Pentazocine

(+/-)-Vesamicol, (+)-pentazocine and DTG were purchased from Sigma-Aldrich Co. (St. Louis, MO). Radioisotopes were purchased from PerkinElmer, Inc. (Waltham, MA), unless otherwise noted.
The HPLC column (Chiralpak IA, 9.6 mm × 250 mm) was purchased from DAICEL Co. (Osaka, Japan). The reverse phase HPLC column (Zorbax-ODS RX-18, 9.6 mm × 250 mm) was purchased from Agilent Technologies Inc. (Santa Clara, CA). [¹²⁵I]NaI was purchased from PerkinElmer Inc. (Waltham, MA)). [¹²³I]NaI was supplied by Nihon Medi-Physics Co (Nishinomiya Japan). Specific rotation was obtained on a Nippon Bunko DIP-181 digital polarimeter. Sprague-Dawley (SD) rats were purchased from Sankyo Labo Service Co. (Tokyo, Japan). Animal experiments were performed in compliance with the Guidelines for the Care and Use of Laboratory Animals at the Takara-machi Campus of Kanazawa University. The animal experimental protocols used were approved by the Committee on Animal Experimentation of Kanazawa University (Permit Number: AP-153454). In animal studies, the animals were sacrificed by decapitation under ether anesthesia. The structures of the two OIDV optical isomers were determined by X-ray crystallographic analysis (APEX II ULTRA, Bruker AXS K. K. Japan) performed by Bruker AXS K. K. company (Fig 1) (S1 and S2 Files).

Reagents and antibodies were obtained from the following sources: anti-OPRS1 (Sig1R) antibody (Abcam, UK), anti-IP3 receptor antibody (Abcam, UK), Anti-GRP78 (BiP) antibody (Abcam, UK), anti-mitofusin 2 antibody (Abcam, UK), anti-Rac1 antibody (Santa Cruz, USA), anti-Bcl 2 antibody (Santa Cruz, USA), GppNHp (Sigma-Aldrich, USA), GDP (Sigma-Aldrich, USA), pentazocine (Sigma-Aldrich, USA), haloperidol (Sigma-Aldrich, USA).

Drug-induced BRET is conducted as optimized previously (i.e., transfection and experimental conditions) (Urizar et al., 2011 (link); Yano et al., 2018 (link)). Briefly, cells were prepared in 96-well plates. 5 μM coelenterazine h was added to each well. Three minutes after addition of coelenterazine h (Nanolight), ligands [(+)-pentazocine (Sigma), PD 144418 (Tocris), and haloperidol (Tocris)] were added to each well in serial dilution. BRET was measured as a ratio between measurements at 535 nm for fluorescence (gain 2500, interval time 0.9) and at 485 nm for luminescence (gain 2500, interval time 0.9) using a PHERastar FSX reader (BMG). Results are calculated for the BRET change (BRET ratio for the corresponding drug minus BRET ratio in the absence of the drug). E_max values are expressed as the basal subtracted BRET change in the dose-response graphs.

The standard drugs such as aspirin, pentazocine, and indomethacin were purchased from Sigma-Aldrich (Bengaluru branch, Karnataka, India). All other chemicals like carrageenan, carboxymethyl cellulose, and so forth were obtained from Himedia (Mumbai, Maharashtra, India) and of the highest purity and analytical grade.

(+)-Pentazocine, haloperidol and buffer components (Tris-HCl, inhibitors), 1,3-di(2-tolyl)guanidine were purchased from Sigma-Aldrich Kft. (Budapest, Hungary). The radioligand [³H]-(+)-Pentazocine (s.a., 1.98 TBq/mmol) and [³H]-DTG (s.a., 363 Gbq/mmol) were prepared in the Laboratory of Chemical Biology (BRC, Hungary). Tritium labeling was carried out in a self-designed vacuum manifold [70 (link)] and radioactivity was measured with a Packard Tri-Carb 2100 TR liquid scintillation analyzer using Insta Gel scintillation cocktail of PerkinElmer. Drugs were dissolved at 1 mM in dimethyl sulfoxide (DMSO) and were stored at −20 °C, and then diluted in the binding buffer.
The sigma receptor ligands in Table 3 were synthesized with the common methods of the preparative organic chemistry; details of the syntheses and the characterization of the compounds are summarized in Supplementary Information.

CM compounds were synthesized in the McCurdy Lab (University of Mississippi, University, MS). [³H]-(+)-1,3-ditolyl guanidine (DTG) and [³H]-progesterone were obtained from PerkinElmer, (Waltham, MA). Nonradioactive DTG, haloperidol, (+)-pentazocine and progesterone were obtained from Sigma-Aldrich (St. Louis, MO). Rabbit anti-PGRMC1 antibody was purchased from Proteintech (Chicago, IL; Cat. No. 12990-1-AP). PGRMC1 cDNA was obtained from Origene (Rockville, MD). All other reagents were from Sigma-Aldrich or Thermo-Fisher unless specifically stated.