Role of water and electrolyte influxes in anoxic plasma
membrane disruption
Jing
Chen and
Lazaro J.
Mandel
Division of Physiology and Cellular Biophysics, Department of Cell
Biology, Duke University Medical Center, Durham, North Carolina
27710
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ABSTRACT |
The role of water and electrolyte influxes in anoxia-induced
plasma membrane disruption was investigated using rabbit proximal tubule suspension. The results indicated that normal proximal tubule
(PT) cells have a great capacity for expanding cell volume in response
to water influx, whereas anoxia increases the susceptibility to water
influx-induced disruption, and this was attenuated by glycine. However,
resistance of anoxic plasma membranes to water influx-induced stress is
not lost, although their mechanical strength was diminished, compared
with normoxic membranes. Anoxic membranes did not disrupt under an
intra-to-extracellular osmotic difference as great as 150 mosM.
Potentiating or attenuating water influx by incubating PT cells in
hypotonic or hypertonic medium, respectively, during anoxia, did not
affect anoxia-induced membrane disruption. After the transmembrane
electrolyte concentration gradient was eliminated by a
"intracellular" buffer or by permeabilizing the plasma membrane
to molecules <4 kDa using
-toxin, anoxia still caused further
membrane disruption that was prevented by glycine or low pH. These
results demonstrate that 1) water or
net electrolyte influxes are probably not a primary cause for
anoxia-induced membrane disruption and
2) glycine could prevent the plasma
membrane disruption during anoxia independently from its effect on
transmembrane electrolyte or water influxes. The present data support a
biochemical rather than a mechanical alteration of the plasma membrane
as the underlying cause of membrane disruption during anoxia.
glycine;
-toxin; renal tubule; osmotic pressure; lactate
dehydrogenase release
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INTRODUCTION |
PLASMA MEMBRANE INTEGRITY is a prerequisite for cell
viability and is maintained normally in an ATP-dependent manner.
Depletion of ATP during anoxia or ischemia causes reversible cell
injury as long as membrane integrity is maintained. Once the membrane disrupts due to prolonged ATP depletion, irreversible cell injury results, probably due to the loss of cellular contents. An example of
this is found in our previous work with the microvillar cytoskeletal protein, ezrin. This protein became dissociated from the cytoskeleton and released into the extracellular medium after 30 min of anoxia due
to plasma membrane disruption. Prevention of the membrane disruption by
glycine retained the dissociated ezrin within the cell, permitting its
reassociation with the microvillar cytoskeleton during the subsequent
reoxygenation (4).
The mechanisms underlying the permeabilization of the plasma membrane
during ATP depletion remain incompletely understood. Various
alterations to the plasma membrane during ATP depletion have been
observed, including degradation of membrane phospholipids (12, 17, 19,
23, 26, 31), aggregation of intramembrane proteins (27, 29), cell
swelling (2, 13, 16, 21), membrane blebbing (3, 9, 13, 36), the loss of
membrane cholesterol (32), less asymmetric distribution of
phospholipids (24, 27, 28), and decreased cytoskeletal support (3, 9).
However, none of these has proved to be the direct cause of membrane
disruption. Furthermore, it is unclear whether the membrane disrupts
gradually or suddenly after a threshold has been reached.
A decade ago, glycine was found to inhibit anoxia-induced membrane
disruption (18, 33). This finding was very important for two reasons:
1) it demonstrated that plasma
membrane integrity could be maintained without ATP and
2) it made more likely that a
breakthrough would occur in the understanding of anoxia-induced membrane disruption through the investigation of the mechanisms for
membrane protection by glycine (11, 34, 35). It has been found that
glycine protects plasma membrane without preventing the degradation of
membrane phospholipids (31). The protective effect of glycine also does
not depend on an intact cytoskeletal support for the plasma membrane
(3). It was recently reported that the membrane protection by glycine
correlated with its effect of blocking increased
Cl
and water influxes
across the plasma membrane during anoxia (20, 21), with the suggestion
that these influxes might initiate membrane disruption, possibly by
osmotic stress. However, osmotic stress-induced cell swelling by itself
would not be expected to cause membrane disruption if the mechanical
strength of the membrane is maintained during anoxia. On the other
hand, alterations in physiochemical properties of the plasma membrane
or decreased cytoskeletal support might weaken the mechanical strength
of the plasma membrane and this would make it more susceptible to
osmotic stress-induced disruption.
