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Forum The Role of Ca2+ in Cell Killing Pierluigi Nicotera,' Giorgio Bellomo,* and Sten Orrenius*tt Department of Toxicology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden, and Department of Internal Medicine, Clinica Medica 1, University of Pavia, I-27100 Pavia, Italy Received September 12, 1990
Introduction The involvement of Ca2+ in the regulation of a large number of physiological processes is now well established. Along with this knowledge has come the understanding that Ca2+can also play a determinant role in a variety of pathological and toxicological processes. It has long been recognized that Ca2+accumulates in necrotic tissue ( I , 2), and subsequent work has revealed that a disruption of intracellular Ca2+homeostasis is frequently associated with the early development of cell injury ( 3 , 4 ) . This led to the formation of the Ca2+hypothesis of cell injury, proposing that disruption of the intracellular Ca2+homeostasis may be a common step in the development of cytotoxicity (3). The possible involvement of changes in intracellular Ca2+ homeostasis in cell killing by toxic agents has since been debated. During the past several years it has become progressively clear that sustained increases in intracellular Ca2+ can activate cytotoxic mechanisms associated with irreversible injury in various cell systems and organs (Figure 1). Thus, Ca2+ appears to mediate the neurotoxicity of cyanide, chlordecone, and heavy metals, including lead, mercury, and organotin compounds (see ref 5 for review). In addition, intracellular Ca2+accumulation due to excessive stimulation of excitatory amino acid receptors (6) and enhanced Ca2+ influx through membrane channels (7) appears to play an important role in brain ischemia. An elevation of intracellular Ca2+also seems to contribute to cell killing by several hepatotoxic agents, such as acetaminophen (8-IO), diquat and CC14 ( l o ) ,quinones ( l l ) , cyanide (12), and maitotoxin, a polyhydroxy polyether compound that stimulates intracellular Ca2+accumulation (13). An increased intracellular Ca2+level has also been reported to be the determinant factor in the development of cardiotoxicity following exposure to the environmental contaminant TCDD (14) and in thymocyte killing by TCDD and tributyltin (15, 16). Moreover, intracellular Ca2+overload is involved in the development of reperfusion injury in the heart (17) and oxidative damage to kidney membranes during cold ischemia (18). There is also increasing evidence that Ca2+plays an important role in cell killing in the immune system. Thus, the killing of immature thymocytes by glucocorticoids (19,20),the killing of target cells by cytotoxic T lymphocytes (21) and by natural killer cells (22),and complement-mediated cell killing (23) all appear to be Ca2+-dependent. Finally, re-
* To whom correspondence should be addressed. 'Karolinska Institutet.
* University of Pavia.
cent studies have suggested that both bacterial toxins (24) and viral components such as the HIV envelope glycoprotein gp 120 (25) can promote killing of infected cells by increasing intracellular Ca2+levels. Normally, intracellular Ca2+homeostasis is maintained by the concerted operation of cellular transport and compartmentalization systems. However, alteration of these processes during cell injury can result in enhanced Ca2+ influx, release of Ca2+from intracellular stores, and inhibition of Ca2+extrusion at the plasma membrane. This can lead to an uncontrolled, sustained rise in intracellular Ca2+concentration (Figure 2) and subsequent loss of cell viability (26). In the following sections, we will describe the mechanisms involved in the regulation of intracellular Ca2+homeostatis and summarize existing evidence for the involvement of Ca2+and Ca2+-dependentprocesses in cell killing.
Regulatlon of Intracellular Ca2+ Homeostasis Recent studies using selective indicators have shown that the Ca2+concentration in the cytosol ([Ca2+Ii)of unstimulated cells is maintained between 0.1 and 0.2 gM (see ref 27 for review). Consequently, there is a concentration difference of about 4 orders of magnitude between the extracellular Ca2+level (-1.3 mM) and the cytosolic Ca2+ concentration. This produces a large electrochemical driving force which is balanced primarily by active Ca2+ extrusion through the plasma membrane and by the coordinated activity of Ca2+sequestering systems located in the endoplasmic reticular, mitochondrial, and nuclear membranes (Figure 3). Ca2+ Transport by the Plasma Membrane. The cellular influx of Ca2+ is driven by its electrochemical gradient. In excitable tissue, at least three types of voltage-dependent Ca2+ channels with different pharmacological properties have been identified (28). In contrast, there is no clear evidence for the existence of voltage-operated Ca2+channels in most nonexcitable cells. Recent studies in this and other laboratories have shown that Ca2+ inflow during agonist stimulation in hepatocytes occurs via receptor-operated Ca2+channels (29). However, it is not yet clear whether Ca2+ influx under resting conditions involves these channels or others yet to be identified. The continuous inflow of Ca2+ through the plasma membrane is balanced by specific Ca2+-ATPases which extrude Ca2+from cells. In addition, in excitable tissues various ion exchangers can function in a coordinated fashion to extrude calcium ions (27). In hepatocytes, the plasma membrane Ca2+-ATPaseis the predominant Ca2+
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Figure 1. The ea2+hypothesis of cell killing. Involvement of Ca2+and Ca2+-activatedprocesses in cell killing by various agents.
extrusion systems (30). It has a molecular mass of around 110 kDa and a very high affinity for Ca2+ = 14 nM) (31). In contrast with the erythrocyte enzyme, the liver plasma membrane Ca2+-ATPase does not appear to be stimulated by calmodulin. Ca2+Sequestration by the Endoplasmic Reticulum. The endoplasmic reticulum sequesters Ca2+ through a Ca2+-ATPasewith a molecular mass of about 125 kDa (32). This enzyme has a high affinity for Ca2+ = 0.2-1 pM), and isolated liver microsomes can accumulate 1&20 nmol of Ca2+/mgof protein in the presence of ATP. The regulation of Ca2+sequestration by the hepatic endoplasmic reticulum is still poorly understood. Although the in-
volvement of calmodulin in this process has been suggested (33), convincing evidence for this assumption is still missing. More recently, a role for glucose-6-phosphatase in the regulation of Ca2+ sequestration by the hepatic endoplasmic reticulum has been proposed in an attempt to couple the hydrolysis of glucose 6-phosphate to the termination of the Ca2+transient by enhancing Ca2+sequestration through the intraluminal accumulation of inorganic phosphate (34). Ca2+Sequestration by Mitochondria. The inner mitochondrial membrane possesses a uniport carrier which allows the electrogenic entry of Ca2+in response to the negative transmembrane potential (35). This Ca2+uni= 5-10 pM), and porter has a low affinity for Ca2+ it is inhibited by ruthenium red, a hexavalent ammonium complex of Ru. Mitochondria also possess a pathway for Ca2+efflux which is separate from the uniporter. In heart and brain, Ca2+efflux from mitochondria is Na+-dependent, whereas in liver mitochondria the predominant Ca2+ efflux is a Na+-independent and electroneutral Ca2+/H+ antiporter (27). The existence of two independent routes for Ca2+uptake and Ca2+release has led to the proposal of the mitochondrial Ca2+cycle. At high intramitochondrial [Ca2+],the efflux pathway becomes saturated, and under these conditions isolated mitochondria act as efficient buffers of extramitochondrial Ca2+. When isolated mitochondria contain little Ca2+,the activity of the efflux pathway increases with matrix [Ca2+];hence, under these
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Figure 2. Menadione-induced increase in cytosolic free ea2+in individual hepatocytes. Rat hepatocytes were grown on cover slips and loaded with the fluorescent Ca2+indicator, fura 2-AM. After loading, cover slips were washed free of extracellular fura 2, mounted on a flow-throughchamber, and placed on a stage of a Nikon Diaphot microscope coupled with a Spex Fluorolog-2fluorometer, operating with the excitation wavelengths set at 352-385 nm. Fluorescence images were filtered a t 410 nm, recorded by a MTI SIT66 camera, and processed by an IT1 150 image processing system. A map of the intracellular free ea2+concentration was obtained by a computer-driven analysis of the ratioed 352/385-nm images, using Spex' Imaging 2.50 software. Fluorescence from individual cells or a group of cells was recorded at 0 min (a), 10 min (b), 30 min (c), and 45 min (d) after the addition of 150 pM menadione. The ea2+concentrations were as follows: green, 89-150 nM; yellow, 151-250 nM; orange, 251-450 nM; red, 451-700 nM. This experiment is representative of at least three different experiments in which 60-100 cells were analyzed.
