Neurotransmitter Metabolism in Rat Brain Synaptosomes: Effect of Anoxia and pH

Synaptosomes isolated from the rat cerebral cortex by means of a discontinuous Ficoll gradient carry out net, sodium-dependent, veratridine-sensitive accumulation of gamma-aminobutyric acid (GABA), serotonin, norepinephrine, and dopamine. The intrasynaptosomal contents of the four neurotransmitters are: 30.4 nmol/mg protein, 17.4 pmol/mg protein, 13.5 pmol/mg protein, and 21.2 pmol/mg protein, respectively. Anaerobic preincubation of synaptosomes causes an irreversible decrease in the rates of neurotransmitter accumulation but does not affect the rates of their release. The inhibitory effect of anaerobiosis is enhanced by increased concentration of [H+] (decreased pH) in the medium. The most sensitive is the uptake of dopamine, the least that of serotonin. The rates of neurotransmitter efflux are unaffected by anaerobiosis. Synaptosomes leak catecholamines, GABA, and serotonin into the medium when subjected to anaerobiosis, and reintroduction of oxygen is accompanied by a rapid reaccumulation of all four neurotransmitters. It is concluded that: (1) Responses of synaptosomes to anaerobiosis are remarkably similar to the behavior of intact brain in hypoxia and ischemia. (2) Neurotransmitter uptake systems are more sensitive to short periods of anaerobiosis than either the energy metabolism or ion transport. (3) Some neurotransmitter uptake systems are more easily damaged by anaerobiosis than others.     

Effects of Polyamines on the Uptake of Neurotransmitters by Rat Brain Synaptosomes

Abstract: The influence of putrescine, spermidine, spermine, and some aliphatic α,ω-diamines on the uptake of neurotransmitters by rat forebrain synaptosomes was investigated. Choline uptake was most effectively inhibited by spermine (IC₅₀= 0.22 m M ), less so by spermidine (IC₅₀= 4.0 m M ), but not by putrescine (IC₅₀ > 100 m M ). At 10 m M, 1,3-diaminopropane, cadaverine, and 1,8-diaminooctane all inhibited choline uptake by 50% or more. Spermine and spermidine inhibited the uptake of dopamine with IC₅₀ values of 2.7 and 2.2 m M , respectively. Putrescine was only slightly inhibitory (IC₅₀= 17.3 m M ) and the other diamines were inactive. The uptake of γ-aminobutyrate (GABA) was only slightly inhibited (15–40%) by the polyamines at 10 m M . With the exception of inhibition of glycine uptake by 1,8-diaminooctane (60%) and of glutamate uptake by cadaverine (35%) none of the polyamines, tested at 10 m M , affected the uptake of adenosine, glutamate, and glycine significantly. A possible modulatory role for polyamines in synaptic transmission through interaction by negatively charged groups of the synaptic membrane with the polycationic compounds is discussed.     

Effect of Oleate on Neurotransmitter Transport and Other Plasma Membrane Functions in Rat Brain Synaptosomes

The effects of fatty acids, oleate and palmitate, on gamma-aminobutyric acid (GABA), aspartate, and 3,4- dihydroxyphenylethylamine (dopamine) transport and a variety of other membrane functions were studied in rat brain synaptosomes at a constant lipid-to-protein ratio. Under the conditions utilized oleate, but not palmitate, caused statistically significant changes in synaptosomal functions. Oleic acid inhibited the uptake of the amino acid neurotransmitters and dopamine in a tetrodotoxin-insensitive manner; it also induced the release of neurotransmitters from synaptosomes. The synaptosomal membrane potential decreased and the maximum GABA accumulation ratio [( GABA]i/[GABA]o) declined in parallel. The same depolarizing effect was seen in the presence of 50 microM verapamil or when chloride was replaced by propionate. The rate of respiration was stimulated by the unsaturated fatty acid; neither verapamil (50 microM) nor ouabain (100 microM) was effective in preventing the increase in oxygen consumption. By contrast, ruthenium red substantially decreased the stimulatory effect of oleate. The intrasynaptosomal [Ca2+] was increased by 40%, whereas [Na+]i remained unaltered. It is postulated that under the conditions used the inhibition of neurotransmitter uptake and the decrease in their accumulation caused by oleate result from the depolarization of synaptosomes that arises, at least in part, from increased permeability of the plasma membrane to calcium ions.     

