Myriocin, an inhibitor of serine palmitoyl transferase, impairs the uptake of transferrin and low-density lipoprotein in mammalian cells.

The role of sphingolipids in clathrin-mediated endocytosis is only poorly understood in mammalian cells. Thus the relationship between sphingolipid de novo synthesis and clathrin-mediated endocytosis of transferrin were studied in L929 fibroblasts and two other cell lines. Endocytosis was measured using live cell imaging with fluorescent transferrin or (125)I-transferrin. Lipids were primarily measured using electrospray ionization tandem mass spectrometry. At physiological temperature, transferrin uptake was significantly decreased by the inhibitor of serine palmitoyl transferase myriocin. Myriocin inhibited also the uptake of low-density lipoproteins. The endocytosis inhibition by myriocin could be released by the addition of sphingoid base and by the protein phosphorylation effectors phorbol-12-myristate, 13-acetate (PMA) and okadaic acid. Myriocin influenced not only sphingolipids but also the glycerophospholipid profile. The study of phosphatidylcholine species shows adaptations to more saturated, alkylated and longer fatty acid moieties. The reported results imply that in mammalian cells, at 37°C, sphingolipid de novo synthesis is required for clathrin-mediated endocytosis.

Disruption of skeletal myocytes initiates superoxide release: contribution of NADPH oxidase.

Generation of reactive oxygen species (ROS) as an early local reaction to muscle crush injury has frequently been predicted. However, although it is known that severe inflammatory reactions occurring after major muscle trauma originate mainly from early local incidents within the injured tissue, no detailed studies exist on the local generation of ROS in response to myocyte destruction thus far. Therefore, in this study, ROS formation after lethal mechanical damage was examined using a model of scraping injury to cultured C2C12 skeletal myocytes and superoxide detection by lucigenin chemiluminescence, nitrotetrazolium blue chloride reduction, or electron spin resonance spectroscopy. Mechanical rupture of myocytes resulted in an immediate release of superoxide from the damaged cells that could be substantially blocked by the superoxide scavengers superoxide dismutase (51%), tiron (95%), and MAMA/NO (93%) and by hypoxia (83% inhibition). Superoxide generation was primarily confined to the myocytes' membrane fraction and 7- to 8-fold enhanced by the addition of NADH or NADPH. The NADPH-enhanced superoxide generation could largely be diminished by the NAD(P)H oxidase inhibitors diphenyleneiodonium and apocynin in cell lysates (97% and 35% inhibition, respectively) and in isolated membrane fractions (61% and 63% inhibition). We thus conclude that immediately after myocyte damage, large amounts of superoxide are formed that predominantly originate from membrane-bound electron-transferring enzymes, especially NAD(P)H oxidase. This suggests a decisive role of ROS in the pathogenesis of tissue trauma, with superoxide being an initiator of the signaling mechanism from injured myocytes to the surrounding tissue and, potentially, to the whole body.

Enhanced oxidation of NAD(P)H by oxidants in the presence of dehydrogenases but no evidence for a superoxide-propagated chain oxidation of the bound coenzymes.

Recently we demonstrated that lactate dehydrogenase (LDH)-bound NADH is oxidized by O2, H2O2, HNO2 and peroxynitrite predominantly via a chain radical mechanism which is propagated by superoxide. Here we studied both whether other dehydrogenases also increase their coenzymes' reactivity towards these oxidants and whether a chain radical mechanism is operating. Almost all dehydrogenases increased the oxidation of their physiological coenzymes by at least one of the oxidants. The oxidation of NADH or NADPH depended both on the binding dehydrogenase and the applied oxidant and in some cases the reactions were remarkably fast. The highest rate constant (k = 370 M-1 s-1) was found for the reaction of HNO2 with NADH bound to alcohol dehydrogenase. Regardless of the applied oxidant, superoxide dismutase failed to inhibit the oxidation of protein-bound NADH and NADPH. We therefore conclude that several dehydrogenases increase the oxidation of NADH and/or NADPH by the employed set of oxidants in bimolecular reactions, but, unlike LDH, do not mediate a O2*(-) dependent chain radical mechanism.

Cold-induced injury to porcine corneal endothelial cells and its mediation by chelatable iron: implications for corneal preservation.

