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Cardiovascular Research 2004 63(4):662-672; doi:10.1016/j.cardiores.2004.05.014
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Carvedilol selectively inhibits oscillatory intracellular calcium changes evoked by human {alpha}1D- and {alpha}1B-adrenergic receptors

Taka-aki Koshimizu*,a, Gozoh Tsujimotob, Akira Hirasawaa, Yoko Kitagawaa and Akito Tanouea

aDepartment of Molecular Pharmacology, National Research Institute for Child Health and Development, 3-35-31, Taishido, Setagaya, Tokyo 154-8567, Japan
bDepartment of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University Faculty of Pharmaceutical Sciences, Kyoto University, Yoshida Shimoadachi-cho, Sakyo, Kyoto 606-8501, Japan

* Corresponding author. Tel.: +81-3-3419-2476; fax: +81-3-3419-1252. Email address: tkoshi{at}nch.go.jp

Received 13 January 2004; revised 19 May 2004; accepted 21 May 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
 References
 
Background: Increasing evidence from clinical trials indicates that carvedilol, an antagonist of {alpha}1- and β-adrenergic receptors (ARs), provides an effective treatment for chronic heart failure, whereas nonselective {alpha}1-AR blockade has an adverse outcome in this disease. It is, however, not clear whether carvedilol exhibits a subtype-dependent impact on three distinct {alpha}1-adrenergic receptors ({alpha}1-ARs). Methods and results: We determined binding properties of human ARs for carvedilol using HEK293 human embryonic kidney cells expressing a single AR subtype. Our results showed that the affinities of {alpha}1D-AR and {alpha}1B-AR for carvedilol are higher than that of the β1-AR subtype, a major target in heart failure treatment. The affinity rank order and pKi values of ARs for carvedilol were as follows: {alpha}1D-AR (8.9)>{alpha}1B-AR (8.6)>β1-AR (8.4)>β2-AR (8.0)>{alpha}1A-AR (7.9)>>{alpha}2C-AR (5.9)>{alpha}2B-AR (5.5)>{alpha}2A-AR (5.3). Furthermore, temporal kinetics of intracellular calcium signaling mediated via {alpha}1D- and {alpha}1B-ARs, but not via {alpha}1A-AR (P<0.01), showed oscillatory patterns with frequencies ranging from 0.3 to 3 per minute in human smooth muscle and HEK293 cells, which were inhibited by the therapeutic concentrations of carvedilol (10 nM) in a subtype-dependent manner. When oscillatory {alpha}1B-AR and non-oscillatory {alpha}1A-AR were co-expressed and heteromer receptors were detected with bioluminescence resonance energy transfer and co-immunoprecipitation, carvedilol suppressed only oscillatory component of global cytosolic free calcium change. Conclusions: These results indicate that in addition to β-ARs, receptor inhibition by carvedilol is directed to {alpha}1-ARs, preferably to {alpha}1D- and {alpha}1B-AR-mediated signaling events, including intracellular calcium oscillations in vascular smooth muscle.

KEYWORDS Adrenergic antagonists; Calcium (cellular); Heart failure; Smooth muscle; Vasoactive agents

Abbreviations: [125I]HEAT, (±)-β-([125I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone • [3H]RX821002, (1,4-[6,7(n)-3H]benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride • Gpp(NH)p, 5'-guanylylimidodiphosphate


   1. Introduction
Top
 Abstract
 1. Introduction
2. Experimental procedures
 3. Results
 4. Discussion
 References
 
The increased sympathetic activity in chronic heart failure (CHF) is a compensatory adaptation to the initial disease stage that eventually damages the myocardium and vascular smooth muscle via stimulation of adrenergic receptors (ARs) and other neurohormonal and inflammatory signaling pathways [1]. The ARs consist of three distinct subclasses, namely, β- (β1, 2, and 3), {alpha}1- ({alpha}1A, 1B, and 1D), and {alpha}2- ({alpha}2A, 2B, and 2C) ARs [2,3]. Several randomized clinical studies on CHF using subtype nonselective {alpha}1-adrenergic blockers, such as prazosin and doxazosin, failed to show efficacy compared to placebo or low-dose diuretics [4,5]. Because both prazosin and doxazosin were shown to have proapoptotic properties on cardiac myocytes [6], the therapeutic efficacy of blocking {alpha}1-ARs without the apoptotic effect has not been determined.

