4.2. Biological Assay

The protocol of the biological assay was according to the previous report [16]. The membrane preparations of Chinese hamster ovary (CHO) cells, which expressed recombinant human histamine H₃ or H₄ receptors, were purchased from Euroscreen (Brussels, Belgium). The binding assay of the H₃ and H₄ receptors was performed using [³H]N^α-methylhistamine (Perkin-Elmer, Boston, MA) and [³H]histamine (Perkin-Elmer), respectively. Briefly, the membrane preparations (7.5–15 µg protein) were incubated with different concentrations of [³H]N^α-methylhistamine (0.1–3 nM) and of [³H]histamine(1–30 nM) for 30 min at 25 °C in 50 mM Tris/5 mM MgCl₂ buffer (pH 7.4). The reaction was terminated by rapid filtration (Cell Harvester, Brandel Co., Gaithersburg, MD) through Whatman GF/B glass fiber filters presoaked for 2 h in 0.5% polyethyleneimine, and the filters were rinsed three times with an ice-cold buffer (2 mL). Membrane-bound radioactivity was extracted from filters overnight in scintillation fluid (toluene, 2 L; Triton X-100, 1 L; 2,5-diphenyloxazole, 15 g; 1,4-bis[2-(5-phenyloxazolyl)]benzene, 0.3 g) and determined in a liquid scintillation counter. The specific binding of each radioligand was determined experimentally from the difference between counts in the presence of 10 µM thioperamide. The apparent dissociation constants (K_d) for each radioligand was determined by nonlinear regression analysis of the curve generated by plotting specific binding concentration versus concentration of the radioligand with GraphPad Prism (GraphPad Software, San Diego, CA) using a one-site binding curve equation. The ability of each compound to inhibit the specific binding of [³H]N^α-methylhistamine (1.5 nM) and [³H]histamine (25 nM) was estimated by IC₅₀ values, which are the molar concentrations of unlabeled drugs necessary for displacing 50% of specific binding (estimated by log probit analysis). The inhibition constant, K_i, was calculated from the equation, K_i = IC₅₀/(1 + L/K_d), where L equals the concentration of each radioligand. The data were presented as mean ± SE (n = 3–5).