Estrogen receptor-mediated enhancement of venous relaxation in female rat: Implications in sex-related differences in varicose veins Joseph D. Raffetto,

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Presentation transcript:

Estrogen receptor-mediated enhancement of venous relaxation in female rat: Implications in sex-related differences in varicose veins Joseph D. Raffetto, MD, Xiaoying Qiao, MD, PhD, Katie G. Beauregard, BS, Raouf A. Khalil, MD, PhD Journal of Vascular Surgery Volume 51, Issue 4, Pages 972-981 (April 2010) DOI: 10.1016/j.jvs.2009.11.074 Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 1 Effect of phenylephrine (PHE) on male and female rat inferior vena cava (IVC) incubated in normal Krebs (2.5 mM Ca2+). After two control 96 mM KCl contractions, followed by washing in Krebs, IVC segments of (A) male and (B) female rats were stimulated with the α-adrenergic agonist PHE (10−9 to 10−5 M). The contractile response was recorded and presented as (C) mg/mg tissue or as (D) percentage of maximal contraction. The unlabeled arrow represents a threefold greater PHE concentration than the preceding arrow. Data represent the means ± standard error of the mean for 16 to 20 vein segments. *Significantly different (P < .05). Journal of Vascular Surgery 2010 51, 972-981DOI: (10.1016/j.jvs.2009.11.074) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 2 Angiotensin II (AngII), potassium chloride (KCl) and calcium (Ca2+)-dependent contraction in male and female rat inferior vena cava (IVC) segments. IVC segments from male (shaded bars) and female rats (white bars) were incubated in normal Krebs (2.5 mM Ca2+). The tissues were stimulated with the angiotensin type 1 receptor agonist AngII (10−6 M) and the (A) peak contractile response was measured or (B) with 96 mM KCl depolarizing solution and the initial maximal contractile response and steady-state contraction were recorded. C, Other IVC segments were incubated in Ca2+-free (2 mM ethyleneglycotetraacetic acid) Krebs for 5 minutes, then nominally 0 Ca2+ Krebs for 5 minutes, then stimulated with phenylephrine (PHE; 10−5 M) and the transient contraction was measured. Increasing concentrations of extracellular CaCl2 (0.1, 0.3, 0.6, 1, 2.5 mM) were added, and the contractile response at different [Ca2+]e was recorded. Data represent the means ± standard error of the mean for 8 to 20 vein segments. *Significantly different (P < .05). Journal of Vascular Surgery 2010 51, 972-981DOI: (10.1016/j.jvs.2009.11.074) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 3 Acetylcholine (Ach)-induced relaxation is shown in male and female rat inferior vena cava (IVC) segments. IVC segments were stimulated with phenylephrine PHE (10−5 M), and the steady-state contraction was recorded. Increasing concentrations of Ach (10−9 to 10−5 M) were added, and the percentage relaxation of PHE contraction was compared in male and female IVC. Data represent the means ± standard error of the mean for 20 to 23 vein segments. *Significantly different (P < .05). Journal of Vascular Surgery 2010 51, 972-981DOI: (10.1016/j.jvs.2009.11.074) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 4 Expression of estrogen receptor (ER)α, ERβ, and G protein-coupled receptor 30 (GPR30)is shown in male and female rat inferior vena cava (IVC). Tissue homogenates of male and female IVC were prepared for Western blot analysis and the amount of ERs were measured using antibodies to ERα (1:100), ERβ (1:1000), and GPR30 (1:100). The intensity of the immunoreactive bands was analyzed using optical densitometry and normalized to the housekeeping protein actin. Data represent the means ± standard error of the mean for three experiments.*Significantly different (P < .05). Journal of Vascular Surgery 2010 51, 972-981DOI: (10.1016/j.jvs.2009.11.074) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 5 Representative traces of estrogen receptor (ER)-mediated relaxation in female rat inferior vena cava (IVC) are shown. Segments of female rat IVC were stimulated with phenylephrine (PHE; 10−5 M), and the steady-state contraction was recorded. IVC segments were (A) nontreated or were treated with increasing concentrations (10−12 to 10−5 M) of (B) 17β-estradiol (E2, activator of most ERs), (C) the ERα agonist 4,4,′4′′-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT), (D) the ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), or (E) ICI 182,780, a ERα/ERβ antagonist and G protein-coupled receptor 30 stimulant. The effect on PHE contraction was observed. The vertical bars show 0.5 g, and the horizontal bars show 5 minutes. Journal of Vascular Surgery 2010 51, 972-981DOI: (10.1016/j.jvs.2009.11.074) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 6 Estrogen receptor (ER)-mediated venous relaxation was tested in female rat inferior vena cava (IVC) segments. Segments of female rat IVC were stimulated with phenylephrine (PHE; 10−5 M), and the steady-state contraction was recorded. Increasing concentrations (10−12 to 10−5M) of 17β-estradiol (E2, activator of most ERs), the ERα agonist 4,4,′4′′-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT), the ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) or ICI 182,780, the ERα/ERβ antagonist and G protein-coupled receptor 30 stimulant, were added and the percentage relaxation of PHE contraction was measured. Data represent the means ± standard error of the mean for six vein segments. *Significantly different (P < .05). Journal of Vascular Surgery 2010 51, 972-981DOI: (10.1016/j.jvs.2009.11.074) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 7 Effect of nitric oxide synthase (NOS) blockade on estrogen receptor (ER)-mediated venous relaxation was tested in female rat inferior vena cava (IVC). Segments of female rat IVC were nontreated or pretreated with the NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME; 3 × 10−4 M) for 15 minutes and then stimulated with phenylephrine (PHE; 10−5 M). The steady-state contraction was recorded. Increasing concentrations (10−12 to 10−5M) of (A) 17β-estradiol (E2), (B) 4,4,′4′′-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT), (C) 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), or (D) ICI 182,780 were added and the percentage relaxation of PHE contraction was compared in L-NAME-treated and nontreated IVC segments. Data represent the means ± standard error of the mean for five to six vein segments. Journal of Vascular Surgery 2010 51, 972-981DOI: (10.1016/j.jvs.2009.11.074) Copyright © 2010 Society for Vascular Surgery Terms and Conditions