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Inhibition of neutrophil-myocyte adhesion and adhesion-dependent myocyte injury by adenosine was evaluated using isolated TNF-alpha-activated canine cells. Adenosine inhibited adhesion of activated neutrophils to cardiac myocytes with an IC50 of 11 +/- 4 nM. Inhibition of neutrophil adhesion (92 +/-3% by 100 nM adenosine) led to inhibition of myocyte injury (by 90 +/- 6%, as assessed by dye exclusion). Inhibition of cell adhesion by adenosine was blocked by the A2 antagonist, 1,3-dimethyl-1-propylxanthine, but not by the A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine. Moreover, the A2 agonist, CGS21680 (2-[4-(2-carboxymethyl)phenethylamino]-5'-N-ethylcarboxamido adenosine), but not the A1 agonist, N6-cyclopentyladenosine, mimicked adenosine in preventing cell adhesion. These observations implicate the A2 receptor in the mechanism of inhibition of cell adhesion. pretreatment and washing of neutrophils, but not cardiac myocytes, with adenosine or CGS21680 led to inhibition of adhesion, suggesting that the neutrophil A2 receptor is the target of adenosine's action. In contrast, inhibition of cell adhesion by adenosine was poteniated by 8-cyclopentyl-1,3-dipropylxanthine (IC50 = 4 +/- 1 nM) and attenuated by N6-cyclopentyladenosine, suggesting that occupancy of A1 receptors can conversely increase cell adhesion. Neutrophil-myocyte adhesion was inhibited by acadesine (IC50 = 12 +/- 2 microM) also via an adenosine-dependent mechanism because it was blocked by 1,3-dimethyl-1-propylxanthine or adenosine deaminase, an enzyme that degrades any adenosine that is formed. Acadesine-induced inhibition if cell adhesion (83 +/- 4% by 100 microM) resulted in inhibition of myocyte injury (by 76 +/- 6%). Other adenosine-regulating agents, including the acadesine analogue, GP531 (5-amino-1 beta-D-(5-benzylamino-5-deoxyribofuranosyl) imidazole-4-carboxamide), and inhibitors of adenosine transport and intracellular metabolism also inhibited cell adhesion. These results indicate that exogenous or endogenous adenosine can inhibit neutrophil-myocyte adhesion and injury in cells activated with TNF-alpha by an A2-mediated mechanism. Although the predominant activity of adenosine is to attenuate cell adhesion, stimulation of A1 receptors has the opposite effect, i.e., to augment adhesive interactions.

Adenosine exhibits potent anti-inflammatory activities but its therapeutic use is limited by cardiovascular side effects. Inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (EC 2.7.1.20), were evaluated for their ability to enhance endogenous adenosine production. One novel adenosine kinase inhibitor, GP-1-515, was studied in two models of septic shock to assess its protective effects. GP-1-515 significantly decreased mortality in mice that received a lethal i.v. injection of endotoxin. The beneficial effect was accompanied by decreased neutrophil accumulation in the lungs and was reversed by an adenosine receptor antagonist, implying that the effects were mediated by endogenous adenosine. Plasma levels of TNF-alpha, but not IL-1 alpha or IL-6, were lower in the GP-1-515-treated animals. In a second model of sepsis, GP-1-515 increased survival in bacterial peritonitis in rats. The mechanism of action in both models was likely multifactorial, including adenosine-mediated inhibition of neutrophil adhesion, cytokine production, and oxygen radical generation. Adenosine kinase inhibitors have potent anti-inflammatory effects in vitro and in vivo and represent a novel therapeutic approach to the treatment of inflammatory diseases.

The effects of repeated episodes of demand-induced ischemia on regional myocardial wall thickening, endocardial electrogram, and regional myocardial blood flow are not well delineated. We studied the cumulative effects of six periods of pacing-induced ischemia in 35 chloralose-anesthetized dogs with circumflex coronary stenosis. Repetitive ischemia of the posterior left ventricular free wall was induced with six 5-min pacing periods separated by 15-min recovery periods. The three groups of dogs studied were 1) saline control, 2) the purine precursor 5-aminoimidazole 4-carboxamide riboside (AICA-r), and 3) nitroglycerin (NTG). During the initial pacing period (before treatment), thickening of the posterior wall declined in the saline group (43 +/- 5% of control), the AICA-r group (47 +/- 8% of control), and the NTG group (55 +/- 3% of control), associated with endocardial S-T segment elevation and a decrease in subendocardial blood flow. Wall thickening continued to decrease in each group with each successive pacing episode. However, during the sixth pacing period wall thickening was significantly (P less than 0.05) less in the saline group (2 +/- 5% of control) than in the AICA-r (31 +/- 7% of control) or NTG (61 +/- 7% of control) group. The progressive decline in wall thickening was accompanied by a further decrease in subendocardial blood flow and a rise in S-T segment in the saline group but not in the AICA-r or NTG group (P less than 0.05). These results demonstrate that sequential periods of ischemia and reperfusion cause a progressive decline in regional wall motion, coincident with a progressive decrease in subendocardial blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Computerized reaction time (RT) tasks are sensitive measures of subclinical HIV-related mental slowing. We previously reported that nondemented HIV-seropositive patients on antiretroviral therapy at the time of testing had faster choice RTs compared to matched untreated seropositive participants. In the present study, we evaluated the performance of 163 nondemented HIV-seropositive participants on a reaction time version of the Stroop task as a function of antiretroviral status. Persons on antiretroviral therapy at the time of testing had significantly faster reaction times than untreated individuals, although treated asymptomatic participants showed significantly less Stroop interference than treated symptomatic participants. These effects could not be attributed to differences in demographic variables, disease status, substance abuse, or psychological distress. These data indicate that central information processing is faster for patients treated with antiretroviral compounds compared to untreated patients, and suggest that reaction time tasks may have significant potential utility in clinical trials of neuroprotective compounds.

