Hypothermia causes a marked injury to rat proximal tubular cells that is aggravated by all currently used preservation solutions

Cold preservation results in cell death via iron-dependent formation of reactive oxygen species, leading to apoptosis during rewarming. We aimed to study cold-induced damage (i.e., injury as a consequence of hypothermia itself and not cold ischemia) in proximal tubular cells (PTC) in various preservation solutions presently applied and to clarify the role of mitochondria in this injury. Primary cultures of rat PTC were incubated at 4 degrees C for 24 h in culture medium, UW, Euro-Collins or HTK solution with and without the iron chelator desferal and rewarmed at 37 degrees C in culture medium. Cell damage, morphology, and apoptosis were studied and mitochondrial membrane potential was assessed by fluorescence microscopy. Cold incubation of PTC in culture medium followed by rewarming caused marked cell damage compared to warm incubation alone (LDH release 39+/-10% vs. 1.6+/-0.3%). Cold-induced damage was aggravated in all preservation solutions (LDH release 85+/-2% for UW; similar in Euro-Collins and HTK). After rewarming, cells showed features suggestive for apoptosis. Desferal prevented cell injury in all solutions (e.g., 8+/-2% for UW). Mitochondrial membrane potential was lost during rewarming and this loss could also be inhibited by desferal. Trifluoperazine, which is known to inhibit mitochondrial permeability transition (MPT), was able to prevent cold-induced injury (LDH 85+/-5% vs. 12+/-2%). We conclude that cold-induced injury occurs in PTC and is aggravated by UW, Euro-Collins, and HTK solution. Iron-dependent MPT is suggested to play a role in this damage. Strategies to prevent cold-induced injury should aim at reducing the availability of "free" iron.     

Ischemic preconditioning in a rodent hepatocyte model of liver hypothermic preservation injury

Ischemic preconditioning (IPC) is a phenomenon of protection in various tissues from normothermic ischemic injury by previous exposure to short cycles of ischemia-reperfusion. The ability of IPC to protect hepatocytes from a model of hypothermic transplant preservation injury was tested in this study. Rat hepatocytes were subjected to 30min of warm ischemia (37 degrees C) followed by 24 or 48h of hypothermic (4 degrees C) storage in UW solution and subsequent re-oxygenation at normothermia for 1h. Studies were performed with untreated control cells and cells treated with IPC (10min anoxia followed by 10min re-oxygenation, 1 cycle). Hepatocytes exposed to IPC prior to warm ischemia released significantly less LDH and had higher ATP concentrations, relative to untreated ischemic hepatocytes. IPC significantly reduced LDH release after 24h of cold storage before reperfusion and after 48h of cold storage and after 60min of warm re-oxygenation, relative to the corresponding untreated hepatocytes. ATP levels were also significantly higher when IPC was used prior to the warm and cold ischemia-re-oxygenation protocols. In parallel studies, IPC increased new protein synthesis and lactate after cold storage and reperfusion compared to untreated cells but no differences in the patterns of protein banding were detected on electrophoresis between the groups. In conclusion, IPC significantly improves hepatocyte viability and energy metabolism in a model of hypothermic preservation injury preceded by normothermic ischemia. These protective effects on viability may be related to enhanced protein and ATP synthesis at reperfusion.     

Actin cytoskeleton derangement induces apoptosis in renal ischemia/reperfusion

This study evaluated whether cytoskeletal alterations during the ischemic conditions associated with kidney preservation could determine apoptosis. Cytoskeletal alterations are among the main effects of ischemia and may induce apoptosis. Rat kidneys were preserved in University of Wisconsin (UW) solution for 24 h. Some groups of animals underwent 45 min of warm ischemia (WI) to evaluate its effect on both the actin cytoskeleton and apoptosis (assessed by caspase-3 activity and TUNEL staining). Swinholide A (SwinA) and Latrunculin B (LB), two actin cytoskeleton-targeted agents, were administered to assess the effect of direct actin disruption on apoptosis. Jasplakinolide (JP), a compound that stabilizes actin filaments, was administered to evaluate the effect of actin stabilization. Apoptosis was evaluated at 3 h of ex vivo reperfusion using the isolated perfused rat kidney (IPK) model. Results: Apoptosis increased during reperfusion with WI or administration of actin disruptor agents. Administration of stabilizing agents reversed apoptosis in kidneys that had previously undergone WI or had received an actin disruptor agent. Conclusion: The disruption of the actin cytoskeleton during ischemic conditions associated with kidney preservation induces apoptosis upon reperfusion through caspase-3 activation.     

