The protective role of adenosine in inducing nitric oxide synthesis in rat liver ischemia preconditioning is mediated by activation of adenosine A2 receptors

The Protective Role of Adenosine in Inducing Nitric OxideSynthesis in Rat Liver Ischemia Preconditioning Is Mediated

by Activation of Adenosine A2 Receptors

CARMEN PERALTA, GEORGINA HOTTER, DANIEL CLOSA, NEUS PRATS,1 CARME XAUS, EMILIO GELPı, AND JOAN ROSELLO-CATAFAU

This study aims to determine if the protective role ofadenosine in liver ischemic preconditioning is mediated bythe activation of adenosine receptors and to ascertain whichof these receptors is implicated in the process. Administra-tion of adenosine A1 and A2 receptor antagonists to precon-ditioned animals indicates that hepatic preconditioning ismediated by the activation of adenosine A2 receptors.Propentofylline (an inhibitor of adenosine transport intocells) in the preconditioned group, subjected to previousadministration of an adenosine A2 receptor antagonist,prevented the negative effect of the latter on the protectionoffered by preconditioning. An increase of NO productionwas detected just immediately after hepatic precondition-ing, and the administration of an adenosine A2 receptorantagonist to the preconditioning group prevented thisincrease, thus abolishing the protective effect of precondi-tioning. However, the administration of a NO donor to thepreconditioned group subjected to previous administrationof the adenosine A2 receptor antagonist was able to main-tain the preconditioning effects. In conclusion, these resultsindicate that, in preconditioning, the protective effect ofadenosine could be a result of an increase in extracellularadenosine. This in turn would induce the activation ofadenosine A2 receptors, which, by eliciting an increase inNO generation, would protect against the injury associatedwith hepatic ischemia-reperfusion. (HEPATOLOGY 1999;29:126-132.)

Ischemic preconditioning, first shown in the myocardiumhas become a phenomenon described in the intestine,1 brain,2

and liver.3 We have previously shown that ischemic precondi-tioning, induced by brief ischemia and reperfusion periods,

renders the liver more tolerant to subsequent sustainedischemia-reperfusion.4 We have also shown that the adminis-tration of adenosine mimics the effect of preconditioning inischemic livers, and that the metabolization of endogenousadenosine by adenosine deaminase abolished the protectiveeffect of preconditioning. We have further shown the benefi-cial effect of adenosine in inducing NO synthesis in ischemictissue,4 although the exact mechanism by which adenosineoffered protection was not determined in that study. Recentwork in heart preconditioning has shown that precondition-ing-induced protection may require the activation of adeno-sine receptors. Thus, an adenosine receptor agonist cansimulate the preconditioning whereas an adenosine receptorantagonist blocks its beneficial effect.5 Also, recent studies incerebral ischemia, have obtained evidence that the responseto adenosine persists or is enhanced by nucleoside transportinhibitors such as propentofylline. Inhibition of adenosineuptake would increase its concentration in the extracellularspace and hence potentiate its effects, particularly if adeno-sine binds to a specific receptor site on the external surface ofthe membrane.6

Adenosine receptors have been divided into three majorsubclasses: A1, A2, and A3,5,7 and it is known that adenosinemainly stimulates the A1 and A2 receptor subtypes.8 Despiteevidence in the heart that adenosine receptors could beinvolved in the preconditioning process, the specific contribu-tions of individual receptor subtypes are less well defined.Accordingly, the aim of this study was to determine if thebeneficial effect of adenosine in liver ischemic precondition-ing is mediated by the activation of adenosine receptors and ifso, to determine the possible receptors implicated in thisprocess.

MATERIALS AND METHODS

Surgical Procedure

The study was performed with male Wistar rats (six for eachgroup) weighing between 250 g and 300 g. All animals (includingcontrols) were anesthetized with urethane (10 mg/kg, ip) and placedin a supine position on a heating pad for maintenance of bodytemperature between 36°C and 37°C. To induce hepatic ischemia,laparotomy was performed, and the blood supply to the right lobe ofthe liver was interrupted by placement of a bulldog clamp at thelevel of the hepatic artery and portal vein. Reflow was initiated byremoval of the clamp.3 All studies performed comply with theEuropean Union regulations for experimental animals (EC-guideline 86/60/CEE).

