Effect Of IB-MECA Research Articles

Targeting the inflammasome and adenosine type-3 receptors improves outcome of antibiotic therapy in murine anthrax

To establish whether activation of adenosine type-3 receptors (A3Rs) and inhibition of interleukin-1β-induced inflammation is beneficial in combination with antibiotic therapy to increase survival of mice challenged with anthrax spores. DBA/2 mice were challenged with Bacillus anthracis spores of the toxigenic Sterne strain 43F2. Survival of animals was monitored for 15 d. Ciprofloxacin treatment (50 mg/kg, once daily, intraperitoneally) was initiated at day +1 simultaneously with the administration of inhibitors, and continued for 10 d. Two doses (2.5 mg/kg and 12.5 mg/kg) of acetyl-tyrosyl-valyl-alanyl-aspartyl-chloromethylketone (YVAD) and three doses (0.05, 0.15 and 0.3 mg/kg) of 1-[2-Chloro-6-[[(3-iodophenyl) methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-β-D- ribofuranuronamide (Cl-IB-MECA) were tested. Animals received YVAD on days 1-4, and Cl-IB-MECA on days 1-10 once daily, subcutaneously. Human lung epithelial cells in culture were challenged with spores or edema toxin and the effects of IB-MECA on phosphorylation of AKT and generation of cAMP were tested. We showed that the outcome of antibiotic treatment in a murine anthrax model could be substantially improved by co-administration of the caspase-1/4 inhibitor YVAD and the A3R agonist Cl-IB-MECA. Combination treatment with these substances and ciprofloxacin resulted in up to 90% synergistic protection. All untreated mice died, and antibiotic alone protected only 30% of animals. We conclude that both substances target the aberrant host signaling that underpins anthrax mortality. Our findings suggest new possibilities for combination therapy of anthrax with antibiotics, A3R agonists and caspase-1 inhibitors.

Adenosine A 2A receptor agonists inhibit lipopolysaccharide-induced production of tumor necrosis factor-α by equine monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes by activating one or more adenosine receptor subtypes, namely A 1, A 2A, A 2B and A 3. The results of previous studies indicate that adenosine analogues inhibit lipopolysaccharide (LPS)-induced production of reactive oxygen species (ROS) by equine neutrophils primarily through activation of A 2A receptors. Because peripheral blood monocytes produce cytokines that are responsible for many of the deleterious effects of LPS, the current study was performed to evaluate the effects of an array of novel adenosine receptor agonists on LPS-induced production of tumor necrosis factor-α (TNF-α), and to assess the selectively of these agonists for equine adenosine A 2A over the A 1 receptor. Radioligand binding studies performed with equine tissues expressing adenosine A 1 and A 2A receptor subtypes yielded a rank order of affinity for the equine A 2A receptor of ATL307 > ATL309 ≈ ATL310 ≈ ATL313 > ATL202 ≈ ATL361 ≈ ATL376 > ATL372 > CGS21680 > NECA. Co-incubation of equine peripheral blood monocytes with LPS and these agonists resulted in inhibition of TNF-α production with a rank order of potency that strongly correlated with their binding affinities for equine adenosine A 2A receptors. Results of experiments performed with one of the adenosine receptor agonists (ATL313) and selective adenosine receptor antagonists confirmed that inhibition of LPS-induced production of TNF-α occurred via stimulation of A 2A receptors. Although incubation of monocytes with IB-MECA, a compound purported to act as an adenosine A 3 receptor agonist, reduced LPS-induced TNF-α production, this effect of IB-MECA was inhibited by the A 2A selective antagonist ZM241385 but not by the A 3 receptor antagonist MRS1220. These results indicate that the adenosine receptor subtype responsible for regulation of LPS-induced cytokine production by equine monocytes is the A 2A receptor. To address the signal transduction mechanism responsible for the anti-inflammatory effects of ATL313 in equine monocytes, production of cAMP was compared in the presence and absence of either the adenosine A 2A receptor antagonist ZM241385 or the adenosine A 2B receptor antagonist MRS1706. In the absence of the antagonists, ATL313 increased production of cAMP; ZM241385 inhibited this effect of ATL313, whereas MRS1706 did not. Furthermore, incubation of monocytes with either the stable analogue of cAMP, dibutyryl cAMP, or forskolin, an activator of adenylyl cyclase, also inhibited LPS-induced production of TNF-α production by equine monocytes. Collectively, the results of the current study indicate that adenosine analogues inhibit LPS-induced production of TNF-α by equine monocytes primarily via activation of adenosine A 2A receptors and do so in a cAMP-dependent manner. The results of this study indicate that stable adenosine analogues that are selective for adenosine A 2A receptors may be suitable for development as anti-inflammatory drugs in horses.

N6-(3-Iodobenzyl)-adenosine-5′-N-methylcarboxamide Confers Cardioprotection at Reperfusion by Inhibiting Mitochondrial Permeability Transition Pore Opening via Glycogen Synthase Kinase 3β

