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Anesth Analg 2001;93:1472-1479
© 2001 International Anesthesia Research Society

CARDIOVASCULAR ANESTHESIA

Cariporide (HOE 642) Attenuates Leukocyte Activation in Ischemia and Reperfusion

Mathias Redlin, MD*, Joachim Werner, MD{dagger}, Helmut Habazettl, MD*{dagger}, Wanja Griethe{dagger}, Hermann Kuppe, MD{dagger}, and Axel R. Pries, MD*{dagger}

*Institute of Anesthesiology, Deutsches Herzzentrum Berlin; and {dagger}Department of Physiology, University Hospital Benjamin Franklin, Freie Universität Berlin, Berlin, Germany

Address correspondence and reprint requests to Axel R. Pries, MD, Freie Universität Berlin, Dept. of Physiology, Arnimallee 22, D-14195 Berlin, Germany. Address e-mail to pries{at}zedat.fu-berlin.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cariporide (HOE 642) ameliorates myocardial ischemia/reperfusion (I/R) injury, by the well established reduction of cytosolic [Ca2+] in cardiac myocytes through inhibition of Na+/H+ exchange. However, postischemic inflammation also contributes to I/R injury. We tested the hypothesis that cariporide also modulates the inflammatory response. The effect of cariporide on L-selectin expression by human leukocytes in vitro and leukocyte adhesion and emigration in the reperfused rat cremaster muscle in vivo were studied. The rat cremaster muscle was exteriorized for intravital videomicroscopy, induction of ischemia (90 min), and reperfusion (90 min). Eleven rats were pretreated with cariporide (9 mg/kg body weight IV) whereas 11 rats received saline. Leukocyte adhesion was quantified offline. Human venous blood was incubated with cariporide (3 µmol/L) or saline, stimulated with formyl- methionine-leucine-phenylalanine (10-10–10-6 mol/L), and granulocyte L-selectin expression was analyzed by flow cytometry. Cariporide reduced leukocyte rolling and adhesion by approximately 35% and 45%, respectively, after 30 min of reperfusion. Leukocyte extravasation was decreased by approximately 85% after 90 min. Cariporide increased L-selectin shedding at each formyl-methionine-leucine-phenylalanine concentration, reducing the 50% effective dose from 9.95 to 4.68 nmol/L. Thus, cariporide may ameliorate I/R injury not only by the known reduction of cytosolic [Ca2+] in cardiomyocytes, but also by attenuating leukocyte-dependent inflammatory responses. Promotion of L-selectin shedding from activated leukocytes may present a mechanism underlying this newly detected effect.

IMPLICATIONS: This study provides evidence that inhibition of Na+/H+ exchange by cariporide (HOE 642) attenuates the postischemic inflammatory response. Leukocyte adhesion and emigration, assessed by in vivo microscopy, were markedly reduced in rat cremaster muscle, possibly because of increased L-selectin shedding of activated leukocytes as demonstrated by flow cytometry.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ischemia/reperfusion (I/R) injury is a common clinical problem that occurs in various situations including coronary artery vasospasm, thrombolytic therapy, coronary angioplasty, and vascular or heart surgery (1). The major mechanisms that initiate I/R injury include intracellular Ca2+ overload, generation of reactive oxygen species, and the inflammatory response during reperfusion (2). Although many drugs under investigation for treatment or prevention of I/R injury are specifically designed to inhibit one of these mechanisms, the possible effects via other pathways of I/R injury often remain unclear.

Inhibition of Ca2+ overload may also attenuate the Ca2+-induced conversion of xanthine dehydrogenase to xanthine oxidase and the generation of superoxide anions. In addition, Ca2+ as well as reactive oxygen species can serve as intracellular second messengers, contributing to the generation and release of proinflammatory cytokines. Thus, drugs aimed at reducing the intracellular Ca2+ accumulation, such as inhibitors of the sodium/hydrogen exchanger (NHE), may also attenuate inflammatory responses during reperfusion.

The protective effects of a specific inhibitor of the NHE, cariporide (HOE 642, 4-isopropyl-3-methylsulfonyl-benzoyl-guanidine-methanesulfonate; Hoechst Marion Roussel, Frankfurt, Germany), were demonstrated in a few experimental studies and confirmed in clinical reports, e.g., for the reperfused myocardium in humans (1,3).

