Introduction

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Considerable experimental evidence suggests that hepatic ischemia-reperfusion activates the complement system (Liu et al., 1994) and primes neutrophils to generate superoxide (Liu et al., 1995). Active episodes of inflammation are associated with increases in tissue-derived cytokines, such as tumor necrosis factor, interleukin-1 (Hibbs et al., 1992), which potentially prime the inflammatory cells to produce ROS and NO. NO formation from the guanidine nitrogen group of L-arginine is catalyzed by specific NOS. Recent data indicate that at least two major isoforms of NOS, i.e., constitutive and inducible forms, catalyze L-arginine to L-citrulline and NO. The constitutive isoform (cNOS) is Ca⁺⁺/calmodulin and NADPH-dependent, and catalyzes the production of small amounts of NO (picomoles) for a short period in response to certain receptor and/or physical stimulation. The inducible isoform (iNOS), expressed in macrophages, neutrophils and smooth muscle cells, catalyzes the production of high levels of NO (nanomoles) for a long period in response to lipopolysaccharide and cytokine stimulation. In the vascular system, the biosynthesis of NO regulates organ blood flow, inhibits platelet aggregation (Radomski et al., 1990) and attenuates neutrophil adherence (Gauthier et al., 1994; Kubes et al., 1991). During reperfusion after ischemia, there is endothelial cell dysfunction that is characterized primarily by a marked decrease of NO release (Tsao et al., 1990; Wang et al., 1993). The decrease in NO release is the initial triggering mechanism that allows neutrophil adherence and diapedesis into the ischemic area, which augments postreperfusion injury (Tsao et al., 1990; Wang et al., 1993).

NO is involved in the relaxation of smooth muscle, the inhibition of platelet aggregation and adhesion and the modulation of leukocyte-endothelium interactions, all believed to be key factors in myocardial ischemia-reperfusion injury. On the other hand, detrimental effects of excessive production of NO are possible because high concentrations of NO are cytotoxic and contribute to cell injury in a variety of disease states including acute lung injury (Kooy et al., 1995), endotoxic shock (Minnard et al., 1994) and ischemia-reperfusion injury (Liu et al., 1996, 1997). Furthermore, NO can react with superoxide to form peroxynitrite, a potentially toxic molecule that may play a pivotal role in the pathophysiology of ischemia-reperfusion injury (Beckman et al., 1993; Liu et al., 1997: Wang et al., 1994). Indeed some of the cytotoxic effects of NO have now been attributed to peroxynitrite, a potent oxidant and nitrating agent (Szabo et al., 1996; Hausladen and Fridovich, 1994). In contrast, NO gas and organic nitrates, which act as NO donors, have been shown to attenuate the severity of reperfusion injury, resulting in functional preservation (Rossaint et al., 1993). Furthermore, the administration of NOS inhibitors such as N^G-monomethyl-L-arginine or L-NAME increased mortality and decreased tissue perfusion (Meakin 1990; Minnard et al., 1994; Mulder et al., 1994) in models of endotoxin shock. Thus the role of NO in ischemia-reperfusion injury is complex. Excessive production of NO can be cytotoxic or lead to the formation of the highly cytotoxic peroxynitrite molecule. On the other hand, inhibition of NO formation may lead to tissue hypoperfusion, increased neutrophil adhesion and infiltration.

In our previous study, inhibition of NOS by L-NAME resulted in the progressive injury seen in the liver and lung in the hepatic I/R with endotoxemia model (Liu et al., 1996). The hepatic injury observed in our experiments with L-NAME treatment was associated with enhanced neutrophil infiltration and superoxide production. In the present study conducted in an in vivo model of hepatic ischemia-reperfusion injury, we hypothesize that NO regulates neutrophil accumulation through the expression of specific adhesion molecules. We further postulate that the detrimental effects of NO inhibition (e.g., neutrophil accumulation) will outweigh the potential benefit of suppressing the production of cytotoxic molecules such as peroxynitrite.

	Materials and Methods

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Male Fischer rats 344 (245-290 g body weight) were purchased from Taconic Farm (Germantown, NY). The animals were given free access to food (Purina Rodent chow J001) and water. The experimental protocols followed the criteria of the Public Health Service "Guide for the Care and Use of Laboratory Animals" and were approved by the Institutional Animal Care and Use Committee.

