Liver Biology and Pathobiology (Pathobiology)
Susceptibility of murine periportal hepatocytes to hypoxia-reoxygenation: Role for NO and Kupffer cell–derived oxidants
Article first published online: 27 MAY 2004
Copyright © 2004 American Association for the Study of Liver Diseases
Volume 39, Issue 6, pages 1544–1552, June 2004
How to Cite
Taniai, H., Hines, I. N., Bharwani, S., Maloney, R. E., Nimura, Y., Gao, B., Flores, S. C., McCord, J. M., Grisham, M. B. and Aw, T. Y. (2004), Susceptibility of murine periportal hepatocytes to hypoxia-reoxygenation: Role for NO and Kupffer cell–derived oxidants. Hepatology, 39: 1544–1552. doi: 10.1002/hep.20217
- Issue published online: 27 MAY 2004
- Article first published online: 27 MAY 2004
- Manuscript Accepted: 13 FEB 2004
- Manuscript Received: 27 AUG 2003
- NIH. Grant Number: DK 43785
Ischemia/reperfusion (I/R) is an important problem in liver resection and transplantation that is associated with hepatocellular dysfunction and injury. This study was designed to investigate whether a difference in hepatocyte susceptibility occurs in the periportal (PP) and/or perivenous (PV) zones in response to hypoxia/reoxygenation (H/R), and to delineate the mechanisms underlying this susceptibility. H/R was induced in an in situ perfused mouse liver model with deoxygenated Krebs-Henseleit buffer followed by oxygenated buffer. Selective destruction of PP or PV sites was achieved by digitonin perfusion into the portal or inferior vena cava, and was confirmed by histological evaluations and zone-specific enzymes. Hepatocellular injury was assessed by alanine aminotransferase (ALT) release. In whole liver, H/R significantly increased perfusate ALT. H/R of PP-enriched zones caused ALT release that was similar to that of whole liver (80 + 10 vs. 70 + 12 U/mg protein), consistent with significant PP hepatocyte injury. Minimal ALT release occurred in PV zones (10 + 5 U/mg protein). Administration of N-acetyl L-cysteine or a chimeric superoxide dismutase (SOD)—SOD2/3, a genetically engineered SOD—abrogated ALT release in H/R-perfused PP zones, implicating a role for superoxide (O). This elevated ALT release was attenuated by gadolinium chloride pretreatment, indicating that Kupffer cells are the O source. Enzymatic inhibition of cellular nitric oxide synthase (NOS) or genetic depletion of endothelial nitric oxide synthase (eNOS) aggravated hypoxia injury while exogenous NO and inducible nitric oxide synthase (iNOS) deficiency abolished reoxygenation injury. In conclusion, PP hepatocytes are more vulnerable to H/R; this injury is mediated directly or indirectly by Kupffer cell derived O and is limited by eNOS-derived NO. (HEPATOLOGY 2004;39:1544–1552.)
Previous studies in animal models show that ischemia/reperfusion (I/R) injury to the liver occurs in 2 distinct phases. The early reperfusion injury response occurs between 1 and 6 hours, and reactive oxygen species (ROS), such as superoxide (O), hydrogen peroxide, and/or hydroxyl radical produced during reperfusion,1–5 have been implicated in this acute hepatic injury process that is independent of leukocyte involvement. Because hepatic proliferation, which is an important determinant of a patient's survival after major hepatic resection, occurs from the periportal (PP) to perivenous (PV) zones,6 an understanding of the differential vulnerability of hepatic zones to postischemic injury would provide an important basis for preventing liver failure caused by alcoholic addiction or after major hepatectomy and liver transplantation. Given that metabolic heterogeneity of hepatic parenchymal cells occurs along the sinusoids in the acinus wherein zonal differences have been described for gradients of oxygen, hormones, and xenobiotic detoxification enzymes,7–9 the PP and PV regions of the liver are likely to exhibit differential vulnerability to I/R. Previous studies in rat liver showed that low-flow I/R induced oxidative stress and impaired midzonal regions.10, 11 In these studies, I/R were induced by changing flow rate that altered both perfusion rate and oxygen concentration. In other studies, Kato et al. found that hepatocytes from PP zones but nonparenchymal cells from PV zones were more susceptible to hepatic I/R in the rat.12 However, the mechanisms underlying differences in zonal susceptibility and tolerance to hepatic I/R remain poorly understood.
