Chemical hypoxia triggers apoptosis of cultured neonatal rat cardiac myocytes: Modulation by calcium-regulated proteases and protein kinases

Myocardial infarctions and stroke arise primarily as a result of hypoxia/ischemia-induced cell injury. However, the molecular mechanism of cardiac cell death due to hypoxia has not been elucidated. We showed here that chemical hypoxia induced by 1 mM azide triggered apoptosis of isolated neonatal rat ventricular cardiac myocytes but had no effect on cardiac fibroblasts. The azide-induced cardiomyocyte apoptosis could be characterized by a reversible initiation phase (0-6 h after azide exposure) during which cytosolic ATP levels remained little affected. This was followed by an irreversible execution phase (12-18 h) exhibiting prominent internucleosomal DNA fragmentation, cell membrane leakage, mitochondrial dysfunction, and increased calpain messenger RNA. Blocking extracellular calcium influx or intracellular calcium release was each effective in suppressing myocyte apoptosis. Cell death was also found to be mediated by calcium sensitive signal transduction events based on the use of specific antagonists. Consistent with the induction of calpain expression during apoptosis, blocking de novo protein synthesis and calpain activity inhibited cell death. These regulatory features coupled with the ease of the cell system suggest that the myocyte apoptosis model described here should be useful in the study of events leading to the demise of the myocardium.     

Protection of mature oligodendrocytes by inhibitors of caspases and calpains

Mature mouse oligodendrocytes (OLs) are susceptible to death in demyelinating diseases such as multiple sclerosis and in brain injury following neurotrauma, ischemia, or stroke. To understand mechanisms leading to death of mature OLs and develop strategies for protection, we utilized cultures of mature mouse OLs to investigate the role of caspases and calpains in OL cell death mediated by different mechanisms. The agents used were (i) staurosporine, which induces apoptotic death via inhibition of protein kinases; (ii) kainate, which activates non-NMDA glutamate receptors; (iii) thapsigargin, which releases intracellular calcium stores; and (iv) SNAP, which releases active NO species and causes necrotic cell death. Inhibitors blocking primary effector caspases (including caspase 3), the FAS (death receptor)-mediated initiator caspases (including caspase 8), and stress-induced caspases (including caspase 9), were tested for their protective effects. Inhibition of caspases 3, 8, and 9 each robustly protected OLs following insult with staurosporine, thapsigargin, or kainate when added at optimal times. The time of addition of the inhibitors for maximal protection varied with the agent, from 1 h of preincubation before addition of staurosporine to 6 h after addition of kainate. Much less protection was seen for the NO generator SNAP under any condition. The role of calcium in OL death in each model was investigated by chelating extracellular Ca⁺⁺ with EGTA, and by inhibiting the Ca⁺⁺-activated calpain proteases. Calcium chelation did not protect against staurosporine, but decreased OL death initiated by kainate, thapsigargin, or NO. The calpain inhibitors PD150606 and calpain inhibitor I protected from cell death initiated by staurosporine, kainate, and thapsigargin, but not from cell death initiated by the NO donor SNAP.     

Calpain-mediated proteolytic cleavage of troponin I induced by hypoxia or metabolic inhibition in cultured neonatal cardiomyocytes

While ischemic damage to myofibrillar proteins is thought to be responsible in part for depressed cardiac function, the relation between myofilament protein breakdown and chronic hypoxia has not been defined. We previously characterized a chemical hypoxia model of neonatal cardiomyocytes mediated by 1 mM azide that exhibits features of calpain activation (Mol Cell Biochem 178:141-149, 1998). We here show that both hypoxia and azide-mediated metabolic inhibition induced heme oxygenase-1 expression, and caused cell death associated with lipid peroxidation. While blocking calcium influx or inhibiting calpain activity efficiently attenuated hypoxia-induced cell injury, it failed to prevent cell injury caused by adenoviral overexpression of the tumor suppressor protein p53. Inhibitors of caspases, on the other hand, suppressed cell injury caused by p53 overexpression. Hypoxia caused selective cleavage of troponin I (TnI), which could be suppressed by either nifedipine or calpeptin. Other myofilament proteins such as troponin T, myosin heavy chain, and actin appeared to remain largely intact. p53-mediated cell injury exhibited proteolysis of the caspase protein substrate lamin B without appreciable breakdown of TnI. We suggest that calpain-induced TnI breakdown may constitute a unique biochemical marker associated with chronically hypoxic cardiomyocytes.     

Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

Glucose deprivation and chemical hypoxia: neuroprotection by P2 receptor antagonists

In this work we investigate cell survival after glucose deprivation and/or chemical hypoxia and we analyse the neuroprotective properties of selected antagonists of P2 ATP receptors. We find that in rat cerebellar granule neurones, the antagonist basilen blue prevents neuronal death under hypoglycaemia. Basilen blue acts through a wide temporal range and it retains its efficacy under chemically induced hypoxic conditions, in the presence of the respiratory inhibitors of mitochondria electron transport chain complexes II (3-nitropropionic acid) and III (antimycin A). In spite of the presence of these compounds, basilen blue maintains normal intracellular ATP levels. It furthermore prevents neuronal death caused by agents blocking the mitochondrial calcium uptake (ruthenium red) or discharging the mitochondrial membrane potential (carbonyl cyanide m-chlorophenylhydrazone). Inhibition of poly (ADP-ribose) polymerase, modulation of the enzyme GAPDH and mitochondrial transport of mono-carboxylic acids are not conceivable targets for the action of basilen blue. Survival is sustained by basilen blue also in CNS primary cultures from hippocampus and in PNS sympathetic-like neurones. Partial neuroprotection is furthermore provided by three additional P2 receptor antagonists: suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium and 4,4'-diisothiocyanatostilbene-2,2'disulphonic acid. Our data suggest the exploitation of selected P2 receptor antagonists as potential neuroprotective agents.     

The Ability of Diphenylpiperazines to Prevent Neuronal Death in Dorsal Root Ganglion Neurons In Vitro After Nerve Growth Factor Deprivation and In Vivo After Axotomy

Abstract: The mechanism of neuroprotection by the calcium channel antagonist flunarizine against neuronal death is unknown. We investigated the ability of other calcium channel antagonists (cinnarizine, nimodipine, nicardipine, diltiazem, and verapamil), calmodulin antagonists, and calpain inhibitors to prevent neuronal death in rat dorsal root ganglion neurons in vitro after nerve growth factor (NGF) deprivation and the ability of cinnarizine and diltiazem to protect in vivo after axotomy. In vitro, only neurons treated with cinnarizine or flunarizine were protected from death after withdrawal. In vivo, cinnarizine, but not diltiazem, protected dorsal root ganglion neurons in rats after unilateral sciatic nerve crush. Intracellular calcium concentration ([Ca²⁺],) was evaluated with fura 2 after NGF deprivation In vitro. Neurons "committed to die" 24 h after NGF deprivation displayed a decline in [Ca^a+], before visible morphological deterioration consistent with cell death. The influx of extracellular calcium was not necessary to produce neuronal death. Neurons deprived of NGF gradually lost the ability to respond to elevated external potassium with an increase in [Ca²⁺], during the first 24 h after trophic factor deprivation. After 24 h, neurons deprived of NGF could not be rescued by readministration of NGF. Neurons protected from cell death with diphenylpiperazines maintained their response to high external potassium, suggesting continued membrane integrity. We speculate that diphenylpiperazines may protect sensory neurons via an unknown mechanism that stabilizes cell membranes.     

Death effector activation in the subventricular zone subsequent to perinatal hypoxia/ischemia

Perinatal hypoxia/ischemia (H/I) is the leading cause of neurological injury resulting from birth complications and pre-maturity. Our studies have demonstrated that this injury depletes the subventricular zone (SVZ) of progenitors. In this study, we sought to reveal which cell death pathways are activated within these progenitors after H/I. We found that calpain activity is detected as early as 4 h of reperfusion and is sustained for 48 h, while caspase 3 activation does not occur until 8 h and peaks at 24 h post-insult. Activated calpains and caspase 3 co-localized within precursors situated in the lateral aspects of the SVZ (which coincides with progenitor cell death), whereas neither enzyme was activated in the medial SVZ (which harbors the neural stem cells that are resilient to this insult). These studies reveal targets for neuroprotective agents to protect precursors from cell death towards the goal of restoring normal brain development after H/I.     

