Protective effect of glutamine on endothelial cell ATP in oxidant injury

Endothelial cell dysfunction following exposure to H2O2 is associated with rapid inhibition of glucose-dependent pathways of ATP synthesis. The role other substrates for ATP synthesis (e.g., amino acids) may play in the metabolism of H2O2-injured cells is unclear. The effect of glutamine, a precursor of the Kreb's cycle intermediate alpha-ketoglutarate on ATP levels in bovine pulmonary artery endothelial cells exposed to H2O2 was examined. The presence of glutamine during H2O2 injury significantly enhanced ATP levels in the injured cells. Concentrations of glutamine as low as 50 microM produced significant improvement of ATP levels in endothelial cells exposed to 5 mM H2O2. The 2 mM concentration of glutamine produced the greatest benefit, while greater concentrations of glutamine (5-20 mM) were actually associated with progressive decrements of the maximal benefit seen with the 2 mM concentration. The 2 mM concentration of glutamine produced similar enhancement of ATP with 1 and 10 mM H2O2 injury as well. Short-term viability following 5 mM H2O2 injury was significantly improved by the presence of 2 mM glutamine. The most effective concentration of glutamine (2 mM) did not scavenge H2O2 in a fluorometric assay. These observations suggest that mitochondrial substrates, such as glutamine, that bypass glucose-dependent pathways of ATP synthesis may be useful therapeutic agents for maintenance of ATP levels in oxidant-injured cells.     

Metabolic inhibition potentiates oxidant injury

Toxic oxygen species have been implicated as important mediators of injury after reperfusion of an ischemic organ. The aim of this study was to determine if prior metabolic inhibition, such as that which occurs during ischemia, potentiates oxidant injury in vitro. Bovine pulmonary artery endothelial cells were metabolically inhibited for various periods of time with or without the mitochondrial inhibitor oligomycin (650 nM). The cells were rescued from metabolic inhibition by a wash step and subsequent addition of 5.5 mM glucose. At the same time that metabolic inhibition was relieved the cells were subjected to doses of H2O2 ranging from 0 to 100 microM. ATP levels were monitored over a 2-hr time course after rescue from metabolic inhibition by the luciferin-luciferase assay. Cell viability at 2 hr after relief of metabolic inhibition was assessed by trypan blue exclusion. Intracellular pH during metabolic inhibition was determined with the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein tetraacetomethoxymethyl ester. H2O2 consumption, a measure of H2O2 scavenging capability, was determined by a fluorescent assay. The viability and ATP levels of cells not subjected to metabolic inhibition were unaffected by these low concentrations of H2O2. Cells metabolically inhibited with glucose depletion and oligomycin were exquisitely sensitive to H2O2. Cells that were only deprived of glucose demonstrated no potentiation of injury, while cells subjected to mitochondrial inhibition with oligomycin alone also showed significant potentiation of oxidant injury. H2O2 consumption was not affected by metabolic inhibition. Conditions associated with mitochondrial inhibition consistently resulted in a decrease in intracellular pH. These experiments suggest that a synergism exists between metabolic inhibition and subsequent oxidant exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of endothelial cell shape change in oxidant injury

Changes in endothelial cell morphology induced by neutrophil-generated hydrogen peroxide (H2O2) may account for the capillary leak of the adult respiratory distress syndrome (ARDS). The relationship of H2O2 effects on the concentration of intracellular Ca2+ [( Ca2+]i) and ATP to changes in microfilaments and microtubules, important determinants of cell shape, was examined. Bovine pulmonary artery endothelial cells were injured over a 2-hr time course with a range of H2O2 doses (0-20 mM). The higher concentrations of H2O2 consistently produced contraction and rounding of greater than 50-75% of cells by 1-2 hr. The range of 1-20 mM H2O2 produced rapid, significant reductions in endothelial ATP levels over the time course of injury. Although there were significant increases in mean endothelial [Ca2+]i in response to 5, 10, and 20 mM H2O2, 1 mM H2O2 did not affect the [Ca2+]i. Fluorescence microscopy revealed that microfilament disruption occurred as ATP levels fell and preceded depolymerization of microtubules which developed after [Ca2+]i approached 1 X 10(-6) M. H2O2 at 1 mM injury caused microfilament disruption but did not depolymerize microtubules. Microfilament disruption occurred without oxidant exposure, when ATP levels were reduced by glucose depletion and mitochondrial inhibition with oligomycin (650 nM). If a Ca2+ ionophore, ionomycin (5 microM), was then added, [Ca2+]i rose to greater than 1 X 10(-6) M, microtubules fragmented and depolymerized, and cell contraction and rounding very similar to that induced by H2O2 occurred. These results suggest that endothelial cell dysfunction and capillary leak in ARDS may be due to H2O2-mediated changes in cellular ATP and [Ca2+]i.     

