Targeted Disruption of the β2 Adrenergic Receptor Gene
1999; Elsevier BV; Volume: 274; Issue: 24 Linguagem: Inglês
10.1074/jbc.274.24.16694
ISSN1083-351X
AutoresAndrzej Chruscinski, Daniel K. Rohrer, Eric Schauble, Kavin Desai, Daniel Bernstein, Brian K. Kobilka,
Tópico(s)Adipose Tissue and Metabolism
Resumoβ-Adrenergic receptors (β-ARs) are members of the superfamily of G-protein-coupled receptors that mediate the effects of catecholamines in the sympathetic nervous system. Three distinct β-AR subtypes have been identified (β1-AR, β2-AR, and β3-AR). In order to define further the role of the different β-AR subtypes, we have used gene targeting to inactivate selectively the β2-AR gene in mice. Based on intercrosses of heterozygous knockout (β2-AR +/−) mice, there is no prenatal lethality associated with this mutation. Adult knockout mice (β2-AR −/−) appear grossly normal and are fertile. Their resting heart rate and blood pressure are normal, and they have a normal chronotropic response to the β-AR agonist isoproterenol. The hypotensive response to isoproterenol, however, is significantly blunted compared with wild type mice. Despite this defect in vasodilation, β2-AR −/− mice can still exercise normally and actually have a greater total exercise capacity than wild type mice. At comparable workloads, β2-AR −/− mice had a lower respiratory exchange ratio than wild type mice suggesting a difference in energy metabolism. β2-AR −/− mice become hypertensive during exercise and exhibit a greater hypertensive response to epinephrine compared with wild type mice. In summary, the primary physiologic consequences of the β2-AR gene disruption are observed only during the stress of exercise and are the result of alterations in both vascular tone and energy metabolism. β-Adrenergic receptors (β-ARs) are members of the superfamily of G-protein-coupled receptors that mediate the effects of catecholamines in the sympathetic nervous system. Three distinct β-AR subtypes have been identified (β1-AR, β2-AR, and β3-AR). In order to define further the role of the different β-AR subtypes, we have used gene targeting to inactivate selectively the β2-AR gene in mice. Based on intercrosses of heterozygous knockout (β2-AR +/−) mice, there is no prenatal lethality associated with this mutation. Adult knockout mice (β2-AR −/−) appear grossly normal and are fertile. Their resting heart rate and blood pressure are normal, and they have a normal chronotropic response to the β-AR agonist isoproterenol. The hypotensive response to isoproterenol, however, is significantly blunted compared with wild type mice. Despite this defect in vasodilation, β2-AR −/− mice can still exercise normally and actually have a greater total exercise capacity than wild type mice. At comparable workloads, β2-AR −/− mice had a lower respiratory exchange ratio than wild type mice suggesting a difference in energy metabolism. β2-AR −/− mice become hypertensive during exercise and exhibit a greater hypertensive response to epinephrine compared with wild type mice. In summary, the primary physiologic consequences of the β2-AR gene disruption are observed only during the stress of exercise and are the result of alterations in both vascular tone and energy metabolism. β-Adrenergic receptors (β-ARs) 1The abbreviations used are: β-AR(s), β-adrenergic receptor(s); RER, respiratory exchange ratio; kb, kilobase pair; ES, embryonic stem; FFA, free fatty acid; 125I-CYP, [125I]iodocyanopindolol are members of the superfamily of G-protein-coupled receptors that are stimulated by the naturally occurring catecholamines, epinephrine and norepinephrine. As part of the sympathetic nervous system, β-ARs have been shown to have important roles in cardiovascular, respiratory, metabolic, central nervous system, and reproductive functions. Using techniques of molecular cloning, three distinct β-AR subtypes have been identified (β1-AR, β2-AR, and β3-AR) (1Dixon R.A. Kobilka B.K. Strader D.J. Benovic J.L. Dohlman H.G. Frielle T. Bolanowski M.A. Bennett C.D. Rands E. Diehl R.E. Mumford R.A. Slater E.E. Sigal I.S. Caron M.G. Lefkowitz R.J. Strader C.D. Nature. 1986; 321: 75-79Crossref PubMed Scopus (928) Google Scholar, 2Emorine L.J. Marullo S. Briend S.M. Patey G. Tate K. Delavier K.C. Strosberg A.D. Science. 