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(Journal of Leukocyte Biology. 2003;73:74-81.)
© 2003 by Society for Leukocyte Biology

Fructose-1,6-biphosphate and nucleoside pool modifications prevent neutrophil accumulation in the reperfused intestine

Anna Sola*, Julián Panés{dagger}, Carme Xaus* and Georgina Hotter*

* Department of Medical Bioanalysis, Instituto de Investigaciones Biomédicas de Barcelona (IIBB-CSIC-IDIBAPS), Spain; and
{dagger} Gastroenterology Department, Hospital Clínic, Barcelona, Spain

Correspondence: Dr. Georgina Hotter, Department of Medical Bioanalysis, IIBB-CSIC-IDIBAPS, C/ Rosselló, 161, 7ª Planta, 08036 Barcelona, Spain. E-mail: ghcbam{at}iibb.csic.es


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ABSTRACT
 
Fructose-1,6-biphosphate (F16BP) attenuates ischemia/reperfusion (I/R) injury by inhibiting microvascular leukocyte adhesion or reducing neutrophil-derived oxygen free-radical production, but the causes of this action, the mechanisms in vivo, and the possible implication of nucleoside pool modifications are still controversial issues. We explored whether F16BP’s inhibition of free-radical production and neutrophil recruitment is a result of its effect on adenosine (Ado) accumulation during intestinal I/R injury. The effects of F16BP administration were tested on the nucleotide/nucleoside metabolism at the end of the ischemic period and on microvascular neutrophil recruitment and free-radical production after reperfusion in vivo, in the presence or absence of Ado deaminase (ADA). Infusion of F16BP markedly increased endogenous Ado, decreased xanthine accumulation during the ischemic period, and inhibited neutrophil recruitment and subsequent neutrophil free-radical generation during reperfusion. Administration of ADA reversed these processes. The results provide strong evidence that F16BP prevents neutrophil accumulation and neutrophil free-radical generation during intestinal I/R by a key mechanism that modifies the nucleoside pool, leading to an endogenous accumulation of Ado and to a reduction of xanthine during ischemia.

Key Words: F16BP • xanthine • adenosine • adenosine deaminase • small intestine


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INTRODUCTION
 
The mortality rate for intestinal disorders remains high, even when the diagnosis is made early in the course of the disease. The progressive intestinal ischemia leads to infarction, sepsis, and death in a high proportion of patients [1 2 3 ]. As a result, there is an urgent need to find ways of minimizing ischemic or hypoxic injury in this area.

Over the years, many attempts have been made in clinical and experimental settings to protect tissues during hypoxia and ischemia and to facilitate metabolic recovery after these episodes. Much attention has been focused on the protective role of fructose-1,6-biphosphate (F16BP), and its administration has been shown to be therapeutically effective in shock and in ischemia/reperfusion (I/R) injury in various organs [4 5 6 7 8 9 10 ]. Previous studies have proposed that F16BP may exert its protective role by acting as a glycolytic intermediate that provides adenosine (Ado) 5'-triphosphate (ATP) anaerobically [11 12 13 14 ], by inhibiting neutrophil-derived free-radical generation [15 ], by chelating extracellular calcium, or via a membrane-stabilizing action [16 ]. However, although the majority of studies of F16BP have focused on ATP preservation, no information is available regarding the effects of its administration on nucleoside pool alterations.

Inside the nucleotide pool, accumulation of Ado in ischemic tissues has a recognized, protective effect [17 18 19 ]. More precisely, it is well known that its accumulation protects tissues from hypoxic or ischemic damage through multiple receptor subtypes and that this nucleotide can regulate a number of specific, physiological roles [20 21 22 23 ].

The beneficial effects reported for F16BP are very similar to those of Ado accumulation. For example, Ado, like F16BP, has been shown to improve energy use during ischemia [20 , 24 ] and to reduce leukocyte adhesion during reperfusion [25 26 27 28 ]. So, it seems clear that any compound that can enhance Ado levels during the I/R process may directly modify a neutrophil-recruitment response to the benefit of the organism in question. However, exogenous Ado administration is not optimal for therapeutic use, as it produces hypotension, bradycardia, and atrioventricular block [29 ]. This is a handicap for its direct application in the clinical setting, and new scenarios have been tested to raise Ado concentrations by other mechanisms, including inhibition of Ado kinase activity or Ado transport [29 , 30 ].

