Heat shock protein 60 or 70 activates nitric-oxide synthase (NOS) I- and inhibits NOS II-associated signaling and depresses the mitochondrial apoptotic cascade during brain stem death

The cellular and molecular basis of brain stem death remains an enigma. As the origin of a "life-and-death" signal that reflects the progression toward brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate for mechanistic delineation of this phenomenon. Here, we evaluated the hypothesis that heat shock proteins (HSPs) play a neuroprotective role in the RVLM during brain stem death and delineated the underlying mechanisms, using a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult. In Sprague-Dawley rats, proteomic, Western blot, and real-time PCR analyses demonstrated that Mev induced de novo synthesis of HSP60 or HSP70 in the RVLM without affecting HSP90 level. Loss-of-function manipulations of HSP60 or HSP70 in the RVLM using anti-serum or antisense oligonucleotide potentiated Mev-elicited cardiovascular depression alongside reduced nitric-oxide synthase (NOS) I/protein kinase G signaling, enhanced NOS II/peroxynitrite cascade, intensified nucleosomal DNA fragmentation, elevated cytoplasmic histone-associated DNA fragments or activated caspase-3, and augmented the cytochrome c/caspase-3 cascade of apoptotic signaling in the RVLM. Co-immunoprecipitation experiments further revealed a progressive increase in the complex formed between HSP60 and mitochondrial or cytosolic Bax or mitochondrial Bcl-2 during Mev intoxication, alongside a dissociation of the cytosolic HSP60-Bcl-2 complex. We conclude that HSP60 and HSP70 confer neuroprotection against Mev intoxication by ameliorating cardiovascular depression via an anti-apoptotic action in the RVLM. The possible underlying intracellular processes include enhancing NOS I/protein kinase G signaling and inhibiting the NOS II/peroxynitrite cascade. In addition, HSP60 exerts its effects against apoptosis by blunting Mev-induced activation of the Bax/cytochrome c/caspase-3 cascade.     

NADPH oxidase- and mitochondrion-derived superoxide at rostral ventrolateral medulla in endotoxin-induced cardiovascular depression

We evaluated the contribution of superoxide anion (O2*-) generated by NADPH oxidase or mitochondria in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for arterial pressure maintenance are located, on cardiovascular depression induced by inducible nitric oxide synthase-derived NO after Escherichia coli lipopolysaccharide (LPS) treatment. In Sprague-Dawley rats maintained under propofol anesthesia, microinjection of LPS bilaterally into the RVLM induced progressive hypotension, bradycardia, and reduction in sympathetic vasomotor outflow over our 240-min observation period. This was accompanied by an increase in O2*- production (60-240 min) in the RVLM, alongside phosphorylation of p47(phox) or p67(phox), upregulation of gp91(phox) or p47(phox) protein, and increase in Rac-1 or NADPH oxidase activity (60-120 min), and a depression of mitochondrial respiratory enzyme activity (120-240 min). Whereas inhibition of NADPH oxidase or knockdown of the gp91(phox) or p47(phox) gene blunted the early phase (60-150 min), coenzyme Q10 or mitochondrial K(ATP) channel inhibitor antagonized the delayed phase (120-240 min) of LPS-induced increase in O2*- production in RVLM and cardiovascular depression. We conclude that, whereas NADPH oxidase-derived O2*- in RVLM participates predominantly in the early phase, O2*- generated by depression in mitochondrial respiratory enzyme activity or opening of mitoK(ATP) channels mediates the delayed phase of LPS-induced cardiovascular depression.     

Hypoxia sensitizes cells to nitric oxide-induced apoptosis

Nitric oxide (NO) can induce apoptosis in a variety of cell types. A non-toxic concentration of nitric oxide under normal oxygen conditions triggered cell death under hypoxic conditions (1.5% O(2)) in fibroblasts. Nitric oxide administered during hypoxia induced the release of cytochrome c, caspase-9 activation, and the loss of mitochondrial membrane potential followed by DNA fragmentation and lactate dehydrogenase release (markers of cell death). Bcl-X(L) protected cells from nitric oxide-induced apoptosis during hypoxia by preventing the release of cytochrome c, caspase-9 activation, and by maintaining a mitochondrial membrane potential. Murine embryonic fibroblasts from bax(-/-) bak(-/-) mice exposed to nitric oxide during hypoxia did not die, indicating that pro-apoptotic Bcl-2 family members are required for NO-induced apoptosis during hypoxia. The nitric oxide-induced cell death during hypoxia was independent of cGMP and peroxynitrite. Cells devoid of mitochondrial DNA (rho secondary-cells) lack a functional electron transport chain and were resistant to nitric oxide-induced cell death during hypoxia, suggesting that a functional electron transport chain is required for nitric oxide-induced apoptosis during hypoxia.     

