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J J Lemasters

Publications and source records attributed to J J Lemasters.

At least 163 records · Page 9Linked to original sources
PubMed1990

Cytosolic free magnesium, ATP and blebbing during chemical hypoxia in cultured rat hepatocytes.

Cytosolic free Mg2+ concentration was determined in 1-day cultured rat hepatocytes using Multiparameter Digitized Video Microscopy (MDVM) of the fluorescent probe, mag-fura-2. Chemical hypoxia with KCN (5 mM) and iodoacetate (1 mM), a model which mimics the ATP depletion and reductive stress of hypoxia, caused a rapid increase of free Mg2+ from 1.1 +/- 0.2 to 1.6 +/- 0.2 mM within 4 min. Concurrently, numerous small plasma membrane blebs formed and ATP levels dropped from 13.24 to 1.32 nmol/10(6) cells. Removal of KCN and iodoacetate resulted in recovery of ATP to 60-70% of pre-exposure levels, a concomitant decrease in cytosolic free Mg2+ back toward basal levels, and reversal of blebbing (bleb resorption). These results indicate that changes of cytosolic free Mg2+ inversely reflect changes of ATP in a model of hypoxia and reoxygenation. Bleb formation and resorption were dependent on the fall and rise of ATP.

Adenosine Triphosphate↗
PubMed1990

Protection by acidotic pH and fructose against lethal injury to rat hepatocytes from mitochondrial inhibitors, ionophores and oxidant chemicals.

The importance of mitochondrial ATP formation and extracellular acidosis was evaluated in hepatocyte suspensions after different toxic treatments. Acidotic pH was protective against cell killing from all toxic treatments examined except for pronase, a toxic protease. Fructose, a substrate for glycolytic ATP formation, provided good protection against toxicity from cyanide, oligomycin, t-butyl hydroperoxide, menadione and cystamine. Protection by fructose against CCCP, gramicidin and Br-A23187 required oligomycin. This indicated that these ionophores were causing cytotoxicity by uncoupling oxidative phosphorylation. Fructose provided little protection against pronase and HgCl2, the latter compound being a potent inhibitor of glycolysis. In conclusion, disruption of mitochondrial ATP formation was a common event contributing to the toxicity of chemical oxidants and ionophores. Acidotic pH was generally protective under these conditions of impaired ATP generation.

Animals↗
PubMed1990

A new method to monitor Kupffer-cell function continuously in the perfused rat liver. Dissociation of glycogenolysis from particle phagocytosis.

In order to study particle phagocytosis and glycogenolysis simultaneously, this study was designed to develop a direct-read-out method to monitor Kupffer-cell function continuously, based on the uptake of colloidal carbon by the isolated perfused rat liver. Livers were perfused for 20 min with Krebs-Henseleit buffer saturated with O2/CO2 (19:1). Colloidal carbon (1-2 mg/ml) was added to the buffer, and absorbance of carbon was monitored continuously at 623 nm in the effluent perfusate. Since colloidal-carbon uptake was proportional to A623, rates of uptake were determined from the influent minus effluent concentration difference, the flow rate and the liver wet weight. Rates of colloidal-carbon uptake were 50-200 mg/h per g and were proportional to the concentration of carbon infused. Data from light-microscopy and cell-separation studies demonstrated that carbon was taken up exclusively by non-parenchymal cells and predominantly by Kupffer cells. Further, the amount of colloidal carbon detected histologically in non-parenchymal cells increased as the concentration of colloidal carbon in the perfusate was elevated. When Kupffer cells were activated or inhibited by treatment with endotoxin or methyl palmitate, carbon uptake was increased or decreased respectively. Taken together, these results indicate that Kupffer-cell function can be monitored continuously in a living organ. This new method was utilized to compare the time course of phagocytosis of carbon by Kupffer cells and carbohydrate output by parenchymal cells. Carbohydrate output increased rapidly by 69 +/- 9 mumol per g within 2-4 min after addition of carbon and returned to basal values within 12-16 min. However, carbon uptake by the liver did not reach maximal rates until about 15 min. Infusion of a cyclo-oxygenase inhibitor, aspirin (10 mM), caused a progressive decrease in carbohydrate output and blocked the stimulation by carbon completely. Aspirin neither altered rates of carbon uptake nor prevented stimulation of carbohydrate release by addition of N2-saturated buffer. The data from these experiments are consistent with the hypothesis that output of mediators by Kupffer cells, presumably prostaglandin D2 and E2, occurs transiently as Kupffer cells begin to phagocytose foreign particles in the intact organ, a process which continues at high rates for hours.

