ROLE OF INTRACELLULAR THlOL STATIUS AND CALCIUM HOMEOSTASIS IN MYOCARDIAL CELL INJURY

The primary biochemical mechanisms involved in chemically-induced cell injury remain to be elucidated. Elevation of intracellular ea2+ is a common feature to cell death due to a wide array of toxic chemicals, and on this basis hypotheses have been put forth suggesting that the chemically-induced elevation of cytosolic ea2+ is responsible for the onset of cell death. The mechanism by which elevated ea2+ causes cell damage may involve activation of ea2+-dependent proteases, phospholipases and endonucleases (2). Previous evidence suggests that a link between intracellular thiol status and ea2+ homeostasis exists (1, 2). Based on these observations, it has been speculated that thiol depletion may lead to an elevation of intracellular ea2+ to cytotoxic levels (1, 2, 4). Glutathione, the major cellular thiol, is primarily a defense mechanism against cytotoxic reaction to oxidative stress or alkylating agents. Glutathione also plays an important role in maintaining protein thiols in a reduced state, which is required for their normal enzymatic activity (6, 7). Such enzymes include the sarcolemmal and sarcoplasmic reticular ea2+-A TPase's, which are involved in the maintenance of low levels of cytosolic ea2+. Therefore depletion of intracellular glutathione may limit the capacity of these enzymes with modified thiol groups to maintain low levels of cytosolic ea2+ (10, 11 ). The loss of GSH as an antioxidant may promote oxidative stress and the resultant peroxidative damage to plasma membrane may be an alternate cause of cell death by a ea2+-independent mechanism. The relative importance of elevated cytosolic free ea2+ or oxidative stress in cell death, in the face of a chemical challenge that alters intracellular thiol status is the subject of this thesis. Our approach towards this problem was to create a chemical model of oxidative stress in cardiomyocytes using ethacrynic acid. Ethacrynic acid depletes thiols by alkylation with a subsequent increase in cytosolic free ea2+, thereby permitting us to examine lethal cell injury due to thiol depletion, including the proposed link to ea2+ homeostasis. Exposure

of primary rat myocardial cells to ethacrynic acid (150 µM ) resulted in a rapid (within 7 min) loss of glutathione and protein thiols that preceded an increase in cytosolic free ea 2 + levels (within 45 min), as detected by the activation of phosphorylase a. The leakage of cytosolic lactate dehydrogenase due to loss of membrane integrity was used as a criterion of loss of cell viability. All of these biochemical events preceded the loss of cell viability, thus permitting us to examine whether thiol depletion or changes in cytosolic free ea 2 + had the primary effect on the loss of cell viability.
Pretreatment of cells with specific intracellular ea 2 + chelators, Quin-2acetoxymethylester and EGTA-acetoxymethylester, were used in an attempt to sequester ea2+, in order to prevent an ethacrynic acid-induced elevation of intracellular ea 2 +. Both intracellular chelators reduced lactate dehydrogenase leakage, protected against lipid peroxidation, but failed to reduce the marked elevation of intracellular ea 2 +. The latter observation required examination of the mechanism of protection afforded by the putative chelators. The antioxidant N,N'-Diphenyl-p-phenylenediamine was employed to investigate the importance of lipid peroxidation in ethacrynic acid-induced cell death. N,N'-Diphenyl-p-phenylenediamine reduced lipid peroxidation and lethal cell injUI)' to control levels but had no effect on intracellular glutathione and ea 2 + levels. Thus, it would appear that the antioxidant activity of the putative chelators might account for their protection. The possibility that cytotoxicity was due to an ethacrynic acid-induced alteration of cellular energy status was also examined. Ethacrynic acid had no significant effect on cellular ATP levels or mitochondrial membrane potential.
In our model of myocardial cell injUI)' the temporal relationship observed between the loss of intracellular thiol status and ea 2 + homeostasis supports the hypothesis that thiol status is linked to ea2+. However elevated ea2+ levels alone, had no effect on cell viability over the time course we observed, further supporting that peroxidative damage is a requisite event for cell death in our model of myocardial cell injUI)'.
iii ACKNOWLEDGEMENT The author would like to thank Dr. John R. Babson for his utmost patience, moral support, and guidance throughout the course of this study.
I would like to dedicate this thesis in loving memory of my uncle Dr   .,

ABSTRACT
Ethacrynic acid, was used to deplete intracellular thiols to create a model of oxidative stress in order to examine the ensuing events leading to myocardial cell injury.
