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.     

Protection against hydrogen peroxide induced injury in renal proximal tubule cell lines by inhibition of poly(ADP-ribose) synthase

Radicals including superoxide anions, hydrogen peroxide or hydroxyl radicals and NO or peroxynitrite cause the breakage of DNA strands and activation of poly-(ADP-ribose) synthase (PARS). Recent studies showed that inhibition of PARS activity reduces the tissue injury after exposure to oxidative stress. However, the role of PARS in renal injury by oxidants has not been examined. In this study effect of a PARS inhibitor, 3-aminobenamide (3-AB), on injury of opossum kidney or LLC-PK(1) cells by hydrogen peroxide or tert-butyl hydroperoxide (t-BHP) was examined. The exposure of opossum kidney cells to hydrogen peroxide activated PARS and decreased cellular adenosine triphosphate levels in a concentration-dependent manner. Inhibition of PARS with 3-AB prevented the cell death induced by hydrogen peroxide and also prevented adenosine triphosphate depletion. 3-AB did not have hydroxyl radical scavenging effect. In contrast, t-BHP did not affect the PARS activity. The decrease in cellular adenosine triphosphate levels by t-BHP was less than that by hydrogen peroxide. 3-AB failed to prevent the cell death induced by t-BHP. PARS activation after exposure of hydrogen peroxide was inhibited by addition of t-BHP. However, t-BHP showed an additive effect on cell death with hydrogen peroxide. These results indicate that activation of PARS plays an important role in hydrogen peroxide induced injury in opossum kidney cells and that hydrogen peroxide and t-BHP induce cell injury by different mechanisms.     

Role of cytochrome P4502E1 activation in proximal tubular cell injury induced by hydrogen peroxide

BACKGROUND: There is now good evidence to suggest that cytochrome P450 (CYP450) may act as an iron-donating catalyst for the production of hydroxyl ion (OH*), which contributes to proximal tubular cell injury. However, it remains unclear which isoform of CYP450 is involved in this process. Cytochrome P4502E1 (CYP2E1) is a highly labile isoform which is not only involved in free radical generation, but has also been shown to be a source of iron in cisplatin-induced renal injury. This study investigates the role of CYP2E1 in the proximal tubular cell injury induced by hydrogen peroxide (H2O2). METHODS: Porcine proximal tubular cells (LLC-PK1) were incubated with H2O2 (1 mM) for 4 h in the presence or absence of 0.1 mM of two CYP2E1 inhibitors; diallyl sulfide (DAS), or disulfiram (DSF), desferrioxamine (DFO) (0.1-0.4 mM), or catalase (CT) (78, 150, 300 U/mL). Cell death was determined by measuring LDH release. CYP2E1 activity was determined by p-nitrophenol hydroxylation after 2 h incubation with H2O2. RESULTS: Exposure of LLC-PKI to H2O2 significantly increased cell death. CT, DFO, DAS and DSF significantly reduced H2O2-mediated cell death. Incubation with H2O2 increased CYP2EI activation in time- and dose-dependent manner, which was significantly reduced by CT, DFO, DAS and DSF. CONCLUSION: We propose that CYP2E1 activation occurs possibly due to OH* and contributes to H2O2-mediated LLC-PK1 cell necrosis by acting as a source of iron and perpetuating the generation of OH* via the Fenton reaction. Inhibition of CYP2E1 may be a novel approach for the prevention of tubular injury caused by oxidative stress.     

The role of poly(ADP-ribose) synthetase inhibition on the intestinal mucosal barrier after thermal injury

Oxidative and nitrosative stressor agents can trigger DNA strand breakage, which then activates the nuclear enzyme poly(ADP-ribose) synthetase (PARS). Activation of the enzyme depletes the intracellular concentration of energetic substrates such as nicotinamide adenine dinucleotide (NAD). This process can result in cell dysfunction and cell death. PARS inhibitors have been successfully used in ischemia–reperfusion injury, inflammation and sepsis in several experimental models. In our experimental study, we investigated the role of 3-aminobeanzamide (3-AB), a non-specific PARS inhibitor, on the intestinal mucosal barrier after burn injury. Twenty-four Wistar rats were randomly divided into three groups. The sham group (n = 8) was exposed to 21 °C water while the burn group (n = 8) and the burn + 3-AB group (n = 9) were exposed to boiling water for 12 s to produce a full thickness burn in 35–40% of total body surface area. In the burn + 3-AB group, 10 mg/kg of 3-AB was given intraperitoneally 10 min before thermal injury. Twenty-four hours later, tissue samples from mesenteric lymph nodes (MLN), spleen and liver were obtained under sterile conditions for microbiological analysis and ileum samples were obtained for biochemical and histopathological analysis. In burn group, the incidence of bacteria isolated from MLN and spleen was significantly higher than other groups (P < 0.05). 3-AB pre-treatment prevented burn induced bacterial translocation and it significantly reduced burn induced intestinal injury. Tissue malondialdehyde and 3-nitrotyrozine levels were found significantly lower than that of the burn group. These data suggest that the relationship between PARS pathway and lipid peroxidation in intestinal tissue and PARS has a role in intestinal injury caused by thermal injury.     

