CLINICAL RESEARCH: CORONARY ARTERY PHYSIOLOGY
The Alpha-1D Is the Predominant Alpha-1-Adrenergic Receptor Subtype in Human Epicardial Coronary Arteries
Brian C. Jensen, MD*, ,
Philip M. Swigart, MS*,
Marie-Eve Laden, MD*,
Teresa DeMarco, MD,
Charles Hoopes, MD and
Paul C. Simpson, MD*,,*
* Cardiology Section, San Francisco VA Medical Center, San Francisco, California
Division of Cardiology, University of California, San Francisco, San Francisco, California
Division of Cardiothoracic Surgery, University of California, San Francisco, San Francisco, California
Manuscript received July 8, 2008;
revised manuscript received May 13, 2009,
accepted May 19, 2009.
* Reprint requests and correspondence: Dr. Paul C. Simpson, VA Medical Center (111-C-8), 4150 Clement Street, San Francisco, California 94121 (Email: paul.simpson{at}ucsf.edu).
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Abstract
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Objectives: The goal was to identify alpha-1-adrenergic receptor (AR) subtypes in human coronary arteries.
Background: The  1-ARs regulate human coronary blood flow. The 1-ARs exist as 3 molecular subtypes, 1A, 1B, and 1D, and the 1D subtype mediates coronary vasoconstriction in the mouse. However, the 1A is thought to be the only subtype in human coronary arteries.
Methods: We obtained human epicardial coronary arteries and left ventricular (LV) myocardium from 19 transplant recipients and 6 unused donors (age 19 to 70 years; 68% male; 32% with coronary artery disease). We cultured coronary rings and human coronary smooth muscle cells. We assayed 1- and β-AR subtype messenger ribonucleic acid (mRNA) by quantitative real-time reverse transcription polymerase chain reaction and subtype proteins by radioligand binding and extracellular signal-regulated kinase (ERK) activation.
Results: The 1D subtype was 85% of total coronary 1-AR mRNA and 75% of total 1-AR protein, and 1D stimulation activated ERK. In contrast, the 1D was low in LV myocardium. Total coronary 1-AR levels were one-third of β-ARs, which were 99% the β2 subtype.
Conclusions: The 1D subtype is predominant and functional in human epicardial coronary arteries, whereas the 1A and 1B are present at very low levels. This distribution is similar to the mouse, where myocardial 1A- and 1B-ARs mediate beneficial functional responses and coronary 1Ds mediate vasoconstriction. Thus, 1D-selective antagonists might mediate coronary vasodilation, without the negative cardiac effects of nonselective 1-AR antagonists in current use. Furthermore, it could be possible to selectively activate beneficial myocardial 1A- and/or 1B-AR signaling without causing coronary vasoconstriction.
Key Words: adrenergic • alpha and beta • arteries • coronary disease • receptors
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Abbreviations and Acronyms
| | AR = adrenergic receptor | | CAD = coronary artery disease | | CTDN = California Transplant Donors Network | | CYP = cyanopindolol | | EF = ejection fraction | | ERK = extracellular signal-regulated kinase | | LV = left ventricle/ventricular | | MLC = myosin light chain | | mRNA = messenger ribonucleic acid | | NE = norepinephrine | | qRT-PCR = quantitative real-time reverse transcription polymerase chain reaction | | RNA = ribonucleic acid | | SMC = smooth muscle cell | | UCSF = University of California, San Francisco |
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Adrenergic receptors (ARs) play an important role in coronary arterial blood flow regulation. Coronary -ARs cause vasoconstriction, whereas β-ARs cause vasodilation. The 1-ARs constrict primarily epicardial coronary arteries and large arterioles, whereas 2-ARs act mostly on the coronary microcirculation (1,2). Stimulation of 1-ARs by endogenous catecholamines produces little constriction of normal coronary arteries (2–4), but causes pronounced vasoconstriction in coronary arteries with atherosclerotic endothelium (1,2,4).
The 1-ARs exist as 3 molecular subtypes, 1A, 1B, and 1D. All 3 subtypes are activated by norepinephrine (NE) and epinephrine but differ in amino acid sequence, tissue expression, and signaling (5). In the mouse heart, cardiac myocytes express the 1A and 1B subtypes, whereas the 1D subtype is functional in coronary arteries (6–8). However, very few data exist on 1-AR subtypes in the human heart. A single small study of post-mortem tissue identified the 1A as the predominant 1-subtype in epicardial coronaries (9). The 1A is also thought to be the predominant 1-AR subtype in the human myocardium, on the basis of messenger ribonucleic acid (mRNA) assay (10). Taken together, these previous results suggest that 1-AR subtype expression is different in the human heart than the mouse heart.
