br Materials and methods Manganese chloride MnCl H O

Materials and methods Manganese chloride (MnCl2·4H2O), glutamate, glutamine, Lucifer Yellow, polyclonal anti-GFAP and MTT assay kits were purchased from Sigma Chemical Co. (St. Louis, MO). Dulbecco's Modified Eagle's Medium-F12 (DMEM-F12) with Earle's salts, foetal bovine serum, penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA). Trizol reagent was purchased from Qiagen(Qiagen, German). PrimeScript 1st strand cDNA Synthesis Kit and SYBR Premix Dimer Eraser were obtained from TaKaRa (TaKaRa Bio, Japan). Monoclonal anti-Connexin43 antibody and monoclonal anti-GAPDH antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). IRDye-conjugated goat anti-rabbit secondary antibody and IRDye-conjugated goat anti-mouse secondary antibody were purchased from LI-COR Biosciences. The kits of Annexin V-FITC & PI Apoptosis Detection were bought from Roche Corporation.
Results
Discussion In this study, we evaluated the toxic effects of excessive Mn by measuring cell viability and cell apoptosis. We found that Mn exposure induced astrocyte viability reduction and increased apoptosis in a time- and dose-dependent manner. Our results are similar to other studies (Stephenson et al., 2013; Zhao et al., 2012). Meanwhile, elevated manganese exposure significantly disrupted intracellular/extracellular glutamate homeostasis, accompanied by gap junctional intercellular communication inhibition and decreased irak1 australia of gap junction’s major protein, Cx43. Astrocytes play a pivotal role in balancing the glutamate-glutamine cycle. However, the exact nature of glutamate excitotoxicity induced by manganese is elusive. The present study showed that excessive manganese exposure disrupted both the intracellular and extracellular glutamate balance, with more susceptibility of intracellular glutamate content. In vivo and in vitro studies have discovered that manganese exposure disrupts the glutamate-glutamine balance (Burton et al., 2009; Milatovic et al., 2007; Lee et al., 2009). Considering the complexity of glutamate metabolism, any disruption of the glutamate metabolic pathway might imbalance intracellular and extracellular glutamate levels. It has been reported that Mn down regulated the activity of glia-specific GS and increased the PAG enzyme in the CNS (Sidoryk-Wegrzynowicz et al., 2009; Yu et al., 2012), thus damaging glutamate and its metabolites. A more serious adverse sequence of glutamate accumulation was that GSH, a ubiquitous antioxidant formed from Glu, was significantly decreased followed by high levels of Mn exposure (Park and Park, 2010). Additionally, several astrocytic glutamate transporters, including GLT-1 and GLAST, were found to play a crucial role in maintaining normal glutamate levels. Multiple lines of evidence have indicated that Mn caused glutamate transporter expression on astrocyte membranes in vitro and in vivo (Karki et al., 2014; Sidoryk-Wegrzynowicz et al., 2009; Erikson et al., 2008). However, Wang et al. proposed that the main reason for glutamate-dependent neuronal death is not an over-activation of NMDARs but rather the expression of neuronal gap junctions (Wang et al., 2012). There are two types of channels existing on the astrocytic membranes: gap junctions and hemi-channels. Both of these channels are responsible for second signalling molecules, such as glutamate and ATP exchange. Up to now, more than 20 subfamilies of channel formed proteins are confirmed in astrocytes. Death-survival factors are secreted to the extracellular space or spread to neighbouring cells through gap junctions and hemi-channels, which are thus termed as bystander effectors upon cell death. It was demonstrated that gap junctions were one of the major determinants governing the susceptibility of renal tubular cells and enhanced gap junctions could increase cell viability against aminoglycoside stimulation (Yao et al., 2010). The function of gap junctions was determined by Connexin43 (Cx43), which is the most highly expressed gap junction protein on astrocyte membranes and has been linked to programmed cell death. Cells transfected with Cx43 have also been capable of transducing survival signals in response to extracellular cues (Plotkin et al., 2002). We currently found that manganese induced astrocyte apoptosis with gap junction inhibition, as well as decreased Cx43 expression, which might be unable to dilute excessive glutamate to remote astrocytes via gap junctions. A contradictory study demonstrated that Cx43 hemi-channel blockers prevented NMDA neurotoxicity induced by pro-inflammatory cytokine mixtures (Froger et al., 2010). The protection might be related to the potential role of hemi-channels formed by Cx43 proteins that govern survival factor exchange across the intracellular and the extracellular space (Decrock et al., 2009). It seems that the types of channels formed by Cx43 are relative to the functions. To our knowledge, this is the first time to report the involvement of gap junction intercellular communication in glutamate excitotoxicity induced by high levels of manganese exposure. Figiel et al. (2007) reported that reducing gap junctions/connexins in astrocytes suppressed transcriptional activity of GLT-1 promoters (Figiel et al., 2007). The precise mechanisms underlying gap junction/connexins governing glutamate levels upon manganese exposure require further investigation.