High-level inhibition of mitochondrial complexes III and IV is required to increase glutamate release from the nerve terminal

Background

The activities of mitochondrial complex III (ubiquinol-cytochrome c reductase, EC 1.10.2.2) and complex IV (cytochrome c oxidase EC 1.9.3.1) are reduced by 30-70% in Huntington's disease and Alzheimer's disease, respectively, and are associated with excitotoxic cell death in these disorders. In this study, we investigated the control that complexes III and complex IV exert on glutamate release from the isolated nerve terminal.

Results

Inhibition of complex III activity by 60-90% was necessary for a major increase in the rate of Ca²⁺-independent glutamate release to occur from isolated nerve terminals (synaptosomes) depolarized with 4-aminopyridine or KCl. Similarly, an 85-90% inhibition of complex IV activity was required before a major increase in the rate of Ca²⁺-independent glutamate release from depolarized synaptosomes was observed. Inhibition of complex III and IV activities by ~ 60% and above was required before rates of glutamate efflux from polarized synaptosomes were increased.

Conclusions

These results suggest that nerve terminal mitochondria possess high reserves of complex III and IV activity and that high inhibition thresholds must be reached before excess glutamate is released from the nerve terminal. The implications of the results in the context of the relationship between electron transport chain enzyme deficiencies and excitotoxicity in neurodegenerative disorders are discussed.     

Adenosine A1 Receptor Inhibition of Glutamate Exocytosis and Protein Kinase C-Mediated Decoupling

Abstract: The adenosine modulation of glutamate exoeytosis from guinea pig cerebrocortical synaptosomes is investigated. Endogenously leaked adenosine is sufficient to cause a partial tonic inhibition of 4-aminopyridine-evoked glutamate release, which can be relieved by adenosine deaminase. The adenosine A₁ receptor is equally effective in mediating inhibition of glutamate exocytosis evoked by 4-aminopyridine (where K⁺-channel activation would inhibit release) and by elevated KC1 (where K⁺-channel activation would have no effect), arguing for a central role of Ca²⁺-channel modulation. In support of this, the plateau phase of depolarization-evoked free Ca²⁺ elevation is decreased by adenosine with both depolarization protocols. No effect of adenosine agonists is seen on membrane potential in polarized or KC1- or 4-aminopyridine-stimulated synaptosomes. The interaction of protein kinase C with the A₁ receptormediated inhibition is examined. Activation of protein kinase C by 4β-phorbol dibutyrate has been shown previously by this laboratory to modulate glutamate release via K⁺-channel inhibition, and is shown here to have an additional action of decoupling the adenosine inhibition of glutamate exocytosis.     

Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

Abstract: During K⁺ -induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 m M Ba²⁺ could substitute for 1 m M Ca²⁺ in evoking the release of endogenous glutamate. In addition, Ba²⁺ was found to evoke glutamate release in the absence of K⁺-induced depolarization. Ba²⁺ (1–10 m M ) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [³H]-tetraphenylphosphonium cation distribution. Ba²⁺ partially inhibited the increase in synaptosomal K⁺ efflux produced by depolarization, as reflected by the redistribution of radiolabeled ⁸⁶Rb⁺. The release evoked by Ba²⁺ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca²⁺] increased during stimulation by approximately 200 n M , but cytosolic [Ba²⁺] increased by more than 1 μ M . Taken together, our results indicate that Ba²⁺ initially depolarizes synaptosomes most likely by blocking a K⁺ channel, which then activates TTX-sensitive Na⁺ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca²⁺channels to evoke neurotransmitter release directly. Though Ba²⁺-evoked glutamate release was comparable in level to that obtained with K⁺-induced depolarization in the presence of Ca²⁺, the apparent intrasynaptosomal level of Ba²⁺ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca²⁺.     