Conversely, treatment with carvedilol, a blocker of both β- and {alpha}1-ARs with antioxidant effects, was significantly beneficial for CHF in several randomized clinical trials [7–10]. Especially, a recently completed Carvedilol or Metoprolol European Trial (COMET) study, which was the first mortality study comparing two β blocking agents in patients with CHF, showed a significant improvement in survival rate in the carvedilol arm when compared to metoprolol, a β1-selective blocker [11].

Although efficacy of carvedilol might be mainly attributable to its β-blocking effect [12,13], it has not been conclusively demonstrated whether the {alpha}1-AR blockade by carvedilol could have an additional benefit for CHF. Our focus in this study is to evaluate the subtype-specific inhibitory potencies of carvedilol against recombinant human ARs to clarify the preferential receptor subtypes for this drug. Specifically, emphasis is on the subtype selectivity of carvedilol relative to the {alpha}1-ARs. The three {alpha}1-AR subtypes, which couple to Gq type of G protein and the phospholipase C pathway, have different tissue and subcellular distributions, intracellular signaling cascades, and transcriptional profiles [14]. In arterial smooth muscle cells, these subtypes are frequently co-expressed and formation of homo- and hetero-oligomer {alpha}1-ARs is proposed [15–17]. Our results revealed that {alpha}1D- and {alpha}1B-AR-dependent intracellular calcium signaling, which causes oscillatory changes in the intracellular calcium concentration ([Ca2+]i), is more susceptible to inhibition by carvedilol than the calcium signaling caused by the {alpha}1A-AR. Therefore, {alpha}1-ARs show subtype-dependent sensitivity to carvedilol.


   2. Experimental procedures
Top
 Abstract
 1. Introduction
 2. Experimental procedures
3. Results
 4. Discussion
 References
 
2.1. Materials
cDNAs for human β1-, β2-, {alpha}2A-, {alpha}2B-, and {alpha}2C-ARs were kind gifts from Dr. Robert J. Lefkowitz (Duke university). The cDNAs for β1- and β2-ARs that have an amino-terminal epitope tag for hemagglutinin protein were kindly provided by Dr. Hitoshi Kurose (Kyushu university) [18]. Characterization of cDNAs for human {alpha}1A-, {alpha}1B-, and {alpha}1D-ARs was described previously [19]. Carvedilol and KMD-3213 dihydrobromide were kindly provided by Daiichi Pharmaceutical (Tokyo, Japan) and Kissei Pharmaceutical (Matsumoto, Japan), respectively.

2.2. Expression constructs
Receptor cDNAs were subcloned into a mammalian expression vector, pcDNA1.3 (Invitrogen, Carlsbad, CA), at XhoI/KpnI ({alpha}1A-, {alpha}1B-, {alpha}1D-, {alpha}2A-, {alpha}2B-, and {alpha}2C-ARs) or at EcoRI/BamHI (β1- and β2-ARs) sites.

2.3. Cell culture and transfection
HEK293 human embryonic kidney cells were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Human aortic smooth muscle cells (AoSM cells) were cultured according to the supplier's instruction (Cambrex, East Rutherford, NJ). The expression constructs (4.5 µg DNA) were transfected into the cells grown in a 10-cm tissue culture dish by cationic lipid method using Lipofectamine Plus (Invitrogen). To obtain stable cell lines expressing each {alpha}1-AR, transfected cells were dispersed at 5 x 105 cells/10 cm tissue culture dish, and Zeocin (0.5 mg/ml) was included in the culture medium. Cloned stable cell lines were maintained in culture medium containing 0.2 mg/ml Zeocin.

2.4. Membrane preparation and binding study
Cellular membrane fraction was prepared as described [20] and stored at –80 °C in buffer B (50 mM Tris–HCL, 1 mM EDTA, pH 7.4). For detection of {alpha}1-, {alpha}2-, and β-ARs, [125I]HEAT (2200 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA), [3H]RX821002 (54 Ci/mmol, Amersham Biosciences, Piscataway, NJ), and [3H]CGP12177 (33 Ci/mmol, Amersham Biosciences) were used, respectively. There was no detectable specific binding of these radioligands to wild-type HEK cells.