Neonatal oxidative liver metabolism: effects of hydrogen peroxide, a putative mediator of septic damage.

BACKGROUND/PURPOSE: Surgical neonates are at risk for sepsis and liver dysfunction. These complications are more common in preterm neonates and in those who receive total parenteral nutrition. Elevated levels of reactive oxygen species (eg, hydrogen peroxide) have been reported in these "at-risk" patients and may be the mediators of liver impairment via their effect on oxidative energy metabolism. The aim of this study was to test the hypothesis that elevated levels of hydrogen peroxide (H2O2) impair neonatal liver oxidative energy metabolism. METHODS: An in vitro model to test this hypothesis was developed in hepatocytes isolated from neonatal (11-day to 15-day) rats. The cells, respiring on palmitate (0.5 mmol/L in 2% bovine serum albumin), were exposed to H2O2. Oxygen consumption was measured polarographically. In experiment A, H2O2 was added to the cell preparation at different concentrations (0.5 mmol/L, 1 mmol/L, 1.5 mmol/L, 2 mmol/L) to assess the effect on oxygen consumption. In experiment B, H2O2 (2 mmol/L) was added to hepatocytes in the presence of inhibitors of mitochondrial respiration to define the site of action of H2O2. In experiment C, electron microscopy was performed on hepatocytes after incubation with 1 mmol/L and 2 mmol/L of H2O2. RESULTS: In experiment A, H2O2 significantly reduced hepatocyte oxygen consumption at 1.5 and 2 mmol/L. In experiment B, in the presence of inhibitors of mitochondrial respiration, myxothiazol (inhibitor of substrate oxidation), and oligomycin (inhibitor of adenosine triphosphate (ATP) synthase), no further inhibition by H2O2 occurred, indicating that the effect of H2O2 was intramitochondrial and affecting the synthesis of ATP. In experiment C, microscopic alterations of mitochondria were noticed exclusively in hepatocytes incubated with 2 mmol/L H2O2. CONCLUSIONS: Results of this study demonstrate that H2O2 impairs neonatal liver oxidative metabolism. H2O2 probably directly inhibits ATP synthase. The authors hypothesize that H2O2 may play a role in the biochemical pathogenesis of liver dysfunction associated with sepsis. Identification of the precise target site of H2O2 may be valuable in directing therapy in septic neonates.     

氯离子通道阻断剂对氧化剂诱导的肾小管上皮细胞损伤的保护作用

目的研究氯离子（Cl-）通道阻断剂在氧化剂诱导的肾小管上皮细胞损伤中的作用。方法以H2O2诱导肾小管上皮细胞株（LLC-PK1）损伤，观察Cl-通道阻断剂对受损细胞LDH释放量、ATP含量和DNA降解程度的影响。结果Cl-通道阻断剂可使受损细胞的LDH释放量下降、ATP含量回升和DNA降解程度减轻。结论Cl-通道参与了氧化剂对细胞损伤的病理过程，Cl-通道阻断剂对肾小管上皮细胞具有保护作用。     

Tempol, a membrane-permeable radical scavenger, reduces oxidant stress-mediated renal dysfunction and injury in the rat

