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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Ivy leaf dry extract EA 575® is used to improve complaints of chronic inflammatory bronchial diseases and acute inflammation of the respiratory tract accompanied by coughing. Its mechanism of action has so far been explained by influencing β2-adrenergic signal transduction. In the present study, we investigated a possible influence on adenosine receptor A2B (A2BAR) signalling, as it has been described to play a significant and detrimental role in chronic inflammatory airway diseases. The influence of EA 575® on A2BAR signalling was assessed with measurements of dynamic mass redistribution. Subsequently, the effects on A2BAR-mediated second messenger cAMP levels, β-arrestin 2 recruitment, and cAMP response element (CRE) activation were examined using luciferase-based HEK293 reporter cell lines. Lastly, the impact on A2BAR-mediated IL-6 release in Calu-3 epithelial lung cells was investigated via the Lumit™ Immunoassay. Additionally, the adenosine receptor subtype mediating these effects was specified, and A2BAR was found to be responsible. The present study demonstrates an inhibitory influence of EA 575® on A2BAR-mediated general cellular response, cAMP levels, β-arrestin 2 recruitment, CRE activation, and IL-6 release. Since these EA 575®-mediated effects occur within a time frame of several hours of incubation, its mode of action can be described as indirect. The present data are the first to describe an inhibitory effect of EA 575® on A2BAR signalling. This may offer an explanation for the beneficial clinical effects of the extract in adjuvant asthma therapy.

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