Animals↗
PubMed1990

Toxic injury from mercuric chloride in rat hepatocytes.

The relationship between cytosolic free Ca2+, mitochondrial membrane potential, ATP depletion, pyridine nucleotide fluorescence, cell surface blebbing, and cell death was evaluated in rat hepatocytes exposed to HgCl2. In cell suspensions, 50 microM HgCl2 oxidized pyridine nucleotides between 1/2 and 2 min, caused ATP depletion between 2 and 5 min, and produced an 89% loss of cell viability after 20 min. Rates of cell killing were identical in high (1.2 mM) and low (2.6 microM) Ca2+ buffers. Cytosolic free Ca2+ was determined in 1-day cultured hepatocytes by ratio imaging of Fura-2 employing multiparameter digitized video microscopy. In high Ca2+ medium, HgCl2 caused a 3-4-fold increase of free Ca2+ beginning after 6-7 min, but free Ca2+ did not change in low Ca2+ medium. Bleb formation occurred after about 4-5 min in both buffers prior to any increase of free Ca2+. Subsequently, in high Ca2+ medium, blebs became hot spots of free Ca2+ (greater than 600 nM). After about 2 min of exposure to HgCl2, rhodamine 123 fluorescence redistributed from mitochondrial to cytosolic compartments signifying collapse of the mitochondrial membrane potential. The results taken together demonstrate that bleb formation, ATP depletion, and the onset of cell death are not dependent on an increase of cytosolic free Ca2+. HgCl2 toxicity appears to be a consequence of inhibition of oxidative phosphorylation leading to ATP depletion and cell death.

Adenosine Triphosphate↗
PubMed1990

Lack of metabolic effects of cholecystokinin on hepatocytes.

We previously reported that the liver was the major organ that extracts small, biologically active, circulating forms of cholecystokinin. Although our work indicated extensive degradation of cholecystokinin extracted from plasma during its transit across the hepatocyte, it was unclear whether cholecystokinin might also have a physiological effect on this cell before its intracellular degradation. Therefore we tested the hypothesis that cholecystokinin has a direct biological effect on hepatocytes. Using freshly isolated or cultured hepatocytes, we studied whether cholecystokinin-octapeptide alters protein synthesis, affects amino acid transport or influences cytosolic free calcium concentrations. Using liver slices, we also determined the effect of cholecystokinin-octapeptide on cyclic nucleotide levels. Cholecystokinin-octapeptide, up to a concentration of 1 mumol/L, had no effect on the incorporation of radiolabeled amino acids into total hepatocyte protein; in contrast, comparable molar amounts of insulin stimulated protein synthesis by as much as 37% (ED50 = 1.5 x 10(-10) mol/L). Although insulin and glucagon stimulated the transport into hepatocytes of 14C-alpha-aminoisobutyric acid, a nonmetabolizable amino acid analog, cholecystokinin-octapeptide had no affect Cholecystokinin-octapeptide also did not affect either the concentration of calcium in individual hepatocytes, as measured by digitized video microscopy using Fura-2, or the levels of cyclic AMP or cyclic GMP in liver slices. Our results show that cholecystokinin has no effect on protein synthesis, on amino acid transport or on hepatocyte calcium and cyclic nucleotide levels. These and our previous data suggest that the primary outcome of hepatic extraction of cholecystokinin is hormone degradation.

