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Am J Physiol Heart Circ Physiol 292: H326-H332, 2007. First published August 25, 2006; doi:10.1152/ajpheart.00744.2006 Free Article
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5'-AMP-activated protein kinase activation prevents postischemic leukocyte-endothelial cell adhesive interactions

F. Spencer Gaskin, Kazuhiro Kamada, Mozow Yusof, and Ronald J. Korthuis

Department of Medical Pharmacology and Physiology, and the Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri

Submitted 11 July 2006 ; accepted in final form 24 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preconditioning (PC) with nitric oxide (NO) donors or agents that increase endothelial NO synthase (eNOS) activity 24 h before ischemia-reperfusion (I/R) prevents postischemic leukocyte rolling (LR) and stationary leukocyte adhesion (LA). Since 5'-AMP-activated protein kinase (AMPK) phosphorylates eNOS at Ser1177, resulting in activation, we postulated that AMPK activation may trigger the development of a preconditioned anti-inflammatory phenotype similar to that induced by NO donors. Wild-type (WT) C57BL/6J and eNOS–/– mice were treated with the AMPK agonist 5-aminoimidazole-4-carboxamide 1-beta-D-furanoside (AICAR) 30 min (early AICAR PC) or 24 h (late AICAR PC) before I/R; LR and LA were quantified in single postcapillary venules in the jejunum using intravital microscopy. I/R induced comparable marked increases in LR and LA in WT and eNOS–/– mice relative to sham-operated (no ischemia) animals. Late AICAR PC prevented postischemic LR and LA, whereas early AICAR PC prevented LA in WT mice. Late AICAR PC was ineffective in preventing I/R-induced LR but not LA in the eNOS–/– mice, and the same pattern was seen in WT animals treated with the NOS inhibitor N{omega}-nitro-L-arginine. Early AICAR PC remained effective in preventing LA in eNOS–/– mice. Our results indicate that both early and late PC with an AMPK agonist produces an anti-inflammatory phenotype in postcapillary venules. Since the protection afforded by late AICAR PC on postischemic LR was prevented by NOS inhibition in WT mice and absent in eNOS-deficient mice, it appears that eNOS triggers this protective effect. In stark contrast, antecedent AMPK activation prevented I/R-induced LA by an eNOS-independent mechanism.

ischemia; reperfusion; leukocyte rolling and adhesion; endothelial nitric oxide synthase-deficient mice

SINCE ISCHEMIC PRECONDITIONING (IPC) was first described by Murry et al. (30) in 1986, this intrinsic protective mechanism has been studied extensively. This seminal work demonstrated that subjecting the myocardium to brief periods of vascular occlusion followed by reperfusion just before induction of a prolonged ischemic insult (index ischemia) significantly reduced myocardial infarct size. Subsequent work demonstrated that increasing the reperfusion time interval between the preconditioning (PC) stimuli and the onset of index ischemia resulted in a progressive decline and eventual loss of the infarct-sparing effects of IPC. However, a second less powerful phase of protection emerged 24 h after PC, an observation that gave rise to the concept that IPC induced biphasic (early vs. late phase) preconditioned responses (2, 40, 42). Because of the impracticality of inducing IPC in the clinical arena, an intensive research effort has been directed at identifying other interventions that trigger the development of innate PC mechanisms. As a result of this work, several pharmacological agents are now known to induce a protective phenotype similar to that seen with IPC, such as PC with ethanol, adenosine receptor agonists, bradykinin, nitric oxide (NO) donors, and exogenous calcitonin gene-related peptide (4, 9, 26, 41, 43). Indeed, our own work (13, 14, 23, 24, 36, 37, 47) has established that these agents induced the development of a protective anti-inflammatory phenotype in postcapillary venules, such that these vessels fail to support adhesion molecule expression, leukocyte rolling and adhesion, and increased vascular permeability when the small bowel is subsequently exposed to prolonged ischemia-reperfusion (I/R).

Most of the agents that induce the development of preconditioned states do so by a triggering mechanism that involves the formation of NO by endothelial NO synthase (eNOS). AMP-activated protein kinase (AMPK) is a ubiquitously expressed heterotrimeric serine/threonine kinase that regulates a diverse array of enzymes and substrates. Among its many enzymatic targets for phosphorylation, AMPK-mediated eNOS activation plays a prominent role in regulating downstream activities of a variety of therapeutic agents (12, 29, 33). Interestingly, the glucose-lowering agent metformin has been shown to activate AMPK and prevent the signaling of inflammatory cytokines through nuclear factor-{kappa}B and tumor necrosis factor-{alpha} (18). This study also demonstrated that adhesion molecule expression was reduced by metformin or 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR) administration, a protective effect that was abrogated by the addition of an small interfering RNA directed toward AMPK{alpha}1. These activities have previously been attributed to eNOS by others (3, 46).