The goals of the present study were to test whether
1) the limit of cellular
distensibility before plasma membrane disruption was decreased by
anoxia, 2) glycine could prevent
this decrease if it occurs, 3) water
and electrolyte influxes are required for anoxic membrane disruption,
and 4) the membrane protection by glycine occurs by inhibition of transmembrane electrolyte and water
influxes during anoxia.
All the present experiments were conducted in freshly isolated rabbit
renal proximal tubules (PTs). The results indicated that normal PT
cells have a great capacity for expanding cell volume before the plasma
membrane disrupts, whereas anoxia increases the susceptibility to
osmotic stress-induced disruption, and this was attenuated by glycine.
The resistance of anoxic PT cells against water influx-induced injury
remained quite high, although it was significantly decreased in a
temperature-dependent manner. Potentiating or attenuating water
influxes by incubating PT cells in hypotonic or hypertonic medium,
respectively, during anoxia did not affect the membrane disruption.
After the transmembrane electrolyte gradient was eliminated by
incubating PT cells in an "intracellular" buffer, or by
permeabilizing the plasma membrane to molecules <4 kDa using
-toxin, anoxia still caused further membrane disruption that was
prevented by glycine or low pH. These results demonstrate that
1) net water and electrolyte
influxes are probably not a primary cause for anoxia-induced membrane
disruption and 2) glycine could
prevent the plasma membrane disruption during anoxia independently from
its effect on transmembrane electrolyte or water fluxes.
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METHODS |
PT isolation.
Rabbit PTs were isolated and purified as previously described (7).
Briefly, female New Zealand White rabbits (1-2 kg; Robinson, Winston-Salem, NC) were injected with heparin and anesthetized. The
cortices were trimmed from the excised kidneys, minced, and digested
for 60 min at 37°C in 300 mosM Dulbecco's modified Eagle's medium
(DMEM) containing 150 U/ml collagenase (Worthington Biochemical, Freehold, NJ) and 1 U/ml deoxyribonuclease, gassed with 100%
O2. The resulting isolated tubules
were washed free of collagenase, and the PTs were separated from other
segments by centrifugation on a self-generating 50% Percoll gradient
for 30 min at 36,000 g. The PT cells
were then washed and resuspended at 2 mg protein/ml. The protein in PT
suspension was measured using the bicinchoninic assay (Pierce
Biochemicals). Purified PT suspensions were gassed with 100%
O2 in DMEM and preincubated at
37°C for 30 min. After the preincubation period, the PT cells were
subjected to normoxia or anoxia. For the normoxic treatment, PT cells
were gassed with 100% O2. For the
anoxic treatment, PT cells were gassed with 100% N2.
Hypotonic swelling response after 30 min of normoxia or anoxia.
This experiment was designed to determine whether the plasma membrane
of anoxic cells that have been injured but have not yet lost their
membrane integrity is mechanically weaker or more vulnerable to osmotic
stress. As shown in Fig. 1, PT cells were initially exposed to normoxia or anoxia in 300 mosM DMEM at 37°C for 30 min, pelleted (110 g, 2 min),
and resuspended in NaCl-H2O of
various osmolarities (from 300 to 0 mosM) on ice for 30 min, or at
37°C for 10 min while exposed to room air. Finally, the changes in
cell volume and membrane integrity were measured as described below.

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Fig. 1.
A flow chart describing how swelling test was conducted after proximal
tubule (PT) cells were exposed to anoxia or normoxia for 30 min. LDH,
lactate dehydrogenase.
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The relative increase in cell volume at each osmolarity was estimated
by two methods: 1) direct
visualization under differential interference contrast (DIC) microscopy
(relative volume was estimated from ratio of cellular radii cubed,
assuming a spherical geometry, and only cells that retained their
integrity during hypotonic treatment were measured; Fig.
