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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 3, 2005 as doi:10.1096/fj.04-3220fje. |
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,1
* Academic Division of Surgical and Interventional Sciences, University College London, London, UK;
Department of Surgery and Liver Transplantation Unit, Royal Free Hampstead NHS Trust Hospital, London, UK; and
Institute of Anatomy, University of Leipzig, Leipzig, Germany
1 Correspondence: Academic Division of Surgical and Interventional Sciences, University College London, Rowland Hill St., London NW3 2PF, UK. E-mail: a.seifalian{at}medsch.ucl.ac.uk
SPECIFIC AIM
The aim of this study was to identify nitric oxide synthase isoforms responsible for generation of the cytoprotective effect of nitric oxide during liver ischemic preconditioning in a rat model of lobar ischemia reperfusion injury.
PRINCIPAL FINDINGS
1) Ischemia reperfusion (IR) results in decreased eNOS expression and increased hepatocellular injury
To determine extent of liver injury with ischemia and reperfusion, rats were subjected to 45 min of lobar ischemia followed by 2 h of reperfusion (IR). This resulted in decreased NO production [3.4±0.4 µM (IR) vs. 15.5±1.7 µM (sham) P<0.05]. IR was associated with decline in L-arginine levels (Fig. 1
) and decreased eNOS expression (Fig. 2
). INOS expression was absent. The local depletion of L-arginine substrate around eNOS may decrease or derange eNOS expression or activity leading to overproduction of reactive oxygen species. This may contribute to the hepatocellular injury indicated by the significant increase in ALT in IR group [3173±215 u/L (IR) vs. 214±168 u/L (sham) P<0.05]. The increased ornithine levels in this group (Fig. 1)
would suggest an increased hepatic arginase activity.
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2) Preconditioned livers had significantly decreased hepatocellular injury and were associated with up-regulation of eNOS expression
To determine the effect of preconditioning on ischemia reperfusion injury, rats had 5 min lobar ischemia and 10 min reperfusion (IPC) prior to 45 min ischemia and 2 h of reperfusion. The preconditioned livers had significantly decreased hepatocellular injury [ALT, 980±220 u/L (IPC) vs. 3173±215 u/L (IR) P<0.05] and were associated with up-regulation of eNOS expression (Fig. 2)
. The eNOS immunostaining was localized to hepatocytes and vascular endothelium. iNOS expression was absent. Measurements of plasma amino acids (Fig. 1)
show that with preconditioning, L-arginine levels were increased and ornithine levels were decreased, indicating increased substrate availability with preconditioning. Citrulline levels were not significantly different between the groups indicating that arginine synthesis was not significantly modified by preconditioning.
3) Cytoprotective NO is produced by eNOS during ischemic preconditioning of the liver
The NO generation with IPC was specific to the liver since hepatic vein NO levels were higher than circulating NO levels [hepatic vein NOx 40.3±2.7 (group 3) vs. circulating NOx 28.6±2.1 (group 3), P<0.05]. Since iNOS expression was not detected, eNOS is the likely potential enzymatic source for the increased NO production in the ischemically preconditioned livers. To determine the role of NO, L-arginine was administered before IR and N
-nitro-L-arginine methyl ester (L-NAME) administered before IPC + IR. L-Arginine administration increased its plasma levels (Fig. 1)
and also increased eNOS expression (Fig. 2)
and NO production. The similar effect to IPC following the stimulation of NO formation by supplementation with L-arginine suggests that NO might inhibit the down-regulation of eNOS during reperfusion. The aggravated hepatocellular injury with L-NAME administration additionally substantiates a role for eNOS derived NO in the preconditioning effect.
CONCLUSIONS AND SIGNIFICANCE
This study has addressed the relationship of endothelial isoform (eNOS) and inducible isoform (iNOS) of nitric oxide synthase with the ischemic preconditioning effect on early (<2 h) ischemia reperfusion injury of the liver. The major finding of the study is that cytoprotective nitric oxide is produced by eNOS during ischemic preconditioning of the liver. The study has shown that eNOS expression was up-regulated and iNOS induction was absent in the ischemically preconditioned livers during early ischemia reperfusion injury. The results also demonstrate that IPC was associated with an increased availability of the L-arginine substrate.