BACKGROUND: The generation of reactive oxygen species (ROS) contributes to the pathogenesis of renal ischemia-reperfusion injury. The aim of this study was to investigate the effects of tempol in (1) an in vivo rat model of renal ischemia/reperfusion injury and on (2) cellular injury and death of rat renal proximal tubular (PT) cells exposed to oxidant stress in the form of hydrogen peroxide (H2O2). METHODS: Male Wistar rats underwent bilateral renal pedicle clamping for 45 minutes followed by reperfusion for six hours. Tempol (30 mg/kg/h), desferrioxamine (DEF; 40 mg/kg/h), or a combination of tempol (30 mg/kg/h) and DEF (40 mg/kg/h) were administered prior to and throughout reperfusion. Plasma concentrations of urea, creatinine, Na+, gamma-glutamyl transferase (gammaGT), aspartate aminotransferase (AST), and urinary Na+ and N-acetyl-beta-D-glucosaminidase (NAG) were measured for the assessment of renal function and reperfusion injury. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of polymorphonuclear (PMN) cell infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of nitrotyrosine and poly(ADP-ribose) synthetase (PARS). Primary cultures of rat PT cells were incubated with H2O2 (1 mmol/L for 4 h) either in the absence or presence of increasing concentrations of tempol (0.03 to 10 mmol/L), DEF (0.03 to 10 mmol/L), or a combination of tempol (3 mmol/L) or DEF (3 mmol/L). PT cell injury and death were determined by evaluating mitochondrial respiration and lactate dehydrogenase (LDH) release, respectively. RESULTS: In vivo, tempol significantly reduced the increase in urea, creatinine, gammaGT, AST, NAG, and FENa produced by renal ischemia/reperfusion, suggesting an improvement in both renal function and injury. Tempol also significantly reduced kidney MPO activity and MDA levels, indicating a reduction in PMN infiltration and lipid peroxidation, respectively. Tempol reduced the histologic evidence of renal damage associated with ischemia/reperfusion and caused a substantial reduction in the staining for nitrotyrosine and PARS, suggesting reduced nitrosative and oxidative stress. In vitro, tempol significantly attenuated H2O2-mediated decrease in mitochondrial respiration and increase in LDH release from rat PT cells, indicating a reduction in cell injury and death. Both in vivo and in vitro, the beneficial actions of tempol were similar to those obtained using the Fe2+ chelator DEF. However, coadministration of DEF and tempol did not produce any additional beneficial actions against renal ischemia/reperfusion injury or against oxidative stress-mediated PT cell injury/death. CONCLUSION: Our results suggest that the membrane-permeable radical scavenger, tempol, reduces the renal dysfunction and injury associated with ischemia/reperfusion of the kidney.     

Reactive oxygen molecule-mediated injury in endothelial and renal tubular epithelial cells in vitro

To investigate renal tubular epithelial cell injury mediated by reactive oxygen molecules and to explore the relative susceptibility of epithelial cells and endothelial cells to oxidant injury, we determined cell injury in human umbilical vein endothelial cells and in four renal tubular epithelial cell lines including LLC-PK1, MDCK, OK and normal human kidney cortical epithelial cells (NHK-C). Cells were exposed to reactive oxygen molecules including superoxide anion, hydrogen peroxide and hydroxyl radical generated by xanthine oxidase and hypoxanthine. We determined early sublethal injury with efflux of 3H-adenine metabolites and a decline in ATP levels, while late lytic injury and cell detachment were determined by release of 51chromium. When the cells were exposed to 25, 50, and 100 mU/ml xanthine oxidase with 5.0 mM hypoxanthine, ATP levels were significantly lower (P less than 0.001) in LLC-PK1, NHK-C and OK cells compared to MDCK cells while ATP levels were significantly lower (P less than 0.01) in endothelial cells compared to all tubular cell lines. A similar pattern of injury was seen with efflux of 3H-adenine metabolites. When the cells were exposed to 50 mU/ml xanthine oxidase with 5.0 mM hypoxanthine for five hours, total 51chromium release was significantly (P less than 0.001) greater in LLC-PK1, NHK-C and OK cells compared to MDCK cells, while total 51chromium release was significantly (P less than 0.001) greater in endothelial cells compared to all tubular cells. However, lytic injury was the greatest in LLC-PK1 cells and NHK-C cells while cell detachment was the greatest in endothelial cells. MDCK cells were remarkably resistant to oxidant-mediated cell detachment and cell lysis. In addition, we determined ATP levels, 3H-adenine release and 51chromium release in LLC-PK1, NHK-C and endothelial cells in the presence of superoxide dismutase to dismute superoxide anion, catalase to metabolize hydrogen peroxide, DMPO to trap hydroxyl radical and DMTU to scavenge hydrogen peroxide and hydroxyl radical. We found that catalase and DMTU (scavengers of hydrogen peroxide) provided significant protection from ATP depletion, prevented efflux of 3H-adenine metabolites and cell detachment while DMPO (scavenger of hydroxyl radical) prevented lytic injury. In addition, we found that the membrane-permeable iron chelator, phenanthroline, and preincubation with deferoxamine prevented cell detachment and cell lysis, confirming the role of hydroxyl radical in cell injury.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin prevents oxidant-induced endothelial cell barrier dysfunction via nitric oxide-dependent pathway

