Skip Navigation

Cardiovascular Research 2003 57(3):694-703; doi:10.1016/S0008-6363(02)00720-4
© 2003 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 Bartel, S.
Right arrow Articles by Karczewski, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bartel, S.
Right arrow Articles by Karczewski, P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2003, European Society of Cardiology

New insights into β2-adrenoceptor signaling in the adult rat heart

Sabine Bartel*, Ernst-Georg Krause, Gerd Wallukat and Peter Karczewski

Max Delbrück Center for Molecular Medicine, Robert Rössle Strasse 10, D-13125 Berlin, Germany

s.bartel{at}mdc-berlin.de

* Corresponding author. Tel.: +49-30-9406-2519; fax: +49-30-9406-2110.

Received 13 May 2002; accepted 9 October 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The role of cAMP in β2-adrenoceptor signaling and its functional relevance in adult rat heart has been the subject of considerable controversy. Therefore, we investigated the β2-adrenoceptor pathways in both adult cardiomyocytes and in the intact hearts of Wistar rats with respect to protein kinase A (at Ser16)-, the key event in shortening of relaxation time, and CaM kinase II (at Thr17)-dependent phospholamban phosphorylation. Methods: Contractile and cellular β1/β2-adrenergic responses were studied in parallel on the same perfused rat heart. (–)Isoproterenol and the β2-adrenergic agonists zinterol and procaterol were used to discriminate the β-adrenoceptor subtype-related actions. Results: β2-Adrenoceptor stimulation induces protein kinase A-dependent phospholamban phosphorylation in both adult cardiomyocytes and in adult hearts of rats. The β2-adrenoceptor-mediated shortening of relaxation time in the heart correlates with Ser16 phosphorylation. Adenosine elicited antiadrenergic action on both β1- and β2-adrenergic signaling cascades by reducing the phosphorylation status of phospholamban. Only β1-adrenoceptor stimulation produced significant CaM kinase II-related Thr17 phosphorylation, troponin I phosphorylation and activation of phosphorylase a. Conclusions: Our findings clearly show that β2-adrenoceptor signaling is coupled to phospholamban phosphorylation and shortening of relaxation time in the adult rat heart.

KEYWORDS Adrenergic (ant)agonists; Myocytes; Receptors; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
β1-Adrenoceptors (ARs) modulate cardiac contractility and relaxation time, mainly through protein kinase A (PKA)-dependent mechanisms involving phosphorylation of phospholamban (PLB) [1–3]. However, there is an incomplete understanding of the β2-AR-dependent modulation of contractile function and its underlying mechanism [4–7]. In general, the signaling cascades from β1- and β2-ARs indicate important differences in their ability to affect downstream cellular events, for example cAMP formation, PKA activation and phosphorylation of PLB at Ser16 (PKA-specific) and Thr17 (Ca2+/calmodulin kinase II (CaMKII)-specific) [4,5]. Moreover, cAMP-independent effects of β2-ARs on heart contractility have also been discussed [4,8–11]. Additionally, despite the similar extent of cAMP accumulation in response to β1- and β2-AR stimulation, marked differences in signal transduction have been observed [8,12–15]. Recent reports have suggested that activation of β2-AR may have significant inotropic effects in cardiomyocytes of adult rats and dogs without eliciting phosphorylation of PLB, the key event in reducing relaxation time [8,12]. However, other investigators have reported that activation of β2-ARs influences both contraction and relaxation by cAMP-dependent mechanisms in human heart preparations and in neonatal rat heart cells [16–18]. Furthermore, there are reports on age- and species-related alterations in the linkage of β2-AR-mediated PLB phosphorylation and relaxation time [12,13,17,18]. Recent studies on the myocardial β2-AR pathway provided evidence for local cAMP signaling linked to the L-type Ca2+ channel in adult rat cardiomyocytes [5,14,19,20]. There are reports indicating β-AR subtype-specific interplay with Gi protein(s), suggesting that β1-AR are predominantly coupled to Gs proteins, whereas β2-AR-mediated effects are modulated by both Gs and Gi proteins [4,5,14,15]. Moreover, pertussis toxin (PTX) switches the profile of β2-AR stimulation to a β1-AR-like response with cAMP-mediated PLB phosphorylation and reduces the relaxation time in adult rat heart cells [14,15]. In contrast, β2-AR responses were not influenced by PTX in embryonic mouse heart cells [21]. Collectively, these results, mostly derived from heart cell preparations treated with zinterol (β2-AR agonist), demonstrate the complexity of the myocardial β2-AR signaling cascade(s), a discussion of the controversial results of which are reviewed in Refs. [4,5]. Furthermore, previous studies have established that adenosine interacts with PTX-sensitive Gi-protein-dependent pathway(s) counteracting the β-AR-induced responses [22]. But it has not yet been determined whether adenosine affects the phosphorylation status of PLB at Ser16 and Thr17 in a β-AR-subtype-dependent manner in the intact adult rat heart. Therefore, respective experiments were included in this study. There are only few data available concerning the underlying mechanisms of β2-AR stimulation in intact adult rat hearts. Moreover, it is unknown whether the β2-adrenergic effects observed in isolated rat cardiomyocytes are also typical for the intact heart. Therefore, one goal of the present study was to investigate PLB phosphorylation in adult rat cardiomyocytes in response to β-AR subtype-specific intervention. Furthermore, studies were designed to assess components of the cAMP-signaling cascade involving PLB phosphorylation in intact isolated rat hearts exposed to selective β1- or β2-adrenergic stimulation. The β-AR agonist (–)isoproterenol and the β2-AR-specific agonists zinterol and procaterol were used as tools to discriminate the β-AR subtype-dependent responses.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
Male Wistar rats (3–4 months old) were used for all experiments. The experiments were performed in accordance with the recommendations of the Declaration of Helsinki and the internationally accepted principles for care and use of laboratory animals.