Binding studies were performed as described previously [20–23]. For saturation binding study, 50–1000 pM of [125I]HEAT, 25–3200 pM of [3H]RX821002, or 12.5–1600 pM of [3H]CGP12177 were used. Because buffer composition, especially concentrations of MgCl2 and NaCl, affects antagonist binding to {alpha}2-ARs [24], binding studies of {alpha}2-ARs were performed in buffer B containing 140 mM NaCl and 100 µM Gpp(NH)p [21]. Specific radioligand binding was determined from the difference between counts in the presence or absence of 10 µM phentolamine ({alpha}1- and {alpha}2-ARs) or of 10 µM propranolol (β-ARs).

2.5. Measurement of intracellular calcium transients
HEK 293 cells stably expressing {alpha}1-ARs were tested for their ability to mobilize calcium using a fluorescent intracellular calcium assay kit (Molecular Devices, Sunnyvale, CA) and a fluorescence-imaging plate reader (FLIPR) system (Molecular Devices) according to the manufacturer's instructions. For dye loading cells in 100 µl Hank's/HEPES buffer (145 mM NaCl, 5 mM KCl, 0.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4) containing FLIPR calcium assay reagents were incubated for 1 h at 37 °C in black 96-well Viewplates (Polyfiltronics, Rockland, ME). Basal [Ca2+]i conditions were monitored for 10 s before adding 50 µl of the agonist with an integrated 96-well pipetter. Fluorescence was measured from all 96 wells simultaneously using a charge-coupled device camera. Concentration–response curves were calculated as percentage of maximum response (Emax) induced by 10 µM phenylephrine. Agonist potency was expressed as pD2 (–log EC50) value.

Single-cell [Ca2+]i measurements were performed as described previously [25]. The wild-type HEK293 cells and AoSM cells did not show any [Ca2+]i change when stimulated with 10 µM phenylephrine (data not shown).

2.6. Measurement of biological resonance energy transfer
The cDNAs for the {alpha}1-ARs were connected in-frame with either yellow fluorescent protein (YFP) or Renilla luciferase (Rluc) gene to detect receptor–receptor interactions. The native stop codons of {alpha}1-ARs were changed to KpnI restriction enzyme site and genes for a variant YFP, "Venus" (F64L/M153T/V163A/S175G) [26], and for Rluc with optimized codons for mammalian expression (Promega) were connected, resulting in two amino acid insertions of glycine and asparagine between the receptor and YFP or luciferase. All fusion constructs were sequenced and transferred to pcDNA3.1 (Invitrogen). The additions of amino- or carboxy-terminal tags to the {alpha}1-ARs had no significant effect on their binding and intracellular signalings [27].

For biological resonance energy transfer (BRET) assays, transfected cells growing on a 10-cm culture dish were collected in PBS containing 1 mM EDTA and suspended in Hank's/HEPES buffer at 1 x 106/ml. After addition of coelenterazine h (Promega) at a final concentration of 5 µM, luminescence was detected with a fluorescence spectrophotometer (F-2500, Hitachi, Tokyo, Japan). The BRET signal was determined by calculating the ratio of the light emitted by the receptor-YFP at 535 nm to the light emitted by the receptor-Rluc at 480 nm. Background signal determined prior to the addition of coelenterazine h was always less than 1% of measured values and was subtracted.

2.7. Immunoprecipitation and Western blot analysis
The FLAG epitope was inserted between the initial methionine residue and the second amino acids of the {alpha}1-ARs by PCR. The 5'-primer sequence contained 6 bases of XhoI site, optimized translation sequence, 3 bases for methionine, 24 bases encoding the FLAG-peptide sequence, and 21 bases encoding 7 amino acids next to initiator methionine. Immunoprecipitation experiments were performed as described previously [25], using anti-FLAG M2 antibody (Kodak, Rochester, NY) and anti-GFP antibody (MBL, Nagoya, Japan) at a concentration of 4 µg/ml. For Western blot detection, monoclonal anti-FLAG M2 or anti-GFP antibodies were used at a dilution of 1:2000 and anti-extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and anti-phospho-ERK1/2 antibodies (New England Biolabs) at a dilution of 1:1000. For secondary antibody, peroxidase-conjugated anti-mouse and anti-rabbit antibodies were diluted to 1:5000, and signals were visualized with enhanced chemiluminescence ECL (Amersham Biosciences).

2.8. Calculations
All values in the text are reported as mean±S.D. Significant differences, with P<0.05 were determined by one-way ANOVA with Newman–Keuls multiple comparison test. Concentration–response relationships were fitted to a four-parameter logistic equation using a nonlinear curve-fitting program, which derives the EC50 and Hill's values (Igor, WaveMetrics, Lake Oswego, OR). Calcium recordings were done in 10–50 cells simultaneously, and each experiment was repeated three or more times to ensure the reproducibility of the findings.


