Skip Navigation

Cardiovascular Research 1998 40(1):211-222; doi:10.1016/S0008-6363(98)00101-1
© 1998 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Poller, U.
Right arrow Articles by Brodde, O.-E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poller, U.
Right arrow Articles by Brodde, O.-E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1998, European Society of Cardiology

Terbutaline-induced desensitization of human cardiac β2-adrenoceptor-mediated positive inotropic effects: attenuation by ketotifen

Ulrike Pollera, Birgit Fuchsb, Antje Gorfa, Jens Jakubetzb, Joachim Radkeb, Klaus Pönickea and Otto-Erich Broddea,*

aInstitute of Pharmacology and Toxicology, Martin-Luther-University of Halle-Wittenberg, D-06097 Halle/Saale, Germany
bDepartment of Anaesthesiology, Martin-Luther-University of Halle-Wittenberg, D-06097 Halle/Saale, Germany

* Corresponding author: Tel.: +49-345-557 1773; Fax: +49-345-557 1835.

Received 2 October 1997; accepted 17 March 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Background: In patients with chronic heart failure cardiac β1-adrenoceptors are desensitized whereas β2-adrenoceptors are only marginally affected. The mechanism underlying this differential regulation is not known. Objectives: To find out whether or not human cardiac β2-adrenoceptors might be ‘resistant’ to agonist-induced desensitization and whether or not the antiallergic drug ketotifen might attenuate possible desensitization. Methods: We investigated, in a single blinded, randomised, placebo-controlled, cross-over study of ten healthy male volunteers (mean age, 25.3±0.7 years), the effects of two weeks treatment with the β2-adrenoceptor agonist terbutaline (3x5 mg/day p.o.) with and without simultaneous treatment with ketotifen (2x1 mg/day p.o. for three weeks) or placebo on β-adrenoceptor-mediated cardiovascular effects. Cardiovascular effects were assessed as isoprenaline (3.5–35 ng/kg/min)- and terbutaline (25–150 ng/kg/min)-infusion-induced increases in heart rate and systolic blood pressure, decreases in diastolic blood pressure and shortening of the systolic time intervals (STIs), heart rate corrected duration of electromechanical systole (QS2c) and pre-ejection period (PEP; as a measure of inotropism). Results: Ketotifen did not significantly affect basal haemodynamics in the volunteers. Isoprenaline- and terbutaline-infusion caused dose-dependent increases in systolic blood pressure and heart rate, decreases in diastolic blood pressure and shortening of QS2c and PEP, whereby isoprenaline effects were more pronounced. After two weeks of treatment with terbutaline p.o., isoprenaline- and terbutaline-infusion-induced increases in heart rate, shortening of QS2c and PEP were significantly reduced whereby terbutaline-infusion effects were markedly more attenuated than isoprenaline-infusion effects. Ketotifen significantly reduced terbutaline p.o. treatment-induced attenuation of all terbutaline-infusion effects (largely β2-adrenoceptor-mediated) and the isoprenaline-infusion-induced increase in heart rate (β1- and β2-adrenoceptor-mediated), but did not (or only marginally) affect reduction in isoprenaline-induced shortening of QS2c and PEP (largely β1-adrenoceptor-mediated). Conclusion: Human cardiac β2-adrenoceptors are not ‘resistant’ to agonist-induced desensitization: Ketotifen might prevent such β2-adrenoceptor-agonist-evoked desensitization.

KEYWORDS Human cardiac β1-adrenoceptors; Human cardiac β2-adrenoceptors; Positive inotropic effect; Desensitization; Terbutaline; Ketotifen


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
A large body of evidence has accumulated indicating that long-term exposure of β-adrenoceptors to β-adrenoceptor agonists leads to desensitization of the functional responsiveness and finally to down-regulation of the density of β-adrenoceptors (for reviews, see [1, 2]). This holds true not only for exogenously applied agonists (for example in the treatment of asthma [3]) but also for chronically elevated endogenous catecholamines, for example in pheochromocytoma [4, 5]or in chronic heart failure (for recent reviews, see [6, 7]).

In the human heart, both β1- and β2-adrenoceptors coexist; both β-adrenoceptor subtypes mediate positive ino- and chronotropic effects (for review, see [8]). In chronic heart failure patients with elevated catecholamines, it has consistently been found that β1-adrenoceptors were down-regulated while β2-adrenoceptors were only marginally affected [6–8], and it has been assumed that this is due to chronically elevated cardiac-derived noradrenaline [9]that, in the human heart, is a rather selective agonist of β1-adrenoceptors [8, 10]. In fact, we have recently shown that, in healthy volunteers, endogenous noradrenaline released by tyramine infusion acts in the heart predominantly at β1-adrenoceptors [11]. On the other hand, in various in vivo animal studies, it has been found that chronic β-adrenoceptor stimulation very rapidly down-regulates cardiac β2-adrenoceptors, while cardiac β1-adrenoceptors were down-regulated much more slowly; this held true not only for the non-selective β-adrenoceptor agonists isoprenaline and adrenaline but also for the rather selective β1-adrenoceptor (see above), noradrenaline ([7, 12]). Moreover, in isolated human coronary arteries in vitro, a 16-h incubation with noradrenaline led to β2-adrenoceptor desensitization without affecting β1-adrenoceptors [13]. Thus, the selective down-regulation of cardiac β1-adrenoceptors in human chronic heart failure is somewhat unique and the question arises as to whether or not human cardiac β2-adrenoceptors might be relatively resistant to agonist-induced desensitization. If this were the case, stimulation of cardiac β2-adrenoceptors could be an alternative possibility for improving cardiac performance in patients with end-stage heart failure (and down-regulated β1-adrenoceptors, see above).

