Cardiovascular and Metabolic Alterations in Mice Lacking Both β1- and β2-Adrenergic Receptors
1999; Elsevier BV; Volume: 274; Issue: 24 Linguagem: Inglês
10.1074/jbc.274.24.16701
ISSN1083-351X
AutoresDaniel K. Rohrer, Andrzej Chruscinski, Eric Schauble, Daniel Bernstein, Brian K. Kobilka,
Tópico(s)Ion channel regulation and function
ResumoThe activation state of β-adrenergic receptors (β-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by β-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of β1- and/or β2-AR expression on these homeostatic control mechanisms. Despite total absence of β1- and β2-ARs, the predominant cardiovascular β-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of β-AR function by β-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in β1/β2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single β1- and β2-AR knockouts as well as the β1-/β2-AR double knock-out suggest that in the mouse, β-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the β1-AR, whereas vascular relaxation and metabolic rate are controlled by all three β-ARs (β1-, β2-, and β3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular β3-AR responsiveness are also observed in β1-/β2-AR double knockouts. In addition to its ability to define β-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed. The activation state of β-adrenergic receptors (β-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by β-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of β1- and/or β2-AR expression on these homeostatic control mechanisms. Despite total absence of β1- and β2-ARs, the predominant cardiovascular β-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of β-AR function by β-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in β1/β2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single β1- and β2-AR knockouts as well as the β1-/β2-AR double knock-out suggest that in the mouse, β-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the β1-AR, whereas vascular relaxation and metabolic rate are controlled by all three β-ARs (β1-, β2-, and β3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular β3-AR responsiveness are also observed in β1-/β2-AR double knockouts. In addition to its ability to define β-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed. The β-adrenergic receptors (β1-, β2-, and β3-AR) 1The abbreviations used are: β-Ar(s), β-adrenergic receptor(s); GTE, graded treadmill exercise; Iso, isoproterenol belong to the superfamily of G-protein-coupled receptors (1Strader C.D. Fong T.M. Graziano M.P. Tota M.R. FASEB J. 1995; 9: 745-754Crossref PubMed Scopus (330) Google Scholar). Both sequence comparisons and functional studies suggest that these three receptors share many structural and mechanistic features (2Pepperl D.J. Regan J.W. Peroutka S.J. Handbook of Receptors and Channels. CRC Press, Inc., Boca Raton, FL1994: 45-78Google Scholar). Agonist stimulation of cloned and exogenously expressed β-ARs has demonstrated that all three subtypes can couple through Gαs to stimulate adenylate cyclase activity (3Dixon 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 (864) Google Scholar, 4Frielle 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 (517) Google Scholar, 5Emorine L.J. Marullo S. Briend-Sutren M. Patey G. Tate K. Delavier-Klutchko C. Strosberg A.D. Science. 1989; 245: 1118-1121Crossref PubMed Scopus (958) Google Scholar). Despite these common structural and functional properties, however, individual β-AR subtypes in vivo remain as distinct therapeutic targets due to a number of factors that actually serve to distinguish them. These distinctions include tissue-specific expression patterns, the ability to couple to different G-proteins, pharmacological heterogeneity, and differences in agonist-dependent desensitization (6Strosberg A.D. Obes. Res. 1995; : 501S-505SCrossref PubMed Google Scholar, 7Rohrer D.K. Bohm M. Laragh J.H. Zehender M. From Hypertension to Heart Failure. Springer-Verlag, Berlin1998: 129-158Crossref Google Scholar). β-AR subtypes can be distinguished pharmacologically by synthetic as well as natural ligands. The β1-AR subtype shows little preference for epinephrine or norepinephrine, whereas the β2-AR preferentially interacts with epinephrine (8Lands A.M. Luduena F.P. Buzzo H.J. Life Sci. 1967; 6: 2241-2249Crossref PubMed Scopus (407) Google Scholar, 9Lands A.M. Arnold A. McAuliff J.P. Luduena F.P. Brown T.J. Nature. 