   3. Results
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
4. Discussion
 References
 
3.1. Affinities of human adrenergic receptors for carvedilol
Specific radioligand binding sites of {alpha}1-, {alpha}2-, and β-ARs were detected in the transfected HEK293 cells, but not in the wild-type cells, by [125I]HEAT, [3H]RX821002, and [3H]CGP12177, respectively. The Kd values obtained in saturation binding experiments were in good agreement with those previously reported for recombinant and native human ARs and listed in Table 1 [20–23,28]. In competition binding experiments using membrane preparations from cells expressing each {alpha}1-AR, carvedilol was moderately selective for the {alpha}1B- and {alpha}1D-AR subtypes compared to the {alpha}1A-AR (Fig. 1A). Thus, a tenfold difference was detected in the Ki values between the {alpha}1D- and {alpha}1A-ARs (1.2 and 12 nM, respectively, Fig. 1 and Table 1). Notably, the affinities of {alpha}1B- and {alpha}1D-ARs for carvedilol were higher than any other ARs including β1-AR and β2-AR (Fig. 1B and Table 1). In contrast, {alpha}2-ARs showed weak binding to carvedilol with Ki values in the micromolar range (Fig. 1C).


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  		Table 1 Radioligand binding characteristics of human adrenergic receptors expressed in HEK293 cells

 

Figure 1
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  		Fig. 1 Competition binding experiments in membrane preparations from HEK293 cells expressing human {alpha}1-ARs (A), β-ARs (B), and {alpha}2-ARs (C). Inhibition curves of specific radioligand bindings by carvedilol (A–C) and phentolamine (A) are shown. Each point represents the mean±S.D. from three to six experiments performed in duplicate.

 
3.2. Inhibition of {alpha}1-AR-mediated intracellular responses by carvedilol
The effects of high affinity binding of carvedilol to {alpha}1D- and {alpha}1B-AR subtypes were further examined by stimulating cells in the presence and absence of therapeutic concentrations of carvedilol (1–10 nM) and measuring averaged [Ca2+]i with a fluorescence-imaging plate reader. When HEK cells stably expressing {alpha}1-ARs were stimulated with phenylephrine or norepinephrine, [Ca2+]i increased in a concentration-dependent manner with pD2 values listed in Table 2. The relationships between agonist concentrations and peak amplitudes in [Ca2+]i were shifted toward the right in the presence of carvedilol or phentolamine, keeping same maximum amplitudes (Fig. 2A and B). The calculated pA2 values of carvedilol upon norepinephrine stimulation were 9.0, 10, and 10.2 for {alpha}1A-, {alpha}1B-, and {alpha}1D-ARs, respectively (Table 2). Therefore, carvedilol is a competitive inhibitor with graded inhibitory potencies against three {alpha}1-ARs. When expressed in HEK cells, each {alpha}1-AR tagged with GFP shows specific patterns of cellular localization, as seen in Fig. 2C; {alpha}1A-AR-GFP localized at both cell surface and intracellular part, while {alpha}1B-AR-GFP and {alpha}1D-AR-GFP were seen mostly at cell surface and at intracellular part, respectively. In spite of the distinct cellular localizations, activities of all {alpha}1-ARs were effectively controlled by carvedilol.


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  		Table 2 Inhibition of intracellular calcium responses by carvedilol in HEK293 cells expressing human {alpha}1-ARs

 

Figure 2
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  		Fig. 2 Carvedilol inhibited [Ca2+]i responses in a concentration-dependent manner in cells expressing {alpha}1-ARs. HEK293 cells stably expressing each {alpha}1-AR were stimulated with increasing concentrations of phenylephrine (A) and norepinephrine (B) in the absence (open circles) and presence of carvedilol (closed circles) or phentolamine (closed triangles). Before stimulations, antagonists at concentrations indicated were incubated with cells for 10 min. Each point represents the mean±S.D. from four to six measurements performed in triplicate. Cellular localizations of {alpha}1-AR-GFP constructs transiently expressed in HEK cells were examined by confocal microscopy (C). Representative pictures from four experiments were presented.

