EFFECT OF DELTA-9-TETRAHYDROCANNABINOL ADMINISTRATION ON THE LIPID CONSTITUENTS OF RAT BRAIN SUBCELLULAR FRACTIONS

An investigation on the effects of acute (10 mg/kg) and chronic (10 mg/kg for 15 days) treatment with Δ⁹-THC administration by the intraperitoneal route, on the cholesterol, cerebroside and individual phospholipid contents in microsomal, synaptosomal, mitochodrial and myelin fractions from adult rat brain, is reported. The drug has been found to affect the different subcellular membranous lipid and phospholipid components in a characteristic manner.     

SUBCELLULAR FRACTIONATION OF GANGLIOSIDE SIALIDASE FROM HUMAN BRAIN

—A subcellular fractionation was performed on forebrain cortex from three human brains and the fractions obtained were assayed for ganglioside sialidase and four p-nitrophenyl glycohydrolases. Differences in the sedimentation patterns of the enzymes were observed. From 53 to 77 per cent of the recovered sialidase activity was found in the synaptosomal fraction, while the p-nitrophenyl glycosidases were mainly recovered in the lysosome-enriched fraction. Three possible interpretations of the sialidase sedimentation pattern are suggested: (1) The ganglioside sialidase is bound to the limiting membrane structure of the nerve ending. (2) The ganglioside sialidase is lysosomal, although bound to lysosomes of low density. (3) The enzyme occurs mainly in lysosomes primarily located in the nerve endings, being trapped under the formation of the synaptosomes.     

Regional and Subcellular Localization of Li+ and Other Cations in the Rat Brain Following Long-Term Lithium Administration

Abstract: Rats were given LiCl in their diet (40 mmol/kg dry weight) for at least 3 months to elucidate the regional and subcellular localization of Li⁺ in the brain as well as the effect of chronic lithium administration on the distribution of other cations. At steady-state the mean concentrations of Li⁺ were 0.66 mmol/kg wet weight in the whole brain and 0.52 mM in plasma. The tissue/plasma concentration ratio exceeded unity in all anatomical regions. No region showed excessive accumulation of Li⁺. Whole brain or regional contents of Na⁺ or K⁺ were unaffected by lithium treatment. Subcellular Li⁺ localization was demonstrated in nuclear, crude mitochondrial, and microsomal fractions of whole brain homogenate. Subfractionation of the crude mitochondrial fraction revealed energy-independent intrasynaptosomal and intramitochondrial Li⁺ and K⁺ localization at 0–4°C. Li⁺ administered in vivo disappeared within 10 min from synaptosomes incubated at 37°C. Li⁺ added in vitro at 1 mM attained a synaptosomal steady-state concentration within 30 min at 37°C. In control rats, synaptosomal concentrations and synaptosomal/medium concentration gradients of cations paralleled their respective in vivo concentrations and gradients. Lithium treatment caused synaptosomal depletion of K⁺ and Mg²⁺ and hence probably partial membrane depolarization. Addition of 1 mM Li⁺ in vitro also caused synaptosomal Mg²⁺ depletion. The results indicate that Li⁺ is “accumulated” in brain sediments and synaptosomes following its long-term treatment. The estimated intracellular and intrasynaptosomal Li⁺ concentrations are lower than predicted by passive distribution according to the Nernst equation, evidencing active extrusion of Li⁺.     

Gangliosides associated with microsomal subfractions of brain: Comparison with synaptic plasma membranes

