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reperfusion injury of the liver
Life Sciences, Vol. Printed in the U S A
50, pp.
1797-1804
Pergamon
Press
ROLE OF NITRIC OXIDE IN THE OXIDANT STRESS DURING ISCHEMIA/REPERFUSION INJURY OF THE LIVER
H. Jaeschke, 1 V.B. Schini and A. Farhood # Center for Exp.#rimental Therapeutics, Department of Medicine, Baylor College of Medicine, and ~Department of Pathology, The University of Texas Medical School, Houston, Texas 77030, USA (Received
in final
form March 27,
1992)
Summary The potential role of nitric oxide (NO) and its reaction product with superoxide, peroxynitrite, was investigated in a model of hepatic ischemiareperfusion injury in male Fischer rats in vivo. Pretreatment with the NO synthase inhibitor nitro-L-arginine (10 mg/kg) did neither affect the postischemic oxidant stress and liver injury during the initial reperfusion phase nor the subsequent infiltration of neutrophils into the liver and the later, neutrophil-induced injury phase. Furthermore, no evidence was found for a postischemic increase of the urinary excretion of nitrite, a stable oxidation metabolite of NO. In contrast, the administration of Salmonella enteritidis endotoxin (1 mg/kg) induced a significant diuresis in Fischer rats and an 800-folcl enhancement of the urinary nitrite excretion. Nitro-L-arginine pretreatment inhibited the endotoxin-induced nitrite formation by 97%. Hepatic cGMP levels, as index of NO formation in the liver, were only increased significantly after endotoxin administration but not after ischemia and reperfusion. Our results provide no evidence for any enhanced generation of NO or peroxynitrite either systemically or locally during reperfusion and therefore it is unlikely that any of these metabolites are involved in the oxidant stress and liver injury during reperfusion after hepatic ischemia. Ischemia-reperfusion injury of the liver is involved in the pathogenesis of shock, and it can occur after hepatic surgery and liver transplantation. Many pharmacological intervention studies have implicated reactive oxygen species as toxic mediators in hepatic reperfusion injury (for review: 1), but studies with the isolated perfused liver and in vivo could not find evidence for an intracellular reactive oxygen formation high enough to cause liver injury (2,3). In contrast, investigations documented the high antioxidant capacity of the liver and the ability to resist substantially higher levels of intracellular reactive oxygen than ever formed under pathophysiological conditions (2,4-6). On the other hand, the significant increase of plasma levels of glutathione disulfide (GSSG) in vivo during reperfusion (3) was shown to reflect an extracellular oxidant stress in the hepatic sinusoids (7) with Kupffer cells as the principal source of reactive oxygen (8). In support of these findings, an enhanced reduction of cytochrome c in the perfusate was found in the postischemic liver in vitro indicating the extracellular formation of superoxide during reperfusion (9). Isolation of Kupffer cells and polymorphonuclear leukocytes (neutrophils, PMN) from the postischemic liver demonstrated directly that only Kupffer cells but not neutrophils generated enhanced amounts of superoxide during the initial reperfusion period (10). Despite the strong evidence for an extracellular, Kupffer cell-derived oxidant stress, the nature of the oxidizing species responsible for the enhanced oxidation of plasma glutathione is not known. 1To whom correspondence should be addressed Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1798
Nitric Oxide and Reperfusion Injury
Vol. 50, No. 23, 1992
Nitric oxide (NO) is a major form of the endothelium-derived relaxing factor (EDRF). It is enzymatically synthesized from L-arginine by NO synthase (11). Its major function is a relaxation of vascular smooth muscle and inhibition of platelet aggregation (11) but NO can also have direct cytotoxic effects if generated in large quantities (12-14). Beckman et al. demonstrated recently in vitro that NO and superoxide can react to form peroxynitrite, which can oxidize sulfhydryl groups to the disulfide and, during spontaneous decomposition, can generate the highly reactive hydroxyl radical (15-17). Endothelial cells (18) smooth muscle cells (19), macrophages (12,13,20) and hepatocytes (21) can generate and release large amounts of NO. S nce there is evidence for the generation of tumor necrosis factor during ischemia-reperfusion injury of the liver (22) and endotoxin and cytokines are known inducers of NO generation in various cell types (19,20,22-24), we tested the hypothesis whether NO or peroxynitrite could be involved in the postischemic, Kupffer cell-derived oxidant stress and therefore be responsible for the initial reperfusion injury in the liver in vivo. Methods Male Fischer 344 rats (240 -270 g) were purchased from Harlan Sprague Dawley Inc. (Houston, Texas). The animals were pretreated with 10 mg/kg body wt. nitro-Larginine (Aldrich Chem., Milwaukee, Wisconsin) or saline (4 ml/kg) iv 1 h before ischemia or endotoxin. All other chemicals were obtained from Sigma Chemical Co. (St. Louis, Missouri). Short-term reperfusion experiments were performed as described (8): After anesthetizing the animal with pentobarbital, the carotid artery was cannulated with PE-50 tubing. The blood vessels supplying the median and left lateral hepatic lobes were occluded with an atraumatic Glover bulldog clamp for 45 min. Reflow was initiated by removal of the clamp. Blood samples (500 pl) were obtained before ischemia, at the end of the ischemic period and at various times during reperfusion. The blood samples were processed as decribed in detail (8) to determine total plasma glutathione (GSH and GSSG) and glutathione disulfide (GSSG) (25) as well as plasma alanine aminotransferase (ALT) activities (Sigma test kit DG 159-UV). At the end of the experiment pieces of the postischemic liver lobes were fixed in phosphate-buffered Formalin, 5-pm thick sections were cut and PMNs were stained using the naphthol AS-D chloroacetate esterase technique (26). PMNs were identified by positive staining and morphology and were counted in 50 high power fields (x400). Long-term reperfusion experiments were performed as described (27): Partial hepatic no-flow ischemia and reperfusion was performed as mentioned above. After initiating reperfusion, the abdominal incission was closed with 3-0 silk and wound clips and the animals were allowed to recover. Twenty-four hours later the animals were reanesthetized, a blood sample obtained from the vena cava for ALT determination and samples of the postischemic lobes were fixed in Formalin. PMNs were stained in 5-pm sections as described above and the area of necrosis was estimated by evaluating parallel sections stained with hematoxilin and eosin. Nitrite assay: Untreated animals were placed individually in metabolic cages with free access to food and water for 24 h. Then groups of 4 animals were treated as follows: a) endotoxin (1 mg/kg Salmonella enteritidis, iv); b) nitro-L-arginine (10 mg/kg, iv) followed by endotoxin 30 min later; c) hepatic no-flow ischemia for 45 min. Immediately after endotoxin injection or after initiating reperfusion and the end of surgery, the animals were taken back to the metabolic cages and monitored for another 24 h. Urine samples were diluted with water and analyzed for nitrite with Griess reagent (23,28) (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2% phosphoric acid): 400 pl of diluted urine and 400/JI of Griess reagent were mixed and incubated for 10-15 rain at room temperature and then the absorbance measured spectrophotometrically at 540 nm. Urine nitrite concentrations were determined by comparison with a calibration curve (0.5-10 pM) of sodium nitrite in water.
