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J J Lemasters

Publications and source records attributed to J J Lemasters.

At least 91 records · Page 5Linked to original sources
PubMed1996

Lung retrieval from cadaver donors with nonbeating hearts: optimal preservation solution.

BACKGROUND: We have previously studied the time course of pulmonary cell viability, ultrastructural damage, and adenine nucleotide metabolites after circulatory arrest in a rat model to investigate the feasibility of lung retrieval for transplantation from cadavers. This study was designed to investigate the effect of hypothermic flush and subsequent 4-hour storage with either modified Euro-Collins or University of Wisconsin solution on lungs retrieved 4 hours after death. METHODS: Ninety-six Sprague-Dawley rats were sacrificed by intraperitoneal injection of pentobarbital. Control lungs were flushed immediately after sacrifice and stored for 4 hours. Rats in the experimental groups were sacrificed, and then their lungs were either ventilated with 100% oxygen or not ventilated for 4 hours before flushing with either Euro-Collins or University of Wisconsin solution followed by 4-hour hypothermic storage. At the end of the storage period, all right lungs were maintained at -70 degrees C and used to determine wet-to-dry weight ratios and adenine nucleotide levels with high-pressure liquid chromatography. Left lungs were assessed for viability with trypan blue dye exclusion. The effect on viability of flushing with Carolina rinse solution after storage was also assessed. RESULTS: The percentage of viable cells in the control group after 4-hour hypothermic storage was 74% +/- 2% in Euro-Collins solution-flushed lungs and 78% +/- 2% in University of Wisconsin solution-flushed lungs. This result was virtually identical to that of lungs retrieved after 4 hours of in situ oxygen ventilation followed by 4 hours of hypothermic storage. Nonventilated cadaver lungs had substantially less viability. Adenosine triphosphate levels were significantly higher in the control group than in the oxygen-ventilated group, which were higher still than those in the nonventilated group. Adenosine triphosphate levels were consistently higher in University of Wisconsin solution-flushed lungs compared with Euro-Collins solution-flushed lungs in all groups. Total adenine nucleotide levels had a similar pattern. Wet-to-dry ratios were significantly lower in the control group (Euro-Collins = 6.27 +/- 0.46, University of Wisconsin = 4.63 +/- 0.07) compared with the oxygen-ventilated (Euro-Collins = 9.80 +/- 0.44, University of Wisconsin = 10.96 +/- 0.60) and nonventilated (Euro-Collins = 9.44 +/- 0.26, University of Wisconsin = 11.54 +/- 1.16; p < 0.0001) groups. CONCLUSIONS: Four hours of circulatory arrest before 4 hours of hypothermic storage had no additional adverse impact on lung viability compared with lungs subjected to 4 hours of hypothermic storage alone, provided nonperfused lungs were ventilated with 100% oxygen. Adenine nucleotide levels were well maintained in oxygen-ventilated cadaver lungs, more so in University of Wisconsin solution-flushed lungs compared with Euro-Collins solution-flushed lungs.

Adenine Nucleotides↗
PubMed1996

Donor lungs from ventilated cadavers: impart of a free radical scavenger.

BACKGROUND: The shortage of donors for lung transplantation may be alleviated with the use of lungs retrieved from cadavers. The purpose of this study was to determine whether a free radical scavenger, dimethylthiourea, would improve the function of lungs retrieved from ventilated cadavers. METHODS: Left lung transplantation was performed in 21 dogs. Donors were sacrificed then ventilated with 100% oxygen. After 2 hours, donor lungs were flushed in a blinded fashion with 2 L of modified Euro-Collins solution, with either dimethylthiourea (n = 10) or saline solution (n = 11) added, then harvested. A donor right lung lobe was perfused with trypan blue vital dye to assess cell viability at harvest and after the transplantation. Percentage of nonviability was similar in the dimethylthiourea and control groups (13 versus 20 at retrieval and 38 versus 41 at graft reperfusion). After transplantation, the right pulmonary artery and bronchus were occluded, rendering the recipient on the pulmonary graft. The recipient's lungs were ventilated for 8 hours, with the inspired oxygen fraction maintained at 0.4. RESULTS: Seven of ten dogs in the dimethylthiourea group survived the 8-hour period, compared with 4 of 11 dogs in the control group. Compared with the control survivors (n = 4) at 8 hours after the operation, the dimethylthiourea survivors (n = 7) had a higher mean arterial oxygen pressure (144 +/- 21 versus 98+/- 12 mm Hg) and cardiac output (2.2 +/- 0.2 versus 1.6 +/- 0.2 L/min) and a lower mean pulmonary vascular resistance (946 +/- 96 versus 1414 +/- 128 dynes.sec-1.cm5, p < 0.05) and extravascular lung water (10.6 +/- 1.2 versus 12.3 +/- 3.2 ml/kg). Differences between groups during the 8-hour period were usually insignificant. CONCLUSIONS: This model imposes a rigorous challenge to the single transplanted lung, and yet cadaver lungs still supported life in half of the recipients. Dimethylthiourea may confer a benefit to recipients of cadaver lungs.

