Damgo

The following reagents were used: DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt) (Sigma-Aldrich, Saint Louis, MO, USA); morphine hydrochloride (Takeda Pharmaceutical, Tokyo, Japan); pertussis toxin (PTX), an inhibitor of the G protein (Gi, Go and Gt) heterotrimer interaction with receptors (Tocris, Bio-Techne Japan, Tokyo, Japan); Actinomycin D (Act D), a transcriptional inhibitor (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); U0126, a potent, selective inhibitor of MEK1 and 2 (Tocris, Bio-Techne Japan, Tokyo, Japan); and Pyridone 6, a potent pan-JAK inhibitor (Tocris, Bio-Techne Japan, Tokyo, Japan). Morphine was dissolved in saline for animal injection. For in vitro experiments, DAMGO, morphine and PTX were diluted with H₂O, while the other reagents were diluted with dimethyl sulfoxide.

The following chemicals were used in this study: [D-Ala2]-Deltorphin II (Sigma T0675), [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO, Sigma E7384), CTOP (Sigma P5296), Naloxone (Sigma N7758), CYM51010 (Sigma SML0980), SNC80 (Tocris Cat. No. 0764), Fluorogold (Thermo Fisher Scientific, H22845), Lucifer Yellow CH (Thermo Fisher Scientific, L453), Cholera Toxin Subunit B (Recombinant) Alexa Fluor 555 Conjugate (Thermo Fisher Scientific, C34776). For inducing DOR internalization, SNC80 (10 mg/kg) was injected subcutaneously to lightly restrained unanesthetized mice using a 30 G needle attached to a microsyringe inserted through the back skin. For behavioral experiments, deltorphin II (1 μg), CTOP (100 ng) or a vehicle solution (0.9% sodium chloride, Hospira NDC 0409-4888-10) were injected intrathecally. A 30 G needle attached to a microsyringe was inserted between the L4/L5 vertebrae, puncturing through the dura (confirmed by the presence of a reflexive tail flick), and then 5 μl of drug was injected.

We used the HTRF^® Phospho-ERK kit (Catalog number 64ERKPEG) recently launched by Cisbio Bioassays. Chinese hamster ovary (CHO) cells stably expressing the different GPCRs were generated by Euroscreen (Gosselies, Belgium), except the CHO–muscarinic M1 receptor (M1) cell line that was kindly provided by Dr. Denis Servent (CEA–DSV, Saclay, France). NIH-3T3, A431, HEK293, HeLa, and SKOV3 cell lines were from American Type Culture Collection (Manassas, VA, USA). HEK293FT cells were from Life Technologies (Burwood, VIC, Australia). All the plasmids coding for the GPCRs used in Figure 4 were generated and/or purchased by Kevin D. G. Pfleger’s laboratory from Missouri S&T cDNA Resource Center¹ and AT1R and NMU2R plasmids were gifts from Prof. Walter Thomas (University of Queensland, Australia) and Dr. Gary B. Willars (University of Leicester, UK), respectively. All the agonists (EGF, MCP-1, MIP1β, SDF1α, DAMGO, Endorphin-2, Norepinephrine, Isoproterenol, ACEA, PGE2, ITAC, FTY720, VIP, arginine-vasopressin (AVP), carbachol, Ang II, and human Neuromedin U-25), the antagonist CTOP and Pertussis toxin were from Sigma. The anti-Phospho-ERK1/2 antibody used for western blot was purchased from Cell Signaling Technology.

For the in vitro assays, NP-5497-KA, nalfurafine hydrochloride, the MOP-selective agonist DAMGO, the KOP-selective agonist U-69,593 (Sigma Aldrich, St. Louis, MO, USA), and the DOP agonist SNC80 (Toronto Research Chemicals, Toronto, ON, Canada) were dissolved in dimethylsulfoxide. NP-5497-KA and nalfurafine were synthesized by Nippon Chemiphar according to patent-related information.