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Figure 3. Schematic representation of intracellular Ca2+compartmentalization in hepatocytes. conditions the mitochondria will not act as Ca2+buffers, but matrix [Ca2+] will reflect the extramitochondrial [Ca2+]. Ca2+Sequestration by Liver Nuclei. The existence of relatively large pores in the nuclear envelope has generally been taken as evidence for the free diffusion of ions and small molecules in and out of the nucleus. However, using a suspension of isolated liver nuclei, we have recently identified and partially characterized a Ca2+pump which promotes Ca2+uptake into the nuclei and an increase in the intranuclear free Ca2+concentration (36). The Ca2+ uptake is coupled to ATP hydrolysis, and it appears to be regulated by calmodulin. Additional studies have shown that liver nuclei can sequester about 2 nmol of Ca2+/mg of protein and have suggested a potential role for the nucleus in the regulation of the cytosolic free Ca2+concentration (37). We have also found that the intracellular messenger inositol 1,4,5-trisphosphatestimulates the release of a fraction of the nuclear Ca2+ and transiently lowers the intranuclear free Ca2+ concentration. Control of Ca2+Homeostasis in Hepatocytes and Role of Ca2+in Cell Signaling. As described above, the low [Ca2+Iiof 0.1-0.2 pM is achieved by the concerted action of the plasma membrane Ca2+pump and active Ca2+ sequestration by the mitochondria, endoplasmic reticulum, and nucleus (Figure 3). Although isolated mitochondria can accumulate large amounts of Ca2+,the affinity for Ca2+ of the uniport carrier is low, which implies that under resting conditions mitochondria play a minor role in buffering cytosolic Ca2+. In addition, electron probe X-ray microanalysis of rapidly frozen liver sections has demonstrated that mitochondria contain little Ca2+in situ (about 1 nmol of Ca2+/mg of protein), whereas the endoplasmic reticulum represents the major intracellular Ca2+store (38). Our recent finding that the nucleus has a high Ca2+buffering capacity opens the possibility that this compartment may also be involved in the regulation of the cytosolic free Ca2+concentration. Stimulation of hepatocytes with Ca2+-mobilizinghormones, such as vasopressin, angiotensin 11, or phenylephrine, produces a transient elevation of [Ca2+Iito 0.4-1 pM (39). Such hormone-induced [Ca2+Iitransients in populations of hepatocytes are comprised of two separate phases, an initial rapid increase in [Ca2+Iifollowed by a slow return to basal, or near basal, levels. With the recent development of digital imaging techniques, it has become possible to study [Ca2+Iitransients in individual cells, and this has led to the observation that, at low, close to threshold, concentrations of Ca2+-mobilizinghormones, the hepatocytes respond to these agents with oscillating [Ca2+Ii spikes (40).
The mechanisms by which Ca2+-mobilizinghormones produce [ Ca2+Iitransients have been extensively studied in recent years (41). The signal transduction pathway leading to the elevation of [Ca2+Iican be summarized as follows. Upon binding of the hormone to its plasma membrane receptor, a specific phospholipase C becomes activated via stimulation of a G protein, resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. Diacylglycerol is a potent activator of protein kinase C (42),and inositol 1,4,5-trisphosphate is the mediator for Ca2+ release from a nonmitochondrial intracellular store; this release of Ca2+is responsible for the initial rapid elevation of [Ca2+Ii(41). The exact intracellular localization of the inositol 1,4,5trisphosphate sensitive Ca2+pool is not known. Subcellular fractionation and immunocytochemical studies of the localization of the specific receptor for inositol 1,4,5-trisphosphate have suggested that at least part of this pool is located within the endoplasmic reticulum (43, 44). In addition, as discussed above, recent work from our laboratory has produced evidence for the existence of a second inositol 1,4,5-trisphosphate sensitive Ca2+store located in the hepatocyte nucleus (37). The possible contribution of this pool to the agonist-induced elevation of cytosolic Ca2+ has, however, yet to be elucidated. In addition to mobilizing Ca2+from intracellular stores, hormones can promote Ca2+influx from the extracellular compartment. Thus, the duration of the second phase of the [Ca2+Iitransient depends on the presence of extracellular Ca2+and involves the stimulation of Ca2+influx through specific receptor-operated Ca2+channels (45). Disruption of Intracellular Ca2+Homeostasis by Toxic Agents. Cellular Ca2+ overload can result from either an enhanced influx of extracellular Ca2+or an impairment of Ca2+ extrusion from the cell. In addition, interference with individual Ca2+translocases can compromise the ability of the cell to buffer cytosolic Ca2+ changes and contribute to an increase in cytosolic Ca2+ level. Over a decade ago, Moore and co-workers showed that Ca2+sequestration by liver microsomes prepared from carbon tetrachloride intoxicated rats was substantially inhibited (46). Since then, a number of hepatotoxins have been found to impair Ca2+sequestration by the isolated microsomal fraction or by the endoplasmic reticulum in intact cells. These findings suggested that an impairment of Ca2+sequestration, whether it occurred through inhibition of the Ca2+-ATPaseor through alterations of the permeability of the endoplasmic reticulum, was the mechanism by which many hepatotoxins caused liver cell death. However, recent work in our laboratory has shown that the antioxidant 2,5-di-tert-butyl-l,4-benzohydroquinone (tBuBHQ), which selectively inhibits both the microsomal Ca2+-ATPaseand Ca2+uptake by the endoplasmic reticulum (47, 48), rapidly releases endoplasmic reticular Ca2+ without producing hepatotoxicity in the isolated perfused rat liver (49). Hence, interference with Ca2+sequestration by the endoplasmic reticulum does not appear to play a major role in the development of acute hepatotoxicity. The role of mitochondria in toxin-induced perturbation of Ca2+homeostasis is still unclear. As discussed above, mitochondria contain only little Ca2+under physiological conditions (38). However, they have the capacity to sequester large quantities of Ca2+and could therefore act as efficient buffers of [ Ca2+Ii under toxic conditions. Unfortunately, this potentially important line of defense does not appear to be operational in many instances be-
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T a b l e I. Cvtourotection bv I n t r a c e l l u l a r Ca2+ Chelators chemical toxicity by oxidative stress (11) acetaminophen (9) TCDD (15) styrene oxide (unpublished observation) tributyltin (16) chemical anoxia (12) programmed cell death induced by glucocorticoids (20) anti-CDB antibodies (107) tumor necrosis factor (Y (112) target cell killing by cytotoxic T lymphocytes (unpublished observation) NK cells (22) cold shock (115) ~~
cause several toxins, such as tert-butyl hydroperoxide, menadione, and N-acetyl-p-benzoquinone imine, cause rapid oxidation of mitochondrial pyridine nucleotides, which in turn stimulates Ca2+release through the antiporter (50). In addition, the release and reuptake of Ca2+ through separate routes result in Ca2+cycling, which can lead to membrane damage, mitochondrial swelling, uncoupling of respiration, and loss of intracellular ATP. This, in turn, will further compromise cell survival. There is compelling evidence that many hepatotoxins interfere with Ca2+uptake and extrusion mechanisms (51). Inhibition of Ca2+efflux will result in the net accumulation of Ca2+and in a pathological elevation of [Ca2+Ii. In addition, it has become clear that chemical toxins can stimulate Ca2+entry by interacting with existing Ca2+channels or by increasing the plasma membrane permeability to Ca2+. The resulting Ca2+ overload can activate several cytotoxic mechanisms that can cause cell death. The main evidence for the importance of Ca2+overload in cell killing comes from experiments in which removal of extracellular Ca2+ (2), or loading of cells with intracellular Ca2+ chelators (12), has prevented cell death. Recently, intracellular Ca2+chelators, such as quin-2 or BAPTA, have been employed to buffer increases in cytosolic Ca2+in a variety of experimental systems, and such treatment has prevented, or delayed, cell killing induced by various agents (Table I). In addition to Ca2+chelators, Ca2+channel blockers have also been used to prevent Ca2+ overload and cell death in several experimental systems (7, 25, 52).
