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.

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.

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.

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.

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.

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'.

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.

Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes.

To simulate ischemia and reperfusion, cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4.

Role of free radicals in primary nonfunction of marginal fatty grafts from rats treated acutely with ethanol.

Acute treatment with one large dose of ethanol, which mimics binge drinking, causes marginal fatty liver and decreases survival significantly after liver transplantation in rats, yet mechanisms remain unclear. Therefore, we evaluated the possible role of free radicals in primary nonfunction caused by acute ethanol. Female donor rats were administered ethanol (5 g/kg orally) 20 hr before explantation, and grafts were stored in UW cold storage solution for 24-42 hr before implantation.

The mitochondrial permeability transition in toxic, hypoxic and reperfusion injury.

Opening of a non-specific, high conductance permeability transition pore or megachannel in the inner mitochondrial membrane causes onset of the mitochondrial permeability transition, which is characterized by mitochondrial swelling, depolarization and uncoupling. Inducers of the permeability transition include Ca2+, oxidant stress and a permissive pH greater than 7.0.

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.

When does the lung die? Kfc, cell viability, and adenine nucleotide changes in the circulation-arrested rat lung.

Lungs harvested from cadaveric circulation-arrested donors may increase the donor pool for lung transplantation. To determine the degree and time course of ischemia-reperfusion injury, we evaluated the effect of O2 ventilation on capillary permeability [capillary filtration coefficient (Kfc)], cell viability, and total adenine nucleotide (TAN) levels in in situ circulation-arrested rat lungs. Kfc increased with increasing postmortem ischemic time (r = 0.

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.

Adaptive Kupffer cell alterations after femur fracture trauma in rats.

Because Kupffer cells constitute the largest fixed macrophage population and reside at a strategic position in hepatic sinusoids, interacting with hepatocytes, circulating cells, and mediators from the gut, they may be important in the inflammatory response after injury. This study examined the effect of remote tissue injury on Kupffer cell function. Femurs of Sprague-Dawley rats were fractured under anesthesia.

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.

Mitochondrial permeability transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species.

Tert-butyl hydroperoxide (t-BuOOH) induces the mitochondrial permeability transition (MPT) in hepatocytes, leading to cell death. Using confocal microscopy, we visualized pyridine nucleotide oxidation and reactive oxygen species (ROS) formation induced by t-BuOOH. Reduced mitochondrial pyridine nucleotides (NADH and NADPH) were imaged by autofluorescence.

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.

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.

Reperfusion injury after liver preservation for transplantation.

Preservation injury remains an obstacle to greater utilization of liver transplantation therapy. Livers can be preserved a maximum of 24 h in University of Wisconsin solution. After longer times, reperfusion precipitates endothelial cell killing and activation of Kupffer cells (liver macrophages).

Protection by Carolina rinse solution, acidotic pH, and glycine against lethal reperfusion injury to sinusoidal endothelial cells of rat livers stored for transplantation.

The critical injury causing graft failure after prolonged liver storage involves reperfusion-induced killing of sinusoidal endothelial cells and activation of Kupffer cells. Treatment of stored livers with Carolina rinse solution (CRS) prevents endothelial cell killing, reduces Kupffer cell activation, and improves graft survival. Accordingly, our aim was to evaluate the components of CRS and other agents for protection against reperfusion injury to rat livers stored 24 hr in University of Wisconsin solution.

Cyclosporin A delays mitochondrial depolarization induced by N-methyl-D-aspartate in cortical neurons: evidence of the mitochondrial permeability transition.

N-Methyl-D-aspartate causes a rapid increase in intracellular Ca2+ leading to collapse of the mitochondrial membrane potential and eventually cell death in cortical neurons. The aim of this study was to investigate the mechanism responsible for mitochondrial depolarization using laser scanning confocal microscopy of single cultured rate cortical neurons. To monitor mitochondrial membrane potential, neuronal mitochondria were labeled with tetramethylrhodamine methyl ester, a cationic fluorophore that accumulates in polarized mitochondria.

Endocytosis and Ca2+ are required for endotoxin-stimulated TNF-alpha release by rat Kupffer cells.

Endotoxin [lipopolysaccharide (LPS)] is a cell wall polymer derived from Gram-negative bacteria that stimulates macrophages to produce a variety of inflammatory mediators. In these studies, we examined LPS-stimulated formation of tumor necrosis factor-alpha (TNF-alpha) by cultured rat Kupffer cells. Cytochalasin B and methylpalmitate, blockers of endocytosis, decreased LPS-stimulated TNF-alpha release by > 92%.

Studies of rat lung viability and adenine nucleotide metabolism after death.

Background: Prior studies from our laboratory have supported the use of cadaveric lungs for transplantation. In this study we investigated different preservation strategies for lungs retrieved from cadavers 4 hours after circulatory arrest.

Methods: Seventy-two Sprague-Dawley rats were sacrificed and then ventilated with 100% oxygen for 4 hours.