1989; 245: 1118-1121Crossref PubMed Scopus (1008) Google Scholar, 3Frielle T. Collins S. Daniel K.W. Caron M.G. Lefkowitz R.J. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7920-7924Crossref PubMed Scopus (567) Google Scholar). All three of these β-AR subtypes are believed to signal by coupling to the stimulatory G-protein Gsα leading to activation of adenylyl cyclase and accumulation of the second messenger cAMP (1Dixon R.A. Kobilka B.K. Strader D.J. Benovic J.L. Dohlman H.G. Frielle T. Bolanowski M.A. Bennett C.D. Rands E. Diehl R.E. Mumford R.A. Slater E.E. Sigal I.S. Caron M.G. Lefkowitz R.J. Strader C.D. Nature. 1986; 321: 75-79Crossref PubMed Scopus (928) Google Scholar, 2Emorine L.J. Marullo S. Briend S.M. Patey G. Tate K. Delavier K.C. Strosberg A.D. Science. 1989; 245: 1118-1121Crossref PubMed Scopus (1008) Google Scholar, 3Frielle T. Collins S. Daniel K.W. Caron M.G. Lefkowitz R.J. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7920-7924Crossref PubMed Scopus (567) Google Scholar). Because of the diverse physiological functions mediated by β-ARs, much effort has been spent in understanding the roles of individual β-AR subtypes. In the past, researchers have relied on pharmacological tools such as subtype-selective agonists and antagonists to probe the function of the different β-AR subtypes. The presence of multiple β-AR subtypes was first suggested by Lands and co-workers (4Lands A.M. Arnold A. McAuliff J.P. Luduena F.P. Brown Jr., T.G. Nature. 1967; 214: 597-598Crossref PubMed Scopus (1662) Google Scholar, 5Lands A.M. Luduena F.P. Buzzo H.J. Life Sci. 1967; 6: 2241-2249Crossref PubMed Scopus (413) Google Scholar) who divided β-ARs into β1-ARs and β2-ARs. According to Lands' classification, β1-ARs mediate cardiac stimulation, and β2-ARs mediate smooth muscle relaxation in the peripheral vasculature and respiratory system. The presence of a third β-AR subtype was suggested when some of the effects of β-AR agonists could not be efficiently blocked by typical β-AR antagonists. This third β-AR subtype is now known as the β3-AR and has been shown to have important roles in adipose tissue and the gastrointestinal tract (6Strosberg A.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 421-450Crossref PubMed Scopus (249) Google Scholar). Although both β1-ARs and β2-ARs are expressed in the heart of most mammalian species, β1-ARs are expressed at higher levels and are recognized as playing the major role in regulating cardiac function. Functional studies have confirmed that activation of β1-ARs leads to increased heart rate and force of contraction (7Brodde O.E. Pharmacol. Rev. 1991; 43: 203-242PubMed Google Scholar). Although they represent a smaller population in the heart than β1-ARs, β2-ARs have also been shown to play a role in regulating cardiac function in a variety of species (7Brodde O.E. Pharmacol. Rev. 1991; 43: 203-242PubMed Google Scholar, 8Takei M. Furukawa Y. Narita M. Murakami M. Ren L.M. Karasawa Y. Chiba S. Jpn. J. Pharmacol. 1992; 59: 23-30Crossref PubMed Scopus (8) Google Scholar, 9Kaumann A.J. Naunyn-Schmiedebergs Arch. Pharmacol. 1986; 332: 406-409Crossref PubMed Scopus (75) Google Scholar). In studies using subtype-selective agonists and antagonists in the human heart, β2-AR stimulation leads to activation of adenylyl cyclase and contributes to both inotropic and chronotropic responses (7Brodde O.E. Pharmacol. Rev. 1991; 43: 203-242PubMed Google Scholar). In the murine heart, however, β2-ARs do not appear to couple to inotropic or chronotropic responses. When isolated cardiac muscle from β1-AR knockout mice is stimulated with the non-subtype-selective β-AR agonist isoproterenol, neither inotropic nor chronotropic responses are observed (10Rohrer D.K. Desai K.H. Jasper J.R. Stevens M.E. Regula Jr., D.P. Barsh G.S. Bernstein D. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7375-7380Crossref PubMed Scopus (243) Google Scholar). In addition to their roles in the heart, β-ARs also regulate peripheral vascular tone. Stimulation of peripheral β-ARs leads to relaxation of vascular smooth muscle, thereby controlling the distribution of blood flow to different tissues. During exercise, for example, stimulation of β-ARs contributes to the increased blood flow to skeletal muscle. Based on the studies of Lands and co-workers (4Lands A.M. Arnold A. McAuliff J.P. Luduena F.P. Brown Jr., T.G. Nature. 