Nitric Oxide Causes Glutamate Release from Brain Synaptosomes

Abstract: We determined the ability of pathological levels of nitric oxide (NO) to cause glutamate release from isolated rat brain nerve terminals using a fluorometric assay. It was found that NO (0.7 and 2 µ M ) produced (4 and 10 nmol/mg of synaptosomal protein) Ca²⁺-independent glutamate release from synaptosomes (after 1 min of exposure). Spermine/NO complex (spermine NONOate; a slow NO donor) and potassium cyanide (an inhibitor of cytochrome oxidase) also caused Ca²⁺-independent glutamate release. Preincubation of synaptosomes with 5 µ M 1 H -[1,2,4]oxadiazole[4,3- a ]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) had no effect on NO-induced Ca²⁺-independent glutamate release. Ca²⁺-independent glutamate release produced by NO was greater in a low-oxygen medium. NO, spermine NONOate, and potassium cyanide inhibited synaptosomal respiration with a similar order of potency with respect to their ability to cause glutamate release. Because NO has been shown previously to inhibit reversibly cytochrome oxidase in competition with oxygen, our findings in this study suggest that NO (and cyanide) causes glutamate release following inhibition of mitochondrial respiration at the level of cytochrome oxidase. Thus, elevated NO production leading to mitochondrial dysfunction, glutamate release, and excitotoxicity may contribute to neuronal death in neurological diseases.     

Inhibition of Ca2+-dependent glutamate release from cerebral cortex synaptosomes of rats with experimental autoimmune encephalomyelitis

Several pathological studies have revealed a prominent involvement of the cerebral cortex in patients with multiple sclerosis (MS). In order to better understand the events that lead to the progressive neuronal dysfunction in MS, herein we explore the contribution of the glutamatergic release in cerebral cortex synaptosomes isolated from rats with experimental autoimmune encephalomyelitis, an animal model reproducing many features of MS. We found that the Ca²⁺-dependent but not the Ca²⁺-independent glutamate release induced by KCl and 4-aminopyridine was significantly decreased during the acute stage of the disease. This inhibited release coincides with the onset of the clinical signs and after 24 h tends to recover the level of the control animals. The results also showed an inhibition of the glutamate release stimulated by ionomycin. When the animals were totally recovered from clinical signs, the neurotransmitter release stimulated by the different inductors was similar to the controls. Examination of the cytosolic Ca²⁺ using fura-2-acetoxymethyl ester revealed that the inhibition of glutamate release could not be attributed to a reduction in voltage-dependent Ca²⁺ influx. However, this inhibition was concomitant with a lower phosphorylation of synapsin I at P-site1. Our results show that the inhibition observed on the Ca²⁺-dependent neurotransmitter release from cerebral cortex synaptosomes in experimental autoimmune encephalomyelitis is specific and correlates with the beginning of the clinical disease. Moreover, they suggest an alteration in the metabolism of proteins involved in the vesicular glutamate release more than a deregulation in the influx of cytosolic Ca²⁺.     

Presynaptic GABAB Receptor Modulation of Glutamate Exocytosis from Rat Cerebrocortical Nerve Terminals: Receptor Decoupling by Protein Kinase C

Abstract: GABA and the GABA_B receptor agonist (−)-baclofen inhibited 4-aminopyridine (4AP)- and KCl-evoked, Ca²⁺-dependent glutamate release from rat cerebrocortical synaptosomes. The GABA_B receptor antagonist CGP 35348, prevented this inhibition of glutamate release, but phaclofen had no effect. (−)-Baclofen-mediated inhibition of glutamate release was insensitive to 2 µg/ml pertussis toxin. As determined by examining the mechanism of GABA_B receptor modulation of glutamate release, (−)-baclofen caused a significant reduction in 4AP-evoked Ca²⁺ influx into synaptosomes. The agonist did not alter the resting synaptosomal membrane potential or 4AP-mediated depolarization; thus, the inhibition of Ca²⁺ influx could not be attributed to GABA_B receptor activation causing a decrease in synaptosomal excitability. Ionomycin-mediated glutamate release was not affected by (−)-baclofen, indicating that GABA_B receptors in this preparation are not coupled directly to the exocytotic machinery. Instead, the data invoke a direct coupling of GABA_B receptors to voltage-dependent Ca²⁺ channels linked to glutamate release. This coupling was subject to regulation by protein kinase C (PKC), because (−)-baclofen-mediated inhibition of 4AP-evoked glutamate release was reversed when PKC was stimulated with phorbol ester. This may therefore represent a mechanism by which inhibitory and facilitatory presynaptic receptor inputs interplay to fine-tune transmitter release.     