Involvement of calpain in hypoxia-induced damage in rat retina in vitro

Our previous study suggested that calpain isoforms played an important role in retinal ganglion cell death induced by ischemia-reperfusion in rats [Curr. Eye Res. 21 (2000) 571]. The purpose of the present study was to further establish the direct involvement of calpain in hypoxia-induced damage by administering calpain inhibitor SJA6017 to oxygen-starved, cultured retinas. Retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. To induce a hypoxic condition, retinas were incubated with 95% N2/5% CO2. Leakage of LDH in the medium was measured to assess retinal cell damage. Activation of calpain and proteolysis of calpain substrate alpha-spectrin were analyzed by casein zymography and immunoblotting. Large amounts of LDH leaked into the medium from retinas under hypoxic conditions for 12 h, and SJA6017 significantly reduced LDH leakage. Caseinolytic activity of mu- and m-calpains decreased with hypoxia for 5 and 12 h, suggesting calpain activation followed by autolytic degradation. SJA6017 partially inhibited decreased calpain activities. Proteolysis of 230 kDa alpha-spectrin to 150 and 145 kDa breakdown products was observed in retinas with hypoxia. SJA6017 completely inhibited production of the 145 kDa breakdown product and partially inhibited production of the 150 kDa breakdown product. These results confirm the direct involvement of calpains in retinal cell damage induced by hypoxia in vitro.     

Influence of cytosolic and mitochondrial Ca2+, ATP,mitochondrial membrane potential,and calpain activity on the mechanism of neuron death induced by 3-nitropropionic acid

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, induces both rapid necrotic and slow apoptotic death in rat hippocampal neurons. Low levels of extracellular glutamate (10 microM) shift the 3NP-induced cell death mechanism to necrosis, while NMDA receptor blockade results in predominantly apoptotic death. In this study, we examined the 3NP-induced alterations in free cytosolic and mitochondrial calcium levels, ATP levels, mitochondrial membrane potential, and calpain and caspase activity, under conditions resulting in the activation of apoptotic and necrotic pathways. In the presence of 10 microM glutamate, 3NP administration resulted in a massive elevation in [Ca(2+)](c) and [Ca(2+)](m), decreased ATP, rapid mitochondrial membrane depolarization, and a rapid activation of calpain but not caspase activity. In the presence of the NMDA receptor antagonist MK-801, 3NP did not induce a significant elevation of [Ca(2+)](c) within the 24h time period examined, nor increase [Ca(2+)](m) within 1h. ATP was maintained at control levels during the first hour of treatment, but declined 64% by 16h. Calpain and caspase activity were first evident at 24h following 3NP administration. 3NP treatment alone resulted in a more rapid decline in ATP, more rapid calpain activation (within 8h), and elevated [Ca(2+)](m) as compared to the results obtained with added MK-801. Together, the results demonstrate that 3NP-induced necrotic neuron death is associated with a massive calcium influx through NMDA receptors, resulting in mitochondrial depolarization and calpain activation; while 3NP-induced apoptotic neuron death is not associated with significant elevations in [Ca(2+)](c), nor with early changes in [Ca(2+)](m), mitochondrial membrane potential, ATP levels, or calpain activity.     

Calpain activation by the Shigella flexneri effector VirA regulates key steps in the formation and life of the bacterium's epithelial niche

The enteropathogen Shigella flexneri invades epithelial cells, leading to inflammation and tissue destruction. We report that Shigella infection of epithelial cells induces an early genotoxic stress, but the resulting p53 response and cell death are impaired due to the bacterium's ability to promote p53 degradation, mainly through calpain protease activation. Calpain activation is promoted by the Shigella virulence effector VirA and dependent on calcium flux and the depletion of the endogenous calpain inhibitor calpastatin. Further, although VirA-induced calpain activity is critical for regulating cytoskeletal events driving bacterial uptake, calpain activation ultimately leads to necrotic cell death, thereby restricting Shigella intracellular growth. Therefore, calpains work at multiple steps in regulating Shigella pathogenesis by disrupting the p53-dependent DNA repair response early during infection and regulating both formation and ultimate death of the Shigella epithelial replicative niche.     