The β2-adrenoceptor agonist terbutaline evokes positive chrono- and inotropic effects in healthy volunteers, predominantly via stimulation of cardiac β2-adrenoceptors [14, 15], while isoprenaline increases heart rate through β1- and β2-adrenoceptor stimulation to about the same extent and induces positive inotropic effects, mainly through stimulation of cardiac β1-adrenoceptors [15]. Thus, experimental in vivo models are available to study human cardiac β1- and β2-adrenoceptor function. In the present study, we have used these models in healthy volunteers in order to find out (a) if chronic terbutaline p.o. treatment desensitizes β2-adrenoceptor (by terbutaline-infusion)- and/or β1-adrenoceptor (by isoprenaline-infusion)-mediated positive inotropic effects and (b) if the antiallergic drug ketotifen, which has been shown to prevent (or at least to blunt) β2-adrenoceptor desensitization ([16, 17]and references therein), might attenuate possible terbutaline-induced cardiac β2- and/or β1-adrenoceptor desensitization. For this purpose, we assessed, in ten healthy volunteers, the effects of isoprenaline- and terbutaline-infusion on heart rate and STIs (as a measure of positive inotropism [18]) before and after two weeks of treatment with terbutaline (3x5 mg/day p.o.) with and without concomitant treatment of ketotifen (2x1 mg/day p.o.).


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Subjects
Ten healthy male volunteers (aged 25.3±0.7 years, range 23 to 29 years; body weight, 74±2 kg, range 69 to 81 kg) participated in this randomised, placebo-controlled, single blinded, cross-over study, having given informed written consent. All subjects were drug-free; they were of average physical fitness and none exercised regularly. Normal health was established by medical history, physical examination, biochemical and haematologic screening and electrocardiogram to exclude asthma, diabetes mellitus, chronic pulmonary disease, hypertension, cardiac disease and other symptoms pertaining to the cardiovascular system. The study protocol was approved by the Ethical Committee of the University of Halle-Wittenberg.

2.2 Study protocol
We compared the effects of either placebo or ketotifen on β-adrenoceptor-mediated cardiovascular effects before and after two weeks p.o. treatment with the β2-adrenoceptor agonist terbutaline. The study protocol is summarized in Fig. 1. At day 1, i.e. prior to the administration of any drug, volunteers underwent a baseline isoprenaline (at 8.00 a.m.)- and terbutaline (at 11.30 a.m.)-infusion test. Isoprenaline was infused at doses of 3.5, 7, 17 and 35 ng/kg/min each for 10 min in a cumulative manner, terbutaline at doses of 25, 50, 100 and 150 ng/kg/min each for 15 min in a cumulative manner. The order of administration of isoprenaline (8.00 a.m.) and terbutaline (11.30 a.m.) was kept constant throughout the whole study period. Previous studies had shown that 10 (isoprenaline) and 15 min (terbutaline) of infusion are necessary to reach steady-state levels of the drugs [19]. Thereafter, volunteers were randomised and treated with either ketotifen (2x1 mg/day at 7.00 a.m. and 7.00 p.m.) or placebo for three weeks, i.e. for the whole study period. At day 8, i.e. after one week of ketotifen or placebo treatment, the isoprenaline (8.00 a.m.)- and terbutaline (11.30 a.m.)-infusion tests were repeated. Thereafter, volunteers were treated for two weeks with 3x5 mg/day terbutaline p.o. (7.00 a.m., 2.00 and 9.00 p.m.) and, on day 22, with the volunteers still on terbutaline treatment, the isoprenaline- (8.00 a.m.) and terbutaline- (11.30 a.m.) infusion tests were again repeated. All tests were performed with the subjects in the supine position in a quiet air-conditioned room and following an almost identical time schedule: After arrival in the clinical laboratory at 7.15 a.m. and after affixment of the instruments, indwelling polythene catheters were positioned in an anticubital vein of each arm. Blood samples were drawn from the left arm, drugs were administered into the right arm. After at least 30 min of rest in the supine position, the infusion tests were started. In the 2 h break between the two infusions, volunteers rested in the supine position and a small breakfast was given. There was a washout period of at least four weeks duration between the two study periods (placebo, ketotifen).


Figure 1
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Study protocol. There was a washout period of at least four weeks duration between the two study periods (placebo and ketotifen).

 
2.3 Haemodynamic parameters
Baseline haemodynamic values were assessed immediately before the start of each infusion, and are summarized in Table 1. Cardiovascular effects of i.v. isoprenaline and i.v. terbutaline were assessed by determination of the systolic and diastolic blood pressure, the heart rate and systolic time intervals (STIs). The systolic and diastolic blood pressure was measured manually by means of a mercury spyghmomanometer (DIPLOMAT Presameter) five times in the last 5 min of each dose-step of isoprenaline- and terbutaline-infusion, respectively. The mean from these recordings was chosen for analysis of the dose–response curves. Measurements were performed by the same individual throughout the whole study period. The STIs were recorded in the last minute of each dose step of isoprenaline- and terbutaline-infusion, respectively. Measurements of STIs were obtained noninvasively from simultaneous recordings from an ECG lead, a phonocardiogram and a carotid pulse tracer at high paper speed (100 mm/s) using a Bioset 8000 multichannel recorder (Hörmann Medizintechnik, Zwönitz, Germany). Measurements were performed by the same individual throughout the whole study period. The following parameters were measured: RR intervals (ms) of the ECG from which heart rate (beats/min) was calculated; the duration of the electromechanical systole (QS2, ms); the duration of the left ventricular ejection time (LVET, ms) and the duration of the pre-ejection period (PEP, ms), which was derived mathematically by subtracting LVET from QS2. For details, especially for correction of QS2 to determine changes in heart rate, see refs. [11, 15]. The QS2 corrected for heart rate is herein referred to as QS2c.


