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J J Lemasters

Publications and source records attributed to J J Lemasters.

At least 37 records · Page 2Linked to original sources
PubMed2000

Proinflammatory kupffer cell alterations after femur fracture trauma and sepsis in rats.

This study examined effects of trauma and sepsis on Kupffer cell function. When CBA/J mice had femur fracture (FFx), no deaths occurred. After cecal ligation and puncture (CLP), 44% died. Following combined injuries (FFx + CLP), mortality increased to 60%, suggesting a deleterious effect between FFx + CLP. Kupffer cell ablation with GdCI3 decreased mortality to 13% after CLP and 5% after FFx + CLP. After FFx, CLP, and FFx + CLP, Kupffer cells isolated from Sprague-Dawley rats produced 720%, 1,100%, and 2,130% more O2. than sham, respectively. Phagocytosis increased 320%, 610%, and 150%. Kupffer cell PGE2 production also increased 300%, 510%, and 300% over sham. After FFx alone, TNF-alpha production decreased 40%. By contrast, CLP and FFx + CLP increased TNF-alpha release 25% and 100%, respectively. After FFx, NO. production decreased 44%, whereas NO increased 280% and 260% after CLP and FFx + CLP. These findings indicate that Kupffer cells mediate mortality after CLP and FFx + CLP. Increased mortality is associated with a more proinflammatory and less antimicrobial Kupffer cell phenotype.

Animals↗
PubMed1999

Protection of sinusoidal endothelial cells against storage/reperfusion injury by prostaglandin E2 derived from Kupffer cells.

BACKGROUND: In clinical liver transplants, grafts are frequently exposed to endotoxin (lipopolysaccharide, LPS) before harvest and may be predisposed to dysfunction. Because graft failure is linked to sinusoidal endothelial cell injury after storage/reperfusion, we investigated the effect of donor exposure to LPS on graft survival in relation to sinusoidal endothelial cell injury after storage/reperfusion in rats. METHODS: Rats were injected with 0.5 mg/kg LPS. In some rats, 20 mg/kg GdCl3 or 5 mg/kg indomethacin was injected before LPS to ablate Kupffer cells and inhibit prostaglandin (PG) synthesis, respectively. Other rats were injected with 100 microg/kg dimethyl PGE2, a stable PGE2 analog. Rat livers were harvested, stored in cold UW solution and transplanted to non-treated rats for determination of survival and liver injury in recipients. Otherwise, after cold storage, the livers were reperfused briefly with physiological buffer containing trypan blue for determination of sinusoidal endothelial cell injury by counting trypan blue-positive nuclei in histological sections. RESULTS: Donor treatment with LPS increased hepatic PGE2 production before storage and decreased recipient survival, but paradoxically decreased killing of sinusoidal endothelial cells after storage and reperfusion. Pretreatment of donors with GdCl3 or indomethacin prevented the protective preconditioning of sinusoidal endothelial cells by LPS, whereas pretreatment with dimethyl PGE2 protected sinusoidal endothelial cells to the same extent as LPS. Unlike LPS, however, PGE2 attenuated graft injury after liver transplants. CONCLUSION: PGE2 derived from LPS-stimulated Kupffer cells protects sinusoidal endothelial cells against storage/reperfusion injury. Unlike LPS, PGE2 improves graft function after liver transplants. Thus, donor preconditioning with PGE2 may be beneficial in liver transplants.

Animals↗
PubMed1999

The mitochondrial permeability transition mediates both necrotic and apoptotic death of hepatocytes exposed to Br-A23187.

A23187 and related Ca2+ ionophores are widely used to study Ca2+-dependent cell injury. Here, using laser scanning confocal microscopy and parameter-indicating fluorophores, we investigated the role of the mitochondrial permeability transition (MPT) in Br-A23187 toxicity to cultured rat hepatocytes. After 10 microM Br-A23187, over 60% of hepatocytes lost viability within 1 h. This necrotic cell killing was preceded by increased mitochondrial free Ca2+, mitochondrial depolarization, and onset of the MPT. Cyclosporin A (CsA), a blocker of the permeability transition pore, prevented the MPT and cell killing but had no effect on increased mitochondrial free Ca2+ and depolarization after Br-A23187. To determine whether Br-A23187-induced cell killing was linked to loss of cellular ATP supply, hepatocytes were incubated with fructose and oligomycin, a source of glycolytic ATP and an inhibitor of the uncoupler-stimulated mitochondrial ATPase, respectively. Fructose plus oligomycin prevented cell killing after Br-A23187 but not the MPT. When fructose plus oligomycin prevented necrotic cell killing, apoptosis developed after 10 h. When cells were treated additionally with CsA, these apoptotic changes were prevented. In conclusion, the MPT mediates Br-A23187 cytotoxicity. Acutely, the MPT causes mitochondrial uncoupling and profound ATP depletion, which leads to necrotic cell death. However, when glycolytic ATP generation is available, the MPT induces apoptosis. CsA blocks the MPT and prevents both necrotic and apoptotic cell killing after Br-A23187.