Materials. ALT activity was measured with a Sigma diagnostics kit. Nitrate reductase (from Aspergillus species), o-dianisidine, -nicotinamide adenine dinucleotide phosphate, reduced form (-NADPH), L-NAME and sodium nitrite were purchased from Sigma (St. Louis, MO). [2,3,4,5-³H]arginine monohydrochloride was purchased from Amersham Life Science (Buckinghamshire, England). Monoclonal anti-nitrotyrosine antibody and Vectastain Universal Quick kit were purchased from the Upstate Biotechnology (Lake Placid, NY) and Vector Lab (Burlingame, CA). The RNA Stat-60 reagent, SuperScript II, Taq DNA polymerase, and GelMarker were purchased from GIBCO BRL (Gaithersburg, MD) and Tel-Test, Inc. (Friendswood, TX).

Experimental protocol. The experimental protocol for partial no-flow hepatic ischemia has been described (Liu et al., 1994, 1995). Under pentobarbital anesthesia (60 mg/kg i.p.), the trachea was cannulated (PE-240) to maintain a patent airway. The carotid artery was cannulated with PE-50 tubing, a laparotomy was performed and the blood supply to the left lateral and median lobes of the liver was occluded with an atraumatic Glover bulldog clamp for 30 min. The remaining caudal three lobes retained an intact portal and arterial blood supply, as well as venous outflow preventing the development of intestinal venous hypertension. Reperfusion was initiated by removal of the clamp, the animal received 1 ml of the sterile saline i.p. and the wound was closed with 4-0 silk and wound staples. In some rats, L-NAME (10 mg/kg i.v. through the penile vein) was given 10 min before the start of reperfusion (I/R + L-NAME), and control rats were given saline (I/R). Animals in the sham control group were subjected to the same surgical operation without occlusion of the artery supplying the left and lateral lobes of liver. Blood samples (300-500 µl) were obtained at 3 and 4 h of reperfusion for determination of ALT activities, nitrite/nitrate concentration (as an index of NO production) and quantification of white blood cell and differential counts. Ischemic and nonischemic lobes of liver tissue at 4 h after reperfusion were taken, snap-frozen in liquid nitrogen and stored at 70°C for measurement of nitrite/nitrate and superoxide. Parts of liver tissues from rats were saved in 4% neutral buffered formalin or 4% formaldehyde for histological or immunohistochemical study, respectively.

ALT activity. Plasma ALT activities were measured with Sigma test kit, DG 159-UV, and expressed as international units per liter.

Quantification of white blood cell and differential counts. Heparin-anticoagulated blood samples were obtained after 4 h of reperfusion. Blood sample (50 µl) was diluted 20-fold with 1% acetic acid solution to lyse red cells. Quantitation of white blood cells was performed using a microscope. Two drops of blood were smeared on a slide, air-dried and stained with Giemsa stain solution (Sigma). The differential counts were performed microscopically.

Nitrite/nitrate assay. The nitrite was measured by the Nitric Oxide Analyzer (model 270B, Sievers Instruments, Denver, CO) (Liu et al., 1996). The NOA measures nitric oxide in biological fluids by a modified gas stripping technique with high sensitivity (<10 pmol/ml of solution). Pieces of ischemic and nonischemic lobes of liver were homogenized in physiological saline (10% w/w tissue suspension) in ice. This suspension was centrifuged at 10,000 × g, 4°C for 10 min. Tissue supernatant (100 µl) was then incubated in the presence of nitrate reductase (0.05 U/ml) and NADPH (0.1 mM) at 37°C for 15 min to convert all the nitrate to nitrite. Sample (20 µl) was then injected into the purge vessel of the NOA which contained 2 ml of 1% sodium iodide in acetic acid to convert the nitrite to NO gas. A stream of nitrogen was passed through the purge vessel under vacuum to eliminate any oxygen. The amount of nitrite was calculated from a standard curve of sodium nitrite (0-400 pmol) (r > 0.99). Blood samples were centrifuged at 1,000 × g for 5 min to obtain plasma. Plasma sample (100 µl) were incubated with nitrate reductase and NADPH for 15 min before injection into the NOA.

