eNOS Gene Therapy Exacerbates Hepatic Ischemia-Reperfusion Injury in Diabetes
http://www.100md.com
John W. Elrod, Mark R. Duranski, Will La
参见附件。
the Departments of Medicine (J.W.E., M.R.D., D.J.L.) and Pathology (J.W.E., D.J.L.), Division of Cardiology, Albert Einstein College of Medicine, Bronx, NY
Department of Molecular and Cellular Physiology (W.L., J.J.M.G.)
Department of Pharmacology, Toxicology and Neuroscience (T.R.D.)
Department of Pathology (C.G.K.), Louisiana State University Health Sciences Center, Shreveport
Department of Emergency Medicine (L.T.), Thomas Jefferson University, Philadelphia, Pa
Department of Cardiology (H.C.C.), Johns Hopkins Hospital, Baltimore, Md.
Abstract
Previous studies indicate that endothelial nitric oxide synthase (eNOS) function is impaired in diabetes as a result of increased vascular generation of reactive oxygen species. We hypothesized that eNOS gene therapy would augment NO· bioavailability and protect against hepatic ischemia-reperfusion (I-R) injury in type 2 diabetes mellitus. We developed a transgenic (Tg) diabetic mouse in which eNOS is systemically overexpressed. We also examined the effects of hepatic eNOS adenovirus therapy in diabetic mice. Diabetic (db/db) and nondiabetic mice were subjected to hepatic I-R injury. In nondiabetic mice, genetic overexpression of eNOS (both eNOS-Tg and eNOS adenovirus) resulted in hepatoprotection. In contrast, hepatic I-R injury was significantly increased in the db/db eNOS-Tg mouse, as serum alanine aminotransaminase (ALT) levels were increased by 3.3-fold compared with diabetic controls. Similarly, eNOS adenovirus treatment resulted in a 3.2-fold increase in serum ALT levels as compared with diabetic controls. We determined that hepatic eNOS was dysfunctional in the db/db mouse and increased genetic expression of eNOS resulted in greater production of peroxynitrite. Treatment with the eNOS cofactor tetrahydrobiopterin (BH4) or the BH4 precursor sepiapterin resulted in a significant decrease in serum ALT levels following I-R injury. We present clear examples of the protective and injurious nature of NO· therapy in I-R. Our data indicate that eNOS exists in an "uncoupled" state in the setting of diabetes and that "recoupling" of the eNOS enzyme with cofactor therapy is beneficial.
Key Words: diabetes mellitus tetrahydrobiopterin eNOS phosphorylation sepiapterin peroxynitrite
Introduction
Nitric oxide (NO·) generated by endothelial nitric oxide synthase (eNOS) is critical for the maintenance of cardiovascular homeostasis. NO· exerts potent vasodilator,1 antiplatelet,2 antiinflammatory, and cytoprotective effects3 in healthy animals. However, the role of NO· in ischemic disease remains controversial, with a number of studies citing protective and deleterious effects of NO· therapy.4 In the liver, eNOS appears to play a central role in protecting against ischemia-reperfusion (I-R) injury.5,6
Experimental studies have revealed that eNOS-derived NO· bioavailability is diminished in diabetic animals.7,8 The quenching of NO· by advanced glycosylation products in diabetic patients is also thought to play a significant role in the pathogenesis of diabetes mellitus.9 Reports10 have implicated glycosylated and oxidized low-density lipoprotein as potent mediators in the downregulation of eNOS. Additionally, the generation of reactive oxygen species (ROS) has been shown to significantly inhibit eNOS production of NO· and its downstream physiological actions.11,12 These pathogenic factors may impair eNOS function via a number of mechanisms including: decreased eNOS production, altered phosphorylation events at various residues, decreased substrate and/or cofactor availability, increased association with inhibitory proteins, and inhibition of dimerization allowing production of superoxide (uncoupling).13 Given that eNOS dysfunction and reduced NO· bioavailability contribute to the pathogenesis of diabetes mellitus, we hypothesized that eNOS gene therapy, in the setting of diabetes, would increase nitric oxide bioavailability and thus ameliorate the severity of hepatic I-R injury.
Materials and Methods
Chemicals and Reagents
DETA NONOate (DETA/NO) was purchased from Alexis Biochemicals (San Diego, Calif) and administered (100 μg/kg) IP 15 minutes before reperfusion dissolved in normal saline. Sodium nitrite was obtained from Sigma Chemical Co (St Louis, Mo). Sodium nitrite was dissolved in phosphate buffered saline and the pH was adjusted to 7.4. In all experiments, 165 μg/kg sodium nitrite or vehicle was administered IP 15 minutes before reperfusion.
L-Sepiapterin was purchased from Alexis Biochemicals. Sepiapterin (5 mg/kg) was administered IP 30 minutes before ischemia and dissolved in normal saline. Tetrahydrobiopterin (BH4) was purchased from Alexis Biochemicals and administered at (5 mg/kg) IP 30 minutes before ischemia, dissolved in normal saline. N-nitro-L-arginine methyl ester (L-NAME) was purchased from Sigma-Aldrich (St Louis, Mo) and administered at (50 mg/kg) 30 minutes before ischemia.
MnTMPyP a cell-permeable superoxide dismutase (SOD) mimetic was purchased from Calbiochem (San Diego, Calif). MnTMPyP was administered at (1 mg/kg) 30 minutes before ischemia, dissolved in normal saline.
Adenovirus Vectors
Replication-deficient recombinant adenoviruses serotype 5–encoding nuclear-targeted -galactosidase (AdCMVgal) and eNOS (AdCMVeNOS), driven by a cytomegalovirus (CMV) promoter, were prepared as previously described.14 Adenovirus was delivered 48 hours before liver I-R experiments via IP injection of AdCMVgal (1x1010 parts), AdCMVeNOS (1x1010 parts/mL), or AdCMV-Null (1x1010 parts) diluted in normal saline.
Diabetic Mice
Cg-m+/+Leprdb/J (C57BL6/J background db/db diabetic) mice were purchased from The Jackson Laboratory and maintained on a normal rodent chow diet. Age-matched nondiabetic littermate animals were used as controls.
Diabetic eNOS Transgenic Mice
We developed a diabetic eNOS transgenic mouse (db/db eNOS-Tg) exhibiting systemic overexpression of the bovine eNOS gene driven by the native eNOS promoter. This was accomplished by breeding the eNOS transgenic mouse (eNOS-Tg)15 onto the db/db diabetic mouse (Jackson Laboratory). Genotype was confirmed by PCR analysis of the leptin receptor mutation and bovine eNOS transgene in all mice.
eNOS Knockout Mice were generated in our breeding colony as described previously.16
eNOS Transgenic Mice
The eNOS transgenic mouse (eNOS-Tg) was developed on a C57BL6 background and has been characterized previously.17
In all studies, male mice were used at 8 to 10 weeks of age. All experimental procedures complied with the NIH Guide for the Care and Use of Laboratory Animals (Publication No. 86-23, revised 1985).
Blood Glucose Determination
Blood obtained via tail snip was screened using a Sure Step glucose-monitoring system (Lifescan).
Hepatic I-R Protocol
The hepatic I-R protocol has been previously described.18 Mice were anesthetized with the combination of ketamine (100 mg/kg) and xylazine (8 mg/kg). The left lateral and median lobes of the liver were rendered ischemic by completely clamping the hepatic artery and the portal vein. The duration of hepatic ischemia was 45 minutes, and reperfusion time was 5 hours in the studies of serum alanine aminotransaminase (ALT) levels.
Hepatic Enzyme Levels
Serum samples were analyzed for ALT using a spectrophotometric method (Thermo Electron Corp, Melbourne, Australia).
Measurement of Nitrotyrosine
Nitrotyrosine was determined by Western blotting and ELISA methods. Western blot was performed as described by Gao et al.19 To determine protein nitration in a fully quantitative manner, liver tissue nitrotyrosine levels were determined by using an ELISA procedure reported by Tanaka et al,20 with modification as previously described.21 The amount of nitrotyrosine content in tissue samples was calculated using standard curves generated from nitrated BSA containing known amounts of nitrotyrosine and expressed as picomoles per milligram protein.
Western Blot Analysis of eNOS
Standard Western blotting procedure was used to analyze total eNOS and eNOS phospho residues as previously reported.16 Low-temperature SDS-PAGE blots were performed as previously described22 to assess eNOS monomer/dimer ratio.
High-Performance Liquid Chromatography Analysis of BH4
Hepatic BH4 content was determined by modification of the method described by Howells et al.23 At the time of analysis, the samples were diluted 1:10 in 0.1 mol/L perchloric acid containing 1 mg/mL dithioerythritol and 1 mg/mL diethylenetriaminepentaacetic acid. BH4 levels were determined using a Waters 2695 (Milford, Mass), interfaced to a Coularray (ESA Inc, Chelmsford, Mass) electrochemical detector.
Statistical Analysis
Data were analyzed where appropriate by Student’s t test or 1-way ANOVA, with post hoc Tukey analysis using JMP IN statistical software (SAS Institute). Data are reported as mean±SEM. Probability values of <0.05 were considered significant.