However, it is unknown whether these protective effects of cariporide are entirely attributable to the direct effects of reduced Ca2+ overload in parenchymal cells or also to an attenuated inflammatory response. The findings that NHE inhibitors prevented hydrogen peroxide-mediated myocardial dysfunction (4) and reduced infarct size as well as neutrophil-specific myeloperoxidase accumulation in reperfused myocardium (5) suggest such additional protective mechanisms. The interaction between leukocytes and endothelium constitutes a critical event in the development of the inflammatory reaction during reperfusion. Thus, several authors have been able to show that accumulation and emigration of leukocytes contribute to I/R injury (6,7), whereas the extent of I/R injury can be diminished by a reduction in leukocyte activation (8).

The present study was, therefore, designed to investigate potential effects of NHE inhibition by cariporide on the inflammatory response during ischemia and reperfusion by intravital video microscopic quantification of leukocyte/endothelium interaction in the rat cremaster muscle preparation. In addition, putative direct effects of cariporide on the activation of circulating leukocytes were evaluated by flow cytometry analysis of stimulated human leukocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Intravital Microscopy
Animal care and experimental design were in accordance with the rules of German animal protection laws, approved by local authorities, and conform with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Male Sprague-Dawley rats (n = 22; body weight [bw] approximately 300 g) were anesthetized by intraperitoneal application of urethane (0.15 g/100 g bw; Sigma-Aldrich, Deisenhofen, Germany) followed by IM ketamine (5 mg/100 g bw; Pharmacia & Upjohn GmbH, Erlangen, Germany). A constant level of anesthesia was maintained throughout the experiment by intraperitoneal injection of supplemental doses of urethane as needed. This anesthetic has no or very little depressant effect on respiratory function (9). The trachea was surgically cannulated to ensure patent airways in the spontaneously breathing animals. Polyethylene catheters were inserted into a jugular vein for infusion of drugs and saline solution (NaCl 0.9%, 2 mL/h) and into a carotid artery for online monitoring of arterial blood pressure (Statham P23 Db; Statham, Hato Rey, Puerto Rico) and pulse rate (Servomed monitor, Hellige, Germany). Body temperature was maintained at 37°C by using a thermistor-controlled electrical heating pad (Elmedex Elektronic HB, Björklinge, Sweden). The right cremaster muscle was prepared for intravital microscopy as described originally by Baez (10) in a modification by Hill et al. (11). The exteriorized cremaster muscle was continuously superfused with Tyrode solution (35°C; 131.9 mM NaCl, 4.7 mM KCl, 2.0 mM CaCl2, 1.2 mM MgCl2, 18.0 mM NaHCO3) equilibrated with 5% CO2 and 95% N2 (pH 7.35–7.4).

After an initial recording period (see below) the cremaster muscle was subjected to 90 min of complete ischemia. Interruption of complete blood flow was achieved by lowering a crossbar covered with a polyvinylchloride tube across the proximal portion of the cremaster muscle. A pressure just sufficient to stop arterial blood flow was used. In addition, a bulldog clip (Aesculap, Tuttlingen, Germany) was attached to the mesorchial ligament to compress deferential vessels. To avoid any contact with atmospheric oxygen, a gas mixture of 95% N2 and 5% CO2 was suffused into a hood made of Parafilm® (American National Can, Chicago, IL) covering the exteriorized muscle. Forty minutes before the initiation of ischemia, leukocytes were fluorescently labeled in vivo by an IV bolus (0.5 mL/kg bw) of rhodamine 6G (0.2 g/L saline; Sigma), followed by a continuous infusion (0.6 mL · h-1 · [kg bw]-1) for the duration of the experiment.

The animals were assigned randomly to the study group (n = 11) or the control group (n = 11). Animal preparation and infusions were performed by technical personnel. Investigators involved in observation and evaluation were blinded to the group assignment of animals.

Vessels were observed with a modified intravital microscope system (12) using a 25x salt water immersion objective (SW 25/0.60; Leitz, Wetzlar, Germany). The cremaster muscle microcirculation was viewed (S-VHS recorder AG 5700; Panasonic, Osaka, Japan) under different illuminations according to the measurement to be performed. Hemodynamic and leukocyte activation variables were recorded under baseline conditions before inducing ischemia and 5, 10, 20, 30, 45, 60, and 90 min after release of the clamps allowing muscle reperfusion: (a) diameter of arterioles and postcapillary venules, blood flow velocity (continuous light transillumination; video camera CV-50 CCD; Philips, Amsterdam, The Netherlands), (b) leukocyte rolling, adhesion, and extravasation (fluorescence microscopy, epillumination; camera SIM ICCD-05 S; SES GmbH, Neustadt a.d.W., Germany). All recorded observations were analyzed offline, except for arteriolar blood flow velocity, which was measured online with a computerized analysis system based on the dual-window method, as described in detail previously (13).