Kupffer cells (KCs) are favored as the critical cellular component in hepatic I/R injury because of their ability to release ROS, cytokines, and chemokines.13–15 Evidence that KC-derived ROS contribute to hepatic I/R injury comes from studies showing that I/R enhances KC phagocytic activity16 and that inactivation of KCs with gadolinium chloride (GdCl3) attenuated liver oxidative stress and plasma alanine aminotransferase (ALT) levels.13 Of note is a preferential localization of KCs to hepatic periportal zones, as well as a preferential activation of PP KCs during oxidative challenges like I/R17 and endotoxemia.18 Given this distinct and predominant PP distribution and activation of KCs along the sinusoid, a zonal sensitivity to I/R can be expected. The current study was designed to investigate whether a difference in susceptibility occurs in the PP and PV zones in response to hepatic hypoxia/reoxygenation (H/R) and to delineate the mechanisms underlying this susceptibility. To address these questions, we have adapted a previously established rat liver model of digitonin perfusion7 to develop a novel in situ mouse model of H/R to assess the sensitivity of PP and PV zones. This model will also enable us to determine the contribution of KC-derived ROS and nitric oxide (NO) to the injury response. The results indicate that H/R injury to hepatocytes primarily occurs in the PP zones and that this hepatocellular injury response is mediated by O derived from KCs. Our data further support a role for endothelial nitric oxide synthase (eNOS)-derived endogenous NO and for exogenous NO supplementation in protection against this H/R-induced PP injury.
Materials and Methods
Age-matched male wild-type (+/+) C57BL/6 mice and mice deficient in eNOS (-/-) and inducible nitric oxide synthase (iNOS [-/-]), 8 to 10 weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME) and allowed free access to laboratory chow and tap water. The animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg), and the livers were perfused with hemoglobin- and albumin-free Krebs-Henseleit solution (pH 7.4, 37°C) and gassed with carbogen (95% O2, 5% CO2) via 22-gauge intravenous catheters cannulated into the portal vein. Venous perfusates were collected from the inferior vena cava cannulated in the same manner.19, 20 Normal grade perfusion was conducted using a peristaltic pump at a constant rate of 3.0 mL/min/g liver in a single-pass mode. All animal protocols have been reviewed and approved by the institutional animal review committee in accordance with the animal use guidelines of the National Institutes of Health.
To obtain PP or PV zone-specific livers, a 3 mg/mL digitonin-Krebs Henseleit buffer was introduced into the inferior vena cava or portal vein at a flow rate of 0.3 mL/min/g liver. Selective zonal cell destruction was achieved within 30 seconds of digitonin perfusion. Washout of digitonin was achieved by perfusion with digitonin-free buffer into the opposite end to the initial digitonin perfusion. We have verified that this procedure provided efficient digitonin clearance and washout. After a -20-minute stabilization time after digitonin washout, the PP- or PV-enriched zones were subjected to H/R as described in Hypoxia/Reoxygenation Protocol.