BACKGROUND: The rigorous maintenance of normoglycemia by the administration of insulin is beneficial to critically ill patients. Because insulin induces endothelial nitric oxide (NO) release, and the constitutive release of NO maintains normal microvascular permeability, the authors postulated that insulin would prevent peroxide (H(2)O(2))-induced endothelial barrier dysfunction, an effect dependent on endothelial NO synthase (eNOS) activity. METHODS: Murine lung microvascular endothelial cells (LMEC) grown to confluence on 8 micro pore polyethylene filters were exposed to media (control), H(2)O(2) (20 to 500 micromol/L), insulin (1 to 1,000 nmol/L) or insulin (100 nmol/L) + H(2)O(2) (10(-4)mol/L). Endothelial monolayer permeability was quantitated by measuring the transendothelial electrical resistance at 15-minute intervals for 120 minutes. Other cells were exposed to H(2)O(2) and insulin after pretreatment with a NO scavenger (PTIO), an eNOS inhibitor (L-NIO), or a phosphoinositol-3-kinase inhibitor (LY-294002). RESULTS: H(2)O(2) caused a concentration- and time-dependent reduction in electrical resistance consistent with an increase in monolayer permeability. This effect was prevented by insulin. Inhibiting NO release (L-NIO, LY-294002) or scavenging NO (PTIO) abolished this protective effect. CONCLUSIONS: These data suggest that insulin may modulate endothelial barrier function during oxidant stress by inducing the release of NO.     

Effect of Volatile Anesthetics on Hydrogen Peroxide-induced Injury in Aortic and Pulmonary Arterial Endothelial Cells

Background: Oxidant damage to endothelial cells occurs during inflammation and reperfusion after ischemia, mediated in part by endogenously produced hydrogen peroxide (H2 O2). Previous studies have established a role for increased cytosolic calcium in the mechanism of endothelial oxidant injury, and have suggested that volatile anesthetics may exacerbate oxidant injury in pulmonary endothelium. However, the effect of volatile anesthetics on oxidant injury to systemic arterial endothelial cells, and their effect on oxidant-related changes in cytosolic calcium homeostasis, have not been reported previously.

Methods: Primary cultures of human aortic and pulmonary arterial endothelial cells were studied. The rate of cell death after H2 O2 exposure was determined in cell suspension by propidium iodide fluorimetry and lactate dehydrogenase release. The final extent of cell death 24 h after H2 O2 exposure was determined in monolayer cultures by methyl thiazolyl tetrazolium reduction. Cytosolic calcium and cell death were determined in single cells using fura-2 and propidium iodide imaging with digitized, multiparameter, fluorescent video microscopy.

Results: In aortic endothelial cells, clinical concentrations of halothane (1.0%) and isoflurane (1.5%) decreased both the rate of cell death and the final extent of cell death after H2 O2 exposure, with halothane being more protective. Supraclinical concentrations of halothane (2.7%) and isoflurane (4.0%) were less protective. In pulmonary arterial endothelial cells, halothane and isoflurane had essentially no effect on H2 O2 -mediated cell death. The protective effect of anesthetic in aortic endothelial cells was not due to an enhanced removal of H2 O2 by endogenous enzymes. Hydrogen peroxide exposure caused a large increase in cytosolic calcium well before cell death, and this was moderated by anesthetic treatment.     

Glutamine and transforming growth factor-alpha stimulate extracellular regulated kinases and enhance recovery of villous surface area in porcine ischemic-injured intestine

BACKGROUND: Epidermal growth factor (EGF) signals enterocyte proliferation via extracellular regulated kinases (ERKs). Because glutamine is required for EGF-stimulated proliferation and stimulates ERKs in intestinal cell culture, we hypothesized that glutamine and the EGF-related peptide transforming growth factor-alpha (TGF-alpha) would synergistically enhance repair associated with stimulation of ERKs. METHODS: Thiry-Vella loops were created in juvenile pigs. One half of the loop was subjected to 2 hours of ischemia, and the other half served as control. Loops were infused daily with Ringer's solution containing 140 mmol/L glucose, 140 mmol/L glutamine, 140 mmol/L glucose plus 60 micrograms/L TGF-alpha, or 140 mmol/L glutamine plus 60 micrograms/L TGF-alpha. RESULTS: After 2 hours of ischemia, complete villous epithelial sloughing was present. By 18 hours, villous epithelium had fully restituted, but villi remained stunted until 144 hours after injury. Glutamine + TGF-alpha triggered sustained increases in ERK activity compared with glucose-treated tissues (maximal at 18 hours), whereas glutamine alone or glucose + TGF-alpha caused only transient elevations in ERK activity. By 72 hours, villous surface area had increased to normal values with glutamine plus TGF-alpha treatment, whereas villi remained stunted with glucose alone, glutamine alone, or glucose plus TGF-alpha. CONCLUSIONS: Glutamine plus TGF-alpha treatment restored mucosal architecture within 72 hours of severe ischemic injury associated with sustained elevations in ERK activity.     