1967; 214: 597-598Crossref PubMed Scopus (1662) Google Scholar,5Lands A.M. Luduena F.P. Buzzo H.J. Life Sci. 1967; 6: 2241-2249Crossref PubMed Scopus (413) Google Scholar), the β-AR in the peripheral vasculature have been classified as the β2-AR. Some reports, however, have shown roles for the other β-AR subtypes, β1-ARs and β3-ARs, in the peripheral vasculature (11Shen Y.T. Zhang H. Vatner S.F. J. Pharmacol. Exp. Ther. 1994; 268: 466-473PubMed Google Scholar, 12Shen Y.T. Cervoni P. Claus T. Vatner S.F. J. Pharmacol. Exp. Ther. 1996; 278: 1435-1443PubMed Google Scholar, 13Vatner S.F. Knight D.R. Hintze T.H. Am. J. Physiol. 1985; : H49-H56PubMed Google Scholar). Although much has been learned about the role of individual β-AR subtypes using classical pharmacological techniques, these studies are complicated by the fact that subtype-selective ligands are never perfectly selective. Moreover, at the doses required to block β-ARsin vivo, most β-AR ligands lose much of their subtype selectivity and may bind to other G-protein-coupled receptors such as serotonin receptors and dopamine receptors. Studies with β-AR ligands are especially difficult to interpret in vivo where it is hard to estimate the concentration of ligands and their metabolites in target tissues. In order to further investigate the roles of the different β-AR subtypes in physiology, we have selectively inactivated the β2-AR gene in mice using gene-targeting techniques. The knockout (β2-AR −/−) mice appear grossly normal and are fertile. Resting cardiovascular physiology is remarkably unperturbed in β2-AR −/− mice. The major effects of β2-AR gene disruption were observed only during the stress of exercise. β2-AR −/− mice were able to exercise farther and with a lower respiratory exchange ratio at any given workload than wild type controls. However, they are hypertensive during exercise, suggesting an imbalance between the vasoconstrictive and vasorelaxant effects of endogenous catecholamines. The targeting vector was constructed using sequence that had been cloned from a C57BL/6 mouse genomic library (14Allen J.M. Baetge E.E. Abrass I.B. Palmiter R.D. EMBO J. 1988; 7: 133-138Crossref PubMed Scopus (46) Google Scholar). In total, the targeting vector contained 11.4 kb of homology to the endogenous β2-AR genomic locus. The gene for the β2-AR was disrupted in the targeting vector by placing a neomycin (neo) resistance gene cassette into the coding sequence at a uniqueClaI site (15Soriano P. Montgomery C. Geske R. Bradley A. Cell. 1991; 64: 693-702Abstract Full Text PDF PubMed Scopus (1842) Google Scholar). This insertion disrupts the β2-AR at the end of the fourth transmembrane segment and should produce a nonfunctional receptor. The short arm of the targeting vector was a 2.6-kb fragment from a 5′ EcoRI site to the ClaI in the receptor. The long arm of the targeting vector (8.8 kb) extended from the ClaI site in the receptor to a downstreamSalI site. Also included in the vector was the herpes simplex virus thymidine kinase cassette to allow for negative selection when isolating ES cell clones (15Soriano P. Montgomery C. Geske R. Bradley A. Cell. 1991; 64: 693-702Abstract Full Text PDF PubMed Scopus (1842) Google Scholar). In order to screen for homologous recombinants a 5′ external probe was used. This probe is a 300-base pair BamHI/EcoRI fragment that detects a 4.9-kb fragment after mouse genomic DNA is digested with BamHI and then subjected to Southern blot analysis. In cases where the targeting vector has homologously recombined with the endogenous locus, the same probe would detect an additional band at 6.6 kb. R1 embryonic stem (ES) (16Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8424-8428Crossref PubMed Scopus (2006) Google Scholar) cells were transfected using standard techniques (17). ES cells were grown on a monolayer of mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium (UCSF tissue culture facility, San Francisco) supplemented with 20% fetal bovine serum (HyClone, Logan, UT), 1 mm sodium pyruvate (Life Technologies, Inc.), non-essential amino acids, and penicillin/streptomycin (UCSF Cell Culture Facility, San Francisco, CA), 10−4m β-mercaptoethanol (Specialty Media, Lavallette, NJ), and 2,000 