At 12 h post-transfection, the culture media were replaced with reduced inositol Media 99 (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 1% FCS and 0.5 μCi/well [
3H]myo-inositol (PerkinElmer, Waltham, MA, USA). Cells were then incubated for a further 12 h at 37 °C before replacing the media with Buffer I (DMEM supplemented with 20 mM HEPES, 10 mM LiCl (to inhibit IP breakdown) and 0.1% bovine serum albumin) and incubating for 30 min at 37 °C. Media were then aspirated, and cells were incubated with ligands prepared in Buffer I.
For the measurement of receptor activation, cells were incubated for 1 h at 37 °C in the presence of vehicle (0.1% DMSO), a range of concentrations of DOTA-[Thi
8,Met(O
2)
11]SP or native ligands for dose–response analyses. For single-concentration analyses, cells were incubated with 1 µM [NK1R, NK2R, NK3R and empty vector) or 10 µM [MRGPRX2 and empty vector] DOTA-[Thi
8, Met(O
2)
11]-SP or native ligands. For the measurement of receptor antagonism, cells were incubated for 30 min at 37 °C in the presence of vehicle (0.1% DMSO), 1 µM DOTA-[Thi
8,Met(O
2)
11]SP or 1 µM talnetant ((S)-N-(1-phenylpropyl)-3-hydroxy-2-phenylquinoline-4-carboxamide, also known as SB-223412; a non-peptide antagonist of the NK3R [49 (
link)]) prior to stimulation for 1 h at 37 °C with native ligands at close to their EC
50 concentration (
Table 1; MRGPRX2, 10 µM SP; NK2R, 10 nM NKA and NK3R, 30 nM NKB).
Following ligand incubation, cells were lysed using incubation in 10 mM formic acid for 60 min at 4 °C. Lysates were transferred to tubes containing 100–200 mesh Dowex 1X8 resin (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 5 min. The resin was washed twice with water and twice with wash buffer (60 mM ammonium formate, 5 mM sodium tetraborate) before elution of the radiolabelled inositol phosphates from the beads in 1 mL elution buffer (1 M ammonium formate/0.1 M formic acid). A total of 1.5 mL scintillation fluid (OptiPhase HiSafe3) (PerkinElmer, Waltham, MA, USA) was then added and radioactivity (decays per minute; dpm) was determined with liquid scintillation counting using a Packard Tri-Carb 4810TR Liquid Scintillation Analyser (PerkinElmer, Waltham, MA, USA).
Suthiram J., Pieters A., Mohamed Moosa Z., Zeevaart J.R., Sathekge M.M., Ebenhan T., Anderson R.C, & Newton C.L. (2023). Tachykinin Receptor-Selectivity of the Potential Glioblastoma-Targeted Therapy, DOTA-[Thi8,Met(O2)11]-Substance P. International Journal of Molecular Sciences, 24(3), 2134.
