A possible involvement of plasma membrane NAD(P)H oxidase in the switch mechanism of the cell death mode from apoptosis to necrosis in menadione-induced cell injury

The effects of inhibitors of plasma membrane NADPH oxidase on menadione-induced cell injury processes were studied using human osteosarcoma 143B cells. The intracellular level of superoxide in the cells treated with menadione for 6 h reached a maximum followed by an abrupt decrease. The population of apoptotic cells detected by Annexin V and propidium iodide double staining also reached its maximum at 6 h of menadione-treatment while that of necrotic cells increased continuously reaching 90% of the total population at 9 h of the treatment. Pretreatment of the cells with inhibitors of NADPH oxidase, including diphenyliodonium chloride, apocynin, N-vanillylnonanamide and staurosporine was effective in lowering the menadione-induced elevations of superoxide, and also in the suppression of the switch of the cell death mode from apoptosis to necrosis in menadione-treated cells except for the case of staurosporine. These results strongly suggest that superoxide generated by NADPH oxidase, besides that generated by the mitochondria, may contribute to the remarkable increase in the intracellular level of superoxide in the cells treated with menadione for 6 h resulting in the switch from apoptosis to necrosis, although a direct evidence of the presence of active and inactive forms of NADPH oxidase in control and menadione-treated 143B cells is lacking at present.     

ATP depletion alters the mode of cell death induced by benzyl isothiocyanate

Pro-inflammatory death is presumably an undesirable event in cancer prevention process, thus biochemical comprehension and molecular definition of this process could have important clinical implications. In the present study, we examined the cytophysiological conversion of cell death mode by benzyl isothiocyanate (BITC) in human cervical cancer HeLa cells. The detailed studies using flow cytometric and morphological analyses demonstrated that the cells treated with appropriate concentration (25 microM) of BITC showed apoptotic feature, such as chromatin condensation, DNA fragmentation, and preserved plasma membrane integrity, whereas these features were disappeared by treatment with higher concentration (100 microM). The treatment with 2-deoxyglucose, an inhibitor of ATP synthesis, drastically increased in the ratio of necrotic dead cells, while it influences little that of apoptotic cells. Moreover, an analysis using the mitochondrial DNA-deficient HeLa cells demonstrated that the rho degrees cells were more susceptible to the BITC-induced necrosis-like cell death compared to the wild-type (rho(+)) cells, whereas the ROS production was significantly inhibited in the rho degrees cells. It is likely that the BITC-induced ROS is derived from mitochondrial respiratory chain and ruled out the contribution to the mechanism of cell death mode switching. In addition, the BITC treatment resulted in a more rapid depletion of ATP in the rho degrees cells than in the rho(+) cells. Furthermore, a caspase inhibitor, Z-VAD-fmk counteracted not only apoptosis, but also necrosis-like cell death induced by BITC, suggesting that increment in this cell death pattern might be due to the interruption of events downstream of a caspase-dependent pathway. The obtained data suggested that the decline in the intracellular ATP level plays an important role in tuning the mode of cell death by BITC.     

Separation of cytochrome c-dependent caspase activation from thiol-disulfide redox change in cells lacking mitochondrial DNA

Release of mitochondrial cytochrome c (cyt c) is an early and common event during apoptosis. Previous studies showed that the loss of cyt c triggered superoxide production by mitochondria and contributed to the oxidation of cellular thiol-disulfide redox state. In this study, we tested whether loss of the functional electron transport chain due to depleting mitochondrial DNA (mtDNA) would affect this redox-signaling mechanism during apoptosis. Results showed that cyt c release and caspase activation in response to staurosporine treatment were preserved in cells lacking mitochondrial DNA (rho0 cells). However, unlike the case with rho+ cells, in which a dramatic oxidation of intracellular glutathione (GSH) occurred after mitochondrial cyt c release, the thiol-disulfide redox state in apoptotic rho0 cells remained largely unchanged. Thus, mitochondrial signaling of caspase activation can be separated from the bioenergetic function, and mitochondrial respiratory chain is the principal source of ROS generation in staurosporine-induced apoptosis.     