Exposure of primary rat myocardial cells to ethacrynic acid resulted in a rapid loss of glutathione and protein thiols that preceded an increase in cytosolic free ea 2 + levels, as detected by the activation of phosphorylase a. The magnitude of lethal cell injury, using leakage of lactate dehydrogenase as a criterion, was dependent on the ethacrynic acid concentration used. The loss of cellular thiols and the elevation of intracellular ea2+ preceded the onset of cell death. Pretreatment of cells with specific intracellular ea 2 + chelators, Quin-2acetoxymethylester and EGTA-acetoxymethylester, were used in an attempt to sequester ea 2 + and thereby prevent an ethacrynic acid-induced elevation of intracellular Ca2+, in this model system of chemically-induced cell killing. Both intracellular chelators reduced leakage of lactate dehydrogenase, protected against lipid peroxidation, but failed to reduce the marked elevation of intracellular Ca 2 +. The latter observation required examination of the mechanism of protection afforded by the putative chelators. The antioxidant N,N'-Diphenyl-p-phenylenediamine was employed to investigate the importance of lipid peroxidation in ethacrynic acid-induced cell death.
N,N'-Diphenyl-p-phenylenediamine reduced lipid peroxidation and lethal cell injury to control levels but had no effect on intracellular glutathione and ea 2 + levels. Thus, one could postulate that the antioxidant activity of the putative chelators might account for their protective properties. An alternative possibility that cytotoxicity was due to an ethacrynic acid-induced alteration of cellular energy status was also examined. Ethacrynic acid had no significant effect on cellular ATP levels or release of the triphenylmethylphosphonium cation, a measure of mitochondrial membrane potential. These results support previous observations that a loss of intracellular thiols is followed by a rise in ea 2 +. and the perturbation of these homeostases may result in the loss of cell viability. However the elevated ea 2 + alone was not responsible for cell death over the time course we observed, and oxidative damage was seen to be a primary requisite for myocardial cell injury. '

INTRODUCTION
An elevation of intracellular ca2+ is a response that is frequently observed following exposure to a wide variety of toxic chemicals (1). This observation led to the hypothesis that chemically-induced elevation of cytosolic ea2+ is a causative event in the cytotoxic mechanism of many chemicals (2). Attempts to understand how chemicals perturb intracellular ea 2 + homeostasis have provided evidence that suggests the elevation of cytosolic ea 2 + is a direct result of thiol depletion (1,2). However, it may be the oxidative stress that results from thiol depletion, and not the accompanying rise in ea2+, that is responsible for cell death (2,4,6). Based on all of these observations, two different mechanisms have been proposed to explain chemically-induced cell injury: 1. A ea 2 + -dependent mechanism of cytotoxicity involving a perturbation of ea 2 + homeostasis that triggers ea 2 + -activated processes that are ultimately responsible for cell death. 2. A ea 2 +-independent mechanism in which cell death is caused by peroxidative damage that results from the loss of glutathione (GSH) as an antioxidant.
GSH, a thiol tripeptide, is a major component of intracellular thiol status and is also a primary cellular defense mechanism against toxic chemical insult (1,2,4 ). GSH is distributed intracellularly in two main pools. The mitochondrial pool contains 15% and the cytosolic pool contains 85% of the total intracellular GSH. GSH protects the cell against toxic challenges via two distinct mechanisms. First, it detoxifies alkylating agents by its ability to form conjugates either directly or enzymatically via GSH-S-transferases (8,9).
Second, GSH protects cells against oxidative stress by its ability to reduce chemical oxidants through the GSH redox cycle (6). Some studies have suggested that the depletion of mitochondrial GSH is a key factor in the onset of cell death (7).
GSH is also involved in the normal functioning of cells, including the maintenance of protein thiol status (6). The reducing equivalents provided by GSH maintain the activity of many cellular enzymes, which require that certain protein thiols be in the reduced state for enzymatic activity (10 ). Included in this group of enzymes are the sarcolemmal and sarcoplasmic reticular ea2+ _A TPase's believed to be involved in the sequestration and extrusion of cytosolic free ea2+ (10,11). Since protein thiols may also be alkylated by toxic chemicals, GSH provides a nucleophilic barrier that protects protein thiols from alkylation. Accordingly, any event resulting in GSH depletion may lead to an alteration of key enzyme thiols, and subsequently limit the capacity of these enzymes to regulate intracellular ea 2 + levels (7).
