Angiotensin receptor II is present in dopaminergic cell line of rat substantia nigra and it is down regulated by aminochrome

Angiotensin receptor II mRNA was found to be expressed in dopaminergic neuronal cell line RCSN3 of rat substantia nigra using RT-PCR reaction. Aminochrome (150 M), a metabolite of the dopamine oxidative pathway, was found to down regulate the expression of angiotensin receptor mRNA in RCSN3 cells by 83% (p < 0.05).     

Persistent MDMA-induced dopaminergic neurotoxicity in the striatum and substantia nigra of mice

Acute administration of repeated doses of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) dramatically reduces striatal dopamine (DA) content, tyrosine hydroxylase (TH), and DA transporter-immunoreactivity in mice. In this study, we show for the first time the spatiotemporal pattern of dopaminergic damage and related molecular events produced by MDMA administration in mice. Our results include the novel finding that MDMA produces a significant decrease in the number of TH-immunoreactive neurons in the substantia nigra (SN). This decrease appears 1 day after injection, remains stable for at least 30 days, and is accompanied by a dose-dependent long-lasting decrease in TH- and DA transporter-immunoreactivity in the striatum, which peaked 1 day after treatment and persisted for at least 30 days, however, some recovery was evident from day 3 onwards, evidencing sprouting of TH fibers. No change is observed in the NAc indicating that MDMA causes selective destruction of DA-containing neurons in the nigrostriatal pathway, sparing the mesolimbic pathway. The expression of Mac-1 increased 1 day after MDMA treatment and glial fibrillary acidic protein increased 3 days post-treatment in the striatum and SN but not in the NAc, in strict anatomical correlation with dopaminergic damage. These data provide the first evidence that MDMA causes persistent loss of dopaminergic cell bodies in the SN.     

Expression of BDNF mRNA in substantia nigra is dependent on target integrity and independent of neuronal activation

We have analyzed the regulation of brain-derived neurotrophic factor (BDNF) mRNA expression in the nigrostriatal system following neurotoxin ablation of striatal targets by means of kainate (KA) or quinolinic acid (QA) injections. Loss of nigral target cells in the striatum was accompanied by significant induction of BDNF mRNA levels in the ipsilateral substantia nigra (SN) at 12 and 24 h post lesion. Dual tyrosine hydroxylase (TH) and BDNF mRNA in situ hybridization (ISH) confirmed the dopaminergic nature of the BDNF mRNA expressing cells. Analysis of neuronal activity in terms of cFos mRNA expression demonstrated intense induction of this marker in the ipsilateral SN pars reticulata (SNPR), but not in SN pars compacta. Dual glutamic acid decarboxylase (GAD) and cFos mRNA ISH confirmed this view. Colchicine injections into the medial forebrain bundle to specifically disrupt neuronal trafficking between SN and striatum induced BDNF mRNA levels in the ipsilateral SNPC, thus demonstrating that nigral expression of BDNF mRNA is dependent of striatal target tissue. In addition, we found significant elevations of BDNF in the subthalamic nucleus following striatal excitotoxic lesion, which may bring novel roles of BDNF in the basal ganglia complex.     

Striatal glutamate release evoked in vivo by NMDA is dependent upon ongoing neuronal activity in the substantia nigra, endogenous striatal substance P and dopamine

The aim of the present microdialysis study was to investigate whether the increase in striatal glutamate levels induced by intrastriatal perfusion with NMDA was dependent on the activation of extrastriatal loops and/or endogenous striatal substance P and dopamine. The NMDA-evoked striatal glutamate release was mediated by selective activation of the NMDA receptor-channel complex and action potential propagation, as it was prevented by local perfusion with dizocilpine and tetrodotoxin, respectively. Tetrodotoxin and bicuculline, perfused distally in the substantia nigra reticulata, prevented the NMDA-evoked striatal glutamate release, suggesting its dependence on ongoing neuronal activity and GABA(A) receptor activation, respectively, in the substantia nigra. The NMDA-evoked glutamate release was also dependent on striatal substance P and dopamine, as it was antagonized by intrastriatal perfusion with selective NK(1) (SR140333), D(1)-like (SCH23390) and D(2)-like (raclopride) receptor antagonists, as well as by striatal dopamine depletion. Furthermore, impairment of dopaminergic transmission unmasked a glutamatergic stimulation by submicromolar NMDA concentrations. We conclude that in vivo the NMDA-evoked striatal glutamate release is mediated by activation of striatofugal GABAergic neurons and requires activation of striatal NK(1) and dopamine receptors. Endogenous striatal dopamine inhibits or potentiates the NMDA action depending on the strength of the excitatory stimulus (i.e. the NMDA concentration).     

