Vasorelaxing Effects of Estetrol in Rat Arteries
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97 Vasorelaxing effects of estetrol in rat arteries Rob H P Hilgers, Suzanne Oparil, Wout Wouters1 and Herjan J T Coelingh Bennink1 Division of Cardiovascular Disease, Department of Medicine, Vascular Biology and Hypertension Program, University of Alabama at Birmingham, 1034 Zeigler Research Building, 703 19th Street South, Birmingham, Alabama 35294-0007, USA 1Pantarhei Bioscience, Zeist, The Netherlands (Correspondence should be addressed to S Oparil; Email: [email protected]) Abstract This study compared ex vivo relaxing responses to the hyperpolarization did not alter relaxation to either E4 or E2 naturally occurring human hormone estetrol (E4)vs in uterine arteries. Combined blockade of NO release and 17b-estradiol (E2) in eight different vascular beds. Arteries action with L-NAME and the soluble guanylate cyclase were mounted in a myograph, contracted with either (sGC) inhibitor ODQ resulted in greater inhibition of the phenylephrine or serotonin, and cumulative concentration- relaxation response to E4 compared with E2 in uterine response curves (CRCs) to E4 and E2 (0.1–100 mmol/l) were arteries. Endothelium denudation inhibited responses to both constructed. In all arteries tested, E4 had lower potency than E4 and E2, while E4 and E2 concentration-dependently 2C C E2, although the differential effect was less in larger than blocked smooth muscle cell Ca entry in K -depolarized 2C smaller arteries. In uterine arteries, the nonselective estrogen and Ca -depleted uterine arteries. In conclusion, E4 relaxes receptor (ER) blocker ICI 182 780 (1 mmol/l) caused a precontracted rat arteries in an artery-specific fashion. significant rightward shift in the CRC to both E4 and E2, In uterine arteries, E4-induced relaxations are partially indicating that the relaxation responses were ER dependent. mediated via an endothelium-dependent mechanism invol- C Pharmacological blockade of nitric oxide (NO) synthases ving ERs, sGC, and inhibition of smooth muscle cell Ca2 u by N -nitro-L-arginine methyl ester (L-NAME) blunted entry, but not NO synthases or endothelium-dependent E2-mediated but not E4-mediated relaxing responses, while hyperpolarization. inhibition of prostaglandins and endothelium-dependent Journal of Endocrinology (2012) 215, 97–106 Introduction not bind to SHBG (Hammond et al. 2008). As a result of these favorable metabolic and protein binding properties, The hormone estetrol (estra-1,3,5(10)-trien-3,15a- E4 has excellent oral potency despite its low-to-moderate 16a,17b-tetrol or E4) was discovered in 1965 (Hagen et al. affinity for ERs. 1965). E4 is produced in nature from 17b-estradiol (E2) E4 has been studied in many validated models and has been and estrol (E3) only by the human fetal liver during pregnancy shown to behave as a full ER agonist, similar to E2, in most of via 15a- and 16a-hydroxylase (Schwers et al. 1965, Gurpide these (Coelingh Bennink et al. 2008b, Heegaard et al. 2008, et al. 1966). The concentration of E4 increases during Holinka et al. 2008, Visser & Coelingh Bennink 2009). pregnancy in both the fetus and the mother, but 24 h after However, important differences between E4 and E2 were delivery, the level is undetectable. noted. E4 did not induce synthesis of SHBG in vitro as did E2 The chemical structure of E4 is closely related to that of E2, (Hammond et al. 2008). Recent studies showed that E4 but the pharmacokinetic, metabolic, and endocrinological behaved as an ER antagonist in in vitro and in vivo models of profile of E4 is substantially different from that of E2.E2 has estrogen-dependent breast tumors in the presence of E2.E2 high affinity for the estrogen receptor a (ERa) and ERb behaved as full agonist in these models (Coelingh Bennink (Ki values: 0.2 and 0.05 nmol/l respectively), while E4 has et al. 2008c). E4 is now undergoing clinical testing as the low-to-moderate affinity for ERa and ERb (Ki values: 4.9 estrogenic component of a combined oral contraceptive and and 19 nmol/l respectively; Coelingh Bennink et al. 2008a). for its effects on breast tumors. E2 has very low oral availability because over 99% of an oral The physiological function of E4 is not completely dose is converted into estrone (E1) and E1 sulfate. Most of the understood, although a role in regulating uterine blood circulating E2 is bound to either albumin or sex hormone flow has been suggested. Unilateral intrauterine injection of binding globulin (SHBG), further decreasing plasma levels E4 in nonpregnant oophorectomized ewes has been shown to of free E2 by a factor of about 30. By contrast, E4 does not increase uterine blood flow, although with a 15- to 30-fold undergo phase I metabolism by human HepG2 cells and does lower potency than E3 (Levine et al. 1984). Similarly, Journal of Endocrinology (2012) 215, 97–106 DOI: 10.1530/JOE-12-0009 