Calpain and calpastatin regulate neutrophil apoptosis

The average polymorphonuclear neutrophil (PMN) lives only a day and then dies by apoptosis. We previously found that the calcium-dependent protease calpain is required for apoptosis in several mouse models of cell death. Here we identify calpain, and its endogenous inhibitor calpastatin, as regulators of human neutrophil apoptosis. Cell death triggered by the translation inhibitor cycloheximide is calpain-dependent, as evidenced using either a calpain active site inhibitor (N-acetyl-leucyl-leucyl-norleucinal) or agents that target calpain's calcium binding sites (PD150606, PD151746). No significant effect on cycloheximide-triggered apoptosis was found by using inhibitors of the proteasome or of other papain-like cysteine proteases, providing further evidence that the active site calpain inhibitor prevents apoptosis via its action on calpain. In addition, we find that potentiation of calpain activity by depleting its endogenous inhibitor, calpastatin, is sufficient to cause apoptosis of neutrophils. Nevertheless, apoptosis signalled via the Fas antigen proceeds regardless of the presence of calpain inhibitor. These experiments support a growing body of work, indicating an upstream regulatory role for calpain in many, but not all, forms of apoptotic cell death. They also identify calpastatin as a participant in apoptotic cell death and suggest that for at least one cell type, a decrease in calpastatin is a sufficient stimulus to initiate calpain-dependent apoptosis. J. Cell. Physiol. 178:311–319, 1999. © 1999 Wiley-Liss, Inc.     

Specific vulnerability of mouse spinal cord motoneurons to membrane depolarization

Intracellular calcium (Ca²⁺) concentration determines neuronal dependence on neurotrophic factors (NTFs) and susceptibility to cell death. Ca²⁺ overload induces neuronal death and the consequences are thought to be a probable cause of motoneuron (MN) degeneration in neurodegenerative diseases. In the present study, we show that membrane depolarization with elevated extracellular potassium (K⁺) was toxic to cultured embryonic mouse spinal cord MNs even in the presence of NTFs. Membrane depolarization induced an intracellular Ca²⁺ increase. Depolarization-induced toxicity and increased intracellular Ca²⁺ were blocked by treatment with antagonists to some of the voltage-gated Ca²⁺ channels (VGCCs), indicating that Ca²⁺ influx through these channels contributed to the toxic effect of depolarization. Ca²⁺ activates the calpains, cysteine proteases that degrade a variety of substrates, causing cell death. We investigated the functional involvement of calpain using a calpain inhibitor and calpain gene silencing. Pre-treatment of MNs with calpeptin (a cell-permeable calpain inhibitor) rescued MNs survival; calpain RNA interference had the same protective effect, indicating that endogenous calpain contributes to the cell death caused by membrane depolarization. These findings suggest that MNs are especially vulnerable to extracellular K⁺ concentration, which induces cell death by causing both intracellular Ca²⁺ increase and calpain activation.     

Analysis of Events Associated with Serum Deprivation-Induced Apoptosis in C3H/Sol8 Muscle Satellite Cells

Satellite cells are the source of new muscle fibers in postnatal skeletal muscle growth and regeneration. Regulation of satellite cell survival is of fundamental importance in maintaining normal muscle function. Here we describe and characterize a tissue culture model of satellite cell apoptosis. Kinetic studies indicate that serum deprivation triggers a set of sequential events: early cell death, transient cell cycle traverse, and delayed cell death. The satellite cell death occurs by apoptosis based on the internucleosomal DNA laddering,in situDNA end-labeling, and the requirements forde novoprotein synthesis and extracellular calcium influx. The transient period of cell cycle progression (7–11 h after serum withdrawal) is accompanied by temporal induction of members of the immediate early gene family, such as c-myc,c-fos,and SRF, and appears to precede the delayed phase of cell death. Satellite cell apoptosis can be suppressed by several growth factors and by blocking the activity of calpain, a calcium-regulated protease. The late phase of apoptosis is marked by selective activation of ubiquitin-mediated protein conjugation and degradation. This study defines several control points where satellite cell apoptosis may be genetically or pharmacologically intervened.     

