Molecular Mechanism Study On Manganese Chloride Caused The Dysfunction Of Glutamate Metabolism And Transport And The Protective Effects Of Riluzole

Manganese (Mn) is an essential trace element that can take part in many biochemical reactions. However, if exposed to excessive Mn, human beings would have a neurodegenerative disease closely resembled Parkinson syndrome termed manganism. Mn poisoning is mainly associated with occupational and environmental exposure. In the central nervous system (CNS), Mn mostly accumulates in the basal ganglia, especially in striatum and globus pallidus. In brain, the uptake of Mn is accomplished by astrocytes, and Mn would accumulate in them.Up to now, the studies on the mechanisms of Mn neurotoxicity were mostly used to be on the depletion of dopamine. Recently, several researches found that manganism would cause the increasing release of Glu and occurrence of excitotoxicity. However, the mechansims about it were still unknown. So the clearance of Glu in synaptic cleft will be accomplished by glutamate transporters (GluTs), and synaptic transmission will be ended up. Up to the present, there were five kinds of GluTs were found by the clone technique. Both glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are expressed in astrocytes. In the astrocytes, Glu is converted to glutamine (Gln) by glutamine synthetase (GS), Gln come back to neurons and is catalyzed to Glu by phosphate activated glutaminase (PAG). Then, and the Glu-Gln cycle is formed. Therefore, Glu metablism and transport may play an important role in the occurrence and development of manganism.Riluzole is a Glu modulator, which is the voltage-dependent sodium channel blocker. It is the only drug currently approved for amyotrophic lateral sclerosis (ALS) treatment. It was confirmed that riluzole had some neuroprotective effects such as inhibiting Glu release from nerve terminals; enhancing Glu uptake to astrocytes; increasing the affinity of Glu to GluTs. However, data regarding its possible effects on Glu metablism and transport in manganism are still few.In the present study, both in vivo and in vitro models were used to determine (1) the dysfunction of striatal Glu metablism and transport caused by MnCl2; (2) the cytotoxicity on astrocytes induced by MnCl2; (3) the dysfunction of astrocytic Glu metablism and transport after MnCl2 exposure. And the protective effects of riluzole on it.Materials and Methods1. MnCl2 caused on the dysfunction of Glu metablism and transport in rat's striatum and the protective effects of riluzoleThe study was performed on wistar rats (180±10g body weight at the beginning of experiment; N=100; equal numbers of male and female), which were purchased for Experimental Animal Center, China Medical University, Shenyang, China. All the rats were divided into five groups by weight at random,20 animals for each group. The first group was the control group. The second to fourth groups were the low, median and high levels of MnCl2 groups. The fifth group was the riluzole pretreatment group. At first, the rats in the first to the fourth groups were subcutaneously (sc) injected with 0.9% NaCl. The rats in the fifth group were sc injected with 21.35μmol/kg riluzole. After two hours, the rats of the first group were given intraperitoneally (ip) of 0.9% NaCl. The rats of the second and third groups were ip given 50 and 100μmol/kg MnCl2 respectively. The rats of both the fourth and fifth groups were given ip injection of 200μmol/kg MnCl2. The volume of injection is 5 ml/kg. All the administration was given five times a week, and pretreatment was given every two days lasting for 4 weeks. At 24h after the last injection, all the rats in each group were sacrificed by decaptation after anesthetized. Firstly, eight of the rats in each group were sacrificed and dissected to take the striatum. Using part of striatum of each rat, the content of Mn was determined by flame atomic absorption. Secondly, two of the rats in each group were perfused with buffered 4% paraformaldehyde solution. The striatum tissue was removed. Some of them were stained by HE to observe morphological changes. Others of them were fixed with 2.5% glutaraldehyde, and the ultrastruactural changes in the neuroglials of striatum were observed by transmission electron microscope. Thirdly, six rats in each group were sacrificed and the striatum were made into homogenate and the levels of Glu and Gln were analyzed by the kit. GS activity was determined by the formation of y-glutamyl hydroxamate as Renis et al described. PAG activity was determined by the method of Curi et al. Na+-K+-ATPase activity was measured according to the method of Murali et al. Finally, the rest striatum of the four rats in each group were used for RT-PCR to determine GS, GLAST and GLT-1 mRNA expression. And the other four rat's striatum were used for western blotting to determine