2.2 Preparation of adult rat cardiomyocytes
Cardiomyocytes of Wistar rats were isolated using a collagenase perfusion adapted from Ref. [23]. Briefly, after anesthesia of the rats, the hearts were removed and perfused with 1.7 mmol/l CaCl2 containing Krebs–Henseleit medium at 37 °C for 5–8 min followed by Ca2+-free perfusion for 5 min. After 25–30 min perfusion with collagenase-containing medium, both atria were discarded. The ventricular tissues were cut into small pieces and suspended in oxygenated collagenase solution. The residue was filtered through a 200 µm mesh screen. After separation of cardiomyocytes from the non-muscle cells by percoll-gradient centrifugation, the heart cells were suspended in modified HEPES medium for studying PLB phosphorylation.

2.3 Heart perfusion
The rats were heparinized and anesthetized (sodium pentobarbital, 25 mg/kg, i.p.). Hearts were perfused in the Langendorff mode as described previously [3]. All hearts were perfused for 30 min before drug application. Left ventricular developed pressure (dLVP), and maximal rate of contraction (+dP/dt) and relaxation (–dP/dt) were monitored during the entire experiment. At selected times the hearts were freeze-clamped and stored at –80 °C until use.

2.4 Drug administration
Hearts were perfused with (–)isoproterenol for 2 min and with zinterol or procaterol for 8 min at the indicated concentrations. CGP 20712A (300 nmol/l; β1-blocker) or ICI 188.551 (200 nmol/l; β2-blocker) were given 10 min before AR agonists. Stock solutions of β-adrenergic agonists were freshly prepared on each experimental day. {alpha}-Chloro adenosine (adenosine) application was for 3 min.

2.5 cAMP-dependent protein kinase A
Soluble and particulate PKA activities were analyzed as described previously [3] and expressed as the ratio of activity in the absence of cAMP to that in the presence of 2 µmol/l.

2.6 Detection of PLB phosphorylation and troponin I phosphorylation by back-phosphorylation
Untreated, zinterol- or (–)isoproterenol-perfused rat hearts were freeze-clamped at the indicated time and the left ventricular tissue (approximately 50 mg) was homogenized with a histidine-containing buffer. Back-phosphorylation performed with the c-subunit of PKA was initiated by [{gamma} 32P]-labeled ATP and processed as described in Ref. [24]. The 32P-incorporation, catalyzed by PKA in vitro, of preparations from control tissue minus 32P-incorporation in fractions from drug-treated tissue was considered to reflect phosphate incorporation in the intact heart and is expressed as pmol phosphate/mg protein.

2.7 Western blot analysis of PKA- and CaMKII-dependent PLB phosphorylation
The analysis of site-specific PLB phosphorylation was performed as described previously [3] using antibodies specific for Ser16 and Thr17 phosphorylated PLB [25]. For the electrophoretic separation, usually 40 µg solubilized tissue protein was loaded per lane. The immunoreaction was visualized by enhanced chemiluminescence, exposed to X-ray film, and quantified by scanning densitometry (PDI, New York, NY, USA) The proteins were determined by the method of Lowry [26] using ovalbumin as standard.

2.8 Statistics
Data are expressed as mean±S.E.M. of n separate hearts. Using GraphPad Prism software, data were checked for normal distribution, and statistical significance was determined by the Student's t-test or nonparametric test as appropriate. P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 β2-Adrenoceptor-related phospholamban phosphorylation in adult rat cardiomyocytes
β2-AR stimulation was found to be associated with PLB phosphorylation in isolated adult rat cardiomyocytes (Fig. 1). PKA-specific Ser16 phosphorylation was significantly increased in response to β1-AR or β2-AR intervention. Thr17 phosphorylation of PLB was enhanced significantly by β1-AR activation with (–)isoproterenol, but only a minor increase was observed with the β2-AR agonist zinterol.


Figure 1
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 β2-Adrenoceptor-related site-specific phospholamban phosphorylation in adult rat cardiomyocytes. Adult rat heart cells were treated with zinterol (Zin; 0.5 µmol/l, 10 min) in the presence of CGP 20712A (300 nmol/l) or with (–)isoproternol (Iso; 10 nmol/l, 5 min) in the presence of ICI 188.551 (200 nmol/l). The heart cells were fixed with trichloroacetic acid and processed for phospholamban (PLB) phosphorylation as described in Methods. PSer16-PLB (Ser16 phosphorylated PLB); PThr17-PLB (Thr17 phosphorylated PLB). (A) Western blots. (B) Densitometric evaluation of data presented in (A). Values are given as means±S.E.M. of arbitrary units of optical density (OD). Ctr, control. *P<0.05 vs. Ctr; {dagger}P<0.05 vs. Iso.