To study ganglioside distribution within subcellular components and test the hypothesis that they are localized at the nerve ending, microsomes and synaptic plasma membranes were isolated from young adult rat brains and compared with respect to ganglioside composition. These were shown to be heterogeneous preparations by fractionation on a discontinuous sucrose gradient into subfractions which had differing ganglioside concentrations. The highest ganglioside concentrations occurred in membranes banding at the 0.8M/1.0M and 1.0M/1.3M interfaces for both microsomes and synaptic plasma membranes. These subfractions had closely similar ganglioside concentrations and pattern distributions. In addition, the kinetics of ganglioside labeling following administration of [³H]-glucosamine were similar for the two preparations. The fact that microsomal subfractions representing heterogeneous mixtures of brain cell membranes showed close similarity to synaptosomal plasma membranes argues against localization of gangliosides at the nerve ending. These results, together with other lines of evidence, support the concept that gangliosides are distributed over large portions of the neuron (and perhaps other brain cells). Data concerning the labeling of gangliosides in different microsomal subfractions indicated a movement of label over time from the more dense to the less dense membranes, as was also noted for the glycoproteins in the same subfractions. Specific radioactivity of the gangliosides increased relative to that of the glycoproteins with time.     

UPTAKE OF [3H-METHYL]CHOLINE BY MICROSOMAL, SYNAPTOSOMAL, MITOCHONDRIAL AND SYNAPTIC VESICLE FRACTIONS OF RAT BRAIN

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [³H-methyl]choline by a similar carrier-mediated transport system. The apparent K_m for the uptake of [³H-methyl]choline in these subcellular fractions was about 5 × 10^?5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [³H-methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [³H-methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC-3 was a competitive inhibitor of the uptake of [³H-methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for K_i of 4.0, 2.1 and 2.3 × 10^?5 M. HC-15 was a competitive inhibitor of the transport of [³H-methyl]choline in the synaptosomal fraction, with a K_i of 1.7 × 10^?4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid. Choline acetyltransferase (EC 2.3.1.6) was assayed with [¹⁴C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11-fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC-3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC-3. Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.     

THE SUBCELLULAR DISTRIBUTION OF β-PHENYLETHYLAMINE, p-TYRAMINE AND TRYPTAMINE IN RAT BRAIN

—The quantitative subcellular distribution of β-phenylethylamine, p-tyramine and tryptamine in rat brain was investigated using the mass spectrometric integrated ion current technique. More of the total cellular tryptamine was found to be associated with paniculate fractions than was the case for phenyiethylamine and p-tyramine but a significant amount of this tryptamine was found to be labile. Analysis of the particulate fractions indicated that each of the amines was localized predominantly in the crude P₂ pellet and that the bulk of this was associated with the synaptosomal (P₂B) fraction. Inhibition of monoamine oxidase systems with pargyline caused an increase in the level of all three amines in all fractions, but the increase was greater in the supernatant than in the combined particulate fractions. This treatment produced changes in the distribution of β-phenylethylamine and p-tyramine between the various particulate subcellular fractions but did not markedly alter the distribution of tryptamine between the same fractions.     

Incorporation of N-Acetylmannosamine into Rat Brain Subcellular Gangliosides: Effect of Pentylenetetrazol-Induced Convulsions on Brain Gangliosides1

Abstract: The labeling pattern of the major individual gangliosides from the microsomal and synaptosomal fractions of rat brain was determined following intracerebral injection of the radioactive sialic acid precursor, N-acetylmannosamine. Microsomal gangliosides initially had a higher specific radioactivity than synaptosomal gangliosides, with both fractions reaching similar specific radioactivities 18 h after precursor injection. In both subcellular fractions, the polysialogangliosides G_T1b and G_Q1b were initially more highly labeled than all other gangliosides. With the establishment of the labeling pattern, the effect of the convulsant pentylenetetrazol on brain gangliosides was examined in detail. Significant decreases in radioactive label were noted in the polysialogangliosides, G_T1b and G_Q1b, from the synaptosomal and microsomal fractions of the convulsed animals. The decreases may be due to activation of the membrane-bound neuraminidase present with the gangliosides in neuronal tissue. Prior to experimentation, a methodology was developed to insure quantitative isolation of small amounts of ganglioside free of other lipids and water-soluble contaminants. Combination of this isolation procedure with quantitative densitometry of thin-layer chromatograms permits accurate distributional analyses for individual gangliosides. In applications involving radioactive gangliosides, the method allows the determination of both radioactivity and sialic acid distributions from the same thin-layer chromatogram.     

