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J J Lemasters

Publications and source records attributed to J J Lemasters.

At least 55 records · Page 3Linked to original sources
PubMed1998

Confocal microscopy of the mitochondrial permeability transition in necrotic cell killing, apoptosis and autophagy.

Onset of the cyclosporin-A-sensitive mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by laser scanning confocal microscopy. The MPT is a causative event in many types of necrotic and apoptotic cell death, including oxidative stress, ischemia/reperfusion injury, Ca2+ ionophore toxicity and tumor necrosis factor alpha (TNF alpha) induced apoptosis, and may contribute to Reye's-related drug toxicity. Pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and increased mitochondrial Ca2+ and pH can each promote onset of the MPT in situ. The MPT can also be directly visualized during TNF alpha-induced apoptosis to hepatocytes. Mitochondria spontaneously depolarize in situ after nutrient deprivation before entering an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. We propose a model in which onset of the MPT to increasing numbers of mitochondria leads progressively to autophagy, apoptosis and necrotic cell death.

Animals↗
PubMed1998

Mitochondrial dysfunction and cytoskeletal disruption during chemical hypoxia to cultured rat hepatic sinusoidal endothelial cells: the pH paradox and cytoprotection by glucose, acidotic pH, and glycine.

We investigated mechanisms underlying death of cultured rat liver sinusoidal endothelial cells exposed to chemical hypoxia with KCN (2.5 mmol/L) to simulate the adenosine triphosphate (ATP) depletion and reductive stress of anoxia. During chemical hypoxia, acidotic pH prevented cell death. Glucose (0.3-10 mmol/L) also prevented cell killing. Cytoprotection by glucose but not acidosis was associated with prevention of ATP depletion. After 4 hours of chemical hypoxia at pH 6.2 (simulated ischemia), rapid cell death occurred when pH was restored to pH 7.4 with or without washout of KCN (simulated reperfusion). This pH-dependent reperfusion injury (pH paradox) was prevented after KCN washout at pH 6.2. Glycine (0.3-3 mmol/L) also prevented the pH paradox, but glucose did not. The initial protection by acidotic pH and glycine during simulated reperfusion was lost when pH was later restored to 7.4 or glycine was subsequently removed. Mitochondria depolarized during chemical hypoxia. After washout of cyanide, mitochondrial membrane potential (delta psi) did not recover in cells that subsequently lost viability. Conversely, those cells that repolarized after cyanide washout did not subsequently lose viability. The actin cytoskeleton and focal adhesions became severely disrupted during chemical hypoxia at both pH 6.2 and 7.4 and did not recover after cyanide washout under any condition. Glucose during chemical hypoxia prevented cytoskeletal disruption. In conclusion, endothelial cell damage during simulated ischemia/reperfusion involves mitochondrial dysfunction, ATP depletion, and ATP-dependent cytoskeletal disruption. Glycine and acidotic pH prevented cell killing after reperfusion but did not reverse mitochondrial injury or the profound disruption to the cytoskeleton.

Adenosine Triphosphate↗
PubMed1998

Lipopolysaccharide-stimulated TNF-alpha release from cultured rat Kupffer cells: sequence of intracellular signaling pathways.

We recently hypothesized that lipopolysaccharide (LPS) stimulation of rat Kupffer cells to induce tumor necrosis factor alpha (TNF-alpha) release requires internalization of LPS, acidification of endosomes, elevation of intracellular calcium, protein kinase C (PKC) activation, and protein tyrosine kinase (PTK) activation. This study uses inhibitors in pulse-chase experiments to determine the sequence of events of intracellular signals required for LPS-stimulated TNF-alpha release from Kupffer cells. Inhibitors of internalization (cytochalasin B, monodansylcadaverine) prevented LPS-stimulated TNF-alpha release when added simultaneously with LPS but when added 10 min after LPS, no significant inhibition occurred. The inhibitor of PTK, tyrphostin AG, blocked TNF-alpha release by only 39 +/- 4% (P < 0.001 compared with TNF-alpha release when added simultaneously with LPS) when added 10 min after LPS. Inhibitors of endosomal acidification (bafilomycin A, monensin) inhibited LPS-stimulated TNF-alpha release by 92 +/- 11% (P < 0.001 when no inhibitor was used) when added 10 min after LPS and their effect was totally abrogated when added 45 min after LPS. The PKC inhibitor, H-7, blocked TNF-alpha release by 94 +/- 9% (P < 0.001 when no inhibitor was used) when added 30 min after LPS. The calcium channel blocker, nisoldipine, still inhibited LPS-stimulated TNF-alpha release when added 45 min after LPS. These data support the hypothesis that for LPS-stimulated TNF-alpha release in Kupffer cells, LPS must first be internalized, which may stimulate PTK activation. An intermediate step of signaling involves endosomal acidification. Elevation of intracellular calcium and PKC activation occur as late intracellular signaling events.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

