1 Pulmonary Disease Division, Department of Medicine, Veterans Affairs Medical Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and 2 Pulmonary/Critical Care Section, Department of Medicine, Veterans Affairs Medical Center, Brown University School of Medicine, Providence, Rhode Island 02908
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ABSTRACT |
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We investigated the role of intracellular pH (pHi) and Na/H exchange in cell death in human pulmonary artery endothelial cells (HPAEC) following a metabolic insult (inhibition-oxidative phosphorylation, glycolysis). Metabolic inhibition in medium at pH 7.4 decreased viability (0-15% live cells) over 6 h. Cell death was attenuated by maneuvers that decreased pHi and inhibited Na/H exchange (acidosis, Na/H antiport inhibitors). In contrast, cell death was potentiated by maneuvers that elevated pHi or increased Na/H exchange (monensin, phorbol ester treatment) before the insult. HPAEC demonstrated a biphasic pHi response following a metabolic insult. An initial decrease in pHi was followed by a return to baseline over 60 min. Maneuvers that protected HPAEC and inhibited Na/H exchange (acidosis, Na+-free medium, antiport inhibitors) altered this pattern. pHi decreased, but no recovery was observed, suggesting that the return of pHi to normal was mediated by antiport activation. Although Na/H antiport activity was reduced (55-60% of control) following a metabolic insult, the cells still demonstrated active Na/H exchange despite significant ATP depletion. Phorbol ester pretreatment, which potentiated cell death, increased Na/H antiport activity above the level observed in monolayers subjected to a metabolic insult alone. These results demonstrate that HPAEC undergo a pH-dependent loss of viability linked to active Na/H exchange following a metabolic insult. Potentiation of cell death with phorbol ester treatment suggests that this cell death pathway involves protein kinase C-mediated phosphorylation events.
sodium/hydrogen exchange; intracellular pH; intracellular cytosolic calcium
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INTRODUCTION |
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ENDOTHELIAL CELLS are vulnerable to injury related to their unique location at the blood-organ interface in most organ systems. They are exposed to decreases in oxygen tension, cytotoxic stimuli in the blood, and metabolic changes after an ischemic insult (4, 39, 41). These exposures initiate a series of events, including neutrophil adhesion, oxidant production, altered membrane permeability, and intracellular ion homeostasis, which culminate in cell injury and death (38, 41). Prior work on endothelial cell injury has focused primarily on the cytotoxic effects of external factors (4), whereas the intracellular events and changes in membrane function linked to loss of endothelial cell viability remain poorly defined. Changes in the normal selective permeability of the cell membrane, involving altered ion transport and intracellular ion homeostasis, are key events that precede cell death in many cell types. These changes represent final common pathways leading to loss of cell viability (2, 9, 16, 18, 38), but many details remain poorly defined.
Specifically, the role of changes in intracellular pH (pHi) and the activity of the ion transport sites, which regulate this parameter in modifying lung cell viability, have not been systematically investigated. The rationale for focusing on the link between pHi and viability is that this parameter modifies many critical aspects of normal cell function (7, 32). Recent work has demonstrated an important role for changes in pHi and the activity of Na/H exchangers (NHEs) in modulating loss of cell viability following ischemic cell injury (13, 19, 24). In this scheme, a primary event ("ischemia") can lead to cell death immediately if the insult is severe or to altered cell function (decreased ATP, pHi, and altered intracellular ion homeostasis) following a sublethal insult. Under these latter conditions, cells may be predisposed to injury when pHi returns to normal. It is the return of pHi to normal, mediated by specific ion transport systems such as NHEs, that is a critical factor that paradoxically initiates and accelerates cell injury (24, 38).
We postulated that this form of pH-dependent cell death plays a role in altered endothelial cell viability under conditions associated with lung injury, such as ischemia-reperfusion. In prior work, we defined the Na/H antiport isoform subtype (NHE1) present in human pulmonary artery endothelial cells (HPAEC) and demonstrated the effects of prolonged hypoxic exposure on NHE1 activity and isoform expression in these cells (14). In the present study, we used a well-defined model of cellular ischemia (metabolic inhibition) to investigate the role of changes in pHi and Na/H exchange on endothelial cell viability. Our objectives were to define the basic features of pH-dependent cell death in pulmonary endothelial cells and to determine the effect of changes in pHi and Na/H antiport activity on the loss of cell viability following a metabolic insult.
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METHODS |
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Cell culture and reagents. Large-vessel HPAEC were obtained from a commercial vendor (Clonetics) and grown in endothelial cell growth medium containing 10% fetal bovine serum as previously described (14). The cells were maintained in a humidified atmosphere (21% O2-5% CO2, 37°C), fed twice a week with endothelial cell growth medium-10% fetal bovine serum, and passaged when confluent (3-5 days). In preparation for the measurement of pHi and Na/H antiport activity, cells were seeded onto glass coverslips coated with CellTak (4 µg/slide; Collaborative Biomedical Products) as previously described (14). The coverslips were incubated in a humidified atmosphere (21% O2-5% CO2, 37°C) until near confluence (~90%) was reached. All experiments were conducted with cells matched for cell line, passage number, time to confluence, and cell density between experimental groups. No differences were observed in the results utilizing different cell lines or passage numbers.