Differential Transmitter Release from Nerve Terminals Isolated from Basal Ganglia and Substantia Nigra

Abstract: The K⁺-induced release of amino acids and dopamine from synaptosomes of basal ganglia and substantia nigra of sheep was studied. K⁺ (56 mM) caused an increase in the release of GABA from caudate, putamen, globus pallidus, and substantia nigra, the increased release being 227, 171, 198, and 366%, respectively, compared with samples incubated without stimulation. The release of glutamate was also increased by 56 mM-K⁺ (136–183%) from all regions except the globus pallidus, and a significant release of aspartate was only seen in response to K⁺ stimulation of synaptosomes from putamen (50%). Veratrine (75 μM) also stimulated a similar pattern of amino acid release from these regions. Regional correlation was shown between the presence of an uptake system for an amino acid and its evoked release. [¹⁴C]Dopamine formed from L-[U-¹⁴C]tyrosine was released only from caudate and putamen synaptosomes by K⁺ stimulation, the increases being 105% and 74%, respectively. Synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine occurred only in synaptosomes prepared from these two regions and was not detected in synaptosomes from substantia nigra or globus pallidus although whole-tissue homogenates of substantia nigra were able to synthesise dopamine.     

Age-related changes in H2O2 production and bioenergetics in rat brain synaptosomes

Detrimental changes to mitochondrial function have been shown to occur with age. In this study we examined the levels of H(2)O(2) production, in situ mitochondrial membrane potential (Deltapsi(m)), oxygen consumption (JO(2)) and electron transport chain (ETC) enzyme activities in synaptosomes isolated from rats of two age groups, 6 and 18 months. The rate of H(2)O(2) production in synaptosomes was found to be higher in the 18-month old group compared to that of 6-month old. Deltapsi(m) was found to be significantly lower in synaptosomes from the older rats, which also correlated with a reduction in JO(2). Measurement of the individual electron transport chain enzyme activities revealed that reduced complex II/III and complex IV activities were the possible contributors to the reduced bioenergetic function in synaptosomes from the older rats. These data suggest that ageing may lead to increased nerve terminal H(2)O(2) production while simultaneous deleterious effects on bioenergetic function occur in in situ synaptosomal mitochondria. In addition, Ca(2+)-independent glutamate release was found to be increased at lower levels of complex I inhibition in the synaptosomes from older rats, suggesting that reduction of mitochondrial function may potentiate excitotoxic conditions in the ageing brain.     

Complex I Controls Mitochondrial and Plasma Membrane Potentials in Nerve Terminals

Reductions in the activities of mitochondrial electron transport chain (ETC) enzymes have been implicated in the pathogenesis of numerous chronic neurodegenerative disorders. Maintenance of the mitochondrial membrane potential (Δψ_m) is a primary function of these enzyme complexes, and is essential for ATP production and neuronal survival. We examined the effects of inhibition of mitochondrial ETC complexes I, II/III, III and IV activities by titrations of respective inhibitors on Δψ_m in synaptosomal mitochondria. Small perturbations in the activity of complex I, brought about by low concentrations of rotenone (1–50 nM), caused depolarisation of Δψ_m. Small decreases in complex I activity caused an immediate and partial Δψ_m depolarisation, whereas inhibition of complex II/III activity by more than 70% with antimycin A was required to affect Δψ_m. A similarly high threshold of inhibition was found when complex III was inhibited with myxothiazol, and inhibition of complex IV by more than 90% with KCN was required. The plasma membrane potential (Δψ_p) had a complex I inhibition threshold of 40% whereas complex III and IV had to be inhibited by more than 90% before changes in Δψ_p were registered. These data indicate that in synaptosomes, both Δψ_m and Δψ_p are more susceptible to reductions in complex I activity than reductions in the other ETC complexes. These findings may be of relevance to the mechanism of neuronal cell death in Parkinson’s disease in particular, where such reductions in complex I activity are present.

