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The Role of Calcium in Lethal Cell Injury John L. Farber* Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 Received August 2, 1990 There has been considerable interest recently in the role that calcium ions play in toxic cell injury. Whereas much of the work addressing this problem has employed isolated rat hepatocytes, either freshly suspended or cultured, the results of these studies have led to seemingly conflicting conclusions. In turn, there exists some confusion as to which interpretation is more relevant to the action of the same or similar agents in the intact animal. We have been using primary cultures of adult rat hepatocytes to study the role of calcium in the genesis of the lethal cell injury that results from exposure to a variety of environmental hazards. The present report offers an overview of these studies and the conclusions stemming from them. It also attempts to assess the bases for the differing conclusions derived from the study of suspended hepatocytes. Finally, an attempt is made to relate the use of cultured hepatocytes to the concerns of whole animal toxicology.
Advantages of the Use of Cultured Hepatocytes Cultured rat hepatocytes have several important advantages as an experimental model. Hepatocytes are readily isolated in high yield and with good viability. Freshly isolated rat hepatocytes promptly attach to plastic culture dishes with essentially 100% plating efficiency. Within 2 h, only dead cells remain unattached and floating in the culture medium. They are easily removed by simply discarding the culture medium, washing the cells, and adding fresh medium. A t this point, all the hepatocytes in the culture are viable and begin to spread out over the surface of the plastic dish. After 24 h in culture, the hepatocytes are firmly attached to the plastic substratum. Adjacent cells have formed bile canaliculi, and the surface area of individual hepatocytes is sufficient to readily allow digital imaging fluorescent microscopy of single hepatocytes loaded with fura-2 for the determination of the cytosolic free calcium concentration. Importantly, the medium in which the hepatocytes are cultured can be manipulated in a number of ways that have proven quite helpful for the analysis of the mechanisms of toxic cell injury. In particular, the hepatocytes can be cultured for many hours in a medium devoid of calcium ions and serum without any loss of viability. Thus, the relative roles of extracellular and intracellular calcium ions in the genesis of lethal cell injury can be readily assessed. Role of Extracellular Calcium Like most other cell types, cultured hepatocytes maintain a cytosolic calcium ion concentration, [Ca2+Ii,around 100-200 nM (I). An increase in this basal [Ca2+Iioccurs in one of two ways: as a result of an increased net influx (influx minus efflux) of calcium ions across the plasma membrane from the millimolar concentrations that exist in the extracellular fluids bathing all cells; or as a result * Address correspondence to this author a t the Department of Pathology, Room 203A, Main, Thomas Jefferson University, Philadelphia, PA 19107. 0893-228~/90/2703-0503$02.50/0
of a release into the cytosol of sequestered stores of intracellular calcium, primarily those in the cisternae of the endoplasmic reticulum and to a lesser extent in the mitochondria. An elevated [Ca2+Iithat results from an increase in the net influx of calcium across the plasma membrane can reflect an increase in the permeability of the plasma membrane to extracellular calcium ions. In this situation, there is an increased passive diffusion of calcium ions into the cell. Alternatively, an inhibition of the active extrusion of calcium ions from the cytosol, against the very large gradient that exists across the plasma membrane, may also result in an elevated cytosolic calcium ion concentration. Increase in Permeability to Calcium of the Plasma Membrane. The permeability of the plasma membrane to calcium ions can be readily increased by treating the cells with a calcium ionophore, such as A23187 or ionomycin. Within 1min of treating cultured hepatocytes with micromolar concentrations of either ionophore, [Ca2+Iirises an order of magnitude from 150-200 nM to 1.5-2.0 NM (2). Cell death follows within 10-15 min, as evidenced by a generalized loss of the permeability barrier function of the plasma membrane (2-4). Importantly, concentrations of either ionophore can be used such that the cell killing is totally dependent on the presence of extracellular calcium ions (2-4). In