units/ml of leukemia inhibitory factor (ESGRO; Life Technologies, Inc.). Cells were grown in an incubator at 37 °C in 95% air, 5% CO2. For the transfection, a 10-cm2 dish of ES cells was transfected via electroporation with 20 μg of targeting vector previously linearized with NotI. After selecting ES cells for 9 days in media containing G418 (Life Technologies, Inc.) and gancyclovir (Syntex, Palo Alto, CA), individual clones were picked and subcloned in 96-well plates. BamHI-digested DNA from clones was analyzed by Southern blot analysis with the 5′ external probe. Nine homologous recombinants were isolated from 300 ES cell clones. Homologous recombinants were also screened with a neo probe to confirm that a single integration of the targeting vector had occurred. Chimeric mice were generated using the morula aggregation technique described previously (18Wood S.A. Allen N.D. Rossant J. Auerbach A. Nagy A. Nature. 1993; 365: 87-89Crossref PubMed Scopus (227) Google Scholar). Briefly, embryos at morula stage (2.5 days pc) were isolated from oviducts of superovulated CD-1 mice by flushing the oviducts with M2 medium (Specialty Media, Lavallette, NJ). After removing the zona pellucida with an acidic Tyrode's solution (Specialty Media, Lavallette, NJ), the embryos were placed in depressions in a 6-cm tissue culture dish and covered with a droplet of M16 medium (Specialty Media, Lavallette, NJ). A protective layer of mineral oil (Sigma) was placed over the droplets. Clumps of ES cells with the targeted disruption (10–20 cells) were then seeded into the depression and placed in contact with the embryos. After an overnight incubation at 37 °C in 95% air, 5% CO2, the chimeric embryos were transferred to the uteri of pseudopregnant CD-1 hosts (20–25 embryos per host). Chimeric mice were identified in the resulting offspring by the presence of dark coat color patches. Chimeric males were then mated to FVB/N female mice to screen for germ line transmission of the ES cell DNA. After achieving germ line transmission, β2-AR +/− mice were intercrossed to generate β2-AR +/+ and −/− mice for use in binding studies. For in vivo studies, the knockout allele was placed on a FVB/N background by backcrossing β2-AR +/− mice to wild type FVB/N mice for four additional generations (5 backcrosses to FVB/N in total). Whole lungs were dissected from wild type and knockout littermates, placed in a lysis buffer (10 mmTris-HCl, 1 mm EDTA, pH 7.4), and homogenized with a Polytron (4 × 20-s bursts). The membrane fraction was isolated by centrifugation at 10,000 × g and resuspended in binding buffer (75 mm Tris-HCl, 12.5 mmMgCl2, 1 mm EDTA, pH 7.4). Binding reactions were carried out by incubating membranes with the radioligand [125I]iodocyanopindolol (125I-CYP) (NEN Life Science Products) in 500-μl volumes. After a 2-h incubation at room temperature, vacuum filtration was performed, and the filters were counted in a gamma counter. For saturation experiments, 3 μg of membrane protein was incubated with increasing amounts of125I-CYP (1–300 pm). Nonspecific binding was determined in the presence of 1 μmdl-propanolol (Sigma). For competition experiments, binding reactions were set up with 50 pm125I-CYP, 3–6 μg of membrane protein, and varying concentrations (50 pm-13 μm) of the β2-AR-selective antagonist ICI 118,551 (Tocris Cookson, Ballwin, MO). Saturation and competition data were analyzed with GraphPAD software (GraphPAD Software Inc., San Diego, CA). In vivostudies were carried out as described previously (19Desai K.H. Sato R. Schauble E. Barsh G.S. Kobilka B.K. Bernstein D. Am. J. Physiol. 1997; : H1053-H1061PubMed Google Scholar). Adult male mice (12–16 weeks of age) were anesthetized with isofluorane using a vaporizer (Airco Inc., Madison, WI), and a stretched Intramedic PE10 polyethylene catheter (Clay Adams, Parsippany, NJ) was inserted into the left carotid artery. The catheter was tunneled through the neck and then placed in a subcutaneous pouch in the back. After a minimum of 16 h recovery, the saline-filled catheter was removed from the pouch and connected to a Spectramed DTX Plus pressure transducer (Spectramed, Oxnard, CA). Output from the pressure transducer was amplified using a Gould 8-channel recorder and digitized using a Data Translation Series DT2801 