Protective effects of a carbon monoxide-releasing molecule (CORM-3) during hepatic cold preservation

There is increasing evidence that carbon monoxide (CO), a signaling molecule generated during the degradation of heme by heme oxygenase-1 (HO-1) in biological systems, has a variety of cytoprotective actions, including anti-hypoxic effects at low temperatures. However, during liver cold preservation, a direct effect needs to be established. Here, we designed a study to analyze the role of CO, delivered via a carbon monoxide-releasing molecule (CO-RM) in the maintenance of liver function, and integrity in rats during cold ischemia/reperfusion (CI/R) injury. We used an isolated normothermic perfused liver system (INPL) following a clinically relevant model of ex vivo 48 h cold ischemia stored in a modified University of Wisconsin (UW) solution, to determine the specific effects of CO in a rat model. CO was generated from 50 μM tricarbonylchloro ruthenium-glycinato (CORM-3), a water-soluble transition metal carbonyl that exerts pharmacological activities via the liberation of controlled amounts of CO in biological systems. The physiological effects of CORM-3 were confirmed by the parallel use of a specific inactive compound (iCORM-3), which does not liberate CO in the cellular environment.CORM-3 addition was found to prevent the injury caused by cold storage by improving significantly the perfusion flow during reperfusion (by almost 90%), and by decreasing the intrahepatic resistance (by 88%) when compared with livers cold preserved in UW alone. Also, CORM-3 supplementation preserved good metabolic capacity as indicated by hepatic oxygen consumption, glycogen content, and release of lactate dehydrogenase. Liver histology was also partially preserved by CORM-3 treatment.

Conclusions

These findings suggest that CO-RM could be utilized as adjuvant therapeutics in UW solutions to limit the injury sustained by donor livers during cold storage prior to transplantation, as has been similarly proposed for the heart, and kidney.     

A subset of 26S proteasomes is activated at critically low ATP concentrations and contributes to myocardial injury during cold ischemia

Molecular mechanisms leading to myocardial injury during warm or cold ischemia are insufficiently understood. Although proteasomes are thought to contribute to myocardial ischemia-reperfusion injury, their roles during the ischemic period remain elusive. Because donor hearts are commonly exposed to prolonged global cold ischemia prior to cardiac transplantation, we evaluated the role and regulation of the proteasome during cold ischemic storage of rat hearts in context of the myocardial ATP content. When measured at the actual tissue ATP concentration, cardiac proteasome peptidase activity increased by 225% as ATP declined during cold ischemic storage of hearts in University of Wisconsin (UW) solution for up to 48 h. Addition of the specific proteasome inhibitor epoxomicin to the UW solution inhibited proteasome activity in the cardiac extracts, significantly reduced edema formation and preserved the ultrastructural integrity of the cardiomyocyte. Utilizing purified 20S/26S proteasome enzyme preparations, we demonstrate that this activation can be attributed to a subset of 26S proteasomes which are stable at ATP concentrations far below physiological levels, that ATP negatively regulates its activity and that maximal activation occurs at ATP concentrations in the low μmol/L range. These data suggest that proteasome activation is a pathophysiologically relevant mechanism of cold ischemic myocardial injury. A subset of 26S proteasomes appears to be a cell-destructive protease that is activated as ATP levels decline. Proteasome inhibition during cold ischemia preserves the ultrastructural integrity of the cardiomyocyte.     

The influence of short-term fasting on the quality of small bowel graft preservation

