Comparison of Mitochondrial Incubation Media for Measurement of Respiration and Hydrogen Peroxide Production.

High-Resolution FluoRespirometry is a well-established and versatile approach to study mitochondrial oxygen uptake amperometrically in combination with measurement of fluorescence signals. One of the most frequently applied fluorescent dyes is Amplex UltraRed for monitoring rates of hydrogen peroxide production. Selection of an appropriate mitochondrial respiration medium is of crucial importance, the primary role of which is to support and preserve optimum mitochondrial function.

High-Resolution FluoRespirometry and OXPHOS Protocols for Human Cells, Permeabilized Fibers from Small Biopsies of Muscle, and Isolated Mitochondria.

Protocols for High-Resolution FluoRespirometry of intact cells, permeabilized cells, permeabilized muscle fibers, isolated mitochondria, and tissue homogenates offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques, small needle biopsies of muscle, and mitochondrial preparation methods. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation (OXPHOS) improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protocols with stepwise modulation of coupling control and electron transfer pathway states.

Molecular structural diversity of mitochondrial cardiolipins.

Current strategies used to quantitatively describe the biological diversity of lipids by mass spectrometry are often limited in assessing the exact structural variability of individual molecular species in detail. A major challenge is represented by the extensive isobaric overlap present among lipids, hampering their accurate identification. This is especially true for cardiolipins, a mitochondria-specific class of phospholipids, which are functionally involved in many cellular functions, including energy metabolism, cristae structure, and apoptosis.

Mis-targeting of the mitochondrial protein LIPT2 leads to apoptotic cell death.

Lipoyl(Octanoyl) Transferase 2 (LIPT2) is a protein involved in the post-translational modification of key energy metabolism enzymes in humans. Defects of lipoic acid synthesis and transfer start to emerge as causes of fatal or severe early-onset disease. We show that the first 31 amino acids of the N-terminus of LIPT2 represent a mitochondrial targeting sequence and inhibition of the transit of LIPT2 to the mitochondrion results in apoptotic cell death associated with activation of the apoptotic volume decrease (AVD) current in normotonic conditions, as well as over-activation of the swelling-activated chloride current (IClswell), mitochondrial membrane potential collapse, caspase-3 cleavage and nuclear DNA fragmentation.

The cyanobacterial metabolite nocuolin a is a natural oxadiazine that triggers apoptosis in human cancer cells.

Oxadiazines are heterocyclic compounds containing N-N-O or N-N-C-O system within a six membered ring. These structures have been up to now exclusively prepared via organic synthesis. Here, we report the discovery of a natural oxadiazine nocuolin A (NoA) that has a unique structure based on 1,2,3-oxadiazine.

High-Resolution Respirometry for Simultaneous Measurement of Oxygen and Hydrogen Peroxide Fluxes in Permeabilized Cells, Tissue Homogenate and Isolated Mitochondria.

Whereas mitochondria are well established as the source of ATP in oxidative phosphorylation (OXPHOS), it is debated if they are also the major cellular sources of reactive oxygen species (ROS). Here we describe the novel approach of combining high-resolution respirometry and fluorometric measurement of hydrogen peroxide (H2O2) production, applied to mitochondrial preparations (permeabilized cells, tissue homogenate, isolated mitochondria). The widely used H2O2 probe Amplex Red inhibited respiration in intact and permeabilized cells and should not be applied at concentrations above 10 µM.

Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production.

Mitochondrial respiration is associated with the formation of reactive oxygen species, primarily in the form of superoxide (O2 (•-)) and particularly hydrogen peroxide (H2O2). Since H2O2 plays important roles in physiology and pathology, measurement of hydrogen peroxide has received considerable attention over many years. Here we describe how the well-established Amplex Red assay can be used to detect H2O2 production in combination with the simultaneous assessment of mitochondrial bioenergetics by high-resolution respirometry.

Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry.

The mitochondrial transmembrane potential (Δψmt or mtMP) is directly influenced by oxidative phosphorylation (OXPHOS). The exact nature of the interactions between respiration (flux) and mtMP (force) under various physiological and pathological conditions remains unclear, partially due to methodological limitations. Here, we describe a combination of high-resolution respirometry and fluorometry based on the OROBOROS Oxygraph-2k and the widely applied mtMP indicator safranin.

