Endogenous Adenosine Modulation of 22Na Uptake by Rat Brain Synaptosomes

To evaluate if endogenous extracellular adenosine influences sodium channel activity in nerve terminals, we investigated how manipulations of extracellular adenosine levels influence ²²Na uptake by rat brain synaptosomes stimulated with veratridine (VT). To decrease extracellular adenosine levels, adenosine deaminase (ADA) that converts adenosine into an inactive metabolite was used. To increase extracellular adenosine levels, we used the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA), as well as the inhibitor of adenosine transport, nitrobenzylthioinosine (NBTI). ADA (0.1–5U/ml) caused an excitatory effect on ²²Na uptake stimulated by veratridine, which was abolished in the presence of the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 25M). Both the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1–10M) and the adenosine deaminase inhibitor EHNA (10–25M) inhibited ²²Na uptake by rat brain synaptosomes. It is suggested that adenosine is tonically inhibiting sodium uptake by rat brain synaptosomes.     

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 Nitric Oxide Donors on GABA Uptake by Rat Brain Synaptosomes

The effect of nitric oxide donors and L-arginine on the uptake of GABA was studied in synaptosomes purified from rat brain. The neurotransmitter uptake was significantly reduced by S-nitrosoacetylpenicillamine and by sodium nitroprusside, although in this case to a lesser extent. A slight inhibitory effect was found preincubating rat brain synaptosomes with 1 mM L-arginine as well. The S-nitrosoacetylpenicillamine effect gradually disappeared with decomposition of the substance by exposure to light. The nitric oxide effect appears to be mainly due to a decrease in the V for synaptosomal GABA uptake and seems to be related to a partial collapse of nerve endings ionic gradients. Functionally, it could result over time in a reduced availability of GABA at the synapses involved.     

Exposure to Ebselen Changes Glutamate Uptake and Release by Rat Brain Synaptosomes

We investigated effects of Ebselen, diphenyl diselenide (PhSe)₂ and diphenyl ditelluride (PhTe)₂ on [³H]glutamate uptake and release by brain synaptosomes. Ebselen after acute exposure inhibited K⁺-stimulated [³H]glutamate release by brain synaptosomes. (PhSe)₂ and (PhTe)₂ did not change [³H]glutamate release by brain synaptosomes. Ebselen, (PhSe)₂ and (PhTe)₂ had no significantly effects on [³H]glutamate uptake after acute exposure. In vitro, Ebselen (100 M) inhibited [³H]glutamate release and uptake. (PhSe)₂ had no significant effect, while (PhTe)₂ (100 M) inhibited [³H]glutamate uptake by brain synaptosomes. In vitro, (PhSe)₂, (PhTe)₂ and Ebselen caused a significant inhibition of [³H]glutamate uptake by brain synaptic vesicles in vitro. The results demonstrated that organochalcogenides have a rather complex effect on glutamate homeostasis depending on the compound and the schedule of exposition. We propose that the neuroprotective action of Ebselen can be related, in addition to its glutathione peroxidase-like and antilipoperoxidative activity, to a direct interaction with the glutamatergic system by reducing K^ï-evoked glutamate release.     

Competitive Inhibition of Catecholamine Uptake in Synaptosomes of Rat Brain by Rigid Bicyclo-Octanes

Abstract: Cis-3-(3, 4-dichlorophenyl)-2- N , N -dirnethylaminomethyl-bi-cyclo-[2, 2, 2]-octane hydrochloride (LR5182) inhibited the uptake of dopamine by a synaptosomal fraction of corpus striatum of rat brain with an inhibitor constant (K_i value) of 6 nM. Kinetics analysis according to the methods of Lineweaver-Burk and Dixon revealed a competitive inhibition of dopamine uptake of LR5182. When the uptake of dopamine by the noradrenergic terminals in the synaptosomal fraction of cerebral cortex was selectively abolished by desipramine, LR5182 competitively inhibited the uptake of dopamine by the dopaminergic terminals with a K_i value of 19 nM. LR5182 was also a competitive inhibitor of noradrenaline uptake by synaptosomes of hypothalamus and cerebral cortex with, however, one-third to one-tenth the effectiveness, as indicated by the larger K_i values, 52 and 58 nM, respectively. Structure-activity studies with LR5182 and related compounds supported the idea that the uptake sites of dopamine favored a gauche conformer.     

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...     

Circadian Variation in the Uptake of Tryptophan by Cortical Synaptosomes of the Rat Brain

The kinetics of the high affinity uptake system for L-tryptophan (L-Try)have been measured over 24 hr in cortical synaptosome preparations of rat brain. Both the K_m and V_max, of the uptake process showed a statistically significant 24 hr variation. The highest K_m value, 6.71 ± 10^-5 M, was measured at the beginning of the light phase and the lowest value, 4.23 ± 10^-5 M, 6 hr into the dark phase. V_max was highest at the end of the dark phase (10.43 nmol/mg/5 min) and lowest (4.80 nmol/mg/5 min) 3 hr into the dark phase. In contrast, there was no variation over 24 hr in the V_max/K_m ratio. These results suggest that the high affinity uptake process serves to ensure a constant rate of L-tryptophan entry into the neuron in the face of circadian or ultradian variations in extracellular concentration of tryptophan.     

