Effect of ethanol on phospholipid turnover and calcium mobilization in chick embryos

Effects of ethanol on [3H]inositol and [14C]choline incorporation into phosphatidylinositol (PI) and phosphatidylcholine (PC), free intrasynaptosomal Ca2+ ([Ca2+]i) and synaptosomal 45Ca2+ uptake, were investigated in the brain and heart of 17-day-old chick embryos to which a 10% ethanol solution had been injected on the 3rd day of embryogenesis. In brain synaptosomes, ethanol increased the incorporation of [3H]inositol and [14C]choline into PI and PC, increased [Ca2+]i, and decreased 45Ca2+ uptake. On the other hand, in heart synaptosomal membrane, ethanol decreased the incorporation of [3H]inositol and [14C]choline into PI and PC, decreased [Ca2+]i, and increased 45Ca2+ uptake. Ethanol stimulated in vitro [3H]inositol and [14C]choline incorporation into PI and PC in the brain and heart in both the control and ethanol-treated groups. However, addition of ethanol did not affect the release of 45Ca2+ from the synaptosomal membrane of either organ in either group. Addition of ethanol inhibited 45Ca2+ uptake in a dose-dependent manner in the brain but not in the heart. In both organs, there was a relationship between phospholipid turnover and [Ca2+]i after ethanol.     

Aluminum impairs glucose utilization and cholinergic activity in rat brain in vitro

The effects of AlCl3 on the production of 14CO2 from [U-14C]glucose and high affinity choline transport in rat brain synaptosomes, and on carbachol-stimulated hydrolysis of phosphoinositides in cortical slices were studied. In buffer containing either high K+ (50 mM) or low K+ (4.9 mM), 1 mM AlCl3 significantly depressed the synaptosomal production of 14CO2 from [U-14C]glucose to 54% and 44% of control rates, respectively. At a concentration of 0.1 mM, AlCl3 depressed the evolution of 14CO2 from [U-14C]glucose from synaptosomes incubated in the high K+ buffer, but did not significantly change 14CO2 production from synaptosomes in the low K+ buffer. Aluminum chloride also inhibited high affinity choline transport in synaptosomes prepared from rat cortex and from hippocampus with an IC50 of approximately 0.5 mM. In brain slices the carbachol-stimulated hydrolysis of phosphoinositides was inhibited by AlCl3 in a dose-dependent manner. One millimolar, 0.5 mM and 0.1 mM AlCl3 inhibited the carbachol-stimulated release of inositol phosphates by 75%, 44% and 33%, respectively. These same concentrations of AlCl3 inhibited the incorporation of [3H]inositol into phospholipids. This inhibitory effect was not dose-dependent as all 3 concentrations of AlCl3 inhibited phospholipid labelling to the same extent (27-37%). These results are discussed in relation to the in vivo neurotoxicity of aluminum.     

The effect of ethanol on phospholipid metabolism in rat pancreas

The phospholipid effect involves agonist-induced breakdown of phosphatidyl inositol (or polyinositides) generating second messengers followed by increased incorporation of 32P during the resynthetic phase of the cycle. Ethanol, an aetiological factor in pancreatitis, has been shown to have various effects on pancreatic secretion. In this study ethanol decreased the incorporation of 32P into phosphatidyl inositol but had no effect on the stimulated breakdown of prelabelled phosphatidyl inositol. However, in addition to recycling of phosphatidyl inositol stimulation of pancreatic tissue results in increased incorporation of precursors into other phospholipids. Cholecystokinin increased the incorporation of both [U-14C] glucose and 32P into phosphatidyl ethanolamine 3-fold but had no effect on 32P incorporation into phosphatidyl choline. As well as increased incorporation of 32P into phosphatidyl inositol (8-fold) cholecystokinin also increased the incorporation of [U-14C] glucose into phosphatidyl inositol (4-5-fold) implying significant de novo synthesis of 1,2 diacyl glycerol in addition to the currently accepted recycling of the 1,2 diacyl glycerol back to phosphatidyl inositol. Ethanol caused an inhibition of 32P incorporation into total phospholipid of rat pancreas during basal and stimulated conditions. When individual phospholipids were separated ethanol was found to decrease the incorporation of 32P into phosphatidyl choline under basal conditions and into all phospholipids during cholecystokinin stimulation. With [U-14C] glucose as the precursor, ethanol inhibited its incorporation into phosphatidyl choline only. Ethanol did not alter the total 32P radioactivity in the aqueous phase of the pancreatic extract nor the percent incorporated into nucleotides. This excluded decreased uptake of 32P and incorporation into nucleotides as a mechanism for the differential inhibition of 32P versus [U-14C] glucose incorporation into phospholipids other than phosphatidyl choline under stimulated conditions.     

