α-Hederin from Hedera helix affects the binding behaviour of β2-adrenergic receptors as revealed by fluorescence correlation spectroscopy

Live cell imaging experiments with α-hederin pretreated cells revealed an inhibition of β₂-adrenergic receptor (β2AR) internalization after agonistic stimulation with terbutaline. Additionally, such treated cells also showed an increased intracellular cAMP level. These effects were not found for hederacoside C and hederagenin, which are also saponins of dry extracts obtained from ivy leaves (Hedera helix L.). Our findings have clearly demonstrated that α-hederin is able to influence regulatory processes affecting the β₂AR activity [1]. Therefore, the dynamics of β₂AR-ligand complexes in the plasma membrane of human alveolar type II cells (A549) were investigated using fluorescence correlation spectroscopy (FCS).

After incubation of A549 cells with 5 nM Alexa-NA (arterenol, labelled with Alexa Fluor 532) for 15 minutes, the laser was focused on the upper plasma membrane surface. Evaluation of the autocorrelation curve revealed an average fast diffusion time constant τ_bound1 of 1.4 (±1.1)ms and a slow diffusion time constant τ_bound2 of 34.7 (±14.1)ms found for receptor-ligand complexes with unrestricted and hindered lateral mobility, respectively. By contrast, freely diffusing Alexa-NA showed a diffusion time constant τ_free of 0.06 (±0.004)ms. Diffusion time constants in these control experiments were distributed as follows: 67 (±4.5) % for τ_free, 24.3 (±2.5) % (equal to 1.21 (±0.12) nM) for τ_bound1, and 8.7 (±4.3) % (equal to 0.43 (±0.22) nM) for τ_bound2. The total binding was 33 (±6.8) % (equal to 1.65 (±0.34) nM). From τ_bound1 and τ_bound2 corresponding diffusion coefficients of D₁=9.09 (±5,09)µm²/s and D₂=0.28 (±0,07)µm²/s were calculated for the different receptor-ligand complexes. Pretreatment of A549 cells with 1µM α-hederin for 24h clearly altered the distribution of diffusion time constants of Alexa-NA with 58.8 (±5.6) % for τ_free, 37.1 (±5.5) % (equal to 1.90 (±0.27) nM) for τ_bound1, and 4.1 (±1.1) % (equal to 0.20 (±0.06) nM) for τ_bound2. A dose-dependent decrease in receptor-ligand complexes with τ_bound2 was found in FCS experiments. From an analysis of the autocorrelation curves, 0.01µM α-hederin does not alter the population with τ_bound2 significantly, whereas a decrease of 25% and of 50% in τ_bound2 was found for α-hederin concentrations of 0.1µM and 1µM, respectively. At the same time, τ_bound1 showed a statistically significant dose-dependent increase of 58% in the presence of 1µM α-hederin. Moreover, A549 cells pretreated with 1µM α-hederin showed increased Alexa-NA binding, i.e., 1.65 (±0.34) nM to 2.1 (±0.2) nM compared to the control cells. The increase in Alexa-NA binding was found to be statistically significant for concentrations between 2 nM and 50 nM. Saturation experiments revealed a B_max value of 46.1 (±1.4) nM and a K_D value of 36.1 (±9.2) nM for control cells. α-Hederin treated cells showed a similar value for B_max of 50.2 (±1.1) nM, whereas the dissociation constant decreased to K_D=24.3 (±11.1) nM. Thus, α-hederin did not influence the number of β₂AR on the plasma membrane of A549 cells, however, an increased binding affinity for Alexa-NA was found. By contrast, pretreatment of the cells with 1µM hederacoside C or hederagenin did not influence either the binding of Alexa-NA or the occurrence and relative distribution of τ_free, τ_bound1, and τ_bound2.

Inhibition of β₂AR internalization of α-hederin treated cells revealed by live cell imaging is also expressed by the observed decrease in τ_bound2, which is typical for a more hindered mobility and for receptor-ligand complexes during the internalization process. Obviously, an enhanced binding affinity rather then an increase in receptor density is responsible for the higher ligand binding after α-hederin pretreatment. These findings showed a clear dose-response-relationship. The main result is an increased β₂-adrenergic responsiveness of A549 after α-hederin treatment, but not after treatment with hederacoside C or hederagenin. Therefore, treatment of upper respiratory lung diseases with dried extracts obtained from ivy leaves leads to a higher β₂-adrenergic responsiveness to endogeous adrenergic ligands, like adrenaline. These findings clearly explain both the secretolytic and broncholytic effects of dried ivy leaf extracts caused by an increased secretion of surfactant for alveolar type II cells and a decrease in the intracellular Ca²⁺ concentration for bronchial smooth muscle cells. Controlled clinical studies with Prospan® have proven both, the secretolytic and broncholytic efficacy of the dried ivy leaf extract (5–7,5 : 1) [2].

References: [1] Sieben A, Prenner L, Sorkalla T et al. Biochemistry 2009; 48, 3477–3482

[2] Mansfeld HJ, Höhre H, Repges R, Dethlefsen U. MMW 1998; 140 (3): 26–30