Targeted Disruption of the β2 Adrenergic Receptor Gene

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) 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, 125Iiodocyanopindolol 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. PubMed Scopus Google Scholar). In the targeting vector of to the endogenous β2-AR genomic The gene for the β2-AR was in the targeting vector by a gene into the sequence at a P. A. 1991; PubMed Scopus Google Scholar). This the β2-AR at the of the and a The of the targeting vector was a from a to the in the The of the targeting vector from the in the to a in the vector was the to for when P. A. 1991; PubMed Scopus Google Scholar). In order to for a probe was This probe is a that a mouse genomic is with and to In where the targeting vector has with the endogenous the probe an at embryonic A. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) were using techniques were on a of mouse embryonic in tissue with and and of Life were in an at in the a of was with of targeting vector with for in and individual were and in from was by with the were isolated from were also with a probe to that a of the targeting vector had mice were using the J. A. A. Nature. PubMed Scopus Google Scholar). at were isolated from of mice by the with the with an the were in in a tissue and with a of of was the of with the disruption were into the and in with the an at in the were to the of mice were identified in the by the presence of were to mice to for of the β2-AR mice were to β2-AR and −/− mice for in in vivo the knockout was on a by β2-AR mice to wild type mice for to in were from wild type and knockout in a and with a The was isolated by at and in were by with the 125Iiodocyanopindolol Life in a at was and the were in a of was with was in the presence of were with of and of the and were with In were as K.H. E. Barsh G.S. Kobilka B.K. Bernstein D. Am. J. Physiol. 1997; : Google Scholar). Adult mice of were with using a and a was into the The was the and in a in the a of the was from the and to a pressure from the pressure was using a and using a The signal was using on a heart rate and blood pressure were a when the were not In order to were the and epinephrine were from and in for In order to heart rate and blood pressure during exercise, mice were with a exercise K.H. E. Barsh G.S. Kobilka B.K. Bernstein D. Am. J. Physiol. 1997; : Google Scholar) on a was at and increased to and were increased by and was the mice had at that to the exercise were from the In order to responses to exercise and exercise mice were with the exercise was the mice had in the exercise for During the exercise and were with an in and were the mice from capacity was as the total by the during the exercise mice, were used for these were in the of was from the at the of the and mice were mouse was and with for in an The mouse was from the and blood was by cardiac with a The mouse was The of the mouse was by a to the mouse and the was as the divided by the the both were from the and The of the was by the by the total the blood had in the were at for to the fatty levels were with an levels were with an of mice, was by a were in with a β2-AR mouse and a β2-AR −/− mouse in individual was were in the at and their was as the of in a The mice used for these studies had not been used for any Using techniques, the was with the β2-AR targeting vector shown in A. were identified by using the were with a probe to the gene to that a of the targeting vector had not mice were with the using the of the knockout heterozygous knockout were to β2-AR β2-AR and β2-AR −/− mice. in is a using from the of a β2-AR β2-AR mice to wild type mice for β2-AR mice were at β2-AR mice, β2-AR mice, and β2-AR −/− mice were are with the ratio by there is no embryonic or lethality associated with disruption of the β2-AR gene in mice. into β2-AR −/− mice appear grossly normal and do not exhibit β2-AR −/− and are fertile. In order to that the of the β2-AR were using tissue isolated from β2-AR and −/− with the 125Iiodocyanopindolol a in total in the β2-AR −/− mice is from in β2-AR mice to in the β2-AR −/− mice of the wild type were using the to the in the β2-AR −/− mice In from wild type mice, the were by a with and The from β2-AR −/− mice were with a that has for the in β2-AR −/− are to β1-ARs, the of in β2-AR −/− mice. also that there has not been a in β1-AR in the as a result of the β2-AR gene β1-AR in β2-AR −/− mice is in β2-AR mice, β1-AR is In order to the effects of the gene disruption on physiology, β2-AR and β2-AR −/− mice were with to of blood pressure and heart rate in and mice. resting blood pressure and heart rate were not significantly different between β2-AR and β2-AR −/− mice In order to the effects of β-AR stimulation, β2-AR and β2-AR −/− mice were given an of of the β-AR agonist isoproterenol, a shown to in heart rate and in blood pressure in wild type mice. the