Adenosine Triphosphate↗
PubMed1999

Contribution of increased mitochondrial free Ca2+ to the mitochondrial permeability transition induced by tert-butylhydroperoxide in rat hepatocytes.

Previously, we showed that the oxidant chemical, tert-butylhydroperoxide (t-BuOOH), induces a mitochondrial permeability transition (MPT) in intact hepatocytes, causing lethal cell injury. Here, we investigated the role of mitochondrial free Ca2+ in t-BuOOH cytotoxicity to 1-day-cultured rat hepatocytes using confocal microscopy of autofluorescence and parameter-indicating fluorophores. t-BuOOH (100 micromol/L) caused an early increase of mitochondrial free Ca2+, as assessed by confocal microscopy of Rhod-2 fluorescence. Increased mitochondrial Ca2+ was followed by onset of the MPT, as evidenced by permeation of cytosolic calcein into mitochondria and loss of the mitochondrial membrane potential-indicating dye, tetramethylrhodamine methylester. Preincubation with an intracellular Ca2+ chelator (BAPTA-AM and its derivatives) partially blocked the late phase of mitochondrial NAD(P)H oxidation after t-BuOOH, but failed to prevent the early oxidation of mitochondrial NAD(P)H. Ca2+ chelation also prevented the increase of mitochondrial Ca2+, generation of mitochondrial reactive oxygen species (ROS), onset of the MPT, and subsequent cell death. Confocal images showed that protection occurred when loading of the Ca2+ chelator was predominantly mitochondrial. The antioxidant, desferal, also diminished increased mitochondrial Ca2+ after t-BuOOH and prevented cell death. We conclude that oxidative stress induced by t-BuOOH enhances mitochondrial Ca2+ uptake, leading to increased matrix Ca2+, increased ROS formation, onset of the MPT, and cell death.

Aminoquinolines↗
PubMed1999

Amino acids in rinse effluents as a predictor of graft function after transplantation of fatty livers in rats.

There are too few reliable markers by which one can predict future function of a liver before implantation. Consequently, the purpose of this study was to test the hypothesis that amino acids in rinse-effluents could predict transplant outcome in marginal fatty livers from rats. Amino acids were measured in the rinse effluent from the livers immediately after harvest and graft preparation or cold storage. Amino acids in the effluent were twice as high in ethanol-treated animals compared to those in nonfatty controls. Ethanol-treated fatty livers survived for no longer than 7 days after transplantation while 83% of nonfatty controls survived (P < 0.05). In subsequent studies, the cold-storage time was decreased to 6 h to determine whether failing fatty livers released more amino acid than grafts that would function normally. There was a significant increase in amino acids in the effluent of fatty grafts compared to controls. Moreover, the sum of the four selected amino acids (alanine, valine, histidine, leucine) was lower than 23 nmol/g liver in functional livers, whereas failing grafts had totals significantly higher than 25 nmol/g liver. The sum of the four amino acids correlated well with 24 h post-transplant serum AST levels (r = 0.78, P < 0.0001). So we can conclude that amino acid release can serve as a useful marker of graft viability and reliably predicts survival.

Alanine Transaminase↗
PubMed1999

Gentle organ manipulation during harvest as a key determinant of survival of fatty livers after transplantation in the rat.