     

Effects of Kainic Acid in Rat Brain Synaptosomes: The Involvement of Calcium

Abstract: The effects of kainic acid were investigated in preparations of rat brain synaptosomes. It was found that kainic acid inhibited competitively the uptake of d -[³H]aspartate, with a K _i of approximately 0.3 m m . Kainic acid also caused release of two excitatory amino acid neurotranstnitters, aspartate and glutamate, in a time- and concentration-dependent manner, but had no effect on the content of γ-aminobutyric acid. Concomitant with the release of aspartate and glutamate, depolarization of the synaptosomal membrane and an increase in intracellular calcium were observed, with no measurable change in the concentration of internal sodium ions. The increase in intrasynaptosomal calcium and decrease in transmem-brane electrical potential were prevented by the addition of glutamate, whereas the kainate-induced release of ra-dioactive aspartate was substantially inhibited by lowering the concentration of calcium in the external medium. It is postulated that kainic acid reacts with a class of glutamate receptors located in a subpopulation of synaptosomes, presumably derived from the glutamatergic and aspartatergic neuronal pathways, which possesses high-affinity uptake system(s) for glutamate and/or aspartate. Activation of these receptors causes opening of calcium channels, influx of calcium into the synaptosomes, and depolarization of the synaptosomal plasma membrane with consequent release of amino acid neurotransmitters.     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Inhibition of Glutamate Transport in Synaptosomes by Dopamine Oxidation and Reactive Oxygen Species

Abstract: Dopamine can form reactive oxygen species and other reactive metabolites that can modify proteins and other cellular constituents. In this study, we tested the effect of dopamine oxidation products, other generators of reactive oxygen species, and a sulfhydryl modifier on the function of glutamate transporter proteins. We also compared any effects with those on the dopamine transporter, a protein whose function we had previously shown to be inhibited by dopamine oxidation. Preincubation with the generators of reactive oxygen species, ascorbate (0.85 m M ) or xanthine (500 µ M ) plus xanthine oxidase (25 mU/ml), inhibited the uptake of [³H]glutamate (10 µ M ) into rat striatal synaptosomes (−54 and −74%, respectively). The sulfhydryl-modifying agent N -ethylmaleimide (50–500 µ M ) also led to a dose-dependent inhibition of [³H]glutamate uptake. Preincubation with dopamine (100 µ M ) under oxidizing conditions inhibited [³H]glutamate uptake by 25%. Exposure of synaptosomes to increasing amounts of dopamine quinone by enzymatically oxidizing dopamine with tyrosinase (2–50 U/ml) further inhibited [³H]glutamate uptake, an effect prevented by the addition of glutathione. The effects of free radical generators and dopamine oxidation on [³H]glutamate uptake were similar to the effects on [³H]dopamine uptake (250 n M ). Our findings suggest that reactive oxygen species and dopamine oxidation products can modify glutamate transport function, which may have implications for neurodegenerative processes such as ischemia, methamphetamine-induced toxicity, and Parkinson's disease.     

Phorbol ester translocation of protein kinase C in guinea-pig synaptosomes and the potentiation of calcium-dependent glutamate release

