Mitochondrial permeability transition in pH-dependent
reperfusion injury to rat hepatocytes
Ting
Qian,
Anna-Liisa
Nieminen,
Brian
Herman, and
John J.
Lemasters
Department of Cell Biology and Anatomy, School of Medicine,
University of North Carolina at Chapel Hill, Chapel Hill, North
Carolina 27599-7090
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ABSTRACT |
To simulate
ischemia and reperfusion, cultured rat hepatocytes were incubated in
anoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. During
anoxia, intracellular pH (pHi)
decreased to 6.3, mitochondria depolarized, and ATP decreased to <1%
of basal values, but the mitochondrial permeability transition (MPT)
did not occur as assessed by confocal microscopy from the
redistribution of cytosolic calcein into mitochondria. Moreover, cell
viability remained >90%. After reperfusion at pH 7.4, pHi returned to pH 7.2, the MPT
occurred, and most hepatocytes lost viability. In contrast, after
reperfusion at pH 6.2 or with
Na+-free buffer at pH 7.4, pHi did not rise and cell
viability remained >80%. After acidotic reperfusion, the MPT did not
occur. When hepatocytes were reperfused with cyclosporin A (0.5-1
µM) at pH 7.4, the MPT was prevented and cell viability remained
>80%, although pHi increased to
7.2. Reperfusion with glycine (5 mM) also prevented cell killing but
did not block recovery of pHi or
the MPT. Retention of cell viability was associated with recovery of
30-40% of ATP. In conclusion, preventing the rise of
pHi after reperfusion blocked the
MPT, improved ATP recovery, and prevented cell death. Cyclosporin A
also prevented cell killing by blocking the MPT without blocking recovery of pHi. Glycine prevented
cell killing but did not inhibit recovery of
pHi or the MPT.
cyclosporin A; dimethyl amiloride; glycine; ischemia; pH paradox
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INTRODUCTION |
CELLS AND TISSUES SUBJECTED to ischemia eventually lose
their viability. Often cells can withstand prolonged periods of
ischemia, only to die after reperfusion. The mechanisms allowing
prolonged cell survival during ischemia and causing lethal cell injury
after reperfusion remain incompletely understood. Previously, studies from our laboratory and others showed that the naturally occurring acidosis of ischemia strongly protects renal cells, myocytes, and
hepatocytes against hypoxic cell killing (5, 6, 12, 16, 17, 22, 39,
47). In contrast, the return of extracellular pH
(pHo) to physiological levels is
an event that actually precipitates lethal cell injury, a pH paradox
(4, 5, 12, 21, 27, 47). In neonatal cardiac myocytes, the pH paradox is
linked to the recovery of intracellular pH
(pHi) after reperfusion (4, 21,
27). However, the role of pHi in
reperfusion injury to cultured hepatocytes is not known.
Recently, onset of the mitochondrial permeability transition (MPT) was
implicated in lethal cell injury associated with anoxia, reperfusion,
and oxidative stress to heart and liver cells (7, 14, 18, 19, 24, 25,
28, 32, 36, 38, 42). Opening of high-conductance pores in the
mitochondrial inner membrane initiates onset of the MPT (20). These
pores conduct both positively and negatively charged solutes of up to
1,500 Da. Pore opening induces mitochondrial depolarization, swelling,
and uncoupling of oxidative phosphorylation. Cyclosporin A, an
immunosuppressive cyclic oligopeptide, specifically blocks conductance
of the permeability transition pore (20) and has been shown to prevent
cell injury caused by anoxia and oxidative stress in a number of models
(7, 14, 18, 19, 24, 25, 28, 32, 36, 38, 42). The MPT pore is also
strongly inhibited by pH <7 (3, 20, 34). Thus the pH dependency of
reperfusion injury may be the consequence of the pH dependency of the
MPT.
Glycine is a cytoprotective amino acid that protects renal tubular
cells and hepatocytes against lethal hypoxic injury (29, 33, 46).
Recently, glycine was also shown to protect against pH-dependent
posthypoxic injury to renal tubular cells and endothelial cells of
livers stored for transplantation surgery (2, 11, 47). Importantly,
glycine protected when used only during reperfusion. However, the
mechanism of cytoprotection by glycine remains controversial.
Accordingly, the goals of the present study were to assess the role of
pH in reperfusion injury to cultured hepatocytes and to determine how
pHi, cyclosporin A, glycine, and
other treatments affect the onset of lethal reperfusion injury. Our
data show that a Na+-dependent pH
paradox linked to pHi plays a key
role in reperfusion-induced killing of hepatocytes. Onset of the MPT is
a crucial step in this cell-killing process. Overall, our results
indicate that the protection by acidotic pH and cyclosporin A against
reperfusion injury to hepatocytes involves inhibition of the
pH-dependent onset of the MPT, whereas glycine exerts its protective
effect downstream to the MPT.
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MATERIALS AND METHODS |
Hepatocyte isolation and culture.
