Original Article NECA and bradykinin at reperfusion reduce infarction in rabbit hearts by signaling through PI3K, ERK, and NO Xi-MingYang a , Thomas Krieg a , Lin Cui a , James M. Downey a , Michael V. Cohen a, * ,b a Department of Physiology, College of Medicine, University of South Alabama, MSB 3074, Mobile, AL 36688, USA b Department of Medicine, College of Medicine, University of South Alabama, Mobile, AL 36688, USA Received 20 August 2003; received in revised form 24 November 2003; accepted 12 December 2003 Abstract The adenosine A 1 /A 2 adenosine agonist 5′-(N-ethylcarboxamido) adenosine (NECA) and bradykinin both limit infarction when adminis- tered at reperfusion in rabbits. This study compares the signal transduction pathways responsible for their anti-infarct effect. Receptor agonists were administered to isolated rabbit hearts starting 25 min after the onset of a 30-min period of ischemia and continued into the 2-h reperfusion period. Infarct size was measured. Both NECA and bradykinin decreased infarction from 31.5 ± 2.4% of the risk zone in untreated hearts to 11.8 ± 2.0% and 15.4 ± 2.4%, respectively (P < 0.05). Protection from both agents was blocked by PD98059, wortmannin, and N x -nitro-L- arginine methyl ester (L-NAME), thus demonstrating dependence on activation of extracellular regulated kinase (ERK) and phosphatidyli- nositol 3-kinase (PI3K) and stimulation of nitric oxide synthase (NOS). Both wortmannin and PD98059 prevented phosphorylation of ERK 1/2 in NECA-treated hearts, whereas only wortmannin and not PD98059 blocked Akt phosphorylation. These data suggest Akt is upstream of ERK 1/2. In addition, 8-(3-chlorostyryl) caffeine blocked NECA’s protection indicating that A 2 adenosine receptors trigger NECA’s anti-infarct effect. Of note, both bradykinin and acetylcholine (ACh) administered before ischemia to trigger preconditioning’s cardioprotec- tion use PI3K and NOS in their signaling pathway. Curiously, however,ACh, unlike bradykinin, was not protective when administered at reperfusion. Hence, both NECA and bradykinin administered at reperfusion protect through a common signaling pathway that includes PI3K, NO, and ERK. © 2003 Elsevier Ltd. All rights reserved. Keywords: Bradykinin; NECA; Nitric oxide; PI3 kinase; Reperfusion injury 1. Introduction Although ischemic preconditioning and pharmacological preconditioning-mimetics are very effective at limiting in- farction during an ischemic insult, the requirement for pre- treatment has greatly limited their clinical relevance since patients with acute myocardial infarction mainly present after the onset of coronary occlusion. What is needed is a cardioprotective intervention that can be applied after is- chemia has begun. Adenosine, the first preconditioning- mimetic identified, was initially examined in this setting with very mixed results. While some reports claimed cardiopro- tection when adenosine was administered only at reperfusion [1–9], other quite credible studies failed to see protection when adenosine or its specific receptor agonists were admin- istered at the time of coronary reflow [10–16]. In contrast to the inconsistent data with adenosine, AMP579, an adenosine agonist with nearly equivalent affini- ties for A 1 and A 2A receptors, has without exception salvaged ischemic myocardium when administered at the time of re- perfusion. AMP579 infused at or shortly before reperfusion has decreased infarct size in pigs [13], dogs [14], rabbits [17–20], and rats [17]. Despite these dramatic effects, little is known about the mechanism of its protection other than a dependence on A 2A activation [13,19] and possible involve- ment of extracellular regulated kinase (ERK) 1/2 [17]. Un- fortunately, AMP579 is no longer available for study. How- ever, 5′-(N-ethylcarboxamido) adenosine (NECA) is a closely related compound having a structure and pharmacol- ogy almost identical to that of AMP579. Recently Bell and Yellon [21] reported that bradykinin could limit infarct size when applied at reperfusion in mouse * Corresponding author. Tel.: +1-251-460-6812; fax: +1-251-460-6464. E-mail address: mcohen@usouthal.edu (M.V. Cohen). Journal of Molecular and Cellular Cardiology 36 (2004) 411–421 www.elsevier.com/locate/yjmcc © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2003.12.008