title: Flow cytometric analysis with a fluorescently labeled formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2 receptor.
abstract: The histamine H2 receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models, or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMP-dependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca(2+)]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression, and potencies and efficacies of fMLP-induced increases in [Ca(2+)]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive, and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression.
authors.affiliation: Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Werner
authors.forename: Solveig
authors.initials: S
authors.lastname: Kälble
authors.forename: Sabine
authors.initials: S
authors.lastname: Wolter
authors.forename: Erich H
authors.initials: EH
authors.lastname: Schneider
authors.forename: Armin
authors.initials: A
authors.lastname: Buschauer
authors.forename: Detlef
authors.initials: D
authors.lastname: Neumann
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000494:pharmacology; D005786:Gene Expression Regulation; D006632:Histamine / Q000008:administration & dosage / Q000378:metabolism; D017442:Histamine Agonists / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D009842:Oligopeptides / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D044042:Receptors, Formyl Peptide / Q000235:genetics / Q000378:metabolism*; D011970:Receptors, Histamine H2 / Q000187:drug effects* / Q000378:metabolism; D020298:U937 Cells
chemical_list: D005452:Fluoresceins; D017442:Histamine Agonists; D008024:Ligands; D009842:Oligopeptides; D012333:RNA, Messenger; D044042:Receptors, Formyl Peptide; D011970:Receptors, Histamine H2; C061989:F-chemotactic peptide-fluorescein; D006632:Histamine; D000242:Cyclic AMP; D002118:Calcium