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1Department of Medical Pharmacology and Physiology and the Dalton Cardiovascular Research Center, University of Missouri-Columbia School of Medicine, Columbia, Missouri; 2Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, School of Medicine, Shreveport, Louisiana; and 3First Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan
Submitted 17 March 2006 ; accepted in final form 6 November 2006
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ABSTRACT |
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ischemia; reperfusion; adenosine; nitric oxide; leukocyte rolling; endothelial nitric oxide synthase knockout mice
The mechanisms that underlie the protective actions of ethanol ingestion have not been extensively investigated, but there is evidence implicating adenosine and nitric oxide (NO) as key triggers for the development of the anti-inflammatory phenotype that occurs in response to ethanol. For example, pharmacological inhibition of NO synthase (NOS) or blockade of adenosine A2 receptors during the first hour after ethanol ingestion prevented the antiadhesive effects of ethanol ingestion noted during I/R 24 h later (9, 15, 34). Moreover, preconditioning with a NO donor or an adenosine A2 receptor agonist in lieu of ethanol 24 h before I/R also prevented postischemic P-selectin expression, leukocyte rolling, and stationary leukocyte adhesion. These results indicate that entrance into this protected or preconditioned state was triggered by adenosine and NO formed during the period of ethanol exposure. However, identification of the NOS isoform that participates in inaugurating the development of the anti-inflammatory phenotype in response to ethanol is uncertain. Nor is it clear whether adenosine acts in parallel with NO to stimulate downstream effectors or as an upstream signaling element that activates endothelial NOS (eNOS) after ingestion, resulting in the production of the NO that triggers entrance into this preconditioned state. Thus the aims of this study were to 1) evaluate the role of eNOS in the development of ethanol preconditioning (EPC) and 2) determine whether ethanol-induced adenosine A2 receptor activation was an important upstream signaling element that acts to stimulate the formation of eNOS-derived NO.
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METHODS |
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Wild-type (WT) male C57BL/6J and eNOS-deficient mice (eNOS–/–, B6.129P2-Nos3tm1Unc) on a C57BL/6 background were obtained from Jackson Laboratories (Bar Harbor, ME). All of the mice were maintained on a purified laboratory diet and used at 8–10 wk of age. The experimental procedures described herein were performed in accordance with the criteria outlined in the National Institutes of Health guidelines and were approved by the Louisiana State University Health Sciences Center-Shreveport and University of Missouri-Columbia Institutional Animal Care and Use Committees.
EPC on Day 1
EPC was induced by instillation of ethanol into the stomach by gavage 24 h before the experiments, as previously described (8, 9, 15, 16, 34, 35). The volume of 95% ethanol to be instilled (in µl) was calculated as follows: [body wt (in g) x 0.6] + 0.3. This volume of ethanol was mixed in 0.3 ml of sterilized-distilled water just before gavage. For example, 15.3 µl of 95% ethanol would be placed in 0.3 ml of sterilized-distilled water for a 25 g mouse. Our previous work demonstrated that this protocol produced an increase in plasma ethanol concentration that peaks at 
45 mg/dl (10 mM) within 30 min of instillation and was no longer detectable in the plasma 60 min after gavage (34). Mice in the sham and I/R alone groups received sterilized-distilled water (i.e., no ethanol) by gavage.
Surgical Procedures and Induction of I/R on Day 2
Twenty-four hours after EPC, mice were anesthetized by intramuscular injection of ketamine (150 mg/kg body wt) and xylazine (7.5 mg/kg body wt). After a surgical plane of anesthesia was attained, a tracheotomy was performed to facilitate breathing during the experiment. The right carotid artery was cannulated, and systemic arterial pressure was measured with a pressure transducer (model P23A; Statham, Oxnard, CA) 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 to label leukocytes. CFDASE was dissolved previously in DMSO at a concentration of 5 mg/ml, aliquoted, and stored at –20°C until use. At all times, CFDASE was protected from exposure to light. After these procedures, a midline abdominal incision was performed and the superior mesenteric artery was occluded with a microvascular clip for 0 (sham) or 45 min. After the ischemic period, the clip was gently removed, and CFDASE from the stock solution was diluted into 100 µl saline to a final concentration of 250 µg/ml and injected intravenously at 20 µl/min for fluorescent microscopic study (16, 34, 35).