 
3.2 β2-Adrenoceptor signaling in isolated rat hearts
3.2.1 Responses to zinterol
3.2.1.1 Contractile responses
Contractile changes induced by zinterol are summarized in Table 1. Zinterol enhanced concentration dependently both the maximal rate of contraction (+dP/dt) and relaxation (–dP/dt) as well as the developed ventricular pressure (dLVP). The zinterol (50 nmol/l)-related effects on +dP/dt and –dP/dt elicited about 48 and 50% of the values observed in the hearts challenged with 5 nmol/l isoproterenol (+dP/dt, 6803±224 mmHg/s; –dP/dt, –5114±127 mmHg/s; n=8; P<0.05 vs. control). The β2-AR-selective action of zinterol was verified in rat hearts by pretreatment with the respective selective β-AR blockers. In the absence of CGP 20712A, zinterol (100 nmol/l) elicited responses (+dP/dt, 4382±66 mmHg; –dP/dt, 3716±80 mmHg/s, n=4) no different from those in the presence of a β1-AR blocker. In contrast, ICI 188.551, a β2-AR blocker, prevented the zinterol-mediated contractile effects (+dP/dt, 2289±108 mmHg/s; –dP/dt, 1622±98 mmHg/s, n=3). The stimulatory effects of (–)isoproterenol were completely inhibited by CGP 20712A (+dP/dt, 2114±123; –dP/dt, –1456±89, n=4), indicating that the drug acts mainly via β1-ARs. The β2-AR-related effects on relaxation time were studied (Fig. 2). In hearts exposed to 100 nmol/l zinterol the relaxation time was 33.8±0.6 ms (n=6) (P<0.05 vs. pre-drug value of 41.8±0.6 ms; n=6). For comparison, (–)isoproterenol (10 nmol/l) reduced the relaxation time to 28.3±0.4 ms (n=5).


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

  		Table 1 Contractile effects of zinterol in isolated adult rat hearts

 

Figure 2
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Effect of zinterol on heart contractility and relaxation. Isolated rat hearts were exposed to 100 nmol/l zinterol (Zin) in the presence of β1-AR-blockade. Top: original registration. Bottom: relaxation time. Data are expressed as means±S.E.M. of six separately perfused hearts. dLVP, developed left ventricular pressure; Ctr, control. *P<0.05 vs. Ctr.

 
3.2.1.2 cAMP level and PKA activation
Since activation of β-AR is coupled to cAMP via stimulation of adenylyl cyclase, we measured the potency of β1- and β2-AR-mediated cAMP generation in left ventricular tissue of rats at drug concentrations dissecting specific receptor subtypes. 100 nmol/l zinterol enhanced cAMP (pmol/mg protein) from a baseline value of 3.1±0.2 (n=3) to 7.2±0.8 (n=3; P<0.05). For comparison, (–)isoproterenol increased cAMP (pmol/mg protein) to 6.9±0.9 (n=4), 9.6±1.3 (n=3), and 12.7±1.8 (n=3), at concentrations of 5, 10 and 20 nmol/l, respectively. Next, we determined the PKA activation in subcellular fractions of rat hearts exposed to zinterol or (–)isoproterenol (Fig. 3). Both drugs promoted PKA activation in the supernatant fraction. Zinterol-generated PKA activation elicited a ratio of 0.35±0.02 at 100 nmol/l zinterol, corresponding to 50% of the value observed with (–)isoproterenol (10 nmol/l). Whereas the β1-AR-linked pathway was associated with a concentration-dependent activation of PKA in the pellet fraction, β2-AR-linked signaling was not.


Figure 3
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 β1- and β2-AR-related protein kinase A activation. Rat hearts were pre-exposed to the respective β1- or β2-AR blocker, perfused with the indicated drug concentrations, freeze-clamped and processed as described in Methods. Values are means±S.E.M. from four to six separate hearts.*P<0.05 vs. pre-drug value (Ctr, control).

 
3.2.1.3 Phosphorylase a activation
Phosphorylase a activation (expressed as percent of total activity) was found to be coupled to the β1-AR signal cascade in isolated rat hearts (pre-drug value, 14.7±1.2 (n=9); (–)isoproterenol, 5 nmol/l, 32.8±3.8 (n=7); P<0.05). In zinterol (100 nmol/l)-treated hearts, no significant phosphorylase a activation was observed (17.8±0.9; n=5).

3.2.1.4 cAMP-dependent PLB phosphorylation and troponin I phosphorylation
In a first set of experiments we assessed the β1/β2-AR-mediated cAMP-dependent PLB phosphorylation in isolated rat hearts using the back-phosphorylation method. 32P-incorporation catalyzed by the c-subunit of PKA with 32P-labeled ATP as substrate in vitro is inversely correlated to the extent of phosphorylation in intact heart. (–)Isoproterenol and zinterol induced phosphate incorporation into PLB in a concentration-dependent manner (Fig. 4A). The β2-AR-linked in vivo phosphorylation of PLB observed at 100 nmol/l zinterol reached about 54% of the level obtained in response to 10 nmol/l (–)isoproterenol (pmol P/mg protein: 15.0±1.0 vs. 27.8±1.7; n=4, P<0.05). Fig. 4B shows the close correlation between drug-related cAMP-specific PLB phosphorylation and relaxation time (correlation coefficient r2=0.93). Additionally, troponin I phosphorylation was analyzed in zinterol- and (–)isoproterenol-exposed rat hearts. In control rat hearts, PKA-catalyzed 32P-incorporation (pmol/mg protein) in vitro was 38.8±.7 (n=5). (–)Isoproterenol (5 nmol/l) reduced this 32P-incorporation to 29.6±1.2 (n=4; P<0.05 vs. control), indicating drug-induced phosphorylation in the intact heart. The 32P-incorporation into troponin I was measured to be 36.8±2.6 pmol/mg protein (n=4; n.s. vs. control) in hearts challenged with 100 nmol/l zinterol.


