Peroxynitrite treatment reduces adenosine uptake via the equilibrative nucleoside transporter in rat astrocytes

In the oxidative stress-loaded brain, extracellular adenosine levels are elevated and thereby neuronal damage is attenuated, but mechanisms underlying alteration of the extracellular kinetics of adenosine remain unclear. Here we investigated whether oxidative stress might alter functional expression of nucleoside transporters (NTs), a predominant regulatory system for nucleoside kinetics, in cultured rat astrocytes. Treatment of astrocytes with 0.5 mM SIN-1 for 3 h caused apparent cellular accumulation of nitrotyrosine, but had no effect on their viability, indicating load of oxidative stress to astrocytes without any change in their viability. Under the condition, [³H]adenosine uptake was significantly less than that by control cells. This decreased uptake was due to decrease in adenosine uptake mediated by an equilibrative NT (ENT) 1 which was inhibited by low concentrations (≤0.1 μM) of nitrobenzylthioinosine (NBMPR), but not by sodium-dependent or high concentrations (≥1 μM) of NBMPR-inhibitable nucleoside transporters. The expression level of ENT1 was not altered, while the Michaelis constant, but not the maximum rate, of adenosine uptake was increased. These findings suggest that under oxidative stress-loaded conditions, decreased adenosine clearance via astrocytic ENT1 might involve, at least in part, in an elevated extracellular adenosine level in the brain.     

The equilibrative nucleoside transporter family,SLC29

The human SLC29 family of proteins contains four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterised family member, hENT1. They belong to the widely-distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporters and are distantly related to a lysosomal membrane protein, CLN3, mutations in which cause neuronal ceroid lipofuscinosis. A predicted topology of 11 transmembrane helices with a cytoplasmic N-terminus and an extracellular C-terminus has been experimentally confirmed for hENT1. The best-characterised members of the family, hENT1 and hENT2, possess similar broad substrate specificities for purine and pyrimidine nucleosides, but hENT2 in addition efficiently transports nucleobases. The ENT3 and ENT4 isoforms have more recently also been shown to be genuine nucleoside transporters. All four isoforms are widely distributed in mammalian tissues, although their relative abundance varies: ENT2 is particularly abundant in skeletal muscle. In polarised cells ENT1 and ENT2 are found in the basolateral membrane and, in tandem with concentrative transporters of the SLC28 family, may play a role in transepithelial nucleoside transport. The transporters play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis, and are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. In addition, by regulating the concentration of adenosine available to cell surface receptors, they influence many physiological processes ranging from cardiovascular activity to neurotransmission.     

Immunolocalisation of nucleoside transporters in human placental trophoblast and endothelial cells: evidence for multiple transporter isoforms

Polyclonal antibodies raised against the human erythrocyte nucleoside transporter were used to investigate the distribution of the nucleoside transporters in the placenta. Immunoblots of brush-border membranes isolated from the human syncytiotrophoblast revealed a cross-reactive species that co-migrated with the erythrocyte nucleoside transporter as a broad band of apparent M _r 55,000. In contrast, no labelling was detected in basal membranes containing a similar number of equilibrative nucleoside transporters as assessed by nitrobenzylthioinosine (NBMPR)-binding. The absence of cross-reactive epitopes in basal membranes and their presence in brush-border membranes was confirmed by confocal immunofluorescence microscopy. These results suggest that at least two isoforms of the NBMPR-sensitive nucleoside transporter are present in the human placenta. The lumenal surfaces of fetal capillaries, small placental vessels and umbilical vein were also strongly labelled by the antibody, a finding that suggests that the high fetal-placental adenosine uptake previously reported is due to endothelial transporters.Abbreviations NT The human erythrocyte nucleoside transporter - Anti-NT Polyclonal antibody against the nucleoside transporter - NBMPR Nitrobenzylthioinosine {6-[(4-nitrobenzyl)thio]-9--D-ribofuranosylpurine}. - GLUTl The human erythrocyte glucose transporter - PBS Phosphate-buffered saline - TBS TRIS-buffered saline - TTBS TRIS-buffered saline containing 0.2% Tween-20     