Animals↗
PubMed1996

The mitochondrial permeability transition: a new pathophysiological mechanism for Reye's syndrome and toxic liver injury.

Aspirin, Neem oil, valproic, adipic, benzoic, isovaleric, 3-mercaptopropionic and 4-pentenoic acids are implicated in the pathogenesis of Reye's syndrome, Jamaican vomiting sickness, and related chemical toxicities. These disorders are characterized by hyperammonemia, hypoglycemia, microvesicular steatosis and encephalopathy. The goal of this study was to determine whether chemicals implicated in Reye's-related disorders induce the mitochondrial permeability translation (MPT). The MPT is induced by opening of a high-conductance, cyclosporin-sensitive pore in the mitochondrial inner membrane, causing swelling, depolarization and uncoupling of oxidative phosphorylation. In freshly isolated rat liver mitochondria, unhydrolyzed aspirin (300 microM) did not induce the MPT in the presence of 50 microM CaCl2. Salicylate, the hydrolysis product of aspirin and its active metabolite, was much more potent causing dose-dependent onset of the MPT in a therapeutic range of concentrations (37.5-300 microM). Similarly, Neem oil and valproic, adipic, benzoic, isovaleric, 3-mercaptopropionic and 4-pentenoic acids induced onset of the MPT. In all cases, cyclosporin A (200 nM), a specific inhibitor of the permeability transition pore, blocked the MPT caused by these inducers. Induction of the MPT by these agents was not caused by mitochondrial depolarization because concentrations of valproic acid and salicylate inducing the MPT had little effect on mitochondrial delta psi. Moreover, equivalent uncoupling caused by 5 nM carbonyl cyanide p-trifluoromethoxyphenylhydrazone did not induce an MPT. These data suggest that induction of the MPT is a common pathophysiological mechanism causing mitochondrial injury in Reye's syndrome and Reye's-related drug toxicities.

Animals↗
PubMed1995

Cloning of a rat cDNA encoding a novel LIM domain protein with high homology to rat RIL.

A complementary DNA (1392 bp) encoding a protein with high homology to rat reversion-induced LIM (RIL) protein was cloned from rat hepatocytes by differential screening of a subtractive (normoxic minus hypoxic) lambda GEM-2 cDNA library. This cDNA clone, denoted CLP-36, encodes a 327-amino-acid (aa) protein that contained a restrictively conserved LIM (a Cys-rich domain with consensus aa sequence C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-20-C-X2-C/H/D that was initially identified in homeodomain proteins, Lin-11 [Freyd et al., Nature 344 (1990) 876-879], Isl-1 [Karlsson et al., Nature 344 (1990) 879-882] and Mec-3 [Way et al., Cell 54 (1988) 5-16] in the C-terminal portion (aa 192-327). It shared an overall 45.1% identity to a rat LIM domain RIL protein [Kiess et al., Oncogene 10 (1995) 61-68], with 62.0% identity in the N terminus (aa 1-89) and 50.0% identity in the C terminus (aa 188-327). The rat CLP-36 mRNA is expressed most abundantly in heart, lung and liver, moderately in spleen and skeletal muscle, and at extremely low levels (if at all) in testis and brain tissues. Northern blotting analysis of total RNA extracted from normoxic or hypoxic rat hepatocytes, with a fragment of clone CLP-36 as probe, demonstrated a single band with a mobility corresponding to a size of 1.4 kb, whose level was significantly decreased during chemical hypoxia.