In animal studies, cariporide (Hoechst Marion Roussel) was administered repetitively because of its short half-life in rats, t1/2 = 40 min (14). The first dose (3 mg in 3 mL of NaCl 0.9%/kg bw) was injected IV 30 min before onset of ischemia, and 4 dosages of 1.5 mg/kg bw each were applied every 40 min during I/R, resulting in a total dosage of 9 mg/kg bw. Control animals received saline solution (NaCl 0.9%) at the same times and volumes. Drug administration was performed before ischemia to achieve maximal protection, because a recent study on myocardial low-flow ischemia suggests that cariporide may be most protective during ischemia as compared with reperfusion (15).

Vessel diameters and the numbers of rolling, adherent, and extravasated leukocytes as well as leukocyte rolling velocity were determined offline by using a digital image processing system (16,17). Rolling leukocytes were defined as fluorescently labeled cells moving along the venular wall with velocities below 150 µm/s. Individual rolling velocities were obtained from video recordings by determining the time leukocytes required to travel a distance of 100 µm. Adherent leukocytes were defined as remaining fixed to a given position of the vessel wall for at least 30 s. The counts of rolling and adherent leukocytes are given as number in the respective category per 100 µm venular length during 60 s of observation. Only cells in the focal plane adjusted to the vessel centerline were taken into account. Emigrated leukocytes were quantified by counting fluorescently labeled cells in the paravascular tissue area over 100-µm venular length and to a distance of 100 µm on both sides of the venule. The animals were killed at the end of the experiments.

Flow Cytometry
In flow cytometry, single cells pass a set of light sources and light detectors which allows for rapid measurements in many cells of variables representing cell size (forward scatter of light) and surface granularity (side scatter of light). By incubating cells with fluorescence-labeled antibodies specifically recognizing distinct surface structures such as adhesion molecules, the relative density of these molecules (i.e., expression) can be estimated by the amount of light emitted by the fluorescent dye after appropriate excitation.

The investigation conformed to the principles outlined in the Declaration of Helsinki. Whole blood samples (2 mL) were obtained from healthy volunteers (n = 5) by puncture of an antecubital vein. After adding heparin (20 IU) to each sample, 1 mL was incubated (37°C) with cariporide (3 µM) for 10 min, and 1 mL served as control. Leukocyte stimulation was induced by adding N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (Sigma) in concentrations from 0.1 to 1000 nM. Reactions were stopped after 5 min by cooling the aliquots to a temperature of 4°C. Samples were kept at this temperature during the following procedures. To analyze cell surface expression of L-selectin, aliquots were incubated for 1 h in the dark with the phycoerythrin-conjugated monoclonal antibody Coulter Clone TQ1-RD1 (Coulter International Corp., Miami, FL) against the CD62L antigen. Nonspecific phycoerythrin binding was measured by using an antimouse control antibody against CD14 antigen conjugated with the same label (rmC5-3; PharMingen, San Diego, CA). Probes were washed and centrifuged twice (Dulbecco’s phosphate-buffered saline [PBS]; 1200 rpm for 5 min). Erythrocytes were hemolyzed by fluorescence-activated cell sorter lysing solution (dilution 1:10, 10 min; Becton Dickinson, Heidelberg, Germany). To remove the lysate, probes were washed again with PBS and centrifuged twice (1200 rpm, 5 min). After resuspending in PBS, probes were subjected to flow cytometry (FACScanTM; Becton Dickinson, San José, CA). In each probe, 104 cells were counted. The subgroup gated by the forward and side scatter values characteristic for granulocytes was analyzed for L-selectin surface expression by using CELL QuestTM software (Becton Dickinson, 1997). Loss of fluorescence intensity was normalized (FLN) with respect to the difference between the positive control (log fluorescence intensity with specific antibody against CD62L antigen, "specAB") and the negative control (log fluorescence intensity with nonspecific antibody, "nonspecAB") according to the following equation:

equation


where specAB[fMLP] is the log fluorescence intensity with the specific antibody after stimulation with a given fMLP concentration. FLN indicates the extent of L-selectin shedding.