analog-digital converter (Marlboro, MA). The digital signal was analyzed using Crystal Biotech Dataflow data acquisition software (Crystal Biotech, Hopkinton, MA) on a Gateway 2000 486DX2 microcomputer (Sioux City, SD). Baseline heart rate and mean arterial blood pressure were recorded after a 1-h equilibration period when the animals were awake but not active. In order to examine drug responses, drugs were administered through the carotid artery catheter. (−)-Isoproterenol hydrochloride (3 μg/kg) and epinephrine bitartrate (3 μg/kg) were purchased from Sigma and dissolved in saline for injection. In order to measure heart rate and blood pressure during exercise, cannulated mice were challenged with a graded treadmill exercise protocol (19Desai K.H. Sato R. Schauble E. Barsh G.S. Kobilka B.K. Bernstein D. Am. J. Physiol. 1997; : H1053-H1061PubMed Google Scholar) on a Simplex II rodent treadmill (Columbus Instruments, Columbus, OH). Treadmill activity was initiated at 3.5 m/min, 0° inclination, and increased to 5 m/min, 2° inclination 3 min later. Treadmill speed and inclination were then increased by 2.5 m/min and 2° inclination every 3 min thereafter. Exercise was terminated after the mice had completed 3 min at 20 m/min, 14° inclination. Mice that failed to complete the exercise protocol were excluded from the study. In order to measure metabolic responses to exercise and exercise capacity, non-instrumented mice were challenged with the graded treadmill exercise protocol described above. Treadmill activity was initiated after the mice had equilibrated in the exercise chamber for 30–60 min. During the exercise protocol, oxygen consumption and carbon dioxide production were continuously monitored with an Oxymax gas analyzer (Columbus Instruments, Columbus, OH). Stepwise increases in treadmill speed and inclination were made every 3 min until the mice stopped running from exhaustion. Exercise capacity was calculated as the total distance run by the animals during the exercise protocol. Male mice, 12–13 weeks old, were used for these studies. Mice were maintained in 12-h light/dark cycles. On the day of study, food was removed from the cage at the beginning of the light cycle, and mice were studied 3–5 h later. Each mouse was weighed and then anesthetized with 5% isofluorane for 45 s in an anesthesia induction box. The mouse was quickly removed from the box, and blood was collected by cardiac puncture with a 22-guage needle. The mouse was then sacrificed via cervical dislocation. The volume of the mouse was determined by attaching a weight to the mouse and measuring the water displacement. Density was calculated as the body weight divided by the volume. After the volume measurement, both epididymal fat pads were dissected from the animal and weighed. The proportional weight of the fat pads was calculated by dividing the fat pad weight by the total body weight. After the blood samples had clotted in serum separator tubes (Becton Dickinson, Franklin Lakes, NJ), the samples were spun at 17,000 × g for 5 min to isolate the serum. Free fatty acid levels were determined with an enzymatic colorimetric kit (Wako Chemicals, Germany). Glycerol levels were determined with an enzymatic colorimetric kit (Roche Molecular Biochemicals). Locomotor activity of male mice, 12–13 weeks old, was measured by a photobeam cage system (San Diego Instruments, San Diego, CA). Mice were studied in pairs with a β2-AR +/+ mouse and a β2-AR −/− mouse placed in individual cages (30 × 50 cm). A frame containing 4 × 6 infrared photobeams was placed around each cage. Mice were placed in the cages at 5 p.m., and their activity was monitored as the number of beam breaks in a 48-h period. The mice used for these studies had not been used for any previous experiments. Using standard ES cell techniques, the R1 ES cell line was transfected with the β2-AR targeting vector shown in Fig. 1 A. Homologous recombinants were identified by performing Southern blot analysis using the 5′ external probe. Targeted clones were rescreened with a probe to the neomycin resistance gene to ensure that a single integration of the targeting vector had occurred (data not shown). Chimeric mice were generated with the targeted ES clones using the morula aggregation technique. Following germ line transmission of