Enhancement of morphine-induced antinociception against mechanical nociceptive stimuli in σ 1 -receptor knockout mice

C Sánchez-Fernández1, JM Entrena2, R González-Cano1, L Romero1, D Zamanillo3, JM Baeyens1. 1University of Granada, Department of Pharmacology (18012), Biomedical Research Center(18071) and Institute of Neurosciences (18012)., Spain, 2University of Granada, Scientific Instrumentation Center, Spain, 3Laboratorios Esteve, Drug Discovery and Preclinical Development, 08032, Spain

Antecedents

Modulation of morphine-induced antinociception against heat-induced pain by σ1-receptor ligands is well-documented. The subcutaneous (s.c.) administration of (+)-pentazocine, a selective σ1-receptor agonist, antagonized morphine antinociception in the tail-flick test. In addition, the σ1 antagonist haloperidol not only reversed the effect of (+)-pentazocine, but also increased morphine-induced antinociception. However, it is unknown whether the antinociceptive effect of morphine is modified in σ1-receptor knockout (σ1-KO) mice. It is also unknown whether the potentiation of morphine antinociception produced by σ1-receptor antagonists happens when mechanical (instead of thermal) nociceptive stimuli are applied. To test it is interesting because morphine produces differential effects against nociceptive mechanical and thermal stimuli in several studies.

Methods

Experiments were performed in CD-1 wild-type (WT) and σ1-KO mice weighing 25-30 g. Non-punctate nociceptive mechanical stimuli (450 g pressure) were applied alternatively to both hind-paws with a rounded tip cone-shaped paw-presser Analgesimeter (Ugo Basile, Italia) until the mouse showed a struggling behaviour or 50 s had passed (cut-off time). Morphine was administered 30 minutes before evaluation and the σ1-receptor antagonists, BD-1063 (32 mg/kg), BD-1047 (32 mg/kg), NE-100 (4 mg/kg) and S1RA (32 mg/kg), were s.c. injected 5 min before morphine. The σ1-receptor agonist PRE-084 (4-16 mg/kg) was used to reverse the effects of σ1-receptor antagonists, it was s.c. injected immediately before the σ1 antagonists. The density of µ-opioid receptors was measured with [3H]DAMGO (0.25-45 nM) saturation binding assays in brain and spinal cord synaptosomes both in WT and σ1-KO mice.

Results

Morphine (0.5-16 mg/kg, s.c.) produced a dose-dependent antinociception (in both hind-paws) with a higher potency and efficacy in σ1-KO than in WT mice, yielding ED50 (mg/kg) values of 2.16 ± 0.01, and 13.89 ± 0.85 respectively. This was not due to changes in µ-opioid receptors in σ1-KO mice, since the KD of [3H]DAMGO binding were similar in WT and σ1-KO mice (2.02 ± 0.33 vs 1.88 ± 0.26 nM, respectively). The antinociceptive effect of morphine (4 mg/kg, s.c.) was potentiated by all the σ1-receptor antagonists evaluated in WT mice (struggle response latencies were: BD-1063 42.89 ± 2.70, BD-1047 37.94 ± 1.72, NE-100 35.39 ± 2.56 and S1RA 32.11 ± 2.4 s). Statistically significant differences between the values obtained in the groups treated with each σ1 antagonist and their solvent. Moreover, this potentiation was dose-dependently reversed by PRE-084. In contrast, none of the σ1-receptor antagonists modified morphine-induced antinociception in σ1-KO mice. Statistical analysis was carried out using one and two-way ANOVA followed by the Bonferroni test, depending on the experiment. The differences between values were considered to be significant when the value of p was below 0.05.

Conclusion

These results suggest that genetic inactivation and pharmacological antagonism of σ1 receptors increase the antinociceptive effect of morphine against non-punctuate mechanical stimuli.

Supported by Junta-Andalucía (CTS-109), MEC (SAF2006-06122) and MEC-FPU (CSF & RGC).