大鼠创伤性脑损伤中内皮性脂酶表达变化 

目的 探讨内皮性脂酶(EL)在创伤性脑损伤(TBI)后的表达变化及定位情况.方法 应用健康成年SD大鼠40只,成功制备TBI模型31只.通过Western blotting检测脑损伤后EL表达的时相变化,免疫荧光化学方法检测EL在脑组织中的细胞定位.结果 Western blotting显示,大鼠脑损伤后,EL表达逐步增高,伤后3d升至最高点,之后逐渐下降,于损伤后2周时降至最低水平;免疫荧光双标记结果显示EL与神经元的标记物NeuN共定位.与凋亡相关的蛋白Caspase-3和Bcl-2在TBI后表达发生变化,并且细胞凋亡趋势与EL表达变化趋势基本一致.结论大鼠TBI后,EL表达增加,推测EL可能参与脑损伤后神经再生及神经元凋亡过程.     

Transport activities involved in intracellular pH recovery following acid and alkali challenges in rat brain microvascular endothelial cells

Transport activities involved in intracellular pH (pH(i)) recovery after acid or alkali challenge were investigated in cultured rat brain microvascular endothelial cells by monitoring pH(i) using a pH-sensitive dye. Following relatively small acid loads with pH(i) approximately 6.5, [Formula: see text] influx accounted for most of the acid extrusion from the cell with both Cl(-)-independent and Cl(-)-dependent, Na(+)-dependent transporters involved. The Cl(-)-independent component has the same properties as the NBC-like transporter previously shown to account for most of the acid extrusion near the resting pH(i). Following large acid loads with pH(i) < 6.5, most of the acid extrusion was mediated by Na(+)/H(+) exchange, the rate of which was steeply dependent on pH(i). Concanamycin A, an inhibitor of V-type ATPase, had no effect on the rates of acid extrusion. Following an alkali challenge, the major component of the acid loading leading to recovery of pH(i) occurred by [Formula: see text] exchange. This exchange had the same properties as the AE-like transporter previously identified as a major acid loader near resting pH(i). These acid-loading and acid-extruding transport mechanisms together with the Na(+), K(+), ATPase may be sufficient to account not only for pH(i) regulation in brain endothelial cells but also for the net secretion of [Formula: see text] across the blood-brain barrier.     

Genomic organization and functional characterization of the human concentrative nucleoside transporter-3 isoform (hCNT3) expressed in mammalian cells

Human CNT3 encodes the concentrative nucleoside transport N3 system. Previous expression studies in oocytes showed that the K_m values for nucleosides of the cloned hCNT3 were 7- to 25-fold lower than the endogenous N3 transporter in HL60 cells. Therefore, in the present study we re-examined the kinetic properties of the cloned hCNT3 using mammalian cell expression systems by transient expression in Cos7L cells and stably expression in nucleoside transporter deficient PK15NTD cells. We demonstrated that hCNT3 is a Na-dependent, broadly-selective nucleoside transporter with affinities (<11 M) for nucleosides closely resembling the endogenous N3 transporter. Pharmacological studies showed that phloridzin is a mixed-type inhibitor of hCNT3 (K_i=15 M), and the dideoxyuridine analogs are poor substrates. By epitope-tagging, we further demonstrated that hCNT3 is N-glycosylated as PNGase F and Endo H deglycosylated hCNT3 from 67 kDa to 58 kDa. Searching the human genome database, we identified the genomic organization of hCNT3. This gene contains 19 exons and its exon-intron boundaries within the coding sequence exactly match with those of hCNT1 and hCNT2 with one additional exon in the N-terminus. Our data suggest that hCNT3 gene is evolutionarily conserved with hCNT1 and hCNT2. Physiologically, hCNT3 is a glycoprotein, which transports purine and pyrimidine nucleosides in a Na-dependent manner with high affinities.     

Distribution of N-methyl-D-aspartate receptors (NMDARs) in the developing rat brain

The N-methyl-D-aspartate receptor (NMDAR), which is one of the glutamate receptors, is considered to have a close relationship to synaptic plasticity in the developing brain. In addition, it is known that the excessive stimulation of NMDARs can trigger neuronal apoptosis. In this study, we examined the distribution of NMDAR subunits [anti-NR1, NR2(A-C)] in the developing rat brain immunohistochemically. As a result, NR1, an essential subunit for the formation of a functional NMDAR complex, was mildly expressed in the restricted areas such as the temporal region of the cerebral cortex and the hippocampus in the fetal brain at Embryonal Days 18 and 20. On the other hand, in neonates, NR1 was expressed widely throughout the whole brain. The distributions of NR2A and NR2C showed temporal and spatial similarities to that of NR1, while the expression of NR2B showed differences in the intensity and distribution. A progressive change in subunit expression seen prenatally and postnatally could contribute to variation of NMDARs and synaptic plasticity during the developing period.     

