Regulation of human male germ cell death by modulators of ATP production

The understanding of testicular physiology, pathology, and male fertility issues requires knowledge of male germ cell death and energy production. Here, we induced human male germ cell apoptosis (detected by Southern blot analysis of DNA fragmentation, TUNEL, activation of caspases-3 and -9, and electron microscopy) by incubating seminiferous tubule segments under hormone- and serum-free conditions. Inhibitors of complexes I to IV of mitochondrial respiration, exposure to anoxia, and inhibition of F0F1-ATPase (with oligomycin) decreased the ATP levels (analyzed by HPLC) and suppressed apoptosis at 4 h. Uncoupler 2,4-dinitrophenol (DNP) and oligomycin combination also suppressed death at 4 h, as did the DNP alone. Inhibition of glycolysis by 2-deoxyglucose neither suppressed nor further induced apoptosis nor altered the antiapoptotic effects of the mitochondrial inhibitors. Furthermore, Fas system activation did not modify the effects of mitochondrial modulators. After 24 h, delayed male germ cell apoptosis was observed despite the presence of the mitochondrial inhibitors. We conclude that the mitochondrial ATP production machinery plays an important role in regulating in vitro-induced primary pathways of human male germ apoptosis. The ATP synthesized by the F0F1-ATPase seems to be the crucial death regulator, rather than any of the complexes (I-IV) alone, the functional electron transport chain, or the membrane potential. We also conclude that there seem to be secondary pathways of human testicular cell apoptosis that do not require mitochondrial ATP production. The present study emphasizes the role of the main catabolic pathways in the complex network of regulating events of male germ cell life and death.     

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.     

Hypoxia-induced cell death in human malignant glioma cells: energy deprivation promotes decoupling of mitochondrial cytochrome c release from caspase processing and necrotic cell death

Hypoxia induces apoptosis in primary and transformed cells and in various tumor cell lines in vitro. In contrast, there is little apoptosis and predominant necrosis despite extensive hypoxia in human glioblastomas in vivo. We here characterize ultrastructural and biochemical features of cell death in LN-229, LN-18 and U87MG malignant glioma cells in a paradigm of hypoxia with partial glucose deprivation in vitro. Electron microscopic analysis of hypoxia-challenged glioma cells demonstrated early stages of apoptosis but predominant necrosis. ATP levels declined during hypoxia, but recovered with re-exposure to normoxic conditions unless hypoxia exceeded 8 h. Longer hypoxic exposure resulted in irreversible ATP depletion and delayed cell death. Hypoxia induced mitochondrial release of cytochrome c, but there was no cleavage of caspases 3, 7, 8 or 9, and no DNA fragmentation. Ectopic expression of BCL-XL conferred protection from hypoxia-induced cell death, whereas the overexpression of the antiapoptotic proteins X-linked-inhibitor-of-apoptosis-protein and cytokine response modifier-A had no effect. These findings suggest that glioma cells resist adverse effects of hypoxia until energy stores are depleted and then undergo necrosis rather than apoptosis because of energy deprivation.     

Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria

Nitric oxide (NO) can trigger either necrotic or apoptotic cell death. We have used PC12 cells to investigate the extent to which NO-induced cell death is mediated by mitochondria. Addition of NO donors, 1 mM S-nitroso-N-acetyl-DL-penicillamine (SNAP) or 1 mM diethylenetriamine-NO adduct (NOC-18), to PC12 cells resulted in a steady-state level of 1-3 microM: NO, rapid and almost complete inhibition of cellular respiration (within 1 min), and a rapid decrease in mitochondrial membrane potential within the cells. A 24-h incubation of PC12 cells with NO donors (SNAP or NOC-18) or specific inhibitors of mitochondrial respiration (myxothiazol, rotenone, or azide), in the absence of glucose, caused total ATP depletion and resulted in 80-100% necrosis. The presence of glucose almost completely prevented the decrease in ATP level and the increase in necrosis induced by the NO donors or mitochondrial inhibitors, suggesting that the NO-induced necrosis in the absence of glucose was due to the inhibition of mitochondrial respiration and subsequent ATP depletion. However, in the presence of glucose, NO donors and mitochondrial inhibitors induced apoptosis of PC12 cells as determined by nuclear morphology. The presence of apoptotic cells was prevented completely by benzyloxycarbonyl-Val-Ala-fluoromethyl ketone (a nonspecific caspase inhibitor), indicating that apoptosis was mediated by caspase activation. Indeed, both NO donors and mitochondrial inhibitors in PC12 cells caused the activation of caspase-3- and caspase-3-processing-like proteases. Caspase-1 activity was not activated. Cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) decreased the activity of caspase-3- and caspase-3-processing-like proteases after treatment with NO donors, but was not effective in the case of the mitochondrial inhibitors. The activation of caspases was accompanied by the release of cytochrome c from mitochondria into the cytosol, which was partially prevented by cyclosporin A in the case of NO donors. These results indicate that NO donors (SNAP or NOC-18) may trigger apoptosis in PC12 cells partially mediated by opening the mitochondrial permeability transition pores, release of cytochrome c, and subsequent caspase activation. NO-induced apoptosis is blocked completely in the absence of glucose, probably due to the lack of ATP. Our findings suggest that mitochondria may be involved in both types of cell death induced by NO donors: necrosis by respiratory inhibition and apoptosis by opening the permeability transition pore. Further, our results indicate that the mode of cell death (necrosis versus apoptosis) induced by either NO or mitochondrial inhibitors depends critically on the glycolytic capacity of the cell.     

Benzo(a)pyrene-induced mitochondrial dysfunction and cell death in p53-null Hep3B cells

Benzo(a)pyrene (BaP) has been shown to induce apoptosis and necrosis in various cell types. However, the effect of BaP on mitochondria function and p73, and their possible roles in BaP-induced cell death have not been well studied. This study focused on mitochondria-mediated cell death and the occurrence of p73 protein accumulation in BaP-treated human hepatoma Hep3B (p53-null) cells. We found that BaP (8, 16, 32 and 64μM) induced early necrosis at 12h and delayed apoptosis at 24h. BaP dramatically induced ethoxyresorufin-O-deethylase activity and led to significant increase in oxidative stress at early time points (6 and 12h). Necrotic cell death was concurrent with loss of mitochondrial membrane potential, decrease in the ATP level and activities of Na(+)/K(+)-ATPase and Ca(2+)/Mg(2+)-ATPase. However, these changes were reversed in the process of apoptosis. In addition, after BaP treatment, c-Jun N-terminal kinase (JNK) and Bax were activated during apoptosis and no change in p73 protein level was observed. These results revealed that the cells with mitochondria dysfunction and ATP depletion underwent necrosis at early time point and apoptosis afterward when they recovered from mitochondrial dysfunction and ATP depletion. Activation of JNK and Bax possibly contributed to BaP-induced apoptosis.     

Nitric oxide inhibition of mitochondrial respiration and its role in cell death

Nitric oxide (NO) or its derivatives (reactive nitrogen species, RNS) inhibit mitochondrial respiration in two different ways: (i) an acute, potent, and reversible inhibition of cytochrome oxidase by NO in competition with oxygen; and, (ii) irreversible inhibition of multiple sites by RNS. NO inhibition of respiration may impinge on cell death in several ways. Inhibition of respiration can cause necrosis and inhibit apoptosis due to ATP depletion, if glycolysis is also inhibited or is insufficient to compensate. Inhibition of neuronal respiration can result in excitotoxic death of neurons due to induced release of glutamate and activation of NMDA-type glutamate receptors. Inhibition of respiration may cause apoptosis in some cells, while inhibiting apoptosis in other cells, by mechanisms that are not clear. However, NO can induce (and inhibit) cell death by a variety of mechanisms unrelated to respiratory inhibition.     

