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A pH-dependent phospholipase A2 contributes to loss of plasma membrane integrity during chemical hypoxia in rat hepatocytes
Vol.
174,
No.
January
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
31, 1991
A pH-DEPENDENT MEMBRANE
654-659
PHOSPHOLIPASE A2 CONTRIBUTES TO LOSS OF PLASMA INTEGRITY DURING CHEMICAL HYPOXIA IN RAT HEPATOCYTES’
D. Corinne Harrison, John J. Lemasters and Brian Herman2 Laboratories for Cell Biology, Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27.599-7090 Received
November
5, 1990
Previous studies have suggestedthat alterations in phospholipid composition of plasma membranes may underlie lethal cell injury due to hypoxic and ischemic injury. The present study was designed to determine if such alterations are due to the activation of a pHdependent phospholipase AZ. Loss of cell viability and phospholipase A2 activity measured by arachidonic acid release increased in parallel during metabolic inhibition with KCN and iodoacetate (chemical hypoxia). Acidosis (pH 6.5) and the phospholipase inhibitors, dibucaine and mepacrine, delayed lossof cell viability and release of arachidonic acid to a similar extent. These findings suggest that a pH-dependent phos holipase A2 causes alterations in plasma membrane phospholipid composition after R TP-depletion which contribute to lethal cell injury. B 1991 Academic Press, Inc.
We have previously reported that ATP-depleted rat hepatocytes sustain plasma membrane damage characterized by the formation, growth, and coalescence of membrane blebs with eventual rupture leading to lethal cell injury (l-5).
However, the mechanisms
which lead to plasma membrane injury during hypoxia remain to be identified. Exposure of hepatocytes to KCN and iodoacetate, a treatment which mimics the ATP-depletion and reductive stressof hypoxic injury, causesa >95% lossof cellular ATP within 10 minutes yet cells survive for up to 60 minutes (1,3-5). Thus, the formation, enlargement and subsequent rupture of plasma membrane blebs which occurs over this period is largely ATPindependent, Accompanying ATP depletion is a drop of intracellular pH from pH 7.1-7.4 to pH 6.0-6.3 (3-5). Intracellular pH remains acidotic for 30-40 minutes but abruptly rises just before the onset of cell death. Low extracellular pH and manipulations which deepen
‘This work was supported by Grants AGO7218 and DK30874 from the National Institutes of Health, the Gustavas and Louise Pfeiffer Foundation and Grant J-1433 from the Office of Naval Research. 2To whom correspondence should be addressed. Abbreviations used are: ATP, adenosine tris hosphate; HEPES-KHB, Krebs-Heinseleit buffer containing 118 mM NaCl, 25 mM NaH e 03, 1.2 mM KH2PO4, 1.2 mM MgSO4, 4.7 mM KCl, 1.3 mM CaC12,20 mM Na- HEPES, pH 7.4., MES-KHB, KHB containing MES (Z(N-morpholino) ethanosulfonic acid, pH 6.5, instead of HEPES. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
654
Vol.
174,
No.
2, 1991
intracellular
BIOCHEMICAL
AND
BIOPHYSICAL
acidosis delay the onset of cell death (3-6).
for membrane physiological
damage appears
to be pH-dependent
RESEARCH
COMMUNICATIONS
Thus the mechanism with
maximal
responsible
activity at or near
pH and minimal activity at acidotic pH.
Several studies lysophospholipids
have demonstrated
following
account for the pH-dependence after hypoxic
injury
release
ATP depletion
of fatty acids and accumulation
(7-10). One potential
mechanism
of cell killing and the changes of phospholipid
is phospholipase
AZ activation
(4-10). Phospholipase
of
which could composition A2 is a pH-
dependent enzyme with maximal activity at neutral or slightly alkaline pH (11). Here we examined the activity of phospholipase and cell viability phospholipase
during chemical A2 activity
A2 in relation to extracellular
hypoxia in hepatocytes.
increases
after
ATP
pH
Our findings indicate that 1)
depletion,
2) acidosis
inhibits
this
phospholipase A2 activity and 3) phospholipase inhibitors delay the onset of lethal cell injury. Taken together, these data suggest that a pH-dependent phospholipase A2 contributes
MATERIALS
to irreversible
AND
hypoxic injury.