Mechanisms of Ca2+-MedlatedCell Killing Both the duration and the extent of the increase in [Ca2+Iiappear to be critical for the development of cytotoxicity. Even moderate increases in cytosolic Ca2+can impair the ability of the cell to respond adequately to agonist stimulation and thereby inhibit cell control by hormones and growth factors. Another early effect of a sustained elevation of the cytosolic free Ca2+concentration is the impairment of mitochondrial functions. In addition, more prolonged and intense increases in cytosolic Ca2+will result in the disruption of cytoskeletal organization and in the activation of a number of Ca2+-stimulatedcatabolic processes, such as proteolysis, membrane degradation, and chromatin fragmentation. In the following sections we shall briefly discuss the involvement of these Ca2+-dependent alterations in cell killing. Alterations of Ca2+Signaling by Toxicants. Calcium ions are required for many phsiological functions, including the control of metabolic processes, cell differentiation and proliferation, and secretory functions (23). Many of these Ca2+-dependentprocesses are tightly controlled by hormones and growth factors. Not only will the loss of the
ability of a cell to respond to such hormones and growth factors deprive the cell of a trophic stimulus but, as recent evidence (53) clearly indicates, it may also result in the activation of a suicide process. The inability of cells to respond to Ca2+-mobilizing hormones can result from the selective depletion of the intracellular agonist-sensitive Ca2+ pool by compounds such as 2,5-di-tert-butyl-l,4-benzohydroquinone (48) or bromotrichloromethane (54). Another possible target for toxicants are Ca2+channels, as recently demonstrated with maitotoxin (52). This potent dinoflagellate toxin opens verapamil-sensitive Ca2+channels in the myocyte plasma membrane and causes cell death by inducing a sustained elevation of [Ca2+Iiin these cells. Finally, prolonged increases in [Ca2+Iimay obliterate the Ca2+transients normally evoked by physiological agonists, thereby resulting in an impairment of cell signaling. Mitochondrial Damage. Work from several laboratories has indicated that mitochondrial damage may represent a common event in cell injury caused by a variety of toxic agents (55-58). Mitochondrial damage is initially manifested by a decrease in the mitochondrial membrane potential followed by a decline in ADP phosphorylation, eventually resulting in ATP depletion. Protons are constantly pumped from the matrix into the intermembrane space in mitochondria of living cells (59). Since the inner mitochondrial membrane is relatively impermeable to anions, a considerable proportion of the energy resulting from the proton concentration gradient is stored as membrane electric potential (60). The proton gradient and the transmembrane potential represent the electrochemical forces that are employed for ATP synthesis as well as for other metabolic activities, including the maintenance of Ca2+ homeostasis within mitochondria. As described above, Ca2+can be actively transported into mitochondria via an electrophoretic uniporter. The driving force for the continuous Ca2+pumping is provided by the transmembrane potential. However, studies performed in isolated mitochondria have demonstrated that during Ca2+ uptake the membrane potential decreases and the extent of the decrease is proportional to the amount of Ca2+taken up by the mitochondria (61). Thus it appears that, under conditions which cause massive amounts of Ca2+to accumulate in the mitochondria, their membrane potential would collapse. To test this assumption, isolated rat hepatocytes were incubated with sodium orthovanadate, which inhibits both the endoplasmic reticular and the plasma membrane Ca2+-ATPases and causes Ca2+ accumulation in mitochondria. Concomitant with the accumulation of Ca2+in the mitochondrial compartment, the mitochondrial membrane potential decreased proportionally to the amount of Ca2+accumulated; ATP depletion then followed (62). Another demonstration that Ca2+overload can cause the collapse of the mitochondrial membrane potential in intact cells is provided by the findings illustrated in Figure 4. In these experiments, primary hepatocyte cultures were incubated with the fluorescent cationic dye rhodamine 123, which accumulates in the mitochondria according to the membrane potential. The effect of the Ca2+ ionophore ionomycin on the intracellular distribution of rhodamine fluorescence was then recorded, and video images of individual cells were stored and processed by computer analysis. Prior to the addition of ionomycin, the intracellular pattern of rhodamine fluorescence was consistent with the accumulation of the dye within the mitochondrial compartment (Figure 4a). The addition of ionomycin caused a 10-fold increase in the cytosolic free Ca2+con-
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E Figure 4. Effects of Ca2+overload on rhodamine 123 distribution in cultured hepatocytes. Rat hepatocytes were grown on quartz cover slips and incubated with 1pM rhodamine 123, in a Krebs-Henseleit buffer supplemented with 12.5 mM Hepes, pH 7.4, at 37 "C for 10 min. The cover slips where then washed, mounted in a flow-through chamber, and placed on a stage of a Nikon Diaphot microscope coupled with a Spex Fluorolog-2 fluorometer and a video camera which was interfaced with a computer running Spex' Imaging v. 2.50 software for video image analysis. When indicated in the text, fluorescence images (excitation 490 nm and filter at 510-560 nm) were recorded. Panel a, control; panel b, 1 min after the addition of 5 pM ionomycin; panel c, 10 min after the addition of 5 pM ionomycin; panel d, control in Ca2+-freemedium, panel e, 10 min after addition of 5 pM ionomycin in Ca2+-freemedium.
centration (from -100 nM to -1 pM). One minute later, rhodamine fluorescence had been lost from the mitochondrial compartment and now appeared diffusely in the cytosol, indicating a collapse of the mitochondrial membrane potential (Figure 4b). After 10 min, rhodamine fluorescence was very faint as a result of the loss of the dye from the cell (Figure 4c). Removal of Ca2+from the extracellular medium prevented the ionomycin-induced redistribution of rhodamine fluorescence (Figure 4d,e), indicating that the loss of mitochondrial membrane potential caused by ionomycin was a direct consequence of the intracellular Ca2+increase. The existence of different Ca2+ uptake and release pathways in mitochondria provides a basis for Ca2+cycling (61).This process continuously utilizes energy which is supplied by the membrane potential. A number of investigations, using mitochondria isolated from different sources, have demonstrated that the oxidation of intramitochondrial NAD(P)H can activate the release route and accelerate Ca2+cycling across the mitochondrial membrane (63-68).This condition is associated with a decrease in the mitochondrial membrane potential that parallels the rate of Ca2+cycling. Moreover, chelation of extramitochondrial Ca2+with EGTA or inclusion of ruthenium red in the incubation medium, to abolish the reuptake of the released Ca2+,completely prevents the collapse of the membrane potential (69,70). Evidence that this mechanism is operational also in intact cells has recently been obtained by using cultured hepatocytes loaded with rhodamine 123 and video imaging analysis. After loading with
rhodamine 123, hepatocytes where incubated with the prooxidant menadione, whose metabolism was associated with a marked loss of rhodamine 123 fluorescence, indicating the collapse of the mitochondrial membrane potential. The decline in mitochondrial membrane potential was partially prevented in hepatocytes preloaded with the intracellular Ca2+chelator quin-2, suggesting that the Ca2+ increase caused by menadione contributed to the mitochondrial damage (G. Bellomo et al., unpublished results). The mechanisms of Ca2+-dependent mitochondrial damage have been extensively investigated by Pfeiffer and co-workers (61,71-74). They have proposed that phospholipase A2 activation may induce permeability changes in mitochondria exposed to Ca2+ and oxidizing agents. According to this view, Ca2+uptake, or Ca2+cycling, would result in phospholipase A2 activation and the accumulation of deacylated phospholipids, whose reacylation would be inhibited by the concomitant depletion of intramitochondrial GSH and the increase in glutathione disulfide. The demonstration that deacylated phospholipids could indeed accumulate and that the process was inhibited by dibucaine supports the involvement of phospholipase A2. As a consequence of the overall process, a proteinaceous pore would open, leading to the release of ions, small molecules, and even proteins into the extramitochondrial environment (74).This process has also been demonstrated to occur in heart mitochondria exposed to Ca2+,inorganic phosphate, and oxidizing agents and has been claimed to represent a mechanism for mitochondrial dysfunction in the ischemic-reperfused heart (75).
