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PubMed1986

Effect of bilirubin on the membrane potential of rat brain synaptosomes.

The effect of the neurotoxic pigment bilirubin on the membrane potential of rat brain synaptosomes was studied by using the tetraphenylphosphonium ion (TTP+) technique. Bilirubin induces a rapid depolarization of synaptosomes, as reflected by an efflux of previously accumulated [3H]TTP+. This phenomenon persisted when the membrane potential across either the plasma membrane of the synaptosome or the inner membrane of the entrapped mitochondria was selectively depressed, thus indicating that both components of the synaptosomal membrane potential were affected by bilirubin. Bovine serum albumin, used at a albumin/bilirubin molar ratio of 1:1, had the capacity to completely prevent and reverse the effect of bilirubin. This fact demonstrates that the bilirubin-induced TPP+ release from synaptosomes is a reversible process that requires the presence of bilirubin interacting with the synaptosomal membranes. These results, together with the inhibition by bilirubin of [3H]TPP+ and [2-14C]acetate uptake by synaptosomal plasma membrane vesicles isolated from rat brain, suggest that bilirubin depresses the membrane potential across the synaptosomal plasma membrane by a mechanism involving alterations in ion permeability. This effect could be of relevance in the pathogenesis of bilirubin encephalopathy.

Animals↗

PubMed1990

Release of immunoreactive insulin from rat brain synaptosomes under depolarizing conditions.

Synaptosome preparations were utilized to characterize the release and compartmentalization of immunoreactive insulin (IRI) in the adult rat brain. Depolarization of synaptosomes by elevation of the external potassium ion concentration elicited release of IRI from the synaptosomes into the incubation medium. This release was reduced or eliminated under three conditions known to prevent depolarization-induced Ca2+ flux: elevating the external MgCl2, adding CoCl2, and eliminating external Ca2+ with EGTA. Depolarization of synaptosomes by veratridine also elicited release of synaptosomal IRI. This release was inhibited by tetrodotoxin. The amount of IRI released under depolarizing conditions represented 3-7% of that contained in the synaptosomes. High levels of IRI release also were observed upon removal of external Na+ to allow depolarization-independent influx of external Ca2+ into the synaptosomal compartment. The Ca2+ dependency of synaptosomal IRI release suggests IRI is stored in the adult rat brain in synaptic vesicles within nerve endings from which it can be mobilized by exocytosis in association with neural activity.

Animals↗

PubMed1986

The regulation of cytosolic calcium in rat brain synaptosomes by sodium-dependent calcium efflux.

1. When pinched-off presynaptic nerve endings (synaptosomes) isolated from rat brain are incubated in a low-Na (24-36 mM) medium, they take up 45Ca in a time-dependent manner. In a medium containing 1 mM-Ca, this Na-dependent 45Ca uptake amounts to approximately 10 nmol/mg protein at 1 min, and to approximately 40 nmol/mg protein at 20 min. The Na-dependent Ca uptake is not reduced when the synaptosomes are loaded with concentrations of quin 2 as high as 2 mM. 2. The increase in 45Ca uptake is paralleled by an increase in the free cytosolic Ca concentration [Ca]i, as monitored with the fluorescent Ca indicators quin 2 or fura 2. [Ca]i increases from the value of approximately 200 to approximately 500 nM within 3-5 min, and thereafter, remains at this elevated level. 3. When synaptosomes that have been loaded with 45Ca (for 1 min, in a low-Na medium) are diluted into an Na-containing medium, there is a rapid efflux of the Ca load. After correcting for Ca that is taken up during the efflux period, calculations show that the total Ca in the synaptosomes returns to the control level within 1 min. Measurements of total chemical Ca parallel the measurements made with radiotracer Ca, and confirm that the Ca loaded into the nerve terminals during a 5 min incubation in a low-Na medium is extruded from the nerve terminals within 1 min in a normal-Na medium. 4. The efflux of Ca from the synaptosomes is paralleled by a drop of [Ca]i to its basal level, also within 1 min. 5. The mitochondrial uncoupler, carbonyl cyanide p-trifluoromethyloxy-phenyl-hydrazone (FCCP, 1 microM), has no effect on either Na-dependent Ca uptake or efflux in synaptosomes. FCCP causes a slight (100-200 nM) increase in [Ca]i in synaptosomes resuspended in either a Na or a low-Na medium. This indicates that little of the Ca that is taken up by the synaptosomes in a low-Na medium is sequestered by the mitochondria. 6. These results suggest that Na-dependent Ca efflux (probably Na-Ca exchange) plays an important role in allowing nerve terminals to recover rapidly from a Ca load.