Mitochondrial Ca2+ transients in cardiac myocytes during the excitation-contraction cycle: effects of pacing and hormonal stimulation.

Using laser scanning confocal microscopy, our objective was to measure mitochondrial, nuclear, and cytosolic free ionized Ca2+ in adult rabbit cardiac myocytes loaded with Ca2+-indicating fluorophores. When myocytes were loaded with Fluo 3 at 37 degrees C, the fluorophore was loaded extensively into the cytosol and nucleus, but poorly into mitochondria, and Fluo 3 fluorescence transients after field stimulation were confined to the cytosol and nucleus. In contrast, after loading at 4 degrees C, Fluo 3 also entered mitochondria, and large transients of mitochondrial Fluo 3 fluorescence then occurred after stimulation. Isoproterenol (1 microM) increased the magnitude of Ca2+ transients and their subsequent rate of decay, an effect more marked in the cytosol and nucleus than in mitochondria. As pacing frequency was increased from 0.5 to 2 Hz, diastolic mitochondrial Ca2+ rose markedly in the absence but not in the presence of isoproterenol. Resting Ca2+ estimated by Indo 1 ratio imaging using UV/visible laser scanning confocal microscopy was about 200 nM in all compartments. During field stimulation, Ca2+ transiently increased to 671, 522, and 487 nM in cytosol, interfibrillar mitochondria, and perinuclear mitochondria, respectively. Isoproterenol increased these respective peak values to 1280, 750, and 573 nM. These results were consistent with those obtained in Fluo 3 experiments. We conclude that rapid mitochondrial Ca2+ transients occur during excitation-contraction coupling in adult rabbit cardiac myocytes, which may be important in matching mitochondrial metabolism to myocardial ATP demand during changes in cardiac output.

Aniline Compounds↗
PubMed1998

The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release.

This study assesses the controversial role of the mitochondrial permeability transition (MPT) in apoptosis. In primary rat hepatocytes expressing an IkappaB superrepressor, tumor necrosis factor alpha (TNFalpha) induced apoptosis as shown by nuclear morphology, DNA ladder formation, and caspase 3 activation. Confocal microscopy showed that TNFalpha induced onset of the MPT and mitochondrial depolarization beginning 9 h after TNFalpha treatment. Initially, depolarization and the MPT occurred in only a subset of mitochondria; however, by 12 h after TNFalpha treatment, virtually all mitochondria were affected. Cyclosporin A (CsA), an inhibitor of the MPT, blocked TNFalpha-mediated apoptosis and cytochrome c release. Caspase 3 activation, cytochrome c release, and apoptotic nuclear morphological changes were induced after onset of the MPT and were prevented by CsA. Depolarization and onset of the MPT were blocked in hepatocytes expressing DeltaFADD, a dominant negative mutant of Fas-associated protein with death domain (FADD), or crmA, a natural serpin inhibitor of caspases. In contrast, Asp-Glu-Val-Asp-cho, an inhibitor of caspase 3, did not block depolarization or onset of the MPT induced by TNFalpha, although it inhibited cell death completely. In conclusion, the MPT is an essential component in the signaling pathway for TNFalpha-induced apoptosis in hepatocytes which is required for both cytochrome c release and cell death and functions downstream of FADD and crmA but upstream of caspase 3.