We studied the integrity of working memory operations in 38 HIV-seropositive and 20 seronegative drug users, using a modified version of the Tower of London task. This new task, the Tower of London-Working Memory version (TOL-WM), includes a delayed-response component in addition to the planning required for successful performance of the standard TOL. Symptomatic HIV-seropositive participants solved significantly fewer TOL-WM problems compared to matched seronegative controls. However, seropositive and seronegative subjects showed similar overall levels of planning efficiency, suggesting that the TOL-WM deficit may be associated primarily with failure to encode or maintain an adequate online memory representation. The results of this study confirm our previous report of a possible working memory deficit in HIV-1 infection and suggest that measures of working memory have particular utility in the evaluation of HIV-related cognitive deficits.

Acadesine (AICAr: 5-amino-4-imidazole carboxamide riboside) has been shown to afford sustained protection against injury during ischemia and reperfusion. The present studies used the heterotopically transplanted rat heart to assess the protective properties of two new acadesine analogs: GP-1-468 and GP-1-668.Hearts were excised, arrested with a 2-minute infusion of cardioplegic solution, and subjected to 4 hours of global ischemia (20 degrees C) with cardioplegic reinfusion for 2 minutes every 30 minutes. The hearts were then transplanted (1 hour of additional ischemia) into the abdomens of recipient rats and reperfused in situ for 30 minutes or 24 hours. The hearts were then excised, perfused aerobically for 20 minutes, and contractile function was assessed. GP-1-468 or GP-1-668 was administered to donor rats (20 mg/kg intravenously, 30 minutes before excision). They were also added to the cardioplegic solution (10 mumol/L for GP-1-468, 5 mumol/L for GP-1-343, the active metabolite of GP-1-668) and were also given to recipient rats (20 mg/kg intravenously, 30 minutes before transplantation, so that the drugs were present during reperfusion). Nine groups of hearts were studied. Three groups of studies were carried out (n = 24 transplants for each group). The first group of hearts was reperfused for 30 minutes, the second group was reperfused for 24 hours, and the third group was transplanted but not reperfused; instead, they were frozen at the end of 5 hours of ischemia and taken for metabolite analysis. Within each group were three subgroups (n = 8 per group) receiving GP-1-468, GP-1-668, or saline solution. In the 30-minute reperfusion group the recoveries of left ventricular developed pressure were 88 +/- 4, 87 +/- 7, and 50 +/- 9 mm Hg, respectively (p0.05 versus saline-treated controls); left ventricular volumes (recorded at 12 mm Hg) were 112 +/- 20, 132 +/- 28, and 41 +/- 9 microliters, respectively (p0.05 versus saline-treated controls), and coronary flows were 13.1 +/- 0.7, 13.4 +/- 1.0, and 9.9 +/- 0.5 ml/min, respectively (p0.05 versus saline-treated controls). In addition to improving functional recovery, the two analogs increased the tissue content of adenosine at the end of the ischemic period (5.4 +/- 0.6 and 7.3 +/- 0.5 mumol/gm dry weight, respectively, versus 2.7 +/- 0.4 mumol/gm dry weight in the saline-treated controls; p0.05); however, they did not influence adenosine triphosphate or its catabolites. In the 24-hour reperfusion group the corresponding values were 77 +/- 6 and 88 +/- 6 versus 35 +/- 4 mm Hg for left ventricular developed pressure (p0.05), 111 +/- 9 and 121 +/- 11 versus 41 +/- 8 microliters for left ventricular volume (p0.05), and 13.7 +/- 0.7 and 13.0 +/- 0.6 versus 11.7 +/- 0.7 ml/min for coronary flow (no significant difference). Thus both analogs afforded an early and comparable degree of protection of contractile function that was sustained even after 24 hours of reperfusion.Both GP-1-468 and GP-1-668 increase the rate and extent of early postischemic recovery, and this protection is sustained for at least 24 hours. These beneficial actions were associated with an increase of the tissue content of adenosine during ischemia, but they appeared to be independent of the status of the high-energy metabolism.