View this table:
[in this window]
[in a new window]

  		Table 1 Baseline haemodynamics of the ten healthy volunteers during the study

 
2.4 Blood samples
Immediately before the start and at the end of the last dose step of the isoprenaline-infusion, blood samples for catecholamine determination were withdrawn through the indwelling forearm venous catheter and were collected in ice-cold EDTA-tubes. Samples were centrifuged at 600xg for 10 min at 4°C, the plasma was removed and quickly frozen at –80°C. Plasma catecholamine levels were measured by HPLC with fluorometric detection, as detailed elsewhere [11]. In order to keep the blood loss for the volunteers to a minimum, we did not assess i.v. terbutaline effects on plasma catecholamines.

2.5 Data analysis
For each subject and each isoprenaline- and terbutaline-infusion, respectively, cumulative dose–response curves were constructed by plotting changes in heart rate, blood pressure and STIs from baseline versus the dose of the agonist, whereby ‘change’ is defined as the effect measured during infusion minus the baseline level (measured immediately before infusion). The individual dose–response relations were fitted by log–linear regression analysis (for details, see ref. [15]) to calculate the effective doses (ED) of isoprenaline and terbutaline required to increase the heart rate by ten beats/min (ED-10) and systolic blood pressure by 10 mmHg (ED-10), to decrease diastolic blood pressure by 10 mmHg (ED-10) and to shorten the QS2c and PEP by 30 (ED-30, isoprenaline) and 15 ms (ED-15, terbutaline), respectively. The doses for isoprenaline and terbutaline were expressed in ng/kg/min throughout. ED-10 values for terbutaline-induced increases in systolic blood pressure and decreases in diastolic blood pressure on study day 22 could not be calculated, because terbutaline-induced blood pressure changes in most volunteers were less than 10 mmHg (cf. Fig. 2); thus, the fits were not linear (r2<0.7). Differences in ED values for each parameter on study days 1, 8 and 22 were tested for statistical significance by one-way ANOVA repeated measure analysis of variance (Tukey–Cramer–multiple comparison test).


Figure 2
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Terbutaline-infusion-induced changes in systolic blood pressure (Psyst, upper panel) and diastolic blood pressure (Pdiast, lower panel) on the control day (day 1, {blacktriangleup}, {triangleup}), on day 8 (i.e. after one week placebo or ketotifen, {diamondsuit}, {diamond}) and on day 22 (i.e. after two weeks treatment with 3x5 mg/day terbutaline, administered orally, bullet, {circ}). Filled symbols, placebo group; open symbols, ketotifen group. Ordinate, changes in Psyst (upper panel) and Pdiast (lower panel) in {Delta} mmHg. Abscissa, dose of terbutaline (in ng/kg/min) for 15 min each. Results are expressed as the mean±S.E.M. of ten experiments each. (*) p<0.05 vs. the corresponding value at day 1; a p<0.05 vs. the corresponding value at day 8. ED-10 values (ng/kg/min) for increases in Psyst were 100.1±17.7 (day 1) and 107.1±7.4 (day 8) in the placebo group; in the ketotifen group, they were 99.5±13.2 (day 1) and 103.3±12.1 (day 8). ED-10 values (ng/kg/min) for decreases in Pdiast were 91.7±15.8 (day 1) and 97.1±20.2 (day 8) in the placebo group; in the ketotifen group, they were 81.8±10.6 (day 1) and 84.3±7.4 (day 8).

 
The reproducibility of the isoprenaline- and terbutaline-infusion-induced changes in cardiovascular parameters was analyzed by calculating ED ratios of ED values for day one of treatment phase one (placebo) over ED values for day one of treatment phase two (ketotifen). If there was no systemic change in cardiovascular responsiveness to i.v. isoprenaline or i.v. terbutaline with time (i.e. during the washout period of at least four weeks), the ED ratios should not have been significantly different from 1.0. This was tested using a one sample t-test (hypothetical mean=1.0).

In order to determine if ketotifen might affect the influence of terbutaline p.o. treatment on cardiovascular effects, the ED ratios during the placebo phase were compared with the ED ratios during the ketotifen phase. This was done by dividing the ED ratio (day 22/day 8) of the placebo phase by the ED ratio (day 22/day 8) of the ketotifen phase; if ketotifen had a significant influence on terbutaline p.o. treatment-induced changes in cardiovascular effects, this ratio should be significantly different from 1.0. This was tested using a one sample t-test (hypothetical mean=1.0).

For statistical analysis of differences in the baseline haemodynamic values, plasma catecholamine levels and changes from the baseline between days 1, 8 and 22, a one-way ANOVA analysis for repeated measure (Tukey–Cramer–multiple comparison test) was used.

A p-value <0.05 was considered to be significant. Experimental data are expressed as the mean±S.E.M. of ten experiments. Statistical calculations were performed using the InSTAT program (GraphPAD software, San Diego, CA, USA).

2.6 Drug used
Isoprenaline sulfate (AleudrinaR), for infusion, was purchased from Boehringer (Ingelheim, Germany), terbutaline sulfate injection solution and terbutaline tablets (BricanylR) were from Pharmastern (Wedel, Germany) and ketotifen tablets (ZaditenR) were from Wander Pharma (Nürnberg, Germany). Placebo tablets (to ketotifen; content, lactose) were produced by the local hospital pharmacy.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Baseline haemodynamic values
Baseline haemodynamic values (i.e. values assessed after 30 min of rest in the supine position immediately before isoprenaline- or terbutaline infusion) are given in Table 1. As can be seen from Table 1, treatment with ketotifen for eight days did not significantly affect baseline haemodynamics (comparison between day 1 and day 8). After intake of ketotifen, all subjects complained about tiredness; after intake of terbutaline, the subjects suffered from tachycardia and finger tremor, but the intensity of these symptoms waned with continuous treatment. Chronic terbutaline p.o. treatment (day 22) caused slight increases in heart rate and systolic blood pressure, slight decreases in diastolic blood pressure and a shortening of STIs.