Intravital Fluorescent 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 portion of the gut was superfused at 1.5 ml/min using a peristaltic pump (model M312, Gilson) with bicarbonate-buffered saline (BBS, pH 7.4) bubbled with a mixture of 5% CO2-95% N2 to reduce the oxygen tension to physiological intraperitoneal levels (40–50 mmHg). The exteriorized region of the small bowel was covered with BBS-soaked gauze to minimize the 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 with 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 lamp. The 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. The fluorescent image of the microcirculation (excitation wavelength, 420–490 nm; emission wavelength, 520 nm) was detected with a charge-coupled device (CCD) camera (XC-77, Hamamatsu Photonics), a CCD camera control unit (C2400, Hamamatsu Photonics), and an intensifier head (M4314, Hamamatsu Photonics), connected to the camera. Microfluorographs were projected on a color television monitor (PVM-1953MD, Sony) and recorded on videotape using a videocassette recorder (HR-S4600U, JVC) for off-line during analysis of the videotaped image. A video time-date generator (WJ810, Panasonic) displayed the stopwatch function onto the monitor.
The intravital microscopic measurements described below were obtained at minutes 30–40 and 60–70 of reperfusion or at equivalent time points in the control groups. The intestinal segment was scanned from the oral to aboral section, and 10 single, unbranched venules (20–50 µm diameter, 100 µm length), were observed, each for at least 30 s (16, 34, 35). The numbers of rolling and 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. Leukocytes were considered to be firmly adherent if they did not move or detach from the venular wall for a period 
30 s. Rolling leukocytes were defined as cells crossing an imaginary line in the microvessel at a velocity that is significantly lower than center line velocity, and 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 millimeter squared of vessel area.
Assay for NOS Activity
Determination of NOS activity was accomplished by measuring the formation of L-[3H]citrulline from L-[3H]arginine in duplicate in four experiments per group, and each determination was derived from a pooled sample of jejunal tissue obtained from four to five animals (25). Briefly, jejunal samples were harvested from control animals receiving drug vehicle (0.3 ml saline) by intraperitoneal injection 10 min before gavage with ethanol vehicle (0.3 ml distilled water) and from mice that were preconditioned with ethanol in the absence or presence of treatment with the adenosine A2 receptor antagonist 3,7-dimethyl-1-propargyl-xanthine [DMPX, administered by intraperitoneal injection (10 nM, 0.3 ml) 10 min before ethanol gavage]. Tissues were obtained 15 min after ethanol gavage, homogenized, and assayed for total NOS activity, using a liquid scintillation counter, as described previously (25). EGTA (2 mM) was added to the incubation buffer to estimate inducible NOS (iNOS) activity. Calcium-dependent NOS activity [includes eNOS and neuronal NOS (nNOS)] was determined from the difference between total NO activity and iNOS activity. To standardize results, total protein was assessed and enzyme activities were expressed as picomoles of L-[3H]citrulline produced per minute per milligram total protein (25).
Experimental Protocols
The number of animals was six in each group (groups 1–17). WT mice were used in groups 1–11, and eNOS–/– animals were used in groups 12–17.
Group 1: sham (WT). As a time control for the effects of experimental duration in WT mice, animals in this group were administered sterilized-distilled water alone (without ethanol) by gavage on day 1. These mice also received an intraperitoneal injection of 0.3 ml saline (which was used as the drug vehicle in groups 4–11 and 15–17) 10 min before gavage on day 1. Twenty-four hours later (day 2), the superior mesenteric artery was exposed but I/R was not produced. Leukocyte/endothelial cell adhesive interactions were observed at time points comparable with those described for the I/R alone studies outlined for group 2 below.
Group 2: I/R (WT). WT mice in this group were administered 0.3 ml sterile saline vehicle [for the drugs administered in groups 4–9, 13, 14 by intraperitoneal injection 10 min before administration of 0.3 ml distilled water (no ethanol) by gavage on day 1, as described for group 1]. Twenty-four hours later, the intestine was prepared for intravital microscopy and subjected to I/R, with assessment of leukocyte rolling and adhesion during minutes 30–40 and 60–70 of reperfusion.
Group 3: EPC + I/R (WT). To confirm the effects of EPC to prevent I/R-induced leukocyte rolling and adhesion, WT mice in this group were treated as described for group 2, except that ethanol was administered by gavage on day 1. We previously demonstrated that EPC alone (no I/R) has no effect on baseline leukocyte rolling and adhesion, when assessed 24 h later (34), and thus did not repeat this control group as part of these studies.