Circadian Variation in the Uptake of Tryptophan by Cortical Synaptosomes of the Rat Brain

The kinetics of the high affinity uptake system for L-tryptophan (L-Try)have been measured over 24 hr in cortical synaptosome preparations of rat brain. Both the K_m and V_max, of the uptake process showed a statistically significant 24 hr variation. The highest K_m value, 6.71 ± 10^-5 M, was measured at the beginning of the light phase and the lowest value, 4.23 ± 10^-5 M, 6 hr into the dark phase. V_max was highest at the end of the dark phase (10.43 nmol/mg/5 min) and lowest (4.80 nmol/mg/5 min) 3 hr into the dark phase. In contrast, there was no variation over 24 hr in the V_max/K_m ratio. These results suggest that the high affinity uptake process serves to ensure a constant rate of L-tryptophan entry into the neuron in the face of circadian or ultradian variations in extracellular concentration of tryptophan.     

Rat Brain Synaptosomes Prepared by Phase Partition

Synaptosomes from rat forebrain can easily be isolated by combining centrifugation with partition in an aqueous two-phase system composed of dextran T500 and polyethylene glycol 4000 in which synaptosomes have an extreme affinity for the upper phase. The fraction thus obtained has been characterized by electron microscopy and biochemical markers for synaptosomes and some other cell components. The contamination by microsomes, free mitochondria, and myelin was 4.4, 3.2, and 0.1%, respectively. The morphometric analysis of the electron micrographs shows that greater than 60% of the structures are synaptosomes. This preparation of the isolation procedure is remarkably short (less than 1 h), formance as assayed by their respiratory activities and ATP level in the absence and presence of depolarizing agents. Synaptosomes prepared by phase partition release the neurotransmitter glutamate in a Ca2(+)-dependent manner. The duration of the isolation procedure is remarkably short (less than 1 h), no ultracentrifuge is required, and the method can be applied for small- or large-scale preparations.     

Some Biochemical Properties of the Rapid Adenosine Uptake System in Rat Brain Synaptosomes

Abstract: The rapid uptake of adenosine into rat brain cortical synaptosomes is mediated by a facilitated diffusion process. The carrier mediated uptake is sensitive to pH and temperature. The average Q₁₀ value for the system is approximately 1.77 and the necessary activation energy ( E _a) is estimated to be 8870 cal/mol. These values are essentially in agreement with values reported for adenosine uptake carriers of other tissues. Substrate specificity of the uptake system in the CNS demonstrates that nucleotides do not interact with the carrier until they have been hydrolyzed to nucleosides. Structural modification of the purine moiety at the "2" position did not have a profound effect on the ability of the molecule to serve as a substrate for the uptake system. Competitive inhibition by sulfhydryl reagents, p -chloromercuribenzoate, and N- ethylmaleimide on adenosine uptake suggests a direct involvement of sulfhydryl group(s) in the uptake mechanism. Other purines and pyrimidines also inhibited adenosine uptake, suggesting that a variety of nucleosides can interact with a common carrier system.     

Uptake of Agmatine into Rat Brain Synaptosomes: Possible Role of Cation Channels

Abstract: Agmatine (decarboxylated arginine), an endogenous ligand for imidazoline receptors, has been identified in brain where it is synthesized from arginine by arginine decarboxylase. Here we report a mechanism for the transport of agmatine into rat brain synaptosomes. The uptake of agmatine was energy- and temperature-dependent and saturable with a K _m of 18.83 ± 3.31 m M and a V _max of 4.78 ± 0.67 nmol/mg of protein/min. Treatment with ouabain (Na⁺,K⁺-ATPase inhibitor) or removal of extracellular Na⁺ did not attenuate the uptake rate. Agmatine transport was not inhibited by amino acids, polyamines, or monoamines, indicating that the uptake is not mediated by any amino acid, polyamine, or monoamine carriers. When we examined the effects of some ion-channel agents on agmatine uptake, only Ca²⁺-channel blockers inhibited the uptake, whereas a reduction in extracellular Ca²⁺ increased it. In addition, some imidazoline drugs, such as idazoxan and phentolamine, were strong noncompetitive inhibitors of agmatine uptake. Thus, a selective, Na⁺-independent uptake system for agmatine exists in brain and may be important in regulating the extracellular concentration of agmatine.     