Both in situ organ manipulation during harvest and steatosis reduce survival after liver transplantation via mechanisms involving Kupffer cells; thus, their effect on survival was compared here. Moderate steatosis was induced by a single dose of ethanol to Lewis rats, while long-term administration of ethanol yielded severe steatosis in donor animals. After minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 min subsequently. Orthotopic liver transplantation was performed after 1 h of cold storage in UW solution. Ethanol increased hepatic lipid content to a level of moderate or severe steatosis that reduced survival after transplantation from 100% to approximately 70% (P < 0.05). However, gentle manipulation decreased survival to approximately 30% (P < 0.05) in livers from normal, saline-treated rats and in livers from rats fed a high-fat control diet. Moreover, after short- or long-term ethanol administration, manipulation of fatty livers decreased survival from 70% to approximately 13% (P < 0.05). Further, manipulation elevated serum transaminases, total bilirubin, and necrosis significantly about 2- to 20-fold in fatty grafts after transplantation. At the end of harvest, trypan blue distribution time and hypoxia assessed from 2-nitroimidazole binding were elevated significantly about two- to fourfold by manipulation of fatty grafts. Gadolinium chloride, a Kupffer cell toxicant, blocked the detrimental effects of manipulation. These data demonstrate for the first time that, while steatosis is detrimental for survival, organ manipulation plays a much greater role than fat in mechanisms of primary nonfunction.

Animals↗
PubMed1999

Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death.

Mitochondria are frequently the target of injury after stresses leading to necrotic and apoptotic cell death. Inhibition of oxidative phosphorylation progresses to uncoupling when opening of a high conductance permeability transition (PT) pore in the mitochondrial inner membrane abruptly increases the permeability of the mitochondrial inner membrane to solutes of molecular mass up to 1500 Da. Cyclosporin A (CsA) blocks this mitochondrial permeability transition (MPT) and prevents necrotic cell death from oxidative stress, Ca2+ ionophore toxicity, Reye-related drug toxicity, pH-dependent ischemia/reperfusion injury, and other models of cell injury. Confocal fluorescence microscopy directly visualizes onset of the MPT from the movement of green-fluorescing calcein into mitochondria and the simultaneous release from mitochondria of red-fluorescing tetramethylrhodamine methylester, a membrane potential-indicating fluorophore. In oxidative stress to hepatocytes induced by tert-butylhydroperoxide, NAD(P)H oxidation, increased mitochondrial Ca2+, and mitochondrial generation of reactive oxygen species precede and contribute to onset of the MPT. Confocal microscopy also shows directly that the MPT is a critical event in apoptosis of hepatocytes induced by tumor necrosis factor-alpha. Progression to necrotic and apoptotic cell killing depends, at least in part, on the effect the MPT has on cellular ATP levels. If ATP levels fall profoundly, necrotic killing ensues. If ATP levels are at least partially maintained, apoptosis follows the MPT. Cellular features of both apoptosis and necrosis frequently occur together after death signals and toxic stresses. A new term, necrapoptosis, describes such death processes that begin with a common stress or death signal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmed cellular resorption (apoptosis) depending on modifying factors such as ATP.

Animals↗
PubMed1999

Confocal microscopy of the mitochondrial permeability transition in necrotic and apoptotic cell death.

Opening of a high-conductance pore in the mitochondrial inner membrane induces onset of the mitochondrial permeability transition (mPT). Cyclosporin A and trifluoperazine inhibit this pore and block necrotic cell death in oxidative stress, Ca2+ ionophore toxicity, Reye-related drug toxicity, pH-dependent ischaemia/reperfusion injury and other models of cell injury. Confocal fluorescence microscopy directly visualizes the increased mitochondrial membrane permeability of the mPT from the movement of calcein from the cytosol into the matrix space. Pyridine nucleotide oxidation, increased mitochondrial Ca2+ and mitochondrial generation of reactive oxygen species (ROS) all contribute to the onset of the mPT in situ. Confocal microscopy also shows directly that the mPT is a critical link in apoptotic signalling by tumour necrosis factor-alpha at a point downstream of caspase 8 and upstream of caspase 3. Cyclosporin A blocks this mPT, preventing release of pro-apoptotic cytochrome c from mitochondria and subsequent apoptotic cell killing. Progression to necrosis or apoptosis after the mPT depends on the availability of ATP, which blocks necrosis but promotes the apoptotic programme. Given the pathophysiological importance of the mPT, development of agents to modulate the mPT represents an important new goal for pharmaceutical drug discovery.

Animals↗
PubMed1999

Role of cytokines and cytokine-producing cells in reperfusion injury to the liver.

We have categorized reperfusion injury into (1) warm ischemia/reperfusion injury and (2) cold storage/reperfusion injury. Warm ischemia/reperfusion injury is relevant to shock and hepatic surgery, whereas cold storage/reperfusion injury occurs when the liver is procured and transported for transplantation. Different stages of reperfusion injury; cells that mediate each stage; and cytokines involved are described. Understanding the specific cells and cytokines involved in causing reperfusion injury and their timing will encourage the development of therapeutic strategies aimed at preventing and diminishing the consequences of reperfusion injury.