Hepatocytes were isolated from 24-h-fasted male Sprague-Dawley rats
(200-300 g) by collagenase digestion, as described previously
(23). Viability of isolated hepatocytes routinely exceeded 95%, as
determined by trypan blue exclusion. For culture, hepatocytes were
resuspended in Waymouth's medium MB-752/1 containing 2 mM
L-glutamine, 27 mM
NaHCO3, 10% fetal calf serum, 100 nM insulin, and 100 nM dexamethasone. For cell viability assay,
aliquots (1 ml) of 1.5 × 105
cells were plated onto 24-well microtiter plates (Falcon, Lincoln Park,
NJ) or glass coverslips, both coated with type I rat tail collagen. For
ATP measurement, cell culture density was 1.5 × 106 cells in 60 × 15 mm
tissue culture dishes (Falcon). Hepatocytes were used after overnight
incubation in 5% CO2-95% air at
37°C. All experiments were carried out in
Krebs-Ringer-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (KRH) containing (in mM) 115 NaCl, 5 KCl, 2 CaCl2, 1 KH2PO4,
1.2 MgSO4, and 25 NaHEPES (pH 7.4 or 6.2). In some experiments, Na+
was substituted with choline or
Cl
was substituted with
gluconate in the buffer.
Model of ischemia and reperfusion in cultured
hepatocytes. To simulate the anoxia and acidosis of
tissue ischemia, hepatocytes were incubated in KRH at pH 6.2 in an
anaerobic chamber (Coy Laboratory Products, Ann Arbor, MI). To simulate
the reoxygenation and return to physiological pH after reperfusion,
anaerobic KRH at pH 6.2 was replaced with aerobic KRH at pH 7.4. In
many experiments, the conditions of this simulated reperfusion were
varied by changing the composition of the reperfusion buffer. Anoxia in
the anaerobic chamber was maintained in an atmosphere of 90%
N2-10%
H2. Any oxygen entering the
chamber by back diffusion was continuously converted to water vapor by
reaction with hydrogen catalyzed by a heated palladium catalyst. Oxygen
tension in the chamber was monitored by a gas analyzer (model 10; Coy
Laboratory Products) and was routinely less than one part per million
(<0.001 Torr).
Cell viability assay. Cell viability
was assessed by propidium iodide fluorometry using a multiwell
fluorescence scanner (CytoFluor 2300; Millipore, Bedford, MA), as
described previously (35). Briefly, hepatocytes were incubated in KRH
containing 30 µM propidium iodide. Fluorescence from each well was
measured using excitation and emission wavelengths of 546 nm (40-nm
band pass) and 620 nm (50-nm band pass), respectively. For each
experiment, an initial fluorescence measurement (A) was made 20 min
after addition of propidium iodide-containing buffer and then at
intervals thereafter. Individual experiments were terminated by
addition of 375 µM digitonin to permeabilize all cells, and a final
fluorescence measurement (B) was obtained 20 min later. The percentage
of viable cells (V) was calculated as V = 100(B
X)/(B
A), where X is the fluorescent intensity at any given
time.
pHi measurement.
Carboxyseminapthorhodofluor-1 (SNARF-1) was used to measure
pHi. SNARF-1 is a dual-emission
fluorophore with a large emission spectral shift in response to pH
changes (10, 41). To load SNARF-1, overnight cultured hepatocytes were
incubated at 37°C for 30 min in KRH with 10 µM
SNARF-1-acetoxymethyl ester (SNARF-1-AM) diluted from a 1 mM stock
solution in dimethyl sulfoxide. The loading buffer was then removed and
replaced with fresh KRH. Before each measurement of SNARF-1
fluorescence, the incubation buffer was replaced. This washing removed
any extracellular fluorophore that may have leaked from the cells,
leaving only intracellular fluorophore as an indicator of
pHi. Fluorescence was measured with a multiwell fluorescence scanner using 530-nm (30-nm band pass)
excitation light. Emission at 590 nm (40-nm band pass) and 620 nm
(50-nm band pass) were collected consecutively in two scans ~18 s
apart. After subtracting background fluorescence from wells not loaded
with SNARF-1, the 590/620 nm fluorescence ratio was calculated. A
calibration curve was generated for each experiment by incubating
SNARF-1-loaded cells with 10 µM nigericin to equilibrate pHi and
pHo. The calibration buffer
contained (in mM) 135 KCl, 15 NaCl, 1 CaCl2, 1 mM
KH2PO4,
0.5 mM MgSO4, and 10 mM HEPES, pH
5.6-7.5. Because all fluorescence measurements were performed outside the anaerobic chamber, pH values during the anoxic period were
obtained from separate plates to avoid oxygen interference. At each
time point during anoxia, a plate was tightly sealed with 3M sealing
tape (Model 471; 3M, St. Paul, MN) after the buffer was first replaced
with fresh anaerobic medium and fluorescence was then
measured immediately (within 1 min) after the plate was removed from
the anoxic chamber. In this way, cells were maintained anaerobic while
SNARF-1 fluorescence was measured.
ATP measurements. Hepatocytes were
incubated at a density of 1.5 × 106 cells/dish in KRH buffer. To
extract ATP, KRH buffer was replaced with 1 ml of cold 0.6 M
HClO4, and cells were scraped with
a disposable cell scraper (Baxter Healthcare, McGow Park, IL). After
centrifugation (9,000 g, 1 min), 0.8 ml of the supernatants was neutralized with 5 M KOH and 0.4 M
imidazole. After one more centrifugation, supernatants were diluted
200-fold with deionized distilled water. ATP was then measured with a
commercial luciferin/luciferase kit (Promega Enliten, Madison, WI)
using an MGM instruments Optocomp I luminometer (Hamden, CT) and was
converted to units of nanomoles per
106 cells.
Laser-scanning confocal microscopy.
The green fluorescence of calcein and red fluorescence of
tetramethylrhodamine methyl ester (TMRM) and propidium iodide were
excited with the 488- and 568-nm lines of an argon-krypton laser.