The distribution of protein kinase C activity and specific phorbol ester binding sites between soluble and particulate fractions of isolated guinea-pig cerebral cortical synaptosomes is examined following preincubation with phorbol esters. Half-maximal decrease in cytosolic activity requires 10 nM 4 beta-phorbol myristoyl acetate. Specific [3H]phorbol dibutyrate binding sites are translocated from cytoplasmic to particulate fractions in parallel with protein kinase C activity. Depolarization of the plasma membrane by 30 mM KCl does not cause translocation of protein kinase C. 1 microM 4 beta-phorbol myristoyl acetate and 1 microM 4 beta-phorbol didecanoate (but not 1 microM 4 alpha-phorbol didecanoate) enhance the release of glutamate from synaptosomes partially depolarized by 10 mM KCl; however, 4 beta-phorbol myristoyl acetate is ineffective at 20 nM. 1 microM 4 beta-phorbol myristoyl acetate slightly increases the cytosolic free Ca2+ concentration of polarized synaptosomes, but not that following partial depolarization. 4 beta-Phorbol myristoyl acetate causes a concentration-dependent increase in the Ca2+-dependent glutamate release induced by sub-optimal ionomycin concentrations, but is without effect on the release induced by maximal ionomycin. It is concluded that phorbol esters stereospecifically enhance the Ca2+-sensitivity of glutamate release, but that higher concentrations may be required than for protein kinase C translocation in the same preparation. Instead the enhancement may be related to the rapid inactivation of protein kinase C which occurs with phorbol esters.     

Berberine Inhibits the Release of Glutamate in Nerve Terminals from Rat Cerebral Cortex

Berberine, an isoquinoline plant alkaloid, protects neurons against neurotoxicity. An excessive release of glutamate is considered to be one of the molecular mechanisms of neuronal damage in several neurological diseases. In this study, we investigated whether berberine could affect endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes) and explored the possible mechanism. Berberine inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP), and this phenomenon was prevented by the chelating extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate. Inhibition of glutamate release by berberine was not due to it decreasing synaptosomal excitability, because berberine did not alter 4-AP-mediated depolarization. The inhibitory effect of berberine on glutamate release was associated with a reduction in the depolarization-induced increase in cytosolic free Ca²⁺ concentration. Involvement of the Ca_v2.1 (P/Q-type) channels in the berberine action was confirmed by blockade of the berberine-mediated inhibition of glutamate release by the Ca_v2.1 (P/Q-type) channel blocker ω-agatoxin IVA. In addition, the inhibitory effect of berberine on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Berberine decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synapsin I, the main presynaptic target of ERK; this decrease was also blocked by the MEK inhibition. Moreover, the inhibitory effect of berberine on evoked glutamate release was prevented in nerve terminals from mice lacking synapsin I. Together, these results indicated that berberine inhibits glutamate release from rats cortical synaptosomes, through the suppression of presynaptic Cav2.1 channels and ERK/synapsin I signaling cascade. This finding may provide further understanding of the mode of berberine action in the brain and highlights the therapeutic potential of this compound in the treatment of a wide range of neurological disorders.     

Inhibition of Phosphorylation of Synapsin I and Other Synaptosomal Proteins by β-Bungarotoxin, a Phospholipase A2 Neurotoxin

Some snake venom neurotoxins, such as beta-bungarotoxin (beta-BuTX), which possess relatively low phospholipase A2 (PLA2) activity, act presynaptically to alter acetylcholine (ACh) release both in the periphery and in the CNS. In investigating the mechanism of this action, we found that beta-BuTX (5 and 15 nM) inhibited phosphorylation, in both resting and depolarized synaptosomes, of a wide range of proteins, including synapsin I. Naja naja atra PLA2, which has higher PLA2 activity, also inhibited phosphorylation but was less potent than beta-BuTX. At 1 nM, beta-BuTX and N. n. atra PLA2 inhibited phosphorylation of synapsin I only in depolarized synaptosomes. Synaptosomal ATP levels were not affected by 5 or 15 nM beta-BuTX or by 5 nM N. n. atra PLA2. Limited proteolysis, using Staphylococcus aureus V-8 protease, indicated that beta-BuTX inhibited phosphorylation of synapsin I in both the head and the tail regions. The inhibition of phosphorylation was not antagonized by nordihydroguaiaretic acid or indomethacin, suggesting that arachidonic acid derivatives do not mediate this inhibition. Furthermore, inhibition of phosphorylation by beta-BuTX and N. n. atra PLA2 was not altered in the presence of the phosphatase inhibitor okadaic acid, suggesting that stimulation of phosphatase activity is not responsible for this inhibition. Inhibition of protein phosphorylation by PLA2 neurotoxins and enzymes may be associated with an inhibition of ACh release.     