Animals↗
PubMed1999

V. Necrapoptosis and the mitochondrial permeability transition: shared pathways to necrosis and apoptosis.

Opening of a high-conductance pore conducting solutes of molecular mass <1,500 Da causes onset of the mitochondrial permeability transition (MPT). Cyclosporin A blocks this pore and prevents acute necrotic cell death in several models. Confocal microscopy directly visualizes onset of the MPT during acute cytotoxicity from the movement of the green-fluorescing fluorophore, calcein, into the mitochondria from the cytosol. The MPT also plays a causative role in tumor necrosis factor-alpha-induced apoptosis in hepatocytes. Progression to apoptosis or necrosis after the MPT may depend on the presence or absence, respectively, of ATP. Often, features of both apoptotic and necrotic cell death develop after death signals and toxic stresses. The term "necrapoptosis" is introduced to emphasize the shared pathways leading to both forms of cell death.

Animals↗
PubMed1999

Intravenous glycine improves survival in rat liver transplantation.

In situ manipulation by touching, retracting, and moving liver lobes gently during harvest dramatically reduces survival after transplantation (P. Schemmer, R. Schoonhoven, J. A. Swenberg, H. Bunzendahl, and R. G. Thurman. Transplantation 65: 1015-1020, 1998). The development of harvest-dependent graft injury upon reperfusion can be prevented with GdCl3, a rare earth metal and Kupffer cell toxicant, but it cannot be used in clinical liver transplantation because of its potential toxicity. Thus the effect of glycine, which prevents activation of Kupffer cells, was assessed here. Minimal dissection of the liver for 12 min plus 13 min without manipulation had no effect on survival (100%). However, gentle manipulation decreased survival to 46% in the control group. Furthermore, serum transaminases and liver necrosis were elevated 4- to 12-fold 8 h after transplantation. After organ harvest, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased about twofold, indicating disturbances in the hepatic microcirculation. Pimonidazole binding, which detects hypoxia, increased about twofold after organ manipulation, and Kupffer cells isolated from manipulated livers produced threefold more tumor necrosis factor-alpha after lipopolysaccharide than controls. Glycine given intravenously to the donor increased the serum glycine concentration about sevenfold and largely prevented the effect of gentle organ manipulation on all parameters studied. These data indicate for the first time that pretreatment of donors with intravenous glycine minimizes reperfusion injury due to organ manipulation during harvest and after liver transplantation.

Adenosine↗
PubMed1999

Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration.

The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichlorofluorescein were excreted into and concentrated in the bile canalicular lumen of day 5 sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca(2+)-free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca(2+)-free buffer. The cumulative uptake of [(3)H]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca(2+)-free buffer, due to accumulation of taurocholate in canalicular spaces. When [(3)H]taurocholate was preloaded in the day 5 sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca(2+)-free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from approximately 8% at day 0 to approximately 60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.

Animals↗
PubMed1999

Binge drinking disturbs hepatic microcirculation after transplantation: prevention with free radical scavengers.

Disturbances in hepatic microcirculation increase graft injury and failure; therefore, this study evaluates the effects of ethanol on microcirculation after liver transplantation. Donor rats were given one dose of ethanol (5 g/kg) by gavage 20 h before explantation, and grafts were stored in University of Wisconsin solution for 24 h before implantation. Acute ethanol treatment decreased 7-day survival of grafts from about 90 to 30%, increased transaminase release nearly 4-fold, and decreased bile production by 60%. Moreover, portal pressure increased significantly and liver surface oxygen tension decreased about 50%, indicating that ethanol disturbs hepatic microcirculation. Pimonidazole, a 2-nitroimidazole hypoxia marker, was given i.v. to recipients 30 min after implantation, and grafts were harvested 1 h later. Ethanol increased hepatic pimonidazole binding about 3-fold, indicating that ethanol led to hypoxia in fatty grafts. Ethanol also significantly increased free radicals in bile. Catechin (30 mg/kg i.v. upon reperfusion), a free radical scavenger, and Carolina Rinse solution, which contains several agents that inhibit free radical formation, minimized disturbances in microcirculation and prevented pimonidazole adduct formation significantly. These treatments also blunted increases in transaminase release and improved survival of fatty grafts. Destruction of Kupffer cells with GdCl(3) (20 mg/kg i.v. 24 h before explantation) or inhibition of formation of leukotrienes with MK-886 (50 microM in University of Wisconsin or rinse solution) also minimized hypoxia and improved survival after transplantation. Taken together, these results demonstrate that ethanol disturbs hepatic microcirculation, leading to graft hypoxia after transplantation, most likely by activating Kupffer cells and increasing free radical production.