Necrosis-like death can engage multiple pro-apoptotic Bcl-2 protein family members.

Necroptosis is a physiologically relevant mode of cell death with some well-described initiating events, but largely unknown executioners. Here we investigated necrostatin-1 (Nec-1) sensitive death elicited by different necroptosis stimuli in L929 mouse fibrosarcoma cells, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages. We found that TNFα- or zVAD-induced necroptosis occurs independently of the recently implicated executioners Bmf or PARP-2, but can involve the Bcl-2 family proteins Bid and Bak.

Regulatory volume decrease and P receptor signaling in fish cells: mechanisms, physiology, and modeling approaches.

For animal cell plasma membranes, the permeability of water is much higher than that of ions and other solutes, and exposure to hyposmotic conditions almost invariably causes rapid water influx and cell swelling. In this situation, cells deploy regulatory mechanisms to preserve membrane integrity and avoid lysis. The phenomenon of regulatory volume decrease, the partial or full restoration of cell volume following cell swelling, is well-studied in mammals, with uncountable investigations yielding details on the signaling network and the effector mechanisms involved in the process.

Apoptosis of leukocytes triggered by acute DNA damage promotes lymphoma formation.

Apoptosis triggered by p53 upon DNA damage secures removal of cells with compromised genomes, and is thought to prevent tumorigenesis. In contrast, we provide evidence that p53-induced apoptosis can actively drive tumor formation. Mice defective in p53-induced apoptosis due to loss of its proapoptotic target gene, puma, resist gamma-irradiation (IR)-induced lymphomagenesis.

Perspective--from describing to understanding environment-physiology relations: 50th birthday of a branch in ecophysiology.

Animals generally show various adaptation features that render them fit for survival in their specific environment or, turned the other way round, specific environments can only be inhabited by animals that have developed corresponding adaptations. While this seems obvious nowadays to every biologist, 50years ago this concept still needed to be validated for each specific case. In a brief historical perspective we highlight an outstanding example of an article where such environment-physiology relations have been examined in detail and where in fact the foundations of a new branch in ecophysiology have been established, the Ecophysiology of the Marine Meiofauna.

Apoptosis and necroptosis are induced in rainbow trout cell lines exposed to cadmium.

Cadmium is an important environmental toxicant that can kill cells. A number of studies have implicated apoptosis as well as necrosis and, most recently, a form of programmed necrosis termed necroptosis in the process of cadmium-mediated toxicity, but the exact mechanism remains ill-defined and may depend on the affected cell type. This study investigated which mode of cell death may be responsible for cell death induction in cadmium-exposed trout cell lines from gill and liver and if this cell death was sensitive to inhibitors of necroptosis or apoptosis, respectively.

Vertebrate cell death in energy-limited conditions and how to avoid it: what we might learn from mammalian hibernators and other stress-tolerant vertebrates.

Dormancy in vertebrates may expose cells to acidosis, hypoxia/anoxia, oxidative damage, and extremes in temperature. All of these insults are known to be pro-apoptotic in typical vertebrate cells, especially mammals. Since dormancy is presumably the result of a need for energy conservation, the inherent energetic demand of replenishing cells that underwent apoptosis seems at odds with this strategy.

A volume regulatory response can be triggered by nucleosides in human erythrocytes, a perfect osmometer no longer.

Human erythrocytes have been regarded as perfect osmometers, which swell or shrink as dictated by their osmotic environment. In contrast, in most other cells, swelling elicits a regulatory volume decrease (RVD) modulated by the activation of purinic and pyrimidinic receptors (P receptors). For human erythrocytes this modulation has not been tested, and we thus investigated whether P receptor activation can induce RVD in these cells.

Caspase-2 activation in the absence of PIDDosome formation.

PIDD (p53-induced protein with a death domain [DD]), together with the bipartite adapter protein RAIDD (receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a DD), is implicated in the activation of pro-caspase-2 in a high molecular weight complex called the PIDDosome during apoptosis induction after DNA damage. To investigate the role of PIDD in cell death initiation, we generated PIDD-deficient mice. Processing of caspase-2 is readily detected in the absence of PIDDosome formation in primary lymphocytes.