Central nervous system myelination in rats exposed to ethanol in utero

Pregnant rats were pair-fed using isocaloric control or 6.6% (v/v) ethanol liquid diets. At 18, 25, and 53 days, the in vivo incorporation of [3H] leucine and [14C] glucose into proteins and lipids of three central nervous system (CNS) myelin subfractions was examined in the offspring (control and ethanol pups) of control and ethanol-treated females. With few exceptions, the pattern of CNS myelination appeared near-normal in the ethanol pups. The ethanol pups had brain weights and total myelin protein content within the normal range. In addition, the amount of protein in light, medium, and heavy myelin was normal at 18 and 25 days. However, the ethanol pups had an excess of the chemically and morphologically immature heavy fraction at 53 days. The incorporation of [3H] leucine and [14C] glucose into myelin subfractions and separated proteins and lipids was similar in control and ethanol pups. The findings of near-normal CNS myelination in the offspring of female rats fed an ethanol liquid diet during gestation differ from our previous findings of a premature onset and slowdown of active CNS myelination in the offspring of female rats that consumed an ethanol liquid diet for one month prior to conception as well as during gestation.     

Influence of mercury on uptake of [3H]dopamine and [3H]norepinephrine by rat brain synaptosomes

Mercuric compounds have been shown to alter several membrane-bound enzymes and associated receptor activities. The present studies were initiated to investigate the in vitro effects of mercuric chloride (HgCl2) and methylmercury chloride (CH3HgCl) on the uptake of [3H]dopamine (3HDA), [3H]norepinephrine (3HNE), and Na+, K+-ATPase in rat brain synaptosomes. Brain synaptosomes were prepared by the ficoll-sucrose gradient method from normal, adult male Sprague-Dawley rats, weighing approx. 200 g. The effect of mercury on Na+, K+-ATPase was determined by using a coupled enzymatic method. Uptake of DA and NE by brain synaptosomes was determined by filtration in the presence and absence of 0-30 microM HgCl2 and 0-100 microM CH3HgCl. A parallel inhibition in the synaptosomal uptake of 3HDA and 3HNE, and the activity of the synaptosomal membrane Na+, K+-ATPase, was observed in both mercuric chloride and methylmercury treatments. The mercury compounds also significantly inhibited the mitochondrial ATPase (Mg2+-oligomycin-sensitive ATPase). The inhibitory influences of the toxins were concentration-dependent. The results suggest that the mercury compound mediated decrease in DA and NE uptake in brain synaptosomes may be related to the inhibition of Na+, K+-ATPase by the same toxins.     

Influence of chronic ethanol consumption on muscarinic cholinergic receptors and their linkage to phospholipid metabolism in mouse synaptosomes

C57/BL mice rendered physically dependent to ethanol exhibited an increase in specific binding of [3H]QNB to a cholinergically enriched synaptosomal fraction of brain without a parallel increase in muscarinic receptor-stimulated incorporation of [32P] into phosphatidylinositol and phosphatidic acid. Receptor-enhanced phospholipid labeling was reduced by the addition of ethanol (100mM) in vitro. In chronically treated mice, tolerance developed to the inhibitory effects of ethanol on receptor-enhanced phospholipid labeling. It is suggested that the inhibition of this response by ethanol added in vitro may be associated with alterations in neuronal function during alcohol intoxication.     