The distribution of cardiac 1-AR subtypes has significant physiological impact in the mouse, where the myocardial 1A and 1B mediate adaptive and beneficial effects, including positive inotropy, physiological hypertrophy, and protection from myocyte death (6,11–13). The coronary 1D mediates vasoconstriction (7,8). In humans, nonselective blockade of all 1-subtypes can be associated with heart failure (14,15). These results and others raise the possibility that the human heart 1A and 1B subtypes should not be blocked and might even be targets for selective agonists to treat myocardial disease (12,16). Thus, it could be significant clinically if human coronary arteries express predominantly the 1D subtype, as in the mouse (7,8), rather than the 1A subtype, as reported previously (9).
In this study, we re-examined the 1-AR subtypes in human epicardial coronary arteries, and measured β-ARs for comparison. Our results show that the 1D is the predominant and functional coronary 1-AR subtype, whereas the 1A and 1B are expressed at very low levels. We contrast this finding with the minimal expression of the 1D in human ventricular myocardium. We also find that 1-AR levels in coronary arteries are approximately one-third the level of β-ARs, most of which are the β2 subtype.
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Methods
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Patients.
With the approval of the University of California, San Francisco (UCSF) Committee for Human Research and with full informed consent, we obtained heart tissue from transplant recipients or unused donors provided by the California Transplant Donors Network (CTDN).
Tissue collection.
The heart was explanted after cold cardioplegia, under anesthesia and analgesia with fentanyl, midazolam, rocuronium, and isoflurane at UCSF, and with varied agents at the CTDN hospitals. The explanted heart was placed immediately in ice-cold physiologic solution, epicardial coronary arteries were dissected, cleaned rapidly of fat, flash frozen in liquid nitrogen, and stored at –80°C.
Ribonucleic acid (RNA) preparation.
Coronaries were pulverized in a liquid nitrogen-cooled mortar and homogenized (Polytron) in TRIzol reagent (Invitrogen, Gibco BRL, Gaithersburg, Maryland). Myocardium was homogenized directly in TRIzol. The RNA was extracted with chloroform and isopropanol, purified on Qiagen Mini-Prep columns, and treated with DNase (Turbo DNAfree, Ambion, Austin, Texas). We found no significant RNA degradation (Agilent 2100 BioAnalyzer, Palo Alto, California).
Quantitative real-time reverse transcription (RT) polymerase chain reaction.
The RT reactions used 1 µg RNA, SuperScript III Reverse Transcriptase (Invitrogen), random hexamers (Invitrogen), and oligo-dT (Roche, Nutley, New Jersey). Quantitative real-time RT polymerase chain reaction (qRT-PCR) was done in triplicate in an ABI PRISM 7900HT Sequence Detection System with 5% of the RT product, primers at 125 nmol/l, and SYBR Green Master (Roche) with ROX reference dye. Data were analyzed with SDS software version 2.3 (Applied Biosystems, Foster City, California).
Relative quantitation of PCR products used the  CT method, where arbitrary units were 2–CT x 1,000, CT = cycles to threshold, and CT = (mean target gene CT) – (mean CT of 2 reference genes, β-actin, and TATA-binding protein, for improved accuracy).
Radioligand binding.
Multiple protocols for membrane preparation from single arteries did not yield sufficient protein for reliable binding. Therefore, 15 epicardial coronaries totaling 10.2 g wet weight were pooled from 11 patients, pulverized in a liquid nitrogen-cooled mortar, homogenized in lysis buffer (5 mmol/l Tris-hydrochloride, 5 mmol/l ethylenediaminetetraacetic acid, 250 mmol/l sucrose pH 7.4 plus phenylmethylsulfonyl fluoride), and centrifuged at 1,000 g for 15 min. The supernatant was saved, and the pellet containing insoluble material was washed in lysis buffer and recentrifuged. The combined supernatants were centrifuged at 100,000 g for 1 h, and the resulting pellet was homogenized in lysis buffer and centrifuged at 100,000 g for 1 h. The resulting final membrane pellet, containing both plasma and intracellular membranes, was resuspended (50 mmol/l Tris pH 7.4, 1 mmol/l ethylenediaminetetraacetic acid) and used for 1- and β-AR binding.
