Downregulation of β1-adrenergic receptors in rat C6 glioblastoma cells by hyperforin and hyperoside from St John's wort

Objectives While the use of St John's wort extracts as treatment for mild to moderate depression is well established the mode of action is still under investiation. Individual constituents of St John's wort extract were tested for possible effects on the 1AR density and a subsequent change in downstream signalling in rat C6 glioblastoma cells. Methods The effect of compounds from St John's wort extract on the downregulation of 1-adrenergic receptor-GFP fusion proteins (1AR-green fluorescent protein (GFP)) of transfected rat C6 gliobastoma cells (C6-1AR-GFP) was investigated by means of confocal laser scanning microscopy (LSM). The influence on the lateral mobility of 1AR-GFP in C6-1AR-GFP was investigated by fluorescence correlation spectroscopy. The formation of second messenger was determined by c-AMP-assay. Key findings Confocal LSM revealed that pretreatment of cells with 1m of hyperforin and hyperoside for 6 days, respectively, led to an internalization of 1AR-GFP under non-stimulating conditions. Observation by fluorescence correlation spectroscopy showed two diffusion time constants for control cells, with diff1=0.78 +/- 0.18ms and diff2=122.53 +/- 69.41ms, similarly distributed. Pretreatment with 1m hyperforin or 1m hyperoside for 3 days did not alter the diff values but decreased the fraction of diff1 whereas the fraction of diff2 increased significantly. An elevated level of 1AR-GFP with hindered lateral mobility was in line with 1AR-GFP internalization induced by hyperforin and hyperoside, respectively. A reduced 1-adrenergic responsiveness was assumed for C6 gliobastoma cells after pretreatment for 6 days with 1m of both hyperforin and hyperoside, which was confirmed by decreased cAMP formation of about 10% and 5% under non-stimulating conditions. Decrease in cAMP formation by 23% for hyperforin and 15% for hyperoside was more pronounced after stimulation with 10m dobutamine for 30min. Conclusions The treatment of C6 gliobastoma cells with hyperforin and hyperoside results in a reduced 1AR density in the plasma membrane and a subsequent reduced downstream signalling.