Adaptor Proteins, Signal Transducing↗
PubMed1998

LPS receptor CD14 participates in release of TNF-alpha in RAW 264.7 and peritoneal cells but not in kupffer cells.

Lipopolysaccharide (LPS) is a bacterial polymer that stimulates macrophages to release tumor necrosis factor-alpha (TNF-alpha). In macrophages (RAW 264.7 and peritoneal cells), LPS binds to the CD14 surface receptor as the first step toward signaling. Liver macrophages, Kupffer cells, are the most numerous fixed-tissue macrophage in the body. The presence of CD14 on Kupffer cells and its role in LPS stimulation of TNF-alpha were examined. TNF-alpha release by Kupffer cells after LPS stimulation was the same in the presence and absence of serum. RAW 264.7 and peritoneal cells, which utilize the CD14 receptor, released significantly less TNF-alpha after LPS stimulation in the absence of serum because of the absence of LPS-binding protein. Phosphatidylinositol-phospholipase C treatment, which cleaves the CD14 receptor, decreased LPS-stimulated TNF-alpha release by RAW 264.7 cells but not by Kupffer cells. Deacylated LPS (dLPS) competes with LPS at the CD14 receptor when incubated in a ratio of 100:1 (dLPS/LPS). Such competition blocked LPS-stimulated TNF-alpha release from RAW 264.7 cells but not from Kupffer cells. Western and fluorescence-activated cell sorter analysis directly demonstrated the presence of CD14 on RAW 264.7 cells and murine peritoneal cells but showed only minimal amounts of CD14 in murine Kupffer cells. LPS stimulation did not increase the amount of CD14 detectable on mouse Kupffer cells. CD14 expression is very low in Kupffer cells, and LPS-stimulated TNF-alpha release is independent of CD14 in these cells.

Animals↗
PubMed1998

A superfusion system to study border zones in confluent cultures of neonatal rat heart cells.

We present a new experimental method to study intracellular ion regulation in cultured cardiomyocytes at a border zone separating two different and distinct environments. Our system uses a dual-flow superfusion chamber to produce two different but adjacent environments over a monolayer of cardiomyocytes. Fluorescent microscopy of fluorescein showed that the transition between the two environments was nearly linear and was 220-320 micron wide depending on fluid viscosity and velocity. We superfused cultured monolayers on one side with a solution at pH 6.5 and on the other side with a solution at pH 7.4. We observed a sharply demarcated difference in intracellular pH (pHi) between the two halves of the cell monolayer as measured with the fluorescent pHi indicator carboxy-seminaphthorhodafluor-1. The demarcation of pHi corresponded well with the demarcation of the border measured with fluorescein. We conclude that our superfusion system will facilitate the study of intercellular communication and interactions across boundaries of cardiac tissue where different ionic or metabolic conditions are present, for example, between ischemic and nonischemic myocardium.

Animals↗
PubMed1998

pHi and pHo at different depths in perfused myocardium measured by confocal fluorescence microscopy.

Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pHi) or extracellular pH (pHo) in isolated, arterially perfused rabbit papillary muscles. Single-excitation, dual-emission fluorescent images of the endocardial surface and underlying myocardium to a depth of 300 micron were simultaneously recorded from perfused cylindrical muscles suspended in a controlled atmosphere oriented oblique to the focal plane. Contraction was inhibited by the addition of butanedione monoxime. In separate muscles, pHo was measured during continuous perfusion of SNARF-1 free acid. pHi measurements were made after the muscle was loaded with SNARF-1/AM and the extracellular space was cleared of residual fluorophore. Initial experiments demonstrated the uniformity of ratiometric measurements as a function of pH, image depth, and fluorophore concentration, thereby establishing the potential feasibility of this method for quantitative intramural pH measurements. In subsequent experiments, the method was validated in isolated, arterially perfused rabbit papillary muscle during normal arterial perfusion and as pHi and pHo were altered by applying CO2 externally, exchanging HEPES and bicarbonate buffers, and changing pHi with NH4Cl washout. We conclude that in situ confocal fluorescent microscopy can measure pHi and pHo changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pHi regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pHi.

Acidosis↗
PubMed1998

Effects of chronic ethanol exposure on oxidation and NMDA-stimulated neuronal death in primary cortical neuronal cultures.