The 1-AR saturation binding used 200 µg membrane protein in 1 ml/tube with 3H-prazosin (0.04 to 1.2 nmol/l, PerkinElmer, Waltham, Massachusetts); phentolamine (10 µmol/l, Sigma #P7561) defined nonspecific binding. The β-AR binding used 50 µg membrane protein/tube with 125I-cyanopindolol (CYP) (0.04 to 1.0 nmol/l, NEN Life Sciences, PerkinElmer); L-propranolol (1 µmol/l, Sigma #P8688) defined nonspecific binding (17). The 1-AR subtype proteins were assayed with competition for 3H-prazosin binding (0.5 nmol/l) by BMY-7378 (0.05 nmol/l to 500 µmol/l, Sigma #B134), an 1D-selective antagonist (18). All incubations were 60 min at 30°C. Binding data were analyzed by Prism 4.0b (GraphPad Software, Inc., San Diego, California).
Coronary artery smooth muscle cell culture and immunoblots.
Human coronary arteries were digested 20 min in Hanks buffer with collagenase Type II (1 mg/ml, Worthington, Lakewood, New Jersey) and elastase (0.5 mg/ml, Worthington), and intima and adventitia were removed mechanically. Rings of media (approximately 2 mm) were cut free hand and cultured. Other rings were minced and digested for 2 h in collagenase and elastase; enzymes were inhibited with serum; and smooth muscle cells (SMCs) grew out of the minces (19). Clonetics normal human coronary artery SMCs were from Lonza (#CC2583, Walkersville, Maryland). All cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum for 8 to 48 h (rings) or 3 to 11 passages (cells). For experiments, cultures were incubated in Dulbecco's modified Eagle's medium without serum with 5 mg/ml bovine serum albumin (Sigma #A7030) for at least 12 h (rings) or 48 h (cells), pretreated without or with 1-antagonists (10 nmol/l BMY-7378; 1 µmol/l prazosin, Sigma #P7791) for 90 min (rings) or 15 min (cells) and treated with 1-agonists (1-200 nmol/l L-NE, Sigma #N5785; 10 nmol/l A61603, Tocris #1052, Ellisville, Missouri), in the presence of the β-AR antagonist L-propranolol (1 µmol/l). After 30 min (rings) or 15 min (cells), homogenates were made in radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors. Ring homogenates were centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant (rings) or total lysates (cells) were used (10 to 20 µg protein/lane) for immunoblotting with antibodies from Cell Signaling for total–extracellular signal-regulated kinase (ERK) 1/2 (rabbit pAb #9102) and phospho-ERK1/2 (rabbit mAb Thr202/Tyr204 #4370) and antibodies for phospho-S19/20-myosin light chain 2 (MLC), including Sigma #M6068, Cell Signaling #3671, and abcam #ab2480.
Data analysis.
Results are mean ± SEM. Significant differences (p < 0.05) were tested with analysis of variance and Tukey's multiple comparison for more than 2 groups and Student unpaired t test for 2 groups. The F test was used to compare goodness-of-fit for competition binding analysis (GraphPad Prism version 4.0). Multivariate linear regression (Stata version 9, Stata Corp., College Station, Texas) was used to determine whether clinical variables were independently associated with 1- and β-AR density. Multivariate model assumptions were checked for all regression analyses.
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Results
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Patients.
To test 1-AR subtype expression in human coronary arteries, we collected hearts from 19 transplant recipients and 6 unused donors. Average age was 46 years (range 19 to 70 years), and 68% were male (Table 1). Coronary artery disease (CAD) was present in 32%, who were older (p < 0.005) and had higher ejection fractions (EFs) (p < 0.05) (Table 1).
1-AR subtype mRNA levels.
To quantify 1-subtype mRNAs, we validated a qRT-PCR approach, with primers that span the single intron in each 1-AR subtype gene. Primer pairs were designed with Primer3 (version 0.4.0) and BLAST (Basic Local Alignment Search Tool) and chosen for comparable reaction efficiencies (20). Specificity of the 1-subtype primers was confirmed with: 1) PCR with human 1-AR complementary deoxyribonucleic acid; 2) a dissociation step in all PCR reactions; and 3) sequencing of the PCR products. Amplification of genomic deoxyribonucleic acid was excluded by: 1) use of intron-spanning primers; 2) DNase treatment of RNA; and 3) end point PCR reactions with no-RT templates as negative controls.