2A) and
2) weighing the PT cells after
centrifugation at 3,000 g for 10 min
at 4°C. If specific gravity and intercellular water are assumed to
be constant, the relative volume of PT cells after exposure to
hypotonic solutions is the ratio of their respective weights. The
relative volumes were expressed as percentage of the total volume of
the PT cells exposed to isotonic
NaCl-H2O and calculated according
to the following formula: percent isotonic volume = 100 × [(total weight after exposure to hypotonic solution)/(total
weight after exposure to isotonic solution)]. Plasma membrane
disruption was measured by lactate dehydrogenase (LDH) assay, as
described below.

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Fig. 2.
A: after 30 min of incubation under
normoxia, PT cells were exposed to various hypotonic NaCl solutions on
ice, fixed in 1% glutaraldehyde, and then examined retrospectively
using differential interference contrast (DIC) microscopy. Numbers
under each image were osmotic differences (mosM) between intra- and
extracellular compartments. These differences were used in all
following figures to indicate driving force for water influx.
Intracellular osmotic pressure was considered to be 300 mosM because it
should be equilibrated with 300 mosM DMEM before exposure to hypotonic
solutions. Bar in top left of each DIC
image is 10 µm. B: groups of
normoxic PT cells with equal volume were exposed to isotonic or
hypotonic solutions on ice. Volume of PT cells after being exposed to
hypotonic solutions was expressed as percentage of PT cells exposed to
isotonic solution. It represents volume of both intact and disrupted
cells in each group. Results indicate an exceptionally great capacity of PT cells to expand their cell volume before membrane disrupts. All
groups were significantly different from one another as indicated by
numbers of asterisks.
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LDH release.
LDH release, an index of plasma membrane integrity, was measured by the
method of Bergmeyer et al. (1). Extracellular LDH was measured by
layering the tubule suspension on 2:1
n-butyl-dioctyl pthalate followed by
centrifugation at 14,000 g for 30 s.
This procedure pelleted the cellular contents and left the
extracellular contents in suspension above the pthalate. Samples of 300 µl of extracellular medium as well as 300 µl of the total
suspension were mixed with 60 µl of 2% Triton X-100 before analysis.
The LDH activities were converted to "percent release" by
dividing the supernatant activity by the total activity.
Potentiation or attenuation of water influx during anoxia.
These experiments were designed to determine whether potentiating or
attenuating water influx during anoxia could affect the process of
membrane disruption. The water influx during 30 min of anoxia was
potentiated by incubating PT cells in a 150 mosM hypotonic buffer that
was made by diluting 300 mosM DMEM with deionized water. Attenuation of
water influx during anoxia was made by incubating PT cells in a 450 mosM hypertonic buffer that was made by adding sucrose to DMEM. Osmotic
concentrations of the hypo- and hypertonic buffer were measured with an
osmometer. The pH of these buffers were adjusted to pH 7.4. After PT
cells were incubated in the iso-, hypo-, or hypertonic buffer under anoxia for 30 min, total weight of live and dead cells was measured, and membrane disruption was determined by LDH assay.
Permeabilization of PT cells by
-toxin.
This experiment was designed to eliminate electrolyte concentration
gradient across the plasma membrane of PT cells during anoxia.
-Toxin has been shown to permeabilize the plasma membrane only to
molecules <4 kDa (6, 14, 15, 22, 25, 30). Molecules >4 kDa,
including
-toxin (34 kDa) itself and LDH (~140 kDa), could not
penetrate the permeabilized plasma membrane. Furthermore, this
permeabilization is selective for the plasma membrane, leaving intracellular organelles intact, and therefore plasma membrane disruption could still be measured by the LDH assay. Under these conditions, the plasma membranes are readily permeable to small electrolytes, eliminating the primary driving forces for the entry of
water and small ions into PT cells. In the present study, PT cells
(~6 mg protein) were incubated with 200 U
-toxin (Calbiochem, La
Jolla, CA) in 1 ml of "intracellular" (Intra) buffer (135 mM potassium glutamate, 10 mM
piperazine-N,N'-bis(2-ethanesulfonic acid), 4 mM ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 1 mM heptanoic acid, pH 7.2) plus 3 mM ATP, at 37°C for 10 min. The permeability of the plasma membranes to large and small
molecules was measured by LDH release and intracellular eosin (624 mol
wt) or propidium iodide (668 mol wt) staining, respectively. The
permeabilized PT cells were then subjected to normoxia or anoxia under
various conditions.