NOS activity assay. NOS activity was assayed as described by Balligand et al. (1994). Samples of ischemic and nonischemic lobes of liver were obtained from the rats after 4 h reperfusion and were frozen at 70°C until assay. The frozen samples were thawed and homogenized on ice with a polytron homogenizer for 20 s in 10% (wt/wt) of buffer composed of (mM): Tris-HCl, 50; EDTA, 0.1; ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid, 0.1; 2-mercaptoethanol, 12 mM; phenylmethylsulfonyl fluoride, 1 mM; and leupeptin, 2 µM (pH 7.4). The homogenates were centrifuged at 10,000 × g for 15 min at 4°C. Conversion of L-[³H]arginine to L-[³H]citrulline was measured in the supernatant. Tissue supernatant (50 µ1) was incubated in the reactive mixture of L-[³H]arginine (10 µM, 5 kBq/tube), NADPH (1 mM), calmodulin (30 nM) and calcium (2 mM) at 37°C for 10 min in HEPES buffer (pH 7.5). The reaction was terminated by adding 1.5 ml of stop buffer [30 mmol/l HEPES and 3 mmol/l EDTA (pH 5.5)] at 4°C. L-[³H]citrulline was eluted on chromatography columns (resin AG-50 WX-8, Na⁺ form, pH 7.0) and quantified by liquid scintillation counting (Mark V, TmAnalytic, Elk Grove Village, IL). To ascertain that L-citrulline production resulted from NO synthase activity, parallel samples were processed in the presence of 100 µmol/l of L-NAME. To measure the contribution of iNOS, tissues were incubated in the absence of calcium. The values were expressed in cpm/mg protein.

Superoxide assay. Superoxide anion production in the ischemic and nonischemic lobes of liver was measured by the method of Cherry et al. (1990). Tissue samples (60-170 mg) were incubated in Krebs-bicarbonate buffer (pH 7.4) consisting of (in mM): NaCl, 118; KCI, 4.7; CaCl₂, 1.5; NaHCO₃, 25; MgSO₄, 1.1; KH₂PO₄, 1.2; and glucose, 5.6. The tissues were gassed with 95% O₂, and 5% CO₂ for 30 min and were placed in plastic scintillation vials containing 0.25 mM lucigenin in 1 ml of Krebs-bicarbonate buffer containing HEPES (pH 7.4). The chemiluminescence elicited by superoxide in the presence of lucigenin was measured with a Mark 5303 scintillation counter (TmAnalytic, Elk Grove Village, IL). After 3 min of dark adaptation, vials containing only the cocktail (blanks) were counted three times for 6 s each time. The tissue samples were subsequently added to vials, allowed 3 min of dark adaptation and counted twice (6 s each time).

Staining with anti-nitrotyrosine monoclonal antibody. Tissue samples of the ischemic and nonischemic lobes of liver were fixed in 4% paraformaldehyde solution for 4 h, washed with PBS (0.1 M, pH 7.2) and immersed in 10% sucrose solution at 4°C before staining. The tissues were embedded with Cryo-Gel medium (Instrumedics Inc., Hackensack, NJ) and frozen at 70°C for 30 min. At 25°C, 8-µm-thick sections were cut, placed on slides and fixed with acetone. The sections were treated with blocking solution (Vectastain Universal Quick kit, Vector Lab, Burlingame, CA) for 30 min, then incubated with monoclonal anti-nitrotyrosine antibody (Upstate Biotechnology, 2 mg/ml, in 0.1 M PBS pH 7.2, contained 0.1% bovine serum albumin) for 30 min. Sections were incubated with biotinylated universal secondary antibody (Vectastain Universal Quick kit, Vector Lab) for 20 min, then incubated with streptavidin/peroxidase preformed complex (Vectastain Universal Quick kit, Vector Lab) for 20 min. A 5-min washing in 0.1 M PBS (pH 7.2) was performed between each step. Some sections were incubated with mouse nonspecific IgG (Vector Lab). A solution of 3,3-diaminobenzidine (Sigma, 0.5 mg/ml in 0.1 M PBS, pH 7.2) was used as the chromogen.