Vol. 50, No. 23, 1992
Nitric O x i d e and Reperfusion
Injury
1799
Cyclic GMP measurements: Liver slices, obtained from sham-operated controls, animals subjected to hepatic ischemia (45 min) and reperfusion (24 h) or endotoxemia (1 mg/kg), were incubated in Krebs-Henseleit bicarbonate buffer (pH 7.4) gassed with 95% 0?/5% CO? at 37°C for 30 min. Subsequently, the incubation was continued for another 3CTmin with-either no addition or in the presence of 100/JM nitro-L-arginine, 300/JM Larginine, a combination of nitro-L-arginine and L-arginine, or 100/JM SIN-1 (a gift from Hoechst, France). At the end of the incubation period, the tissues were dropped in 1 ml 5% TCA and frozen in liquid nitrogen until analysis. Cyclic GMP levels were determined after homogenization and extraction with a cGMP RIA kit (Biomedical Technologies Inc., Stoughton, MA). Statistics: Data are given as means _+ SE. Comparison of data sets were performed with paired and unpaired Student's t-test or Wilcoxon rank sum test. Results The effect of the nitric oxide synthase inhibitor nitro-L-arginine (29) was tested in an established model of hepatic ischemia-reperfusion injury in vivo. We demonstrated previously (7,8) that 45 min of normothermic, no-flow ischemia of the liver induced a postischemic oxidant stress (indicated by the increased plasma GSSG concentrations) and hepatocellular injury (indicated by the increase of plasma ALT activities) during the initial reperfusion period of 1 h. As shown in Figure 1, pretreatment with nitro-L-arginine did neither significantly affect the postischemic increase of plasma GSSG concentrations nor plasma ALT activities in this model. Moreover, the number of neutrophils accumulating in the liver during this initial reperfusion phase was not significantly different in nitro-Larginine-treated animals (53 _+ 9 PMNs/50 HPF) compared to controls (63 -+ 5 PMNs/50 HPF). Animals treated only with nitro-L-arginine did not show any increase of plasma GSSG levels or evidence for liver injury (Figure 1); drug treatment alone had also no effect on the hepatic neutrophil count (NoArg: 16 _ 2 PMNs/50 HPF; Controls: 13 _ 3). These results obtained with a single dose of 10 mg/kg nitro-L-arginine did not change significantly when the animals received two additional intravenous injections of 10 mg/kg, one at the end of the ischemic period and one after 30 min of reperfusion (data not shown). 15-
10-
1200.
P~osrno GSSG
I/RP
0--0= e--e
Plosma
/!
T
= NoArg +
I/RP
/k--Z~ = NoArg
/ / T
/
• /
600,
5"
o-------
Pre-I
I
.
15
30
45
Reperfus~on (rain)
o
60
Pre-I
I
15
30
45
Reperfuslon (rain)
FIG. 1 Plasma ALT activities (right graph) and plasma concentrations of GSSG (left graph) in hepatic ischemia-reperfusion injury. Samples were taken before ischemia, at the end of 45 min ischemia and at various times during reperfusion. Ischemia-reperfusion experiments were performed in untreated (I/RP) and nitro-L-arginine (10 mg/kg) pretreated animals (NoARG + I/RP). A sham-operated control group with only nitro-L-arginine pretreatment (NoArg) is also shown. Data are given as means ___SE of n =4 animals per group. *p<0.05 (NoArg versus NoArg + I/RP)
60
1800
Nitric Oxide and Reperfusion
Injury
Vol. 50, No. 23, 1992
To test whether any direct evidence for the enhanced production of nitric oxide can be found in vivo during reperfusion, the urinary excretion of nitrite (NO2-), a known metabolite of nitric oxide (20), was measured before and after hepatic ischemia. The total amount of NO~- excreted by animals during a 24 h observation period was very low in untreated anir~als and did not change significantly after hepatic ischemia (Figure 2). As a positive control experiment, animals excreted about 800-times more NO~" after administration of 1 mg/kg Salmonella enteritidis endotoxin (Figure 2). Pretreatmen'f with 10 mg/kg nitro-Larginine reduced the excretion of NOg- by more than 97%. Basal urine production (8.8 ml/24 h) increased after endotoxin injection by 226% (p <0.05); nitro-L-arginine treatment reduced the endotoxin-induced diuresis to 21% (p < 0.05; paired Student's t-test). Despite the high efficacy of nitro-L-arginine in the endotoxin model, the same treatment regimen was also not effective in preventing the later reperfusion injury phase (Table 1), which is thought to be mainly neutrophil-dependent (27). Nitro-L-arginine did neither affect significantly the accumulation of neutrophils in the postischemic liver nor the hepatocellular injury, as judged by the plasma ALT activities and histological assessment of liver necrosis (Table 1).
