Fig. 8.

Histamine dose-dependently potentiates the efficacy of PAMs on mGlu₄-mediated calcium mobilization in cells co-expressing mGlu₄ and H₁ receptors. Results of traces in Fig. 7 were plotted in concentration-response curve format. Increasing concentrations of the mGlu₄ PAMs PHCCC, 4PAM-2, ADX88178 or VU0155041 (A-D, respectively) were applied either alone (■) or together with 100 nM (▲) or 300 nM (●) histamine in the first add. After 150 s, a 1 µM glutamate concentration was applied in the second add. Calcium responses were measured as the fluorescence ratio, which involves dividing all fluorescence data for each point in the kinetic trace by the fluorescence value obtained in the first baseline sample read, which corrects for differences in dye loading and cell plating. Data were further normalized by taking the maximum calcium response minus the minimum response measured 3 s prior to either the first or second addition. For PHCCC (A), 4PAM-2 (B), and ADX88178 (C) responses, the effect on the second addition (“Glutamate Add”) window is shown. For VU0155041 (D), the effect on the first addition (“Compound Add”) is shown. Potencies for the different conditions were: PHCCC alone, no fit, PHCCC + 100 nM histamine, 8.2 ± 5.1 µM, PHCCC + 300 nM histamine, 7.6 ± 1.4 µM; 4PAM-2 alone, no fit, 4PAM-2 + 100 nM histamine, 54.2 ± 29.0 nM, 4PAM-2 + 300 nM histamine, 40.8 ± 10.0 nM; ADX88178 alone, no fit, ADX88178 + 100 nM histamine, 37.2 ± 15.1 nM, ADX88178 + 300 nM histamine, 30.0 ± 5.2 nM; VU0155041 alone, no fit, VU0155041 + 100 nM histamine, 9.5 ± 3.9 µM, VU0155041 + 300 nM histamine, 6.5 ± 1.6 µM. Maximal responses in the absence or presence of 100 nM or 300 nM histamine were: for PHCCC, 0.016 ± 0.003, 0.089 ± 0.010, 0.259 ± 0.018 (*p < 0.0001; One-way ANOVA); for 4PAM-2, 0.017 ± 0.002, 0.212 ± 0.013, 0.460 ± 0.022 (*p < 0.0001; One-way ANOVA); for ADX88178, 0.016 ± 0.002, 0.197 ± 0.003, 0.438 ± 0.020 (*p < 0.0001; One-way ANOVA); and for VU0155041, 0.018 ± 0.003, 0.221 ± 0.032, 0.864 ± 0.043 (*p < 0.0001; One-way ANOVA). Data shown were performed in triplicate; Mean ± SEM. Statistical analysis was performed using GraphPad Prism (La Jolla, CA).