Excessive oxidative radical formation has been implicated in a number of neurodegenerative processes, including glutamate-N-methyl-D-aspartate (NMDA)-mediated excitotoxicity. Previous studies have suggested that the formation of reactive oxygen species (ROS) during NMDA exposure is involved in triggering the excitotoxic cascade. Chronic exposure of primary neuronal cultures to ethanol has been shown to potentiate NMDA-mediated processes, such as nitric oxide formation and excitotoxicity. It was the objective of this study to investigate the role of NMDA and ROS formation in the development of NMDA supersensitivity after chronic ethanol exposure. The fluorescent dyes dichlorofluorescein diacetate (H2DCF) and propidium iodide, which have been shown to be reliable markers for the detection of cellular oxidation and cell death, respectively, in neuronal culture preparations, were used to examine the relationship between ROS production and NMDA-mediated neuronal death after chronic ethanol administration. Cultures treated chronically with ethanol (100 mM) for 96 hr displayed significantly less H2DCF oxidation when exposed to various concentrations of FeSO4 for 25 min. However, this decrease in intracellular oxidation did not have any apparent inhibitory effects on the amount of cell death observed 12 hr after the 25-min exposure to FeSO4. When NMDA-FeSO4-mediated oxidation was examined in cultures treated chronically with ethanol, dose-dependent increases in H2DCF oxidation were observed, but only in control-treated cultures. This blunting of intracellular H2DCF oxidation did not attenuate the potentiation of NMDA-mediated excitotoxicity observed after chronic ethanol exposure. These results suggest that the observed supersensitivity to NMDA is not due to increases in intracellular ROS formation and that chronic ethanol may induce neuronal factors that reduce ROS formation, but do not protect against normal death.

Animals↗
PubMed1998

Kupffer cell-dependent reperfusion injury in liver transplantation: new clinically relevant use of glycine.

Kupffer cell-dependent reperfusion injury occurs to the liver following transplantation, most often in fatty livers which fail most frequently due to primary nonfunction. Failure was largely blocked with Carolina rinse solution, which contains glycine, and prevents the activation of Kupffer cells. Furthermore, gentle in situ organ manipulation, which cannot be prevented using standard harvesting techniques, has a detrimental effect on survival. These effects were also prevented by glycine. Since proteolytic activity is increased in both fatty and manipulated livers, amino acids were measured in rinse effluents collected at harvest. A combination of four amino acids correlated with graft function. It is concluded that glycine could be beneficial in clinical liver transplantation to prevent reperfusion injury, and that amino acids measured at harvest may predict graft function.

Fatty Liver↗
PubMed1997

Selective loading of Rhod 2 into mitochondria shows mitochondrial Ca2+ transients during the contractile cycle in adult rabbit cardiac myocytes.

A strategy of cold loading of the Ca2+-indicating fluorophore Rhod 2-AM followed by warm incubation was developed to selectively label mitochondria of adult rabbit cardiac myocytes. After electrical stimulation, mitochondrial Rhod 2 fluorescence observed by confocal microscopy increased and then rapidly decayed to baseline. In regions between mitochondria, the fluorescent transients were small or absent. Subsequent addition of calcium ionophore increased mitochondrial but not cytosolic fluorescence, confirming the mitochondrial localization of Rhod 2. These experiments directly demonstrate rapid mitochondrial free Ca2+ transients during the contractile cycle in rabbit cardiac myocytes.

Adenosine Triphosphate↗
PubMed1997

Role of ICE-like proteases in endothelial cell hypoxic and reperfusion injury.