Copper reduction by yeast cell wall materials and its role on copper uptake in Debaryomyces hansenii

Copper binding reducing activities of cell wall materials (CWM) prepared from cells of the yeast Debaryomyces hamsenii were examined. When CWM was treated with copper sulfate (0.1 mM CuSO₄), the copper was partially reduced from Cu (II) to Cu (I) and bound to CWM (below 10 nmol per mg dry wt.). The bound copper was mostly in the fraction of mannan-protein. Both copper-binding ability and protein content decreased with protease treatments. Mannan-protein prepared from CWM bound more copper than mannan did. This suggests that Cu (II) bound to the protein portion in CWM and was reduced to Cu (I). The optimum pH of copper reduction by CWM was about 5.0. The amount of copper bound to CWM increased with reducing agents and decreased with oxidizing agents. On the other hand, the copper uptake by yeast whole cells and spheroplasts was also stimulated by reducing agents, but inhibited by oxidizing agents. Furthermore, copper uptake by spheroplasts was stimulated in the presence of CWM. The optimum pH of copper uptake coincided with that of copper reducing activity. These results suggest that yeast cell wall not only supplies copper binding but also reduces copper, and the reduced copper is transported into yeast cells. The yeast cells may have copper-reducing proteins in the cell wall.     

Fas-mediated apoptosis in a rat acinar cell line is dependent on caspase-1 activity

Apoptosis plays an important role in the dysfunction of exocrine glands. Fas is a death-inducing receptor found on many types of cells including epithelial acinar cells. To elucidate the intracellular mechanism of Fas-mediated cell death in exocrine glands, an epithelial acinar cell line, SMG-C6, was studied. Caspase-1, -3, -8, and -9 activities were elevated in SMG-C6 cells after the induction of apoptosis by soluble Fas ligand (FasL). The activation of caspase-1 and -8 occurred prior to caspase-3 and -9 activation. The caspase-1 inhibitor, zYVAD-fmk, was effective in preventing cell death, whereas the caspase-3 and -8 inhibitors (ac-DEVD-CHO and ac-IETD-CHO, respectively) were not. zYVAD-fmk was able to inhibit caspase-3 activation indicating that caspase-1 is upstream to caspase-3. Furthermore, kinetic studies show that caspase-1 is an early event in the Fas apoptotic pathway. This study shows that caspase-1 participates in Fas-mediated apoptosis of epithelial cells by initiating the caspase cascade.     

Effects of copper ions on the free radical-scavenging properties of reduced gluthathione: implications of a complex formation

The interaction between glutathione (GSH) and copper ions was investigated in vitro to determine whether such interaction could affect the free-radical scavenging properties of the tripeptide. To this end, the bleaching (decrease in OD734 nm) of a coloured solution containing the stable free-radical cation ABTS+, (which results from the addition of thiols to such a solution) was employed as an in vitro indication of the ability of the tripeptide to scavenge free radicals. While GSH bleached concentration-dependently (1.0-7.5 gM) the ABTS+-containing solution, its prior incubation (5 microM) in the presence of Cu+1 or Cu+2 ions (1-7.5 M) led to a metal concentration-dependent decrease of the bleaching capacity. At a ratio equal to one (5 microM each), the bleaching capacity of the copper plus GSH mixture was 50% of that seen for GSH alone. Further additions of copper (reaching ratios up to 2) did not result in greater decreases in the GSH-bleaching capacity. Noteworthy at the ratio of onewas that the copper plus GSH solutions maintained their bleaching capacity despite the lack of any DTNB-reactivity, i. e., the complete absence of thiols in the mixture. Mixtures of increasing concentrations of a fixed ratio (equal to 2) of copper plus GSH, which were found not to exhibit any DTNB-reactivity, showed a linear and concentration-dependent increase in bleaching capacity. The bleaching capacity remained unaltered when TRIEN, EDTA or histidine were added to pre-incubated (1:1) mixtures of copper plus GSH. However, the incubation of copper with TRIEN or EDTA (but not histidine) prior to GSH addition, totally prevented the loss of the original GSH-bleaching capacity. The present data supports the formation of a copper-glutathione complex which is stable to the presence of some copper-chelators, lacks all thiol reactivity, but fully conserves the free-radical scavenging properties of GSH.     