PURPOSE: During hypothermic storage of the cornea, corneal endothelial damage restricts storage times. We previously reported a new, iron-dependent mechanism of cold-induced injury to cultured liver cells. In this study, we sought to evaluate whether corneal endothelial cells incur a similar kind of injury. METHODS: Cultured porcine corneal endothelial cells were exposed to 4 degrees C in either cell culture medium, Krebs-Henseleit buffer, Optisol-GS solution, or McCarey-Kaufman medium for 5 hours to 14 days and then rewarmed under cell culture conditions (3 hours). The cultures were assessed for lethal cell injury (LDH release); cellular, nuclear, and mitochondrial morphologic alterations; lipid peroxidation; and mitochondrial membrane potential. RESULTS: Corneal endothelial cells sustained substantial injury following cold storage and rewarming in cell culture medium (47% +/- 8% and 64% +/- 20% cell death after 2 and 5 days cold storage, respectively). The injury displayed some apoptotic features, and cells lost mitochondrial membrane potential before cell death occurred. The iron chelators deferoxamine, 1,10-phenanthroline, and 2,2'-dipyridyl and the antioxidant butylated hydroxytoluene completely inhibited this cell injury. Marked iron-dependent cell injury and lipid peroxidation also occurred during and after cold incubation in Krebs-Henseleit buffer and, most importantly, iron-dependent cell injury was also observed after cold incubation in Optisol solution and in McCarey-Kaufman medium. CONCLUSIONS: Cultured porcine corneal endothelial cells incur a strong iron-dependent injury elicited by hypothermia. This cold-induced injury might provide an explanation for the known corneal endothelial susceptibility to hypothermic preservation injury, which thus might be amenable to therapeutic interventions (ie, by iron chelators).

ATP-induced calcium increase as a potential first signal in mechanical tissue trauma. A laser scanning microscopic study on cultured mouse skeletal myocytes.

Although it is known that after major tissue trauma, local incidents in the mechanically destroyed muscle tissue form the basis of subsequently occurring severe inflammatory reactions, the very first events taking place immediately after myocyte destruction have not been studied on the single cell level thus far. Therefore, in this study, the reaction of cultured C2C12 mouse skeletal myocytes to lethal injury was examined using laser scanning microscopy. Mechanical rupture of one single myocyte induced an immediate accumulation of calcium in its cytosol and nuclei, as detected by an increase in the fluorescence intensity of the intracellular calcium-sensitive dye Fluo-3. The intracellular calcium elevation propagated further to the adjacent, noninjured myocytes in a wave-like fashion within seconds. The calcium increase detected in these neighboring cells was higher and up to 1000 times more extended than the physiological calcium spike that induces C2C12 myocyte contraction. Wave propagation did not depend on gap junctional communication but occurred via liberation of nucleotides, mainly ATP, but presumably also UTP and others, from the destroyed cell and subsequent calcium release from the sarcoplasmic reticulum via a purinoceptor-mediated mechanism in the adjacent cells. These findings suggest a decisive role of ATP and related nucleotides in the pathogenesis of tissue trauma because they appear to initiate the signaling mechanism from injured myocytes to the surrounding tissue and potentially to the whole body.

Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron.

We previously described that the cold-induced apoptosis of cultured hepatocytes is mediated by an increase in the cellular chelatable iron pool. We here set out to assess whether a mitochondrial permeability transition (MPT) is involved in cold-induced apoptosis. When cultured hepatocytes were rewarmed after 18 h of cold (4 degrees C) incubation in cell culture medium or University of Wisconsin solution, the vast majority of cells rapidly lost mitochondrial membrane potential. This loss was due to MPT as assessed by confocal laser scanning microscopy and as evidenced by the inhibitory effect of the MPT inhibitors trifluoperazine plus fructose. The occurrence of the MPT was iron-dependent: it was strongly inhibited by the iron chelators 2,2'-dipyridyl and deferoxamine. Addition of trifluoperazine plus fructose also strongly inhibited cold-induced apoptosis, suggesting that the MPT constitutes a decisive intermediate event in the pathway leading to cold-induced apoptosis. Further experiments employing the non-site-specific iron indicator Phen Green SK and specifically mitochondrial iron indicators and chelators (rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester, RPA, and rhodamine B-[(2,2'-bipyridin-4-yl)aminocarbonyl]benzyl ester, RDA) suggest that it is the cold-induced increase in cytosolic chelatable iron that triggers the MPT and that mitochondrial chelatable iron is not involved in this process.