Influence of Hypotonic Shock on Glutamate and GABA Uptake in Rat Brain Synaptosomes

Hyponatremia leads to hyperexcitability of neurons, seizures, and coma. It is well established that uptake of neurotransmitters is a sodium-dependent process. Therefore, we suggest that inhibition of neurotransmitter uptake can lead to the clinical manifestations of hyponatremia. Decreasing of sodium concentration down to 92 mM in incubation medium, which corresponds to lowering the osmolarity down to 230 mOsm/l, leads to a 45% decrease in glutamate uptake and a 46% decrease in gamma-aminobutyric acid (GABA) uptake. However, this effect was mediated by the nonspecific lowering of osmolarity rather than by decreasing sodium concentration. Hypotonic shock was able to reduce glutamate uptake in the presence of protein kinase inhibitors staurosporine and genistein, the phosphatase inhibitor okadaic acid, the phosphatidylinositol 3-kinase inhibitor wortmannin, and cytoskeleton modulators colchicine and cytochalasin B. Therefore, we suggest that intracellular signaling is not mediating the effect of osmolarity reduction on neurotransmitter uptake.     

Localization of Thiamine-Binding Protein in Synaptosomes from the Rat Brain

Using preparations of synaptosomes and subsynaptosomal fractions from the rat brain, we studied the localization of thiamine-binding protein (TBP) in the subcellular structures of the neurons. In addition, we studied the distribution in synaptosomes of two types of activity typical of TBP (thiamine triphosphatase and thiamine-binding activities), as well as the effects of factors destroying the plasma membrane of synaptosomes on binding of [¹⁴C]thiamine with the latter. We found that the thiamine-associated activity of TBP was the highest in fractions of the synaptic vesicles and plasma membranes. Hydrolysis of thiamine triphosphate was also most active in these structures. Our results allow us to conclude that TBP is localized mostly in the synaptic vesicles and plasma membranes of synaptosomes.     

N 6-Cyclohexyladenosine Inhibits Veratridine-Stimulated Na Uptake by Rat Brain Synaptosomes

The effect of the stable adenosine analogue, N⁶-cyclohexyladenosine, on ²²Na uptake by rat brain synaptosomes stimulated by veratridine was investigated. In the presence of N⁶-cyclohexyladenosine, both the initial rate and the maximum sodium uptake were decreased. The inhibitory effect of N⁶-cyclohexyladenosine on sodium uptake by synaptosomes after 5 s of incubation with ²²Na was concentration-dependent, antagonized by 1,3-dipropyl-8-p-sulfophenylxanthine, and attenuated by increasing the concentration of veratridine. The possibility that the adenosine analogue, by activating a xanthine-sensitive adenosine receptor, can operate inhibition of the voltage-dependent sodium channels is discussed.     

Isolation of Monoaminergic Synaptosomes from Rat Brain by Immunomagnetophoresis

Monoaminergic synaptosomes have been isolated and purified from rat brain by immunomagnetophoresis. This novel technique uses magnetic beads to which Protein A is bound. Noradrenergic, dopaminergic, and serotonergic synaptosomes (previously cell-surface labelled with anti-dopamine-beta-hydroxylase, anti-tyrosine hydroxylase, and anti-tryptophan hydroxylase, respectively) may be isolated in a highly purified state. The synaptosomal subpopulations are recovered in a viable metabolic state and show glucose-stimulated respiration and Ca2(+)-dependent neurotransmitter release. A novel subtype of dopamine-beta-hydroxylase was found in dopaminergic terminals. No evidence for glutamate corelease from monoaminergic synaptosomes was obtained.     

Histidine Transport into Rat Brain Synaptosomes

Abstract: Histidine transport and metabolism in rat brain synptosomes were investigated to study the possible role of histidine uptake in the synthesis of the putative neurotransmitter histamine (HA). Histidine uptake was found to be regionally distributed and temperature sensitive and was not totally independent of sodium or possium ions. Transport was inhibited by metabolic inhibitors, as well as by promethazine and quinacrine. A number of other HA-related agents and several histidine metabolites had no effect. Kinetic analyses of histidine transport revealed the presence of both high- and lowaffinity systems in cerebral cortex. Histidine uptake increased following preexposure of synaptosomes to depolarizing concentrations of potassium. This effect was dependent on the presence of calcium ions during the preincubation. No newly formed [³H]HA was detectable in rat brain synaptosomes following [³H]histidine transport. Lesions of the medial forebrain bundle did not alter histidine uptake in the hippocampus or cerebral cortex. Ontogenic studies indicated that the histidine uptake system developed rapidly and reached a peak during postnatal days 12–17. Overall, the present findings do not support a role for histidine transport in the regulation or maintenance of neurotransmitter pools of HA in rat brain.     