1967; 214: 597-598Crossref PubMed Scopus (1649) Google Scholar). More recent experiments demonstrate that the β3-AR (previously termed “atypical”) preferentially interacts with norepinephrine over epinephrine. Synthetic subtype-selective agents have been developed which display much greater selectivity than these endogenous catecholamines. Some typical examples of these would include the antagonists CGP20712A (β1-AR-selective) and ICI118551 (β2-AR-selective) and the agonist CL316243 (β3-AR-selective). Such synthetic compounds have proven invaluable for studying β-AR pharmacology and function (2Pepperl D.J. Regan J.W. Peroutka S.J. Handbook of Receptors and Channels. CRC Press, Inc., Boca Raton, FL1994: 45-78Google Scholar, 10Malinowska B. Schlicker E. Br. J. Pharmacol. 1997; 122: 1307-1314Crossref PubMed Scopus (38) Google Scholar). In vivo, β-ARs are known to modulate a wide range of physiological processes, from cardiac chronotropy and inotropy to vascular and smooth muscle tone, metabolism, and behavior. Functional assignment of β-AR subtype functions using pharmacological tools suggests that the β1-AR is the predominant subtype regulating heart rate and contractility, although at least in the human, β2-ARs are also thought to participate. β2-ARs have been thought to be the predominant subtype mediating the vascular smooth muscle relaxant properties of β-AR agonists. The β3-AR was initially identified and proposed to be the major β-AR subtype controlling lipolysis in adipose tissue. Although these functional divisions are not absolute, they appear to be well conserved across species and serve as a convenient framework for β-AR classification. However, defining β-AR subtype-specific functions in vivo can present significant challenges. Some subtype-selective agents display non-ideal behavior in vivo, either due to poor biodistribution or cross-reactivity with unrelated receptors. Gene disruption, or “knockout” experiments, has proven to be a useful approach in defining adrenergic receptor function in vivo. To date, this technique has been used to disrupt expression of all three α2-AR subtypes, the α1b-AR, the β1-, and the β3-ARs (11Link R.E. Stevens M.S. Kulatunga M. Scheinin M. Barsh G.S. Kobilka B.K. Mol. Pharmacol. 1995; 48: 48-55PubMed Google Scholar, 12Susulic V.S. Frederich R.C. Lawitts J. Tozzo E. Kahn B.B. Harper M.E. Himms-Hagen J. Flier J.S. Lowell B.B. J. Biol. Chem. 1995; 270: 29483-29492Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 13Rohrer D.K. Desai K.H. Jasper J.R. Stevens M.E. Regula D.J. Barsh G.S. Bernstein D. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7375-7380Crossref PubMed Scopus (236) Google Scholar, 14Link R.E. Desai K. Hein L. Stevens M.E. Chruscinski A. Bernstein D. Barsh G.S. Kobilka B.K. Science. 1996; 273: 803-805Crossref PubMed Scopus (428) Google Scholar, 15MacMillan L.B. Hein L. Smith M.S. Piascik M.T. Limbird L.E. Science. 1996; 273: 801-803Crossref PubMed Scopus (449) Google Scholar, 16Cavalli A. Lattion A. Hummler E. Nenninger M. Pedrazzini T. Aubert J. Michel M.C. Yang M. Lembo G. Vecchione C. Mostardini M. Schmidt A. Beermann F. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11589-11594Crossref PubMed Scopus (270) Google Scholar), and most recently, the β2-AR (17Chruscinski A.J. Rohrer D.K. Schauble E. Desai K.H. Bernstein D. Kobilka B.K. J. Biol. Chem. 1999; 274 (acc art 568292): 16694-16700Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). When the pharmacologic tools outlined above are used in conjunction with genetic techniques, the power to reveal novel functions and mechanisms of action can be greatly enhanced. Given the prominent role of β-AR signaling in the maintenance of normal physiology in vivo, we sought to test the functional consequences of β-AR gene disruption via a combinatorial approach. In the companion article (17Chruscinski A.J. Rohrer D.K. Schauble E. Desai K.H. Bernstein D. Kobilka B.K. J. Biol. Chem. 1999; 274 (acc art 568292): 16694-16700Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar), the functional consequences of β2-AR disruption are described. We have previously described the functional consequences of β1-AR gene disruption (13Rohrer D.K. Desai K.H. Jasper J.R. Stevens M.E. Regula D.J. Barsh G.S. Bernstein D. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7375-7380Crossref PubMed Scopus (236) Google Scholar, 18Rohrer D.K. Schauble E.H. Desai K.H. Kobilka B.K. Bernstein D. Am. J. Physiol. 1998; 274: H1184-H1193PubMed Google Scholar). We have now produced mice that lack both β1- and β2-ARs. The role of these two β-AR subtypes and the inferred role of the remaining β3-AR subtype in cardiovascular physiology and metabolism are reported here. The generation of β1-AR knock-out mice has been previously described (13Rohrer D.K. Desai K.H. Jasper J.R. Stevens M.E. Regula D.J. Barsh G.S. Bernstein D. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7375-7380Crossref PubMed Scopus (236) Google Scholar). Briefly, disruption of the β1-AR gene was achieved using a positive-negative selection strategy to effect homologous recombination in the R1 embryonic stem cell line, using a targeting construct in which over 90% of the coding sequence was deleted. The strain background of β1-AR knockout mice was a mixture of 129SvJ, C57Bl6/J, and DBA/2 which is less prone to the prenatal mortality previously described (13Rohrer D.K. Desai K.H. Jasper J.R. Stevens M.E. Regula D.J. Barsh G.S. Bernstein D. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7375-7380Crossref PubMed Scopus (236) Google Scholar). The targeting strategy used to create β2-AR knockout mice is described in the companion article (17Chruscinski A.J. Rohrer D.K. Schauble E. Desai K.H. Bernstein D. Kobilka B.K. J. Biol. Chem. 1999; 274 (acc art 568292): 16694-16700Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar) and is based on a similar positive-negative selection scheme and homologous recombination in the R1 embryonic stem cell line. Combination β1/β2-AR double knockouts were generated by mating β2-AR homozygous knockouts (on a combined 129SvJ and FVB/N mouse strain background) to homozygous β1-AR knockouts. The resulting F1 generation of compound heterozygotes was subsequently intercrossed to generate F2 mice with all possible combinations of β1- and β2-AR gene disruptions. According to Mendelian inheritance, 1/16 of progeny were predicted to be homozygous-deficient for β1- and β2-AR, and 1/16 of progeny were predicted to be wild type for both β1- and β2-AR (see Table I). The F2 β1/β2-AR double knockouts were bred to produced to double knockouts used in our experiments. The wild type F2 mice were bred to produce wild type controls. Thus, the overall strain contributions between wild type and β1/β2-AR double knockouts were equivalent. Mice were genotyped for both β1- and β2-AR disruptions by Southern blotting of mouse tail biopsies (13Rohrer D.K. Desai K.H. Jasper J.R. Stevens M.E. Regula D.J. Barsh G.S. Bernstein D. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7375-7380Crossref PubMed Scopus (236) Google Scholar, 17Chruscinski A.J. Rohrer D.K. Schauble E. Desai K.H. Bernstein D. Kobilka B.K. J. Biol. Chem. 1999; 274 (acc art 568292): 16694-16700Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar).Table IFrequency of viable pups at weaning from β1 +/−:β2 +/− × β1 +/−:β2 +/− intercrossesβ1+/++/++/++/−+/−+/−−/−−/−−/−Totalsβ2+/++/−−/−+/++/−−/−+/++/−−/−Observed99513191417683Expected5.1910.385.1910.3820.7510.385.1910.385.19χ22.800.180.010.660.151.273.381.100.139.67The nine possible genotypes are listed together with the