Nitric oxide and cell signaling pathways in mitochondrial-dependent apoptosis

Nitric oxide, generated by endogenous nitric oxide synthases or nitric oxide donors, can promote or prevent apoptosis induced by diverse pro-apoptotic stimuli in cell culture models. Both mitochondrial-dependent and -independent apoptotic signaling pathways mediate this dichotomous cellular response to nitric oxide. The molecular mechanisms behind these effects are complex and involve a number of nitrogen oxide-related species that are more reactive than nitric oxide itself. The local cellular environment plays a dynamic role in determining the nature and concentration of these species. Important components of the microenvironment include: the cellular redox state, glutathione, transition metals and the presence of other oxygen- and nitrogen-centered radicals. In particular, redox-sensitive nitrosating species are favorably generated under physiological conditions and capable of modifying multiple cell signaling pathways through reversible S-nitrosation reactions. Cytochrome c release from mitochondria is an important mechanism for the activation of caspase-3 and the initiation of cell death in response to 'intrinsic' pro-apoptotic stimuli, including oxidative and nitrosative stress. In turn, caspases and mitogen associated protein kinases may modulate cytochrome c release through their effects on the Bcl-2 family of proteins. This review will focus on (i) the importance of the cellular environment in determining the fate of nitric oxide and (ii) the ability of S-nitrosation to regulate mitochondrial-dependent apoptosis at the level of mitochondrial bioenergetics, cytochrome c release, caspases, mitogen associated protein kinases, and the Bcl-2 family of proteins.     

Overexpression of SOD1 protects vulnerable motor neurons after spinal cord injury by attenuating mitochondrial cytochrome c release

Defective Cu,Zn-superoxide dismutase (SOD1) is responsible for some types of amyotrophic lateral sclerosis, and ventral horn motor neurons (VMN) have been shown to die through a mitochondria-dependent apoptotic pathway after chronic exposure to high levels of reactive oxygen species (ROS). VMN are also selectively vulnerable to mild spinal cord injury (SCI); however, the involvement of SOD1, ROS, and apoptosis in their death has not been clarified. Mild compression SCI was induced in SOD1-overexpressing transgenic rats and wild-type littermates. Superoxide production, mitochondrial release of cytochrome c, and activation of caspase-9 were examined, and apoptotic DNA injury was also characterized. In the wild-type animals, increased superoxide production, mitochondrial release of cytochrome c, and cleaved caspase-9 were observed exclusively in VMN after SCI. Subsequently, a majority of VMN (75%) selectively underwent delayed apoptotic cell death. Transgenic animals showed less superoxide production, mitochondrial cytochrome c release, and caspase-9 activation, resulting in death of only 45% of the VMN. These results suggest that the ROS-initiated mitochondrial signaling pathway possibly plays a pivotal role in apoptotic VMN death after SCI and that increased levels of SOD1 in VMN reduce oxidative stress, thereby attenuating the activation of the pathway and delayed cell death.     

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis is dependent on activation of cysteine and serine proteases

We examined the role of caspases and serine protease(s) in cell death induced by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). After incubation of adenocarcinoma cells with TRAIL, caspase-3, -8 were activated and the cleavage of Bid induced the release of cytochrome c, from the mitochondria to the cytosol. Tetrapeptide inhibitors of caspase-1, -2, -3, and -8 suppressed DNA fragmentation and attenuated the release of cytochrome c, whereas inhibitors of caspase-5 did not. Interestingly, the general serine protease(s) inhibitor 4-(2-aminoethyl)benzylsulfonyl fluoride (AEBSF) resulted in the arrest of apoptosis. However, the AEBSF did not prevent the release of mitochondrial cytochrome c during TRAIL-induced apoptosis. From these results, we postulate that serine protease(s) may be involved in post-mitochondrial apoptotic events, that lead to the activation of the initiator, caspase-9.     