Accumulation of neutrophils is one of the characteristic microvascular alterations of I/R injury. The generation of oxygen-free radicals (OFR) during I/R and the infiltration of tissue by activated neutrophils are known causative factors of tissue injury [31 ]. It is also known that the conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO) and the increase in concentrations of hypoxanthine (substrate of XO) during ischemia in the small intestine [32 33 34 ] are prerequisites for OFR production during reperfusion. In this context, some studies have demonstrated the protective role of exogenous F16BP through its modulation of leukocyte adhesion [8 , 35 ], but very little is currently known regarding the relationship between this modulation and modifications in the nucleoside pool.

Accordingly, we hypothesized that the modulation of Ado and xanthine/hypoxanthine levels by F16BP exogenously administered to the ischemic intestine is responsible for F16BP’s reported action on vascular-neutrophil recruitment and OFR production. Specifically, we investigated whether the exogenous supply of F16BP inhibits OFR production and microvascular-neutrophil recruitment (both assessed by intravital microscopy in vivo) through nucleoside pool modifications during the ischemic period.


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MATERIALS AND METHODS
 
The study was performed with male Sprague Dawley rats (Ifa Credo, Barcelona, Spain) weighing between 200 and 210 g. Animals were fasted for 24 h before surgery. All animals (including controls) were anesthetized with thiobarbital (100 mg/Kg intraperitoneally; Inactin, Research Biochemical International, Natick, MA) and were placed in a supine position on a heating pad to maintain body temperature between 36°C and 37°C. To induce intestinal ischemia, laparotomy was performed, and a silicone tube was placed around the superior mesenteric artery and exteriorized through the lateral abdominal wall. Traction was applied to induce ischemia.

All studies performed were in accordance with European Union regulations for experimental animals.

Experimental groups
Effect of F16BP on nucleotide metabolism at the end of the ischemic period
Group I: Control (C). Animals subjected to anesthesia and laparotomy for 2, 10, or 90 min.

Group II: Ischemia (I). Three subgroups of seven animals each underwent different periods of ischemia (2, 10, and 90 min).

Group III: I + F16BP. Three subgroups of seven animals each underwent different periods of ischemia (2, 10, and 90 min) during which F16BP (5 mM) was administered by superfusion over the small intestine throughout the process. This dose has been shown to be effective in previous studies [36 ].

Group IV: I + F16BP + Ado deaminase (ADA). The protocol described for group III was used but included superfusion over the mesentery with ADA (0.25 IU/ml) as described previously [37 ].

All animals received continuous superfusion over the small intestine of bicarbonate-buffered saline (BBS; pH 7.4) at 37°C and at 2.5 ml/min with or without pharmacological treatment. At the end of each period, tissue samples were obtained, immediately frozen, and maintained at -80°C to evaluate nucleotide and nucleoside content in tissue.

Effect of F16BP administration on microvascular-neutrophil recruitment after I/R
To study the effect of F16BP on leukocyte adherence and emigration across the postcapillary venular endothelium in vivo, as well as the generation of oxidants by these cells, we used intravital microscopy in the presence or absence of F16BP during the I/R process. To determine whether the beneficial effects of F16BP administration are mediated by alterations in Ado, we tested whether the addition of ADA (an enzyme that converts Ado to its inactive metabolite) reversed the protection.

Animals were distributed in the following groups (n=7):

Group V: Control (C). Animals subjected to anesthesia and laparotomy for 120 min.

Group VI: I/R. Animals subjected to 90 min of ischemia followed by 30 min of reperfusion.

Group VII: I/R + F16BP. As in group VI but with superfusion over the small intestine of 5 mM F16BP throughout the process.

Group VIII: I/R + F16BP + ADA. The protocol described for group VII was used but with superfusion over the mesentery with ADA (0.25 IU/ml).

All groups received continuous superfusion over the small intestine of BBS (pH 7.4) at 37°C and at 2.5 ml/min with or without pharmacological treatment.

After a stabilization period of 10 min, images from the mesenteric preparation were recorded for 5 min in baseline conditions in all the studied groups (baseline). Ischemia was then induced in the corresponding groups by pulling the silicone tubing. For the reperfusion, the tube was removed, allowing blood to recirculate. In all animals studied, a decrease in centerline red blood cell (RBC) velocity of 90% was induced during the period of ischemia. Images were then recorded for 5-min periods, at 5 and 30 min of reperfusion.

At the end of 30 min of the reperfusion period, tissue samples were obtained, immediately frozen, and maintained at -80°C to evaluate myeloperoxidase (MPO) activity.