Amino Acids↗
PubMed1990

Increase in survival time of liver transplants by protease inhibitors and a calcium channel blocker, nisoldipine.

Kupffer cells are activated by calcium and release a variety of toxic mediators, including proteases. The purpose of these studies, therefore, was to determine if protease inhibitors and a calcium channel blocker could increase survival time in the rat model of orthotopic liver transplantation. Survival for 30 days was greater than 90% in this model when livers were stored for 1 hr in Ringer's solution (survival conditions)--however, grafts stored for 4 hr in Euro-Collins solution or 8 hr in University of Wisconsin (UW) solution survived postoperatively only 1.2 and 0.7 days, respectively (nonsurvival conditions). When livers were stored for 4 hr in Euro-Collins containing a cocktail of protease inhibitors (leupeptin, pepstatin A, phenylmethylsulfonyl fluoride, 20 ng/ml each; diisopropyl fluorophosphate, 100 microM) and subsequently transplanted, however, survival time was increased significantly to 11.5 days. Inclusion of a calcium channel blocker, nisoldipine (1.4 microM), in the protease inhibitor cocktail increased survival time to 23 days. Actually, nisoldipine alone increased survival time to 25 days. Nisoldipine alone also increased survival time in livers stored for 8 or 16 hr in UW solution to between 15 and 20 days. Serum transaminase levels reached peak values greater than 2400 U/L one day postoperatively in the nonsurvival groups, and liver injury assessed histologically was apparent. Under these conditions, pulmonary infiltration of inflammatory cells was observed in about 60% of the lungs examined and was associated with massive bleeding. Inclusion of the protease cocktail, nisoldipine, or both in the storage solutions decreased maximal SGOT levels and injury to both liver and lung significantly by about 50% postoperatively. Nisoldipine also decreased phagocytosis of carbon particles by the perfused liver 2- to 3-fold following storage under nonsurvival conditions (half-maximal effect = 0.3-0.4 microM nisoldipine). Moreover, nisoldipine improved hepatic microcirculation. It accelerated blood flow into the liver, as indexed by hemoglobin reflectance from the liver surface. These data support the hypothesis that Kupffer cells are activated early in the sequence of events that causes graft failure leading to endothelial cell-mediated alterations in the microcirculation. This work demonstrates clearly that dihydropyridine-type calcium channel blockers such as nisoldipine may be clinically useful in storage solutions for liver prior to transplantation.

Animals↗
PubMed1990

Multiparameter digitized video microscopy of toxic and hypoxic injury in single cells.

There is no clear picture of the critical events that lead to the transition from reversible to irreversible injury. Many studies have suggested that a rise in cytosolic free Ca2+ initiates plasma membrane bleb formation and a sequence of events that lead ultimately to cell death. In recent studies, we have measured changes in cytosolic free Ca2+, mitochondrial membrane potential, cytosolic pH, and cell surface blebbing in relation to the onset of irreversible injury and cell death following anoxic and toxic injury to single hepatocytes by using multiparameter digitized video microscopy (MDVM). MDVM is an emerging new technology that permits single living cells to be labeled with multiple probes whose fluorescence is responsive to specific cellular parameters of interest. Fluorescence images specific for each probe are collected over time, digitized, and stored. Image analysis and processing then permits quantitation of the spatial distribution of the various parameters with the single living cells. Our results indicate the following: The formation of plasma membrane blebs accompanies all types of injury in hepatocytes. Cell death is a rapid event initiated by rupture of a plasma membrane bleb, and it is coincident with the onset of irreversible injury. An increase of cytosolic free Ca2+ is not the stimulus for bleb formation or the final common pathway leading to cell death. A decrease of mitochondrial membrane potential precedes the loss of cell viability. Cytosolic pH falls by more than 1 pH unit during chemical hypoxia. This acidosis protects against the onset of cell death.

