Measurement of concentration-response relationships by concentration-ramp application of agonists

Dose-response relationships of agonist- induced membrane currents were obtained during accumulation of agonist and a simultaneously applied dye in the culture dish. The concentration of the dye was continuously measured using a laboratory-made photometer. When the superfusate was switched from the control solution to a test solution containing an agonist and an inert dye (e.g. Fast Green) both accumulated in the bath together and the change of dye concentration altered the intensity of light falling through the objective. The change in light intensity in an area directly surrounding the cell under study was measured by the photometer. The normal microscope lamp was used as the light source and a colour filter was placed between the microscope lamp and the condenser in order to enhance the signal-to-noise ratio of the photometer output. Assuming that the rates of accumulation and clearance of dye are similar to those of the agonist, it is possible to continuously measure the membrane current at given dye and agonist concentrations. To calculate the dose-response curves we calibrated the relationship between the dye concentration and the photometer voltage output.