Under normal physiological conditions the cytosolic free ea 2 + concentrations are quite low, ranging between 50 and 200 nM (14). It has been proposed that many toxic chemicals may elevate cytosolic free ea2+ by a combination of two processes. First, they are believed to promote the release of intracellular ea 2 + stores or the influx of extracellular ea 2 + by mechanisms that have yet to be determined. Second, by inhibiting ea 2 + -A TPase activities responsible for removing ea2+ from the cytosol, the chemical challenges are believed to elevate cytosolic free ea 2 + (12,15,24 ). Whether extracellular or intracellular ea2+ is the primary source of elevated cytosolic free ea 2 + observed in lethal cell injury (18,19), is beyond the scope of this study. Regardless of the source or mechanism, any chemical insult that promotes a sustained elevation of cytosolic Ca 2 +, including those that deplete intracellular thiols, may ultimately cause lethal cell injury due to a variety of ea 2 + activated processes (2) . Elevated cytosolic ea 2 + could be expected to activate several ea2+ -dependent enzymes, including ea2+ -activated proteases, ea2+ -activated phospholipases, and ea 2 +-activated endonucleases (1,2,20). Prolonged activation of these enzymes is believed to contribute to cell death (1,3).
A number of studies conducted on hepatocytes, and some on myocardial cells, suggest that cell injury due to thiol depletion results from the consequent elevation of intracellular ea 2 + (1,2,25). However, there is equally persuasive evidence to suggest that thiol depletion alone may cause cell death, by a ea2+ _independent mechanism, through resultant oxidative damage (16,17). Although lethal cell injury accompanying oxidative stress is associated with elevation in cytosolic free ea2+, the loss of cell viability may not result from the observed changes in intracellular ea 2 + levels (32). The loss of GSH as an antioxidant may play a more important role in loss of cell viability, through oxidative damage, and the resultant lipid peroxidation (3,21).
In this study our approach towards this problem was to create a model of oxidative stress, using ethacrynic acid (EA), a selective sulfbydryl alkylating agent (3,40,41), to deplete intracellular thiols. This model permitted us to examine the key biochemical events affected by thiol depletion, including penurbations of ca2+ homeostasis, and the contribution of these events to cell death. A major goal of this study was directed towards attempting to elucidate the role of thiol status in chemically-induced lethal cell injury and to determine the relative importance of elevated intracellular Ca 2 levels and oxidative stress to cell death.

METI:IODS
Cardiomyocyte Isolation and Culturing. Myocardial cells were isolated and cultured essentially according to the method of Bollon et al (26) with several modifications aimed at increasing the yield of beating myocytes over that of non-muscle cells (27)(28)(29). Cells were plated in separate 35 mm culture dishes, incubated in Eagles MEM medium buffered with 25 mM Hepes, pH 7.4 and containing 10% horse serum, 5% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 1 U/mL insulin at 37°C and 95% humidity . Protein Thiol and GSH Analyses. The original method of Sedlak and Lindsay (35), as modified by Orrenius ~ (36) was followed. Briefly, cell incubate was removed and plates were treated with 6.5% TCA, scraped and centrifuged at low speed, and the resultant supematants analyzed for GSH levels by the HPLC method of Reed~ (37).
Lipid Peroxidation Assay. Lipid peroxidation was measured by monitoring the formation of a colored complex between malondialdehyde and thiobarbituric acid (TBA), by the method of Stacey and Klaassen (38) as modified by Thomas and Reed (17). Briefly, at the end of incubation 100% TCA was added ~o the cells to a final concentration of 12%, and the cell suspension was treated with 0.67% thiobarbituric acid for 20 minutes at 90°C.
TBA-reactants formed were mea5ured at 532 run. Results were expressed in terms of percent of EA-treated cell values.
Mitochondrial Membrane Potential. The distribution of [ 3 H]-triphenylmethylphosphonium ion (TPMP) between the cardiomyocytes and the incubation solutions was used to determine the collapse of mitochondrial membrane potential. Changes in mitochondrial membrane potential were monitored according to the procedure of Hoeke '1.fil (23). Cells were radiolabeled by incubation with 0.25 µCi/ml of [3H]-TPMP for 1 hr at 37° C. At end of incubation cells were exposed to 150 µM EA or solvent control for 30-75 min. The results were expressed as percent [ 3 H]-TPMP released into the culture medium at the end of incubation time.