Fluorescence was divided by a 560-nm emission dichroic reflector and
was measured by separate photomultipliers through 515- to 565-nm
band-pass and 590-nm long-pass barrier filters using a Zeiss LSM-410
inverted laser-scanning confocal microscope (Carl Zeiss, Oberkochen,
Germany). A Zeiss numerical aperture 1.4, 63× planapochromat
objective lens was used, and pinholes were set to Airy units of 0.9 in
both channels.
Cell loading and incubation.
Hepatocytes cultured on glass coverslips were coloaded in KRH with 500 nM TMRM and 1 µM calcein-AM for 15 min at 37°C as described in
Ref. 36. To simulate ischemia, hepatocytes were incubated at pH 6.2 in
KRH containing 100 nM TMRM and 3 µM propidium iodide in the anoxic
chamber for 4 h. During the last 15 min of anoxic incubation, cells
were reloaded with 0.5 µM calcein-AM to improve cellular calcein
loading, since preliminary experiments showed a nonspecific leakage of
calcein during the 4 h of anoxic incubation. Inside the anoxic chamber, coverslips containing cultured hepatocytes were mounted in a closed gas-tight FCS2 chamber (Bioptechs, Butler, PA). After 4 h, the sealed
FCS2 chamber was mounted on the microscope stage to view the anoxic
cells. Subsequently, reperfusion was simulated by infusion of aerobic
KRH containing 500 nM TMRM and 3 µM propidium iodide.
Materials. SNARF-1-AM, calcein-AM, and
TMRM were purchased from Molecular Probes (Eugene, OR); the
luciferase/luciferin ATP measurement kit was from Promega Enliten
(Madison, WI); propidium iodide and type I collagen were from Sigma
(St. Louis, MO); HEPES and collagenase D were from Boehringer Mannheim
Biochemicals (Indianapolis, IN); Waymouth's medium MB-252/1 was from
GIBCO Laboratories (Grand Island, NY); insulin was from Squibb-Novo
(Princeton, NJ); and dexamethasone sodium phosphate was from LyphoMed
(Rosemont, IL). Cyclosporin A, a cyclic endecapeptide, was the gift of
Sandoz Pharmaceuticals (East Hanover, NJ). Other chemicals of
analytical grade were obtained from the usual commercial sources.
Statistics. Differences between
treatment groups and the control group were compared by Student's
t-test or Fisher's exact test, using
P < 0.05 as the criterion of
statistical significance. Error bars shown on graphs represent SE. When
error bars are not shown, they fall within the diameter of the symbol.
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RESULTS |
pH dependency of ischemia-reperfusion injury to
cultured hepatocytes. To study ischemia-reperfusion
injury to cultured hepatocytes under conditions relevant to tissue
ischemia in vivo, we simulated the anoxia and acidosis of tissue
ischemia by placing sealed microtiter plates of aerobic cultured
hepatocytes in an anaerobic chamber and then replacing the still
aerobic KRH at pH 7.4 with preequilibrated anaerobic KRH at pH 6.2. Under these conditions, cell viability remained >85% after 4 h (Fig.
1, A and
B). This result is consistent with
our previous findings showing that acidotic pH strongly protects against loss of hepatocyte viability in models of hypoxia (16, 17).
After subjecting cultured hepatocytes to simulated ischemia, we
simulated the reoxygenation and restoration of normal
pHo after reperfusion by replacing
anaerobic KRH at pH 6.2 with aerobic KRH at pH 7.4. After reperfusion
in this way, cell viability decreased to <40% in 2 h (Fig.
1A). By contrast, when the
hepatocytes were reoxygenated with KRH at pH 6.2, little additional
cell killing occurred (Fig. 1A).

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Fig. 1.
pH paradox of ischemia-reperfusion injury to cultured hepatocytes. To
simulate anoxia and acidosis of tissue ischemia, 1-day cultured
hepatocytes were incubated in anaerobic Krebs-Ringer-HEPES (KRH) at pH
6.2, as described in MATERIALS AND
METHODS. A: cells were
reoxygenated after 4 h by replacing anaerobic medium with aerobic KRH
at pH 7.4 or 6.2. B: pH was changed to
pH 7.4 after 4 h of anoxia at pH 6.2 or was left unchanged, but
hepatocytes were not reoxygenated. Cell viability was assessed by
propidium iodide fluorometry. Values are means ± SE of triplicate
determinations from 3 experiments with 3 cell isolations.
* P < 0.01 vs. pH 7.4.
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After 4 h of anoxia at pH 6.2, we also raised pH without reoxygenation.
When pH was increased without reoxygenation, cell viability decreased
to ~30% within 2 h (Fig. 1B). In
contrast, viability remained >75% after a total of 6 h of anoxia at
pH 6.2. Thus lethal reperfusion injury was initiated by increased pH
rather than by reoxygenation, namely, reperfusion injury in this model was a pH paradox rather than an oxygen paradox. These results confirmed
our previous findings in perfused rat livers (12) and established a
model to study intracellular events during pH-dependent ischemia-reperfusion injury.
Protection by
Na+-free buffer
and lack of protection by
Ca2+-free buffer
and dimethyl amiloride against pH-dependent reperfusion injury.