Although the adenosine A(3) receptor agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) has been reported to be cardioprotective at reperfusion, little is known about the mechanisms underlying the protection. We hypothesized that IB-MECA may protect the heart at reperfusion by preventing the opening of mitochondrial permeability transition pore (mPTP) through inactivation of glycogen synthase kinase (GSK) 3beta. IB-MECA (1 microM) applied during reperfusion reduced infarct size in isolated rat hearts, an effect that was abrogated by the selective A3 receptor antagonist 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)-methyl]ester (MRS1334) (100 nM). The effect of IB-MECA was abrogated by the mPTP opener atractyloside (20 microM), implying that the action of IB-MECA may be mediated by inhibition of the mPTP opening. In cardiomyocytes, IB-MECA attenuated oxidant-induced loss of mitochondrial membrane potential (DeltaPsim), which was reversed by MRS1334. IB-MECA also reduced Ca2+-induced mitochondrial swelling. IB-MECA enhanced phosphorylation of GSK-3beta (Ser9) upon reperfusion, and the GSK-3 inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) (3 microM) mimicked the protective effect of IB-MECA by attenuating both infarction and the loss of DeltaPsim. In addition, the effect of IB-MECA on GSK-3beta was reversed by wortmannin (100 nM), and IB-MECA was shown to enhance Akt phosphorylation upon reperfusion. In contrast, rapamycin (2 nM) failed to affect GSK-3beta phosphorylation by IB-MECA, and IB-MECA did not alter phosphorylation of either mTOR (Ser2448) or 70s6K (Thr389). Taken together, these data suggest that IB-MECA prevents myocardial reperfusion injury by inhibiting the mPTP opening through the inactivation of GSK-3beta at reperfusion. IB-MECA-induced GSK-3beta inhibition is mediated by the PI3-kinase/Akt signal pathway but not by the mTOR/p70s6K pathway.

A3 adenosine receptor agonist IB-MECA reduces myocardial ischemia-reperfusion injury in dogs.

We examined the effect of the A3 adenosine receptor (AR) agonist IB-MECA on infarct size in an open-chest anesthetized dog model of myocardial ischemia-reperfusion injury. Dogs were subjected to 60 min of left anterior descending (LAD) coronary artery occlusion and 3 h of reperfusion. Infarct size and regional myocardial blood flow were assessed by macrohistochemical staining with triphenyltetrazolium chloride and radioactive microspheres, respectively. Four experimental groups were studied: vehicle control (50% DMSO in normal saline), IB-MECA (100 microg/kg iv bolus) given 10 min before the coronary occlusion, IB-MECA (100 microg/kg iv bolus) given 5 min before initiation of reperfusion, and IB-MECA (100 microg/kg iv bolus) given 10 min before coronary occlusion in dogs pretreated 15 min earlier with the ATP-dependent potassium channel antagonist glibenclamide (0.3 mg/kg iv bolus). Administration of IB-MECA had no effect on any hemodynamic parameter measured including heart rate, first derivative of left ventricular pressure, aortic pressure, LAD coronary blood flow, or coronary collateral blood flow. Nevertheless, pretreatment with IB-MECA before coronary occlusion produced a marked reduction in infarct size ( approximately 40% reduction) compared with the control group (13.0 +/- 3.2% vs. 25.2 +/- 3.7% of the area at risk, respectively). This effect of IB-MECA was blocked completely in dogs pretreated with glibenclamide. An equivalent reduction in infarct size was observed when IB-MECA was administered immediately before reperfusion (13.1 +/- 3.9%). These results are the first to demonstrate efficacy of an A3AR agonist in a large animal model of myocardial infarction by mechanisms that are unrelated to changes in hemodynamic parameters and coronary blood flow. These data also demonstrate in an in vivo model that IB-MECA is effective as a cardioprotective agent when administered at the time of reperfusion.

Positive coupling of atypical adenosine A 3 receptors on human eosinophils to adenylyl cyclase

Adenosine A 3 receptors are reported to couple negatively to adenylyl cyclase (AC) but their mediation of anti-inflammatory effects in human eosinophils prompted us to investigate their coupling to AC. The A 3-selective agonists IB-MECA and Cl-IB-MECA evoked a concentration-dependent generation of cAMP (EC 50, 3.2 and 1.8 μM, respectively) and were more potent than the A 2A agonist CGS 21680 ( EC 50=15.4 μM ) and adenosine ( EC 50=19.2 μM ). The cAMP response was additive to that produced by forskolin (10 μM). The effect of IB-MECA was insensitive to A 1 and A 2A receptor antagonists, but was antagonized by the A 3-selective antagonist MRS 1220 (0.1–2.5 μM) in a competitive manner. The estimated K B of 190 nM was, however, atypical. The cyclo-oxygenase inhibitor, indomethacin, had no effect on the cAMP response. A general inverse relationship between cAMP generation and inhibition of degranulation was seen. We conclude that in human eosinophils, an atypical form of A 3 receptors positively coupled to AC may exist. The resulting cAMP generation may underlie the anti-inflammatory actions of A 3 agonists in eosinophils.

Adenosine A3receptor-mediated potentiation of mucociliary transport and epithelial ciliary motility

To examine the effect of adenosine A(3) receptor stimulation on airway mucociliary clearance, we measured transport of Evans blue dye in rabbit trachea in vivo and ciliary motility of epithelium by the photoelectric method in vitro. Mucociliary transport was enhanced dose dependently by the selective A(3) agonist N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) and to a lesser extent by the less-selective N(6)-2-(4-amino-3-iodophenyl)ethyladenosine, whereas the A(1) agonist N-cyclopentyladenosine (CPA) and the A(2) agonist CGS-21680 had no effect. The effect of IB-MECA was abolished by pretreatment with the selective A(3) antagonist MRS-1220 but not by the A(1) antagonist 1,3-dipropyly-8-cyclopentylxanthine or the A(2) antagonist 3,7-dimethyl-L-propargylxanthine. Epithelial ciliary beat frequency was increased by IB-MECA in a concentration-dependent manner, the maximal increase being 33%, and this effect was inhibited by MRS-1220. The IB-MECA-induced ciliary stimulation was not altered by the Rp diastereomer of cAMP but was greatly inhibited by Ca(2+)-free medium containing BAPTA-AM. Incubation with IB-MECA increased intracellular Ca(2+) contents. Therefore, A(3) agonist enhances airway mucociliary clearance probably through Ca(2+)-mediated stimulation of ciliary motility of airway epithelium.