typical response of a β2-AR and a β2-AR −/− mouse to isoproterenol. In β2-AR mice, a and In β2-AR −/− mice, the response to was the hypotensive response was significantly blunted and at and in cardiovascular and epinephrine in β2-AR and −/− at for β2-AR and −/− mice. at for β2-AR and −/− mice. shown represent the The of mice is shown in The represent the in blood pressure and heart rate The represent the blood pressure and the heart rate The was used for between at for β2-AR and −/− at for β2-AR and −/− mice. in a shown represent the The of mice is shown in The represent the in blood pressure and heart rate The represent the blood pressure and the heart rate The was used for between to the endogenous epinephrine and β-AR were also significantly different between β2-AR −/− and wild type mice In both β2-AR −/− and wild type mice, of epinephrine a hypertensive response pressure to However, the hypertensive response was significantly greater in β2-AR −/− mice than in wild and rate responses in both β2-AR −/− and wild type mice to epinephrine were Although there was a for wild type mice to heart rate β2-AR −/− heart rate these heart rate responses were not significantly different between The effects of exercise on heart rate and blood pressure are shown in these mice were using a exercise β2-AR −/− and wild type mice heart rate during the exercise however, was observed in the blood pressure response to exercise. During the exercise β2-AR −/− mice hypertensive compared with wild type mice. At the exercise of β2-AR −/− mice had a blood pressure of wild type mice had a blood pressure of In a of responses to exercise and exercise capacity were in mice. and were the mice to a exercise and were not significantly different between the However, there was a for β2-AR −/− mice to have greater levels of at any given β2-AR −/− mice had a significantly lower respiratory exchange ratio during exercise than wild type mice was also a difference between β2-AR mice and β2-AR −/− mice in exercise β2-AR −/− mice significantly than wild type mice type mice β2-AR −/− mice during the exercise investigate for the greater exercise capacity in β2-AR −/− mice, we and levels of free fatty and in wild type and β2-AR −/− mice. As shown in β2-AR −/− mice significantly than wild type mice. from β2-AR −/− mice also represent a smaller of total than from wild type mice. studies have shown that the as a of total is with total in mice P. J. 43: PubMed Scopus Google Scholar, J.M. Google Scholar). and levels were not significantly different between the of the free fatty and levels in β2-AR and β2-AR −/− at for β2-AR to −/− mice. at for β2-AR and −/− mice. shown represent the for β2-AR and β2-AR −/− mice. The was used for between The in the of mice used for the at for β2-AR to −/− at for β2-AR and −/− mice. in a shown represent the for β2-AR and β2-AR −/− mice. The was used for between The in the of mice used for the studies were to the observed in exercise and in β2-AR and β2-AR −/− mice can be by in the of As shown in there was no difference in between the a were observed when the was into and not Using gene targeting techniques we have mice that have a disruption in the β2-AR Based on this of the β2-AR. β2-ARs do not appear to play a role in prenatal there is no embryonic lethality associated with the mutation. β2-AR −/− mice appear grossly normal and are that β2-ARs are not required for or for normal reproductive function. is no of β1-ARs in the of β2-AR −/− mice. of the in β2-AR −/− mice was to define further the roles of the three different β-AR subtypes in the cardiovascular system. has been used to that β1-AR and not β2-AR stimulation cardiac and in the murine heart (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). β2-ARs are in the of β1-AR −/− mice, stimulation of β2-ARs not to in cardiac function in in In in β2-AR −/− mice, normal heart rate responses are observed in response both to and to exercise, further that the β1-AR the major role in regulating cardiac function in the pharmacological studies have suggested that the β2-AR is the β-AR subtype that vascular smooth muscle relaxation (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). relaxation leads to a in total peripheral and is by a hypotensive blood pressure In β2-AR −/− mice, the hypotensive response to is significantly blunted compared with wild type mice, that β2-ARs play a role in peripheral vascular The fact that hypotensive responses are still in β2-AR −/− mice, however, that other β-AR subtypes also play a a smaller in regulating peripheral vascular tone in the This response could also be to an of other β-AR subtypes in response to of the β2-AR however, there was no for of the β1-AR in the the tissue with the of β2-ARs in wild type mice. Although β1-ARs are to be the cardiac some studies that β1-ARs can mediate vascular β1-ARs have been shown to be in vascular relaxation in the isolated and J. Pharmacol. Exp. Ther. Google Scholar). β1-ARs have also been in vascular relaxation in the from a variety of species D.J. R.E. J. Proc. Pharmacol. Google Scholar, J. Pharmacol. 