Histology. Ischemic and nonischemic lobes of liver of tissue were fixed by immersion in 10% formalin solution. The tissues were dehydrated in ethanol and embedded in paraffin. Sections (8 µm) were cut with a microtome, adhered to glass slides with poly-L-lysine and stained with hematoxylin and eosin for light microscopic examination. PMNs were counted in 50 HPF at magnification, ×40.

RT-PCR amplification of mRNA. The liver tissues from rats of different groups were snap frozen in liquid nitrogen and stored at 70°C until analysis. Total cellular RNA was isolated by homogenizing tissues with a polytron homogenizer in RNA Stat-60 reagent (Tel-Test, Inc., Friendswood, TX). Total RNA was extracted by chloroform and then centrifuged at 12,000 × g for 15 min at 4°C. The RNA was precipitated by isopropanol and the pellet dissolved in diethyl pyrocarbonate water (Sigma, St. Louis, MO). Total RNA concentration was determined by spectrophotometric analysis at 260 nm wavelength, and 4 µg of total RNA was reverse transcribed into cDNA in 30 µl reaction mixture containing Superscript II (Gibco BRL, Gaithersburg, MD) and oligo (dT)12-18 primers. The cDNA was amplified by specific primers with Perkin-Elmer DNA Thermal Cycler 480. The amplification mixture contained 1 µl of 10 µM forward primer, 1 µl of 10 µM reverse primer, 5 µl of 10× buffer, 1.5 µl of 15 mM Mg⁺⁺, 5 µl of the reverse-transcribed cDNA samples and 1 µl of Taq polymerase. Primers were designed from the published cDNA sequences by the Oligo Primer Detection Program. The cDNA was amplified after determining the optimal number of cycles. The mixture was first incubated for 5 min at 94°C, then cycled 29 times at 94°C for 45 s, 60°C for 60 s and elongated at 72°C for 90 s. After amplification, the sample (10 µl) was separated on a 2% agarose gel containing .3 µg/ml (0.003%) of ethidium bromide, and bands were visualized and photographed by ultraviolet transillumination, and the size of each PCR product was determined by comparing to the standard DNA size marker. Relative quantification of gene expression was performed with the Image Master VDS program (Pharmacia Biotech, Piscataway, NJ). The designed primer sequences are shown as below:

Statistical analysis. Data were analyzed using one-way analysis of variance through the Sigma Stat program (Jandel Scientific Software, Jandel Corp., San Rafael, CA). Differences between groups were then determined using the Neuman-Keuls test. If test of normality failed, Dunn's test of analysis of variance by ranks was used. Groups were deemed to be significantly different from one another when P < .05.

	Results

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Plasma ALT activity. Partial hepatic no-flow ischemia for 30 min induced significant cell injury in the liver during the first 4 h of reperfusion as indicated by the greater than 13-fold increase of ALT activity in plasma of the I/R group. Inhibition of NOS by L-NAME (10 mg/kg) exacerbated the liver injury as evidenced by a 61% increase in ALT activity at 4 h of reperfusion compared with I/R (fig. 1).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Plasma ALT activities after 30 min of hepatic ischemia and 3 and 4 h of reperfusion in the sham control (blank), I/R with administration of saline (solid) or with L-NAME (cross) before reperfusion. Data represent means ± S.E. of five animals per group. *P < .05 compared with sham control; #P < .05 compared with I/R + L-NAME.

NOS activity. Total NOS activity in the ischemic lobes increased by approximately 2-fold compared to both nonischemic lobes and sham-operated animals. The activity of iNOS increased by more than 5-fold in the ischemic lobe compared with nonischemic lobes, and by approximately 8-fold compared with sham-operated control animals. The activity of iNOS accounted for 81% of total NOS activity (2, A and B). L-NAME administration markedly attenuated total NOS activity and iNOS activity compared with I/R animals.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   (A) Total NOS activity was measured in ischemic and nonischemic lobes of I/R (solid), I/R with L-NAME animals (cross) and sham-operated animals (blank). The data are presented as mean ± S.E. of six animals, and * P < .05 compared with sham-operated animals; # P < .05 compared with I/R with L-NAME. (B) The activity of iNOS was measured in ischemic and nonischemic lobes of I/R (solid), I/R with L-NAME (cross) and sham-operated animals (blank). The data are presented as mean ± S.E. of six animals, * P < .05 compared with sham-operated animal; # P < .05 compared with I/R with L-NAME. IL, ischemic lobe; NIL, nonischemic lobe.