other words, the same concentrations of the ionophore in a culture medium that contains less than 20 pM total calcium are not accompanied by any loss of viability over the same time course in which virtually all of the cells are killed in the presence of extracellular calcium (2-4). Furthermore, there is no loss of viability of control hepatocytes maintained for hours in the presence or absence of extracellular calcium without exposure to either ionophore (2-4). Thus, flooding the cytosol of a cultured hepatocyte with calcium ions is toxic to the cell. Cell killing is evidenced by a profound loss of the integrity of the plasma membrane with the release into the medium of constituents normally kept within the cell, e.g., cytosolic enzymes, and failure to prevent entry of agents normally excluded, such as trypan blue. However, the mechanism(s) by which the elevated cytosolic calcium ion concentration results in this generalized loss of plasma membrane integrity is still debated. It is generally held that activation of calcium-dependent degradative processes mediates the loss of viability. In particular, calcium-dependent proteases, phospholipases, and endonucleases may be activated by a sustained increase in [Ca2+Ii. Which of these activities is primarily responsible for the cell killing seems to depend more on the cell type, e.g., hepatocyte or lymphocyte, rather than on the mechanisms by which [Ca2+Iiis increased. With respect to the calcium-dependent killing of cultured hepatocytes, we have reached several conclusions. The cells die in the absence of any evidence of DNA fragmentation.' Protease inhibitors do not protect ( 5 ) . Inhibition of the activation of phospholipases by a variety of means reduces the extent of cell killing (2). Cytochalasin B, an agent that K. Yamamoto and J. L. Farber, unpublished data.
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depolymerizes actin microfilaments and thereby prevents an activation of actin polymerization by an increased [Ca2+Ii,has no protective effect against the toxicity of A23187 (2,3). Thus, in the cultured hepatocyte system, phospholipase activation remains the only mechanism to date that can be implicated in the lethal cell injury that results from a sustained elevation in the cytosolic calcium ion concentration. Importantly, this conclusion does not necessarily exclude other as yet unidentified mechanisms by which a sustained elevation of [Ca2+Iiin a cultured hepatocyte leads to a loss of viability. These results with cultured hepatocytes differ from those initially reported from the use of suspensions of freshly isolated hepatocytes (6, 7). When suspended hepatocytes were treated with A23187 in a Krebs-Henseleit buffer (2.6 mM Ca2+)or in Ca2+-and Mg2+-freeHank’s solution, loss of viability principally occurred in the absence of extracellular calcium (6, 7). Interestingly, such a dependence of toxicity on the absence of extracellular calcium was also seen with a number of other agents (6). One in particular, bromobenzene, is a potent hepatotoxin in intact animals. Cell killing under these conditions was attributed to a redistribution of intracellular calcium stores (7). These results with suspended hepatocytes (6, 7) are difficult to reconcile with the opposite findings discussed above from the study of cultured cells (2-4). Clearly, cells in the intact animal never exist in the absence of extracellular calcium. Thus, an experimental model that reports toxicity only in the absence of extracellular calcium (6, 7) must have questionable relevance to the intact animal. In the absence of extracellular calcium, suspensions of freshly isolated hepatocytes exposed to 95% 02-5% C02 rapidly manifest significant cell injury in the absence of any exogenous toxicant (8, 9). Thus, it is likely that the suspended hepatocytes used in the above studies (6, 7) were injured sufficiently by the absence of extracellular calcium to render them susceptible to the toxic effects of the agents employed. In the presence of extracellular calcium, the hepatocytes were healthier and, thus, not susceptible to similar consequences of exposure to the toxicants studied. Such an interpretation would cast further doubt on the relevance to intact animals of the previously reported data (6, 7). Interestingly, 5 years later, the same group now reported killing of suspended hepatocytes by A23187 in the presence of extracellular calcium (10). One can only suspect that the problems with maintaining suspended hepatocytes with 95% 02-5% COPin the absence of calcium were now apparent. However, this still does