Figure 4
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Protein kinase A-dependent phospholamban phosphorylation in response to β-adrenoceptor subtype specific activation. Rat hearts were perfused with drugs in the presence of the respective β-AR antagonists as described in Methods. The analysis of phospholamban (PLB) phosphorylation was performed using the back-phosphorylation technique. (A) Phosphorylation data. (B) Relationship between relaxation time and PLB phosphorylation resulting from zinterol- (Zin) and (–)isoproterenol (Iso)-treated hearts. Data are means±S.E.M. of three to five separate perfused hearts.

 
3.2.1.5 PKA- and CaMKII-linked PLB phosphorylation
The back-phosphorylation as reported above reflects specifically the cAMP-linked PLB phosphorylation. To dissect site-specific PLB phosphorylation, we investigated in a second subset of experiments the Ser16 (PKA specific) and Thr17 (CaMKII specific) PLB phosphorylation in response to β-AR subtype selective interventions with antibodies specific for each phosphorylated site. Representative Western blots and their densitometric evaluation are given in Fig. 5. β2-AR stimulation induced significant Ser16 phosphorylation, resulting in 33.7±4.1% (n=4) of the level reached with 5 nmol/l (–)isoproterenol (100±9.7%, n=5, P<0.05). The Ser16 phosphorylation measured by Western blotting and the corresponding relaxation time resulted in a correlation coefficient of 0.89 in β-AR-subtype-activated perfused rat hearts. Interestingly, only a weak CaMKII-associated Thr17 phosphorylation was observed in rat hearts exposed to 100 nmol/l zinterol, reaching 7.8±1.6% of the phosphorylation elicited by 5 nmol/l (–)isoproterenol.


Figure 5
View larger version (44K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Ser16 and Thr17 phospholamban phosphorylation in response to selective β-AR subtype stimulation. Isolated rat hearts were challenged with zinterol (in the presence of β1-AR blockade) or (–)isoproterenol (in the presence of β2-AR blockade) and analyzed for Ser16 and Thr17 phosphorylated phospholamban (PLB) as outlined in Methods. (A) Western blots. (B) Densitometric evaluation of data presented in (A). Values are given as means±S.E.M. of arbitrary units of optical density (OD). Ctr, control.

 
3.2.2 Responses to procaterol
To verify the above β2-AR-specific responses observed with zinterol we included procaterol, another β2-AR-selective agonist, in our investigations.

3.2.2.1 Contractile responses
The maximal rates of left ventricular pressure development (mmHg/s) and decline (mmHg/s), measured in the presence of β1-AR blockade, were increased significantly in response to 50 nmol/l procaterol (control value: +dP/dt, 2204±76; –dP/dt, –1456±51 versus +dP/dt, 3993±154; –dP/dt, –3172±14; n=10). Furthermore, the relaxation time was significantly reduced (%) to 85.5±1.4 of the control level (100.0±1.0; P<0.05) in hearts challenged with procaterol (Fig. 6A). The procaterol-mediated contractile effects were prevented by ICI 118.551 (+dP/dt, 2078±11 mmHg/s; –dP/dt, –1252±29 mmHg/s; n=3).


Figure 6
View larger version (45K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Procaterol-mediated effects on phospholamban phosphorylation and relaxation time. Rat hearts were exposed to procaterol (50 nmol/l; in the presence of CGP 20712A) or isoproterenol (Iso, 5 nmol/l; in the presence of ICI 188.551) and processed for site-specific phospholamban (PLB) phosphorylation as described in Methods. (A) Relaxation time. (B) Representative Western blots. PSer16-PLB (Ser16 phosphorylated PLB); PThr17-PLB (Thr17 phosphorylated PLB). *P<0.05 vs. Ctr (control).

 
3.2.2.2 PKA activation
Similar to the pattern of zinterol-elicited effects, procaterol affected PKA activity only in the soluble compartment. The activity ratio of PKA was changed from 0.09±0.01 in controls to 0.22±0.02 (n=4) and 0.34±0.03 (n=4) in hearts exposed to 10 and 50 nmol/l procaterol, respectively. There was no significant procaterol-mediated PKA activation in the pellet fractions (control, 0.24±0.09, n=4; procaterol (50 nmol/l), 0.27±0.04, n=4).

3.2.2.3 PKA- and CaMKII-linked phosphorylation
As shown in Fig. 6B, procaterol markedly increased Ser16 phosphorylation to 36.7±7.0% (n=6; P<0.05) of the level obtained in (–)isoproterenol (5 nmol/l) exposed hearts (100±7.9%; n=6). Only a moderate effect of procaterol on Thr17 PLB phosphorylation was obtained (5 nmol/l (–)isoproterenol, 100±12%; 50 nmol/l procaterol, 6.5±2.6%, n=6).