 
3.3. Single-cell calcium responses of {alpha}1-ARs
We next searched for an intracellular signaling pathway in AoSM cells, which could show specificity among {alpha}1-AR subtypes and exhibit a different sensitivity to carvedilol. In AoSM cells, specific binding of [125I]HEAT or [3H]RX821002 was not detectable prior to transfection procedure. Thus, no functional expression of native {alpha}-AR subtypes was observed in our culture condition. When each {alpha}1-AR subtype was transiently transfected, total binding sites of [125I]HEAT for {alpha}1A-AR, {alpha}1B-AR, and {alpha}1-AR were 256±20 (n=3), 138±8 (n=3), and 271±47 (n=3) fmol/mg protein, respectively, while no specific binding sites of [3H]RX821002 were detected in these transfected cells.

In fura-2 loaded AoSM cells expressing {alpha}1A-AR, a submaximal concentration of 10 µM phenylephrine evoked a [Ca2+]i increase of spike and plateau pattern (Fig. 3A). In contrast, oscillatory patterns of [Ca2+]i changes were observed in 78% (n=119) and 91% (n=156) of cells expressing {alpha}1B- and {alpha}1D-ARs, respectively, while only 4% (n=203) in {alpha}1A-AR expressing cells showed oscillation in [Ca2+]i. The oscillation frequencies in the cells expressing {alpha}1B-AR and {alpha}1D-AR had no significant difference, ranging between 0.3 and 3 per minute. The {alpha}1A-AR-mediated repetitive [Ca2+]i change maintained a high basal [Ca2+]i level during oscillations, while those seen in {alpha}1B- and {alpha}1D-AR-expressing cells returned to basal [Ca2+]i between oscillations (Fig. 3A). The differences in the [Ca2+]i response patterns among the {alpha}1-ARs were well preserved in HEK293 cells separately transfected with each {alpha}1-AR subtype (Fig. 3B). Therefore, distinct oscillatory and non-oscillatory [Ca2+]i changes induced by {alpha}1-ARs are not specific to the smooth muscle cells and can occur in a nonexcitable cell type such as HEK cells. Phenylephrine-induced [Ca2+]i changes in both AoSM cells and HEK cells were efficiently suppressed by preincubation of cells with 10 µM phentolamine, while ATP (100 µM)-induced calcium responses were not affected (Fig. 3C and D).


Figure 3
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  		Fig. 3 Single-cell [Ca2+]i responses by {alpha}1-AR subtypes expressed in primary culture of human AoSM cells (A and C) and HEK293 cells (B and D). Cells were stimulated with 10 µM phenylephrine and subsequently with 100 µM ATP (C and D) in the absence (A and B) and presence of 10 µM phentolamine (C and D). The inset in panel A shows [Ca2+]i response with fast fluctuations in small fraction (4%) of cells expressing {alpha}1A-AR. Representative traces from three to six experiments are presented.

 
3.4. The effect of carvedilol on the subtype-dependent single-cell calcium responses of {alpha}1-ARs
We next examined an effect of carvedilol treatment on the single-cell calcium response patterns. The peak [Ca2+]i amplitudes induced by 10 µM phenylephrine gradually decreased with increasing carvedilol concentrations used in all cell types (Fig. 4A). In {alpha}1B- and {alpha}1D-AR expressing cells, spiking frequencies, in addition to the amplitudes, were significantly lowered by carvedilol (Fig. 4A). These inhibitory effects were partially overcome by increasing agonist concentration to 100 µM in cells expressing {alpha}1B-AR (Fig. 4B) and {alpha}1D-AR (not shown). In contrast to phenylephrine, a partial agonist at all of the human {alpha}1-ARs [29], stimulation with submaximal concentration (10 µM) of norepinephrine, a full agonist for {alpha}1-ARs, resulted in high oscillatory frequencies in both {alpha}1B- and {alpha}1D-AR-expressing cells and [Ca2+]i did not decrease to the basal resting level during oscillations (Fig. 4C). Preincubation of the cells with 10 nM carvedilol and stimulation with norepinephrine resulted in a return to the large oscillatory pattern (Fig. 4C). In contrast, carvedilol treatment in {alpha}1A-AR-expressing cells did not increase fraction of cells showing the oscillatory [Ca2+]i response. These results indicate that {alpha}1D- and {alpha}1B-ARs evoke oscillatory patterns of [Ca2+]i mobilization, the frequencies of which could be modulated by both agonist potencies and antagonist concentrations at single-cell level.