not account for the initially reported (6, 7) absence of cell killing in the presence of extracellular calcium with even higher concentrations of A23187. No explanation was given (10) for the change in the calcium dependency of the toxicity of A23187, and these data (6, 7, 10) remain difficult to understand. Recent studies have elucidated the mechanism of cell injury that occurs when isolated hepatocytes are maintained in suspension with 95% 02-5% COPin the absence of extracellular calcium (8,9). Under such conditions, the cells are injured by an oxidative stress. An increased formation of partially reduced oxygen species accompanies the stimulation of O2 consumption that, in turn, results from the cycling of calcium ions across the mitochondrial inner membrane. The oxidative cell injury can be reduced by antioxidants or by ruthenium red, an inhibitor of mitochondrial calcium cycling. -423187 presumably augments such cycling and potentiates the cell injury that occurs in
Farber
the absence of extracellular calcium. Two points need emphasis here. First, such a mechanism of cell injury that depends on the absence of extracellular calcium, and the presence of 95% 02,has no immediate correlate in any situation occurring in an intact animal. Second, the effect of A23187 in this circumstance is not relevant to the killing of cultured hepatocytes in the presence of extracellular calcium, as ruthenium red ( I I ) , or antioxidants, have no protective effect on the toxicity of A23187 in the presence of extracellular calcium.2 It remains to be considered whether mitochondrial calcium cycling in cells exposed to other hazards, and under more relevant physiologic circumstances, is a mechanism that has relevance to toxic cell injury. It has been speculated that such calcium cycling may be a general mechanism of oxidative cell injury (12). Whereas one cannot necessarily dismiss this hypothesis, there is little evidence to support it. In particular, ruthenium red does not protect against the cell injury produced by a wide variety of hazards in the presence of extracellular calcium (11).2
Returning to our main theme, it can be asked whether the calcium-dependent killing of cultured hepatocytes by an ionophore is a model for the action of any other toxic agents. In other words, is disruption of the permeability barrier function of the plasma membrane with the consequent influx and accumulation of calcium a mechanism of lethal cell injury in circumstances other than the treatment with a calcium ionophore? We reported previously that treatment of cultured hepatocytes with a variety of agents that act directly on the plasma membrane produced lethal cell injury only in the presence of extracellular calcium (3). Such a result needs to be interpreted carefully. It does not imply, as some may have thought, that cell death under any condition is necessarily dependent on extracellular calcium. This is clearly false. The assays of viability of hepatocytes in culture reflect the integrity of the plasma membrane. In turn, the permeability barrier function of the plasma membrane can be disrupted, and the cells scored as dead, without extracellular calcium being required. Simply treating the cells with a detergent, such as Triton X-100 or deoxycholate, will produce such a result. Our data (3)would suggest that some toxic agents, under specific conditions of dosage and time of exposure, act to render the plasma membrane permeable to the large concentrations of extracellular calcium. The influx and accumulation of calcium then mediates the actual lethal cell injury. A quite different interpretation (13-16) of our data (3) argued that the expression of chemical toxicity under the conditions we reported depended not on the influx of extracellular calcium but rather on the cellular content of vitamin E. This conclusion derived from the observation that addition of 25 pM vitamin E succinate to suspensions of isolated hepatocytes maintained under 95% 02-5% CO, in the absence of extracellular calcium prevented cell injury by selected drugs. Consistent with previous reports (6, 3, the toxicity of these drugs in the absence of vitamin E supplementation was less when extracellular calcium was present. Furthermore, in cells supplemented with vitamin E, injury only occurred in the presence of extracellular calcium, a result consistent with our observations ( 3 ) . Since the medium we use to culture hepatocytes contains vitamin E phosphate (20 nM), it was argued that our system was analogous to suspended hepatocytes supple-
* D. Nakae and J. L. Farber, unpublished data.