Apoptosis supercedes necrosis in mitochondrial DNA-depleted Jurkat cells by cleavage of receptor-interacting protein and inhibition of lysosomal cathepsin

In the present study, we used mitochondrial DNA-depleted Jurkat subclones (rho0 cells) to demonstrate that Fas agonistic Ab (CH-11), at the concentrations that evoke apoptotic death of the parental Jurkat cells, induced necrosis mainly through generation of excess reactive oxygen species, lysosomal rupture, and sequential activation of cathepsins B and D, and in minor part through activation of receptor-interacting protein (RIP). In the rho0 cells treated with CH-11, ATP supplementation converted necrosis into apoptosis by the formation of the apoptosome and subsequent activation of procaspase-3. In these ATP-supplemented rho0 cells (ATP-rho0), generation of excess ROS and lysosomal rupture were still seen, yet cathepsins B and D were inactivated and RIP was degraded. The conversion of necrosis to apoptosis, RIP degradation, and cathepsin inactivation in ATP- rho0 cells were blocked by caspase-3 inhibitors. Activities of cathepsins B and D in the lysate of necrotic rho0 cells were inhibited by the addition of apoptotic parental Jurkat cell lysate. Thus, apoptosis may supercede necrosis.     

Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved.

Mitochondria serve as a pivotal component of the apoptotic cell death machinery. However, cells that lack mitochondrial DNA (rho(0) cells) retain apparently normal apoptotic signaling. In the present study, we examined mitochondrial mechanisms of apoptosis in rho(0) osteosarcoma cells treated with staurosporine. Immunohistochemistry revealed that rho(0) cells maintained a normal cytochrome c distribution in mitochondria even though these cells were deficient in respiration. Upon staurosporine treatment, cytochrome c was released concomitantly with activation of caspase 3 and loss of mitochondrial membrane potential (Deltapsi(m)). After mitochondrial loss of cytochrome c, rho(0) cells underwent little change in glutathione (GSH) redox potential whereas a dramatic oxidation in GSH/glutathione disulfide (GSSG) pool occurred in parental rho(+) cells. These results show that mitochondrial signaling of apoptosis via cytochrome c release was preserved in cells lacking mtDNA. However, intracellular oxidation that normally accompanies apoptosis was lost, indicating that the mitochondrial respiratory chain provides the major source of redox signaling in apoptosis.     

Phagocytosis of necrotic cells by macrophages is phosphatidylserine dependent and does not induce inflammatory cytokine production

Apoptotic cells are cleared by phagocytosis during development, homeostasis, and pathology. However, it is still unclear how necrotic cells are removed. We compared the phagocytic uptake by macrophages of variants of L929sA murine fibrosarcoma cells induced to die by tumor necrosis factor-induced necrosis or by Fas-mediated apoptosis. We show that apoptotic and necrotic cells are recognized and phagocytosed by macrophages, whereas living cells are not. In both cases, phagocytosis occurred through a phosphatidylserine-dependent mechanism, suggesting that externalization of phosphatidylserine is a general trigger for clearance by macrophages. However, uptake of apoptotic cells was more efficient both quantitatively and kinetically than phagocytosis of necrotic cells. Electron microscopy showed clear morphological differences in the mechanisms used by macrophages to engulf necrotic and apoptotic cells. Apoptotic cells were taken up as condensed membrane-bound particles of various sizes rather than as whole cells, whereas necrotic cells were internalized only as small cellular particles after loss of membrane integrity. Uptake of neither apoptotic nor necrotic L929 cells by macrophages modulated the expression of proinflammatory cytokines by the phagocytes.     