METHODS
Heputocyte Isolation and Culture- Hepatocytes were isolated from the livers of male Sprague-Dawley rats (200-300g) by collagenase perfusion as previously described (2). Cell viability was >90% as determined by trypan blue exclusion. lo6 hepatocytes were cultured on 35 mm petri plates (Falcon in 1.5 ml Waymouth’s MB 152/l containing 27 mM NapC03, 2 mM L-glutamine, 10d/o heat-inactivated fetal calf serum (Colorado Serum Co.), 10 U/ml pemctlhn, 10 pg/ml streptomycm, 100 nM insulin, and 10 nM dexamethasone at 37’ in 5% CO2/95% air at pH 7.4. Measurement of 14C Arachidonic Acid Release- [14C]-arachidonic acid (20 pCi/ml) (Amersham) in toluene was dried under N2 and reconstituted in an equal volume of dimethyl sulfoxide which was then added to Waymouth’s media to a final concentration of 0.267 pCi/ml. Cultured hepatocytes were washed with HEPES-KHB at 4’C and incubated in 0.75 ml of 14C arachidonic acid-containing media at 37’C for 11 to 20 hours. Under the labeling conditions used, approximately 94% of the labeled arachidonic acid is incorporated into phospholipids with no more than 3% remaining as free arachidonic acid (12). Following labeling, hepatocytes were washed 3 times in HEPES-KHB or MES-KHB and incubated under various experimental conditions. Aliquots of 0.5 ml were removed at 30 minute intervals, acidified to pH 4.5 with 1 N metaphosphoric acid, and microfuged for 1 minute. Supernatants were further acidified with 35 ~1 1N HCl and extracted with 1.8 ml ethyl acetate. The organic phase was dried over N2 and redissolved in 10 ml Scintiverse cocktail for liquid scintillation counting in a Beckman CS 5000 CE Scintillation counter. After the final aliquot wft removed, cells were scraped from the dish and processed as described to determine the C arachidonic acid remaining . Cell ViubiZity Asay- In parallel experiments, cell viability was assessed by trypan blue exclusion. 0.6% trypan blue was added to cultures for 20 seconds, and the percentage of trypan blue positive cells determined after washing with HEPES-KHB. RESULTS Loss of Cell Viability and 14C Arachidonic Acid Release at pH 7.4- Phospholipase
A2
cleaves phospholipids at the sn2 position causing release of free arachidonic acid (11). Accordingly, phospholipase A2 activity was monitored by labeling cultured hepatocytes with r4C arachidonic acid and quantifying release of radioactivity. In parallel experiments, cell 655
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Figure l- Viability and 14C Arachidonic Acid ReleTf in Cultured Rat Hepatocytes during Chemical Hypoxia- Hepatocyteswere labeledwith C arachidonicacid, and exposedto KCN (2.5 mM) and iodoacetic acid (9. rnM) asdescribedin the materialsand methodsand
incubatedin HEPES-KHB at 37°C. C arachidonicacid releasewasmeasuredin aliquots of supernatantat 30 minute intervals (open circles). In paralle experiments,hepatocytes were quantifiedfor viability by trypan blue exclusion(crosses).’ aC arachidonicacid release was adjusted for the remaining amount of incubation medium after removal of sequential aliquot volumes. Data points represent means + SEM from 12 trials.
viability was monitored by trypan blue exclusion. After exposure of cultured hepatocytes to 2.5 mM KCN and 0.5 mM iodoacetate, viability decreased 10% after 30 minutes and 90% after 60 minutes (Figure 1). Release of arachidonic acid closely paralleled loss of cell viability. Rates of cell killing and arachidonic acid release were both maximal between 30 and 60 minutes of chemical hypoxia (Figure 1). These data suggestthat arachidonic acid release and lossof cell viability are simultaneousevents.
80
60
Tme
(Minutes)
Figure 2 Effect of Acidosis and PhospMipase Inhibitors on 14C Arachidonic Acid Cultured Hepakxytes During Chemical Hypoxia- Conditions were as described in Cells were incubated in HEPES-KHB (pH 7.4; plus marks), HEPES-KHB plus (100 PM; closed triangles), or mepacrine (30 PM; open triangles), or MES-KHB open circles). Data points represent means + SEM from 10 or more trials. 656
Release in Figure 1. dibucaine (pH 6.5;
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No.