Effect of fructose-1,6-bisphosphate in the cold storage solution after 12 and 36 hours of rat liver preservation

Fructose-1,6-bisphosphate (FBP) has been reported to have a protective effect on liver injury following ischemic/reperfusion periods because it maintains ATP levels during cold preservation. In the present study, we evaluated the effects of addition of FBP to storage solutions for cold liver preservation during 12 or 36 hours. Adult male Wistar rats were randomly divided into three experimental groups. The hepatic perfusion and preservation were performed with these solutions: UW; UW plus 10 mmol/L FBP; and FBP 10 mmol/L (FBPS) alone. The biochemical measurements of AST and ALT were performed on samples of the cold storage solution after 12- or 36-hour preservation. UW and FBPS solutions showed similar preservation grades at 12 hours. Addition of 10 mmol/L of FBP to UW solution induced liver injury and a poor preservation grade during 12 or 36 hours. UW solution was better than FBPS after 36 hours preservation. UW solution continues to offer a superior performance for liver preservation during long times; however, FBPS may be an alternative for short cold preservation times.     

Effect of stable glutamine compounds on porcine islet culture

BACKGROUND: Pig islets are characterized by significant fragility, preventing successful islet culture prior to xenotransplantation. To improve outcome after culture, we compared the effects of glutamine supplementation on survival and viability of isolated pig islets during culture. METHODS: Pig islets were suspended in CMRL 1066 supplemented either with 2.5 mmol/L N-acetyl-L-alanyl-L-glutamine (NALG), a stable compound of L-glutamine, or with 2.5 or 5.0 mmol/L of free L-glutamine (L-Glu). After 24 hours of preincubation, islets were stressed for additional 48 hours with H2O2, DETA, or a cytokine mix. RESULTS: Twenty-four-hour survival of unstressed controls precultured with 2.5 mmol/L NALG was significantly decreased compared with islets pretreated with 2.5 or 5.0 mmol/L L-Glu (P < .01). Fresh islets, viability decreased significantly after NALG preincubation, but was maintained after preincubation in 2.5 or 5.0 mmol/L L-Glu (not significant vs fresh; P < .05 vs NALG). Compared with NALG pretreatment L-Glu did not significantly ameliorate the relative survival (related to cultured controls) of islets during proinflammatory treatment. Nevertheless, the beneficial effect of L-Glu preculture on absolute survival (related to freshly isolated islets) of stressed islets was still present in contrast to NALG pretreatment (P < .01). Viability of stressed islets was significantly protected by L-Glu but not by NALG. CONCLUSIONS: Pig islet culture is significantly improved if L-glutamine is administered in an unbound form compared with the stable compound NALG. Stress resistance of pig islets seems to be increased by free L-glutamine as well.     

The immune-enhancing enteral agents arginine and glutamine differentially modulate gut barrier function following mesenteric ischemia/reperfusion

BACKGROUND: Immune-enhancing enteral diets have been shown to improve patient outcome. One contributing mechanism may be via maintenance of gut barrier function. While recent data has shown that glutamine is beneficial, arginine may be harmful. We therefore hypothesized that the immune-enhancing agents, glutamine and arginine, differentially modulate gut barrier function. METHODS: At laparotomy, rats had jejunal sacs filled with 10 mmol/L glutamine, arginine, fructose, or magnesium sulfate (osmotic control) followed by 60 minutes of superior mesenteric artery occlusion and 2 hours of reperfusion. Jejunum was harvested for histology, deconvolution microscopy, F:G actin, ATP, and permeability measurements. RESULTS: Glutamine and fructose minimized mucosal injury compared with controls and arginine. Deconvolution microscopy confirmed that glutamine and fructose preserved the actin cytoskeleton but there was disruption by arginine which correlated with F:G actin ratios and tissue ATP levels. Permeability was enhanced by arginine compared with the other groups. CONCLUSION: Arginine resulted in worsened mucosal injury, disruption of the actin cytoskeleton, decreased tissue ATP and enhanced permeability compared with glutamine which appeared protective. The immune-enhancing agent arginine results in breakdown of gut barrier function which may have important implications for critically injured patients.     