Differential effects of bcl-2 on cell death triggered under ATP-depleting conditions

The intracellular ATP concentration decides on the onset of either apoptosis or necrosis in Jurkat cells exposed to death stimuli. Bcl-2 can block apoptotic demise, which occurs preferably under conditions of high cellular ATP levels. Here, we investigated the effects of Bcl-2 on the necrotic type of cell demise that prevails under conditions of energy loss. ATP levels were modulated by using mitochondrial inhibitors, such as rotenone or S-nitrosoglutathione, in medium either lacking glucose or supplemented with glucose to stimulate glycolytic ATP generation. Under conditions of ATP depletion, staurosporine (STS) induced >90% necrosis in vector control-transfected cells, whereas bcl-2-transfected cells were protected. Thus, the antiapoptotic protein Bcl-2 can reduce the overall amount of cell death in ATP-depleted cells regardless whether it occurs by apoptosis or necrosis. Cytochrome c release, normally preceding STS-induced necrosis, was also inhibited by Bcl-2. However, Bcl-2 did not prevent an initial STS-induced drop of the mitochondrial membrane potential (DeltaPsi(m)). Therefore, the mechanisms whereby Bcl-2 prevents cell death and favors retention of cytochrome c in the mitochondria require neither the maintenance of mitochondrial DeltaPsi nor the maintenance of normal ATP levels.     

Cigarette smoke-induced blockade of the mitochondrial respiratory chain switches lung epithelial cell apoptosis into necrosis

Increased lung cell apoptosis and necrosis occur in patients with chronic obstructive pulmonary disease (COPD). Mitochondria are crucially involved in the regulation of these cell death processes. Cigarette smoke is the main risk factor for development of COPD. We hypothesized that cigarette smoke disturbs mitochondrial function, thereby decreasing the capacity of mitochondria for ATP synthesis, leading to cellular necrosis. This hypothesis was tested in both human bronchial epithelial cells and isolated mitochondria. Cigarette smoke extract exposure resulted in a dose-dependent inhibition of complex I and II activities. This inhibition was accompanied by decreases in mitochondrial membrane potential, mitochondrial oxygen consumption, and production of ATP. Cigarette smoke extract abolished the staurosporin-induced caspase-3 and -7 activities and induced a switch from epithelial cell apoptosis into necrosis. Cigarette smoke induced mitochondrial dysfunction, with compounds of cigarette smoke acting as blocking agents of the mitochondrial respiratory chain; loss of ATP generation leading to cellular necrosis instead of apoptosis is a new pathophysiological concept of COPD development.     

Glutamate dehydrogenase requirement for apoptosis induced by aristolochic acid in renal tubular epithelial cells

Ingestion of aristolochic acids (AA) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adduct formation, is well-documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. Epithelial cell death is a critical characteristic of these pathological conditions. To elucidate the mechanisms of AA-induced cytotoxicity, we explored AA-interacting proteins in tubular epithelial cells (TEC). We found that AA interacts with a mitochondrial enzyme glutamate dehydrogenase (GDH) and moderately inhibits its activity. We report that AA induces cell death in GDH-knockdown TEC preferentially via non-apoptotic means, whereas in GDH-positive cells, death was executed by both the non-apoptotic and apoptotic mechanisms. Apoptosis is an energy-reliant process and demands higher adenosine 5′-triphosphate (ATP) consumption than does the non-apoptotic cell death. We found that, after AAI treatment, the ATP depletion is more pronounced in GDH-knockdown cells. When we reduced ATP in TEC cells by inhibition of glycolysis and mitochondrial respiration, cell death mode switched from apoptosis and necrosis to necrosis only. In addition, in cells incubated at low glucose and no glutamine conditions, oxaloacetate and pyruvate reduced AAI-induced apoptosis our data suggest that AAI-GDH interactions in TEC are critical for the induction of apoptosis by direct inhibition of GDH activity. AA binding may also induce changes in GDH conformation and promote interactions with other molecules or impair signaling by GDH metabolic products, leading to apoptosis.     