Cold-induced apoptosis of rat liver endothelial cells: contribution of mitochondrial alterations.

BACKGROUND: Maintenance of the integrity of the vascular endothelium is a critical issue in liver preservation, but hypothermia, applied for cellular protection, induces apoptotic cell death in liver endothelial cells. This cold-induced apoptosis is mediated by an iron-dependent formation of reactive oxygen species. Here, we study the involvement of mitochondria in this process. METHODS: Cultured rat liver endothelial cells were incubated in cold University of Wisconsin solution for 18 hr and subsequently rewarmed in cell culture medium. Mitochondrial morphology and membrane potential were evaluated using laser scanning microscopy. RESULTS: During cold incubation in University of Wisconsin solution, a marked, progressive mitochondrial shortening and a reduction in mitochondrial membrane potential occurred. Rewarming of the cells led to mitochondrial ultracondensation, complete loss of the mitochondrial membrane potential, and subsequent apoptotic cell death. The inhibitors of mitochondrial permeability transition, trifluoperazine and fructose, or iron chelation with deferoxamine did not affect mitochondrial shortening during cold incubation but inhibited ultracondensation, loss of mitochondrial membrane potential, and loss of viability during rewarming. Moreover, in these protected cells, an almost complete reestablishment of the mitochondrial membrane potential and morphology could be observed; the few mitochondria that were irreversibly damaged were incorporated into autophagosomes during cellular recovery. CONCLUSION: Two apparently independent mitochondrial alterations take place during cold incubation and subsequent rewarming of liver endothelial cells. Cold-induced mitochondrial shortening represents a reversible process, whereas iron-mediated mitochondrial permeability transition and ultracondensation during rewarming are irreversible and constitute an important mediator of cold-induced apoptosis.

Population pharmacokinetic analysis of circadian rhythms in hepatic CYP3A activity using midazolam.

Diurnal changes in the activity of drug metabolizing enzymes may contribute to the variability in drug disposition and drug effects. The aim of this study was to quantify the circadian rhythmicity exhibited by hepatic CYP3A. A 10 µg/kg intravenous bolus dose, followed by a 30-hour 4 µg/kg/h intravenous infusion of midazolam, used as a probe substrate for hepatic CYP3A activity, was administered to 16 healthy volunteers (8 males and 8 females). Blood samples were drawn hourly for 24 hours after achieving steady state, and plasma concentrations of midazolam and its main metabolite 1-OH midazolam were determined. Population pharmacokinetic analysis was performed using nonlinear mixed effects modeling. One-compartment pharmacokinetic models best described midazolam and 1-OH midazolam pharmacokinetic disposition. An unequivocal but minor diurnal pattern was identified in the midazolam plasma concentration profiles, which was described using a cosine function with a 24-hours period. The fluctuation in the relative CYP3A activity ranged between 10% above average around 15:00, and 10% below average around 03:00. None of the covariates tested had a significant impact on the parameters estimated. Although a diurnal pattern in hepatic CYP3A activity was identified, its magnitude suggests that it is small and without clinical significance for drug therapy.

Activation of hypoxia-inducible factor 1 in skeletal muscle cells after exposure to damaged muscle cell debris.