For all experiments, the following standard H/R protocol was used: liver hypoxia was induced by perfusion with buffer equilibrated with 95% N2/5% CO2 mixture via the portal vein for 40 minutes, followed by perfusion with oxygenated buffer for an additional 40 minutes for the reoxygenation period.19, 21 Perfusates were sampled at times before hypoxia initiation, at the end of the hypoxic period, and at every 10 minutes during reoxygenation for determination of hepatic release of ALT. Animals were divided into controls and 10 experimental groups. In the control group, livers were from wild-type mice subjected to H/R to establish the baseline hepatocellular injury response to H/R without treatment with digitonin or other agents. In the experimental groups, livers from mice in groups 1 and 2 were perfused with digitonin to obtain enrichment of PP and PV zones and thereafter subjected to H/R to determine zonal injury responses. In the remaining experimental groups (3 to 10), H/R studies were performed in PP-enriched livers, and the roles of ROS and NO were examined. To test the role of ROS, livers from mice in groups 3 and 4 were treated with 5 mmol/L of N-acetyl-L-cysteine (NAC) or 1 unit/g body weight of a genetically engineered chimeric superoxide dismutase (SOD2/3). NAC was present throughout the H/R periods, and SOD2/3 was administered at the time of reoxygenation. To determine whether KCs were the ROS source, mice from group 5 were injected intraperitoneally with 10 mg/kg body weight of GdCl3 for 24 hours before prepping the livers for PP enrichment and H/R. Mice from groups 6 to 10 were used to test the role of NO. In group 6, livers were treated with 1 mmol/L N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of cellular NOS activity. To specifically inhibit iNOS, group 7 livers were treated with 5 mmol/L aminoguanidine. These inhibitors were administered at the time of hypoxia and were present throughout reoxygenation. To further explore the roles of eNOS and iNOS, livers from eNOS−/− or iNOS−/− mice were prepped for digitonin perfusion to acquire PP-enriched zones and then subjected to the standard H/R protocol in groups 8 and 9. Livers from group 10 mice were subjected to the standard H/R protocol in the presence of spermine NO (10 μmol/L), an NO donor. In some experiments, the role of NO was also evaluated in livers enriched with PV hepatocytes.
Measurements of Zone-Specific Enzymes in Venous Perfusates.
In each experiment, venous perfusate samples were collected before hypoxia, after 40 minutes of hypoxia, and at every 10 minutes during reoxygenation to determine the activities of ALT as an index of overall hepatocellular injury. ALT was quantified using a commercially available assay kit (Sigma, St. Louis, MO).7, 8 At the end of the H/R perfusion, livers were excised, and tissue ALT activities were determined. Pyruvate kinase (PK) activities were determined as previously described in venous perfusates and tissues as a PV zone-specific enzyme.7, 8, 22
In some experiments, liver histology was performed to determine the hepatic microarchitecture in PP- and PV-enriched livers after digitonin perfusion. Tissues were fixed in formalin, embedded in JB-4 plastic (Polysciences, Warrington, PA), and 5 μm sections were cut and stained with hematoxylin-eosin (HE).
All data were expressed as the mean ± SEM. Differences between the control and experimental groups were determined using one-way ANOVA with Fisher multiple-comparison test. P less than .05 was considered significant.
Establishment of PP- and PV-Enriched Zones by Digitonin Perfusion and Their Responses to H/R.
As shown in Fig. 1, complete and uniform clearance of blood from all the hepatic lobes was achieved with normal grade perfusion of the liver via the portal vein at a flow rate of 3 mL/min/g (Fig. 1A). Subsequent perfusion with digitonin via the portal vein (Fig. 1B) or the inferior vena cava (Fig. 1C) resulted in selective destruction of parenchymal cells uniformly throughout the PP and PV regions of the liver. Notably, the surfaces of the livers exhibited characteristic reticular patterns: destruction of PV zones appear as pale center cores of damaged PV cells surrounded by dark brown rings of undamaged PP cells; destruction of PP zones appear as pale rings of damaged PP cells surrounding dark brown center cores of undamaged PV cells.7 To validate that digitonin treatment did not compromise the hepatic microarchitecture, liver histology was performed. Control (nondigitonin-perfused) livers exhibited normal hepatic architecture with equally well-stained PP and PV areas (Figs. 2A–C). In digitonin-perfused livers, the hepatic architecture is well preserved, with demarcated regions of PP and PV hepatocytes and minimal hepatocellular necrosis and alterations in hepatic sinusoids (Figs. 2D–I). PP zones perfused by digitonin were evidenced by blanching and some sinusoidal dilatation; intact PV zones retained normal HE stain and architecture (Figs. 2E and F). Retention of normal stain in PP hepatocytes and blanched PV hepatocytes and some sinusoidal dilatation were evident in livers perfused via the inferior vena cava (Figs. 2H and I). Liver wet-to-dry weight ratios were determined to assess the effect of digitonin and H/R on tissue swelling. Livers perfused with digitonin (PP or PV zones) without H/R exhibited a small (≈10%) but significant increase in wet-to-dry weight ratios as compared to nondigitonin-treated (whole) livers (Fig. 3A), indicating that digitonin induced some fluid retention. However, the wet-to-dry weight ratios of sinusoidal endothelial cells, KCs, and hepatocytes prepared from elutriation of PP-enriched livers were not different from those isolated from untreated livers (Fig. 3B), showing a lack of cell swelling; thus, the digitonin-induced increase in hepatic fluid accumulation was largely extracellular. H/R exposure caused a significantly greater increase in wet-to-dry weight ratio in whole and PP-enriched livers as compared to the corresponding livers perfused with normoxic buffer (Fig. 3C), consistent with H/R-induced edema. Taken together, these results demonstrate that the digitonin method does provide a reasonable experimental approach for PP and PV enrichment to investigate zonal tolerance to hepatic I/R.