Aminoquinolines↗

PubMed1991

Reduced phospholipid contents of brain synaptosomes in phosphate depletion.

The effects of 6 wk phosphate depletion (PD) and pair-weight feeding (PW) without and with treatment with verapamil (PD-V and PW-V, respectively) on phospholipid (PL) and cholesterol content and on resting levels of cytosolic calcium concentration ([Ca2+]i) of brain synaptosomes of rats were examined. PD was associated with significantly (P less than 0.01) lower synaptosomal content of total PL, phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylethanolamine (PE) and with significant (P less than 0.01) elevation in [Ca2+]i. Verapamil treatment of PD rats prevented the rise in [Ca2+]i of brain synaptosomes and the fall in PI and caused significant (P less than 0.01) improvement in the synaptosomal content of PS and PE; values of PS and PE in PD-V rats, however, were still significantly (P less than 0.01) lower than those of PD rats. Verapamil treatment of PW rats did not affect synaptosomal content of the various PL or resting levels of [Ca2+]i. Cholesterol content of brain synaptosomes of the various groups was not significantly different, but cholesterol-to-total PL ratios were significantly (P less than 0.01) higher in PD and PD-V rats than in PW or PW-V animals. Results indicate that PD affects metabolism of total phospholipids, PI, PS, and PE of brain synaptosomes, and these derangements are due to reduced availability of phosphorus and to the rise in [Ca2+]i. The data are consistent with the proposition that PD-induced changes in PL render the synaptosomal membrane more permeable to calcium, an event that may lead to a rise in [Ca2+]i.

Animals↗

PubMed1986

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.

Animals↗

PubMed2003

Contribution of lipid dynamics on the inhibition of bovine brain synaptosomal Na+-K+-ATPase activity induced by 4-hydroxy-2-nonenal.

The effects of lipid hydroperoxide degradation products, such as 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA), on bovine brain synaptosomal ATPase activities and their membrane lipid organization were examined. When the synaptosomes were treated with HNE, this resulted in the decrease of Na(+)-K(+)-ATPase activity with the loss of sulfhydryl (SH) groups in the membrane proteins. In contrast, MDA treatment of the synaptosomes did not induce an appreciable decrease in the ATPase activity or a loss of SH groups. The decreases in ATPase activity and SH content by treatment with HNE were also observed, as a Na+-K+-ATPase preparation was used in place of the synaptosomes. On the other hand, HNE had very little effect on synaptosomal Ca2+- and Mg2+-ATPase activities. The results of the kinetic analysis of the Na+-K+-ATPase activity indicated that the decrease in the activity by HNE-modification is due to a decreased affinity for the substrate. ATP completely protected the ATPase from the HNE attack. Modification of the synaptosomes with HNE caused a decrease in the membrane lipid fluidity near the lipid/water interface, not the lipid layer interior. In addition, it was found that there is a good relationship between the lipid fluidity and the Na+-K+-ATPase activity under the presence of various concentrations of HNE, suggesting that the lipid dynamics are closely related to HNE-induced inhibition of the ATPase activity. On the other hand, MDA did not induce change in the membrane lipid fluidity. HNE and MDA are mainly incorporated into the lipid and protein fractions in the synaptosomal membranes, respectively. Based on these results, we proposed a possible mechanism of HNE-induced inhibition of synaptosomal Na+-K+-ATPase activity associated with alterations in the membrane lipid organization.