Neurochemical characterization of the release and uptake of dopamine in ventral tegmental area and serotonin in substantia nigra of the mouse

In the present report, fast-scan cyclic voltammetry was used to identify the monoamines that were released by electrical stimulation in mouse brain slices containing ventral tegmental area (VTA), substantia nigra (SN) -pars compacta (SNc) and -pars reticulata (SNr). We showed that voltammograms obtained in mouse VTA were consistent with detection of a catecholamine, while those in both subregions of the SN were consistent with detection of an indolamine, based on the reduction peak potentials. We used pharmacological blockade and genetic deletion of monoamine transporters to further confirm the identity of released monoamines in mouse midbrain and to assess the control of monoamines by their transporters in each brain region. Inhibition of dopamine and norepinephrine transporters by nomifensine (1 and 10 microm) decreased uptake rates in the VTA, but did not change uptake rates in either subregion of the SN. Serotonin transporter inhibition by fluoxetine (10 microm) decreased uptake rates in the SNc and SNr, but was without effect in the VTA. Selective inhibition of the norepinephrine transporter by desipramine (10 microm) had no effect in any brain region. Using dopamine transporter- and serotonin transporter-knockout mice, we found decreased uptake rates in VTA and SN subregions, respectively. Peak signals recorded in each midbrain region were pulse number dependent and exhibited limited frequency dependence. Thus, dopamine is predominately detected by voltammetry in mouse VTA, while serotonin is predominately detected in mouse SNc and SNr. Furthermore, active uptake occurs in these areas and can be altered only by specific uptake inhibitors, suggesting a lack of heterologous uptake. In addition, somatodendritic dopamine release in VTA was not mediated by monoamine transporters. This work offers an initial characterization of voltammetric signals in the midbrain of the mouse and provides insight into the regulation of monoamine neurotransmission in these areas.     

Copper(II) import and reduction are dependent on His-Met clusters in the extracellular amino terminus of human copper transporter-1

Copper(I) is an essential metal for all life forms. Though Cu(II) is the most abundant and stable state, its reduction to Cu(I) via an unclear mechanism is prerequisite for its bioutilization. In eukaryotes, the copper transporter-1 (CTR1) is the primary high-affinity copper importer, although its mechanism and role in Cu(II) reduction remain uncharacterized. Here we show that extracellular amino-terminus of human CTR1 contains two methionine-histidine clusters and neighboring aspartates that distinctly bind Cu(I) and Cu(II) preceding its import. We determined that hCTR1 localizes at the basolateral membrane of polarized MDCK-II cells and that its endocytosis to Common-Recycling-Endosomes is regulated by reduction of Cu(II) to Cu(I) and subsequent Cu(I) coordination by the methionine cluster. We demonstrate the transient binding of both Cu(II) and Cu(I) during the reduction process is facilitated by aspartates that also act as another crucial determinant of hCTR1 endocytosis. Mutating the first Methionine cluster (⁷Met-Gly-Met⁹) and Asp¹³ abrogated copper uptake and endocytosis upon copper treatment. This phenotype could be reverted by treating the cells with reduced and nonreoxidizable Cu(I). We show that histidine clusters, on other hand, bind Cu(II) and are crucial for hCTR1 functioning at limiting copper. Finally, we show that two N-terminal His-Met-Asp clusters exhibit functional complementarity, as the second cluster is sufficient to preserve copper-induced CTR1 endocytosis upon complete deletion of the first cluster. We propose a novel and detailed mechanism by which the two His-Met-Asp residues of hCTR1 amino-terminus not only bind copper, but also maintain its reduced state, crucial for intracellular uptake.     

Changes in extracellular levels of glutamate and aspartate in rat substantia nigra induced by dopamine receptor ligands: In vivo microdialysis studies

The microdialysis technique was utilized to study the local effects of D₁ and D₂ family type dopamine (DA) receptor (R) ligands on the in vivo release of endogenous glutamate (GLU) and aspartate (ASP) from rat substantia nigra (SN). Addition to the dialysis perfusion solution of either D₁-R and D₂-R agonists, such as SKF-38393 (50 and 100 M) and Quinpirole (5 and 10 M), resulted in dose-dependent increases in extracellular concentrations of GLU and ASP, respectively. The SKF-38393 and Quinpirole-induced effects were reduced by SCH-23390 (0.5 M), a D₁-R antagonist, and by Spiperone (1.0 M), a D₂-R antagonist, respectively. However, SCH-23390 and Spiperone did increase GLU and ASP extracellular concentrations. Local infusion with Tetrodotoxin (TTX) (1.0 M), a blocker of voltage-dependent Na⁺ channels, increased basal extracellular levels of GLU. In addition, co-infusion of TTX and SKF-38393 evoked increases in extracellular GLU levels higher than those observed after SKF-38393 alone. Finally, chemical lesions of nigral DA cells with 6-OH-DA increased the basal extracellular levels of GLU. It is proposed that the release of GLU and ASP from SN may be regulated by D₁- and D₂-receptors present in this basal ganglia structure. In addition, part of the D₁ receptors present in SN might be located presynaptically on GLU-containing nerve endings.     