To verify that the targeted digitonin perfusion did, in fact, cause enrichment of the desired specific zones within the liver, we monitored the time course of release of PK, (a PV-zone–specific enzyme) with respect to ALT, an enzyme that is present in both the PP and PV zones but that is generally higher in PP regions.7, 8, 22 Digitonin administration into the inferior vena cava resulted in significant release of PK and ALT (Fig. 4A), consistent with selective destruction of PV cells. In contrast, digitonin perfusion into the portal vein caused release of ALT, but not PK (Figure 4A), consistent with selective destruction of PP cells. To validate that the digitonin perfusion into the portal vein or inferior vena cava yielded PV- or PP-enriched hepatocytes, we measured total hepatocellular activities of PK and ALT post–digitonin perfusion and washout. The results (Fig. 4B) show that PP-enriched hepatocytes exhibited lower PK and higher ALT enzyme activities, and higher PK and lower ALT activities were found in PV-enriched hepatocytes. Accordingly, the ratio of PK –to ALT—an index of zonal enrichment—revealed a significantly greater PK/ALT in PV-enriched zones compared to PP-enriched zones. Collectively, these results demonstrate that the procedure of using targeted digitonin perfusion via the portal vein or inferior vena cava provided satisfactory establishment of select hepatic zones enriched in PV or PP hepatocytes. In subsequent experiments, we have utilized this digitonin-perfusion approach to generate zone-specific hepatocytes to investigate the responses of PP or PV zones to H/R and to define the determinants of the injury response, namely, ROS and NO.
Figure 5A illustrates the kinetics of ALT release from untreated whole livers after a 40-minute perfusion with deoxygenated buffer followed by a 40-minute perfusion with oxygenated buffer. In some experiments, we determined the release of lactate dehydrogenase as an index of cellular damage, and the results were similar to those of ALT release from both PP- and PV-enriched livers. Because ALT is a more specific PP enzyme, we subsequently assayed perfusate ALT as the index of PP hepatocyte injury. There was no detectable increase in perfusate ALT activity during the hypoxia period. However, reoxygenation induced a time dependent increase in ALT that approached approximately 20% of total hepatic ALT activity over the 40-minute reoxygenation period. The effect of H/R on hepatocyte injury in PV- and PP-enriched livers is shown in Fig. 5B. Exposure of PP livers to H/R resulted in a time-dependent increase in ALT release that reached a value 6- to 8-fold greater at 40 minutes than at 0, indicating an enhanced vulnerability of PP hepatocytes to H/R. In comparison, H/R caused a small but insignificant increase in ALT in PV livers at 40 minutes of reoxygenation. There was no detectable release of PK in PV livers under these conditions (data not shown), indicating that the PV regions of the liver are relatively tolerant of the H/R insult. Notably, the magnitude of H/R-induced ALT release in PP livers was quantitatively similar to that of whole liver (Fig. 5A), suggesting that the H/R-induced hepatocellular injury in whole liver was predominantly attributed to hepatocyte damage in the PP zones.