Uptake of taurine into subcellular fractions of C-6 glioma cells.

Subcellular fractions from cultured C-6 glioma cells prepared by methods similar to those for crude synaptosomal fractions of rat cerebral cortex accumulated [³⁵S]taurine as did intact glioma cells. Thus, the accumulation of taurine was dependent on temperature and sodium concentration and sensitive to osmotic shock. The kinetic properties of this uptake are characterized by an apparent K_m, of about 25 μm, The properties of taurine uptake into subcellular fractions from C-6 glioma cells were compared with those of crude synaptosomal fractions and differences could be observed in temperature sensitivity and with metabolic inhibitors, which were less potent in the glioma preparation. Equilibrium density gradient centrifugation of subcellular fractions from glioma cells revealed that particles containing [³⁵S]taurine sediment to a lower buoyant density than mitochondria. But on co-sedimentation of subcellular fractions from glioma cells with synaptosomal fractions derived from cerebral cortex, differences in the buoyant density between these two preparations could be found. The findings support the possibility of a contamination of synaptosomal fractions with subcellular fractions derived from glial origin.     

THE ORIGIN OF THE ACETYLCHOLINE RELEASED FROM THE SURFACE OF THE CORTEX

—The specific radioactivity of acetylcholine liberated from the surface of the rabbit occipital cortex has been compared with that of the underlying cortical synaptosomal and vesicular acetylcholine at varying times after the administration of [N-Me-³H]choline. Choline was administered by diffusion from solutions placed in cups formed by Perspex cylinders applied to the surface of the cortex. Acetylcholine was collected by diffusion into these cups. The specific radioactivity of the acetylcholine declined progressively. The effect of stimulation of afferent cholinergic pathways was to cause a fall in the specific radioactivity of the released acetylcholine. However this was always higher than that of the synaptosomal or vesicular acetylcholine as represented by fractions P₂ and D of the authors’fractionation scheme. It is concluded that acetylcholine released from the cortex must come from a store or stores more recently synthesized than the endogenous acetylcholine of these subcellular fractions.     

Age-Related Changes in the Ceramide Composition of the Major Gangliosides Present in Rat Brain Subcellular Fractions Enriched in Plasma Membranes of Neuronal and Myelin Origin

Abstract: Age-related changes of the ceramide composition of gangliosides were studied in the synaptosomal and myelin fractions from rat brain, carrying plasma membranes of neuronal and glial origin, respectively. The five major gangliosides (GM1, GD1 a, GD1 b, GT1 b, and GQ1 b) present in these fractions were separated and quantitated by normal-phase HPLC. Each ganglioside was then fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base (LCB). The largely preponderant LCBs in the synaptosomal and myelin fractions were the C18:1 and C20:1. The content of C20.1 LCB, generally low at 1 month, increased with age in all analyzed gangliosides and in all subcellular fractions and was greater in the "b series" than in the "a series" gangliosides. Remarkably, GM1 was the only ganglioside where the proportion of LCB 20:1 was higher in the synaptosomal fraction than in the myelin fraction. The fatty acid composition of the C18:1 or C20:1 LCB species of the different gangliosides in the synaptosomal and myelin fractions did not undergo appreciable changes with age. Stearic acid was largely predominant in all the gangliosides of the synaptosomal fraction, more in the C18:1 than in the C20:1 LCB species (80–90% vs. 60–70%). The gangliosides of the myelin fraction were characterized by a lower content of 18:0 and a much higher content of 16:0 and 18:1 fatty acids than those of the synaptosomal fraction. Thus, the ceramide composition is different in the gangliosides of neuronal and myelin origin and appears to be subjected to an age-related control.     

