Impact of St. John's wort extract Ze 117 on stress induced changes in the lipidome of PBMC.

BACKGROUND: Membrane lipids have an important function in the brain as they not only provide a physical barrier segregating the inner and outer cellular environments, but are also involved in cell signaling. It has been shown that the lipid composition effects membrane fluidity which affects lateral mobility and activity of membrane-bound receptors. METHODS: Since changes in cellular membrane properties are considered to play an important role in the development of depression, the effect of St. John's wort extract Ze 117 on plasma membrane fluidity in peripheral blood mononuclear cells (PBMC) was investigated using fluorescence anisotropy measurements. Changes in fatty acid residues in phospholipids after treatment of cortisol-stressed [1 µM] PBMCs with Ze 117 [10-50 µg/ml] were analyzed by mass spectrometry. RESULTS: Cortisol increased membrane fluidity significantly by 3%, co-treatment with Ze 117 [50 µg/ml] counteracted this by 4.6%. The increased membrane rigidity by Ze 117 in cortisol-stressed [1 µM] PBMC can be explained by a reduced average number of double bonds and shortened chain length of fatty acid residues in phospholipids, as shown by lipidomics experiments. CONCLUSION: The increase in membrane rigidity after Ze 117 treatment and therefore the ability to normalize membrane structure points to a new mechanism of antidepressant action of the extract.

Effect of St. John's wort extract Ze 117 on the lateral mobility of ß1-adrenergic receptors in C6 cells.

Depression has been associated with altered signal transduction of serotonergic, dopaminergic and adrenergic neurotransmitter systems in the brain. Signaling relies on receptor-ligand interactions and subsequent regulatory processes, but also on lateral receptor mobility. The aim of this study was to investigate the effect of the St. John's wort extract Ze 117 on the lateral mobility of SNAP-tagged ß1-adrenergic receptors (ß1AR) in the plasma membrane of C6 cells under both, non-stimulating and isoprenaline-stimulating conditions. Single particle tracking (SPT) was used, whereby the registered trajectories were evaluated by variational Bayesian treatment of a hidden Markov model (vbSPT) and packing coefficient (Pc) analysis with respect to diffusion coefficients, receptor state occupancies and confinement. Three different diffusion states were identified, differing in their diffusion coefficients. Treatment with Ze 117 [25 µg/ml] decreased the mobility of the ß1AR, which was manifested by a relative increase in the slow-diffusing state S1 (0.21-0.30) compared to control and by an increase in receptor confinement (79.4-68.1 nm). After isoprenaline stimulation of control cells, the slow-diffusing state was more pronounced, whereas confinement was not affected. In summary, SPT has been shown to be a powerful method to analyze lateral receptor mobility. Furthermore, the present study identified a correlation between Ze 117 treatment and ß1AR mobility.

A comparative study on the influence of an ivy preparation and an ivy/thyme combination on the ß2-adrenergic signal transduction.

The ß2-adrenergic receptor (ß2AR) 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 ß2AR 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 ß2-adrenergic signal transduction. For receptor binding studies and characterization of the lateral mobility of ß2AR 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 ß2AR we have generated luciferase based reporter cell lines, which produce a cAMP-dependent luciferase in the cytosol and express ß2AR with extracellular luciferase moiety in the plasma membrane. While both preparations increased the ß2AR 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 ß2AR under stimulating conditions. In contrast, isoprenaline-mediated internalization of HiBiT-tagged ß2AR of ivy/thyme combination pre-treated cells was not inhibited. Cells comparatively pre-treated with a thyme preparation did not show inhibition of ß2AR internalization either. Furthermore, SNAP-tagged ß2AR of ivy preparation pre-treated cells, which were not internalized after isoprenaline stimulation, showed a redistribution from fast-to-slowly diffusing ß2AR. 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 ß2AR 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 ß2-adrenergic signal transduction.

St John's wort extract influences membrane fluidity and composition of phosphatidylcholine and phosphatidylethanolamine in rat C6 glioblastoma cells.

BACKGROUND: Chronic stress, an important factor in the development of depressive disorders, leads to an increased formation of cortisol, which causes a hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, cortisol mediates an adaptive effect on plasma membrane fluidity which may affect signal transduction of membrane-bound receptors and contribute to pathophysiological changes. METHODS: Membrane fluidity was measured by fluorescence anisotropy using DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene). Changes in cellular content of phosphatidylcholine species was determined by pulse-chase experiments using deuterated choline and mass spectrometry. Single molecule tracking was used to examine the lateral mobility of ß1-adrenoceptors and changes in cAMP formation were measured by ELISA. RESULTS: Chronic exposure (6 - 8 days) of C6 cells to cortisol dose-dependently decreased DPH and TMA-DPH fluorescence anisotropy, reflecting increased membrane fluidity. In contrast, cells pretreated with St. John's wort extract Ze117 showed increased DPH and TMA-DPH fluorescence anisotropy values, indicating a membrane rigidification effect which was mediated at least by the constituents hypericin, hyperforin, quercetin, amentoflavone and biapigenin. The observed membrane fluidizing effect of cortisol could be reversed by cotreatment with Ze117. The membrane rigidification of Ze117 was in line with the in parallel observed decrease in the phosphatidylcholine/phosphatidylethanolamine ratio determined in whole cell lipid extracts. Interestingly, pulse-chase experiments demonstrated, that Ze117 inhibited the incorporation of choline-D9 in phosphatidylcholine species with saturated or monounsaturated fatty acids compared to control cells, while the synthesis of phosphatidylcholine species with polyunsaturated fatty acids was not affected. C6 cells whose membranes have become more rigid by Ze117 showed altered lateral mobility of ß1-adrenoceptors as well as reduced cAMP formation after stimulation with the ß1-adrenoceptor agonist dobutamine. CONCLUSION: Obviously, the signaling of ß1-adrenoceptors depends on the nature of the membrane environment. It can therefore be assumed that Ze117 has a normalizing effect not only on the membrane fluidity of "stressed" cells, but also on lateral mobility and subsequently on the signal transduction of membrane-associated receptors.