Aldehydes↗

PubMed1990

Phase partitioning of beta-bungarotoxin-treated cerebrocortical synaptosomes: quantitation of the toxin-vulnerable population.

beta-Bungarotoxin induces changes in the partition of cerebrocortical synaptosomes in Dextran-poly(ethylene glycol) two-phase systems. The toxin action promotes an increase of the partition percentage of synaptosomes in the upper poly(ethylene glycol) rich phase. This effect was observed in both Dextran T500 and Dextran T40 two-phase systems. In both cases an increase of partition in this upper phase of a discrete amount of the whole synaptosomal population accounts for almost 100% of choline acetyltransferase activity, a marker of cholinergic synaptosomes, appearing in this same phase. This effect is specifically dependent on the toxin action since a control experiment shows that extraction into the upper phase of the synaptosomal preparation carried out by addition of an unspecific ligand (hexaethonium) bound to poly(ethylene glycol), could induce the partition of both the whole choline acetyltransferase activity and synaptosomal material in this upper phase. These results suggest that beta-bungarotoxin specifically induces the partition into the upper phase of a toxin-vulnerable synaptosomal population. Therefore, it has been estimated that 30 or 32% of the total synaptosomal preparation is sensitive to the toxin action, as calculated from the maximal increase in the toxin-treated particles partition obtained in the Dextran T500 or Dextran T40 two-phase systems, respectively.

Animals↗

PubMed1987

Modulation of levels of free calcium within synaptosomes by organochlorine insecticides.

Effects of the organochlorine insecticides chlordecone, mirex, 1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane on free intrasynaptosomal Ca2+ [( Ca++]i), synaptosomal 45Ca uptake and synaptosomal plasma and mitochondrial membrane potentials in vitro were studied. Chlordecone (10-50 microM) increased [Ca++]i from the resting level of 370 nM in a dose- and time-dependent manner to above 1.5 microM. This took place in the presence of 1 mM extrasynaptosomal Ca++ but not in nominally Ca++-free medium. Verapamil, a voltage sensitive Ca++ channel blocker, inhibited the initial increase of [Ca++]i caused by chlordecone, by 40%. Chlordecone also elevated [Ca++]i in synaptosomes in which mitochondrial Ca++ uptake had been abolished by valinomycin. Chlordecone depolarized partially the synaptosomal plasma membrane and, to a lesser extent, the potential of mitochondria within synaptosomes. However, chlordecone appeared to inhibit synaptosomal K+-stimulated and unstimulated 45Ca++ uptake by 20 to 30%. Inasmuch as chlordecone also stimulated release of 45Ca++ and the fluorescent dye fura-2 from preloaded synaptosomes, the apparent inhibition of uptake might be due to lysis of some synaptosomes by chlordecone. The effect of chlordecone on [Ca++]i decreased when the total amount of tissue in incubations was increased. [Ca++]i was only elevated marginally by mirex at the same concentration range. The results suggest that chlordecone increases free intrasynaptosomal Ca++ mainly by increasing influx of extrasynaptosomal Ca++. The principal mechanism appears to be a nonspecific leakage of Ca++ through the plasma membrane but some Ca++ may pass through voltage-sensitive Ca++ channels due to chlordecone-induced membrane depolarization.

Animals↗

PubMedDate not supplied

[Binding of latrotoxin to synaptosomes and synaptic membranes of the rat brain].

The binding of [125I] alpha-latrotoxin to synaptosomes from the rat brain is studied. It is shown that the constant rate of toxin association with the synaptosome receptor at 37 degrees C is equal to 8.2 +/- 1.3 x 10(7) M-1.s-1, while that of synaptosomal membrane -7.6 +/- 2.7 x 10(6) M-1 s-1. Depolarization of the synaptosome membrane induced by 55 mM KCl decreases the binding rate of toxin to the receptor, the rate constant being equal to 3.9 +/- 1.5 x 10(7) m-1 s-1. The pattern of the dissociation process of the toxin-receptor complex of synaptosomes and of synaptosomal membrane is different. In the first case dissociation follows two stages with the rate constants 3.6 x 10(-3) s-1 and 1.2/10(-4) s-1, in the second case it follows one stage with the constant equalled 2.0 x 10(-5) s-1. The quantity of the toxin binding sites on synaptosomes may vary under the action of agents modifying the activity of calcium fluxes which are induced by alpha-latrotoxin. It is supposed that a decrease in the ATP level in synaptosomes as well as deenergy of the surface membrane leads to a change in the state of the alpha-latrotoxin receptor.

Animals↗

PubMed1977

Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.

Animals↗

PubMed1983

Inhibition of fast- and slow-phase depolarization-dependent synaptosomal calcium uptake by ethanol.