Hypertonicity Induces Injury to Cultured Human Endothelium: Attenuation by Glutamine

Background. Although most preservation solutions as well as some cardioplegic solutions used for organ storage and transplantation are hypertonic, the effects of extracellular hypertonicity on endothelium are not well established. Aims of this study were to evaluate the response of cultured human saphenous vein endothelial cells to extracellular hypertonicity and to investigate the role of the amino acid glutamine in preventing endothelial damage in vitro.

Methods. Eight distinct strains of human saphenous vein endothelial cells were studied. Hypertonic (350 and 400 mosm/kg) media were obtained by supplementing culture medium with sucrose. Cell viability was assessed in the absence or the presence of glutamine through the determination of cell number and protein content of the cultures. Confocal microscopy of cells loaded with the fluorescent dye calcein was also performed.

Results. Exposure of human saphenous vein endothelial cells to hypertonic media without glutamine caused significant cell loss within 30 minutes. Cell loss progressed steadily during incubation and after 6 hours reached 50% at 350 mosm/kg and 65% at 400 mosm/kg. In the presence of 2 mmol/L glutamine, endothelial damage was completely prevented at 350 mosm/kg and significantly lessened at 400 mosm/kg compared with glutamine-free media. Confocal microscopy showed that most hypertonicity-treated cells exhibited the typical features of an apoptotic death and confirmed the osmoprotective effect of glutamine.

Conclusions. These results indicate that the supplementation of hypertonic storage solutions with glutamine might exert a partial osmoprotective effect and suggest that the relationship between endothelial damage and tonicity of storage and cardioplegic solutions should be carefully investigated.     

Oxidation of albumin is enhanced in the presence of uremic toxins

Albumin has been considered a "sacrificial plasma antioxidant" due to the high reactivity of the protein sulfhydryl groups with oxidants such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). Based on its large quantity and high turnover. It is considered as one of the most important plasma antioxidants for protecting key cellular and regulatory proteins. Since hemodialysis patients have lower overall levels of albumin and possible protein modifications due to uremic toxins, we investigated whether modifications by various uremic toxins would affect the susceptibility of albumin to an oxidative challenge. We incubated bovine serum albumin in the presence of carboxymethyllysine (CML) (10 micromol/L(-1) mmol/L), methyl glyoxal (50 micromol/L(-5) mmol/L), p-cresol (100 micromol/L-10 mmol/L) or hippuric acid (200 micromol/L-20 mmol/L) for 16 hours at 37 degrees C and then subsequently added 0.5 mmol/L(-1) mmol of H2O2/HOCl. We measured the extent of protein modification by the loss of protein sulfhydryl groups, dityrosine formation and the formation of advanced oxidation protein products (AOPP). Incubation of albumin with the uremic toxins caused a loss of protein sulfhydryl groups and an increase in dityrosines and AOPP. The presence of uremic toxins had no effect on the loss of protein sulfhydryl groups after addition of H2O2/HOCl; however, low levels of CML, p-cresol and methyl glyoxal inhibited the formation of AOPP and dityrosines. We suggest that uremic toxins may possibly play a role in mediating free radical initiated protein damage.     

Activation of ERK or inhibition of JNK ameliorates H(2)O(2) cytotoxicity in mouse renal proximal tubule cells

BACKGROUND: Our previous studies suggest that the balance between the activation of extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal/stress-activated protein kinase (JNK) might determine cell fate following oxidant injury in vivo. METHODS: The mouse proximal tubule cell line (TKPTS) was used to study hydrogen peroxide (H(2)O(2))-induced death and survival. The role of ERK and JNK in this process was studied by using adenoviruses that contain either a constitutively active mitogen-activated protein kinase kinase 1 (MEK1) or a dominant-negative JNK. Acridine orange plus ethidium bromide staining was applied to distinguish between viable, apoptotic, and necrotic cells following H(2)O(2) treatment. We analyzed cell cycle events by fluorescence-activated cell sorter (FACS) analysis and the phosphorylation status of ERK and JNK by Western blotting. RESULTS: TKPTS cells survived a moderate level of oxidative stress (0.5 mM/L H(2)O(2)) via temporary growth arrest, while high dose of H(2)O(2) (1 mM/L) caused extensive necrosis. Survival was associated with activation of both ERK and JNK, while death was associated with JNK activation only. Prior adenovirus-mediated up-regulation of ERK or inhibition of JNK function increased the survival (8- or 7-fold, respectively) of TKPTS cells after 1 mmol/L H(2)O(2) treatment. Interestingly, ERK activation and, thus, survival was associated with growth arrest not proliferation. CONCLUSION: We demonstrate that oxidant injury-induced necrosis could be ameliorated by either up-regulation of endogenous ERK or by inhibition of JNK-related pathways. These results directly demonstrate that the intracellular balance between prosurvival and prodeath mitogen-activated protein kinases (MAPKs) determine proximal tubule cell survival from oxidant injury and reveal possible mediators of survival.     