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30
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Figure 3- Effect of Acidosis and Phospholipase Inhibitors on Cell fiability during Chemical Hypoxia- Cells were incubated as described in Figure 2 and cell viability was determined by trypan blue exclusion. Symbols are as described in Figure 2. Data points represent means + SEM from 10 or more trials.
The data in Figure 1 suggeststhat the 14Carachidonic acid release ceasesonce cells lose viability. To establish further that dead cells do not release arachidonic acid, 14C arachidonic acid-labeled cells were treated with KCN and iodoacetate until all cells were trypan blue positive. The cells were washed with HEPES-KHB,
reincubated in 1 ml
HEPES-KHB, and returned to the incubator. 14Carachidonic acid release into the medium was then measured after another 30 minutes. The cellswere then scraped from the dish and the amount of 14Carachidonic acid associatedwith the cells was determined as described in the materials and methods. The results indicated that after cell viability was lost, little 14C arachidonic acid release occurred (409 cpm released from treated hepatocytes w 368 cpm for untreated cells), although greater than 65% of radioactivity remained with the cells. To determine whether release of intracellular material at bleb rupture might account for release of 14C arachidonic acid associated with cell death, labeled hepatocytes were exposed to 10 /.LM digitonin to permeabilize the plasma membranes of all cells. 14C arachidonic acid release after 25 minutes exposure to digitonin was low and no different than that from untreated cells (1003 vs 957 cpm respectively). Taken together, these data indicate that release of arachidonic acid precedes loss of cell viability and is not the consequenceof cell death. Efects of PhospholipaseInhibitors and Acidotic Extracellular pH on 14CArachidonic Acid Releaseand Loss of Viability- In preliminary experiments the phospholipase inhibitors, dibucaine and mepacrine, at concentrations greater than 100 PM and 50 PM respectively, were toxic to hepatocytes (data not shown).
Therefore we employed nontoxic concentrations of dibucaine (100 ,uM) and mepacrine (30 FM) to determine whether these phospholipase inhibitors decreased 14C arachidonic acid release during chemical hypoxia and delayed the onset of cell death. Both mepacrine and dibucaine substantially delayed release of r4C arachidonic acid (Figure 2). Incubation of hepatocytes at a extracellular pH 657
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BIOCHEMICAL
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of 6.5 in the absence of the phospholipase inhibitors also delayed release of 14C arachidonic acid to a similar extent. Phospholipase inhibitors also delayed loss of cell viability during chemical hypoxia (Figure 3). The extent of protection was similar to that afforded by acidosis (pH 6.5). Moreover, the delay of cell killing with either treatment paralleled the delay of 14C arachidonic acid release. DISCUSSION The plasma membrane is a key site of injury due to hypoxia. Morphological, biochemical and biophysical changes in the form of surface blebs (3), changes of lipid composition (9,10,13)
(9,10), accelerated phospholipid all
phospholipase
occur
following
hypoxic
degradation injury
in
(S), and an increase in lipid order
hepatocytes.
A
membrane-bound
A2 is known to be active during hypoxic injury (1 l), and may account, at least
in part, for alterations
to the plasma membrane
after ATP-depletion.
The data presented
here confirm that phospholipid hydrolysis is activated during hypoxic injury and show further that acidotic pH, which protects against lethal cell injury during chemical hypoxia, also slows phospholipid
degradation.
The similar delay of cell killing and r4C arachidonic
acid release caused by acidotic pH and by phospholipase dependent
phospholipid
degradation
inhibitors
makes an important
strongly argues that pH-
contribution
of lethal hypoxic
injury. The time course of r4C arachidonic consequence of changes of intracellular intracellular
acid release during chemical hypoxia may be a pH (4). At the onset of chemical hypoxia,
pH drops by one pH unit in parallel with the hydrolysis of ATP and remains at
this level for 30 to 40 minutes. The results presented here indicate that during this period of intracellular
acidosis, little phospholipid
hydrolysis
occurs. Subsequently,
begins to rise and loss of cell viability follows closely. Therefore, appears to increase phospholipase integrity.