Isofurans,but not F2-isoprostanes,are increased in the substantia nigra of patients with Parkinson's disease and with dementia with Lewy body disease

F2-isoprostanes (F2-IsoPs) are well-established sensitive and specific markers of oxidative stress in vivo. Isofurans (IsoFs) are also products of lipid peroxidation, but in contrast to F2-IsoPs, their formation is favored when oxygen tension is increased in vitro or in vivo. Mitochondrial dysfunction in Parkinson's disease (PD) may not only lead to oxidative damage to brain tissue but also potentially result in increased intracellular oxygen tension, thereby influencing relative concentrations of F2-IsoPs and IsoFs. In this study, we attempted to compare the levels of F2-IsoPs and IsoFs esterified in phospholipids in the substantia nigra (SN) from patients with PD to those of age-matched controls as well as patients with other neurodegenerative diseases, including dementia with Lewy body disease (DLB), multiple system atrophy (MSA), and Alzheimer's disease (AD). The results demonstrated that IsoFs but not F2-IsoPs in the SN of patients with PD and DLB were significantly higher than those of controls. Levels of IsoFs and F2-IsoPs in the SN of patients with MSA and AD were indistinguishable from those of age-matched controls. This preferential increase in IsoFs in the SN of patients with PD or DLB not only indicates a unique mode of oxidant injury in these two diseases but also suggests different underlying mechanisms of dopaminergic neurodegeneration in PD and DLB from those of MSA.     

Effect of medium copper concentration on the growth,uptake and intracellular balance of copper and zinc in Menkes' and normal control cells

The precise nature of the variation in cellular copper load against medium copper concentration is defined using a comprehensive logarithmically incremented series of medium copper concentrations ranging from low levels (4.8 p.p.b.) through normal to toxic levels (40 p.p.m.) in which fibroblasts were grown followed by determination of intracellular content. Menkes' fibroblasts showed an unexpected plateau region of stable intracellular copper content against a change in medium concentration of over 100-fold, albeit only when sufficient copper was present in the medium (0.08–8.0 p.p.m.). Thus, Menkes' cells are clearly capable of balancing uptake/efflux providing copper availability allows. Simultaneous analysis of cellular copper and zinc load at various medium copper concentrations shows an indistinguishable intracellular copper:zinc ratio between the two cell lines. The nature of non-labeled copper uptake by fibroblasts over a 40 min and 7 day period is reported. During the 40 min period copper uptake (20 p.p.m.) was essentially the same in both cell lines. However, copper absorbed was superimposed upon large pre-existing copper pools in the case of Menkes' cells only. Advantages of techniques determining non-labeled copper in copper uptake/efflux experiments are discussed in the light of these results. Fibroblast growth studies showed that, compared with normal cells, Menkes' cells are significantly (P < 0.01) more growth sensitive to extended exposure to low copper concentrations. Thus, Menkes' disease appears to be not only a result of copper maldistribution but also a direct result of an inability of Menkes' cells to function normally in low copper environments.     

狮子山铜尾矿植物对铜的吸收及土壤特的影响

我国铜矿储藏丰富,铜矿开采带来巨大经济利益的同时,也对生态环境造成极大的破坏,这种恶劣的环境严重阻碍了植物的定居,但是自然界物种繁多,总有一些植物能适应这种环境而生存下来.本文通过对狮子山优势植物吸收和积累铜的分析,发现这些植物均能富集较多的铜,在土壤铜含量很高的情况下,依然生长旺盛,没有出现受害症状,成为尾矿上的优势种,并形成了单优群落或多优小群落.这些植物的存在改变了土壤的理化特性,降低了土壤中的重金属的含量,提高了土壤的全N、全P、全K和有机质含量,一定程度上改善了土壤的不良环境,在尾矿的植被恢复和土壤修复中起着非常重要的作用.     