INTRODUCTION: Donor nutritional status may be a determinant of small bowel (SB) quality following storage. In this study, we investigated the effect of donor nutritional status and a proven nutrient-rich preservation solution on graft quality following cold storage. METHODS: Rats were fasted (12-14 h) or non-fasted. SB (n=6) was flushed vascularly with modified University of Wisconsin (UW) solution and flushed luminally with UW or an amino acid-rich (AA) solution as follows: Fasted. UWV, none; UWL, UW solution; AAL, AA solution. Non-fasted. UWV, none; UWL, UW solution; AAL, AA solution. Energetics, peroxidation (malondialdehyde; MDA), glutathione and histology were assessed over 24 h at 4 degrees C. RESULTS: Energetics (ATP, ATP/ADP, and energy charge) were significantly higher in AAL (fasted and non-fasted) groups than other groups. However, there were no differences in energetics parameters between fasted and non-fasted animals in all groups. MDA was higher in fasted groups than non-fasted tissues; interestingly, AAL values were up to 10-fold lower than other groups. Higher glutathione levels were detected in non-fasted AAL tissues. Mucosal integrity was markedly superior in luminally treated tissues (UWL and AAL) in fasted and non-fasted states. Most noteably, AAL tissues from fasted animals exhibited grade 2 injury (villus clefting), whereas normal mucosa was observed in non-fasted tissues (grade 0). CONCLUSION: Luminal flushing and a nutrient-rich preservation solution improve energetics, oxidative stress, and mucosal integrity during storage. Poorer donor nutritional status does not affect energetics throughout storage, but causes mucosal injury as a result of increased oxidative stress, even after a brief period of donor fasting.     

Improvement of renal preservation by adding lidoflazine to University of Wisconsin solution. An experimental study in the rat.

The purpose of this study was to investigate the possibility of improving the organ preservation properties of the University of Wisconsin (UW) solution by adding the calcium entry blocker lidoflazine. We also investigated the possibility of decreasing the cold ischemia and reperfusion damage by pretreatment with lidoflazine of the donor and/or recipient. The protective effects of lidoflazine treatment were estimated by measuring the amount of trapped erythrocytes in the rat renal medulla after 48 h of cold storage, subsequent transplantation, and 20 min of reperfusion. Lidoflazine (20 mg/liter) added to the UW solution decreased the amount of erythrocyte trapping from 14.8 +/- 3.1% in controls to 8.6 +/- 1.7% (P less than 0.01). The flow rate of the flush-out solution during the harvesting procedure was also significantly (P less than 0.01) increased when lidoflazine was included in the UW solution (1.10 +/- 0.21 ml/min vs 0.75 +/- 0.22 ml/min). Administration of lidoflazine (0.28 mg/kg body wt) to the donor and/or the recipient did not further reduce the postischemia/reperfusion damage as estimated by the degree of erythrocyte trapping. In conclusion, the results indicate that the preservation properties of the UW solution can be significantly improved by adding lidoflazine to the solution.     

Noninvasive monitoring of citrate, acetate, lactate, and renal medullary osmolyte excretion in urine as biomarkers of exposure to ischemic reperfusion injury

Injury during the transplant process affects the alloantigen-dependent factors and the alloantigen-independent processes of "chronic" rejection. Consequently, the determination of reliable parameters for the assessment of ischemic damage is essential for the prediction of renal changes after ischemia/reperfusion injury. The aim of this study was to assess the ability of (1)H NMR spectroscopy to predict the early graft dysfunction in an ischemia/reperfusion model after preservation in two standard preservation solutions, Euro-Collins (EC) and University of Wisconsin (UW). The second aim was to specify the role of the UW solution in preventing renal medullary injury. Urine and plasma samples from three experimental groups were examined during 2 weeks: control group (n = 5), EC group (cold flushed and 48-h cold storage of kidney in EC and autotransplantation, n = 12), and UW group (cold flushed and 48-h cold storage of kidney in UW and autotransplantation; n = 12). We also examined these kidneys 30-40 min after implantation and on the sacrifice day. Creatinine clearance was significantly reduced in the EC group during the second week. Fractional excretion of sodium and urine N-acetyl-beta-d-glucosaminidase activity were improved but not significantly different in the preserved groups. Urinary concentrations of the alpha-class glutathione S-transferase were significantly greater in the EC group during the first week after transplantation. The most relevant resonances for evaluating renal function after transplantation determined by (1)H NMR spectroscopy were those arising from citrate, dimethylamine (DMA), lactate, and acetate in urine and trimethylamine-N-oxide (TMAO) in urine and plasma. These findings suggest that graft dysfunction is associated with damage to the renal medulla determined by TMAO release in urine and plasma associated with DMA and acetate excretion. Citrate is also a urinary marker that can discriminate kidneys with a favorable evolution. Our results suggest that (1)H NMR spectroscopy is an efficient technique for detecting ischemic damage when accurate and precise data on graft injury is required. In addition, this study outlines the specific impact of the UW solution against injury to the renal medulla.     

