Calcium Dependence of Phalloidin-Induced Liver Cell Death (Toxin/Plasma Membrane/Cytochalasin B/Microfilaments/Scanning Electron Microscopy) AGNES B
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Proc. Nati. Acad. Sci. USA Vol. 77, No. 2, pp. 1177-1180, February 1980 Medical Sciences Calcium dependence of phalloidin-induced liver cell death (toxin/plasma membrane/cytochalasin B/microfilaments/scanning electron microscopy) AGNES B. KANE, ELLORA E. YOUNG, FRANCIS A. X. SCHANNE, AND JOHN L. FARBER* Department of Pathology and the Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Communicated by Hans Popper, December 10, 1979 ABSTRACT The role of Ca2+ in toxic liver cell death was Phalloidin is a toxic bicyclic heptapeptide isolated from the studied with primary cultures of adult rat hepatocytes. Within mushroom Amanita phalloides (16, 17). Treated animals die 1 hr of exposure to phalloidin, a bicyclic heptapeptide isolated within a few hours with a hemorrhagic necrosis of the liver from the mushroom Amanita phalloides, at 50 ,g/ml, 60-70% of the cells were dead (trypan blue stainable). There was no loss characterized by numerous nonfatty vacuoles (18). Phalloidin of viability of the same cells exposed to phalloidin in culture is active on rat hepatocytes in uitro, where it produces easily medium devoid of Ca2+. A marked structural alteration of the observed deformations of the cell surface that accompany the surface of the phalloidin-treated hepatocytes characterized by death of the cells (14, 15). The protrusions or evaginations of innumerable evaginations seen by scanning electron microscopy the plasma membrane seen by scanning electron microscopy occurred in the presence or absence of Ca2+. Pretreatment of are felt to to the of the the cells with cytochalasin B at Ig/ml10 prevented the surface correspond invaginations plasma alteration and the death of the cells in Ca2+ medium. Exposure membrane and subsequent vacuolization of the cells in the in- of the cells to phalloidin in the absence of Ca2+ followed by tact liver as seen by transmission electron microscopy (14, 15, exposure to cytochalasin B and then to Ca2+ also prevented the 18). The molecular basis of this injury most likely lies in the cell death. These results suggest a two-step mechanism by which interaction of phalloidin with actin filaments intimately asso- phalloidin causes liver cell death. Initially phalloidin interacts ciated with the plasma membrane (19-21). Phalloidin accel- in a Ca2+-independent process with cell membrane-associated erates the polymerization of G-actin to filamentous structures actin. The second step is a Ca2+-dependent process that most likely represents an increased influx of Ca2+ across a compro- (Ph-actin) that are more resistant than F-actin to the destabi- mised cell membrane permeability barrier and down the steep lizing effects of 0.6 M KI (22-25). Liver plasma membranes concentration gradient that exists between the outside and in- isolated from phalloidin-treated rats contain increased fila- side of the cell. These results strengthen the hypothesis that mentous structures (26). [3H]Desemethylphalloin, a phallotoxin disturbances in Ca2+ homeostasis induced in vivo by a variety chemically similar to phalloidin, binds to isolated rat liver of hepatotoxins are causally related to liver cell death. plasma membranes with a dissociation constant identical to that of the binding of the same toxin to muscle actin (25, 26). These A possible role for calcium in the production of liver necrosis data suggest that phalloidin-binding sites in the rat liver plasma was suggested by investigations of Judah and colleagues membrane are predominantly actin isolated together with the showing that some anti-inflammatory drugs give considerable membrane fragments. protection to the liver against the action of hepatotoxins (1-6). In the present report we have used this ability of phalloidin Although calcium had been known for many years to accu- to kill rat hepatocytes in culture to assess the role of Ca2+ in toxic mulate in necrotic tissues, these studies showed an early increase liver cell death. It is shown that phalloidin interacts with isolated in the calcium content of the cells that was prevented by pro- hepatocytes to produce alterations of the cell surface inde- methazine and that could be related to inhibition of various pendently of extracellular Ca2+. However, the death of the cells enzyme activities. Antihistamines such as promethazine were is known to block ion movements, and it was suggested that the absolutely dependent upon the presence of Ca2+ in the cul- is ture medium. These results suggest that the death of liver cells primary lesion a loss of the semipermeable properties of the in the intact animal is very likely to be similarly dependent upon cells with consequent entry of lethal concentrations of Ca2+. calcium ions. Subsequent studies documenting alteration in calcium ho- meostasis with carbon