Uptake of 45Ca++ by synaptosomes isolated from cerebral cortex, cerebellum, midbrain and brain stem of male, Sprague-Dawley rats was measured at 1-, 3-, 5-, 15-, 30- and 60-sec time periods. At 1 sec, the Ca++ uptake rate by cerebrocortical synaptosomes was 1.45 mumol/sec/g of protein, whereas the 60-sec rate was 0.03 mumol/sec/g of protein. In vitro addition of ethanol, 80 mM, inhibited depolarization-dependent (65 mM KCl) 45Ca++ uptake by synaptosomes but the time-response relationships varied depending upon the brain region studied. In cerebrocortical synaptosomes, ethanol significantly inhibited only the fast-phase component of 45Ca++ uptake (1 and 3 sec). Ethanol inhibited 45Ca++ uptake by midbrain synaptosomes at all measurement times studied (1, 3, 5 and 15 sec), whereas in cerebellum and brain stem ethanol inhibited 45Ca++ uptake at 3- and 5-sec time periods. Ethanol at concentrations of 25, 50, 100 and 150 mM inhibited 45Ca++ uptake by 9.0, 15.9, 24.8 and 30.7%, respectively, in cerebrocortical synaptosomes. In vitro ethanol, 80 mM, added to cerebrocortical synaptosomes isolated from rats fed a nutritionally adequate liquid ethanol diet did not significantly inhibit depolarization-dependent 45Ca++ uptake. The results of this study show that pharmacologically relevant ethanol concentrations inhibit voltage-dependent 45Ca++ uptake into synaptosomes. This inhibitory action may, at least in part, underlie some of the intoxicating effects of ethanol. In addition, chronic administration of ethanol resulted in an apparent adaptive response such that addition of ethanol no longer blocked 45Ca++ uptake. This adaptive response involving the calcium channel may represent a cellular mechanism for functional tolerance development.

Animals↗

PubMed1981

[Quantitative determination of neurospecific protein S-100 in mouse brain cortical synaptosomes].

Using monospecific antibodies against protein S-100 labelled with horseradish peroxidase (a-S-100-HP) or iodine (a-S-100-125I) two types of protein S-100 bound to the synaptosomal membrane were obtained. The first type accessible for a-S-100-HP (6.4% of total protein S-100 in the synaptosomes) and the second type accessible for a-S-100-125I (22.4%) differ in their location on the membrane surface. The total content of protein S-100 in the synaptosomes determined by solid phase radioimmune analysis is 284 +/- 0.84 ng per mg of total synaptosomal protein. Coprecipitation of the protein with the synaptosomal fraction is insignificant. After treatment of the synaptosomal membranes with 1% glutaric aldehyde or 4% paraformaldehyde, 20 and 50% of membrane-bound protein S-100 are found in the synaptosomal membrane, respectively. The immunohistochemical localization of protein S-100 in synaptosomes was established.

Animals↗

PubMed1993

Fewer PGE2 and PGF2 alpha receptors in brain synaptosomes of newborn than of adult pigs.

PGE2 and PGF2 alpha decrease cerebral metabolism in the adult but increase it in the newborn. The present study was done using synaptosomes from newborn and adult pigs to find out whether these diverse cerebral effects of prostanoids are a consequence of newborn synaptosomes containing fewer or no EP2 receptors for PGE2 (mediating cAMP increase and in turn cerebral metabolism decrease) and PGF2 alpha receptors [mediating IP3 (inositol 1,4,5-triphosphate) increase and in turn cerebral metabolism decrease]. It was found that maximum binding values (fmol/mg protein) of [3H]PGE2 and [3H]PGF2 alpha were, respectively, only 11.6 +/- 0.8 and 8.6 +/- 1.4 on newborn synaptosomes compared with 41.8 +/- 1.8 and 31.2 +/- 4.8 on adult tissues. EP1 receptors were virtually absent in newborn and adult preparations. Practically all the PGE2 receptors on newborn synaptosomes were EP3 subtype, whereas adult brain synaptosomes contained both EP2 and EP3 subtypes; this was indicated by the ability of M&B 28,767, a specific EP3 receptor agonist, to cause 100% displacement of [3H]PGE2 binding to synaptosomes of the newborn but only 51% in the case of the adult. Also, PGE2, 11-deoxy PGE1 (a nonselective EP2 and EP3 agonist) and M&B 28,767 (an EP3 agonist) caused comparable decreases in cAMP formation in synaptosomes from the newborn, whereas in those from the adult, PGE2 and 11-deoxy PGE1 increased and M&B 28,767 decreased cAMP production. PGF2 alpha markedly increased IP3 synthesis in synaptosomes of the adult but not of the newborn.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1976

Effects of neonatal hypothyroidism on cerebral and cerebellar synaptosome development.

