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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

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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 ([Ca2+]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 [Ca2+]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.

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Abbreviations

AC:

Adenylyl cyclase

ACTB:

ß-actin

AM:

Amthamine

[Ca2+]i :

Intracellular calcium concentration

cAMP:

Adenosine-3′,5′-cyclic monophosphate

C5aR:

C5a receptor

DB-cAMP:

N 6,2′-O-dibutyryladenosine-3′,5′-cyclic monophosphate

DI:

Dimaprit

FAM:

Famotidine

fMLP:

N-formyl-l-methionyl-l-leucyl-l-phenylalanine

fNleLFNleYK-Fl:

Fluorescein-labeled formyl-Nle-Leu-Phe-Nle-Tyr-Lys

FPR:

Formyl peptide receptor

GPCR:

G protein-coupled receptor

GUSB:

ß-glucuronidase

HA:

Histamine

H2R:

Histamine H2 receptor

IBMX:

3-Isobutyl-1-methylxanthine

IM:

Impromidine

JNJ:

1-[(5-Chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120)

MEP:

Mepyramine

Nle:

Norleucine

UR-NK22:

(1-(Amino{[3-(2-amino-4-methyl-thiazol-5-yl)propyl]amino}methylene)-3-{6-[3-(amino{[3-(2-amino-4-methyl-thiazol-5-yl)propyl]amino}methylene)ureido]hexyl}urea)

PCR:

Polymerase chain reaction

PROB:

Probenecid

ROL:

Rolipram

ROS:

Reactive oxygen species

TIO:

Tiotidine

5-MHA:

5-Methylhistamine

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Acknowledgments

We thank Mrs. A. Garbe (Research Core Unit Metabolomics, Hannover Medical School) for performing the HPLC-MS/MS analyses. Many thanks also go to Mrs. N. Kagermeier (Institute of Pharmacy, University of Regensburg) for the synthesis of UR-NK22, which was supported by the Graduate Training Program (Graduiertenkolleg) GRK1910 of the Deutsche Forschungsgemeinschaft. Thanks are also due to the reviewers for their helpful critique.

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Authors and Affiliations

  1. Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany

    Kristin Werner, Solveig Kälble, Sabine Wolter, Erich H. Schneider, Detlef Neumann & Roland Seifert

  2. Department of Pharmaceutical and Medicinal Chemistry II, University of Regensburg, 93040, Regensburg, Germany

    Armin Buschauer

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  1. Kristin Werner
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  2. Solveig Kälble
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Correspondence to Roland Seifert.

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Werner, K., Kälble, S., Wolter, S. et al. 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. Naunyn-Schmiedeberg's Arch Pharmacol 388, 1039–1052 (2015). https://doi.org/10.1007/s00210-015-1133-2

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