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Functional alpha-1B adrenergic receptors on human epicardial coronary artery endothelial cells.

Jensen BC, Swigart PM, Montgomery MD, Simpson PC - Naunyn Schmiedebergs Arch. Pharmacol. (2010)

Bottom Line: Functionally, NE and EPI through the α1B subtype activated extracellular signal-regulated kinase (ERK) in ECs, stimulated phosphorylation of EC endothelial nitric oxide synthase (eNOS), and increased deoxyribonucleic acid (DNA) synthesis.These results are the first to demonstrate α1-ARs on human coronary ECs and indicate that the α1B subtype is predominant.Our findings provide another potential mechanism for adverse cardiac effects of drug antagonists that nonselectively inhibit all three α1-AR subtypes.

View Article: PubMed Central - PubMed

Affiliation: Cardiology Division, VA Medical Center, University of California, San Francisco, San Francisco, CA, USA.

ABSTRACT
Alpha-1-adrenergic receptors (α1-ARs) regulate coronary arterial blood flow by binding catecholamines, norepinephrine (NE), and epinephrine (EPI), causing vasoconstriction when the endothelium is disrupted. Among the three α1-AR subtypes (α1A, α1B, and α1D), the α1D subtype predominates in human epicardial coronary arteries and is functional in human coronary smooth muscle cells (SMCs). However, the presence or function of α1-ARs on human coronary endothelial cells (ECs) is unknown. Here we tested the hypothesis that human epicardial coronary ECs express functional α1-ARs. Cultured human epicardial coronary artery ECs were studied using quantitative real-time reverse transcription polymerase chain reaction, radioligand binding, immunoblot, and (3)H-thymidine incorporation. The α1B-subtype messenger ribonucleic acid (mRNA) was predominant in cultured human epicardial coronary ECs (90-95% of total α1-AR mRNA), and total α1-AR binding density in ECs was twice that in coronary SMCs. Functionally, NE and EPI through the α1B subtype activated extracellular signal-regulated kinase (ERK) in ECs, stimulated phosphorylation of EC endothelial nitric oxide synthase (eNOS), and increased deoxyribonucleic acid (DNA) synthesis. These results are the first to demonstrate α1-ARs on human coronary ECs and indicate that the α1B subtype is predominant. Our findings provide another potential mechanism for adverse cardiac effects of drug antagonists that nonselectively inhibit all three α1-AR subtypes.

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The α1B-AR subtype activates ERK in coronary ECs. Cultured ECs were treated for 15 min with the drugs indicated, and total and phosphorylated ERK were quantified by immunoblot. The nonselective β-AR antagonist propranolol (1 μM) was present throughout in all groups. a Representative blot with NE, EPI, and the α1A-selective agonist A61603. b ERK activation by NE (mean 1 μM, range 0.2–2 μM) is inhibited by the nonselective α1-antagonist prazosin (1 μM, N = 6–10). c The α1D-selective antagonist, BMY-7378, inhibited NE (1 μM)-stimulated phospho-ERK with a log IC50 of −6.5 (N = 5). This value is much closer to the known low-affinity BMY-7378 site (log IC50 -5.6) than the high-affinity site (−10.9)
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Fig2: The α1B-AR subtype activates ERK in coronary ECs. Cultured ECs were treated for 15 min with the drugs indicated, and total and phosphorylated ERK were quantified by immunoblot. The nonselective β-AR antagonist propranolol (1 μM) was present throughout in all groups. a Representative blot with NE, EPI, and the α1A-selective agonist A61603. b ERK activation by NE (mean 1 μM, range 0.2–2 μM) is inhibited by the nonselective α1-antagonist prazosin (1 μM, N = 6–10). c The α1D-selective antagonist, BMY-7378, inhibited NE (1 μM)-stimulated phospho-ERK with a log IC50 of −6.5 (N = 5). This value is much closer to the known low-affinity BMY-7378 site (log IC50 -5.6) than the high-affinity site (−10.9)

Mentions: Saturation binding identified 34 fmol/mg protein of total α1-ARs in coronary ECs, with a Kd 0.07 nM, and specific binding 90% of total at the 3H-prazosin Kd (Fig. 1b). For comparison, in parallel experiments, we found that Lonza human coronary SMCs contained 17 fmol/mg protein of total α1-ARs. We did not perform competition radioligand binding on ECs given the marked predominance of α1B mRNA in our qPCR assays.