Hyperbranched Polyglycerol as a Colloid in Cold Organ Preservation Solutions

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation.     

Evaluation of early regenerative processes in a preclinical pig model of acute kidney injury

Renal failure due to ischemic injury is a common denominator of various clinical situations in critically ill patients. This study was designed to characterize the TPSO/Cholesterol synthesis and cell division pathways in response to different levels of ischemia. Porcine kidneys were subjected to either 60 min-warm ischemia (WI) or auto-transplanted after cold storage for 24 h at 4°C (CS), or both conditions (WI+CS), pathway activation and function were evaluated at 3 h, 3 and 7 days after reperfusion. CS combined to WI affects renal functions indicating a high degree of injury. During the first week of reperfusion, renal levels of free and esterified cholesterol, major cellular components, increased in CS group with an attenuated production when WI was associated. CS and WI+CS groups exhibited an elevated expression of cell cycle induction markers such as PCNA and stathmin. TSPO expression was highest in groups with the lowest injury, and correlated with kidney outcome, revealing its potential for diagnosis.     

Inherent toxicity of organ preservation solutions to cultured hepatocytes

Organ preservation solutions have been designed to protect grafts against the injury inflicted by cold ischemia. However, toxicity of University of Wisconsin (UW) solution during rewarming has been reported. Therefore, we here assessed the toxicity of UW, histidine-tryptophan-ketoglutarate (HTK), Euro-Collins, histidine-lactobionate (HL), sodium-lactobionate-sucrose and Celsior solutions in cultured hepatocytes under hypothermic (4 °C), intermediate (21 °C) and physiological (37 °C) conditions. Marked toxicity of UW, HTK, HL and Euro-Collins solutions was observed at both 37 and 21 °C. With the exception of UW solution, these solutions also increased cell injury during cold incubation (LDH release after 18 h at 4 °C: HTK 76 ± 2%, Euro-Collins 78 ± 17%, HL 81 ± 15%; control: Krebs-Henseleit buffer 20 ± 6%). Testing of individual components using modified Krebs-Henseleit buffers suggested that histidine and phosphate are responsible for (part of) this toxicity. These potential toxicities should be taken into account in the development of future preservation solutions.     

Effect of cold storage on tissue and cellular glutathione

One of the mechanisms thought to cause injury in preserved organs is the formation of oxygen free radicals. The cell is protected from oxidative stress by many defense mechanisms. A major defense mechanism involves glutathione and glutathione-dependent enzymes. During organ preservation by simple cold storage the loss of glutathione may sensitize the organ to free radical damage after transplantation. In this study we show that glutathione is depleted from the rabbit liver, kidney, and heart cold-stored (5 degrees C) for up to 72 h in the UW solution without glutathione. In the first 24 h kidney glutathione decreased to 84 +/- 3% of control values, liver glutathione decreased to 49 +/- 3% of control values, and heart glutathione decreased to 73 +/- 3% of control values. After 48 h of storage the kidney and liver lost an additional 30 and 20%, respectively, whereas heart glutathione changed very little. By 72 h all three organs had lost more than 50% of the glutathione found in freshly obtained tissue. To determine if glutathione added to the UW solution can effectively prevent this loss of glutathione during preservation, hepatocytes were cold-stored for up to 72 h in a preservation solution with and without glutathione. We found that adding glutathione to the preservation solution slowed the rate of loss of glutathione from the cells. These data suggest that at hypothermia the cell may be permeable to GSH. Methods to suppress the loss of glutathione during preservation of organs may be an important factor in suppressing oxygen free radical injury.     

IGL-1 solution reduces endoplasmic reticulum stress and apoptosis in rat liver transplantation