Source of chemicals.
Unless noted in the text, chemicals were purchased from Sigma Chemical
(St. Louis, MO).
Data analysis.
Data are shown as means ± SE from at least four preparations.
Unless noted in the figure legends, analysis of variance was used to
compare the means of various experimental groups, tested for
significance with Fischer's protected least significance difference test and P < 0.05. All experiments
were repeated four to six times.
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RESULTS |
Cell volume and lysis as a function of osmotic pressure.
Normoxic PT cells swelled in response to applied hypotonic osmotic
gradients across the plasma membrane (Fig. 2,
A and
B). The results are expressed as a
function of the driving osmotic force. To calculate the difference
between intra- and extracellular osmolarity, it was assumed that the
intracellular osmolarity was 300 mosM at the beginning of the swelling
test, because it should have equilibrated with the 300 mosM DMEM buffer
during the 30-min incubation under normoxia (see
METHODS). The exposure of PT cells to low temperature for 30 min by itself did not significantly alter
cell volume (data not shown). After being exposed to water (osmotic
difference of 300 mosM), the average volume of normoxic PT cells
increased to more than seven times their isotonic volume (Fig.
2B), although under DIC it was
observed that individual live PT cells could swell to >2,000% of
their original volume (Fig. 2A).
These results suggest that the relative volumes calculated in Fig.
2B were the average of lysed and live
cells. Nevertheless, it can be seen that PT cells have a great capacity
for expanding their cell volume before the loss of membrane integrity.
Cell lysis, measured as LDH release from the PT cells, increased as the
osmotic difference between intra- and extracellular media widened,
indicating that more PT cells lost membrane integrity (Fig.
3). Figure 3 also shows that significantly
more cell lysis occurred when the swelling test was performed at
37°C compared with 4°C.

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Fig. 3.
Normoxic PT cells were incubated in various osmotic
NaCl-H2O solutions on ice or at
37°C. Greater LDH release at 37°C indicates that resistance of
normoxic plasma membranes against water influx-induced tension was
weaker at 37°C than at ~4°C.
* P < 0.05 compared with counterpart at ~4°C.
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Effects of anoxia on osmotic stress-induced cell lysis.
PT cells were incubated in various hypotonic NaCl solutions on ice
after being exposed to normoxia or anoxia for 30 min at 37°C. As
seen in Fig. 4, there was no significant
difference between the two groups when the osmotic stress test was
performed at 4°C; however, a significant difference was observed
when this test was performed at 37°C (Fig.
5). Anoxic PT cells were more susceptible to osmotic stress.

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Fig. 4.
After being exposed to normoxia or anoxia for 30 min, PT cells were
incubated in various osmotic
NaCl-H2O solutions on ice for 30 min. To express membrane disruption only due to hypotonic insults, LDH
release during swelling tests shown in Figs. 3-6 was normalized
according to following formula: %LDH release = 100 × (actual LDH
release LDH release in 300 mosM NaCl)/(100 LDH release
in 300 mosM NaCl). Results shown here revealed no significant difference between mechanic strength of normoxic and anoxic plasma membranes at low temperature.
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Fig. 5.
After being exposed to normoxia or anoxia for 30 min, PT cells were
incubated in various osmotic NaCl solutions at 37°C for 10 min. In
contrast to Fig. 4, more LDH was released from anoxic PT cells when
swelling test was performed at 37°C. Results indicate that plasma
membranes were indeed mechanically weaker after anoxia. * P < 0.05 compared with
normoxic counterparts.
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Glycine protection of membrane disruption during anoxia.
The presence of 4 mM glycine during anoxia significantly ameliorated
the increase in LDH release obtained at the two largest osmotic
differences tested (Fig. 6). This
protective effect was only observed if glycine was present during
anoxia. PT cells subjected to anoxia in the absence of glycine and
subsequently subjected to the swelling test in the presence or absence
of glycine showed no protection by glycine (Table
1).

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Fig. 6.