Because of its location between blood and tissue, the endothelium is particularly vulnerable to hypoxic/reperfusion injury, but the mechanisms responsible for this injury are not known. A number of recent findings suggest that hypoxia and reperfusion injures neuronal cells via apoptosis. Apoptosis has recently been shown to depend on the activation of a class of proteases with homology to Interleukin-1 beta converting enzyme (ICE) protease. Therefore, we examined the effect of specific inhibitors of ICE-like proteases on hypoxic and reperfusion injury in cultured EAhy926 endothelial cells. Pretreatment of cells with ICE inhibitor II (Ac-YVAD-CMK), ICE inhibitor III (Z-Asp-2,6-dichlorobenzoyloxy-methylketone-Z-Asp-CH2-DCB+ ++), or ICE inhibitor IV (Ac-YVKD-CHO) (all at 10-100 microM) did not protect cells from hypoxic injury. However, pretreatment of cells with ICE inhibitor III and to a lesser extent with ICE inhibitor II, but not with ICE inhibitor IV, protected cells from reperfusion injury. The protective effect of ICE inhibitor III was not dependent upon pH, but was associated with decreased release of arachidonic acid from cells. These findings suggest that reperfusion injury to EAhy926 endothelial cells involves ICE-like proteases. The identity of the protease(s) is not known but it does not appear to be a YAMA-type protease based upon ICE inhibitor specificity. Our data also indicate that a potential target of this protease is phospholipase A2 (PLA2).

Arachidonic Acid↗
PubMed1997

Use of a multiwell fluorescence scanner with propidium iodide to assess NMDA mediated excitotoxicity in rat cortical neuronal cultures.

Glutamate mediated excitotoxicity is a major area of experimentation due to the potential for prevention of morbidity and brain damage associated with stroke and brain trauma. We have developed a simple rapid method to study excitotoxicity in primary cortical neuronal cultures using propidium iodide (PI) fluorescence read by a multiwell fluorescence scanner. Transient (25 min) or continuous N-methyl-D-aspartate (NMDA) treatment led to progressive neuronal death over 24 h that was blocked by 1 microM MK-801, 10 microM ifenprodil, and 200 mM ethanol. Results with PI fluorescence were identical to those found using the lactate dehydrogenase (LDH) release and trypan blue staining assays of excitotoxicity. This method provides a simple rapid means to test the effects of drugs during glutamate excitotoxicity and to do accurate time course experiments of delayed neuronal death.

Animals↗
PubMed1997

Reperfusion after liver transplantation in rats differentially activates the mitogen-activated protein kinases.

The injury resulting from cold ischemia and warm reperfusion during liver transplantation is a major clinical problem that limits graft success. Kupffer cell activation plays a pivotal role in reperfusion injury, and Kupffer cell products, including free radicals and tumor necrosis factor alpha (TNF-alpha), are implicated as damaging agents. However, the second messengers and signaling pathways that are activated by the stress of hepatic ischemia/reperfusion remain unknown. The purpose of this study is to assess the activation of the three known vertebrate mitogen activated protein kinase (MAPKs) and the activating protein 1 (AP-1) transcription factor in response to ischemia and reperfusion in the transplanted rat liver. There was a potent, sustained induction of c-jun N-terminal kinase (JNK), but not of the related MAPKs extracellular signal-regulated kinases (ERK) or p38, upon reperfusion after transplantation. TNF-alpha messenger RNA (mRNA) levels and transcription factors AP-1 and nuclear factor-kappaB (NF-kappaB) were induced in the liver after 60 minutes of reperfusion. Finally, there was an elevation of ceramide, but not diacylglycerol or sphingosine, in the transplanted liver. Ceramide is a second messenger generated by TNF-alpha treatment and is an activator of JNK. Because JNK activation preceded the elevations in ceramide and TNF-alpha mRNA, these results suggest that increased hepatic TNF-alpha and ceramide may perpetuate JNK induction, but that they are not the initiating signals of JNK activation during reperfusion injury in the transplanted liver.

Animals↗
PubMed1997

Role of the mitochondrial permeability transition in salicylate toxicity to cultured rat hepatocytes: implications for the pathogenesis of Reye's syndrome.