31P NMR and saturation transfer studies of the effect of Pb2+ on cultured osteoblastic bone cells

The mechanism of lead toxicity at the cellular level remains unknown, although an effect of lead on intracellular Ca2+ has been described. Since bone is a major target for lead, we have investigated the effect of lead on bioenergetic rates and on the intracellular free Mg2+ concentration in cultured osteoblastic bone cells. Using 31P NMR and the saturation transfer technique we have detected a sizable (18%) transfer of saturation from gamma ATP to Pi in a perfused osteoblastic osteosarcoma bone cell line, Ros 17/2.8, and have found a large (greater than 82%) reduction in the Pi----ATP rate upon treatment with 10 microM Pb2+. The NMR-measured unidirectional rate was much greater than the net rate of ATP synthesis through glycolysis and oxidative phosphorylation. By using iodoacetate we investigated the mechanism of the saturation transfer and found that it is catalyzed by the glycolytic enzyme couple glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. The net rate of glycolysis as measured by lactate production and that of oxidative phosphorylation as measured by O2 consumption were found to be significantly decreased by 18 and 74%, respectively, with lead treatment. In addition, from the chemical shifts of intracellular ATP resonances, we found a significant reduction of 21% in the intracellular free Mg2+ concentration upon Pb2+ treatment. The observed lead-induced reduction in ATP synthesis/utilization and the decrease in intracellular free Mg2+ may contribute to the impairment of bone formation during lead intoxication.     

Apoptotic and necrotic blebs in epithelial cells display similar neck diameters but different kinase dependency

Apoptotic and necrotic blebs elicited by H(2)O(2) were compared in terms of dynamics, structure and underlying biochemistry in HeLa cells and Clone 9 cells. Apoptotic blebs appeared in a few minutes and required micromolar peroxide concentrations. Necrotic blebs appeared much later, prior to cell permeabilization, and required millimolar peroxide concentrations. Strikingly, necrotic blebs grew at a constant rate, which was unaffected throughout successive cycles of budding and detachment. At 1 microm diameter, the necks of necrotic and apoptotic blebs were almost identical. ATP depletion was discarded as a major factor for both types of bleb. Inhibition of ROCK-I, MLCK and p38MAPK strongly decreased apoptotic blebbing but had no effect on necrotic blebbing. Taken together, these data suggest the existence of a novel structure of fixed dimensions at the neck of both types of plasma membrane blebs in epithelial cells. However, necrotic blebs can be distinguished from apoptotic blebs in their susceptibility to actomyosin kinase inhibition.     

Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium

Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment. Simultaneously, elevation of intracellular [Ca²⁺]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells.     

Mechanisms of internalization of apoptotic and necrotic L929 cells by a macrophage cell line studied by electron microscopy

Rapid and efficient phagocytic removal of dying cells is a key feature of apoptosis. In necrotic caspase-independent modes of death, the role and extent of phagocytosis is not well documented. To address this issue, we studied at the ultrastructural level the phagocytic response to dying cells in an in vitro phagocytosis assay with a mouse macrophage cell line (Mf4/4). As target cells, murine L929sAhFas cells were induced to die by TNFR1-mediated necrosis or by Fas-mediated apoptosis. Apoptotic L929sAhFas cells are taken up by complete engulfment of apoptotic bodies as single entities forming a tight-fitting phagosome, thus resembling the "zipper"-like mechanism of internalization. In contrast, primary and secondary necrotic cells were internalized by a macropinocytotic mechanism with formation of multiple ruffles by the ingesting macrophage. Ingestion of necrotic cellular material was invariably taking place after the integrity of the cell membrane was lost and did not occur as discrete particles, in contrast to apoptotic material that is surrounded by an intact membrane. Although nuclei of necrotic cells have been observed in the vicinity of macrophages, no uptake of necrotic nuclei was observed. The present report provides a basis for future studies aimed at discovering molecular pathways that precede these diverse mechanisms of uptake.     

