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2-adrenoceptor exhibits blunted desensitization of cardiac functional responses in vivo
1Departments of Pathophysiology and Nephrology, University of Essen Medical School, D-45147 Essen; and 2Institute of Pharmacology and 3Department of Anesthesiology, Martin-Luther-University of Halle, D-06097 Halle, Germany
Submitted 10 April 2003 ; accepted in final form 8 July 2003
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ABSTRACT |
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2-adrenoceptor (2AR) polymorphism, cardiac responses to 2AR agonist stimulation are blunted. In this study, we investigated agonist-induced desensitization of Thr164Ile 2ARs. For this purpose, we assessed in six subjects with heterozygous Thr164Ile 2ARs and in 10 subjects with homozygous wild-type (WT) 2ARs the effects of 2-wk oral treatment with 3 x 5 mg/day terbutaline on terbutaline infusion-induced increases in heart rate (HR) and contractility [measured as shortening of HR-corrected duration of electromechanical systole (QS2c)]. Compared with WT 2AR subjects, Thr164Ile subjects exhibited a blunted terbutaline-induced maximum increase in HR (WT 32 ± 4 beats/min, Thr164Ile 19 ± 3 beats/min, P < 0.05) and contractility (WT –54 ± 2 ms, Thr164Ile –37 ± 6 ms, P < 0.05). Two-week oral terbutaline treatment desensitized cardiac 2AR responses to terbutaline infusion (increase in HR: WT 10 ± 2 beats/min, Thr164Ile 8 ± 4 beats/min; increase in contractility: WT –22 ± 5 ms Thr164Ile: –17 ± 6 ms); however, the extent of desensitization was larger in WT than Thr164Ile 2AR subjects. Thus, after 2-wk oral terbutaline treatment cardiac, 2AR responses did not differ anymore between WT and Thr164Ile 2AR subjects. We conclude that agonist-induced desensitization of cardiac 2ARs is more pronounced in WT than Thr164Ile subjects. Thus cardiac Thr164Ile subjects appear to be somewhat protected against agonist-induced desensitization.
genetic polymorphism; heart rate; contractility
2-ADRENOCEPTORS (2ARs) are polymorphic (13). In vitro studies have shown that Thr164Ile 2AR polymorphism exhibits decreased affinity for 2AR agonists, a 50% reduction in agonist-induced adenylyl cyclase activity, and uncoupling of the receptor from the G protein (10). In humans, this polymorphism has been detected in 
4% of the population, but only in the heterozygous form (13). We (4) have recently shown that in healthy volunteers heterozygous for Thr164Ile 2AR polymorphism, increases in heart rate and contractility evoked by cardiac 2AR agonist stimulation were blunted compared with volunteers carrying the wild-type (WT) 2AR.
There are two other major coding sequence polymorphisms of the 2AR: Arg16Gly and Gln27Glu (13). In vitro studies have shown that the functional properties of these variants do not differ from those of the WT 2AR; however, both polymorphisms differ from the WT 2AR in their susceptibility to agonist-induced downregulation: Arg16Gly polymorphism shows increased, and Gln27Glu polymorphism shows reduced, agonist-induced downregulation (11). In vivo, however, in healthy volunteers, we have recently found that the extent of cardiac 2AR desensitization after a 2-wk oral treatment with 3 x 5 mg/day terbutaline was nearly identical in subjects with the Arg16Gly or Gln27Glu 2AR polymorphism (7), in contrast to what has been observed in vitro (11).
It is not known whether and to what extent the Thr164Ile 2AR (which exhibited blunted cardiac 2AR responses, see above) undergoes agonist-induced desensitization. To answer this question, in the present study, we assessed in 6 healthy volunteers heterozygous for Thr164Ile 2AR polymorphism and in 10 volunteers homozygous WT 2AR terbutaline infusion induced increase in heart rate (HR) and shortening of the HR-corrected duration of the electromechanical systole [QS2c; an established measure of contractility (2)] before and after 2-wk treatment with 3 x 5 mg/day terbutaline, a protocol that has been shown to evoke desensitization of 2AR responses in vivo (3, 5, 7, 16).
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METHODS |
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2AR polymorphisms (i.e., Arg16Gly, Gln27Glu, and Thr164Ile). All were Caucasians; their haplotypes have been described in detail elsewhere (7). Six volunteers heterozygous for Thr164Ile 2AR polymorphism (2 men and 4 women, mean age: 27 ± 3 yr) agreed to participate in the study (for detailed haplotypes, see Table 1). Effects of infused terbutaline were compared with those obtained in 10 randomly selected volunteers with the WT 2AR (Arg16Arg, Gln27Gln, and Thr164Thr; 5 men and 5 women, mean age: 24 ± 2 yr).
