Sensitization of rat hepatocytes to hyperthermia by calcium

JOURNAL OF CELLULAR PHYSIOLOGY 128:279-284 (1986)

Sensitization of Rat Hepatocytes to yperthermia by Calcium

ARUN MALHOTRA, jACK KRUUV, AND jAMES R. kEPOCK* Guelph-Waterloo Program for Graduate Work in Physics and Department of Biology,

University of Waterloo, Waterloo, Ontario N2L 3G1, Canada

The viability of isolated rat hepatocytes, as assayed by trypan blue exclusion, decreases in a dose-dependent fashion during exposure to hyperthermia (Do [43OC] = 105 & 10 min, Do [45OC] = 24 & 4 min). Hyperthermic sensitivity varies as a function of extracellular Ca2+ concentration in a biphasic manner; optimum survival occurs at 1-5 mM Ca2+, with sensitization in the absence of Ca+ and increasing sensitization at Ca2+ concentrations greater than 10 mM. Ca influx does not correlate well with loss of viability for hepatocytes in 4 mM extracellular Ca2+; influx does not occur until viability decreases to less than 4%. Under sensitizing conditions, Ca2+ influx preceeds loss of viability. Influx begins within 15 min at 45OC in 15 mM Ca2+, and the ionophore A23187 is a potent hyperthermic sensitizer in the presence of extracellular Ca2+. Thus, Ca2+ influx, whether caused by high extracellular Ca2+ or A23187, increases cellular damage caused by supraoptimal temperatures, although some Ca2+ is necessary for maximum resistance, probably because of stabilization of Ca2+ binding proteins against thermal denaturation or possibly to Ca2+-induced decrease in lipid fluidity.

The basic mechanisms of cell killing by supraoptimal temperatures are not understood, although there is rea- son to believe that cellular membranes are involved in some way. Local anesthetics, which are known to be membrane active agents, are potent hyperthermic sensitizers in both bacteria (Yatvin, 1977) and mammalian cells (Yau, 1979). Membrane lipid composition modu- lates the hyperthermic sensitivity of cells. Supplemen- tation of bacteria (Overath et al., 1970; Yatvin, 1977) or mammalian cells (Hidvegi et al., 1980) with fatty acids of greater unsaturation than are normally present in cell membranes both increases membrane fluidity and sensitizes cells to high temperatures.

Damage to membranes from mammalian cells, in the form of membrane protein denaturation, occurs over the same temperature range as hyperthermic killing. Most mammalian cells are killed by temperatures in excess of 41-42°C (Leith et al., 1977). The erythrocyte membrane protein spectrin denatures above approximately 40°C (Brandts et al., 1977). Also, both mitochondria1 and plasma membranes isolated from Chinese hamster lung V79 cells experience protein denaturation, with an onset of approximately 40°C, detectable by fluorescence and electron spin resonance (ESR) probes and differential scanning calorimetry (DSC) (Lepock, 1982; Lepock et al., 1983). Thus, there is a good correlation between the onset temperatures of membrane protein denaturation and cell killing (Lepock, 1982).

Many specific membrane functional alterations have been detected in mammalian cells after heating. There are changes in permeability and transport of many compounds; for example, inhibition of Na f -dependent amino acid transport has been observed (Lin et al., 1978), the

active transport of Na+ and K+ appears to be inhibited and there is loss of K+ (Burdon and Cutmore, 1982; Ruifrok et al., 1985), and the permeability of the plasma membrane to some drugs is altered (Brown et al., 1983). The binding of insulin (Calderwood and Hahn, 1983), epidermal growth factor (EGF) Magun and Fennie, 1981), and some antibodies (Medhi et al., 1984) to cell surface receptors is reduced in a dose-dependent manner by hyperthermia.

Damage of the type discussed above might be expected to cause changes in the permeability of other compounds and ions, particularly @a2+. An influx of Ca2+ across the plasma membrane has been implicated in the killing of cells, particularly hepatocytes, by several different stresses. Ischemia (Trump et al., 1980) and some toxic chemicals, e.g. phalloidin (Kane et al., 1980), cause an increased uptake of Ca2+ that correlates well with killing. High levels of cytoplasmic Ca2', in addition to altering metabolism, activate lipases and proteases that may irreversibly program a cell to death (Trump et al., 1980). Accumulation of cytoplasmic calcium ions appears to be a universal, and possibly final, step in cell death CFarber, 1981).

MATERIALS AND METHODS Sources

Chemicals were obtained from the following sources: collagen, Flow Laboratories; heparin, Fisher Scientific; A23187, Calbiochem Behring; collagenase, Boehringer Manheim; and 45CaC12, New England Nuclear.

