2-(4-(2-carboxyethyl)phenethylamino)-5--n-ethylcarboxamidoadenosine and Reperfusion-Injury

Ischemia-reperfusion (IR) of liver results in hepatocytes (HP) and sinusoidal endothelial cells (LSEC) irreversible damage. Ischemic preconditioning protects IR damage upon adenosine A2a receptor (A2aR) stimulation. Understanding the phenotypic changes that underlie hepatocellular damage and protection is critical to optimize strategies against IR.. The proteome of HP and LSEC, isolated from sham or IR exposed mice, receiving or not the A2aR agonist CGS21680 (0.5mg/kg b.w.), was analyzed by 2-D DIGE/MALDI-TOF.. We identified 64 proteins involved in cytoprotection, regeneration, energy metabolism and response to oxidative stress; among them, 34 were associated with IR injury and A2aR protection. The main pathways, downregulated by IR and upregulated by CGS21680 in HP and LSEC, were related to carbohydrate, protein and lipid supply and metabolism. In LSEC, IR reduced stress response enzymes that were instead upregulated by CGS21680 treatment. Functional validation experiments confirmed the metabolic involvement and showed that inhibition of pyruvate kinase, 3-chetoacylCoA thiolase, and arginase reduced the protection by CGS21680 of in vitro hypoxia-reoxygenation injury, whereas their metabolic products induced liver cell protection. Moreover, LSEC, but not HP, were sensitive to H2O2-induced oxidative damage and CGS21680 protected against this effect.. IR and A2aR stimulation produces pathological and protected liver cell phenotypes, respectively characterized by down- and upregulation of proteins involved in the response to O2 and nutrients deprivation during ischemia, oxidative stress, and reactivation of aerobic energy synthesis at reperfusion. This provides novel insights into IR hepatocellular damage and protection, and suggests additional therapeutic options.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Animals; Antioxidants; Cytoprotection; Endothelial Cells; Hepatocytes; Lipid Metabolism; Liver; Male; Mice; Mice, Inbred C57BL; Phenethylamines; Proteome; Receptor, Adenosine A2A; Reperfusion Injury

Postconditioning is a procedure based on the induction of intracellular protective reactions immediately after the onset of reperfusion. Because of the growing need to prevent ischemia/reperfusion (I/R) injury during liver surgery and transplantation, we investigated the possibility of pharmacologically inducing hepatic postconditioning. The effects of the adenosine A2A receptor agonist 2p-(2-carboxyethyl)-phenyl-amino-5'-N-ethylcarboxyamido-adenosine (CGS21680; 5 μmol/L) and the phosphatase and tensin homologue deleted from chromosome 10 (PTEN) inhibitor dipotassium bisperoxo-(5-hydroxypyridine-2-carboxyl)-oxovanadate [bpV(HOpic); 250 nmol/L] were investigated in primary rat hepatocytes during reoxygenation after 24 hours of cold storage and in an in vivo model of rat liver warm I/R. The addition of CGS21680 at reoxygenation significantly reduced hepatocyte death through the activation of the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB)/Akt signal pathway and through the reduction of the intracellular level of PTEN. PTEN lowering was associated with the increased generation of reactive oxygen species after A2A receptor-mediated stimulation of β-nicotinamide adenine dinucleotide phosphate oxidase (NOX). The inhibition of PI3K or NOX with wortmannin or diphenyleneiodonium chloride, respectively, and the addition of the antioxidant N,N'-diphenyl-p-phenylenediamine reversed the effects of CGS21680. The PTEN inhibitor bpV(HOpic) mimicked the protection provided by CGS21680 against reoxygenation damage. An in vivo rat treatment with CGS21680 or bpV(HOpic) during reperfusion after 1 hour of partial hepatic ischemia also promoted PKB/Akt activation and ameliorated alanine aminotransferase release and histological lesions induced by 2 hours of reperfusion. We conclude that adenosine A2A receptor agonists and PTEN inhibitors are possibly useful agents for the pharmacological induction of postconditioning in the liver.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Animals; Liver; Male; Oxidative Stress; Phenethylamines; PTEN Phosphohydrolase; Rats; Rats, Wistar; Reperfusion Injury