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All participants of the study gave written informed consent. The Ethical Committee of the University of HalleWittenberg approved the study protocol. All subjects were in normal health based on cardiovascular and other medical history, physical examination, and biochemical, hematological, and electrocardiographic screening. None of the subjects took any medication. Smokers and nonsmokers were equally distributed in both groups. Subjects were blinded for the genotype. The investigators were aware of the alleles of the volunteers because the participants were recruited according to their genotype. All subjects were studied in the morning after an overnight fast and were tested in the supine position on a comfortable bed. The volunteers were advised to avoid caffeine, alcohol, nicotine, and physical exercise before each experiment. Room temperature was kept stable between 24 and 26°C.
The cardiovascular effects of infused terbutaline were investigated twice, before and after the volunteers were treated for 2 wk with 3 x 5 mg/day oral terbutaline.
After 1 h of rest in the supine position and when a stable resting HR was reached, volunteers were infused intravenously with terbutaline (Bricanyl, Astra Stern-Pharma) at increasing doses of 25, 50, 100, and 150 ng · kg–1 · min–1, each for 15 min. Hemodynamics were assessed immediately before the beginning of terbutaline infusion and during the last 5 min of each dosing interval. Cardiovascular effects of intravenous terbutaline were assessed by determination of HR, systolic time intervals, and systolic (BPsyst) and diastolic blood pressure (BPdiast) (4, 7, 16, 17). BPsyst and BPdiast (phase V) were measured with a standard mercury sphygmomanometer (Erkameter, Richard Kallmeyer; Bad Tölz, Germany). Measurements of systolic time intervals were obtained noninvasively from simultaneous recordings of an ECG lead, a phonocardiogram, and a carotid pulse tracing at high paper speed (100 mm/s) using a Bioset 8000 multichannel recorder (Hörmann Medizintechnik; Zwönitz, Germany). At each dose step of the terbutaline infusion, systolic time interval recordings were performed during quiet respiration after the last blood pressure measurement. The following parameters were measured: 1) 20 R-R intervals (in ms) of the ECG from which HR (in beats/min) was calculated; and 2) duration of the electromechanical systole (QS2; in ms) from the beginning of the Q wave of the ECG to the first high-frequency vibrations of the second heart sound. For further details, especially for correction of QS2 for changes in HR, see Refs. 4, 16, and 17. The QS2 corrected for HR is referred to as QS2c; throughout this article, only data for QS2c are shown because this is the most sensitive parameter for changes in contractility (2, 8).
Statistics. All data are means ± SE; n is the number of experiments. The doses of terbutaline in the figures were log transformed. For comparison of dose-response curves of terbutaline infusion-induced increases in HR and contractility, we performed a two-way ANOVA with factors genotype, oral terbutaline treatment, and terbutaline dose with a Bonferroni post test. Basal resting values for HR, QS2c, BPsyst, and BPdiast and maximum terbutaline infusion-induced changes in HR, QS2c, BPsyst, and BPdiast were compared between genotype groups by ANOVA with the Bonferroni correction for multiple comparisons. Effects of oral terbutaline treatment on resting hemodynamics and maximum terbutaline infusion-induced changes in HR, QS2c, BPsyst, and BPdiast were assessed by ANOVA with the Bonferroni correction for multiple comparisons.
A P value of <0.05 was considered to be statistical significant.
All statistical calculations were performed with GraphPad Prism 3.0 software.
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RESULTS |
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2AR or those with the Thr164Ile 2AR (Table 2).
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Terbutaline infusion dose dependently increased HR (Fig. 1) and shortened QS2c (Fig. 2). In Thr164Ile 2AR volunteers, however, dose-response curves for both effects were significantly shifted to the right to higher terbutaline doses; the terbutaline-induced maximum HR increase (WT 32 ± 4 beats/min, Thr164Ile 19 ± 3 beats/min, P < 0.05) and QS2c shortening (WT –54 ± 2 ms, Thr164Ile –37 ± 6 ms, P < 0.05) were in the Thr164Ile 2AR volunteers significantly lower than in the WT 2AR volunteers. Thus, in agreement with our recently published data (4), in Thr164Ile 2AR volunteers cardiac responses to 2AR stimulation were blunted compared with those in WT 2AR volunteers.
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Baseline values of BPsyst and BPdiast (Table 2) and the terbutaline infusion-induced maximum increase in BPsyst (WT +19 ± 3 mmHg, Thr164Ile +20 ± 4 mmHg) and the maximum decrease in BPdiast (WT –26 ± 4 mmHg, Thr164Ile –22 ± 4 mmHg) were not significantly different between the two groups.
Two-week treatment with 3 x 5 mg/day oral terbutaline increased resting HR and shortened resting QS2c, whereby, however, the changes in resting QS2c did not reach statistical significance (Table 2). Resting BPsyst and BPdiast were not significantly affected by 2-wk oral terbutaline treatment (Table 2).