Received November 14, 1985; accepted April 8, 1986. *To whom reprint requestskorrespondence should be addressed.

0 1986 ALAN R. LISS, INC.

280 MALHOTRA, KRUUV, AND LEPOCK

Hepatocyte isolation and maintenance Hepatocytes were isolated from nonfasted male Wistar

rats, 10-12 weeks old (250-300 g), by minor modification of the two-step collagenase perfusion technique (19). Briefly, the animals were mildly anesthetized under ether, and the liver was perfused in vivo with a Ca2+- free Hepes-buffered salt solution (NaC1137 mM, KCI 5.4 mM, Hepes 25 mM, pH 7.4) at a flow rate of 20 mVrnin for 10 min. The perfusate was then changed to the same buffer containing 0.025% collagenase and 5 mM Ca" at a flow rate of 10 mVmin for 10 min. Perfusion solutions were bubbled with oxygen, and the temperature was maintained at 38°C.

The liver was surgically removed, and the cells were incubated in collagenase solution at 38°C for 10 min. After filtration of the cell suspension through a sterile gauze, hepatocytes were washed in basal Eagle's Me- dium (BME) supplemented with L-glutamine, the anti- biotics penicillin and streptomycin, and 1% fetal calf serum. The cells were seeded into colla en coated 25 cm2 plastic flasks at a density of 1-2 x 10 celldflask in 5 ml of preequilibrated BME. They were maintained in culture as monolayers at 37°C in a humidified incubator in an atmosphere of 95% air and 5% COz.

The medium was changed 2 hr postseeding to elimi- nate unattached (nonviable) hepatocytes. After 8 hr at 37 "C, the cells were exposed to experimental conditions. (Preparations with at least 95% viability were used for all experimentation.)

Hyperthermia For the heating experiments in the presence or ab-

sence of calcium, the cells were rinsed twice with warm Ca2+-free solution (NaC1 137 mM, KC1 5.4 mM, MgClz 0.5 mM, dextrose 1%, Hepes 25 mM, pH 7.4) and placed in the above solution containing various concentrations of Ca2+ plus 0.1 mM EGTA. The flasks were tightly sealed and placed in a waterbath at the desired temperature. Viability was measured immediately after heating.

Viability The viability was assessed in hepatocytes by trypan

blue exclusion. Trypan blue (pH 7.4) was added directly to the flasks (0.04% final concentration), which were incubated for 3 min, and the attached dye-excluding (viable) and the stained cells (nonviable) were counted. Approximately 700 cells from each flask were counted for each survival point.

A23187 experiments The calcium ionophore A23187 was added to the cells,

either in the presence or absence of calcium, from a stock solution of 2 mM A23187 in dimethylsulfoxide (DMSO). The resulting concentrations of DMSO had no effect on cell viability at 37°C or 43°C.

Calcium uptake The uptake of calcium was measured after incubation

of cells at 37°C or 45°C for various lengths of time in calcium-containing solutions (4 or 15 mM Ca") with 1.7 pCi/ml of 45Ca. At each time interval, cells were thor- oughly washed to remove external, surface bound iso- tope. Cells were rinsed twice with a cold (4'0,

8

nonradioactive calcium solution (NaCl 137 mM, KC15.4 mM, MgC12 0.5 mM, dextrose 1%, Hepes 25 mM, pH 7.4) followed by two washes with a cold lanthanum chloride solution (LaCI3 80.8 mM, glucose I1 mM, Hepes 25 mM, pH 7.2). The cells were then left on ice for 60 min (a high concentration of La3+ at low temperature decreases the slow component of 45Ca ef'flux and increases the rate of loss of more superficial, surface-bound Ca2'). After the last wash, cells were solubilized in sodium deoxycholate (0.1% in 0.1 N NaOH). 45Ca was counted in "Picofluor 15" in a Mark III liquid scintillation counter.

RESULTS When hepatocytes are exposed to a hyperthermic

treatment (43"C, 135 min) at various extracellular calcium concentrations, a distinct biphasic response of ther- mosensitivity is observed (Fig. 1). Viability as a function of extracellular Ca2' concentrations peaks at 1-5 mM Ca2+, with some hyperthermic sensitization in Ca2'- free solution and increasingly greater sensitization above 5 mM Ca2+. Numerous full survival curves re- producibly demonstrate greater viability when the cells are heated in 4 compared to 0 mM Ca2' (See Figs. 4,5). Heating of hepatocytes in 0.1 mM EGTA in the absence of extracellular Ca2+ did not increase the number of cells that released from the plate. This was assayed by counting the cells released with a Celloscope cell counter.