Results are given as mean values with standard deviation (SD) or standard error (SEM). Group values were compared by using one-way analysis of variance followed by post hoc correction for multiple comparisons (Bonferroni test). To describe data exhibiting asymmetric distributions (e.g., leukocyte rolling velocity or L-selectin shedding), the median with its standard error (SE) was used. The significance of differences was tested here by the median test and the Kruskal-Wallis nonparametric analysis of variance. SPSS software (SPSS, Chicago, IL) was used for all statistical analyses. Values of P < 0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intravital Microscopy
Mean arterial blood pressure and heart rate did not change significantly throughout the experiments (Table 1). The effects of cariporide treatment on microcirculatory hemodynamic variables were minimal. Upon reperfusion, arteriolar and venular vessel diameters in cariporide-treated animals slightly increased relative to their respective controls (arterioles approximately +4% and venules approximately +6%), whereas arteriolar blood flow velocity in the Cariporide group decreased by approximately 8% (all not significant, Table 1).


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  		Table 1. Macro- and Microhemodynamic Data of Rats Undergoing 90 Min of Cremaster Ischemia and Reperfusion
 
Leukocyte rolling, adhesion, and extravasation induced by I/R were significantly reduced upon cariporide treatment (Fig. 1). The attenuation of leukocyte rolling and adhesion was already apparent in the early reperfusion period, reaching -35% and -45%, respectively, after approximately 30 min, and became statistically significant at this time. In contrast, the reduction in the number of extravasated leukocytes by cariporide became apparent only approximately 30 min after reperfusion and was maintained until the end of the observation time when it reached a maximum of approximately -85%.



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  		Figure 1. The sequential steps of leukocyte recruitment into the tissue, i.e., slow, selectin-mediated rolling along vessel walls, firm, integrin-mediated adhesion to the vessel wall, and, eventually, transendothelial migration were simultaneously recorded and quantified in collecting venules of the rat cremaster preparation. Numbers of rolling, adherent, and extravasated leukocytes are shown in the control state and at different time points in the reperfusion phase. Mean values (±SEM) are given for the untreated group ({square}, n = 11) and for the cariporide-treated group (•, n = 11). The values of leukocyte rolling, adhesion, and extravasation in the control state are not significantly different between groups. Cariporide treatment led to a significant reduction of all three leukocyte activation variables for the integrated time interval from 30 to 90 min after onset of reperfusion.

 
Preischemic leukocyte rolling velocities (Fig. 2) were not different between control and cariporide-treated animals (26.3 and 29.0 µm/s, respectively). In the Cariporide group, leukocyte rolling velocity increased from approximately 20 µm/s at 5 min to approximately 50 µm/s at 90 min after onset of reperfusion. In contrast, leukocyte rolling velocity in untreated animals was approximately 30 µm/s at 5 min of reperfusion and decreased to approximately 20 µm/s until the end of the reperfusion phase.



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  		Figure 2. Leukocyte rolling velocities in postcapillary venules in the untreated ({square}) and cariporide-treated (•) group (n = 11) before onset of ischemia and at different time points during reperfusion (median ± SE). *P < 0.05 versus untreated group; #P < 0.05 versus respective control value.

 
L-Selectin Expression
The loss of L-selectin expression was analyzed in fMLP-activated human leukocytes by measuring fluorescence intensity using flow cytometry. A reduction of average fluorescence intensity relative to the specific control indicates a loss of the CD62L antigen by L-selectin shedding. As demonstrated in an example of fluorescence histograms in Figure 3, L-selectin shedding is increased after cariporide treatment. Concentration-response curves (Fig. 4) show a dose-dependent sigmoidal increase of L-selectin shedding induced by fMLP in both groups. Cariporide treatment led to a left shift of the concentration-response curve with a reduced 50% effective dose value (4.68 nM relative to 9.95 nM) and an increased asymptotic maximal value (0.84 nM relative to 0.75 nM). These differences are significant. The strongest differences between both groups were seen for fMLP concentrations in the range of the 50% effective dose values, specifically of 1, 3, and 10 nM (P < 0.05).



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  		Figure 3. Representative original recordings of fluorescence histograms obtained by flow cytometry of human leukocytes. Upper left, unstimulated leukocytes incubated with a fluorescence-labeled nonspecific antibody (ab) present with a distribution curve on the left of the histogram with relative fluorescent units between 1 and 10, indicating that very few antibodies bound to the cells. Upper right, in contrast, after incubation with a monoclonal antibody against the CD62L antigen, the distribution curve is shifted to higher relative fluorescence units between 200 and 2000, indicating that almost all cells carry the CD62L surface molecule, i.e., L-selectin. Lower left, after stimulation with formyl-methionine-leucine-phenylalanine (fMLP), the distribution curve is shifted to the left with two distinct peaks representing two populations of leukocytes, one with most CD62L lost (relative fluorescence approximately 10) and another population with only partial loss of CD62L (relative fluorescence approximately 200). Lower right, fMLP stimulation of leukocytes after incubation with the sodium/hydrogen exchanger blocker cariporide results in a further left shift of the distribution curve with a single peak (relative fluorescence approximately 7), indicating that now most leukocytes lost their CD62L.