Improvement of the cold storage of blood vessels with a vascular preservation solution. Study in porcine aortic segments

BACKGROUND: Cold-induced injury to various cell types has been shown to be mediated predominantly by chelatable iron. For endothelial cells, this type of injury has so far only been shown in cultured cells. Hypothesizing that this iron-dependent cold-induced injury might also occur in the endothelium of intact vessels, we here set out to optimize the hypothermic storage of blood vessels. METHODS: Segments of porcine aorta were stored for 2 to 21 days in histidine-tryptophan-ketoglutarate (HTK) solution or in modified solutions with or without the iron chelators deferoxamine or LK 614 at 4 degrees C. Parts of the segments were assayed immediately after cold storage, the other parts after subsequent rewarming. The percentage of dead (propidium iodide-positive) endothelial cells was assessed by "intravital" fluorescence microscopy, mitochondrial membrane potential was assessed by laser scanning microscopy after staining with tetramethylrhodamine methyl ester (TMRM) and thrombocyte adhesion was studied using 5-(and -6)-carboxy SNARF-1-stained thrombocytes. RESULTS: The endothelium of porcine aortic segments sustained moderate injury during the cold incubation itself, but major injury during rewarming. The addition of the iron chelator deferoxamine (1 mmol/L) significantly inhibited cold-induced endothelial cell injury irrespective of the solution used for cold storage (eg, 14 days of cold storage + 3 hours rewarming: HTK 66 +/- 7%, HTK + 1 mmol/L deferoxamine 40 +/- 10% propidium iodide-positive endothelial cells). An amino acid (glycine, alanine, aspartate)-containing base solution with N-acetylhistidine as buffer was optimized. The optimized base solution with pH 7.0 and potassium and chloride as main ions yielded a further decrease of endothelial cell injury. Combination of deferoxamine (in lower concentration, ie, 0.1 mmol/L) with the new, more membrane-permeable iron chelator LK 614 (20 mumol/L) further improved preservation so that even after 3 weeks of cold storage plus 3 hours rewarming only 10 +/- 1% of endothelial cells were propidium iodide positive. In this optimized solution, both endothelial cell survival and mitochondrial membrane potential were significantly better preserved than in the clinically used solutions HTK, University of Wisconsin (UW) and Perfadex, or in physiological saline. Thrombocyte adhesion was also significantly reduced after cold storage in the optimized solution compared with HTK solution. CONCLUSION: Cold-induced injury to the endothelium of porcine aortic segments is, as the injury to cultured endothelial cells, mediated by chelatable iron. Thus, iron chelators, but also optimized base solutions, are options to improve the storage of vascular endothelium. The optimized solution should now be tested in in vivo animal experiments.     

Glutamine and glutathione counteract the inhibitory effects of mediators of sepsis in neonatal hepatocytes