This study has suggested that the preconditioned livers (group 3) had significantly decreased hepatocellular injury and were associated with up-regulation of eNOS expression. This suggests that IPC prevents the decline of eNOS or restores eNOS expression during ischemia reperfusion. The decreased eNOS expression and decline in L-arginine levels observed with ischemia reperfusion may be caused by inhibition of NO synthases by reactive oxygen species, local depletion of L-arginine substrate around eNOS, or impaired delivery of L-arginine to eNOS and, if L-arginine becomes rate limiting for eNOS activity, the decreased eNOS expression results in decreased NO production during ischemia and reperfusion. Under these conditions, IPC reversed the reduction in eNOS expression. Since iNOS expression was not detected, eNOS is the likely potential enzymatic source for the increased NO production in the preconditioned livers. The results show that the NO generation with IPC was specific to the liver since the hepatic vein NO levels were higher than circulating NO levels. An increased NO production immediately after the preconditioning treatment is well documented in previous studies. The increased L-arginine levels with IPC would indicate increased substrate availability for eNOS. Circulating citrulline maintains endogenous synthesis of arginine in rats and humans. Since the citrulline levels were not significantly different between the groups, this would suggest that the arginine synthesis was not significantly modified. Arginine is degraded by arginase to ornithine and sufficient quantities of arginase can limit the availability of arginine for NO synthesis and hepatic ischemia reperfusion has been shown to be associated with an increased arginase I activity. The low ornithine levels in the preconditioned livers could suggest the possibility that the effect of IPC may involve modulation of arginase to increase substrate availability for NO biosynthesis. Arginase has two distinct isoenzymes and the interplay between arginase and NOS isoenzymes is more complex than the fact that they use a common substrate (the mechanisms by which IPC may modulate this interplay is beyond the scope of this study). Although eNOS up-regulation at the end of preconditioning treatment has not been demonstrated in the present study, our data support the notion that IPC prevents the decline of eNOS during early ischemia reperfusion injury and this together with increased L-arginine substrate availability leads to increased cytoprotective NO production. The aggravated hepatocellular injury with L-NAME administration additionally substantiates a role for eNOS derived NO in the preconditioning effect.
The effects of L-arginine administration were similar to those evident following IPC. L-Arginine has been shown to increase eNOS expression and NO production in cells and in vivo in humans and in mice. L-Arginine supplementation modulates the NO-dependent vascular functions and clinical studies have reported that L-arginine administration reduces the symptoms of coronary heart disease in patients. The similar effect to IPC following the stimulation of NO formation by supplementation with L-arginine suggests that NO might inhibit the down-regulation of eNOS during reperfusion. This is consistent with the observation that the prevention of eNOS down-regulation by exogenous adenosine attenuated hepatic ischemia reperfusion injury. However, the mechanism by which adenosine prevents the down-regulation of eNOS is not explained. In this study, the administration of exogenous L-arginine not only significantly increased the plasma level but also led to increased ornithine levels. The higher production of ornithine would indicate arginase activity. This raises the possibility that some of the NO generation after L-arginine administration may be independent of the L-arginine/NOS pathway. A nonenzymatic pathway for NO generation by the reaction of hydrogen peroxide and arginine has been proposed. In a previous study, it was shown that continuous L-arginine infusion not only reverted effects of NO inhibition but decreased hepatocyte injury induced by ischemia reperfusion. The NOS inhibitor L-NAME can, to variable levels, inhibit arginine binding and metabolism as well as its transport across the hepatocyte plasma membrane. Cellular arginine may be shunted into different metabolic pathways including the ornithine cycle and this may explain the reduction in plasma arginine and increase in ornithine in the L-NAME group in this study.
The sustained protection shown by the results raises the possibility of other molecules in addition to eNOS contributing to the preconditioning signal. NO is not the sole product of eNOS and free radicals may be generated and recent evidence suggests that free radicals were essential for preconditioning. The possibility of compensatory up-regulation of iNOS isoform is excluded by the results. The possibility of free radicals synthesized as a by-product of eNOS activity contributing to the preconditioning effect cannot be excluded in this study.
The NOS inhibition used in this study was not isoform specific. It was assumed that the role of iNOS would be negligible since the enzyme takes several hours to be induced and the results indicate that NOS inhibition was complete and effective for eNOS as well as iNOS. However, the inhibition of eNOS expression with L-NAME is likely a result of the aggravated injury in this group rather than assuming a direct effect of L-NAME on eNOS transcription. The observation from this study that eNOS may contribute to early preconditioning may have potential importance in the clinical application of preconditioning in humans. Patients who have developed acute myocardial infarction have increased eNOS polymorphisms. Such polymorphism may adversely affect the nitric oxide generation resulting from a transient ischemic insult and may therefore attenuate protection potentially afforded by IPC. There is no evidence that such polymorphism exists in liver ischemia reperfusion injury so far and this area warrants further investigation. Nonetheless, eNOS may prove to be a useful target in hepatoprotection from ischemia reperfusion injury.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3220fje; doi: 10.1096/fj.04-3220fje
This article has been cited by other articles:
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  S. Bertuglia Intermittent hypoxia modulates nitric oxide-dependent vasodilation and capillary perfusion during ischemia-reperfusion-induced damage Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1914 - H1922. [Abstract] [Full Text] [PDF]
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M. R. Duranski, J. W. Elrod, J. W. Calvert, N. S. Bryan, M. Feelisch, and D. J. Lefer Genetic overexpression of eNOS attenuates hepatic ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H2980 - H2986. [Abstract] [Full Text] [PDF]
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