Inhibition of neutrophil-myocyte adhesion and adhesion-dependent myocyte injury by adenosine was evaluated using isolated TNF-alpha-activated canine cells. Adenosine inhibited adhesion of activated neutrophils to cardiac myocytes with an IC50 of 11 +/- 4 nM. Inhibition of neutrophil adhesion (92 +/-3% by 100 nM adenosine) led to inhibition of myocyte injury (by 90 +/- 6%, as assessed by dye exclusion). Inhibition of cell adhesion by adenosine was blocked by the A2 antagonist, 1,3-dimethyl-1-propylxanthine, but not by the A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine. Moreover, the A2 agonist, CGS21680 (2-[4-(2-carboxymethyl)phenethylamino]-5'-N-ethylcarboxamido adenosine), but not the A1 agonist, N6-cyclopentyladenosine, mimicked adenosine in preventing cell adhesion. These observations implicate the A2 receptor in the mechanism of inhibition of cell adhesion. pretreatment and washing of neutrophils, but not cardiac myocytes, with adenosine or CGS21680 led to inhibition of adhesion, suggesting that the neutrophil A2 receptor is the target of adenosine's action. In contrast, inhibition of cell adhesion by adenosine was poteniated by 8-cyclopentyl-1,3-dipropylxanthine (IC50 = 4 +/- 1 nM) and attenuated by N6-cyclopentyladenosine, suggesting that occupancy of A1 receptors can conversely increase cell adhesion. Neutrophil-myocyte adhesion was inhibited by acadesine (IC50 = 12 +/- 2 microM) also via an adenosine-dependent mechanism because it was blocked by 1,3-dimethyl-1-propylxanthine or adenosine deaminase, an enzyme that degrades any adenosine that is formed. Acadesine-induced inhibition if cell adhesion (83 +/- 4% by 100 microM) resulted in inhibition of myocyte injury (by 76 +/- 6%). Other adenosine-regulating agents, including the acadesine analogue, GP531 (5-amino-1 beta-D-(5-benzylamino-5-deoxyribofuranosyl) imidazole-4-carboxamide), and inhibitors of adenosine transport and intracellular metabolism also inhibited cell adhesion. These results indicate that exogenous or endogenous adenosine can inhibit neutrophil-myocyte adhesion and injury in cells activated with TNF-alpha by an A2-mediated mechanism. Although the predominant activity of adenosine is to attenuate cell adhesion, stimulation of A1 receptors has the opposite effect, i.e., to augment adhesive interactions.

Ischaemic preconditioning protects myocardium from infarction if the reperfusion interval between the brief and prolonged ischaemic intervals is less than 1 h. In anaesthetised rabbits acadesine (5-amino-4-imidazolecarboxamide riboside, AICAR), an adenosine enhancer which increases tissue adenosine during ischaemia, prolongs the window of protection to 2 h. The aim of this study was to try to determine the maximum extension of this window of protection, using chronically instrumented, unsedated rabbits.Rabbits were instrumented with a balloon occluder around a major branch of the left coronary artery for reversible coronary occlusion. Five to seven days after surgery all animals underwent a 30 min coronary occlusion. Animals were randomised to one of seven groups: (1) No additional treatment (control); (2) Ischaemic preconditioning with 5 min regional ischaemia followed by 10 min reperfusion before the 30 min coronary occlusion; (3) and (4) Ischaemic preconditioning followed by 2 or 4 h of reperfusion before the 30 min occlusion, respectively; (5) Treatment with acadesine (2.5 mg.kg-1.min-1 intravenously for 5 min and then 0.5 mg.kg-1.min-1 beginning 45 min before and continuing until 30 min after release of the 30 min occlusion) without ischaemic preconditioning; (6) and (7) Treatment with the higher dose of acadesine for 5 min beginning 35 min before the 5 min ischaemic period, and then the lower dose continuing until 30 min after release of the 30 min coronary occlusion in rabbits with 4 or 6 h reperfusion intervals, respectively.Rabbits with ischaemic preconditioning with 10 min reperfusion preceding the 30 min coronary occlusion (group 2) had only 5.6(SEM 1.1)% infarction of the ischaemic zone. Ischaemic preconditioning followed by 2 h reperfusion (group 3) offered continued protection [18.2(2.2)% infarction] as compared to control animals [37.7(2.6)% infarction]. However, protection waned if ischaemic preconditioning was followed by 4 h reperfusion (group 4) [36.7(3.0)% infarction]. Additionally, treatment with acadesine alone did not modify infarct size (group 7) [39.5(4.0)%], but acadesine largely restored the protection of ischaemic preconditioning despite a 4 h reperfusion interval (group 5) [20.4(3.0)% infarction, P0.01 v control]. However, when reperfusion was extended to 6 h (group 6) acadesine could no longer restore protection [36.2(0.9)% infarction].The protection afforded by a 5 min ischaemic preconditioning period lasts from 2 to 4 h in the awake, unsedated rabbit, and acadesine can extend the duration of this window of protection to at least 4 h but not to 6 h.