Measurement of ATP. Following various chemical exposures, ATP levels of cell lysates
were determined using the luciferin-luciferase bioluminescent method of Wulf and Doppen (39).
Glyceraldehyde 3-phosphate Dehydrogenase Activity. Myocyte glyceraldehyde 3-phosphate dehydrogenase activity was measured by monitoring the conversion of 3-phosphoglycerol phosphate and NADH to D glyceraldehyde-3-phosphate and NAD+, spectrophotometrically according to the method of Birkett et al (13).
Protein Estimation. Certain data as mentioned in the methods was normalized on the basis of per mg protein.
All protein values were obtained by the Bradford protein assay (44).
STATISTICAL ANALYSIS: All data were summarized as the mean± SD. Significance of the difference between the groups was determined by the 2-tailed Student's t-test.

RESULTS
LDH leakage was used as criterion for assessing the loss of cell viability. As shown in Fig. 1 The results in Fig. 3 show that there was a rapid depletion of intracellular GSH levels in cells treated with 150 µM EA. GSH dropped rapidly to 50% of the control levels at 7 min and to less than 1 % of controls by 60 min. The status of intracellular protein thiols in the face of an EA challenge was examined over the same time period and illustrated in Fig.   4. We observed a time-dependent depletion in protein thiols, which corresponds to 32% of the control values at 60 minutes. Our data suggests that in EA-induced myocardial cell injury.a significant decrease in GSH precedes the depletion of intracellular protein thiols, and both these events occur well before the onset of LDH leakage is observed.
Intracellular ea 2 + levels were monitored by measurement of the ea 2 + -dependent conversion of phosphorylase b to phosphorylase a. As shown in Fig. 5, there was a time-dependent increase in phosphorylase a activity when the cells were treated with 150 µM EA. The elevation of phosphorylase a activity, expressed as nmoles Pi I min· mg protein, reached a maximum at 60 min and the value was 124 ± 43 as compared to 18±11 for the controls. This indicates that EA-induced increase in intracellular ea 2 + reached a maximum by 60 min. Again, as with thiol depletion this maximum was reached well before I.DH leakage was observed.
The intracellular ea 2 + chelators, Quin-2-AM and EGTA-AM were used to assess the importance of EA-induced Ca 2 + increase in cell injury. These specific intracellular ea 2 + chelators were used to attempt buffering the rise in intracellular ea2+ levels, and their effect on subsequent cell injury. Results show I.DH leakage after 2 hr incubation with EA (150 µM ). Cells preincubated 45 min with Quin-2-AM or EGTA-AM showed leakage of 25 ± 8% and 11 ± 2%, upon subsequent treatment with EA, respectively (Fig.   6). These data indicate that Quin-2-AM and EGTA-AM decreased I.DH leakage, which was 58% at 2 hr, by 57 and 81 %, respectively. Pretreatment of cells with just DMSO did not alter EA toxicity (I.DH release, 58 ± 5% as compared to 54 ± 6% ). Surprisingly, while both the intracellular ea 2 + chelators reduced cell injury, they failed to reduce the magnitude of EA-induced increase in intracellular free ea 2 +, based on phosphorylase a activity, as shown in Fig.7. Phosphorylase a activity in cells treated with 150 µM EA in the presence of DMSO, at the end of lhr incubation, was found to be 120 ± 27 nmoles Pi I min • mg protein. In comparison, phosphorylase a activity of cells preincubated with Quin-2 AM or EGTA AM, preceding an EA challenge was found to be 129 ± 40 and 171 ± 40 nmoles Pi/mg protein·min, respectively. These values were not significantly different from the phosphorylase a activity of cells treated with EA, which was 120 ± 27 nmoles Pi I min• mg protein at 1 hr. Furthermore, pretreatment of cells with Quin-2-AM or EGTA-AM, and in the absence of EA, did not alter the the phosphorylase a activity in comparison to the DMSO control (67 ± 21 and 68 ± 13, respectively as compared to 50 ± 24 nmoles Pi I min • mg protein at 1 hr). Phosphoinositide hydrolysis initiated by ea 2 + -dependent phospholipase c activation, was used as an alternative method to verify the observed increase in intracellular ea 2 + levels . Fig. 8 shows that phosphoinositide hydrolysis, initiated by ea 2 + -dependent phospholipase c activation, is 6% in the face of 150 µM EA challenge. The intracellular ea2+ chelators Quin-2-AM and EGTA-AM have no effect in reducing the extent of phosphoinositide hydrolysis.
