Endothelin receptor subtype distribution predisposes coronary arteries to damage

Abstract

Several vasoactive drugs that lower blood pressure and increase heart rate induce regional cardiotoxicity in the dog, most frequently of right coronary arteries and right atrium. The basis for this selective damage is thought to result from local changes in vascular tone and blood flow. Administration of an endothelin receptor antagonist (ETRA, SB 209670) to dogs induced damage most frequent and severe in the right coronary artery and right atrium. Because site predisposition may correlate with distribution of vasoactive receptors, the objectives of this study were to map endothelin (ET) receptor distribution and density within regions of dog heart using both gene (mRNA) and protein expression endpoints for dog ET(A) and ET(B) receptors, and, additionally, correlate ET receptor subtype density with regional cardiac blood flow. A 10- to 15-mmHg reduction in mean arterial pressure with a concomitant increase in heart rate (10-20%), a six- and twofold increase in regional blood flow to the right and left atrium, respectively, and acute hemorrhage, medial necrosis, and inflammation were observed in the right coronary arteries and arteries of the right atrium after ETRA infusion for 5 days. Radioligand protein binding to quantify both ET receptors in normal dog heart indicated a twofold greater density of ET receptors in atrial regions versus ventricular regions. Importantly, ET receptor density in coronary arteries was markedly (about five- to sixfold) increased above that in atrial or ventricular tissues. ET receptor subtype characterization indicated ET(B) receptors were three times more prevalent in right coronary arteries compared to left coronary arteries and in situ hybridization confirmed localization of ET(B) in vascular smooth muscle. ET(A) receptor density was comparable in right and left coronary arteries. Quantitative real-time polymerase chain reaction for ET(A) and ET(B) receptor mRNA transcripts supported the site prevalence for message distribution. Consequently, the composite of protein and message expression profiles for ET(A) and ET(B) receptors indicated a disproportionate distribution of ET(B) receptors within right coronary artery of dog and this, along with functional measures of blood flow after ETRA infusion indicated a predisposition for exaggerated pharmacological responses and subsequent damage to right coronary arteries by ET and/or ETRAs.

Figure 1.

Partial nucleotide and amino acid sequences for the dog ET_A receptor is shown aligned with homologous region of the human ET_A receptor, the non-identical region at the amino acid level are shaded.

Figure 2.

Mean arterial pressure (MAP; A) and heart rate (HR; B) from control and ETRA infused dogs. *P < 0.05 (statistically significant increase).

Figure 3.

Regional blood flow from vehicle- and ETRA-infused dogs. Note marked increase in right atrial blood flow after ETRA infusion for 5 days. *P < 0.05 (statistically significant increase). No change was observed in vehicle-infused dogs (inset).

Figure 4.

Heart from ETRA-infused dog. Note areas of hemorrhage in right atria (A) and right atrial appendage (B). Periarterial inflammation and fibrinoid necrosis of right extramural coronary arteries with inflammation extending into pericardial adipose tissue of the coronary groove (C and D). Multifocal areas of hemorrhage in tunica media of right coronary artery (E and F). H&E stain; original magnifications, ×24 (C), ×120 (D and E), ×240 (F).

Figure 5.

ET_A and ET_B receptor density in different regions of the normal dog heart. RCA, right coronary artery; LCA, left coronary artery; RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle.

Figure 6.

Quantitative analysis of ET_A (A) and ET_B (B) receptors mRNA by RT-PCR (Taqman) in normal dog heart and coronary arteries (n ≥ 3). The normal dog femoral artery (FA) was use as a reference base and values for all sites are expressed relative to ET_A or ET_B receptor mRNA abundance in FA.

Figure 7.

Localization of ET_B receptor mRNA expression in extramural coronary arteries of the dog. ET_B anti-sense hybridization signal was localized predominantly to the vascular intima and media of the RCA (A). Hybridization signal was weak or absent in the LCA (C) as compared to the RCA (A), suggesting a lower density of ET_B receptors in the LCA. Using ET_B sense probes, hybridization signal was not observed in either LCA (B) or RCA (not shown). Dark field illumination; original magnifications, ×130.