Previous experiments in cardiac myocytes showed that protection by
acidotic pH against reperfusion injury was mediated by maintenance of
intracellular acidosis after reperfusion. In particular, when the
Na+/H+
exchanger, a major pHi regulator,
was inhibited with dimethyl amiloride,
pHi did not rise after reperfusion
at pH 7.4, and lethal reperfusion injury was prevented (4, 21, 27). In
contrast to this earlier finding in myocytes, dimethyl amiloride (50 µM) did not prevent lethal reperfusion injury to cultured hepatocytes (Fig. 2). Indeed, dimethyl amiloride in a
concentration range between 5 and 1,000 µM failed to improve
viability of reperfused hepatocytes (data not shown). However,
substitution of Na+ with choline
in the reperfusion buffer protected completely against cell killing
after reoxygenation at pH 7.4 (Fig. 2). To test the hypothesis that
pH-dependent injury was mediated by
Ca2+ influx as suggested by Carini
et al. (9), we also reoxygenated hepatocytes with
Ca2+-free KRH.
Ca2+-free buffer failed to produce
any protective effect (Fig. 2).

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Fig. 2.
Protection against reperfusion injury by
Na+-free buffer but not by
Ca2+-free buffer or dimethyl
amiloride (DMA)-containing buffer. Hepatocytes were incubated in anoxic
KRH at pH 6.2 for 4 h as described in Fig. 1. Hepatocytes were then
reoxygenated at pH 7.4 with KRH, with KRH containing 50 µM dimethyl
amiloride, with Na+-free KRH, and
with Ca2+-free KRH. Cell viability
was assessed by propidium iodide fluorometry. Values are means ± SE
of triplicate determinations from 3 experiments with 3 cell isolations.
* P < 0.001 vs. KRH.
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pHi during reperfusion injury.
To measure pHi during simulated
ischemia and reperfusion, we loaded hepatocytes with SNARF-1 and used
an emission fluorescence ratio technique to monitor
pHi. Under the conditions
employed, the 590/620 nm emission ratio was almost linear with
pHi when pHi was clamped to
pHo using nigericin in the pH
range of 5.6-7.5 (Fig. 3,
inset). Although SNARF-1 was well
retained by aerobic hepatocytes, we replaced the buffer before each
fluorescence measurement to avoid interference by dye that may have
leaked into the extracellular medium from cells damaged by
ischemia-reperfusion. From ratiometric measurements of SNARF-1
fluorescence, we observed a progressive recovery of
pHi during reoxygenation at pH 7.4 after 4 h of anoxia at pH 6.2. Half-maximal recovery occurred after
~30 min, and full recovery was achieved after 2 h. Reoxygenation with
buffer at pH 6.2 or with Na+-free
buffer at pH 7.4 blocked the recovery of
pHi. By contrast, reoxygenation
with Ca2+-free buffer or with
dimethyl amiloride-containing buffer at pH 7.4 did not prevent recovery
of pHi (Fig. 3).

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Fig. 3.
Intracellular pH (pHi) during
simulated ischemia and reperfusion to cultured hepatocytes. Hepatocytes
were subjected to simulated ischemia and reperfusion as described in
Fig. 2. pHi was measured from
carboxyseminapthorhodofluor-1 (SNARF-1) fluorescence, as described in
MATERIALS AND METHODS. Data are means ± SE from triplicate determination.
Inset: calibration curve relating
590/620 nm fluorescence ratio to
pHi.
**P < 0.001 vs. KRH, pH 7.4.
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Protection by cyclosporin A against reperfusion
injury. To test the hypothesis that onset of a
cyclosporin A-sensitive MPT was contributing to reperfusion injury, we
reoxygenated hepatocytes with buffer containing cyclosporin A. Because
cyclosporin A may not penetrate cells rapidly, we added cyclosporin A
to the cells during the last 20 min of 4 h of anoxia at pH 6.2. As
shown in Fig. 4, cyclosporin A protected
against lethal reperfusion injury in a biphasic fashion. Lower
concentrations (0.5-1 µM) protected, whereas higher
concentrations (2 µM) did not. Maximal protection occurred with
~0.5 µM cyclosporin A.

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Fig. 4.
Protection by cyclosporin A against reperfusion injury. Hepatocytes
were subjected to simulated ischemia and reperfusion as described in
Fig. 2. Cyclosporin A (0.1-2 µM) was added 15 min before
reperfusion. Cell viability was determined by propidium iodide
fluorometry, as described in MATERIALS AND
METHODS. Values are means ± SE of triplicate
determinations from 3 experiments with 3 cell isolations.
* P < 0.01 vs. no
cyclosporin.
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Protection by glycine against reperfusion
injury. To assess the effect of glycine, a
cytoprotective amino acid, on reperfusion injury to cultured
hepatocytes, we subjected hepatocytes to 4 h of anoxia at pH 6.2 followed by reoxygenation at pH 7.4 in the presence of various
concentrations of glycine. Glycine at low concentrations (0.05-0.5
mM) partially prevented lethal reperfusion injury (data not shown),
whereas 5 mM glycine blocked essentially all reperfusion-induced cell
killing (Fig. 5). To test the hypothesis that the cytoprotection of glycine was mediated by regulation of
Cl
influx as suggested by
Schnellmann and associates (30, 31, 45), we reperfused cells in
Cl
-free KRH buffer with and
without glycine. As shown in Fig. 5, Cl
-free reperfusion did not
protect against reperfusion injury to cultured hepatocytes or alter the
cytoprotection by glycine. Similarly, cell killing and glycine
protection against cell killing were also not changed when
Cl
concentration in the
reperfusion buffer was titrated to 25% and 50% of values in KRH (data
not shown). We also determined the effects of cyclosporin A and glycine
on the recovery of pHi after reperfusion. Although both agents prevented cell killing, neither cyclosporin A nor glycine prevented the recovery of
pHi to pH 7.1-7.2 after
reperfusion (Fig. 6). Also, dimethyl
amiloride in the presence of glycine did not prevent recovery of
pHi during reperfusion (Fig. 6).