Effect of toluene on rat synaptosomal phospholipid methylation and membrane fluidity

This study investigated the effects of toluene (1 g/kg, 1 hr, i.p.) on rat synaptosomal phospholipid methylation (PLM), phospholipid composition, and membrane fluidity. Toluene significantly decreased basal PLM (35%) in studies using [3H]methionine [( 3H]Met) as the methyl donor; this was reflected by similar decreases in phosphatidylmonomethylethanolamine (PME) (30%). No effects were observed in either PLM reactions that used [3H]adenosylmethionine [( 3H]AdoMet) as methyl donor, or AdoMet synthetase, suggesting that toluene preferentially affects PLM reactions that derive methyl groups from [3H]Met. Also, toluene decreased synaptosomal phosphatidylethanolamine (PE) (24%), the initial substrate for PLM, and the addition of PE back to PE-depleted synaptosomes restored methyltransferase activity. Agonist-stimulated PLM using norepinephrine (NE) demonstrated that agonist-receptor coupling returned PLM to control values in synaptosomes from toluene-treated rats. NE-stimulated PLM was also blocked by propranolol (PRO), suggesting a role for toluene in receptor-mediated events. Membrane fluidity studies demonstrated that in vivo administration of toluene increased the outer synaptosomal membrane fluidity, whereas in vitro administration of toluene had no effect. Our observations support a positive relationship between increased PLM activity and increased outer, not core, membrane fluidity. These data demonstrate that specific toluene-phospholipid interactions occur in synaptosomes, resulting in altered membrane composition, function and fluidity.     

Inhibition of mouse brain synaptosomal ATPases and ouabain binding by chlordecone

The effect of chlordecone on the mouse brain synaptosomal Na+-K+ ATPase, Mg2+ ATPase and p-nitrophenyl phosphatase (PNPPase) activities was determined. In addition, the effect of chlordecone on [3H] ouabain binding to synaptosomes was also investigated. The in vitro data show that chlordecone inhibits PNPPase, Na+-K+ ATPase, and Mg2+ ATPase activities with ID50 values of 4, 5 and 7 micrograms respectively. Treatment of mice with symptomatogenic doses of chlordecone resulted in a decreased synaptosomal Na+-K+ and oligomycin-sensitive (mitochondrial) Mg2+ ATPases. The decrease was dose-dependent. The oligomycin-insensitive Mg2+ ATPase activity was unaffected either in vitro or in vivo. The binding of [3H] ouabain to synaptosomal membranes was inhibited by chlordecone in a dose-dependent manner in both in vitro and in vivo experiments. The binding of [14C] chlordecone to synaptosomes occurs even at nanomolar concentrations. The marked inhibition of brain synaptosomal ATPases and ouabain binding by chlordecone suggests that chlordecone may impair active transport mechanisms in synaptosomal membranes.     

Purification and characterization of iotrochotin, a novel toxin from the Caribbean sponge Iotrochota birotulata, which selectively permeabilizes synaptosomes.

A protein termed iotrochotin (IOT) was isolated from the exudate of the Caribbean sponge Iotrochota birotulata using as an assay its stimulation of the release of radioactivity from synaptosomes preloaded with [3H]choline. Sephadex G-50 chromatography of the exudate produced one peak, with a mol. wt of approximately 18,000, which was further resolved into two active fractions by anion exchange chromatography. The more tightly bound of the two fractions was characterized and referred to as IOT. The action of IOT was essentially complete by 0.5-1.0 min and was independent of the Ca2+ or Na+ content of the incubation mixture. Released radioactivity included about 50% each of [3H]acetylcholine and [3H]choline. Release of radioactivity increased as a function of IOT concentration and then reached a maximum. Extrapolated asymptotic release was nearly equal to that obtained by lysing the synaptosomes. IOT also released radioactivity from synaptosomes which had been preincubated with other tritiated neurotransmitters or with 2-[3H]deoxy-D-glucose. Lactate dehydrogenase and choline acetyltransferase activities were not released from synaptosomes by treatment with IOT, but were released by digitonin. IOT therefore releases some of the smaller molecular weight components of synaptosomes, but does not permeabilize the synaptosomal membrane in the same way as digitonin treatment.     