0022–0795/12/0215–097 q 2012 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/27/2021 03:01:35PM via free access 98 R H P HILGERS and others . Vasodilator effects of estetrol administration of E2 to ovariectomized ewes results in a rapid both uterine horns were cleared of adipose and connective uterine vasodilation leading to a rise in uterine blood flow tissues. The thoracic aorta and left common carotid artery were within 30–45 min (Killam et al. 1973). This rise in uterine cleaned of connective tissue and cut into 2.5–3 mm segments. blood flow is partially mediated via the release of nitric oxide A fourth-order branch segment of the superior mesenteric (NO) and resultant increases in cGMP secretion, as artery was dissected from the mesentery; a small (200–300 mm shown by local infusion of the NO synthase blocker in diameter) pulmonary arterial segment was dissected from u N -nitro-L-arginine methyl ester (L-NAME; Van Buren the lung; a segment of the left main renal artery was dissected, et al. 1992, Rosenfeld et al. 1996). The dilating effect of E2 and a segment of the middle cerebral artery was dissected from on ovine uterine arteries is ER dependent, as shown by local the brain. The atria and the left ventricle were removed to infusion of the nonselective ER blocker ICI 182 780 in expose the interventricular septum of the heart, and a segment nonpregnant ewes (Magness et al. 2005). of the septal coronary artery was dissected. Aortic and carotid To further profile E4, this study was designed to study the artery segments were mounted between two stainless steel pins, in vitro vasorelaxing effects of E4 and to compare them with whereas the other segments (all 2 mm long) were mounted those of E2. The objectives of this study were 1) to determine between two stainless steel jaws connected with two wires the ex vivo relaxing effects of E4 compared to E2 in rat uterine, (40 mm in diameter) through the lumen of the segment in a thoracic aortic, carotid, mesenteric, pulmonary, renal, middle myograph chamber (Danish Myo Technology, Inc., Aarhus, cerebral, and septal coronary arterial segments; 2) to assess the Denmark) filled with 5 ml KRB solution, maintained at 37 8C, involvement of ERs in the vasorelaxing response to E4 in and continuously aerated with 95% O2 and 5% CO2. Four uterine arteries; and 3) to assess the endothelium-dependent arterial segments were analyzed in parallel. The remaining and endothelium-independent vasorelaxing effects of E4 in segments were temporarily stored at 4 8C. uterine arteries. The uterine artery was chosen for assessment of the acute vasoactive properties of E4 and for comparison of these to the effects of E2 because the uterine arterial Determination of optimal diameters circulation is known to be highly sensitive to the vasodilator Thoracic aortae and carotid and pulmonary arteries were effects of E2. The uterine arteries are exposed to high passively stretched according to a procedure first described by physiological levels of E2 during the follicular and luteal Mulvany & Halpern (1977). Briefly, the segments were phases of the menstrual cycle and during pregnancy, resulting distended stepwise in 100 mm increments measured with a in major increases in uterine blood flow (Magness 1998). built-in micrometer and the wall tension (N/m) was recorded using data acquisition (Powerlab 8/35, ADInstruments, Materials and Methods Colorado Springs, CO, USA) and recording Software (ChartLab7, Colorado Springs, CO, USA). Thoracic aortic Animals segments and carotid arteries were stretched at wall tension corresponding to a pressure of 90 mmHg, whereas A total of 40 female nulliparous Sprague Dawley rats (age pulmonary arteries were stretched at a wall tension 12 weeks) were obtained from Charles River Breeding corresponding to 40 mmHg. At this passive wall tension, G C Laboratories, maintained at constant humidity (60 5%), segments were contracted with high K KRB (60 mmol/l G temperature (24 1 8C), and light cycle (0600–1800 h), and KCl in KRB solution; replacing equimolar NaCl with KCl), fed a standard rat pellet diet (2016 Teklad Global 16% Protein thus generating active wall tension, which was set to a Rodent Diet (Harlan Laboratories, Teklad Diets, Madison, 100% contraction level. WI, USA) ad libitum. All protocols were approved by the All other arterial segments were progressively and actively Institutional Animal Care and Use Committee at the stretched to the internal diameter at which the largest University of Alabama at Birmingham and were consistent contractile response to 10 mmol/l norepinephrine (NE) with the Guide for the Care and Use of Laboratory Animals C or 60 mmol/l K KRB (middle cerebral arteries) was published by the National Institutes of Health. obtained. This internal diameter was referred to as the optimal diameter and the corresponding active wall tension Vessel preparation was set to a 100% contraction level.