Adenosine and adenosine analogues exhibit anti-inflammatory effects in vitro and in vivo, but their usefulness is limited by profound cardiovascular side effects. Therefore, we synthesized inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (AK) (EC 2.7.1.20), to enhance endogenous adenosine concentrations at sites of inflammation. GP-1-515 (4-amino-1-(5-amino-5-deoxy-1-beta-D- ribofuranosyl)-3-bromo-pyrazolo[3,4-d]pyrimidine), a novel AK inhibitor, decreased adhesion of activated human neutrophils to cultured endothelial cell monolayers by increasing local adenosine levels. The mechanism of inhibition in this assay seemed to involve selectin blockade and was independent of the beta 2 integrins. GP-1-515 and 2-chloroadenosine (a nonmetabolizable adenosine analogue) had no effect on the surface expression or shedding of adhesion molecules. An agent that disrupts the cytoskeleton, cytochalasin B, mimicked the effect of adenosine on cell adhesion. Interactions between L-selectin and the neutrophil cytoskeleton might be altered by adenosine and could contribute to adenosine-mediated adhesion inhibition.

Adenosine exhibits potent anti-inflammatory activities but its therapeutic use is limited by cardiovascular side effects. Inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (EC 2.7.1.20), were evaluated for their ability to enhance endogenous adenosine production. One novel adenosine kinase inhibitor, GP-1-515, was studied in two models of septic shock to assess its protective effects. GP-1-515 significantly decreased mortality in mice that received a lethal i.v. injection of endotoxin. The beneficial effect was accompanied by decreased neutrophil accumulation in the lungs and was reversed by an adenosine receptor antagonist, implying that the effects were mediated by endogenous adenosine. Plasma levels of TNF-alpha, but not IL-1 alpha or IL-6, were lower in the GP-1-515-treated animals. In a second model of sepsis, GP-1-515 increased survival in bacterial peritonitis in rats. The mechanism of action in both models was likely multifactorial, including adenosine-mediated inhibition of neutrophil adhesion, cytokine production, and oxygen radical generation. Adenosine kinase inhibitors have potent anti-inflammatory effects in vitro and in vivo and represent a novel therapeutic approach to the treatment of inflammatory diseases.

Acadesine is a cardioprotective nucleoside that can attenuate postischemic contractile dysfunction in the isolated heart even if administered at reperfusion, thereby implying an effect on a reperfusion-induced component of injury. Consequently, the effects of acadesine on the maintenance of cardiac function were evaluated in isolated guinea pig hearts in which the perfusion buffer was subjected to electrolysis to produce a mixture of free radicals and oxidants that have been implicated in reperfusion injury. A reduction of left ventricular developed pressure to 38 +/- 3% at 10 min after electrolysis was prevented by acadesine in a concentration-dependent manner (EC50 1 microM). Hypochlorous acid (HOCl) is a principal oxidizing species implicated in electrolysis-induced myocardial damage, and it is a major oxidant produced by neutrophils. Isolated hearts, perfused with HOCl to induce damage, were also protected by acadesine (EC50 1-3 microM). In addition, acadesine protected alpha 1-antiproteinase (alpha 1-AP) against inactivation by 30 microM HOCl with an EC50 of approximately 10 microM, demonstrating that acadesine reacts rapidly enough with HOCl to protect important biological targets. Additionally, acadesine scavenged the hydroxyl radical with a second-order rate constant of 5.0 x 10(9) M-1 s-1. In contrast, acadesine had no effect on superoxide anions generated from either xanthine-xanthine oxidase or hydrogen peroxide-mediated peroxidation. The free base of acadesine 5-amino-4-imidazole carboxamide (AICA) also reduced HOCl and attenuated the electrolysis-induced cardiac injury. However, unlike the parent molecule acadesine, AICA was not protective in the isolated heart subjected to ischemia and reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND Although there are many factors that might contribute to tissue injury during ischemia and reperfusion, the loss of adenine nucleotides has long been considered to be of importance. This has led to the study of interventions designed to limit the loss of nucleotides or to enhance the rate of nucleotide resynthesis during reperfusion. Alternatively, the breakdown of adenosine triphosphate to adenosine might represent a protective response of the ischemic heart because adenosine is considered an anti-injury autocoid. Augmentation of endogenous adenosine levels might be beneficial. For these reasons, the protective properties of acadesine (AICAr: 5-amino-4-imidazole carboxamide riboside) were assessed in a rat model of myocardial ischemia and reperfusion. METHODS AND RESULTS The protective properties of acadesine were studied in the isolated, perfused rat heart subjected to global hypothermic (20 degrees C) ischemia and reperfusion. When acadesine was given as an in vivo pretreatment (100 mg/kg i.v. 15 minutes before study) followed by being administered as an additive (20 mumol/l) to the St. Thomas' Hospital cardioplegic solution (single dose) and then as an additive (20 mumol/l) to the initial reperfusion (15 minutes) solution, the recovery of aortic flow after 2.5 hours of ischemia was improved from its control value of 16.5 +/- 3.9 ml/min to 28.9 +/- 4.1 ml/min (n = 8 per group; p less than 0.05). Similar protection was seen with other indexes of cardiac function. Analysis of hearts obtained at the end of 2.5 hours of ischemia and 35 minutes of reperfusion revealed no significant differences in metabolite content between control and drug-treated hearts with the exception of inosine monophosphate, which was increased from its drug-free control value of 0.10 +/- 0.01 mumol/g dry wt to 0.86 +/- 0.06 mumol/g dry wt (p less than 0.05). In further studies (n = 8 per group), with multidose (every 30 minutes) cardioplegia and extended periods (6 hours) of hypothermic ischemia, acadesine consistently led to higher mean recoveries of function and lower levels of creatine kinase leakage. Again, the only significant metabolic effect was an increase in tissue inosine monophosphate content. In studies (n = 12 per group) to determine whether acadesine was acting before, during, or after ischemia, the drug was given 1) only as pretreatment (100 mg/kg i.v.), 2) only during single-dose cardioplegia (20 mumol/l), or 3) only during reperfusion (20 mumol/l). Significant protection was observed in the first two groups (recovery of aortic flow increased from 10.6 +/- 2.6 ml/min in the acadesine-free control to 22.6 +/- 2.8 and 23.6 +/- 3.1 ml/min, respectively; p less than 0.05). No significant protection was observed when acadesine was given only during reperfusion. In dose-response studies, acadesine (0, 5, 20, 50, 200, and 1,000 mumol/l; n = 12 per group) was given only as a cardioplegic additive; the postischemic recoveries of aortic flow were 15.4 +/- 2.8, 16.9 +/- 3.6, 29.5 +/- 3.8, 27.4 +/- 3.8, 26.7 +/- 4.2, and 27.1 +/- 2.7 ml/min, respectively. CONCLUSIONS Acadesine improves the ability of the heart to recover from ischemia and reperfusion when administered before ischemia or with cardioplegia. The mechanism underlying the protection remains to be resolved.