Abbreviations: NO, nitrous oxide; AST, aspartate aminotransferase; ALT, alanineaminotransferase; I/R, ischemia reperfusion; PC, preconditioning; ADA, adenosinedeaminase; ip, intraperitoneally; iv, intravenously; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; DMPX, 3,7-dimethyl-1-propargyxanthine; Ado, adenosine.

From the Department of Medical Bioanalysis, Instituto de Investigaciones Biomedicasde Barcelona, CSIC-IDIBAPS, Barcelona, Spain; and the 1Department of AnimalPathology, Veterinary School, Universitat Autonoma de Barcelona, Bellaterra, Spain.

Received April 6, 1998; accepted August 7, 1998.This work was supported by the Fondo de Investigaciones de la Seguridad Social

(FIS) through the project 97/2076.Address reprint requests to: Dr. Carmen Peralta Uroz, Department of Medical

Bioanalysis, Instituto de Investigaciones Biomedicas de Barcelona, CSIC, C/JordiGirona, 18-26, 08034-Barcelona, Spain. E-mail: [email protected]; fax: 34-932045904.

Copyright r 1999 by the American Association for the Study of Liver Diseases.0270-9139/99/2901-0018$3.00/0

126

Experimental Design

Effect of Preconditioning on the Liver Injury Associated to IschemiaReperfusion. A control group (n 5 6) was subjected to anesthesia andlaparotomy. To evaluate the effectiveness of preconditioning versusthe length of the ischemic period, the following experimental groupswere studied: a group of animals (n 5 24 in 4 groups of 6) wassubjected to different ischemic periods (15, 30, 60, and 90 minutes).A second group (n 5 24) was subjected to the same ischemicperiods with previous preconditioning induced by 10 minutes ofischemia and 10 minutes of reperfusion. Blood samples werecollected at 90 minutes post-reperfusion and processed to determineplasma aminotransferases (aspartate transaminase, AST; alaninetransaminase, ALT), used herein as markers of hepatic injury.

To evaluate the effectiveness of preconditioning versus the lengthof the reperfusion period, the following experimental groups werestudied: A group of animals (n 5 18 in 3 groups of 6) was subjectedto 90 minutes of ischemia followed by different reperfusion periods(30, 60, and 90 minutes). A second group (n 5 18) was subjected tothe same reperfusion periods but with previous preconditioning.Blood samples were collected after reperfusion to determine plasmaaminotransferases (AST, ALT).

Role of Adenosine in Hepatic Preconditioning. To study the role ofadenosine, the following experimental groups (n 5 6 each) were setup:

Group 1) Control: Animals subjected to anesthesia and lapa-rotomy.

Group 2) Ischemia-reperfusion (I/R): Animals subjected to 90minutes of right-lobe hepatic ischemia, followed by 90 minutes ofreperfusion.

Group 3) Preconditioning (PC): Previous to the ischemic period(as in Group 2), animals were subjected to 10 minutes of ischemiaand 10 minutes of reperfusion.

Group 4) I/R 1 adenosine (Sigma Chemical (St Louis, MO))(I/R 1 Ado): Animals subjected to 90 minutes of ischemia (as inGroup 2) were treated with a continuous infusion of adenosinedissolved in bicarbonate-buffered saline (pH 7.4) (0.066 mL/min,iv) during 20 minutes previous to ischemia.4

Group 5) PC 1 adenosine deaminase (ADA) (Sigma Chemical):Animals subjected to 90 minutes of ischemia with previous precon-ditioning (as in Group 3) were treated with a continuous infusion ofADA dissolved in bicarbonate-buffered saline (pH 7.4; 0.066 mL/min, iv) 20 minutes before and throughout preconditioning.4 At theend of the protocol blood flow was measured.

Implication of Adenosine Receptors in Hepatic Preconditioning. Thestudy of the implication of adenosine receptors in the protectiveeffect of ischemic preconditioning was performed on the followingexperimental groups (n 5 6 each):

Group 6) PC 1 adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; RBI, Natick, MA) (PC 1 A1 antago-nist): Animals (n 5 18 in 3 subgroups of 6) subjected to 90 minutesischemia with previous preconditioning (as in group 3) were treatedwith DPCPX at doses 0.1, 1, and 10 mg/kg in dimethylsulfoxide, ip,5 minutes before preconditioning.