Figure 4
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  		Fig. 4 The {alpha}1-AR-mediated single-cell [Ca2+]i responses are dependent on antagonist concentrations. (A) HEK cells expressing {alpha}1-ARs were stimulated with 10 µM of phenylephrine in the presence of increasing concentrations of carvedilol. (B) Oscillatory responses were recovered by higher concentration (100 µM) of phenylephrine and norepinephrine even in the presence of 100 nM carvedilol in cells expressing {alpha}1B-AR. (C) Stimulation with 10 µM norepinephrine in the absence (upper) and presence (lower) of 10 nM carvedilol in HEK cells expressing {alpha}1B-and {alpha}1D-ARs. The frequency of oscillation varied in each cell and traces from three separate single cells are presented (lower panels).

 
3.5. Inhibition of ERK1/2 phosphorylation by carvedilol
We further examined the effects of blocking each {alpha}1-AR on phenylephrine-induced ERK1/2 phosphorylation, which are related to cellular growth and hypertrophy. As shown in Fig. 5, carvedilol (100 nM), as well as phentolamine (1 µM), significantly inhibited these ERK1/2 phosphorylations. When signals with phospho-ERK1/2 specific antibody were normalized by total ERK1/2 amount, ERK1/2 was phosphorylated more effectively in smooth muscle cells transiently expressing {alpha}1B- or {alpha}1D-ARs than in cells expressing {alpha}1A-AR (Fig. 5B).


Figure 5
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  		Fig. 5 Inhibitions of phenylephrine-induced ERK phosphorylation by carvedilol. (A) Transiently transfected AoSM cells were stimulated with 10 µM phenylephrine for 5 min and collected in lysis buffer. Equal amounts (20-µg protein) of samples were subjected to SDS-PAGE and immunoblotting for the analysis of ERK1/2 phosphorylation using phosphospecific (Thr202/Tyr204) antibody. The blots were then stripped and reprobed with ERK1/2 antibody. Carvedilol (100 nM) and phentolamine (1 µM) were incubated for 10 min prior to the stimulations. Representative data from four experiments are presented. (B) Signal intensities of phosphorylated ERK1/2 were normalized with intensities of total ERK1/2 and the mean±S.D. are shown (n=4). *P<0.05, compared to cells treated with carvedilol or phentolamine.

 
3.6. The effect of carvedilol on cells co-expressing {alpha}1-AR subtypes
Because different {alpha}1-AR subtypes are frequently co-expressed in the same vascular smooth muscle [14], we examined subtype selectivity of carvedilol on cells co-expressing different {alpha}1-ARs. When YFP-tagged {alpha}1B-AR and Rluc-tagged receptors were co-expressed, significant increases of the BRET signal occurred in all combinations of {alpha}1-AR subtypes, compared to the cells co-expressing receptor-YFP and Rluc or to the cells expressing YFP and receptor-Rluc (Fig. 6A). These results suggested that when co-expressed, {alpha}1-ARs could form homomers and hetero-oligomers. The co-immunoprecipitation experiments confirmed interaction between {alpha}1B-AR and other subtypes (Fig. 6B). In both BRET and co-immunoprecipitation experiments, {alpha}1B-AR interacted to YFP-tagged and Rluc-tagged {alpha}1-ARs with preference to {alpha}1D-AR and {alpha}1B-AR over {alpha}1A-AR (Fig. 6A and B).


Figure 6
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  		Fig. 6 Oligomerization of {alpha}1-AR subtypes. (A) BRET signals were measured in cells co-expressing YFP-tagged {alpha}1B-AR and Rluc-tagged receptors. Asterisks (*) indicate significant increase in the emission intensity at 535 nm compared to the negative control cells co-expressing {alpha}1B-AR-YFP and Rluc or cells co-expressing YFP and {alpha}1B-AR-Rluc (P<0.05). Values were the mean±S.D. from four independent experiments performed in triplicates. (B) Co-immunoprecipitation of YFP-tagged {alpha}1-ARs with FLAG-tagged {alpha}1B-AR. Each YFP-tagged {alpha}1-AR was co-transfected with FLAG-tagged {alpha}1B-AR, immunoprecipitated with anti-FLAG antibody, and detected by Western blotting with anti-GFP antibody (left panel) and anti-FLAG antibody (right upper panel). Expression levels of GFP-tagged receptors were also examined (right lower panel). Representative results from the five experiments are shown.