Interactions between platelets and erythrocytes can influence platelet aggregation and hemostasis. Adenosine inhibits platelet aggregation and the levels of adenosine can be modified by the presence of erythrocytes. Acadesine (AICA riboside; 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide) is a nucleoside which enters cells and is phosphorylated by adenosine kinase to ZMP, an intermediate in de novo purine biosynthesis. Some of the pharmacological properties of acadesine are attributed to it’s ability to increase local adenosine concentrations. Consequently we investigated whether acadesine inhibits platelet aggregation in whole blood using an impedance aggregometer. Acadesine induced a time- and concentration-dependent inhibition of aggregation elicited by ADP, collagen or arachadonic acid. The IC50 for acadesine against ADP-induced aggregation decreased from greater than 5mM after 5 min incubation to 0.47 ±0.05 mM (n=5) after 60 minutes at 37°C. Dipyridamole showed an IC50 of 0.20 ± 0.05 mM (n=6) independent of the incubation period. Inhibition of adenosine kinase by 20μM 5’-deoxy 5-iodotubercidin suppressed the antithrombotic activity of acadesine by 74 ± 7%. Similarly, addition of adenosine deaminase (3.6 U/ml) reversed the antiaggregatory effects by 78 ± 5%. A background of exogenous adenosine (10μM), insufficient to modify platelet behavior directly, decreased the concentration of acadesine required to inhibit aggregation 5-fold. Thus, acadesine inhibits human platelet aggregation in whole blood. The response is dependent upon the metabolism of acadesine and is mediated by adenosine.

Acadesine (AICA riboside; 5-amino-1-β-D-ribofuranosyl imidazole-4-carboxamide) has beneficial effects in reducing ischemic damage which are mediated, at least in part, by its ability to increase local adenosine concentration (Gruber et al. Circulation, 80:1400, 1989). Adenosine also protects the heart from the deleterious effects of post-ischemic reperfusion. Therefore in this study acadesine was administered solely at reperfusion to isolated guinea pig hearts perfused in a Langendorff mode and subjected to ischemia for 45 min followed by reperfusion for 30 min. Addition of acadesine (10–50μM) to the perfusion buffer at the onset of reperfusion improved the recovery of left ventricular developed pressure (LVDP) at 30 min from 68 ± 3% in controls (n=12) to e. g. 82 ± 3% at 20μM (P < 0.01, n=6) implying prevention of reperfusion injury. Acadesine also attenuated myocardial damage induced by electrolysis of the perfusion buffer (1mA for 1 min) to produce a mixture of free radicals and oxidants that have been implicated in reperfusion injury. The 38 ± 4% (n=8) decrease of LVDP after 10 min in untreated hearts subjected to electrolysis was completely prevented by 20μM acadesine. Hypochlorous acid (HOCI), implicated in the electrolysis-induced damage and a major oxidant formed by neutrophils, was reduced by acadesine to H2O and CI− in vitro suggesting that its beneficial effects are at least partly related to its antioxidant activity. Other antioxidants tested reduced HOCI and attenuated electrolysis-induced damage but had no beneficial effect in the ischemia/reperfusion model. This suggests that the antioxidant activity may not be sufficient to account for the protection from reperfusion injury and that acadesine has additional protective properties.