Taken together with the fact that AMPK has been shown to stimulate eNOS, the aforementioned observations led us to hypothesize that the direct activation of AMPK should result in the development of an anti-inflammatory phenotype similar to that seen in tissues preconditioned with ethanol or brief periods of ischemia and reperfusion. Moreover, we postulated that such beneficial effects would occur by an eNOS-dependent mechanism. To test these hypotheses, we examined the effects of an AMPK activator AICAR, as an early- and late-phase PC stimulus, observing postischemic leukocyte-endothelial cell adhesive interactions in single postcapillary venules of the small intestine of wild-type (WT) C57BL/6J mice by intravital microscopy. In addition, we evaluated the role of NO in AMPK PC via NO synthase (NOS) inhibition with N{omega}-nitro-L-arginine methyl ester (L-NAME) before AICAR administration in WT animals and by evaluating the effectiveness of AICAR PC in mice that were genetically deficient in eNOS (eNOS–/–).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

WT male C57BL/6J and eNOS–/– mice (6–7 wk of age) were obtained from the Jackson Laboratories (Bar Harbor, ME). All mice were maintained on standard mouse chow and water ad libitum with 12-h:12-h light-dark cycle and used at 8–10 wk of age. The experimental procedures have been described previously (24) and were performed according to the criteria outlined in the National Institutes of Health guidelines and were approved by the University of Missouri-Columbia Institutional Animal Care and Use Committee.

AICAR PC, Surgical Procedures, and Induction of I/R

Mice were preconditioned with the AMPK agonist AICAR (100mg/kg, 0.5 ml ip; Sigma, St. Louis, MO) either 30 min or 24 h before induction of I/R in C57BL/6J and eNOS–/– mice. Early- and late-phase preconditioned mice were subsequently anesthetized initially with a mixture of ketamine (150 mg/kg body wt im) and xylazine (7.5 mg/kg body wt im). After a surgical plane of anesthesia was attained, a midline abdominal incision was performed, and the superior mesenteric artery was occluded with a microvascular clip for 0 (sham) or 45 min. After these procedures, the right carotid artery was cannulated and the systemic arterial pressure was measured with a pressure transducer connected to the carotid artery catheter. Systemic blood pressure was recorded continuously with a personal computer (Power Macintosh 8600; Apple) equipped with an analog-to-digital converter (MP 100; Biopac Systems). The left jugular vein was also cannulated for administration of carboxyfluorescein diacetate, succinimidyl ester (CFDASE, Molecular Probes, Eugene, OR), a fluorescent dye that labels leukocytes. CFDASE was dissolved in DMSO at a concentration of 5 mg/ml, divided into 25-µl aliquots, and stored in light-tight containers at –20°C until use. During the preparation and storage of CFDASE, care was taken to minimize light exposure. After the 45-min ischemic period, the clip was gently removed, and leukocytes were labeled with CFDASE by intravenous administration of the fluorochrome solution (250 µg/ml saline) at a rate of 20 µl/min for 5 min. The sham-operated group had an equivalent 45-min period without occlusion of the superior mesenteric artery before CFDASE administration. Leukocyte/endothelial cell adhesive interactions were observed over minutes 30–40 and 60–70 of reperfusion via intravital fluorescence microscopy.

Intravital Fluorescence Microscopy

The mice were positioned on a 20 x 30-cm Plexiglas board in a manner that allowed a selected section of small intestine to be exteriorized and placed carefully and gently over a glass slide covering a 4 x 3-cm hole centered in the Plexiglas. The exposed small intestine was superfused with warmed (37°C) bicarbonate-buffered saline (pH 7.4) at 1.5 ml/min using a peristaltic pump (Model M312; Gilson). The exteriorized region of the small bowel was covered with bicarbonate-buffered saline-soaked gauze to minimize tissue dehydration, temperature changes, and the influence of respiratory movements. The superfusate was maintained at 37 ± 0.5°C by pumping the solution through a heat exchanger warmed by a constant temperature circulator (model 1130; VWR). Body temperature of the mouse was maintained between 36.5° and 37.5°C by use of a thermostatically controlled heat lump. The Plexiglas board was mounted on the stage of an inverted microscope (Diaphot TMD-EF; Nikon), and the intestinal microcirculation was observed through a x20 objective lens. Fluorescence images of the microcirculation (excitation wavelength, 420–490 nm; and emission wavelength, 520 nm) were detected with a charge-coupled device camera (XC-77; Hamamatsu Photonics), a charge-coupled device camera control unit (C2400; Hamamatsu Photonics), and an intensifier head (M4314; Hamamatsu Photonics) attached to the camera. Microfluorographs were projected on a television monitor (PVM-1953MD; Sony) and recorded on DVD using a DVD video recorder (DMR-E50; Panasonic) for off-line quantification of measured variables during playback of the recorded images. A video time-date generator (WJ810; Panasonic) displayed the stopwatch function onto the monitor.