Mort programmée des cellules germinales testiculaires: causes et mécanismes mis en jeu

Spermatogenesis results from a balance between proliferation and apoptosis. An alteration in this balance could lead to testicular diseases such as testicular tumour or infertility. Apoptosis seem to be important in regulating the processes of spermatogenesis since 60 to 75% of germ cells do not reach the spermatozoa stage. The various molecules of the apoptotic cascade have been detected in rodent or human germ cells, such as effector caspases and upstream proteins from cell death receptor or mitochondrial pathways. One or several different pathways may be involved in the germ cell apoptotic process triggered physiologically, by hormonal deprivation, or by chemical or physical inducers. Finally, caspases appear to play a role in various testicular diseases (particularly infertility).     

Adenine nucleotide (ADP/ATP) translocase 3 participates in the tumor necrosis factor induced apoptosis of MCF-7 cells

Mitochondrial adenine nucleotide translocase (ANT) is believed to be a component or a regulatory component of the mitochondrial permeability transition pore (mtPTP), which controls mitochondrial permeability transition during apoptosis. However, the role of ANT in apoptosis is still uncertain, because hepatocytes isolated from ANT knockout and wild-type mice are equally sensitive to TNF- and Fas-induced apoptosis. In a screen for genes required for tumor necrosis factor alpha (TNF-alpha)-induced apoptosis in MCF-7 human breast cancer cells using retrovirus insertion-mediated random mutagenesis, we discovered that the ANT3 gene is involved in TNF-alpha-induced cell death in MCF-7 cells. We further found that ANT3 is selectively required for TNF- and oxidative stress-induced cell death in MCF-7 cells, but it is dispensable for cell death induced by several other inducers. This data supplements previous data obtained from ANT knockout studies, indicating that ANT is involved in some apoptotic processes. We found that the resistance to TNF-alpha-induced apoptosis observed in ANT3 mutant (ANT3(mut)) cells is associated with a deficiency in the regulation of the mitochondrial membrane potential and cytochrome c release. It is not related to intracellular ATP levels or survival pathways, supporting a previous model in which ANT regulates mtPTP. Our study provides genetic evidence supporting a role of ANT in apoptosis and suggests that the involvement of ANT in cell death is cell type- and stimulus-dependent.     

Nitric oxide promotes germ cell necrosis in the delayed phase after experimental testicular torsion of rat

The purpose of this study is to determine whether inducible nitric oxide synthase (iNOS) is involved in the pathogenesis of testicular ischemia-reperfusion (I/R) injury in association with germ cell death, through either necrosis or apoptosis. Western blot analysis showed that iNOS expression was markedly increased 1 h after ischemia, and was accompanied by a huge nitric oxide (NO) production, as measured by the Griess method, with a peak at 48 h of reperfusion. Immunohistochemistry showed that iNOS was expressed predominantly in the macrophage-like cells infiltrated in the interstitial tissues of the testis. Intraperitoneal injection of aminoguanidine (AMG) (400 mg/day), the inhibitor of iNOS, reduced NO production by 57.7% at 96 h of reperfusion. Calpain activation and proteolysis of alpha-fodrin induced by I/R were inhibited by AMG. Germ cell apoptosis was demonstrated by in situ TUNEL and DNA fragmentation on agarose gel electrophoresis. Germ cell apoptosis was maximally induced at 24 h of reperfusion, and was not inhibited by AMG. NO produced by iNOS in the delayed phase of reperfusion promoted alpha-fodrin proteolysis, which is closely associated with necrosis. Inducible NOS inhibition combined with calpain inhibition may improve impaired spermatogenesis after testicular torsion.     

Harpin-induced hypersensitive cell death is associated with altered mitochondrial functions in tobacco cells

