Expression, Function, and Regulation of E-Type Prostaglandin Receptors (EP 3 ) in the Nonischemic and Ischemic Pig Heart

Abstract

Abstract The action of prostacyclin, prostaglandin E 1 (PGE 1 ), and their mimetics on myocardial function includes changes in contractility, electrophysiological properties, and protection from injury caused by transient myocardial ischemia. This study was undertaken to investigate the basic properties of myocardial E-type prostaglandin (EP) receptors. Ligand binding studies using an enriched preparation of sarcolemmal membranes prepared from pig hearts revealed a single class of binding sites for [ 3 H]PGE 1 , with a K d of 3.7 nmol/L and a B max of 92 fmol/mg protein. Competition experiments indicated highest affinity for EPs, suggesting an EP receptor. In addition, the EP receptor subtype–selective agonists sulprostone (EP 1 and EP 3 ) and M&B 28.767 (EP 3 ) were active, suggesting the presence of an EP 3 receptor subtype. PGE 1 stimulated sarcolemmal GTPase and inhibited sarcolemmal adenylyl cyclase activity, indicating EP 3 receptor coupling to an inhibitory G protein (G i ). Additional in vivo experiments showed that intracoronary infusion of PGE 1 (1 nmol/min) decreased isoprenaline-stimulated left ventricular contractile activity without altering systemic vascular resistance. This inhibition of β-adrenergic effects is compatible with the known myocardial anti-ischemic action of prostaglandins. Further experiments examined EP 3 receptor density and G-protein coupling in sarcolemma from ischemic and reperfused ischemic myocardium. In anesthetized open-chest minipigs, occlusion of the left anterior descending coronary artery for 60 minutes increased EP 3 receptor density by 50%, whereas receptor affinity was unchanged. This upregulation was prevented by pretreatment with colchicine (2 mg/kg IV), indicating microtubule-dependent receptor externalization. Northern hybridization showed comparable EP 3 receptor mRNA expression in control and ischemic myocardium. The increase of receptor protein was reversed during 60 minutes of reperfusion. G-protein coupling proved to be intact in ischemic and reperfused ischemic myocardial tissue, as shown by preserved GTP-γ-S–induced decrease of [ 3 H]PGE 1 binding. These data demonstrate for the first time that myocardial receptors for PGE 1 belong to the EP 3 subtype. The properties of this receptor include inhibition of adenylyl cyclase and upregulation during regional myocardial ischemia, suggesting an involvement in the anti-ischemic activity of E- and I-type prostaglandins.