The alpha-1D Is the predominant alpha-1-adrenergic receptor subtype in human epicardial coronary arteries

Abstract

Objectives: The goal was to identify alpha-1-adrenergic receptor (AR) subtypes in human coronary arteries.

Background: The alpha1-ARs regulate human coronary blood flow. The alpha1-ARs exist as 3 molecular subtypes, alpha1A, alpha1B, and alpha1D, and the alpha1D subtype mediates coronary vasoconstriction in the mouse. However, the alpha1A 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 alpha1- and beta-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 alpha1D subtype was 85% of total coronary alpha1-AR mRNA and 75% of total alpha1-AR protein, and alpha1D stimulation activated ERK. In contrast, the alpha1D was low in LV myocardium. Total coronary alpha1-AR levels were one-third of beta-ARs, which were 99% the beta2 subtype.

Conclusions: The alpha1D subtype is predominant and functional in human epicardial coronary arteries, whereas the alpha1A and alpha1B are present at very low levels. This distribution is similar to the mouse, where myocardial alpha1A- and alpha1B-ARs mediate beneficial functional responses and coronary alpha1Ds mediate vasoconstriction. Thus, alpha1D-selective antagonists might mediate coronary vasodilation, without the negative cardiac effects of nonselective alpha1-AR antagonists in current use. Furthermore, it could be possible to selectively activate beneficial myocardial alpha1A- and/or alpha1B-AR signaling without causing coronary vasoconstriction.

Figure 1. α1-AR Subtype mRNA Levels in Coronary Arteries and Left Ventricle

qRT-PCR of α1-AR subtypes in (A) human coronary arteries; (B) left ventricle free wall.

Figure 2. α1-AR and β-AR Protein Levels by Saturation Binding

Saturation RLB was done in membranes pooled from 15 epicardial coronary arteries of 11 patients. (A) Binding with ³H-prazosin for total α1-ARs; (B) Binding with ¹²⁵I-CYP for total β-ARs.

Figure 3. α1-AR Subtype Protein Levels by Competition Binding

Competition for ³H-prazosin binding by the α1D-selective antagonist BMY-7378 yielded a two-site binding curve with (A) predominantly high-affinity sites in coronary artery membranes; (B) a one-site low affinity curve in ventricular myocardium from 3 patients.

Figure 4. α1-AR-induced ERK Activation in Human Coronary Artery SMCs

Cultured human epicardial coronary SMCs and coronary media rings were treated for 15–30 min with low concentrations of NE (1–200 nM, mean 27 nM), and the nonselective β-AR antagonist propranolol (1µM), in the absence or presence of the α1D-selective antagonist, BMY-7378 (10nM), or the non-selective α1-antagonist, prazosin (1µM). (A) Western blot showing ERK activation in duplicate dishes from a Lonza SMC culture; (B) Summary data for 8 Lonza SMC preparations from 2 patients, a ring preparation from 1 patient, and 2 primary SMC cultures from 1 patient.

Figure 5. α1-Subtype mRNA Levels by Clinical Variables

qRT-PCR for α1-subtype mRNAs and all α1 mRNA are displayed according to (A) CAD, (B) β-blocker use, all carvedilol, except metoprolol (circled) and nadolol (squared), (C) β-agonist exposure, (D) age, and (E) EF. p values are for multivariate analysis.