Figure 5.

Effects of HA antagonists/inverse agonists and HA agonists on the [Ca²⁺]i response to ACh/HA, ACh or A23187/HA (age of culture > 1 year). Fluorescence enhancement was measured 10 s after addition of 200 µl medium containing ACh or A23187 (final concentrations as described below) with or without HA, methimepip, 4-methylhistamine or JNJ-7777120 (all added to final concentrations of 10 µM if not indicated otherwise). For preincubations with HA antagonists/inverse agonists the indicated substances or medium alone were added in 50 µl volume 2 min before taking the first time point for determination of baseline fluorescence, e.g. 10 s before addition of ACh or ACh/HA. In this case mepyramine, BF2.649, JNJ-7777120 were added to final concentrations of 1 µM and ranitidine to a final concentration of 10 µM. Statistical significance compared to the treatment group indicated on top of the bar was calculated by univariate analysis with the Mann-Whitney U-test. Mean values and standard deviations were calculated from at least 4 experiments. (A) Left panel: Inhibition of the [Ca²⁺]i response to ACh/HA (final concentrations 1 and 10 µM, respectively) by preincubation with HA receptor antagonists/inverse agonists (6 experiments), middle panel: effect of a higher concentration (10 µM) of JNJ-7777120 simultaneously added with ACh/HA (concentrations as in left panel, 6 experiments), right panel: inverse agonist effects of preincubation with HA receptor antagonists/inverse agonists on the [Ca²⁺]i response evoked by ACh (final concentration 2 µM, 8 experiments). The inset calculated with IBM SPSS Statistics shows the frequency distribution of the magnitude of [Ca²⁺]i response to treatment with ACh with and without preincubation with JNJ-7777120 derived from the same data. (B) Left panel: effect of methimepip and 4-methylhistamine on the [Ca²⁺]i response evoked by ACh/HA (final concentrations 1 and 10 µM, respectively, 4 experiments), middle panel: effect of methimepip and 4-methylhistamine on the [Ca²⁺]i response to ACh (final concentration 1 µM, 6 experiments), right panel: effect of methimepip on the [Ca²⁺]i response to ACh (final concentration 2 µM, 9 experiments) and A23187/HA (final concentrations 0.1 and 10 µM, respectively, 6 experiments). ^1,2 p = 0.035 (¹) and p = 0.049 (²) when calculated from ranked data by the unpaired two-tailed t-test assuming unequal variances.