INCORPORATION OF LIPIDS INTO SUBCELLULAR FRACTIONS OF BRAIN OF QUAKING MUTANT

The synthesis of lipids and their assembly into subcellular membrane fractions of the myelin deficient Quaking mutant and control brains was studied in 18-, 24- and 41-day-old animals using a double label methodology with¹⁴C and ³H acetate as precursors. As a general procedure, Quaking mutants were injected intracranially with 50 μCi [¹⁴C]acetate and their littermate controls with 300 μCi [³H]acetate. The animals were killed 3 h post-injection, their brains were pooled and subcellular fractions prepared from the common homogenate. An 80-90% decrease in the incorporation of acetate into eleven lipids of myelin in the Quaking mutant was found. This occurred in the face of apparent normal incorporation (relative to microsomes) into lipids of the other main subcellular fractions (nuclear. mitochondrial and synaptosomal) with the exception of decreased incorporation into the myelin-like fraction at 18 and 24 days. Cholesterol and cerebroside were less readily incorporated into Quaking myelin than the other lipids. Although the microsomal synthesis of cholesterol and cerebroside was depressed by about 30% in the Quaking mutant, the incorporation of cholesterol into nuclear, synaptosomal and mitochondrial fractions was unaffected in the mutant. This indicates that sufficient cholesterol is synthesized for the normal assembly of these organelles. In contrast the incorporation of acetate into cholesterol and cerebroside of Quaking myelin was decreased much more than microsomal synthesis. This latter result is consistent with a defect in the process of myclin membrane assembly     

GANGLIOSIDE COMPOSITION AND CONTENT OF RAT-BRAIN SUBCELLULAR FRACTIONS

Abstract— The composition and content of gangliosides from rat-brain microsomal, synaptosomal, mitochondrial and myelin fractions were studied. Outer membranes of synaptosomes were also isolated, separated into subfractions and investigated. Of all the fractions studied the outer membranes of synaptosomes are richest in gangliosides, in one of their sub-fractions the concentration of gangliosides per mg of protein is five times higher than in the homogenate. Microsomes are rich in gangliosides as well, but to a lesser degree, whereas the mitochondrial fraction contains considerably smaller amounts of gangliosides per mg of protein than does the homogenate. The ganglioside pattern of outer membranes of synaptosomes and of their subfractions is somewhat different from that of the homogenate; the outer membranes contain approximately one-third less monosialogangliosides. On the contrary a very high content of monosialogangliosides is characteristic of the ganglioside pattern of the myelin fraction. In this fraction monosialoganglioside G_MI (nomenclature of Svennerholm, 1963) constitutes 60–63 per cent of ganglioside sialic acid, or 75–80 molar per cent of gangliosides, the content of di- and trisialogangliosides being much lower than in other fractions. Fatty acid and long chain base composition of gangliosides from synaptosomal and microsomal fractions and homogenate is very similar, almost identical. In gangliosides from myelin fractions the relaitve content of palmitic and monoenoic acids is higher and that of arachinic acid and C₂₀-sphingosine—lower than in other fractions studied. The difference in ganglioside composition of synaptosomes and their outer membranes and on the other hand of myelin appears to reflect the difference in ganglioside composition of neuronal and oligodendroglial plasma membranes.     

DIFFERENCES IN THE SUBCELLULAR AND SUBSYNAPTOSOMAL DISTRIBUTION OF THE PUTATIVE ENDOPLASMIC RETICULUM MARKERS, NADPH-CYTOCHROME c REDUCTASE, ESTRONE SULFATE SULFOHYDROLASE AND CDP-CHOLINE DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE IN RAT BRAIN

