The Roles Of DMT1and Calcium Channels In Lead Absorption In Astrocytes And Choroid Plexus Epithelial Cells

Background:Lead (Pb) is one of the well-known environmental heavy metal pollutants which canbe taken up by ways of air, dust, water, soil and food chains. Currently, no physiologicalfunctions of Pb were found in the human body. Lead can enter the human body throughthe respiratory tract, gastrointestinal tract, and skin into the blood, and then re-distributedto the blood circulation through various organizations, damages multiple organs andsystems, especially, the central nervous system and lead to growth retardation, mentaldecline, unresponsive, hyperactivity, attention deficit disorder and other symptoms. Themechanisms of Pb-induced neurotoxicity have not been fully understood. The transport,distribution, and the tissue affinity of Pb in the brain affect the toxicity of Pb. Therefore,the transport and distribution of Pb is an important basis and a clue for the investigationof Pb neurotoxicity. The knowledge about Pb transport and distribution is poorly known. Previous studiessuggested that divalent metal transporter1(DMT1), Ca2+channels, endocytosis, andanion exchange may be involved in Pb transport. However, the way of Pb transport indifferent organs and cell types, and under different ways of exposure, are much different.Thus, to investigate the mechanism of Pb transport in different neural cells, indentify keymolecules, and explore possible preventive measures, are big issues for preventivemedicine.Aims:In this study, SD rats, rat glioma cell line C6, Z310rat choroid plexus epithelial cellswere employed to investigate the roles of DMT1and calcium channel in Pb transport inastrocytes and choroid plexus epithelial cells. And to provide basis for clarifying themechanisms of Pb transport and distribution in the brain.Methods1. Establishment of chronic Pb exposure rat model. PND21SD male rats weighing65±15g were randomly divided into control and Pb exposure groups. Pb exposure groupwere given100ppm lead acetate in drinking water freely for8weeks, and meantime thecontrol group were given100ppm sodium acetate.2. Establishment of Pb exposure cell model. C6glioma cells, immortalized rat choroidplexus epithelial cells Z310cells and primary cultured rat astrocytes were exployed inthis study. The dose of Pb exposure is5μmol/L.3. The Pb levels in rat hippocampus, choroid plexus and C6cells, Z310cells weredetermined by atomic absorption spectrophotometry. Immunofluorescence was used todetect the expression level of DMT1in hippocampus, choroid plexus, C6cells, andZ310cells.4. Western blot, Real-time RT-PCR were used to detect the expression level of DMT1inC6and Z310cell.5. Laser scanning confocal microscopy was used to investigate the impact of leadexposure on calcium ion uptake by Fluo-3/AM calcium fluorescent probe labeling. Results:1. The Pb levels in blood, CSF, and brain tissue were significantly increased following Pbexposure.2. Pb effected the DMT1’s expression:(1) Immunofluorescence showed that DMT1expressed in both hippocampus and choroid plexus. DMT1’s expression level was notaffected by low-level Pb exposure, and the DMT1’s level in Z310cells is much higherthan that in C6cells;(2) The results from Western blot and RT-PCR showed that theprotein and mRNA levels of DMT1were not changed by Pb.3. The DMT1acted on both astrocyte and choroid plexus in the Pb absorption:(1) Theresults from atomic absorption spectrophotometry showed that the Pb uptake dropped inboth C6and Z310cells following DMT1siRNA knockdown, especially in Z310cells.(2) The Pb uptake increased in both C6and Z310cells under Fe diffcient condition. In aFe-overload status, the Pb uptake decreased in Z310cells but not in C6cells. When Feand Pb were added to the cells at the same time, the cellular Pb uptake significantlydecreased in both cell lines.4. The results from laser scanning confocal microscopy showed that:(1) calciumfluorescence intensity in C6cells increased following the addition of Bay K8644.Pretreatment with Pb did not affect the Bay K8644-induced increase of intracellularfluorescence; however, the time of fluorescence fading was shotened.(2)When Ca andPb co-exist in the cell culture medium, the Ca uptake was significantly inhibited, andwith a rightward shift of the curve.(3)The impact of Bay K8644on Z310cells wasmuch weaker than that of the C6cells. Pretreatment with Pb not resulted in a slightlyincreased in Ca uptake.(4)Co-existence of Ca and Pb did not significantly affectted thecalcium fluorescence intensity in Z310cells.5. Calcium channel acted on both astrocyte and choroid plexus in the Pb absorption:(1)Bay K8644affected the Pb uptake in C6, but not Z310cells.(2) Nimodipine affectedthe Pb uptake in C6, but not Z310cells.(3) Pretreatment of CaCl2has little effect on thecellular Pb uptake in C6and Z310cells. When10mmol/L CaCl2and Pb co-exist in thecell culture medium, the cellular Pb uptake increased in both cell lines. Conclution:1. The contribution of DMT1and Ca2+channels in C6and Z310cells are not the same.Ca2+channels play a major role in Pb uptake in C6cells, while the Pb uptake in Z310cells is mainly done by DMT1.2. Fe supplementation reduced the cellular uptake of Pb, especially when Fe and Pbco-exist in the cell culture medium, the cellular Pb uptake significantly decreased. Themechanism may be related to cellular iron levels by negative feedback effects of DMT1expression levels, as well as competitive inhibition between iron and lead ions relevant.3. The addition of calcium in the culture medim had little effect on the Pb absorption inboth cell lines. And the co-existence of calcium and Pb ions did not show anycompetitive inhibition in the cellular Pb absorption.