Coelenterazine 400a

HEK293T cells (4 × 10⁴) were plated in each well
of 96-well white-walled, clear-bottom polystyrene microplates coated
with poly-d-lysine. The following day, cells were transfected
with WT or variant OXTR-GFP10 and β-arrestin-1-Rluc8 or β-arrestin-2-Rluc8
at a ratio of 15:1 (w/w). For transfections, 50 ng of DNA and 0.5
μL of Lipofectamine 2000 reagent (Thermo Fisher Scientific),
both diluted in Opti-MEM reduced-serum media, were added to each well.
After 24 h, media was removed and replaced with 100 μL of Hank’s
buffered salt solution (HBSS) supplemented with 20 mM HEPES. A Synergy2
plate reader was used to add 100 μL of assay buffer containing
10 μM coelenterazine 400a (Biotium, Fremont, CA) and the indicated
concentrations of oxytocin to 10 wells at a time. Luminescence at
520 and 400 nm was read every 26 s for a total of 182 s. The BRET
ratio was calculated as the average ratios of emission at 520 nm/400
nm at the five time points from 78 to 182 s. WT controls were tested
on each plate in parallel with variants.

Standard opioids were purchased from Cedarlane (Burlington, Canada) and Sigma-Aldrich (St. Louis, MO, USA). Fifteen novel compounds were provided by Pfizer Inc. (Worldwide Research and Development). (−)-Isoproterenol hydrochloride, (−)-norepinephrine, DL-propranolol hydrochloride, ( ± ) metoprolol ( + )-tartrate salt, carvedilol, and salmeterol xinafoate were purchased from Sigma-Aldrich (St Louis, MO). ICI 118,551 and salbutamol hemisulfate were purchased from Tocris Bioscience (Ellisville, MO). Coelenterazine 400a was purchased from Biotium.

HEK293T cells transiently expressing the AT₁ receptor and RlucII-βarr1-GFP₁₀ or RlucII-βarr2-GFP₁₀ were washed once with PBS, detached and seeded in 96-well white plates (OptiPlate; PerkinElmer, Waltham, MA, USA). BRET was monitored in a Victor™ X Light Luminescence microplate reader (PerkinElmer) equipped with different donor/acceptor emission filter sets, after addition of 5 μM coelenterazine-400A (Biotium, Hayward, CA, USA) to the cells. BRET signals were derived from the emission detected with the energy acceptor filter (515 ± 15 nm) divided by the emission detected using the energy donor filter (410 ± 40 nm). Five independent experiments with full concentration-response curves and time-course assays were performed in cells stimulated with AngII, SII or TRV027. Concentrations used for time-course assays were AngII 100 nM, SII 30 μM, and TRV027 100 nM. Agonist concentrations were chosen based on their differences in affinities, where SII has lower affinity as compared to AngII and TRV027.

G protein activation was evaluated in HEK293T cells transiently expressing H₃R or H₄R and a BRET-based biosensor composed of Gα_i fused to RLucII (in this case, G_i1-RLucII) and GFP10-Gγ2, in the presence of the untagged Gβ₁ subunit (Galés et al., 2005 (link)). Cells were transfected in suspension, seeded in 96-well white plates (OptiPlate; PerkinElmer) and grown for 48 h. BRET values were monitored using Victor^TM X Light Luminescence microplate reader (PerkinElmer) equipped with BRET400-GFP2/10 filter set (acceptor, 515 ± 20 nm; and donor, 400 ± 70 nm filters), 5 min after the addition of 2.5 μM of coelenterazine 400-a (Biotium, Hayward, CA, United States) and the analyzed ligands. In the antagonism assays, the different ligands were incubated 30 min prior to stimulation with histamine and addition of coelenterazine, according to the procedure described earlier.

HEK293T cells transiently expressing the AT1R (or AT₁R-GFP) and BRET-based biosensors (Gαq-RLucII or Gαi3-RLucII, Gβ1 and Gγ1-GFP₁₀; RLucII-β-arr1-GFP₁₀; RLucII-β-arr2-GFP₁₀; β-arr1-RLucII; β-arr2-RLucII) were washed once with PBS, detached, and seeded in 96-well white plates (OptiPlate – PerkinElmer, Waltham, MA, USA). Following agonist stimulation, cells were incubated at 37 °C and the luciferase substrate (coelenterazine H for BRET1 or coelenterazine 400a for BRET2 – Biotium, Hayward, CA, USA) was added 5 min before reading BRET in a Victor^TM X Light Luminescence microplate reader (PerkinElmer) equipped with different donor/acceptor emission filter sets. The BRET signal was determined as the ratio of light emitted by fluorescently-labeled biosensors, detected with energy acceptor filters (530 ± 20 nm for BRET1 or 515 ± 20 nm for BRET2), and light emitted by RLucII-tagged biosensors, detected with energy donor filters (480 ± 20 nm for BRET^{1 (link)} or 410 ± 40 nm for BRET2). The specific BRET signal was defined as the difference between the total BRET signals and the one obtained with RLucII alone. Three independent experiments with full concentration-response curves were performed in cells stimulated with AngII or Ang-(1-7).