number of recovered viable pups. Expected values are derived from Mendelian inheritance patterns; χ2 = Σd 2/E, where d is expected number − observed number, and E is expected number, with 8 degrees of freedom. Open table in a new tab The nine possible genotypes are listed together with the number of recovered viable pups. Expected values are derived from Mendelian inheritance patterns; χ2 = Σd 2/E, where d is expected number − observed number, and E is expected number, with 8 degrees of freedom. Catheters were surgically implanted in either the left carotid artery or the left carotid artery plus the left jugular vein under isoflurane anesthesia. Briefly, anesthesia was induced with 3% (v/v) isoflurane in oxygen using an isoflurane vaporizer (Airco Inc., Madison, WI), and then induction was maintained at 1.25–1.75% while monitoring the responsiveness of the animal. The vessels were cannulated with a stretched Intramedic PE10 polyethylene catheter (Clay Adams, Parsippany, NJ), which was filled with heparinized normal saline, sutured in place, and tunneled to the back. Blood pressure was measured using a DTX Plus pressure transducer (Spectramed, Oxnard, CA) amplified with a Gould 8-channel recorder, and the analog pressure was digitized using a Data Translation Series DT2801 analog-digital converter (Marlboro, MA). Digital signals were analyzed and stored using Crystal Biotech Dataflow data acquisition software (Crystal Biotech, Hopkington, MA). Heart rate measurements were determined on-line and were derived from the pressure recordings. Drugs were infused through the arterial catheter as a bolus in a volume of 1–3 μl/g. (−)-Isoproterenol hydrochloride (3 μg/kg), atropine sulfate (1 mg/kg), epinephrine bitartrate (3 μg/kg), and sodium nitroprusside (30 μg/kg) were purchased from Sigma. CL316243 (100 μg/kg) was a kind gift of Wyeth Ayerst Laboratories (Philadelphia, PA). A 1.4 French micromanometer-tipped Millar pressure transducer (Millar Instruments, Houston, TX) was advanced into the left ventricle via the right carotid artery under isoflurane anesthesia (see above). Correct placement of the catheter in the ventricle was judged by loss of the arterial waveform and transition to a waveform with similar peak systolic pressure, but diastolic pressures with minima in the 0–5 mm Hg range. Following correct placement, a jugular venous catheter was placed via the left jugular vein and advanced ∼1 cm. The surgical incision was then sutured closed with 4-0 silk, and the mouse was allowed to stabilize for 10–15 min at 2.5% isoflurane. Pressure recordings were measured using a MacLab 8S digitizer/amplifier (MacLab, Milford, MA), recorded, and analyzed using MacLab/s version 3.5 software on a Macintosh 3400c. “Anesthetized” recordings of ventricular function were taken during a 1-min interval at the end of this 10–15-min equilibration period, at 2.5% isoflurane. Isoflurane anesthesia was then reduced in a stepwise fashion, from 2.5 to 1.25%, and mice were allowed to stabilize for 10 min. Following this period, isoflurane anesthetic was turned off, and the mouse was removed from the anesthetic nose cone and placed on its back. Upon self-righting (or the “awakening” state), mice were quickly euthanized with an intravenous dose of avertin. Awakening recordings of ventricular function were taken in the 30–60 s prior to the righting response. Mice were subjected to either constant or graded treadmill exercise, using a Columbus Instruments Simplex II metabolic rodent treadmill, fitted with Oxymax oxygen and carbon dioxide gas analyzers (Columbus Instruments, Columbus, OH). For graded exercise, mice were placed in the exercise chamber and allowed to equilibrate (usually 30–60 min). 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. Pre-operatively, mice were initially subjected to this protocol, with regular stepwise increases until mice stopped running from exhaustion. Post-operatively, mice were run to a final end point of 20 m/min and 14° inclination. We have previously shown linear relationships between heart rate, VO2 and VCO2during graded treadmill exercise in mice (19Desai K.H. Sato R. Schauble E. Barsh G.S. Kobilka B.K. Bernstein D. Am. J. Physiol. 