Group 7) PC 1 adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargyxanthine (DMPX; RBI, Natick) (PC 1 A2 antagonist):Animals (n 5 18 in 3 subgroups of 6) subjected to 90 minutes ofischemia with previous preconditioning (as in Group 3) were treatedwith DMPX at doses 0.1, 1, and 10 mg/kg, in distilled water, ip, 15minutes before preconditioning. Blood samples were collected afterreperfusion to determine plasma aminotransferases (AST, ALT).

Extracellular Adenosine and Adenosine A2 Receptors. To study whetherthe protective effect of adenosine could be a result of increased levelsof extracellular adenosine capable of eliciting the activation ofadenosine A2 receptors, the following additional groups werestudied:

Group 8) (n 5 24): Animals subjected to 90 minutes of ischemiawith previous preconditioning (as in Group 3), were divided into 4

subgroups in which 0.1, 0.5, 1, and 5 mg/kg of DMPX was ipadministered 15 minutes before the start of the protocol.

Group 9) (n 5 24): Same as Group 7, but animals were treatedwith an adenosine transport inhibitor, propentofylline (3-methyl-1-[5-oxohexyl]-7-propylxanthine; Sigma Chemical; 10 mg/kg, inphosphate-buffered saline, ip), administered 40 minutes before thestart of the protocol.

Control experiments were performed with the vehicles used forthe different drugs. The doses and pretreatment times for DMPX andDPCPX were as described by Malhotra8 and, for propentofylline asdescribed by Phillis.9

Blood and liver samples were obtained at 90 minutes of reperfu-sion. Blood samples were processed to determine plasma aminotrans-ferases (AST, ALT), and liver samples were taken for histopathology.

Adenosine A2 Receptors and NO Production. To study whether theactivation of adenosine A2 receptors by adenosine was capable ofinducing an increase in NO production to confer cytoprotection, thefollowing experimental groups were studied.

In a first series of animals, tissue levels of NO were determined. Acontrol group (n 5 6) was subjected to anesthesia and laparotomy. Asecond group of animals (n 5 12 in 2 groups of 6) was subjected to10 minutes of ischemia followed by 10 minutes of reperfusion(preconditioning period) with or without previous administration ofadenosine A2 receptor antagonist, DMPX (1 mg/kg in distilled water,ip) 15 minutes before the start of protocol. Tissue samples wereobtained immediately after the preconditioning period and pro-cessed to determine the tissue accumulation of nitrites and nitrates.To evaluate if the increase in NO production by previous activationof adenosine A2 receptors conferred cytoprotection, a second seriesof experiments was performed: Animals subjected to 90 minutes ofischemia with previous preconditioning (as in Group 3) were treatedwith DMPX (1 mg/kg in distilled water, ip) 15 minutes beforepreconditioning and a NO donor spermine NONOate (10 mg/kg inphosphate buffered saline, pH 7.4, iv) 5 minutes before precondition-ing. Blood samples were obtained at 90 minutes of reperfusion andprocessed to determine plasma aminotransferases (AST, ALT).

Enzymatic Determinations

The evaluation of hepatic injury was performed by enzymaticdeterminations of AST and ALT in blood plasma by using acommercial kit from Boehringer Mannheim (Munich, Germany).

Hepatic Blood Perfusion Measurement

Hepatic microcirculation was analyzed with use of a laser-Doppler blood flowmeter (model LD 5000, Transonic Systems Inc.,Ithaca, NY). A fiberoptic probe was positioned against the surface ofthe right lobe of the liver for monitoring hepatic capillary bloodflow. The laser-Doppler flowmeter provides continuous measure-ment of relative changes in microcirculatory blood flow in varioustissues.10

