H2r specific agonist dimaprit

The medium of cultured parietal cells was replaced with DMEM/F-12 medium containing 10 μM RO-201724 (Sigma-Aldrich Co.) prior to treatment with test materials. The parietal cells were pretreated with C. tricuspidata extracts or 10 μM of ranitidine hydrochloride for 5 min, followed by 10 μM of the H2R specific agonist dimaprit (Sigma-Aldrich Co.) for 30 min. After 30 min of dimaprit treatment, parietal cells were treated with cAMP assay cell lysis buffer (R&D Systems) and immediately frozen. The frozen cells were disrupted by repeatedly freezing and thawing three times, centrifuged at 800 g for 15 min, and cAMP production was measured in the supernatant. cAMP assays were performed using the Rat/Mouse cAMP assay kit (R&D Systems).

U937 cells were cultured in RPMI-1640 medium, centrifuged at 200 g, and suspended in RPMI-1640 medium containing 10 μM RO-20-1724 (Sigma, St. Louis, MO, USA). Cell suspensions were pretreated with C. tricuspidata extracts or 10 μM of ranitidine hydrochloride (Sigma) for 5 min and then treated with 10 μM of H₂R specific agonist dimaprit (Sigma) for 20 min. After 20 min of dimaprit treatment, U937 cells recovered by centrifugation (1,800 g) were treated with cAMP assay cell lysis buffer (R&D Systems, Minneapolis, MN, USA) and immediately frozen. The frozen cells were disrupted by repeatedly freezing and thawing three times, centrifuged at 2,000 g for 15 min, and cAMP production was measured in the supernatant. The cAMP assay was performed using a cAMP assay kit from R&D Systems.