Taken together, these results indicate that α-hederin acts as an indirect GRK2 inhibitor leading to a reduced homologous desensitization of β2AR-GFP in HEK293 cells.

EA 575 is an ivy leaves dry extract (DER 5-7.5:1, 30% ethanol) used against diseases of the lower respiratory tract associated with productive cough. EA 575 improves symptoms associated with chronic inflammatory bronchial conditions. Compared to its bronchospasmolytic and secretolytic properties, the anti-inflammatory effects of EA 575 are mostly untried. Therefore, we addressed the question of whether the anti-inflammatory effect of EA 575 is due to an impact on the NFκB pathway. NFκB nuclear translocation was visualized by immunofluorescence in J774.2 as well as HEK293 cells. In the latter, a luciferase-based reporter was used to monitor NFκB transcriptional activity. Phosphorylation of RelA and its inhibitor IκB was measured by Western blot analysis. Additionally, changes in the stability of NFκB:IκB complex were shown by protein fragment complementation. Decreased transcriptional activity of NFκB under treatment with EA 575 was also shown for a human monocytic as well as a human lung epithelial cell line. EA 575 is able to inhibit NFκB transcriptional activity by partially inhibiting its translocation to the nucleus after stimulation with TNFα. Furthermore, phosphorylation of IκBα is reduced while phosphorylation of RelA is enhanced after pre-incubation with EA 575, leading to an enhanced stability of NFκB:IκBα complex. EA 575 has an regulatory impact on the NFκB pathway, possibly by switching specificity of IKK from IκBα to RelA, resulting in enhanced stability of NFκB:IκBα complex and reduced RelA translocation into the nucleus.

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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