After being exposed to normoxia or anoxia in presence (anoxia + glycine) or absence (anoxia) of 4 mM glycine for 30 min, PT cells
were incubated in various osmotic NaCl solutions at 37°C for 10 min. Results indicate that glycine prevented plasma membrane mechanic
strength from decreasing during anoxia.
* P < 0.05 compared with
normoxia counterpart. # P < 0.05 compared with anoxia counterpart.
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Manipulation of osmotic stress did not affect membrane disruption
during anoxia.
Potentiating or attenuating water influx by incubating PT cells in a
150 mosM hypotonic medium or a 450 mosM hypertonic medium during anoxia
altered cell volume as expected but did not affect LDH release from
anoxic PT cells, compared with anoxic incubation in an isotonic medium.
(Fig. 7). This result demonstrates that water influx does not play an important role in anoxia-induced membrane
disruption.

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Fig. 7.
Potentiation or attenuation of water influx during anoxia was conduced
by subjecting equal amount of PT cells to 30 min of anoxic incubation
in a 150 mosM hypotonic medium, a 300 mosM isotonic DMEM, or a 450 mosM
hypertonic medium (see METHODS).
A: total (live + dead) cell volume is
expressed as percentage of volume of normoxic cells after 30 min of
incubation. PT cells swelled after being incubated in isotonic buffer
under anoxia. Hypotonic medium potentiated cell swelling during anoxia;
hypertonic medium attenuated cell swelling.
* P < 0.05 compared with cell
volume of normoxic PT cells;
** P < 0.05 compared with cell
volume of anoxic PT cells incubated in isotonic buffer.
B: LDH release from these PT cells was
compared with that from PT cells subjected to 30 min of normoxic
incubation in 300 mosM DMEM. Neither incubation in hypotonic nor in
hypertonic extracellular medium significantly altered LDH release
during anoxia. Results demonstrate that swelling does not play a
critical role in membrane disruption during anoxia. * P < 0.05 compared with LDH
release from normoxic PT cells.
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Use of
-toxin permeabilization to eliminate driving
force for net water and electrolyte movements across plasma membrane
during anoxia.
-Toxin permeabilized PT cells to eosin and propidium iodide but not
to LDH (Table 2, Fig.
8). This permeabilization would be
expected to eliminate the driving forces for the net movement of water
and small electrolytes across the plasma membrane. The permeabilized PT
cells (6 mg protein) were then incubated at 37°C for 30 min under
the following conditions: 1) in 3 ml
of Intra buffer, gassed with 100%
O2, containing initially 3 mM
MgATP and supplied subsequently with 50 µl of 30 mM MgATP every 5 min
during 30 min of incubation (ATP);
2) in 3 ml of Intra buffer, gassed with 100% N2 (Anox);
3) in 3 ml of Intra buffer, gassed
with 100% N2 plus 4 mM glycine
(Anox + Gly); and 4) in 3 ml of pH
6.8 Intra buffer, gassed with 100%
N2 (Anox + pH 6.8). LDH release
from these 4 groups of PT cells is shown in Fig.
9. The ATP group displayed an LDH release
of only 21 ± 3%, significantly lower than the 74 ± 4%
LDH release from the Anox group. Both glycine and pH 6.8 completely
protected the plasma membrane from the disruption caused by anoxia that
lead to LDH release. This result clearly demonstrates that net
transmembrane movement of electrolytes is not the primary cause of the
membrane disruption elicited by anoxia.

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Fig. 8.
PT cells were incubated in a permeabilization buffer [200 U
-toxin/ml, 135 mM potassium glutamate, 20 mM
piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 4 mM ethylene glycol-bis( -aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), and 3 mM ATP, pH 7.2] at 37°C for 10 min. PT
cells were then washed once with Intra buffer (135 mM potassium
glutamate, 20 mM PIPES, 4 mM EGTA, and 3 mM ATP, pH 7.2) and incubated
with 50 µM propidium iodide (PI) in Intra buffer on ice for 10 min. PT cells were checked with DIC (A ) and fluorescent (PI
stain; B ) microscopy. Staining of nuclei by PI indicates
that -toxin-treated PT cells were permeable to PI (668 mol wt).
However, these -toxin-treated PT cells were not permeable to LDH, as
indicated in Table 2.
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Fig. 9.