Aspirin is strongly implicated in the pathogenesis of Reye's syndrome, a childhood disorder characterized by hyperammonemia, microvesicular steatosis, and encephalopathy. Previously, we showed that salicylate, the active metabolite of aspirin, induces the mitochondrial permeability transition (MPT) in isolated mitochondria, as do several other chemicals implicated in Reye's-related disorders. Opening of a high conductance, cyclosporin A-sensitive pore in the mitochondrial inner membrane causes the MPT, leading to swelling, depolarization, and uncoupling of oxidative phosphorylation. The goal of this study was to characterize the role of the MPT in salicylate toxicity to cultured rat hepatocytes. Salicylate (0.3-5 mM) caused concentration-dependent cell killing. In Krebs-Ringer buffer, half-maximal cell killing occurred 150 min after 3 mM salicylate. Increasing Ca2+ enhanced salicylate lethality. Salicylate-dependent cell killing was blocked by 0.5-5 microM cyclosporin A and its nonimmunosuppresive analog, 4-methylvaline cyclosporin, implicating the MPT in the pathogenesis of cell killing. The contribution of the MPT to lethal cell injury was confirmed by laser scanning confocal microscopy, which demonstrated the redistribution of the fluorophore calcein from the cytosol into mitochondria prior to cell killing, an event blocked by cyclosporin A. Salicylate toxicity was enhanced at high extracellular Ca2+. In the range of 10-100 microM, several chemically diverse calcium antagonists blocked or reduced salicylate toxicity including verapamil, diltiazem, chlorpromazine, nifedipine, and nisoldipine. Calcium antagonists also blocked the increase of mitochondrial free Ca2+ in high Ca2+ buffer, as determined by confocal imaging of the fluorophore Rhod-2. These data with salicylate suggest that onset of the MPT may be the common pathophysiologic mechanism causing mitochondrial injury in Reye's syndrome and Reye's-related drug toxicities. Further, elevated intramitochondrial Ca2+ may be a predisposing condition promoting onset of the MPT by Reye's-related chemicals.

Animals↗
PubMed1997

Effect of perfusion pH on rat lung viability of non-heart beating donors.

OBJECTIVE: Non-heart beating donors could be an important source of lungs for transplantation. In prior experiments, trypan blue exclusion was used to assess the percentage of viable lung cells after different intervals following circulatory arrest. In this study, we assessed the importance of the trypan blue perfusate pH because in liver preservation studies, pH of the perfusate appears to be very important with the presence of a 'pH paradox'. METHODS: Forty Sprague-Dawley rats (n = 5 each group) were sacrificed, left at room temperature, and ventilated with 100% O2 (tidal volume 0.01 cc/g, rate 30/min) for 4 or 8 h after death. Lungs were then flushed with 150 cc of trypan blue in Krebs-Heinseleit buffer via the pulmonary artery, then with 150 cc of fixative (2%) paraformaldehyde + 2% gluteraldehyde). The pH of the trypan blue solution was varied from 6.5 to 7.8. Lungs were sectioned, and cell viability was quantified based on exclusion of trypan blue. RESULTS: Longer cadaver time was associated with less viability, but pH had no effect on viability. The results were similar in groups ventilated for the same period of time and then flushed with solutions at different pH (4 h, viability 80-84%; 8 h, viability 68-72%). Over the range investigated, the pH of the trypan blue solution had no impact on lung viability in non-heart beating donors and the lungs did not show the 'pH paradox'. CONCLUSION: Lung tissue is not susceptible to the 'pH paradox' observed in liver preservation studies. Because more than 80% of lung cells are still viable 4 h after death if the lungs are ventilated with 100% O2 during the cadaveric period, retrieval and transplantation of cadaveric lungs may be feasible and might alleviate the paucity of lung donors.

Animals↗
PubMed1997

Mitochondrial oxygen radical formation during reductive and oxidative stress to intact hepatocytes.

After simple respiratory inhibition, glycolytic substrates prevent cell death by providing an alternate source of cellular ATP. When mitochondrial uncoupling ensues, the uncoupler-stimulated mitochondrial ATPase hydrolyzes ATP formed by glycolysis and protection is lost. Electron transfer components abnormally reduced by respiratory inhibition, especially ubisemiquinone, react directly with oxygen to form toxic radicals. Mitochondria also generate reactive oxygen species after exposure to oxidant chemicals. A consequence is onset of the mitochondrial permeability transition, which leads to uncoupling, cellular ATP depletion and loss of viability. Thus, mitochondria are both a source and a target of toxic oxygen radicals in cell injury.

Animals↗