100000-
NO2--Excretion nrnol
100004 ,4
iiiiii
1000
100 50 I/RP
N
El" + No~g
FIG. 2 Cumulative urinary nitrite excretion after 45 min of hepatic ischemia (I/RP) or SaL enteritidis injection (1 mg/kg) in untreated animals (ET) and nitro-Larginine (10 mg/kg) pretreated animals (ET + NoArg). The nitrite excretion of each animal was determined for a 24 h period before treatment (solid bars) and for 24 h after treatment (diagonal bars). Data are given as means _+ SE of n =5 animals per group. *p<0.05 (before versus after treatment) #p<0.05 (ETversus ET + NoArg) TABLE I Effect of Nitro-L-Arginine Pretreatment on Hepatic Reperfusion Injury Control
Nitro-L-Arginine
5390 _+ 560
5980 _+ 1280
806 _+ 79 81 _+ 5
847 _+ 140 72 _+ 9
Plasma
ALT (U/I) Liver
Neutrophil (50HPF) Necrosis (%)
Animals were pretreated with nitro-L-arginine (10 mg/kg body wt.; i.v.) or saline 30 min before hepatic ischemia of 45 min. All animals were killed after 24 h of reperfusion. Liver injury was determined by measurement of alanine aminotransferase (ALT) activities in plasma and histological assessment of the area of necrosis (given as % of the total field). The accumulated neutrophils in the liver were counted in 50 high power fields (HPF; x400). Data are given as means -+ SE of n =4 animals per group.
Vol. 50, No. 23, 1992
Nitric Oxide and Reperfusion Injury
1801
To obtain more direct evidence for a potential induction of NO synthase and enhanced NO production in the liver, cGMP levels were measured in hepatic tissue. Since NO activates the soluble enzyme guanylyl cyclase, an increased generation of NO in the liver is expected to induce higher tissue cGMP levels. As shown in Figure 3, hepatic cGMP levels in tissue subjected to 45 min of ischemia and 24 h of reperfusion were not significantly different from those of sham-operated control animals. On the other hand, the significantly higher cGMP content in endotoxin-treated animals is indicative of the induction of NO synthase in the liver. To exclude a difference in the cGMP synthesis capacity in the tissue between the various experimental groups, liver slices were also incubated with SIN-l, a direct source of NO. As shown in Figure 3, liver tissue from all three groups had a similar capacity to generate cGMP. When tissue slices were incubated in vitro in the presence of 100/~M nitro-L-arginine, cGMP levels were reduced by 70% (Figure 4). This block could be partially overcome by high levels of L-arginine (Figure 4). These results support the conclusion that nitro-L-arginine is able to inhibit the hepatic NO synthase. A similar inhibitory effect of nitro-L-arginine in vitro was also seen in liver tissue obtained from animals after ischemia and reperfusion or endotoxemia (data not shown). 400
Hepot;¢ ¢GMP pr~ol/9
300. 20Q 100, 0
c
t/RP
ET
C L
I/RP + SIN 1
£'T J
FIG. 3 Cyclic GMP content in liver tissue obtained from sham-operated controls, animals subjected to 45 min of hepatic ischemia and 24 h of reperfusion or 24 h after endotoxin treatment (1 mg/kg SaL enteritidis). Liver slices were incubated in Krebs-Henseleit buffer for 30 min with either no addition (diagonal bars) or in the presence of 100/~M SIN-1 (cross-hatched bars). Data are given as means ___SE of n = 5 animals per group. * p<0.05 (sham-operated controls versus treated groups). 60
Hepotic cGMP pmol/9
4.0
20
C
N~At9
No~"9 L-At 9
L-At 9
FIG. 4 Cyclic GMP content in liver tissue obtained from sham-operated controls. Liver slices were incubated in Krebs-Henseleit buffer for 30 rain with either no addition (C) or in the presence of 100/IM nitro-L-arginine (NoArg), 300 /IM L-arginine (L-Arg) or a combination of NoArg and L-Arg. Data are given as means _ SE of n = 5 animals per group. *p<0.05 (C versus treated slices) #p<0.05 (NoArg versus NoArg + L-Arg).