The Effects of Chronic Manganese Chloride Treatment Expressed as Age-Dependent, Transient Changes in Rat Brain Synaptosomal Uptake of Amines

Abstract: As a result of chronic manganese treatment of rats from conception onwards, a decrease was observed in the uptake of dopamine, but not of noradrenaline or serotonin, by synaptosomes isolated from hypothalamus, striatum, and midbrain and in choline uptake by hypothalamic synaptosomes obtained from 70–90-day-old animals. In 100–120-day-old manganese-treated rats the only difference observed was increased choline uptake by striatal synaptosomes. All comparisons were with age-matched controls. These results, which are consistent with views of a dopaminergic and cholinergic involvement in manganese encephalopathy, point out that changes in these systems are observable only at specific times during manganese intoxication.     

Effects of Taurine on Calcium Ion Uptake and Protein Phosphorylation in Rat Retinal Membrane Preparations

The effects of taurine on ATP-dependent calcium ion uptake and protein phosphorylation of rat retinal membrane preparations were investigated. Taurine (20 mM) stimulates ATP-dependent calcium ion uptake by twofold in crude retinal homogenates. In contrast, it inhibits the phosphorylation of specific membrane proteins as shown by acrylamide gel electrophoresis and autoradiography. The close structural analogue of taurine, 2-aminoethylhydrogen sulfate, demonstrates similar effects in both systems, i.e., stimulation of ATP-dependent calcium ion uptake and inhibition of protein phosphorylation, whereas isethionic acid and guanidinoethanesulfonate have no effect on either system. A P1 subcellular fraction of the retinal membrane preparation that contains photoreceptor cell synaptosomes has a higher specific activity for the uptake of calcium ions. Phosphorylation of specific proteins in the P1 fraction is also inhibited by the addition of 20 mM taurine. Taurine has no effect on retinal ATPase activities or on phosphatase activity, thus suggesting that it directly affects a kinase system.     

Chronic Manganese Treatment of Rats Alters Synaptosomal Uptake of Dopamine and the Behavioural Response to Amphetamine Administration

Abstract: Chronic manganese treatment from conception onward resulted in increased striatal synaptosomal uptake of dopamine, but not of a variety of other neurotransmitters/precursors in 80-day-old rats. The open-field behaviour of these manganese-treated 80-day-old rats was no different from that of untreated age-matched rats. However, amphetamine administration (1 mg/kg body weight) increased activity to a significantly lower extent in manganese-treated rats. These observations indicate that chronic manganese treatment results in marked alterations of activities associated with the dopaminergic system.     

Synaptosomal Calcium Uptake Systems: Prostaglandins Are Probably Not Involved in the Regulation of Calcium Fluxes Into and Within the Nerve Endings

Abstract: ⁴⁵Ca²⁺ uptake measurements were performed on intact and osmotically lysed synaptosomes from rat brain to study the possible influence of prostaglandins (PGs) on Ca²⁺ movements into and within the nerve endings. The K⁺-induced ⁴⁵Ca²⁺ uptake of intact synaptosomes was not influenced by several inhibitors of PG synthesis. ⁴⁵Ca²⁺ uptake in lysed synaptosomal preparations was promoted by ATP and seemed to be largely attributable to mitochondria, as it was inhibited by mitochondrial poisons. This Ca²⁺ uptake was strongly reduced by PG synthesis inhibitors but also by PG precursor fatty acids. Both PG synthesis inhibitors and precursors, according to their relative efficacy in blocking Ca²⁺ uptake, were able to induce Ca²⁺ efflux from preloaded intrasynaptosomal organelles. The PGs E₂, F_2α, D₂, and thromboxane B₂ were without effect on ⁴⁵Ca²⁺ uptake in lysed synaptosomal preparations. On the basis of our results it does not seem likely that PGs influence Ca²⁺ availability by modulating Ca²⁺ fluxes into or within the nerve endings. The observed inhibitory effects of PG synthesis inhibitors and precursors on the intrasynaptosomal Ca²⁺ uptake might be due to unspecific impairment of mitochondrial functions.     