Abstract— A comprehensive study has been undertaken on the subcellular and subsynaptosomal distribution of a number of markers for subcellular organelles in preparations from rat brain. Although the activity of most enzymatic markers was decreased by freezing and storage at - 70^oC, no significant changes were noted in the distribution of these activities. This demonstrates that contamination of brain fractions by subcellular organelles can be accurately assessed after freezing and thawing. A marked discrepancy was noted between the distribution of three putative markers for endoplasmic reticulum. CDP-choline-diacylglycerol cholinephosphotransferase (EC 2.7.8.1) activity was mainly limited to the microsomal fraction and was present to a lesser extent in the synaptosomal fraction than the other putative markers for endoplasmic reticulum. Estrone sulfate sulfohydrolase (EC 3.1.6.2) activity demonstrated a bimodal distribution between the crude nuclear and microsomal fractions. However, considerable activity was associated with the synaptosomal fraction. NADPH-cytochrome c reductase (EC 2.3.1.15) activity sedimented in the microsomal and the synaptosomal fractions. Calculations based on the relative specific activities of the microsomal and synaptic plasma membrane fraction indicated that the contamination of the synaptic plasma membranes by endoplasmic reticulum was 44.5% (NADPH-cytochrome c reductase), 38.0% (estrone sulfatase) and 9.0% (cholinephosphotransferase). Since it is believed that virtually all of the synthesis of phosphatidylcholine by cholinephosphotransferase occurs in the neuronal and glial cell bodies, it was concluded that cholinephosphotransferase is a satisfactory marker for the endoplasmic reticulum derived from these sources. The results suggest that NADPH-cytochrome c reductase and estrone sulfatase may be present in the smooth endoplasmic reticulum system responsible for the fast transport of macromolecules along the axon to the nerve endings as well as in the endoplasmic reticulum of the cell bodies. The possible relation between that portion of the smooth endoplasmic reticulum involved in fast axonal transport and the GERL (Golgi, Endoplasmic Reticulum, Lysosomes) complex discovered by Novikoff and his coworkers (Novikoff , 1976) is discussed.     

Effects of chronic ethanol administration on the activities and relative synthetic rates of myelin and synaptosomal plasma membrane-associated sialidase in the rat brain

In an attempt to understand the possible mechanism of chronic ethanol-induced generation of asialoconjugates in the brain and consequent behavioral abnormalities, we have studied the effects of chronic ethanol feeding to rats on the plasma membrane sialidase status in the various subcellular fractions of the brain. We determined sialidase activity using 3H-monosialoganglioside (3H-GM3), 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4-MU-NeuAC) substrates and Amplex Red (Sialidase) kit. We determined the plasma membrane sialidase protein by Western blot using the anti-plasma membrane sialidase. We also determined its relative synthetic rate (RSR) by the 60 min incorporation of intracranially infused [35S]-methionine (50 microCi/100 g) into immunoprecipitable plasma membrane sialidase. Chronic ethanol administration stimulated the sialidase activity in the total brain homogenate as well as the myelin and synaptosomal membrane fractions, respectively, in all the three experimental models. Chronic ethanol also increased the concentration of the rat brain plasma membrane sialidase protein relative to that of glyceraldehyde-3-phosphate dehydrogenase by 2.4-, 1.62- and 1.51-fold in the total brain homogenate, myelin and synaptosomal membrane fractions, respectively. These increases in plasma membrane sialidase activity and its protein content were due to concomitant increases in their relative synthetic rates by 115% (p < 0.01) and 72% (p < 0.01) in the myelin and synaptosomal membrane fractions, respectively. Thus, our studies clearly show that chronic ethanol induced deglycosylation of brain gangliosides is in part, due to specific up-regulation of plasma membrane sialidase in the myelin and synaptosomal membrane fractions of the brain. This increase in plasma membrane sialidase may be responsible for chronic-ethanol-induced physiological and neurological impairment in the brain, presumably due to deglycosylation of gangliosides that are essential for crucial cellular and metabolic activities.     