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Nitric Oxide and Reperfusion Injury
Vol. 50, No. 23, 1992
Discussion The objective of the present paper was to test in an in vivo model the hypothesis that peroxynitrite, derived from NO and superoxide, could be involved in the postischemic oxidant stress and therefore be at least partially responsible for the initial reperfusion injury in the liver. Alternatively, NO could also be involved in the later, neutrophil-dependent injury phase (27). The hypothesis is based on in vitro studies demonstrating that NO and superoxide react very rapidly in aqueous solution to form peroxynitrite, a compound which was shown to oxidize sulfhydryl groups faster than hydrogen peroxide and, after protonation, generate the highly reactive hydroxyl radical (16). It was therefore hypothesized that this mechanism may play a role in the pathogenesis of inflammation and ischemia-reperfusion injury (15-17). Inhibition of NO synthesis in vivo with the NO synthase inhibitor nitro-L-arginine did neither affect GSSG formation nor hepatocellular injury during the initial reperfusion period. Nitro-L-arginine did also not attenuate reperfusion injury of the liver at a later time. Since there is strong evidence that Kupffer cells cause the postischemic oxidant stress and contribute to the initial injury (8,10) while neutrophils are mainly responsible for the later injury phase (27), it seems unlikely that NO and peroxynitrite are involved in the oxidant stress or the development of the reperfusion injury in the liver. This conclusion is supported by the lack of any increase of urinary nitrite excretion suggesting that neither NO nor peroxynitrite synthesis was enhanced during the entire 24 h reperfusion period. As a positive control experiment nitrite excretion was determined after endotoxin injection. In support of previous findings in vitro (12,20,23,24) and in vivo (24,30,31) endotoxin caused a dramatic increase of NO production as documented by an 800-fold enhancement of the urinary nitrite excretion, which was almost completely suppressed in animals pretreated with nitro-L-arginine. These experiments document the high sensitivity of the urinary nitrite measurement as index for NO formation in vivo as well as the high efficacy of the drug treatment. A potential criticism of these experiments could be that urinary nitrite excretion may not accurately reflect NO synthesis in the liver, i.e., systemic NO production may obscure more subtle local effects in the liver. We therefore determined cGMP levels in the liver. Any induction of NO synthase and the resulting increase of NO synthesis during ischemia and reperfusion should cause an activation of the soluble guanylyl cyclase and thus higher cGMP levels in the tissue. Similar to the urinary nitrite excretion, no significant increase of cGMP levels could be detected in the postischemic tissue while endotoxin substantially enhanced the hepatic cGMP content. Since SIN-1 induced similar cGMP levels in tissues of all three experimental groups, guanylyl cyclase activity was not impaired under these conditions. Thus, the hepatic cGMP content is a valid index of NO production. Furthermore, nitro-L-arginine was shown to be a very effective inhibitor for the hepatic NO synthase. Since the block could be overcome by L-arginine the data support the conclusion that NO is indeed responsible for the increase of tissue cGMP levels. Thus, neither urinary nitrite excretion as index for more systemic NO production, nor hepatic cGMP levels as indicator for local NO formation, provide evidence for any relevant increase of the NO generation in the liver, which could be sufficient to contribute to the postischemic oxidant stress and reperfusion injury. Evidence for a different role of NO was presented in a recent paper. Kubes et al. (32) demonstrated that inhibition of NO synthesis in the postcapillary venules of the cat mesentery in vivo increased leukocyte adherence and suggested that NO may be an endogenous modulator of leukocyte adherence to the endothelium. Our data do not support such a hypothesis for the liver. Nitro-L-arginine did neither cause hepatic neutrophil infiltration in untreated animals nor did it modulate neutrophil accumulation during reperfusion after hepatic ischemia. Thus, the findings in the postcapillary venules do not directly apply to the liver sinusoid. In summary, we demonstrated in this study that the effective inhibition of NO synthesis in vivo did not affect the Kupffer cell-induced oxidant stress and initial reperfusion
Vol. 50, NO. 23, 1992
Nitric Oxide and Reperfusion
Injury
1803
injury nor did it influence neutrophil accumulation and the neutrophil-dependent injury phase. No evidence was found for enhanced NO production either systemically (urinary NO2- ) or locally in the liver (tissue cGMP). It is therefore concluded that NO and peroxynitrite are unlikely to be involved in the postischemic oxidant stress and reperfusion injury after hepatic ischemia. Acknowledgment The authors thank Michael Fisher for expert technical assistance. This work was supported by National Institutes of Health grant GM-42957. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
H. JAESCHKE, Chem. Biol. Interact. 7._99115-136(1991). H. JAESCHKE, C.V. SMITH and J.R. MITCHELL, J. Clin. Invest. 8__11240-1246 (1988). J. METZGER, S.P. DORE and B.H. LAUTERBURG, Hepatology 8580-584 (1988). H. JAESCHKE, A.E. BENZICK, C.V. SMITH and J.R. MITCHELL, Biological Reactive Intermediates IV C.M. WlTMER, R.R. SNYDER, D.J. JOLLOW, G.F. KALF, J.J. KOCSIS and I.G. SIPES p. 295-298. Plenum Publ. Corp., New York (1990). H. JAESCHKE, C.V. SMITH and J.R. MITCHELL, Biochem. Biophys. Res. Comm. 150568-574 (1988). H. JAESCHKE and J.R. MITCHELL, Biochem. Biophys. Res. Commun..160140147 (1989). H. JAESCHKE, Free Rad. Res. Commun. 12-13737-743 (1991). H. JAESCHKE and A. FARHOOD, Am. J. Physiol. 260 G355-G362 (1991). T.R. WALSH, P.N. RAO, L. MAKOWKA and T.E. STARZL, J. Surg. Res. 4__9918-22 (1990). H. JAESCHKE, A.P. BAUTISTA, Z. SPOLARICS and J.J. SPITZER, Free Rad. Res. Commun. _15277-284 (1991). S. MONCADA, R.M.J. PALMER and E.A. HIGGS, Biochem. Pharmacol. 3.8817091715 (1989). J.B. HIBBS, R.R. TAINTOR, Z. VAVRIN and E.M. RACHLIN, Biochem. Biophys. Res. Commun. 15787-94 (1988). D.J. STUEHR and C.F. NATHAN, J. Exp. Med. 1691543-1553 (1989). T.R. BILLIAR, R.D. CURRAN, M.A. WEST, K. HOFMANN and R.L. SIMMONS, Arch. Surg. 1241416-1421 (1989). J.S. BECKMAN, T.W. BECKMAN, J. CHEN, P.A. MARSHALL and B.A. FREEMAN, Proc. Natl. Acad. Sci. (USA) 8! 1620-1624 (1990). R. RADI, J.S. BECKMAN, K.M. BUSH and B.A. FREEMAN, J. Biol. Chem. 266 4244-4250 (1991). R. RADI, J.S. BECKMAN, K.M. BUSH AND B.A. FREEMAN, Arch. Biochem. Biophys. 288481-487 (1991) R.M.J. PALMER, A.G. FERRIGE and S. MONCADA, Nature Lond. 327524-526 (1987). V.B. SCHINI, D.C. JUNQUERO, T. SCOI-F-BURDEN and P.M. VANHOUTFE, Biochem. Biophys. Res. Commun. 176114-121 (1991). M.A. MARLETrA, P.S. YOON, R. IYENGAR, C.D. LEAF and J.S. WISHNOK, Biochem. 278706-8711 (1988). R.D. CURRAN, T.R. BILLIAR, D.J. STUEHR, K. HOFMANN and R.L SIMMONS, J. Exp. Med. 1701769-1774 (1989). L.M. COLLETTI, D.G. REMICK, G.D. BURTCH, S.L. KUNKEL, R.M. STRIETER and D.A. CAMPBELL, J. Clin. Invest. 8__551936-1943(1990). R.G. KILBOURN and P. BELLONI, J. Natl. Cancer Inst. 8_22772-776 (1990). D.J. STUEHR and M.A. MARLETTA, Proc. Natl. Acad. Sci (USA) 8_227738-7742 (1985). H. JAESCHKE and J.R. MITCHELL, Methods Enzymol. 186 752-759 (1990).