In both groups, the terbutaline infusion-induced increase in HR (Fig. 1) and shortening of QS2c (Fig. 2) were significantly attenuated after 2-wk oral terbutaline treatment. However, after the 2-wk oral terbutaline treatment, dose-response curves for the terbutaline infusion-induced increase in HR and contractility were nearly superimposable in Thr164Ile and WT 2AR volunteers; thus the maximum HR increase (WT 10 ± 2 beats/min, Thr164Ile 8 ± 4 beats/min) and QS2c shortening (WT –22 ± 5 ms, Thr164Ile –17 ± 6 ms) were nearly identical in both groups, indicating that the extent of desensitization was larger in WT 2AR volunteers than in Thr164Ile 2AR volunteers (Figs. 1 and 2). After the 2-wk oral terbutaline treatment, the terbutaline infusion-induced maximum increase in BPsyst (WT 9 ± 3 mmHg, Thr164Ile 8 ± 2 mmHg) and the maximum decrease in BPdiast (WT –17 ± 2 mmHg, Thr164Ile –14 ± 6 mmHg) were also not significantly different between the two groups.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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2AR responses are blunted in volunteers with the Thr164Ile 2AR and 2) long-term agonist treatment of the volunteers causes desensitization not only of cardiac WT but also of Thr164Ile 2ARs. Interestingly, in the present study, after the 2-wk terbutaline treatment, dose-response curves for terbutaline-induced increases in HR and contractility were nearly identical in volunteers with the WT or Thr164Ile 2AR.
These results confirm our recently published data showing that, in volunteers heterozygous for Thr164Ile 2AR polymorphism, terbutaline infusion-induced increases in HR and contractility are blunted when compared with WT 2AR volunteers (4). It is important to note that in the present study only three of the Thr164Ile 2AR volunteers had also participated in our previous study, whereas three volunteers had not been investigated before. Thus we can be quite sure that, in humans heterozygous for Thr164Ile 2AR polymorphism, cardiac responses to 2AR stimulation are blunted. Moreover, very recently, Barbato et al. (1) also found in subjects heterozygous for Thr164Ile 2AR polymorphism that terbutaline infusion-induced increases in HR and contractility are blunted when compared with WT 2AR subjects. In addition, a blunted cardiac response in patients with the Thr164Ile 2AR has been also obtained by Wagoner et al. (18), who found in patients with congestive heart failure carrying this 2AR polymorphism blunted exercise capacity. Furthermore, also in human adipose tissue, the lipolytic effect of the 2AR agonist terbutaline, but not that of the 1AR agonist dobutamine, was reduced in patients with Thr164Ile 2AR polymorphism (12).
It should be mentioned, however, that Thr164Ile polymorphism is closely associated with the existence of Gly at position 16 and Gln at position 27, resulting in distinct haplotypes (9). This also holds true for the Thr164Ile 2AR volunteers of the present study (see Table 1). Thus it might be possible that these different haplotypes might have divergent influences on the cardiac responses of the Thr164Ile 2AR to terbutaline. This is, however, quite unlikely because we have recently shown in volunteers that polymorphisms at positions 16 and 27 did neither affect cardiac responses (increases in HR and contractility) to terbutaline infusion (7) nor isoprenaline-induced increases in lymphocyte cAMP content (6); i.e., independent of polymorphisms at positions 16 and/or 27, dose-response curves for terbutaline-induced cardiac responses and concentration-response curves for isoprenaline-induced increases in lymphocyte cAMP content were nearly superimposable (6, 7).
We and others have shown in several studies that, in humans, chronic oral treatment with the 2AR agonist terbutaline causes desensitization of 2AR-mediated effects (3, 5, 7, 14–16). Very recently, we found that, in contrast to in vitro observations (11), the extent of terbutaline-induced desensitization of cardiac 2AR responses after a 2-wk oral treatment with 3 x 5 mg/day terbutaline was not different in volunteers carrying the Gly16Gly or Glu27Glu allele; however, volunteers homozygous Glu27Glu exhibited a delayed onset in desensitization (7). Moreover, the pattern of downregulation of lymphocyte 2ARs after 2-wk oral treatment with 3 x 5 mg/day terbutaline was in the same volunteers very similar: the extent of downregulation was not different between volunteers homozygous Gly16Gly or Glu27Glu versus WT 2AR volunteers; however, the time course of lymphocyte 2AR downregulation was in volunteers homozygous Glu27Glu significantly slower than in volunteers homozygous Gly16Gly or in WT 2AR volunteers (6).
