Differential Distribution of -Adrenergic Receptor Subtypes in Blood Vessels of Knockout Mice Lacking

-Adrenergic receptors ( -AR) are essential regulators of cardiovascular homeostasis. In addition to their prominent function in the heart, -AR are located on vascular smooth muscle cells, where they mediate vasodilating effects of endogenous catecholamines. In this study, we have investigated in an isometric myograph different types of blood vessels from mice lacking 1and/or 2-adrenergic receptor subtypes ( 1-KO, 2-KO, 1 2-KO). In wild-type mice, isoproterenol induced relaxation of segments from thoracic aorta, carotid, femoral and pulmonary arteries, and portal vein. The relaxant effect of -receptor stimulation was absent in femoral and pulmonary arteries from 1-KO mice. In aortic and carotid arteries and in portal veins, the vasodilating effect of isoproterenol was reduced in mice lacking 1or 2-receptors. However, in these vessels the vasodilating effect was only abolished in double KO mice lacking both 1and 2-receptors. Vessel relaxation induced by forskolin did not differ between wild-type and KO mice. Similar contributions of 1and 2-receptors to isoproterenol-induced vasorelaxation were found when vessels from KO mice were compared with wild-type arteries in the presence of subtypeselective -receptor antagonists. These studies demonstrate that 1-adrenergic receptors play a dominant role in the murine vascular system to mediate vasodilation. Surprisingly, 2-receptors contribute to adrenergic vasodilation only in a few major blood vessels, suggesting that differential distribution of -adrenergic receptor subtypes may play an important role in redirection of tissue perfusion. -Adrenergic receptors ( -ARs), members of the G-proteincoupled receptor superfamily, mediate the effects of catecholamines in the sympathetic nervous system. Using techniques of molecular cloning, three distinct -AR subtypes have been identified ( 1-AR, 2-AR, 3-AR) (for reviews, see Benovic et al., 1988; Bylund et al., 1994). One of the important functions of -ARs is the regulation of blood pressure and vascular smooth muscle tone. Activation of -ARs in the peripheral vasculature leads to vascular smooth muscle relaxation, which is manifested as a hypotensive blood pressure response in humans and in animals (Allwood et al., 1963). During times of stress, -AR mediated vascular relaxation may help redirect the cardiac output to tissues that have an increased oxygen demand (Goldenberg et al., 1950). Based on early pharmacological studies, the 2-AR was shown to be the major vascular -AR subtype (Lands et al., 1967). Additional pharmacological studies, however, demonstrated a role for the other -AR subtypes in the vasculature. Pharmacological experiments in dogs have revealed the presence of 1-ARs in the vasculature (Taira et al., 1977; Vatner et al., 1985; Nakane et al., 1988). In addition, the rat coronary and mesenteric arteries have been shown to possess functional 1-ARs (Abdelrahman et al., 1990; Zwaveling et al., 1996). Recent reports also demonstrate that 3-AR activation can lead to hypotensive responses caused by peripheral vasodilation (Enoksson et al., 1995). Most importantly, one report suggests that also in the human vascular system, 1-adrenergic receptors may play a dominant role over the 2-mediated effects (Wellstein et al., 1988). Further insights into the roles of individual -AR subtypes in cardiovascular homeostasis have resulted from studies on genetically engineered mice (Rohrer et al., 1996, 1999; Chruscinski et al., 1999). In vivo studies on 1-AR knockout, 2-AR knockout, and 1-/ 2-AR double knockout mice have implicated all three -AR subtypes in mediating hypotensive responses to exogenous catecholamines. In 2-AR knockout mice the hypotensive blood pressure response to the -recepThis study was supported by grants from the Deutsche Forschungsgemeinschaft SFB355 (to L.H. and M.J.L.) and the Howard Hughes Medical Institute (to B.K.K.) and by the Leibniz award (to M.J.L.). 1 Current address: Institut für Galenische Pharmazie ETH, Zürich, Switzerland. ABBREVIATIONS: -AR, -adrenergic receptor; KO, knockout; PG, prostaglandin. 0026-895X/01/6005-955–962$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 5 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 955/936174 Mol Pharmacol 60:955–962, 2001 Printed in U.S.A. 955 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from tor agonist isoproterenol was significantly blunted, demonstrating a role for 2-ARs in mediating vascular relaxation (Chruscinski et al., 1999). The fact that a hypotensive response remained in 2-AR knockout mice, however, suggests that additional -AR subtypes can mediate vascular relaxation. In 1 2-AR double knockout mice, the hypotensive response to isoproterenol was further attenuated, demonstrating a role for the 1-AR in mediating vascular relaxation (Rohrer et al., 1999). Residual hypotensive responses to isoproterenol in 1 2-AR double knockout mice are presumably caused by 3-AR activation. Interestingly, hypotensive responses to the 3-receptor agonist CL316243 were exaggerated in 1 2-AR double-knockout mice, suggesting up-regulation of 3-ARs as part of a compensatory process (Rohrer et al., 1999). To further define the roles of individual -AR subtypes in the peripheral vasculature, we have studied -AR mediated relaxation in isolated blood vessels from the various -AR knockout models. Using a small vessel myograph, we studied the function of adrenergic receptor subtypes in isolated segments of mouse large conduit arteries, smaller muscular arteries and veins. The results demonstrate that the 1adrenergic receptor subtype dominates over the 2-subtype in mediating vasorelaxation in the murine vasculature. Materials and Methods Generation of Knockout Mice. Mice lacking functional 1and/or 2-adrenergic receptors have been generated previously (Rohrer et al., 1996, 1999; Chruscinski et al., 1999). All mice were maintained under specified pathogen-free conditions and animal studies were in accordance with the University and government authorities guidelines. Mice were genotyped by Southern blot analysis as described previously (Rohrer et al., 1996; Chruscinski et al., 1999). 1-Receptor KO chimeric mice were originally crossed with C57BL/6J DBA/2 F1 hybrid mice (Rohrer et al., 1996), whereas the 2-receptor deletion was crossed onto an FVB/N background (Chruscinski et al., 1999). Wild-type mice for the present studies were from the C57BL/6J DBA/2 background as well as from the inbred C57BL/6J strain. Initial experiments had demonstrated that isoproterenol-induced vasorelaxation did not differ between these and the