Results
Phenotypic Profile of Diabetic and Transgenic Mice
Analysis of body weight, blood glucose, and glycosylated hemoglobin (HbA1C) are presented in Table I in the online data supplement, available at http://circres.ahajournals.org. The nondiabetic mice (wild type, eNOS–/–, eNOS-Tg) all displayed similar weights, blood glucose, and HbA1C. In contrast, the db/db diabetic mouse is obese with an average weight &90% greater than the wild-type control. Both diabetic mice strains showed significantly elevated body weights, blood glucose, and HbA1c.
The Diametric Role of Genetic eNOS Overexpression in Hepatic I-R Injury
The genetic overexpression of bovine eNOS in nondiabetic mice dramatically attenuated hepatic tissue injury, as assessed by serum ALT levels, following hepatic I-R injury (Figure 1A). This overexpression of eNOS in the nondiabetic mouse resulted in a 3.8-fold decrease in mean serum ALT level. To the contrary, overexpression of the same bovine eNOS transgene in the db/db diabetic mouse profoundly increased hepatic injury (Figure 1B). The db/db eNOS-Tg mouse displayed a 3.25-fold increase in serum ALT following hepatic I-R, suggesting that overexpression of eNOS in the setting of diabetes exacerbates hepatic I-R injury.
To further examine the role of eNOS gene therapy in I-R and rule out the possibility that some compensatory modification resulting from chronic overexpression was responsible for the increase in injury seen in the db/db eNOS-Tg mouse, we next used an adenoviral gene therapy approach to increase eNOS expression. Intraperitoneal delivery of AdCMVeNOS or AdCMV-Null was performed 48 hours before hepatic I-R. At 48 hours following eNOS adenovirus delivery eNOS expression increased &7-fold as compared with mice receiving the null adenovirus (supplemental Figure I). eNOS adenoviral therapy in the nondiabetic (Figure 1C) significantly attenuated serum ALT levels by a 3.9-fold decrease similar to that observed in the nondiabetic eNOS-Tg animals. The protective action of the eNOS adenovirus was abrogated in the diabetic db/db mouse (Figure 1D), as assessed by serum ALT, increasing from 2312±260 in the adeno-null-treated animals to 9519±3428 U/L in adeno-eNOS mice. This represented a 4.1-fold increase in serum ALT following eNOS gene therapy mimicking the results found in the db/db eNOS-transgenic animal. These results clearly suggest that although genetically increasing eNOS in nondiabetic mice during I-R injury is advantageous, in the setting of diabetes mellitus eNOS gene therapy exacerbates hepatic injury.
Nitric Oxide Donors Are Protective in the Nondiabetic and Injurious in the Diabetic
DETA NONOate (100 μg/kg) reduced serum ALT from 482±65 U/L in nondiabetic mice subjected to hepatic I-R to 61±10 U/L (Figure 2A), however diabetic mice demonstrated a 1.7-fold increase in serum ALT post I-R injury (Figure 2B).
To validate these results a second NO· source, sodium nitrite (165 μg/kg), was used. Figure 2C shows the protective nature of nitrite in the nondiabetic mouse subjected to I-R injury (2.3-fold decrease in serum ALT). Yet in the db/db diabetic mouse, sodium nitrite increased the severity of hepatic I-R injury (Figure 2D).
The Diabetic Liver Displays Altered Phosphorylation of Key eNOS Regulatory Residues
To more clearly understand the state of eNOS in the diabetic liver Western blots were performed. Figure 3A shows representative blots using antibodies against total eNOS, threonine 495 (Thr495) phospho residue, and the serine 1177 (Ser1177) phospho residue. Total eNOS expression was semiquantitatively assessed by densitometric analysis. Figure 3B demonstrates no significant difference in total eNOS protein expression in the diabetic liver as compared with nondiabetic liver. Densitometric analysis of the ratio of phospho:total eNOS revealed a 2-fold decrease in liver eNOS Thr495 phosphorylation in the diabetic versus the nondiabetic (Figure 3C). We also found that diabetic mice had a 2.9-fold reduction in liver eNOS Ser1177 phosphorylation versus nondiabetic mice (Figure 3D). Low-temperature SDS-PAGE Western blots were also performed to assess the monomer/dimer ratio of eNOS in diabetic mice (supplemental Figure II). Nondiabetic mice were found to have a 3.9-fold increase in the ratio of eNOS monomer/dimer ratio versus diabetic mice, suggesting eNOS may exist in an altered state in diabetic mice.
Nitrated Tyrosine Is Increased in the Diabetic and eNOS Overexpression Augments Nitrotyrosine Formation in the Liver
We first performed Western blots to qualitatively assess the amount of nitrotyrosine formation (Figure 4A). To quantitatively assess nitrated tyrosine residues, we performed nitrotyrosine ELISAs. Figure 4B shows low nitrotyrosine levels in wild-type and eNOS transgenic mice liver samples. However, the livers of diabetic (db/db) eNOS-Tg mice demonstrated a significant increase in nitrotyrosine formation over those of nondiabetic control and eNOS-Tg mice.
Inhibition of NOS Further Exacerbates Liver I-R Injury
First, we subjected eNOS–/– animals to the hepatic I-R protocol and found that deletion of the eNOS gene in nondiabetic animals resulted in a 3.1-fold increase in serum ALT levels (Figure 5A). Next, we pharmacologically inhibited eNOS in the db/db diabetic and nondiabetic control mice with the NOS inhibitor L-NAME. Nondiabetic mice receiving L-NAME exhibited increased serum ALT levels 7.6-fold higher than mice receiving the vehicle normal saline (Figure 5A). Following hepatic I-R, serum ALT levels increased more than 5-fold higher in diabetic mice receiving L-NAME, rather than vehicle, before hepatic I-R (Figure 5B).
The eNOS Cofactor BH4 Is Reduced in the Diabetic Liver
We examined the eNOS cofactor BH4. We found that BH4 levels in the nondiabetic liver averaged 332.91±40.29 pmol/mg protein (Figure 6). Likewise, BH4 levels in the eNOS-Tg mice averaged 362.41±86.05 pmol/mg. However, BH4 levels in db/db mouse liver samples were significantly reduced by 2.2-fold to 149.61±17.19 pmol/mg protein. These results suggest that decreased levels of BH4 may be responsible for eNOS dysfunction and help to explain the deleterious effects of eNOS gene therapy in the diabetic liver.
Increasing Bioavailability of BH4 or Sepiapterin Protects Against Hepatic I-R Injury in the Diabetic
In nondiabetic mice, acute administration of sepiapterin, an intermediate in the pterin salvage pathway, was used to increase BH4 biosynthesis. Sepiapterin administered before ischemia reduced serum ALT levels by 4.5-fold compared with mice receiving vehicle (Figure 7A). Injection of BH4 also very significantly reduced serum ALT levels in nondiabetic mice from 678±61 U/L to 225±45 U/L. Figure 7B displays the 3.7-fold reduction in serum ALT in the diabetic receiving sepiapterin versus vehicle. BH4 delivery mimicked these results decreasing serum ALT levels post I-R injury from 2986±274 U/L in the vehicle to 1419±275 U/L in mice given BH4.
BH4 Supplementation Plus the Addition of Nitrite Further Protects Against Hepatic I-R Injury
We next treated both nondiabetic and diabetic mice with BH4 before ischemia, followed by nitrite administration 15 minutes before reperfusion. In the nondiabetic mouse administration of BH4 before the delivery of nitrite lowered serum ALT by 3.2-fold following hepatic I-R (Figure 7C). In the db/db diabetic mouse, we observed the greatest protection of all therapeutic interventions with a reduction of serum ALT from 2933±490 U/L in the vehicle-treated group to 768±121 U/L in the BH4+nitrite group. This represents a 3.8-fold decrease in ALT injury (Figure 7D).
A Cell-Permeable SOD Mimetic With the Addition of a Nitric Oxide Donor Protects Against Hepatic I-R Injury in the Diabetic
Experiments in the nondiabetic revealed a significant reduction in serum ALT with the administration of MnTMPyP (Figure 8A). In diabetic mice, SOD therapy lowered serum ALT levels, although not significantly, from 3173±694.3 U/L in vehicle-treated mice to 2088±490.6 U/L following hepatic I-R (Figure 8B). Next, db/db diabetic mice were given MnTMyP before ischemia followed by the administration of the NO donor DETA NONOate. This combination of therapy significantly lowered serum ALT following hepatic I-R by 2.4-fold.
Discussion
There have been numerous reports citing protective effects of eNOS gene therapy.24,25 In the present study, we found that eNOS gene therapy protects the nondiabetic liver against I-R injury. In stark contrast, and disproving our initial hypothesis, we established that genetic overexpression of eNOS in diabetic (db/db) mice significantly exacerbates hepatic I-R injury. These results were validated by 2 different approaches. We first developed a diabetic (db/db) mouse with systemic overexpression of the bovine eNOS transgene. Secondly, to account for possible compensatory mechanisms in the transgenic mouse, we used an adenoviral approach.24 In both instances, we observed a 3- to 4-fold increase in serum ALT levels following hepatic I-R in the db/db diabetic mouse.