Determination of Nitrous Oxide

NO production in hepatic tissue was determined by tissueaccumulation of nitrite and nitrate, by using a modification of amethod described previously.11 Briefly, frozen tissue specimens werehomogenized in 2 mL of 100 mmol/L TRIS-HCl, pH 7.4 at 4°C, thena protein precipitation with 100 µL of 1N HCl in 500 µL homog-enate was performed. After centrifugation, the supernatant wasadjusted to pH 7.6 with 100 µL of 1N NaOH and 300 µL of 100mmol/L TRIS-HCl. The tissue nitrite and nitrate was measured withuse of a commercial kit from Cayman Chemical (Ann Arbor, MI).Nitrate was reduced to nitrite by incubation during 3 hours withnitrate reductase in the presence of nicotinamide adenine dinucleo-tide 3-phosphate (NADPH) and flavin-adenin-dinucleotide (FAD).Nitrite was determined with Griess reagent, reading the absorbanceat 540 nm.

HEPATOLOGY Vol. 29, No. 1, 1999 PERALTA ET AL. 127

Histopathologic Analyses

Liver samples were taken for histopathology. These were fixed in10% neutral buffered formalin, embedded in paraplast and 5-µmsectioned, and stained with hematoxylin and eosin, according tostandard procedures. Sections were evaluated by light microscopicexamination.

Statistics

Data are expressed as means 6 standard errors of the mean (SEM)and were statistically evaluated by ANOVA, followed by the Student-Newman-Kleus test. An associated probability of P , .05 wasconsidered to be significant.

RESULTS

Effect of Preconditioning on the Injury AssociatedWith Ischemia Reperfusion

As shown in Fig. 1, significant differences in AST (Fig. 1A)and ALT (Fig. 1B) levels were observed at 60 and 90 minutesof ischemia, with respect to those found in the control group.At 15 or 30 minutes of ischemia, significant differences wereobserved in ALT but not in AST levels. When ischemia waspreceded by 10 minutes of ischemia and reperfusion periods(preconditioning), the increases in AST observed in thegroup subjected to 90 minutes of ischemia were preventedand those in ALT were attenuated. At 60 minutes of ischemia,preconditioning attenuated only the increases in ALT levels.

In the case of 15 and 30 minutes of ischemia, no differences inliver enzyme levels were observed.

AST and ALT levels in the animals subjected to 90 minutesof ischemia followed by increasing reperfusion periods (30,60, and 90 minutes) with and/or without previous precondi-tioning are shown in Fig. 2. Significant increases in AST (Fig.2A) and ALT (Fig. 2B) levels were observed in the I/R groupsubjected to 60 and 90 minutes of reperfusion. At 30 minutesof reperfusion, significant differences were observed in ALT,but no differences were obtained in AST levels. However,when ischemia was preceded by preconditioning, theseincreases were significantly lower at 90 minutes of reperfu-sion.

Taking these results into account, animals subjected to 90minutes of ischemia followed by 90 minutes of reperfusionwere selected for a study on the effect of hepatic precondition-ing at the level of adenosine and NO roles.

Role of Adenosine in Hepatic Preconditioning

Figure 3 shows the hepatic blood perfusion measured ingroups I/R, PC, I/R 1 Ado, and PC 1 ADA. Blood flowin PC was higher than that observed in I/R. Administration ofadenosine to the I/R group increased the blood flow whereasthe metabolization of endogenous adenosine with ADA(PC 1 ADA), leads to blood flow values lower than thosefound in the PC group.

FIG. 1. AST (A) and ALT (B) levels (U/L) in the animals subjected todifferent ischemic periods (15, 30, 60, and 90 minutes) followed each one by90 minutes of reperfusion, with/without previous preconditioning. s 5 P ,.05 versus Control; * 5 P , .05 versus I/R.

FIG. 2. AST (A) and ALT (B) levels (U/L) in the animals subjected to 90minutes of ischemia followed by different reperfusion periods (30, 60, and 90minutes) with and/or without previous preconditioning. s 5 P , .05 versusControl; * 5 P , .05 versus I/R.

128 PERALTA ET AL. HEPATOLOGY January 1999

Implication of Adenosine Receptors in Hepatic Preconditioning

To study the implication of adenosine receptors in theprotective effect of ischemic preconditioning, adenosine A1

and A2 receptor antagonists were administered to precondi-tioned animals (Fig. 4). Administration of adenosine A1

receptor antagonist (squares) at all doses used (0.1, 1, and 10mg/kg) leads to results comparable with those found in thePC group. However, administration of adenosine A2 receptorantagonist (triangles) at a dose of 1 mg/kg leads to similarincreases in AST and ALT levels as observed for the I/R group.At a dose of 10 mg/kg, the hepatic injury is exacerbated evenrelative to that observed in the I/R.