Intravital microscopy
In the groups described above, a section of the mesentery was exteriorized and observed through a glass slide centered on a steel microscope board. The exposed intestine was covered with a BBS (pH 7.4)-soaked gauze, and the mesentery was continuously superfused with BBS at 37°C and at 2.5 ml/min. The board was placed on the stage of an inverted microscope (Diaphot 300, Nikon, Tokyo, Japan) equipped with a CF Fluor x40 objective lens (Nikon). The preparation was transilluminated with a 12-V, 100-W, DC-stabilized light source. A 3CCD camera (DXC-930P, Sony, Tokyo, Japan) mounted on the microscope projected the image onto a color monitor (Trinitron KX-14CP1, Sony), and the images were captured on videotape (SR-S368E, JVC, Tokyo, Japan) with superimposed time and date (KPM Systems, Barcelona, Spain). Rectal temperature was maintained between 36.5°C and 37.5°C through the use of an infrared heat lamp. Single unbranched venules with diameters ranging between 25 and 35 µm and lengths 100 µm were studied. Venular diameter was measured on-line using a video caliper (Microcirculation Research Institute, Texas A&M University, College Station). Centerline RBC velocity was measured using an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M University) to assess venular blood flow and shear rate according to standard equations [38 ]. The number of adherent and emigrated leukocytes was measured off-line during playback of videotaped images using criteria described previously [39 ].

"In vivo" assessment of free-radical generation
To quantify the generation of oxidants by the cells in the area under study, the oxidant-sensitive fluorochrome dihydrorhodamine (DHR)-123 (Molecular Probes, Eugene, OR) was superfused (10 µmol/L) onto the mesentery as described previously [38 ]. During an initial 10-min stabilization period, the mesenteric preparation was superfused with DHR-free BBS, and a background autofluorescence image was recorded. Fluorescence intensity (excitation wavelength, 500 nm; emission wavelength, 536 nm) was detected using a 3CCD camera (DXC-930P, Sony). The fluorescence intensity of the venule under study, the fluorescence intensity of the contiguous perivenular interstitium in the segment 10–50 µm from the venule wall, and the background fluorescence before superfusing DHR-123 were measured with a Power Macintosh computer (Apple, Cupertino, CA) equipped with a 24STV graphics display board with the public domain NIH Image digital image processor. An index of free-radical generation in the intravascular and interstitial areas was obtained after subtracting background fluorescence from fluorescence intensity in the area of interest.

Nucleotide and nucleoside determination
Intestinal samples, weighing 100–150 mg, were placed in 1.0 ml 3.6% perchloric acid solution and then immediately homogenized and processed by high-pressure liquid chromatography as described previously [40 ].

MPO activity
MPO was measured spectrophotometrically as described previously [41 ].

Protein concentration
Total protein concentration in homogenates was determined using a commercial kit from BioRad (Munich, Germany).

Statistics
Data were analyzed using ANOVA with Bonferroni correction as a post hoc test and Student’s paired or unpaired t-test when appropriate. All values are reported as mean ± SEM. Statistical significance was set at P< 0.05.


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RESULTS
 
Effect of F16BP on nucleotide metabolism and XO system at the end of the ischemic period
Figure 1 summarizes the tissue levels of nucleotides [ATP+Ado 5'-diphosphate (ADP)+Ado 5'-monophosphate (AMP)] and nucleosides (Ado and hypoxanthine+xanthine) after the various ischemic periods evaluated in this study. As shown in Figure 1A , no significant differences were observed with respect to baseline conditions or the control group when tissue samples were obtained at 2 min of ischemia. High-energy nucleotides began to decrease significantly at 10 min of ischemia; F16BP administration only reached significance at 90 min. The Ado pattern is shown in Figure 1B . In this case, the levels were similar for the three experimental groups, but at 90 min of sustained ischemia, accumulation was significantly higher in the F16BP group than in the nontreated animals. In the case of ADA administration, the enzyme showed the desired effect, as a decrease in Ado levels was detected after 10 min of the sustained ischemia. Figure 1C shows the accumulation of xanthine. As expected, xanthine increased during the ischemic period, reaching significance at 90 min of sustained ischemia. However, the increase in animals without F16BP was significantly higher than in the treated ones.



