Dopamine levels were quantitated by using an enzyme-linked immunosorbent assay kit from Rocky Mountain Diagnostics (Denver, CO), with the manufacturer’s protocols. peroxide and RNS. Exposure to methamphetamine also stimulated the release of dopamine from NG108-15 cells into the culture medium. AC927 attenuated methamphetamine-induced apoptosis, necrosis, ROS/RNS generation, and dopamine release in NG108-15 cells. Together, the data suggest that modulation of receptors can mitigate methamphetamine-induced cytotoxicity, ROS/RNS generation, and dopamine release in cultured cells. == Introduction == Methamphetamine (METH) is a highly abused psychostimulant drug IX 207-887 that results in neurotoxicity with high or repeated drug administration (Krasnova and Cadet, 2009). This neurotoxicity is seen as dopamine nerve terminal degeneration and apoptosis in several brain regions (Krasnova and Cadet, 2009), the mechanism of which is not completely understood. However, some of the neurotoxic cascades involved are excessive release of dopamine, IX 207-887 oxidative/nitrosative stress, endoplasmic reticulum (ER) stress, and activation of mitochondrial death cascades (Cadet and Brannock, 1998;Krasnova and Cadet, 2009). Among its various targets, IX 207-887 METH interacts with receptors at physiologically relevant micromolar concentrations (Nguyen et al., 2005;Han and Gu, 2006). Two subtypes of receptors, 1and 2, are expressed in almost all types of mammalian cells (Guitart et al., 2004). 1Receptors are highly conserved, 223-amino acid, membrane-bound proteins (Kekuda et al., 1996). They function as chaperones at the mitochondria-ER membrane and regulate Ca2+signaling via inositol trisphosphate receptors (Hayashi and Su, 2007). In addition, 1receptors can undergo protein-protein interactions with several other cellular proteins, such as binding immunoglobulin protein, ankyrin-B, inositol trisphosphate receptors, and dopamine D1receptors (Hayashi and Su, 2007;Navarro et al., 2010;Su et al., 2010). 1Receptors are also capable of translocation to different subcellular compartments upon stimulation through ligand binding and changes in intracellular conditions (Hayashi and Su, 2003). In contrast, 2receptors are less well characterized. However, their levels are elevated in immortalized cell lines and tumor cells (Vilner et al., 1995), where they have been implicated in the control of cell cycle functions (Wheeler et al., 2000), alterations in cellular Ca2+levels (Vilner and Bowen, 2000), and cell death signaling involving sphingolipid products (Crawford et al., 2002). Previous in vivo studies showed that pretreatment withN-phenethylpiperidine oxalate (AC927), a receptor ligand, attenuates the neurotoxic effects of METH in male Swiss-Webster mice (Matsumoto et al., 2008;Seminerio et al., 2011). The ability of AC927 to mitigate neurotoxic cascades is consistent with the location of receptors in various subcellular organelles, such as ER, mitochondria, lysosomes, cell membranes, and nuclei (McCann et al., 1994;Zeng et al., 2007), as well as the functional roles of receptors in cells, as described above. The role of receptors in neurotoxicity resulting from drugs of abuse such as METH remains unclear because of various limitations, including the lack of a suitable in vitro model system. Previous IX 207-887 studies on the mechanisms of METH-induced toxicity used either primary neuronal cultures or immortalized cell lines of neuronal origin (Cadet et al., 1997;Wu et al., 2007). Millimolar concentrations of METH were required to induce cell death/damage, but such concentrations are rarely achieved in vivo (Melega et al., 2008). Therefore, a model system suitable for identification of potential mechanisms leading to neurotoxicity is needed. To elucidate the cellular mechanisms of METH-induced neurotoxicity at which AC927 may intervene via receptors, a neuronal/glial cell culture model (neuroblastoma glioma hybridoma; NG108-15 cells) was used in the present study. When they are differentiated, NG108-15 cells display neurite outgrowth and electrical excitability and express proteins with neuronal and glial properties (Ma et al., 1998). These cells have been used extensively as a sensitive cell line for studies of the toxic effects of various drugs and chemicals (Caete and Diogne, 2008). In addition, NG108-15 cells have commonly been used to study the functions of receptors (Hayashi and Su, 2003). In the current study, the endpoints Rabbit polyclonal to GAPDH.Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) is well known as one of the key enzymes involved in glycolysis. GAPDH is constitutively abundant expressed in almost cell types at high levels, therefore antibodies against GAPDH are useful as loading controls for Western Blotting. Some pathology factors, such as hypoxia and diabetes, increased or decreased GAPDH expression in certain cell types measured were reactive oxygen species (ROS) IX 207-887 and reactive nitrogen species (RNS) generation, dopamine release, apoptosis, and necrosis. Finally, AC927 was tested against these cytotoxic endpoints and mediators for insights into the mechanisms of receptor-mediated neuroprotection. == Materials and Methods == == == == Drugs and Chemicals. == Phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin solution, hypoxanthine/aminopterin/thymidine (HAT), trypsin/EDTA (0.05% trypsin, 0.43 mM EDTA), 5-(and 6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), dithiothreitol, and TRIzol lysis reagent.