intracellular
pH
this rise of intracellular
pH
AZ activity which in turn causes loss of plasma membrane
Thus, release of r4C arachidonic
acid during chemical hypoxia may be a direct
consequence of changes of intracellular pH. In addition, in isolated hepatocytes, perfused livers and sinusoidal endothelial cells of livers stored for transplantation surgery, lethal injury is greatly accelerated physiological
when the acidotic pH of hypoxic cells is adjusted back to a
range (14,15). The abrupt activation
of phospholipase
A2 brought
about by
increasing pH may be responsible for this ‘pH paradox’. In summary, our data suggest that during hypoxic injury, phospholipase inhibited by acidotic intracellular
pH. Subsequently,
as intracellular
A2 activity is
pH rises, phospholipase
A2 activity increases leading to release of arachidonic acid and an increase of permeability of the plasma membrane. Any intervention which delays the return intracellular pH to physiological levels or prolongs intracellular acidosis should likewise delay phospholipid degradation and the onset of irreversible injury. Such a process might explain the protective effects of amiloride and its analogues on ischemic injury in cardiac muscle (16) and the ‘pH paradox’ in isolated hepatocytes and perfused rat livers (.5,14,15). 658
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REFERENCES 1. Lemasters, J.J., DiGuiseppi,
J., Nieminen,
A.-L., and Herman,B.,
(1987) Nature 325,78-
2. !&man, B., Nieminin, A.-L., Gores, G.J., and Lemasters, J.J., (1988) F’SEBJ. 2, 146151. 3. Nieminien, A.-L,, Gores. G.J., Wray, B.E., Tanaka, Y., Herman, B., and Lemasters J.J., 1988) Cell Calcium, 9,237-246. 4. I ores, G.J., Nieminin, A.-L., Dawson, T.L., Herman, B., and Lemasters, J.J., (1988) Am. J. Physiol. 25$, C315-C322. 5. Gores, G.J., Nieminen, A.-L., Wray, B.E., Herman, B., and Lemasters, J.J., (1989)J. Clin. Invest, 83,386-396.
6. Nieminen, A.-L., Dawson, T.L., Gores, G.J., Kawanishi, T., Herman, B., and Lemasters, J.J., (1990) Biochem. Biophys. Res. Comm. 2,600-606. 7. Gunn, M.D., Sen, A., Chang, A., WilIerson, J.T., Buja, L.M., and Chein, K.R., (1985) Arch. Bioch. Biophys., 2& 312-320. 8. Jones, R.L., Miller, J.C., Hagler, H.K., Willerson, J.T., and Buja, L.M., (1989)Am. J. Path -7-7 135 541-556. 9. Farber, J.L. and Young, E.E., (1981) Arch. Biochem. Biophys, 2& 312-320. 10. Chein, K.R., Abrams, J., Serroni, A., Martin, J.T., and Farber, J.L., (1978)J. Biol. Chem., =,4809-4817. 11. Shirazi, Y. and Mergner, W.J., (1990) In Cell Death, Mergner, W.J., Jones, R.T., $r. Trump, B.F., eds. Field and Wood Medical Publishers Inc., N.Y., N.Y. pp.210-220. 12. Sheir, W.T., and Durkin, J.P., (1986)J. Biol. Chem., 261, 14628-14635. 13. Florine-Casteel, K., Lemasters, J.J., and Herman, B. (1990) in Optical Micrscoov for Biology, eds. B. Herman and K. Jacobson, Wiley/Liss, pp.559-573. 14. Currin, R.T., Gores, G.J., Thurman, R.G., and Lemasters,J.J., (1990) FASEBJ. in press. 15. Currin, R.T., Thurman, R.G., Toole, J.G., and Lemasters, J.J., (1990) Transplutution in press.
16. Karmazyn, M., (1988). Am. J. Physiol. 255, H608-H615.
659
174,
No.
January
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
31, 1991
A pH-DEPENDENT MEMBRANE
654-659
PHOSPHOLIPASE A2 CONTRIBUTES TO LOSS OF PLASMA INTEGRITY DURING CHEMICAL HYPOXIA IN RAT HEPATOCYTES’
D. Corinne Harrison, John J. Lemasters and Brian Herman2 Laboratories for Cell Biology, Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27.599-7090 Received
November
5, 1990
Previous studies have suggestedthat alterations in phospholipid composition of plasma membranes may underlie lethal cell injury due to hypoxic and ischemic injury. The present study was designed to determine if such alterations are due to the activation of a pHdependent phospholipase AZ. Loss of cell viability and phospholipase A2 activity measured by arachidonic acid release increased in parallel during metabolic inhibition with KCN and iodoacetate (chemical hypoxia). Acidosis (pH 6.5) and the phospholipase inhibitors, dibucaine and mepacrine, delayed lossof cell viability and release of arachidonic acid to a similar extent. These findings suggest that a pH-dependent phos holipase A2 causes alterations in plasma membrane phospholipid composition after R TP-depletion which contribute to lethal cell injury. B 1991 Academic Press, Inc.