Polymorphonuclear leukocytes (PMNs) have been implicated in postischemic myocardial injury and associated derangements in contractile function. To examine the direct effects of PMNs on cardiac function, isolated right ventricular papillary muscles of the rabbit were exposed to increasing concentrations of purified rabbit PMNs in the presence of cimetidine. PMNs induced a significant concentration-dependent decrease in contractile function, where 5 x 10(5) PMNs/ml reduced contractile force to 75 +/- 2.1% of control (vs. 95 +/- 5% for time control; P less than 0.005). Similar decreases were also observed for peak positive and negative first derivatives of contractile force. The degree of PMN-induced contractile dysfunction correlated with the activity of the PMNs in an aggregation assay (r = 0.82, P less than 0.01). The loss of contractile function in response to PMNs was attenuated by catalase, which metabolizes H2O2, but not by superoxide dismutase, a scavenger of the superoxide anion. PMNs can convert H2O2 to either the hypochlorite anion or the hydroxyl radical, which are removed by methionine or mannitol, respectively. However, these scavengers did not ameliorate the PMN-induced loss of cardiac function. Exposure of papillary muscles to H2O2 resulted in a concentration-dependent decrease in contractile function where 100 microM reduced contractile force to 78 +/- 4%, an effect prevented by catalase. Thus PMNs reduce the contractile function of isolated papillary muscles probably by the release of H2O2.

Neutrophils accumulate in myocardium rendered ischemic and reperfused. Activated neutrophils release mediators such as metabolites of oxygen that can compromise myocellular integrity and provoke cardiac dysfunction. Although it is established that leukopenia reduces infarct size, the role of leukocytes and the source of free radicals in postischemic contractile dysfunction is unresolved. A carotid left anterior descending coronary-artery extracorporeal circuit without (n = 8) or with a Leukopak filter (n = 6) to deplete the leukocytes and platelets from blood entering the left anterior descending artery was established in the anesthetized, open-chest dog 30 minutes before ischemia. Subendocardial segmental function was monitored by sonomicrometry, and ischemia was produced by stopping flow for 15 minutes followed by 3 hours of reperfusion. Depleting leukocytes by 90 +/- 3.2% and platelets by 100% improved segmental function (from 30.5 +/- 7% to 74.1 +/- 12.7% for control versus leukocyte-depleted dogs, respectively) at 15 minutes of reperfusion. In the leukopenic group, however, there was a progressive decline in contractility to 32.5 +/- 13.8% by 3 hours of reperfusion that was associated with a return of leukocytes and, to a lesser extent, a return of platelets in the extracorporeal blood to 70.2 +/- 21.9% and 15.5 +/- 4.3% of systemic values, respectively. Removal of leukocytes and platelets from blood perfusing the coronary vascular bed only at reperfusion improved contractile function to 67.7 +/- 6.9% at 15 minutes and 54.7 +/- 12.1% at 3 hours (n = 6). Scanning electron microscopy revealed adherent leukocytes in the epicardial coronary arteries of control animals after 3 hours of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

In the early 1970s Maroko, Braunwald, and others demonstrated the im­ portance of myocardial oxygen supply and demand as a major determinant of the extent of myocardial injury 0-3). A variety of agents that reduced myocar­ dial oxygen demand, B-adrenoceptor antagonists and calcium channel blockers for example, were shown to be beneficial in animal models of myocardial ischemia. The time for clinical testing of agents that improve myocardial oxygenation was said to have come (4). A decade later, however, the limited clinical results have been far from exciting. This approach to reducing isch­ emia-induced myocardial injury suffers from three major problems. Implicit in the concept of salvaging ischemic myocardium by altering myocardial oxygen supply and demand is the assumption that myocardial tissue injury results solely from an inability of the coronary vasculature to deliver necessary oxygen and nutrients to maintain myocyte viability. However, we believe that other pathophysiologic processes contribute to irreversible myocar­ dial damage. It is also assumed that the restoration of coronary blood flow to the ischemic myocardium would, of itself, prevent the development of irreversible damage. However, reperfusion of jeopardized myocardium may lead to cellular pro­ cesses that extend the area of cell necrosis beyond that which could be attributed

Cytochrome P-450 is the terminal oxidase of the electron transport chain in the endoplasmic reticulum. Arachidonic acid (AA) can be oxidatively metabolized by cytochrome P-450 hemoproteins to an array of compounds identified as constituents of tissues and biological fluids. Their synthesis can be manipulated by pharmacologic probes and are altered in pathophysiologic conditions. These novel eicosanoids stimulate release of peptide and steroid hormones, inhibit platelet and leukocyte aggregation, influence Na+-K+-ATPase and alter vasomotor tone. They also participate in fluid and electrolyte homeostasis, stimulus-secretion coupling, and regulation of tissue blood flow. Since these novel AA metabolites may participate in receptor-mediated signal transduction, and have been implicated in the generation of second messengers and changes in intracellular calcium, these studies have important implications for understanding basic biological mechanisms.

1. Studies were made of the effects on responses to vasoconstrictor agents of prostaglandins released from Krebs perfused isolated kidneys of genetic hypertensive and normotensive rats. 2. Prostaglandin E-like activity, detected by bio-assay, was released from kidneys of both groups of rats during the vasoconstriction produced by noradrenaline, angiotensin or prostaglandin F2α. 3. In preparations obtained from hypertensive rats, responses to higher doses of noradrenaline or angiotensin were initially greater than those from normotensive rats and these were then reduced to a greater extent by infusion of indomethacin, which abolished release of prostaglandin E-like activity. Thereafter, in kidneys of either group, vasoconstriction to noradrenaline was potentiated by infusion of prostaglandin E2. 4. We conclude that, in rats, renal prostaglandins released in response to vasoconstrictor agents could augment the effect of such agents and in genetic hypertensive rats release of renal prostaglandins could contribute to the disease.