To further investigate the deleterious nature of eNOS gene therapy in the diabetic mouse, we examined direct NO· therapy using various nitric oxide donor compounds. In the nondiabetic animals DETA/NO significantly reduced I-R induced hepatic injury. This result is predictable given the numerous experimental studies citing the protective effects of NO· donor therapy in hepatic I-R.26,27 In a second series of experiments, db/db diabetic mice received DETA/NO before reperfusion. The NO· donor significantly increased serum ALT levels in diabetic mice. A second agent that has been shown to increase NO· following ischemic insult, sodium nitrite, was examined to further evaluate the role of NO· therapy on hepatic I-R injury in diabetes.18 Nitrite is a biological storage form of NO· that releases NO· following hypoxia or ischemic insult.18,28 Nitrite significantly increased serum ALT levels following I-R in the db/db diabetic mouse. This further corroborated the injurious nature of increasing NO· levels in the diabetic mouse experiencing liver I-R injury. Although these results were quite surprising, we can only speculate that a direct bolus of NO· just before reperfusion interacted with superoxide anion and formed the potent free radical peroxynitrite, thereby resulting in increased hepatic injury. This seems a plausible explanation given the high levels of reactive oxygen species, such as superoxide, found in the diabetic, especially during I-R injury. These data are supported by our findings that peroxynitrite (ONOO–) levels are significantly elevated in the db/db mouse at baseline and that a SOD mimetic decreased hepatic I-R injury.
To evaluate the molecular status of eNOS in the diabetic liver, we examined various phospho residues. We found that although there was no difference in total hepatic eNOS protein between nondiabetic and diabetic animals, there was a significant alteration in the phosphorylation of key regulatory residues. We discovered decreased phosphorylation of both threonine 495 and serine 1177 residues in diabetic mice as compared with nondiabetic control animals. It has been suggested that phosphorylation status of Thr495 is critical to the coupling of the eNOS enzyme for efficient production of nitric oxide. Lin et al29 have shown that Thr495 may be the "intrinsic switch mechanism" that determines whether eNOS produces either NO· (phospho 495) or superoxide (dephospho 495). The phosphorylation state of Ser1177 is indicative of eNOS enzyme activity. Decreased phosphorylation of Ser1177 is associated with a decrease in enzyme activity, whereas increased phosphorylation of Ser1177 has been shown by many to increase catalytic activity.30,31 In a study of diabetic animals,32 it has also been shown that this regulatory site may be subject to posttranslational modification, thereby decreasing eNOS activity. We also performed low-temperature SDS-PAGE Western blots to assess the monomer/dimer ratio of the eNOS enzyme in diabetic mice. We found a striking difference in the monomer/dimer ratio in the diabetic mice as compared with nondiabetic controls, further suggesting that eNOS exists in an altered state in the diabetic mice.
Our present findings that eNOS is "uncoupled" in the diabetic mouse support our data demonstrating that eNOS gene therapy enhanced hepatic I-R injury in db/db diabetic mice. We therefore sought to further this notion by examining the possibility that increases in eNOS in the diabetic mouse resulted in increased ONOO– via interactions between superoxide and NO·. To indirectly identify peroxynitrite formation, we examined hepatic nitrotyrosine formation using 2 distinct methods. Qualitative assessment of nitrotyrosine via Western blot analysis and quantitative assessment with an ELISA revealed an increase in nitrotyrosine in the diabetic as compared with nondiabetic animals. Remarkably, these levels were increased to an even greater extent in the db/db eNOS-Tg animals under baseline conditions. These results are very suggestive that eNOS therapy in the diabetic further increased oxidative stress in these animals via the formation of ONOO–. Current studies22,33 have cited ONOO– as a mediator of eNOS uncoupling. This presents a possible positive feedback in the db/db diabetic mouse whereby increased superoxide production further increases ONOO– formation, which in turn acts to further uncouple eNOS perpetuating superoxide production. Similarly, the addition of exogenous NO· (DETA/NO or nitrite), which we found to increase hepatic injury, could result in increased ONOO– formation by interaction with high levels of superoxide anion following I-R.
To further implicate eNOS as a pathogenic mediator of I-R injury in the diabetic, we next pharmacologically inhibited eNOS with L-NAME. First, nondiabetic controls and eNOS–/– animals were subjected to hepatic I-R. We found that genetic deletion of the eNOS enzyme in the nondiabetic increased serum ALT by 3.1-fold following I-R. This result was anticipated given previous studies16,34 demonstrating increased I-R injury in eNOS–/– animals. Pharmacological inhibition with L-NAME was also found to increase serum ALT levels following I-R in nondiabetic mice. However, the administration of L-NAME to the nondiabetic caused ALT levels to rise more than 2-fold higher than those seen in the eNOS–/–, suggesting L-NAME was not simply inhibiting eNOS but was also exerting additional effects that resulted in enhanced hepatic I-R injury. The administration of L-NAME in the diabetic was the most injurious intervention in the present study and given the results noted in the nondiabetic, clearly L-NAME is not solely specific for the inhibition of eNOS.
In an effort to further our understanding of eNOS function in the db/db diabetic mouse, we next measured the eNOS cofactor BH4 levels in these animals. BH4 availability has been shown to be important for both catalytic activity and the coupling of the enzyme for efficient production of nitric oxide.35,36 High-performance liquid chromatography (HPLC) analysis revealed a substantial 2-fold decrease in BH4 levels in the diabetic liver. This result supports other published studies8,37,38 and provides another possible mechanism by which eNOS may be uncoupled in the diabetic liver. Decreases in BH4 availability has been suggested37,39 to be directly linked to superoxide production in diabetes. We administered either BH4 or sepiapterin before induction of hepatic I-R injury. Sepiapterin is a precursor in the BH4 salvage pathway40 and has been shown to increase BH4 levels and augment eNOS function in vivo.38 The acute administration of either BH4 or sepiapterin decreased serum ALT levels in the both the nondiabetic and diabetic following I-R injury. These data suggest that the restoration of BH4 levels in the diabetic liver is beneficial via augmenting eNOS function. Working on the hypothesis that increasing BH4 levels in the diabetic "recouples" eNOS and thereby reduces superoxide production, we again attempted NO· donor therapy following the supplementation of BH4. Treatment of diabetic mice with BH4 before sodium nitrite therapy significantly reduced hepatic injury. ALT levels with this combination therapy decreased hepatic injury to the lowest seen in the current study in db/db diabetic mice. This was in stark contrast to the previous finding that nitrite administration alone trended toward an increase in hepatic injury in the db/db mouse. A possible mechanism for this protection is that increased levels of BH4 recoupled eNOS, thus decreasing the production of superoxide. The subsequent administration of NO· thereby did not yield the potent oxidizing species ONOO– but rather increased NO· bioavailability, leading to protection against I-R injury.
To examine the possibility that superoxide anion modulates the deleterious effects of NO· therapy we investigated a cell-permeable SOD mimetic. Treatment of nondiabetic mice with the SOD mimetic before ischemia resulted in a decrease in serum ALT. This finding supports work by Hines et al,41 who found significant protection against hepatic I-R injury following treatment with an SOD mimetic. Treatment of diabetic mice with SOD therapy before ischemia trended to lower serum ALT levels, although not significantly. However, cotreatment with SOD therapy followed by the administration of the NO· donor DETA/NO significantly lowered ALT levels following I-R injury. This result further supports the likelihood that decreasing superoxide levels in the setting of diabetic I-R injury allows for nitric oxide therapy to be beneficial rather than injurious. Taken together, these data suggest that recoupling eNOS provides protection in an additive fashion by both decreasing superoxide production and increasing NO· production.
The controversy of nitric oxide therapy has been at the forefront of the I-R injury field for more than 2 decades. We present a clear example of both the protective and injurious nature of NO· therapy in an in vivo model of hepatic I-R injury. We show that eNOS gene therapy and direct nitric oxide therapy are very protective in nondiabetic animals subjected to I-R Injury. In contrast, in the setting of type 2 diabetes mellitus, these therapies are pathogenic. The injurious nature of NO· therapy could however be reversed with prior administration of BH4 or a SOD mimetic. These results suggest that superoxide may play a pivotal role by combining with NO· to form the potent free radical ONOO– and that eNOS is a major source of superoxide anion generation in the diabetic liver. Our study suggests that eNOS cofactor therapy may restore eNOS function in diabetes mellitus and may be beneficial in the setting of ischemic disorders.
Acknowledgments
We thank Jeff Szot, Kyle Aaron, and Michael Hicks for invaluable technical expertise in conducting these studies.
Sources of Funding
These studies were supported by a grant from the NIH (2R01 HL-6049 to D.J.L.) and by a grant from the American Diabetes Association (7-04-RA-59 to D.J.L.).
Disclosure
D.J.L. is a participant on a pending U.S. patent, filed on October 14, 2003 through NIH (patent no. 60/511, 244), regarding the use of sodium nitrite in cardiovascular disease.
Footnotes
This manuscript was sent to H. Michael Piper, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received September 13, 2005; resubmission received March 23, 2006; revised resubmission received May 22, 2006; accepted May 30, 2006.