Dysfunction of the mitochondrial respiratory chain in the rostral ventrolateral medulla during experimental endotoxemia in the rat

We investigated the functional changes in the mitochondrial respiratory chain at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor tone, in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration ofEscherichia coli lipopolysaccharide (LPS; 30 mg/kg) induced a reduction (Phase I), followed by an augmentation (Phase II) and a secondary decrease (Phase III) in the power density of vasomotor components (0–0.8 Hz) in systemic arterial pressure signals. LPS also elicited progressive hypotension, and death ensued within 4 h. Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochromec reductase (Complexes I + III) and cytochromec oxidase (Complex IV) in the RVLM during all three phases of endotoxemia. On the other hand, the activity of succinate cytochromec reductase (Complexes II + III) remained unaltered. We conclude that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM, whose neuronal activity is intimately related to the death process, is closely associated with fatal endotoxemia in the rat.     

Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

Treatment of elicited peritoneal macrophages or the macrophage cell line RAW 264.7 with high concentrations of nitric oxide donors is followed by apoptotic cell death. Analysis of the changes in the mitochondrial transmembrane potential (DeltaPsi(m)) with specific fluorescent probes showed a rapid and persistent increase of DeltaPsi(m), a potential that usually decreases in cells undergoing apoptosis through mitochondrial-dependent mechanisms. Using confocal microscopy, the release of cytochrome c from the mitochondria to the cytosol was characterized as an early event preceding the rise of DeltaPsi(m). The cytochrome c from cells treated with nitric oxide donors was modified chemically, probably through the formation of nitrotyrosine residues, suggesting the synthesis of peroxynitrite in the mitochondria. These results indicate that nitric oxide-dependent apoptosis in macrophages occurs in the presence of a sustained increase of DeltaPsi(m), and that the chemical modification and release of cytochrome c from the mitochondria precede the changes of DeltaPsi(m).-Hortelano, S., Alvarez, A. M., Boscá, L. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.     

Early release of arachidonic acid prevents an otherwise immediate formation of toxic levels of peroxynitrite in astrocytes stimulated with lipopolysaccharide/interferon-gamma

Addition of bacterial lipopolysaccharides (LPS) and interferon-gamma (IFN-gamma) to rat astrocytes in primary culture promotes an early release of arachidonic acid (ARA) associated with an immediate inhibition of neuronal nitric oxide synthase (nNOS). Preventing the release of constitutive nitric oxide (NO) is indeed critical for activation of the nuclear factor kappa B, and for the expression of inducible nitric oxide synthase responsible for the formation of large amounts of NO. LPS/IFN-gamma also promotes an early release of superoxide, via activation of NADPH oxidase, but the generation of peroxynitrite (ONOO-) is prevented by the different timing of superoxide (minutes) and NO (hours) formation. Upstream inhibition of the ARA-dependent nNOS inhibitory signaling, however, caused the parallel release of superoxide and constitutive NO, thereby leading to formation of ONOO- levels triggering loss of ATP and mitochondrial membrane potential followed by the mitochondrial release of cytochrome c, activation of caspase 3 and morphological evidence of apoptosis. Nanomolar levels of exogenous ARA prevented all these events via inhibition of early ONOO- formation. Thus, the ARA-dependent nNOS inhibition observed in astrocytes exposed to pro-inflammatory stimuli, as LPS/IFN-gamma, is critical for both the expression of nuclear factor kappa B-dependent genes and for survival.     

Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVADfmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.     

Phosphorylation of p38 MAPK induced by oxidative stress is linked to activation of both caspase-8- and -9-mediated apoptotic pathways in dopaminergic neurons

We evaluated the contribution of p38 mitogen-activated protein kinase and the events upstream/downstream of p38 leading to dopaminergic neuronal death. We utilized MN9D cells and primary cultures of mesencephalic neurons treated with 6-hydroxydopamine. Phosphorylation of p38 preceded apoptosis and was sustained in 6-hydroxydopamine-treated MN9D cells. Co-treatment with PD169316 (an inhibitor of p38) or expression of a dominant negative p38 was neuroprotective in death induced by 6-hydroxydopamine. The superoxide dismutase mimetic and the nitric oxide chelator blocked 6-hydroxydopamine-induced phosphorylation of p38, suggesting a role for superoxide anion and nitric oxide in eliciting a neurotoxic signal by activating p38. Following 6-hydroxydopamine treatment, inhibition of p38 prevented both caspase-8- and -9-mediated apoptotic pathways as well as generation of truncated Bid. Consequently, 6-hydroxydopamine-induced cell death was rescued by blockading activation of caspase-8 and -9. In primary cultures of mesencephalic neurons, the phosphorylation of p38 similarly appeared in tyrosine hydroxylase-positive, dopaminergic neurons after 6-hydroxydopamine treatment. This neurotoxin-induced phosphorylation of p38 was inhibited in the presence of superoxide dismutase mimetic or nitric oxide chelator. Co-treatment with PD169316 deterred 6-hydroxydopamine-induced loss of dopaminergic neurons and activation of caspase-3 in these neurons. Furthermore, inhibition of caspase-8 and -9 significantly rescued 6-hydroxydopamine-induced loss of dopaminergic neurons. Taken together, our data suggest that superoxide anion and nitric oxide induced by 6-hydroxydopamine initiate the p38 signal pathway leading to activation of both mitochondrial and extramitochondrial apoptotic pathways in our culture models of Parkinson's disease.     