In the present study, we tested the efficacy of treatment with the selective adenosine A2A receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-50-ethylcarboxamidoadenosine (CGS 21680) on ischemia and reperfusion injury of the multivisceral organs. Ischemia and reperfusion injury was induced in mice by clamping both the superior mesenteric artery and the celiac artery for 30 min, followed thereafter by reperfusion. Sixty minutes after reperfusion, animals were killed for histological examination and biochemical studies. Injured vehicle-treated mice developed a significant increase of ileum TNF-alpha levels, myeloperoxidase activity, and marked histological injury and apoptosis. Ischemia and reperfusion injury of the multivisceral organs was also associated with significant mortality. Reperfused ileum sections from injured vehicle-treated mice showed positive staining for P-selectin and intercellular adhesion molecule 1. The intensity and degree of P-selectin and intercellular adhesion molecule 1 were markedly reduced in tissue sections from injured CGS 21680-treated mice. Ischemia and reperfusion-injured mice that have been treated with CGS 21680 showed also a significant reduction of neutrophil infiltration into the intestine, a reduction of apoptosis, and improved histological status of the intestine and survival. Taken together, our results clearly demonstrate that selective activation of adenosine A2A receptors plays an important role in the regulation of ischemia and reperfusion injury and results put forward the hypothesis that selective activation of adenosine A2A receptors may represent a novel and possible strategy.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Fas Ligand Protein; Intercellular Adhesion Molecule-1; Intestine, Small; Male; Mice; Mitogen-Activated Protein Kinases; NF-kappa B; P-Selectin; Peroxidase; Phenethylamines; Poly Adenosine Diphosphate Ribose; Proto-Oncogene Proteins c-bcl-2; Receptor, Adenosine A2A; Reperfusion Injury; Signal Transduction; Tumor Necrosis Factor-alpha; Tyrosine

Although adenosine exerts cardio-and vasculoprotective effects, the roles and signaling mechanisms of different adenosine receptors in mediating skeletal muscle protection are not well understood. We used a mouse hindlimb ischemia-reperfusion model to delineate the function of three adenosine receptor subtypes. Adenosine A(3) receptor-selective agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IBMECA; 0.07 mg/kg ip) reduced skeletal muscle injury with a significant decrease in both Evans blue dye staining (5.4 +/- 2.6%, n = 8 mice vs. vehicle-treated 28 +/- 6%, n = 7 mice, P < 0.05) and serum creatine kinase level (1,840 +/- 910 U/l, n = 13 vs. vehicle-treated 12,600 +/- 3,300 U/l, n = 14, P < 0.05), an effect that was selectively blocked by an A(3) receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191; 0.05 mg/kg). The adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 0.05 mg/kg) also exerted a cytoprotective effect, which was selectively blocked by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.2 mg/kg). The adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.07 mg/kg)-induced decrease in skeletal muscle injury was selectively blocked by the A(2A) antagonist 2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e] [1,2,4]triazolo[1,5-C]pyrimidin-5-amine (SCH-442416; 0.017 mg/kg). The protection induced by the A(3) receptor was abrogated in phospholipase C-beta2/beta3 null mice, but the protection mediated by the A(1) or A(2A) receptor remained unaffected in these animals. The adenosine A(3) receptor is a novel cytoprotective receptor that signals selectively via phospholipase C-beta and represents a new target for ameliorating skeletal muscle injury.

Topics: Adenosine; Animals; Dihydropyridines; Disease Models, Animal; Hindlimb; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Phenethylamines; Phospholipase C beta; Pyrazoles; Pyrimidines; Receptor, Adenosine A1; Receptor, Adenosine A2A; Receptor, Adenosine A3; Reperfusion Injury; Signal Transduction; Xanthines