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A more detailed analysis of mitochondrial damage induced by the combined effects of Ca2+and active oxygen species has been performed by Malis and Bonventre (76). In the presence of Ca2+,oxygen radicals caused a marked increase in mitochondrial membrane permeability and a major functional breakdown in the electron transport chain. Furthermore, there was a 50% reduction of ATPase activity, a decrement in ADP translocase, and uncoupling of respiration, reflecting the inability of the mitochondria to synthesize ATP. It is important to stress the role of Ca2+in triggering these alterations, since the omission of this ion from the incubation medium minimized the damaging effects of oxygen free radicals. A different type of mitochondrial damage induced by Ca2+has been described in a series of papers by Reed and co-workers (see refs 77 and 116, the second of three Forum papers in this issue, for a review). They have reported that the removal of Ca2+from the incubation medium of freshly isolated rat hepatocytes resulted in the collapse of the mitochondrial membrane potential and in the appearance of signs of oxidative cell injury which were abolished by inhibitors of mitochondrial Ca2+cycling, such as ruthenium red and lanthanum. These findings have led to the hypothesis that mitochondrial Ca2+cycling by itself could generate oxidative stress and cell injury. However, the mechanism by which Ca2+-freemedium stimulates mitochondrial Ca2+cycling and the relationship between the latter and the onset of oxidative stress remain to be elucidated. Cytoskeletal Alterations. One of the early signs of cell injury caused by a variety of toxic agents is the appearance of multiple surface protrusions (blebs) (3, 78). The events leading to bleb formation have not yet been fully elucidated, and several mechanisms may independently contribute to their formation. However, it is generally accepted that a perturbation of cytoskeletal organization and of the interaction between the cytoskeleton and the plasma membrane plays an important role. Evidence for this assumption is provided by the observation that agents which modify the cytoskeleton, such as cytochalasins and phalloidin, stimulate bleb formation and by the recent demonstration that the bundles of actin microfilaments present a t the base of the bleb appear to be completely dissociated from the bleb-forming portion of the plasma membrane (79). The finding that treatment of cells with a Ca2+ionophore was able to induce similar blebbing and that this was prevented by the omission of Ca2+from the incubation medium led to the proposal that Ca2+ is involved in the cytoskeletal alterations associated with the formation of surface blebs during cell injury ( 3 ) . The cytoskeleton is organized into a complex array of fibers, which belong to three main classes: microfilaments, microtubules, and intermediate filaments (see ref 80 for review). Microfilaments are mainly composed of actin and several actin-binding proteins (81). Many of the actinbinding proteins require Ca2+to be able to interact with other cytoskeletal constituents. Typical examples include caldesmon (which binds to actin and prevents myosin binding), gelsolin (which severs actin microfilaments), and villin (which severs actin microfilaments into short fragments) (82). Moreover, Ca2+regulates the function of three other actin-binding proteins which are directly involved in the association of microfilaments with the plasma membrane. Among these proteins, a-actinin is involved in the normal organization of actin filaments into regular, parallel arrays. However, in the presence of micromolar Ca2+concentrations (e.g., after exposure of cells to Ca2+ ionophores), cu-actinin dissociates from the actin filaments
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(83). The other two actin-binding proteins, vinculin and ABP (actin-binding protein, in platelets), are substrates for Ca2+-dependentproteases (84). Thus, an increase in the cytosolic free Ca2+concentration to micromolar levels results in the proteolysis of these two polypeptides. This can also occur under physiological conditions, Le., during platelet activation (85). Another example of Ca2+-mediated modification of actin-binding proteins is found in the work by Harris and Morrow on fodrin (86). Fodrin is a ubiquitous cytoskeletal protein which is able to link integral membrane proteins to cortical actin filaments. Thus, it is involved in the organization of receptor domains and in the control of vesicular traffic a t the plasma membrane. When the cytosolic Ca2+level increases, either fodrin can bind calmodulin or it can be cleaved by a Ca2+-dependent protease. Both processes result in the loss of the ability of fodrin to bind actin and in the dissociation of microfilaments from membrane integral proteins. Recent work has provided ample evidence for the involvement of Ca2+in the toxic alterations of actin microfilaments and actin-binding proteins. For example, the incubation of human platelets with menadione resulted in the dissociation of a-actinin from the whole cytoskeleton and in the proteolysis of the ABP (87) (Figure 5). These changes were largely prevented in cells preloaded with intracellular Ca2+chelators. Furthermore, immunocytochemical investigations using anti-a-actinin antibodies and NBD-phallacidin to stain actin revealed that dissociation of the a-actinin from the actin filaments may be responsible for bleb formation (88). Other studies by Steenbergen and co-workers, utilizing anti-vinculin antibodies in canine heart during the development of ischemia and reperfusion injury, have revealed a progressive loss of vinculin staining along the lateral margin of myocytes (89). This loss was associated with the appearance of subsarcolemmal blebs and breaks in the plasma membrane. Since vinculin is a substrate for Ca2+-dependentproteases and since the cytosolic Ca2+concentration during ischemia and reperfusion rises well above the level necessary for protease activation, it appears that Ca2+-activatedproteases may be responsible for the loss of vinculin. Microtubule structure and distribution are also controlled by Ca2+. This assumption is validated by the simple demonstration that GTP-dependent microtubule polymerization, obtained "in vitro" by warming tubulin solutions a t 37 "C, is abolished by the presence of micromolar Ca2+ concentrations in the incubation medium (90).In addition, the activity of microtubule-associated proteins (MAPS), which control the turnover and the distribution of microtubules, is modulated by phosphorylation catalyzed by a Ca2+-and calmodulin-dependent protein kinase (91).