Antioxidant defences and homeostasis of reactive oxygen species in different human mitochondrial DNA-depleted cell lines.

Three pairs of parental (rho+) and established mitochondrial DNA depleted (rho0) cells, derived from bone, lung and muscle were used to verify the influence of the nuclear background and the lack of efficient mitochondrial respiratory chain on antioxidant defences and homeostasis of intracellular reactive oxygen species (ROS). Mitochondrial DNA depletion significantly lowered glutathione reductase activity, glutathione (GSH) content, and consistently altered the GSH2 : oxidized glutathione ratio in all of the rho0 cell lines, albeit to differing extents, indicating the most oxidized redox state in bone rho0 cells. Activity, as well as gene expression and protein content, of superoxide dismutase showed a decrease in bone and muscle rho0 cell lines but not in lung rho0 cells. GSH peroxidase activity was four times higher in all three rho0 cell lines in comparison to the parental rho+, suggesting that this may be a necessary adaptation for survival without a functional respiratory chain. Taken together, these data suggest that the lack of respiratory chain prompts the cells to reduce their need for antioxidant defences in a tissue-specific manner, exposing them to a major risk of oxidative injury. In fact bone-derived rho0 cells displayed the highest steady-state level of intracellular ROS (measured directly by 2',7'-dichlorofluorescin, or indirectly by aconitase activity) compared to all the other rho+ and rho0 cells, both in the presence or absence of glucose. Analysis of mitochondrial and cytosolic/iron regulatory protein-1 aconitase indicated that most ROS of bone rho0 cells originate from sources other than mitochondria.     

Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture

Cadmium is a toxic agent that it is also an environmental contaminant. Cadmium exposure may be implicated in some humans disorders related to hyperactivity and increased aggressiveness. This study presents data indicating that cadmium induces cellular death in cortical neurons in culture. This death could be mediated by an apoptotic and a necrotic mechanism. The apoptotic death may be mediated by oxidative stress with reactive oxygen species (ROS) formation which could be induced by mitochondrial membrane dysfunction since this cation produces: (a) depletion of mitochondrial membrane potential and (b) diminution of ATP levels with ATP release. Necrotic death could be mediated by lipid peroxidation induced by cadmium through an indirect mechanism (ROS formation). On the other hand, 40% of the cells survive cadmium action. This survival seems to be mediated by the ability of these cells to activate antioxidant defense systems, since cadmium reduced the intracellular glutathione levels and induced catalase and SOD activation in these cells.     

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

A laser scanning confocal microscopy method. Simultaneous detection of intracellular Ca2+ and apoptosis using Fluo-3 and Hoechst 33342

OBJECTIVE: To develop a simple and direct method to simultaneously determine apoptotic cells from a treated population of cells and detect the changes of intracellular Ca2+ in these apoptotic cells, in particular single ones, by confocal microscopy. STUDY DESIGN: MGC-803 cells treated with As2O3 were used as the double-staining cell model with Hoechst 33342 as a DNA probe and Fluo-3AM as a Ca2+ indicator. MGC-803 cell apoptosis induced by As2O3 was first demonstrated by DNA ladder in gel electrophoresis. Based on the difference in DNA stainability with Hoechst 33342 and corresponding fluorescence intensity between live and apoptotic cells, apoptotic cells and the changes in intracellular Ca2+ were detected at the same time by confocal microscopy. No necrotic cells in the group treated with As2O3 were found by the trypan blue exclusion test. RESULTS: The results from confocal microscope detection showed that intact and apoptotic cells were successfully recognized and the changes of intracellular Ca2+ in apoptotic and intact cells were simultaneously detected in the same sample. CONCLUSION: We provided a useful method to exactly detect changes in intracellular Ca2+ in apoptotic cells, especially in single ones, by confocal microscopy and to exclude the artifact effect of necrotic and intact cells.     