Prevention of myocardial reperfusion injury by poly(ADP-ribose) synthetase inhibitor, 3-aminobenzamide, in cardioplegic solution: in vitro study of isolated rat heart model.

OBJECTIVE: Cardioplegic arrest remains the method of choice for myocardial protection in cardiac surgery. Poly(adenosine 5'-diphosphate-ribose) synthetase (PARS) inhibitor has been suggested to attenuate the ischemia-reperfusion injury in myocardial infarction by preventing energy depletion associated with oxidative stress. We investigated the efficacy of a cardioplegic solution containing a PARS inhibitor, 3-aminobenzamide (3-AB), for myocardial protection against ischemia-reperfusion injury caused by cardioplegic arrest. METHODS: Isolated hearts were set on a Langendorff apparatus and perfused. The hearts were arrested for 90 min with a cardioplegic solution given at 30-min intervals and then reperfused for 20 min. The hearts of rat in the 3-AB(-) group (n = 8) were perfused with a standard cardioplegic solution and terminal warm cardoplegia, whereas the 3-AB(+) group (n = 8) received these solutions supplemented with 3-AB (100 microM). Left ventricular function and release of cardiac enzymes were monitored before and after cardioplegic arrest. After reperfusion, NAD+ (nicotinamide-adenine dinucleotide) levels were assessed, and the tissues were examined immunohistochemically for oxidative stress and apoptosis. RESULTS: During reperfusion, the 3-AB(+) group showed significantly higher (P = 0.005)dp/dt and lower creatine phosphokinase (CPK) level and glucotamic-oxaloacetic transaminase (GOT) in the effluent (CPK; P = 0.003 GOT; P < 0.001) The cardiomyocytes of the 3-AB(+) group also preserved a higher NAD+ level (P < 0.001). Immunohistochemical study of oxidative stress revealed a lesser extent (P = 0.007) of nuclear staining and a lower fraction of apoptosis in the 3-AB(+) group. CONCLUSION: Cardioplegic solution supplemented with 3-AB provides efficient myocardial protection in cardioplegic ischemic reperfusion by suppressing oxidative stress and overactivation of PARS.     

Apoptosis inhibition plays a greater role than necrosis inhibition in decreasing bacterial translocation in experimental intestinal transplantation

BACKGROUND: During small-bowel transplantation, necrosis and apoptosis are involved in the destruction of intestinal epithelial cells. This study was conducted to assess which mode of cell death plays a greater role as a trigger of the bacterial translocation (BT) associated with intestinal transplantation. METHODS: The following experimental groups were studied: sham, Tx (intestinal transplantation), Tx + poly (ADP-ribose) synthetase (PARS) inhibitor 3-aminobenzamide (3-AB), and Tx + caspase inhibitor Z-VAD-fmk. Histological analysis, caspase-3 activity, DNA fragmentation, and BT were measured in tissue samples after transplantation. RESULTS: During intestinal transplantation, apoptosis and necrosis both increased, showing graft injury and high levels of BT. Rats treated with 3-AB showed histological protection of the transplanted graft and a tendency toward lower BT despite the existence of high apoptosis levels. The rats treated with Z-VAD showed histological protection of the transplanted graft and decreased levels of caspase-3 and DNA fragmentation. The Tx + Z-VAD group showed the lowest levels of BT in all tissues. CONCLUSIONS: In small intestinal transplantation, both apoptosis and cell necrosis give rise to histological injury and BT. Apoptosis inhibition and necrosis inhibition treatments protect intestinal grafts from ischemia/reperfusion injury; Z-VAD supplementation has a greater effect on BT prevention than does administration of the PARS inhibitor 3-AB.     