the expression of GS, GLAST and GLT-1 protein levels.2. MnCl2 induced the cytotoxicity on astrocytes and the protective effects of riluzoleThe procedure for isolation and culturing of astrocytes was according to the method of Hertz et al. The cells would be used after more than 95% of cells stained positively for glial fibrillary acidic protein (GFAP) immunocytochemically. The cytotoxicity of Mn and preventive effects of riluzole about it was observed by MTT assay and LDH leakage assay after exposed to MnCl2 (0-1000μmol/L) for 6-48h and pretreated with riluzole (25～200μmol/L) for 1～12h. And then, the appropriate and representive levels or time of Mn and riluzole were chosen. At that levels and time, the morphological changes of astrocytes were observed by inverted phase-contrast microscope. The cell apoptosis rates and cell cycle were determined by flow cytometry (FCM). 3. MnCl2 exposure caused the dysfunction of astrocytic Glu metablism and transport and the protective effects of riluzoleAfter exposure to 0,125,250, and 500μmol/L MnCl2 for 24h, and prevention with 100μmol/L riluzole for 6h on the level of 500μmol/L MnCl2. According to the methods mentioned above, the activity of GS and Na+-K+-ATPase, and GS, GLAST and GLT-1 mRNA expression and protein levels were determined.4. Statistic analysisAll the results were expressed as mean±standard deviation and treated in SPSS 11.5 software by one-way ananlysis of variance (ANOVA) to test the significant differences among groups followed by q test (Student-Newman-Keuls, SNK) to find the differences between the groups.Results1. MnCl2 caused the dysfunction of Glu metablism and transport in rat's striatum and protective effects of riluzoleCompared with the control group, with the increased injection levels of MnCl2, Mn content increased; morphological and ultrastructural changes of striatum was aggravated; the cell apoptosis rates were increased; Glu levels increased and Gln levels decreased; activity of PAG elevated, activities of GS and Na+-K+-ATPase decreased; GS, GLAST and GLT-1 mRNA expression and protein levels decreased concentration-dependently. Compared with 200μmol/kg MnCl2 group, Mn content decreased; morphological and ultrastructural changes of striatum reduced; the cell apoptosis rates decreased; Glu levels decreased and Gln levels increased; activity of PAG reduced, activities of GS and Na+-K+-ATPase increased; GS, GLAST and GLT-1 mRNA expression and protein levels elevated concentration-dependently.2. MnCl2 induced the cytotoxicity on astrocytes and the protective effects of riluzoleThe cultured astrocytes would be used after more than 95% of the cells stained positively for GFAP immunocytochemically. The cytotoxicity of MnCl2 was determined by MTT assay and LDH release assay. It was found that with increasing of MnCl2 concentration (0～1000μmol/L) and time (6-48h), the cell viability decreased and LDH leakage increased concentration-dependently. After exposed to 500μmol/L MnCl2 for 24h, the cell viability rate decreased to 51.81%. It was suggested that 500μmol/L MnCl2 might be about 50% inhibitory concentrations (IC50). After prevented with 25-200μmol/L riluzole for 1～12h, the MTT assay and LDH leakage assay were taken. Compared with 500μmol/L MnCl2 group, the cell viability increased and LDH release decreased in 100 and 200μmol/L riluzole after prevention for 6h and 12h. However, there was no notable difference between the 100 and 200μmol/L riluzole group at the same prevented time. And there was no significant difference between 6h and 12h group at the same prevented concentration. After treated to 0,125,250, and 500μmol/L MnCl2 for 24h, the astrocyte morphological impairment increased; cell apoptosis rates elevated; G0/G1 phase cells increased, S phase cells decreased but G2/M phase cells did not reduced obviously. Compared with 500μmol/L MnCl2 group, the degree of morphological impairment decreased; cell apoptosis rates reduced; G0/G1 phase cells decreased, S phase cells increased however G2/M phase cells did not elevated obviously in the riluzole group.3. MnCl2 exposure caused the dysfunction of astrocytic Glu metablism and transport and the protective effects of riluzoleCompared with the control group, with the elevating concentration of MnCl2 exposure, the activities of GS and Na+-K+-ATPase, GS, GLAST and GLT-1 mRNA expression and protein levels decreased concentration-dependently. Compared with 500μmol/L MnCl2 group, the activities of GS and Na+-K+-ATPase, GS, GLAST and GLT-1 mRNA expression and protein levels increased obviously in the riluzole group. Conclusion1. MnCl2 caused the dysfunction of Glu metabolism and transport in the rat's striatum and riluzole could prevent it.2. MnCl2 induced cytotoxicity on astrocytes and riluzole might antagonize it.3. Riluzole might antagonize the dysfunction of astrocytic Glu metabolism and transport induced by Mn exposure.