3.3 β2-Adrenoceptor-related signaling and its modulation by adenosine
The next series of studies were designed to assess the modulation of β2-AR-elicited effects in the presence of adenosine, known to interact with PTX-sensitive Gi-protein-dependent pathway(s).

3.3.1 Effect of adenosine on β2-AR-linked contractile responses
Table 2 summarizes contractile parameters from β1- or β2-AR-stimulated rat hearts without and with subsequent coperfusion with adenosine. Adenosine significantly depressed heart contractility independent of the maintained subtype-specific β-AR stimulation. The zinterol-related effects on +dP/dt (188.8±12.6% vs. 100±5.2%, control) or –dP/dt (212.9±15.9% vs. 100±5.4%, predrug value) were reduced by adenosine to 123.5±9.5 and 132.1±12.7%, respectively. In procaterol pretreated hearts, similar contractile responses were observed upon adenosine application. The β1-AR-mediated contractile alterations (–adenosine vs. +adenosine; 100%=pre-drug value) were measured to be +dP/dt, 289.0±14.% vs. 144.7±9.9%; and –dP/dt, 328±9.1% vs. 145.2±16.2%. The β2-AR-mediated effect on the relaxation time was altered from 34.2±0.8 to 39.1±0.4 ms (P<0.05; n=5) by adenosine (41.5±0.3 ms; pre-drug value). The (–)isoproterenol-induced decrease of relaxation time was diminished from 30.7±0.4 ms in the absence of adenosine to 38.2±0.8 ms in the presence of adenosine (n=5).


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

  		Table 2 Effect of adenosine on β1- and β2-adrenergically induced contractile responses in isolated adult rat hearts

 
3.3.2 Effect of adenosine on β2-AR-linked PLB phosphorylation
As Fig. 7 indicates, adenosine applied with persistent β-AR stimulation significantly attenuated the PKA-specific PLB phosphorylation at Ser16 independent of whether it was induced by β1- or β2-AR stimulation in isolated rat hearts. The β-AR-mediated CaMKII-dependent PLB phosphorylation at Thr17 was also substantially attenuated by adenosine. In preliminary experiments we observed that β2-AR-induced PKA activation (soluble fraction, 0.36±0.3; particulate fraction, 0.27±0.03, n=3, versus values in the absence of adenosine as presented in Fig. 3) was not influenced by adenosine. Adenosine did not alter the baseline values of the PKA activation quotient in the respective tissue fractions (soluble fraction, 0.11±003; particulate fraction, 0.23±0.03, n=3, versus values in the absence of adenosine as presented in Fig. 3).


Figure 7
View larger version (33K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Effect of adenosine on phospholamban phosphorylation in rat heart pre-stimulated by selective β-AR subtype activation. (–)Isoproterenol (Iso, 5 nmol/l), zinterol (Zin, 100 nmol/l) and procaterol (Pro, 50 nmol/l) were applied for 5 min followed by a coperfusion with adenosine (1 µmol/l, 3 min). The hearts were pre-perfused with the respective β1-AR or β2-AR blocker as mentioned in Methods. After freeze-clamping of the hearts at the indicated time the site-specific phospholamban (PLB) phosphorylation was analyzed in the ventricular tissue as described in Methods. Data are means±S.E.M. of four to six separate perfused hearts. Insets: representative Western blots. PSer16-PLB (Ser16 phosphorylated PLB); PThr17-PLB (Thr17 phosphorylated PLB). *P<0.05 vs. Iso; {dagger}P<0.05 vs. –adenosine.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Although the myocardial β2-AR density is low in the adult rat heart we observed an effective β2-AR signaling cascade involving PLB phosphorylation and relaxation. We provide evidence that β2-AR stimulation induces PKA-related PLB phosphorylation at Ser16 in both adult rat cardiomyocytes and in intact adult rat hearts. Furthermore, we demonstrate that β2-AR-mediated Ser16 PLB phosphorylation correlates with shortening of relaxation time in the intact rat heart.

4.1 β2-Adrenoceptor-modulated contractile properties
The functional profile of β2-AR-modulated myocardial contractility is presently under controversial discussion (for reviews, see Refs. [4,5]). In the present report we clearly show by systematic studies that selective β2-AR intervention evoked by two independent specific β2-AR agonists increases contractility and reduces relaxation time in a concentration-dependent manner in intact rat hearts. Thus, our findings demonstrate that the β2-AR-related signaling in the adult rat heart has similarities to that observed in the human heart. In human heart preparations, both β1- and β2-AR stimulation enhances inotropy and reduces relaxation time by cAMP-dependent signaling. However, these results are not compatible with those obtained for other species [16,17]. In intact dog heart, β2-AR-associated relaxation was found without coupling to cAMP-dependent PLB phosphorylation, probably realized by a spatial cAMP-dependent interaction with the L-type Ca2+ channel [13]. A zinterol-induced reduction of relaxation time by a cAMP-dependent pathway was found in neonatal rat cardiomyocytes, but not in adult rat cardiomyocytes [4,9,14]. Our findings do not support the notion that β2-AR-mediated shortening of relaxation time is absent or masked in the normal adult rat heart.