The effect of hypothyroidism on cerebral and cerebellar synaptosome development has been studied. Neonatal hypothyroidism was induced following addition of 0.3% propylthiouracil to the diet of nursing mothers. Maturation profiles of total synaptosome fraction and specific activities of lactate dehydrogenase, Na+K ATPase, cytochrome c oxidase, and protein were obtained from days 6 to 32 on synaptosomes isolated from Ficoll-sucrose gradients. The greatest changes were found in the total activities of enzymes isolated from the cerebellum. Hypothyroidism induced a retardation of LDH and cytochrome c oxidase in cerebellar synaptosomes, but no change in corresponding specific activities. Maximum rates of 14C-leucine incorporation into cerebellar synaptosome protein was found at 16-20 days, after which a rapid decline occurred to adult levels at 32 days. In neonatal hypothyroidism, synthesis was significantly reduced at 8 and 14 days, but reached control levels or above at 21--32 days. In the cerebrum, maximum rates of 14C-leucine incorporation into synaptosome protein were identified at 8--12 days in normal with a rapid drop to adult levels at approximately 20 days. In neonatal hypothyroidism, peak activities were identified at 14 days and increased activities over control were noted at 14, 20 and 30 days. These observations demonstrate the sensitivity of the developing cerebellar synaptic apparatus to neonatal hypothyroidism, with a protraction in the peak levels of synaptosome protein synthesis in cerebrum and cerebellum.

Adenosine Triphosphatases↗

PubMed1987

Glycolipid sialosyltransferase activity in synaptosomes exhibits a product specificity for (2-8)disialosyl lactosyl ceramide (ganglioside GD3).

Intact synaptosomes prepared from 28-day-old rat brains were incubated with CMP-N-acetyl-(14C) neuraminic acid in Krebs-Henseleit buffer in an atmosphere of 95% O2: 5% CO2, at 37 degrees C. The activity of CMP-NANA:ganglioside sialosyltransferase using endogenous acceptors was 0.84 pmoles NANA transferred/mg synaptosomal protein/hr. Analysis of the distribution of labeled sialic acid revealed that GD3 ganglioside (alpha 2----8 disialosyl, alpha 2----3 galactosyl, beta 1----4 glucosyl, beta 1----1-ceramide) was the major product in the membrane carrying 32% of the total lipid bound label. Treatment of the reaction products with Clostridium neuraminidase liberated labeled sialic acid from GD3 and yielded labeled GM3, then unlabeled lactosyl ceramide. Lac-cer and GM3 are present in small amounts in synaptosomes, and GD3 represents less than 2% of the total ganglioside. Our findings indicate that the sialosyltransferase activity of synaptosomes exhibits a preferential product specificity for the small pool of synaptosomal membrane GD3 ganglioside that may be formed in situ, via sialosylation of its precursor (GM3 or lactosyl ceramide) which pre-exists in the synaptosomal plasma membrane. The second major labeled product quantitatively was GD1a whose precursor substrate, GM1, is quite abundant in the membrane, so that the conversion rate of GM1 to GD1a was low in comparison with GD3 formation. Sialosylation of other synaptosomal membrane gangliosides was negligible.

Animals↗

PubMed1991

Characterization of the distribution of G alpha o in rat striatal synaptosomes and its colocalization with tyrosine hydroxylase.