Effect of pentobarbital on regional brain phospholipid synthesis

Rats were given 45 mg/kg i.p. sodium pentobarbital 15 min prior to the intraventricular injection of 200 μCi [³²P]phosphoric acid and 50 μCi [³H]glycerol. The animals were sacrificed 1 hr later, subcellular fractions were prepared from four subcortical brain regions and phospholipids were extracted. Pentobarbital significantly increased the ratio of [³H]- and [³²P]-triphosphatidylinositol (TPI) to diphosphatidylinositol (DPI) in the microsomal but not synaptosomal fractions. The possible relationship of this change to nicotinic receptor activity is discussed. Pentobarbital specifically decreased ³²Pi but not [³H]glycerol incorporation into synaptosomal phosphatidylinositol (PI). Thus, pentobarbital induced the opposite of the “neurotransmitter effect” on PI turnover. Pentobarbital either decreased or had no effect on the incorporation of ³²Pi and [³H]glycerol into phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylethanolamine (PE).     

The Effect of Long-term Lithium Treatment on the Incorporation and Distribution of 32P-Orthosphosphate into the Phospholipids from Rat Synaptosomes

Abstract: Rats were treated with lithium added to their diet for five weeks (40 mmol LiCl/kg diet). Mean plasma concentration was 0.45 mmol Li⁺/plasma. The investigation was divided into two sections. I) In an in vivo experiment in which the rats were injected with ³²P-orthosphosphate for 20 hours, and with carbamoylcholine for 20 min. prior to their death, the distribution of ³²P in the synaptosomal phospholipids, phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylcholine (PC), was investigated. II) An in vitro experiment which was carried out in order to establish the effect of carbamoylcholine on the incorporation of ³²P into total phospholipids from extracted synaptosomes. In I) the incorporation of ³²P-orthosphosphate into PI from carbamoylcholine-stimulated rats was significantly lower than from unstimulated rats, whereas there was no difference between the incorporation of ³²P into PI from synaptosomes from control animals and lithium-treated animals. In II) the incorporation of ³²P-orthosphosphate was significantly lower in unstimulated synaptosomes from lithium-treated rats than from control rats, while the increase in the ³²P-incorporation after stimulation followed the same pattern with regard to synaptosomes from control rats and lithium-treated rats. The results support the idea of lithium exerting an effect on basal synaptosomal activity but not on stimulated synaptosomal activity.     

Comparison of postnatal changes in alpha 1-adrenoceptor binding and adrenergic stimulation of phosphoinositide hydrolysis in rat cerebral cortex

Adrenergic stimulation of phosphoinositide hydrolysis is mediated by the alpha 1-adrenoceptor subtype in many tissues including the brain. We have investigated the coupling of alpha 1-adrenoceptors to phosphoinositide hydrolysis during ontogeny. Alpha 1-adrenoceptor number and affinity were measured using [3H]prazosin binding in crude membranes of cerebral cortex and compared to the ability of the adrenergic agonists norepinephrine (NE) and phenylephrine (PE) to stimulate the formation of [3H] inositol phosphates from [3H]myo-inositol in brain slices at various ages. The greatest changes in the developmental expression of both the Bmax for [3H]prazosin binding and maximal (10(-4)M) NE- or PE-stimulated [3H]inositol phosphates were observed during the period of 7-21 days of age. No changes in the KD for [3H]prazosin were observed. However, at 14 days of age the EC50 for NE but not PE stimulation of [3H]inositol phosphates was slightly but significantly lower than at later ages. To quantitatively compare these two parameters during ontogeny, data were expressed as a percentage of the adult (greater than 65 day) value. At early ages (7 and 14 days) but not at later ages (21 and 37 days) the percent expression of [3H]prazosin binding sites was significantly greater than the maximal NE-stimulated [3H]inositol phosphates. This suggests that early in neonatal development alpha 1 adrenoceptors in brain are not tightly coupled to phosphoinositide hydrolysis.     

Differential inhibition of synaptosomal accumulation of [3H]-monoamines by cocaine, tropacocaine and amphetamine in four inbred strains of mice

