Differential thermodynamic driving force of first- and second-generation antihistamines to determine their binding affinity for human H1 receptors

Differential Regulation of Thermodynamic Binding Forces of Levocetirizine and (S)-Cetirizine by Lys191 in Human Histamine H1 Receptors

“…3.0 (Applied Biosystems, Tokyo, Japan). The cells expressing HA-WT and HA-K191A were cultured in 150 cm 2 culture flasks, as described previously [18]. The dissociated cells were homogenized with Polytron PT-10 (Kinematica, Lucerne, Switzerland) in an ice-cold lysis buffer (Tris, 1 mM; EDTA, 2 mM; pH 7.4 at 37 °C).…”

“…The receptor binding assay with [ 3 H]mepyramine, a radioligand for H 1 receptors, was performed in accordance with the methods described previously [18], as follows: Aliquots (0.1 mL) of membrane preparations (approximately 50 µg of membrane proteins) were incubated with 3 nM [ 3 H]mepyramine in the presence or absence of various concentrations of levocetirizine or ( S )-cetirizine (displacement experiments) for 3 h at 37 °C, 24 h at 25 °C, and 3 days at 14 °C in a normal HEPES buffer (final volume 1 mL). The actual concentration of [ 3 H]mepyramine present was determined by the analysis of an aliquot of the [ 3 H]mepyramine/HEPES medium.…”

“…Thermodynamic analyses are useful methods for the evaluation of the electrostatic and hydrophobic binding forces of ligands to determine their binding affinity for receptors [15,16,17,18]. Binding enthalpy (∆ H º) is usually associated with electrostatic binding forces via the formation of new bonds between receptors and ligands, such as hydrogen bonds and van der Waals interactions, whereas binding entropy (∆ S º) is usually characterized as hydrophobic binding forces via the displacement of ordered water molecules coupled to the formation of new hydrophobic interactions.…”

C‐terminal of human histamine H1receptors regulates their agonist‐induced clathrin‐mediated internalization and G‐protein signaling

“…) and the regulatory mechanisms underlying its binding affinity for ligands are currently being explored (Hishinuma et al . ). However, the regulatory mechanisms responsible for the intracellular trafficking of H 1 receptors have not been examined in as much detail.…”

Ca2+‐dependent down‐regulation of human histamine H1receptors in Chinese hamster ovary cells

“…In experiments to evaluate the direct effects of Ca 2+ on the [ 3 H]mepyramine binding to H 1 receptors, membranes were prepared from ionomycin‐non‐treated cells and incubated with 0.1–10 nM [ 3 H]mepyramine in the presence or absence of 10 μM mepyramine in 25 mM HEPES buffer simply containing either 0.2 mM EGTA (control) or 1.8 mM CaCl 2 according to the methods described previously (Hishinuma et al . ). The cell suspension or the membrane preparation was filtered through Whatman GF/B glass fiber filters (pre‐soaked for at least 3 h in 0.3% polyethylenimine), using a 24‐place cell harvester (Brandel, Gaithersburg, MD, USA).…”