In human epicardial coronary arteries, the 1D was 85% of total 1-mRNA (Fig. 1A). The 1B (11%) and the 1A (4%) were markedly less abundant than the 1D (p < 0.001 for each). In LV myocardium, by contrast, the 1D was only 21% of the total 1-mRNA, and the 1A (63%) was the most abundant (Fig. 1B). The absolute level of 1D mRNA in coronary arteries was almost twice that in LV (p = 0.01), whereas the absolute level of the 1A was 30-fold higher in LV than in coronary arteries. As a control, there was no difference between coronary arteries and myocardium in the qRT-PCR cycles-to-threshold for the reference genes, β-actin, and TATA-binding protein. Levels of 1-subtype mRNAs were the same in 4 right and 4 left anterior descending coronaries (data not shown). There were no differences in 1-subtype mRNA levels in coronaries collected at UCSF versus the CTDN hospitals, suggesting no important effects due to anesthetic and analgesic agents (data not shown).
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Figure 1 1-AR Subtype mRNA Levels in Coronary Arteries and Left Ventricle
Quantitative real-time reverse transcription polymerase chain reaction of 1-adrenergic receptor (AR) subtypes in (A) human coronary arteries and (B) left ventricle free wall. mRNA = messenger ribonucleic acid; TBP = TATA-binding protein.
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In summary, the 1D is 85% of total 1-AR subtype mRNA in human epicardial coronary arteries but is significantly less abundant in human LV myocardium.
1-AR subtype protein levels.
To test 1-AR subtype protein levels we used radioligand binding with 3H-prazosin. We could not use immunohistochemistry or immunoblot, because none of the 10 1-AR antibodies that we tested is specific for 1-ARs (21).
We used pooled membranes from 11 patients for binding. Patient characteristics were similar in these 11 patients and the entire patient population (Table 1), and the 1-subtype mRNA levels in the pooled samples were similar to the levels of the entire group (data not shown). Saturation binding identified 8.7 fmol/mg protein of total 1-ARs in coronary artery membranes, with a Kd 0.03 nmol/l, and specific binding 70% of total at the 3H-prazosin Kd (Fig. 2). The level of 1-AR binding in coronaries was roughly twice that in LV myocardium (20).
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Figure 2 1- and β-AR Protein Levels by Saturation Binding
Saturation radioligand binding was done in membranes pooled from 15 epicardial coronary arteries of 11 patients. (A) Binding with 3H-prazosin for total 1-adrenergic receptors (ARs); (B) binding with 125I-CYP for total β-ARs. CYP = cyanopindol; DPM = disintegrations/minute.
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To test whether the 1D subtype protein was also predominant in coronaries, as with mRNA, we did competition binding with the 1D-selective antagonist BMY-7378. The BMY-7378 competition yielded a 2-site binding curve (p = 0.002 vs. 1-site model), with 75% high-affinity sites (Kd 13 pmol/l) and 25% low-affinity (2.6 µmol/l) (Fig. 3A, Table 2). In ventricular myocardium, BMY competition gave a 1-site curve with low BMY-7378 affinity, indicating minimal or no 1D binding (Fig. 3B).
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Figure 3 1-AR Subtype Protein Levels by Competition Binding
Competition for 3H-prazosin binding by the 1D-selective antagonist BMY-7378 yielded (A) a 2-site binding curve with predominantly high-affinity sites in coronary artery membranes; (B) a 1-site low affinity curve in ventricular myocardium from 3 patients. AR = adrenergic receptor.
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In summary, the 1D is 75% of total 1-AR subtype protein in human epicardial coronary arteries but is much less abundant in myocardium. The coronary levels of 1D mRNA and protein agree very well (Table 2).
1-AR signaling in coronary SMCs.
To test whether the 1D was functional in human coronary SMCs, we used immunoblot to quantify phosphorylation (activation) of ERK, which is a target for the 1D in rat aortic SMCs (22) and is involved in activation of MLC kinase in smooth muscle (23). We used SMCs from Lonza, coronary media rings, and primary isolates from the coronary medial SMC layer (Fig. 4). A qRT-PCR for -smooth muscle actin and smooth muscle myosin heavy chain confirmed SMC identity, and 1D mRNA was the predominant 1-subtype, with less 1B and no 1A (data not shown). Low concentrations of NE (mean 27 nmol/l), in the presence of propranolol to block β-ARs, induced a 1.8-fold increase in phospho-ERK, and activation was abrogated to an equal extent by a low concentration of BMY-7378 (10 nmol/l), the 1D-selective antagonist, and prazosin, the nonselective 1-antagonist (Fig. 4). The 1A-selective agonist, A61603 (10 to 100 nmol/l), did not activate ERK (data not shown). Phospho-MLC was barely detectable with 3 different antibodies and was not useful as a read-out (data not shown).