Plasma and tissue nitrite/nitrate. Plasma nitrite/nitrate levels were increased in rats with hepatic ischemia-reperfusion injury compared with sham controls (fig. 3). L-NAME decreased the plasma levels of nitrite/nitrate in I/R group of rats. Nitrite/nitrate levels in the ischemic lobes of I/R group were markedly lower than that of the livers of sham control animals. On the other hand, the nitrite/nitrate level in the ischemic and nonischemic lobes of livers in the I/R + L-NAME group were decreased significantly compared with the livers of sham control animals (fig. 4).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Plasma nitrite/nitrate concentration after 30 min of hepatic ischemia and 3 and 4 h of reperfusion in the sham control (blank), I/R with administration of saline (solid) or with L-NAME (cross) before reperfusion. Data represent means ± S.E. of five animals per group. *P < .05 compared with sham control; #P < .05 compared with I/R with L-NAME.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Nitrite/nitrate concentration in the liver tissues after 30 min of hepatic ischemia and 4 h of reperfusion in the sham control (blank), I/R with administration of saline (solid) or with L-NAME (cross) before reperfusion. Data represent means ± S.E. of five animals per group. *P < .05 compared with sham control.

Superoxide generation. Figure 5 illustrates the superoxide generation in sham-operated control animals and in the ischemic and nonischemic lobes of the I/R and I/R + L-NAME groups of rats. At 4 h of reperfusion, the superoxide generation in the ischemic lobes of I/R rats significantly increased by 41% compared with the sham-operated animals. L-NAME administration enhanced superoxide generation as seen by a 2.6-fold increase in superoxide generation compared with sham control animals.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Superoxide generation in the liver tissues after 30 min of hepatic ischemia and 4 h of reperfusion in the sham control (blank), ischemic/reperfusion (I/R) with administration of saline (solid) or with L-NAME (cross) before reperfusion. Data represent means ± S.E.M. of 5 animals per group. *P < .05 compared with sham control.

Neutrophil quantitation. Quantitative evaluation of neutrophil infiltration and accumulation in the sinusoid of ischemic lobes of liver increased from 9 ± 2 in sham control animals to 215 ± 27 PMN/50 HPF in animals subjected to hepatic ischemia-reperfusion. Treatment with L-NAME drastically increased PMN accumulation in ischemic lobes of livers (436 ± 48, P < .05 compared with sham control and I/R) and in the nonischemic lobes (34 ± 3.2, P < .05 compared with sham control animals). However, despite the significant increase in accumulation of PMN in nonischemic liver lobes of I/R + L-NAME, there was no significant difference in neutrophil infiltration between nonischemic lobes of the I/R group and the livers of sham control animals (fig. 6).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Neutrophil accumulation in the liver was determined histologically after 30 min of hepatic ischemia and 4 h of reperfusion in the sham control (blank), I/R with administration of saline (solid) or with L-NAME (cross) before reperfusion. Neutrophils were counted in 50 high-power fields (HPF, ×400). Data represent means ± S.E. of n = 5 animals per group. *P < .05 compared with sham control; #P < .05 compared with I/R with L-NAME animals.

Circulating neutrophils. Hepatic I/R not only induced regional inflammatory responses, but also induced a systemic inflammatory response, as shown by a 4.4-fold increase in circulating neutrophils in the I/R group compared with sham-operated animals (P < .05) at 4 h of reperfusion. Administration of L-NAME did not affect the number of circulating neutrophils (fig. 7).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   I/R (solid) induced a systemic inflammatory response manifested as a 4.43-fold increase in circulating neutrophils compared with sham-operated animals (blank). Application of L-NAME did not affect the circulating neutrophils between the I/R (solid) and I/R with L-NAME (cross) groups. The results are presented as mean ± S.E. of six animals. * P < .05 compared with sham-operated group.