-Toxin-permeablized PT cells (see
METHODS) were incubated in 3 ml of
Intra buffer (135 mM potassium glutamate, 20 mM PIPES, 4 mM EGTA pH
7.2) under following experimental conditions at 37°C for 30 min:
1) gassed with 100%
O2 in presence of initial 3 mM MgATP, 50 µl of 30 mM ATP was added every 5 min during 30 min of
incubation (ATP), 2) gassed with
100% N2 (Anox),
3) gassed with 100%
N2 in presence of 4 mM glycine
(Anox+Gly), and 4) gassed with 100%
N2, in pH 6.8 Intra buffer (Anox + pH 6.8). LDH release from these PT cells was expressed as percentage of
total LDH. * P < 0.05 compared
with ATP. # P < 0.05 compared
with Anox. These results indicate that
1) anoxia caused membrane disruption
in absence of transmembrane Na+
and Cl influxes and
2) glycine could protect anoxic
membrane disruption in absence of transmembrane
Na+ and
Cl influxes.
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Plasma membrane disruption in a
Na+- and
Cl
-free buffer during anoxia.
Nonpermeabilized PT cells were incubated in a
Na+- and
Cl
-free Intra buffer
mimicking intracellular enviroment (see
METHODS) under normoxia or anoxia in
the presence or absence of 4 mM glycine. Under this condition, the
concentration gradient of Na+,
Cl
, and
K+ across the plasma membrane is
expected to be eliminated. As seen in Fig.
10, LDH release during anoxia was the
same as in DMEM and so was the protection by glycine. Surprisingly,
there was significant LDH release during normoxia in the Intra buffer.
These results demonstrate that net
Na+ and
Cl
influxes are not
required for anoxic membrane disruption, and the protection by glycine
is not based on a block of those influxes.

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Fig. 10.
PT cells were incubated in DMEM or a
Na+- and
Cl -free medium (135 mM
potassium glutamate, 20 mM PIPES, 4 mM EGTA, pH 7.4) under normoxia or
anoxia in presence or absence of 4 mM glycine.
* P < 0.05 compared with ** or
#; ** P < 0.05 compared with
#. Results demonstrate that Na+
and Cl influxes were not
cause of anoxia-induced membrane disruption and that glycine protects
anoxia-induced plasma membrane disruption without depending on blocking
Cl or
Na+ influx. It is not clear why
Na+- and
Cl -free medium caused LDH
release under normoxia.
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DISCUSSION |
By investigating osmotic stress-induced lysis in proximal renal
tubules, we determined that anoxia increases their susceptibility to
this type of injury. Nevertheless, water and electrolyte influxes are
not required for anoxic membrane disruption.
Cell lysis in response to hypotonic stress may be viewed as a two-step
process.
The first step is swelling, which necessitates an increase in membrane
surface area. Normoxic renal proximal tubular cells have a great
capacity for expanding their cell volumes without disruption. The cell
radius (measured from Fig. 2A) was a
function of
P, the osmotic pressure difference across the cell
membrane. At low
P, the cell radius does not change much due to
cytoskeletal constraints. On the other hand, the cell radius increases
rapidly at
P of >200 mosM (Fig. 2). Some proximal tubular cells
could maintain their membrane integrity even when they were incubated in deionized water (
P of 300) and swelled to >20 times their normal cell volume (Fig. 2A). This
exceptional property of PT cells might be due to its great plasma
membrane surface area-to-cell volume ratio. The microvillar membranes
on the apical side and membrane invaginations on the basal and
basolateral sides could be expected to contribute their "excess"
membrane. In addition, intracellular vesicles would also be expected to
migrate to the surface under these conditions, contributing to the
expansion of the plasma membrane.
The second step is membrane disruption, which occurs when the tension
in the plane of the plasma membrane exceeds its mechanical strength.
The full osmotic pressure is not felt across the plasma membrane until
maximal volume is reached. At that time, the membrane tension would be
given by Laplace's law for a sphere as follows: T =
P · r/4, where T
is tension in the plane of the membrane,
P is the osmotic pressure
difference across the cell membrane, and
r is the cell radius. The predicted
T-
P relationship for the data shown in Fig.
2A is displayed in Fig.