4.2 β2-Adrenoceptor-coupled cAMP signaling
Steinberg proposed a cAMP-independent cascade in β2-adrenergically stimulated adult rat heart cells [4]. Others obtained evidence for highly localized cAMP signaling restricted to augment the L-type Ca2+ current [5,14,27,28]. Based on these observations it has been argued that the differences in the β1- and β2-AR signaling may be attributed to distinct coupling of their pathways to PLB phosphorylation [5,14,29]. Because most studies were performed in isolated rat cardiomyocytes it therefore remains uncertain whether the same profile of β2-AR-dependent signaling is present in the adult rat heart. In contrast to the present view, we provide evidence that β2-adrenergically induced PLB phosphorylation occurs in adult rat cardiomyocytes. In consequence, we demonstrate that β2-AR intervention induces cAMP-dependent PLB phosphorylation at Ser16 also in the intact adult rat heart. Obviously, there is no difference in β2-AR signaling between these two experimental models in terms of PLB phosphorylation. Whereas the phosphorylation status of Ser16 correlates with the drug-affected relaxation time in isolated rat hearts the functional importance of Thr17 phosphorylated PLB is not clear [3,30–33]. Thus, our results do not confirm the notion that PLB is not a target of β2-AR signaling in the adult rat heart. In contrast, our data support the conclusion that PLB phosphorylation plays a crucial role in β2-AR-modified relaxation. Additionally, recent data have shown that phosphorylation of both PLB and troponin I contribute to the relaxant effect in isoproterenol-stimulated mouse hearts [34,35]. These results are clearly consistent with a major role of PLB in regulating relaxation as well as contractility. In contrast, previous studies demonstrate that troponin I phosphorylation was not accompanied by a shortening of relaxation time [33]. We found that β2-AR vs. β1-AR signaling is not linked to significant troponin I phosphorylation in the intact rat heart, indicating a β-AR subtype-dependent phosphorylation profile of regulatory proteins. Based on these observation we suggest that the β2-AR-mediated lusitropic action is due to phosphorylation of PLB at Ser16. Furthermore, our data indicate local β2-AR-dependent cAMP signaling, which differs in some aspects from previous reports [4,14,26–28]. In contrast to recently published data [14], our observations indicate no qualitative differences of β-AR subtype signaling with respect to cAMP generation and activation of soluble PKA. But we obtained significant activation of particulate PKA only in hearts exposed to β1-adrenergic stimulation. The compartment-selective PKA activation upon β2-AR stimulation may determine, at least in part, the distinct β2- versus β1-AR signaling, as demonstrated, for example, by its inability to significantly activate phosphorylase a and to produce significant PLB phosphorylation at Thr17. These findings may reflect different β-AR subtype interactions with the Ca2+ influx system. Recent observations from our group and others support this notion [13,14,30]. In line with this assumption are reports demonstrating that β2-AR activation of the L-type Ca2+ channel differs qualitatively from the classical cAMP mechanism [35–37]. Taken together, our findings clearly demonstrate that the β2-AR-subtype-activated pathway is associated with PKA-dependent PLB phosphorylation at Ser16 in both isolated adult rat ventricular cells and in the intact rat heart, resulting in a decrease of the relaxation time without significant CaMKII-catalyzed PLB phosphorylation at Thr17. Therefore, we propose that troponin I is not a relevant target in β2-AR signaling.

4.3 β2-Adrenoceptor signaling and its modulation by adenosine
There are reports indicating a β-AR-subtype-specific interplay with Gi protein(s), suggesting that β1-AR are predominantly coupled to Gs proteins, whereas β2-AR-mediated effects are modulated by both Gs and Gi proteins [4,5,14,15]. β-AR-subtype-distinct mechanisms with respect to muscarinergic agonists were observed in neonatal rat heart cells [18]. Whereas the effects of carbachol interaction with β1-ARs may be explained by reduced cAMP generation, the action on β2-AR-related responses seems to be cAMP-independent. These observations and results of recent studies support the assumption that Gi protein-coupled protein phosphatases may contribute to reduced PLB phosphorylation [14]. Now we add results regarding the interplay between selective β-AR stimulation, adenosine and site-specific PLB phosphorylation in the intact rat heart. Our data clearly show adenosine-elicited dephosphorylation of PLB independent of selective β-AR subtype activation in rat hearts. Thus, adenosine is a potent antiadrenergic agent of both the β1- and β2-AR signaling cascades at the level of the Ca2+ uptake system of the sarcoplasmic reticulum by depressing the phosphorylation status of Ser16. Furthermore, adenosine also counteracts the CaMKII system related to PLB phosphorylation at Thr17 in hearts exposed to β-AR stimulation. In preliminary experiments we observed that PKA activation was not under the control of adenosine, supporting the hypothesis that the adenosine-mediated dephosphorylation of Ser16 and Thr17 phosphorylated PLB is associated with the activation of phosphatases. However, the underlying mechanisms responsible for the adenosine-dependent regulation of PLB phosphorylation with maintained β-AR-subtype-specific stimulation remains to be elucidated.

