Vascular Reactivity of Isolated Thoracic Aorta of the C57BL/6J Mouse AMBER RUSSELL and STEPHANIE WATTS

We characterized the thoracic aorta from the C57BL/6J mouse, a strain used commonly in the generation of genetically altered mice, in response to vasoactive substances. Strips of aorta were mounted in tissue baths for measurement of isometric contractile force. Cumulative concentration-response curves to agonists were generated to observe contraction, or relaxation in tissues contracted with phenylephrine or prostaglandin F2a (PGF2a). In endothelium-denuded strips, the order of agonist contractile potency (2log EC50 [M]) was norepinephrine . phenylephrine 5 5-hydroxytryptamine . dopamine . PGF2a . isoproterenol . KCl. Angiotensin II and endothelin-1 were weakly efficacious (15% of maximum phenylephrine contraction), as were UK14,304, clonidine, histamine, and adenosine. In endothelium-intact strips, agonists still caused contraction and both angiotensin II and endothelin-1 remained ineffective. In experiments focusing on angiotensin II, angiotensin II-induced contraction was abolished by the AT1 receptor antagonist losartan (1 mM) but was not enhanced in the presence of the AT2 receptor antagonist PD123319 (0.1 mM), tyrosine phosphatase inhibitor orthovanadate (1 mM) or when angiotensin II was given noncumulatively. Prazosin abolished isoproterenolinduced contraction and did not unmask isoproterenol-induced relaxation. Angiotensin II and endothelin-1 did not cause endothelium-dependent or -independent relaxation in phenylephrineor PGF2a-contracted tissues. Acetylcholine but not histamine, dopamine, or adenosine caused an endotheliumdependent vascular relaxation. These experiments provide information as to the vascular reactivity of the normal mouse thoracic aorta and demonstrate that the mouse aorta differs substantially from rat aorta in response to isoproterenol, angiotensin II, endothelin-1, histamine, and adenosine. For years, the rat has served as a valuable model for studies in cardiovascular disease. With the advent of genomic manipulation, the mouse is at the forefront of use in scientific investigation. Herein, we establish normal vascular responses to a group of vasoactive substances in the thoracic aorta isolated from the C57BL/6J mouse, a mouse used commonly in the creation of genetically altered mice. Although significant effort has been made previously to examine the role of the endothelium and endothelial cell-derived vasoactive factors in mouse vasculature (Abe et al., 1998, Akishita, 1999; Faraci and Sigmund, 1999), a study of mouse vascular reactivity to contractile and relaxant agonists has not been previously performed. It should be noted that this series of studies was not meant to be nor is it exhaustive in terms of investigating all substances that can alter arterial smooth muscle tone. However, the group of agonists examined are representative of several important vasoactive systems. We found that the mouse aorta contracted to a-adrenergic, serotonergic, dopaminergic, and prostaglandin (PG) receptor agonists and, as has been observed in tissues from the rat, relaxed to acetylcholine in an endothelium-dependent manner. Suprisingly, the mouse aorta contracted with significantly weak efficacy to angiotensin II and endothelin-1, two peptide hormones with significant potency in the cardiovascular system of the rat. Because of our laboratory’s interest in hypertension, we performed a preliminary investigation in to some of the mechanisms that might explain the lack of response to angiotensin II. Our focus on this particular agonist should not detract from the finding that other agonists, such as isoproterenol, histamine, and adenosine, did not act in the mouse aorta in a fashion similar to that observed in the rat aorta. Thus, there are significant differences in the vascular responsiveness of the rat and mouse aorta. Materials and Methods Isolated Tissue Bath Protocol. All animal procedures were followed in accordance with institutional guidelines established by Michigan State University. Male C57BL/6J mice (16–18 g; Jackson Laboratories, Bar Harbor, ME; carbon dioxide) or male SpragueDawley rats [250–300 g; Charles River Laboratories, Indianapolis, IN; pentobarbital (60 mg/kg i.p.)] were euthanized and thoracic aortae removed. Arteries were dissected into helical strips (mouse: Received for publication January 18, 2000. 1 This study was supported in part by Grant HL58489 from the National Institutes of Health. ABBREVIATIONS: PG, prostaglandin; 5-HT, 5-hydroxytryptamine; AT, angiotensin; SNAP, S-nitroso-N-acetylpenicillamine. 0022-3565/00/2942-0598$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 294, No. 2 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 294:598–604, 2000 /2506/836494 598 at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from 0.15 3 0.75 cm; rat: 0.2 3 1.0 cm) and, in some experiments, the endothelial cell layer removed by rubbing the luminal side of the vessel with a moistened cotton swab. Tissues were placed in physiological salt solution for measurement of isometric contractile force with standard bath procedures. Physiological salt solution contained 130 mM NaCl, 4.7 mM KCl, 1.18 mM KH2PO4, 1.17 mM MgSO4z7H2O, 1.6 mM CaCl2z2H2O, 14.9 mM NaHCO3, 5.5 mM dextrose, and 0.03 mM CaNa2EDTA. One end of the preparation was attached to a stainless steel rod, the other was attached to a force transducer (FT03; Grass Instruments, Quincy, MA). Muscle baths were filled with warmed (37°C), aerated (95% O2, 5% CO2) physiological salt solution. Changes in isometric force were recorded on a Grass polygraph (Grass Instruments). Determination of Optimal Resting Tension. Mouse aortic strips were placed under a particular tension by means of a rack and pinion, allowed to equilibrate for 30 min with buffer exchanges every 10 min, and then challenged with a maximal concentration of KCl (100 mM). Active force generation was recorded, tissues were washed for 30 min, and the passive tension placed on the tissues was increased. This procedure was repeated multiple times from passive tensions of 50 to 400 mg so as to generate a passive-active tension curve for determination of the optimal passive tension under which tissues should be placed. Such an experiment had been done previously for rat aortic strips and 1500 mg of tension was determined as optimal for this tissue. Determination of Agonist-Induced Mouse Thoracic Aortic Contraction. Tissues equilibrated for 1 h under optimal tension. Tissues were challenged with a maximal concentration of phenylephrine (10 M). This contraction to phenylephrine within each experimental grouping was not different (;100 mg), and thus this response to phenylephrine was used to normalize contractile data. Tissues were washed and the status of the endothelium was examined by observing arterial relaxation to the endothelium-dependent agonist acetylcholine (1 3 10 M) in tissues contracted by a halfmaximal concentration of the a1-adrenergic receptor agonist phenylephrine (1 3 10 M). Tissues were then washed multiple times and one of the following agonists was added in a cumulative fashion (from 10 pM to 30 mM) to generate a concentration-response curve: angiotensin II, endothelin-1, norepinephrine, phenylephrine, 5-hydroxytryptamine (5-HT), clonidine, UK14,304, dopamine, isoproterenol, PGF2a, KCl, histamine, or adenosine. After the cumulative response had reached a maximum, tissues were washed for 1 h (washes every few minutes) and a cumulative concentration-response curve was performed to a second agonist. The order in which agonists were tested was random except for endothelin-1. Because endothelin-1-induced contraction, even though small, was difficult to wash out, a second curve was not generated in these tissues. Tissues in which no contraction to the test agonist was observed were rechallenged with phenylephrine (10 mM) to ensure that the tissues were