References
Katsuki S, Arnold WP, Murad F. Effects of sodium nitroprusside, nitroglycerin, and sodium azide on levels of cyclic nucleotides and mechanical activity of various tissues. J Cyclic Nucleotide Res. 1977; 3: 239–247. [Order article via Infotrieve]
Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, Kadowitz PJ. Evidence for the inhibitory role of guanosine 3', 5'-monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood. 1981; 57: 946–955.
Ignarro LJ, Napoli C, Loscalzo J. Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide: an overview. Circ Res. 2002; 90: 21–28.
Kim PK, Zuckerbraun BS, Otterbein LE, Vodovotz Y, Billiar TR. ‘Til cell death do us part: nitric oxide and mechanisms of hepatotoxicity. Biol Chem. 2004; 385: 11–15. [Order article via Infotrieve]
Wang Y, Vodovotz Y, Kim PK, Zamora R, Billiar TR. Mechanisms of hepatoprotection by nitric oxide. Ann N Y Acad Sci. 2002; 962: 415–422.
Taniai H, Hines IN, Bharwani S, Maloney RE, Nimura Y, Gao B, Flores SC, McCord JM, Grisham MB, Aw TY. Susceptibility of murine periportal hepatocytes to hypoxia-reoxygenation: role for NO and Kupffer cell-derived oxidants. Hepatology. 2004; 39: 1544–1552. [Order article via Infotrieve]
Pieper GM. Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction. Hypertension. 1998; 31: 1047–1060.
Pannirselvam M, Verma S, Anderson TJ, Triggle CR. Cellular basis of endothelial dysfunction in small mesenteric arteries from spontaneously diabetic (db/db -/-) mice: role of decreased tetrahydrobiopterin bioavailability. Br J Pharmacol. 2002; 136: 255–263. [Order article via Infotrieve]
Bucala R, Tracey KJ, Cerami A. Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in experimental diabetes. J Clin Invest. 1991; 87: 432–438. [Order article via Infotrieve]
Napoli C, Lerman LO, de Nigris F, Loscalzo J, Ignarro LJ. Glycoxidized low-density lipoprotein downregulates endothelial nitricoxide synthase in human coronary cells. J Am Coll Cardiol. 2002; 40: 1515–1522.
Aslan M, Ryan TM, Adler B, Townes TM, Parks DA, Thompson JA, Tousson A, Gladwin MT, Patel RP, Tarpey MM, Batinic-Haberle I, White CR, Freeman BA. Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease. Proc Natl Acad Sci U S A. 2001; 98: 15215–15220.
Zou MH, Cohen R, Ullrich V. Peroxynitrite and vascular endothelial dysfunction in diabetes mellitus. Endothelium. 2004; 11: 89–97. [Order article via Infotrieve]
Sessa WC. eNOS at a glance. J Cell Sci. 2004; 117: 2427–2429.
Ooboshi H, Rios CD, Chu Y, Christenson SD, Faraci FM, Davidson BL, Heistad DD. Augmented adenovirus-mediated gene transfer to atherosclerotic vessels. Arterioscler Thromb Vasc Biol. 1997; 17: 1786–1792.
Ohashi Y, Kawashima S, Hirata K, Yamashita T, Ishida T, Inoue N, Sakoda T, Kurihara H, Yazaki Y, Yokoyama M. Hypotension and reduced nitric oxide-elicited vasorelaxation in transgenic mice overexpressing endothelial nitric oxide synthase. J Clin Invest. 1998; 102: 2061–2071.
Sharp BR, Jones SP, Rimmer DM, Lefer DJ. Differential response to myocardial reperfusion injury in eNOS-deficient mice. Am J Physiol Heart Circ Physiol. 2002; 282: H2422–H2426.
Jones SP, Greer JJ, Kakkar AK, Ware PD, Turnage RH, Hicks M, van Haperen R, de Crom R, Kawashima S, Yokoyama M, Lefer DJ. Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury. Am J Physiol Heart Circ Physiol. 2004; 286: H276–H282.
Duranski MR, Greer JJ, Dejam A, Jaganmohan S, Hogg N, Langston W, Patel RP, Yet SF, Wang X, Kevil CG, Gladwin MT, Lefer DJ. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J Clin Invest. 2005; 115: 1232–1240.
Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, Ma XL. Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation. 2002; 105: 1497–1502.
Tanaka S, Choe N, Hemenway DR, Zhu S, Matalon S, Kagan E. Asbestos inhalation induces reactive nitrogen species and nitrotyrosine formation in the lungs and pleura of the rat. J Clin Invest. 1998; 102: 445–454.
Gao F, Yao CL, Gao E, Mo QZ, Yan WL, McLaughlin R, Lopez BL, Christopher TA, Ma XL. Enhancement of glutathione cardioprotection by ascorbic acid in myocardial reperfusion injury. J Pharmacol Exp Ther. 2002; 301: 543–550.
Zou MH, Shi C, Cohen RA. Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin Invest. 2002; 109: 817–826.
Howells DW, Smith I, Hyland K. Estimation of tetrahydrobiopterin and other pterins in cerebrospinal fluid using reversed-phase high-performance liquid chromatography with electrochemical and fluorescence detection. J Chromatogr. 1986; 381: 285–294. [Order article via Infotrieve]
Champion HC, Bivalacqua TJ, Hyman AL, Ignarro LJ, Hellstrom WJ, Kadowitz PJ. Gene transfer of endothelial nitric oxide synthase to the penis augments erectile responses in the aged rat. Proc Natl Acad Sci U S A. 1999; 96: 11648–11652.
Lake-Bruse KD, Faraci FM, Shesely EG, Maeda N, Sigmund CD, Heistad DD. Gene transfer of endothelial nitric oxide synthase (eNOS) in eNOS-deficient mice. Am J Physiol. 1999; 277: H770–H776.
Ricciardi R, Foley DP, Quarfordt SH, Saavedra JE, Keefer LK, Wheeler SM, Donohue SE, Callery MP, Meyers WC. V-PYRRO/NO: an hepato-selective nitric oxide donor improves porcine liver hemodynamics and function after ischemia reperfusion. Transplantation. 2001; 71: 193–198. [Order article via Infotrieve]
Aiba M, Takeyoshi I, Ohwada S, Kawashima Y, Iwanami K, Sunose Y, Yamada T, Tsutsumi H, Matsumoto K, Morishita Y. Novel nitric oxide donor (FK409) ameliorates liver damage during extended liver resection with warm ischemia in dogs. J Am Coll Surg. 2001; 193: 264–271. [Order article via Infotrieve]
Webb A, Bond R, McLean P, Uppal R, Benjamin N, Ahluwalia A. Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage. Proc Natl Acad Sci U S A. 2004; 101: 13683–13688.
Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA Jr, Sessa WC. Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem. 2003; 278: 44719–44726.
Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999; 399: 601–605. [Order article via Infotrieve]
McCabe TJ, Fulton D, Roman LJ, Sessa WC. Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation. J Biol Chem. 2000; 275: 6123–6128.
Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee M. Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest. 2001; 108: 1341–1348.
Kuzkaya N, Weissmann N, Harrison DG, Dikalov S. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase. J Biol Chem. 2003; 278: 22546–22554.
Kawachi S, Hines IN, Laroux FS, Hoffman J, Bharwani S, Gray L, Leffer D, Grisham MB. Nitric oxide synthase and postischemic liver injury. Biochem Biophys Res Commun. 2000; 276: 851–854. [Order article via Infotrieve]
Rodriguez-Crespo I, Gerber NC, Ortiz de Montellano PR. Endothelial nitric-oxide synthase. Expression in Escherichia coli, spectroscopic characterization, and role of tetrahydrobiopterin in dimer formation. J Biol Chem. 1996; 271: 11462–11467.
Berka V, Wu G, Yeh HC, Palmer G, Tsai AL. Three different oxygen-induced radical species in endothelial nitric-oxide synthase oxygenase domain under regulation by L-arginine and tetrahydrobiopterin. J Biol Chem. 2004; 279: 32243–32251.
Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, Goh N, Rockett KA, Channon KM. Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression. J Clin Invest. 2003; 112: 725–735.
Meininger CJ, Marinos RS, Hatakeyama K, Martinez-Zaguilan R, Rojas JD, Kelly KA, Wu G. Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency. Biochem J. 2000; 349: 353–356. [Order article via Infotrieve]
Shinozaki K, Kashiwagi A, Nishio Y, Okamura T, Yoshida Y, Masada M, Toda N, Kikkawa R. Abnormal biopterin metabolism is a major cause of impaired endothelium-dependent relaxation through nitric oxide/O2- imbalance in insulin-resistant rat aorta. Diabetes. 1999; 48: 2437–2445.
Milstien S, Kaufman S. Tetrahydro-sepiapterin is an intermediate in tetrahydrobiopterin biosynthesis. Biochem Biophys Res Commun. 1983; 115: 888–893. [Order article via Infotrieve]
Hines IN, Harada H, Flores S, Gao B, McCord JM, Grisham MB. Endothelial nitric oxide synthase protects the post-ischemic liver: potential interactions with superoxide. Biomed Pharmacother. 2005; 59: 183–189
the Departments of Medicine (J.W.E., M.R.D., D.J.L.) and Pathology (J.W.E., D.J.L.), Division of Cardiology, Albert Einstein College of Medicine, Bronx, NY
Department of Molecular and Cellular Physiology (W.L., J.J.M.G.)