tetrachloride supported this hypothesis MATERIALS AND METHODS (7-11). The antihistamine compounds also protected against CCI4-induced liver necrosis (6). With galactosamine a corre- Isolated hepatocytes were prepared from the livers of nonfasted lation was shown among early plasma membrane injury, an female Wistar rats (150-200 g) by the method of collagenase increased Ca2+ content, and the ability of uridine to prevent perfusion as described by Laishes and Williams (27). Colla- and to reverse these changes while preventing the liver cell genase (type 1, Sigma) at 100 units/ml in 250 ml of Hanks' death (12, 13). balanced salt solution at pH 7.4 (28) (Flow Laboratories, Such accumulations of Ca2+ could always be explained, McLean, VA) was recirculated through the liver for 15 min at however, as simply a passive equilibration of Ca2+ concentra- 37°C at a flow rate of 32 ml/min. A yield of 2-4 X 108 cells with tions in cells lethally injured by different and as yet unexplained 85-90% viability (trypan blue exclusion) was obtained. mechanisms. The ability to maintain isolated hepatocytes in The hepatocytes were plated in plastic multiwell chambers culture for several days has provided a means for more directly (Costar, Cambridge, MA) with wells 1.6 cm in diameter at a exploring the role of Ca2+ in toxic liver cell death. In particular, density of 5 X 104/cm2 in Williams' medium E (29) (Flow the effects of phalloidin on such cells (14, 15) provide an ideal Laboratories) containing 10% inactivated (560C for 10 min) experimental model. fetal calf serum (Flow Laboratories), garamycin at 50 Mg/ml, and insulin at 0.02 units/ml. Williams' medium E plus serum, The publication costs of this article were defrayed in part by page garamycin, and insulin is referred to below as complete Wil- charge payment. This article must therefore be hereby marked "ad- liams' medium. After incubation at 370C in a humidified at- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom correspondence should be addressed. 1177 Downloaded by guest on September 24, 2021 1178 Medical Sciences: Kane et al. Proc. Natl. Acad. Sci. USA 77 (1980) mosphere of 5% C02/95% air for 90 min to allow the cells to In the presence of 3.6 mM CaCl2, phalloidin at 50 ug/ml attach, the cultures were rinsed three times with pre-warmed killed 60-70% of the cells within 1 hr as evidenced by their Hanks' balanced salt solution and incubated for 15-20 hr in inability to exclude trypan blue (Table 1). This represents complete Williams' medium. Prior to treatment of the cultures 60-90% of the hepatocytes, because at least 20% of the cells are with phalloidin, cytochalasin B, or the Ca2+ ionophore A23187, probably Kupffer cells as evidenced by the presence of Fc re- the cells were rinsed four times with prewarmed Ca2+-free ceptors (unpublished data). In the absence of extracellular Ca2 , Hanks' balanced salt solution and placed in Williams' medium the same concentration of phalloidin had no effect on cell via- E without CaC12 (made under special order by Flow Labora- bility (Table 1). Without any calcium in the medium the cells tories) and serum. Where indicated CaCI2 was added to give remained viable in the presence of phalloidin for at least 8 hr. a final concentration of 3.6 mM. If the cells were exposed to phalloidin for 1 hr in the absence Phalloidin (Boehringer Mannheim) was dissolved in Ca2 - of Ca2+, washed six times, and then exposed to medium con- free Hanks' solution and added to the cultures at a concentration taining Ca2+, 54% of the cells took up trypan blue within 2 hr of 50 Mg/ml. A23187 was a gift from R. L. Hammill (Eli Lilly (Table 1). We have shown previously (32) that this use of trypan Research Laboratories, Indianapolis, IN). It was dissolved in blue to assess the viability of cultured hepatocytes correlates absolute ethanol (1 mg/ml) and added to the cultures at a final exactly with two independent assays, the hydrolysis of fluo- concentration of 10 yg/ml. Ethanol alone did not affect cell rescein diacetate and the plating efficiency. viability. Cytochalasin B (Sigma) was dissolved in Ca2 -free In order to interpret these results it is necessary to know Hanks' solution and added to the cultures at a concentration of whether the interaction of phalloidin with the liver cells is de- 10 Yg/ml. pendent upon extracellular Ca2+. This interaction produces Cell viability was assayed by trypan blue exclusion. Trypan morphological alterations in the surface of the hepatocytes blue (0.4% in 0.15% NaCl, GIBCO) was added directly to the easily appreciated with scanning electron microscopy (14, 15). cultures at a final concentration of 0.01%. Within 10 min the Fig. 1A illustrates the appearance by scanning electron mi- attached cells that excluded the dye were counted by using a croscopy of a representative hepatocyte 1 hr after exposure to 10-mm2 eyepiece grid in an inverted microscope at X200 phalloidin at 50 Mg/ml in the presence of extracellular Ca2+.