Functional alpha-1B adrenergic receptors on human epicardial coronary artery endothelial cells.

Jensen BC, Swigart PM, Montgomery MD, Simpson PC - Naunyn Schmiedebergs Arch. Pharmacol. (2010)

The α1B-AR subtype activates ERK in coronary ECs. Cultured ECs were treated for 15 min with the drugs indicated, and total and phosphorylated ERK were quantified by immunoblot. The nonselective β-AR antagonist propranolol (1 μM) was present throughout in all groups. a Representative blot with NE, EPI, and the α1A-selective agonist A61603. b ERK activation by NE (mean 1 μM, range 0.2–2 μM) is inhibited by the nonselective α1-antagonist prazosin (1 μM, N = 6–10). c The α1D-selective antagonist, BMY-7378, inhibited NE (1 μM)-stimulated phospho-ERK with a log IC50 of −6.5 (N = 5). This value is much closer to the known low-affinity BMY-7378 site (log IC50 -5.6) than the high-affinity site (−10.9)
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991196&req=5

Fig2: The α1B-AR subtype activates ERK in coronary ECs. Cultured ECs were treated for 15 min with the drugs indicated, and total and phosphorylated ERK were quantified by immunoblot. The nonselective β-AR antagonist propranolol (1 μM) was present throughout in all groups. a Representative blot with NE, EPI, and the α1A-selective agonist A61603. b ERK activation by NE (mean 1 μM, range 0.2–2 μM) is inhibited by the nonselective α1-antagonist prazosin (1 μM, N = 6–10). c The α1D-selective antagonist, BMY-7378, inhibited NE (1 μM)-stimulated phospho-ERK with a log IC50 of −6.5 (N = 5). This value is much closer to the known low-affinity BMY-7378 site (log IC50 -5.6) than the high-affinity site (−10.9)
Mentions: Saturation binding identified 34 fmol/mg protein of total α1-ARs in coronary ECs, with a Kd 0.07 nM, and specific binding 90% of total at the 3H-prazosin Kd (Fig. 1b). For comparison, in parallel experiments, we found that Lonza human coronary SMCs contained 17 fmol/mg protein of total α1-ARs. We did not perform competition radioligand binding on ECs given the marked predominance of α1B mRNA in our qPCR assays.

Bottom Line: Functionally, NE and EPI through the α1B subtype activated extracellular signal-regulated kinase (ERK) in ECs, stimulated phosphorylation of EC endothelial nitric oxide synthase (eNOS), and increased deoxyribonucleic acid (DNA) synthesis.These results are the first to demonstrate α1-ARs on human coronary ECs and indicate that the α1B subtype is predominant.Our findings provide another potential mechanism for adverse cardiac effects of drug antagonists that nonselectively inhibit all three α1-AR subtypes.

View Article: PubMed Central - PubMed

Affiliation: Cardiology Division, VA Medical Center, University of California, San Francisco, San Francisco, CA, USA.

ABSTRACT
Alpha-1-adrenergic receptors (α1-ARs) regulate coronary arterial blood flow by binding catecholamines, norepinephrine (NE), and epinephrine (EPI), causing vasoconstriction when the endothelium is disrupted. Among the three α1-AR subtypes (α1A, α1B, and α1D), the α1D subtype predominates in human epicardial coronary arteries and is functional in human coronary smooth muscle cells (SMCs). However, the presence or function of α1-ARs on human coronary endothelial cells (ECs) is unknown. Here we tested the hypothesis that human epicardial coronary ECs express functional α1-ARs. Cultured human epicardial coronary artery ECs were studied using quantitative real-time reverse transcription polymerase chain reaction, radioligand binding, immunoblot, and (3)H-thymidine incorporation. The α1B-subtype messenger ribonucleic acid (mRNA) was predominant in cultured human epicardial coronary ECs (90-95% of total α1-AR mRNA), and total α1-AR binding density in ECs was twice that in coronary SMCs. Functionally, NE and EPI through the α1B subtype activated extracellular signal-regulated kinase (ERK) in ECs, stimulated phosphorylation of EC endothelial nitric oxide synthase (eNOS), and increased deoxyribonucleic acid (DNA) synthesis. These results are the first to demonstrate α1-ARs on human coronary ECs and indicate that the α1B subtype is predominant. Our findings provide another potential mechanism for adverse cardiac effects of drug antagonists that nonselectively inhibit all three α1-AR subtypes.

Show MeSH
Related in: MedlinePlus