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  		Figure 1. Nucleotide (ATP+ADP+AMP; A) and nucleoside (Ado and xanthine) levels (B and C) in intestinal samples subjected to different periods of ischemia (2, 10, and 90 min) and F16BP-treated and untreated animals with or without ADA administration. *, P < 0.05 versus control; +, P < 0.05 versus ischemia (I).

Effect of F16BP administration on microvascular-neutrophil recruitment after I/R
Figure 2 depicts MPO tissue activity in control, small intestine as well as in I/R tissue with or without F16BP administration. The enzyme activity was increased in the I/R animals and significantly reduced with F16BP administration. ADA administration to F16BP-treated rats reversed this reduction.



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  		Figure 2. Intestinal tissue MPO activities (U/mg protein) in control, I/R animals, I/R + F16BP, and I/R + F16BP + ADA. *, P < 0.05 versus control; +, P < 0.05 versus I/R + F16BP.

Figures 3 4 and 5 present intravital microscopy findings. As shown in Figure 3A , the number of vascular rolling leukocytes increased with the reperfusion period in all ischemic animals, although in animals receiving F16BP (cross-hatched bars), the increase was significantly lower. ADA administration eliminated the effects of F16BP on the recruitment of rolling leukocytes. In addition, the microvascular leukocyte rolling velocity (Fig. 3B) increased significantly when F16BP was administered, reaching significance at 30 min after reperfusion. I/R did not show this increase in rolling velocity, and in ADA-administered animals, the effects of F16BP administration were again reversed.



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  		Figure 3. Alterations in number of rolling leukocytes (A) and rolling velocity (B) in baseline conditions and during reperfusion in control and F16BP-treated and untreated animals with or without ADA administration. *, P < 0.05 versus control; &, P < 0.05 versus I/R + F16BP.



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  		Figure 4. Changes in number of adherent (A) and emigrated (B) leukocytes observed under baseline conditions and at 5 and 30 min of reperfusion in control and F16BP-treated and untreated animals with or without ADA administration. *, P < 0.05 versus control; &, P < 0.05 versus I/R + F16BP.



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  		Figure 5. DHR-123 oxidation in rat mesenteric postcapillary venules (A) and in the interstitium (B) under baseline conditions and at 5 and 30 min of reperfusion in control and F16BP-treated and untreated animals with or without ADA administration. *, P < 0.05 versus control; &, P < 0.05 versus I/R + F16BP.

Figure 4 shows the number of adherent (A) and emigrated (B) leukocytes observed after different periods of reperfusion. A significant increase in both types was observed following the reperfusion periods. Treatment with F16BP completely eliminated leukocyte recruitment, whereas ADA administration reversed the effects of F16BP and showed a response that was similar to or higher than that recorded in I/R animals.

The adhesion and emigration of leukocytes through the vascular wall were accompanied by the generation of oxidants (Fig. 5) . Under baseline conditions, no difference was found between controls and treated or untreated animals. However, following reperfusion, the rhodamine fluorescence intensity increased significantly in all animals compared with controls. F16BP administration without ADA significantly reduced oxidant production, whereas in animals treated with ADA, oxidant production was similar to that observed in I/R-untreated animals. Taken together, these findings indicate that in the small intestine, F16BP administration reduces microvascular-neutrophil recruitment and subsequent neutrophil OFR generation through Ado-linked action.


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DISCUSSION
 
F16BP, a high-energy, glycolytic intermediate, is a physiological product that exhibits pharmacological properties. It has been shown to be therapeutically effective in shock and in I/R injury in various organs [5 6 7 8 ]. The mechanisms by which F16BP may exert its protective vascular effects include the inhibition of neutrophil free-radical production [15 , 35 ], the maintenance of the correct XD/XO ratio [42 ], the prevention of changes in intracellular calcium [43 ], and the increase in peristaltic activity [8 ]. In the clinical setting, addition of F16BP improves the efficacy of preservation solutions in renal transplantation [44 ]. However, the most important and most widely studied of its effects has been its regulation of glycolysis, particularly the enzyme phosphofructokinase. The protection provided by the exogenous administration of F16BP has been associated with the prevention of intracellular ATP depletion and increased glycolytic activity [10 11 12 13 14 ]. Indeed, F16BP prevents ATP decreases in ischemic muscle [10 ]. In intestinal I/R, administration of F16BP may enhance energy production from carbohydrates, thereby attenuating ischemic damage and accelerating regeneration of intestinal villi after reperfusion [8 ].