The intravital microscopic measurements described below were obtained over minutes 30–40 and 60–70 of reperfusion or at equivalent time points in the sham-operated control groups, as described in Experimental Protocol. The intestinal segment was scanned from the oral to aboral section, and 10 single, unbranched venules (20–50 µm in diameter, and 100 µm in length) were observed for at least 30 s. Leukocyte-endothelial cell interactions (the numbers of rolling and firmly adherent leukocytes) were quantified in each of the 10 venules, followed by calculation of the mean value, which was used in the statistical analysis of the data. Circulating leukocytes were considered to be firmly adherent if they did not move or detach from the venular wall for at least 30 s. Rolling cells are defined as cells crossing an imaginary line in the microvessel at a velocity that is significantly lower than centerline velocity; their numbers were expressed as rolling cells per minute. The numbers of rolling or adherent leukocytes were normalized by expressing each as the number of cells per squared millimeter vessel area.

Experimental Protocol

Figure 1 illustrates the general design of the experimental protocols for the study. The number of animals used in each group is described below, with the protocols for groups 1–4 being conducted in both WT C57BL/6J and eNOS–/– mice. Drug doses were selected based on previous experiments in our laboratory and reports in the literature (7, 31, 37).


Figure 1
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  		Fig. 1. Schematic illustration of the experimental protocols assigned to each group. The numbers at the top of the diagram refer to minutes in the time line for the protocol on days 1 and 2 (24 h between both 0s). Gray bars: when the 10-min video recordings were obtained in the protocol. Solid bars: the 45-min period of ischemia. Triangles: when administration of saline vehicle or drugs was accomplished in the protocol time line. I/R, ischemia-reperfusion; AICAR, 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside; L-NAME, N{omega}-nitro-L-arginine methyl ester; eNOS–/–, endothelial nitric oxide (NO) synthase-deficient mice. See the text for further details.

 
Group 1: sham-operated mice. As a time control for the effects of experimental duration, WT C57BL/6J (n = 7) or eNOS–/– (n = 6) mice in this group received an intraperitoneal injection of 0.5 ml saline, which was used as a vehicle for AICAR and L-NAME in groups 3–5. Twenty-four hours later, the superior mesenteric artery was exposed but not subjected to occlusion, with leukocyte/endothelial cell adhesive interactions quantified at time points comparable with those described for mice subjected to 45 min of intestinal ischemia followed by 70 min of reperfusion (see Group 2: I/R alone).

Group 2: I/R alone. C57BL/6J (n = 7) and eNOS–/– (n = 6) mice in this group were treated as described for group 1, except that I/R was induced 24 h after the intraperitoneal injection of saline vehicle on day 1. Leukocyte rolling and adhesion were quantified during minutes 30–40 and 60–70 of reperfusion, following 45 min of ischemia on day 2.

Group 3: late AICAR PC + I/R. To determine whether AMPK activation with AICAR could initiate late-phase PC and prevent postischemic leukocyte rolling and adhesion on exposure to I/R 24 h later, WT (n = 6) or eNOS–/– (n = 6) mice were treated with AICAR (100 mg/kg, 0.5 ml ip; Sigma) on day 1. Twenty-four hours later (day 2), the intestine was exposed to I/R, and leukocyte rolling and adhesion were quantified as for Group 2.

Group 4: early AICAR PC + I/R. The aim of the studies outlined for this group was to determine whether administration of AICAR (100 mg/kg, 0.5 ml ip; Sigma) 30 min before I/R would induce an early phase of PC in WT C57BL/6J (n = 9) and eNOS–/– (n = 6) mice. Thirty minutes after AICAR administration, the intestine was exposed to I/R, and leukocyte rolling and adhesion were quantified as described for Group 2.

Group 5: L-NAME + late AICAR PC + I/R. To further substantiate whether direct activation of AMPK with AICAR produces late PC via a NO-dependent mechanism, a specific NOS inhibitor L-NAME (100 mg/kg, 0.5 ml ip; Sigma) was administered 10 min before AICAR on day 1 to WT C57BL/6J (n = 9) mice. Twenty-four hours later, the intestine was exposed to I/R, and leukocyte rolling and adhesion were quantified as for Group 2.

Statistical Analysis

The data were analyzed with standard statistical analysis, i.e., ANOVA with Scheffé's (post hoc) test for multiple comparisons. All values are expressed as means ± SE. Statistical significance was defined at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Figure 2 illustrates the effects of early- and late-phase PC with AICAR on postischemic leukocyte rolling and adhesion determined after 30 and 60 min of reperfusion in WT C57BL/6J mice. I/R markedly increased leukocyte rolling (Fig. 2, top) and adhesion (Fig. 2, bottom) compared with those in sham-operated (no ischemia) control mice. Whereas AICAR administration 24 h before induction of I/R (late AICAR PC) prevented postischemic leukocyte rolling and adhesion, early-phase PC with this AMPK activator (early AICAR PC) at the same dose was only effective in preventing I/R-induced leukocyte adhesion, although there was a tendency for postischemic leukocyte rolling to be reduced after early AICAR PC.


