Involvement of Endothelin in Morphine Tolerance in Neuroblastoma (SH-SY5Y) Cells

Drugs and Chemicals.

Morphine sulfate (Mallinckrodt Chemical Co., St. Louis, MO) was dissolved in distilled deionized pyrogen-free water. BMS182874 (Tocris Pharmaceuticals Inc., Ellisville, MO) was dissolved in 20% dimethylsulfoxide prepared in sterile saline. ET-1 (American Peptide Company Inc., Sunnyvale, CA) was dissolved in 0.1% bovine serum albumin. [³⁵S]GTPγS (specific activity, 1000 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) was diluted in 50 mM Tris buffer, pH 7.4. Guanosine-5′-diphosphate (GDP) and unlabeled GTPγS (Sigma, St. Louis, MO) were dissolved in 50 mM Tris buffer, pH 7.4.

Estimation of [³⁵S]GTPγS Binding in SH-SY5Y Neuroblastoma Cells.

Human neuroblastoma SH-SY5Y cells (American Type Culture Collection, Manassas, VA) were used for this study because this cell line expresses both opioid and ET_A receptors. This is a prototypical model system frequently used to study cellular aspects of opioid tolerance (11). Cells were cultured in monolayers in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin, and maintained at 37°C in a humidified 5% CO₂ atmosphere. For acute treatment, separate cell plates were treated with different concentrations of morphine, ET-1, BMS182874, and morphine plus BMS182874 for 1 hr. For chronic treatment, cells were treated for 48 hrs with morphine, ET-1, BMS182874, or morphine plus BMS182874. Cells were harvested after drug treatments in ice-cold phosphate-buffered saline containing 1 mM EDTA and dispersed using a glass homogenizer. After centrifugation at 500 g, the cell pellet was suspended in 50 mM Tris-HCl buffer, pH 7.4; 100 mM NaCl; and 10 mM MgCl₂·H₂O (Buffer A) and homogenized again using a glass homogenizer. The resultant homogenate was centrifuged at 20,000 g, and the pellet was collected, washed in Buffer A, and centrifuged again. The pellet was finally resuspended in Buffer A, to give a protein concentration of 100–200 μg/ml, and the membrane preparation was prepared as described previously (12). All procedures were performed in triplicate. The final pellet was resuspended to a protein concentration of approximately 50–75 μg. Membranes (50–75 μg protein) were incubated in Buffer A containing 80 pM [³⁵S]GTPγS, 30 μM GDP, and varying concentrations of ET-1 and BMS182874, in a volume of 1 ml for 60 mins at 30°C. Nonspecific binding was determined using unlabeled 10 μM GTPγS. Bound and free [³⁵S]GTPγS were separated by vacuum filtration using GF/B filters and quantified by liquid scintillation counting. Specific binding (determined by subtracting nonspecific binding from total binding) was expressed as femtomoles per milligram of protein (mean ± SEM). The concentration of protein in the samples was determined by Lowry’s method (13).

Statistical Analysis.

Data was analyzed by one-way analysis of variance followed by a Bonferroni test. A level of P < 0.05 was considered significant.

Stimulation of [³⁵S]GTPγS Binding After Acute Treatment of Neuroblastoma Cells with Morphine, ET-1, and BMS182874.

Morphine increased [³⁵S]GTPγS binding in SH-SY5Y cells in a concentration-dependent manner. The median effective concentration (EC₅₀) values for the different treatments with morphine indicate that the curve shifted to the left with increasing concentrations (Fig. 1a). The EC₅₀ values of morphine for GTP binding after acute treatment with 100 nM, 1 μM, and 10 μM morphine were 426.67 nM, 186.61 nM, and 106.45 nM, respectively, indicating that acute treatment with morphine may increase the sensitivity of the opioid receptors and produce a higher G-protein activation. ET-1–induced GTP stimulation also increased in a concentration-dependent manner (Fig. 1b). The EC₅₀ values for acute treatment with ET-1 did not differ significantly (P > 0.05) between treatments (126.98 nM with 100 nM ET-1; and 95.47 nM with 1 μM ET-1). These findings indicate that ET-1 did not cause sensitization of ET receptors. The EC₅₀ values after acute treatment with increasing concentrations of BMS182874 (1 nM, 100 nM, and 1 μM) were greater than 1000 nM. This indicates that BMS182874 did not activate GTP binding in SH-SY5Y cells.

Maximal stimulation after combined acute treatment with 1 μM morphine plus 100 nM BMS182874 (76.55 ± 1.08%) was similar (P > 0.05) to control-treated cells (79.07 ± 2.86%; Fig. 1c). The EC₅₀ value was significantly lower in morphine plus BMS182874–treated cells (73.95 nM), compared with control treatment (416.81 nM). Activation of G-proteins was observed even with lower concentrations of morphine. Therefore, BMS182874 enhanced the morphine-induced activation of G-proteins in SH-SY5Y cells.