Neuroprotective Effect of Acute and Chronic Administration of Copper (II) Sulfate against MPP+ Neurotoxicity in Mice

Neurodegenerative effects of MPP⁺, the main metabolite of MPTP include dopamine (DA) depletion and enhanced lipid peroxidation (LPO) in mice striata, both associated to free radicals overproduction. Since copper is related to several antioxidant enzymes, we tested its neuroprotective effect against MPP⁺-induced neurotoxicity (20 g/3 l). CuSO₄ pretreatment was administrated by either acute (2.5 mg/kg, i.p) or chronic (350 or 700 mg/l doses through drinking water, for 30 days) schemes. Acute administration blocked MPP⁺-induced striatal LPO only when administered 16 or 24 hours before MPP⁺, and prevented the DA-depleting effect only at 24 hours. Chronic CuSO₄ prevented the LPO increase, and blocked the DA depletion only at the higher dose used (700 mg/l). Neuroprotective effect of CuSO₄ was dependent on the dose and the time of pretreatment, which suggest that this lag could be related with mechanisms of activation or synthesis of copper-dependent proteins responsible of cellular defense against MPP⁺.     

Intrastriatal administration of human immunodeficiency virus‐1 glycoprotein 120 reduces glial cell‐line derived neurotrophic factor levels and causes apoptosis in the substantia nigra

Uninfected neurons of the substantia nigra (SN) degenerate in human immunodeficiency virus (HIV)‐positive patients through an unknown etiology. The HIV envelope glycoprotein 120 (gp120) causes apoptotic neuronal cell death in the rodent striatum, but its primary neurotoxic mechanism is still under investigation. Previous studies have shown that gp120 causes neurotoxicity in the rat striatum by reducing brain‐derived neurotrophic factor (BDNF). Because glial cell line‐derived neurotrophic factor (GDNF) and BDNF are neurotrophic factors crucial for the survival of dopaminergic neurons of the SN, we investigated whether gp120 reduces GDNF and BDNF levels concomitantly to induce apoptosis. Rats received a microinjection of gp120 or vehicle into the striatum and were sacrificed at various time intervals. GDNF but not BDNF immunoreactivity was decreased in the SN by 4 days in gp120‐treated rats. In these animals, a significant increase in the number of caspase‐3‐ positive neurons, both tyrosine hydroxylase (TH)‐positive and ‐negative, was observed. Analysis of TH immunoreactivity revealed fewer TH‐positive neurons and fibers in a medial and lateral portion of cell group A9 of the SN, an area that projects to the striatum, suggesting that gp120 induces retrograde degeneration of nigrostriatal neurons. We propose that dysfunction of the nigrostriatal dopaminergic system associated with HIV may be caused by a reduction of neurotrophic factor expression by gp120. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Effect of Salmonella typhimurium enterotoxin (S-LT) on lipid peroxidation and cell viability levels of isolated rat enterocytes

Reactive oxygen species (ROS) are potent mediators of inflammatory disorders and may be of pathophysiological importance in S. typhimurium induced tissue damage. This study was carried out to investigate if ROS play a role in mediating the enterocyte damage during in vitro exposure to Salmonella typhimurium enterotoxin (S-LT). The ROS generation was detected by measuring the changes in the enterocyte arachidonic acid (AA) metabolism (measured indirectly by estimating the level of enterocyte damage in the absence and presence of the cyclooxygenase inhibitor, indomethacin) and xanthine oxidase activity. The enterocyte damage was estimated by measuring the changes in the level of lipid peroxidation and cell viability. The results obtained showed that the exposure of isolated rat enterocytes to S-LT resulted in an increased XO activity; an increased arachidonic acid metabolism, dose and time dependent increase in the level of lipid peroxidation and decreased cell viability. Lipid peroxidation decreased and cell viability increased in the presence of the antioxidant enzymes superoxide dismutase (SOD) or catalase. Thus the in vitro exposure of the enterocytes to S-LT is accompanied by an increased generation of ROS which may induce the lipid peroxidation of the enterocyte membrane thereby leading to a loss of cell viability.     

Functional consequences of RNA interference targeting COMMD1 in a canine hepatic cell line in relation to copper toxicosis

A deletion in the copper metabolism (Murr1) domain containing 1 (COMMD1) gene is associated with hepatic copper toxicosis in dogs, yet evidence of copper retention in COMMD1-depleted hepatic cells has not been shown. In a dog hepatic cell line, we analysed the copper metabolic functions after an 80% (mRNA and protein) COMMD1 reduction with COMMD1-targeting siRNAs. Exposure to 64Cu resulted in a significant increase in copper retention in COMMD1-depleted cells. COMMD1-depleted cells were almost three times more sensitive to high extracellular copper concentrations. Copper-mediated regulation of metallothionein gene expression was enhanced in COMMD1-depleted cells. Based on the increased copper accumulation and enhanced cellular copper responses upon COMMD1 reduction, we conclude that COMMD1 has a major regulatory function for intracellular copper levels in hepatic cells.