Figure 2
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  		Fig. 2. Effects of preconditioning (PC) with AICAR 30 min (early AICAR PC + I/R) or 24 h (late AICAR PC + I/R) before I/R on postischemic leukocyte rolling (top) and adhesion (bottom) determined after 30 and 60 min of reperfusion in wild-type C57BL/6J mice. L-NAME + late AICAR PC + I/R refers to experiments in which the NO synthase inhibitor was administered 10 min before PC with AICAR in animals subsequently exposed to I/R 24 h later. Open and solid bars represent data obtained at minutes 30–40 and 60–70 of reperfusion respectively. *P < 0.05, values statistically different from I/R; #P < 0.05, values statistically different compared with control.

 
The data depicted in Fig. 3 show the effects of PC with AICAR on I/R-induced leukocyte rolling and adhesion after 30 and 60 min of reperfusion in eNOS-deficient mice. I/R increased leukocyte rolling and adhesion in eNOS–/– mice (Fig. 3, top and bottom, respectively) to levels that were similar to that noted in WT animals (Fig. 2, top and bottom, respectively). Early- and late-phase PC with the AMPK activator (early and late AICAR PC, respectively) failed to limit postischemic rolling (Fig. 3, top) in eNOS–/– mice, a result that supports the concept that AMPK-dependent eNOS activation is essential to the beneficial effect of late-phase AICAR PC to prevent postischemic leukocyte rolling. However, the ability of early and late AICAR PC to prevent postischemic leukocyte adhesion that was noted in WT animals (Fig. 2, bottom) persisted in mice genetically deficient in eNOS (Fig. 3, bottom). Thus, the effect of PC with the AMPK activator to prevent postischemic leukocyte adhesion appears to occur by an eNOS-independent mechanism.


Figure 3
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  		Fig. 3. Effects of PC with AICAR 30 min (early AICAR PC + I/R) or 24 h (late AICAR PC + I/R) before I/R on postischemic leukocyte rolling (top) and adhesion (bottom) determined after 30 and 60 min of reperfusion in eNOS–/– mice. Open and solid bars represent data obtained at minutes 30–40 and 60–70 of reperfusion, respectively. *P < 0.05, values statistically different from I/R; #P < 0.05, values statistically different compared with control.

 
To further substantiate the role for NOS-derived NO in the beneficial action of AMPK activation 24 h before induction of I/R (late AICAR PC) to prevent postischemic leukocyte rolling, WT mice were treated with L-NAME before AICAR administration on day 1 (Fig. 2). Pharmacological NOS inhibition abrogated the effect of late AICAR PC to limit postischemic leukocyte rolling but did not prevent the reduction in leukocyte adhesion induced by antecedent AMPK activation. There were no significant differences in mean arterial blood pressures between treatment groups and their respective controls (data not shown).


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
AMPK is a ubiquitously expressed heterotrimeric serine/threonine kinase that is composed of an {alpha}-, a beta-, and a {gamma}-subunit (1, 48). The {alpha}-subunit, which exists as either the {alpha}1 or {alpha}2 isoform, is responsible for the catalytic activity of the enzyme. The regulatory beta- and {gamma}-subunits occur in either the beta1 or beta2 and the {gamma}1, {gamma}2, or {gamma}3 isoforms, respectively. AMPK is often referred to as a metabolic "master switch" due to its high sensitivity for the AMP-to-ATP ratio and its centralized role in both short- and long-term metabolic signaling pathways (6, 8, 17, 22). When cellular energy levels decrease as ATP is converted to AMP in response to stressful stimuli such as exercise or hypoxia, AMPK is activated by binding AMP via its {gamma}-subunit (22, 48). The ability of AMPK activation to protect cellular energy levels and maintain the integrity of the mitochondrial membrane potential makes it an essential prosurvival signaling mediator in protecting cells and tissues from I/R injury (20).

Recent observations support the possibility that AMPK activation may induce the development of preconditioned states that render tissues resistant to the deleterious effects of I/R. For example, treatment with AICAR or antidiabetic drugs of the biguanide class (e.g., metformin) has been shown to activate AMPK, prevent the signaling of inflammatory cytokines through nuclear factor-{kappa}B and tumor necrosis factor-{alpha}, and decrease adhesion molecule expression, effects that were abrogated by the addition of an small interfering RNA directed toward AMPK{alpha}1 (18). AMPK also phosphorylates and activates eNOS and the CFTR, both of which have been implicated as essential triggering elements in the development of IPC (10, 16). Furthermore, antecedent ethanol ingestion, which induces late-phase PC and prevents postischemic leukocyte rolling and adhesion by an eNOS-dependent mechanism, also activates AMPK (21). These observations led us to postulate that direct pharmacological activation of AMPK should induce development of an anti-inflammatory state similar to that seen with ethanol or IPC. In addition, we sought to determine whether AMPK-agonist PC is eNOS-dependent.

