Ca2+−dependent contraction by the saponoside escin in rat vena cava: Implications in venotonic treatment of varicose veins

BackgroundSaponosides (horse chestnut seed extract, escin) and flavonoids (diosmin, Daflon 500, Servier, France) exhibit venotonic properties that have been utilized in treatment of varicose veins. However, the cellular mechanisms underlying the venotonic properties of escin and diosmin are unclear. Because Ca2+ is a major regulator of venous smooth muscle (VSM) function, we tested the hypothesis that escin and diosmin promote Ca2+−dependent venous contraction.MethodsRings of inferior vena cava (IVC) from male rats were suspended in a tissue bath for measurement of isometric contraction. Following control contraction to 96 mM KCl, the effects of escin and diosmin (10−10 to 10−4 M) on vein contraction were measured. To test the role of intracellular Ca2+ release, the vein response to escin and diosmin was measured in Ca2+−free (2mM EGTA) Krebs. To test for Ca2+−dependent effects, IVC segments were pretreated with escin or diosmin (10−4 M) in 0 Ca2+ Krebs, then extracellular CaCl2 (0.1, 0.3, 0.6, 1, 2.5 mM) was added and the [Ca2+]e−contraction relationship was constructed. To test for synergistic effects of diosmin, IVC segments were pretreated with diosmin (10−4 M), then stimulated with KCl (16-96 mM) or escin (10−10 to 10−4 M) and vein contraction was measured. Contraction data were presented as mg/mg tissue (means ± SEM).ResultsIn IVC segments incubated in normal Krebs (2.5 mM Ca2+), escin caused concentration-dependent contraction (max 104.3 ± 19.6 at 10−4 M). Escin-induced contraction was not a rigor state, because after washing with Krebs, the veins returned to a relaxed state. In Ca2+−free Krebs, there was essentially no contraction to escin. In escin-treated veins incubated in 0 Ca2+ Krebs, stepwise addition of extracellular CaCl2 caused corresponding increases in contraction (max 80.0 ± 11.1 at 2.5 mM). In the absence of escin, the α-adrenergic agonist phenylephrine (PHE, 10−5 M), angiotensin II (AngII, 10−6 M), and membrane depolarization by KCl (96 mM) caused significant contraction (122.5 ± 45.1, 114.2 ± 12.2 and 221.7 ± 35.4, respectively). In IVC segments pretreated with escin (10−4 M), the contractile response to PHE (9.7 ± 2.6), AngII (36.0 ± 9.1), and KCl (82.3 ± 10.2) was significantly reduced. Diosmin (10−4 M) caused small contractions in normal Krebs (11.7 ± 1.9) and Ca2+−free Krebs (4.2 ± 2.2). In diosmin-treated veins incubated in 0 Ca2+ Krebs, addition of extracellular CaCl2 caused minimal contraction. Diosmin did not enhance the IVC contraction to PHE, AngII, or escin, but enhanced the contractile response to KCl (24-51 mM).ConclusionIn rat IVC, escin induces extracellular Ca2+−dependent contraction, but disrupts α-adrenergic and AT1R receptor-mediated pathways and depolarization-induced contraction. The initial venotonic benefits of escin may be offset by disruption of vein response to endogenous venoconstrictors, limiting its long-term therapeutic benefits in varicose veins. Diosmin does not cause venous contraction or potentiate the venotonic effects of endogenous venoconstrictors or escin ex vivo, and its use as venotonic may need to be further evaluated.

Clinical RelevanceVenotonic agents are used in clinical practice for treatment of varicose veins. Saponosides such as escin (horse chestnut seed extract) and flavonoids such as disomin (active ingredient in Daflon 500, Servier, France) are commonly prescribed venotonic agents. Although escin is known to form pores in the plasma membrane, the mechanisms of its venotonic action are less clear. Also, while diosmin may affect the venous tissue inflammatory response and lymphatic drainage, its direct effects on vein function have not been fully examined. The present results suggest that escin has significant Ca2+−dependent venotonic effects but also reduces the effects of endogenous venoconstrictors, which could limit its long-term therapeutic benefits in varicose veins. Disomin has negligible direct venotonic effects and does not potentiate the effects of endogenous venoconstrictors or escin, and its use as a venotonic agent may need further evaluation.