Beta-arrestins have been suggested to regulate μ-, δ-, and κ-opioid receptor-mediated responses. In the present study, we examined the effects of pretreatment with β-arrestin-2 antibody on tail-flick inhibition induced by opioid receptor agonists in the mouse spinal cord. Intrathecal (i.t.) pretreatment with β-arrestin-2 antibody potentiated the antinociception induced by i.t.-administered μ-opioid receptor agonists [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO) and endomorphin-1, but not endomorphin-2, the δ-opioid receptor agonist [D-Ala2]deltorphin II or the κ-opioid receptor agonist U50,488H. The present result suggests that β-arrestin-2 may tonically down-regulate a selected population of μ-opioid receptors activated by endomorphin-1 or DAMGO in the mouse spinal cord.

The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of μ-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated μ-opioid peptide endomorphins can activate G proteins through μ-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS). An autoradiographic [35S]GTPγS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 μM increased [35S]GTPγS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6 ± 3.8 and 72.3 ± 4.0%, respectively, at 10 μM. In contrast, the synthetic selective μ-opioid receptor agonist [D-Ala2,NHPhe4,Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6 ± 5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [35S]GTPγS binding was verified by coincubating membranes with endomorphins in the presence of specific μ-, δ- or κ-opioid receptor antagonists. Coincubation with selective μ-opioid receptor antagonists β- funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2 (CTOP) blocked both endomorphin-1 and-2-stimulated [35S]GTPγS binding. In contrast, neither δ- nor κ-opioid receptor antagonist had any effect on the [35S]GTPγS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [35S]GTPγS binding by selectively stimulating μ-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for μ-opioid receptors in the mouse PAG.