Lack of Apaf-1 expression confers resistance to cytochrome c-driven apoptosis in cardiomyocytes

Apoptosis plays a role in cardiomyocyte death in several cardiovascular disorders. Here, we show that primary postnatal cardiomyocytes did not die upon activation of the intrinsic (cytochrome c-dependent) apoptotic pathway. Release of cytochrome c from mitochondria to the cytosol occurred, but did not activate the effector phase of apoptosis. Myocardial cells did not express apoptotic protease-activating factor-1 (Apaf-1), the allosteric activator of caspase-9 acting downstream of cytochrome c release. Forced expression of Apaf-1 restored the competence to complete the cytochrome c-induced apoptotic program and this effect was prevented by overexpression of Bcl-X(L). However, cardiomyocytes were able to enter the apoptotic program when it was initiated by activation of death receptors, as observed during serum deprivation and metabolic inhibition. Our results indicate that regulation of Apaf-1 expression may be a new regulatory mechanism developed in postmitotic cells in order to prevent irreversible commitment to die after release of cytochrome c.     

c-Abl Tyrosine Kinase Mediates Neurotoxic Prion Peptide-Induced Neuronal Apoptosis via Regulating Mitochondrial Homeostasis

Prion diseases are neurodegenerative disorders characterized by the accumulation of a disease-associated prion protein and apoptotic neuronal death. Previous studies indicated that the ubiquitous expression of c-Abl tyrosine kinase transduces a variety of extrinsic and intrinsic cellular signals. In this study, we demonstrated that a synthetic neurotoxic prion fragment (PrP106-126) activated c-Abl tyrosine kinase, which in turn triggered the upregulation of MST1 and BIM, suggesting the activation of the c-Abl-BIM signaling pathway. The peptide fragment was found to result in cell death via mitochondrial dysfunction in neuron cultures. Knockdown of c-Abl using small interfering RNA protected neuronal cells from PrP106-126-induced mitochondrial dysfunction, production of reactive oxygen species, and apoptotic events inducing translocation of Bax to the mitochondria, cytochrome c release into the cytosol, and activation of caspase-9 and caspase-3. Blocking the c-Abl tyrosine kinase also prevented neuronal cells from PrP106-126-induced apoptotic morphological changes. This is the first study reporting that c-Abl tyrosine kinase as a novel upstream activator of MST1 and BIM plays an important role in prion-induced neuron apoptosis via mitochondrial dysfunction. Our findings suggest that c-Abl tyrosine kinase is a potential therapeutic target for prion disease.     

Mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury: protection by cyclosporin A

Hyperoxia is known to induce extensive alveolar cell death by still poorly defined mechanisms. In this study, the mitochondria-dependent cell death pathway was explored during hyperoxia-induced lung injury in mice. We observed a progressive release of cytochrome c from the mitochondria into the cytosol of alveolar cells. This release was accompanied by the translocation of the proapoptotic protein Bax from cytosol to mitochondria without detectable activation of caspase-3. As cytochrome c release can be induced by mitochondrial membrane alteration and permeability transition (MPT), mice were treated with cyclosporin A, which specifically inhibits MPT. Cyclosporin A treatment prevented mitochondrial release of cytochrome c during hyperoxia and concomitantly preserved mitochondria from extensive swelling and crista disorganization, as assessed by electron microscopy analysis of alveolar epithelial cells. These morphological and biochemical observations correlated with decreased lung tissue damage, as evaluated by morphological score and lung weight. In conclusion, mitochondrial damage and cytochrome c release are important linked events in hyperoxia-induced lung injury and can be efficiently blocked by cyclosporin A.     

