3h 1 3 di o tolylguanidine 3h dtg

Brain and liver homogenates for S1R and S2R receptor binding assays were prepared from male Dunkin-Hartley guinea pigs and Sprague Dawley rats, respectively (ENVIGO RMS S.R.L., Udine, Italy; Italian Minister of Health, authorization for animal experimentation—Project acronym 335/1984F.N.JLT). [³H](+)-pentazocine (26.9 Ci/mmol) and [³H]1,3-di-o-tolylguanidine ([³H]DTG, 35.5 Ci/mmol) were purchased from PerkinElmer (Zaventem, Belgium). Ultima Gold MV Scintillation cocktail was from PerkinElmer (Milan, Italy). All the other materials were obtained from Merck Life Science S.r.l. (Milan, Italy). UV absorbance was measured using a microplate spectrophotometer reader (Synergy HT, Biotec). The bound radioactivity has been determined using a Beckman LS 6500 liquid scintillation counter (Beckman Coulter, Brea, CA, USA).

[³H] (+)-Pentazocine (26.9 Ci/mmol) and [³H]1,3-di-o-tolylguanidine ([³H]DTG, 35.5 Ci/mmol) were purchased from PerkinElmer (Zaventem, Belgium). Ultima Gold MV Scintillation cocktail was from PerkinElmer (Milan, Italy). All the other materials were obtained from Merck Life Science S.r.l. (Milan, Italy). The test compound solutions were prepared by dissolving approximately 10 µmol of the test compound in DMSO so that a 10 mM stock solution was obtained. The required test concentrations for the assay (from 10⁻⁵ to 10⁻¹¹ M) have been prepared by diluting the DMSO stock solution with the respective assay buffer. All experiments were performed using ultrapure water obtained with a Millipore Milli-Q Reference Ultrapure Water Purification System. All the laboratory glassware was first washed with a 6 M HCl water solution and then rinsed with ultrapure water.