The results of the present study also show that cardiac Thr164Ile 2ARs undergo agonist-induced desensitization. Thus, in volunteers heterozygous for Thr164Ile 2AR polymorphism, 2-wk oral treatment with terbutaline evoked a significant reduction in terbutaline infusion-induced increases in HR and contractility. Interestingly, the differences in cardiac responses to 2AR stimulation between WT and Thr164Ile 2AR volunteers observed before terbutaline treatment did not occur any more after the 2-wk agonist treatment; i.e., in the desensitized state, both genotype groups exhibited nearly identical responses to agonist stimulation. This could mean that after the 2-wk oral terbutaline treatment, maximum desensitization of the cardiac 2AR has been reached, i.e., the remaining response is somewhat like a minimum effect of terbutaline. It could, however, also indicate that desensitization was more pronounced in WT than Thr164Ile 2AR subjects. This would be compatible with the view that cardiac Thr164Ile 2ARs are somewhat protected against agonist-induced desensitization possibly to maintain an essential rest of functional responsiveness during chronic agonist treatment. However, it might also be possible that Thr164Ile 2ARs are already somewhat desensitized under basal conditions, and hence the extent in terbutaline treatment-induced desensitization should be less in these subjects than in subjects with the WT 2AR that is not desensitized under basal conditions.
As mentioned above, the extent of agonist-induced 2AR desensitization does not differ between volunteers homozygous for Arg16Gly or Gln27Glu 2AR polymorphisms and WT 2AR volunteers (6, 7). Therefore, it is rather unlikely that polymorphisms at positions 16 and/or 27 in the Thr164Ile 2AR volunteers might have affected the extent of 2AR desensitization. Accordingly, the Thr164Ile 2AR variant exhibits a different pattern of agonist-induced desensitization than the Gly16Gly or Glu27Glu 2AR variants: the extent of long-term agonist-induced desensitization of cardiac Thr164Ile 2AR is less than that of the cardiac WT 2AR, in contrast to cardiac Gly16Gly or Glu27Glu 2ARs, which are long-term desensitized to a very similar extent as cardiac WT 2ARs (7).
It should be noted that in the present study we did not determine cardiac contractility by echocardiography, but we assessed systolic time intervals (STIs) and QS2c as a parameter to determine changes in contractility. Heart rate-corrected STIs are sensitive to changes in pre- and afterload (for a review, see Ref. 2). It might be possible, therefore, that terbutaline infusion-induced changes in QS2c reflect not only increases in contractility due to direct 2AR stimulation but might also involve indirect effects due to afterload reduction. However, it has been clearly demonstrated that among the STIs, changes in QS2c predominantly reflect changes in contractility, whereas changes in afterload have, if any, only marginal influences (2). Moreover, it has been shown that shortening of QS2c was the most sensitive method to detect changes in contractility due to isoprenaline infusion when compared with impedance cardiography, dual-beam Doppler echoaortography, and left ventricular echocardiography (8). Therefore, it is quite likely that, in the present study, shortening of QS2c induced by terbutaline infusion reflects rather precisely terbutaline-induced increases in contractility.
2AR agonists are often used in the treatment of pulmonary diseases and to prevent preterm labor during pregnancy. The clinical implications of the present results are that in patients carrying Thr164Ile 2AR polymorphism, initially the therapeutic efficacy of such treatment with 2AR agonists might be lower than in patients with the WT 2AR; however, with ongoing treatment (and thus 2AR desensitization), this disadvantage might disappear because desensitization in these patients is less than in WT 2AR patients.
In conclusion, in subjects heterozygous for Thr164Ile 2AR polymorphism, cardiac responses to 2AR stimulation are blunted when compared with WT 2AR subjects. Cardiac Thr164Ile 2ARs undergo in vivo agonist-induced desensitization as do WT 2ARs; however, the extent of desensitization for Thr164Ile 2ARs was less than for WT 2ARs, so that in the desensitized state cardiac responses in both genotype groups are nearly identical.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. Proc Natl Acad Sci USA 97: 10483–10488, 2000.2-adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor. J Biol Chem 268: 23116–23121, 1993.2-adrenergic receptor impart distinct agonist-promoted regulatory properties. Biochemistry 33: 9414–9419, 1994.[Medline]
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  D. Rosskopf and M. C. Michel Pharmacogenomics of G Protein-Coupled Receptor Ligands in Cardiovascular Medicine Pharmacol. Rev., December 1, 2008; 60(4): 513 - 535. [Abstract] [Full Text] [PDF]
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 E. Barbato, M. Penicka, L. Delrue, F. Van Durme, B. De Bruyne, M. Goethals, W. Wijns, M. Vanderheyden, and J. Bartunek Thr164Ile polymorphism of {beta}2-adrenergic receptor negatively modulates cardiac contractility: implications for prognosis in patients with idiopathic dilated cardiomyopathy Heart, July 1, 2007; 93(7): 856 - 861. [Abstract] [Full Text] [PDF]
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