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Forum Studies by Shelanski have demonstrated that microinjection of Ca2+/calmodulincomplexes in 3T3 fibroblasts results in the complete depolymerization of microtubules which is spatially limited to the site of injection (92). Although the depolymerization of microtubules during toxic cell injury has been reported (93),the possible involvement of Ca2+in this process has not been clarified. Little is also known about the role of Ca2+in toxic modifications of intermdiate filaments. In fact, our knowledge of the composition of intermediate filaments and of the role of these structures in cell physiology is still fragmentary. It has been shown that several intermediate filament proteins, including vimentin and cytokeratins, are substrates for a Ca2+-activatedprotease (94). However, direct evidence is still missing for a physiological role of proteolysis in the control of intermediate filament function and for a possible contribution of cleavage of intermediate filaments to the cytoskeletal alterations occurring during cell injury. Ca2+-DependentDegradative Enzymes. The catabolism of phospholipids, proteins, and nucleic acids involves enzymes most of which require Ca2+for activity. Ca2+overload can result in a sustained activation of these enzymes and in the degradation of cell constituents, which may ultimately lead to cell death. Phospholipases catalyze the hydrolysis of membrane phospholipids. They are widely distributed in biological membranes and generally require Ca2+for activation. A specific subset of phospholipases, collectively designated phospholipase A2, have been proposed to function in the detoxication of phospholipid hydroperoxides by releasing fatty acids from peroxidized membranes (95). However, phospholipase activation can also mediate pathophysiological reactions by stimulating membrane breakdown or by generating toxic metabolites. Therefore, phospholipase activation has been proposed as an important mechanism of cell killing. Phospholipase A, is Ca2+-and calmodulin-dependent, and thus it is susceptible to activation following an increase in cytosolic Ca2+concentration. Hence, it has been suggested that a sustained increase in cytosolic Ca2+can result in enhanced breakdown of membrane phospholipids and, in turn, in mitochondrial and cell damage. Although a number of studies have indicated that accelerated phospholipid turnover occurs during anoxia or toxic cell injury (96-99), the importance of phospholipase activation in the development of cell damage remains to be established. During the past 10 years the involvement of nonlysosoma1 proteolysis in several cell processes has become progressively clear. Proteases that have a neutral pH optimum include the ATP- and ubiquitin-dependent proteases and the calcium-dependent proteases, or calpains. Calpains are present in virtually all mammalian cells (26) and appear to be largely associated with membranes in conjunction with a specific inhibitory protein (calpastatin) (100). The extralysosomal localization of this proteolytic system allows the proteases to participate in several specialized cell functions, including cytoskeletal and cell membrane remodeling, receptor cleavage and turnover, enzyme activation, and modulation of cell mitosis. Cellular targets for these enzymes include cytoskeletal elements and membrane integral proteins (84-87, 101). Thus, the activation of ea2+proteases has been shown to cause modification of microfilaments in platelets (85,87) and to be involved in cell degeneration during muscle dystrophy (102) and in the development of ischemic injury in nervous tissue (6). Studies from our and other laboratories have suggested the involvement of ea2+-activated
proteases in the toxicity of certain agents in liver (99,103), myocardial cells (104), and platelets (83). Although the substrates for protease activity during cell injury remain largely unidentified, it appears that cytoskeletal proteins may be a major target for Ca2+-activatedproteases during chemical toxicity. During physiological cell killing a suicide process is activated in affected cells which is known as "apoptosis" or programmed cell death. Several early morphological changes occur within apoptotic cells, including widespread plasma and nuclear membrane blebbing, compacting of organelles, and chromatin condensation (105). The most reliable and characteristic marker for this process is the activation of a Ca2+-dependentendonuclease which results in the cleavage of cell chromatin into oligonucleosomelength fragments (105,106). Endonuclease activation has been implicated in the killing of target cells by cytotoxic T lymphocytes (21) and natural killer cells (221, and in thymocytes exposed to glucocorticoid hormones (20,105, 106) or to an antibody to the CD3/T cell-receptor complex (107).
The results of several recent studies have shown that Ca2+overload can trigger endonuclease activation. The Ca2+ionophore A23187 stimulates apoptosis in thymocytes (108), and characteristic endonuclease activity in isolated nuclei is dependent on Ca2+(109) and sensitive to inhibition by zinc (105). In addition, Ca2+-mediatedendonuclease activation appears to be involved in the cytotoxicity of TCDD and tributytin in thymocytes (15, 16). Although Ca2+-dependent endonuclease activation has been most extensively studied in thymocytes, it appears that this process may also be important in a variety of other tissues. For example, we have identified a constitutive endonuclease in liver nuclei that is activated by submicromolar Ca2+concentrations in intact nuclei incubated in the presence of ATP to stimulate Ca2+uptake (109). Endonuclease activation has also been implicated in cell killing by bacterial toxins (110) and in damage to macrophages caused by oxidative stress (111). More recently, we have found that exposure of human adenocarcinoma cells to tumor necrosis factor causes intracellular Ca2+ accumulation and endonuclease activation (112). Interestingly, in many cells the initial Ca2+ increase is found in the nucleus. This suggests that selective elevation of the nuclear Ca2+ concentration may be sufficient to stimulate DNA fragmentation. However, although the responsible endonuclease requires Ca2+ for activity, its regulation appears to be more complex and to involve additional signals (113). Ca2+overload may also stimulate other enzymatic processes that result in DNA damage. Elevated Ca2+levels can lock topoisomerase I1 in a form that cleaves, but does not religate, DNA and topoisomerase I1 mediated DNA fragmentation has been implicated in the cytotoxic action of some anticancer drugs (114). DNA single-strand breaks in cells exposed to oxidative stress can also be generated through a Ca2+-dependentmechanism (11). Thus, further work is required not only to identify other situations in which endogenous endonuclease activation can mediate Ca2+-dependentcell killing but also to identify additional Ca2+-dependentcatabolic processes which may generate DNA damage.
Concluding Remarks Thus, it appears safe to conclude that calcium ions play an important role in both toxic cell killing and programmed cell death. Recent research has revealed some of the biochemical mechanisms by which intracellular Ca2+ov-
492 Chem. Res. Tonicol., Vol. 3, No. 6, 1990
erload can cause cytotoxicity. However, the relative importance of the various Ca2+-dependentprocesses in toxic cell killing needs to be further clarified. Finally, it should be emphasized that cell death can occur without any apparent early change in intracellular Ca2+homeostasis and that mechanisms other than Ca2+overload are also important in toxic cell killing.
Acknowledgment. Studies from our laboratories were supported by grants from the Swedish Medical Research Council (Project 03)3-2471), the CFN (Project L-90-08), and by a C.N.R. Special Project Grant “Physiology and Pathology of Calcium” to G.B. Registry No. ea2’, 7440-70-2.
References (1) Fleckenstein, A., Frey, M., and Fleckenstein-Grun, G. (1983)
Cellular injury by cytosolic calcium overload and its prevention by calcium antagonists-a new principle of tissue protection. In Mechanisms of Hepatocyte Injury a n d Death (Keppler, D., Popper, H., Bianchi, L., and Reutter, W., Eds.) up . _321-335, M T P Press Ltd., Lancaster. (2) Schanne. F. A. X.. Kane. A. B.. Young. E. E.. and Farber. J. L. (1979) Calcium dependence of toxic ceb death: a final common pathway. Science 206, 700-702. (3) Jewell, S. A., Bellomo, G., Thor, H., Orrenius, S., and Smith, M. T . (1982) Bleb formation in hepatocytes during drug metabolism is associated with alterations in intracellular thiol and calcium ion homeostasis. Science 217, 1257-1259. (4) Trump, B. F., Berezesky, I. K., and Cowley, R. A. (1982) in Pathophysiology of Shock, Anoxia and Ischemia (Cowley, R. A., and Trump, B. F., Eds.) pp 6-46, Williams and Wilkins, Baltimore. (5) Komulainen, H., and Bondy, S. C. (1988) Increased free intracellular CaZt by toxic agents: an index of potential neurotoxicity? Trends Pharmacol. Sci. 9,154-156. (6) Manev, H., Costa, E., Wroblewski, J. T., and Guidotti, A. (1990) Abusive stimulation of excitatory amino acids receptors: a strategy to limit neurotoxicity. FASEB J . 4, 2789-2797. (7) Pauwels, P. J., Van Assouw, H. P., Leysen, J. E., and Janssen, P. A. J. (1989) Ca2+-mediatedneuronal death in rat brain neuronal cultures by veratridine: protection by flunarizine. Mol. Pharmacol. 36, 525-531. (8) Moore, M., Thor, H., Moore, G., Nelson, S., MoldBus, P., and Orrenius, S. (1985) The toxicity of acetaminophen and N-acetylp-benzoquinoneimine in isolated hepatoxytes is associated with thiol depletion and increased cytosolic Ca2+. (1985) J. Biol. Chem. 260, 13035-13040. (9) Boobis, A. R., Seddon, C. E., Nasseri-Sina, P., and Davies, D. S. (1990) Evidence for a direct role of intracellular CaZ+in paracetamol toxicity. Biochem. Pharmacol. 39, 1277-1281. (10) Tsokos-Kuhn, J. 0.(1989) Evidence in vivo for elevation of intracellular CaZCin the liver after diquat, acetaminophen and CC1,. (1989) Biochem. Pharmacol. 38,3061-3065. (11) Dypbukt, J. M., Thor, H., and Nicotera, P. (1990) Intracellular CaZt chelators prevent DNA damage and protect hepatoma lclc7 cells from quinone-induced cell killing. Free Radical Res. Commun. 8,347-354. (12) Nicotera, P., Thor, H., and Orrenius, S. (1989) Cytosolic free CaZCand cell killing in hepatoma lclc7 cells exposed to chemical anoxia. FASEB J . 3, 59-64. (13) Kutty, K. R.,Singh, Y., Santostasi, G., and Krishna, G. (1989) Maitotoxin-induced liver cell death involving loss of ATP following influx of calcium. Toxicol. Appl. Pharmacol. 101, 1-10. (14) Canga, L.,Levi, R., and Rifkind, A. B. (1988) 2,3,7,8-Tetrachlorodibenzo-p-dioxin toxicity: decreased &adrenergic responsiveness and evidence of increased intracellular calcium. Proc. Natl. Acad. Sci. U.S.A. 85,905-909. (15) McConkey, D. J., Hartzell, P., Duddy, S. K., HPkansson, H., and Orrenius, S. (1988) 2,3,7,8-Tetrachlorodibenzo-p-dioxin kills immature thymocytes by Ca2+-mediatedendonuclease activation. Science 242, 256-259. (16) Aw, T. Y., Nicotera, P., Manzo, L., and Orrenius, S. (1990) Tributyltin stimulates apoptosis in rat thymocytes. Arch. Biochem. Riophys. (in press). (17) Tani, M. (1990) Mechanisms of Ca2+overload in reperfused ischemic myocardium. Annu. Reu. Physiol. 52, 543-559.
