The A₃ Adenosine Receptor Agonist CP-532,903 [N⁶-(2,5-Dichlorobenzyl)-3′-aminoadenosine-5′-N-methylcarboxamide] Protects against Myocardial Ischemia/Reperfusion Injury via the Sarcolemmal ATP-Sensitive Potassium Channel

Abstract

We examined the cardioprotective profile of the new A 3 adenosine receptor (AR) agonist 903 [N 6 -(2,5-dichlorobenzyl)-3Ј-aminoadenosine-5Ј-N-methylcarboxamide] in an in vivo mouse model of infarction and an isolated heart model of global ischemia/reperfusion injury. In radioligand binding and cAMP accumulation assays using human embryonic kidney 293 cells expressing recombinant mouse ARs, CP-532,903 was found to bind with high affinity to mouse A 3 ARs (K i ϭ 9.0 Ϯ 2.5 nM) and with high selectivity versus mouse A 1 AR (100-fold) and A 2A ARs (1000-fold). In in vivo ischemia/reperfusion experiments, pretreating mice with 30 or 100 g/kg CP-532,903 reduced infarct size from 59.2 Ϯ 2.1% of the risk region in vehicle-treated mice to 42.5 Ϯ 2.3 and 39.0 Ϯ 2.9%, respectively. Likewise, treating isolated mouse hearts with CP-532,903 (10, 30, or 100 nM) concentration dependently improved recovery of contractile function after 20 min of global ischemia and 45 min of reperfusion, including developed pressure and maximal rate of contraction/relaxation. In both models of ischemia/reperfusion injury, CP-532,903 provided no benefit in studies using mice with genetic disruption of the A 3 AR gene, A 3 knockout (KO) mice. In isolated heart studies, protection provided by CP-532,903 and ischemic preconditioning induced by three brief ischemia/ reperfusion cycles were lost in Kir6.2 KO mice lacking expression of the pore-forming subunit of the sarcolemmal ATPsensitive potassium (K ATP ) channel. Whole-cell patch-clamp recordings provided evidence that the A 3 AR is functionally coupled to the sarcolemmal K ATP channel in murine cardiomyocytes. We conclude that CP-532,903 is a highly selective agonist of the mouse A 3 AR that protects against ischemia/reperfusion injury by activating sarcolemmal K ATP channels.A 3 adenosine receptor (AR) agonists have been shown to effectively limit infarct size and reduce contractile dysfunction in several different animal models of ischemia/reperfusion injury (Auchampach et al., 1997b(Auchampach et al., , 2003Tracey et al., 1997Tracey et al., , 1998Tracey et al., , 2003Jordan et al., 1999;Thourani et al., 1999;Ge et al., 2004Ge et al., , 2006. A 3 AR agonists are attractive as cardioprotective agents because they do not alter systemic hemodynamic parameters in nonrodent species and are effective if administered before the ischemic event or only during reperfusion (Auchampach et al

KATPChannels in the Cardiovascular System

“…Postischemic functional recovery is largely unaffected by glibenclamide in mice and rats (653,722,723). Contrary to the notion that K ATP channel block is devastating to ischemic mouse hearts, infarct size and/or functional recovery after ischemia are not markedly in- (771,856). Collectively, these data indicate that species with an inherently short cardiac action potential (such as mice and rats) are not more dependent on K ATP channels to protect against stress than other species with inherently longer action potentials (such as rabbits, dogs, and perhaps humans).…”

“…As noted above, the preconditioning-like cardioprotective effect of diazoxide is similarly lost in the Kir6.2 Ϫ/Ϫ mice (770,876). Moreover, the protective effect of late preconditioning, induced by pretreatment with the A 3 AR agonist CP-532,903, is absent in the hearts of Kir6.2 Ϫ/Ϫ mice (856). Knockout of Kir6.2 worsens myocardial Ca 2ϩ load in vivo and impairs functional recovery after ischemia (305).…”

International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and Classification of Adenosine Receptors—An Update

“…gov identifier NCT00612521). Adenosine acting at A 2A or A 3 receptors has been found to reduce reperfusion injury after coronary artery occlusion Wan et al, 2008). The aim of this study is to assess whether the use of intracoronary adenosine given directly into the coronary artery before stenting can reduce the incidence of myocardial necrosis and achieve better outcomes at 30-day follow-up.…”

Activation of the A3Adenosine Receptor Suppresses Superoxide Production and Chemotaxis of Mouse Bone Marrow Neutrophils

“…5A). It is noteworthy, however, that pretreating neutrophils with the selective A 3 AR agonist CP-532,903 [100 nM (Tracey et al, 2003;Wan et al, 2008)] also effectively inhibited fMLP-induced neutrophil superoxide generation regardless of priming with TNF-␣ (Fig. 5A).…”

“…In this study, we have used bone marrow neutrophils isolated from A 2A and A 3 AR gene knockout mice, as well as the newly developed, highly selective mouse A 3 AR agonist CP-532,903, to explore the possibility that the A 3 AR mediates some of the suppressive effects of adenosine on neutrophil function. CP-532,903 binds potently to the murine A 3 AR (K i ϭ 9.0 nM) with greater than 100-and 1000-fold selectivity versus murine A 1 -and A 2A /A 2B ARs, respectively (Wan et al, 2008). Our results demonstrate that, like the A 2A AR, activation of the A 3 AR subtype inhibits neutrophil superoxide production.…”