To test these hypotheses, we examined the effects of an AMPK activator AICAR as a PC stimulus. AICAR is first metabolized to ZMP, an AMP analog, which initially activates AMPK by binding to its {gamma}-subunit (32). This allosteric activation increases both AMPK activity and its affinity for upstream AMPK kinases, which, in turn, phosphorylate AMPK at threonine-172 on the {alpha}-subunit to further activate the enzyme. This latter process is responsible for the majority of AMPK activity and is known to be catalyzed by at least two kinases: the tumor suppressor LKB1 and Ca2+-calmodulin-dependent kinase kinase-beta (19, 35, 39, 44, 45). The binding of AMP or ZMP also increase AMPK activity by increasing the duration of the active state, which is accomplished by decreasing the affinity of phosphorylated AMPK for deactivating phosphatases such as protein phosphatase 2C (11).

Our results indicate that early- and late-phase PC with an AMPK activator (early and late AICAR PC, respectively) produce an anti-inflammatory phenotype in postcapillary venules. However, whereas late AICAR PC prevented both postischemic leukocyte rolling and adhesion, only I/R-induced leukocyte adhesion was abrogated during the early phase (early AICAR PC). These observations suggest that late AICAR PC may prevent expression of adhesion molecules that mediate leukocyte rolling (e.g., P-selectin) and adhesion (e.g., ICAM-1), whereas the signaling mechanisms activated by early AICAR PC may selectively target adhesive structures that specifically mediate stationary adhesion without influencing P-selectin expression. Alternatively, it is possible that early- and late-phase AMPK activation may exert differential effects on the production of inflammatory mediators that preferentially influence leukocyte rolling versus adhesion.

Although the aforementioned results clearly establish that AMPK activation can induce early and late PC, the identity of downstream effectors that contribute to the development of the anti-inflammatory phenotype is unknown. However, a myriad of downstream signaling molecules are phosphorylated secondary to AMPK activation and include: glycogen synthase, 6-phosphofructo-2-kinase, insulin receptor substrate-1, carbohydrate response element-binding protein, acetyl-CoA carboxylase-1/{alpha} and -2/beta, 3-hydroxy-3-methylgluatryl coenzyme A reductase (HMG-CoA reductase), hormone-sensitive lipase, transcription factor nuclear respiratory factor 1, uncoupling protein-3, peroxisome proliferator-activated receptor {gamma} co-activator 1{alpha}, CFTR, eNOS, tumor suppressor gene tuberous sclerosis complex-2, target of rapamycin, and co-activator p300 (20). Of the effectors listed here, we were most interested in initially testing for a role for eNOS as a downstream triggering event because 1) it is well established that AMPK phosphorylates eNOS at Ser1177, resulting in activation and increased NO production (10, 27); and 2) activation of eNOS has been implicated as a major triggering event for the development of late-phase PC in response to adenosine A2 receptor agonist treatment, antecedent exposure to short bouts of ischemia, and ethanol ingestion (47). Thus we sought to evaluate the role of eNOS in the beneficial anti-inflammatory effects of antecedent AICAR by employing a pharmacological inhibitor approach in WT mice and molecular genetic evidence obtained using an eNOS knockout model.

Given that the pattern of postischemic leukocyte/endothelial cell adhesive interactions in response to early AICAR PC was similar in WT and eNOS–/– mice, it does not appear that eNOS activation plays a role in the antiadhesive effect noted in this phase. However, the ability of AICAR treatment 24 h before I/R (late phase) to prevent postischemic leukocyte rolling was completely absent in eNOS–/– mice, whereas the abrogation of stationary leukocyte adhesion remained effective in these mice. A similar pattern of response was noted in WT animals treated coincidentally with the NOS inhibitor L-NAME at the time of AICAR PC 24 h before I/R. These observations suggest that the ability of late-phase AICAR PC to prevent postischemic leukocyte rolling was triggered by eNOS-derived NO that was formed during the period of AMPK activation 24 h earlier. Interestingly, late-phase AMPK activation prevented leukocyte adhesion by an NO-independent mechanism.

These results are intriguing due to their divergence from the canonical view that NO release prevents both leukocyte rolling and adhesion (34) and that PC stimuli, such as antecedent ethanol ingestion, treatment with exogenous adenosine or adenosine A2 receptor agonists, calcitonin gene-related peptide, bradykinin, or NO donors, which utilize NO to trigger entrance into an inflammatory state, prevent both types of adhesive interactions (12, 21, 22, 33, 43). Kubes and coworkers (25) have presented evidence that adherent leukocytes are almost exclusively recruited from the rolling cell population and that reductions in rolling by >90% are required before a significant effect on leukocyte adhesion is noted. Thus it may be that the eNOS-dependent effect of late AICAR PC to reduce postischemic leukocyte rolling, despite being quite dramatic, is not sufficient to elicit a significant change in leukocyte adhesion. Such a scenario implies that other AICAR-induced effects contribute to the antiadhesive responses of this AMPK activator. Indeed, it is well established that in addition to activating AMPK, AICAR administration also increases tissue levels of the potent antiadhesive purine nucleoside adenosine, which has been implicated as a trigger for the development of other forms of PC (15). However, this hypothesis seems unlikely in view of the fact that late-phase PC with adenosine or adenosine receptor agonists prevents both leukocyte rolling and stationary leukocyte adhesion (14, 47). Clearly, much additional work will be required to elucidate the mechanisms underlying the differential role of AICAR-induced eNOS activation to prevent leukocyte rolling without influencing postischemic leukocyte adhesion.

