Des arg9 bradykinin

Peptides were separated by reverse-phase high-performance liquid chromatography using an EASY-nLC system (Proxeon, now Thermo Fisher Scientific) with mobile phase A: 0.1% trifluoroacetic acid (TFA) in water; mobile phase B: 0.1% TFA in acetonitrile; flow rate of 300 nl/min. Lyophilised peptides were dissolved in 23 μl of buffer A, of which 18 μl were loaded onto the column (inner diameter = 75 μm; length = 15 cm; filled with ReproSil-Pur C18 AQ, 3 μm, 120 Å beads; Dr. Maisch GmbH). The peptides were eluted with a gradient of 5–33% B for 62 min, 33–48% B for 15 min, 48–100% B for 2 min and 100% B for 10 min. The column was then equilibrated with 5% B for 20 min before the next sample was analysed. Eluting fractions were mixed with a solution of 3 mg/ml α-cyano-4-hydroxycinnamic acid, 187.5 pmol/ml of each of the four internal standard peptides ([des-Arg⁹]-bradykinin, neurotensin, angiotensin I and adrenocorticotropic hormone fragment 1–17; all from Sigma), 0.1% TFA and 70% acetonitrile in water and deposed on a blank MALDI target plate (416 spots per sample) using an online SunCollect system (SunChrom). The final concentration of each internal peptide standard was 50 fmol per spot.

Cisplatin (cis-diamminedichloridoplatinum II, C-Platin^®; Blau, SP, Brazil), bradykinin (Bk; kinin B₂ receptor agonist), Icatibant (peptide kinin B₂ receptor antagonist), des-Arg⁹-bradykinin (DABk; kinin B₁ receptor agonist), and des-Arg⁹-[Leu⁸]-bradykinin (DALBK; peptide kinin B₁ receptor antagonist) were purchased from Sigma Chemical Company (St. Louis, MO, USA). FR173657 (non-peptide kinin B₂ receptor antagonist) and SSR240612 (non-peptide kinin B₁ receptor antagonist) were obtained from Sanofi-Aventis (Berlin, Germany). Antisense oligonucleotides targeting the kinin B₁ receptor (5′-AGG TTC CTG TGG ATG GCG TCCC-3′), kinin B₂ receptor (5′-AGA ATT CTG TTC ACT GTT TCT TCC CTG-3′), and nonsense oligonucleotides (5′-GGT GGA T TTG AGG ATT TCG GC-3′) were acquired from GenOne Biotechnologies (Rio de Janeiro, Brazil). Cisplatin and antagonists were prepared in a saline solution (0.9%). Phosphate-buffered saline (PBS; 10 mM) was used to dilute reagents administered via the intraplantar route (kinin B₁ and B₂ receptor agonists). The control groups (vehicles) received the vehicles where the treatments were solubilized. All the intraperitoneal treatments were administered in mice in a volume of 10 mL/kg, while intraplantar injections were administered in a volume of 20 µL.