We conclude that the 1D is functional in human epicardial coronary SMCs, whereas there is no evidence for the 1A.
β-AR mRNA and protein levels.
We measured β-AR mRNAs and protein in the same coronary arteries, to compare with 1-ARs. By qRT-PCR (20) the β2 was the predominant β-subtype mRNA (99% of total β-AR mRNA) (Table 2). Total 1-AR mRNA was 37% of total β-AR mRNA (p < 0.0001) (Table 2).
The β-AR proteins were quantified by saturation binding, because the available β-AR antibodies are not specific in our hands (12). Saturation binding with 125I-CYP, a nonselective β-AR antagonist, identified 25.2 fmol/mg protein of total β-ARs in coronary artery membranes, with a Kd 0.16 nmol/l and specific binding 41% of total at the 125I-CYP Kd (Fig. 2B). Given the preponderance of β2-mRNA in coronary tissue, β-AR competition binding was not done. Total 1-AR binding was 35% of total β-AR binding, in excellent agreement with the mRNA values (Table 2).
In summary, the β2 was the predominant β-AR subtype in epicardial coronary arteries, and 1-AR mRNA and binding were only one-third of β-ARs.
Impact of clinical variables on coronary 1- and β-AR mRNA levels.
The qRT-PCR results were analyzed to determine whether clinical variables affected the expression of AR subtypes in human coronaries. Human noncoronary arterial and prostate 1-ARs are said to increase with age (9,24), and 1-mediated vasoconstriction is more prominent in CAD (1–4). However, we found that age, EF, β-agonist exposure, CAD (Fig. 5), and sex (data not shown) had no effect on coronary artery 1-subtype mRNA levels. Interestingly, 1D and total 1-mRNA levels were 35% lower in coronary arteries of patients using β-blockers (p = 0.04). This association persisted after adjusting for age, sex, CAD, and EF (p = 0.03) (Fig. 5B). Among β-blockers, 1D mRNA levels appeared similar among patients taking metoprolol (1 patient), nadolol (1 patient), or carvedilol (8 patients) (Fig. 5B).
The levels of coronary β-AR mRNAs, which were almost entirely β2, did not vary with age, EF, β-blocker or β-agonist use, or sex (data not shown).
In summary, age, sex, CAD, EF, and β-agonists had no significant effect on 1- or β-subtype mRNA levels in coronary arteries. Beta-blocker use was associated with a significant decrease in 1D and total 1-mRNA levels.
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Discussion
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This study reports that the 1D subtype is the predominant 1-AR in human epicardial coronary arteries, comprising approximately 80% of total 1-AR mRNA and protein. These data also reveal that coronary 1-AR levels are only one-third of β-ARs. This is the most extensive characterization of 1-AR subtypes in coronary tissue in any species.
Our results disagree with those of a previous investigation that identified the 1A as the predominant 1-subtype in human coronary arteries (9). In our study, the 1A in the coronaries was only 4% of total 1-mRNA in coronaries and was absent in isolated SMCs, and the combined 1A and 1B were only 25% of total 1-binding. The discrepancy might be explained by the prior study's small sample size (5 arteries from an unspecified number of patients), the use of post-mortem tissue, or a qualitative RNA assay (RNase protection) (9).
Of particular importance is our finding that the 1-AR subtype profiles in the human coronary arteries and ventricular myocardium were quite different. Previous limited evidence suggests that 1A subtype mRNA is predominant in both coronary arteries and myocardium (9,10). In contrast, we found that the 1D was predominant in the coronary arteries but was much less abundant in the myocardium, where 1A mRNA was predominant. In the mouse heart, myocardium has the 1A and 1B subtypes (6), whereas the 1D subtype is functional in coronary arteries (7,8). Thus, rodents and humans might have similar 1-AR subtype expression in coronary arteries and myocardium (7,8,17,20,25–27) (Table 3), contrary to prior claims (9), and studies done in mouse models might therefore be applicable to human cardiac 1-AR biology.
Technical aspects of this study warrant emphasis. We studied coronaries from a large number of patients of both sexes without and with CAD or heart failure and assessed the effects of these variables on 1-AR expression. We took extensive measures to validate our qRT-PCR approach. We quantified 1-subtype proteins by competition binding, because binding is for now the only accurate method to measure 1-AR proteins (21).