Crithidia fasciculata adenosine transporter 1 (CfAT1), a novel high-affinity equilibrative nucleoside transporter specific for adenosine

Most eukaryotic organisms including protozoans like Crithidia, Leishmania, and Plasmodium encode a repertoire of equilibrative nucleoside transporters (ENTs). Using genomic sequencing data from Crithidia fasciculata, we discovered that this organism contains multiple ENT genes of highly similar sequence to the previously cloned and characterized adenosine transporter CfNT1: CfAT1 and CfNT3, and an allele of CfAT1, named CfAT1.2. Characterization of CfAT1 shows that it is an adenosine-only transporter, 87% identical to CfNT1 in protein sequence, with a 50-fold lower K_m for adenosine. Site directed mutation of a key residue in transmembrane domain 4 (TM4) in both CfNT1 and CfAT1 shows that lysine at this position results in a high affinity phenotype, while threonine decreases adenosine affinity in both transporters. These results show that C. fasciculata has at least two adenosine transporters, and that as in other protozoan ENTs, a lysine residue in TM4 plays a key role in ligand affinity.     

地塞米松对癫痫大鼠脑组织内IgG免疫反应阳性细胞分布的影响

目的：研究地塞米松对大鼠脑免疫反应的影响,探讨其对某些类型癫痫的治疗机理。方法：美解眠致痫及致痫前后分别给予地塞米松的大鼠用免疫细胞化学法观察ＩｇＧ免疫反应阳性（ＩｇＧＩＲ）细胞在大鼠脑组织内分布。结果：致痫前给予地塞米松大鼠脑皮层和海马ＩｇＧＩＲ细胞数比单纯致痫组减少（Ｐ＜００１,Ｐ＜００５）;致痫后给予地塞米松组大鼠,只有海马ＩｇＧＩＲ细胞数比单纯致痫组增多（Ｐ＜００１）。结论：地塞米松对脑免疫反应的调节在某些类型癫痫的治疗中可能有一定意义。     

Fluorescence histochemical observations on the distribution of exogenous dihydroisoquinoline in the rat brain

Summary The distribution of fluorescence in the rat brain after i.p. or intracerebral (i.c.) injections of a fluorescent dihydroisoquinoline derivate of dopamine was studied. After low i.p. doses (50 mg/kg of body weight) the fluorescence was totally confined to the capillary endothelial cells in the cerebral cortex, neostriatum, and substantia nigra (SN). After large i.p. doses (500 mg/kg) fluorescent material was also present in the neuropil of all the regions studied and some cells of the cerebral cortex and SN. After injections to the neostriatum or SN fluorescence was observed in the endothelial cells and some small to medium-sized rounded cells in both regions. A conspicious dark area contrasting with the background fluorescence was constantly present around capillaries, and this area was in contact with nonfluorescent multibranched cells of astrocytic type. In fluorescent cells the fluorescence was present both in the cytoplasm and the nucleus.     

Age-dependent decrease in the activity of protein-synthesis initiation factors in rat brain

The age-dependent reduction of brain protein synthesis was examined at the initiation step of translation in the rat. Activity of brain initiation factor 2, as well as that of other eukaryotic initiation factors that contribute to the binding of initiator aminoacyl-tRNA to ribosomes, was found to decrease with age and to decline parallel to the decrease in total protein synthesis and brain elongation factor 1 activity. Change in the activity of initiation factors was demonstrated by the results of two different assays which compared favorably with each other. Decreased activity of preparations of initiation factors derived from older animals was observed in: (1) saturating conditions; (2) mixtures of preparations from different ages; and (3) both major cell fractions, i.e. cell sap and microsomes.     