Estrogen-induced spermatogenic cell apoptosis occurs via the mitochondrial pathway: role of superoxide and nitric oxide

The detrimental effects of estrogen on testicular function provide a conceptual basis to examine the speculative link between increased exposure to estrogens and spermatogenic cell death. Using an in vitro model, we provide an understanding of the events leading to estrogen-induced apoptosis in cells of spermatogenic lineage. Early events associated with estrogen exposure were up-regulation of FasL and increased generation of H(2)O(2), superoxide, and nitric oxide. The ability of anti-FasL antibodies to prevent several downstream biochemical changes and cell death induced by 17beta-estradiol substantiates the involvement of the cell death receptor pathway. Evidence for the amplification of the death-inducing signals through mitochondria was obtained from the transient mitochondrial hyperpolarization observed after estradiol exposure resulting in cytochrome c release. A combination of nitric oxide and superoxide but not H(2)O(2) was responsible for the mitochondrial hyperpolarization. Mn(III) tetrakis(4-benzoic acid)porphyrin chloride, an intracellular peroxynitrite scavenger, was able to reduce mitochondrial hyperpolarization and cell death. Although nitric oxide augmentation occurred through an increase in the expression of inducible nitric-oxide synthase, superoxide up-regulation was a product of estradiol metabolism. All of the above changes were mediated through an estrogen receptor-based mechanism because tamoxifen, the estrogen receptor modulator, was able to rescue the cells from estrogen-induced alterations. This study establishes the importance of the independent capability of cells of the spermatogenic lineage to respond to estrogens and most importantly suggests that low dose estrogens can potentially cause severe spermatogenic cellular dysfunction leading to impaired fertility even without interference of the hypothalamo-hypophyseal axis.     

Apoptosis: checkpoint at the mitochondrial frontier.

Apoptosis, an evolutionarily conserved form of cell death, requires a regulated program. Central to the apoptotic program is a family of cysteine proteases, known as caspases, that cleave a subset of cellular proteins, resulting in the stereotypic morphological changes of apoptotic cell death. In living cells caspases are present as inactive zymogens and become activated in response to pro-apoptotic stimuli. Mitochondria participate in the activation of caspases by releasing cytochrome c into the cytosol where it binds to the adaptor molecule Apaf-1 (apoptotic protease activating factor 1) and causes its oligomerization. This renders Apaf-1 competent to recruit and activate the cell death initiator caspase, pro-caspase-9. Once caspase-9 is activated, it cleaves and activates downstream cell death effector caspases. Bcl-2, an apoptosis inhibitor localized to mitochondrial outer membranes, prevents cytochrome c release, caspase activation and cell death. This review discusses recent advances on the role of mitochondria and cytochrome c in the central pathway leading to apoptotic cell death.     

Reovirus-induced apoptosis requires mitochondrial release of Smac/DIABLO and involves reduction of cellular inhibitor of apoptosis protein levels

Many viruses belonging to diverse viral families with differing structure and replication strategies induce apoptosis both in cultured cells in vitro and in tissues in vivo. Despite this fact, little is known about the specific cellular apoptotic pathways induced during viral infection. We have previously shown that reovirus-induced apoptosis of HEK cells is initiated by death receptor activation but requires augmentation by mitochondrial apoptotic pathways for its maximal expression. We now show that reovirus infection of HEK cells is associated with selective cytosolic release of the mitochondrial proapoptotic factors cytochrome c and Smac/DIABLO, but not the release of apoptosis-inducing factor. Release of these factors is not associated with loss of mitochondrial transmembrane potential and is blocked by overexpression of Bcl-2. Stable expression of caspase-9b, a dominant-negative form of caspase-9, blocks reovirus-induced caspase-9 activation but fails to significantly reduce activation of the key effector caspase, caspase-3. Smac/DIABLO enhances apoptosis through its action on cellular inhibitor of apoptosis proteins (IAPs). Reovirus infection is associated with selective down-regulation of cellular IAPs, including c-IAP1, XIAP, and survivin, effects that are blocked by Bcl-2 expression, establishing the dependence of IAP down-regulation on mitochondrial events. Taken together, these results are consistent with a model in which Smac/DIABLO-mediated inhibition of IAPs, rather than cytochrome c-mediated activation of caspase-9, is the key event responsible for mitochondrial augmentation of reovirus-induced apoptosis. These studies provide the first evidence for the association of Smac/DIABLO with virus-induced apoptosis.     

p38 MAP kinase mediates nitric oxide-induced apoptosis of neural progenitor cells