In summary, the present study clearly demonstrates that despite the low β2-AR density, selective β2-AR adrenergic stimulation activates a cAMP-dependent pathway linked to PLB phosphorylation at Ser16 in both isolated adult rat cardiomyocytes and intact adult rat hearts. The results provide evidence that β2-AR activation contributes to the lusitropic response by increasing the phosphorylation status of PLB at Ser16 in the adult rat heart. However, stimulation of β2-AR, in contrast to β1-AR stimulation, failed to promote phosphorylase a activation, significant CaMKII-catalyzed PLB phosphorylation at Thr17, troponin I phosphorylation and to significantly activate particulate PKA in the adult rat heart. Thus, in the rat heart, β2-AR signaling differs qualitatively from β1-AR mechanisms except for the cAMP-dependent pathway coupled to phosphorylation of PLB and lusitropy.

Time for primary review 22 days.


   Acknowledgements
 
This study was support by the Sonnenfeld Stiftung, Berlin, and by ESF Project 20010019. We thank Dr. Rosemarie Morwinski for preparing the adult rat cardiomyocytes, and Donathe Vetter, Inge Beyerdörfer, Monika Wegener and Wolfgang-Peter Schlegel for expert technical assistance.


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

  1. Luo W., Grupp I.L., Harrer J., et al. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of β-agonist stimulation. Circ Res (1994) 75:401–409.[Abstract/Free Full Text]
  2. Brittsan A.G., Carr A.N., Schmidt A.G., Kranias E.G. Maximal inhibition of SERCA2 Ca2+ affinity by phospholamban in transgenic hearts overexpressing a non-phosphorylatable form of phospholamban. J Biol Chem (2000) 275:12129–12135.[Abstract/Free Full Text]
  3. Kuschel M., Karczewski P., Hempel P., et al. Ser16 prevails over Thr17 phospholamban phosphorylation in the β-adrenergic regulation of cardiac relaxation. Am J Physiol (1999) 276:H1625–H1633.[ISI][Medline]
  4. Steinberg S.F. The molecular basis for distinct β-adrenergic receptor subtype actions in cardiomyocytes. Circ Res (1999) 85:1101–1111.[Free Full Text]
  5. Xiao R.P., Cheng H., Zhou Y.Y., Kuschel M., Lakatta E.G. Recent advances in cardiac β2-adrenergic signal transduction. Circ Res (1999) 85:1092–1100.[Abstract/Free Full Text]
  6. Zhou Y.Y., Cheng H., Song L.S., et al. Spontaneous β2-adrenergic signaling fails to modulate L-type Ca2+ current in mouse ventricular myocytes. Mol Pharmacol (1999) 56:485–493.[Abstract/Free Full Text]
  7. Zhou Y.Y., Song L.S., Lakatta E.G., Xiao R.P., Cheng H. Constitutive β2-adrenergic signalling enhances sarcoplasmic reticulum Ca2+ cycling to augment contraction in mouse heart. J Physiol (1999) 521(2):351–361.[Abstract/Free Full Text]
  8. Xiao R.P., Ji X., Lakatta E.G. Functional coupling of the β2-adrenoceptor to a pertussis toxin-sensitive G protein in cardiac myocytes. Mol Pharmacol (1995) 47:322–329.[Abstract]
  9. Pepe S., Xiao R.P., Hohl C., Altschuld R., Lakatta E.G. Cross talk between opioid peptide and adrenergic receptor signaling in isolated rat heart. Circulation (1997) 95:2122–2129.[Abstract/Free Full Text]
  10. Jiang T., Steinberg S.F. β2-Adrenergic receptors enhanced contractility by stimulating HCO3-dependent intracellular alkalization. Am J Physiol (1997) 273:H1044–H1047.[ISI][Medline]
  11. Pavoine C., Magne S., Sauvadet A., Pecker F. Evidence for a β2-adrenergic/arachidonic acid pathway in ventricular cardiomyocytes: regulation by the β1-adrenergic/cAMP pathway. J Biol Chem (1999) 274:628–637.[Abstract/Free Full Text]
  12. Altschuld R.A., Starling R.C., Hamlin R.L., et al. Response of failing canine and human heart cells to β2-adrenergic stimulation. Circulation (1995) 92:1612–1618.[Abstract/Free Full Text]
  13. Kuschel M., Zhou Y.Y., Spurgeon H., et al. β2-Adrenergic cAMP signaling is uncoupled from phosphorylation of cytoplasmic proteins in canine heart. Circulation (1999) 99:2458–2465.[Abstract/Free Full Text]
  14. Kuschel M., Zhou Y.Y., Cheng H., et al. Gi protein-mediated functional compartmentalization of cardiac β2-adrenergic signaling. J Biol Chem (1999) 274:22048–22052.[Abstract/Free Full Text]
  15. Xiao R.P., Avdonin P., Zhou Y.Y., et al. Coupling of β2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes. Circ Res (1999) 84:43–52.[Abstract/Free Full Text]
  16. Kaumann A.J., Bartel S., Molenaar P., et al. Activation of β2-adrenergic receptors hastens relaxation and mediates phosphorylation of phospholamban, troponin I, and C-protein in ventricular myocardium from patients with terminal heart failure. Circulation (1999) 99:65–72.[Abstract/Free Full Text]