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Injury
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W.C. MOLONEY, K. MCPHERSON and L. FLIEGELMAN, J. Histochem. Cytochem. 8200-207 (1960). H. JAESCHKE, A. FARHOOD, and C.W. SMITH, FASEB. J. 43355-3359 (1990). L.C. GREEN, D.A. WAGNER, J. GLOGOWSKI, P.L. SKIPPER~ J.S. WlSHNOK and S.R. TANNENBAUM, Anal. Biochem. 126131-138 (1982). P.K. MOORE, O.A. AL-SWAYEH, N.W.S. CHONG, R.A. EVANS and A. GIBSON, Br. J. Pharmacol. 99408-412 (1990). D.A. WAGNER, V.R. YOUNG and S.R. TANNENBAUM, Proc. Natl. Acad. Sci (USA) 8__04518-4521 (1983). T.R. BILLIAR, R.D. CURRAN, B.G. HARBRECHT, D.J. STUEHR, A.J. DEMETRIS and R.J. SIMMONS, J. Leukocyte Biol. 4__88565-569 (1990). P. KUBES. M. SUZUKI and D.N. GRANGER, Proc. Natl. Acad. Sci (USA) 8846514655 (1991).
50, pp.
1797-1804
Pergamon
Press
ROLE OF NITRIC OXIDE IN THE OXIDANT STRESS DURING ISCHEMIA/REPERFUSION INJURY OF THE LIVER
H. Jaeschke, 1 V.B. Schini and A. Farhood # Center for Exp.#rimental Therapeutics, Department of Medicine, Baylor College of Medicine, and ~Department of Pathology, The University of Texas Medical School, Houston, Texas 77030, USA (Received
in final
form March 27,
1992)
Summary The potential role of nitric oxide (NO) and its reaction product with superoxide, peroxynitrite, was investigated in a model of hepatic ischemiareperfusion injury in male Fischer rats in vivo. Pretreatment with the NO synthase inhibitor nitro-L-arginine (10 mg/kg) did neither affect the postischemic oxidant stress and liver injury during the initial reperfusion phase nor the subsequent infiltration of neutrophils into the liver and the later, neutrophil-induced injury phase. Furthermore, no evidence was found for a postischemic increase of the urinary excretion of nitrite, a stable oxidation metabolite of NO. In contrast, the administration of Salmonella enteritidis endotoxin (1 mg/kg) induced a significant diuresis in Fischer rats and an 800-folcl enhancement of the urinary nitrite excretion. Nitro-L-arginine pretreatment inhibited the endotoxin-induced nitrite formation by 97%. Hepatic cGMP levels, as index of NO formation in the liver, were only increased significantly after endotoxin administration but not after ischemia and reperfusion. Our results provide no evidence for any enhanced generation of NO or peroxynitrite either systemically or locally during reperfusion and therefore it is unlikely that any of these metabolites are involved in the oxidant stress and liver injury during reperfusion after hepatic ischemia. Ischemia-reperfusion injury of the liver is involved in the pathogenesis of shock, and it can occur after hepatic surgery and liver transplantation. Many pharmacological intervention studies have implicated reactive oxygen species as toxic mediators in hepatic reperfusion injury (for review: 1), but studies with the isolated perfused liver and in vivo could not find evidence for an intracellular reactive oxygen formation high enough to cause liver injury (2,3). In contrast, investigations documented the high antioxidant capacity of the liver and the ability to resist substantially higher levels of intracellular reactive oxygen than ever formed under pathophysiological conditions (2,4-6). On the other hand, the significant increase of plasma levels of glutathione disulfide (GSSG) in vivo during reperfusion (3) was shown to reflect an extracellular oxidant stress in the hepatic sinusoids (7) with Kupffer cells as the principal source of reactive oxygen (8). In support of these findings, an enhanced reduction of cytochrome c in the perfusate was found in the postischemic liver in vitro indicating the extracellular formation of superoxide during reperfusion (9). Isolation of Kupffer cells and polymorphonuclear leukocytes (neutrophils, PMN) from the postischemic liver demonstrated directly that only Kupffer cells but not neutrophils generated enhanced amounts of superoxide during the initial reperfusion period (10). Despite the strong evidence for an extracellular, Kupffer cell-derived oxidant stress, the nature of the oxidizing species responsible for the enhanced oxidation of plasma glutathione is not known. 1To whom correspondence should be addressed Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1798
Nitric Oxide and Reperfusion Injury
Vol. 50, No. 23, 1992
Nitric oxide (NO) is a major form of the endothelium-derived relaxing factor (EDRF). It is enzymatically synthesized from L-arginine by NO synthase (11). Its major function is a relaxation of vascular smooth muscle and inhibition of platelet aggregation (11) but NO can also have direct cytotoxic effects if generated in large quantities (12-14). Beckman et al. demonstrated recently in vitro that NO and superoxide can react to form peroxynitrite, which can oxidize sulfhydryl groups to the disulfide and, during spontaneous decomposition, can generate the highly reactive hydroxyl radical (15-17). Endothelial cells (18) smooth muscle cells (19), macrophages (12,13,20) and hepatocytes (21) can generate and release large amounts of NO. S nce there is evidence for the generation of tumor necrosis factor during ischemia-reperfusion injury of the liver (22) and endotoxin and cytokines are known inducers of NO generation in various cell types (19,20,22-24), we tested the hypothesis whether NO or peroxynitrite could be involved in the postischemic, Kupffer cell-derived oxidant stress and therefore be responsible for the initial reperfusion injury in the liver in vivo. Methods Male Fischer 344 rats (240 -270 