Korean J Anesthesiol Introduction Polyphenol (-)-epigallocatechin gallate (EGCG), a major catechin of green tea, by targeting ischemia [1] and reperfusion [2] provides cardioprotection against ischemia-reperfusion injury. We recently reported that the cardioprotective effect by EGCG was mediated via the ATP-sensitive potassium (KATP) channels [2]. On the other hand, homeostatic regulation and stress responses are mainly regulated by the extracellular signals transduced by guanine nucleotide binding protein (G-protein)- coupled receptor (GPCR) in the heart [3]. Adenosine receptor (ADR) and opioid receptor (OPR) belong to the GPCR family and activation of these upstream receptors might protect the heart by triggering second messengers [4,5]. We hypothesized that ADR or OPR might be activated by EGCG-induced cardioprotection. We therefore investigated the infarct reducing effect with ADR or OPR antagonists in EGCG- induced cardioprotection in isolated rat hearts. Materials and Methods The experimental procedures and protocols used in this study were reviewed and approved by our Institutional Animal Care and Use Committee. Drugs and chemicals EGCG, 8-(p-sulfophenyl)theophylline hydrate (8-SPT), and 2,3,5-triphenyltetrazolium chloride (TTC) were ob- tained from Sigma-Aldrich Chemical, St. Louis, MO, USA. Naloxone was purchased from Reyon Pharmaceutical Co., Seoul, Republic of Korea. Fluorescent polymer microspheres were purchased from Duke Scientific, Palo Alto, CA, USA. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), 4-(2-[7-amino- 2-(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5-ylamino] ethyl)phenol (ZM241385), N-(4-acetylphenyl)-2-[4-(2,3,6,7- tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy] acetamide (MRS1706), and 1,4-dihydro-2-methyl-6-phenyl-4- (phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3- nitrophenyl)-methyl]ester (MRS1334) were purchased from Tocris Bioscience, Ellisville, MO, USA. Other chemicals were obtained from Sigma-Aldrich Chemical. EGCG, 8-SPT and naloxone were dissolved in distilled water. DPCPX, ZM241385, MRS1706 and MRS1334 were dissolved in dimethyl sulfoxide. Stock chemicals were stored at -20oC and were diluted with Krebs-Henseleit (KH) solution to the required final concentrations on the day of each experiment. Lee, et al. Langendorff isolated heart perfusion preparation Male Sprague-Dawley rats, weighing 280-330 gm obtained from KOATECH Co., Cheongwon-gun, Republic of Korea, were used. They received 50 mg/kg of pentobarbital sodium and 300 IU of heparin intraperitoneally. Hearts were isolated and perfused with modified KH solution containing (in mM) 118.5 NaCl, 4.7 KCl, 1.2 MgSO4, 1.8 CaCl2, 24.8 NaHCO3, 1.2 KH2PO4, and 10 glucose, as described previously [6]. Regional ischemia was induced by pulling the snare which was made at the level of the proximal length of the left coronary artery (LCA) and its major branches and confirmed by regional cyanosis and a substantial decrease in left ventricular developed pressure (LVDP). Reperfusion was started by releasing the snare. Experimental protocol All hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. Infusion of EGCG and antagonists was started 10 min before the onset of reperfusion and continued for 40 min (Fig. 1). To assess the involvement of ADR or OPR in EGCG-induced cardioprotection, ADR and OPR antagonists were perfused via 2nd port 10 min before EGCG perfusion. The concentrations of all chemicals were based on our and other previous studies on isolated working rat hearts that had no effect on infarct size in hearts subjected to ischemia and reperfusion [5,7-11]. Determination of area at risk and infarct size At the end of each experiment, the LCA perfusion circuit was precluded, and diluted fluorescent polymer microspheres (Duke Scientific Corp., Palo Alto, MA, USA) were infused to demarcate the area at risk (AR). The hearts were cut into 2-mm Fig. 1. Experimental protocols. Hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Polyphenol (-)-epigallo- catechin gallate (EGCG) was perfused from 5 min before reperfusion to 30 min after reperfusion. Adenosine or opioid receptor antagonists were pre-treated 10 min before EGCG perfusion. www.ekja.org 341