Since thiol depletion is known to correlate with oxidative damage, we examined whether EA-induced cell death involved peroxidative damage. Towards this end, cells were treated with 150 µM EA, in the presence or absence of the antioxidant DPPD (5 µM), then examined for cell viability at the end of 2 hr incubation. Our results, illustrated in Fig.   9, show that 5 µM DPPD was capable of reducing damage by 89% for cells treated with 150 µM EA, as measured by LDH leakage. DPPD treated cells showed 7 ± 1 % leakage as compared to 61 ± 5% for EA treated cells. The solvent control for DPPD showed no significant amount of leakage at 2 hr. We also assessed the role of DPPD on elevated intracellular ea 2 + levels monitored by ea2+ _dependent activation of phosphorylase a. Fig.   10, shows that DPPD had no effect in reducing the elevated levels of intracellular Ca 2 + in the presence of 150 µM ethacrynic acid No changes in phosphorylase a activity was observed in the appropriate controls (65 ± 13 nmoles Pi I min· mg protein for DMSO and 60 ± 9 nmoles Pi I min • mg protein for DPPD) suggesting that there was no interference in our assay by solvents employed. Furthermore, 5 µM DPPD had no effect on EA-induced depletion of intracellular GSH levels ( Table 1).
The possibility that EA-induced cell injury involved impairment of cellular energy status was assessed by examining the integrity of mitochondrial membrane potential and cellular ATP levels as biochemical indices.Changes in mitochondrial membrane potential were expressed as the percent [ 3H]-TPMP released into the culture medium. Results of previous studies have observed that 90% of [ 3H]-TPMP is localized in the mitochondria, and the total cellular content closely corresponds to changes in mitochondrial membrane potential (32). Fig. 12, shows the results of various chemical exposures on the mitochondrial membrane potential, for varying pericxls of time ranging from 30-75 min.
The cells treated with 150 µM EA did not vary significantly from the solvent control values even at 75 min. As a positive control, cells were exposed to an uncoupler of oxidative phosphorylation, CCCP (25 µM). A rapid collapse of mitochondrial membrane potential was observed ( 24 ± 1 % at 30 min and 14 ± 3% at 7 5 min). ATP levels in the cell lysates were determined for cells treated with 150 µM EA over a time range of 7-7 5 min. As shown in Table 2

DISCUSSION
Using exposure to EA as a mcxlel of chemically-induced cytotoxicity, we examined the relationship between intracellular thiol status, Ca 2 + homeostasis, and myocardial cell death. The maintenance of intracellular GSH has been suggested to play a pivotal role in the maintenance of cell viability (1,2,4). Results of previous studies suggest that depletion of GSH levels to less than 10-15% of initial levels is generally observed to correlate with the loss of cell viability (6). These results have been interpreted to suggest that the extent of cell death following the chemical depletion of GSH corresponds to depletion of the mitochondrial pool, which comprises 15% of the total intracellular GSH ( 4 ). We observed a rapid depletion of intracellular GSH levels to less than 1 % within 1 hr in the presence of 150 µM EA. These data demonstrate that the EA challenge in our model system was of sufficient magnitude to deplete almost all of the intracellular GSH, including the mitochondrial pool.
Recent studies have led to the suggestion that the alteration of protein thiols in concert with the depletion of intracellular GSH may be responsible for the loss of protection against a chemically-induced oxidative stress and subsequent loss of cell viability (43). EA has been effectively used to deplete intracellular thiols in isolated rat hepatocytes (3).
Modification of these protein thiol groups may limit the enzymatic capacity of many critical enzymes. Such enzymes include sarcoplasmic reticular and sarcolemmal Ca 2 +-A TPases, and modification of these enzymes result in the perturbation of intracellular ea 2 + homeostasis preceding cell injury (10,11 ). Results described herein, demonstrate a time-dependent depletion of protein thiols (30% depletion by 1 hr), which follows the depletion of intracellular GSH. This pool of depleted protein thiols may include thiol containing enzymes responsible for maintaining intracellular Ca 2 + homeostasis. Thus this alteration of critical thiol groups may be linked to the perturbation of Ca 2 + homeostasis, and the resultant elevation of cytosolic free ea 2 + may contribute to cell death.