Effects of inhibition of poly(adenosine diphosphate-ribose) synthase on acute cardiac allograft rejection

BACKGROUND: Nitric oxide synthase (NOS)-2 is expressed during acute cardiac allograft rejection in association with death of heart muscle cells. The nuclear enzyme poly(adenosine diphosphate [ADP]-ribose) synthase (PARS) is activated by agonists such as NO and peroxynitrite, which cause single-strand DNA breaks; PARS, in turn can promote both necrosis and apoptosis. To investigate the hypothesis that NO produced by NOS-2 in cardiomyocytes activates PARS and contributes to heart muscle cell death by apoptosis, experiments were performed using a heterotopic rat abdominal heart transplant model and cytokine-stimulated heart muscle cells in tissue culture. METHODS: Cardiac allografts were treated after transplantation with either the PARS inhibitor 5-aminoisoquinolinone at 3 mg/kg subcutaneously daily or with vehicle. Isolated purified adult rat cardiomyocytes incubated with cytokines to induce NOS-2 were treated in vitro with another PARS inhibitor, 3-aminobenzamide (3AB). RESULTS: PARS inhibition increased cardiac-allograft survival from 6 +/- 2 to 10 +/- 3 days (n=6, n=6, P<0.05). The inflammatory infiltrate, NOS-2-positive macrophages, myocyte apoptosis, and myocyte content of nitrotyrosine and poly(ADP-ribose) were significantly decreased in PARS inhibited allografts at day 5 posttransplantation. Similarly, apoptosis and PARS activity were diminished in cytokine-stimulated adult rat cardiomyocytes when either 3AB or L-NMMA were applied. CONCLUSIONS: The data indicate that PARS activation occurs during acute cardiac-allograft rejection and contributes significantly to the inflammatory response and to the death of cardiac muscle cells by apoptosis. They suggest that PARS inhibition combined with immunosuppression might enhance salvage of heart-muscle cells during acute cardiac-allograft rejection.     

5-Aminoisoquinolinone reduces renal injury and dysfunction caused by experimental ischemia/reperfusion

BACKGROUND: Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the development of ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of a water-soluble and potent PARP inhibitor, 5-aminoisoquinolinone (5-AIQ), on the renal injury and dysfunction caused by oxidative stress of the rat kidney in vitro and in vivo. METHODS: Primary cultures of rat renal proximal tubular cells, subjected to oxidative stress caused by hydrogen peroxide (H2O2), were incubated with increasing concentrations of 5-AIQ (0.01 to 1 mmol/L) after which PARP activation, cellular injury, and cell death were measured. In in vivo experiments, anesthetized male Wistar rats were subjected to renal bilateral ischemia (45 minutes) followed by reperfusion (6 hours) in the absence or presence of 5-AIQ (0.3 mg/kg) after which renal dysfunction, injury and PARP activation were assessed. RESULTS: Incubation of proximal tubular cells with H2O2 caused a substantial increase in PARP activity, cellular injury, and cell death, which were all significantly reduced in a concentration-dependent by 5-AIQ [inhibitory concentration 50 (IC50) approximately 0.03 mmol/L]. In vivo, renal I/R resulted in renal dysfunction, injury, and PARP activation, primarily in the proximal tubules of the kidney. Administration of 5-AIQ significantly reduced the biochemical and histologic signs of renal dysfunction and injury and markedly reduced PARP activation caused by I/R. CONCLUSION: This study demonstrates that 5-AIQ is a potent, water soluble inhibitor of PARP activity, which can significantly reduce (1) cellular injury and death caused to primary cultures of rat proximal tubular cells by oxidative stress in vitro, and (2) renal injury and dysfunction caused by I/R of the kidney of the rat in vivo.     

Proximal tubules from caspase-1-deficient mice are protected against hypoxia-induced membrane injury.