Dopamine Uptake by Rat Striatal Synaptosomes: A Compartmental Analysis

Abstract: Dopamine (DA) uptake into synaptosomes from rat corpus striatum was studied in the presence of a monoamine oxidase (MAO) inhibitor and dithiothreitol, by means of a filtration technique. Under these conditions a steady state develops rapidly in which the synaptosomal DA content remains constant while the continuing DA uptake is counterbalanced by DA efflux from the synaptosome. Exchange of synaptosomal [³H]DA and [¹⁴C]DA was measured under these conditions. In timecourse experiments it was found that exchange could be described significantly better by a three-compartment model than by a two-compartment model. However, if synaptosomes from reserpine-pretreated animals were used, analysis according to a three-compartment model did not result in a significantly better fit compared with a two-compartment model. Subsequently, kinetic transfer parameters describing DA fluxes between compartments at different DA concentrations were calculated from the fitted exchange curves. A Michaelis-Menten kinetic analysis indicated that only the in-series three-compartment configuration, in which DA is taken up from the medium into one synaptosomal compartment, from which it can subsequently be transferred to a second compartment without direct access to the medium, gave kinetically acceptable results. Transfer parameters in synaptosomes from reserpine-treated rats were comparable to those parameters describing DA transport between the medium and the first intrasynaptosomal compartment as measured under control conditions. Morover, it was found that potassium depolarization of synaptosomes resulted in a release of DA in a quantity similar to that found in the second intrasynaptosomal compartment. It is suggested that the two intrasynaptosomal compartments found correspond to a cytoplasmatic and vesicular DA pool, respectively. The functional significance of these findings is discussed in terms of the regulation of DA levels within the nerve terminal.     

Pretreatment of Rat Brain Synaptosomes with GABA Increases Subsequent GABA Uptake Via GABAB Receptor Activation

Pretreatment with 100 M GABA of synaptosomes purified from rat brain results in an increased uptake of the labelled neurotransmitter in subsequent incubations. The effect is blocked by a GABA_B receptor antagonist, 2-hydroxy-saclofen. The effect is mimicked by baclofen and the baclofen effect is blocked by saclofen too. Lower GABA concentrations (up to 50 M) do not result in an increase of subsequent GABA uptake. Treatment of synaptosomes with 8-Br-cAMP results in a decreased GABA uptake. Since the uptake incubations were run with saturating concentrations of labelled GABA, the data indicates that GABA_B receptor activation in brain synaptosomes up-regulates their GABA uptake capacity by an increase in V_max. This mechanism appears of physiological relevance under conditions of sustained GABA release and substantial increase of its extracellular concentration.     

Inhibition by Methylmalonate of Glycine Uptake by Synaptosomes from Rat Spinal Cord

Methylmalonate is accumulated in the genetically linked deficiency of methylmalonyl-CoA mutase (methylmalonic acidemia). In this condition is also observed an elevation of the glycine levels. This communication reports the inhibition of the synaptosomal glycine uptake by methylmalonate, when present at similar concentrations to those found in methylmalonic acidemia. This inhibition could be responsible, at least in part, for the neurological damage characteristic of this disease, by increasing the glycine levels in the synaptic cleft and thus interfering with the normal function of the inhibitory glycinergic synapsis in the spinal cord.     

Lipid Peroxidation-Induced Inhibition of γ-Aminobutyric Acid Uptake in Rat Brain Synaptosomes: Protection by Glucocorticoids

Incubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) resulted in an inhibition of gamma-aminobutyric acid (GABA) uptake. The inhibitory effects of X/XO were temperature- and time-dependent, and were characterized by an increased Km for GABA and a decreased Vmax. Inhibition of GABA uptake by X/XO was associated with both the formation of malonyldialdehyde (MDA) and conjugated dienes, indicating that lipid peroxidation was involved. Studies with catalase, superoxide dismutase (SOD), mannitol, and chelated iron suggested that hydroxyl radical (OH X) was probably responsible for the initiation of lipid peroxidation. Both the peroxidation of synaptosomal membranes and the inhibition of GABA uptake by X/XO were enhanced by the addition of ADP and FeCl2. The X/XO-induced inhibition of GABA uptake by synaptosomes could be prevented by preincubation of synaptosomes with certain glucocorticoids prior to X/XO exposure. Methylprednisolone sodium succinate (MPSS), dexamethasone sodium phosphate (DMSP), and prednisolone sodium succinate (PSS) all prevented the inhibition of GABA uptake by X/XO. MPSS was most effective at concentrations around 100 microM, DMSP was slightly more potent, and PSS was optimal at around 300 microM. On the other hand, hydrocortisone sodium succinate (HCSS) was ineffective at preventing X/XO-induced inhibition of GABA uptake at concentrations up to 3 mM. The steroids are presumed to work through a mechanism that blocked the formation of lipid peroxides, as MPSS inhibited the formation of conjugated dienes in synaptosomes exposed to X/XO at a concentration that also protected GABA uptake.