g) were purchased from Harlan Sprague Dawley Inc. (Houston, Texas). The animals were pretreated with 10 mg/kg body wt. nitro-Larginine (Aldrich Chem., Milwaukee, Wisconsin) or saline (4 ml/kg) iv 1 h before ischemia or endotoxin. All other chemicals were obtained from Sigma Chemical Co. (St. Louis, Missouri). Short-term reperfusion experiments were performed as described (8): After anesthetizing the animal with pentobarbital, the carotid artery was cannulated with PE-50 tubing. The blood vessels supplying the median and left lateral hepatic lobes were occluded with an atraumatic Glover bulldog clamp for 45 min. Reflow was initiated by removal of the clamp. Blood samples (500 pl) were obtained before ischemia, at the end of the ischemic period and at various times during reperfusion. The blood samples were processed as decribed in detail (8) to determine total plasma glutathione (GSH and GSSG) and glutathione disulfide (GSSG) (25) as well as plasma alanine aminotransferase (ALT) activities (Sigma test kit DG 159-UV). At the end of the experiment pieces of the postischemic liver lobes were fixed in phosphate-buffered Formalin, 5-pm thick sections were cut and PMNs were stained using the naphthol AS-D chloroacetate esterase technique (26). PMNs were identified by positive staining and morphology and were counted in 50 high power fields (x400). Long-term reperfusion experiments were performed as described (27): Partial hepatic no-flow ischemia and reperfusion was performed as mentioned above. After initiating reperfusion, the abdominal incission was closed with 3-0 silk and wound clips and the animals were allowed to recover. Twenty-four hours later the animals were reanesthetized, a blood sample obtained from the vena cava for ALT determination and samples of the postischemic lobes were fixed in Formalin. PMNs were stained in 5-pm sections as described above and the area of necrosis was estimated by evaluating parallel sections stained with hematoxilin and eosin. Nitrite assay: Untreated animals were placed individually in metabolic cages with free access to food and water for 24 h. Then groups of 4 animals were treated as follows: a) endotoxin (1 mg/kg Salmonella enteritidis, iv); b) nitro-L-arginine (10 mg/kg, iv) followed by endotoxin 30 min later; c) hepatic no-flow ischemia for 45 min. Immediately after endotoxin injection or after initiating reperfusion and the end of surgery, the animals were taken back to the metabolic cages and monitored for another 24 h. Urine samples were diluted with water and analyzed for nitrite with Griess reagent (23,28) (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2% phosphoric acid): 400 pl of diluted urine and 400/JI of Griess reagent were mixed and incubated for 10-15 rain at room temperature and then the absorbance measured spectrophotometrically at 540 nm. Urine nitrite concentrations were determined by comparison with a calibration curve (0.5-10 pM) of sodium nitrite in water.
Vol. 50, No. 23, 1992
Nitric O x i d e and Reperfusion
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Cyclic GMP measurements: Liver slices, obtained from sham-operated controls, animals subjected to hepatic ischemia (45 min) and reperfusion (24 h) or endotoxemia (1 mg/kg), were incubated in Krebs-Henseleit bicarbonate buffer (pH 7.4) gassed with 95% 0?/5% CO? at 37°C for 30 min. Subsequently, the incubation was continued for another 3CTmin with-either no addition or in the presence of 100/JM nitro-L-arginine, 300/JM Larginine, a combination of nitro-L-arginine and L-arginine, or 100/JM SIN-1 (a gift from Hoechst, France). At the end of the incubation period, the tissues were dropped in 1 ml 5% TCA and frozen in liquid nitrogen until analysis. Cyclic GMP levels were determined after homogenization and extraction with a cGMP RIA kit (Biomedical Technologies Inc., Stoughton, MA). Statistics: Data are given as means _+ SE. Comparison of data sets were performed with paired and unpaired Student's t-test or Wilcoxon rank sum test. Results The effect of the nitric oxide synthase inhibitor nitro-L-arginine (29) was tested in an established model of hepatic ischemia-reperfusion injury in vivo. We demonstrated previously (7,8) that 45 min of normothermic, no-flow ischemia of the liver induced a postischemic oxidant stress (indicated by the increased plasma GSSG concentrations) and hepatocellular injury (indicated by the increase of plasma ALT activities) during the initial reperfusion period of 1 h. As shown in Figure 1, pretreatment with nitro-L-arginine did neither significantly affect the postischemic increase of plasma GSSG concentrations nor plasma ALT activities in this model. Moreover, the number of neutrophils accumulating in the liver during this initial reperfusion phase was not significantly different in nitro-Larginine-treated animals (53 _+ 9 PMNs/50 HPF) compared to controls (63 -+ 5 PMNs/50 HPF). Animals treated only with nitro-L-arginine did not show any increase of plasma GSSG levels or evidence for liver injury (Figure 1); drug treatment alone had also no effect on the hepatic neutrophil count (NoArg: 16 _ 2 PMNs/50 HPF; Controls: 13 _ 3). These results obtained with a single dose of 10 mg/kg nitro-L-arginine did not change significantly when the animals received two additional intravenous injections of 10 mg/kg, one at the end of the ischemic period and one after 30 min of reperfusion (data not shown). 15-
10-
1200.
P~osrno GSSG
I/RP
0--0= e--e
Plosma
/!
T
= NoArg +
I/RP
/k--Z~ = NoArg
/ / T
/
• /
600,
5"
o-------
Pre-I
I
.