Skeletal muscle damage provokes complex repair mechanisms including recruitment of leukocytes as well as activation of myogenic precursor cells such as satellite cells. To study muscle cell repair mechanisms after muscle fiber damage, we used an in vitro model of scrape-injured myotubes. Exposing vital C2C12 myoblasts and myotubes to cell debris of damaged myotubes revealed mRNA upregulation of adrenomedullin (ADM), insulin-like growth factors 1 and 2, metallopeptidase 9, and monocyte chemoattractant protein11. When cell debris was treated with ultrasound, frozen in liquid nitrogen, or heat inactivated before addition to C2C12 cells, gene expression was drastically reduced or completely absent. Moreover, incubations of myoblasts with debris separated by transwell inserts indicated that direct cell contact is required for gene induction. Incubation with albumin and PolyIC ruled out that ADM induction by cell debris simply results from increased protein or nucleic acid concentrations in the supernatant. Because the genes, which were upregulated by cell debris, are potential target genes of hypoxia-inducible factor (HIF), cells were analyzed for HIF-1α expression. Western blot analysis showed accumulation of the α-subunit upon contact to cell debris. Knockdown of HIF-1α in C2C12 cells proved that activation of HIF-1 in response to cell debris was responsible for upregulating ADM and monocyte chemoattractant protein 1. Furthermore, by incubating cells on gas-permeable culture dishes, we excluded a reduced pericellular pO2 induced by cell debris as the cause for ADM upregulation. Our data suggest that damaged myofibers activate HIF-1 in neighboring myotubes and precursor myoblasts by direct contact, concomitantly upregulating factors necessary for angiogenesis, tissue regeneration, and phagocyte recruitment.

Release of redox-active iron by muscle crush trauma: no liberation into the circulation.

After major skeletal muscle trauma, the iron-containing protein myoglobin and diverse other intracellular metabolites are liberated into the circulation from injured myocytes. Because chelatable iron should also be present in skeletal muscle cells, this redox-active, not tightly bound iron should be released from injured muscle tissue in addition to myoglobin and potentially account for oxidative tissue damage. The current study demonstrates in vitro the existence of 5 muM chelatable iron within the supernatant of a 1:10 homogenate of rat gastrocnemius muscle. This iron was almost exclusively associated with macromolecules greater than 30 kDa, most likely proteins. Presumably because of this association, only part of the chelatable iron could be scavenged by added apotransferrin. The chelatable iron was redox-active and thus responsible for the formation of thiobarbituric acid-reactive substances (TBARS) within the muscle homogenate. Correspondingly, using an in vivo model of closed trauma to the rat gastrocnemius muscle, a local TBARS formation in the damaged muscle tissue could be detected. Muscle trauma significantly increased plasma creatine kinase and myoglobin levels; however, no increase in serum non-transferrin-bound iron could be observed. Likewise, the serum parameters of iron-induced oxidative damage, TBARS, and protein carbonyls did not significantly increase after trauma. In conclusion, chelatable, redox-active iron is locally released by muscle destruction and responsible for lipid peroxidation within the damaged tissue. However, the liberation of chelatable iron into the circulation and its contribution to oxidative alterations of serum lipids and proteins could not be confirmed.

Iron-dependent vs. iron-independent cold-induced injury to cultured rat hepatocytes: a comparative study in physiological media and organ preservation solutions.

We previously described the entity of cold-induced apoptosis to rat hepatocytes and characterized its major, iron-dependent pathway. However, after cold incubation in some solutions, e.g. cell culture medium, hepatocytes show an additional, yet uncharacterized component of cold-induced injury. We here assessed the effects of organ preservation solutions on both components of cold-induced injury and tried to further characterize the iron-independent component. None of the preservation solutions (University of Wisconsin, histidine-tryptophan-ketoglutarate, Euro-Collins, histidine-lactobionate, sodium-lactobionate-sucrose and Celsior solutions) provided significant protection against cold-induced cell injury (LDH release after 24-h cold incubation/3h rewarming >65% for all solutions); three solutions even enhanced cold-induced injury. However, when the predominant iron-dependent mechanism was eliminated by the addition of iron chelators, all preservation solutions yielded hepatocyte protection that was clearly superior to the one obtainable in cell culture medium or Krebs-Henseleit buffer with iron chelators (LDH release after 24-h cold incubation/3h rewarming

The HIF-1 response to simulated ischemia in mouse skeletal muscle cells neither enhances glycolysis nor prevents myotube cell death.