Actions of Tremorgenic Fungal Toxins on Neurotransmitter Release

The neurochemical effects of the tremorgenic mycotoxins Verruculogen and Penitrem A, which produce a neurotoxic syndrome characterised by sustained tremors, were studied using sheep and rat synaptosomes. The toxins were administered in vivo, either by chronic feeding (sheep) or intraperitoneal injection 45 min prior to killing (rat), and synaptosomes were subsequently prepared from cerebrocortical and spinal cord/medullary regions of rat, and corpus striatum of sheep. Penitrem A (400 mg mycelium/kg) increased the spontaneous release of endogenous glutamate, GABA (gamma-aminobutyric acid), and aspartate by 213%, 455%, and 277%, respectively, from cerebrocortical synaptosomes. Verruculogen (400 mg mycelium/kg) increased the spontaneous release of glutamate and aspartate by 1300% and 1200%, respectively, but not that of GABA from cerebrocortical synaptosomes. The spontaneous release of the transmitter amino acids or other amino acids was not increased by the tremorgens in spinal cord/medullary synaptosomes. Penitrem A pretreatment reduced the veratrine (75 microM) stimulated release of glutamate, aspartate, and GABA from cerebrocortical synaptosomes by 33%, 46%, and 11%, respectively, and the stimulated release of glycine and GABA from spinal cord/medulla synaptosomes by 67% and 32% respectively. Verruculogen pretreatment did not alter the veratrine-induced release of transmitter amino acids from cerebrocortex and spinal cord/medulla synaptosomes. Penitrem A pretreatment increased the spontaneous release of aspartate, glutamate, and GABA by 68%, 62%, and 100%, respectively, from sheep corpus striatum synaptosomes but did not alter the synthesis and release of dopamine in this tissue. Verruculogen was shown to cause a substantial increase (300-400%) in the miniature-end-plate potential (m.e.p.p.) frequency at the locust neuromuscular junction. The response was detectable within 1 min, rose to a maximum within 5-7 min, and declined to the control rate over a similar period. No change in the amplitude of the m.e.p.p.'s was observed. These effects of the tremorgens on transmitter release are interpreted in terms of their mode of action.     

Effect of Impact Trauma on Neurotransmitter and Nonneurotransmitter Amino Acids in Rat Spinal Cord

N-Methyl-D-aspartate (NMDA) administration exacerbates neurological dysfunction after traumatic spinal cord injury in rats, whereas NMDA antagonists improve outcome in this model. These observations suggest that release of excitatory amino acids contributes to secondary tissue damage after traumatic spinal cord injury. To further examine this hypothesis, concentrations of free amino acids were measured in spinal cord samples from anesthetized rats subjected to various degrees of impact trauma to the T9 spinal segment. Levels of excitatory and inhibitory neurotransmitter amino acids [gamma-aminobutyric acid (GABA), glutamate, aspartate, glycine, taurine] and levels of nonneurotransmitter amino acids (asparagine, glutamine, alanine, threonine, serine) were determined at 5 min, 4 h, and 24 h posttrauma. Uninjured surgical (laminectomy) control animals showed modest but significant declines in aspartate and glutamate levels, but not in other amino acids, at all time points. In injured animals, the excitatory amino acids glutamate and aspartate were significantly decreased by 5 min posttrauma, and remained depressed at 4 h and 24 h as compared with corresponding laminectomy controls. In contrast, the inhibitory amino acids, glycine, GABA, and taurine, were decreased at 5 min postinjury, had partially recovered at 4 h, and were almost fully recovered at 24 h. The nonneurotransmitter amino acids were unchanged at 5 min posttrauma and significantly increased at 4 h, with partial recovery at 24 h. At 4 h postinjury, severe trauma caused significantly greater decreases in aspartate and glutamate than did either mild or moderate injury. These findings are consistent with the postulated role of excitatory amino acids in CNS trauma.     

The Decay of the ATPase Activity of Light Plus Thiol-Activated Thylakoid Membranes in the Dark

Oxidized ATP synthase of spinach thylakoid membranes catalyzes high rates of ATP synthesis in the light, but very low rates of ATP hydrolysis in the dark. Reduction of the disulfide bond in the γ subunit of the ATP synthase in the light enhances the rate of Mg²⁺-ATP hydrolysis in the dark. The light plus thiol-activated state decays in a few minutes in the dark after illumination in Tris buffer, but not when Tricine was used in place of Tris. In this paper, it is shown that Tris in the assay mixture is an inhibitor of the light plus thiol-activated ATPase activity of thylakoids, but only after the activated membranes had incubated in the dark. Aminopropanediols and diethanolamine, also selectively inhibited ATPase activity of activated membranes after storage in the dark, whereas NH₄Cl and imidazole inhibit the ATPase activity of activated thylakoids almost equally whether they are added directly after the illumination or several minutes later. The fluorescence of 9-amino-6-chloro-2-methoxyacridine (ACMA) is quenched by the establishment of proton gradients by ATP-dependent proton uptake. Addition of ATP to activated membranes results in rapid quenching of ACMA fluorescence. If the activated membranes were incubated in the dark prior to ATP addition, a lag in the ATP-dependent ACMA fluorescence quenching as well as a similar lag in the rate ATP hydrolysis were seen. It is concluded that ADP rebinds to CF1 in the dark following illumination and inhibits the activity of the ATP synthase. Reactivation of the ATP synthase in the dark can occur by the slow generation of proton gradients by ATP hydrolysis in the dark. This reactivation takes place in Tricine buffer, but not in Tris because of its uncoupling action. Whether ADP binding plays a role in the regulation of the activity of the ATP synthase in situ remains to be established.     