Our results may have important implications regarding the cardioprotective actions of therapeutic agents that activate AMPK, such as the glucose-lowering agent metformin; that is, in addition to its salutary metabolic actions in diabetes, metformin may induce significant anti-inflammatory effects in afflicted patients. AMPK activation has also been shown to augment IPC, suggesting that AICAR might represent a useful adjunctive therapy when administered with other agents that produce a preconditioned phenotype (5, 38). As another example, Xenos et al. (46) demonstrated that the HMG-CoA reductase inhibitor fluvastatin increases AMPK expression and activation in addition to increasing eNOS expression and phosphorylation. They also showed that fluvastatin was able to decrease ICAM-1 and PECAM-1 expression in an NO-dependent manner. An earlier study (28) showed that fluvastatin was also able to decrease expression of E-selectin and ICAM-1 in addition to increasing eNOS activity. Taken together with our work in the present study, these intriguing findings suggest that anti-inflammatory phenotype induced by AMPK activation may contribute to the well-known cardioprotective effects of widely prescribed statin drugs and metformin, in addition to their actions to inhibit HMG-CoA reductase and reduce plasma glucose, respectively.

Studies conducted to date showing that AICAR is cardioprotective in the setting of I/R have utilized protocols that involve treatment coincident with the onset of reperfusion or throughout I/R. Our study is the first to demonstrate that PC with this AMPK activator induces the development of early- and late-phase protection in the microcirculation, such that postcapillary venules fail to support leukocyte rolling and adhesion following subsequent exposure to I/R. However, important mechanistic differences exist since late-phase AICAR PC prevents both leukocyte rolling and adhesion induced by I/R, whereas early-phase AICAR limits only postischemic leukocyte adhesion. In addition, the ability of late AICAR PC to prevent leukocyte rolling involves eNOS-derived NO, whereas the salutary effects on leukocyte adhesion occur by an eNOS-independent mechanism. A role for eNOS in the early phase appears unlikely because the pattern of response to AICAR PC 30 min before I/R was identical in both WT and eNOS-deficient mice.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants DK-43785 and AA-14945.


   ACKNOWLEDGMENTS
 
K. Kamada has returned to Japan and is currently with the Department of Inflammation and Immunology, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kyoto 602–8566, Japan