Similarly, reperfusion in
Ca2+-free buffer in the presence
of glycine did not alter pHi
recovery (data not shown).

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Fig. 5.
Lack of Cl dependence of
cell killing and glycine cytoprotection after ischemia-reperfusion.
Hepatocytes were subjected to simulated ischemia for 4 h, as described
in Fig. 1. Hepatocytes were then reperfused at pH 7.4 in KRH or
Cl -free KRH with or without
glycine (5 mM). Cell viability was assessed by propidium iodide
fluorometry, as described in MATERIALS AND
METHODS. Values are means ± SE of triplicate
determinations from 3 experiments with 3 cell isolations.
**P < 0.001 vs. no glycine.
Difference between
Cl -containing and
Cl -free KRH was not
statistically significant at any time point
(P > 0.1).
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Fig. 6.
pHi during reperfusion with
cyclosporin A and glycine. Hepatocytes were exposed to simulated
ischemia and reperfusion, as described in Fig. 1. Cyclosporin A (1 µM), glycine (5 mM), or glycine + dimethyl amiloride (50 µM) were
added at or just before reperfusion.
pHi was measured by SNARF-1
fluorescence as described in Fig. 3. Data are means ± SE from
triplicate determinations.
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Onset of the MPT after reperfusion.
Because cyclosporin A prevented pH-dependent reperfusion injury to
hepatocytes, we applied laser-scanning confocal microscopy to directly
monitor the changes of mitochondrial membrane permeability and membrane
potential (
) in relation to cell killing during reperfusion.
Hepatocytes were coloaded with TMRM and calcein. TMRM is a cationic
fluorophore that accumulates electrophoretically in mitochondria in
response to their highly negative 
. Calcein is taken up into the
cytosol when loaded at 37°C but is excluded by normal mitochondria.
Thus, during normal aerobic incubation, confocal microscopy reveals mitochondria as bright spots in the red fluorescence channel and as
dark voids in the green fluorescence channel, as described previously
(36).
After 4 h of simulated ischemia (anoxia at pH 6.2), TMRM labeling of
mitochondria was almost completely lost, indicating mitochondrial depolarization (Fig. 7,
top; compare with the baseline image
in Fig. 4 of Ref. 36). By contrast, the calcein image was unchanged and
mitochondria remained as dark voids, indicating that they continued to
exclude the cytosolic fluorophore. Additionally, the calcein image
showed development of plasma membrane blebbing and general cellular
swelling, characteristic features of ATP depletion injury (23).

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Fig. 7.
Onset of mitochondrial permeability transition (MPT) after simulated
ischemia and reperfusion. Cultured hepatocytes were loaded with 500 nM
tetramethylrhodamine methyl ester (TMRM) and 1 µM calcein
acetoxymethyl ester (calcein-AM) for 15 min at 37°C and subjected
to 4 h of simulated ischemia (anoxia at pH 7.4). During the last 15 min
of ischemia, cells were reloaded with calcein-AM (0.5 µM) to replace
calcein that had leaked from cells during the ischemic period. Red
fluorescence of TMRM and propidium iodide (PI;
top) and green fluorescence of
calcein (bottom) were imaged by
laser-scanning confocal microscopy, as described in
MATERIALS AND METHODS. Images were
collected at end of 4 h of anoxia at pH 6.2 and after 5, 20, and 25 min
of reoxygenation at pH 7.4. Top (red
fluorescence): note that TMRM fluorescence was lost at end of 4 h of
simulated ischemia. Subsequently, mitochondrial TMRM fluorescence
recovered after 5 min of reperfusion in both cells in the field. After
20 min, 1 cell lost TMRM fluorescence and went on to lose viability
after 25 min, as indicated by nuclear labeling with propidium iodide
(arrow). The other cell in the field continued to accumulate TMRM.
Bottom (green fluorescence): calcein
was excluded from mitochondria at end of ischemia. Calcein remained
excluded after 5 min of reperfusion, but, after 20 min, calcein
fluorescence filled most mitochondria in the cell whose mitochondria
lost TMRM fluorescence. This cell went on to lose virtually all
intracellular calcein fluorescence as viability was lost after 25 min
of reperfusion. A total of 20 cells were examined in 5 different
experiments. Of these, MPT occurred in 19 cells within 20 min after
reoxygenation. Cell death then occurred within 30 min. Only the cell
not having onset of MPT survived for >50 min.
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When hepatocytes were reperfused (reoxygenated at pH 7.4) after 4 h of
simulated ischemia (anoxia at pH 6.2), mitochondria in hepatocytes
began to repolarize and to take up TMRM within 5 min (Fig. 7). After 20 min of reperfusion, however, one of the two cells in the field lost
TMRM fluorescence. Simultaneously, calcein fluorescence filled the dark
mitochondrial voids. These events signify onset of the MPT (36).