Animals↗
PubMed1990

Calcium and pH in anoxic and toxic injury.

The critical events that lead to the transition from reversible to irreversible injury remain unclear. Studies are reviewed that have suggested that a rise in cytosolic free Ca2+ initiates plasma membrane bleb formation and a sequence of events that leads ultimately to cell death. In recent studies, we have measured changes in cytosolic free Ca2+, mitochondrial membrane potential, cytosolic pH, and cell surface blebbing in relation to the onset of irreversible injury and cell death following anoxic and toxic injury to single hepatocytes utilizing multiparameter digitized video microscopy (MDVM). MDVM is an emerging new technology that permits single living cells to be labeled with multiple probes whose fluorescence is responsive to specific cellular parameters of interest. Fluorescence images specific for each probe are collected over time, and then digitized and stored. Image analysis and processing then permits quantitation of the spatial distribution of the various parameters within the single living cells. Our results indicate the following: (1) formation of plasma membrane blebs accompanies all types of injury in hepatocytes; (2) cell death is a rapid event, initiated by rupture of a plasma membrane bleb, and is coincident with the onset of irreversible injury; (3) an increase of cytosolic free Ca2+ is not the stimulus for bleb formation or the final common pathway leading to cell death; (4) a decrease of mitochondrial membrane potential precedes loss of cell viability; (5) cytosolic pH falls by more than 1 pH unit during chemical hypoxia. This acidosis protects against the onset of cell death.

Animals↗
PubMed1989

Reperfusion injury to endothelial cells following cold ischemic storage of rat livers.

Storage of donor livers in Euro-Collins solution for human transplantation surgery is limited to about 8 hr. Here, tissue damage to isolated rat livers stored under the same conditions as human livers was characterized following reperfusion. The purpose of this work was to determine the importance of nutritional status on injury due to cold storage and reperfusion, to establish whether lethal injury occurs during cold storage or only after reperfusion, and to identify the cell types most vulnerable to damage. Rat livers were perfused with Krebs-Henseleit-bicarbonate buffer, stored 8 to 48 hr in Euro-Collins solution and reperfused with warm, oxygenated Krebs-Henseleit buffer for 15 min. Nuclear trypan blue uptake and lactate dehydrogenase release were used as indices of cell death. After 8 hr of cold storage and reperfusion, little loss of parenchymal or nonparenchymal viability occurred. After 24 or 48 hr, virtually all parenchymal cells remained viable. However, severe damage to nonparenchymal cells was observed, and about 40% of nonparenchymal cells were trypan blue positive. Nutritional status (fed vs. fasted) did not affect the extent of cell damage. Nonparenchymal cell killing was accompanied by cellular rounding, nuclear pyknosis and protrusion of cells into sinusoidal lumens. Scanning electron micrographs demonstrated denudation of the sinusoidal lining. Rounding and pyknosis were not observed in 24-hr-stored livers which were not reperfused, and trypan blue uptake did not occur in stored livers infused with cold, anoxic Euro-Collins solution. Based on cytochemistry and electron microscopy, lethal cell injury occurred predominantly to endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Evidence that graft survival is not related to parenchymal cell viability in rat liver transplantation. The importance of nonparenchymal cells.