We also measured β-AR subtypes in the coronaries and found that 1-AR levels were only one-third of β-ARs. However, these lower 1-AR levels do not negate the physiological significance of the 1D. The contractile response to NE in isolated human epicardial coronary arteries is constriction at low concentrations (nmol/l) and relaxation at high concentrations (µmol/l) (28). The 1D has the highest NE affinity of any subtype (29), and thus constriction at low NE concentrations is consistent with an 1D-response. The larger population of coronary β-ARs could mediate relaxation with µmol/l NE.
Indeed, our experiments in epicardial coronary SMCs revealed that the 1D mediated activation of ERK by low concentrations of NE (Fig. 4). ERK is activated by the 1D in rat aortic SMCs (22), and ERK phosphorylation facilitates activation of MLC kinase in smooth muscle, thus contributing to the adrenergic contractile response (23). These findings suggest that the 1D is both abundant and functional in human epicardial coronary arteries.
We analyzed coronary 1- and β-AR subtype mRNA levels by age, CAD, EF, β-blockers, and β-agonists (Fig. 5) and sex (data not shown). The only association we found was a decrease in the 1D and total 1-ARs in patients treated with β-blockers, possibly implying that 1D expression in coronary vascular cells is increased by β-stimulation. In fact, in human monocytes, β2-stimulation induces 1D mRNA and protein (30).
Coronary 1-subtype mRNA levels did not differ in patients with CAD versus without CAD. This was noteworthy, because 1-ARs cause pronounced vasoconstriction in atherosclerotic coronary arteries but have little effect in normal coronaries (1–4). Thus, increased 1D levels in coronary vascular SMCs might not explain augmented 1-vasoconstriction in CAD. Instead, a small population of endothelial cell 1-ARs mediating endothelium-dependent vasodilation (31) could be lost in CAD.
Clinical implications.
Important clinical implications derive from the predominance of the 1D subtype in human coronary arteries. Cell and animal models show that the cardiac myocyte 1A and 1B subtypes have significant adaptive and protective roles (6,11–13,16). Clinical trials also suggest 1-mediated cardioprotection, because nonselective antagonism of all 1-subtypes was associated with a 2-fold excess of heart failure in the doxazosin arm of the ALLHAT (Antihypertensive and Lipid-Lowering treatment to prevent Heart Attack Trial), and a trend towards increased mortality in the prazosin arm of the V-HeFT (Vasodilator-Heart Failure Trial) (14,15).
Despite the ALLHAT trial results, 13.4 million prescriptions were dispensed in 2002 for mostly nonselective -blockers (32), primarily to treat symptoms from prostate hypertrophy. However, the 1D-selective antagonist, naftopidil, is effective in relieving prostate symptoms (33). In light of our results, it seems possible that more selective antagonism of the 1D subtype might relax both coronary and prostate smooth muscle, without blocking beneficial signaling mediated by the myocardial 1A and 1B subtypes. Furthermore, the adaptive and protective roles of the myocardial 1A and 1B raise the intriguing possibility of activating one or both of these subtypes selectively to treat myocardial disease (16). The low levels of the 1A and 1B in human coronary arteries would make 1A- or 1B-agonist–induced coronary vasoconstriction unlikely.
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Conclusions
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We present here the most extensive characterization of human coronary ARs to date. We show that the 1D is the predominant 1-AR subtype in human epicardial coronary arteries—not the 1A as reported previously (9)—and that the 1D is low in myocardium. The 1A and 1B are present in coronaries at very low levels, and total 1-AR levels are one-third the level of β-ARs, most of which are the β2 subtype. The tissue distribution of 1-subtypes in the human heart is similar to the rodent heart (Table 3). These results are clinically relevant to the widespread use of 1-antagonists and to the potential development of 1A- and/or 1B-AR–selective agonists.
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Acknowledgments
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The authors thank the CTDN for unused donor hearts and Celia Rifkin and the staff in UCSF operating rooms 9 and 10 for help with transplant hearts. Sanjiv Shah, MD, did the multivariate analysis.
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Footnotes
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Dr. Simpson received funding from the Veterans Administration and the National Institutes of Health. Dr. Jensen was the recipient of a Young Investigators Award from the GlaxoSmithKline Research and Education Foundation for Cardiovascular Disease and has received support from the University of California, San Francisco Foundation for Cardiac Research. Dr. Laden received a fellowship from the Sarnoff Cardiovascular Research Foundation, and was a medical student at Duke University, Durham, North Carolina, when this work was done. Dr. DeMarco has served as a speaker/consultant for Actelion, Gilead, Boston Scientific, Cardiokinetics, and Medtronic. Dr. Jensen is currently affiliated with the Division of Cardiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina. Dr. Laden is currently affiliated with the Department of General Surgery, Stanford University, Stanford, California.