BACKGROUND: Caspase-1 is a proinflammatory caspase via activation of the cytokine IL-18. We have recently demonstrated that the caspase-1-mediated production of IL-18 plays a deleterious role in ischaemic acute renal failure (ARF) which is independent of neutrophils and CD4+ T cells. The role of caspase-1 in hypoxia-induced membrane injury of proximal tubules (PT) in vitro is unknown. METHODS: Freshly isolated mouse PT exposed to 25 min of hypoxia were used to study the role of caspases, caspase-1 and IL-18 in hypoxia-induced membrane injury. Lactate dehydrogenase (LDH) release into the PT medium was used as a biochemical parameter of cell membrane damage. IL-18 was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: PT pre-incubated with the novel pancaspase inhibitor IDN-8050 were protected; LDH release (%) was 35+/-3 in vehicle-treated hypoxic PT and 21+/-2 in IDN-8050-treated hypoxic PT (P<0.01, n=6). To investigate the mechanism of protection and examine the role of caspase-1 specifically, PT were isolated in parallel from wild-type and caspase-1- deficient (-/-) mice. PT from caspase-1-/-mice demonstrated less hypoxia-induced membrane injury. LDH release was 37+/-2 in wild-type hypoxic PT and 28+/-2 in caspase-1-/-hypoxic PT (P<0.01, n=12). IL-18 was detected in PT by immunoblotting and ELISA. PT pre-incubated with IL-18 binding protein, an inhibitor of IL-18, were not protected. CONCLUSIONS: These studies demonstrate a deleterious effect of the proinflammatory caspase, caspase-1, on PT in vitro in the absence of inflammatory cells and vascular effects.     

Efficacy of poly(adenosine diphosphate-ribose) polymerase inhibition in extracorporeal shock wave-induced renal injury

Objectives: Extracorporeal shock wave lithotripsy (ESW) induces renal damage by excessive production of free oxygen radicals. Free Oxygen radicals cause cellular injury by inducing nicks in DNA. The enzyme poly(adenosine diphosphate-ribose) polymerase (PARP) involved in the process of repair of DNA in damaged cells. However, its activation in damaged cells can lead to adenosine triphosphate depletion and death. Thus, we designed a study to evaluate the efficacy of 3-aminobenzamide (3-AB), a PARP inhibitor, against extracorporeal shock wave induced renal injury. Methods: Twenty-four Sprague-Dawley rats were divided into three groups: control, ESW, ESW?+?3-AB groups. All groups except control group were subjected to ESW procedure. ESW?+?3-AB group received 20?mg/kg/day 3-aminobenzamide intraperitoneally at 2?h before ESW and continued once a day for consecutive 3 days. The surviving animals were sacrificed at the 4th day and their kidneys were harvested for biochemical and histopathologic analysis. Blood samples from animals were also obtained. Results: Serum ALT and AST levels, serum neopterin and tissue oxidative stress parameters were increased in the ESW group and almost came to control values in the treatment group (p?p?Conclusion: Our data showed that PARP inhibition protected renal tissue against ESW induced renal injury. These findings suggest that it would be possible to improve the outcome of ESW induced renal injury by using PARP inhibitors as a preventive therapy.     

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.     

A small dose of hydrogen peroxide enhances tumor necrosis factor-alpha toxicity in inducing human vascular endothelial cell apoptosis: reversal with propofol

We designed the present study to test the hypothesis that oxygen free radicals can enhance tumor necrosis factor (TNF)-alpha cellular toxicity, which might be reversed by propofol, an anesthetic with antioxidant properties, in human vascular endothelial cell line ECV304. Cultured ECV304 were either not treated, treated with 10 muM of hydrogen peroxide (H2O2), treated with TNF-alpha (40 ng/mL) alone, TNF-alpha in the presence of 10 microM of H2O2 (H+T), or propofol plus H2O2 for 24 h. Cell viability was measured by lactate dehydrogenate (LDH) assay. Cell apoptosis was assessed by flow cytometry and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labeling. The antiapoptotic Bcl-2 and pro-apoptotic Bax protein expressions were measured by immunocytochemical analysis. Increases in apoptosis, Bax, lipid peroxidation product malondialdehyde, LDH, and decreases in Bcl-2, superoxide dismutase, and glutathione peroxidase were observed in TNF-alpha-treated cells. H2O2 10 microM did not cause significant lipid peroxidation (0.75 +/- 0.03 nmol/mg of malondialdehyde protein) as compared with control (0.70 +/- 0.04 nmol/mg of malondialdehyde protein) (P > 0.05) but further enhanced TNF-alpha-induced lipid peroxidation, upregulated Bax, and down-regulated Bcl-2 expression and enhanced TNF-alpha-induced cell apoptosis (P < 0.05). Propofol 50 microM attenuated TNF-alpha and H2O2-induced cell apoptosis, accompanied by decreases in malondialdehyde and LDH production and restoration of Bcl-2 expression. Propofol exerts protective effects against H2O2-enhanced TNF-alpha cell toxicity by reducing oxidative injury.     

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.     

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.     