Antioxidants and Depletion of KCs Abolish H/R-Induced PP Injury.
To determine a role for ROS in mediating PP injury, two strategies were used. First, ROS production was measured in perfusates using oxidation of dihydrorhodamine. However, while there was a trend for increased dihydrorhodamine oxidation with H/R (data not shown), these quantitative measurements in the perfusates (at 3ml/min/g single-pass mode) were at the limit of detection. Second, H/R studies were performed in the presence of antioxidants, namely, NAC, a thiol antioxidant, or SOD2/3, a genetically engineered chimeric SOD that combines the mature human manganese SOD2 with the heparin-binding C-terminal region of the human extracellular SOD3 that has an affinity for endothelial cell surfaces. Figure 6A shows that NAC treatment completely prevented the time-dependent increase in ALT induced by H/R in PP livers. Similarly, the presence of SOD2/3 was equally effective in preventing reoxygenation-induced ALT release (Fig. 6B). To determine whether KCs were the source of ROS, we pretreated mice with GdCl3 (10 mg/kg body weight) to inactivate KCs. Figure 7 shows that GdCl3 pretreatment completely abrogated the elevation in ALT caused by H/R. Taken together, these results implicate a role for ROS in mediating H/R-induced PP injury and are consistent with KCs being the source of the ROS.
eNOSDerived NO and Exogenous NO Attenuate H/R-Induced PP Injury.
Since NO is known to rapidly interact with and decompose O to nitrate and is an endogenous anti-inflammatory mediator,23 we determined whether H/R-induced PP injury is modulated by NO. Administration of L-NAME (1 mmol/L) to wild-type mice elicited marked PP hepatocellular injury during the hypoxia phase as evidenced by a significant increase in ALT release after 40-minute perfusion with deoxygenated buffer prior to reoxygenation (Fig. 8A). This enhanced ALT activity was maintained at an elevated level without further increases during the 40-minute reoxygenation period (open square) compared to that of wild-type mice (open circle). A role for eNOS was evaluated using PP-enriched livers from eNOS-deficient mice (eNOS−/−). The PP injury response during hypoxia in eNOS-deficient animals was similar to those after L-NAME treatment; this elevated ALT activity was likewise maintained throughout the 40-minute reoxygenation without further increases (Fig. 8B), indicating that a deficiency in the eNOS gene per se elicited marked PP injury during the hypoxia phase. In comparison, eNOS deficiency was without injurious effects on PV-enriched livers during hypoxia or reoxygenation (Fig. 8B, open triangle). That inhibition of cellular NOS or loss of eNOS is deleterious is consistent with a protective effect of endogenously generated NO in maintaining PP integrity during the hypoxia phase. Because a deleterious role of iNOS has been implicated in inflammatory processes, we evaluated its role in H/R-induced PP injury using iNOS-deficient mice (iNOS−/−). Figure 9A shows that iNOS deficiency afforded complete protection against H/R-induced ALT release, suggesting that iNOS upregulation during I/R promotes tissue damage. Similarly, treatment with aminoguanidine (5 mmol/L), a specific iNOS inhibitor, resulted in complete protection against H/R-induced PP injury (Fig. 9B), thus confirming a deleterious role for iNOS in postischemic injury. To investigate whether exogenously administered NO afforded protection against reoxygenation injury, mice were given spermine NO (10 μmol/L), an NO donor that produces 2 moles of NO per mole of parent compound. Administration of the NO donor was without effect in normoxic livers (Fig. 10A). However, the H/R-induced ALT release from PP livers was prevented in the presence of spermine NO (Fig. 10B). Measurements of nitrate and nitrite in liver perfusates were at their limit of detection; thus, the production of NO under the different pharmacological and genetic conditions cannot be readily assessed. Notwithstanding, the collective results demonstrate that (1) eNOS-derived NO is critical for maintaining PP hepatocyte integrity during hypoxia, (2) iNOS-derived NO is deleterious, and (3) exogenous NO contributes to protection against reoxygenation injury.