the knockout allele, heterozygous knockout pairs were intercrossed to generate β2-AR +/+, β2-AR +/−, and β2-AR −/− mice. Shown in Fig. 1 B is a Southern blot using DNA from the offspring of a β2-AR +/− intercross. After backcrossing β2-AR +/− mice to wild type FVB/N mice for 5 generations, β2-AR +/− mice were intercrossed. From 171 intercross progeny screened at weaning, 36 β2-AR +/+ mice, 91 β2-AR +/− mice, and 44 β2-AR −/− mice were identified. These results are consistent with the ratio predicted by Mendelian genetics (χ-squared = 1.45, p > 0.4). Thus, there is no embryonic or postnatal lethality associated with disruption of the β2-AR gene in mice. After maturing into adults, β2-AR −/− mice appear grossly normal and do not exhibit overtly abnormal behavior. Both β2-AR −/− males and females are fertile. In order to verify that the genetic modification prevents expression of the β2-AR gene, ligand binding experiments were performed using lung tissue isolated from β2-AR +/+ and −/− littermates. Saturation binding experiments with the radioligand [125I]iodocyanopindolol (125I-CYP) demonstrate a reduction in total binding in the β2-AR −/− mice (Fig. 2 A). TheB max is reduced from 990 fmol/mg in β2-AR +/+ mice to 360 fmol/mg in the β2-AR −/− mice (36% of the wild type value). Competition binding experiments were performed using the β2-AR-selective antagonist ICI 118,551 to characterize the residual125I-CYP binding in the β2-AR −/− mice (Fig.2 B). In lung membranes from wild type mice, the data were best fit by a biphasic curve with 62% high affinity ICI 118,551 binding (β2-AR) sites and 38% low affinity (β1-AR) sites. The competition binding data from β2-AR −/− mice were best fit with a one-site curve that has low affinity for ICI 118,551. Thus, the residual 125I-CYP-binding sites in β2-AR −/− lung are due to β1-ARs, confirming the loss of β2-AR-binding sites in β2-AR −/− mice. These data also demonstrate that there has not been a compensatory change in β1-AR expression in the lung as a result of the β2-AR gene disruption; β1-AR expression in β2-AR −/− mice is 330 fmol/mg protein, whereas in β2-AR +/+ mice, β1-AR expression is 380 fmol/mg protein (0.38 × 990 fmol/mg protein). In order to examine the effects of the gene disruption on whole animal physiology, β2-AR +/+ and β2-AR −/− mice were instrumented with carotid catheters to allow measurements of mean arterial blood pressure and heart rate in awake, non-anesthetized, and non-restrained mice. Under baseline resting conditions, mean blood pressure and heart rate were not significantly different between β2-AR +/+ and β2-AR −/− mice (TableI). In order to examine the effects of β-AR stimulation, β2-AR +/+ and β2-AR −/− mice were given an intra-arterial bolus of 3 μg/kg of the non-selective β-AR agonist isoproterenol, a dose previously shown to produce maximal increases in heart rate and maximal reductions in blood pressure in wild type mice. Fig. 3 shows the typical response of a β2-AR +/+ and a β2-AR −/− mouse to isoproterenol. In β2-AR +/+ mice, isoproterenol produced a rapid onset tachycardia and hypotension. In β2-AR −/− mice, the tachycardic response to isoproterenol was preserved, but the hypotensive response was significantly blunted (Fig.3 and Table I).Table ICardiovascular indices at rest and changes in cardiovascular indices after isoproterenol (Iso) and epinephrine (Epi) administration in β2-AR +/+ and −/− miceGenotypeBasal HRBasal BPΔHR Iso stimulatedΔBP Iso stimulatedΔHR Epi stimulatedΔBP Epi stimulatedbpmmm Hgbpmmm Hgbpmmm Hgβ2-AR +/+641 ± 20113.5 ± 2.4141 ± 4023.6 ± 1.719 ± 3213.2 ± 1.9(10)(10)(7)(7)(7)(7)β2-AR −/−602 ± 42114.2 ± 1.9163 ± 5612.6aSignificance at p = 0.0009 for comparing β2-AR +/+ and −/− mice. ± 1.9−22 ± 1727.1bSignificance at p = 0.0027 for comparing β2-AR +/+ and −/− mice. ± 3.2(9)(9)(7)(7)(7)(7)Values shown represent the mean ± S.E. The number of mice studied is shown in parentheses. The isoproterenol-stimulated values represent the maximum changes in blood pressure (BP) and heart rate (HR) after drug administration. The epinephrine-stimulated values represent the maximum blood pressure change and the corresponding heart rate change after drug administration. The unpaired t test was used for statistical comparison between groups.a Significance at p = 0.0009 for