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References
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1. Jones CJ, DeFily DV, Patterson JL, Chilian WM. Endothelium-dependent relaxation competes with alpha 1- and alpha 2-adrenergic constriction in the canine epicardial coronary microcirculation Circulation 1993;87:1264-1274.[Abstract/Free Full Text]2. Heusch G, Baumgart D, Camici P, et al. Alpha-adrenergic coronary vasoconstriction and myocardial ischemia in humans Circulation 2000;101:689-694.[Abstract/Free Full Text] 3. Chilian WM, Boatwright RB, Shoji T, Griggs Jr DM. Evidence against significant resting sympathetic coronary vasoconstrictor tone in the conscious dog Circ Res 1981;49:866-876.[Abstract/Free Full Text] 4. Baumgart D, Haude M, Gorge G, et al. Augmented alpha-adrenergic constriction of atherosclerotic human coronary arteries Circulation 1999;99:2090-2097.[Abstract/Free Full Text] 5. Graham RM, Perez DM, Hwa J, Piascik MT. Alpha 1-adrenergic receptor subtypes. Molecular structure, function, and signaling. Circ Res 1996;78:737-749.[Free Full Text] 6. O'Connell TD, Ishizaka S, Nakamura A, et al. The alpha(1A/C)- and alpha(1B)-adrenergic receptors are required for physiological cardiac hypertrophy in the double-knockout mouse J Clin Invest 2003;111:1783-1791.[CrossRef][Web of Science][Medline] 7. Turnbull L, McCloskey DT, O'Connell TD, Simpson PC, Baker AJ. Alpha 1-adrenergic receptor responses in alpha 1AB-AR knockout mouse hearts suggest the presence of alpha 1D-AR Am J Physiol Heart Circ Physiol 2003;284:H1104-H1109.[Abstract/Free Full Text] 8. Chalothorn D, McCune DF, Edelmann SE, et al. Differential cardiovascular regulatory activities of the alpha 1B- and alpha 1D-adrenoceptor subtypes J Pharmacol Exp Ther 2003;305:1045-1053.[Abstract/Free Full Text] 9. Rudner XL, Berkowitz DE, Booth JV, et al. Subtype specific regulation of human vascular alpha(1)-adrenergic receptors by vessel bed and age Circulation 1999;100:2336-2343.[Abstract/Free Full Text] 10. Price DT, Lefkowitz RJ, Caron MG, Berkowitz D, Schwinn DA. Localization of mRNA for 3 distinct alpha 1-adrenergic receptor subtypes in human tissues: implications for human alpha-adrenergic physiology Mol Pharmacol 1994;45:171-175.[Abstract] 11. McCloskey DT, Turnbull L, Swigart P, O'Connell TD, Simpson PC, Baker AJ. Abnormal myocardial contraction in alpha(1A)- and alpha(1B)-adrenoceptor double-knockout mice J Mol Cell Cardiol 2003;35:1207-1216.[CrossRef][Web of Science][Medline] 12. O'Connell TD, Swigart PM, Rodrigo MC, et al. Alpha1-adrenergic receptors prevent a maladaptive cardiac response to pressure overload J Clin Invest 2006;116:1005-1015.[CrossRef][Web of Science][Medline] 13. Huang Y, Wright CD, Merkwan CL, et al. An alpha1A-adrenergic-extracellular signal-regulated kinase survival signaling pathway in cardiac myocytes Circulation 2007;115:763-772.[Abstract/Free Full Text] 14. ALLHAT Collaborative Research Group Major cardiovascular events in hypertensive patients randomized to doxazosin vs chlorthalidone: the antihypertensive and lipid-lowering treatment to prevent heart attack trial (ALLHAT) JAMA 2000;283:1967-1975.[Abstract/Free Full Text] 15. Cohn JN. The Vasodilator-Heart Failure Trials (V-HeFT). Mechanistic data from the VA Cooperative Studies. Introduction. Circulation 1993;87:VI1-VI4.[Medline] 16. Chan T, Dash R, Simpson PC. An alpha-1A-adrenergic receptor subtype agonist prevents cardiomyopathy without increasing blood pressure (abstr) Circulation 2008;118:S533. 17. Rokosh DG, Stewart AF, Chang KC, et al. Alpha1-adrenergic receptor subtype mRNAs are differentially regulated by alpha1-adrenergic and other hypertrophic stimuli in cardiac myocytes in culture and in vivo. Repression of alpha1B and alpha1D but induction of alpha1C. J Biol Chem 1996;271:5839-5843.