Extracellular Adenosine and Adenosine A2 Receptors

Figure 5 shows the AST (Fig. 5A) and ALT (Fig. 5B) levelsin the PC group with previous administration of the adeno-sine A2 receptor antagonist at doses 0.1, 0.5, 1, and 5 mg/kgwithout (squares) or with (triangles) previous administrationof propentofylline. The administration of this receptor antago-nist (squares) at a dose 0.1 mg/kg did not modify thepreconditioning effect on liver enzyme levels. However, adoses of 0.5, 1, and 5 mg/kg, it abolished the beneficial effectof preconditioning, leading to similar results in terms ofhepatic injury as observed in the I/R group. On the otherhand, the previous propentophylline administration (tri-angles) to these A2 antagonized groups leads to results at alldoses that are similar as those obtained when only PC wascarried out. The histological study of the liver (Fig. 6) showsin both I/R (Fig. 6A) or (PC 1 A2 antagonist, 1 mg/kg) (Fig.6C) multiple and extensive areas of hepatocyte necrosisrandomly distributed through the parenchyma. On the con-trary, in the liver of animals subjected either to precondition-ing before I/R (Fig. 6B) or to administration of propentofyl-line previous to PC 1 A2 antagonist, 1 mg/kg (Fig. 6D)minimal lesions of incipient necrosis (cytoplasmatic eosino-philia and nuclear picnosis) in scattered hepatocytes wereobserved.

Adenosine A2 Receptors and NO Production

A significant increase in NO production (reflected in thevalues of tissue nitrites and nitrates) (Fig. 7) was foundimmediately after the hepatic preconditioning period withrespect to that observed in the control group. This increasewas prevented by the administration of adenosine A2 receptorantagonist to the preconditioning group (PC 1 A2 antagonist,1 mg/kg). In addition, the administration of adenosine A2

receptor antagonist to the PC group (Fig. 8) abolished theprotective effect of preconditioning, showing AST and ALTlevels comparable with those in the I/R group. However, theadministration of NO donors to the PC 1 A2 antagonistgroup (1 mg/kg) (Fig. 8) prevented the injurious effect of theA2 receptor antagonist on hepatic preconditioning, leading tothe same results in AST and ALT levels as those observed inthe PC group.

DISCUSSION

Ischemic preconditioning, induced by brief periods ofischemia and reperfusion, triggers an endogenous protectivemechanism towards subsequent ischemia-reperfusion injury.This protection depends on the length of the sustainedischemia and the number of cycles of ischemia and reperfu-sion. Some studies of the heart have reported that the longertime of ischemia, the lesser the protection from precondition-ing. In this sense, preconditioning was shown to exert aprotective effect in front of 60 minutes of ischemia but not

FIG. 4. AST (A) and ALT (B) levels (U/L) in the PC group with previousadministration of adenosine A1 receptor antagonist (squares) and adenosineA2 receptor antagonist (triangles) at doses 0.1, 1, and 10 mg/kg. * 5 P , .05versus I/R; 1 5 P , .05 versus PC.

FIG. 3. Blood flow in the following experimental groups: I/R, PC, I/R 1Ado, PC 1 ADA. Values are expressed as percentages of the initial blood flow.Differences between these groups are not significant.


when the sustained ischemia was prolonged to 90 min-utes.12,13 Also, it has been reported that multiple brief cyclesof ischemia and reperfusion are necessary in the heart toobtain a protective effect from preconditioning.14

In the liver, using AST and ALT as biochemical markers ofhepatic injury, we have found evidence of a protective effectof preconditioning when sustained ischemia was extended to90 minutes. In fact, a single preconditioning episode, inducedby 10 minutes of ischemia followed by 10 minutes ofreperfusion is enough to induce the protection (see Fig. 1).These results are in concordance with a preliminary study byLloris et al.,3 which evidenced a protective effect of hepaticpreconditioning towards survival when the animals weresubjected to prolonged ischemic periods after a single precon-ditioning episode.