Peroxynitrite formation. Peroxynitrite formation in the tissue sections was examined by immunohistochemical staining with monoclonal anti-nitrotyrosine antibody. Nitrotyrosine reacts with this highly specific antibody and forms dark deposits when reacted with chromogen. In sham-operated liver sections and in nonischemic liver sections, small amounts of dark deposit formation indicated very little immunoreactivity of specific antibodies with their substrate, nitrotyrosine (data not shown). However, significant dark staining was found in the ischemic lobe of liver, which indicates the presence of nitrotyrosine (fig. 8a). Attenuation of immunoreaction with anti-nitrotyrosine antibody (fig. 8b) was found in liver sections of I/R with L-NAME animals. The section shown in fig. 8c (ischemic lobe of liver) was treated with nonimmune mouse IgG and shows little background staining, which thus confirms the specificity of the immunohistochemical staining reaction.

View larger version (86K): [in this window] [in a new window]   Fig. 8.   Nitrotyrosine formation in the ischemic and nonischemic lobes of liver of rats subjected to 30 min of hepatic ischemia followed by 4 h reperfusion as seen by immunohistochemical staining with monoclonal anti-nitrotyrosine antibody. Significant dark staining was found in the ischemic lobe of liver, which indicates the presence of nitrotyrosine (a). Attenuation of formation of dark deposits was seen in liver of I/R rat with L-NAME (b). Section shown in panel c (ischemic lobe of liver) was treated with nonimmune mouse IgG and shows little background staining, which confirms the specificity of the immunohistochemical staining reaction (magnification, ×400).

RT-PCR of adhesion molecules. The mechanism of enhancement of neutrophil migration in the liver was studied at the molecular level by use of RT-PCR. There was virtually no gene expression of P-selectin or ICAM-1 in nonischemic lobes of the liver. The gene expression of these two adhesion molecules increased substantially in hepatic lobes subjected to I/R. Nonselective inhibition of NOS by L-NAME increased P-selectin (1.6-fold) and ICAM-1 (1.7-fold) gene expression in the ischemic lobes of liver of I/R + L-NAME rats as compared with I/R rats (fig. 9).

View larger version (19K): [in this window] [in a new window]   Fig. 9.   RT-PCR analysis of mRNA for ICAM-1 (lanes 2-6) and P-selectin (lanes 7-11) in I/R rats treated with L-NAME. A gel marker of 100-1,000 base pairs (lane 1, 12) was used to verify that the products were the predicted size. Relative quantification of gene expression was measured with the Image Master VDS program (Pharmacia Biotech). Lanes 1, 12, GelMarker; lanes 2, 7, sham control; lanes 3, 8, I/R ischemic lobe; lanes 4, 9, I/R nonischemic lobe; lanes 5, 10, I/R + L-NAME ischemic lobe; lanes 6, 11, I/R + L-NAME nonischemic lobe.

	Discussion

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Hepatic ischemia-reperfusion injury induced by severe circulatory shock and/or local operation usually is characterized by endothelial dysfunction, i.e., decreased endothelium-derived factors such as NO (Tsao et al., 1990; Wang et al., 1993), and increased leukocyte adhesion (Liu et al., 1994). Local formation of NO is not only proinflammatory where it has been implicated in edema formation and is potentially cytotoxic (possibly through its conversion to peroxynitrite), but NO also has anti-inflammatory properties, such as inhibition of platelet aggregation and neutrophil adherence to endothelium (Gauthier et al., 1994; Kubes et al., 1991). Thus the role of NO in ischemia-reperfusion is unclear because of its dual and conflicting properties.

In our model of hepatic ischemia-reperfusion injury, there was a marked increase in NOS activity in the liver. L-NAME is an L-arginine analog and a nonselective inhibitor of NOS, which directly inhibits L-arginine from interacting with NOS. Inhibition of NOS by administration of L-NAME significantly exacerbated hepatic ischemia-reperfusion injury reflected by an increase in plasma ALT activity in the I/R + L-NAME animals compared with that in I/R group (fig. 1). The mechanism of enhancement of hepatic ischemia-reperfusion injury by L-NAME is probably caused by the increase in generation of superoxide (fig. 5) and neutrophil accumulation in the liver (fig. 6). Our previous work showed that Kupffer cells are the main source of ROS formation at the early phase of reperfusion, but at later periods of reperfusion, neutrophils accumulate in liver sinusoids and also contribute to the postischemic oxidant stress (Liu et al., 1995).