Group 4: NG-nitro-L-arginine + EPC + I/R (WT). To determine the role of NO as an initiator of the anti-inflammatory effects of EPC, the studies outlined for group 2 were repeated, except that WT mice in this group received an intraperitoneal injection of NG-nitro-L-arginine (L-NNA; 650 µg/ml, 0.5 ml; Sigma, St. Louis, MO), a specific, but non-isoform-selective, NOS inhibitor, 10 min before ethanol.
Group 5: PTIO + EPC + I/R (WT). Mice in this group were treated as described for group 4, except that the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 100 µM, 0.3 ml; Calbiochem, San Diego, CA), rather than L-NNA, was administered by intraperitoneal injection 10 min before ethanol. We previously demonstrated that this dose of PTIO was effective in preventing the beneficial effect of antecedent ethanol to prevent P-selectin expression (9).
Group 6: 1400W dihydrochloride + EPC + I/R (WT). Mice in this group were treated as described for group 4, except that the selective iNOS inhibitor, 1400W dihydrochloride [1400W, 1 mg/kg, 0.3 ml, Sigma (Ref. 18)], rather than L-NNA, was administered by intraperitoneal injection 10 min before ethanol.
Group 7: 7-nitroindazole + EPC + I/R (WT). Mice in this group were treated as described for group 4, except that the selective nNOS inhibitor 7-nitroindazole [7-NI, 30 mg/kg, 0.3 ml, Sigma (Ref. 19)], rather than L-NNA, was administered by intraperitoneal injection 10 min before ethanol.
Groups 8–10: SNAP (1, 10, and 100 µM, respectively) + I/R (WT). WT mice in these groups were treated as described for group 2, except that S-nitroso-N-acetylpenicillamine (SNAP; Sigma Chemical) was administered by intraperitoneal injection (0.3 ml of SNAP at concentrations of 1, 10, or 100 µM, groups 8–10, respectively) 24 h before I/R to determine whether this NO donor could substitute for EPC and produce an anti-inflammatory phenotype.
Group 11: DMPX + SNAP (100 µM) + I/R (WT). WT mice in this group were treated as described for group 10, except that 3,7-dimethyl-1-propargyl-xanthine (DMPX) was administered by intraperitoneal injection (10 nM, 0.3 ml) 10 min before SNAP (100 µM, 0.3 ml) administration. We previously demonstrated that treatment with this selective adenosine A2 receptor antagonist at the same dose abrogated the anti-inflammatory effects of EPC (9, 34).
Group 12: sham (eNOS–/–). As a time control for the effects of experimental duration, eNOS–/– animals in this group were treated as described for WT sham animals in group 1.
Group 13: I/R (eNOS–/–). To determine the effect of I/R on postischemic leukocyte rolling and adhesion in eNOS–/– mice, the studies outlined for group 2 were repeated in these animals.
Group 14: EPC + I/R (eNOS–/–). To determine whether EPC would be effective in preventing leukocyte rolling and adhesion in eNOS–/– mice, these animals were subjected to the protocol outlined for WT mice in group 3.
Group 15: PTIO + EPC + I/R (eNOS–/–). To determine whether PTIO would exacerbate the postischemic adhesive responses noted in ethanol-preconditioned, eNOS-deficient mice, the studies outlined for group 5 were repeated in these gene-targeted animals.
Group 16: DPMA + I/R (eNOS–/–). To determine whether preconditioning with an adenosine A2 receptor agonist would prevent postischemic leukocyte rolling and adhesion in eNOS–/– mice, the protocol outlined for group 10 was used, except that N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA; Research Biochemicals International, Natick, MA), was administered to eNOS–/– mice by intraperitoneal injection (100 nM, 0.5 ml), in lieu of SNAP. We previously demonstrated that treatment with this selective adenosine A2 receptor agonist at the same dose induced a preconditioned state that mimicked the anti-inflammatory effects of EPC in WT mice (34).
Group 17: SNAP (100 µM) + I/R (eNOS–/–). To determine whether NO preconditioning would induce a preconditioned anti-inflammatory state in eNOS–/– mice, the studies outlined for WT animals in group 10 were repeated in eNOS–/– mice.
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.