Neuroprotective Mechanism of Taurine due to Up-regulating Calpastatin and Down-regulating Calpain and Caspase-3 during Focal Cerebral Ischemia

Aims Taurine as an endogenous substance possesses a number of cytoprotective properties. In the study, we have evaluated the neuroprotective effect of taurine and investigated whether taurine exerted neuroprotection through affecting calpain/calpastatin or caspase-3 actions during focal cerebral ischemia, since calpain and caspase-3 play central roles in ischemic neuronal death. Methods Male Sprague–Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo), and 22 h of reperfusion. Taurine was administrated intravenously 1 h after MCAo. The dose–responses of taurine to MCAo were determined. Next, the effects of taurine on the activities of calpain, calpastatin and caspase-3, the levels of calpastatin, microtubule-associated protein-2 (MAP-2) and αII-spectrin, and the apoptotic cell death in penumbra were evaluated. Results Taurine reduced neurological deficits and decreased the infarct volume 24 h after MCAo in a dose-dependent manner. Treatment with 50 mg/kg of taurine significantly increased the calpastatin protein levels and activities, and markedly reduced the m-calpain and caspase-3 activities in penumbra 24 h after MCAo, however, it had no significant effect on μ-calpain activity. Moreover, taurine significantly increased the MAP-2 and αII-spectrin protein levels, and markedly reduced the ischemia-induced TUNEL staining positive score within penumbra 24 h after MCAo. Conclusions Our data demonstrate the dose-dependent neuroprotection of taurine against transient focal cerebral ischemia, and suggest that one of protective mechanisms of taurine against ischemia may be blocking the m-calpain and caspase-3-mediated apoptotic cell death pathways.     

Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2.

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress-specific protease activation pathway exists, and to what extent Bcl-2 plays a role in preventing drug-induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase-dependent apoptosis while a dopaminergic neurotoxin, MPP(+) largely induced caspase-independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS- and MPP(+)-induced cell death, Bax was cleaved into an 18-kDa fragment. This 18-kDa fragment appeared only in the mitochondria-enriched heavy membrane fraction of STS-treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP(+)-treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [(35)S]methionine-labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP(+)- and STS-induced Bax cleavage. Intriguingly, overexpression of baculovirus-derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS- but not MPP(+)-induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP(+)-induced but not from STS-induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl-2 prevented both STS- and MPP(+)-induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl-2 may play a protective role by primarily blocking drug-induced caspase or calpain activity in dopaminergic neuronal cells.     

Involvement of NO/cGMP signaling in the apoptotic and anti-angiogenic effects of beta-lapachone on endothelial cells in vitro

Neovascularization is an essential process in tumor development, it is conceivable that anti-angiogenic treatment may block tumor growth. In angiogenesis, nitric oxide (NO) is an important factor which mediates vascular endothelial cell growth and migration. beta-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho-[1,2-b]pyran-5,6-dione), a natural product extracted from the lapacho tree (Tabebuia avellanedae), has been demonstrated to possess anti-cancer and anti-viral effects. Whether beta-lapachone can induce endothelial cell death or has an anti-angiogenic effect is still an enigma. We investigated the in vitro effect of beta-lapachone on endothelial cells, including human vascular endothelial cell line, EAhy926, and human umbilical vascular endothelial cells (HUVEC). Our results revealed that (1) the intracellular cGMP levels and the mitochondria membrane potential (MMP) decreased, and calpain and caspases were activated, during beta-lapachone-induced endothelial cell death; (2) co-treatment with calpain inhibitors (ALLM or ALLN) or the intracellular calcium chelator, BAPTA, but not the general caspase inhibitor, zVAD-fmk, provided significant protection against apoptosis by preventing the beta-lapachone-induced MMP decrease and cytoplasmic calcium increase; (3) addition of NO downregulated the beta-lapachone-induced cGMP depletion and protected the cells from apoptosis by blocking the MMP decrease and the calcium increase; and (4) exogenous NO protects endothelial cells against the cell death induced by beta-lapachone, but not the anti-angiogenic effect. From all the data above, we demonstrated that NO can attenuate the apoptotic effect of beta-lapachone on human endothelial cells and suggest that beta-lapachone may have potential as an anti-angiogenic drug.     