Injury due to cold ischemia reperfusion (I/R) is a major cause of primary graft non-function following liver transplantation. We postulated that I/R-induced cellular damage during liver transplantation might affect the secretory pathway, particularly at the endoplasmic reticulum (ER). We examined the involvement of ER stress in organ preservation, and compared cold storage in University of Wisconsin (UW) solution and in Institute Georges Lopez-1 (IGL-1) solution. In one group of rats, livers were preserved in UW solution for 8 h at 4 °C, and then orthotopic liver transplantation was performed according to Kamada''s cuff technique. In another group, livers were preserved in IGL-1 solution. The effect of each preservation solution on the induction of ER stress, hepatic injury, mitochondrial damage and cell death was evaluated. As expected, we found increased ER stress after liver transplantation. IGL-1 solution significantly attenuated ER damage by reducing the activation of three pathways of unfolded protein response and their effector molecules caspase-12, C/EBP homologous protein-10, X-box-binding protein 1, tumor necrosis factor-associated factor 2 and eukaryotic translation initiation factor 2. This attenuation of ER stress was associated with a reduction in hepatic injury and cell death. Our results show that IGL-1 solution may be a useful means to circumvent excessive ER stress reactions associated with liver transplantation, and may optimize graft quality.     

The functional effects of suppression of hypothermia-induced cell swelling in liver preservation by cold storage

It is known that cellular edema and functional impairment develop during anaerobic cold storage of organs. The extent of both is related to the storage time and the composition of the preservation solution used. We studied hypothermia-induced cell swelling and its effect on liver function after cold storage preservation with either Eurocollins (EC), a number of modified EC solutions in which glucose was replaced by various concentrations of raffinose, or UW solution. After 24 h storage, tissue swelling as determined by total tissue water (TTW) in rat liver tissue slices was most pronounced in slices incubated in Eurocollins, whereas the TTW was only moderately increased in slices stored in modified Eurocollins containing 90 to 120 mM raffinose. In contrast, slices incubated in UW solution had a TTW equal to normal rat liver tissue. Furthermore, intact rabbit livers preserved with Eurocollins had an increase in the whole organ weight, while there was no weight change after preservation with the modified solution containing 120 mM raffinose (M120). In contrast, a pronounced weight loss was observed after preservation with UW solution. After cold storage, the livers were reperfused for 2 h at 38 degrees C in an isolated perfusion circuit (IPL) with an acellular perfusate. Bile flow was significantly greater in livers preserved in M120 than in those preserved with the conventional Eurocollins. However, the bile flow in the livers stored in M120 was inferior to that in the livers preserved with UW solution, which in turn was equal to that in control livers. The release of alanine-aspartate-aminotransferase into the perfusate was higher in livers preserved with Eurocollins, with or without modification, than in the livers preserved with UW solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced energy metabolism during cold hypoxic organ preservation: studies on rat liver after pyruvate supplementation

Previous studies have indicated that pyruvate is able to reduce ischaemia/reperfusion (I/R) injury in a variety of tissues, but a full understanding of the effects is lacking. In this current preliminary study, magnetic resonance spectroscopy (MRS) was used to investigate the biochemical effects of differing concentrations of pyruvate (3 and 15mM) on liver metabolism during the cold hypoxic preservation period itself, in order to gain insight into possible mechanisms. Hepatic lactate, alanine, and succinate levels were increased in livers preserved with 15mM pyruvate added to the University of Wisconsin (UW) solution and were generally elevated (but to a lesser degree) in livers flushed with 3mM pyruvate, compared to those cold stored in UW alone. Further, from enzymatic assays of adenine nucleotides, 15mM levels of pyruvate were found to maintain higher ATP levels during short periods (up to 4h) of cold hypoxic storage than in UW stored livers, whilst energy charge ratios (after 4 and 24h) were also higher (P<0.01 in each case). This may arise from enhanced glycolysis secondary to an improved redox status in the pyruvate-treated livers, as evident by the increase in the levels of lactate.     

A comparison of a sucrose-based solution with other preservation media for cold storage of isolated hepatocytes

A sucrose-based solution has been compared with other preservation solutions (University of Wisconsin (UW) solution and Marshall's citrate solution, with Dulbecco's medium as control) during hypothermic preservation of isolated rat hepatocytes for up to 72 h. Studies on the stability of liver cells at low temperature by exclusion of trypan blue dye and morphological appearance were conducted. During storage beyond 24 h, there was a clear difference between cells stored in Dulbecco's medium and Marshall's citrate and those stored in sucrose-based solution and UW solution, with the former storage groups showing many cells developing large membrane "blebs" and the latter storage groups maintaining a more typical morphology and developing only small membrane protrusions. Dye exclusion was higher in sucrose-based solution (48 h, 75 +/- 7%; 72 h, 65 +/- 6%) and UW solution (48 h, 72 +/- 5%; 72 h, 63 +/- 4%) than in Marshall's citrate (48 h, 31 +/- 5%; 72 h, 10 +/- 1%) and Dulbecco's medium (48 h, 8 +/- 2%; 72 h, 5 +/- 1%). These data suggest that sucrose-based solution should be investigated further as a less complex alternative solution for storage of isolated hepatocytes.     