Nicotera et al. (18) Cotterill, L. A., Gower, J. D., Fuller, B. J., and Green, C. J. (1989) Oxidative damage to kidney membranes during cold ischemia: Evidence of a role for calcium. Transplantation 48, 745-751. (19) Kaiser, N., and Edelman, I. S. (1977) Calcium dependence of glucocorticoid-induced lymphocytolysis. Proc. Natl. Acad. Sci. U.S.A. 74, 638-642. (20) McConkey, D.J., Nicotera, P., Hartzell, P., Bellomo, G., Wyllie, A. H., and Orrenius, S. (1989) Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic CaZ+ concentration. Arch. Biochem. Biophys. 269, 365-370. (21) Berke, G., (1989) The cytolytic T lymphocyte and its mode of action. Immunol. Lett. 20, 169-178. (22) McConkey, D. J., Chow, S. C., Orrenius, S., and Jondal, M. (1990) NK cell-induced cytotoxicity is dependent on a Ca2+increase in the target. FASEB J. 4, 2661-2664. (23) Campbell, A. K. (1983) Intracellular Calcium: Its Uniuersal Role a s Regulator, Wiley, New York. (24) Caspar, M., Florin, I., and Thelestam, M. (1987) Calcium and calmodulin in cellular intoxication with clostridium difficile toxin B. J . Cell. Physiol. 132, 168-172. (25) Dreyer, E. B., Kaiser, P. K., Offerman, J. T., and Lipton, S. A. (1990) HIV-1 coat protein neurotoxicity prevented by calcium channel antagonists. Science 248, 364-367. (26) Nicotera, P., McConkey, D. J., Svensson, S. h.,Bellomo, G., and Orrenius, S. (1988) Correlation between cytosolic Ca2+ concentration and cytotoxicity in hepatocytes exposed to oxidative stress. Toxicology 52, 55-63. (27) Carafoli, E. (1987) Intracellular Ca2+homeostasis. Annu. Reu. Biochem. 56, 395-433. (28) Hosey, M. M., and Lazdunski, M. (1988) Calcium channels. Molecular pharmacology, structure and regulation. J . Membr. Biol. 104,81-105. (29) Kass, G.E. N., Llopis, J., Chow, S. C., Duddy, S. K., and Orrenius, S. (1990) Receptor-operated Ca2+channels in hepatocytes: Identification and characterization using manganese. J. Biol. Chem. (in press). (30) Lotersztajn, S., Hanoune, D., and Pecker, F. (1981) A high affinity calcium stimulated magnesium-dependent ATPase in rat liver plasma membranes: Dependence on an endogenous protein activator distinct from calmodulin. J . Biol. Chem. 256, 11209-11215. (31) Pavoine, C., Lotersztajn, S., Mallat, A., and Pecker, F. (1987) The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membrane is a CaZf pump: Reconstitution of the purified enzyme into phospholipid vesicles. J . Biol. Chem. 262, 5113-5117. (32) Kraus-Friedman, N., Fleshneer, C. R., Zimniak, P., and Moore, P. B. (1988) The hepatic microsomal Ca2+sequestering system. In Cellular Ca2+ regulation (Pfeiffer, D. R., McMillan, J. B., and Little, S., Eds.) pp 59-68, Plenum Press, New York. (33) Moore, P. B., and Kraus-Friedman, N. (1983) Hepatic microsomal Ca2+-ATPase. Biochem. J . 214, 61-75. (34) Benedetti, A., Fulceri, R., Ferro, M., and Comporti, M. (1986) On a possible role for glucose-6-phosphatase in the regulation of liver cell CaZt concentration. Trends Biochem. Sci. 11, 284-285. (35) Nicholls, D. J. (1986) Intracellular Ca2+homeostasis. Br. Med. Bull. 42, 353-358. (36) Nicotera, P., McConkey, D. J., Jones, D. P., and Orrenius, S. (1989) ATP stimulates Ca2+uptake and increases the free Ca2+ concentration in isolation liver nuclei. Proc. Natl. Acad. Sci. U.S.A. 86, 453-457. (37) Nicotera, P., Orrenius, S., Nilsson, T., and Bergreen, P. 0. (1990) An inositol (1,4,5)-trisphosphate-~ensitive Ca2+pool in liver nuclei. Proc. Natl. Acad. Sci. U.S.A. 87, 6858-6862. (38) Somlyo, A. P., Bond, M., and Somlyo, A. V. (1985) Calcium content of mitochondria and endoplasmic reticulum in liver frozen rapidly in uiuo. Nature 314, 622-625. (39) Charest, R., Blackmore, P. F., Berthon, B., and Exton, J. H. (1983) Changes in free cytosolic Ca2+ in hepatocytes following adrenergic stimulation. Studies on quin 2 loaded hepatocytes. J . Biol. Chem. 258, 8769-8773. (40) Woods, N. M., Cuthbertson, K. S. R., and Cobbold, P. E. (1986) Repetitive transient rises in cytoplasmic free calcium in hormone stimulated hepatocytes. Nature 319,600-602. (41) Berridge, M. J. (1987) Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu. Rev. Biochem. 56, 159-193. (42) Nishizuka, Y.(1984) The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 308,693-698.