Animals↗
PubMed1998

Ethanol, not fat accumulation per se, increases free radical production in a low-flow, reflow liver perfusion model.

BACKGROUND: Ethanol increases primary graft failure after liver transplantation, yet whether it acts via mechanisms involving fat accumulation remains unclear. METHODS: Rats were pair-fed a modified Lieber-DeCarli liquid diet containing 35% (high-fat) or 12% (low-fat) of calories as fat combined with 36% of calories as ethanol or isocaloric maltose-dextrin for 4-5 weeks. Reperfusion injury to the liver was studied using a low-flow, reflow perfusion model and a liver transplantation model, and free radicals were detected using electron spin resonance and the spin trapping technique. RESULTS: As expected, basal hepatic triglycerides were similar in livers from rats fed low- and high-fat control diets. Ethanol did not alter triglyceride levels significantly in rats fed a low-fat diet, but increased values about 2.4-fold in rats fed a high-fat diet. Ethanol increased lactate dehydrogenase release during reperfusion from 10 to 26 IU/g/h in rats fed a low-fat diet and from 17 to 34 IU/g/h in rats fed a high-fat diet, respectively. Portal pressure increased from about 3 to 10.5 cm H2O upon reperfusion in livers from high-fat, ethanol-fed rats, but only reached values of 9.1 in the low-fat, ethanol-fed group. A free radical adduct signal was detected in the bile of livers from ethanol-treated rats, and the magnitude of this signal was similar in livers of ethanol-treated rats fed high- or low-fat diets. However, radical adducts could not be detected in either group in the absence of dietary ethanol. Moreover, 67-77% rats given low-fat or high-fat control diets survived after liver transplantation, but only 11% survived if treated with ethanol. CONCLUSIONS: It is concluded that ethanol plays a major role in hepatic reperfusion injury, most likely via mechanisms involving free radicals. Increased hepatic fat content alone plays only a minor role, probably by causing slight disturbances in the hepatic microcirculation.

Animals↗
PubMed1998

The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy.

Using confocal microscopy, onset of the mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by the redistribution of the cytosolic fluorophore, calcein, into mitochondria. Simultaneously, mitochondria release membrane potential-indicating fluorophores like tetramethylrhodamine methylester. The MPT occurs in several forms of necrotic cell death, including oxidative stress, pH-dependent ischemia/reperfusion injury and Ca2+ ionophore toxicity. Cyclosporin A (CsA) and trifluoperazine block the MPT in these models and prevent cell killing, showing that the MPT is a causative factor in necrotic cell death. During oxidative injury induced by t-butylhydroperoxide, onset of the MPT is preceded by pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and an increase of mitochondrial free Ca2+, all changes that promote the MPT. During tissue ischemia, acidosis develops. Because of acidotic pH, anoxic cell death is substantially delayed. However, when pH is restored to normal after reperfusion (reoxygenation at pH 7.4), cell death occurs rapidly (pH paradox). This killing is caused by pH-dependent onset of the MPT, which is blocked by reperfusion at acidotic pH or with CsA. In isolated mitochondria, toxicants causing Reye's syndrome, such as salicylate and valproate, induce the MPT. Similarly, salicylate induces a CsA-sensitive MPT and killing of cultured hepatocytes. These in vitro findings suggest that the MPT is the pathophysiological mechanism underlying Reye's syndrome in vivo. Kroemer and coworkers proposed that the MPT is a critical event in the progression of apoptotic cell death. Using confocal microscopy, the MPT can be directly documented during tumor necrosis factor-alpha induced apoptosis in hepatocytes. CsA blocks this MPT and prevents apoptosis. The MPT does not occur uniformly during apoptosis. Initially, a small proportion of mitochondria undergo the MPT, which increases to nearly 100% over 1-3 h. A technique based on fluorescence resonance energy transfer can selectively reveal mitochondrial depolarization. After nutrient deprivation, a small fraction of mitochondria spontaneously depolarize and enter an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. A model is proposed in which onset of the MPT to increasing numbers of mitochondria within a cell leads progressively to autophagy, apoptosis and necrotic cell death.

Animals↗