 


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  		Figure 4. Normalized loss of fluorescence intensity (FLN) indicating the extent of L-selectin shedding as a function of formyl-methionine-leucine-phenylalanine (fMLP) concentration for human leukocytes with (•) and without ({square}) pretreatment with cariporide (3 µM). Given are median values (±SE; n = 5). Sigmoidal functions (Hill function, 3 parameter) were fitted to the data to obtain concentration-response curves (continuous and dashed lines, respectively). Cariporide pretreatment led to an increase in L-selectin shedding seen by a left shift of the concentration-response curves and an increased asymptotic value reached at high fMLP concentrations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results establish a previously unknown antiinflammatory effect of cariporide in the setting of I/R injury as assessed by reduced venular rolling, adhesion, and extravasation of leukocytes in the reperfused rat cremaster muscle. Increased L-selectin shedding, demonstrated in cariporide pretreated, fMLP-activated human leukocytes, is consistent with the increased rolling velocity of leukocytes in the reperfused cremaster muscle and may present the mechanism of this antiinflammatory action.

The pathophysiology of I/R injury in experimental animal models and humans has recently been reviewed (2,7,18). During ischemia, the lack of oxygen ultimately leads to breakdown of ATP to hypoxanthine and to cytosolic acidosis because of increased glycolytic activity. Normalization of cytosolic pH via exchange of protons for Na+ by the NHE and the simultaneously reduced activity of the Na+/K+ adenosine triphosphatase result in increased cytosolic Na+. Excess cytosolic Na+ then causes the Na+/Ca2+ exchanger that usually exports Ca2+ from the cell to change the direction of its activity, now exporting Na+ in exchange for Ca2+ (19,20). This Ca2+ influx adds to the increase in cytosolic Ca2+ induced by the breakdown of adenosine triphosphate-dependent Ca2+ uptake into the sarcoplasmic reticulum. In skeletal and cardiac myocytes, increased Ca2+ will cause direct injury by activating actin myosin interaction, which results in contracture and mechanical injury of cells (21). Ca2+ also activates an array of cytosolic enzymes, notably a yet unidentified protease that converts xanthine dehydrogenase to xanthine oxidase (22). On reperfusion, the hypoxanthine that accumulates during ischemia is rapidly metabolized by xanthine oxidase with the generation of superoxide anion as a toxic byproduct (23). In addition, superoxide anions are already produced during ischemia by electron transfer errors in the mitochondrial respiration chain. These oxygen radicals and their metabolites, namely hydrogen peroxide and the hydroxyl radical, injure cellular organelles as well as cell membranes. The above-mentioned mechanisms, namely increased cytosolic Ca2+ and reactive oxygen species, contribute to the inflammatory reaction during ischemia and reperfusion by increasing the release of cytokines, and activating endothelial cells as well as leukocytes in the reperfused tissue.

Based on these mechanisms, inhibition of the NHE should reduce the increase of cytosolic Ca2+ and the resultant myocyte injury. And indeed, data from several investigators obtained in a large variety of experimental settings and animal species suggest that the NHE inhibitor cariporide attenuates I/R injury (4,14,24,25).

However, the majority of studies investigating effects of cariporide treatment in I/R have focused on inhibition of the NHE in parenchymal cells and its consequences for cellular Ca2+ homeostasis. Although there is no doubt that NHE blockade during I/R conveys true benefit for cell protection, favorable effects may also result from attenuation of the I/R-induced inflammatory response. There is evidence that the NHE is involved in additional pathways associated with I/R injury, e.g., interference with leukotriene B4 production (26) or platelet activation (27). As major mediators of I/R-induced inflammation, leukocytes contain an extensive cytotoxic potential realized by synergistic effects of exocytosed granule constituents and generation of oxygen-derived free radicals (6). Thus, intravascular activation of leukocytes and their inflammatory recruitment into the tissue play a major role in initiation and development of I/R injury (28–31). In addition, experimental evidence consistently demonstrates a significantly reduced extent of I/R injury because of different means of leukocyte depletion, e.g., filtration, chemotherapy-induced bone marrow suppression, and specific antibodies, or modulation of platelet-neutrophil interaction (6,8,32,33).

