2.1.3. Drug administration
A u-tube system (Fig 1) was used for the rapid application of capsaicin. The u-tube was placed close to a group of cells of interest, and this was attached to a gravity-feed perfusion system through microbore Teflon PTFE tubing (O.D. 0.042”, I.D. 0.022” (Cole Palmer). The tubing had previously been coated with a siliconising agent (sigmacote, Sigma-Aldrich), and the application of capsaicin was controlled by a 3-way solenoid valve (The Lee Company, England) connected to pulse generator unit (High Med, England). The drug application lasted 10s for all experiments involving capsaicin.
capsaicin was applied twice with a 10 min rest period, at the second application the magnitude of the calcium responses was diminished compared to the response obtained following the first capsaicin application (mean ± sem) response ratio which is described as the (F510 self-ratio) of the second response as a percentage of the first response (F510 self-ratio).
The different chemical reagents used to inhibit the sensitisation effect of bradykinin on capsaicin cells were started 10–30 min before the challenge with the first capsaicin and continued until the experiment done. Each chemical was dissolved in the appropriate solvent at the desired concentration, according to the manufacturer’s recommendations. All reagents were diluted to the desired concentration in a normal Ringer’s solution (pH 7.4) before the experiments (table1).
Table 1: The concentrations and solvents applied to investigate their role in capsaicin sensitization by bradykinin.