IGF-1 protects intestinal epithelial cells from oxidative stress-induced apoptosis

BACKGROUND: Reactive oxygen species (ROS) are involved in the pathogenesis of necrotizing enterocolitis (NEC) in premature infants. We have recently found that activation of multiple cellular signaling transduction pathways occurs during ROS-induced intestinal cell apoptosis; the phosphatidylinositol 3-kinase (PI3-K) pathway plays an anti-apoptotic role during this process. Insulin-like growth factor (IGF)-1 activates PI3-K pathway to promote cell survival; however, the effects of IGF-1 treatment during gut injury are not clearly defined. The purpose of this study was to determine whether IGF-1 protects intestinal cells from ROS-induced apoptosis. MATERIALS AND METHODS: Rat intestinal epithelial (RIE)-1 cells were treated with either IGF-1 (100 nm), hydrogen peroxide (H2O2; 500 microm), or combination. Western blotting was performed to assess phosphorylation of Akt, a downstream effector of PI3-K. Cell Death Detection ELISA, DCHF, and JC-1 assays were performed to demonstrate protective effects of IGF-1. Wortmannin, an inhibitor of PI3-K, was used to show PI3-K-dependent mechanism of action for IGF-1. RESULTS: H2O2 treatment resulted in increased intestinal epithelial cell apoptosis with intracellular ROS generation and mitochondrial membrane depolarization; IGF-1 pre-treatment attenuated this response without affecting ROS production. H2O2-induced phosphorylation of Akt was further increased with IGF-1 treatment; wortmannin abolished these effects in RIE-1 cells. CONCLUSIONS: PI3-K pathway is activated during ROS-induced intestinal epithelial cell injury; IGF-1 exerted an anti-apoptotic effect during this response by PI3-K activation. A better understanding of the exact role of IGF-1-mediated activation of PI3-K may allow us to facilitate the development of novel therapy against NEC.     

Membrane stabilizing effects of calcium and taxol during the cold storage of isolated rat hepatocytes.

BACKGROUND: Calcium plays an important role in liver preservation and preservation induces depletion of cellular Ca. This may affect hepatocyte cytoskeleton integrity necessary for maintaining cell shape and organ viability. We tested the effects of a microtubular stabilizer (Taxol) in liver cell preservation. METHODS: Isolated rat hepatocytes were preincubated with or without a microtubule stabilizing agent, 100 microM Taxol, at 37 degrees C for 20 min, then stored in the University of Wisconsin (UW) solution +/-1.5 mM CaC12 at 4 degrees C for up to 48 hr. After storage, the cells were rewarmed in Krebs-Henseleit buffer with air at 37 degrees C for 1 hr. Morphological changes in the plasma membrane (scanning electron microscopy) and cell viability (percentage of lactate dehydrogenase [LDH] release) before and after rewarming were studied. RESULTS: Hepatocytes showed time-dependent increase in bleb formation (cytoskeleton disruption) during cold storage. Rewarming the cells caused even greater bleb formation and increased LDH release (cell death). Pretreatment of cells with Taxol and cold storage in the UW solution with 1.5 mM Ca suppressed both bleb formation and LDH release in 48-hr coldstored cells. CONCLUSIONS: Cold storage of hepatocytes leads to reperfusion injury and cell death. This can be suppressed with Taxol and Ca. This suggests that hypothermia induces changes in cellular Ca and a disruption of the microtubules, leading to loss of cell viability. Improved liver preservation may require suppression of Ca-dependent disruption of the cytoskeleton system of liver cells.     

Propofol attenuates lung endothelial injury induced by ischemia-reperfusion and oxidative stress

Lung dysfunction after cardiopulmonary bypass and lung transplantation results from oxidant-mediated cellular damage. Previously, we observed the shedding of angiotensin-converting enzyme (ACE) from the endothelial cell surface to be a more sensitive and earlier marker of oxidative lung endothelial injury than lung wet-to-dry weight ratio. The aim of this study was to evaluate the potential of the anesthetic propofol, which has antioxidant properties, to prevent oxidative lung injury by measuring ACE shedding. ACE release from isolated perfused rat lungs increased significantly after ischemia-reperfusion (I/R). Propofol significantly decreased I/R-induced ACE release by 23.4% (P < 0.05). Perfusion with 0.75 mM H(2)O(2) also caused ACE release from the lung microvasculature, which was similarly attenuated by propofol. The protective effect of propofol on H(2)O(2)-induced ACE shedding was confirmed in vitro using Chinese Hamster Ovary cells overexpressing human ACE. Thus, propofol can attenuate oxidative injury of the pulmonary endothelium as detected by ACE shedding in I/R and H(2)O(2) models of acute lung injury.