AC927, a σ receptor ligand, blocks methamphetamine-induced release of dopamine and generation of reactive oxygen species in NG108-15 cells

Abstract

Methamphetamine is a highly addictive psychostimulant drug of abuse that causes neurotoxicity with high or repeated dosing. Earlier studies demonstrated the ability of the selective σ receptor ligand N-phenethylpiperidine oxalate (AC927) to attenuate the neurotoxic effects of methamphetamine in vivo. However, the precise mechanisms through which AC927 conveys its protective effects remain to be determined. With the use of differentiated NG108-15 cells as a model system, the effects of methamphetamine on neurotoxic endpoints and mediators such as apoptosis, necrosis, generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and dopamine release were examined in the absence and presence of AC927. Methamphetamine at physiologically relevant micromolar concentrations caused apoptosis in NG108-15 cells. At higher concentrations of methamphetamine, necrotic cell death was observed. At earlier time points, methamphetamine caused ROS/RNS generation, which was detected with the fluorigenic substrate 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescin diacetate, acetyl ester, in a concentration- and time-dependent manner. N-Acetylcysteine, catalase, and l-N(G)-monomethyl arginine citrate inhibited the ROS/RNS fluorescence signal induced by methamphetamine, which suggests the formation of hydrogen 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 σ receptors can mitigate methamphetamine-induced cytotoxicity, ROS/RNS generation, and dopamine release in cultured cells.

Fig. 1.

Expression of σ₁ receptors and TH in NG108-15 cells. A, representative histograms from six independent experiments. NG108-15 cells were fixed, permeabilized, stained, and analyzed as described under Materials and Methods. Histograms are as follows: control chicken IgY (dashed black), chicken anti-σ₁ receptor (solid black), control rabbit IgG (dashed gray), and rabbit anti-TH (solid gray) antibodies. FL1-H (x-axis), fluorescence channel height; counts (y-axis), number of events detected for the corresponding fluorescence channel. B, MFI values for all four stains from six separate experiments plotted against the staining reagent. Ch, chicken; Rab, rabbit.

Fig. 2.

ROS induced in NG108-15 cells after exposure to METH or AC927. The production of ROS capable of oxidizing CM-H₂DCFDA was conducted as described under Materials and Methods, in NG108-15 cells after exposure to 100 μM H₂O₂,100 μM Na₂Cr₂O₇ (Cr), or 0 to 300 μM METH (A) or 0 to 300 μM AC927 (B). Relative fluorescence units from five (A) or two (B) separate experiments for each indicated time point are shown; data were normalized to the mean relative fluorescence unit values for untreated cells to obtain percentage of control values (n = 8 per experiment; mean ± S.E.M.). *, p < 0.05; **, p < 0.01; ***, p < 0.001, versus control treatment.

Fig. 3.

Inhibition of ROS production in NG108-15 cells by NAC, catalase, and l-NMMA. Inhibition of ROS after exposure to 0 to 3 μM METH or 100 μM H₂O₂ was determined in the absence or presence of 10 mM NAC (A), 10,000 units catalase (B), or 20 mM l-NMMA (C), 20 min after exposure to METH. Data represent the average ± S.E.M. of relative fluorescence units from two separate experiments (n = 4 per experiment). *, p < 0.05; **, p < 0.01, control versus METH-treated; #, p < 0.05; ###, p < 0.001, METH alone versus METH with ROS inhibitor.

Fig. 4.

Inhibition of METH-induced ROS generation in NG108-15 cells by AC927. The production of ROS capable of oxidizing CM-H₂DCFDA was assessed in differentiated NG108-15 cells in the presence of 0 to 300 μM METH, 100 μM H₂O₂, or 100 μM Na₂Cr₂O₇ (Cr), in the absence or presence of 0.3 μM AC927, as described under Materials and Methods. Data represent the average ± S.E.M. of relative fluorescence units from two different experiments (n = 8 per experiment), obtained 20 min after the addition of drug. **, p < 0.01, control versus METH-treated; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, METH alone versus METH with AC927.

Fig. 5.

Release of dopamine from NG108-15 cells in response to METH. Values for supernatants harvested from NG108-15 cells cultured in the presence or absence of METH and/or AC927 (x-axis) and quantitated as described under Materials and Methods are represented as picograms of dopamine per 1.5 × 10⁵ cells in 500 μl of medium. Data represent the mean ± S.E.M. from four (METH or KCl alone) or three (METH with AC927) independent experiments. *, p < 0.05; **, p < 0.01, control versus METH- or KCl-treated; #, p < 0.05; ###, p < 0.001, METH alone versus METH with AC927.

Fig. 6.

AC927 protection against METH-induced apoptosis (A) and necrosis (B). NG108-15 cells were pretreated with AC927 (0–30 μM) before exposure to METH (0–1 mM) for 24 h. After 24 h, the wells were incubated with Hoechst 33342 and propidium iodide stains (20 μg/ml each), for determination of proportions of apoptotic and necrotic cells. Data represent the mean ± S.E.M. from two separate experiments (n = 3 per experiment). *, p < 0.05; **, p < 0.01, control versus METH-treated; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, METH alone versus METH with AC927.