title: Ca(2+)-dependent contraction by the saponoside escin in rat vena cava: implications in venotonic treatment of varicose veins.
abstract: BACKGROUND
  Saponosides (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 Ca(2+) is a major regulator of venous smooth muscle (VSM) function, we tested the hypothesis that escin and diosmin promote Ca(2+)-dependent venous contraction.
  
  
  METHODS
  Rings 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 Ca(2+) release, the vein response to escin and diosmin was measured in Ca(2+)-free (2mM EGTA) Krebs. To test for Ca(2+)-dependent effects, IVC segments were pretreated with escin or diosmin (10(-4) M) in 0 Ca(2+) Krebs, then extracellular CaCl(2) (0.1, 0.3, 0.6, 1, 2.5 mM) was added and the [Ca(2+)](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).
  
  
  RESULTS
  In IVC segments incubated in normal Krebs (2.5 mM Ca(2+)), 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 Ca(2+)-free Krebs, there was essentially no contraction to escin. In escin-treated veins incubated in 0 Ca(2+) Krebs, stepwise addition of extracellular CaCl(2) 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 Ca(2+)-free Krebs (4.2 ± 2.2). In diosmin-treated veins incubated in 0 Ca(2+) Krebs, addition of extracellular CaCl(2) 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).
  
  
  CONCLUSIONS
  In rat IVC, escin induces extracellular Ca(2+)-dependent contraction, but disrupts α-adrenergic and AT(1)R 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.
authors.affiliation: Division of Vascular and Endovascular Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Raffetto
authors.forename: Raouf A
authors.initials: RA
authors.lastname: Khalil
mesh_terms: D058646:Adrenergic alpha-1 Receptor Agonists / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects*; D004145:Diosmin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004928:Escin / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D010656:Phenylephrine / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D044140:Receptor, Angiotensin, Type 1 / Q000187:drug effects / Q000378:metabolism; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects / Q000378:metabolism; D014648:Varicose Veins / Q000188:drug therapy* / Q000378:metabolism / Q000503:physiopathology; D014661:Vasoconstriction / Q000187:drug effects*; D014662:Vasoconstrictor Agents / Q000494:pharmacology*; D014682:Vena Cava, Inferior / Q000187:drug effects* / Q000378:metabolism
chemical_list: D058646:Adrenergic alpha-1 Receptor Agonists; D044140:Receptor, Angiotensin, Type 1; D018340:Receptors, Adrenergic, alpha-1; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D010656:Phenylephrine; D011189:Potassium Chloride; D004928:Escin; D004145:Diosmin