15
30
45
Reperfus~on (rain)
o
60
Pre-I
I
15
30
45
Reperfuslon (rain)
FIG. 1 Plasma ALT activities (right graph) and plasma concentrations of GSSG (left graph) in hepatic ischemia-reperfusion injury. Samples were taken before ischemia, at the end of 45 min ischemia and at various times during reperfusion. Ischemia-reperfusion experiments were performed in untreated (I/RP) and nitro-L-arginine (10 mg/kg) pretreated animals (NoARG + I/RP). A sham-operated control group with only nitro-L-arginine pretreatment (NoArg) is also shown. Data are given as means ___SE of n =4 animals per group. *p<0.05 (NoArg versus NoArg + I/RP)
60
1800
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Vol. 50, No. 23, 1992
To test whether any direct evidence for the enhanced production of nitric oxide can be found in vivo during reperfusion, the urinary excretion of nitrite (NO2-), a known metabolite of nitric oxide (20), was measured before and after hepatic ischemia. The total amount of NO~- excreted by animals during a 24 h observation period was very low in untreated anir~als and did not change significantly after hepatic ischemia (Figure 2). As a positive control experiment, animals excreted about 800-times more NO~" after administration of 1 mg/kg Salmonella enteritidis endotoxin (Figure 2). Pretreatmen'f with 10 mg/kg nitro-Larginine reduced the excretion of NOg- by more than 97%. Basal urine production (8.8 ml/24 h) increased after endotoxin injection by 226% (p <0.05); nitro-L-arginine treatment reduced the endotoxin-induced diuresis to 21% (p < 0.05; paired Student's t-test). Despite the high efficacy of nitro-L-arginine in the endotoxin model, the same treatment regimen was also not effective in preventing the later reperfusion injury phase (Table 1), which is thought to be mainly neutrophil-dependent (27). Nitro-L-arginine did neither affect significantly the accumulation of neutrophils in the postischemic liver nor the hepatocellular injury, as judged by the plasma ALT activities and histological assessment of liver necrosis (Table 1).
100000-
NO2--Excretion nrnol
100004 ,4
iiiiii
1000
100 50 I/RP
N
El" + No~g
FIG. 2 Cumulative urinary nitrite excretion after 45 min of hepatic ischemia (I/RP) or SaL enteritidis injection (1 mg/kg) in untreated animals (ET) and nitro-Larginine (10 mg/kg) pretreated animals (ET + NoArg). The nitrite excretion of each animal was determined for a 24 h period before treatment (solid bars) and for 24 h after treatment (diagonal bars). Data are given as means _+ SE of n =5 animals per group. *p<0.05 (before versus after treatment) #p<0.05 (ETversus ET + NoArg) TABLE I Effect of Nitro-L-Arginine Pretreatment on Hepatic Reperfusion Injury Control
Nitro-L-Arginine
5390 _+ 560
5980 _+ 1280
806 _+ 79 81 _+ 5
847 _+ 140 72 _+ 9
Plasma
ALT (U/I) Liver
Neutrophil (50HPF) Necrosis (%)
Animals were pretreated with nitro-L-arginine (10 mg/kg body wt.; i.v.) or saline 30 min before hepatic ischemia of 45 min. All animals were killed after 24 h of reperfusion. Liver injury was determined by measurement of alanine aminotransferase (ALT) activities in plasma and histological assessment of the area of necrosis (given as % of the total field). The accumulated neutrophils in the liver were counted in 50 high power fields (HPF; x400). Data are given as means -+ SE of n =4 animals per group.
Vol. 50, No. 23, 1992
Nitric Oxide and Reperfusion Injury
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To obtain more direct evidence for a potential induction of NO synthase and enhanced NO production in the liver, cGMP levels were measured in hepatic tissue. Since NO activates the soluble enzyme guanylyl cyclase, an increased generation of NO in the liver is expected to induce higher tissue cGMP levels. As shown in Figure 3, hepatic cGMP levels in tissue subjected to 45 min of ischemia and 24 h of reperfusion were not significantly different from those of sham-operated control animals. On the other hand, the significantly higher cGMP content in endotoxin-treated animals is indicative of the induction of NO synthase in the liver. To exclude a difference in the cGMP synthesis capacity in the tissue between the various experimental groups, liver slices were also incubated with SIN-l, a direct source of NO. As shown in Figure 3, liver tissue from all three groups had a similar capacity to generate cGMP. When tissue slices were incubated in vitro in the presence of 100/~M nitro-L-arginine, cGMP levels were reduced by 70% (Figure 4). This block could be partially overcome by high levels of L-arginine (Figure 4). These results support the conclusion that nitro-L-arginine is able to inhibit the hepatic NO synthase. A similar inhibitory effect of nitro-L-arginine in vitro was also seen in liver tissue obtained from animals after ischemia and reperfusion or endotoxemia (data not shown). 400
Hepot;¢ ¢GMP pr~ol/9
300. 20Q 100, 0
c
t/RP
ET
C L
I/RP + SIN 1
£'T J
FIG. 3 Cyclic GMP content in liver tissue obtained from sham-operated controls, animals subjected to 45 min of hepatic ischemia and 24 h of reperfusion or 24 h after endotoxin treatment (1 mg/kg SaL enteritidis). Liver slices were incubated in Krebs-Henseleit buffer for 30 min with either no addition (diagonal bars) or in the presence of 100/~M SIN-1 (cross-hatched bars). Data are given as means ___SE of n = 5 animals per group. * p<0.05 (sham-operated controls versus treated groups). 60
Hepotic cGMP pmol/9
4.0
20
C
N~At9
No~"9 L-At 9
L-At 9
FIG. 4 Cyclic GMP content in liver tissue obtained from sham-operated controls. Liver slices were incubated in Krebs-Henseleit buffer for 30 rain with either no addition (C) or in the presence of 100/IM nitro-L-arginine (NoArg), 300 /IM L-arginine (L-Arg) or a combination of NoArg and L-Arg. Data are given as means _ SE of n = 5 animals per group. *p<0.05 (C versus treated slices) #p<0.05 (NoArg versus NoArg + L-Arg).