We have previously reported that ATP-depleted rat hepatocytes sustain plasma membrane damage characterized by the formation, growth, and coalescence of membrane blebs with eventual rupture leading to lethal cell injury (l-5).
However, the mechanisms
which lead to plasma membrane injury during hypoxia remain to be identified. Exposure of hepatocytes to KCN and iodoacetate, a treatment which mimics the ATP-depletion and reductive stressof hypoxic injury, causesa >95% lossof cellular ATP within 10 minutes yet cells survive for up to 60 minutes (1,3-5). Thus, the formation, enlargement and subsequent rupture of plasma membrane blebs which occurs over this period is largely ATPindependent, Accompanying ATP depletion is a drop of intracellular pH from pH 7.1-7.4 to pH 6.0-6.3 (3-5). Intracellular pH remains acidotic for 30-40 minutes but abruptly rises just before the onset of cell death. Low extracellular pH and manipulations which deepen
‘This work was supported by Grants AGO7218 and DK30874 from the National Institutes of Health, the Gustavas and Louise Pfeiffer Foundation and Grant J-1433 from the Office of Naval Research. 2To whom correspondence should be addressed. Abbreviations used are: ATP, adenosine tris hosphate; HEPES-KHB, Krebs-Heinseleit buffer containing 118 mM NaCl, 25 mM NaH e 03, 1.2 mM KH2PO4, 1.2 mM MgSO4, 4.7 mM KCl, 1.3 mM CaC12,20 mM Na- HEPES, pH 7.4., MES-KHB, KHB containing MES (Z(N-morpholino) ethanosulfonic acid, pH 6.5, instead of HEPES. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
654
Vol.
174,
No.
2, 1991
intracellular
BIOCHEMICAL
AND
BIOPHYSICAL
acidosis delay the onset of cell death (3-6).
for membrane physiological
damage appears
to be pH-dependent
RESEARCH
COMMUNICATIONS
Thus the mechanism with
maximal
responsible
activity at or near
pH and minimal activity at acidotic pH.
Several studies lysophospholipids
have demonstrated
following
account for the pH-dependence after hypoxic
injury
release
ATP depletion
of fatty acids and accumulation
(7-10). One potential
mechanism
of cell killing and the changes of phospholipid
is phospholipase
AZ activation
(4-10). Phospholipase
of
which could composition A2 is a pH-
dependent enzyme with maximal activity at neutral or slightly alkaline pH (11). Here we examined the activity of phospholipase and cell viability phospholipase
during chemical A2 activity
A2 in relation to extracellular
hypoxia in hepatocytes.
increases
after
ATP
pH
Our findings indicate that 1)
depletion,
2) acidosis
inhibits
this
phospholipase A2 activity and 3) phospholipase inhibitors delay the onset of lethal cell injury. Taken together, these data suggest that a pH-dependent phospholipase A2 contributes
MATERIALS
to irreversible
AND
hypoxic injury.