This biphasic response of survival as a function of Ca2' concentration during heating suggests that some Ca2' is necessary for o timal survival, but that an excess of extracellular Ca'+ is toxic. An attractive hypoth- esis is that toxicity in high extracellular Ca2' is due to an influx of Ca2' durin heating, which was further investigated using the Ca!' ionophore A23187. The ionophore A23187 is known to increase the passive flux of Ca" across membranes. The viability of hepatocytes is unaffected by exposure of up to 2 pm A23187 at 37°C

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Ca2+ (mM1 Fig. 1. Viability of rat hepatocytes as a function of Ca2+ concentration during exposure to 43°C (0) or 37°C (0) for 135 min. In this and all subsequent figures, a standard deviation larger than the symbols is represented by error bars.

CALCIUM AND HYPERTHERMIA 281

I I I

I1 I I I 1 0 30 60

TIME (min)

Fig. 2. The viability of rat hepatocytes as a function of time at 37°C during exposure to the Ca" ionophore A23187 at concentrations of 2 (O), 5 ( 0 ) , and 20 (W pm.

(Fig. 2). Higher concentrations of the ionophore cause a time and dose-dependent cytotoxicity during the incubation period. These measurements were made in BME with 1% fetal calf serum, which contains approximately 2.9 mM Ca2' (as determined by atomic absorption spectroscopy).

The toxicity of A23187 rerquires extracellular Ca2'. This can be inferred from Figure 3, which shows that concentrations of A23187 of up to at least 20 pM are not toxic in the absence of Ca2', while much lower concentrations are toxic in the presence of Ca2+.

The ionophore A23187 sensitizes hepatocytes to hyperthermia. Hepatocytes were exposed to 1 pm A23187 at 43°C in the absence or presence of 4 mM Ca2' (Fig. 4). Slight sensitization by A23187 was observed in the absence of Ca2', probably caused either by a direct effect of A23187 on the plasma membrane or by the release of Ca2+ from intracellular stores. However, the presence of external calcium significantly enhanced the sensitization by A23187 under the same conditions, implying that an increased influx of Ca2+ during hyperthermia can be toxic. In addition, this figure demonstrates the increased sensitivity of hepatocytes to hyperthermia in the absence of Ca2+.

In each of these experiments cell viability was assayed by the method of trypan blue exclusion as soon as possible after heating, usually within 5 to 10 minutes. The time after heating that the assay is performed is not crucial, as can be seen in Figure 5, where there is only a slight decrease in viability during the period of 2.5 hr after heating. The difference in viability between the hepatocytes heated in the presence and absence of Ca2+ is maintained over this time.

There is conflicting evidence in the literature regard- ing whether or not extracellular Ca2+ sensitizes cells during exposure to toxic chemicals (for a discussion, see Fariss et al., 1985). Fariss et al. (1985) have suggested that Ca2' sensitization occurs only in the presence of

I I I I 2 0

I1 0 10

A23187 ( g m ) Fig. 3. The viability of rat hepatocytes at 37°C as a function of A23187 concentration with no Ca2+ (0) or with 4 mM Ca" (0) present. The length of exposure was 2 hr.

I I I I I 1 0 60 120 I80

TIME (min) Fig. 4. The viability of rat hepatocytes at 43°C as a function of time in the presence and absence of A23187 (1 pm) and Ca2+ (4 mM). The experimental conditions for each curve are given on the figure.

vitamin E and that Ca2+ is protective in the absence of vitamin E. If this holds for hyperthermic killing, then sensitization in the absence of calcium may be due to the lack of vitamin E (a-tocopherol) in the incubation medium. This possibility was explored by maintaining the hepatocyte culture in medium containing 25 pm a- tocopherol acetate for 8 hr before exposing them to hyperthermia in salt solutions also containing a-tocopherol (Fig. 6). Vitamin E was found to be slightly protective, and to a similar degree, in solutions with 0 and 4 mM Ca2' during hyperthermia. No distinct protective role of a-tocopherol in the absence of Ca2' compared to 4mM Ca2' was observed.

282 MALHOTRA, KRUW, AND LEPOCK

1

I I I I I 0 30 60 90 120 150 180

I [

TIME (min) Fig. 5. Viability of rat hepatocytes as a function of time &r heating for 120 min at 43°C in the absence of Ca2+ (0) and in the presence of 4 mM Ca2' (0). The cells were maintained in the same solutions at 37°C between the end of the exposure to 43°C and the assay for viability.