Sodium-dependent adenosine transport is diminished in brush border membrane vesicles from hypothyroid rat kidney

 Studies of the uptake of [3H]adenosine ([³H]ADO) were performed using brush border membrane vesicles (BBMV) from normal (N) and hypothyroid (Tx) rat kidneys, to test if decreased Na⁺ reabsorption in hypothyroidism might be associated with abnormalities in ADO transport. [³H]ADO uptake (1–10 μmol) for both conditions was measured in the presence of Na⁺ (10–150 mmol/l); the effects of dipyridamole (10 μmol/l) and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX, 10 μmol/l) were also studied. Na⁺-stimulated ADO uptake was decreased in Tx BBMV. Michaelis–Menten constants showed a decreased ADO carrier affinity (K _m 2.46 ± 0.14 in N, vs K _m 4.46 ± 0.88 μmol/l in Tx, P<0.05), with no change in the number of carriers (V _max 295 ± 25 in N, vs 229.2 ± 56 pmol·min^–1·mg protein in Tx). Na⁺ affinity remained unchanged (K _Na+ 11.5 ± 3.5 in N, vs K _Na+ 12.72 ± 0.7 mmol/l in Tx). Inhibition of Na⁺-dependent ADO transport was 50% in N as opposed to 58% in Tx with dipyridamole, and 72% in N versus 47% in Tx with PACPX. These results suggest that decreased Na⁺-dependent ADO cotransport contributes to the diminished tubular reabsorption that occurs in hypothyroidism. Received: 17 June 1996 / Received after revision: 9 September 1996 / Accepted: 16 September 1996     

Vesicular glutamate transporters in the brain

Glutamate is an excitatory amino acid that acts as a major neurotransmitter throughout the brain. Although its neurotransmitter action has been evidenced by the identification of various receptor subtypes at synapses, a cellular mechanism by which this amino acid accumulates in synaptic vesicles has long been in doubt until the discovery in recent years of specific vesicular transporters. Three kinds of transporter isoforms have so far been cloned and their transport properties and distribution in the brain have been studied extensively. In contrast with the apparently similar ability of all transporter isoforms to highly selectively transport glutamate and their presence in synaptic vesicles, their regional distribution of gene expression and immunoreactivity in the rodent or human brain are surprisingly different from one another. This indicates that the glutamatergic neuron system of mammalian brains is substantially comprised of at least three different neuron subpopulations, each of which uses a unique transport system for the vesicular storage of glutamate. Thus, we now have highly useful and reliable tools for a comprehensive understanding of the glutamatergic neuron system in the brain from a new viewpoint different from that of other components, such as receptors. The scope of the present review is to provide an overview of the history and present status of the study of vesicular glutamate transporters and to highlight some unresolved issues requiring clarification for the progress of future brain function research.     

Regional distribution of phosphoinositide-specific phospholipase C activity in rat brain

The phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolytic activities of phosphoinositide-specific phospholipase C (PLC) were measured in membrane and cytosol fractions from 7 discrete areas of the rat brain. Both the PI-PLC and PIP₂-PLC specific activities were found to differ significantly among the 7 discrete brain areas. In the membrane fraction, the PIP₂-PLC activity was higher than that of PI-PLC in each region, suggesting that the PLC in membranes prefers PIP₂ to PI as substrate. The PIP₂-PLC activities in the membrane were high in prefrontal cortex and cerebellum, but rather low in medulla oblongata and hypothalamus. The PI-PLC specific activity in the cytosol was significantly higher than that in the membrane of all brain areas examined. The PI-PLC specific activity in membranes is inversely proportional to its activity in the cytosol. In the cytosol fraction, the distribution pattern of PI-PLC specific activity resembled that of PIP₂-PLC. These results indicate that PLCs are differently distributed in various regions of rat brain, and suggest the regional differences in neuronal transduction.     

Histochemical characteristics of diphosphate nucleoside consumption in cat and rat nervous system.

Consumption of diphosphate nucleosides is investigated by histochemical methods in rat and cat nervous system. Results show NDP-ase is effectively in histological sections from animals previously perfused with glutaraldehyde or formaldehyde. Histochemical reaction is increased in presence of Ca++, Mg++, Mn++ or imidazole and inhibited by L-DOPA and Noradrenaline in incubation medium. A comparative study of TPP-ase and NDP-ase activities to dilucidate the identity of both enzymes is described.     

