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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -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; alpha -toxin; renal tubule; osmotic pressure; lactate dehydrogenase release

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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.

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.

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 alpha -toxin. This experiment was designed to eliminate electrolyte concentration gradient across the plasma membrane of PT cells during anoxia. alpha -Toxin has been shown to permeabilize the plasma membrane only to molecules <4 kDa (6, 14, 15, 22, 25, 30). Molecules >4 kDa, including alpha -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 alpha -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(beta -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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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.

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.

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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Table 1.   Hypotonicity-induced LDH release in presence or absence of 4 mM glycine

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.

Use of alpha -toxin permeabilization to eliminate driving force for net water and electrolyte movements across plasma membrane during anoxia. alpha -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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Table 2.   alpha -Toxin-treated cells were permeable to eosin and PI but not LDH


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Fig. 8.   PT cells were incubated in a permeabilization buffer [200 U alpha -toxin/ml, 135 mM potassium glutamate, 20 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 4 mM ethylene glycol-bis(beta -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 alpha -toxin-treated PT cells were permeable to PI (668 mol wt). However, these alpha -toxin-treated PT cells were not permeable to LDH, as indicated in Table 2.


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Fig. 9.   alpha -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.

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.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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 Delta P, the osmotic pressure difference across the cell membrane. At low Delta P, the cell radius does not change much due to cytoskeletal constraints. On the other hand, the cell radius increases rapidly at Delta P of >200 mosM (Fig. 2). Some proximal tubular cells could maintain their membrane integrity even when they were incubated in deionized water (Delta 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 = Delta P · r/4, where T is tension in the plane of the membrane, Delta P is the osmotic pressure difference across the cell membrane, and r is the cell radius. The predicted T-Delta P relationship for the data shown in Fig. 2A is displayed in Fig. 11. There is a monotonic increase in tension as Delta P increases, with the most rapid increase at Delta 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 Delta 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. Delta P for data shown in Fig. 2A. Membrane tension was given by Laplace's law for a sphere as T = Delta P · r/4, where T is tension in plane of membrane, Delta P is osmotic pressure difference across cell membrane, and r is cell radius.

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 alpha -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.

    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.

    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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Bergmeyer, H. U., E. Bernt, and B. Hess. Lactic dehydrogenase. In: Methods of Enzymatic Analysis, edited by H. U. Bergmeyer. New York: Academic, 1963, p. 736-741.

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4.   Chen, J., R. B. Doctor, and L. J. Mandel. Cytoskeletal dissociation of ezrin during renal anoxia: role in microvillar injury. Am. J. Physiol. 267 (Cell Physiol. 36): C784-C795, 1994[Abstract/Free Full Text].

5.   Currin, R. T., J. C. Caldwell-Kenkel, S. N. Lichtman, S. B. Bachmann, Y. Takei, S. Kawano, R. G. Thurman, and J. J. Lemasters. Protection by Carolina rinse solution, acidotic pH, and glycine against lethal reperfusion injury to sinusoidal endothelial cells of rat livers stored for transplantation. Transplantation 62: 1549-1558, 1996[Medline].

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7.   Dickman, K. R., and L. J. Mandel. Glycolytic and oxidative metabolism in primary renal proximal tubule cultures. Am. J. Physiol. 257 (Cell Physiol. 26): C333-C340, 1989[Abstract/Free Full Text].

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AJP Cell Physiol 273(4):C1341-C1348
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