The relative ability of cocaine, tropacocaine and amphetamine to inhibit the uptake of [3H]norepinephrine (NE), [3H]dopamine (DA) and [3H]5-hydroxytryptamine (5HT) was examined in whole brain synaptosomes from BALB, C3H, C57BL and DBA inbred mouse strains. With inhibition of [3H]NE uptake, synaptosomes from BALB and C57 mice were substantially more sensitive to cocaine inhibition than those from DBA or C3H. Moreover, with BALB and C57 tissue, amphetamine was as potent as cocaine, whereas with C3H and DBA, amphetamine and tropacocaine were much less potent inhibitors of [3H]NE uptake. With respect to [3H]DA accumulation, synaptosomes from BALB, C57 and DBA were equally sensitive to cocaine inhibition, while C3H synaptosomes were significantly less sensitive. In each of the four strains, amphetamine was more potent than cocaine, and tropacocaine far less potent. The relative potencies of the three drugs varied significantly among the four strains. With [3H]5HT accumulation, synaptosomes from DBA were exquisitely sensitive to cocaine inhibition, followed by BALB and lastly by C57 and C3H. In each of these strains, amphetamine and tropacocaine were equipotent at [3H]5HT inhibition, and less potent than cocaine. The results suggest that there are pronounced genetic differences in sensitivity to monoamine uptake inhibition by cocaine, which may arise from genetic differences in either carrier topology or other site of cocaine interaction. The results further suggest that genetic behavioral differences to cocaine and amphetamine may involve complex neurotransmitter interactions.     

GBR 12783, a potent and selective inhibitor of dopamine uptake: biochemical studies in vivo and ex vivo

The effects of GBR 12783, an aryl 1,4-dialk(en)ylpiperazine derivative, were studied on the in vivo and ex vivo neuronal uptake of dopamine (DA), norepinephrine (NE) and serotonin (5HT). The drug inhibited potently (IC50 = 1.8 nM) and competitively the [3H]DA uptake by rat striatal synaptosomes. It produced significant [14C]DA release only at much higher concentrations (in the micromolar range). Depending on the animal species (rat or mouse) and the experimental conditions, GBR 12783 was 18-90 times and 85-300 times less effective against NE and 5HT uptake respectively than against DA uptake. In synaptosomes preloaded with [3H]DA, GBR 12783 added to the superfusion medium prevented the (+)amphetamine-induced DA release. The total binding of [3H]GBR 12783 to a membranal fraction prepared from striatum was lower than the binding to a synaptosomal fraction, suggesting its entry in synaptosomes. In addition, the concentration-dependent release of [3H]DA produced by GBR 12783 from a striatal vesicular fraction may account for the synaptosomal DA release promoted by micromolar concentrations of the drug. In ex vivo experiments, the ID50 for DA uptake inhibition (30 min after i.p. administration) was 8.1 mg/kg. After a dose of 10 mg/kg i.p., the striatal DA uptake inhibition occurred quickly (less than 10 min) and was long-lasting (greater than 5 h). The specificity of the drug for the DA uptake relative to NE and 5HT uptakes was also seen after i.p. administration of GBR 12783.     

Inhibition of inositol phospholipid synthesis and norepinephrine-stimulated hydrolysis in rat brain slices by excitatory amino acids

Inhibition by excitatory amino acid agonists of norepinephrine (NE)-stimulated phosphoinositide hydrolysis was studied in rat brain slices. Inhibition was not observed in cortical slices prelabeled with [3H]inositol but was observed when slices were incubated simultaneously with [3H]inositol, glutamate, and NE. Therefore, we hypothesized that glutamate inhibits the synthesis of inositol phospholipids available to the alpha 1-adrenergic receptor, thereby reducing NE-stimulated phosphoinositide hydrolysis. To test this hypothesis, the distribution of [3H]inositol in cortical slices was measured after 5, 10, 20, 40 and 60 min of incubation, with some slices being exposed to 200 microM NE, 1 mM glutamate, 1 mM N-methyl-D-aspartate (NMDA), 1 mM kainate, 1 mM quisqualate, or to NE in the presence of each of the excitatory amino acid agonists. Glutamate had little effect on the slice content of free [3H]inositol, but it severely reduced the synthesis of [3H]inositol phospholipids, in the presence or absence of NE. Glutamate also abolished NE-induced production of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Quisqualate mimicked the effects of glutamate, whereas NMDA and kainate caused less inhibition of the synthesis of [3H]inositol phospholipids and did not inhibit the response to NE. Glutamate produced similar inhibitory effects in slices from hippocampus and striatum. To test if the inhibitory effect of glutamate was the result of irreversible cell damage, cortical slices were incubated with 1 mM glutamate for 60 min prior to exposure to [3H]inositol and NE. Preincubation with glutamate did not reduce the synthesis of [3H]inositol phospholipids or inhibit NE-stimulated [3H]inositol monophosphate production. These results indicate that glutamate impairs the synthesis of inositol phospholipids. Each of the excitatory amino acid agonists, quisqualate, NMDA and kainate, inhibited [3H]inositol phospholipid synthesis, but only quisqualate affected [3H]inositol phospholipids available to the alpha 1-adrenergic receptor.     