We have also evaluated the effectiveness of precondition-ing in front of shorter reperfusion times (30-60 minutes).However, significant differences in hepatic injury parameters(AST, ALT) were obtained only at 90 minutes of reperfusion(see Fig. 2A and B).

Several theories have been suggested to explain the protec-tive effect of preconditioning; changes in energy metabo-lism,15 antioxidant enzymes,16 or vasoactive mediators includ-ing adenosine and/or NO17 could be implicated in thisphenomenon.

Adenosine is considered to be one of the most likelycandidates for mediating preconditioning.18 Along this line, a

recent study from our group4 has shown the implication ofadenosine in the protective effect of preconditioning againsthepatic ischemia-reperfusion-induced injury. However, thetarget cells for adenosine were not investigated in this study.In the present study, we have ascertained that adenosinecould improve blood flow in ischemic tissues. In this sense,the administration of adenosine to the I/R group (Ado 1 I/R)increased blood flow, whereas the metabolization of endog-enous adenosine with ADA (PC 1 ADA) decreased it (Fig. 3)in the I/R and PC groups, respectively. These results are inline with some reports in heart,19 which have shown a role ofadenosine in the regulation of blood flow.

It is known that NO may play a protective role in I/R.20 Inprevious studies21 we have observed that NO exerted theprotective effect in hepatic preconditioning through an inhibi-tory action on endothelin synthesis. On the other hand, theeffect of adenosine in inducing NO release by endothelialcells is well documented.22 Thus, along these lines, we alsoreported that the protective effect of adenosine in hepaticpreconditioning was mediated by the induction of NOsynthesis.4 However, the exact mechanism by which adeno-sine could induce the NO production was not determined inthat study.

There is evidence that adenosine receptors might beinvolved in ischemic preconditioning in the heart.5 However,it has also been reported that adenosine’s mechanism ofischemic resistance is not mediated by adenosine receptorsand that the ischemic resistance may be related to adenosine,muscarinic and a-adrenergic receptor signal balance.23

Four adenosine receptors have been cloned and designatedA1, A2a, A2b, and A3.5 Of these, the adenosine A1 and A2

receptors have been most extensively implicated in theprotective effects of adenosine and ischemic precondition-ing.2,24 However, recent studies in myocardium precondition-ing have shown that the adenosine A3 receptor25 or the dualactivation of these receptors5 could be involved in thepreconditioning process.

In the liver, our work reveals that an A1 receptor antagonist(Fig. 4, squares) at a dose 1 mg/kg, is not able to modify theprotective effect of preconditioning, whereas an adenosine A2

receptor antagonist (triangles) at the same dose, leads toresults comparable with those found in the I/R group.Increasing the doses of A1 and A2 receptor antagonists to 10mg/kg resulted in exacerbated hepatic injury for the A2

receptor antagonist, however, at the same dose, A1 receptorantagonist did not modify the protective effect of precondition-ing. Consequently, these results indicate that is the binding ofadenosine to the adenosine A2 receptors that mediates theprotective effect of hepatic preconditioning.

In line with the biochemical findings, the histologicalstudy of the liver shows that the administration of adenosineA2 receptor antagonist in the preconditioning group (PC 1 A2

antagonist, 1 mg/kg) (Fig. 6C) abolished the protection offeredby preconditioning (Fig. 6B), showing multiple and extensiveareas of hepatocyte necrosis and a mild sinusoidal neutrophilinfiltration. These lesions were randomly distributed throughthe hepatic parenchyma.

An increase in NO production has been observed immedi-ately after hepatic preconditioning (Fig. 7). This was pre-vented by administration of adenosine A2 receptor antagonist(Fig. 7), which abolished the protective effect of precondition-ing (Fig. 8). Thus, the administration of a NO donor topreconditioned animals with previous administration of aden-

FIG. 5. AST (A) and ALT (B) levels (U/L) in the PC group with previousadministration of adenosine A2 receptor antagonist (DMPX) at doses 0.1, 0.5,1, and 5 mg/kg without (squares) or with (triangles) previous to administra-tion of propentofylline. * 5 P , .05 versus I/R; 1 5 P , .05 versus PC.


osine A2 receptor antagonist, simulated the protective effectof preconditioning (Fig. 8).