Forum mented with 25 WMvitamin E succinate. It needs emphasis that the calcium dependence of the killing of cultured hepatocytes (2-4) is unrelated to either physiologic or pharmacologic levels of vitamin E. Hepatocytes cultured for 24 h and then incubated in Williams’ E medium in an atmosphere of 95% room air-5% C 0 2 are stable for many hours in the absence of extracellular calcium (2-4). Importantly, the hepatocytes remain viable despite the fact that the vitamin E content has fallen to 10-15% of the value at the time the cells were ~ r e p a r e d . ~ It requires at least 1 pM vitamin E phosphate to maintain vitamin E levels in the physiologic range,3 whereas the medium we use (Williams’ E) contains only 20 nM vitamin E phosphate. Thus, the fact that cultured hepatocytes are stable in the absence of extracellular calcium cannot be attributed to an enrichment of vitamin E. Similarly, the cell killing in the presence of extracellular calcium is also unrelated to vitamin E. Suspended hepatocytes supplemented with 25 pM vitamin E succinate and cultured cells maintained with 20 nM vitamin E phosphate differ by an order of magnitude in their content of vitamin E. Thus, the dependence in both systems of the cell killing by A23187 and other agents on extracellular calcium cannot be a consequence of the content of vitamin E. Vitamin E supplementation of suspended hepatocytes (95% 02-5% C02) is necessery in order to prevent the cell injury that occurs in the absence of extracellular calcium. Cultured hepatocytes (95% room a i r d % C02) are not susceptible to such injury in the absence of calcium. We must now consider whether a dependence on extracellular calcium in vitro is relevant to the action of the same or other hazards in the intact animal. In the intact animal, cell death can be assessed by an additional criterion that is not applicable in vitro. When cells die in the intact animal, they manifest a reproducible series of morphological alterations that is termed coagulative necrosis. Necrotic cells contain large accumulations of calcium. Furthermore, it is likely that the morphological changes characteristic of such necrotic cells are produced, at least in part, by the presence of excess calcium ions. It then becomes interesting to consider whether the accumulation of calcium and the resulting necrosis are equivalent to the death of the cell. Alternatively, the cell dies by mechanisms unrelated to calcium accumulation and then becomes necrotic as calcium accumulates. Unfortunately, there are very little data which attempt to distinguish between these two interpretations of the significance of coagulative necrosis. The problem can only be studied in the intact animal, where it is not possible to remove extracellular calcium ions. Thus, one is forced to use inhibitors of calcium fluxes, and the inevitable problem with the use of such inhibitors arises. How does one know that the inhibitor used is indeed acting by inhibiting calcium fluxes, rather than by some as yet poorly characterized mechanism? The study of galactosamine hepatitis provided evidence to link an influx of calcium ions to irreversible liver cell injury in the intact rat. Within 2 h of the administration of galactosamine, the liver cells evidenced changes in their plasma membranes and slight increases in the total liver calcium content (17). However, the liver cells were not necrotic a t this point. The liver cell calcium content continued to rise between 2 and 8 h with the appearance of necrotic cells. Chlorpromazine, an inhibitor of calcium fluxes across biological membranes, given 2 h after the galactosamine prevented any further rise in total liver P. A. Glascott, Jr., and J. L. Farber, unpublished data.