11. There is a monotonic increase in
tension as
P increases, with the most rapid increase at
P of
>200 mosM. Comparison of Fig. 11 with Figs. 3 and 4 suggests that LDH
release occurred after a threshold tension was achieved at
P of
>250 mosM. Differences in LDH release between normoxia and anoxia
could represent a decrease in the mechanical strength of the membrane.
Previous studies from our laboratory (3, 9) have determined that
cytoskeletal support for the plasma membrane was diminished during
anoxia, and this would be expected to cause decreased mechanical
strength in the membrane.

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Fig. 11.
Calculated plasma membrane tension (T) vs. P for data shown in Fig.
2A. Membrane tension was given by
Laplace's law for a sphere as T = P · r/4, where T
is tension in plane of membrane, P is osmotic pressure difference
across cell membrane, and r is cell
radius.
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The fact that anoxic plasma membrane was more resistant to osmotic
stress at 4°C than at 37°C might be related to phase transition of membrane lipids. Membrane lipids are at a more homogeneous physical
state at 4°C than at 37°C. This may enhance mechanical strength
of anoxic plasma membrane at 4°C.
Despite the increased susceptibility of anoxic cells to water
influx-induced stress, further investigation revealed that water and
electrolyte influxes are not a primary cause for anoxic membrane disruption. First, resistance of anoxic plasma membranes to water influx-induced stress is not lost, although their mechanical strength was diminished compared with normoxic membranes. The plasma membranes of those anoxic cells that had been injured during 30 min of anoxia and
would disrupt at any time if anoxia lasted a little longer did not
disrupt under an intra-to-extracellular osmotic difference as great as
200 mosM (Fig. 5), a pressure sufficient to lyse some normal cells
other than PT cells. Second, potentiation or attenuation of water
influx during anoxia did not affect membrane disruption (Fig. 7).
Third, elimination of Na+ and
Cl
influxes using a
Na+- and
Cl
-free extracellular
medium did not protect membrane during anoxia (Fig. 10). Finally,
elimination of transmembrane electrolyte concentration gradient by
permeabilizing plasma membrane to small electrolytes with
-toxin
established definitively that electrolyte flux was not required to
cause anoxic membrane disruption (Fig. 9).
Consistent with the minimal role of water and electrolyte influxes in
anoxic membrane injury, the membrane protection by glycine or low pH is
independent of net water and electrolyte influxes (Figs. 9 and 10). In
addition, the plasma membrane was protected only when glycine was
present during anoxia (Fig. 6). Subsequent addition of glycine during
swelling test after anoxia did not prevent water influx-induced
membrane disruption (Table 1).
Before the present study, Currin et al. (5) also found that omission of
Cl
in extracellular medium
did not affect anoxic membrane injury in hepatic endothelial cells. In
contrast, Carini et al. (2) reported that replacement of NaCl by
choline chloride in extracellular buffer protected hepatocytes from
hypoxic injury. We do not know whether this discrepancy between our
observation and theirs was caused by the difference in cell type. In
the present investigation, osmotic stress contributed by small
molecules was found not as a primary cause of anoxic plasma membrane
disruption. However, the role of osmotic stress contributed by larger
molecules remains to be determined, since Kreisberg et al. (16)
reported that 8% polyethylene glycol (6,000 mol wt) protected cultured
renal tubular epithelial cells from anoxic cell swelling and cell
death. It needs to be pointed out that cultured cells are much more
resistant to anoxic injury than those cells freshly isolated from
kidney, indicating an altered metabolism and its regulation mechanisms in cultured cells.
In conclusion, the present data support a biochemical alteration of the
plasma membrane as the underlying cause of membrane disruption during
anoxia.
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ACKNOWLEDGEMENTS |
This work was supported by postdoctoral grants from the National
Kidney Foundation and National Institutes of Health to J. Chen and
National Institute of Diabetes and Digestive and Kidney Diseases Grant
DK-26816 to L. J. Mandel.
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FOOTNOTES |
Address for reprint requests: L. J. Mandel, Dept. of Cell Biology, Box
3709, Duke University Medical Center, Durham, NC 27710.
Received 31 January 1997; accepted in final form 27 May 1997.
 |
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