Hemorrhagic shock and resuscitation trigger a global ischemia/reperfusion phenomenon, in which various inflammatory processes critically contribute to the ensuing tissue damage. Adenosine is an endogenous nucleoside that is released during shock. Activation of adenosine A(2A) receptors can broadly inactivate inflammatory cascades. The current study was designed to evaluate the effect of A(2A) receptor activation on organ injury and inflammation in the setting of global ischemia/reperfusion elicited by trauma/hemorrhagic shock and resuscitation.. Prospective animal study with concurrent control.. Small animal laboratory.. Adult male Sprague-Dawley rats.. The rats were subjected to a laparotomy (trauma) and 90 mins of hemorrhagic shock or trauma/sham shock. The selective A(2A) receptor agonist CGS-21680 (2-p-(2-carboxyethyl) phenethylamino-5'-N-ethyl-carboxamidoadenosine; 0.5 mg/kg) or its vehicle was injected 30 mins before shock or immediately after resuscitation. At 3 hrs following resuscitation, animals were killed and tissue was harvested for analysis. Lung permeability and pulmonary myeloperoxidase levels were used to quantitate lung injury. Intestinal injury was determined by histologic analysis of terminal ileum. Red blood cell deformability was measured by a laser-assisted ektacytometer. In this assay, a decrease in the elongation index is a marker of decreased red blood cell deformability.. Pretreatment with CGS-21680 protected the lung but not the gut against shock-induced injury and prevented the shock-induced decrease in red blood cell deformability. Posttreatment with CGS-21680 ameliorated shock-induced lung injury but failed to prevent gut injury and preserve red blood cell deformability.. A(2A) receptor agonists may represent a novel therapeutic approach in preventing organ injury following trauma/hemorrhagic shock.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Animals; Inflammation; Lung Diseases; Male; Phenethylamines; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A; Reperfusion Injury; Resuscitation; Shock, Hemorrhagic; Shock, Traumatic

To determine the effect of 2-(6-cyano-1-hexyn-1-yl)adenosine (2-CN-Ado), an adenosine A2A receptor agonist, on retinal ischaemia/reperfusion damage in rats.. Retinal ischaemia/reperfusion damage was induced by elevating the intraocular pressure of one eye to 130 mm Hg for 60 minutes and returning it to normal. 7 days later, retinal ischaemia/reperfusion damage was histologically quantified by measuring the thickness of retinal layers. Intraocular pressure was measured by pressure transducer.. Retinal ischaemia/reperfusion caused cell loss in the ganglion cell layer and thinning of the inner plexiform and nuclear layer. Both ocular topical and intravenous administration of 2-CN-Ado caused a reduction of retinal ischaemia/reperfusion damage. A selective A2A receptor antagonist, 1,3,7-trimethyl-8-(3-chlorostyryl) xanthine (CSC), but not a selective A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), or a selective A2B receptor antagonist, alloxazine, reduced the protective effect of 2-CN-Ado. While ocular topical administration of 2-CN-Ado caused a sustained reduction of intraocular pressure, intravenous administration of 2-CN-Ado showed a transient ocular hypotensive effect.. These results suggest that 2-CN-Ado attenuates retinal ischaemia/reperfusion damage, and at least some of this protective effect of 2-CN-Ado might be mediated via activation of the adenosine A2A receptor.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Administration, Topical; Animals; Antihypertensive Agents; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Intraocular Pressure; Ischemic Preconditioning; Male; Phenethylamines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Retinal Vessels

It has been observed that a cytokine synthesis inhibitor, pentoxifylline, prevents the apoptotic processes taking place in the amygdala following myocardial infarction. However, it is unknown if the cardioprotective effect of A(2A) adenosine receptor agonist, CGS21680, which reduces cytokine synthesis, would lead to such amygdala apoptosis regression. Thus, this study was designed to investigate whether cardioprotective A(2A) adenosine receptor activation reduces apoptosis in the amygdala following myocardial infarction. Anesthetized rats were subjected to left anterior descending coronary artery occlusion for 40 min, followed by 72 h of reperfusion. The A(2A) agonist CGS21680 (0.2 mug/kg/min i.v.) was administered continuously for 120 min, starting (1) five minutes prior to instituting reperfusion (Early) or (2) five minutes after the beginning of reperfusion (Late). After reperfusion, myocardial infarct size was determined and the amygdala was dissected from the brain. Infarct size was reduced significantly in the Early compared to the Control group (34.6 +/- 1.8% and 52.3 +/- 2.8% respectively; p < 0.05), with no difference compared to the Late group (40.1 +/- 6.1%). Apoptosis regression was documented in the amygdala of the Early group by an enhanced phosphatidylinositol 3-kinase-Akt pathway activation and Bcl-2 expression concurrently to a caspase-3 activation limitation and reduction in TUNEL-positive cells staining. On the other hand, amygdala TUNEL-positive cell numbers were not reduced in the Late group. Moreover, TNFalpha was significantly reduced in the amygdala of the Early group compared to the Control and Late groups. These results indicate that A(2A) adenosine receptor stimulation is associated with apoptosis regression in the amygdala following myocardial infarction.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Amygdala; Animals; Antihypertensive Agents; Apoptosis; bcl-2-Associated X Protein; Body Weight; Caspase 3; Caspases; In Situ Nick-End Labeling; Myocardial Infarction; Myocardium; Organ Size; Phenethylamines; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A; Reperfusion Injury; Tumor Necrosis Factor-alpha

