Amthamine dihydrobromide

The Bmal1-luciferase rhythms were analyzed as described (29 (link)) using culture medium supplemented with 50 µM beetle luciferin (Promega, Madison, WI, USA) and a multichannel chemiluminescence analyzer (Kronos-Dio, Model AB-2550; ATTO Co. Ltd., Tokyo, Japan) set at 37°C. The time point with the peak chemiluminescence level in the Bmal1-luciferase rhythms was regarded as circadian time (CT) 20. To analyze phase-response curves (PRCs) against pharmacological stimulations, Kronos recordings were paused for 5 min. During the pause, 10% of culture medium (100 µL) was collected from each dish. Histamine, amthamine dihydrobromide, ketotifen fumarate (Sigma-Aldrich), or d-CPA was added to the collected culture medium and gently returned to the culture dish (final diluted concentration: 50 µM for histamine, 50 µM for amthamine dihydrobromide, 10 µM for d-CPA, and 10 µM for ketotifen). Although hRPE-YC cells represented little sensitivity to light (29 (link)), above medium exchanges were carefully conducted under dim red light (<3 lx). The PRCs were eye fitted by three experienced investigators.

Histamine dihydrochloride, amthamine dihydrobromide, pyrilamine maleate salt, cimetidine, thioperamide maleate salt, indomethacin, atropine, and Nω-Nitro-L-arginine were obtained from Sigma-Aldrich (Missouri, USA), and fexofenadine hydrochloride, αβ-methylene ATP (sodium salt) and cyproheptadine hydrochloride hydrate from Cayman Chemicals (Michigan, USA). Concentrations chosen for the agonists and antagonists were selected based on their selectivity at each receptor and consistent with concentrations used in previous studies^{11 (link)}.