If the hyperthermic sensitizing effect of extracellular Ca2+ is due to increased cytoplasmic levels resulting from an influx of Ca2+ across the plasma membrane, then it should be possible to detect a heat-induced increase in cellular Ca2+. Total influx of extracellular Ca2' during heating was measured by 45Ca uptake (Figs. 7,8). Total uptake at 37°C is constant with time, amounting to 2-3 nmole Ca/106 cells and 6-12 nmol Ca/ lo6 cells in 4 and 15 mM extracellular Ca2+, respec- tively, and appears to be primarily Ca2+ bound to the collagen-coated plastic culture plates that is not removed by the lanthanum washes. Similar background levels of radioactive Ca2+ bound to control plates with- out cells. There is a considerable increase in net influx of Ca2+ during heating, beginning after 150 min and reachin 16 nmole/106 cells at 270 min for the cells in 4 d Ca'+. The influx of extracellular Ca2+ when the cells are heated in 4 mM Ca2' cannot be responsible for the loss of cell viability, as assayed by trypan blue stain- ing, since viability is already less than 1% at the time that Ca2+ influx begins (Fig. 7). The Do (inverse of the inactivation rate) for viability is 24 & 4 min.

The situation is uite different when the cells are heated in 15 mM Ca'+ (Fig. 8). This level of Ca2+ consid- erably sensitizes hepatocytes to elevated temperatures (Do = 5.7 ir: 1.1 min) and increases the rate of Ca2+ influx (Fig. 8). Influx is now detectable at 15 min, ten times sooner than in 4 mM Ca2+ and well before the loss in viability occurs. Uptake appears to occur in two steps, the first beginning immediately upon the increase in temperature, reaching an approximate plateau at 30- 40 nmole Ca/106 cells between 30 and 150 min, and the second beginning after 150 minutes, as in 4 mM Ca2+, and reaching much higher levels of Ca2+ uptake (greater than 100 nmole Ca/106 cells at 270 rnin). Thus, in 15 mM Ca2+, the Ca2+ influx occurs before the loss in viability and may be directly responsible for the increased killing.

I ~

0 SO 120 180 TIME (minl

Fig. 6. The viability of rat hepatocytes at 44°C in the presence (closed symbols) and absence (open symbols) of Ca2+ in salt solutions with no vitamin E (circles) or supplemented with 25 p M vitamin E (squares) during and for 8 hr before beating.

DISCUSSION The principal rationale for this investigation was to

determine if increased permeability of the hepatocyte plasma membrane at elevated temperatures, resulting in an influx of Ca2+, could contribute to hyperthermic killing. Although an influx of Ca2' can sensitize hepatocytes to hyperthermia, under normal conditions (normal extracellular Ca2+ and absence of ionophores) an influx of Ca2+ cannot be responsible for the loss of cell viability, as assayed by trypan blue exclusion, at hyperthermic temperatures. The increase in permeability to trypan blue occurs before the increase in permeability to extracellular Ca2+. Vidair and Dewey (1986) also have recently concluded that an influx of Ca2+ does not play an important role in hyperthermic cell killing of Chinese hamster ovary (CHO) cells. Eventually hepatocytes do become permeable to Ca2', potentially resulting in very high levels of intracellular Ca2+, and this may irreversibly program cells to death, as appears to be the case for other forms of cellular damage (Farber, 1981).

Trypan blue uptake is the most widely used assay for the viability of non-dividing cells, such as hepatocytes, and in general correlates very well with other viability assays, such as lactate dehydrogenase (LDH) release, that are also based on membrane integrity and sensitive only to severe cell injury (Danpure, 1984). If the influx of trypan blue is due to a general loss of membrane integrity, then it is not clear how hepatocytes can stain positive for trypan blue but maintain their impermea- bility to Ca2+ (Fig. 7) unless more subtle forms of cellular damage than a complete loss of membrane integrity can also lead to trypan blue uptake.

Ca2+ can act as a hyperthermic sensitizer. Hepato- cytes are more sensitive to heating in high levels of extracellular Ca2+ (greater than 10 mM) and when ex-

CALCIUM AND HYPERTHERMU 283

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Fi 7 Ca2+ uptake and viability of rat hepatocytes exposed to 4 mM C$+ 'as a function of time at 45°C. Uptake was measured for cells exposed to 37°C (0) and 45°C (0). Viability (0) at 45°C was determined by trypan blue exclusion.