Department of Pharmacology, Toxicology and Neuroscience (T.R.D.)
Department of Pathology (C.G.K.), Louisiana State University Health Sciences Center, Shreveport
Department of Emergency Medicine (L.T.), Thomas Jefferson University, Philadelphia, Pa
Department of Cardiology (H.C.C.), Johns Hopkins Hospital, Baltimore, Md.
Abstract
Previous studies indicate that endothelial nitric oxide synthase (eNOS) function is impaired in diabetes as a result of increased vascular generation of reactive oxygen species. We hypothesized that eNOS gene therapy would augment NO· bioavailability and protect against hepatic ischemia-reperfusion (I-R) injury in type 2 diabetes mellitus. We developed a transgenic (Tg) diabetic mouse in which eNOS is systemically overexpressed. We also examined the effects of hepatic eNOS adenovirus therapy in diabetic mice. Diabetic (db/db) and nondiabetic mice were subjected to hepatic I-R injury. In nondiabetic mice, genetic overexpression of eNOS (both eNOS-Tg and eNOS adenovirus) resulted in hepatoprotection. In contrast, hepatic I-R injury was significantly increased in the db/db eNOS-Tg mouse, as serum alanine aminotransaminase (ALT) levels were increased by 3.3-fold compared with diabetic controls. Similarly, eNOS adenovirus treatment resulted in a 3.2-fold increase in serum ALT levels as compared with diabetic controls. We determined that hepatic eNOS was dysfunctional in the db/db mouse and increased genetic expression of eNOS resulted in greater production of peroxynitrite. Treatment with the eNOS cofactor tetrahydrobiopterin (BH4) or the BH4 precursor sepiapterin resulted in a significant decrease in serum ALT levels following I-R injury. We present clear examples of the protective and injurious nature of NO· therapy in I-R. Our data indicate that eNOS exists in an "uncoupled" state in the setting of diabetes and that "recoupling" of the eNOS enzyme with cofactor therapy is beneficial.
Key Words: diabetes mellitus tetrahydrobiopterin eNOS phosphorylation sepiapterin peroxynitrite
Introduction
Nitric oxide (NO·) generated by endothelial nitric oxide synthase (eNOS) is critical for the maintenance of cardiovascular homeostasis. NO· exerts potent vasodilator,1 antiplatelet,2 antiinflammatory, and cytoprotective effects3 in healthy animals. However, the role of NO· in ischemic disease remains controversial, with a number of studies citing protective and deleterious effects of NO· therapy.4 In the liver, eNOS appears to play a central role in protecting against ischemia-reperfusion (I-R) injury.5,6
Experimental studies have revealed that eNOS-derived NO· bioavailability is diminished in diabetic animals.7,8 The quenching of NO· by advanced glycosylation products in diabetic patients is also thought to play a significant role in the pathogenesis of diabetes mellitus.9 Reports10 have implicated glycosylated and oxidized low-density lipoprotein as potent mediators in the downregulation of eNOS. Additionally, the generation of reactive oxygen species (ROS) has been shown to significantly inhibit eNOS production of NO· and its downstream physiological actions.11,12 These pathogenic factors may impair eNOS function via a number of mechanisms including: decreased eNOS production, altered phosphorylation events at various residues, decreased substrate and/or cofactor availability, increased association with inhibitory proteins, and inhibition of dimerization allowing production of superoxide (uncoupling).13 Given that eNOS dysfunction and reduced NO· bioavailability contribute to the pathogenesis of diabetes mellitus, we hypothesized that eNOS gene therapy, in the setting of diabetes, would increase nitric oxide bioavailability and thus ameliorate the severity of hepatic I-R injury.
Materials and Methods
Chemicals and Reagents
DETA NONOate (DETA/NO) was purchased from Alexis Biochemicals (San Diego, Calif) and administered (100 μg/kg) IP 15 minutes before reperfusion dissolved in normal saline. Sodium nitrite was obtained from Sigma Chemical Co (St Louis, Mo). Sodium nitrite was dissolved in phosphate buffered saline and the pH was adjusted to 7.4. In all experiments, 165 μg/kg sodium nitrite or vehicle was administered IP 15 minutes before reperfusion.
L-Sepiapterin was purchased from Alexis Biochemicals. Sepiapterin (5 mg/kg) was administered IP 30 minutes before ischemia and dissolved in normal saline. Tetrahydrobiopterin (BH4) was purchased from Alexis Biochemicals and administered at (5 mg/kg) IP 30 minutes before ischemia, dissolved in normal saline. N-nitro-L-arginine methyl ester (L-NAME) was purchased from Sigma-Aldrich (St Louis, Mo) and administered at (50 mg/kg) 30 minutes before ischemia.
MnTMPyP a cell-permeable superoxide dismutase (SOD) mimetic was purchased from Calbiochem (San Diego, Calif). MnTMPyP was administered at (1 mg/kg) 30 minutes before ischemia, dissolved in normal saline.
Adenovirus Vectors
Replication-deficient recombinant adenoviruses serotype 5–encoding nuclear-targeted -galactosidase (AdCMVgal) and eNOS (AdCMVeNOS), driven by a cytomegalovirus (CMV) promoter, were prepared as previously described.14 Adenovirus was delivered 48 hours before liver I-R experiments via IP injection of AdCMVgal (1x1010 parts), AdCMVeNOS (1x1010 parts/mL), or AdCMV-Null (1x1010 parts) diluted in normal saline.
Diabetic Mice
Cg-m+/+Leprdb/J (C57BL6/J background db/db diabetic) mice were purchased from The Jackson Laboratory and maintained on a normal rodent chow diet. Age-matched nondiabetic littermate animals were used as controls.
Diabetic eNOS Transgenic Mice
We developed a diabetic eNOS transgenic mouse (db/db eNOS-Tg) exhibiting systemic overexpression of the bovine eNOS gene driven by the native eNOS promoter. This was accomplished by breeding the eNOS transgenic mouse (eNOS-Tg)15 onto the db/db diabetic mouse (Jackson Laboratory). Genotype was confirmed by PCR analysis of the leptin receptor mutation and bovine eNOS transgene in all mice.
eNOS Knockout Mice were generated in our breeding colony as described previously.16
eNOS Transgenic Mice
The eNOS transgenic mouse (eNOS-Tg) was developed on a C57BL6 background and has been characterized previously.17
In all studies, male mice were used at 8 to 10 weeks of age. All experimental procedures complied with the NIH Guide for the Care and Use of Laboratory Animals (Publication No. 86-23, revised 1985).
Blood Glucose Determination
Blood obtained via tail snip was screened using a Sure Step glucose-monitoring system (Lifescan).
Hepatic I-R Protocol
The hepatic I-R protocol has been previously described.18 Mice were anesthetized with the combination of ketamine (100 mg/kg) and xylazine (8 mg/kg). The left lateral and median lobes of the liver were rendered ischemic by completely clamping the hepatic artery and the portal vein. The duration of hepatic ischemia was 45 minutes, and reperfusion time was 5 hours in the studies of serum alanine aminotransaminase (ALT) levels.
Hepatic Enzyme Levels
Serum samples were analyzed for ALT using a spectrophotometric method (Thermo Electron Corp, Melbourne, Australia).
Measurement of Nitrotyrosine
Nitrotyrosine was determined by Western blotting and ELISA methods. Western blot was performed as described by Gao et al.19 To determine protein nitration in a fully quantitative manner, liver tissue nitrotyrosine levels were determined by using an ELISA procedure reported by Tanaka et al,20 with modification as previously described.21 The amount of nitrotyrosine content in tissue samples was calculated using standard curves generated from nitrated BSA containing known amounts of nitrotyrosine and expressed as picomoles per milligram protein.
Western Blot Analysis of eNOS
Standard Western blotting procedure was used to analyze total eNOS and eNOS phospho residues as previously reported.16 Low-temperature SDS-PAGE blots were performed as previously described22 to assess eNOS monomer/dimer ratio.
High-Performance Liquid Chromatography Analysis of BH4
Hepatic BH4 content was determined by modification of the method described by Howells et al.23 At the time of analysis, the samples were diluted 1:10 in 0.1 mol/L perchloric acid containing 1 mg/mL dithioerythritol and 1 mg/mL diethylenetriaminepentaacetic acid. BH4 levels were determined using a Waters 2695 (Milford, Mass), interfaced to a Coularray (ESA Inc, Chelmsford, Mass) electrochemical detector.
Statistical Analysis
Data were analyzed where appropriate by Student’s t test or 1-way ANOVA, with post hoc Tukey analysis using JMP IN statistical software (SAS Institute). Data are reported as mean±SEM. Probability values of <0.05 were considered significant.
