Candesartan

Ligand binding was analyzed using total membranes prepared from COS-1 cells transiently expressing HA-AT₁R (wild type), ΔBRIL-AT₁R (crystallized construct without BRIL), and BRIL-AT₁R (crystallized construct) constructs. Single mutants were constructed by a PCR-based site-directed mutagenesis strategy as previously described (Unal et al., 2010 (link)). Protein concentration was determined by Bio-Rad Protein Assay (Bio-Rad). For both saturation and competition binding assays, 10 µg of homogenous cell membrane was used per well.
Saturation binding assays with ³H-candesartan were performed under equilibrium conditions, with ³H-candesartan (Amersham Pharmacia Biotech) concentrations ranging between 0.125 and 12 nM (specific activity, 16 Ci/mmol) as duplicates in 96-well plates for 1h at room temperature. Nonspecific binding was measured in the presence of 10 µM candesartan (gift from AstraZeneca). The binding kinetics was analyzed by nonlinear curve-fitting program GraphPad Prism 5, which yields the mean ± S.D. for the K_d and B_max values.
Competition binding assays were performed under equilibrium conditions, with 2 nM ³H-candesartan and various concentrations of the ZD7155 ranging between 0.04 and 1000 nM. The binding kinetics was analyzed by nonlinear curve-fitting program GraphPad Prism 5, which yields the mean ± S.D. for the IC₅₀ values.

[¹²⁵I]-Labelled AngII was obtained from ProSearch. HEK293, Chinese hamster ovary (CHO-K1), NIH-3T3, cells were obtained from American Type Culture Collection. HEK293FT were obtained by Thermo Fisher Scientific. HEK-adherent-293 cell line was a generous gift from Dr Dominic Ng, University of Queensland, Australia. AngII was obtained from Auspep or Sigma Aldrich and EGF from R&D Systems or Peprotech. Inhibitors used were YM-254890 (Wako Pure Chemical Industries), Candesartan (AstraZeneca), AG1478 (Merck Millipore) with PP2 and BAPTA obtained from Sigma Aldrich.

Male Wistar rats (290–300 g; Charles River Laboratories, Wilmington, MA) were used according to procedures approved by the Institutional Animal Care and Use Committee (IACUC) of the Charlie Norwood VA Medical Center. The rats were quarantined for at least 5 days before the experiment. The animals were housed in individual cages in a room maintained at 21–25 °C, 45–50 % humidity, and 12-h light/dark cycle with free access to pellet chow and water.
The animals were separated into three groups: group I, sham-operated saline-treated control (S); group II, MCAO and saline-treated stroke (MCAO); and group III, MCAO and candesartan (0.3 mg/kg) (MCAO + cand). Consistent with previous research, candesartan (Astra-Zeneca) was dissolved in saline and given in a dose of 0.3 mg/kg by intravenous injection 90-min postocclusion to ensure rapid delivery following injury. Additional injections of 0.3 mg/kg were administered intraperitoneally every 24 h continuing daily for up to 7 days after MCAO. In this 14-day survival study, we used 90-min MCAO to reduce the mortality. A diagram of the experimental design is shown in Fig. 1. A total of 38 rats were used in the present study; one rat each in the saline and candesartan groups died at 24 h and 3-day post-MCAO, respectively.