Fish as model systems for the study of vertebrate apoptosis.

Apoptosis is a process of pivotal importance for multi-cellular organisms and due to its implication in the development of cancer and degenerative disease it is intensively studied in humans and mammalian model systems. Invertebrate models of apoptosis have been well-studied, especially in C. elegans and D.

Kinetics of ATP release and cell volume regulation of hyposmotically challenged goldfish hepatocytes.

In most animal cells, hypotonic swelling is followed by a regulatory volume decrease (RVD) thought to prevent cell death. In contrast, goldfish hepatocytes challenged with hypotonic medium (180 mosM, HYPO) increase their volume 1.7 times but remain swollen and viable for at least 5 h.

Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling.

Apoptotic cell death in mammalian models is frequently associated with cell shrinkage. Inhibition of apoptotic volume decrease (AVD) is cytoprotective, suggesting that cell shrinkage is an important early event in apoptosis. In salmonid hepatoma and gill cells staurosporine induced apoptosis, as assessed by activation of effector caspases, nuclear condensation, and a decrease of mitochondrial membrane potential (MMP), and these changes were accompanied by cell shrinkage.

Activation and nuclear translocation of ERK in response to ligand-dependent and -independent stimuli in liver and gill cells from rainbow trout.

The mitogen-activated protein kinase ERK is an important signalling molecule involved in the control of cell proliferation, differentiation and cell death, targeting molecules at the cell membrane, in the cytosol, and in the nucleus. This study investigated the activation pattern and subcellular distribution of ERK in liver and gill cells of rainbow trout upon hypo-osmotic shock, addition of epidermal growth factor (EGF) and copper treatment. It further set out to characterize the hypothetical role of nuclear-export signal (NES)-dependent relocation of ERK after nuclear entry and the potential involvement of the ERK activator MEK.

Signalling pathways involved in hypertonicity- and acidification-induced activation of Na+/H+ exchange in trout hepatocytes.

In trout hepatocytes, hypertonicity and cytosolic acidification are known to stimulate Na+/H+ exchanger (NHE) activity, which contributes to recovery of cell volume and intracellular pH (pHi), respectively. The present study investigated the signalling mechanisms underlying NHE activation under these conditions. Exposing trout hepatocytes to cariporide, a specific inhibitor of NHE-1, decreased baseline pHi, completely blocked the hypertonicity-induced increase of pHi and reduced the hypertonicity-induced proton secretion by 80%.

Copper-induced stimulation of extracellular signal-regulated kinase in trout hepatocytes: the role of reactive oxygen species, Ca2+, and cell energetics and the impact of extracellular signal-regulated kinase signaling on apoptosis and necrosis.

The present study investigated if copper (Cu) exposure of trout hepatocytes, which stimulates formation of reactive oxygen species (ROS) and increases intracellular free Ca(2+) (Ca(2+)i), leads to an activation of extracellular signal-regulated kinase (ERK), the mechanisms underlying this activation, and the role of ERK signaling in cell death. Cu stimulated a time- and dose-dependent increase of phosphorylated extracellular signal-regulated kinase (pERK), and preventing the associated Ca(2+) influx or radical formation diminished or inhibited ERK activation, respectively. Furthermore, Cu enhanced caspase 3/7 activity and necrosis, and both effects were inhibited by treatments diminishing radical production and by chelating extracellular Ca(2+).

Extracellular signal regulated MAP-kinase signalling in osmotically stressed trout hepatocytes.

Activation of the extracellular signal-regulated MAP-kinase (ERK) by anisoosmotic conditions, the underlying signalling pathways, and the role of protein kinases in cell volume regulation were investigated in trout hepatocytes. While hyperosmolarity left phosphorylated ERK (pERK) levels unaffected, hypoosmolarity caused a significant increase of pERK within 2 min which peaked at around 30 min. Chelating extracellular Ca2+ to prevent the influx of Ca2+ associated with swelling reduced iso- and abolished hypoosmotic ERK activation.