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Vol. 50, No. 23, 1992
Discussion The objective of the present paper was to test in an in vivo model the hypothesis that peroxynitrite, derived from NO and superoxide, could be involved in the postischemic oxidant stress and therefore be at least partially responsible for the initial reperfusion injury in the liver. Alternatively, NO could also be involved in the later, neutrophil-dependent injury phase (27). The hypothesis is based on in vitro studies demonstrating that NO and superoxide react very rapidly in aqueous solution to form peroxynitrite, a compound which was shown to oxidize sulfhydryl groups faster than hydrogen peroxide and, after protonation, generate the highly reactive hydroxyl radical (16). It was therefore hypothesized that this mechanism may play a role in the pathogenesis of inflammation and ischemia-reperfusion injury (15-17). Inhibition of NO synthesis in vivo with the NO synthase inhibitor nitro-L-arginine did neither affect GSSG formation nor hepatocellular injury during the initial reperfusion period. Nitro-L-arginine did also not attenuate reperfusion injury of the liver at a later time. Since there is strong evidence that Kupffer cells cause the postischemic oxidant stress and contribute to the initial injury (8,10) while neutrophils are mainly responsible for the later injury phase (27), it seems unlikely that NO and peroxynitrite are involved in the oxidant stress or the development of the reperfusion injury in the liver. This conclusion is supported by the lack of any increase of urinary nitrite excretion suggesting that neither NO nor peroxynitrite synthesis was enhanced during the entire 24 h reperfusion period. As a positive control experiment nitrite excretion was determined after endotoxin injection. In support of previous findings in vitro (12,20,23,24) and in vivo (24,30,31) endotoxin caused a dramatic increase of NO production as documented by an 800-fold enhancement of the urinary nitrite excretion, which was almost completely suppressed in animals pretreated with nitro-L-arginine. These experiments document the high sensitivity of the urinary nitrite measurement as index for NO formation in vivo as well as the high efficacy of the drug treatment. A potential criticism of these experiments could be that urinary nitrite excretion may not accurately reflect NO synthesis in the liver, i.e., systemic NO production may obscure more subtle local effects in the liver. We therefore determined cGMP levels in the liver. Any induction of NO synthase and the resulting increase of NO synthesis during ischemia and reperfusion should cause an activation of the soluble guanylyl cyclase and thus higher cGMP levels in the tissue. Similar to the urinary nitrite excretion, no significant increase of cGMP levels could be detected in the postischemic tissue while endotoxin substantially enhanced the hepatic cGMP content. Since SIN-1 induced similar cGMP levels in tissues of all three experimental groups, guanylyl cyclase activity was not impaired under these conditions. Thus, the hepatic cGMP content is a valid index of NO production. Furthermore, nitro-L-arginine was shown to be a very effective inhibitor for the hepatic NO synthase. Since the block could be overcome by L-arginine the data support the conclusion that NO is indeed responsible for the increase of tissue cGMP levels. Thus, neither urinary nitrite excretion as index for more systemic NO production, nor hepatic cGMP levels as indicator for local NO formation, provide evidence for any relevant increase of the NO generation in the liver, which could be sufficient to contribute to the postischemic oxidant stress and reperfusion injury. Evidence for a different role of NO was presented in a recent paper. Kubes et al. (32) demonstrated that inhibition of NO synthesis in the postcapillary venules of the cat mesentery in vivo increased leukocyte adherence and suggested that NO may be an endogenous modulator of leukocyte adherence to the endothelium. Our data do not support such a hypothesis for the liver. Nitro-L-arginine did neither cause hepatic neutrophil infiltration in untreated animals nor did it modulate neutrophil accumulation during reperfusion after hepatic ischemia. Thus, the findings in the postcapillary venules do not directly apply to the liver sinusoid. In summary, we demonstrated in this study that the effective inhibition of NO synthesis in vivo did not affect the Kupffer cell-induced oxidant stress and initial reperfusion
Vol. 50, NO. 23, 1992
Nitric Oxide and Reperfusion
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injury nor did it influence neutrophil accumulation and the neutrophil-dependent injury phase. No evidence was found for enhanced NO production either systemically (urinary NO2- ) or locally in the liver (tissue cGMP). It is therefore concluded that NO and peroxynitrite are unlikely to be involved in the postischemic oxidant stress and reperfusion injury after hepatic ischemia. Acknowledgment The authors thank Michael Fisher for expert technical assistance. This work was supported by National Institutes of Health grant GM-42957. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
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26. 27. 28. 29. 30. 31. 32.
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W.C. MOLONEY, K. MCPHERSON and L. FLIEGELMAN, J. Histochem. Cytochem. 8200-207 (1960). H. JAESCHKE, A. FARHOOD, and C.W. SMITH, FASEB. J. 43355-3359 (1990). L.C. GREEN, D.A. WAGNER, J. GLOGOWSKI, P.L. SKIPPER~ J.S. WlSHNOK and S.R. TANNENBAUM, Anal. Biochem. 126131-138 (1982). P.K. MOORE, O.A. AL-SWAYEH, N.W.S. CHONG, R.A. EVANS and A. GIBSON, Br. J. Pharmacol. 99408-412 (1990). D.A. WAGNER, V.R. YOUNG and S.R. TANNENBAUM, Proc. Natl. Acad. Sci (USA) 8__04518-4521 (1983). T.R. BILLIAR, R.D. CURRAN, B.G. HARBRECHT, D.J. STUEHR, A.J. DEMETRIS and R.J. SIMMONS, J. Leukocyte Biol. 4__88565-569 (1990). P. KUBES. M. SUZUKI and D.N. GRANGER, Proc. Natl. Acad. Sci (USA) 8846514655 (1991).