3.2 Reproducibility of dose–response curves
Effective doses (ED) for i.v. isoprenaline- and i.v. terbutaline-induced increases in systolic blood pressure and heart rate, shortening of QS2c and PEP and decreases in diastolic blood pressure did not differ significantly between day 1 of the two study periods; thus, the corresponding ED ratios were not significantly different from unity (Table 2). It should be noted that there was a period of at least four weeks between the two baseline measurements of the two study periods.


View this table:
[in this window]
[in a new window]

  		Table 2 Reproducibility of dose–response curves for isoprenaline and terbutaline

 
3.3 Effects of two weeks treatment with terbutaline p.o.
3.3.1 Terbutaline-infusion-induced cardiovascular effects
Terbutaline-infusion caused dose-dependent increases in systolic blood pressure (Fig. 2) and heart rate (Fig. 3), decreases in diastolic blood pressure (Fig. 2) and shortening of the STIs, QS2c and PEP (Fig. 4). After one week of treatment of the volunteers with placebo or ketotifen, dose–response curves for all of these parameters were not significantly altered. Two weeks p.o. treatment with terbutaline only slightly affected terbutaline-infusion-induced blood pressure changes (Fig. 2), but significantly shifted the dose–response curves for heart rate increases (Fig. 3) and shortening of QS2c and PEP (Fig. 4) to the right to higher doses. Thus, after two weeks p.o. treatment with terbutaline, i.v. terbutaline-induced effects on heart rate and STIs were significantly reduced at each dose. The simultaneous administration of ketotifen significantly attenuated these desensitizing effects of terbutaline p.o. treatment: In the presence of ketotifen, two weeks of terbutaline p.o. treatment caused a marginal, but not significant, shift to the right of the dose–response curves for i.v. terbutaline-induced shortening of PEP (Fig. 4). For increases in heart rate (Fig. 3) and shortening of QS2c (Fig. 4), only the highest dose of terbutaline was still significantly reduced. Accordingly, the quotients calculated from the ED ratios (day 22/day 8) for terbutaline-infusion-induced increases in heart rate, and shortening of QS2c and PEP in the placebo phase over the ketotifen phase, were significantly different from 1.0 (Table 3).


Figure 3
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Terbutaline-infusion-induced changes in heart rate on the control day (day 1, {blacktriangleup}, {triangleup}), on day 8 (i.e. after one week of placebo or ketotifen, {diamondsuit}, {diamond}) and on day 22 (i.e. after two weeks of treatment with 3x5 mg/day terbutaline, administered orally, bullet, {circ}). Filled symbols, placebo group; open symbols, ketotifen group. Ordinate, changes in heart rate in {Delta} beats/min. Abscissa, dose of terbutaline (in ng/kg/min) for 15 min each. Results are expressed as the mean±S.E.M. of ten experiments each. (*) p<0.05 vs. the corresponding value at day 1; a p<0.05 vs. the corresponding value at day 8. ED-10 values (ng/kg/min) for increases in heart rate were 59.7±3.5 (day 1), 55.4±4.1 (day 8) and 144.9±37.4 (day 22) in the placebo group, and 56.3±5.3 (day 1), 55.6±3.4 (day 8) and 75.5±12.3 (day 22) in the ketotifen group.

 

Figure 4
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Terbutaline-infusion-induced changes in the systolic time intervals QS2c (upper panel) and PEP (lower panel) on the control day (day 1, {blacktriangleup}, {triangleup}), on day 8 (i.e. after one week of placebo or ketotifen, {diamondsuit}, {diamond}) and on day 22 (i.e. after two weeks of treatment with 3x5 mg/day terbutaline, administered orally, bullet, {circ}). Filled symbols, placebo group; open symbols, ketotifen group. Ordinates, shortening in QS2c (upper panel) and PEP (lower panel), in {Delta} ms. Abscissa, dose of terbutaline (in ng/kg/min) for 15 min each. The results are expressed as the mean±S.E.M. of ten experiments each. (*) p<0.05 vs. the corresponding values at day 1; a p<0.05 vs. the corresponding values at day 8. ED-15 values (ng/kg/min) for shortening of QS2c were 50.9±5.7 (day 1), 50.7±5.0 (day 8) and 116.0±42.4 (day 22) in the placebo group, and 53.2±6.0 (day 1), 65.9±6.4 (day 8) and 77.5±15.6 (day 22) in the ketotifen group. ED-15 values (ng/kg/min) for shortening of PEP were 43.8±5.6 (day 1), 47.0±2.8 (day 8) and 133.3±2.5 (day 22) in the placebo group, and 49.5±6.3 (day 1), 55.2±5.0 (day 8) and 84.0±24.1 (day 22) in the ketotifen group.