Forum (43) Guillemette, G., Balla, T., Baukal, A. J., and Catt, K. J. (1988) Characterization of inositol 1,4,5trisphosphate receptors and Ca2+ mobilization in an hepatic plasma membrane fraction. J . Biol. Chem. 264, 15192-15198. (44) Ross, C. A., Meldolesi, J., Milner, T. A., Satoh, T., Supattapone, S., and Snyder, S. H. (1989)Inositol 1,4,5-trisphosphate receptors localized to endoplasmic reticulum in cerebellar Purkinje neurons. Nature 339,468-470. (45) Barritt, G.J., Parker, J. C., and Wadsworth, J. C. (1981)A kinetic analysis of the effect of adrenalin on calcium distribution in isolated rat liver parenchymal cells. J. Physiol. (London) 312, 29-55. (46) Moore, L., Davenport, G. R., and Landon, E. J. (1976)Calcium uptake of a rat liver microsomal subcellular fraction in response to in vivo administration of carbon tetrachloride. J. Biol. Chem. 251, 1197-1201. (47) Moore, G. A., McConkey, D. J., Kass, G. E. N., O'Brien, P. J., and Orrenius, S. (1987)2,5-di-(tert-butyl)-1,4-benzihydroquinone: A novel inhibitor of liver microsomal Ca2+sequestration. FEBS Lett. 224,331-336. (48) Kass, G. E. N., Duddy, S. K., Moore, G. A., and Orrenius, S. (1989) Di- (tert -butyl)-1,4benzohydroquinone rapidly elevates cytosolic Ca2+concentration by mobilizing the inositol 1,4,5-trisphosphate-sensitive CaZt pool. J . Biol. Chem. 264,15191-15198. (49) Farrell, G.C., Duddy, S. K., Kass, G. E. N., Llopis, J., Gahm, A., and Orrenius, S. (1990)Release of Ca2+from the endoplasmic reticulum is not the mechanism for bile acid-induced cholestasis and hepatotoxicity in the intact rat liver. J . Clin. Inuest. 85, 1255-1259. (50) Richter, C., and Frei, B. (1988)Ca2+release from mitochondria induced by prooxidants. Free Radical Biol. Med. 4, 365-375. (51) Orrenius, S., McConkey, D. J., Bellomo, G., and Nicotera, P. (1989)Role of Ca2+ in toxic cell killing. Trends Pharmacol. Sci. 10,281-285. (52) Santostasi, G., Kutty, K. R., Bartorelli, A. L., Yasumoto, T., and Krishna, G. (1990)Maitotoxin-induced myocardial cell injury: calcium accumulation followed by ATP depletion precedes cell death. Toxicol. Appl. Pharmacol. 102,164-173. (53) Barnes, D. S.(1988)Cells without growth factor commit suicide. Science 242,1510-1511. (54) Benedetti, A., Graf, P., Fulceri, R., Romani, A,, and Sies, H. (1989)Ca2+mobilization by vasopressin and glucagon in perfused livers: Effect of prior intoxication with bromotrichloromethane. Biochem. Pharmacol. 38, 1799-1805. (55) Lemasters, J. J., DiGiuseppi, J., Nieminen, A. L., and Herman, B. (1987)Blebbing, free Ca2+ and mitochondrial membrane potential preceding cell death in hepatocytes. Nature 325,78-81. (56) Masaki, N.,Kyle, M. E., Serroni, A., and Farber, J. L. (1989) Mitochondrial damage as a mechanism of cell injury in the killing of cultured hepatocytes by tert-butyl hydroperoxide. Arch. Biochem. Biophys. 270,672-680. (57) Nagelkerke, J. F., Dogterom, P., DeBony, H. J. G. M., and Mulder, G. J. (1989)Prolonged high intracellular free calcium concentrations induced by ATP are not immediately cytotoxic in isolated rat hepatocytes. Biochem. J . 263,347-353. (58) Nieminen, A. L.,Dawson, T. L., Gores, G. J., Kawanishi, T., Herman, B., and Lemaster, J. J. (1990)Protection by acidotic pH and fructose against lethal injury to rat hepatocytes from mitochondrial inhibitors, ionophores and oxidant chemicals. Biochem. Biophys. Res. Commun. 167,600-606. (59) Prince, R. C. (1989)The proton pump of cytochrome oxidase. Trends Biochem. Sci. 13, 159-160. (60) Chen, L. B. (1988)Mitochondrial membrane potential in living cells. Annu. Reu. Cell Biol. 4, 155-181. (61) Gunther, T. E.,and Pfeiffer, D. R. (1990)Mechanisms by which mitochondria transport calcium. Am. J. Physiol. 258,C755-C786. (62) Richelmi, P., Mirabelli, F., Salis, A., Finardi, G., Bert6, F., and Bellomo, G. (1989)On the role of mitochondria in cell injury caused by vanadate-induced Ca2+overload. Toxicology 57,29-44. (63) Lehninger, A. L., Vercesi, A,, and Bababunmi, E. A. (1978) Regulation of Ca2+release from mitochondria by the oxidationreduction state of pyridine nucleotides. Proc. Natl. Acad. Sci. U.S.A. 75, 1690-1694. (64) Lotscher, H., Winterhalter, K. H., and Carafoli, E., and Richter, C. (1980)The energy state of mitochondria during the transport of Ca2+. Eur. J . Biochem. 110,211-216. (65) Lotscher, H.,Winterhalter, K. H., Carafoli, E., and Richter, C. (1980)Hydroperoxide-induced loss of pyridine nucleotides and release of calcium from liver mitochondria. J. Biol. Chem. 255, 9325-9330.
Chem. Res. Toxicol., Vol. 3, No. 6, 1990 493 (66) Bellomo, G., Jewell, S. A,, and Orrenius, S. (1982)The metabolism of menadione impairs the ability of rat liver mitochondria to take up and retain CaZt. J . Biol. Chem. 257, 11558-11562. (67) Bellomo, G., Martino, A. P., Richelmi, P., Moore, G. A,, Jewell, S. A., and Orrenius, S. (1984)Pyridine nucleotide oxidation, Ca2+ cycling and membrane damage during tert-butyl hydroperoxide metabolism by rat liver mitochondria. Eur. J. Biochem. 140,1-6. (68) Masini, A., Ceccarelli, D., Trenti, T., Corongiu, F. P., and Muscatello, U. (1989)Perturbation of liver mitochondrial Ca2+ homeostasis in experimental iron overload: a possible factor in cell injury. Biochim. Biophys. Acta 1014, 133-140. (69) Baumhuter, S., and Richter, C. (1982)The hydroperoxide-induced release of mitochondrial calcium occurs via a distinct pathway and leaves mitochondria intact. FEBS Lett. 148, 271-275. (70) Moore, G. A., Jewell, S. A., Bellomo, G., and Orrenius, S. (1983) On the relationship between Ca2+efflux and membrane damage during tert-butyl hydroperoxide metabolism by liver mitochondria. FEBS Lett. 153,289-292. (71) Pfeiffer, D. R., Schmid, P. C., Beatrice, M. C., and Schmid, H. H. 0. (1979)Intramitochondrial phospholipase activity and the effects of Ca2+plus N-ethylmaleimide on mitochondrial functions. J . Biol. Chem. 254, 11485-11494. (72) Beatrice, M. C., Stiers, D. L., and Pfeiffer, D. R. (1980)The relationship between mitochondrial membrane permeability, membrane potential and retention of Ca2+by mitochondria. J . Biol. Chem. 255,8663-8671. (73) Beatrice, M.C., Stiers, D. L., and Pfeiffer, D. R. (1984)The role of glutathione in the retention of Ca2+by liver mitochondria. J . Biol. Chem. 259, 1279-1287. (74) Igbavoba, U.,Zwizinski, C. W., and Pfeiffer, D. R. (1989)Release of mitochondrial matrix proteins through a Ca2+-requiring, cyclosporin-sensitive pathway. Biochem. Biophys. Res. Commun. 161,619-625. (75) Crompton, M., and Costi, A., (1988)Kinetic evidence for a heart mitochondrial pore activated by Ca2+,inorganic phosphate and oxidative stress. Eur. J. Biochem. 