Light scattering change precedes loss of cerebral adenosine triphosphate in a rat global ischemic brain model

Measurement of intrinsic optical signals (IOSs) is an attractive technique for monitoring tissue viability in brains since it enables noninvasive, real-time monitoring of morphological characteristics as well as physiological and biochemical characteristics of tissue. We previously showed that light scattering signals reflecting cellular morphological characteristics were closely related to the IOSs associated with the redox states of cytochrome c oxidase in the mitochondrial respiratory chain. In the present study, we examined the relationship between light scattering and energy metabolism. Light scattering signals were transcranially measured in rat brains after oxygen and glucose deprivation, and the results were compared with concentrations of cerebral adenosine triphosphate (ATP) measured by luciferin–luciferase bioluminescence assay. Electrophysiological signal was also recorded simultaneously. After starting saline infusion, EEG activity ceased at 108 ± 17 s, even after which both the light scattering signal and ATP concentration remained at initial levels. However, light scattering started to change in three phases at 236 ± 15 s and then cerebral ATP concentration started to decrease at about 260 s. ATP concentration significantly decreased during the triphasic scattering change, indicating that the start of scattering change preceded the loss of cerebral ATP. The mean time difference between the start of triphasic scattering change and the onset of ATP loss was about 24 s in the present model. DC potential measurement showed that the triphasic scattering change was associated with anoxic depolarization. These findings suggest that light scattering signal can be used as an indicator of loss of tissue viability in brains.     

Michaelis-Menten kinetics model of oxygen consumption by rat brain slices following hypoxia

In the present study, we have measured partial pressure of oxygen (pO₂) profiles through rat brain slices before and after periods of hypoxia (5 and 10 min) to determine its effect on tissue oxygen demand. Tissue pO₂ profiles were measured through rat cerebral cortex slices superfused with phosphate buffer using oxygen (O₂)-sensitive microelectrodes at different times in controls [40% O₂ balance nitrogen (N₂)], and at different times before and after 5 or 10 min of hypoxia (100% N₂). A one-dimensional, steady-state model of ordinary diffusion with a Michaelis-Menten model of O₂ consumption where the maximal O₂ consumption (V_max) and the rate at half-maximal O₂ consumption (K_m) were allowed to vary was used to determine the kinetics of O₂ consumption. Actual pO₂ profiles through tissue were fitted to theoretical profiles by a least-squares method. V_max varied among penetrations in a control slice and among slices. V_max seemed to decrease after hypoxic insult, but the change was not statistically significant. The K_m value measured before hypoxia was lower than the first K_m value measured after the end of hypoxia, indicating that hypoxia induced a compensatory change in the metabolic state of the tissue. K_m increased immediately after both 5- and 10-min hypoxic insults and returned to normal after recovery for each case. It seems that during and immediately after short periods of hypoxia, K_m increases from near zero but returns to normal values within a few minutes.     

The effect of an AMPA antagonist (NBQX) on postischemic neuron loss and protein synthesis in the rat brain

Two groups of rats were subjected to 12 min of global cerebral ischemia and 6 days recirculation using the four-vessel occlusion model with hypotension and then treated with the -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) antagonist NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxa-linedione (Honoré et al. 1988). One group was used for routine and quantitative histology and immunostaining for glial fibrillary acidic protein (GFAP). The second group was subjected to autoradiographic studies of regional cerebral protein synthesis, with special emphasis on the hippocampus, the frontal cortex, and the thalamus. It was found that neuroprotective treatment with NBQX normalized cerebral protein synthesis rate (CPSR) in all investigated regions 6 days after ischemia. In untreated ischemic animals CPSR was normalized in all regions except for the CA3 and thalamus, where it had increased by 29% and 41%, respectively. Treatment of controls with NBQX had no effect on CPSR after 6 days. The histological investigations revealed that NBQX did not protect vulnerable cells in the dentate hilus and the reticular thalamic nucleus (RTN). In these regions reactive astrocytosis visualized by GFAP immunostaining was equally pronounced in both ischemic and NBQX-treated animals, and most neurons in the RTN were eosinophilic. The 80–100% pyramidal neuron loss in CA1 was accompanied by a high degree of reactive astrocytosis, whereas the NBQX-treated animals showed no signs of astrocytosis in this region. The ischemic CA1 pyramidal layer was also massively invaded by microglia. Together these observations strongly suggest that the quantitatively normal protein synthesis in this region after ischemia must be attributed to these glial cell populations.