Influence of the saponins from Hedera helix on the β2-adrenergic responsiveness of living cells investigated by β2-adrenergic receptor internalization studies and determination of intracellular cAMP levels

α-Hederin, hederacoside C and hederagenin are saponins of dry extracts obtained from ivy leaves (Hedera helix L.). These extracts are used in the treatment of obstructive airway diseases characterized by hypersecretion of viscous mucus and coughing. Based on spirometric and plethysmographic measurements, clinical relevant improvements in lung function have been observed, and statistically significant efficacy of a dried ivy leaf extract (5–7,5 : 1) has been shown [1]. Moreover, ivy leaf extracts show spasmolytic activity, reducing smooth muscle spasm in vitro [2], as well as bronchodilatory effects shown in a study with asthmatic patients [1], which, as we know today, is mainly due to their triterpene saponin content.

The expectorant activity of saponins in general is thought to be mediated by the gastric mucosa, with reflex stimulation of the bronchial mucous glands via parasympathic pathways. This mode of action, however, cannot explain the broncholytic effects, as shown in-vivo by dose-dependent inhibition of PAF-induced bronchoconstriction in guinea pigs by an ethanolic ivy extract, or in vitro inhibition of acetylcholine-induced spasm in guinea pig ileum by α-hederin and hederagenin [3]. These findings suggest that the ivy leaf extracts have β₂-adrenergic mimetic activities, and this could explain both secretolytic and bronchiolytic effects.

In order to contribute to the mode of action behind the named effects of ivy leaf extracts, we established a HEK293 cell line stably expressing the β₂-adrenergic receptor (β₂AR) as a GFP fusion protein (β₂AR-GFP). Stimulation of these cells with 1µM terbutaline, a specific β₂-agonist, led to a pronounced endocytotic internalization of occupied receptors that occurred after 20min. Compared to control cells, those stimulated showed large intracellular vesicles. This finding is consistent with a decrease in the β₂AR density on cell membranes, mediated by a high agonist concentration. Agonistic stimulation of β₂ARs leads to phosphorylation of the agonist-occupied receptor protein by GRK2 followed by binding of β-arrestin, c-Src, and AP-2, which finally leads to receptor internalization via clathrin-coated pits and formation of early endosomes. Preincubation with 1µM α-hederin for 24h clearly led to an inhibition of receptor internalization even after stimulation with 1µM terbutaline for 20min. Compared to the positive control, similar intracellular vesicles were not observed in α-hederin-treated cells [4]. Using α-hederin-pretreated alveolar type II cells (A549) we were able to confirm the inhibition of the β₂ARs internalization [5]. Remarkably, preincubation with hederagenin and hederacoside C, two saponins structurally related to α-hederin, did not influence this regulatory process at a concentration of 1µM, as shown by live cell imaging studies. Thus, one can expect an elevated β₂-adrenergic responsiveness of α-hederin-treated cells and a subsequently increased signal transduction. In order to confirm an increased second messenger level due to the internalization inhibition, intracellular cAMP levels of human airway smooth muscle cells (HASM) were determined under stimulating conditions (10µM foskolin, 10µM terbutaline, 10min) after pretreatment with 1µM α-hederin for 24h. Compared to control cells, a significantly increased cAMP level of about 13.5 (±7) % was found for α-hederin pretreated cells, whereas hederacoside C and hederagenin did not influence the intracellular concentration of the second messenger. Therefore, an increased transmembrane signalling via β₂ARs after α-hederin incubation was confirmed by augmented intracellular accumulation of the second messenger cAMP, leading to diverse downstream cell regulatory effects.

For alveolar type II cells this could cause increased secretion of surfactant, which could thus explain the secretolytic effect of ivy extracts. In this manner one could also expect both a decrease in the intracellular Ca²⁺ concentration and an increase in phosphorylation of the Ca²⁺/calmodulin-dependent myosin light-chain kinase (MLCK) in bronchial smooth muscle cells, which could itself explain the bronchospasmolytic effect of ivy extracts.

References: [1] Mansfeld HJ et al. MMW 1998; 140(3): 26–30

[2] Trute A et al. Planta Med 1997; 63: 125–129

[3] Haen E. Phytomedicine 1996; Suppl. 1: 144

[4] Sieben A et al. Biochemistry 2009; 48: 3477–3482

[5] Hegener O et al. Biochemistry 2004; 43: 6190–6199.