178,489-501. (76) Malis, C. D., and Bonventre, J. V. (1986)Mechanism of calcium potentiation of oxygen free radical injury to renal mitochondria. J . Biol. Chem. 261, 14201-14208. (77) Pascoe, G. A., and Reed, D. J. (1989)Cell calcium, vitamin E and the thiol redox system in cytotoxicity. Free Radical Biol. Med. 6,209-224. (78) Gores, G. J., Herman, B., and Lemasters, J. J. (1990)Plasma membrane bleb formation and rupture: a common feature of hepatocellular injury. Hepatology 11, 690-698. (79) Phelps, P. C.,Smith, M. W., and Trump, B. F. (1989)Cytosolic ionized calcium and bleb formation after acute cell injury of cultured rabbit renal tubule cells. Lab. Inuest. 60,630-642. (80) Berdshadski, A. D., and Vasiliev, J. M. (1988)Cytoskeleton, Plenum Press, New York. (81) Pollad, T.D. (1986)Assembly and dynamics of the actin filament system in nonmuscle cells. J . Cell. Biochem. 31, 87-95. (82) Pollad, T. D., and Cooper, J. A. (1986)Actin and actin binding proteins. A critical evaluation of mechanisms and functions. Annu. Reu. Biochem. 55,987-1035. (83) Rosemberg, S.,Stracher, A., and Lucas, R. C. (1981)Isolation and characterization of a-actinin from human platelets. J. Cell. Bid. 91,201-211. (84) Dayton, W. R., Shollmayer, J., Lepley, R. A., and Cortes, L. R. (1981)A calcium-activated protease possibly involved in myofibrillar protein turnover. Isolation of a low calcium-requiring form of the protease. Biochim. Biophys. Acta 659,48-61. (85) Fox, J. E. B., and Phillips, D. R. (1983)Stimulus-induced activation of the calcium-dependent protease within platelets. Cell Motil. 3, 579-588. (86) Harris, A. S., and Morrow, J. S. (1990)Calmodulin and calcium-dependent protease I coordinately regulate the interaction of fodrin with actin. Proc. Natl. Acad. Sci. U.S.A. 87,3009-3013. (87) Mirabelli, F., Salis, A., Vairetti, M., Bellomo, G., Thor, H., and Orrenius, S. (1989)Cytoskeletal alterations in human platelets exposed to oxidative stress are modulated by oxidative and Ca*+-dependent mechanisms. Arch. Biochem. Biophys. 270, 478-488. (88) Bellomo, G., Mirabelli, F., Richelmi, P., Malorni, W., Iosi, F., and Orrenius, S. (1990)The cytoskeleton as a target in quinone toxicity. Free Radical Res. Commun. 8,391-399. (89) Steenbergen, C., Hill, M. L., and Jennings, R. B. (1987)Cytoskeletal damage during myocardial ischemia: changes in vinculin immunofluorescence staining during total in vitro ischemia
494 Chem. Res. Toxicol., Vol. 3, No. 6, 1990 in canine heart. Circ. Res. 60, 478-486. (90) Gaskin, F., Cantor, C. R., and Shelanski, M. R. (1974) Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules. J . Mol. Biol. 89, 737-758. (91) Olmsted, J. B. (1986) Microtubule-associated proteins. Annu. Reu. Cell Riol. 2, 421-457. (92) Shelanski, M. L. (1990) Intracellular ionic calcium and the cytoskeleton in living cells. Ann. N.Y. Acad. Sci. 568, 121-124. (93) Bellomo, G., Mirabelli, F., Vairetti, M., Iosi, F., and Malorni, W.(1990) Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells. Biochemical and immunocytochemical features. J . Cell. Physiol. 143, 118-128. (94) Traub, P. (1985) Intermediate Filaments: A Reuiew, Springer-Verlag, Berlin. (95) Van Kuijk, F. J. G. M., Sevanian, A., Handelman, G. J., and Dratz, E. A. (1987) A new role for phospholipase A,: protection of membranes from lipid peroxidation damage. Trends Biochem. Sei. 12, 31-34. (96) Chien, K. R., Pfau, R. G., and Farber, J. L. (1979) Ischemic myocardic injury. Prevention by chlorpromazine of an accelerated phospholipid degradation and associated membrane dysfunction. Am. J . Pathol. 97, 505-530. (97) Farber, J . L., and Young, E. E. (1981) Accelerated phospholipid degradation in anoxic rat hepatocytes. Arch. Biochem. Biophys. 221, 312-320. (98) Glende, E. A,, Jr., and Pushpendran, K. C. (1986) Activation of phospholipase A, by carbon tetrachloride in isolated rat hepatocytes. Riochem. Pharmacol. 35, 3301-3307. (99) Nicotera, P., Rundgren, M., Porubek, D., Cotgreave, J., MoldBus, P., Orrenius, S., and Nelson, S. D. (1989) On the role of Ca2+in the toxicity of alkylating and oxidizing quinone imines in isolated hepatocytes. Chem. Res. Toxicol. 2, 46-50. (100) Murachi, T. (1983) Intracellular Ca2+proteases and its inhibitor protein: calpain and calpastatin. In Calcium and Cell Function (Cheung, W.Y., Ed.) Vol. IV, pp 376-410, Academic Press, Orlando, FL. (101) Mellgren, R. L. (1987) Calcium-dependent proteases: an enzyme system active a t cellular membranes? (1987) FASEB J . 1, 110-115. (102) Imahori, K. (1982) Calcium-dependent neutral protease: its characterization and regulation. In Calcium a n d Cell Function (Cheung, W. Y., Ed.) Vol. 111, Academic Press, Orlando, FL. (103) Nicotera, P., Hartzell, P., Baldi, C., Svensson, S. A., Bellomo, G., and Orrenius, S. (1986) Cystamine induces toxicity in hepa-
Nicotera et al. tocytes through the elevation of cytosolic Ca2+and the stimulation of a non-lysosomal proteolytic system. J . Biol. Chem. 261, 14628-14635. (104) Tzenp.. W. F.. and Chen. Y. H. (1988) Sumression of snakevenom caydiomyocyte degeneration by blockage of Ca2+influx or inhibition of non-lysosomal proteinases. Biochem. J . 256,89-95. (105) Arends, M. J., Morris, R. G., and Wyllie, A. H. (1990) Apoptosis: The role of the endonuclease. Am. J. Pathol. 136,593-608. (106) Wyllie, A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 280, 555-556. (107) McConkey, D. J., Hartzell, P., Amador-PBrez, J. F., Orrenius, S., and Jondal, M. (1989) Calcium-dependent killing of immature thymocytes by stimulation via CD3/T cell receptor complex. J . Immunol. 143, 1801-1806. (108) McConkey, D. J., Hartzell, P., Nicotera, P., and Orrenius, S. (1989) Calcium-activated DNA fragmentation kills immature thymocytes. FASEB J . 3, 1843-1849. (109) Jones, D. P., McConkey, D. J., Nicotera, P., and Orrenius, S. (1989) Calcium-activated DNA fragmentation in rat liver nuclei. J . Biol. Chem. 264, 6398-6403. (110) Chang, M. P., Baldwin, R. L., Bruce, C., and Wisnieski, B. J. (1989) Second cytotoxic pathway of diphteria toxin suggested by nuclease activity. Science 246, 1165-1168. (111) Waring, P., Eichner, R. D., Mullbacher, A., and Sjaarda, A. (1988) Gliotoxin induces apoptosis in macrophages unrelated to its antiphagocytic properties. Science 263, 18493-18499. (112) Bellomo, G., Perotti, M., Taddei, F., Mirabelli, F., Finardi, G., Nicotera, P., and Orrenius, S. (1990) TNF-a kills adenocarcinoma cells by an increase in nuclear free Ca2+concentration and DNA fragmentation. Science (submitted for publication). (113) Brune, B., Hartzell, P., Nicotera, P., and Orrenius, S. (1990) Spermine prevents endonuclease activation and thymocyte apoptosis. J . Biol. Chem. (submitted for publication). (114) Udvardy, A., Schedl, P., Sander, M., and Hsieh, T. (1986) Topoisomerase I1 cleavage in chromatin. J . Mol. Biol. 191, 231-246. (115) Perotti, M., Taddei, F., Mirabelli, F., Vairetti, M., Bellomo, G., McConkey, D. J., and Orrenius, S. (1990) Calcium-dependent DNA fragmentation in human synovial cells exposed t o cold shock. FEBS Lett. 259, 331-334. (116) Reed, D. J. (1990) Review of the current status of calcium and thiols in cellular injury. Chem. Res. Toxicol. (second of three Forum articles in this issue).