Leukocyte recruitment into the tissue requires a few sequential steps (2), namely leukocyte margination toward the vessel wall, then capturing, i.e., first direct contact and interaction between leukocyte and endothelial cells, slow rolling of the leukocyte along the vessel wall, and, finally, firm adhesion and emigration. Leukocyte capturing and rolling are mediated predominantly by the selectin family of adhesion molecules, whereas ß2 integrins (CD11/CD18) are critical for firm adhesion and extravasation (34,35). L-selectin (CD62L) is constitutively represented on almost all circulating leukocytes; P-selectin (CD62P) is expressed on platelets, or quickly up-regulated in cytokine-activated endothelial cells, whereas E-selectin (CD62E) expression on endothelial cells requires de novo synthesis and is more relevant in chronic inflammatory processes. It is hypothesized that L-selectin promotes the initial capturing of leukocytes from the flowing blood, whereas the counteracting L-selectin ligand must be expressed simultaneously on the endothelium. The following synergistic action of L- and P- or E-selectin is required for optimally slow leukocyte rolling preceding permanent attachment and transendothelial migration, penetration of the basement membrane, and extravasation in the interstitial space (35,36). These mechanisms and the contributing adhesion molecules are well preserved among different species including rodents and humans so that the principles developed in animal studies were successfully transferred to humans or helped in understanding human genetic diseases, e.g., leukocyte adhesion deficiency type I or II [for a review see (37)]. Thus, animal models such as the rat cremaster preparation may be used to study the mechanisms of leukocyte endothelial cell interaction in a clinically relevant ischemia and reperfusion protocol.

Our in vivo data show a significant increase of rolling velocity in the Cariporide group up to 50 µm/s compared with only approximately 20 µm/s in untreated animals. Previous findings indicate a causal relationship between rolling velocity and L-selectin shedding. There is evidence that inhibition of L-selectin shedding from the surface of activated leukocytes not only decreases their rolling velocity, but also aids in promoting leukocyte capture from the free blood stream (31). Thus, increased L-selectin shedding as observed in cariporide-treated activated leukocytes in vitro, may mediate the increased leukocyte rolling velocity in the cariporide-treated group in vivo. However, because leukocyte rolling velocity is significantly increased in P-selectin-deficient mice under conditions in which rolling is exclusively L-selectin dependent (34,36,38), the possibility that attenuation of endothelial activation and subsequent P-selectin expression by cariporide contributed to the increased rolling velocity in our study cannot be totally excluded.

An increased rolling velocity decreases the transit time of leukocytes (31). Subsequently, leukocytes are not sufficiently exposed to inflammatory mediators from endothelial cells to induce permanent adhesion, which results in decreased leukocyte recruitment (39). Thus, the increased leukocyte rolling velocity in cariporide-treated animals may well be responsible for the decreased numbers of rolling, adherent, and extravasated leukocytes. The reason for the reduction of leukocyte rolling velocity at five minutes of reperfusion remains unclear. One might speculate that increased L-selectin shedding would occur only over time; however, we did not measure this in our animal model. The time course of leukocyte rolling velocity increasing between 5 and 60 minutes of reperfusion does, however, correspond to the decreasing number of adherent leukocytes in the same time interval, supporting a causal role of leukocyte velocity for leukocyte adhesion.

The cellular mechanism by which cariporide modulates L-selectin shedding in circulating leukocytes has not yet been explored. However, leukocyte reactivity is influenced by their intracellular pH, and intracellular acidification attenuates activation (26,28). Thus, systemic inhibition of the NHE may affect circulating leukocytes by inducing or preserving intracellular acidosis, thereby attenuating leukocyte/endothelium interaction.

Our findings not only describe a new mechanism underlying the effects of the NHE inhibitor cariporide, but may also suggest a new therapeutic concept in the prevention and treatment of I/R injury. The relevance of this concept needs to be confirmed in a clinical setting, e.g., simultaneous collection of arterial and coronary venous blood from myocardial infarct patients undergoing catheter revascularization would allow for determination of transcoronary leukocyte gradients, an indirect measure for leukocyte recruitment, and for flow cytometry measurement of L-selectin expression. If thus confirmed, the principle of NHE inhibition by cariporide could find application in the treatment of patients who manifest ischemic injury to the heart, brain, gut, and other tissues.


   Acknowledgments
 
The authors thank U. Hilse and S. May for technical assistance.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Accepted for publication August 15, 2001.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press