Mitochondria play important roles in animal apoptosis and are implicated in salicylic acid (SA)-induced plant resistance to viral pathogens. In a previous study, we demonstrated that SA induces rapid inhibition of mitochondrial electron transport and oxidative phosphorylation in tobacco cells. In the present study, we report that plant programmed cell death induced during pathogen elicitor-induced hypersensitive response (HR) is also associated with altered mitochondrial functions. Harpin, an HR elicitor produced by Erwinia amylovora, induced inhibition of ATP synthesis in tobacco cell cultures. Inhibition of ATP synthesis occurred almost immediately after incubation with harpin and preceded hypersensitive cell death induced by the elicitor. Diphenylene iodonium, an inhibitor of the oxidative burst, did not block harpin-induced inhibition of ATP synthesis or cell death, suggesting that oxidative burst was not the direct cause for these two harpin-induced processes. Unlike SA, harpin had no significant effect on total respiratory O2 uptake of treated cells. However, respiration of harpin-treated tobacco cells became very sensitive to the alternative oxidase inhibitors salicyl-hydroxamic acid and n-propyl gallate. Thus, harpin treatment resulted in reduced capacity of mitochondrial cytochrome pathway electron transport, which could lead to the observed inhibition of ATP synthesis. Given the recently demonstrated roles of mitochondria in apoptosis, this rapid inhibition of mitochondrial functions may play a role in harpin-induced hypersensitive cell death.     

Aponecrosis: morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis

A rat fibroblastic cell line (rat-1/myc-ERtrade mark) was treated with different concentration of Antimycin A, a metabolic poison that affects mitochondrial respiratory chain complex III. The modes of cell death were analyzed by time-lapse videomicroscopy, in situ end-labeling (ISEL) technique, and ultrastructural analysis. Intracellular ATP levels were also measured in order to detect whether the energetic stores were determinant for the type of cell death. It was found that while apoptosis was the prevalent cell death in the fibroblasts treated with low doses, 100 or 200 microM Antimycin A, a new type of cell demise that shared dynamic, molecular, and morphological features with both apoptosis and necrosis represents the most common cell death when the cells were exposed to high doses, 300 or 400 microM, of the hypoxic stimulus. This new type of cell death has been chimerically termed aponecrosis. The inhibition of caspase 3, an enzyme critical for the apoptotic DNA degradation, caused a clear shift from aponecrosis to necrosis in the cell culture, suggesting that this new type of cell death could account for an incomplete execution of the apoptotic program and the following degeneration in necrosis. After being treated with higher doses, i.e., 1000 microM Antimycin A, almost all of the cells died by true necrosis. The analysis of the cellular energetic stores showed that the levels of ATP were a primary determinant in directing toward active cell death (apoptosis), aponecrosis, or necrosis. We conclude that chemically induced hypoxia produces different types of cell death depending on the intensity of the insult and on the ATP availability of the cell, and that the classic apoptosis and necrosis may represent only two extremes of a continuum of intermediate forms of cell demise.     

Ceramide-induced apoptosis of D283 medulloblastoma cells requires mitochondrial respiratory chain activity but occurs independently of caspases and is not sensitive to Bcl-xL overexpression

Ceramides are potent lipid second messengers that are involved in apoptotic and hypoxic/ischaemic neurone death. We investigated the role of mitochondria and the mitochondrial apoptosis pathway in ceramide-induced cell death using human D283 medulloblastoma cells with a reduced mitochondrial DNA copy number (rho- cells) and a corresponding defect in mitochondrial respiration. Treatment with the complex I inhibitor rotenone, C2- or C8-ceramide induced cell death in D283 control cells, while rho- cells were significantly protected. In contrast, activation of the mitochondrial apoptosis pathway by transient overexpression of the pro-apoptotic Bax protein or exposure to the kinase inhibitor staurosporine induced apoptosis to a similar extent in control and rho- cells. Overexpression of the antiapoptotic protein Bcl-xL failed to inhibit the toxic effect of C2-ceramide in D283 control cells, and no significant increase in caspase-3-like protease activity could be detected during the death process. Despite this, C2-ceramide induced significant chromatin condensation and cell shrinkage in D283 control cells, reminiscent of apoptosis. These morphological alterations were associated with the activation of calpains. Both apoptotic morphology and calpain activation were attenuated in rho- cells. Our data indicate that the apoptosis-inducing effect of C2-ceramide may require mitochondrial respiratory chain activity and can occur independently of the mitochondrial apoptosis pathway, but involves the activation of calpains.     