 

View this table:
[in this window]
[in a new window]

  		Table 3 Effects of ketotifen on terbutaline (p.o.) treatment-induced alterations in the dose–response curves for i.v. isoprenaline and i.v. terbutaline

 
3.3.2 Isoprenaline-infusion-induced cardiovascular effects
Isoprenaline-infusion dose-dependently increased systolic blood pressure (Fig. 5) and heart rate (Fig. 6), decreased diastolic blood pressure (Fig. 5) and shortened the STIs QS2c and PEP (Fig. 7). Treatment with ketotifen for one week did not significantly affect these dose–response curves. After two weeks of treatment with terbutaline p.o., the isoprenaline-induced increase in systolic blood pressure was not affected (Fig. 5), while the dose–response curve for the isoprenaline-induced decrease in diastolic blood pressure was significantly shifted to the right (Fig. 5). Moreover, after two weeks p.o. treatment with terbutaline, the dose–response curves for i.v. isoprenaline-induced increases in heart rate (Fig. 6) and shortening of QS2c and PEP (Fig. 7) were significantly shifted to the right, although to a lesser extent than those of i.v. terbutaline (cf. Figs. 3 and 4Go). Ketotifen attenuated the p.o. terbutaline-evoked desensitization of the isoprenaline-induced decrease in diastolic blood pressure (Fig. 5) and the increase in heart rate (Fig. 6), but did not (or only marginally) affect desensitization of i.v. isoprenaline-induced shortening of QS2c and PEP (Fig. 7). However, the quotients calculated from the ED ratios (day 22/day 8) for isoprenaline-infusion-induced increases in heart rate and systolic blood pressure, decrease in diastolic blood pressure and shortening of QS2c and PEP in the placebo phase over the ketotifen phase were not significantly different from 1.0, although, for increases in heart rate, the quotient (1.48±0.22) just failed to reach statistical significance (p=0.057, cf. Table 3).


Figure 5
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Isoprenaline-infusion-induced changes in systolic blood pressure (Psyst, upper panel) and diastolic blood pressure (Pdiast, lower panel) on the control day (day 1, {blacktriangleup}, {triangleup}), on day 8 (i.e. after one week of placebo or ketotifen, {diamondsuit}, {diamond}) and on day 22 (i.e. after two weeks of treatment with 3x5 mg/day terbutaline, administered orally, bullet, {circ}). Filled symbols, placebo group; open symbols, ketotifen group. Ordinate, changes in Psyst (upper panel) and Pdiast (lower panel), in {Delta} mmHg. Abscissa, dose of isoprenaline (in ng/kg/min) for 10 min each. The results are expressed as the mean±S.E.M. of ten experiments each. a p<0.05 vs. the corresponding values at day 8. ED-10 values (ng/kg/min) for increases in Psyst were 7.3±1.0 (day 1), 6.4±1.1 (day 8) and 12.0±2.5 (day 22) in the placebo group, and 8.6±1.6 (day 1), 8.5±1.2 (day 8) and 11.4±2.8 (day 22) in the ketotifen group. ED-10 values (ng/kg/min) for decreases in Pdiast were 9.1±1.1 (day 1), 7.7±1.0 (day 8) and 18.7±5.0 (day 22) in the placebo group, and 8.2±0.9 (day 1), 9.7±1.8 (day 8) and 18.0±4.2 (day 22) in the ketotifen group.

 

Figure 6
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Isoprenaline-infusion-induced changes in heart rate on the control day (day 1, {blacktriangleup}, {triangleup}), on day 8 (i.e. after one week of placebo or ketotifen, {diamondsuit}, {diamond}) and on day 22 (i.e. after two weeks of treatment with 3x5 mg/day terbutaline, administered orally, bullet, {circ}). Filled symbols, placebo group; open symbols, ketotifen group. Ordinate, changes in heart rate, in {Delta} beats/min. Abscissa, dose of isoprenaline (in ng/kg/min) for 10 min each. The results are expressed as the mean±S.E.M. of ten experiments each. (*) p<0.05 vs. the corresponding values at day 1, a p<0.05 vs. the corresponding values at day 8. ED-10 values (ng/kg/min) for increases in heart rate were 7.2±0.5 (day 1), 6.7±0.6 (day 8) and 17.2±3.1 (day 22) in the placebo group, and 6.9±0.8 (day 1), 7.2±0.6 (day 8) and 14.2±2.3 (day 22) in the ketotifen group.

 

Figure 7
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Isoprenaline-infusion-induced changes in the systolic time intervals, QS2c (upper panel) and PEP (lower panel), on the control day (day 1, {blacktriangleup}, {triangleup}), on day 8 (i.e. after one week of placebo or ketotifen, {diamondsuit}, {diamond}) and on day 22 (i.e. after two weeks of treatment with 3x5 mg/day terbutaline, administered orally, bullet, {circ}). Filled symbols, placebo group; open symbols, ketotifen group. Ordinates, shortening in QS2c (upper panel) and PEP (lower panel), in {Delta} ms. Abscissa, dose of isoprenaline (in ng/kg/min) for 10 min each. The results are expressed as the mean±S.E.M. of ten experiments each. (*) p<0.05 vs. the corresponding values at day 1; a p<0.05 vs. the corresponding values at day 8. ED-30 values (ng/kg/min) for shortening of QS2c were 7.0±0.7 (day 1), 6.2±0.5 (day 8) and 9.8±1.3 (day 22) in the placebo group, and 6.5±0.6 (day 1), 7.1±1.0 (day 8) and 9.7±1.2 (day 22) in the ketotifen group. ED-30 values (ng/kg/min) for shortening of PEP were 7.1±0.9 (day 1), 8.2±1.7 (day 8) and 31.2±8.9 (day 22) in the placebo group, and 9.8±1.4 (day 1), 8.2±1.2 (day 8) and 32.8±6.1 (day 22) in the ketotifen group.