Hydrogen peroxide-induced DNA repair and death of resting human blood lymphocytes

DNA single-strand breaks induced by cell treatment with hydrogen peroxide are repaired and simultaneously trigger programmed cell death in resting human blood lymphocytes. Apoptosis is accompanied by special morphological changes in lymphocytes (15% of total cell number), internucleosomal DNA degradation, and p53 level elevation. According to morphological criteria, a major part (up to 40% of total cell number) displayed necrotic death features. Nicotinamide inhibited repair in cells with 2.5-fold elevation of the apoptotic cell proportion, whereas the fraction of cells with necrotic nuclear morphology decreased 4.5-fold. Both the inhibition of repair and the protective effect of nicotinamide against necrotic death indicate that the repair process and related poly(ADP-ribose)polymerase (PARP) activation induce a decrease in intracellular NAD+ and ATP contents below the threshold at which necrosis becomes the preferential mechanism of cell death. The mixed pattern of cell death induced by hydrogen peroxide observed in resting lymphocytes can be explained in the context of a concept of cell de-energization as a consequence of effective single-stand break repair during the first hours after removing the genotoxic agent.     

Depletion of mitochondrial DNA alters glucose metabolism in SK-Hep1 cells

Maternally inherited mitochondrial DNA (mtDNA) has been suggested to be a genetic factor for diabetes. Reports have shown a decrease of mtDNA content in tissues of diabetic patients. We investigated the effects of mtDNA depletion on glucose metabolism by use of rho(0) SK-Hep1 human hepatoma cells, whose mtDNA was depleted by long-term exposure to ethidium bromide. The rho(0) cells failed to hyperpolarize mitochondrial membrane potential in response to glucose stimulation. Intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were decreased in rho(0) cells compared with parental rho(+) cells. Our results suggest that the quantitative reduction of mtDNA may suppress the expression of nuclear DNA-encoded glucose transporters and enzymes of glucose metabolism. Thus this may lead to diabetic status, such as decreased ATP production and glucose utilization.     

Resistance of mitochondrial DNA-depleted cells against cell death: role of mitochondrial superoxide dismutase

We have shown that mitochondrial DNA-depleted (rho(0)) SK-Hep1 hepatoma cells are resistant to apoptosis, contrary to previous papers reporting normal apoptotic susceptibility of rho(0) cells. We studied the changes of gene expression in SK-Hep1 rho(0) cells. DNA chip analysis showed that MnSOD expression was profoundly increased in rho(0) cells. O(2)(.) contents increased during rho(0) cell derivation but became normalized after establishment of rho(0) phenotypes, suggesting that MnSOD induction is an adaptive process to increased O(2)(.). rho(0) cells were resistant to menadione, paraquat, or doxorubicin, and O(2)(.) contents after treatment with them were lower in rho(0) cells compared with parental cells because of MnSOD overexpression. Expression levels and activity of glutathione peroxidases were also increased in rho(0) cells, rendering them resistant to exogenous H(2)O(2). rho(0) cells were resistant to p53, and intracellular ROS contents after p53 expression were lower compared with parental cells. Other types of rho(0) cells also showed increased MnSOD expression and resistance against ROS. Heme oxygenase-1 expression was increased in rho(0) cells, and a heme oxygenase-1 inhibitor decreased the induction of MnSOD in rho(0) cells and their resistance against ROS donors. These results indicate that rho(0) cells are resistant to cell death contrary to previous reports and suggest that an adaptive increase in the expression of antioxidant enzymes renders cancer cells or aged cells with frequent mitochondrial DNA mutations to resist against oxidative stress, host anti-cancer surveillance, or chemotherapeutic agents, conferring survival advantage on them.     