1997; 272: H1053-H1061Crossref PubMed Google Scholar). The right ventricular free wall was dissected away from the left ventricle and interventricular septum, and silk sutures were tied at both ends of the long axis. Ventricles were placed in an oxygenated 32 °C tissue bath containing modified Krebs solution (118 mm NaCl, 5.4 mm KCl, 2.5 mm CaCl2, 0.57 mmMgSO4, 1.0 mm Na2HPO4, 2.5 mm NaHCO3, 11.1 mmd-glucose). Ventricles were paced at 3.3 Hz by use of a Grass stimulator (30-ms pulse duration, 8–15 V). Signals from isometric force transducers were amplified and digitized with a MacLab 8S series amplifier and fed to MacLab/s version 3.5 software running on a Macintosh 3400c to determine twitch amplitude. For spontaneously beating atria, right and left atria were dissected free of ventricular tissue, and both atrial appendages were tied with 4-0 silk sutures. These were placed in an oxygenated 32 °C tissue bath, where isometric force transduction and rate were monitored as above. Ventricular homogenates were prepared by Polytron homogenization of whole organs in 5 mmTris-Cl, 5 mm EDTA, pH 7.4, followed by centrifugation at 10,000 × g. The resultant pellet was resuspended in 1× binding buffer (for α-adrenergic receptor binding, 150 mm NaCl, 50 mm Tris-Cl, 5 mm EDTA, pH 7.4; for muscarinic receptor binding, 75 mm Tris-Cl, 12.5 mm MgCl2, 1 mm EDTA, pH 7.4), and protein concentration was determined. For saturation binding, 50–100 μg of homogenate protein was used in a 500-μl reaction containing 300 pm125I-2-[β-(4-hydroxyphenyl)-ethyl-aminomethyl]tetralone or 300 pm [3H]N-methyl scopolamine (both from NEN Life Science Products). Nonspecific binding was performed in duplicate with 20 μm prazosin (Research Biochemicals, Natick, MA) or 5 μm atropine sulfate, respectively (Sigma). All binding reactions were carried out at room temperature for ∼2 h prior to vacuum filtration onto Whatman GF-C filters and determination of membrane-bound radioligand. The generation and viability of β1-AR knockout mice (β1-AR −/−) have been described previously (13Rohrer D.K. Desai K.H. Jasper J.R. Stevens M.E. Regula D.J. Barsh G.S. Bernstein D. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7375-7380Crossref PubMed Scopus (236) Google Scholar). Briefly, homozygous β1-AR knockouts derived from heterozygote:heterozygote matings (β1-AR +/− × β1-AR +/−) are recovered at an unexpectedly low frequency as predicted from Mendelian inheritance, although this effect can be ameliorated if the β1-AR gene disruption is bred onto a multiple strain background. As described by Chruscinski et al. (17Chruscinski A.J. Rohrer D.K. Schauble E. Desai K.H. Bernstein D. Kobilka B.K. J. Biol. Chem. 1999; 274 (acc art 568292): 16694-16700Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar), the recovery of homozygous β2-AR knockouts (β2-AR −/−) is in accord with expected Mendelian frequencies. Crosses were carried out between homozygous β1-AR knock-outs and homozygous β2-AR knockouts to generate compound heterozygotes (β1-AR +/−:β2-AR +/−, see “Materials and Methods”), and these in turn were intercrossed to generate homozygous β1- and β2-AR double knockout mice (β1-AR −/−:β2-AR −/−). The expected frequency of recovering double knockout mice from compound heterozygote matings is 1 out of 16 or 6.25%. The observed frequency among weanlings was 7.23%, well within the expected range. TableI lists the expected and observed frequencies among the nine possible genotypes arising from the compound heterozygote intercrosses. The χ2 distribution suggests that there are no significant deviations from Mendelian expectations either among individual genotypes or the group as a whole (χ2 = 9.38 with 8 degrees of freedom, p = 0.29), although β1-AR knockouts (β1-AR −/−:β2-AR +/+) appear to be less well represented, in accord with our previous findings (13Rohrer D.K. Desai K.H. Jasper J.R. Stevens M.E. Regula D.J. Barsh G.S. Bernstein D. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7375-7380Crossref PubMed Scopus (236) Google Scholar). Double knockout:double knockout matings were subsequently performed to generate mice for the studies reported here. Litter size, maternal behavior, and pup viability all appeared to be normal in this group. Basal cardiovascular parameters were measured in awake, unrestrained mice by use of indwelling carotid arterial catheters. As seen in Fig. 1, neither baseline heart rate (range 400–470 beats/min) nor mean arterial blood pressure (range 115–125 mm Hg) are significantly different when comparing wild type mice (β1-AR +/+:β2-AR +/+) to double knockouts (β1-AR −/−:β2-AR −/−). Both isoproterenol and epinephrine were administered to wild type and β1/β2-AR double knockout mice. The grouped response to these agents is shown in Fig.2 A. Whereas the non-selective β-AR agonist isoproterenol (3 μg/kg) elicits robust chronotropic and hypotensive responses in wild types, both of these responses are severely attenuated in β1/β2-AR double knockout mice. Of note, both responses are also significantly time-delayed in β1/β2-AR double knockouts in comparison to wild type responses. Furthermore, the small but significant increase in heart rate seen in double knockout mice in response to isoproterenol was attenuated by 93% in mice pretreated with the muscarinic antagonist atropine (1 mg/kg, data not shown), suggesting that the majority of this effect is due to the baroreflex, mediated by the vagus in response to the drop in blood pressure. The effect of epinephrine (3 μg/kg) on β1/β2-AR double knockouts is seen in the right-hand panel of Fig. 2 A. This endogenous catecholamine is a mixed, non-selective α-AR and β-AR agonist. In wild types, this dose of epinephrine elicits tachycardia and a biphasic blood pressure response consisting of an initial brief hypertension followed by a more prolonged hypotensive response. In contrast, ablation of β1- and β2-AR signaling in the double knockout appears to convert this mixed α- and β-AR agonist into a selective α-AR agonist; these mice display concomitant bradycardia and a monophasic hypertensive blood pressure response. Again, the heart rate response to epinephrine seen in double knockouts appears to be predominantly due to baroreflex stimulation, as atropine pretreatment blocks 60% of this response (data not shown). Fig. 2 B is a compilation of the chronotropic and hemodynamic effects of isoproterenol on conscious and unrestrained β1-AR knockouts, β2-AR knockouts, and β1/β2-AR double knockouts. These are all displayed relative to the response seen in wild type mice (dotted line at 100%) and represent the peak chronotropic and vasodilatory responses obtained in each genotype, respectively. Based on these data, ∼50% of the chronotropic response to isoproterenol is lost when the β1-AR is knocked out, whereas there is no detrimental effect on heart rate in β2-AR knockouts. The combined β1- and β2-AR deficiency reduces the chronotropic response by over 85%. In terms of the vasodilatory response to isoproterenol, there appears to be a graded and additive attenuation of the hypotensive response with loss of the β1-AR (20% reduction), β2-AR (35% reduction), and combined β1-/β2-AR (71% loss). The hemodynamic response to the β3-AR agonist CL316243 was tested in β-AR knockout mice to clarify the role of the β3-AR in the regulation of peripheral vasodilatory responses in vivo. Infusion of the β3-AR-selective agonist was followed by infusion of the non-selective β-AR agonist isoproterenol, to ascertain residual β1- and/or β2-AR responsiveness. Both drugs were used at doses that elicit maximal responses in vivo (19Desai K.H. Sato R. Schauble E. Barsh G.S. Kobilka B.K. Bernstein D. Am. J. Physiol. 