Cytochrome P-450 is the terminal oxidase of the electron transport chain the endoplasmic reticulum. Arachidonic acid (AA) can be oxidatively metabolized by cytochrome P-450 hemoproteins to an array of compounds identified as constituents of tissues and biological fluids. Their synthesis can be manipulated by pharmacologic probes and altered in pathophysiologic conditions. These novel eicosanoids stimulate release of peptide and steroid hormones, inhibit platelet and leukocyte aggregation, influence Na+-K+-atpase and alter vasomotor tone. They also participate in fluid and electrolyte homeostasis, stimulus-secretion coupling, and regulation of tissue blood flow. Since these novel AA metabolites may participate in receptor-mediated signal transduction, and have been implicated in the generation of second messengers and changes in intracellular calcium,these studies have important implications for understanding basic biological mechanisms.

Noradrenaline releases prostaglandins in the kidney, and in rats these augment rather than reduce vasoconstriction produced by the amine. Homogenates of kidneys of New Zealand rats inbred for hypertension exhibit lower prostaglandin inactivation by 15-hydroxydehydrogenase than controls. At the same time, augmentation of noradrenaline vasoconstriction by the released prostaglandin is exaggerated. This biochemical defect could be the inherited abnormality primarily responsible for the development of hypertension in these animals.

The invasion of leukocytes into and around a myocardial infarct was studied in chloralose-anesthetized dogs subjected to 1-hr occlusion of the left anterior descending coronary artery and reperfused for periods up to 5 hr. Polymorphonuclear leukocytes adhering to the endothelium of blood vessels within the ischemic area are evident at the end of the occlusion period. During reperfusion, the leukocytes migrate into the myocardium and large groups of cells can be observed "streaming" toward the irreversibly damaged area after 5 hr reperfusion. Infarcted tissue produces 10 times more 12-hydroxyeicosatetraenoic acid (a metabolite attributed to the invading leukocytes) from arachidonic acid than adjacent "normal" areas of the ventricle. BW755C (10 mg/kg-1 i.v.), which inhibits both the lipoxygenase and cyclooxygenase pathways of arachidonic metabolism, attenuates leukocyte infiltration into the infarcted myocardium, prevents 12-hydroxyeicosatetraenoic acid formation and significantly reduces infarct size (P less than .005). BW755C also significantly diminishes the incidence of cardiac arrhythmias during infarction. In animals where circulating white cells are reduced 60% by treatment with hydroxyurea (20 mg/kg-1 i.v./day for 5 days), there is also a smaller infarct (P less than .01). Indomethacin (5 mg/kg-1 i.v.) and dexamethasone (0.2 mg/kg-1 i.v.), which do not affect leukocyte migration into the ischemic myocardium, do not reduce infarct size. It is proposed that migrating leukocytes contribute to the tissue injury accompanying myocardial ischemia, possibly by the release of proinflammatory mediators such as lipoxygenase products, free radicals (oxygen metabolites) and hydrolytic enzymes. Drugs which reduce the migration and/or activation of leukocytes may be useful in reducing infarct size.

A postischemic contractile dysfunction termed myocardial stunning has been described in vivo and is attributed, in part, to the generation of oxygen-derived free radicals and the presence of neutrophils. An analogous contractile derangement occurs in the posthypoxic heart in vitro. This study determined the role of neutrophils in hypoxia/reoxygenation-induced cardiac dysfunction in the isolated buffer-perfused rabbit heart utilizing a recirculating system with or without neutrophils present. In control hearts perfused with a neutrophil-free buffer, reoxygenation after 20 min of hypoxia was associated with a slow recovery of contractility which returned to prehypoxic values by 30 to 45 min. Although perfusion with buffer-containing neutrophils did not affect the hypoxia-induced decrease in myocardial contractility, the recovery of contractile function during subsequent reoxygenation was significantly diminished (P less than .01 vs. control), remaining depressed by 30 to 35% at 45 min. The myocardial neutrophil content increased approximately 2-fold in response to hypoxia and reoxygenation, as assessed using 51Cr-labeled neutrophils. The deleterious effects of neutrophil perfusion on cardiac function could not be attributed to neutrophil-mediated plugging of coronary vessels or enhanced myocellular damage. These results support the concept that neutrophils contribute to the cardiac dysfunction described in this model.