TAURINE IN DEVELOPING RAT BRAIN: SUBCELLULAR DISTRIBUTION AND ASSOCIATION WITH SYNAPTIC VESICLES OF [35S]TAURINE IN MATERNAL, FETAL AND NEONATAL RAT BRAIN

The concentration of taurine in the brain of the fetus in several species is higher than that found in the mature animal. In order to explore the functional significance of this, we have studied the subcellular distribution of taurine and [³⁵S]taurine in the brain of the mother, the fetus and the neonate after [³⁵S]taurine was administered to pregnant rats. In maternal brain, the distribution of taurine and of radioactivity (all of which was recovered from brain as taurine) in the subcellular fractions of maternal brain were essentially identical and were recovered primarily in two fractions (72% taurine, 71% [³⁵S]taurine was soluble, S₃; 16% and 17%, respectively, was in the crude mitochondrial and synaptosomal fraction, P₂). After further fractionation of P₂, most of the taurine and [³⁵S]taurine were in the cytoplasmic, O, and the synaptosomal, B, fractions. In the neonatal brain, shortly after birth there was a decrease in taurine and [³⁵S]taurine recovered in the supernatant fraction, S₃, accompanied by an increase in the percentage of taurine and [³⁵S]taurine recovered in the crude mitochondrial fraction. A small percentage of taurine and [³⁵S]taurine was consistently recovered in the synaptic vesicle fraction. Fractionation of the synaptic vesicles on a gel column separated the vesicle bound taurine completely from the free taurine: approx 1% of the taurine in the synaptic vesicle fraction was eluted with vesicles and could not be released by hypo-osmotic shock. The pattern of development in subcellular fractions of neonatal rat brain labelled with [³⁵S]taurine via intraperitoneal injections of the pregnant mother may be an indication of maturation or protection of putative taurinergic nerve endings.     

The effect of cold stress on ganglioside fatty acid composition and ganglioside-bound sialic acid content of rat brain subcellular fractions

The effect of cold stress on the ganglioside fatty acid composition and sialic acid content of brain subcellular fractions and homogenate of rats was studied, the animals were kept in a cold room with 12h light-dark cycles at 3 and 10 degrees C for 2 weeks. (1) The rat brain homogenate, synaptosomes and myelin of rats exposed to 3 degrees C contained significantly higher amounts of ganglioside-bound sialic acid per mg of protein than these fractions of control rats kept at 23 degrees C; the differences were less pronounced in rats exposed to 10 degrees C. (2) A small, but significant, diminution of relative palmitic acid content and an increase of stearic acid content was found to take place in gangliosides from rat brain synaptosomes, synaptosomal plasma membranes and homogenate as a result of the exposure of animals to 3 degrees C and to a lesser extent to 10 degrees C. (3) The content of unsaturated fatty acids in gangliosides from brain subcellular fractions was approximately the same in cold exposed and control rats.     

Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions.

—The incorporation of [4,5-³H]lysine and [1-¹⁴C]leucine into the proteins of subcellular fractions of mouse brain was examined following a single electroconvulsive shock (ECS) or following cycloheximide injections. When the [³H]lysine was injected intraperitoneally immediately after the ECS the incorporation into total brain proteins was decreased by more than 50% as compared to sham controls. The proportion of lysine incorporated into the microsomal fraction was increased, but no changes were observed in the other subcellular fractions including the synaptosomal fraction. With extended pulses administered at various times after the ECS there was no change in total incorporation nor were selective effects seen in any subcellular fractions. With intracranial injections of both [³H]lysine and [¹⁴C]leucine the decreased incorporation caused by ECS was not observed, neither were there selective changes in any subcellular fraction. This lack of inhibition occurred because the intracranial injection itself severely inhibited [³H]lysine incorporation. Cycloheximide (30 mg/kg) which depressed [³H]lysine incorporation into brain proteins by 84% caused a selective depression of the incorporation into the cell-sap fraction and selective elevations into the microsomal and synaptosomal fractions. Similar changes were seen with a higher (amnestic) dose of cycloheximide (150 mg/kg) which inhibited incorporation by 94%. These data are interpreted in terms of the diverse mechanisms by which ECS and cycloheximide inhibit protein synthesis.     