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DISCUSSION |
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Our first line of evidence indicating that NO serves as a trigger for EPC was derived from studies in which the bowel of WT mice was exposed to L-NNA (a specific, but non-isoform-selective, NOS inhibitor) or PTIO (a NO scavenger) during the period of ethanol exposure (Fig. 1). These mechanistically different interventions, which effectively decrease the bioavailability of NO, almost completely prevented the development of the antiadhesive phenotype that became apparent during I/R on day 2 in WT mice that had ingested ethanol 24 h earlier. These findings corroborate our earlier work demonstrating that another pan-NOS inhibitor, L-NIO, was equally effective in preventing EPC (9, 34). Our results are also consistent with the observations that ethanol enhances both basal and flow-stimulated NOS activity and NO production in vivo and in cultured endothelial cells (6, 13).
Whereas our pharmacological data (present study and Ref 34) support a role for NO as an initiator of EPC, these results provide little insight regarding the NOS isoform involved. The endothelial (eNOS or NOS 3), inducible (iNOS or NOS 2), and neuronal (nNOS or NOS 1) NOS isoforms can be expressed in the small intestine (20, 25). In contrast to many tissues, where iNOS is not constitutively present and requires several hours to reach full expression in response to appropriate stimuli (1, 2, 33), all three isoforms are constitutively expressed in the bowel wall (25). Since NOS inhibition was only effective when the NOS inhibitors were administered coincident with, but not 1 h after, ethanol ingestion, we postulated that constitutively expressed eNOS is the most likely source of NO that triggers the development of the anti-inflammatory phenotype in postcapillary venules after ethanol ingestion (34). This notion is supported by our observations that treatment with selective iNOS (1400W) or nNOS (7-NI) inhibitors failed to alter the anti-inflammatory actions of EPC in the intestine of WT mice subsequently exposed to I/R, whereas a pan-NOS isoform inhibitor was effective in this regard.
To provide more compelling support for a role for eNOS as the source of NO that induces the development of the anti-inflammatory phenotype in response to antecedent ethanol ingestion, we evaluated the effectiveness of this preconditioning stimulus in eNOS–/– mice. In stark contrast to the profound anti-inflammatory actions of ethanol ingestion noted in WT animals (Fig. 1), antecedent ethanol was largely ineffective in preventing postischemic leukocyte rolling and adhesion in eNOS–/– mice (Fig. 3). Moreover, coincident administration of a NO scavenger (PTIO) with ethanol did not exacerbate the postischemic increases in leukocyte rolling and adhesion noted in eNOS-deficient mice, a result that suggests that compensatory alterations in NO derived from other NOS isotypes does not occur in these gene-targeted mice. These studies provide strong support for the notion that eNOS is the major isoform responsible for generating the NO that triggers the development of the antiadhesive phenotype demonstrated by postcapillary venules exposed to low concentrations of ethanol. As such, these studies also constitute a second line of evidence supporting the view that NO formed during the period of ethanol exposure on day 1 is required for the development of this anti-inflammatory phenotype on day 2.
Leukocyte rolling and adhesion were similar under baseline conditions and increased to similar levels after I/R in WT control and eNOS–/– mice. Sanz et al. (30) have also reported that baseline leukocyte rolling and adhesion were similar in jejunal submucosal (the same preparation used in the present study) and cremasteric postcapillary venules of eNOS–/– mice anesthetized with ketamine and xylazine (which was employed in the present study). On the other hand, Lefer and coworkers (21) have noted increased baseline adhesion in peri-intestinal venules located in the ileal mesentery of eNOS–/– mice anesthetized with pentobarbital sodium. The reasons underlying these disparate reports are not clear but may be related to the region of the bowel examined or anesthetics used. In any event, it would be expected that the postischemic increases in leukocyte rolling and adhesion would be similar in eNOS–/– vs. WT mice because I/R is associated with dramatic reductions in NOS activity and NO bioavailability in the intestine in WT animals (8).
It is important to note that pharmacological preconditioning with a NO donor (SNAP) 24 h before I/R effectively prevented postischemic leukocyte rolling and adhesion in eNOS–/– mice (Fig. 3). This latter observation indicates that the signaling elements downstream from the formation of NO that participate in the development of a preconditioned state are operative in eNOS–/– mice. Thus it appears that the inability to generate eNOS-derived NO accounts for the failure of ethanol to produce an anti-inflammatory phenotype in these knockout animals, rather than potential compensatory alterations in downstream signaling elements that might occur in response to a lack of this enzyme.