Characterization of the Exocytotic Release of Glutamate from Guinea-Pig Cerebral Cortical Synaptosomes

Abstract: A continuous enzyme-linked fluorometric assay was used for determining the characteristics for glutamate exocytosis from guinea-pig cerebrocortical synaptosomes. Ca²⁺-dependent release can be induced not only by K⁺, but also by the Na⁺ channel activator veratridine and the Ca²⁺ ionophore ionomycin. K⁺-induced release can be inhibited by the Ca²⁺ channel inhibitor verapamil. Sr²⁺ and Ba²⁺ substitute for Ca²⁺ in promoting K⁺-induced release. Agents that would be predicted to transform the transvesicular pH gradient into a membrane potential are without effect on glutamate release. However, the protonophore carbonylcy-anide p -trifluoromethoxyphenylhydrazone causes a time-dependent loss of exocytosis that is oligomycin insensitive and may be due to depletion of vesicular glutamate. The Ca²⁺-independent release of glutamate from the cytosol on depolarization is unchanged or promoted by metabolic inhibitors that lower the ATP/ADP ratio. In contrast, Ca²⁺-dependent release is ATP dependent and is blocked by the combined inhibition of oxidative phosphorylation and glycolysis.     

Glutamine--a major substrate for nerve endings

Abstract— Mammalian cortical synaptosomes incubated in the presence of glucose (2.5 MM) plus glutamine (0.5 mM) showed a 30% increase in transmitter amino acid content over controls with glucose alone and a doubling of glutamate release induced by Veratrine or high K⁺. Double-label experiments, i.e. [U-¹⁴C]glucose with [³H]glutamine, and single-label experiments, i.e. [U-¹⁴C]glucose or [U-¹⁴C]-glutamine showed that stimulus-released glutamate was derived principally (80%) from glutamine. Released glutamine-derived glutamate was of higher (x 2) specific radioactivity than its tissue equivalent. Glutamine alone (0.5–0.75 mM) was much less effective than equivalent amounts of glucose alone, in stimulating respiration and maintaining tissue K⁺ levels.     

Identification and Properties of Methyltransferases That Synthesize Phosphatidylcholine in Rat Brain Synaptosomes

Abstract: Rat brain was found to enzymatically methylate phospholipids to form phosphatidylcholine with S -adenosyl- l -methionine serving as the methyl donor. Methyltransferase activity was localized in the microsomes and synaptosomes. In synaptosomes, at least two enzymes were found to be involved in the formation of phosphatidylcholine. The first methyltransferase which catalyzes the methylation of phosphatidylethanolamine to form phosphatidyl- N -monomethylethanolamine was found to have a pH optimum of 7.5, a low K_m for 5-adenosyl- l -methionine and a partial requirement for Mg². Methyltransferase I is tightly bound to membranes. The second methyltransferase (II) catalyzes the successive methylations of phosphatidyl- N -monomethylethanolamine to phosphatidyl- N , N -dimethylethanolamine and then to phosphatidylcholine. In contrast to methyltransferase I, methyltransferase II has a pH optimum of 10.5, a high apparent K_m for S -adenosyl- l -methionine and no requirement for Mg². Methyltransferase II is easily solubilized by sonication. The highest specific activity for both enzymes was found in the synaptosomal plasma membrane.     

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H₂O₂ on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H₂O₂ (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca²⁺-dependent exocytosis of glutamate, induced by KCl or by the K⁺-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H₂O₂, although a transient decrease was observed after the treatment. H₂O₂ did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H₂O₂ did not modify significantly the KCl-induced increase of [Ca²⁺]_i. H₂O₂ inhibited exocytosis also when the latter was induced by increasing [Ca²⁺]_i with the Ca²⁺ ionophore ionomycin. The effects of H₂O₂ were unchanged in the presence of superoxide dismutase and the presence of the Fe³⁺ chelator deferoxamine. These results appear to indicate that H₂O₂, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.