 
We then focused our analysis on the cells co-expressing {alpha}1A-AR and {alpha}1B-AR, which cause two distinct [Ca2+]i kinetics. In cells stably expressing {alpha}1A-AR that transiently expressed {alpha}1B-AR-YFP, [125I]HEAT, a subtype nonselective antagonist for {alpha}1-AR, detected single high affinity site. Bmax value of such co-transfected cells was 13 pmol/mg protein and Kd value 335±89 nM (n=3). Fractions of high and low affinity binding sites, which were detected by an {alpha}1A-AR-specific antagonist, KMD-3213, were 45±7% and 55±7%, respectively (n=4), as shown in Fig. 7A. The pKi values of {alpha}1A- and {alpha}1B-ARs for KMD-3213 were 8.0±0.1 and 6.8±0.1 (n=4), respectively, and those of high and low affinity sites in co-expressed cells were 8.2±0.1 and 7.0±0.1 (n=3), respectively. Stimulation of these cells with 10 µM phenylephrine caused oscillatory calcium response with gradually decreasing basal [Ca2+]i. When 10 nM carvedilol was added in the external buffer and present throughout the measurement, [Ca2+]i response changed to single spike without oscillation (Fig. 7B). Thus, among the homo- and hetero-oligomeric receptors formed in the cells co-expressing {alpha}1A-AR and {alpha}1B-AR subtypes, carvedilol effectively inhibited the receptors responsible for the oscillatory component of complex [Ca2+]i response.


Figure 7
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  		Fig. 7 Calcium responses of cells co-expressing {alpha}1A- and {alpha}1B-ARs. (A) Competition binding studies using KMD-3213, an {alpha}1A-selective antagonist, were performed with membrane preparations from HEK cells expressing {alpha}1A- or {alpha}1B-AR (upper) and both {alpha}1A- and {alpha}1B-ARs (lower). Nonspecific binding was determined in the presence of 10 µM phentolamine. Data presented are the mean from single experiment performed in triplicate. Similar results were obtained from three additional experiments. (B) Co-expressed cells were stimulated with 10 µM phenylephrine in the absence (upper) and presence (lower) of 10 nM carvedilol. Traces are representative of 58 cells (upper) and 71 cells (lower) from three experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
References
 
Recombinant expression of each AR subtype enabled us to delineate a series of inhibitory potencies of carvedilol in the adrenergic system. Thus, the binding affinities of carvedilol for {alpha}1D- and {alpha}1B-ARs were comparable to that for β1-AR and were more potent than those for β2-AR and {alpha}1A-AR subtypes. Several meta-analysis and COMET study for CHF showed consistent benefit of combined {alpha}1- and β-AR blockade by carvedilol [11,30]. It was also reported that carvedilol lowered coronary sinus norepinephrine levels, an index of cardiac adrenergic activity, whereas metoprolol actually increased central venous norepinephrine levels [31]. Although the mechanism whereby carvedilol lowers norepinephrine levels needs to be further investigated, findings from {alpha}1D-AR knockout mice suggested that ablating the function of this subtype attenuates the elevation of plasma norepinephrine level during the development of hypertension accompanied with renal failure [32]. When bucindolol, a nonselective β-AR blocker with only weak {alpha}1-AR blocking activity, was used for CHF in β-Blocker Evaluation of Survival Trial, it increased the combined endpoints of death or heart failure hospitalization within the first 6 months and did not result in overall benefit [33]. Although patient populations and severity of the disease differ in each CHF trial, these results indicate that AR types, which are selected for a pharmacological intervention, could have a significant impact on the final outcome of CHF treatment.

Previous pharmacokinetics data suggested that 25 and 50 mg oral administration of carvedilol produced peak plasma levels of 150 and 300 nM, respectively, and about 95% of carvedilol was found to bind to plasma proteins [34]. These plasma concentrations indicate that blockade of {alpha}1- and β-ARs by carvedilol could occur in a linear range of the competition binding curve and that ligand binding to {alpha}2-ARs could be largely preserved. However, in examining ligand affinities using recombinant receptors and translating these results to efficacy estimation of agonist and antagonist in vivo, care should be taken for potential difference in cellular environment, which might influence receptor functions. In addition, there is species difference in relative affinities of carvedilol to {alpha}1- and β-ARs. By using vascular preparation from pithed rat, Ruffolo et al. [35] found that KB values of β- and {alpha}1-ARs for carvedilol were 0.9 and 11 nM, respectively. On the other hand, Bristow et al. [36] used membrane preparations from human ventricular myocardium for radioligand assay and found that Ki values of β1- and {alpha}1-ARs for carvedilol were 4.5 and 8.1 nM, respectively. Thus, the ratios of β1-AR/{alpha}1-AR blocking activities of carvedilol are 12 and 1.7 for rat and human receptors, respectively. The reason for lower affinity rank order of {alpha}1-AR than the β1-AR in human heart is based on the fact that main cardiac {alpha}1-AR subtype has been reported to be the {alpha}1A-AR subtype, although the exact amount of each {alpha}1-AR protein has not been examined [37]. Therefore, binding affinities calculated in our study using recombinant human ARs should provide basic knowledge for understanding affinities of native ARs against carvedilol.