Topics: Adenosine; Adenosine A2 Receptor Agonists; Animals; Blood Pressure; Erythrocyte Deformability; Humans; Immunologic Factors; Inflammation; Lung Diseases; Phenethylamines; Rats; Reperfusion Injury; Shock, Hemorrhagic; Shock, Traumatic; Time Factors; Toll-Like Receptor 4; Vasodilator Agents

Ischemia/reperfusion injury during liver transplantation is a major cause of primary nonfunctioning graft for which there is no effective treatment other than retransplantation. Adenosine prevents ischemia-reperfusion-induced hepatic injury via its A2A receptors. The aim of this study was to investigate the role of A2A receptor agonist on apoptotic ischemia/reperfusion-induced hepatic injury in rats. Isolated rat livers within University of Wisconsin solution were randomly divided into four groups: (1) continuous perfusion of Krebs-Henseleit solution through the portal vein for 165 minutes (control); (2) 30-minute perfusion followed by 120 minutes of ischemia and 15 minutes of reperfusion; (3) like group 2, but with the administration of CGS 21680, an A2A receptor agonist, 30 microg/100 ml, for 1 minute before ischemia; (4) like group 3, but with administration of SCH 58261, an A2A receptor antagonist. Serum liver enzyme levels were measured by biochemical analysis, and intrahepatic caspase-3 activity was measured by fluorometric assay; apoptotic cells were identified by morphological criteria, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorometric assay, and immunohistochemistry for caspase-3. Results showed that at 1 minute of reperfusion, there was a statistically significant reduction in liver enzyme levels in the animals pretreated with CGS (p < 0.05). On fluorometric assay, caspase-3 activity was significantly decreased in group 3 compared to group 2 (p < 0.0002). The reduction in postischemic apoptotic hepatic injury in the CGS-treated group was confirmed morphologically, by the significantly fewer apoptotic hepatocyte cells detected (p < 0.05); immunohistochemically, by the significantly weaker activation of caspase-3 compared to the ischemic group (p < 0.05); and by the TUNEL assay (p < 0.05). In conclusion, the administration of A2A receptor agonist before induction of ischemia can attenuate postischemic apoptotic hepatic injury and thereby minimize liver injury. Apoptotic hepatic injury seems to be mediated through caspase-3 activity.

Topics: Adenosine; Adenosine A2 Receptor Agonists; Animals; Apoptosis; Caspase 3; Caspases; In Situ Nick-End Labeling; In Vitro Techniques; Liver; Male; Phenethylamines; Rats; Rats, Wistar; Reperfusion Injury