comparing β2-AR +/+ and −/− mice.b Significance at p = 0.0027 for comparing β2-AR +/+ and −/− mice. Open table in a new tab Values shown represent the mean ± S.E. The number of mice studied is shown in parentheses. The isoproterenol-stimulated values represent the maximum changes in blood pressure (BP) and heart rate (HR) after drug administration. The epinephrine-stimulated values represent the maximum blood pressure change and the corresponding heart rate change after drug administration. The unpaired t test was used for statistical comparison between groups. Responses to the endogenous catecholamine, epinephrine (a combined α–AR and β-AR agonist), were also significantly different between β2-AR −/− and wild type mice (Fig.4). In both β2-AR −/− and wild type mice, administration of epinephrine produced a transient hypertensive response (blood pressure typically returned to baseline within 1 min). However, the hypertensive response was significantly greater in β2-AR −/− mice than in wild types (Table I and Fig. 4). Heart rate responses in both β2-AR −/− and wild type mice to epinephrine were variable (Table I). Although there was a trend for wild type mice to show heart rate increases while β2-AR −/− showed heart rate decreases, these heart rate responses were not significantly different between genotypes. The effects of exercise on heart rate and blood pressure are shown in Fig. 5. For these experiments, catheterized mice were tested using a graded exercise treadmill protocol. β2-AR −/− and wild type mice showed similar heart rate increases during the exercise protocol. A significant difference, however, was observed in the blood pressure response to exercise. During the exercise protocol, β2-AR −/− mice became hypertensive compared with wild type mice. At the peak exercise level of 20 m/min, β2-AR −/− mice had a mean blood pressure of 139.3 ± 4.4 mm Hg (mean ± S.E.), whereas wild type mice had a mean blood pressure of 126.3 ± 3.3 mm Hg (mean ± S.E.). In a separate set of experiments, metabolic responses to exercise and exercise capacity were measured in uncatheterized mice. Oxygen consumption and carbon dioxide production were continuously monitored while the mice exercised according to a graded treadmill exercise protocol (Fig.6). Oxygen consumption and carbon dioxide production were not significantly different between the two genotypes. However, there was a trend for β2-AR −/− mice to have greater levels of oxygen consumption at any given workload. β2-AR −/− mice had a significantly lower respiratory exchange ratio during exercise than did wild type mice (Fig. 6 C). There was also a significant difference between β2-AR +/+ mice and β2-AR −/− mice in exercise capacity. Interestingly, β2-AR −/− mice exercised significantly longer than wild type control mice (Fig. 6 D). Wild type mice covered 471 ± 22 meters (mean ± S.E.), whereas β2-AR −/− mice covered 582 ± 15 meters (mean ± S.E.) during the graded exercise protocol. To investigate possible mechanisms for the greater exercise capacity in β2-AR −/− mice, we examined body weight, epididymal fat pad weight, body density, and serum levels of free fatty acid (FFA) and glycerol in wild type and β2-AR −/− mice. As shown in TableII, β2-AR −/− mice weigh significantly less than wild type mice. Epididymal fat pads from β2-AR −/− mice also represent a smaller proportion of total body weight than fat pads from wild type mice. Previous studies have shown that the epididymal fat pad weight as a proportion of total body weight is highly correlated with total body fat in mice (20Rogers P. Webb G.P. Br. J. Nutr. 1980; 43: 83-86Crossref PubMed Scopus (119) Google Scholar, 21Eisen E.J. Leatherwood J.M. Growth. 1981; 45: 100-107PubMed Google Scholar). Body density, serum FFA levels, and serum glycerol levels were not significantly different between the two genotypes under base-line conditions.Table IIBody weight, proportional weight of the epididymal fat pads, density, free fatty acid (FFA) levels, and glycerol levels in β2-AR +/+ and β2-AR −/− mice.GenotypeWeightFat padDensityFFAGlycerolg% weightg/mlμmol/litermg/dlβ2-AR +/+32.31 ± 0.653.01 ± 0.270.942 ± 0.0062190 ± 20739.2 ± 8.4(11)(11)(11)(11)(11)β2-AR −/−29.12aSignificance at p = 0.0008 for comparing β2
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