Degeneration of the midgut epithelium in Allacma fusca L. (Insecta, Collembola, Symphypleona): apoptosis and necrosis

Apoptotic and necrotic changes in the midgut epithelium cells of Allacma fusca (Collembola, Symphypleona) are described at the ultrastructural level. The morphological sign indicating the beginning of the apoptotic process in these cells is their shrinkage and the transformation of their mitochondria. The nucleus assumes a lobular shape and finally undergoes fragmentation. The intercellular junctions between an apoptotic cell and adjacent epithelial cells gradually disappear. Apoptotic cells are discharged into the midgut lumen just beneath the peritrophic membrane, where they are initially distributed singly but ultimately form a single layer. No phagocytosis was observed, so no apoptotic bodies are formed. Only young midgut epithelium shows apoptosis; as cells age, necrosis accompanies apoptosis, and necrosis finally completely replaces apoptosis.     

Quinidine and procainamide inhibit murine macrophage uptake of apoptotic and necrotic cells: A novel contributing mechanism of drug-induced-lupus

A number of mechanisms have been proposed to explain the etiology of drug-induced lupus (DIL) but the effect of apoptotic and necrotic cell handling has not been previously examined.Objective. To evaluate the effect of quinidine and procainamide at therapeutic range concentrations, on the uptake of apoptotic and necrotic thymocytes by murine peritoneal macrophages and on macrophage survival, as a novel mechanism for DIL.Methods. Thymocytes were stained and induced to undergo apoptosis by serum withdrawal. Apoptosis was evaluated using annexin V and propidum iodide (PI) and PI staining. Necrosis was induced by heating. Peritoneal macrophages were treated with quinidine or procainamide at a range of therapeutic concentrations and incubated with stained apoptotic and necrotic thymocytes. Apoptotic and necrotic cell uptake was evaluated by flow cytometry using double staining of thymocytes and macrophages and by confocal microscopy. Green fluorescent latex beads were used as controls for phagocytosis.Results. Significantly decreased uptake of apoptotic and necrotic cells was seen in the presence of quinidine and procainamide. The documented effect was mainly on the number of apoptotic/necrotic cells per macrophage. Uptake of fluorescent latex beads offered to resident macrophages was not significantly affected by quinidine or procainamide. No pro-apoptotic effect of quinidine or procainamide on macrophages was seen.Conclusion. Quinidine and procainamide at therapeutic range concentrations specifically inhibit clearance of apoptotic and necrotic cells by peritoneal macrophages. Altered handling of apoptotic and necrotic cells may represent a contributing mechanism for DIL.     

Bradykinin enhances reactive oxygen species generation, mitochondrial injury, and cell death induced by ATP depletion--a role of the phospholipase C-Ca(2+) pathway

This study aimed to study the effect of bradykinin on reactive oxygen species (ROS) generation, mitochondrial injury, and cell death induced by ATP depletion in cell culture. Renal tubular cells were subjected to ATP depletion. Cell death was evaluated with LDH release, sub-G0/G1 fraction, Hoechst staining, and annexin V binding assay. ROS generation, mitochondrial membrane potential (DeltaPsi(m)), and intramitochondrial calcium were evaluated with flow cytometry. Translocation of cytochrome c and activation of apoptotic protein were analyzed with cell fractionating and Western blotting. Intracellular calcium was measured with a spectrofluorometer. Bradykinin enhanced cellular LDH release, apoptosis, generation of superoxide, and hydrogen peroxide induced by ATP depletion. Bradykinin also enhanced the loss of DeltaPsi(m), translocation of cytochrome c into cytosol, and activation of apoptotic protein. The intracellular/mitochondrial calcium was higher in bradykinin-treated cells. All these effects were reversed by coadministration with bradykinin B2 receptor (B2R) antagonist. Besides, blocking the phospholipase C (PLC) could reverse the synergistic effect of bradykinin with ATP depletion on ROS generation, mitochondrial damage, accumulation of intracellular/mitochondrial calcium, and apoptosis. Activation of B2R aggravates ROS generation, mitochondrial damage, and cell death induced by ATP depletion. These effects may act through the PLC-Ca(2+) signaling pathway.