Metabolic inhibition models. Metabolic inhibition is defined as inhibition of cellular metabolism-oxidative phosphorylation and glycolysis.
These models have been used extensively by other researchers to investigate different aspects of altered cell function and to simulate ischemia in vivo (5, 17, 29). Metabolic inhibition was induced with inhibitors, which act at different specific sites in the mitochondrial electron transport chain. The following regimens were used: 5 mM sodium cyanide-0.5 mM iodoacetic acid (NaCN-IAA), 10 µM myxothiazol (Mx)-0.5 mM IAA, and 20 mM sodium azide (Az)-0.5 mM IAA, and 20 µg/ml antimycin-10 mM 2-deoxyglucose (A-D). Each regimen includes a site-specific inhibitor of the mitochondrial electron transport chain and an inhibitor of glycolysis. The sites of action of the mitochondrial inhibitors were as follows: NaCN and Az inhibit complex IV (cytochrome oxidase), Mx blocks oxidation of ubiquinol by the Rieske iron-sulfur center of complex III, antimycin binds to cytochrome bc1 (complex III). We utilized different sets of inhibitors to verify the findings and address questions related to the potential nonspecific effects of any one agent. In each experiment involving metabolic inhibition, monolayers were incubated in HEPES-buffered MEM without glucose at neutral pH (7.4), unless otherwise indicated, during the experimental protocol. Several agents [antimycin, methylisobutylamiloride (MIA), 5-(N,N-hexamethylene)-amiloride (HMA)] were solubilized in DMSO; the final concentration of this agent never exceeded 0.5% in all experiments.
General experimental protocols. Viability experiments were performed using three metabolic inhibition models (NaCN-IAA, Mx-IAA, and Az-IAA) under several conditions: control, medium at pH 7.4; metabolic inhibition, medium at pH 7.4; metabolic inhibition, medium at pH 6.2; metabolic inhibition, medium at pH 7.4 plus specific Na/H antiport inhibitor; or metabolic inhibition, medium at pH 7.4 plus monensin, a Na/H exchange ionophore. Monensin stimulates Na/H exchange across the cell membrane, leading to an increase in pHi. This agent simulates active Na/H exchange, but it does not actually stimulate the Na/H antiport, an integral membrane protein (14). In separate experiments, we pretreated monolayers with a phorbol ester, phorbol 12-myristate 13-acetate (PMA), for 2 h to upregulate Na/H antiport activity (1), followed by initiation of a metabolic insult 18 h later. This protocol was utilized to investigate the effect of increased Na/H antiport activity on cell viability following a metabolic insult.
Fluorescent viability assay. The assay was performed, as
described previously (11, 15), using monolayers grown to near confluence in 24-well tissue culture plates. Monolayers were inspected before the start of an experiment to confirm normal morphology. Monolayers were incubated with metabolic inhibitors in HEPES-buffered MEM, pH 7.4 (unless otherwise stated) at 37°C and a 3 µM
concentration of the probe propidium iodide. Propidium iodide
intercalates with cellular DNA but is impermeable to the cell membrane
of viable cells. The probe rapidly crosses the cell membrane of dying
cells and binds to nuclear DNA, generating a fluorescent signal. The magnitude of the change in the pH-independent signal from this probe
was used to monitor changes in viability during an experiment. Changes
in the propidium iodide signal were monitored at 30-min intervals with
a microplate fluorimeter (Cambridge Technologies model 7630). The probe
was excited with a 530-nm (25-nm band-pass) filter, and emission was
detected with a 620-nm (35-nm band-pass) filter. Percent cell viability
at each time point was calculated as follows: %viable cells = 100[1 (X
Fmin)/(Fmax
Fmin)], where X is the fluorescent signal in each well at a specific
time point, Fmin is the background signal obtained at the
start of the experimental protocol, and Fmax is the signal
representing 100% cell death. Fmax was obtained by adding
saponin (final concentration 0.1%) to triplicate wells within each
plate. In each experimental condition, a minimum of three readings were
obtained at each time point; n = 1 represents the average of
these triplicate readings. Prior results demonstrated that fluorescent
viability assays have an excellent correlation with other standard
methods used to assess cell viability (11, 15, 19).