The purpose of the current study was to investigate potential differences in hepatocellular susceptibility in the different hepatic zones to I/R and the mechanisms underlying this differential susceptibility. We adopted a normal flow perfusion system by means of anoxic gas saturation (i.e., H/R model) to mimic the I/R model—without the complications caused by changes of blood flow and sheer stress—to evaluate the direct effect of oxygen deficit and oxidative stress.19, 21 To achieve enrichment of selective hepatic zones in mouse liver, we adapted the previously published in situ rat liver perfusion model, using digitonin perfusion for specific destruction of upstream or downstream regions of the liver along the hepatic sinusoid, to obtain PV or PP zones.7 Our results demonstrated for the first time the successful establishment of an in situ mouse model to obtain PP- and PV-enriched hepatic zones by the digitonin perfusion protocol. Histological evaluations showed that digitonin did not compromise the hepatic microarchitecture; clearly delineated PP and PV zones were evident after perfusion of digitonin into the inferior vena cava and portal vein. Despite some extracellular fluid accumulation and sinusoidal dilatation, there was no evidence of intracellular swelling in hepatocytes, KCs, and sinusoidal endothelial cells as a result of digitonin treatment. Zone enrichments were validated by analyses of the distribution of the PV-specific enzyme, PK, relative to ALT.7, 8 We have capitalized on this newly established experimental mouse model to investigate hepatic zonal susceptibility to H/R and to define the contributions of ROS and NO to the injury process.
Our data show that PP hepatocytes sustained greater H/R-induced injury than PV hepatocytes, in agreement with previous studies in rats.12 This enhanced susceptibility of PP hepatocytes to oxygen lack is consistent with a significantly higher oxygen requirement for maximal mitochondrial function and maintenance of cellular homeostasis in zone 1 hepatocytes.8, 24 Moreover, the results implicate KC-derived O in mediating this H/R-induced reoxygenation injury. Our conclusion is supported by several lines of evidence. First, evidence for a role for ROS comes from findings that H/R-induced ALT release is abolished by the administration of NAC25 and SOD2/3; notably, the attenuation by SOD2/3 suggests an involvement for O. Because SOD2/3 was engineered to attach to endothelial cell surfaces by means of its 26-amino-acid heparin-binding tail,26 the enzyme serves to dismutate O generated from within the lumen of the sinusoids (as of KCs) or produced from endothelial cells which then diffuses into the extracellular milieu. These results underscore the importance of the endothelial barrier in O elimination during reoxygenation and are in agreement with previous studies, in which the extent of oxidative damage of hepatic parenchymal cells was found to be a function of the balance between oxidant production from KCs and the antioxidant defenses of endothelial cells.27 Moreover, the results suggest that, clinically, exogenous administration of SOD2/3 and/or NAC may prove to be useful strategies against hepatic I/R injury in perfused storage livers for transplants. That KCs were the likely source of O was evidenced by the finding that inactivation of KCs with GdCl3 pretreatment essentially abolished the reoxygenation injury. The kinetics of reoxygenation injury (i.e., within 40 minutes) are consistent with a rapid activation of KCs and with the production of ROS and with the current paradigm that KC-derived O mediates an early reperfusion injury that is independent of neutrophil involvement.1–5 Notably, the finding that the H/R-induced hepatocyte injury was localized to the PP zones is consistent with the predominant location of KCs as well as the preferential activation of PP KCs during oxidative challenge, including that of I/R17, 18 in hepatic zone 1.