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calcium content and prevented the appearance of liver cell necrosis (18). Ischemic liver necrosis in the intact rat is another model in which there is some evidence implicating an influx of calcium ions in the genesis of irreversible cell injury. The reperfusion of a rat liver that has been ischemic for 2-3 h is accompanied by a rapid increase in the total calcium content of the liver and the development of liver cell necrosis (19). Administration of chlorpromazine just prior to the reestablishment of blood flow reduced the extent of the subsequent liver cell necrosis (19). Again, such a result is consistent but does not establish the point that an influx of calcium ions may be related to the transition from potentially reversible to irreversible cell injury. Inhibition of Active Calcium Efflux. The plasma membrane of all cells does not represent an absolute permeability barrier to the very large extracellular concentrations of calcium. In addition, there are many physiologic processes that depend on a regulated change in the permeability to extracellular calcium. Thus, all cells are endowed with a mechanism to actively extrude calcium ions from the cytosol against a very large concentration gradient. Inhibition of such a calcium pump should lead to a rise in [Ca2+Ii.Indeed, such a situation has been reported (20). Cystamine was reported to inhibit Ca2+efflux in suspensions of isolated hepatocytes, an effect that resulted in the accumulation of intracellular Ca2+and a sustained increase in [Ca2+Ii. This was followed by a stimulation of both phospholipid and protein breakdown. Interestingly, evidence was presented that the activation of a nonlysosomal proteolytic enzyme was more critical than was phospholipase activation for the development of cystamine toxicity (20). Protease inhibitors were also reported to protect suspensions of isolated hepatocytes from the toxicity of A23187 in the presence of extracellular calcium (10). However, no protection by leupeptin of either suspended (11)or cultured hepatocytes (5)treated with A23187 in the presence of extracellular calcium was subsequently reported. The reasons for this failure to confirm protection by protease inhibitors of the calcium-dependent cell killing by A23187 is not entirely clear. We would suggest that protease activation that can mediate, even in part, lethal liver cell injury may require extremely high cytosolic calcium ion concentrations. Such concentrations were probably not achieved under the conditions of treating cultured (11)or suspended hepatocytes (5) with A23187. A quite different situation obtains with the killing by cystamine of cultured (5),as opposed to suspended (20), hepatocytes. Importantly, there is no dependence on extracellular calcium for the killing of cultured hepatocytes by cystamine ( 5 ) . Thus, the sensitivity of the cultured hepatocytes to cystamine was unaffected by the concentration of calcium in the extracellular medium. There was no difference in the extent of the killing by 10 mM cystamine when the calcium concentration of the culture medium was varied from 20 pM to 5 mM CaC12. Furthermore, a rise in cytosolic calcium was prevented without affecting the toxicity of cystamine. Finally, the killing of cultured hepatocytes by cystamine was not accompanied by an accelerated degradation of protein, and the addition of three different protease inhibitors reduced by 4 0 4 0 % the rate of protein turnover without an effect on the extent of cell killing (11). The basis for this difference in the results with cultured as opposed to suspended hepatocytes probably relates again to the fact that suspensions of freshly isolated hepatocytes are more sensitive to extracellular calcium than
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are their cultured counterparts. This can be attributed to damage to the plasma membrane during their preparation. Such cells may be more leaky to calcium than are cultured hepatocytes. Thus, inhibition of calcium efflux from suspended cells (20) would be expected to result in calcium accumulation that is not seen with cultured hepatocytes ( 5 ) .
Role of Alterations in Intracellular Calcium Homeostasis An elevation of the cytosolic calcium concentration can occur by mechanisms that are not dependent on extracellular calcium ions. In particular, hepatocytes, as well as other cell types, sequester calcium ions within the cisternae of the endoplasmic reticulum. Upon either physiologic or pathologic stimulation, these stores can be released into the cytosol with an accompanying rise in [Ca2+Ii. Such an increase in the cytosolic calcium ion concentration attendant on the release of calcium stores has been argued to be an important mechanism in the genesis of lethal cell injury (21-23). Mechanisms of Cell I n j u r y by Activated Oxygen Species. The hypothesis that lethal cell injury can result from a rise in cytosolic calcium following its release from sequestered stores in the endoplasmic reticulum derived from