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. J. Korthuis, Dept. of Medical Pharmacology and Physiology, Univ. of Missouri-Columbia, 1 Hospital Dr., Columbia, MO 65212 (e-mail address: korthuisr{at}health.missouri.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aschenbach WG, Sakamoto K, Goodyear LJ. 5' adenosine monophosphate-activated protein kinase, metabolism and exercise. Sports Med 34: 91–103, 2004.[CrossRef][ISI][Medline]
  2. Baxter GF. Ischaemic preconditioning of myocardium. Ann Med 29: 345–352, 1997.[ISI][Medline]
  3. Blais V, Rivest S. Inhibitory action of nitric oxide on circulating tumor necrosis factor-induced NF-kappaB activity and COX-2 transcription in the endothelium of the brain capillaries. J Neuropathol Exp Neurol 60: 893–905, 2001.[ISI][Medline]
  4. Bullough DA, Magill MJ, Firestein GS, Mullane KM. Adenosine activates A2 receptors to inhibit neutrophil adhesion and injury to isolated cardiac myocytes. J Immunol 155: 2579–2586, 1995.[Abstract]
  5. Burckhartt B, Yang XM, Tsuchida A, Mullane KM, Downey JM, Cohen MV. Acadesine extends the window of protection afforded by ischaemic preconditioning in conscious rabbits. Cardiovasc Res 29: 653–657, 1995.[CrossRef][ISI][Medline]
  6. Carling D. AMP-activated protein kinase: balancing the scales. Biochimie 87: 87–91, 2005.[Medline]
  7. Carrasco-Chaumel E, Rosello-Catafau J, Bartrons R, Franco-Gou R, Xaus C, Casillas A, Gelpi E, Rodes J, Peralta C. Adenosine monophosphate-activated protein kinase and nitric oxide in rat steatotic liver transplantation. J Hepatol 43: 997–1006, 2005.[CrossRef][ISI][Medline]
  8. Chan AY, Dyck JR. Activation of AMP-activated protein kinase (AMPK) inhibits protein synthesis: a potential strategy to prevent the development of cardiac hypertrophy. Can J Physiol Pharmacol 83: 24–28, 2005.[CrossRef][ISI][Medline]
  9. Chen CH, Gray MO, and Mochly-Rosen D. Cardioprotection from ischemia by a brief exposure to physiological levels of ethanol: role of epsilon protein kinase C. Proc Natl Acad Sci USA 96: 12784–12789, 1999.[Abstract/Free Full Text]
  10. Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, Kemp BE. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 443: 285–289, 1999.[CrossRef][ISI][Medline]
  11. Davies SP, Helps NR, Cohen PT, Hardie DG. 5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC. FEBS Lett 377: 421–425, 1995.[CrossRef][ISI][Medline]
  12. Davis BJ, Xie Z, Viollet B, Zou MH. Activation of the AMP-activated kinase by antidiabetes drug metformin stimulates nitric oxide synthesis in vivo by promoting the association of heat shock protein 90 and endothelial nitric oxide synthase. Diabetes 55: 496–505, 2006.[Abstract/Free Full Text]
  13. Dayton C, Yamaguchi T, Kamada K, Carter P, Korthuis RJ. Antecedent ethanol ingestion prevents postischemic leukocyte adhesion and P-selectin expression by a protein kinase C-dependent mechanism. Dig Dis Sci 50: 684–690, 2005.[CrossRef][ISI][Medline]
  14. Dayton C, Yamaguchi T, Kamada K, Carter P, Korthuis RJ. Antecedent ethanol ingestion prevents postischemic P-selectin expression in murine small intestine. Microcirculation 11: 709–718, 2004.[CrossRef][ISI][Medline]
  15. Gruber H, Hoffer M, McAllister D, Laikind P, Lane T, Schmid-Schoenbein G, Engler R. Increased adenosine concentration in blood from ischemic myocardium by AICA riboside. Effects on flow, granulocytes, and injury. Circulation 80: 1400–1411, 1989.
  16. Hallows KR, Raghuram V, Kemp BE, Witters LA, Foskett JK. Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase. J Clin Invest 105: 1711–1721, 2000.[ISI][Medline]
  17. Hardie DG. The AMP-activated protein kinase pathway—new players upstream and downstream. J Cell Sci 117: 5479–5487, 2004.[Abstract/Free Full Text]
  18. Hattori Y, Suzuki K, Hattori S, Kasai K. Metformin inhibits cytokine-induced nuclear factor kappaB activation via AMP-activated protein kinase activation in vascular endothelial cells. Hypertension 47: 1183–1188, 2006.[Abstract/Free Full Text]
  19. Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR, Witters LA. The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J Biol Chem 280: 29060–29066, 2005.[Abstract/Free Full Text]
  20. Ido Y, Carling D, Ruderman N. Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells: inhibition by the AMP-activated protein kinase activation. Diabetes 51: 159–167, 2002.[Abstract/Free Full Text]
  21. Jing M, Ismail-Beigi F. Role of 5'-AMP-activated protein kinase in stimulation of glucose transport in response to inhibition of oxidative phosphorylation. Am J Physiol Cell Physiol 290: C484–C491, 2006.[Abstract/Free Full Text]
  22. Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1: 15–25, 2005.[CrossRef][ISI][Medline]
  23. Kamada K, Dayton CB, Yamaguchi T, Korthuis RJ. Antecedent ethanol ingestion prevents postischemic microvascular dysfunction. Pathophysiology 10: 131–137, 2004.[CrossRef][Medline]
  24. Kamada K, Gaskin FS, Yamaguchi T, Carter P, Yoshikawa T, Yusof M, Korthuis RJ. Role of calcitonin gene-related peptide in the postischemic anti-inflammatory effects of antecedent ethanol ingestion. Am J Physiol Heart Circ Physiol 290: H531–H537, 2006.[Abstract/Free Full Text]