NO recently has been shown to play an important role in regulation of neutrophil adherence in the postcapillary venule (Gauthier et al., 1994; Kubes et al., 1991). Our data are consistent with the finding that neutrophil accumulation was increased by inhibition of NO production with L-NAME (Liu et al., 1995). An important question to be addressed is the mechanism by which NO is able to attenuate neutrophil accumulation in the sinusoids of the liver tissue during reperfusion. Leukocyte-endothelial interactions involve a complex interplay among adhesion glycoproteins (i.e., selectins, integrins, and members of the immunoglobulin superfamily). Leukocyte rolling is the first step in leukocyte-endothelial interaction and facilitates leukocyte activation, adherence and transendothelial migration. One important adhesion molecule involved in early leukocyte-endothelial interaction is P-selectin, which is rapidly translocated from the Weibel-Palade bodies to the endothelial cell surface upon activation by inflammatory mediators such as C3a, C5a, hypoxia-reoxygenation or ROS. Gauthier et al. (1994) demonstrated that effects of exogenous NO on leukocyte-endothelial interaction after ischemia-reperfusion apparently are attenuated partially through the endothelial adhesion molecule P-selectin. Additionally, inhibition of NOS leads to increased expression of CD11/CD18 adhesion molecules on neutrophils, enhanced CD11/CD18-dependent attachment of leukocytes to the vessel wall and increased migration into the extravascular place (Kubes et al., 1991). Our results show that there is increased hepatic P-selectin and ICAM-1 mRNA expression after ischemia-reperfusion. This increase is augmented by L-NAME, which lends support to the postulate that NO attenuates mRNA expression of these adhesion molecules. The importance of NO in ischemia-reperfusion also has been demonstrated by the protective effects of administration of exogenous NO (Rossaint et al., 1993). In this regard, NO gas or NO donors have been shown to attenuate the severity of reperfusion injury. This may be caused partly by the capacity of NO to down-regulate adhesion molecule gene expression.

NO levels in the ischemic lobes of liver were attenuated compared with nonischemic and sham control (fig. 4). This attenuation may be caused by the reaction of NO with superoxide to form peroxynitrite (Beckman et al., 1990; Petros et al., 1991). In addition, NO may react with thiols and iron cysteines to produce stable nitrosothiols and dinitrosyl iron-cysteines, which are more stable than NO (Stamler et al., 1992). GSH is the most important intracellular thiol present in mammalian cells and its concentration varies over a wide range (0.5-10 mM), whereas plasma GSH concentrations are in the micromolar range. One of its well-established functions as a cosubstrate of glutathione peroxidase is to scavenge intracellularly generated ROS. Almost all of N₂O₃ converted from NO at a rate constant of 7 × 10⁶ M^-2 s^-1 (Kharitonov et al., 1995) is consumed in GS-NO formation in cells in which GSH concentrations are 5 mM or higher (DeMaster et al., 1995; Kharitonov et al., 1995). Because the intracellular hepatocyte concentration of GSH is higher than 5 mM (Liu et al., 1994), N₂O₃ reacts with thiols to form RS-NO. At lower concentration of GSH, nitrosation of GSH by N₂O₃ will become progressively less significant. Thus, NO levels would be significantly affected by the intracellular GSH in the liver tissue, but be less affected by the plasma GSH. This could explain, in part, the discrepancy in liver versus plasma NO levels after hepatic ischemia-reperfusion injury.