 
Isoprenaline-infusion led to a significant increase in plasma noradrenaline levels (absolute values are given in Table 4). Immediately after the last dose of isoprenaline (35 ng/kg/min), plasma noradrenaline had increased by 114.9±15.2 pg/ml (n=10). Ketotifen treatment did not affect the i.v. isoprenaline-induced increase in plasma noradrenaline (Table 4). After two weeks of treatment with terbutaline p.o., the i.v. isoprenaline-induced increase in plasma noradrenaline was significantly reduced and amounted to only 27.9±18 pg/ml (n=10, p<0.02). Ketotifen partially restored the i.v. isoprenaline-induced increase in plasma noradrenaline: After two weeks p.o. treatment of the volunteers with terbutaline plus ketotifen, the increase was 63.3±18.9 pg/ml (n=10).


View this table:
[in this window]
[in a new window]

  		Table 4 Effects of ketotifen on terbutaline (p.o.) treatment-induced alterations in the plasma catecholamine response to isoprenaline, administered i.v.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In this study, infusion of isoprenaline and terbutaline led to dose-dependent increases in heart rate and shortening of the STIs, PEP and QS2c (as a measure of positive inotropism [18]), in healthy volunteers; the positive inotropic effects of isoprenaline were more pronounced than those evoked by terbutaline, in agreement with our recently reported data [15]. It is now generally accepted that, in humans, isoprenaline increases heart rate by stimulation of (cardiac) β1- and β2-adrenoceptors to about the same extent (for reviews, see [8, 20]), whereas it shortens STIs, predominantly through stimulation of cardiac β1-adrenoceptors [15, 21]. On the other hand, the β2-adrenoceptor agonist terbutaline induces heart rate increases and positive inotropic effects, predominantly via (cardiac) β2-adrenoceptor stimulation [14, 15, 22]; however, it should be mentioned that, at higher doses, the cardiac effects of terbutaline appear to include a small β1-adrenoceptor component, since β1-adrenoceptor-selective antagonists, such as atenolol [14, 22]or bisoprolol [15, 19], slightly attenuated the positive chrono- and inotropic effects of higher doses of terbutaline. Very recently, a similar antagonistic effect of atenolol on the positive inotropic response to intracoronary artery-infused salbutamol was found [23]; in addition, these authors observed a markedly enhanced cardiac noradrenaline spillover during salbutamol infusion, indicating that prejunctional β2-adrenoceptor activation with subsequent noradrenaline release is involved in the cardiac actions of β2-adrenoceptor agonists. In fact, in vitro studies have clearly demonstrated the existence of prejunctional β2-adrenoceptors that facilitate noradrenaline release in the human heart [24, 25].

In the present study, treatment of healthy volunteers for two weeks with 3x5 mg/day terbutaline p.o. markedly attenuated a terbutaline-infusion-induced increase in heart rate and shortening of the STIs. As discussed above, these cardiac effects of i.v. terbutaline are mediated predominantly by β2-adrenoceptor stimulation; these results, therefore, indicate that, in humans, chronic β2-adrenoceptor stimulation desensitizes not only atrial β2-adrenoceptors mediating positive chronotropic effects [17, 26, 27]but also ventricular β2-adrenoceptors mediating positive inotropic effects.

Previous in vivo studies had shown that the antiallergic drug ketotifen can prevent (or at least attenuate) β2-adrenoceptor agonist-induced desensitization of β2-adrenoceptors in rats [28]and humans [16, 17, 29–31]. In the present study, concomitant treatment of the volunteers with ketotifen significantly attenuated the terbutaline p.o. treatment-induced desensitization of cardiac β2-adrenoceptor responses: In the presence of ketotifen, treatment with terbutaline p.o. caused a marginal, but not significant, shift to the right of the dose–response curves for i.v. terbutaline-induced shortening of PEP (cf. Fig. 4); i.v. terbutaline-induced shortening of QS2c (cf. Fig. 4) and increases in heart rate (cf. Fig. 3) were only significantly reduced at the highest dose of terbutaline. These data, therefore, confirm and extend our previously reported findings [17]. They show that ketotifen not only attenuates (or prevents) agonist-induced desensitization of atrial β2-adrenoceptors mediating heart rate increases, but also that of ventricular β2-adrenoceptors mediating positive inotropic effects. The mechanism of this β2-adrenoceptor desensitization-preventing effect of ketotifen, which has been observed only in in vivo, but not in in vitro, studies [17]is still unknown but might be possibly due to one of the metabolites of ketotifen [17]. However, ketotifen does not appear to have any β-adrenergic (antagonistic) properties [28].

It is interesting to note that in the present study, treatment with terbutaline p.o. significantly attenuated isoprenaline-infusion-evoked increases in heart rate and shortening of the STIs PEP and QS2c, although to a lesser extent than those of terbutaline-infusion-evoked effects. A slight but significant desensitization of isoprenaline-induced tachycardia following terbutaline p.o. treatment could be expected (see also [17, 26]) because this isoprenaline effect is mediated by (cardiac) β1- and β2-adrenoceptor stimulation to about the same extent (see above). The terbutaline p.o. treatment-induced desensitization of isoprenaline-induced positive inotropic effects, however, is somewhat surprising, since results from in vivo studies had suggested that this isoprenaline effect is mediated predominantly via β1-adrenoceptor stimulation (see above). However, numerous studies on isolated human ventricular preparations have shown that, in vitro, the positive inotropic effect of isoprenaline is brought about by β1- and β2-adrenoceptor stimulation (for review, see [8]); thus, the terbutaline p.o. treatment-induced desensitization of the i.v. isoprenaline-evoked positive inotropic effect might be due to the fact that, in vivo, a (small) β2-adrenoceptor component is included in the isoprenaline effect. Since the in vitro studies showed that the β2-adrenoceptor component comprises only 20–30% of the whole isoprenaline effect, this would fit with the smaller amount of desensitization in comparison with the i.v. terbutaline effects (that are mediated predominantly via β2-adrenoceptor stimulation, see above).