Results
Phenotypic Profile of Diabetic and Transgenic Mice
Analysis of body weight, blood glucose, and glycosylated hemoglobin (HbA1C) are presented in Table I in the online data supplement, available at http://circres.ahajournals.org. The nondiabetic mice (wild type, eNOS–/–, eNOS-Tg) all displayed similar weights, blood glucose, and HbA1C. In contrast, the db/db diabetic mouse is obese with an average weight &90% greater than the wild-type control. Both diabetic mice strains showed significantly elevated body weights, blood glucose, and HbA1c.
The Diametric Role of Genetic eNOS Overexpression in Hepatic I-R Injury
The genetic overexpression of bovine eNOS in nondiabetic mice dramatically attenuated hepatic tissue injury, as assessed by serum ALT levels, following hepatic I-R injury (Figure 1A). This overexpression of eNOS in the nondiabetic mouse resulted in a 3.8-fold decrease in mean serum ALT level. To the contrary, overexpression of the same bovine eNOS transgene in the db/db diabetic mouse profoundly increased hepatic injury (Figure 1B). The db/db eNOS-Tg mouse displayed a 3.25-fold increase in serum ALT following hepatic I-R, suggesting that overexpression of eNOS in the setting of diabetes exacerbates hepatic I-R injury.
To further examine the role of eNOS gene therapy in I-R and rule out the possibility that some compensatory modification resulting from chronic overexpression was responsible for the increase in injury seen in the db/db eNOS-Tg mouse, we next used an adenoviral gene therapy approach to increase eNOS expression. Intraperitoneal delivery of AdCMVeNOS or AdCMV-Null was performed 48 hours before hepatic I-R. At 48 hours following eNOS adenovirus delivery eNOS expression increased &7-fold as compared with mice receiving the null adenovirus (supplemental Figure I). eNOS adenoviral therapy in the nondiabetic (Figure 1C) significantly attenuated serum ALT levels by a 3.9-fold decrease similar to that observed in the nondiabetic eNOS-Tg animals. The protective action of the eNOS adenovirus was abrogated in the diabetic db/db mouse (Figure 1D), as assessed by serum ALT, increasing from 2312±260 in the adeno-null-treated animals to 9519±3428 U/L in adeno-eNOS mice. This represented a 4.1-fold increase in serum ALT following eNOS gene therapy mimicking the results found in the db/db eNOS-transgenic animal. These results clearly suggest that although genetically increasing eNOS in nondiabetic mice during I-R injury is advantageous, in the setting of diabetes mellitus eNOS gene therapy exacerbates hepatic injury.
Nitric Oxide Donors Are Protective in the Nondiabetic and Injurious in the Diabetic
DETA NONOate (100 μg/kg) reduced serum ALT from 482±65 U/L in nondiabetic mice subjected to hepatic I-R to 61±10 U/L (Figure 2A), however diabetic mice demonstrated a 1.7-fold increase in serum ALT post I-R injury (Figure 2B).
To validate these results a second NO· source, sodium nitrite (165 μg/kg), was used. Figure 2C shows the protective nature of nitrite in the nondiabetic mouse subjected to I-R injury (2.3-fold decrease in serum ALT). Yet in the db/db diabetic mouse, sodium nitrite increased the severity of hepatic I-R injury (Figure 2D).
The Diabetic Liver Displays Altered Phosphorylation of Key eNOS Regulatory Residues
To more clearly understand the state of eNOS in the diabetic liver Western blots were performed. Figure 3A shows representative blots using antibodies against total eNOS, threonine 495 (Thr495) phospho residue, and the serine 1177 (Ser1177) phospho residue. Total eNOS expression was semiquantitatively assessed by densitometric analysis. Figure 3B demonstrates no significant difference in total eNOS protein expression in the diabetic liver as compared with nondiabetic liver. Densitometric analysis of the ratio of phospho:total eNOS revealed a 2-fold decrease in liver eNOS Thr495 phosphorylation in the diabetic versus the nondiabetic (Figure 3C). We also found that diabetic mice had a 2.9-fold reduction in liver eNOS Ser1177 phosphorylation versus nondiabetic mice (Figure 3D). Low-temperature SDS-PAGE Western blots were also performed to assess the monomer/dimer ratio of eNOS in diabetic mice (supplemental Figure II). Nondiabetic mice were found to have a 3.9-fold increase in the ratio of eNOS monomer/dimer ratio versus diabetic mice, suggesting eNOS may exist in an altered state in diabetic mice.
Nitrated Tyrosine Is Increased in the Diabetic and eNOS Overexpression Augments Nitrotyrosine Formation in the Liver
We first performed Western blots to qualitatively assess the amount of nitrotyrosine formation (Figure 4A). To quantitatively assess nitrated tyrosine residues, we performed nitrotyrosine ELISAs. Figure 4B shows low nitrotyrosine levels in wild-type and eNOS transgenic mice liver samples. However, the livers of diabetic (db/db) eNOS-Tg mice demonstrated a significant increase in nitrotyrosine formation over those of nondiabetic control and eNOS-Tg mice.
Inhibition of NOS Further Exacerbates Liver I-R Injury
First, we subjected eNOS–/– animals to the hepatic I-R protocol and found that deletion of the eNOS gene in nondiabetic animals resulted in a 3.1-fold increase in serum ALT levels (Figure 5A). Next, we pharmacologically inhibited eNOS in the db/db diabetic and nondiabetic control mice with the NOS inhibitor L-NAME. Nondiabetic mice receiving L-NAME exhibited increased serum ALT levels 7.6-fold higher than mice receiving the vehicle normal saline (Figure 5A). Following hepatic I-R, serum ALT levels increased more than 5-fold higher in diabetic mice receiving L-NAME, rather than vehicle, before hepatic I-R (Figure 5B).
The eNOS Cofactor BH4 Is Reduced in the Diabetic Liver
We examined the eNOS cofactor BH4. We found that BH4 levels in the nondiabetic liver averaged 332.91±40.29 pmol/mg protein (Figure 6). Likewise, BH4 levels in the eNOS-Tg mice averaged 362.41±86.05 pmol/mg. However, BH4 levels in db/db mouse liver samples were significantly reduced by 2.2-fold to 149.61±17.19 pmol/mg protein. These results suggest that decreased levels of BH4 may be responsible for eNOS dysfunction and help to explain the deleterious effects of eNOS gene therapy in the diabetic liver.
Increasing Bioavailability of BH4 or Sepiapterin Protects Against Hepatic I-R Injury in the Diabetic
In nondiabetic mice, acute administration of sepiapterin, an intermediate in the pterin salvage pathway, was used to increase BH4 biosynthesis. Sepiapterin administered before ischemia reduced serum ALT levels by 4.5-fold compared with mice receiving vehicle (Figure 7A). Injection of BH4 also very significantly reduced serum ALT levels in nondiabetic mice from 678±61 U/L to 225±45 U/L. Figure 7B displays the 3.7-fold reduction in serum ALT in the diabetic receiving sepiapterin versus vehicle. BH4 delivery mimicked these results decreasing serum ALT levels post I-R injury from 2986±274 U/L in the vehicle to 1419±275 U/L in mice given BH4.
BH4 Supplementation Plus the Addition of Nitrite Further Protects Against Hepatic I-R Injury
We next treated both nondiabetic and diabetic mice with BH4 before ischemia, followed by nitrite administration 15 minutes before reperfusion. In the nondiabetic mouse administration of BH4 before the delivery of nitrite lowered serum ALT by 3.2-fold following hepatic I-R (Figure 7C). In the db/db diabetic mouse, we observed the greatest protection of all therapeutic interventions with a reduction of serum ALT from 2933±490 U/L in the vehicle-treated group to 768±121 U/L in the BH4+nitrite group. This represents a 3.8-fold decrease in ALT injury (Figure 7D).
A Cell-Permeable SOD Mimetic With the Addition of a Nitric Oxide Donor Protects Against Hepatic I-R Injury in the Diabetic
Experiments in the nondiabetic revealed a significant reduction in serum ALT with the administration of MnTMPyP (Figure 8A). In diabetic mice, SOD therapy lowered serum ALT levels, although not significantly, from 3173±694.3 U/L in vehicle-treated mice to 2088±490.6 U/L following hepatic I-R (Figure 8B). Next, db/db diabetic mice were given MnTMyP before ischemia followed by the administration of the NO donor DETA NONOate. This combination of therapy significantly lowered serum ALT following hepatic I-R by 2.4-fold.
Discussion
There have been numerous reports citing protective effects of eNOS gene therapy.24,25 In the present study, we found that eNOS gene therapy protects the nondiabetic liver against I-R injury. In stark contrast, and disproving our initial hypothesis, we established that genetic overexpression of eNOS in diabetic (db/db) mice significantly exacerbates hepatic I-R injury. These results were validated by 2 different approaches. We first developed a diabetic (db/db) mouse with systemic overexpression of the bovine eNOS transgene. Secondly, to account for possible compensatory mechanisms in the transgenic mouse, we used an adenoviral approach.24 In both instances, we observed a 3- to 4-fold increase in serum ALT levels following hepatic I-R in the db/db diabetic mouse.