The relationship between energy metabolism and viability of organs subjected to I/R and/or transplantation has been reported in the kidney, heart, liver, and small intestine [45 46 47 48 ]. The early metabolic event during ischemic and anoxic phenomena is a rapid and pronounced depletion of ATP with a resulting increase in its degradation products, such as ADP, AMP, and Ado. These substances are further degraded to inosine, hypoxanthine, and xanthine, which accumulate in the ischemic tissue. Then, after reperfusion, the presence of xanthine leads to enhanced generation of OFR. Furthermore, it is known that OFR generation induces neutrophils to produce more OFR, a process that in turn attracts more neutrophils to the endothelium [31 ], increasing the inflammatory response and organ damage.

It has been proven that diverse organ systems benefit from pretreatment with F16BP during oxygen deprivation. It has been postulated that the primary action of F16BP is metabolic and that during oxygen deprivation, F16BP improves ATP supply versus demand [49 50 51 ]. Moreover, the studies by Espanol et al. [11 ] indicate that pretreatment with F16BP during severe hypoxia preserves ATP completely. Although the aforementioned studies suggested a link between high-energy nucleotide preservation and F16BP administration, little is known about the effect of F16BP administration on Ado/xanthine content in ischemic tissues.

As Figure 1 shows, our results confirm that the concentration of high-energy metabolites is reduced during ischemia. However, in F16BP-treated animals, high-energy metabolites were preserved for a significantly longer period, attaining levels very similar to those during the baseline period. It is also clear that F16BP treatment significantly reduces xanthine accumulation and increases Ado levels; in other words, the nucleoside pool is modified. These results indicate that nucleotide depletion in ischemic intestines is slower when F16BP is administered, and as a consequence, the accumulation of nucleotide degradation substances is delayed. Therefore, the availability of the substrates for the XO enzyme is also limited, leading probably to a reduction in the generation of substrate-dependent OFR. In this sense, our group has recently demonstrated that oxidative stress in the intestine could be modified by a large decrease in xanthine, which occurs following the degradation of adenine nucleotides during ischemia [40 ]. Consequently, these changes in response to F16BP could result in a reduction in OFR generation.

Like F16BP, the purine nucleoside Ado has previously been shown to reduce I/R-associated injury in a range of organs by a mechanism involving the improvement of energy use during ischemia and reduction in vascular leukocyte adhesion during reperfusion [25 26 27 28 ].

A number of attempts have been made to raise pharmacological Ado concentrations in ischemic tissues and have achieved functional improvement [18 , 19 , 21 , 23 ]. However, as a result of the data of the negative action of exogenous Ado administration [29 ], new approaches are required to obtain the endogenous elevation of Ado. So compounds that endogenously increase Ado concentration could be valuable pharmacological tools in a variety of clinical situations associated with ischemia. However, in spite of the proven, protective properties of endogenous Ado accumulation, the aim of this study was not to focus on new or described effects of Ado in this model by itself but rather to elucidate the mechanism by which F16BP has gained its reputation as a protective agent against ischemia and hypoxia injury. We hypothesized that the mechanism by which F16BP exerts its action could be related to Ado enhancement.

To explore this possibility further, we added ADA to the F16BP-treated groups to directly eliminate the effects associated with the presence of Ado. The dose of the ADA used in this study has been well-tested in the small intestine as a drug solution that degrades extracellular Ado to its inactive metabolite [52 , 53 ].

The process of polymorphonuclear leukocyte (PMN) recruitment at the onset of reperfusion of postichemic tissue is a multistep mechanism that includes initial rolling of PMNs, a progressive reduction in PMN rolling velocity, and ultimately, PMN adhesion and emigration [54 ]. Recruitment of PMNs in postischemic tissue contributes to the development of tissue injury induced by I/R through the production of reactive oxygen metabolites and/or the release of hydrolytic enzymes [31 ]. Moreover, it seems clear that leukocyte adhesion to the postcapillary venular endothelium is also a prerequisite for neutrophils to exert their injurious effects [55 ]. Therefore, depletion of neutrophils from the ischemic tissues decreases tissue injury [56 ], and so any mechanism that could prevent I/R-induced leukocyte adhesion in models in vivo may be able to decrease tissue injury.

Figure 2 shows an increase in tissue neutrophil content in I/R intestine (as assessed by tissue MPO levels). F16BP reduces neutrophil accumulation, a finding consistent with previous evidence of its anti-inflammatory properties in vivo [35 ]. The fact that the application of ADA to F16BP-treated animals reversed the protective effect of F16BP indicates that the effect of F16BP on PMN accumulation is mediated by Ado.