To examine whether adenosine reduces ischemia/reperfusion (I/R)-induced liver injury by inhibiting leukocyte activation via A(2) receptor (A(2)R) stimulation, we investigated the effects of adenosine and selective A(2A) receptor (A(2A)R) agonists (YT-146 and CGS21680C) on I/R-induced liver injury in rats. Adenosine, YT-146, and CGS21680C, in the concentration of 10(-7) to 10(-5) M, significantly inhibited neutrophil elastase release by about 30 to 40% and increased intracellular Ca(2+) concentrations in isolated neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro. Adenosine, YT-146, and CGS21680C, in the concentration of 10(-7) to 10(-5) M, significantly inhibited tumor necrosis factor (TNF)-alpha production by monocytes stimulated with endotoxin by about 50%. Although ZM241385, a selective A(2A)R antagonist, significantly enhanced the increase in neutrophil elastase release and intracellular Ca(2+) concentrations in neutrophils stimulated with fMLP, this agent did not affect the endotoxin-induced TNF-alpha production by monocytes. Rats were subjected to liver ischemia for 60 min. Serum levels of transaminases increased after hepatic I/R, peaking at 12 h after reperfusion. The i.v. infusion of adenosine (1 and 10 mg/kg/h), YT-146 (0.1 and 1 mg/kg/h), and CGS21680C (0.1 and 1 mg/kg/h) significantly inhibited the I/R-induced increase in serum transaminase levels 12 h after reperfusion. The I/R-induced decrease in hepatic tissue blood flow was significantly prevented by adenosine and YT-146. Hepatic levels of TNF-alpha, cytokine-induced neutrophil chemoattractant (equivalent to human interleukin-8), and myeloperoxidase were significantly increased after I/R. These increases were significantly inhibited by the administration of adenosine, YT-146, and CGS21680C. Although the histological neutrophil accumulation in the liver was significantly increased after I/R as evaluated by the naphthol AS-D chloroacetate technique, the administration of adenosine, YT-146, and CGS21680C significantly inhibited this increase. These findings suggest that adenosine reduces I/R-induced liver injury both by inhibiting the synthesis of inflammatory mediators and by inhibiting neutrophil degranulation directly, probably through A(2A)R stimulation.

Topics: Adenosine; Alkynes; Animals; Calcium; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Ischemia; Leukocyte Elastase; Lipopolysaccharides; Liver; Male; Monocytes; Neutrophil Activation; Peroxidase; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; Receptor, Adenosine A2A; Reperfusion Injury; Triazines; Triazoles; Tumor Necrosis Factor-alpha

It has been postulated that the adenosine A1 receptor subtype, but also A2a receptors, are involved in mediating the beneficial properties of adenosine during ischemia and reperfusion. We investigated the effects of the selective A1 adenosine receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), the selective A2A adenosine receptor agonists, 2-[p-(2-carboxyethyl)phenetylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA), and the non selective agonist, 5'-N-ethylcarboxamidoadenosine (NECA), on ischemia-reperfusion injury in Langendorff-perfused rat hearts. Global ischemia was induced for 15 min in paced hearts followed by 60 min reperfusion. Control hearts developed left ventricular dysfunction, as indicated by the increase in end diastolic pressure to 40.8 +/- 5.1 vs 5.9 +/- 1.0 mm Hg baseline, and in coronary perfusion pressure to 57.6 +/- 8.4 vs 28.8 +/- 2.2 mm Hg before ischemia. After 15 min of reperfusion, ventricular function (LVDP) recovered by 83%, but creatine kinase levels were still significantly increased (294 +/- 55 IUl(-1) vs basal), indicating the occurrence of myocardial injury. All adenosine agonists added to the perfusion medium 15 min prior to ischemia exerted protective effects against myocardial dysfunction and reperfusion injury. Thus, 2HE-NECA (100 nM), CGS 21680 (10 nM), CCPA (3 nM) and NECA (100 nM) significantly (P < 0.05) decreased end diastolic pressure by 50-75% as compared with the control group. Similarly, all compounds significantly (P < 0.05) reduced coronary perfusion pressure by 30-45% vs control. For all drugs, recovery of LVDP occurred immediately after restoration of coronary flow. At 15-min reperfusion the adenosine agonists decreased myocardial creatine kinase release by 80-95% (P < 0.05 vs control). These findings indicate that both A1 and A2A adenosine receptors are involved in protecting the myocardium against ischemia and reperfusion in isolated rat heart, even if through different mechanisms.