Necrotizing enterocolitis (NEC) was produced in anesthetized rabbits by transmural injection of intestinal loops with an acidified solution of casein and calcium gluconate, mimicking the luminal milieu of afflicted neonates. Intravenous infusion of superoxide dismutase (SOD) 15 min after NEC induction prevented intestinal damage. In ex vivo perfused intestinal loops, we determined the sites of eicosanoid release and their contribution to the vascular effects of N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) in damaged and SOD-salvaged intestine. The vascular effluent was the primary site of stimulated eicosanoid release. The vascular responses to fMLP (vasoconstriction) and PAF (vasodilation) were not altered by SOD, although vascular resistance was higher in the SOD group. SOD treatment attenuated 1) transmural fluid shifts in ex vivo perfused intestinal preparations, an index of vascular permeability, 2) fMLP-induced prostaglandin E2, 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), and leukotriene B4 (LTB4) release, and 3) PAF-induced release of 6-keto-PGF1 alpha and LTB4. Stimulated thromboxane B2 release was not altered by SOD. Thus NEC can be established by a luminal insult that causes local generation of free radicals and exaggerated release of prostaglandins and leukotrienes.

Oxygen-derived free radicals, particularly superoxide anion, are considered important mediators of intestinal injury induced by ischemia/reperfusion based on the protective effects of superoxide dismutase and allopurinol. A role for free radicals was investigated in a model of necrotizing enterocolitis (NEC) which was initiated by a luminal, as opposed to a vascular, insult. Intestinal loops of weanling rabbits received either saline (control loops) or a solution of 10 mg/ml casein and 50 mg/ml calcium gluconate acidified to pH 4 with proprionic acid (treated loops). When the animals were sacrificed 3 hours later, severe damage was noted in the treated loops, which included blunting of villi and edema, with all animals surviving. At 16 hours only 5 of 8 rabbits survived, and 3 had hemorrhagic necrosis. Control loops were normal in each case. Intravenous infusion of superoxide dismutase (4 mg/kg/hr), commencing 15 minutes after NEC induction, totally prevented intestinal injury. On the other hand, pretreatment with allopurinol, an inhibitor of xanthine oxidase, for 2 days (30 and 60 mg/kg by mouth) was not protective against intestinal damage. A cellular infiltration in treated loops was not histologically evident in the majority of animals at 3 hours after treatment, a finding confirmed by the minimal accumulation of 111In-labeled leukocytes in damaged and intact intestinal tissue. These results suggest that superoxide generated locally from sources other than xanthine oxidase play a critical and early role in experimental NEC and that superoxide dismutase may prove to be an effective therapy in this devastating neonatal disease.

Recently, the metabolism of arachidonic acid to two unidentified products (Peak 1 and Peak 2) by a cytochrome P-450 dependent mixed function oxidase has been described in canine polymorphonuclear leukocytes (PMNs). This study assessed the biologic activity of one of these metabolites, Peak 2, on PMN function. Peak 2 was formed biologically following addition of exogenous arachidonic acid to canine PMNs pretreated with BW755c to inhibit lipoxygenase and cyclooxygenase enzymes, and purified by high performance liquid chromatography following separation by column chromatography. Peak 2 (20-200 ng/ml) inhibited calcium ionophore A23187-induced aggregation and the second phase of LTB4-induced aggregation. Additionally, Peak 2 inhibited A23187-induced PMN adhesion to columns of Sephadex G-25. BW755c (94 microM), which increased the formation of Peaks 1 and 2 by almost 300%, also inhibited A23187-induced PMN adhesion. In contrast, Peak 2 did not inhibit the release of superoxide anions or immunoreactive LTB4, after stimulation of the PMNs with A23187. Thus, Peak 2 may modulate some activities of canine PMNs. Because the biologic activity of Peak 2 is opposite to that of LTB4, which promotes PMN aggregation and adhesion, and because LTB4 may be metabolized by a cytochrome P-450-dependent mixed function oxidase to less active metabolites, this enzyme system may play a central role in the control of PMN function.

Within minutes of occlusion of a major coronary artery the polymorphonuclear leukocytes (PMNs) are activated whereby they adhere to the vascular endothelium and migrate through the endothelial layer. Interactions with the endothelium can promote increased vascular resistance, diminished collateral flow, capillary blockade, and predisposition to vasospasm, as well as enhanced vascular permeability. On subsequent reperfusion entrapped leukocytes contribute to the no-reflow phenomenon, while more leukocytes gain access to the previously ischemic region. The leukocytes infiltrate the myocardium where they exacerbate the process of tissue injury and the development of arrhythmias. The release of leukocyte-derived mediators including arachidonic acid (AA) metabolites and oxygen-derived free radicals probably underlies these activities of the leukocytes. PMNs contain active lipoxygenase enzymes capable of metabolizing AA to products that are not normally found in the myocardium, and can dominate the metabolic profile of that tissue, leading to changes in myocardial integrity and function. Inhibitors of the lipoxygenase enzymes suppress the accumulation of leukocytes into the ischemic myocardium and reduce infarct size. However, because the drugs prevent cell invasion it cannot be inferred that a lipoxygenase metabolite per se is deleterious to the ischemic heart, inasmuch as any leukocyte-dependent mechanism of injury will be attenuated whether it is mediated by eicosanoids or by any other leukocyte-derived product. Additional studies with specific inhibitors/antagonists are required to determine the biochemical mechanisms underlying the different aspects of leukocyte-mediated myocardial injury.