During acute inflammation, NO and superoxide are formed simultaneously in the activated cells at high concentrations (µM) (Freeman et al., 1995). In in vitro systems, peroxynitrite can be converted to hyroxyl radicals (Pryor and Squadrito, 1995), one of the most ROS (Wang et al., 1994). It can also rapidly oxidize sulfhydryl groups and thioethers, as well as nitration and hydroxylation of aromatic amino acids, such as tyrosine, tryptophan and guanine (Salman-Tabcheh et al., 1995). Although tyrosine nitration can occur with Cl-ONO and/or Cl-NO₂, formed by the reaction of NO₂ with HOCl in vitro (Eiserich et al., 1996), the major tyrosine nitration most likely is caused by peroxynitrite, because the reaction of NO with superoxide is a very rapid radical-radical terminal reaction with high reaction rate constant (Beckman et al., 1990). Recently, it has been reported that nitrotyrosine can also be formed from the reaction of nitrite and myeloperoxidase (Eiserich et al., 1996). The study was performed in vitro and required high concentrations of nitrite. Furthermore, in our study, absolute levels of nitrite in the ischemic lobes of the liver were low compared with the nonischemic lobes of liver. Thus, we can state with some degree of certainty that in hepatic ischemia-reperfusion the nitrotyrosine immunostaining was at least in part, a result of peroxynitrite nitration of tyrosine residues, and this formation of nitrotyrosine in the ischemic lobes of liver was greater than in control livers.

Nitrotyrosine is an important marker of peroxynitrite formation (Pryor and Squadrito, 1995). The production of peroxynitrite, demonstrated indirectly by increased nitrotyrosine immunoreactivity, recently has been demonstrated in endotoxin shock and hemorrhagic shock (Szabo et al., 1995a, b), in patients with arthritis (Kaur and Halliwell, 1994), in myocardial ischemia-reperfusion in rats (Liu et al., 1997) and in lung ischemia-reperfusion in vitro (Ischiropoulos et al., 1995; Kooy et al., 1995). In our study, immunohistochemical staining with monoclonal anti-nitrotyrosine antibodies demonstrated the formation of nitrotyrosine, which correlated with the high concentration of superoxide (fig. 8, a and c) in the ischemic tissue. Administration of L-NAME decreased NO production (figs. 3 and 4), and inhibited NOS activity (fig. 2), which were associated with decreased immunohistochemical staining of nitrotyrosine in I/R liver (fig. 8b). Recently, Miles et al. (1996) showed that peroxynitrite forms from equimolar concentration of NO and superoxide and that inequality of either precursor greatly limits peroxynitrite production (Miles et al., 1996). Our studies showing that reduction of NO production by L-NAME decreases peroxynitrite formation is consistent with the report by Miles and co-workers (1996). However, because evidence of peroxynitrite formation is based on correlation analysis and indirect evidence, the development of specific peroxynitrite probes, such as potent and specific peroxynitrite scavengers, is needed for further experiments.

The administration of L-NAME attenuates nitrotyrosine formation immunoreactivity; however, this attenuation did not reduce liver damage. In fact, plasma ALT activity (fig. 1) and superoxide generation in the ischemic lobes of liver (fig. 5) were increased. Significant enhancement of neutrophil infiltration may account for the detrimental effects of L-NAME. Elevation of neutrophil accumulation in the liver (fig. 6) correlated with the increase in superoxide production in the I/R with L-NAME rats (fig. 5), which suggests that the neutrophils are the major source of the increased superoxide. Lefer et al. (1997) have demonstrated that at physiological concentrations (nM), peroxynitrite inhibits leukocyte-endothelial cell interactions and P-selectin expression, and exerts cytoprotective effects in myocardial ischemia-reperfusion injury. Thus, similar to NO, peroxynitrite may have conflicting biological actions, where it can be a highly reactive oxygen species or it may inhibit leukocyte-endothelial cell interaction.

Experimental and clinical studies have demonstrated that use of a NOS inhibitor could be beneficial in terms of hemodynamic parameters (Petros et al., 1991; Kooy et al., 1995; Minnard et al., 1994), such as elevation of vascular resistance and mean arterial pressure in septic shock and trauma patients. However, the effects of NOS inhibitors on the cellular immune-inflammatory functions are unclear. Our results demonstrate that NO modulates/regulates cell interactions and neutrophil migration in hepatic ischemia-reperfusion. Down-regulation of adhesion molecule gene expression may be a key mechanism of the modulatory actions of NO on leukocyte-endothelial cell interaction. This function of NO apparently is critical to maintenance of hepatic function after ischemia-reperfusion and is more important to the extent of the injury than the cytotoxic potential of NO and/or peroxynitrite. Nonselective inhibition of NOS decreases NO and peroxynitrite formation, but aggravates neutrophil infiltration and neutrophil-mediated tissue damage.