To further investigate the deleterious nature of eNOS gene therapy in the diabetic mouse, we examined direct NO· therapy using various nitric oxide donor compounds. In the nondiabetic animals DETA/NO significantly reduced I-R induced hepatic injury. This result is predictable given the numerous experimental studies citing the protective effects of NO· donor therapy in hepatic I-R.26,27 In a second series of experiments, db/db diabetic mice received DETA/NO before reperfusion. The NO· donor significantly increased serum ALT levels in diabetic mice. A second agent that has been shown to increase NO· following ischemic insult, sodium nitrite, was examined to further evaluate the role of NO· therapy on hepatic I-R injury in diabetes.18 Nitrite is a biological storage form of NO· that releases NO· following hypoxia or ischemic insult.18,28 Nitrite significantly increased serum ALT levels following I-R in the db/db diabetic mouse. This further corroborated the injurious nature of increasing NO· levels in the diabetic mouse experiencing liver I-R injury. Although these results were quite surprising, we can only speculate that a direct bolus of NO· just before reperfusion interacted with superoxide anion and formed the potent free radical peroxynitrite, thereby resulting in increased hepatic injury. This seems a plausible explanation given the high levels of reactive oxygen species, such as superoxide, found in the diabetic, especially during I-R injury. These data are supported by our findings that peroxynitrite (ONOO–) levels are significantly elevated in the db/db mouse at baseline and that a SOD mimetic decreased hepatic I-R injury.
To evaluate the molecular status of eNOS in the diabetic liver, we examined various phospho residues. We found that although there was no difference in total hepatic eNOS protein between nondiabetic and diabetic animals, there was a significant alteration in the phosphorylation of key regulatory residues. We discovered decreased phosphorylation of both threonine 495 and serine 1177 residues in diabetic mice as compared with nondiabetic control animals. It has been suggested that phosphorylation status of Thr495 is critical to the coupling of the eNOS enzyme for efficient production of nitric oxide. Lin et al29 have shown that Thr495 may be the "intrinsic switch mechanism" that determines whether eNOS produces either NO· (phospho 495) or superoxide (dephospho 495). The phosphorylation state of Ser1177 is indicative of eNOS enzyme activity. Decreased phosphorylation of Ser1177 is associated with a decrease in enzyme activity, whereas increased phosphorylation of Ser1177 has been shown by many to increase catalytic activity.30,31 In a study of diabetic animals,32 it has also been shown that this regulatory site may be subject to posttranslational modification, thereby decreasing eNOS activity. We also performed low-temperature SDS-PAGE Western blots to assess the monomer/dimer ratio of the eNOS enzyme in diabetic mice. We found a striking difference in the monomer/dimer ratio in the diabetic mice as compared with nondiabetic controls, further suggesting that eNOS exists in an altered state in the diabetic mice.
Our present findings that eNOS is "uncoupled" in the diabetic mouse support our data demonstrating that eNOS gene therapy enhanced hepatic I-R injury in db/db diabetic mice. We therefore sought to further this notion by examining the possibility that increases in eNOS in the diabetic mouse resulted in increased ONOO– via interactions between superoxide and NO·. To indirectly identify peroxynitrite formation, we examined hepatic nitrotyrosine formation using 2 distinct methods. Qualitative assessment of nitrotyrosine via Western blot analysis and quantitative assessment with an ELISA revealed an increase in nitrotyrosine in the diabetic as compared with nondiabetic animals. Remarkably, these levels were increased to an even greater extent in the db/db eNOS-Tg animals under baseline conditions. These results are very suggestive that eNOS therapy in the diabetic further increased oxidative stress in these animals via the formation of ONOO–. Current studies22,33 have cited ONOO– as a mediator of eNOS uncoupling. This presents a possible positive feedback in the db/db diabetic mouse whereby increased superoxide production further increases ONOO– formation, which in turn acts to further uncouple eNOS perpetuating superoxide production. Similarly, the addition of exogenous NO· (DETA/NO or nitrite), which we found to increase hepatic injury, could result in increased ONOO– formation by interaction with high levels of superoxide anion following I-R.
To further implicate eNOS as a pathogenic mediator of I-R injury in the diabetic, we next pharmacologically inhibited eNOS with L-NAME. First, nondiabetic controls and eNOS–/– animals were subjected to hepatic I-R. We found that genetic deletion of the eNOS enzyme in the nondiabetic increased serum ALT by 3.1-fold following I-R. This result was anticipated given previous studies16,34 demonstrating increased I-R injury in eNOS–/– animals. Pharmacological inhibition with L-NAME was also found to increase serum ALT levels following I-R in nondiabetic mice. However, the administration of L-NAME to the nondiabetic caused ALT levels to rise more than 2-fold higher than those seen in the eNOS–/–, suggesting L-NAME was not simply inhibiting eNOS but was also exerting additional effects that resulted in enhanced hepatic I-R injury. The administration of L-NAME in the diabetic was the most injurious intervention in the present study and given the results noted in the nondiabetic, clearly L-NAME is not solely specific for the inhibition of eNOS.
In an effort to further our understanding of eNOS function in the db/db diabetic mouse, we next measured the eNOS cofactor BH4 levels in these animals. BH4 availability has been shown to be important for both catalytic activity and the coupling of the enzyme for efficient production of nitric oxide.35,36 High-performance liquid chromatography (HPLC) analysis revealed a substantial 2-fold decrease in BH4 levels in the diabetic liver. This result supports other published studies8,37,38 and provides another possible mechanism by which eNOS may be uncoupled in the diabetic liver. Decreases in BH4 availability has been suggested37,39 to be directly linked to superoxide production in diabetes. We administered either BH4 or sepiapterin before induction of hepatic I-R injury. Sepiapterin is a precursor in the BH4 salvage pathway40 and has been shown to increase BH4 levels and augment eNOS function in vivo.38 The acute administration of either BH4 or sepiapterin decreased serum ALT levels in the both the nondiabetic and diabetic following I-R injury. These data suggest that the restoration of BH4 levels in the diabetic liver is beneficial via augmenting eNOS function. Working on the hypothesis that increasing BH4 levels in the diabetic "recouples" eNOS and thereby reduces superoxide production, we again attempted NO· donor therapy following the supplementation of BH4. Treatment of diabetic mice with BH4 before sodium nitrite therapy significantly reduced hepatic injury. ALT levels with this combination therapy decreased hepatic injury to the lowest seen in the current study in db/db diabetic mice. This was in stark contrast to the previous finding that nitrite administration alone trended toward an increase in hepatic injury in the db/db mouse. A possible mechanism for this protection is that increased levels of BH4 recoupled eNOS, thus decreasing the production of superoxide. The subsequent administration of NO· thereby did not yield the potent oxidizing species ONOO– but rather increased NO· bioavailability, leading to protection against I-R injury.
To examine the possibility that superoxide anion modulates the deleterious effects of NO· therapy we investigated a cell-permeable SOD mimetic. Treatment of nondiabetic mice with the SOD mimetic before ischemia resulted in a decrease in serum ALT. This finding supports work by Hines et al,41 who found significant protection against hepatic I-R injury following treatment with an SOD mimetic. Treatment of diabetic mice with SOD therapy before ischemia trended to lower serum ALT levels, although not significantly. However, cotreatment with SOD therapy followed by the administration of the NO· donor DETA/NO significantly lowered ALT levels following I-R injury. This result further supports the likelihood that decreasing superoxide levels in the setting of diabetic I-R injury allows for nitric oxide therapy to be beneficial rather than injurious. Taken together, these data suggest that recoupling eNOS provides protection in an additive fashion by both decreasing superoxide production and increasing NO· production.
The controversy of nitric oxide therapy has been at the forefront of the I-R injury field for more than 2 decades. We present a clear example of both the protective and injurious nature of NO· therapy in an in vivo model of hepatic I-R injury. We show that eNOS gene therapy and direct nitric oxide therapy are very protective in nondiabetic animals subjected to I-R Injury. In contrast, in the setting of type 2 diabetes mellitus, these therapies are pathogenic. The injurious nature of NO· therapy could however be reversed with prior administration of BH4 or a SOD mimetic. These results suggest that superoxide may play a pivotal role by combining with NO· to form the potent free radical ONOO– and that eNOS is a major source of superoxide anion generation in the diabetic liver. Our study suggests that eNOS cofactor therapy may restore eNOS function in diabetes mellitus and may be beneficial in the setting of ischemic disorders.
Acknowledgments
We thank Jeff Szot, Kyle Aaron, and Michael Hicks for invaluable technical expertise in conducting these studies.
Sources of Funding
These studies were supported by a grant from the NIH (2R01 HL-6049 to D.J.L.) and by a grant from the American Diabetes Association (7-04-RA-59 to D.J.L.).
Disclosure
D.J.L. is a participant on a pending U.S. patent, filed on October 14, 2003 through NIH (patent no. 60/511, 244), regarding the use of sodium nitrite in cardiovascular disease.
Footnotes
This manuscript was sent to H. Michael Piper, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received September 13, 2005; resubmission received March 23, 2006; revised resubmission received May 22, 2006; accepted May 30, 2006.
