(N)-Methanocarba adenosine 5′-methyluronamides containing known A3 AR (adenosine receptor)-enhancing modifications, i.e. 2-(arylethynyl)adenine and N6-methyl or N6-(3-substituted-benzyl), were nanomolar full agonists of human (h) A3AR and highly selective (Ki ~0.6 nM, N6-methyl 2-(halophenylethynyl) analogues 13, 14). Combined 2-arylethynyl-N6-3-chlorobenzyl substitutions preserved A3AR affinity/selectivity in the (N)-methanocarba series (e.g. 3,4-difluoro full agonist MRS5698 31, Ki 3 nM, human and mouse A3) better than for ribosides. Polyaromatic 2-ethynyl N6-3-chlorobenzyl analogues, such as potent linearly extended 2-p-biphenylethynyl MRS5679 34 (Ki hA3 3.1 nM; A1, A2A: inactive) and fluorescent 1-pyrene adduct MRS5704 35 (Ki hA3 68.3 nM) were conformationally rigid; receptor docking identified a large, mainly hydrophobic binding region. The vicinity of receptor-bound C2 groups was probed by homology modeling based on recent X-ray structure of an agonist-bound A2AAR, with a predicted helical rearrangement requiring an agonist-specific outward displacement of TM2 resembling opsin. Thus, X-ray structure of related A2AAR is useful in guiding design of new A3AR agonists.

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We used pharmacological agents and genetic methods to determine whether the potent A 3 adenosine receptor (AR) agonist 2-chloro-N 6 -(3-iodobenzyl)adenosine-5Ј-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/ reperfusion injury in mice via the A 3 AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IB-MECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 g/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A 3 AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio) To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A 3 AR gene "knock-out" (A 3 KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A 3 KO mice in vivo and did not protect isolated perfused hearts obtained from A 3 KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 [condensation product of p-methoxyphenethyl methylamine with formaldehyde] to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IB-MECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A 3 AR.Several different A 3 adenosine receptor (AR) agonists, including the N 6 -benzyl adenosine-5Ј-N-methylcarboxamide derivatives IB-MECA, Cl-IB-MECA, and CB-MECA, have been shown to be effective at protecting against myocardial ischemia/reperfusion injury in animal models of infarction and myocardial stunning (Auchampach et al., 1997b(Auchampach et al., , 2003Tracey et al., 1997Tracey et al., , 1998Tracey et al., , 2003Jordan et al., 1999; Thourani et al., 1999a,b;Kodani et al., 2001;Takano et al., 2001). However, it remains uncertain whether these agents are effective by activating the A 3 AR or by nonspecific interactions with other AR subtypes. This issue has been difficult to address, because useful A 3 AR antagonists have only recently been developed.The goal of this investigation was to test the cardioprotective effectiveness of Cl-IB-MECA in an in vivo mouse model of infarction and in an isolated mouse heart model of global ischemia and reperfusion. A second goal of this investigation was to establish definitively whether Cl-IB-MECA exerts cardioprotection by activating A 3 ARs. Our experimental approach involved the use of the rodent A 3 AR antagonist MRS 1523 (Li et al., 1998), the potent A 2A AR antagonist ZM This work was supported by National Institutes of Health Grants R01 HL60051, R01 HL07707, and T32 HL73643 and by American Heart Association Research Fellowships 0320019Z and 0315274Z.Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

Small mammals have the ability to enter torpor, a hypothermic, hypometabolic state, allowing impressive energy conservation. Administration of adenosine or adenosine 5'-monophosphate (AMP) can trigger a hypothermic, torpor-like state. We investigated the mechanisms for hypothermia using telemetric monitoring of body temperature in wild type and receptor knock out (Adora1−/−, Adora3−/−) mice. Confirming prior data, stimulation of the A3 adenosine receptor (AR) induced hypothermia via peripheral mast cell degranulation, histamine release, and activation of central histamine H1 receptors. In contrast, A1AR agonists and AMP both acted centrally to cause hypothermia. Commonly used, selective A1AR agonists, including N6-cyclopentyladenosine (CPA), N6-cyclohexyladenosine (CHA), and MRS5474, caused hypothermia via both A1AR and A3AR when given intraperitoneally. Intracerebroventricular dosing, low peripheral doses of Cl-ENBA [(±)-5'-chloro-5'-deoxy-N6-endo-norbornyladenosine], or using Adora3−/− mice allowed selective stimulation of A1AR. AMP-stimulated hypothermia can occur independently of A1AR, A3AR, and mast cells. A1AR and A3AR agonists and AMP cause regulated hypothermia that was characterized by a drop in total energy expenditure, physical inactivity, and preference for cooler environmental temperatures, indicating a reduced body temperature set point. Neither A1AR nor A3AR were required for fasting-induced torpor. A1AR and A3AR agonists and AMP trigger regulated hypothermia via three distinct mechanisms.