1985; PubMed Scopus Google Scholar, J. Pharmacol. PubMed Scopus Google Scholar, T. J. Pharmacol. Exp. Ther. Google Scholar). Although these are not to a to the total peripheral studies on have also suggested that β1-ARs may regulate in the peripheral vasculature S.F. Knight D.R. Hintze T.H. Am. J. Physiol. 1985; : H49-H56PubMed Google Scholar). β3-ARs, are in adipose have been shown to play a role in regulating peripheral (11Shen Y.T. Zhang H. Vatner S.F. J. Pharmacol. Exp. Ther. 1994; 268: 466-473PubMed Google Scholar), this was Y.T. Cervoni P. Claus T. Vatner S.F. J. Pharmacol. Exp. Ther. 1996; 278: 1435-1443PubMed Google Scholar). β1-AR −/− β2-AR −/− mice, further into β3-AR subtype function in the peripheral vasculature has been D.K. A. Bernstein D. Kobilka B.K. J. PubMed Scopus Google Scholar). In β2-AR −/− mice, we were also in the role of β2-ARs in O.E. J. 1992; PubMed Scopus Google Scholar). is a with and vascular relaxation may to this by in total peripheral vascular In of vascular relaxation has been shown to be R.D. J. Physiol. Pharmacol. 1987; PubMed Scopus Google Scholar). is not from these however, β-AR is a primary of the or is a to of β-ARs by catecholamines PubMed Scopus Google Scholar). vascular relaxation is not only in also in with H. M. A.J. J. PubMed Scopus Google Scholar). with have suggesting that β-AR in the and may be in H. M. A.J. J. PubMed Scopus Google Scholar). human population has that of the β2-AR is associated with a to E. A. P. H. J.P. PubMed Scopus Google Scholar). in may be a to the of In the β2-AR −/− mouse we had the to or not β2-AR can to a hypertensive we that β2-AR −/− mice are compared with wild type mice. β2-AR stimulation may not be in regulating resting blood pressure in mice. that β2-AR −/− mice have had the disruption of the β2-AR gene it is also that there have been other that the of resting blood pressure During the stress of exercise, however, β2-AR −/− mice become hypertensive compared with wild type mice. As an endogenous for exercise may to mediated in the β2-AR −/− mice. In of this of epinephrine to the β2-AR −/− mice the hypertensive As a result of the disruption of the β2-AR stimulation may and the to in where endogenous catecholamines are In with the of In the we at of studies be required to the β2-AR −/− mice as they β2-ARs are known to play a role in the response to stress Am. J. 1985; PubMed Scopus Google Scholar). investigate the effects of the knockout mouse on physiologic responses to exercise and exercise capacity were in β2-AR −/− and wild type mice. β2-AR −/− mice for a than wild type mice for this difference in exercise capacity is that there are alterations in energy to of the β2-AR In of this is the that β2-AR −/− mice have a lower and the respiratory exchange ratio was significantly lower in β2-AR −/− mice at any given workload than in wild type mice during exercise. Although was between wild type and β2-AR −/− mice, there was a higher in the β2-AR −/− mice. This for increased during exercise may be by the lower in β2-AR −/− mice. can be to be in can also have an on and when these are to total in however, not the in exercise in the β2-AR −/− mice. is a ratio of to are by a is an of and is required to than a lower in β2-AR −/− mice that they may a greater ratio of to than do wild type mice during exercise. This could also the lower in β2-AR −/− mice. studies G. Am. J. Physiol. 1985; : Google Scholar, J. Physiol. PubMed Google Scholar) have that β-AR stimulation leads to during exercise. the knockout mice were to result in a defect in of β2-AR −/− mice may during exercise. of the during exercise muscle and may be for the of exercise not in fatty and in β2-AR −/− mice however, it is that in during exercise. may also be by the β2-AR gene has been shown that catecholamines β2-AR receptors G. Am. J. Physiol. 1985; : Google Scholar, J. Physiol. PubMed Google Scholar). β2-AR −/− mice may have lower levels for a given may to the in exercise capacity of β2-AR −/− mice. may be in the of cardiac between and peripheral during exercise of alterations in In the β2-AR −/− the defect in may the in flow to thereby a of the cardiac to be to skeletal muscle. The increased exercise capacity and of β2-AR −/− mice could an of in these mice. However, β2-AR and β2-AR −/− mice levels In summary, we have mice that have a disruption in the β2-AR mice have normal resting heart rate and blood pressure in response to epinephrine or to the cardiovascular stress by exercise. β2-AR knockout mice to be a for further the roles of β-AR subtypes in cardiovascular, respiratory, metabolic, central nervous system, and reproductive functions. A. for the embryonic and J. M. for the of the mouse β2-AR genomic