Hederacoside C, alpha-hederin, and hederagenin are saponins of dry extracts obtained from the leaves of ivy (Hedera helix L.). Internalization of beta(2)-adrenergic receptor-GFP fusion proteins after stimulation with 1 microM terbutaline was inhibited by preincubation of stably transfected HEK293 cells with 1 microM alpha-hederin for 24 h, whereas neither hederacoside C nor hederagenin (1 microM each) influenced this receptor regulation. After incubation of A549 cells with 5 nM Alexa532-NA, two different diffusion time constants were found for beta(2)AR-Alexa532-NA complexes by fluorescence correlation spectroscopy. Evaluation of the autocorrelation curve revealed diffusion time constants: tau(bound1) = 1.4 +/- 1.1 ms (n = 6) found for receptor-ligand complexes with unrestricted lateral mobility, and tau(bound2) = 34.7 +/- 14.1 ms (n = 6) for receptor-ligand complexes with hindered mobility. The distribution of diffusion time constants was 24.3 +/- 2.5% for tau(bound1) and 8.7 +/- 4.3% for tau(bound2) (n = 6). A549 cells pretreated with 1 microM alpha-hederin for 24 h showed dose-dependent alterations in this distribution with 37.1 +/- 5.5% for tau(bound1) and 4.1 +/- 1.1% for tau(bound2). Simultaneously, the level of Alexa532-NA binding was significantly increased from 33.0 +/- 6.8 to 41.2 +/- 4.6%. In saturation experiments, alpha-hederin did not influence the beta(2)-adrenergic receptor density (B(max)), whereas the K(D) value for Alexa532-NA binding decreased from 36.1 +/- 9.2 to 24.3 +/- 11.1 nM. Pretreatment of HASM cells with alpha-hederin (1 microM, 24 h) revealed an increased intracellular cAMP level of 13.5 +/- 7.0% under stimulating conditions. Remarkably, structure-related saponins like hederacoside C and hederagenin did not influence either the binding behavior of beta(2)AR or the intracellular cAMP level.
The bronchospasmolytic and secretolytic effects of ivy leaves dry extracts can be explained by an increased β2-adrenergic responsiveness of the bronchi. Recently, it was shown that α-hederin inhibits the internalization of β2-adrenergic receptors (ß2AR) under stimulating conditions. α-Hederin pretreated alveolar type II cells and human airway smooth muscle cells revealed an increased ß2AR binding and an elevated intracellular cAMP level, respectively. In order to identify whether additional compounds also mediate an increased β2-adrenergic responsiveness, we examined the ingredients of an ivy leaves dry extract (EA 575) protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, kaempferol-3-O-rutinoside, 3,4-, 3,5- and 4,5-dicaffeoylquinic acid, hederacoside B, and β-hederin. Within all the tested substances, only β-hederin inhibited the internalization of GFP-tagged ß2AR in stably transfected HEK293 cells. Using fluorescence correlation spectroscopy β-hederin (1 μM, 24 h) pretreated HASM cells showed a statistically significant increase in the ß2AR binding from 33.0 ± 8.9% to 44.1 ± 11.5% which was distributed with 36.0 ± 9.5% for τbound1 and 8.1 ± 2.6% for τbound2, respectively (n = 8, p < 0.05). The increased binding was selectively found for the receptor-ligand complex with unrestricted lateral mobility (τbound1 of 0.9 ± 0.1 ms, D1 = 9.1 ± 0.2 μm(2)/s, n = 8), whereas the binding of ß2AR with hindered lateral mobility (τbound2 of 64.2 ± 47.6 ms, D2 = 0.15 ± 0.02 μm(2)/s, n = 8) was not affected. Compared to control cells, a statistically significant increase of 17.5 ± 6.4% (n = 4, p < 0.05) and 24.2 ± 5.8% (n = 4, p < 0.001) in the cAMP formation was found for β-hederin pretreated HASM cells after stimulation with 10 μM of terbutaline and simultaneous stimulation with 10 μM terbutaline and 10 μM forskolin, respectively. Within this systematic study focusing on the influence of the ingredients of an ivy leaves dry extract on HASM cells it was possible to identify β-hederin as further component presumably responsible for the β2-mimetic effects.
A detailed conception of intranuclear messenger ribonucleoprotein particle (mRNP) dynamics is required for the understanding of mRNP processing and gene expression outcome. We used complementary state-of-the-art fluorescence techniques to quantify native mRNP mobility at the single particle level in living salivary gland cell nuclei. Molecular beacons and fluorescent oligonucleotides were used to specifically label BR2.1 mRNPs by an in vivo fluorescence in situ hybridization approach. We characterized two major mobility components of the BR2.1 mRNPs. These components with diffusion coefficients of 0.3 ± 0.02 μm²/s and 0.73 ± 0.03 μm²/s were observed independently of the staining method and measurement technique used. The mobility analysis of inert tracer molecules revealed that the gland cell nuclei contain large molecular nonchromatin structures, which hinder the mobility of large molecules and particles. The mRNPs are not only hindered by these mobility barriers, but in addition also interact presumably with these structures, what further reduces their mobility and effectively leads to the occurrence of the two diffusion coefficients. In addition, we provide evidence that the remarkably high mobility of the large, 50 nm-sized BR2.1 mRNPs was due to the absence of retarding chromatin.
Cerebroside sulfotransferase (CST) catalyzes the 3‘-sulfation of galactose residues in several glycolipids. Its major product in the mammalian brain is sulfatide, which is an essential myelin component. Using epitope-tagged variants, murine CST was found to localize to the Golgi apparatus, but in contrast to previous assumptions, not to the trans-Golgi network. An examination of enhanced green fluorescent protein (EGFP)-tagged CST suggests that CST forms homodimers and that dimerization is mediated by the lumenal domain of the enzyme, as shown by immunoprecipitation and density gradient centrifugation. In order to verify that dimerization of CST observed by biochemical methods reflects the behavior of the native protein within living cells, the mobility of CST-EGFP was examined using fluorescence correlation spectroscopy. These experiments confirmed the homodimerization of CST-EGFP fusion proteins in vivo. In contrast to full-length CST, a fusion protein of the amino-terminal 36 amino acids of CST fused to EGFP was exclusively found as a monomer but nevertheless showed Golgi localization.
G protein-coupled receptors (GPCRs) comprise a large protein family of transmembrane receptors involved in many physiological processes. They are engaged in various transduction processes of extracellular signals into intracellular responses. Due to their involvement in numerous diseases they represent an important pharmacological target. Fluorescence correlation spectroscopy (FCS) poses a very sensitive analytical technique well-suited for the investigation of GPCRs. It is minimally invasive and operates on a single molecular level. It further provides detailed pharmacological information on receptor kinetics and quantities of activated receptors on the cell membrane. In addition, FCS allows distinguishing between different receptor states based on different diffusion time constants. In order to be applicable for FCS, the molecule of interest has to be fluorescently labeled. This review focuses on the physical requirements for dyes intended for FCS, their influence on the binding characteristics of coupled ligands and strategies to generate dye labeled ligands, exemplified on GPCR ligands.
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