Determination of the sodium,potassium and chloride ion concentrations in the chloroplasts of the halophyteSuaeda maritima by non-aqueous cell fractionation

Summary Leaf material from the halophyteSuaeda maritima L. Dum. grown under both saline and non-saline conditions was fractionated under non-aqueous conditions in order to determine the ion content of various subcellular compartments. Fractions containing cell walls, nuclei and chloroplasts were successfully prepared and contents of DNA, chlorophyll, protein and Na⁺, K⁺, and Cl^– determined. The cell wall fraction was not apparently heavily contaminated by the other fractions and had a low ion content although the nuclear fraction was contaminated by other organelles. The ion contents of chloroplasts were determined and the results discussed in relation to earlier microscopical data.     

RAPID EFFECTS OF VERATRIDINE, TETRODOTOXIN, GRAMICIDIN D, VALINOMYCIN AND NaCN ON THE NA+, K+ AND ATP CONTENTS OF SYNAPTOSOMES

Abstract— The effects of brief exposures of a number of depolarizing agents on ²⁴Na⁺ influx and on the Na⁺, K⁺ and ATP contents of synaptosomes were studied using a Millipore filtration technique to terminate the reaction. When synaptosomes were incubated in normal medium, there was a rapid influx of ²⁴Na⁺ and a gain in Na’contents; neither the ²⁴Na⁺ influx nor the Na⁺ gain were blocked by tetrodotoxin suggesting that this Na⁺ entry did not involve Na⁺-channels. Veratridine markedly increased the rate of ²⁴Na⁺ influx into synaptosomes and also increased the Na⁺ content and decreased the K⁺ content of synaptosomes within the first 10s of exposure. The normal ion contents were reversed by 1 min. The effects of veratridine on Na⁺ influx and on synaptosomal ion contents were prevented by tetrodotoxin and required Na⁺ in the medium. The ionophores gramicidin D and valinomycin also rapidly reversed the Na⁺ and K⁺ contents of synaptosomes, but these effects could not be blocked by tetrodotoxin. The reducing effect of gramicidin D on synaptosomal K⁺ content required Na’in the medium, whereas valinomycin caused a fall in the K⁺ content of synaptosomes in a Na⁺-free medium. Veratridine and gramicidin D, at concentrations known to reverse the synaptosomal ion contents, did not affect synaptosomal ATP levels. In contrast, valinomycin and NaCN caused an abrupt fall in synaptosomal ATP levels. The above findings suggest that veratridine quickly alters synaptosomal Na⁺ and K⁺ contents by opening Na ⁺-channels in the presynaptic membrane, and provide direct evidence for the existence of Na⁺-channels in synaptosomes. In contrast, gramicidin D and valinomycin appear to act independently of Na ⁺-channels, possibly by their ionophoric effects and, in the case of valinomycin, by diminishing synaptosomal ATP contents and hence diminishing Na⁺-pump activity. The rapid reversals of Na⁺ and K⁺ contents by these drugs could affect the resting membrane potentials, Na⁺-Ca²⁺ exchange across the synaptosomal membrane, and the release, synthesis and uptake of neurotransmitters by synaptosomes.     

The biosynthesis of brain gangliosides. Separation of membranes with different ratios of ganglioside sialylating activity to gangliosides.

Brain subcellular fractions were analysed for ganglioside-sialylating activity by measuring the incorporation of N-[3H]acetylneuraminic acid from CMP-N-[3H]acetylneuraminic acid into endogenous ganglioside acceptors (endogenous incorporation) and into exogenous lactosyceramide (haematoside synthetase activity). The ratios of endogenous incorporation to gangliosides and of haematoside synthetase to gangliosides for the synaptosomal and mitochondrial fractions from a washed crude mitochondrial fraction were lower than those obtained for other membrane fractions. The differences appear to reflect intrinsic characteristics of each membrane fraction. The results of labelling in vitro and the time course of labelling of gangliosides of the different subcellular fractions in vivo after injection of N-[3H]acetylmannosamine are consistent with the possibility of a subcellular site for synthesis of gangliosides different from that of ganglioside deposition.