Pancreatic islets isolation using different protocols with in situ flushing and intraductal collagenase injection

Human islet transplantation seems to be a very promising clinical procedure for patients with type I diabetes mellitus. The aim of our study was to investigate the influence of in situ intravascular flushing with University of Wisconsin (UW) solution and intraductal collagenase injection at the time of pancreas procurement on the isolated islets and exocrine tissue injury. Our experiments indicated that in situ perfusion with the UW solution has a beneficial effect on pancreatic islets and intraductal distention results in an increase in the concentration of pancreatic enzymes released into the cold preservation solution during ischemic conditions. Cold ischemia reduced islet yield, but pancreas perfusion with the UW solution showed better ischemic tolerance of isolated islets during glucose static incubation. We conclude that intravascular pancreas flushing has a crucial effect on recovery and yield of pancreatic islets and protects against exocrine tissue injury.     

Cardiac proteasome dysfunction during cold ischemic storage and reperfusion in a murine heart transplantation model

Recent observations suggest that the ubiquitin-proteasome system (UPS) contributes to the pathophysiology of myocardial ischemia-reperfusion injury. Since its regulation during cold ischemia-reperfusion is unknown, we evaluated the cardiac UPS in a model of heart transplantation in mice. Cardiac ubiquitylation rates and ubiquitin-protein conjugates increased after 3 h of cold ischemia (CI) and normalized post-transplant. 20S proteasome content and proteasome peptidase activities were unchanged after CI. 4 h/24 h post-transplant 20S proteasome concentrations decreased and chymotryptic-like but not tryptic-like proteasome peptidase activity was inactivated. Epoxomicin sensitivity of the proteasome increased 5.7-fold during CI and normalized 4 h/24 h post-transplant. This was accompanied by the disappearance of a 13.5 kDa-ubiquitin-conjugate during CI that could be attenuated by addition of epoxomicin to the preservation fluid. We conclude that substrate specificity of the proteasome changes during cold ischemia and that proteasome inhibition preserves the physiological ubiquitin-protein conjugate pool during organ preservation. Reduced proteasome activity during reperfusion is caused by a decrease in proteasome content and enzyme inhibition.     

Effect of phospholipase A2 inhibitors on the release of arachidonic acid and cell viability in cold-stored hepatocytes

We investigated the effect of phospholipase A(2) (PLA(2)) inhibitors on PLA(2) activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [(3)H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA(2) inhibitors. PLA(2) activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m-chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA(2) activity was also suppressed (58% of control) by trifluoperazine (50 microM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 microM) suppressed PLA(2) activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA(2) activity. Thus, the inhibitory activity of these agents appears unrelated to Ca-calmodulin-phospholipid interaction but to have an inhibitory effect on PLA(2) activity. To study the effects of PLA(2) inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs-Henseleit buffer for 2 h at 37 degrees C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA(2) inhibitors blocked PLA(2) activity, there was no suppression of LDH release. PLA(2) may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes.     

Alterations of nitric oxide synthase expression and activity during rat lung transplantation

Decreased nitric oxide (NO) production has been reported during lung transplantation in patients. To study the effects of ischemia and reperfusion on endogenous NO synthase (NOS) expression, both an ex vivo and an in vivo lung injury model for transplantation were used. Donor rat lungs were flushed with cold low-potassium dextran solution and subjected to either cold (4 degrees C for 12 h) or warm (21 degrees C for 4 h) ischemic preservation followed by reperfusion with an ex vivo model. A significant increase in inducible NOS and a decrease in endothelial NOS mRNA was found after reperfusion. These results were confirmed in a rat single-lung transplant model after warm preservation. Interestingly, protein contents of both inducible NOS and endothelial NOS increased in the transplanted lung after 2 h of reperfusion. However, the total activity of NOS in the transplanted lungs remained at very low levels. We conclude that ischemic lung preservation and reperfusion result in altered NOS gene and protein expression with inhibited NOS activity, which may contribute to the injury of lung transplants.