Influence of Endothelin on Human Coronary Arteries and Localization of Its Binding Sites Adrian H. Chester, BSc, Michael R. Dashwood, PhD, John G. Clarke, MB, BCh, BAO, Simon W. Larkin, BSC, Graham J. Davies, MD, Samad Tadjkarimi, MD, Attilio Maseri, MD, and Magdi H. Yacoub, MD Endothelin, a 21 amino acid peptide synthesized by cultured porcine aortic endothelial cells, has recent- ly been identified and shown to produce a potent and prolonged constriction of mammalian blood vessels in vitro. Using tissue obtained from ex- planted hearts at the time of cardiac transplanta- tion, the response of isolated human epicardial cor- onary arteries to endothelin was studied. The pres- ence of endothelin binding sites was demonstrated in these vessels using an autoradiographic tech- nique. Endothelin produced a dose-dependent in- crease of tension in the isolated coronary vessels with a maximal tension achieved equal to 135% of that induced by 90 mM of potassium. The maximal response was slow to develop and had a prolonged duration of 15 to 20 minutes. Nicardipine (4 PM) failed to affect the contraction induced by low doses of endothelin, but decreased the tension ob- tained at high doses. However, adenosine, sub- stance P and glyceryl trinitrate were all effective in reversing the contraction induced by endothelin, while indomethacin and acetylcholine were ineffec- tive. These features differ from those of other en- dogenous constrictor agents and make endothelin a potential candidate for long-term modulation of vascular tone. (Am J Cardiol 1989;63:1395-1398) From the National Heart and Lung Institute, Harefield Hospital, Mid- dlesex, the Division of Cardiovascular Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, and the Department of Physiology, Royal Free Hospital School of Medicine, London, En- gland. This study was supported by a grant from the British Heart Foundation, London, England. Manuscript received December 20, 1988; revised manuscript received and accepted March 13, 1989. Address for reprints: Magdi H. Yacoub, MD, Harefield Hospital, Middlesex, England UB9 6JH. F ollowing reports that endothelial cell culture super- natant exhibits vasoconstrictor activity,1-3 Yana- gisawa et al4 isolated and characterized a novel constrictor peptide, termed endothelin, from the culture supernatant of porcine aortic endothelial cells. They have shown in vitro that the potency of endothelin as a vasoconstrictor in porcine coronary arteries is greater than that of other known constrictor agents including angiotensin II. Endothelin’s structural similarity with other peptides that affect membrane ion channels, and its sensitivity to nicardipine and extracellular calcium, suggested a role for it as an endogenous ligand for volt- age-dependent calcium channels.4 More recently, it has been suggested that endothelin may possess a specific receptor.5 The action of endothelin on the human coro- nary circulation has not been studied before. We inves- tigated the response of isolated fresh human coronary vessels to increasing concentrations of endothelin in the absence and presence of various antagonists and vasodi- lator peptides. We also report on endothelin binding and distribution in human coronary arteries using in vitro autoradiography. METHODS In vitro functional studies: Fifty-six segments of left anterior descending coronary artery were obtained from 10 patients undergoing orthotopic heart transplantation, aged 10 days to 64 years. Diagnoses before transplanta- tion were cardiomyopathy (3 patients) ischemic heart disease (4), congenital heart disease (2) and primary pulmonary hypertension (1). Vessel segments from pa- tients with ischemic heart disease that showed macro- scopic evidence of atherosclerotic plaques were discard- ed. Vessel biopsies were placed immediately in modified Tyrode solution (composition described below), and then dissected free of connective tissue. Specimens were either used within 1 hour of removal from the patient or stored at 4’ C and used within 6 hours. The vesselswere cut into ring segments 3 to 5 mm in length and mounted on L-shaped metal hooks in 5-ml organ baths. The baths contained modified Tyrode solution containing (mM concentration): NaCl 136.8; NaHCOs 11.9; KC1 2.6; Na2 HP04.2HzO 0.42:Mg C12.6HzO 1.05; CaC12.6H20 2.55; and glucose 5.55. The temperature in the bath was maintained at 37O C and the buffer solution was aerated with a mixture of 95% 02 and 5% COz. Each ring segment was attached to a Grass FTO-3C force displacement transducer for measurement of iso- THE AMERICAN JOURNAL OF CARDIOLOGY JUNE 1, 1989 13%