Subsequently, cell death occurred, as shown by nuclear staining with
propidium iodide and release of all cytosolic calcein. In the other
hepatocyte in the field, onset of the MPT and mitochondrial
depolarization did not occur. Rather, mitochondrial 
continued to
recover, as indicated by accumulation of TMRM fluorescence. Moreover,
cell viability was retained. Overall, in 5 separate experiments, 19 of
20 cells showed onset of the MPT and subsequent cell death after
reoxygenation at pH 7.4.
To test the hypothesis that acidotic reperfusion and reperfusion with
cyclosporin A were preventing cell killing by blocking onset of the
MPT, we monitored TMRM and calcein fluorescence as ischemic hepatocytes
were reoxygenated with KRH at pH 6.2 (Fig. 8) or with KRH at pH 7.4 in the presence of
cyclosporin A (Fig. 9). After reperfusion
at acidotic pH, a repolarization occurred as indicated by TMRM uptake.
Additionally, calcein did not redistribute into the mitochondria,
indicating that the MPT had not occurred. Overall, in 3 experiments,
only 1 of 12 cells underwent the MPT after reoxygenation at pH 6.2. This cell subsequently lost viability. Similarly, when cells were
reoxygenated at pH 7.4 in the presence of cyclosporin A, mitochondria
repolarized and mitochondrial impermeability to calcein was retained,
an effect observed in 13 of 13 cells in 4 separate experiments.
Reperfusion at pH 6.2 and at pH 7.4 with cyclosporin A also prevented
loss of cell viability, as indicated by retention of cytosolic calcein
fluorescence and lack of nuclear labeling with propidium iodide.

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Fig. 8.
Inhibition of MPT and cell killing by reperfusion at pH 6.2. Hepatocytes loaded with TMRM and calcein were subjected to ischemia as
described in Fig. 7. After 4 h of ischemia, cells were reoxygenated at
pH 6.2. Images were collected at end of 4 h of ischemia and after 5, 20, and 60 min of reoxygenation. After acidotic reperfusion, note that
mitochondria repolarized (top) and
did not undergo MPT (bottom). Cell
viability was not lost. A total of 12 cells in 3 different experiments
were examined. Only 1 cell in 12 displayed onset of MPT after
reoxygenation, and only this cell subsequently lost viability
(P < 0.001 vs. data of Fig. 7).
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Fig. 9.
Protection by cyclosporin A against MPT and cell killing after
simulated ischemia-reperfusion. Hepatocytes loaded with TMRM and
calcein were exposed to ischemia and reperfusion as described in Fig.
7. Images were collected at end of 4 h of ischemia and after 5, 30, and
60 min of reoxygenation at pH 7.4. Cyclosporin A (CyA; 1 µM) was
added to incubation buffer for last 15 min of ischemia and during
reoxygenation. Note that mitochondria repolarized after reperfusion
with cyclosporin A (top). Onset of
MPT did not occur (bottom), and cell
viability was retained. Thirteen cells in four different experiments
were observed. None of the cells showed onset of MPT or cell killing
within 1 h of reoxygenation (P < 0.001 vs. Fig. 7).
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We also investigated whether glycine could prevent pH-dependent onset
of the MPT after reperfusion (Fig. 10).
In contrast to acidotic reperfusion and cyclosporin A, reperfusion with
glycine did not produce mitochondrial repolarization or block onset of the MPT, although cell viability was maintained. Overall, onset of the
MPT occurred in 23 of 25 cells in 3 experiments, but only 1 cell lost
viability.

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|
Fig. 10.
Protection by glycine against cell killing and lack of protection
against onset of MPT after reperfusion. Hepatocytes loaded with TMRM
and calcein were exposed to simulated ischemia and reperfusion as
described in Fig. 7. Glycine (5 mM) was added to reoxygenation buffer.
Images were collected at end of 4 h of ischemia and after 3, 20, and 30 min of reperfusion. Note that glycine prevented loss of cell viability
but did not promote repolarization of mitochondria
(top) or prevent onset of MPT
(bottom). Twenty-five cells in three
different experiments were examined. Onset of MPT occurred in 23 cells
within 30 min of reoxygenation (not significant vs. Fig 7), but only 1 cell lost viability after 55 min (P < 0.001 vs. Fig. 7).
|
|
ATP during reperfusion injury. We also
measured cellular ATP during simulated ischemia and reperfusion. After
4 h of anoxia at pH 6.2, cellular ATP decreased from 18 to 0.1 nmol/106 cells (Table
1). After reperfusion at pH 7.4, ATP
initially increased to 1.4 nmol/106 cells after 30 min but
then decreased to about one-half that value after 60 and 120 min. When
hepatocytes were reoxygenated at pH 6.2 or 7.4 in the presence of
cyclosporin A or glycine, a greater and sustained recovery of ATP
occurred, and 30-40% of basal ATP was restored within 2 h (Table
1).
 |
DISCUSSION |
pH paradox in ischemia-reperfusion injury to
cultured rat hepatocytes. Anoxia and acidosis are
cardinal features of tissue ischemia. Previously, our laboratory and
others demonstrated that the naturally occurring acidosis of ischemia
protects strongly against hypoxic killing of heart, kidney, and liver
cells (5, 6, 12, 16, 17, 22, 39, 47). In reperfusion, however, the
transition from acidosis to normal pH precipitates cell death (4, 5,
12, 21, 27, 47). Here, we documented such a pH paradox in cultured rat
hepatocytes. We showed that hepatocytes retained viability for as long
as 6 h of anoxic incubation at pHo
6.2 (Fig. 1B). However, when
pHo was returned to 7.4 with reoxygenation, cell killing ensued in a majority of cells within 1-2 h. This injury was not oxygen dependent, since reperfusion with oxygenated buffer at pH 6.2 prevented cell killing (Fig. 1A). Moreover, increasing
pHo to 7.4 without reoxygenation
precipitated cell killing to the same extent as with reoxygenation
(Fig. 1B). In experiments not
reported here, we observed a nearly identical pH paradox in
hepatocytes after washout of 2.5 mM cyanide (chemical hypoxia).
