A comparative study on the influence of an ivy preparation and an ivy/thyme combination on the β2-adrenergic signal transduction

Abstract

The β 2 -adrenergic receptor (β 2 AR) is relevant for surfactant formation in alveolar type 2 cells and reduction of intracellular calcium concentration in bronchial muscle cells and thus for secretolytic and bronchospasmolytic effects. Herbal medicinal products that affect the β 2 AR system are used to treat common cold and bronchitis accompanied with mucus covered and narrowed airways. The present work compares the influence of an ivy preparation and an ivy/thyme combination on the β 2 -adrenergic signal transduction. For receptor binding studies and characterization of the lateral mobility of β 2 AR we have used single molecule detection by fluorescence correlation spectroscopy and single particle tracking. For the determination of both the second messenger cAMP and the internalization of β 2 AR we have generated luciferase based reporter cell lines, which produce a cAMPdependent luciferase in the cytosol and express β 2 AR with extracellular luciferase moiety in the plasma membrane. While both preparations increased the β 2 AR binding, a significant increase of the cAMP level was observed only for the ivy preparation, which can be explained by the inhibited internalization of HiBiT-tagged β 2 AR under stimulating conditions. In contrast, isoprenaline-mediated internalization of HiBiT-tagged β 2 AR of ivy/thyme combination pre-treated cells was not inhibited. Cells comparatively pre-treated with a thyme preparation did not show inhibition of ß 2 AR internalization either. Furthermore, SNAP-tagged β 2 AR of ivy preparation pre-treated cells, which were not internalized after isoprenaline stimulation, showed a redistribution from fast-to-slowly diffusing β 2 AR. A corresponding redistribution of these receptors was not observed after pre-treatment with both the ivy/thyme combination and the thyme preparation. Comparable to the ivy/thyme combination, no decrease in the intratrack transitioning probability ratio (p23/p32) for fast and slow diffusing β 2 AR was found for the thyme preparation, which, however, significantly decreased for control cells and for pre-treatment with the ivy preparation under stimulating conditions. It can therefore be concluded that the thyme fluid extract fraction in the ivy/thyme combination may have in part a negative effect on the β 2 -adrenergic signal transduction.

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Investigation of adenosine A1 receptor mediated β-arrestin 2 recruitment using a split-luciferase assay

“…Establishment of HEK 293 cells expressing a cAMP biosensor as well as measurement of cAMP were performed as described by Bussmann et al (Bussmann et al, 2020). Cells were seeded in a white clear bottom 96-well plate (35.000 cells/well) and incubated at 37°C and 5% CO 2 for 24 hours.…”

Investigation of adenosine A1 receptor-mediated β-arrestin 2 recruitment using a split-luciferase assay

“…The establishment of HEK 293 cells expressing a cAMP biosensor and measurement of cAMP were performed as described by Bussmann et al (2020 ). The cells were seeded in a white clear bottom 96-well plate (35.000 cells/well) and incubated at 37°C and 5% CO 2 for 24 h. The medium was changed to 25 µL substrate solution per well containing 4% GloSensor™ cAMP Reagent stock solution in HEPES-buffered DMEM.…”