TIME ( m i n )

posed to the Ca'" ionophore A23187. The sensitization by increased levels of extracellular Ca2+ cannot be due to hypertonicity, as the tonicity of the solutions was maintained at a constant level by reducing the NaCl content. In 15 mM Ca" at 45"C, influx of Ca2+ occurs immediately and preceeds the uptake of trypan blue. Thus, loss of viability may be accounted for by the increased permeability of the plasma membrane to Ca". This is supported by the Ca2+-dependent sensitization to hyperthermia of A23187, which also increases Ca" flux across the plasma membrane. It has previously been shown that DEAE-dextran both increases uptake of Ca2+ and sensitizes HA1-CHO cells to hyperthermia (Calderwood, 1984).

These measurements of Ca2+ uptake cannot discrimi- nate between high uptake by a few cells or lesser uptake by all the cells. At longer heating times the uptake is so high that most, if not all, of the cells must be involved.

Ca2+ influx in 15 mM Ca2+ occurs in two stages: a lower level of uptake commencing immediately and par- tially stabilizing at 30-40 nmole Ca/106 cells after 30 min exposure and a second stage of uptake beginning after about 150 min. During this latter stage uptake increases linearly, reaching a level of 120 nmole Ca/106 cells at 270 min. Even at these long heating times uptake has not stabilized and is still increasing. Further increase probably occurs with time until the intracellular and extracellular levels are equal.

The mechanism responsible for this early influx of Ca2+ is not clear. Hepatocytes are able to maintain the normal Ca2+ gradient across the plasma membrane in 4 mh4 Ca2+ for 150 min at 45"C, but influx is detectable almost immediately upon exposure to 45°C in 15 mM Ca2+. Thus, high levels of Ca" must destabilize the membrane in some way so as to increase passive CaZf permeability.

The presence or absence of vitamin E cannot explain the toxicity of Ca2+ at hyperthermic temperatures, as may be the case for other forms of injury (Fariss et al.,

I 1 - I - Ih I I I I I I 0

0 60 120 I80 240

TIME (minl Fig. 8. Ca2+ uptake and viability of rat hepatocytes exposed to 15 mM extracellular Ca2+ as a function of time at 45°C. Uptake was determined for cells at 37°C (0) and 45°C (0). Viability (0) at 45°C was determined by trypan blue exlusion.

1986). Sensitization occured in both the presence and absence of vitamin E, although vitamin E confers addi- tional protection.

The response of hepatocytes to heating as a function of Ca" concentration in the medium is biphasic; cells are slightly more sensitive in the absence of Ca2+ than at 1-5 mM Ca". Thus, low levels of Ca2+ may stabilize the plasma membrane against heat damage. This im- plies that at least part of the cellular damage critical for hyperthermic killing is damage at the plasma membrane level, which is consistent with the conclusions of several other studies Gepock, 1982).

There are two possible mechanisms for the stabilization of the plasma membrane by extracellular Ca2+. Many proteins are stabilized against thermal denaturation by Ca2' (Alexandrov, 1977). This appears to occur because of an increase in the denaturation temperature upon binding of Ca2+. The Ca2+-ATPase of rabbit sar- coplasmic reticulum is protected from loss of activity at elevated temperatures by low levels of Ca2+ (McIntosh and Berman, 1978). Thus, one would expect the Ca2+ transport proteins of the plasma membrane, such as the calmodulin-sensitive Ca2+-ATPase (Carafoli, 1983), and the Na+-Ca2+ exchange protein Ganger, 1982) also to be protected.

Ca2+ also decreases membrane lipid fluidity, which may cause a stabilization of the lipid bilayer. This occurs by two distinct mechanisms: 1) direct binding of Ca2' to lipid headgroups (Jacobson and Papahadjopoulos, 1975) and 2) a modulation of the phospholipid acyl chain composition resulting in a reduction in arachidonic acid and the double bond index (Storch and Schachter, 1985).

In summary, Ca2+ influx during heating cannot be directly responsible for hyperthermic killing of hepatocytes, although it may play an important role in irreversibly programming a cell to death. Hepatocytes are sensitized to hyperthermia if Ca" influx is increased during heating by other means, i.e., high extracellular Ca2+ or the ionophore A23187. This may be useful in

284 MALHOTRA, KRUUV, AND LEPOCK

cancer therapy by hvperthermia if a way can be found ture, pH, and concentration of bivalent cations. Biochemistry, 14:152- to differentiaily hc&ase, even slightly,"the Ca2' permeability of cells in a tumor. A question resulting from these observations is whether release of Ca2' from intracellular stores, i.e., endoplasmic reticulum or mitochondria, may be more sensitive to elevated temperature than influx across the plasma membrane and play a more direct role in cell death.

ACKNOWLEDGMENTS This work was supported by the Natural Sciences and

Engineering Research Council of Canada.

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Sensitization of rat hepatocytes to hyperthermia by calcium

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