the study of oxidative cell injury (7,24-26). Alterations in intracellular calcium homeostasis that result from an oxidant-induced depletion of both glutathione and protein thiol groups provided an attractive hypothesis of the sequence of events that mediate the lethal cell injury from an oxidative stress. Release of calcium stores certainly occurs upon the reduction of Hz02(or an organic hydroperoxide such as tert-butyl hydroperoxide) and the accumulation of GSSG as a result of glutathione peroxidase activity. Furthermore, a disruption of intracellular calcium homeostasis by treatment of a variety of cell types with the calcium ionophore A23187 can precipitate cell death. Finally, the calcium activation of a variety of degradative processes is a reasonable explanation of the mechanism by which an elevated cytosolic calcium concentration may mediate lethal cell injury. However, there are significant problems with this hypothesis. The most serious deficiency of this hypothesis is that it ignores alternative pathways for the metabolism of hydrogen peroxide. In particular, it ignores the iron-dependent formation of more potent oxidizing species from H202. When the formation of such oxidizing species is prevented, or the toxic consequences of their interactions with cellular constituents is prevented, there is no effect on the biochemical consequences of the metabolism of hydrogen peroxide by peroxidase: GSH is lost, with the parallel accumulation of GSSG; protein thiols are depleted, and there is an increase in the cytosolic calcium ion concentration. However, despite these changes, the cells do not die. In this manner, the killing of cultured hepatocytes by either hydrogen peroxide or tert-butyl hydroperoxide was dissociated from changes in intracellular calcium homeostasis (3,27). The killing of cultured hepatocytes by these peroxides depends on the formation of hydroxyl or tert-butyl alkoxy1 radicals by Fenton chemistry (27-29). The formation of these radicals was prevented, and the liver cells protected from the toxicity of either peroxide, by chelating a cellular pool of ferric iron with deferoxamine (27,28). However, hepatocytes pretreated with deferoxamine still manifested the same early changes in calcium homeostasis as occurred in cells that were not pretreated with deferoxamine (27, 28).
Furber The radicals that are formed in the reaction of hydrogen peroxide or tert-butyl hydroperoxide with iron initiated the peroxidative decomposition of membrane phospholipids of cultured hepatocytes (27,30). Antioxidants added to the culture medium prevented both this lipid peroxidation and the death of the cells without any effect on the reduction of either peroxide by glutathione peroxidase (27, 30). Thus, changes in calcium homeostasis occur without accompanying irreversible liver cell injury from an oxidative stress. Conversely, irreversible injury can occur without changes in calcium homeostasis (3). Pretreating cultured hepatocytes with EGTA in a calcium-free medium removed over 75% of the cell-associated calcium, lowered the basal cytosolic calcium ion concentration, and eliminated its rise in response to hydrogen peroxide. However, these same cells remained sensitive to the toxicity of hydrogen peroxide (3). The above studies assessed changes in calcium homeostasis indirectly, by measuring changes in glycogen phosphorylase. It might be argued that such an indirect measurement may not accurately reflect the true changes in cytosolic calcium ion concentration. Thus, we utilized digital imaging fluorescence microscopy of fura-2-loaded cultured hepatocytes in order to directly assess the relationship between intracellular calcium homeostasis and lethal cell injury from an oxidative stress (31). An additional benefit of the use of cultured hepatocytes was that these studies could be carried out at the single-cell level, an approach that allowed the temporal relationships between changes in [Ca2+Iiand cell death to be determined in individual cells. Within minutes of the addition of tert-butyl hydroperoxide, individual hepatocytes displayed one or more peaks of increased [Ca2+Iithat promptly returned to the prestimulation level (31). This was followed by a slower increase of [Ca2+Iithat reached a plateau 2-3 times higher than the basal level. Another rise in [Caz+li,now abrupt and much larger, preceded the death of the cells as evidenced by the loss of fura-2 fluorescence. Pretreatment of the hepatocytes with deferoxamine, or the addition to the cultures of an antioxidant, prevented the loss of viability. However, neither the number of hepatocytes displaying the initial [Ca2+Iitransients nor the magnitude of these oscillations was affected by pretreating the cells with deferoxamine. Similar results were obtained by addition to the cultures of the antioxidants DPPD or catechol. Deferoxamine, or the antioxidants DPPD and catechol, prevented both the plateau phase and the abrupt rise in [Ca2+Ii. Treatment of the hepatocytes with tert-butyl hydroperoxide in a low-calcium buffer (