References
Katsuki S, Arnold WP, Murad F. Effects of sodium nitroprusside, nitroglycerin, and sodium azide on levels of cyclic nucleotides and mechanical activity of various tissues. J Cyclic Nucleotide Res. 1977; 3: 239–247. [Order article via Infotrieve]
Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, Kadowitz PJ. Evidence for the inhibitory role of guanosine 3', 5'-monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood. 1981; 57: 946–955.
Ignarro LJ, Napoli C, Loscalzo J. Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide: an overview. Circ Res. 2002; 90: 21–28.
Kim PK, Zuckerbraun BS, Otterbein LE, Vodovotz Y, Billiar TR. ‘Til cell death do us part: nitric oxide and mechanisms of hepatotoxicity. Biol Chem. 2004; 385: 11–15. [Order article via Infotrieve]
Wang Y, Vodovotz Y, Kim PK, Zamora R, Billiar TR. Mechanisms of hepatoprotection by nitric oxide. Ann N Y Acad Sci. 2002; 962: 415–422.
Taniai H, Hines IN, Bharwani S, Maloney RE, Nimura Y, Gao B, Flores SC, McCord JM, Grisham MB, Aw TY. Susceptibility of murine periportal hepatocytes to hypoxia-reoxygenation: role for NO and Kupffer cell-derived oxidants. Hepatology. 2004; 39: 1544–1552. [Order article via Infotrieve]
Pieper GM. Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction. Hypertension. 1998; 31: 1047–1060.
Pannirselvam M, Verma S, Anderson TJ, Triggle CR. Cellular basis of endothelial dysfunction in small mesenteric arteries from spontaneously diabetic (db/db -/-) mice: role of decreased tetrahydrobiopterin bioavailability. Br J Pharmacol. 2002; 136: 255–263. [Order article via Infotrieve]
Bucala R, Tracey KJ, Cerami A. Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in experimental diabetes. J Clin Invest. 1991; 87: 432–438. [Order article via Infotrieve]
Napoli C, Lerman LO, de Nigris F, Loscalzo J, Ignarro LJ. Glycoxidized low-density lipoprotein downregulates endothelial nitricoxide synthase in human coronary cells. J Am Coll Cardiol. 2002; 40: 1515–1522.
Aslan M, Ryan TM, Adler B, Townes TM, Parks DA, Thompson JA, Tousson A, Gladwin MT, Patel RP, Tarpey MM, Batinic-Haberle I, White CR, Freeman BA. Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease. Proc Natl Acad Sci U S A. 2001; 98: 15215–15220.
Zou MH, Cohen R, Ullrich V. Peroxynitrite and vascular endothelial dysfunction in diabetes mellitus. Endothelium. 2004; 11: 89–97. [Order article via Infotrieve]
Sessa WC. eNOS at a glance. J Cell Sci. 2004; 117: 2427–2429.
Ooboshi H, Rios CD, Chu Y, Christenson SD, Faraci FM, Davidson BL, Heistad DD. Augmented adenovirus-mediated gene transfer to atherosclerotic vessels. Arterioscler Thromb Vasc Biol. 1997; 17: 1786–1792.
Ohashi Y, Kawashima S, Hirata K, Yamashita T, Ishida T, Inoue N, Sakoda T, Kurihara H, Yazaki Y, Yokoyama M. Hypotension and reduced nitric oxide-elicited vasorelaxation in transgenic mice overexpressing endothelial nitric oxide synthase. J Clin Invest. 1998; 102: 2061–2071.
Sharp BR, Jones SP, Rimmer DM, Lefer DJ. Differential response to myocardial reperfusion injury in eNOS-deficient mice. Am J Physiol Heart Circ Physiol. 2002; 282: H2422–H2426.
Jones SP, Greer JJ, Kakkar AK, Ware PD, Turnage RH, Hicks M, van Haperen R, de Crom R, Kawashima S, Yokoyama M, Lefer DJ. Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury. Am J Physiol Heart Circ Physiol. 2004; 286: H276–H282.
Duranski MR, Greer JJ, Dejam A, Jaganmohan S, Hogg N, Langston W, Patel RP, Yet SF, Wang X, Kevil CG, Gladwin MT, Lefer DJ. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J Clin Invest. 2005; 115: 1232–1240.
Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, Ma XL. Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation. 2002; 105: 1497–1502.
Tanaka S, Choe N, Hemenway DR, Zhu S, Matalon S, Kagan E. Asbestos inhalation induces reactive nitrogen species and nitrotyrosine formation in the lungs and pleura of the rat. J Clin Invest. 1998; 102: 445–454.
Gao F, Yao CL, Gao E, Mo QZ, Yan WL, McLaughlin R, Lopez BL, Christopher TA, Ma XL. Enhancement of glutathione cardioprotection by ascorbic acid in myocardial reperfusion injury. J Pharmacol Exp Ther. 2002; 301: 543–550.
Zou MH, Shi C, Cohen RA. Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin Invest. 2002; 109: 817–826.
Howells DW, Smith I, Hyland K. Estimation of tetrahydrobiopterin and other pterins in cerebrospinal fluid using reversed-phase high-performance liquid chromatography with electrochemical and fluorescence detection. J Chromatogr. 1986; 381: 285–294. [Order article via Infotrieve]
Champion HC, Bivalacqua TJ, Hyman AL, Ignarro LJ, Hellstrom WJ, Kadowitz PJ. Gene transfer of endothelial nitric oxide synthase to the penis augments erectile responses in the aged rat. Proc Natl Acad Sci U S A. 1999; 96: 11648–11652.
Lake-Bruse KD, Faraci FM, Shesely EG, Maeda N, Sigmund CD, Heistad DD. Gene transfer of endothelial nitric oxide synthase (eNOS) in eNOS-deficient mice. Am J Physiol. 1999; 277: H770–H776.
Ricciardi R, Foley DP, Quarfordt SH, Saavedra JE, Keefer LK, Wheeler SM, Donohue SE, Callery MP, Meyers WC. V-PYRRO/NO: an hepato-selective nitric oxide donor improves porcine liver hemodynamics and function after ischemia reperfusion. Transplantation. 2001; 71: 193–198. [Order article via Infotrieve]
Aiba M, Takeyoshi I, Ohwada S, Kawashima Y, Iwanami K, Sunose Y, Yamada T, Tsutsumi H, Matsumoto K, Morishita Y. Novel nitric oxide donor (FK409) ameliorates liver damage during extended liver resection with warm ischemia in dogs. J Am Coll Surg. 2001; 193: 264–271. [Order article via Infotrieve]
Webb A, Bond R, McLean P, Uppal R, Benjamin N, Ahluwalia A. Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage. Proc Natl Acad Sci U S A. 2004; 101: 13683–13688.
Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA Jr, Sessa WC. Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem. 2003; 278: 44719–44726.
Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999; 399: 601–605. [Order article via Infotrieve]
McCabe TJ, Fulton D, Roman LJ, Sessa WC. Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation. J Biol Chem. 2000; 275: 6123–6128.
Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee M. Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest. 2001; 108: 1341–1348.
Kuzkaya N, Weissmann N, Harrison DG, Dikalov S. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase. J Biol Chem. 2003; 278: 22546–22554.
Kawachi S, Hines IN, Laroux FS, Hoffman J, Bharwani S, Gray L, Leffer D, Grisham MB. Nitric oxide synthase and postischemic liver injury. Biochem Biophys Res Commun. 2000; 276: 851–854. [Order article via Infotrieve]
Rodriguez-Crespo I, Gerber NC, Ortiz de Montellano PR. Endothelial nitric-oxide synthase. Expression in Escherichia coli, spectroscopic characterization, and role of tetrahydrobiopterin in dimer formation. J Biol Chem. 1996; 271: 11462–11467.
Berka V, Wu G, Yeh HC, Palmer G, Tsai AL. Three different oxygen-induced radical species in endothelial nitric-oxide synthase oxygenase domain under regulation by L-arginine and tetrahydrobiopterin. J Biol Chem. 2004; 279: 32243–32251.
Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, Goh N, Rockett KA, Channon KM. Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression. J Clin Invest. 2003; 112: 725–735.
Meininger CJ, Marinos RS, Hatakeyama K, Martinez-Zaguilan R, Rojas JD, Kelly KA, Wu G. Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency. Biochem J. 2000; 349: 353–356. [Order article via Infotrieve]
Shinozaki K, Kashiwagi A, Nishio Y, Okamura T, Yoshida Y, Masada M, Toda N, Kikkawa R. Abnormal biopterin metabolism is a major cause of impaired endothelium-dependent relaxation through nitric oxide/O2- imbalance in insulin-resistant rat aorta. Diabetes. 1999; 48: 2437–2445.
Milstien S, Kaufman S. Tetrahydro-sepiapterin is an intermediate in tetrahydrobiopterin biosynthesis. Biochem Biophys Res Commun. 1983; 115: 888–893. [Order article via Infotrieve]
Hines IN, Harada H, Flores S, Gao B, McCord JM, Grisham MB. Endothelial nitric oxide synthase protects the post-ischemic liver: potential interactions with superoxide. Biomed Pharmacother. 2005; 59: 183–189
您现在查看是摘要介绍页，详见ORG附件。