METHODS
Heputocyte Isolation and Culture- Hepatocytes were isolated from the livers of male Sprague-Dawley rats (200-300g) by collagenase perfusion as previously described (2). Cell viability was >90% as determined by trypan blue exclusion. lo6 hepatocytes were cultured on 35 mm petri plates (Falcon in 1.5 ml Waymouth’s MB 152/l containing 27 mM NapC03, 2 mM L-glutamine, 10d/o heat-inactivated fetal calf serum (Colorado Serum Co.), 10 U/ml pemctlhn, 10 pg/ml streptomycm, 100 nM insulin, and 10 nM dexamethasone at 37’ in 5% CO2/95% air at pH 7.4. Measurement of 14C Arachidonic Acid Release- [14C]-arachidonic acid (20 pCi/ml) (Amersham) in toluene was dried under N2 and reconstituted in an equal volume of dimethyl sulfoxide which was then added to Waymouth’s media to a final concentration of 0.267 pCi/ml. Cultured hepatocytes were washed with HEPES-KHB at 4’C and incubated in 0.75 ml of 14C arachidonic acid-containing media at 37’C for 11 to 20 hours. Under the labeling conditions used, approximately 94% of the labeled arachidonic acid is incorporated into phospholipids with no more than 3% remaining as free arachidonic acid (12). Following labeling, hepatocytes were washed 3 times in HEPES-KHB or MES-KHB and incubated under various experimental conditions. Aliquots of 0.5 ml were removed at 30 minute intervals, acidified to pH 4.5 with 1 N metaphosphoric acid, and microfuged for 1 minute. Supernatants were further acidified with 35 ~1 1N HCl and extracted with 1.8 ml ethyl acetate. The organic phase was dried over N2 and redissolved in 10 ml Scintiverse cocktail for liquid scintillation counting in a Beckman CS 5000 CE Scintillation counter. After the final aliquot wft removed, cells were scraped from the dish and processed as described to determine the C arachidonic acid remaining . Cell ViubiZity Asay- In parallel experiments, cell viability was assessed by trypan blue exclusion. 0.6% trypan blue was added to cultures for 20 seconds, and the percentage of trypan blue positive cells determined after washing with HEPES-KHB. RESULTS Loss of Cell Viability and 14C Arachidonic Acid Release at pH 7.4- Phospholipase
A2
cleaves phospholipids at the sn2 position causing release of free arachidonic acid (11). Accordingly, phospholipase A2 activity was monitored by labeling cultured hepatocytes with r4C arachidonic acid and quantifying release of radioactivity. In parallel experiments, cell 655
Vol.
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No.
2, 1991
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Figure l- Viability and 14C Arachidonic Acid ReleTf in Cultured Rat Hepatocytes during Chemical Hypoxia- Hepatocyteswere labeledwith C arachidonicacid, and exposedto KCN (2.5 mM) and iodoacetic acid (9. rnM) asdescribedin the materialsand methodsand
incubatedin HEPES-KHB at 37°C. C arachidonicacid releasewasmeasuredin aliquots of supernatantat 30 minute intervals (open circles). In paralle experiments,hepatocytes were quantifiedfor viability by trypan blue exclusion(crosses).’ aC arachidonicacid release was adjusted for the remaining amount of incubation medium after removal of sequential aliquot volumes. Data points represent means + SEM from 12 trials.
viability was monitored by trypan blue exclusion. After exposure of cultured hepatocytes to 2.5 mM KCN and 0.5 mM iodoacetate, viability decreased 10% after 30 minutes and 90% after 60 minutes (Figure 1). Release of arachidonic acid closely paralleled loss of cell viability. Rates of cell killing and arachidonic acid release were both maximal between 30 and 60 minutes of chemical hypoxia (Figure 1). These data suggestthat arachidonic acid release and lossof cell viability are simultaneousevents.
80
60
Tme
(Minutes)
Figure 2 Effect of Acidosis and PhospMipase Inhibitors on 14C Arachidonic Acid Cultured Hepakxytes During Chemical Hypoxia- Conditions were as described in Cells were incubated in HEPES-KHB (pH 7.4; plus marks), HEPES-KHB plus (100 PM; closed triangles), or mepacrine (30 PM; open triangles), or MES-KHB open circles). Data points represent means + SEM from 10 or more trials. 656
Release in Figure 1. dibucaine (pH 6.5;
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
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P 0
30
60
90
Tme
120
150
(Minutes)
Figure 3- Effect of Acidosis and Phospholipase Inhibitors on Cell fiability during Chemical Hypoxia- Cells were incubated as described in Figure 2 and cell viability was determined by trypan blue exclusion. Symbols are as described in Figure 2. Data points represent means + SEM from 10 or more trials.