Amino Acid Sequence↗
PubMed1995

Cloning of a rat cDNA encoding a protein with high homology to mouse phospholipase A2-activating protein.

A complementary DNA (2510 bp) encoding a protein with high homology to mouse phospholipase A2-activating protein (PLAP) was cloned from rat hepatocytes by differential screening of a subtractive (normoxic minus hypoxic) lambda GEM-2 cDNA library. This cDNA clone, denoted as HCW9, encodes a 647-amino-acid (aa) protein with 96.1% identity in the N-terminal portion (255 aa) to mouse PLAP [Clark et al., Proc. Natl. Acad. Sci. USA 88 (1991) 5418-5422]. Four cDNA clones were further isolated by screening a primary normoxic lambda GEM-2 cDNA library with a fragment of the HCW9 clone as probe. Partial sequencing of these clones revealed that all of the four clones were identical to clone HCW9. Northern blotting analysis of total RNA extracted from normoxic or hypoxic rat hepatocytes, with two different fragments of clone HCW9 as probes, demonstrated a single band with a mobility corresponding to a size of 2.5 kb, whose level was significantly decreased during chemical hypoxia.

Amino Acid Sequence↗
PubMed1995

Protection by pentoxifylline against normothermic liver ischemia/reperfusion in rats.

Previously, pentoxifylline treatment of graft recipients was shown to protect against liver graft failure from storage/reperfusion injury after orthotopic rat liver transplantation. To determine whether pentoxifylline also protects against normothermic ischemia/reperfusion injury to liver, we induced lobar ischemia in rats followed by reflow and partial hepatectomy of the noninvolved liver. In rats receiving pentoxifylline 2 hr before surgery and then twice daily for 5 days, the 1-week survival rate more than doubled from 25% to 67% (P < 0.05). Liver enzymes (alanine transaminase, aspartate transaminase, and lactate dehydrogenase) in the serum and liver necrosis evaluated histologically were also significantly reduced in the pentoxifylline-treated rats (P < 0.01). Hepatic ischemia/reperfusion increased leukocyte infiltration into the lungs, and pentoxifylline tended to reduce this lung injury (P = 0.06). These results show that pentoxifylline treatment reduces hepatic injury and improves survival after normothermic ischemia and reperfusion.

Animals↗
PubMed1995

Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide.

We have developed a novel method for monitoring the mitochondrial permeability transition in single intact hepatocytes during injury with t-butylhydroperoxide (t-BuOOH). Cultured hepatocytes were loaded with the fluorescence probes, calcein and tetramethylrhodamine methyl ester (TMRM). Depending on loading conditions, calcein labelled the cytosolic space exclusively and did not enter mitochondria or it stained both cytosol and mitochondria. TMRM labelled mitochondria as an indicator of mitochondrial polarization. Fluorescence of two probes was imaged simultaneously using laser-scanning confocal microscopy. During normal incubations, TMRM labelled mitochondria indefinitely (longer than 63 min), and calcein did not redistribute between cytosol and mitochondria. These findings indicate that the mitochondrial permeability transition pore ('megachannel') remained closed continuously. After addition of 100 microM t-BuOOH, mitochondria filled quickly with calcein, indicating the onset of mitochondrial permeability transition. This event was accompanied by mitochondrial depolarization, as shown by loss of TMRM. Subsequently, the concentration of ATP declined and cells lost viability. Trifluoperazine, a phospholipase inhibitor that inhibits the permeability transition in isolated mitochondria, prevented calcein redistribution into mitochondria, mitochondrial depolarization, ATP depletion and cell death. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, also rapidly depolarized mitochondria of intact hepatocytes but did not alone induce a permeability transition. Trifluoperazine did not prevent ATP depletion and cell death after the addition of CCCP. In conclusion, the permeability transition pore does not 'flicker' open during normal incubation of hepatocytes but remains continuously closed. Moreover, mitochondrial depolarization per se does not cause the permeability transition in intact cells. During oxidative stress, however, a permeability transition occurs quickly which leads to mitochondrial depolarization and cell death.