Topics: Adenosine; Analysis of Variance; Animals; Antihypertensive Agents; Creatine Kinase; Male; Phenethylamines; Purinergic P1 Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Reperfusion Injury; Vasodilator Agents

This study tests the hypothesis that cardioprotection exerted by adenosine A2-receptor activation and neutrophil-related events involves stimulation of ATP-sensitive potassium (K(ATP)) channels on neutrophils during reperfusion. The adenosine A2 agonist CGS-21680 (CGS) inhibited superoxide radical generation from isolated rabbit polymorphonuclear neutrophils (PMNs) in a dose-dependent manner from 17.7 +/- 2.1 to 7.4 +/- 1.3 nmol/5 x 10(6) PMNs (P < 0.05). Pinacidil, a K(ATP)-channel opener, partially inhibited superoxide radical production, which was completely reversed by glibenclamide (Glib). Incremental doses of Glib in combination with CGS (1 microM) did not alter CGS-induced inhibition of superoxide radical generation. CGS significantly reduced PMN adherence to the endothelial surface of aortic segments in a dose-dependent manner from 189 +/- 8 to 50 +/- 6 PMNs/mm2 (P < 0.05), which was also not altered by incremental doses of Glib. Infusion of CGS (0.025 mg/kg) before reperfusion reduced infarct size from 29 +/- 2% in the Vehicle group to 15 +/- 1% in rabbits undergoing 30 min of ischemia and 120 min of reperfusion (P < 0.05). Glib (0.3 mg/kg) did not change the infarct size (28 +/- 2%) vs. the Vehicle group and did not attenuate infarct size reduction by CGS (16 +/- 1%). Glib did not change blood glucose levels. Cardiac myeloperoxidase activity was decreased in the ischemic tissue of the CGS group (0.15 +/- 0.03 U/100 mg tissue) compared with the Vehicle group (0.37 +/- 0.05 U/100 mg tissue; P < 0.05). We conclude that adenosine A2 activation before reperfusion partially reduces infarct size by inhibiting neutrophil activity and that this effect does not involve K(ATP)-channel stimulation.

Topics: Adenosine; Adenosine Triphosphate; Animals; Cardiovascular Agents; Cell Adhesion; Creatine Kinase; Female; Glyburide; Hemodynamics; Male; Myocardial Infarction; Myocardium; Neutrophils; Peroxidase; Phenethylamines; Potassium Channels; Rabbits; Reperfusion Injury; Superoxides

The adhesion of leukocytes to the endothelium of postcapillary venules hallmarks a key event in ischemia-reperfusion injury. Adenosine has been shown to protect from postischemic reperfusion injury, presumably through inhibition of postischemic leukocyte-endothelial interaction. This study was performed to investigate in vivo by which receptors the effect of adenosine on postischemic leukocyte-endothelium interaction is mediated. The hamster dorsal skinfold model and fluorescence microscopy were used for intravital investigation of red cell velocity, vessel diameter, and leukocyte-endothelium interaction in postcapillary venules of a thin striated skin muscle. Leukocytes were stained in vivo with acridine orange (0.5 mg kg-1 min-1 i.v.). Parameters were assessed prior to induction of 4 h ischemia to the muscle tissue and 0.5 h, 2 h, and 24 h after reperfusion. Adenosine, the adenosine A1-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the A2-selective agonist CGS 21,680, the non-selective adenosine receptor antagonist xanthine amine congener (XAC), and the adenosine uptake blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) were infused via jugular vein starting 15 min prior to release of ischemia until 0.5 h after reperfusion. Adenosine and CGS 21,680 significantly reduced postischemic leukocyte-endothelium interaction 0.5 h after reperfusion (p less than 0.01), while no inhibitory effect was observed with CCPA. Coadministration of XAC blocked the inhibitory effects of adenosine. Infusion of NBTI alone effectively decreased postischemic leukocyte-endothelium interaction. These findings indicate that adenosine reduces post-ischemic leukocyte-endothelium interaction via A2 receptor and suggest a protective role of endogenous adenosine during ischemia-reperfusion.

Topics: Adenosine; Animals; Cell Adhesion; Cricetinae; Endothelium, Vascular; Ischemia; Leukocytes; Mesocricetus; Muscles; Phenethylamines; Receptors, Purinergic; Reperfusion Injury; Thioinosine; Xanthines