Injury to parenchymal and nonparenchymal cells of livers stored in cold Euro-Collins solution was assessed following reperfusion and compared with graft survival following orthotopic rat liver transplantation. Parenchymal cells maintained their viability nearly completely after up to 24 hr of cold storage as assessed by trypan blue exclusion (97% of cells) and LDH release (4% of total) from livers reperfused for 20 min following storage. Furthermore, hepatic glycolysis (rates of lactate plus pyruvate production), oxygen uptake and NADH redox state (lactate:pyruvate ratio) were in the normal range at all time points studied up to 24 hr of cold storage. In contrast, nonparenchymal cells lost viability as assessed from trypan blue staining beginning after 8 hr of storage: 40% were nonviable after 24 hr of storage. Since injury to nonparenchymal cells occurs only upon reperfusion, oxygen radicals may be involved. Accordingly, xanthine and hypoxanthine, substrates for oxygen radical formation, were measured in perfusate upon reperfusion. Both purines accumulated (up to 80 microM) with time of storage and were washed out rapidly (less than 10 min) upon reperfusion. Although parenchymal cell function was in the normal range in livers stored in the cold for 24 hr, liver grafts stored for 6 hr and longer in Euro-Collins solution could not be transplanted successfully. Thus, we conclude that viability of parenchymal cells in liver grafts prior to transplantation is a poor parameter to predict the outcome of transplantation. Therefore, assessment of parenchymal cell energy state (e.g., with 31P NMR and other methods) most likely will not predict survival reliably. On the other hand, nonparenchymal cells lose their viability significantly earlier following storage and reperfusion. These data suggest that preservation of nonparenchymal cell viability is critical for successful liver transplantation.

Animals↗
PubMed1989

Swelling, reductive stress, and cell death during chemical hypoxia in hepatocytes.

In rat hepatocytes, we examined the relationship between cell volume, bleb formation, and loss of cell viability during chemical hypoxia with KCN plus iodoacetic acid. In hypotonic media (150-200 mosmol/kgH2O), cells swelled to a greater extent during chemical hypoxia than in isotonic media, but rates of cell killing were identical. Sucrose (300 mM) added to isotonic media prevented early cell swelling but actually accelerated cell killing. In contrast, mannitol (300 mM) improved cell survival but did not prevent cell swelling. Bleb formation occurred regardless of buffer tonicity. The antioxidants desferrioxamine and cyanidanol but not superoxide dismutase +/- catalase delayed lethal cell injury. Cell killing was greater during aerobic compared with anaerobic chemical hypoxia. Hydroperoxide formation was measured using a dichlorofluorescin assay and was accelerated during aerobic but not anaerobic chemical hypoxia. The results indicate that cell swelling is not the driving force for bleb formation or lethal cell injury. We conclude that "reductive stress" caused by respiratory inhibition favors formation of toxic oxygen species and may contribute to lethal cell injury during intermittent or incomplete oxygen deprivation.

Anaerobiosis↗
PubMed1989

Intracellular pH during "chemical hypoxia" in cultured rat hepatocytes. Protection by intracellular acidosis against the onset of cell death.

The relationships between extracellular pH (pHo), intracellular pH (pHi), and loss of cell viability were evaluated in cultured rat hepatocytes after ATP depletion by metabolic inhibition with KCN and iodoacetate (chemical hypoxia). pHi was measured in single cells by ratio imaging of 2',7'-biscarboxy-ethyl-5,6-carboxyfluorescein (BCECF) fluorescence using multiparameter digitized video microscopy. During chemical hypoxia at pHo of 7.4, pHi decreased from 7.36 to 6.33 within 10 min. pHi remained at 6.1-6.5 for 30-40 min (plateau phase). Thereafter, pHi began to rise and cell death ensued within minutes, as evidenced by nuclear staining with propidium iodide and coincident leakage of BCECF from the cytoplasm. An acidic pHo produced a slightly greater drop in pHi, prolonged the plateau phase of intracellular acidosis, and delayed the onset of cell death. Inhibition of Na+/H+ exchange also prolonged the plateau phase and delayed cell death. In contrast, monensin or substitution of gluconate for Cl- in buffer containing HCO3- abolished the pH gradient across the plasma membrane and shortened cell survival. The results indicate that intracellular acidosis after ATP depletion delays the onset of cell death, whereas reduction of the degree of acidosis accelerates cell killing. We conclude that intracellular acidosis protects against hepatocellular death from ATP depletion, a phenomenon that may represent a protective adaptation against hypoxic and ischemic stress.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