Adenosine Triphosphate↗
PubMed1995

Primary nonfunction of fatty livers produced by alcohol is associated with a new, antioxidant-insensitive free radical species.

The formation of free radicals after orthotopic liver transplantation in the rat correlates with graft failure. Fatty livers from alcoholics transplant poorly, so these studies were designed to examine the effect of alcohol on free radical formation in a rearterialized rat liver transplantation model. Treatment of rats for 3-5 weeks with either a high-fat or an ethanol-containing liquid diet caused characteristic pericentral lipid accumulation. After storage in University of Wisconsin cold storage solution (UW) and transplantation, a reperfusion injury characterized by increased postoperative AST levels (greater than 1500 U/l in about 3 hours) was observed in rats fed high-fat or alcohol-containing diets, whereas parenchymal cell injury was seen much less in low-fat controls. Survival was around 63% in the low-fat group but decreased to 12 and 18% in the high-fat and alcohol groups, respectively. Furthermore, intracellular lipid content correlated inversely with survival. In untransplanted livers, the spin trap alpha-phenyl N-tert-butylnitrone (PBN) was infused, and blood samples were collected and extracted with chloroform:methanol. Signals indicative of carbon-centered PBN radical adducts were barely detectable in all untransplanted groups studied by electron paramagnetic resonance. In contrast, a robust 6-line complex spectrum was obtained from all groups studied immediately after 48 hours of cold storage in UW solution and transplantation. A mixture of 3 radical species was identified. Two had coupling constants similar to lipid-derived free radicals, whereas the third is a new species with unique coupling constants and is most likely oxygen derived. In low-fat controls, the signal was reduced significantly by superoxide dismutase (SOD)/catalase; however, SOD/catalase had no effect on free radicals in lipid-loaded livers. Thus, both dietary high fat and alcohol exposure produce a unique SOD/catalase-insensitive free radical species that may be involved in the mechanism of failure of fatty livers after orthotopic liver transplantation.

Alcoholism↗
PubMed1995

When does the lung die? Time course of high energy phosphate depletion and relationship to lung viability after "death".

The shortage of lung donors for clinical transplantation could be significantly alleviated if lungs could be retrieved from cadavers hours after death. However, the time course of loss of lung viability after circulatory arrest and organism death remains unclear. To determine postmortem adenine nucleotide tissue levels in the lung and their relationship to lung viability, Sprague-Dawley rats were sacrificed and then ventilated with 100% oxygen (n = 50, O2) or 100% nitrogen (n = 40, N2) or left nonventilated (n = 50). Lungs from control rats (n = 20) were retrieved immediately after sacrifice. Lungs in the three study groups were retrieved at successive intervals postmortem. Adenine nucleotides (ATP, ADP, and AMP) and hypoxanthine and xanthine metabolites of adenosine were extracted from lung tissue and measured using high-performance liquid chromatography. Pulmonary parenchymal cell viability was quantified by pulmonary artery infusion of trypan blue vital dye in the contralateral lung of each animal. By 4 hr postmortem, viability was 85 +/- 1% in the O2-ventilated cadaver rat lungs, significantly higher than in the N2-ventilated (43 +/- 8%) and in the nonventilated (48 +/- 4%) lungs, where the percentage of viable cells was similar. All of the groups showed a time-dependent decrement in ATP levels and total adenine nucleotide (TAN) levels after death, but this was markedly attenuated in O2-ventilated cadaveric rat lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1995

Reperfusion injury to endothelial cells after cold storage of rat livers: protection by mildly acidic pH and lack of protection by antioxidants.