An alternative explanation could be that, during terbutaline p.o. treatment, chronic activation of (cardiac) prejunctional β2-adrenoceptors might cause enhanced release of endogenous noradrenaline. Since this acts in the human heart selectively at β1-adrenoceptors [10, 11], it might desensitize cardiac β1-adrenoceptors and might lead, therefore, to a partial desensitization of the isoprenaline-induced positive inotropic effects. Finally, it might be also possible that, in vivo, isoprenaline-induced positive inotropic effects are composed of two components: Direct activation of cardiac β1- (and β2-) adrenoceptors and indirect activation of cardiac β1-adrenoceptors through endogenous noradrenaline, released upon activation of prejunctional β2-adrenoceptors. In fact, in the present study, isoprenaline-infusion does increase plasma noradrenaline levels (see Section 3Table 4); this effect is mediated by β2-adrenoceptor stimulation [32–35]and is desensitized after terbutaline p.o. treatment. Thus, after the two weeks of treatment with terbutaline p.o., less noradrenaline is released during isoprenaline infusion (cf. Section 3Table 4) and this might lead to the slight reduction in its positive inotropic effects. At present, we do not know which is the right explanation; however, the fact that ketotifen attenuates terbutaline p.o. treatment-induced desensitization of prejunctional β2-adrenoceptors (as indicated by the restoration of the i.v. isoprenaline-induced increase in plasma noradrenaline, see Section 3) while it does not (or only marginally) affect desensitization of isoprenaline-induced positive inotropic effects argues against a considerable involvement of released noradrenaline in the positive inotropic effects of isoprenaline.

In conclusion, the present results clearly demonstrate that, in humans, chronic treatment with β2-adrenoceptor agonists causes desensitization of atrial β2-adrenoceptors, mediating heart rate increases, as well as ventricular β2-adrenoceptors, mediating positive inotropic effects. Ketotifen can prevent (or at least attenuate) this agonist-induced desensitization of cardiac β2-adrenoceptors; the mechanism underlying this effect remains to be elucidated. According to these results, there is no evidence that human cardiac β2-adrenoceptors might be relatively resistant to agonist-induced desensitization; thus, other mechanisms must be responsible for the selective cardiac β1-adrenoceptor desensitization in chronic human heart failure.

Time for primary review 38 days.