There are numerous reports that NO plays a protective role in hepatic I/R injury.28, 29 Our results show that hypoxia alone mediates PP hepatocyte injury in livers deficient in eNOS expression, suggesting that eNOS-derived NO is essential for PP hepatocyte survival during anoxia. It is unclear how eNOS affords protection against hypoxia-induced PP injury. One possibility is that eNOS-derived NO mediates dilation of terminal portal venules and sinusoids to maintain hepatic microcirculation after an ischemic/anoxic perturbation.27, 30 Since vascular smooth muscle cells of terminal portal venule and Ito cells control hepatic microcirculation, and Ito cells are distributed to PP zones,8 NO effects on these vasoresponsive cells could ultimately determine PP oxygenation and the extent of hypoxia-induced injury. The finding that hypoxia did not elicit PV injury provides additional support to the suggestion that eNOS-derived NO, smooth muscle cells, and the PP distribution of Ito cells participate in the hypoxic phase of the PP injury response. Curiously, the enzymatic inhibition of cellular NOS with L-NAME elicited a greater PP hypoxia-induced eNOS deficiency, presumably due to other vascular effects of L-NAME on blood flow.29, 30 Because neutrophils are not involved in early hypoxia/reoxygenation injury, we can rule out an effect of L-NAME on polymorphonuclear leukocytes. Protection of PP hepatocytes by endogenous NO against hypoxic damage may be related to the conservation of cellular bioenergetics for maintenance of tissue homeostasis during hypoxia via NO-mediated inhibition of the use of mitochondrially derived ATP for nonessential metabolic processes.8, 19, 31
It is notable that the hypoxia-induced elevation in ALT in eNOS-deficient mice was sustained throughout the reoxygenation period without additional enzyme release. This suggests that endogenously derived NO plays a minimal role in reoxygenation injury; however, the finding that an exogenous NO donor afforded protection against PP injury during reoxygenation indicates that NO is, in fact, cytoprotective in the reoxygenation phase. The enzymatic or cellular source of NO during reoxygenation is unknown. A potential candidate is iNOS. Sonin et al. have shown that iNOS messenger RNA was induced sixfold following 1 hour of hepatic ischemia and 6 hours of reperfusion.32 Other studies have demonstrated the induction of iNOS in KCs in a variety of inflammatory processes, including I/R, endotoxemia, and multiple organ failure.33–36 In this study, we found no increases in iNOS message or protein expression over the short reoxygenation times (40 minutes, data not shown); this is in agreement with our previous findings that iNOS induction is not detectable following hepatic I/R.28 These results are consistent with the paradigm that injury events in the early phase of reoxygenation were transcriptionally independent. Notwithstanding, we found that the H/R-induced injury was ameliorated in iNOS-deficient animals (Fig. 9). The reason for this curiosity is unclear; one possibility may be that iNOS gene depletion results in a compensatory upregulation of eNOS. Although an interesting possibility, there is no evidence to support it. Another possibility is that there may exist a baseline level of iNOS that can participate in the early initiation of I/R injury that is independent of transcriptional control.28, 36 The fact that NAC can inhibit NO production by iNOS suggests that the protective effect of NAC against H/R-induced PP injury may, in part, be related to its suppression of iNOS function,25 apart from its role as an antioxidant. The possibility that differential NO production resulting from eNOS and iNOS inhibition may explain our results cannot be satisfactorily resolved at present. The large perfusate volumes from single-pass perfusion precluded quantitative measurements of NO metabolites and assessment of NO formation under these conditions.
In summary, we have established an in situ mouse perfusion model to evaluate potential differences in susceptibility of PP and PV hepatocytes to hepatic H/R. Our results demonstrated marked zonal differences in susceptibility to H/R-induced damage, with the PP hepatocytes being more vulnerable than the PV hepatocytes. H/R-induced PP hepatocellular injury was mediated by KC-derived O, while endogenously generated NO by eNOS provided a restraint to the development of the hypoxic injury response.
The authors thank Drs. John Fuseler and Wen Ma and Ms. Carla Brown for assistance with the liver histology.
- 18Superoxide anion generation in the liver during the early stage of endotoxemia in rats. J Leukoc Biol 1990; 49: 123–128., , , .
- 24Metabolic zonation of carbohydrate metabolism in the liver. In: LemastersJJ, HackenbrockCR, ThurmanRG, eds. Integration of Mitochondrial Function. New York: Plenum Press, 1988: 561–579..