Lethal reperfusion injury to sinusoidal endothelial cells occurs after cold ischemic storage of livers and may be responsible for liver graft failure from storage injury. Here, we evaluated potential mechanisms underlying this reperfusion injury. In rat livers stored in Euro-Collins solution for 24 h and reperfused with Krebs-Henseleit bicarbonate buffer, nonparenchymal cell killing showed periportal predominance as assessed by nuclear staining with trypan blue. In livers reperfused in the retrograde direction, the lobular distribution of cell killing was reversed, indicating that cell killing was more rapid in oxygen-rich upstream regions. However, antioxidants, including allopurinol, desferrioxamine, catalase, superoxide dismutase, superoxide dismutase plus catalase, and U74006F, did not reduce cell killing. Similarly, reperfusion with anoxic buffer did not prevent lethal injury. Antioxidants and anoxic reperfusion also did not improve cell viability in livers stored in UW solution. Nevertheless, superoxide generation, as identified by formazan formation from nitroblue tetrazolium, was increased in Kupffer cells after lives storage and reperfusion as compared to unstored livers. Acidification of the reperfusion buffer from pH 7.4 to pH 7.15 reduced overall nonparenchymal cell killing from about 40% to 10%. Moreover, a pH gradient developed across the liver lobule during reperfusion with the effluent 0.2-0.4 pH units more acidic than the influent. This intralobular pH gradient appears to account for the relative sparing of cells in more acidic downstream regions of the lobule. Lower temperatures of reperfusion also reduced lethal injury. In conclusion, Kupffer cells generated superoxide after perfusion of stored rat livers, but formation of oxygen free radicals did not appear to contribute to lethal reperfusion injury to endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Role of Kupffer cells in failure of fatty livers following liver transplantation and alcoholic liver injury.

Kupffer cells have been implicated in mechanisms of pathophysiology following liver transplantation. Recently, postoperative injury in ethanol-induced fatty liver has been evaluated because fatty livers often fail following transplantation. The low-flow, reflow liver perfusion model was used to study the role of Kupffer cells (KC) in reperfusion injury to fatty livers from rats fed a diet containing ethanol for 4-5 weeks. Treatment with GdCl3, which selectively destroys KC, decreased cell death significantly. Thus, destruction of KC minimized hepatic reperfusion injury, most likely by inhibiting free radical formation and improving microcirculation. Since it was demonstrated recently that destruction of KC prevented the hypermetabolic state observed with acute alcohol exposure, their involvement in events leading to alcohol-induced liver disease was investigated. In rats exposed to ethanol continuously via intragastric feeding for up to 4 weeks, GdCl3 treatment prevented elevation of aspartate aminotransferase (AST) and dramatically reduced the average hepatic pathological score. These results indicate that KC participate in the early phases of alcohol-induced liver injury. Endotoxaemia occurs in alcoholics and activates KC; therefore, we evaluated the effect of minimizing bacterial endotoxin by intestinal sterilization with the antibiotics polymyxin B and neomycin. Antibiotics diminished plasma endotoxin levels significantly and prevented ethanol-induced increases in AST values. These results indicate that endotoxin is involved in the mechanism of ethanol-induced liver injury. A six-line radical spectrum was detected with electron paramagnetic resonance spectroscopy in bile from alcohol-treated rats which was blocked by GdCl3. The free radical adducts had hyperfine coupling constants characteristic of lipid-derived free radical products. In conclusion, these studies demonstrate that KC are involved in reperfusion injury to ethanol-induced fatty livers and hepatic injury due to long-term treatment with ethanol.

Animals↗
PubMed1995

Dual role of Kupffer cell activation and endothelial cell damage in reperfusion injury to livers stored for transplantation surgery.

In rat models of liver preservation, the primary event leading to liver graft failure after cold storage is a reperfusion injury causing damage to sinusoidal endothelial cells and activation of Kupffer cells (KC). After storage for longer than 16 h in University of Wisconsin solution, reperfusion induces rapid endothelial cell killing. Kupffer cell activation also occurs as indicated by cell surface ruffling, degranulation, release of hydrolytic enzymes, generation of oxygen radicals, and increased phagocytosis. Down-regulation of KC activity with nisoldipine or pentoxifylline improves graft survival. Moreover, pretreatment of donors with small amounts of endotoxin to activate KC causes a drastic reduction of graft survival. Together, KC activation and endothelial damage cause marked microcirculatory disturbances after transplantation characterized by reduced and uneven blood flow and increased leucocyte and platelet adhesion. Such events culminate in inflammation, necrosis and fulminant graft failure. Modification of reperfusion conditions can reduce the extent of injury. In particular, flushing livers with Carolina rinse solution (CRS) at the end of storage reduces endothelial cell killing, suppresses KC activation, improves the microcirculation, and increases graft survival. Active ingredients in CRS include antioxidants (allopurinol, desferrioxamine and glutathione), adenosine and slightly acidic pH (6.5). Other potentially important ingredients are nicardipine, a calcium channel blocker, and fructose, glucose and insulin to promote glycolysis. The cytoprotective amino acid, glycine, further improves the performance of Carolina rinse solution. Reperfusion-induced changes to nonparenchymal cells play an essential role in damage to livers preserved for transplantation surgery. Understanding the role of sinusoidal endothelial cells and KC in this injury has led to promising new strategies to prolong organ storage and reduce graft failure.