[Abstract/Free Full Text] 18. Goetz AS, King HK, Ward SD, True TA, Rimele TJ, Saussy Jr DL. BMY 7378 is a selective antagonist of the D subtype of alpha 1-adrenoceptors Eur J Pharmacol 1995;272:R5-R6.[CrossRef][Web of Science][Medline] 19. Owens GK, Loeb A, Gordon D, Thompson MM. Expression of smooth muscle-specific alpha-isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation J Cell Biol 1986;102:343-352.[Abstract/Free Full Text] 20. Jensen BC, Swigart PM, DeMarco T, Hoopes C, Simpson PC. Alpha-1-Adrenergic receptor subtypes in nonfailing and failing human myocardium Circ Heart Fail 2009In press. 21. Jensen B, Swigart P, Simpson P. Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific Naunyn Schmiedebergs Arch Pharmacol 2009;379:409-412.[CrossRef][Web of Science][Medline] 22. Xin X, Yang N, Eckhart AD, Faber JE. Alpha1D-adrenergic receptors and mitogen-activated protein kinase mediate increased protein synthesis by arterial smooth muscle Mol Pharmacol 1997;51:764-775.[Abstract/Free Full Text] 23. Wier WG, Morgan KG. Alpha1-adrenergic signaling mechanisms in contraction of resistance arteries Rev Physiol Biochem Pharmacol 2003;150:91-139.[Medline] 24. Kojima Y, Sasaki S, Shinoura H, Hayashi Y, Tsujimoto G, Kohri K. Quantification of alpha1-adrenoceptor subtypes by real-time RT-PCR and correlation with age and prostate volume in benign prostatic hyperplasia patients Prostate 2006;66:761-767.[CrossRef][Web of Science][Medline] 25. Stewart AF, Rokosh DG, Bailey BA, et al. Cloning of the rat alpha 1C-adrenergic receptor from cardiac myocytes. Alpha 1C, alpha 1B, and alpha 1D mRNAs are present in cardiac myocytes but not in cardiac fibroblasts. Circ Res 1994;75:796-802.[Abstract/Free Full Text] 26. Michel MC, Hanft G, Gross G. Radioligand binding studies of alpha 1-adrenoceptor subtypes in rat heart Br J Pharmacol 1994;111:533-538.[Web of Science][Medline] 27. Simpson P. Lessons from knockouts: the alpha1-ARsIn: Perez DM, editor. The Adrenergic Receptors in the 21st Century. Totowa, NJ: Humana Press; 2006. pp. 207-240. 28. Toda N. Isolated human coronary arteries in response to vasoconstrictor substances Am J Physiol 1983;245:H937-H941.[Web of Science][Medline] 29. Schwinn DA, Johnston GI, Page SO, et al. Cloning and pharmacological characterization of human alpha-1 adrenergic receptors: sequence corrections and direct comparison with other species homologues J Pharmacol Exp Ther 1995;272:134-142.[Abstract/Free Full Text] 30. Rouppe van der Voort C, Kavelaars A, van de Pol M, Heijnen CJ. Neuroendocrine mediators up-regulate alpha1b- and alpha1d-adrenergic receptor subtypes in human monocytes J Neuroimmunol 1999;95:165-173.[CrossRef][Web of Science][Medline] 31. Mendez E, Calzada C, Ocharan E, et al. Differential expression of alpha1-adrenergic receptor subtypes in coronary microvascular endothelial cells in culture Eur J Pharmacol 2006;546:127-133.[CrossRef][Web of Science][Medline] 32. Stafford RS, Furberg CD, Finkelstein SN, Cockburn IM, Alehegn T, Ma J. Impact of clinical trial results on national trends in alpha-blocker prescribing, 1996–2002 JAMA 2004;291:54-62.[Abstract/Free Full Text] 33. Nishino Y, Masue T, Miwa K, Takahashi Y, Ishihara S, Deguchi T. Comparison of two alpha1-adrenoceptor antagonists, naftopidil and tamsulosin hydrochloride, in the treatment of lower urinary tract symptoms with benign prostatic hyperplasia: a randomized crossover study BJU Int 2006;97:747-751discussion 751.[CrossRef][Web of Science][Medline]
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