During ischemia, cellular consumption of high energyadenosine phosphate nucleotides leads to accumulation ofintracellular adenosine.18 We investigated if this fact increasesthe level of extracellular adenosine, which could elicit theprotective effect in preconditioning through activation ofadenosine A2 receptors. Modulation of the concentration ofextracellular adenosine could be regulated by various pro-cesses. One of them involves the release of adenosine by thecells, and the other the removal of adenosine from theextracellular space by transport into the cell. Perturbation ofthese processes will alter the steady state extracellular concen-tration of adenosine.26 In this sense, recent studies havereported that adenosine transport inhibitors, such as propen-tofylline, exert a protective effect in rat cerebral ischemia byinhibiting the transport of adenosine into cells, resulting inincreased extracellular concentrations of endogenous adeno-sine and enhanced stimulation of adenosine receptors.6,27

As shown in Fig. 5, the administration of an adenosine A2

receptor antagonist (DMPX) at a dose 0.1 mg/kg to precondi-tioned rats did not modify the protective effect of precondi-tioning. At a dose of 0.5 mg/kg, it partially abolished thepreconditioning effect. However, when the dose was in-creased to 1 or 5 mg/kg, both abolished the protective effect

FIG. 6. Histological lesions. (A) I/R. Extense area of hepatic necrosis. (B) PC. No apparent hepatic lesions. (C) PC 1 adenosine A2 receptor antagonist, 1mg/kg (PC 1 A2 antagonist, 1 mg/kg). Area of hepatic necrosis and mild neutrophil infiltration. (D) Propentofylline 1 (PC 1 A2 antagonist, 1 mg/kg). Noapparent hepatic lesions. Hematoxylin and eosin (3 215).

FIG. 7. NO production, measured by NO32/NO2

2 (nmol/mg prot)immediately after preconditioning period, in the following experimentalgroups: Control, PC, P 1 adenosine A2 receptor antagonist (PC 1 A2

antagonist). s 5 P , .05 versus Control; 1 5 P , .05 versus PC.


of preconditioning, leading to results comparable with thosefound in the I/R group. This would indicate that at least 1mg/kg of antagonist is required to totally abolish the precon-ditioning. By contrast, when propentofylline was adminis-tered to the preconditioning group, previously subjectedadministration of different doses (0.1, 0.5, 1, and 5 mg/kg) ofthe adenosine A2 receptor antagonist, then preconditioningoffered protection at doses of antagonist, which had beenfound to abolish the beneficial effect of preconditioning. Inline with our biochemical findings, the histological study ofthe liver shows that administration of propentofylline (Fig.6D) to the preconditioning group with previous administra-tion of the adenosine A2 receptor antagonist at doses 1 mg/kg(Fig. 6C), showed minimal lesions of incipient necrosis inscattered hepatocytes throughout the hepatic parenchyma.The alterations observed in these hepatocytes were mostlycytoplasmatic eosinophilia and nuclear picnosis.

Propentofylline inhibits the transport of adenosine intocells and increases extracellular adenosine concentration.This increases the amount of adenosine available for bindingto an external receptor, and consequently, was able to preventthe effect of adenosine A2 receptor antagonist on the protec-tion offered by preconditioning.

These results suggest that in ischemic preconditioning anelevated adenosine extracellular concentration induces theactivation of adenosine A2 receptors, which, in turn, by induc-tion of NO synthesis, confers cytoprotection to ischemic tissue.

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FIG. 8. AST (A) and ALT (B) levels (U/L) in the following experimentalgroups. Control; I/R; PC; PC 1 adenosine A2 receptor antagonist; at dose 1mg/kg (PC 1 A2 antag); PC 1 adenosine A2 receptor antagonist 1 NO donors(PC 1 A2 antagonist 1 NO). * 5 P , .05 versus I/R; 1 5 P , .05 versus PC.


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The protective role of adenosine in inducing nitric oxide synthesis in rat liver ischemia preconditioning is mediated by activation of adenosine A2 receptors