  17. Molenaar P., Bartel S., Cochrane A., et al. Both β2- and β1-adrenergic receptors mediate hastened relaxation and phosphorylation of phospholamban and tropinin I in ventricular myocardium of Fallot infants, consistent with selective coupling of β2-adrenergic receptors to Gs-protein. Circulation (2000) 102:1814–1821.[Abstract/Free Full Text]
  18. Aprigliano O., Rybin V., Pak E., Robinson R.B., Steinberg S.F. β1- And β2-adrenergic receptors exhibit differing susceptibility to muscarinic accentuated antagonism. Am J Physiol (1997) 272:H2726–H2735.[ISI][Medline]
  19. Skeberdis V.A., Jurevicius J., Fischmeister R. β2-Adrenergic activation of L-type Ca2+ current in cardiac myocytes. J Pharmacol Exp Ther (1997) 283:452–461.[Abstract/Free Full Text]
  20. Yabana H., Sasaki Y., Narita H., Nagao T. Subcellular fractions of cyclic AMP and cyclic AMP-dependent protein kinase and the positive inotropic effects of selective β1- and β2-adrenoceptor agonists in guinea pig heart. J Cardiovasc Pharmacol (1995) 26:893–898.[ISI][Medline]
  21. Sabri A., Pak E., Alcott S.A., Wilson B.A., Steinberg S. Coupling function of endogenous {alpha}1- and β-adrenergic receptors in mouse cardiomyocytes. Circ Res (2000) 86:1047–1053.[Abstract/Free Full Text]
  22. Gupta R.C., Neumann J., Durant P., Watanabe A.M. A1-adenosine receptor-mediated inhibition of isoproterenol-stimulated protein phosphorylation in ventricular myocytes. Evidence against a cAMP-dependent effect. Circ Res (1993) 72:65–74.[Abstract/Free Full Text]
  23. Nees S., Dendorfer A. Pharmaceutical application of cell and tissue culture. Wilson G., Davis S., Illum S., eds. (1990) New York: Plenum Press. 72–79.
  24. Karczewski P., Bartel S., Krause E.G. Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts. Biochem J (1990) 266:115–122.[ISI][Medline]
  25. Drago G.A., Colyer J. Phosphorylation site-specific antibodies to cardiac phospholamban. J Biol Chem (1994) 269:25073–25077.[Abstract/Free Full Text]
  26. Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.L. Protein measurement with Folin phenol reagent. J Biol Chem (1951) 193:265–275.[Free Full Text]
  27. Kuznetsov V., Pak E., Robinson R.B., Steinberg S.F. β2-Adrenergic receptor actions in neonatal and adult ventricular myocytes. Circ Res (1995) 76:40–52.[Abstract/Free Full Text]
  28. Xiao R.P., Lakatta E.G. β1-Adrenoceptor stimulation and β2-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca2+, and Ca2+ current in single rat ventricular cells. Circ Res (1993) 73:286–300.[Abstract/Free Full Text]
  29. Xiao R.P., Hohl C., Altschuld R., et al. β2-Adrenergic receptor-stimulated increase in cAMP in rat heart cells is not coupled to changes in Ca2+ dynamics, contractility, or phospholamban phosphorylation. J Biol Chem (1994) 269:19151–19156.[Abstract/Free Full Text]
  30. Bartel S., Vetter D., Schlegel W.P., et al. Phosphorylation of phospholamban at threonine-17 in the absence and presence of β-adrenergic stimulation in neonatal rat cardiomyocytes. J Mol Cell Cardiol (2000) 32:2173–2185.[CrossRef][ISI][Medline]
  31. Hagemann D., Xiao R.P. Dual site phospholamban phosphorylation and its physiological relevance in the heart. Trends Cardiovasc Med (2002) 12:51–56.[CrossRef][ISI][Medline]
  32. Vittone L., Mundina-Weilenmann X., Said M., Ferrero P., Mattiazzi A. Time course and mechanisms of phosphorylation of phospholamban residues in ischemia-reperfused rat hearts. Dissociation of phospholamban phosphorylation pathways. J Mol Cell Cardiol (2002) 34:39–50.[CrossRef][ISI][Medline]
  33. Boknik P., Khorchidi S., Bodor G.S., et al. Role of protein phosphatases in regulation of cardiac inotropy and relaxation. Am J Physiol (2001) 280:H786–H794.[ISI]
  34. Li L., Destantiago J., Chu G., Kranias E.G., Bers D.M. Phosphorylation of phospholamban and troponin I in β-adrenergic-induced acceleration of cardiac relaxation. Am J Physiol (2000) 278:H769–H779.[ISI]
  35. Laflamme M.A., Becker P.L. Do β2-adrenergic receptors modulate Ca2+ in adult rat ventricular myocytes? Am J Physiol (1998) 274:H1308–H1314.[ISI][Medline]
  36. Schröder F., Herzig S. Effects of β2-adrenergic stimulation on single-channel gating of rat cardiac L-type Ca2+ channel. Am J Physiol (1999) 276:H834–H843.[ISI][Medline]
  37. Chen-Izu Y., Xiao R.P., Izu L.T., et al. Gi-dependent localization of β2-adrenergic receptor signaling to L-type Ca2+ channels. Biophys J (2000) 79:2547–2556.[Abstract/Free Full Text]

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 Bartel, S.
Right arrow Articles by Karczewski, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bartel, S.
Right arrow Articles by Karczewski, P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?