Calpain inhibitors and antioxidants act synergistically to prevent cell necrosis: effects of the novel dual inhibitors (cysteine protease inhibitor and antioxidant) BN 82204 and its pro-drug BN 82270

Cell death is a common feature observed in neurodegenerative disorders, and is often associated with calpain activation and overproduction of reactive oxygen species (ROS). This study investigated the use of calpain inhibitors and antioxidants in combination to protect cells against necrosis. Maitotoxin (MTX), which induces a massive influx of calcium, was used to provoke neuronal cell death. This toxin increased, in a concentration-dependent manner, both calpain activity and ROS formation. Calpain inhibitors or antioxidants inhibited MTX-induced necrosis only marginally (below 20%), whereas their association protected against cell death by 40-66% in a synergistic manner. BN 82204, which possesses both calpain-cathepsin L inhibitory and antioxidant properties, and its acetylated pro-drug BN 82270, totally protected cells at 100 microm. The pro-drug BN 82270, which had better cell penetration, was twice as effective as the active principle BN 82204 in protecting glioma C6 or neuroblastoma SHSY5Y cells against death. These results suggest the potential therapeutic relevance of using a single molecule with multiple activities (cysteine protease inhibitor/antioxidant), and warrant further in vivo investigations in models of neuronal disorders.     

Effect of hypoxia upon intracellular calcium concentration of human endothelial cells.

Ischemia is a situation occurring in several diseases including myocardial infarction and organ transplantation in which oxygenated blood supply is impaired. Ischemia leads to many cellular and tissue modifications, the most important one being cell death. Several explanations have been proposed to account for these modifications and cell death; among them is calcium overload. However, the influence of calcium concentration on the alteration of endothelial cell functions or viability during ischemia are still unknown. We developed here an in vitro model where human endothelial cell monolayers were submitted to hypoxia with or without reoxygenation and variation in calcium concentration was followed using a specific intracellular probe Fura 2. We observed a significant increase of [Ca2+]i during 2 h hypoxia reaching values similar to those observed during agonist stimulation of endothelial cells but far lower than values toxic for the cells. This increase was constant during the hypoxic incubation and was due mainly to an influx of extracellular calcium. Viability was also followed during hypoxia and using calcium channel blockers, we could show that there was no correlation between viability and the rise in calcium concentration. During the reoxygenation period, [Ca2+]i decreased to reach the normal value of resting cells after 45 min, suggesting that cells were still able to recover their calcium homeostasis. The use of a ketone body (beta-hydroxybutyrate) indicated that an energy deficiency was responsible for the hypoxia-induced increase in [Ca2+]i. We actually observed a 43% decrease in ATP concentration after 2 h hypoxia. This decrease was already significant after 30 min which thus precedes the changes in [Ca2+]i. These results show that during hypoxia, energy deficiency led to an increase in [Ca2+]i which is, however, too low to account for the loss of viability but which is within the range of concentrations observed during stimulation of endothelial cells. We propose that such increased intracellular calcium concentrations could play a role in the synthesis of mediators leading to the development of local inflammation.     

Calpain activation in apoptosis

Programmed cell death is an active process wherein the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and disintegration of the cell into small, membrane-bound apoptotic bodies. Examination of the death program in various models has shown common themes, including a rise in cytoplasmic calcium, cytoskeletal changes, and redistribution of membrane lipids. The calcium-dependent neutral protease calpain has putative roles in cytoskeletal and membrane changes in other cellular processes; this fact led us to test the role of calpain in a well-known model of apoptotic cell death, that of thymocytes after treatment with dexamethasone. Assays for calcium-dependent proteolysis in thymocyte extracts reveal a rise in activity with a peak at about 1 hr of incubation with dexamethasone, falling to background at approximately 2 hr. Western blots indicate autolytic cleavage of the proenzyme precursor to the calpain I isozyme, providing additional evidence for calpain activation. We have also found that apoptosis in thymocytes, whether induced by dexamethasone or by low-level irradiation, is blocked by specific inhibitors of calpain. Apoptosis of metamyelocytes incubated with cycloheximide is also blocked by calpain inhibitors. These studies suggest a required role for calpain in both “induction” and “release” models of apoptotic cell death. © 1994 wiley-Liss, Inc.