The Lipid Peroxidation Product, 4-Hydroxy-2-trans-Nonenal, Alters the Conformation of Cortical Synaptosomal Membrane Proteins

Abstract: Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid β-peptide (Aβ). Aβ-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2- trans -nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of Aβ and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6(2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1–10 µ M HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and Aβ-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 µ M ). The results obtained are discussed with relevance to the hypothesis of Aβ-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.     

The Specific Interaction of S-100 Protein with Synaptosomal Particulate Fractions. Modulation of Binding by Fractional Occupancy of Sites and by the Physical State of Membranes

Abstract: The nonlinearity of single components of the Scatchard plot of S-100 binding to synaptosomal particulate fractions (SYN) and the observation that dilution of the ¹²⁵I-labeled S-100 site complex results in a greater extent of dissociation of the tracer in the presence than in the absence of an excess of unlabeled S-100 suggest that sites change their binding behavior depending on fractional occupancy. To study this aspect of the interaction in more detail, ¹²⁵I-labeled S-100 binding experiments were conducted in the presence of, or after preincubation of SYN with various concentrations of, unlabeled S-100. The results indicate that: (a) S-100 synaptosomal sites do change their binding behavior depending on fractional occupancy; and (b) the nonrapid equilibrium between bound S-100 and the medium, which has been referred to as the formation of a tight complex between S-100 and its binding sites, is related to the activation of high-affinity sites. However, no univocal interpretation of these data in terms of binding model can be offered at present, as the binding models currently employed in the analysis of ligand-site interactions can each account for only part of the results described in this report. In any case, data obtained by studying ¹²⁵I-labeled S-100 binding to untreated SYN at 2°C and to prefixed SYN at 37°C indicate that the physical state of membranes influences both the extent of the interaction and the binding behavior of the sites.     

Effects of Taurine and Mitochondrial Metabolic Inhibitors on ATP-Dependent Ca2+ Uptake in Synaptosomal and Mitochondrial Subcellular Fractions of Rat Retina

ATP-dependent Ca2+ uptake was investigated at low Ca2+ concentrations (10 microM) in rat retinal synaptosomal and mitochondrial preparations obtained by differential centrifugation on Ficoll gradients. Ca2+ uptake in the synaptosomal and mitochondrial subcellular preparations was stimulated by ATP and additionally stimulated by ATP plus taurine. The ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptakes were inhibited by mitochondrial metabolic inhibitors (atractyloside, oligomycin, and ruthenium red). These metabolic inhibitors had a greater effect on the ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptake activities in the mitochondrial preparation than in the synaptosomal preparation. ATP-dependent Ca2+ uptake in a synaptosomal subfraction obtained by osmotic shock was only partially inhibited by atractyloside. ATP-dependent Ca2+ uptake in the synaptosomal subfraction was also stimulated by taurine but to a lesser extent than in either the synaptosomal or mitochondrial preparation. These studies suggest that mitochondria are primarily responsible for taurine-stimulated ATP-dependent Ca2+ uptake in synaptosomal preparations.     

抗体药物免疫原性评估的研究进展

抗体药物引发机体产生的非必要免疫反应,将影响药物的疗效和安全性。因此,有必要对处于不同研发阶段的抗体药物的免疫原性进行评估,包括上市后的监测。免疫原性评价是所有抗体药物研发过程中的关键环节,监管部门对抗体药物的免疫原性评估有严格要求,但是对于证据类型,数量和质量缺少统一标准,也缺少抗体药物免疫原性测定实验设计的指导文件或免疫原性比较的标准。新技术的出现促进了免疫原性评估的发展,免疫原性检出率也有了相应的提高,因此,只能进行“头对头”临床试验,才能对抗体药物的免疫原性进行评估。因此,研究机构,监管机构和临床医生都需要认识到免疫原性分析方法的变化。在这里,讨论了抗体药物免疫原性的相关因素,潜在的临床后果,评估免疫原性的监管指导变化,非临床和临床研究的免疫原性评估方法的发展,以及生物仿制药免疫原性评估需要特别注意的事项。     

Intensified Downstream Processing of Monoclonal Antibodies Using Membrane Technology

The need to intensify downstream processing of monoclonal antibodies to complement the advances in upstream productivity has led to increased attention toward implementing membrane technologies. With the industry moving toward continuous operations and single use processes, membrane technologies show promise in fulfilling the industry needs due to their operational flexibility and ease of implementation. Recently, the applicability of membrane-based unit operations in integrating the downstream process has been explored. In this article, the major developments in the application of membrane-based technologies in the bioprocessing of monoclonal antibodies are reviewed. The recent progress toward developing intensified end-to-end bioprocesses and the critical role membrane technology will play in achieving this goal are focused upon.     