Neural progenitor cells (NPC) can proliferate, differentiate into neurons or glial cells, or undergo a form of programmed cell death called apoptosis. Although death of NPC occurs during development of the nervous system and in the adult, the underlying mechanisms are unknown. Here we show that nitric oxide (NO) can induce death of C17.2 NPC by a mechanism requiring activation of p38 MAP kinase, poly(ADP-ribose) polymerase, and caspase-3. Nitric oxide causes release of cytochrome c from mitochondria, and Bcl-2 protects the neural progenitor cells against nitric oxide-induced death, consistent with a pivotal role for mitochondrial changes in controlling the cell death process. Inhibition of p38 MAP kinase by SB203580 abolished NO-induced cell death, cytochrome c release, and activation of caspase-3, indicating that p38 activation serves as an upstream mediator in the cell death process. The anti-apoptotic protein Bcl-2 protected NPC against nitric oxide-induced apoptosis and suppressed activation of p38 MAP kinase. The ability of nitric oxide to trigger death of NPC by a mechanism involving p38 MAP kinase suggests that this diffusible gas may regulate NPC fate in physiological and pathological settings in which NO is produced.     

Endotoxin induces luteal cell apoptosis through the mitochondrial pathway

The effect of endotoxin (lipopolysacharide, LPS) exposure on luteal cells was studied using an in vitro cell culture system. Buffalo luteal cells were isolated from corpora lutea of the late luteal phase (days 14-16 post estrus) and exposed to various LPS doses (5, 10 and 100 microg/ml) for different time periods (6, 12, 18 or 24 h). The cultured cells were subsequently evaluated for oxidative stress (super oxide, nitric oxide, inducible nitric oxide synthase activity, reduced glutathione depletion and lipid peroxidation) and apoptotic markers (mitochondrial membrane potential, DNA fragmentation, apoptotic cells and cell viability). LPS exposure significantly increased the production of super oxide (P<0.05) and nitric oxide (P<0.01) and increased inducible nitric oxide synthase activity (P<0.01). LPS exposure further depleted reduced glutathione (P<0.05) levels and induced lipid peroxidation (P<0.05). LPS exposure also induced the loss of mitochondrial membrane potential (P<0.05), increased DNA fragmentation (P<0.01) and apoptosis (P<0.01) and decreased cell viability (P<0.01). LPS mediated apoptotic pathway in luteal cells was further characterized using a selected LPS dose (10 microg/ml). It was observed that LPS exposure induced mitochondrial translocation of proapoptotic protein Bax, increased the total Bad expression and down regulated the expression of antiapoptotic proteins Bcl2 and BclXL. LPS exposure further induced cytochrome c release and increased Caspase-9 (P<0.01) and Caspase-3 (P<0.01) activities. LPS exposure also inhibited luteal progesterone secretion (P<0.01). It was evident that the LPS mediated apoptotic effects could be prevented by the coincubation of luteal cells with mitochondrial permeability transition pore blocker Cyclosporine A, inducible nitric oxide synthase inhibitor N-[3-(aminomethyl)benzyl]acetamidine and oxidative stress scavenger N-acetyl cysteine. Our study clearly indicates that LPS induces oxidative stress mediated apoptosis in luteal cells through the mitochondrial pathway.     

Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia.

It has been shown that nitric oxide (NO), synthesized by the inducible NO synthase (iNOS) expressed in the diaphragm during endotoxemia, participates in the development of muscular contractile failure. The aim of the present study was to investigate whether this deleterious action of NO was related to its effects on cellular oxidative pathways. Rats were inoculated with E. coli lipopolysaccharide (LPS) or sterile saline solution (controls) and studied at 3 and 6 h after inoculation. iNOS protein and activity could be detected in the rat diaphragm as early as 3 h after LPS, with a sustained steady-state concentration of 0.5 microM NO in the muscle associated with increased detection of hydrogen peroxide (H(2)O(2)). In vitro, the same NO concentration produced a marked increase in H(2)O(2) production by isolated control diaphragm mitochondria, thus reflecting a higher intramitochondrial concentration of nondiffusible superoxide anion (O(2)(-.)). In a similar way, whole diaphragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H(2)O(2) production associated with uncoupling and decreased phosphorylating capacity. Simultaneous with the maximal impairment in respiration (6 h after LPS), nitration of mitochondrial proteins (a peroxynitrite footprint) was detected and diaphragmatic force was reduced. Functional mitochondrial abnormalities, nitration of mitochondrial proteins, and the decrease in force were significantly attenuated by administration of the NOS inhibitor L-NMMA. These results show that increased and sustained NO levels lead to a consecutive formation of O(2)(-.) that reacts with NO to form peroxynitrite, which in turn impairs mitochondrial function, which probably contributes to the impairment of muscle contractility. during endotoxemia.