The data in Figure 1 suggeststhat the 14Carachidonic acid release ceasesonce cells lose viability. To establish further that dead cells do not release arachidonic acid, 14C arachidonic acid-labeled cells were treated with KCN and iodoacetate until all cells were trypan blue positive. The cells were washed with HEPES-KHB,
reincubated in 1 ml
HEPES-KHB, and returned to the incubator. 14Carachidonic acid release into the medium was then measured after another 30 minutes. The cellswere then scraped from the dish and the amount of 14Carachidonic acid associatedwith the cells was determined as described in the materials and methods. The results indicated that after cell viability was lost, little 14C arachidonic acid release occurred (409 cpm released from treated hepatocytes w 368 cpm for untreated cells), although greater than 65% of radioactivity remained with the cells. To determine whether release of intracellular material at bleb rupture might account for release of 14C arachidonic acid associated with cell death, labeled hepatocytes were exposed to 10 /.LM digitonin to permeabilize the plasma membranes of all cells. 14C arachidonic acid release after 25 minutes exposure to digitonin was low and no different than that from untreated cells (1003 vs 957 cpm respectively). Taken together, these data indicate that release of arachidonic acid precedes loss of cell viability and is not the consequenceof cell death. Efects of PhospholipaseInhibitors and Acidotic Extracellular pH on 14CArachidonic Acid Releaseand Loss of Viability- In preliminary experiments the phospholipase inhibitors, dibucaine and mepacrine, at concentrations greater than 100 PM and 50 PM respectively, were toxic to hepatocytes (data not shown).
Therefore we employed nontoxic concentrations of dibucaine (100 ,uM) and mepacrine (30 FM) to determine whether these phospholipase inhibitors decreased 14C arachidonic acid release during chemical hypoxia and delayed the onset of cell death. Both mepacrine and dibucaine substantially delayed release of r4C arachidonic acid (Figure 2). Incubation of hepatocytes at a extracellular pH 657
Vol.
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No.
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BIOCHEMICAL
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of 6.5 in the absence of the phospholipase inhibitors also delayed release of 14C arachidonic acid to a similar extent. Phospholipase inhibitors also delayed loss of cell viability during chemical hypoxia (Figure 3). The extent of protection was similar to that afforded by acidosis (pH 6.5). Moreover, the delay of cell killing with either treatment paralleled the delay of 14C arachidonic acid release. DISCUSSION The plasma membrane is a key site of injury due to hypoxia. Morphological, biochemical and biophysical changes in the form of surface blebs (3), changes of lipid composition (9,10,13)
(9,10), accelerated phospholipid all
phospholipase
occur
following
hypoxic
degradation injury
in
(S), and an increase in lipid order
hepatocytes.
A
membrane-bound
A2 is known to be active during hypoxic injury (1 l), and may account, at least
in part, for alterations
to the plasma membrane
after ATP-depletion.
The data presented
here confirm that phospholipid hydrolysis is activated during hypoxic injury and show further that acidotic pH, which protects against lethal cell injury during chemical hypoxia, also slows phospholipid
degradation.
The similar delay of cell killing and r4C arachidonic
acid release caused by acidotic pH and by phospholipase dependent
phospholipid
degradation
inhibitors
makes an important
strongly argues that pH-
contribution
of lethal hypoxic
injury. The time course of r4C arachidonic consequence of changes of intracellular intracellular
acid release during chemical hypoxia may be a pH (4). At the onset of chemical hypoxia,
pH drops by one pH unit in parallel with the hydrolysis of ATP and remains at
this level for 30 to 40 minutes. The results presented here indicate that during this period of intracellular
acidosis, little phospholipid
hydrolysis
occurs. Subsequently,
begins to rise and loss of cell viability follows closely. Therefore, appears to increase phospholipase integrity.
intracellular
pH
this rise of intracellular
pH
AZ activity which in turn causes loss of plasma membrane
Thus, release of r4C arachidonic
acid during chemical hypoxia may be a direct
consequence of changes of intracellular pH. In addition, in isolated hepatocytes, perfused livers and sinusoidal endothelial cells of livers stored for transplantation surgery, lethal injury is greatly accelerated physiological
when the acidotic pH of hypoxic cells is adjusted back to a
range (14,15). The abrupt activation
of phospholipase
A2 brought
about by
increasing pH may be responsible for this ‘pH paradox’. In summary, our data suggest that during hypoxic injury, phospholipase inhibited by acidotic intracellular
pH. Subsequently,
as intracellular
A2 activity is
pH rises, phospholipase
A2 activity increases leading to release of arachidonic acid and an increase of permeability of the plasma membrane. Any intervention which delays the return intracellular pH to physiological levels or prolongs intracellular acidosis should likewise delay phospholipid degradation and the onset of irreversible injury. Such a process might explain the protective effects of amiloride and its analogues on ischemic injury in cardiac muscle (16) and the ‘pH paradox’ in isolated hepatocytes and perfused rat livers (.5,14,15). 658
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