Effects of ATP and Taurine on Calcium Uptake by Membrane Preparations of the Rat Retina

Abstract: The effects of ATP and taurine on the kinetics of calcium uptake in rat retinal membrane preparations were determined. ATP increased calcium uptake at low calcium ion concentrations. Addition of ATP plus taurine further increased calcium uptake. Cooperative relationships were observed for calcium uptake in the absence of ATP and taurine. In the presence of phosphate ions reciprocal plots demonstrated upward deflections from linear ty, while in the absence of phosphate ions downward deflections were noted. Addition of ATP plus taurine to the incubation system appeared to obliterate the cooperativity. Two uptake systems for calcium were observed.     

The Specific Interaction of S-100 Protein with Synaptosomal Particulate Fractions. Evidence for the Formation of a Tight Complex Between S-100 and Its Binding Sites

Abstract: The dissociation of the ¹²⁵I-labelled S-100 specifically bound to synaptosomal particulate fractions (SYN) has been studied under a variety of conditions after different association times. The results indicate that after a critical association time of about 20 min at 37°C, the bound protein becomes progressively less accessible to the dissociating agents or conditions employed. These findings support the view that the partial irreversibility of the ¹²⁵I-labelled S-100 binding to SYN could be due to the formation of a tight complex between the protein and its synaptosomal sites. These data are discussed mainly in relation to the particulate-bound fraction of native S-100.     

Hormonal and Neurotransmitter Regulation of Ca++ Influx Through Voltage-Dependent Slow Channels in Cardiac Muscle Membrane

The voltage-and time-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca⁺⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. These slow channels behave kinetically as if their gates open, close, and recover more slowly than those of the fast Na⁺ channels; in addition, the slow channel gates operate over a less negative (more depolarized) voltage range. Tatrodotoxin does not block the slow channels, whereas the calcium antagonistic drugs, Mn⁺⁺, Co⁺⁺, and La⁺⁺⁺ ions do. The slow channels have some special properties, including their functional dependence on metabolic energy, their selective blockade by acidosis, and their regulation by cyclic AMP level. Because of their regulation by cyclic AMP, it is proposed that either the slow channel protein or an associated regulatory protein must be phosphorylated in order for the channel to be made available for voltage activation during excitation. That is, the dephosphorylated channel would be electrically silent.

The requirement for phosphorylation allows the extrinsic control of the slow channels and Ca⁺⁺ influx by neurotransmitters, hormones, and autacoids that affect the cyclic nucleotide levels.     

超氧自由基对谷氨酸摄取的抑制及Ebselen的保护

超氧自由基对谷氨酸摄取的抑制及Ｅｂｓｅｌｅｎ的保护易永杨祥良徐辉碧＊（华中理工大学化学系,武汉４３００７４）赵西龙张亨山秦钰慧（中国预防医学科学院环境卫生监测所毒理室,北京１０００２１）关键词大脑皮层突触体;谷氨酸摄取;Ｎａ＋,Ｋ＋－ＡＴＰａｓｅ;超...     

Inhibition of Glutamate Uptake and Proton Pumping in Synaptic Vesicles by S-Nitrosylation

Abstract: Nitric oxide (NO; including NO^•, NO⁺, and NO⁻) was found to inhibit glutamate uptake by isolated synaptic vesicles of rat brain. This was observed when two unrelated NO donors, S -nitrosogluthathione and S -nitroso- N -acetylpenicillamine, were used. The primary target of NO is the H⁺-ATPase found in the synaptic vesicles, which leads to dissipation of the electrochemical proton gradient and inhibition of glutamate uptake. Oxyhemoglobin (12 µ M ) and, to a much lesser extent, methemoglobin protected the vacuolar H⁺-ATPase from inhibition. Inhibition of H⁺ pumping by NO was reversed by addition of 0.5 m M dithiothreitol. The results indicate that the vacuolar H⁺-ATPase from synaptic vesicles is inhibited by NO by a mechanism that involves S -nitrosylation of critical sulfhydryl groups in the enzyme. The interaction of NO with synaptic vesicles might be of importance for the understanding of the multiple effects of NO in neurotransmission.