sites, more recent biological and pharmacological studies support the notion of BDZ receptor multiplicity. We now propose that two biochemically distinct BDZ receptors exist in brain which are responsible for the mediation of different pharmacological activities. Type I BDZ receptors display a high affinity for both BDZ's and TPZ's, are not coupled to GABA receptors or to chloride ionophores, and are the sites which mediate anxiolytic actions. Type II BDZ receptors display a high affinity for BDZ's display a low affinity for TPZ's, are coupled to GABA receptors and/or chloride ionophores, and are the sites which mediate pharmacological effects other than anxiolytic activity.

Characterization and localization of acid hydrolase activity in the synaptosomal fraction from rat cerebral cortex.

AbstractSynaptosomes were prepared from the cerebral cortex of adult rats by a rapid technique of centrifugation in a Ficoll‐sucrose discontinuous gradient. The synaptosomal fraction contained 40 per cent of the total gradient activity of acid α‐naphthyl phosphatase (EC 3.1.3.2). Quantitative electron microscopy of this fraction revealed rare, typical, extrasynaptosomal dense body lysosomes. pH‐activity profiles of free and Triton X‐100 (total) activities were prepared for α‐naphthyl phosphatase, β‐glucuronidase (EC 3.2.1.31), β‐galactosidase (EC 3.2.1.23), arylsulfatase (EC 3.1.6.1) and N‐acetylglucosaminidase (EC 3.2.1.30). The ratios of total to free activity varied in the order: arylsulfatase > β‐galactosidase > β‐glucuronidase > N‐acetylglucosaminidase > acid phosphohydrolase. Incubation of synaptosomal fractions at pH 5 and 37°C produced significant activation of β‐galactosidase and N‐acetylglucosaminidase but no activation of cryptic lactate dehydrogenase (EC 1.1.1.27). Hyposmotic suspension and subfractionation of the synaptosomal fraction produced considerable solubilization of lactate dehydrogenase, arylsulfatase and β‐galactosidase but only partial liberation of α‐naphthyl phosphatase, the remainder being associated with synaptosomal membrane fragments. Incomplete equilibrium sedimentation of synaptosomes in a continuous sucrose gradient (0·55‐1·5 M) provided a broad lactate dehydrogenase and Na + K ATPase (EC 3.6.1.4) peak (peak I) at low sucrose densities. β‐Glucuronidase, β‐glucosidase and α‐naphthyl phosphatase were significantly present in peak I. Conversely, N‐acetylglucosaminidase, arylsulphatase and β‐galactosidase were predominantly located in denser particles sedimenting through 1·2 M sucrose (peak II). Electron microscopy confirmed the heterogeneity of this second peak and the presence of numerous extrasynapto‐somal dense body lysosomes.

Evidence for Protein Synthesis in Synaptosomal Membranes

Evidence is presented that a brain fraction containing synaptosomal membrane fragments and ghosts prepared from lysed rat forebrain synaptosomes synthesize protein in a cell free system. The protein synthesis was dependent upon the addition of a synaptosomal membrane fraction, Mg++, monovalent cations, ATP, GTP, and enzyme factors prepared from intact synaptosomes. Protein synthesis was inhibited by ribonuclease, cyclohexamide, puromycin, and phosphate ion, but chloramphenicol, erythromycin, ouabain, and KCN had no significant effect. The products of protein synthesis with both the synaptosomal membrane fraction and microsomal fractions from rat forebrain were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The distribution of the synaptosomal membrane fraction product in the polyacrylamide gels differed significantly from the microsomal product. Also the distribution of products following density gradient centrifugation and a comparison of requirements for monovalent cations further differentiated protein synthesis with the synaptosomal membrane fraction from that with microsomal preparations.

Ion effects and protein synthesis in synaptosomal fraction.

AbstractThe capacity of the synaptosomal fraction to synthesize protein in vitro is affected by many ionic environments which also exert effects upon neuronal properties in vivo. Protein synthesis in this fraction is at a maximum in high sodium ion, and low but not zero potassium ion medium. The effects of changes in the ionic composition of the incubation medium upon synaptic fraction protein synthesis resemble the effects observed in vivo on the neuronal membrane from which it is derived. Evidence is obtained for the function of Na‐ATPase in amino acid uptake into the particles.Since the experiments of Weiss and Hiscoe, 1948, it has been generally accepted that pre‐synaptic and axonal proteins of neurons are supplied from the cell‐body to these areas by the process of somato‐axonal flow. However we have argued from the time‐course of incorporation of labelled amino acids into proteins of the synaptosomal fraction in chopped brain tissue, that synaptic protein synthesis occurs in vivo (Austin and Morgan, 1967). This conclusion has been supported by an analysis of the pattern of labelling of brain subcellular fractions in vivo (Von Hungen et al., 1968) which indicated that synaptosomal membrane fragments were capable of protein synthesis.Recently we have shown that synaptosomal fractions isolated from immature rat brain, synthesize protein in a cell‐free system (Morgan and Austin, 1968). This ability of isolated pre‐synaptic material to synthesize protein has been supported by other studies on protein synthesis in synaptosomal preparations (Autilio, et al., 1968; Gordin, et al., 1968) and in accord with the previously demonstrated ability of isolated nerve segments to incorporate labelled amino acid into axonal proteins (Edström, 1966).Although a high rate of protein synthesis has been observed in synaptosomal preparations, the morphological entities with which it is associated have not yet been unequivocally identified. Previous studies (Morgan and Austin, 1968) demonstrated that the properties of the protein synthesis were indicative of the operation of a eukaryotic protein synthesizing system though a small component was attributed to mito‐chondrial protein synthesis. A recent report (Gordon and Deanin, 1968) has suggested that brain mitochondria have similar properties to synaptosomes with regard to protein synthesis but these effects are possibly due to contamination by other subcellular particles (Yellin, personal communication).The functional activity of the nervous system in conducting nerve impulses is extremely sensitive to extracellular ion concentrations (Eccles, 1964) and these effects have been studied in isolated neurons (Edwards et al., 1963). Ion effects upon a biochemical parameter, respiration, have been observed in isolated neurons (Giacobini, 1967). Many of these effects are believed to be mediated by changes in properties of the neuronal membrane.Morphologically the synaptosome appears to be a membrane‐bound sample of axoplasm which typically contains mitochondria and synaptic vesicles (Whittaker, 1965). There is evidence that the synaptosomal membrane may retain some of the in uiuo functions of the neuronal membrane, namely that synaptosomal preparations take up noradrenaline and serotonin (Bogdansky et al., 1968), GABA (Varon et al., 1967), and choline (Potter, 1967). Active transport has not been demonstrated for sodium and potassium ions (Marchbanks, 1967), nor for acetylcholine (Marchbanks, 1968). But these studies were carried out at 0–5° where little enzymic activity would be expected. In view of the possible active functions of the synaptosomal membrane we set out to determine the effects of variations in the incubation medium upon protein synthesis in isolated synaptosomal fractions.