Given the proposed relationship between thiol status and Ca 2 + homeostasis and the possible significance of these events to cell viability, we examined intracellular Ca 2 + homeostasis in the face of an EA challenge. A time and concentration-dependent increase in cytosolic free Ca 2 + levels was observed in myocardial cells, as measured by the elevation of phosphorylase a activity. The elevation of intracellular ea2+ followed the depletion of intracellular GSH and protein thiols, and preceded the onset of cell death. The temporal relationship we observed suggests that an EA challenge may alter thiol groups critical to the regulation of intracellular Ca 2 +, resulting in elevated cytosolic Ca 2 +. This perturbation of Ca 2 + homeostasis may contribute to cell injury due to Ca 2 +-activated processes.
This temporal relationship suggested that intracellular thiol depletion may be linked to the perturbation of Ca 2 + homeostasis that preceded cell death. We next attempted to determine if the observed Ca2+ increase was responsible for the cell injury. Toward this end, we tested the effect of two specific intracellular Ca 2 + chelators, Quin-2-AM and EGTA-AM, on EA toxicity. While both the chelators protected the cells, contrary to expectations, they did not do so by lowering Ca 2 +. These results do not support interpretations from other studies that elevated Ca 2 + levels are a primary cause of cell death.
Besides its proposed importance in Ca 2 + homeostasis, GSH is a major intracellular antioxidant The extent of GSH depletion caused by EA treatment suggested a possible role of peroxidative damage in our system of chemically-induced injury. Indeed, our results indicate that EA-induced thiol depletion results in lipid peroxidation. This rapid depletion of intracellular GSH may incapacitate the effective reduction of free radicals and the resultant generation of partially reduced oxygen species may initiate an iron-dependent mechanism of peroxidative damage to the cellular membrane (22). The antioxidant DPPD was used to investigate the importance of the loss of GSH antioxidant protection in a situation where GSH is depleted due to its alk:ylation by EA. We also observed the effect of DPPD on EA-induced lipid peroxidation, in the absence of GSH as an antioxidant.
DPPD protected cardiomyocytes in the face of an EA challenge that virtually eliminated intracellular GSH, and also reduced the subsequent lipid peroxidation by 90%.
Furthermore, the antioxidant DPPD conferred 89% protection against EA-induced cell death, but had no effect on the elevated phosphorylase a activity. The protective property of DPPD may be attributed exclusively to its ability to prevent lipid peroxidation (9). These data showed that elevated cytosolic Ca2+ alone had no effect on cell viability over the time course of our experiments. However lipid peroxidation appeared to be a key factor in the onset of cell death.
Treatment of the cells with the ea 2 + chelators Quin-2-AM and EGTA-AM, markedly decreased lipid peroxidation by 60% and 62%, respectively. The ability of iron to undergo redox reactions linked to single-electron cycling is well known. This Fenton-type of reaction which generates free radicals, may possibly interact with the cellular membrane and cause lipid peroxidation. The protective action of the Ca 2 + chelators may be more complex to explain. One possibility is that chelation of endogenous iron may protect against lipid peroxidation, which would inhibit a Fenton-type reaction (42).
While our data supports a primary role for peroxidative damage in EA-induced cell death, it was also possible that EA treatment might compromise cellular energy status.
Our results demonstrate that EA inhibits G3PD activity, thus demonstrating a potential to inhibit the enzymatic activity of other thiol containing enzymes. This inhibition of such thiol containing enzymes may, in turn, lead to an alteration in normal cellular ATP production. Results of previous studies conducted on myocardial cells have demonstrated that ATP levels may be depleted to 50% of their initial levels without any corresponding decrease in cell viability (45). We monitored the mitochondrial membrane potential and cellular ATP levels, as an important indicator of cellular energy status. Our data indicate that ATP levels did not decrease appreciably (< 16% by 75 min) and mitochondrial membrane potential is not significantly impaired during EA exposure. These results suggest that the EA-induced alteration of cellular energy status does not play a major role in the onset of cell injury in this model system.
Taken collectively, the results of this study suggest that while elevated cytosolic free ea 2 + may contribute to the cell injury from thiol depletion, we observed, it alone is not the cause of cell death; oxidative damage is a primary requisite. The fact that EA by itself does not generate any free radicals, leads to further speculation regarding the source of oxidative damage in our model system. This study also demonstrates the utility ofEAtreated myocytes as a model to study the biochemical events involved in oxidative stress.