Previously, in our model of chemical hypoxia to hepatocytes, we showed
that protection against cell killing by acidotic pH was mediated by
pHi. Treatments that increased
pHi during chemical hypoxia, such
as monensin, accelerated cell killing, whereas treatments that
decreased pHi, such as the
Na+/H+
exchange inhibitor amiloride, delayed the onset of cell death (17).
Similarly, in models of ischemia-reperfusion to cultured cardiac
myocytes and isolated perfused papillary muscles, dimethyl amiloride
delayed recovery of pHi and
prevented cell killing, whereas monensin accelerated recovery of
pHi and hastened cell death (21,
27). Thus our expectation was that dimethyl amiloride would prevent
lethal reperfusion cell injury in the pH paradox to hepatocytes.
However, dimethyl amiloride over a broad range of concentrations had no
effect on reperfusion-induced cell killing (Fig. 2). Likewise, dimethyl
amiloride did not prevent the recovery of
pHi from acidotic to normal level
after reperfusion (Fig. 3), even in the presence of glycine (Fig. 6).
Thus the likely explanation for the lack of cytoprotection by dimethyl
amiloride is its failure to prevent the recovery of
pHi after reperfusion.
Recently, Carini and co-workers (8) suggested that
protection against cell death by acidosis may be mediated by
suppression of Na+ uptake via
Na+/H+
exchange and
Na+-
cotransport. However, we showed previously that acidosis protects
against chemical hypoxia (KCN + iodoacetate), even when intracellular
and extracellular Na+ are
equilibrated with monensin in 10 mM
Na+ medium. Thus acidosis is a
protective factor independent of
Na+ uptake. Nonetheless, this does
not exclude the possibility that Na+ uptake is an additional factor
promoting cell killing.
pH regulation and cell killing after
reperfusion. Because dimethyl amiloride did not prevent
the recovery of pHi after
reperfusion, we conclude that amiloride-sensitive
Na+/H+
exchange does not mediate this pHi
recovery in cultured hepatocytes. In rat cardiac myocytes, the
Na+-
cotransporter participates in the recovery of
pHi after acidic loading (8). This
occurs even in
-free medium,
presumably by employing endogenously produced
CO2 that is converted to
by carbonic anhydrase on the cell
surface (43). Thus the recovery of
pHi in hepatocytes after
reperfusion may also be mediated by
Na+-
cotransport. Additionally, an amiloride-insensitive and
-independent mechanism may
contribute to pHi recovery, as
recently shown in acid-loaded perfused rat livers (13). Future
experiments will be needed to determine the exact mechanism by which
pHi recovers in our model of
simulated ischemia-reperfusion to cultured hepatocytes.
When recovery of pHi was blocked
by reperfusion with Na+-free or
acidotic buffer, cell killing was prevented. To test the hypothesis that reperfusion injury was the result of cellular
Ca2+ overloading mediated by
concerted
Na+/H+
and
Na+/Ca2+
exchange (9), we also reperfused ischemic hepatocytes with Ca2+-free buffer.
Ca2+-free reperfusion failed to
protect hepatocytes against lethal reperfusion injury or alter the
recovery of pHi. Thus we conclude that Ca2+ overload after
reperfusion is not the mechanism underlying pH-dependent reperfusion
injury in our model.
Contribution of the MPT to pH-dependent reperfusion
injury. Recently, mitochondrial dysfunction associated
with onset of the MPT was implicated in lethal cell injury after
anoxia, oxidative stress, and reperfusion in heart and liver cells (7,
14, 18, 19, 24, 25, 28, 32, 36, 38, 42). In hepatocytes, onset of the
MPT during oxidative stress was directly demonstrated using confocal
microscopy (36). In the present study, we showed by direct confocal
imaging that onset of the MPT and mitochondrial depolarization preceded
cell killing after reperfusion (Fig. 7). Moreover, low pH and
cyclosporin A, treatments that strongly suppress onset of the MPT in
isolated mitochondria, blocked onset of the MPT and mitochondrial
depolarization after reperfusion (Figs. 8 and 9). These results support
our hypothesis that recovery of pHi after reperfusion causes onset
of the MPT and leads to lethal reperfusion injury.
Cytoprotection by cyclosporin A showed a biphasic dose response.
Protection occurred at 0.5-1 µM but was lost at higher
concentrations. This dose-response relationship is similar to that
reported earlier for isolated myocytes and perfused hearts (18, 19,
32). At higher concentrations, cyclosporin A becomes cytotoxic (26, 40), which may overcome its beneficial effect on the MPT.
