Role of the guanine nucleotide binding protein, Gαo, in the development of morphine tolerance and dependence

Abstract

Rationale: The use of morphine and other opioids for chronic pain is limited by the development of analgesic tolerance and physical dependence. Morphine produces its effects by activating the μ opioid receptor, which couples to Gα_i/o-containing heterotrimeric G proteins. Evidence suggests that the antinociceptive effects of morphine are mediated by Gα_o. However, the role of Gα_o in the development of morphine tolerance and dependence is unknown.

Objective: The objective of the study is to evaluate the contribution of Gα_o to the development of morphine tolerance and dependence in mice.

Methods: 129S6 mice lacking one copy of the Gα_o gene (Gα_o +/-) were administered morphine acutely or chronically. Mice were examined for tolerance to the antinociceptive action of morphine using the 52 °C hot plate as the nociceptive stimulus and for dependence by evaluating the severity of naltrexone-precipitated withdrawal. Wild-type littermates of the Gα_o +/- mice were used as controls. Changes in μ receptor number and function were determined in midbrain and hindbrain homogenates using radioligand binding and μ agonist-stimulated [³⁵S]GTPγS binding, respectively.

Results: Following either acute or chronic morphine treatment, all mice developed antinociceptive tolerance and physical dependence, regardless of genotype. With chronic morphine treatment, Gα_o +/- mice developed tolerance faster and displayed more severe naltrexone-precipitated withdrawal in some behaviors than did wild-type littermates. Morphine tolerance was not associated with changes in μ receptor number or function in brain homogenates from either wild-type or Gα_o +/- mice.

Conclusions: These data suggest that the guanine nucleotide binding protein Gα_o offers some protection against the development of morphine tolerance and dependence.

Keywords: Antinociception; G protein; Morphine; Receptor binding; Tolerance; Withdrawal; μ receptor.

Figure 1

Acute morphine tolerance and dependence. (a) Antinociception was measured in the 52°C hot plate test 30 min following a challenge injection of morphine (10 mg/kg) in wild type and Gα_o +/− mice that were treated with either saline (n=6–7) or 128 mg/kg morphine (n=10) 6 h previously. **p<0.01 compared with saline-treated mice of the same genotype by Bonferroni’s post-test. (b) naltrexone (10 mg/kg)-precipitated withdrawal in wild type (n=6) and Gα_o +/− mice (n=7) that were treated with 128 mg/kg morphine and challenged with 10 mg/kg morphine 6 h later. Withdrawal behaviors were scored for a period of 30 min (see Materials and methods). **p<0.01 compared with wild type by unpaired t-test. All data are plotted as the mean ± SEM.

Figure 2

Chronic morphine tolerance. Morphine-mediated antinociception in (a) wild type and (b) Gα_o +/− mice treated twice-daily with saline, 56 mg/kg morphine or 128 mg/kg morphine. Morphine antinociception was evaluated in the 52°C hot plate test using a cumulative dosing procedure in all mice on Day 1 and again in the same mice on Day 8 following chronic administration of saline, 56 mg/kg morphine or 128 mg/kg morphine. See Table 1 for numbers of subjects per group and statistical analysis. Time course of morphine-mediated antinociception in (c) wild type and (d) Gα_o +/− mice treated twice-daily with saline, 56 mg/kg morphine or 128 mg/kg morphine. Antinociception was measured as above, 30 min following each morning injection of morphine. *p<0.05, **p<0.01, ***p<0.001 compared with antinociception on Day 2 within the same treatment group by Bonferroni’s post-test. All data are plotted as the mean ± SEM.

Figure 3

Chronic morphine dependence. Naltrexone (10 mg/kg)-precipitated withdrawal in wild type (n=9–10) and Gα_o +/− mice (n=9) that were treated twice-daily with either 56 mg/kg morphine or 128 mg/kg morphine. Withdrawal behaviors were scored for a period of 30 min (See Materials and methods). *p<0.05 compared with wild type mice that received the same chronic treatment by Bonferroni’s post-test. All data are plotted as the mean ± SEM.

Figure 4

µ-agonist-stimulated G protein activity. Whole midbrain and hindbrain homogenates were prepared from (a) wild type (n=3–4) or (b) Gα_o +/− mice (n=3–4) following twice-daily treatment with saline or 128 mg/kg morphine, and membranes prepared from these sections were evaluated for 10 µM DAMGO- or 10 µM morphine-stimulated [³⁵S]GTPγS incorporation. Agonist-stimulated [³⁵S]GTPγS binding was calculated by subtracting basal binding from binding that occurred in the presence of agonist. Basal [³⁵S]GTPγS binding was as follows (fmol/mg protein): wild type midbrain (saline, 56 ± 6; morphine, 60 ± 3); wild type hindbrain (saline, 42 ± 2; morphine, 45 ± 2); Gα_o +/− midbrain (saline, 38 ± 6; morphine, 38 ± 1); Gα_o +/−hindbrain (saline, 35 ± 4; morphine, 34 ± 1). All data are plotted as the mean ± SEM.