The {alpha}1-ARs in the peripheral vasculature play a major role in positively regulating arterial vascular tone [15]. Gene targeting for each of the {alpha}1-AR subtype further revealed that deletion of one subtype gene was directly correlated to the attenuated pressor response to an {alpha}1-AR agonist administered intravenously [38–40]. Therefore, there was minimal functional redundancy among the three vascular {alpha}1-AR subtypes and blocking a specific fraction of {alpha}1-AR had direct impact on the regulation of systemic blood pressure.

Recent clinical studies, however, indicate that efficacy of carvedilol during CHF treatment might not belong to its ability to reduce peripheral vascular resistance. Chronic administration of carvedilol to CHF patients did not increase calf vascular conductance and did not attenuate vasoconstrictor response of forearm vasculature to adrenergic stimuli [41,42]. From these results, biological process that is suppressed by carvedilol is not likely to be hemodynamic change in microvasulature, but might be related to metabolic or hypertrophic signaling to arterial smooth muscle cells via vascular {alpha}1-ARs [43]. In fact, carvedilol effectively attenuated phosphorylation of ERK1/2. Furthermore, deletion of mouse {alpha}1B-AR gene resulted in a beneficial effect for preventing remodeling of peripheral vessels and cardiac muscle induced by continuous infusion of phenylephrine at subpressor doses [44].

Native arterial smooth muscle cell frequently co-expresses distinct {alpha}1-AR subtypes [43,45], in which heteromeric receptor can be composed of different {alpha}1-AR subtypes [16,17]. Receptor heteromerization between {alpha}1A- and {alpha}1B-ARs increased total receptor binding sites and internalized {alpha}1B-AR in response to stimulation of an {alpha}1A-AR-selective agonist, oxymetazoline [16,17]. Analysis of the heteromer-specific character at single-cell level can be confounded by the concomitant presence of homomeric receptors. We found that in a cell co-expressing non-oscillatory {alpha}1A-AR and oscillatory {alpha}1B-AR subtypes to a similar extent, a pattern of global intracellular [Ca2+]i change caused by norepinephrine or phenylephrine was fluctuating [Ca2+]i levels with gradually decreasing basal [Ca2+]i. Carvedilol was more effective on the oscillatory component than on the non-oscillatory one.

In conclusion, the {alpha}1D- and {alpha}1B-ARs show high affinities for carvedilol, which are comparable to that of β1-AR. The oscillatory [Ca2+]i responses by {alpha}1D- and {alpha}1B-ARs are distinct from the response induced by {alpha}1A-AR and are more susceptible to carvedilol. From the results of clinical studies, it was concluded that subtype-nonselective anti-{alpha}1-AR antagonist is not a first-line drug for the CHF treatment, while an effectiveness of a subtype-selective {alpha}1-blocker directed against {alpha}1D- and/or {alpha}1B-ARs in combination with β-blockade needs to be further addressed in clinical and basic research fields. Thus, obtaining knowledge of the cellular and molecular targets of an effective medication supported with clinical evidence could be valuable and efficient information in developing a better treatment strategy for patients with CHF.


   Acknowledgements
 
We are thankful to Dr. Robert J. Lefkowitz and Dr. Hitoshi Kurose for kindly providing the adrenergic receptor cDNAs and to Dr. Atsushi Miyawaki for kindly providing the Venus gene. We also thank Daiichi Pharmaceutical and Kissei Pharmaceutical for gifts of carvedilol and KMD-3213, respectively.


   Notes
 
Time for primary review 27 days


   References
Top
 Abstract
 1. Introduction
 2. Experimental procedures
 3. Results
 4. Discussion
 References
 

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E. A. Woodcock, X.-J. Du, M. E. Reichelt, and R. M. Graham
Cardiac {alpha}1-adrenergic drive in pathological remodelling
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