1997; 272: H1053-H1061Crossref PubMed Google Scholar, 20Shen Y.T. Cervoni P. Claus T. Vatner S.F. J. Pharmacol. Exp. Ther. 1996; 278: 1435-1443PubMed Google Scholar). As can be seen in the top panel of Fig. 3, administration of CL316243 at 100 μg/kg to a wild type mouse leads to a gradual but sustained hypotensive response. Near-maximal responses to this agonist were observed after 10 min, at which time isoproterenol was infused. These results clearly show that a residual β1- and/or β2-AR vasodilatory response can be elicited by isoproterenol even while β3-ARs are maximally stimulated. The time course of β3-AR-mediated vasodilatation suggests that the duration of action as well as the time interval to peak response is much longer for the β3-AR response to CL316243 than for the β1/β2-AR response to isoproterenol. This appears to be unique to the response mediated by β3-ARs and not specific to CL316243, as isoproterenol given to β1/β2-AR double knockouts also exhibits a similar time lag (Fig. 2 A). The bottom panel of Fig. 3 summarizes identically performed experiments on wild type, β1-, β2-, and combination β1-/β2-AR knockout mice. Interestingly, the response to the β3-AR agonist CL316243 alone was significantly augmented in the β1/β2-AR double knockouts. Furthermore, the effect of isoproterenol following β3-AR stimulation (residual response) revealed that mice lacking β1-ARs showed no deficit in the residual vasodilatory response, whereas loss of β2-ARs had a large impact on further vasodilatory responses. Surprisingly, when both β1- and β2-ARs were lacking, isoproterenol infusion actually had a small hypertensive effect. Such a response can be due to either injection artifact or cross-reactivity to α-ARs. In either case, these results suggest that all three β-ARs can mediate vasodilatory responses in vivo and that an enhancement of β3-AR responsiveness is seen in mice lacking both β1- and β2-ARs. We also tested the role of β1- and β2-AR signaling on the response to the physical stress of exercise. Knowing that β-ARs are recruited during exercise to modulate heart rate, hemodynamics, airway conductance, and metabolic rate, we hypothesized that mice lacking both β1- and β2-ARs would be compromised in both exercise capacity as well as the cardiovascular and metabolic response to exercise. Using graded treadmill exercise (GTE) as a stimulus, where both speed and angle of inclination are progressively increased, both wild type mice and β1/β2-AR double knockouts were tested for total exercise capacity as well as the physiological response to fixed end point GTE. Total exercise capacity was measured as cumulative distance run in non-instrumented mice, with treadmill speed and angle of inclination increasing by 2.5 m/min and 2° every 3 min until mice stopped running from exhaustion. Physiological responses to fixed end point GTE were obtained by running instrumented mice to a final end point of 20 m/min and 14° inclination (see “Materials and Methods”). Experiments designed to test total exercise capacity showed no significant differences between wild types and β1/β2-AR double knockouts with respect to cumulative distance run. Wild type mice ran a total distance of 578.8 ± 33.3 m (n = 7), whereas β1/β2-AR double knockouts ran a total distance of 545.2 ± 30.0 m (n = 5). The metabolic response to GTE in the maximal exercise capacity experiment is shown in Fig. 4B, demonstrating that whereas both wild types and β1/β2-AR double knockouts have virtually identical levels of O2 consumption and CO2 production at rest, consistent deficits in both of these indices are revealed at all exercise levels in the double knockout. This metabolic deficit appears to result from the combined deficiency of β1- and β2-ARs, since neither the β1-AR knockout nor the β2-AR knockout display such deficits (17Chruscinski A.J. Rohrer D.K. Schauble E. Desai K.H. Bernstein D. Kobilka B.K. J. Biol. Chem. 1999; 274 (acc art 568292): 16694-16700Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 18Rohrer D.K. Schauble E.H. Desai K.H. Kobilka B.K. Bernstein D. Am. J. Physiol. 1998
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