Kb r7943

Drugs were made up in dimethyl sulphoxide (DMSO), ethanol, or water depending on solubility. Stock solutions were added to the drug delivery reservoirs containing Hanks solution to make up the final concentrations. Drugs used were as follows: KB‐R7943, Tocris (Bristol, UK); Mibefradil, Sigma (Wicklow, Ireland); Nifedipine, Bayer (Leverkusen, Germany); SEA0400 was synthesized by Taisyo Pharmaceutical Co., Ltd., Saitama, Japan. Mibefradil was water soluble, while SEA0400 and KB‐R7943 were dissolved in DMSO and diluted with Hanks solution to give a final DMSO concentration of 0.1% and 0.05%, respectively. Nifedipine was first dissolved in ethanol and diluted with Hanks solution to give a final ethanol concentration of 0.1%. Control experiments showed that these concentrations of vehicle had no significant effects on the responses measured in this study.
The cell under study was continuously superfused with Hanks solution by means of a close delivery system consisting of a pipette (tip diameter 200 μm) placed approximately 300 μm away. This could be switched, with a dead space time of around 5 sec, to a solution containing a drug. All experiments were carried out at 35–37°C.

TTX and KB-R7943 were obtained from Tocris Biosciences (UK) and culture medium from PromoCell (Germany). All other chemicals and compounds were purchased from Sigma-Aldrich (France). KB-R7943 was dissolved in DMSO, veratridine in 0.1N HCl and the remaining compounds in distilled water with further dilutions made from stock solutions with PSS.

KB-R7943 is an isothiourea derivative, quaternary ammonium-containing molecule, with a molecular weight 372, logP 3,83, logD from 1,41 to 1,52 (for pH from 1,7 to 8,0), HLB 16,31 pKa 10 (data from Chemaxone) (Fig. 1).Fig. 1

KB-R7943 [(2-{4-[(4-nitrophenyl)methoxy]phenyl}ethyl)sulfanyl]methanimidamide.

Fig. 1KB-R7943 was from Tocris Bioscience (Bristol, UK). STZ, citrate buffer, and amitriptyline were from Merck KGaA (Darmstadt, Germany), and OraPlus from Perrigo (Dublin, Ireland). The formalin test used a freshly prepared 0.5% formaldehyde solution in saline. amitriptyline (purity not less than 98%) was obtained from Merck (USA), аcetonitrile (ACN, HPLC grade) manufactured by ITW Group (USA), hexane was obtained from Cryochrom (Russia), formic acid produced by Sigma-Aldrich (USA), purified water was prepared in the laboratory with a Milli-Q system from Millipore (USA), ethylacetate (purity not less than 99,8%) produced by Vekton (Russia), reserpine (99%, HPLC grade) manufactured by “Sigma-Aldrich” (USA) was used as an internal standard.

Cardiotoxins were isolated from Naja oxiana cobra venom as described earlier [40 (link),41 (link)]. Ryanodine and KB-R7943 were from Tocris Bioscience (Bristol, UK). Nifedipine, 2-aminoethoxydiphenyl borate, inorganic salts, and glucose were obtained from Merck KGaA (Darmstadt, Germany). All other reagents obtained from local suppliers were of analytical grade or higher purity.

Human OA chondrocytes or C28I2 cells were seeded on 8-well chamber (Thermo Fisher, 154461) and loaded with 5 µM CoroNa Green (Invitrogen, C36676) or Fluo-8 (Abcam, ab112129) in Hanks’ Balanced Salt Solution for 45 min at 37 °C in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264 with or without 0.5 µM KB-R7943 (Tocris, 1244). CoroNa Green and Fluo-8 were excited at 488 nm and fluorescence images (525–530 nm) were acquired with 25x water-dipping objective on Zeiss 880 confocal microscope every 2 s during the experiment. Na⁺ and Ca²⁺ transients in chondrocytes were induced by 100 nM ATP in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264. Fluorescence was expressed as the ratio of cytosolic fluorescence and initial intensity (F/F₀).