Fig. 5.

Effects of tyrosyl derivatives on P2X₇ receptor-activation in hP2X₇-HEK cells. The adherent cells were preincubated with antagonists for 15 min prior to stimulation for 10 min with 3 mM ATP (final concentration). K⁺ content in these nitric acid extracts was assayed by atomic absorbance spectrophotometry. Duplicate or triplicate wells were run for all test conditions in each separate experiment. A: hP2X₇-HEK cells were preincubated with or without 3 µM 1 prior to stimulation with 3 mM ATP. Data points represent the mean (± SD) K⁺ content from nine separate experiments. B: hP2X₇-HEK cells were preincubated with or without the indicated concentrations of selected antagonists prior to stimulation with 3 mM ATP. Data points represent the mean (± SD) K⁺ contents from triplicate wells in a single experiment. The dashed horizontal lines illustrate the mean K⁺ content in control cells incubated in the absence of antagonist or ATP. The dotted horizontal lines illustrate the mean K⁺ content in ATP-stimulated cells that were not preincubated with antagonist. IC₅₀ values are rough estimates from visual inspection of the concentration-response relationships. Hill coefficients were not determined, since previous studies have shown that the lead compound (KN-62,1) represses P2X₇ receptor function via complex mechanisms that are not readily amenable to standard ligand-binding analyses.