Ceramide-induced formation of ROS and ATP depletion trigger necrosis in lymphoid cells

In lymphocytes, Fas activation leads to both apoptosis and necrosis, whereby the latter form of cell death is linked to delayed production of endogenous ceramide and is mimicked by exogenous administration of long- and short-chain ceramides. Here molecular events associated with noncanonical necrotic cell death downstream of ceramide were investigated in A20 B lymphoma and Jurkat T cells. Cell-permeable, C6-ceramide (C6), but not dihydro-C6-ceramide (DH-C6), induced necrosis in a time- and dose-dependent fashion. Rapid formation of reactive oxygen species (ROS) within 30 min of C6 addition detected by a dihydrorhodamine fluorescence assay, as well as by electron spin resonance, was accompanied by loss of mitochondrial membrane potential. The presence of N-acetylcysteine or ROS scavengers like Tiron, but not Trolox, attenuated ceramide-induced necrosis. Alternatively, adenovirus-mediated expression of catalase in A20 cells also attenuated cell necrosis but not apoptosis. Necrotic cell death observed following C6 exposure was associated with a pronounced decrease in ATP levels and Tiron significantly delayed ATP depletion in both A20 and Jurkat cells. Thus, apoptotic and necrotic death induced by ceramide in lymphocytes occurs via distinct mechanisms. Furthermore, ceramide-induced necrotic cell death is linked here to loss of mitochondrial membrane potential, production of ROS, and intracellular ATP depletion.     

Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling

Death ligands not only induce apoptosis but can also trigger necrosis with distinct biochemical and morphological features. We recently showed that in L929 cells CD95 ligation induces apoptosis, whereas TNF elicits necrosis. Treatment with anti-CD95 resulted in typical apoptosis characterized by caspase activation and DNA fragmentation. These events were barely induced by TNF, although TNF triggered cell death to a similar extent as CD95. Surprisingly, whereas the caspase inhibitor zVAD prevented CD95-mediated apoptosis, it potentiated TNF-induced necrosis. Cotreatment with TNF and zVAD was characterized by ATP depletion and accelerated necrosis. To investigate the mechanisms underlying TNF-induced cell death and its potentiation by zVAD, we examined the role of poly(ADP-ribose)polymerase-1 (PARP-1). TNF but not CD95 mediated PARP activation, whereas a PARP inhibitor suppressed TNF-induced necrosis and the sensitizing effect of zVAD. In addition, fibroblasts expressing a noncleavable PARP-1 mutant were more sensitive to TNF than wild-type cells. Our results indicate that TNF induces PARP activation leading to ATP depletion and subsequent necrosis. In contrast, in CD95-mediated apoptosis caspases cause PARP-1 cleavage and thereby maintain ATP levels. Because ATP is required for apoptosis, we suggest that PARP-1 cleavage functions as a molecular switch between apoptotic and necrotic modes of death receptor-induced cell death.     

Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria

Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in “reverse” mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.     

Inhibition of calpain but not caspase protects the testis against injury after experimental testicular torsion of rat

Testicular torsion requires emergent release of the twisted spermatic cord. Ischemia/reperfusion (I/R) plays an important role in its pathogenesis, and recent data suggest that germ cells undergo apoptosis during I/R. In a model of torsion/detorsion (i.e., I/R) of the rat testis, involvement of calpain and caspase in necrotic and apoptotic cell death was examined. After 1 h of ischemia followed by 0, 0.5, 1, 6, or 24 h of reperfusion, the germ cells positively stained with in situ TUNEL, and DNA fragmentation, activation of caspase-3, and proteolysis of caspase substrates increased with time of reperfusion, demonstrating apoptosis. In addition, m-calpain activation and proteolysis of alpha-fodrin were increased during reperfusion, and its activation is thought to be involved in the necrosis. A calpain inhibitor, acety-leucyl-leucyl-norleucinal, inhibited the phenomena associated with apoptosis and necrosis induced by I/R, although a caspase inhibitor, Z-Val-Ala-Asp-fluoromethlyketone, only inhibited apoptotic changes. The inhibition of calpain but not caspase ameliorated the injury after 60 days of reperfusion following 1 h of ischemia. The calpain inhibitor injected just before reperfusion effectively suppressed alpha-fodrin proteolysis, suggesting its usefulness in the treatment of testicular torsion.