Influence of supidimide on brain neurotransmitter systems of rats and mice

The influence of 2-(2-oxo-3-piperidyl)-1,2-benzisothiazoline-3-one-1, 1-dioxide (supidimide), a representative of a new class of sedative drugs, on the noradrenergic, dopaminergic, serotoninergic and gamma-aminobutyric acid (GABA)ergic neuronal systems of rodent brains was investigated. In each case the brain transmitter levels after administration of supidimide were determined. Utilisation of noradrenaline (norepinephrine, NE), dopamine (DA), and 5-hydroxytryptamine (5-HT) was also investigated ex vivo. The study was complemented with in vitro investigations of biosynthesis, synaptosomal uptake, degradation, and receptor binding of the transmitters. Based on a preliminary study of the distribution of [35S]-supidimide in rat brain, in vitro effects observed at greater than 10(-4) mol/l were considered irrelevant. Similarly, in vivo effects requiring dosages higher than 300 mg/kg i.p. were not regarded adequate to explain the sedative and antiaggressive efficacy of supidimide. With the above restrictions, the following parameters can be rated as not influenced by supidimide: levels of tryptophan in rat brain and serum (free and total); 5-HT biosynthesis in vivo (rat brain; 5-HT accumulation after monoamine oxidase (MAO) blockade); activity of MAO-A and MAO-B (rat brain mitochondria); uptake of 5-HT, NE and DA (rat synaptosomes); 5-HT receptor binding ( [3H]-LSD binding assay in rat cortical membranes); tyrosine hydroxylase activity (rat adrenal glands); catechol-O-methyl transferase (COMT) (rat liver); NE binding to central alpha 1- and alpha 2-receptors (rat brain; radioligand assay with [3H]-dihydroergocryptine, [3H]-prazosin and [3H]-WB 4101 (2',6'-dimethoxy-(G-3H]-phenoxy]-ethylaminomethylbenzo-1,4-dioxane ); DA levels (whole rat brain and striata); dihydroxyphenylacetic acid (DOPAC) levels (whole rat brain without cerebellum and striata); elevated DOPAC levels after pretreatment with haloperidol; DA-dependent adenylate cyclase in vitro (rat striatum); D2 receptor binding ( [3H]-spiperone binding assay, rat striatum); GABA levels (mouse brain); GABA transaminase activity (mouse brain stem); sodium-independent [3H]-GABA receptor binding (rat brain) and benzodiazepine binding (rat cortical membranes, [3H]-diazepam binding assay). Two effects on the GABAergic system were induced by supidimide. Starting at 300 mg/kg i.p., supidimide slowed down the GABA accumulation in brains of aminooxyacetate-treated mice. At 10(-4) mol/l supidimide caused a significant inhibition of GABA uptake (rat synaptosomes).(ABSTRACT TRUNCATED AT 400 WORDS)     

The Ginkgo biloba extract, EGb761, increases synaptosomal uptake of 5-hydroxytryptamine: in-vitro and ex-vivo studies.

The Ginkgo biloba extract (EGb 761) added to a synaptosomal fraction prepared from mice cerebral cortex modified [3H]5-hydroxytryptamine ([3H]5-HT) uptake in a biphasic manner. Between 4 and 16 micrograms mL-1 EGb 761 increased significantly the [3H]5-HT uptake (maximum + 23%). A similar increase was also obtained when synaptosomes were prepared from the cortex of mice treated orally with EGb 761, either acutely (100 mg kg-1, 14 h and 2 h before death) or semi-chronically (2 x 100 mg-1 kg daily for 4 consecutive days). The in-vitro increase in [3H]5-HT uptake induced by EGb 761 was not observed in the presence of 10(-6) M clomipramine, a 5-HT-uptake inhibitor. EGb 761 did not increase [3H]dopamine uptake by synaptosomes prepared from striatum of mice. We investigated different fractions of EGb 761 in order to determine the compounds inducing the increase in [3H]5-HT uptake. The BN 52063 extract (corresponding to the EGb 761 devoid of flavonoid substances) did not increase [3H]5-HT uptake. The Cp 202 extract (corresponding to the EGb 761 devoid of terpenic substances and containing mostly flavonoid substances) increased [3H]5-HT uptake. Among the flavonoids, quercetin has been tested and had no effect on the [3H]5-HT uptake. Since at the usual therapeutic doses of EGb 761, the effective concentrations of the components responsible for this increase are likely to be reached in the brain, one may suggest that this effect could contribute to the therapeutic effect of EGb 761.     