Dopaminergic striatal synaptosomes can be detected and isolated with a fluorescence-activated cell sorter (FACS). In the present study, two antigens were detected simultaneously with primary antisera raised in different species and species-specific fluorescent secondary antibodies with different emission spectra. Double-label FACS analysis was used to determine whether tyrosine hydroxylase (TH) and the alpha subunit of Go (G alpha o) are colocalized in striatal synaptosomes. Rabbit antibodies generated against a synthetic fragment of G alpha o (corresponding to amino acids 22-35) combined with fluorescein-conjugated secondary antibodies were used to detect G alpha o-containing striatal synaptosomes. Preadsorption of G alpha o antiserum with the synthetic peptide antigen reduced labeling to the level obtained with preimmune serum. Approximately 65-75% of striatal synaptosomes were specifically labeled by G alpha o antiserum. Tyrosine hydroxylase-containing synaptosomes were detected with a mouse monoclonal antibody to TH and R-phycoerythrin-conjugated secondary antibody. They comprised 15-17% of total striatal synaptosomes. Double-label studies indicated that at least 50% of TH-containing synaptosomes also contained G alpha o. These findings suggest that G alpha o may not be a protein component of all striatal nerve terminals, and provide a basis for a role for G alpha o in signal transduction within subpopulations of intrinsic and afferent nerve terminals, including those of nigrostriatal dopamine neurons.

Animals↗

PubMed1981

Topographic studies of gangliosides of intact synaptosomes from rat brain cortex.

Gangliosides in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups with galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components fractionated on diatrizoate to give four synaptosomal membrane fractions (A-D) and a mitochondrial pellet (E). Fractions A and B represent synaptosomal plasma membranes. When intact synaptosomes were labeled, the major portion of the total radioactivity incorporated into ganglioside fraction was found to be in GM1 3 species. With isolated membrane fractions little selectivity was seen: (1) more label was present compared to intact synaptosomes, and (2) zones corresponding to GM2, GM1, GD1a, GD1b were the major gangliosides labeled. The results confirm the conclusion that membrane fractions A and B are derived from the exposed synaptosome surface and also show that GM1 is the major ganglioside species available for enzyme oxidation at the surface.

Animals↗

PubMed1976

Carrier-mediated sodium-dependent and calcium-dependent calcium efflux from pinched-off presynaptic nerve terminals (synaptosomes) in vitro.

The influence of external cations on 45Ca2+ efflux from Ca2+ loaded synaptosomes has been examined. The synaptosomes were pre-loaded with 45Ca2+ by incubating the suspensions in potassium-rich media for 2 min. The suspensions were then diluted into "efflux" media containing a "normal" (5mM) K+ concentration; the content of Na+ and Ca2+ was varied, as noted below. Efflux of 45Ca2+ was measured for a 2-min period (except for "zero-time" samples), and was terminated by filtering the suspensions on 0.3 mum cellulose acetate filters. 45Ca2+ retained on the filters was determined by liquid scintillation spectroscopy. The difference between the 45Ca2+ in the "zero-time" samples (="Ca2+ load") and in the samples incubated for 2 min was taken as the 45Ca2+ efflux. 45Ca2+ loss into Ca2+ -free efflux media containing ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) was markedly influenced by the Na+ concentration: nearly 80% of the 45Ca2+ was lost from the synaptosomes if the media contained 132 mM Na+, but only about 7% was lost in 2 min if 97% of the Na+ was replaced mol-for-mol by choline. In media containing 1.2 mM Ca2+ and 132 mM Na+, the 45Ca2+ uptake by synaptosomes previously loaded with 40Ca2+ was significantly less than 45Ca2+ loss from synaptosomes loaded with 45Ca2+. Thus there was a net efflux of Ca2+ from the Ca2+ -loaded synaptosomes; this efflux was, presumably, Na+ dependent. In media containing 1.2 mM 40Ca2+ and only 4 mM Na+, the 45Ca2+ efflux from 45Ca2+ -loaded synaptosomes was significantly greater if most of the external Na+ (128 mM) was replaced isomotically by Li+ rather than by choline, guanidine or glucose. This observation may be evidence for a Ca2+ -Ca2+ exchange which is promoted by Li+. Both the Na+ -dependent and the Ca2+ -dependent Ca2+ effluxes were inhibited by Mn2+. The data are consistent with a Ca2+ carrier mechanism which can extrude Ca2+ in exchange for Na+ or for Ca2+, the latter being activated by Li+. These properties bear a striking resemblance to those of a Ca2+ efflux mechanism which has been characterized in squid axons. This mechanism may there fore have evolved fairly early on in the history of the animal kingdom.

Animals↗