Further support for this notion is provided by the use of intravital microscopy. In the present study, this technique was used to examine the ability of F16BP to reduce neutrophil accumulation or neutrophil-endothelium interactions in vivo. We observed that F16BP administration to mesenteric venules of ischemic animals is able to reduce the rolling, adhesion, and emigration of leukocytes and increases leukocyte-rolling velocity during the reperfusion (Figs. 3 and 4) . This is consistent with the findings of Akimitsu et al. [35] who reported the inhibition of leukocyte adhesion and emigration in postischemic skeletal muscle when F16BP was administered. Like MPO, application of ADA also reversed the protective effect of F16BP (Figs. 3 and 4) . These results indicate that F16BP, through its effect on Ado, eliminates I/R-related microvascular leukostasis.

These figures highlight another important issue: Vascular perfusion (represented by RBC velocity; Fig. 3B ) is improved by F16BP at the end of reperfusion and reversed by ADA administration. This increase in the vascular perfusion is inversely related with the number of roller and adherent cells (Figs. 3A and 4A) . Therefore, we can assume that a decrease in the number of cells obstructing the blood flow leads to an improvement of the vascular perfusion mediated by the increase in Ado acting as an inhibitor of the adherence and extravasation or as a vasodilator helping to increase the venous outflow [57 ].

This study also shows a relationship between neutrophil recruitment and oxygen-radical production (see Fig. 5 ). To monitor the oxidative stress elicited by I/R, we used the oxidant-sensitive fluorescent probe DHR-123, which reacts specifically with oxidants [58 59 60 ]. This technique has been successfully used to monitor in vivo free-radical generation in the mesentery [38 , 61 , 62 ]. As shown in Figure 5A and 5B , animals undergoing I/R with F16BP administration presented significant reductions in DHR-123 oxidation. In the case of ADA administration, the effects of F16BP were reversed.

Taken together, these results demonstrate that F16BP decreases neutrophil recruitment and OFR generation. Our observations support previous studies with canine and human neutrophils, indicating that F16BP inhibits the generation of OFR by stimulated neutrophils. In these two cell types, F16BP elminated the respiratory burst and the generation of superoxide by inactivating the enzyme 6-phosphoglucanate dehydrogenase and thereby inhibiting the activity of the hexose-monophosphate shunt, which is a major source of OFR in neutrophils [63 ]. So, additional support for this notion is provided by our observations that attribute the inhibitory effect of F16BP on OFR generation to Ado accumulation.

In general, our results are consistent with a body of evidence showing that F16BP administration ameliorates tissue injury associated with ischemia. However, our study is the first to provide novel, in vivo data, directly relating the effect of F16BP on nucleosides to the reduction of vascular-neutrophil recruitment and oxidative stress.

The biochemical mechanism by which F16BP exerts its effect on the nucleoside pool could be linked with previous theories that suggested that the beneficial effect was a result of the accumulation of low concentrations of ATP catabolites if F16BP is taken as a starting point during glycolytic metabolization inside the cells. In our opinion, in this model, this reduction in ATP catabolites in the intestinal tissue leads to a lower level of degradation of the nucleoside/nucleotide content and therefore to a decrease in Ado degradation. If the rate of Ado degradation is reduced, then the substrates for XO, such as hypoxanthine and xanthine, are not available, leading to a reduction in OFR generation; at the same time, as explained above, the accumulated Ado may act to reduce vascular leukocyte adhesion by itself. This reduction in OFR generation could also contribute to the decrease in the neutrophil interactions in the endothelium.

In summary, we explain how the administration of F16BP to the ischemic intestine markedly inhibits reperfusion-associated leukostasis. In addition we report two new phenomena related to nucleoside pool modifications by which F16BP protects against intestinal I/R injury: the reduction of xanthine (the OFR-producing agent) and the accumulation of Ado, which as well as F16BP, reduces microvascular leukostasis recruitment. To our knowledge, this is the first study to explain the ability of F16BP supply to allow the endogenous accumulation of Ado, which has been the basis of many approaches in the clinical setting in recent years.


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ACKNOWLEDGEMENTS
 
This work was supported by EU grants QLK6-CT-2000-00064, SAF 2000/3090-CE0057, and FISS 01/1691.

Received June 13, 2002; revised September 30, 2002; accepted October 2, 2002.


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