Hypoxia-inducible factor (HIF) plays an important role in regulating gene expression in response to ischemia. Although activation of HIF-1 in muscle tissue was found during ischemia in vivo, the meaning and mechanisms in isolated cells are still incompletely understood. We studied activation of HIF-1 in skeletal muscle cells cultured in either their undifferentiated myoblast state or differentiated into myotubes. HIF-1 was activated in myoblasts and myotubes by hypoxia and simulated ischemia. Induction of adrenomedullin mRNA and, to a lesser extent, VEGF mRNA correlated well with the induction of HIF-1alpha protein in both cell types. Enzymes of glycolysis-like lactate dehydrogenase and pyruvate kinase showed upregulation of their mRNA only under hypoxic conditions but not during simulated ischemia. Phosphofructokinase mRNA showed no significant upregulation at all. Although HIF-1 was activated in myotubes during simulated ischemia, myotubes died preceded by a loss of ATP. Myoblasts survived simulated ischemia with no decrease in ATP or ATP turnover. Furthermore, pharmacological inhibition of HIF-1 hydroxylases by dimethyloxalylglycine (DMOG) increased HIF-1alpha accumulation and significantly upregulated the expression of adrenomedullin, VEGF, lactate dehydrogenase, and pyruvate kinase in myoblasts and myotubes. However, DMOG provided no protection from cell death. Our data indicate that HIF-1, although activated in myotubes during simulated ischemia, cannot protect against the loss of ATP and cell viability. In contrast, myoblasts survive ischemia and thus may play an important role during regeneration and HIF-1-induced revascularization.

Screening for the formation of reactive oxygen species and of NO in muscle tissue and remote organs upon mechanical trauma to the mouse hind limb.

BACKGROUND: Until now, no systematic surveys exist in the literature on the early local and systemic generation of reactive oxygen species and of nitric oxide in response to muscle crush injury. Therefore, this study aims to evaluate the formation of reactive oxygen species and nitric oxide in different tissues (injured and contralateral muscle, liver, kidney, spleen and blood) that is induced by closed muscle trauma. METHODS: 5, 45 and 180 min after induction of blunt trauma to the mouse gastrocnemius muscle, animals were sacrificed, tissues harvested and homogenized, and analyzed for their content of glutathione, nitrate and thiobarbituric acid-reactive substances. RESULTS: The local formation of reactive oxygen species in the injured muscle started immediately upon induction of the mechanical trauma as indicated by changes in the glutathione redox balance. Liver and kidney did not show any response to trauma; however, a marked and immediate increase in the splenic nitrate content was detected, thus suggesting a specific nitric oxide-dependent response of splenic cells to injury. CONCLUSION: We conclude that immediately after the induction of trauma a formation of reactive oxygen species takes place at the site of crush injury. This might constitute the basis of further damage to the injured tissue by free radical-dependent mechanisms. The immediate formation of nitric oxide within the spleen upon muscle crush appears to represent a specific signalling mechanism of the body in response to distant organ injury.

Cold-induced apoptosis of rat liver cells in University of Wisconsin solution: the central role of chelatable iron.

Although University of Wisconsin (UW) solution aims at the prevention of cold-induced cell injury, it failed to protect against cold-induced apoptosis of hepatocytes and liver endothelial cells: when incubated in UW solution at 4 degrees C for 24 hours and subsequently rewarmed at 37 degrees C, 72% +/- 8% of rat hepatocytes and 81% +/- 5% of liver endothelial cells lost viability. In both cell types, the observed cell damage occurred under an apoptotic morphology; it appeared to be mediated by a rapid increase in the cellular chelatable iron pool by a factor > or =2 (as determined in hepatocytes) and subsequent formation of reactive oxygen species (ROS). Consequently, this cell injury was decreased by iron chelators to 6 to 25% (hepatocytes) and 4% +/- 2% (liver endothelial cells). Deferoxamine nearly completely inhibited the occurrence of apoptotic morphology in both cell types. In liver endothelial cells, cold-induced apoptosis occurring during rewarming after 24 hours of cold incubation in UW solution was far more pronounced than in cell culture medium (loss of viability: 81% +/- 5% vs. 28% +/- 13%), but viability could even be maintained for 2 weeks of cold incubation by use of deferoxamine. In conclusion, this pathological mechanism might be an explanation for the strong endothelial cell injury known to occur after cold preservation. With regard to the extent of this iron-mediated injury, addition of a suitable iron chelator to UW solution might markedly improve the outcome of liver preservation.