Compound 1 specifically inhibits GPR4-mediated responses.

<p>GPR4-expressing CHO cells (A), GPR4-expressing COS7 cells (B), or TDAG8-expressing COS7 cells (C) were incubated for 30 min to measure cAMP accumulation under the indicated pH with or without 1 μM compound 1 (C1) and/or 1 μM forskolin (Fors). Results are expressed as means ± SD of three determinations of the representative experiment in (A) and expressed as means ± SEM of five determinations of three separate experiments in (B and C). The effect of compound 1 was significant (*<i>p</i> < 0.05). HUVECs infected with GPR4 adenovirus were incubated for 6 h in the presence of the indicated concentrations of compound 1 in (D and E) or in the presence of absence of 1 μM compound 1 in (F and G) at the indicated pH to measure mRNAs for VCAM-1 (D), ICAM-1 (E), CXCL2 (F), and IL-8 (G). The mRNA expression normalized to GAPDH was expressed as percentages of the value in the absence of compound 1 at pH 6.8. These values at pH 6.8 were 22.7 ± 6.6 for VCAM1 mRNA, 15.1 ± 4.1 for ICAM1 mRNA, 226 ± 20 for CXCL2 mRNA, 12.9 ± 0.9 for IL-8 mRNA (normalized to GAPDH x 10³). The results are means ± SEM of three to five separate experiments. The effect of compound 1 was significant (*<i>p</i> < 0.05). (H) AoSMCs harvested from 10-cm dish were prelabeled with fura-2/AM. The cells were first incubated with compound 1 (1 μM) and then further incubated under indicated pH with or without LPA (1 μM) and SPC (20 μM) to monitor [Ca²⁺]_i. The net [Ca²⁺]_i change (peak value-basal value) at around 15 s was calculated. Data are means ± SEM from three separate experiments.