Evidence for kappa- and mu-opioid receptor expression in C6 glioma cells

Abstract

The astrocytoma cell line rat C6 glioma has been used as a model system to study the mechanism of various opioid actions. Nevertheless, the type of opioid receptor(s) involved has not been established. Here we demonstrate the presence of high-affinity U69,593, endomorphin-1, morphine, and beta-endorphin binding in desipramine (DMI)-treated C6 cell membranes by performing homologous and heterologous binding assays with [3H]U69,593, [3H]morphine, or 125I-beta-endorphin. Naive C6 cell membranes displayed U69,593 but neither endomorphin-1, morphine, nor beta-endorphin binding. Cross-linking of 125I-beta-endorphin to C6 membranes gave labeled bands characteristic of opioid receptors. Moreover, RT-PCR analysis of opioid receptor expression in control and DMI-treated C6 cells indicate that both kappa- and mu-opioid receptors are expressed. There does not appear to be a significant difference in the level of mu nor kappa receptor expression in naive versus C6 cells treated with DMI over a 20-h period. Collectively, the data indicate that kappa- and mu-opioid receptors are present in C6 glioma cells.

FIG. 1

Homologous competition binding of ¹²⁵I-β-endorphin to DMI-treated C6 cell membranes. Binding was performed in 50 mM Tris buffer supplemented with 0.1% bacitracin and 0.2% bovine serum albumin. ¹²⁵I-β-Endorphin (2 nM) binding was assayed against 0-1 μM unlabeled ligand. Data are mean ± SEM values of three experiments.

FIG. 2

Cross-linking of ¹²⁵I-β-endorphin to DMI-treated and control C6 cell membranes. Representative autoradiograph (± DMI) and densitometric analysis of DMI-treated C6 membranes are shown. Specific densities were calculated as the difference between total and nonspecific binding. Background measurements were determined for each lane and were subtracted from values for both total and nonspecific binding. Densities are expressed as a fraction of 100%. Lanes: T, total binding; NS, nonspecific binding. Data are mean ± SEM values of three experiments.

FIG. 3

Homologous and heterologous competition binding of [³H]morphine to DMI-treated C6 cell membranes. A: Homologous binding with morphine sulfate. Heterologous competition with endomorphin-1 (A), U69,593 (B), naltrindole (C), and norbinaltorphimine (Nor-BNI) (D). Binding was performed on the P₂₀ membranes of 20 μM DMI-treated C6 cells. Specific binding was estimated with 1 nM [³H]morphine. Data are mean ± SEM values of three to eight experiments.

FIG. 4

Homologous competition binding of [³H]U69,593 to DMI-treated C6 cell membranes. Binding was performed on the membrane preparations of control and 20 μM DMI-treated C6 cells. Specific binding was estimated with 1 nM [³H]U69,593. Nonspecific binding was determined in the presence of 10 μM U69,593. Data are mean ± SEM values of seven to 10 experiments.

FIG. 5

RT-PCR analysis of RNA derived from C6 cells. Opioid receptor specific primers were used to amplify RT cDNA generated from total RNA isolated from the indicated cell sources. Southern blotting was performed by using radiolabeled oligonucleotides specific for μ- or κ-opioid receptor-amplified sequences, respectively. A: μ-Opioid receptor-specific primers and probe identifying amplified cDNA originating from C6 cells (C6), C6 cells + DMI (DMI), adult rat brain, C6 cells transfected with μ-opioid receptor (C6μ), and CHO cells transfected with μ-opioid receptor (CHOμ). B: κ-Opioid receptor-specific primers and probe identifying amplified cDNA originating from C6 cells, C6 cells + DMI, adult rat brain, and CHO cells transfected with κ-opioid receptor (CHOκ). Blots shown are representative of the experiments performed four to seven times.

FIG. 6

Semiquantification of opioid receptor mRNA expression after DMI treatment. Expression levels (at a number of thermocycles determined to fall within the linear range for both GAPDH and opioid receptor cDNA amplification) are shown as a function of time of DMI treatment. Product (5 μl) from each PCR reaction (RTs from C6 and DMI-treated C6) was taken over a range of 15-35 thermocycles and analyzed via agarose electrophoresis, followed by Southern blotting using radiolabeled probes specific for GAPDH, and the μ- or κ-opioid receptor cDNA sequence amplified. Each blot was then analyzed with the phosphorimaging program ImageQuant. Normalization of opioid receptor expression is given as the density of the receptor band divided by the density of the GAPDH band. A: Normalized κ expression after 0, 0.5, 1, 1.5, 2, and 20 h of DMI treatment. Shown is the comparison at 24 cycles, a point within the linear part of the amplification curve. B: Normalized μ expression after 0, 0.5, 1, 1.5, 2, and 20 h of DMI treatment. Shown is the comparison at 24 cycles, a point within the linear amplification curve. This experiment was repeated in triplicate, using different preparations of control and treated C6 cells. Data represent the average ± SEM values of three experiments.