Effects of ethanol and pentobarbital on neuronal hexose uptake in inbred mice

The effect of ethanol and pentobarbital narcosis on 2-deoxyglucose uptake into brain synaptosomes prepared from inbred C57BL/6J and DBA/2J mice which exhibit differential central sensitivity to ethanol and heterogeneous ICR mice was examined. A reversible depression of synaptosomal uptake was exhibited in all strains administered ethanol acutely, occurring at 2 min in ICR and C57BL/6J mice and 15 min in DBA/2J. Uptake returned to control values in all strains at 30 min although the mice remained intoxicated. Brain glucose concentration was significantly elevated at this time. Pentobarbital administration was without effect on synaptosomal hexose transport in DBA/2J and C57BL/6J mice but increased it significantly in ICR mice at 30 min. Pentobarbital anesthesia did not alter brain glucose concentration. No correlation was apparent between synaptosomal 2-deoxyglucose uptake and differential CNS sensitivity to ethanol and pentobarbital. The effects of ethanol and pentobarbital on neuronal hexose transport is discussed with respect to reported changes in glycolytic metabolism produced by these agents.     

Stimulatory effect of veratridine on lysophosphatidylethanolamine formation in rat brain synaptosomes

[3H]-Arachidonic acid incorporation into phospholipids of synaptosomal lysates prepared from veratridine-treated synaptosomes was examined. Synaptosomal lysates were shown to acylate exogenously added lysophosphatidylcholine, lysophosphatidylinositol, and lysophosphatidylethanolamine, when incubated with [3H]-arachidonic acid, ATP, CoA and MgCl2, yielding the respective phospholipids. Preincubation of synaptosomes with veratridine for 30 sec gave rise to an increase in [3H]-arachidonic acid incorporation into phosphatidylethanolamine, but not phosphatidylcholine nor phosphatidylinositol, indicating that lysophosphatidylethanolamine might be produced by veratridine. This increase of radioactivity in phosphatidylethanolamine caused by veratridine was completely inhibited by 1 microM tetrodotoxin or in calcium-free condition. These observations show that lysophosphatidylethanolamine was formed in a calcium-dependent manner and accumulated in synaptosomes treated with veratridine, which may relate to its action on the sodium channel and enhanced calcium influx.     

Involvement of phospholipase A2 in the regulation of [3H]hemicholinium-3 binding

We have examined the effects of exogenous phospholipase A2 (PLA2) on the sodium-dependent high-affinity choline uptake mechanism as assessed by the specific binding of [3H]hemicholinium-3 ([3H]HCh-3). Incubation of striatal synaptic membranes with bee venom PLA2 resulted in a concentration-dependent increase in the specific binding of [3H]HCh-3. The effect of PLA2 on [3H]HCh-3 binding was inhibited by quinacrine, a PLA2 inhibitor, and by removal of calcium. Scatchard analysis revealed that the observed changes in binding reflected a 2-fold increase in both the capacity and affinity of [3H]HCh-3 for its binding site. Choline and N-butylcholine inhibited the specific binding of [3H]HCh-3 in both control and PLA2-treated membranes with similar potency. When a low concentration of PLA2 was incubated with the striatal synaptosomes, a small but significant increase in high-affinity [3H]choline uptake was observed. However, higher concentrations of PLA2, which further increased the specific binding of [3H]HCh-3, caused a reduction of [3H]choline uptake, apparently due to disruption of synaptosomal integrity by PLA2. Finally, potassium depolarization- and PLA2-induced increases in specific [3H]HCh-3 binding were not additive. These results suggest a possible role for endogenous PLA2 in the calcium-dependent regulation of sodium-dependent high-affinity choline uptake.