Our results showed that rising pHi
after reperfusion triggers onset of the MPT. Once the MPT occurs,
mitochondria depolarize and the uncoupler-stimulated mitochondrial
ATPase become activated (24, 35). Such ATP hydrolysis may promote cell
killing. To test this hypothesis, we measured ATP during simulated
ischemia-reperfusion to cultured hepatocytes. When ischemic hepatocytes
were reperfused at pH 7.4, we observed little recovery of ATP (Table
1). By contrast, when cells were reperfused at pH 6.2 or at pH 7.4 with
1 µM cyclosporin A, 30-40% of basal ATP was restored in 2 h.
Thus protection against cell killing by acidosis and cyclosporin A was
associated with partial ATP recovery. Because acidosis and cyclosporin
A both act to block onset of the MPT, we conclude that accelerated ATP hydrolysis caused by onset of the MPT is the likely basis for continued
ATP depletion and subsequent cell killing.
Protection of glycine against reperfusion
injury. Glycine is a cytoprotective amino acid that
protects kidney and liver cells against hypoxia and ATP
depletion-induced cell death in various models (29, 46). Glycine also
protects against pH-dependent posthypoxic injury to renal tubular cells
and reperfusion injury to endothelial cells of livers after cold
storage for transplantation (2, 11). In the present study, glycine
strongly protected against pH-dependent reperfusion injury to cultured
hepatocytes. Full protection occurred when glycine was administered
only during reperfusion. Thus glycine acted specifically against those
mechanisms that precipitated reperfusion-induced cell death. However,
glycine did not block recovery of
pHi to normal levels after
reperfusion (Fig. 6). This result indicated that cytoprotection by
glycine was not mediated by intracellular acidification.
Several mechanisms have been suggested to explain cytoprotection by
glycine, including inhibition of proteolysis (33). Studies in renal
tubules also suggest that inhibition of
Cl
influx through
Cl
channels mediates
glycine cytoprotection (31). To investigate the contribution of
Cl
influx to reperfusion
injury in hepatocytes, we used gluconate to substitute for
Cl
in our reperfusion
buffer. We found that glycine cytoprotection was not dependent on
Cl
in the medium (Fig. 5)
and further that cell killing was not diminished in the absence of
Cl
as it was in the absence
of Na+ (Fig. 2). Two very recent
studies also demonstrate that glycine cytoprotection is independent of
medium Cl
and that
Cl
-free medium is not
cytoprotective (11, 44). Earlier, Miller and Schnellmann (31) reported
cytoprotection to renal proximal tubular cells exposed to antimycin
when 50% of medium NaCl was replaced isosmotically with mannitol.
However, mannitol is an antioxidant that protects against injury caused
by mitochondrial oxygen radical generation during respiratory
inhibition (17). Thus protection may be due to mannitol rather than
decreased Cl
concentration.
Nonetheless, a number of agonists and antagonists of amino acid-gated
Cl
channels do protect
against lethal cell injury (44, 45). As suggested by Venkatachalam et
al. (44), the conductance of the putative channel involved in cell
killing may not be specific only to
Cl
. Moreover, recent work
in cultured sinusoidal endothelial cells exposed to cyanide suggests
that glycine protects by inhibiting an organic anion channel, which
opens just before the onset of cell death (37). In any event, glycine
cytoprotection did not prevent the onset of the MPT and mitochondrial
depolarization (Fig. 10). Thus protection by glycine in reperfusion
injury appears to occur at a point downstream to onset of the MPT.
In previous work in cells from kidney and other tissues, glycine
cytoprotection was not associated with increased ATP levels (29, 44).
Thus recovery of ATP after reperfusion of hepatocytes with glycine in
the present work was unexpected (Table 1). One possible source for ATP
after reperfusion with glycine is the glycine cleavage system, which
catalyzes the tetrahydrofolate-dependent oxidation of glycine to
CO2,
NH+4, and formate, the reduction of
NAD+ and
NADP+ to NADH and NADPH, and the
phosphorylation of ADP to ATP (1, 15). Hepatocytes are particularly
enriched in the enzymes of the glycine cleavage system. During
reoxygenation, NADH and NADPH formed during glycine cleavage are
reoxidized by mitochondrial respiration, further driving the overall
reaction to completion. We cannot say from our experiments that ATP
recovery is necessary for glycine cytoprotection against pH-dependent
reperfusion injury, but work in other cell types indicates that glycine
cytoprotection is independent of the recovery of ATP. However, when it
occurs, ATP recovery is most likely beneficial.
In conclusion, our data demonstrate that the return of normal
pHi after reperfusion leads to
onset of the MPT and lethal reperfusion injury to cultured hepatocytes.
Treatments such as reperfusion at low pH inhibited recovery of
pHi, blocked onset of the MPT, and
prevented cell killing. Cyclosporin A also blocked onset of the MPT and
reduced cell killing. In addition, glycine protected against
reperfusion injury, but the mechanism by which glycine prevented cell
killing remains obscure and may be related to inhibition of proteolysis
(33) or another process occurring after recovery of
pHi.
 |
ACKNOWLEDGEMENTS |
This work was supported, in part, by National Institutes of Health
Grants DK-37034, AG-07218, and AG-13318.
 |
FOOTNOTES |
Present address of A.-L. Nieminen: Department of Anatomy, Case Western
Reserve University, Cleveland, OH 44106-4938.
Address for reprint requests: J. J. Lemasters, Dept. of Cell Biology
and Anatomy, School of Medicine, Univ. of North Carolina at Chapel
Hill, Campus Box 7090, 236 Taylor Hall, Chapel Hill, NC 27599-7090.
Received 3 January 1996; accepted in final form 28 July 1997.
 |
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