BACKGROUND/PURPOSE: Surgical neonates are at risk of sepsis-associated liver dysfunction. Hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) are important mediators of sepsis, which impair neonatal hepatic metabolism. Glutamine has been shown to have beneficial effects on hepatocyte metabolism during neonatal sepsis. However, the molecular basis of these effects are unknown. The aim of this study was to test the hypotheses that (1) glutamine and its dipeptides counteract the inhibitory effect of septic mediators on neonatal hepatocyte oxygen consumption and (2) the effects of glutamine are specific and not shared by other amino acids. In addition, we wished to determine the metabolic pathways and mediators involved in the action of glutamine. METHODS: Hepatocytes were isolated from suckling rats, and O(2) consumption measured polarographically. Study A: the ability of 10 mmol/L glutamine to reverse the inhibitory effects of 1.5 mmol/L H(2)O(2) and 300 micromol/L S-Nitroso-N-acetylpenicillamine (SNAP; a nitric oxide donor) on O(2) consumption was examined. Study B: the ability of other amino acids and dipeptides of glutamine to reverse the effects of H(2)O(2) was examined. Study C: various concentrations of glutamine were tested for their ability to reverse the H(2)O(2) inhibition of O(2) consumption. Study D: the mechanism of action of glutamine was examined by incubating hepatocytes with either an inhibitor of entry into the Krebs cycle or an inhibitor of glutathione synthesis. Study E: the ability of glutathione to reverse the inhibitory effects of H(2)O(2) was examined. RESULTS: Study A: glutamine reversed the inhibition of hepatocyte O(2) consumption exerted by either H(2)O(2) or NO. Study B: glutamine dipeptides reversed the inhibition of hepatocyte O(2) consumption by H(2)O(2), but other amino acids did not. Study C: the counteracting effect of glutamine was proportional to the dose administered. Study D: blocking entry of glutamine into the Krebs cycle did not abolish the effects of glutamine, but blocking glutathione synthesis completely abolished the effect of glutamine. Study E: exogenous glutathione reversed the inhibitory effect of H(2)O(2) on hepatocyte O(2) consumption. CONCLUSIONS: This study found that glutamine and its dipeptides are unique in reversing the effects of septic mediators on neonatal rat liver oxidative metabolism. The effectiveness of glutamine appears to be mediated via glutathione synthesis. Addition of glutamine, glutamine dipeptides, or glutathione to total parenteral nutrition (TPN) may be beneficial in preventing liver damage in neonatal sepsis.     

二氮嗪预先给药对大鼠心肌微血管内皮细胞缺氧复氧时细胞凋亡的影响

目的 探讨二氮嗪预先给药对大鼠心肌微血管内皮细胞缺氧复氧时细胞凋亡的影响.方法培养SD大鼠心肌微血管内皮细胞,以1×106个/ml的密度接种于96孔培养板(100 μl/孔)或培养皿(2 ml/皿),采用随机数字表法,将其随机分为4组(n=12),正常对照组(C组)不作任何处理,缺氧复氧组(H/R组)、二氮嗪预先给药组(DZ组)和二氮嗪预先给药+5-羟葵酸组(DZ+5-HD组)均进行缺氧2 h复氧2 h,DZ组和DZ+5-HD组在缺氧前2 h分别加入100 μmol/L二氮嗪和100 μmol/L二氮嗪+100 μmol/L线粒体ATP敏感性钾通道阻断剂5-羟葵酸.于复氧2 h时测定细胞活力和凋亡率.结果 与C组比较,H/R组细胞活力降低,细胞凋亡率升高(P＜0.01);与H/R组比较,DZ组细胞活力升高,细胞凋亡率降低(P＜0.05或0.01);5-羟葵酸可抑制二氮嗪预先给药导致的上述改变(P＜0.05或0.01).结论 二氮嗪预先给药可抑制大鼠心肌微血管内皮细胞凋亡,从而减轻缺氧复氧损伤,其机制与激活线粒体ATP敏感性钾通道有关.

Abstract：

Objective To investigate the effects of diazoxide pretreatment on apoptosis in rat myocardial microvascular endothelial cells exposed to hypoxia-reoxygenation (H/R) . Methods The SD rat myocardial microvascular endothelial cells were cultured. The cells were seeded in 96-well plates (100 μl/hole) or in 6 cm diameter dishes (2 ml/dish) with the density of 1×106/ml and randomly divided into 4 groups ( n = 12 each) : normal con trol group (group C), H/R group, diazoxide pretreatment group (group DZ) and diazoxide pretreatment + 5-hydroxydecanoate (5-HD, a mitochondrial ATP-sensitive potassium channel blocker) group (group DZ + 5-HD) .The cells were exposed to 2 h hypoxia followed by 2 h reoxygenation. Diazoxide 100 μmol/L and diazoxide 100 μmol/L + 5-HD 100 μmol/L were added to the culture medium 2 h before hypoxia in groups DZ and DZ + 5-HD respectively. The cell viability and apoptotic rate were detected at the end of reoxygenation. Results Compared with group C, the cell viability was significantly decreased, while the apoptotic rate increased in group H/R ( P ＜ 0.01) . Compared with group H/R, the cell viability was significantly increased, while the apoptotic rate decreased in group DZ (P ＜ 0.05 or 0.01) . 5-HD could inhibit diazoxide pretreatment-induced changes mentioned above ( P ＜ 0.05 or 0.01). Conclusion Diazoxide pretreatment can reduce H/R injury through inhibiting apoptosis in rat myocardial microvascular endothelial cells, and the mechanism is related to the activation of mitochondrial ATP-sensitive potassium channels.