Animals↗
PubMed1995

Hypercapnic acidosis and dimethyl amiloride reduce reperfusion induced cell death in ischaemic ventricular myocardium.

OBJECTIVE: The aim was to investigate the effects of slowing the recovery of ischaemia induced intracellular acidosis with hypercapnic acidosis or dimethyl amiloride (DMA) on the extent of reperfusion induced cell death. METHODS: Isolated arterially perfused rabbit papillary muscles and septa were suspended in a controlled atmosphere and perfused with a modified Tyrode solution containing erythrocytes and trypan blue (500 microM). Ischaemia was produced by arrest of perfusion and withdrawal of atmospheric O2. Extracellular pH of the muscle during reperfusion was controlled by adjusting the pH of the perfusate (pH 6.6 or pH 7.6 with and without DMA 20 microM) and changing the PCO2 of the chamber atmosphere. After 30 min of reperfusion following 30 min (group A) or 60 min (group B) of ischaemia, papillary muscles were fixed with paraformaldehyde. Cell death was assessed by trypan blue staining of nuclei in histological sections of the papillary muscles. RESULTS: The magnitude of cell death was greatest after reperfusion with pH 7.6 as measured by the percentage of nuclei staining with trypan blue (15.1% in group A; 41.8% in group B). By contrast, reperfusion at pH 6.6 reduced cell killing (group A, 3.6%; group B, 7.2%). Reperfusion at pH 7.6 with DMA (20 microM) also reduced trypan blue uptake (group A, 2.8%; group B, 3.8%). Despite the attenuation of cell death afforded by acidosis or Na+/H+ exchange inhibition, significant swelling of the extracellular space and microvascular injury was noted. CONCLUSIONS: Hypercapnic acidosis and Na+/H+ exchange inhibition during reperfusion attenuate lethal reperfusion injury to ventricular myocardium and extend to the intact myocardium the concept of the "pH paradox" in which recovery of intracellular pH after reperfusion is a precipitating factor in lethal cell injury.

Acidosis↗
PubMed1995

Progression of subcellular changes during chemical hypoxia to cultured rat hepatocytes: a laser scanning confocal microscopic study.

The aim of this study was to evaluate changes in the subcellular organelles of cultured hepatocytes by laser scanning confocal microscopy during chemical hypoxia with cyanide and iodoacetate, inhibitors of mitochondrial respiration and glycolysis, respectively. Parameter-specific fluorophores used were calcein for cell topography and membrane permeability, rhodamine-dextran for lysosomes, rhodamine 123 and tetramethylrhodamine methylester (TMRM) for mitochondrial membrane potential (delta psi) and propidium iodide for loss of cell viability. During the first 30 to 40 minutes of chemical hypoxia to cultured hepatocytes, numerous surface blebs formed and cell volume increased, but delta psi decreased relatively little. Subsequently, the nonspecific permeability of mitochondrial membranes increased, and mitochondria depolarized. These events were followed a few minutes later by disintegration of individual lysosomes. After a few more minutes, viability was lost as indicated by bleb rupture, gross plasma membrane permeability to calcein, and nuclear labeling with propidium iodide. Thus, the following sequence of intracellular events occurred during chemical hypoxia: adenosine triphosphate (ATP) depletion, bleb formation with cellular swelling, onset of a mitochondrial permeability transition, disintegration of lysosomes, plasma membrane failure from bleb rupture, and cell death. Any explanation of the pathophysiology of hypoxic injury must take into account this unique sequence of events.

Animals↗