  25. Kubes P, Jutila M, Payne D. Therapeutic potential of inhibiting leukocyte rolling in ischemia/reperfusion. J Clin Invest 95: 2510–2519, 1995.[ISI][Medline]
  26. Li YJ, Xiao ZS, Peng CF, Deng HW. Calcitonin gene-related peptide-induced preconditioning protects against ischemia-reperfusion injury in isolated rat hearts. Eur J Pharmacol 311: 163–167, 1996.[CrossRef][ISI][Medline]
  27. Morrow VA, Foufelle F, Connell JM, Petrie JR, Gould GW, Salt IP. Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells. J Biol Chem 278: 31629–31639, 2003.[Abstract/Free Full Text]
  28. Mueck AO, Seeger H, Wallwiener D. Further evidence for direct vascular actions of statins: effect on endothelial nitric oxide synthase and adhesion molecules. Exp Clin Endocrinol Diabetes 109: 181–183, 2001.[CrossRef][ISI][Medline]
  29. Murakami H, Murakami R, Kambe F, Cao X, Takahashi R, Asai T, Hirai T, Numaguchi Y, Okumura K, Seo H, Murohara T. Fenofibrate activates AMPK and increases eNOS phosphorylation in HUVEC. Biochem Biophys Res Commun 341: 973–978, 2006.[CrossRef][ISI][Medline]
  30. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124–1136, 1986.
  31. Peralta C, Bartrons R, Serafin A, Blazquez C, Guzman M, Prats N, Xaus C, Cutillas B, Gelpi E, Rosello-Catafau J. Adenosine monophosphate-activated protein kinase mediates the protective effects of ischemic preconditioning on hepatic ischemia-reperfusion injury in the rat. Hepatology 34: 1164–1173, 2001.[CrossRef][ISI][Medline]
  32. Sabina RL, Patterson D, Holmes EW. 5-Amino-4-imidazolecarboxamide riboside (Z-riboside) metabolism in eukaryotic cells. J Biol Chem 260: 6107–6114, 1985.[Abstract/Free Full Text]
  33. Schulz E, Anter E, Zou MH, Keaney JF Jr. Estradiol-mediated endothelial nitric oxide synthase association with heat shock protein 90 requires adenosine monophosphate-dependent protein kinase. Circulation 111: 3473–3480, 2005.
  34. Sethi S, Dikshit M. Modulation of polymorphonuclear leukocytes function by nitric oxide. Thromb Res 100: 223–247, 2000.[CrossRef][ISI][Medline]
  35. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, Cantley LC. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci USA 101: 3329–3335, 2004.[Abstract/Free Full Text]
  36. Shigematsu S, Ishida S, Gute DC, Korthuis RJ. Bradykinin prevents postischemic leukocyte adhesion and emigration and attenuates microvascular barrier disruption. Am J Physiol Heart Circ Physiol 277: H161–H171, 1999.[Abstract/Free Full Text]
  37. Shigematsu S, Ishida S, Gute DC, Korthuis RJ. Postischemic anti-inflammatory effects of bradykinin preconditioning. Am J Physiol Heart Circ Physiol 280: H441–H454, 2001.[Abstract/Free Full Text]
  38. Shinmura K, Tamaki K, Bolli R. Short-term caloric restriction improves ischemic tolerance independent of opening of ATP-sensitive K+ channels in both young and aged hearts. J Mol Cell Cardiol 39: 285–296, 2005.[CrossRef][ISI][Medline]
  39. Soltys CL, Kovacic S, Dyck JR. Activation of cardiac AMP-activated protein kinase by LKB1 expression or chemical hypoxia is blunted by increased Akt activity. Am J Physiol Heart Circ Physiol 290: H2472–H2479, 2006.[Abstract/Free Full Text]
  40. Sun JZ, Tang XL, Knowlton AA, Park SW, Qiu Y, Bolli R. Late preconditioning against myocardial stunning. An endogenous protective mechanism that confers resistance to postischemic dysfunction 24 h after brief ischemia in conscious pigs. J Clin Invest 95: 388–403, 1995.[ISI][Medline]
  41. Takano H, Tang XL, Qiu Y, Guo Y, French BA, Bolli R. Nitric oxide donors induce late preconditioning against myocardial stunning and infarction in conscious rabbits via an antioxidant-sensitive mechanism. Circ Res 83: 73–84, 1998.[Abstract/Free Full Text]
  42. Tang XL, Qiu Y, Park SW, Sun JZ, Kalya A, Bolli R. Time course of late preconditioning against myocardial stunning in conscious pigs. Circ Res 79: 424–434, 1996.[Abstract/Free Full Text]
  43. Wall TM, Sheehy R, Hartman JC. Role of bradykinin in myocardial preconditioning. J Pharmacol Exp Ther 270: 681–689, 1994.[Abstract/Free Full Text]
  44. Woods A, Dickerson K, Heath R, Hong SP, Momcilovic M, Johnstone SR, Carlson M, Carling D. Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab 2: 21–33, 2005.[CrossRef][ISI][Medline]
  45. Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D, Schlattner U, Wallimann T, Carlson M, Carling D. LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 13: 2004–2008, 2003.[CrossRef][ISI][Medline]
  46. Xenos ES, Stevens SL, Freeman MB, Cassada DC, Goldman MH. Nitric oxide mediates the effect of fluvastatin on intercellular adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 expression on human endothelial cells. Ann Vasc Surg 19: 386–392, 2005.[CrossRef][ISI][Medline]
  47. Yamaguchi T, Dayton C, Shigematsu T, Carter P, Yoshikawa T, Gute DC, Korthuis RJ. Preconditioning with ethanol prevents postischemic leukocyte-endothelial cell adhesive interactions. Am J Physiol Heart Circ Physiol 283: H1019–H1030, 2002.[Abstract/Free Full Text]
  48. Young LH, Li J, Baron SJ, Russell RR. AMP-activated protein kinase: a key stress signaling pathway in the heart. Trends Cardiovasc Med 15: 110–118, 2005.[CrossRef][ISI][Medline]



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