Whereas the factors responsible for increasing eNOS activity after ethanol ingestion (or any form of preconditioning for that matter) are unknown, our previous observations that NOS inhibition was as effective as adenosine deaminase or adenosine A2 receptor blockade in abolishing the protective effects of antecedent ethanol ingestion led us to suggest that adenosine and NO may serve as sequential triggering elements in the signaling cascade that induces the development of the protective phenotype rather than acting as independent initiators of this preconditioned state (15, 34). Our current findings that pharmacological preconditioning with an adenosine A2 receptor agonist (DPMA) 24 h before I/R prevented postischemic leukocyte rolling and adhesion in WT (34), but not eNOS–/– mice (Fig. 3, present study), are consistent with this postulate.
The notion that ethanol acts to trigger the formation of adenosine A2 receptor-dependent eNOS-derived NO as essential and sequential initiating events in the development of an anti-inflammatory phenotype is supported by the following observations. It is well known that ethanol increases adenosine concentration in extracellular fluid via inhibition of the nucleoside transporter in cell membranes (10, 11, 23). Moreover, both ethanol and ligation of adenosine A2 receptors increase the activity of cAMP-dependent kinase, which in turn can activate eNOS by phosphorylating Ser1177 (4, 6, 13, 22). Adenosine has also been shown to stimulate L-arginine transport and NO biosynthesis by activation of A2 receptors on human umbilical vein endothelial cells (32). Although NO donor preconditioning was not abrogated by coincident treatment with an A2-receptor antagonist in WT animals (Fig. 2), we demonstrated in a previous study (34) that postischemic anti-inflammatory effects induced by antecedent exposure of the bowel to an adenosine A2 receptor agonist could be prevented by concomitant administration of a NOS inhibitor.
The data presented in Figs. 2 and 3 and our earlier work (34) provide strong, albeit indirect, support for the notion that the triggering mechanism for EPC involves adenosine A2 receptor-dependent stimulation of eNOS, which produces NO as a downstream signaling element. More direct evidence for this concept is provided by our observations that calcium-dependent NOS activity was increased by twofold in jejunal samples obtained from ethanol preconditioned animals, an effect that was prevented by coincident adenosine A2 receptor blockade. Taken together, these data indicate that adenosine and eNOS-derived NO serve as sequential triggering elements in the signaling cascade that induces the development of this protective phenotype, rather than acting as independent initiators of EPC.
Whereas the aforementioned results establish adenosine A2 receptor-dependent eNOS activation as essential triggering elements in the signaling cascade that inaugurates entrance into a preconditioned state by antecedent ethanol ingestion, the downstream mediators of this response are unknown. However, we have recently implicated release of CGRP from capsaicin-sensitive neurons as an important initiating event in the development of EPC (16). Adenosine A2 receptor activation and NO formation both induce the release of CGRP (3, 6, 14, 28, 29, 36, 37). When coupled with our demonstration that adenosine A2 receptor-dependent eNOS activation is required for triggering entrance into a preconditioned state after ethanol ingestion (present study and Refs. 9, 15, and 34), these observations suggest that CGRP release may occur downstream of these events. We and others have also obtained evidence implicating heme oxygenase-1 (HO-1) as a major effector of EPC during I/R 24 h later (unpublished observations and Refs. 15, 27, and 28). However, the links between the ethanol-induced, adenosine A2 receptor-stimulated, eNOS-derived NO-dependent CGRP release on day 1 and increased HO-1 activity during I/R 24 h later are not known. A likely candidate intermediary signaling molecule is protein kinase C, which has been shown to play a role in preconditioning induced by ethanol and exogenous adenosine, NO, and CGRP (5, 8, 23, 26).
In summary, the results of this study establish a critical role for eNOS activation as the major source of the NO that triggers the development of an anti-inflammatory phenotype in intestinal postcapillary venules after ethanol ingestion. Our observations also provide strong support for the notion that adenosine and NO serve as sequential triggering elements in the signaling cascade that induces the development of this protective phenotype rather than acting as independent initiators of EPC. According to this scenario, tissue levels of adenosine increase during the period of ethanol exposure. Subsequent ligation of adenosine A2 receptors leads to activation of eNOS and the formation of NO, which acts as a downstream signaling element to initiate the development of the antiadhesive phenotype that becomes apparent 24 h after ethanol ingestion.
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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.
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protein kinase C correlates with a cardioprotective effect of regular ethanol consumption. Proc Natl Acad Sci USA 95: 8262–8267, 1998.
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Values statistically different from corresponding values in sham, I/R, and EPC + I/R, respectively, at P < 0.05.