   Acknowledgements
 
The skilful technical assistance of Mrs. Annemarie Dunemann and Mrs. Monika Niebisch is gratefully acknowledged. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Br 526/3-3 to O.-E. Brodde).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Hausdorff W.P, Caron M.G, Lefkowitz R.J. Turning off the signal: desensitization of β-adrenergic receptor function. FASEB J (1990) 4:2881–2889.[Abstract]
  2. Lohse M.J. Molecular mechanisms of membrane receptor desensitization. Biochim Biophys Acta (1993) 1179:171–188.[Medline]
  3. Nelson H.S. Adrenergic therapy of bronchial asthma. J Allergy Clin Immunol (1986) 77:771–785.[CrossRef][ISI][Medline]
  4. Snavely M.D, Mahan L.C, O'Connor D.T, Insel P.A. Selective down regulation of adrenergic receptor subtypes in rats with pheochromocytoma. Endocrinology (1983) 113:354–361.[Abstract]
  5. Tsujimoto G, Manger W.M, Hoffman B.B. Desensitization of beta-adrenergic receptors by pheochromocytoma. Endocrinology (1984) 114:1272–1278.[Abstract]
  6. Bristow M.R. Changes in myocardial and vascular receptors in heart failure. J Am Coll Cardiol (1993) 22(4):61A–71A.[Medline]
  7. Brodde O.-E, Michel M.C, Zerkowski H.-R. Signal transduction mechanisms controlling cardiac contractility and their alterations in chronic heart failure. Cardiovasc Res (1995) 30:570–584.[Free Full Text]
  8. Brodde O.-E. β1- and β2-adrenoceptors in the human heart: properties, function and alterations in chronic heart failure. Pharmacol Rev (1991) 43:203–242.[ISI][Medline]
  9. Bristow M.R, Sandoval A.B, Gilbert E.M, et al. Myocardial {alpha}- and β-adrenoceptors in heart failure: is cardiac-derived norepinephrine the regulatory signal? Eur Heart J (1988) 9(H):35–40.
  10. Kaumann A.J, Hall J.A, Murray K.J, Wells F.C, Brown M.J. A comparison of the effects of adrenaline and noradrenaline on human heart: the role of β1- and β2-adrenoceptors in the stimulation of adenylate cyclase and contractile force. Eur Heart J (1989) 10(B):29–37.[Medline]
  11. Schäfers R.F, Poller U, Pönicke K, et al. Influence of adrenoceptor and muscarinic receptor blockade on the cardiovascular effects of exogenous noradrenaline and of endogenous noradrenaline released by infused tyramine. Naunyn-Schmiedeberg's Arch Pharmacol (1997) 355:239–249.[CrossRef][Medline]
  12. Muntz K.H, Zhao M, Miller J.C. Downregulation of myocardial β-adrenergic receptors. Receptor subtype selectivity. Circ Res (1994) 74:369–375.[Free Full Text]
  13. Ferro A, Kaumann A.J, Brown M.J. β-Adrenoceptor subtypes in human coronary artery: desensitization of β2-adrenergic vasorelaxation by chronic β1-adrenergic stimulation in vitro. J Cardiovasc Pharmacol (1995) 25:134–141.[ISI][Medline]
  14. Levine M.A.H, Leenen F.H. Role of β1-receptors and vagal tone in cardiac inotropic and chronotropic responses to a β2-agonist in humans. Circulation (1989) 79:107–115.[Abstract/Free Full Text]
  15. Schäfers R.F, Adler S, Daul A, et al. Positive inotropic effects of the beta2-adrenoceptor agonist terbutaline in the human heart: effects of long-term beta1-adrenoceptor antagonist treatment. J Am Coll Cardiol (1994) 23:1224–1233.[Abstract]
  16. Brodde O.-E, Brinkmann M, Schemuth R, O'Hara N, Daul A. Terbutaline-induced desensitization of human lymphocyte β2-adrenoceptors. Accelerated restoration of β-adrenoceptor responsiveness by prednisolone and ketotifen. J Clin Invest (1985) 76:1096–1101.[ISI][Medline]
  17. Brodde O.-E, Petrasch S, Bauch H.-J, et al. Terbutaline-induced desensitization of β2-adrenoceptor in vivo function in humans: attenuation by ketotifen. J Cardiovasc Pharmacol (1992) 20:434–439.[ISI][Medline]
  18. Belz G.G. Systolic time intervals: a method to assess cardiovascular drug effects in humans. Eur J Clin Invest (1995) 25:35–41.[ISI][Medline]
  19. Daul A, Hermes U, Schäfers R.F, et al. The β-adrenoceptor subtypes mediating adrenaline- and dobutamine-induced blood pressure and heart rate changes in healthy volunteers. J Clin Pharmacol Ther (1995) 33:140–148.
  20. McDevitt D.G. In vivo studies on the function of cardiac β-adrenoceptors in man. Eur Heart J (1989) 10(B):22–28.[Medline]
  21. Wellstein A, Belz G.G, Palm D. Beta adrenoceptor subtype binding activity in plasma and beta blockade by propranolol and beta-1 selective bisoprolol in humans. Evaluation with Schild-plots. J Pharmacol Exp Ther (1988) 246:328–337.[Abstract/Free Full Text]
  22. Strauss M.H, Reeves R.A, Smith D.L, Leenen F.H.H. The role of cardiac beta-1 receptors in the hemodynamic response to a beta-2 agonist. Clin Pharmacol Ther (1986) 40:108–115.[ISI][Medline]
  23. Azevedo E.R, Newton G.E, Parker J.D. Cardiac β2-adrenergic receptor stimulation. J Am Coll Cardiol (1997) 29(1):41A.
  24. Hill R.J, Neligan M, Keenan A.K. Identification of presynaptic β2-adrenoceptors in human atria. Med Sci Res (1987) 15:1463–1465.[ISI]
  25. Rump L.C, Schwertfeger E, Schaible U, Fraedrich G, Schollmeyer P. β2-Adrenergic receptor and angiotensin II receptor modulation of sympathetic neurotransmission in human atria. Circ Res (1994) 74:434–440.[Abstract/Free Full Text]
  26. Brodde O.-E, Daul A, Michel-Reher M, et al. Agonist-induced desensitization of β-adrenoceptor function in humans. Subtype-selective reduction in β1- or β2-adrenoceptor-mediated physiological effects by xamoterol or procaterol. Circulation (1990) 81:914–921.[Abstract/Free Full Text]
  27. Stein M, Deegan R, Wood A.J. Long-term exposure to beta 2-receptor agonist specifically desensitizes beta-receptor-mediated venodilation. Clin Pharmacol Ther (1993) 54:187–193.[ISI][Medline]
  28. Bretz U, Martin U, Mazzoni L, Ney U.M. β-Adrenergic tachyphylaxis in the rat and its reversal and prevention by ketotifen. Eur J Pharmacol (1983) 86:321–328.[CrossRef][ISI][Medline]
  29. Reinhardt D, Ludwig J, Braun D, Kusenbach G, Griese M. Effects of the antiallergic drug ketotifen on bronchial resistance and β-adrenoceptor density of lymphocytes in children with exercise-induced asthma. Dev Pharmacol Ther (1988) 11:180–188.[ISI][Medline]
  30. Pauwels R, Van der Straeten M. The effect of ketotifen on bronchial beta-adrenergic tachyphylaxis in normal human volunteers. J Allergy Clin Immunol (1988) 81:674–680.[CrossRef][ISI][Medline]
  31. Brodde O.-E, Howe U, Egerszegi S, Konietzko N, Michel M.C. Effect of prednisolone and ketotifen on β2-adrenoceptors in asthmatic patients receiving β2-bronchodilators. Eur J Clin Pharmacol (1988) 34:145–150.[CrossRef][ISI][Medline]
  32. Arnold J.M.O, O'Connnor P.C, Riddell J.G, et al. Effects of the β2-adrenoceptor antagonist ICI 118,551 on exercise tachycardia and isoprenaline-induced β-adrenoceptor responses in man. Br J Clin Pharmacol (1985) 19:619–630.[ISI][Medline]
  33. Vincent H.H, Man in'tVeld A.J, Boomsma F, Derkx F.H.M, Schalekamp M.A.D.H. Is β1-antagonism essential for the antihypertensive action of β-blockers? Hypertension (1987) 9:198–203.[Abstract/Free Full Text]
  34. Brodde O.-E, Daul A, Wellstein A. Differentiation of β1- and β2-adrenoceptor-mediated effects in humans. Am J Physiol (1988) 254:H199–H206.[ISI][Medline]
  35. Motomura S, Zerkowski H.-R, Daul A, Brodde O.-E. On the physiologic role of beta-2 adrenoceptors in the human heart: in vitro and in vivo studies. Am Heart J (1990) 119:608–619.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Poller, U.
Right arrow Articles by Brodde, O.-E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poller, U.
Right arrow Articles by Brodde, O.-E.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?