Measurement of pHi and Na/H antiport activity.
pHi and Na/H antiport activity were measured in
monolayers grown on glass coverslips with a fluoroprobe assay with
2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF,
Molecular Probes) as previously described (14). Monolayers were
inspected with phase microscopy before the start of each experiment to
ensure a normal morphology. Measurement of pHi with BCECF
is a dual excitation-to-single emission ratio technique in which the
ratio of the pH-sensitive (490-nm) to the pH-insensitive (440-nm)
fluorescent signal provides a measurement of pHi that is
independent of probe concentration or cell number. Na/H antiport activity is measured as the initial rate of pHi recovery
from intracellular acidosis. The rate of recovery from acidosis is determined from an analysis of the kinetics of the recovery following acidification of monolayers in MEM (pH 6.6) containing the ionophore nigericin (1 µM), in which choline chloride is substituted for NaCl
on an equimolar basis. This maneuver acidifies the monolayers, related
to the ionophore-induced K+ exit and the H+
entry into the cell. Acid recovery is initiated by addition of normal
Na+-containing HEPES-buffered MEM. Because the
experiments are performed in a nominally
HCO3-free, HEPES- buffered
MEM, the activity of other pHi regulating systems is
minimal, and acid recovery reflects Na/H antiport activity under these
conditions. Acid recovery is Na+ dependent and sensitive to
blockade with amiloride analogs, which are specific Na/H antiport
inhibitors (14, 27). Antiport activity is measured as the rate of
recovery from acidosis 10-20 s after the initiation of acid
recovery over a well-defined pH range (6.5-6.75) and expressed as
change in pHi per minute. The fluorescent signal was
calibrated by the H+ equilibration method (14, 40).
Measurement of ATP content. ATP content in control monolayers and cells exposed to metabolic inhibition for different time periods was determined using a fluorimetric assay as previously described (26). The assay is based on ATP-dependent conversion of glucose to 6-phosphogluconate, which generates the fluorescent product NADPH on a equimolar basis. HPAEC monolayers grown on 100-mm tissue culture dishes were subjected to the experimental treatments and then immediately washed (1×) with 5 ml of HEPES-buffered MEM. After removal of the wash solution, each dish was placed on ice and rinsed again with 10 ml of ice-cold PBS. After removal of the PBS, ATP in each monolayer was extracted by scraping the monolayer in 1 ml of ice-cold 0.4 M perchloric acid and then neutralizing the solution with ~0.4 ml of 2 M potassium carbonate. Aliquots (800 µl) of each sample solution were assayed for ATP content by adding the sample solution to 2.1 ml of reaction buffer containing 100 mM Tris, 5 mM MgCl2, 5 mM glucose, 50 µM NADP+, and 1.75 U/ml of glucose-6-phosphate dehydrogenase. This mixture (total volume 2.9 ml) was then placed into the thermostated cuvette compartment (24°C) of the spectrofluorimeter (SPEX Fluorolog II), followed by the addition of hexokinase (5 U) to the cuvette to initiate the reaction. Each reaction was monitored continuously (excitation/emission wavelengths 345/455 nm, respectively) until the fluorescent signal reached a maximum level. Next, calibration of each sample measurement was achieved by monitoring the change in fluorescence after addition of a known quantity of ATP (10 nmol) and additional hexokinase (5 U) to the reaction mixture in the same cuvette. Both the samples and the internal standard signals were read 5 min after the initiation of the reaction, based on preliminary experiments that demonstrated that this was an optimal time point for obtaining the maximal signal from each reaction in this system. ATP content was normalized to the number of live cells in the sample.
Data analysis. Results are presented as the means ± SE. Differences between treatment groups for viability and rate of acid recovery at different time points were first analyzed with ANOVA. If the ANOVA demonstrated a significant overall treatment effect, then the mean values were compared with a post hoc multiple comparison procedure (Student-Newman-Keuls, Bonferroni correction). Differences between means were considered significant if P < 0.01 or the level appropriate for the number of comparisons of interest using the Bonferroni correction.
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RESULTS |
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Cell viability. Figure 1
demonstrates the time-dependent effects of a metabolic insult on HPAEC
viability using three different inhibitor regimens: NaCN-IAA, Mx-IAA,
and Az-IAA. Each model exhibited a qualitatively similar response.
After initiation of a metabolic insult in medium at pH 7.4, there was a
significant loss of viability over the 6-h measurement period compared
with control monolayers incubated in medium at pH 7.4 alone. Loss of
viability in HPAEC consistently began 90-120 min after initiation
of the metabolic insult, with progressive cell death occurring during
the remainder of the experiment.
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The loss of viability in HPAEC was significantly reduced by interventions that modified Na/H exchange in each model. Maneuvers that reduced Na/H exchange protected HPAEC following a metabolic insult. Alteration of the transmembrane pH gradient with extracellular acidosis (incubation in medium at pH 6.2) or pharmacological inhibition of Na/H exchange with a specific Na/H antiport inhibitor attenuated the loss of viability following metabolic inhibition in monolayers incubated medium at pH 7.4. Incubation in acidotic medium was consistently more effective than the presence of Na/H antiport inhibitors in preserving cell viability in each model. In contrast, a maneuver that simulated active Na/H exchange and increased pHi potentiated the loss of viability in each model. Thus addition of the Na/H ionophore monensin at the onset of metabolic inhibition in medium at pH 7.4 increased the loss of cell viability above the level observed following metabolic inhibition alone. Administration of antiport inhibitors and monensin to control monolayers in the absence of metabolic inhibitors had no effect on viability (data not shown).
Collectively, the similar results with each model of metabolic inhibition demonstrate the presence of a pH-dependent cell death pathway in HPAEC and suggest that changes in pHi and Na/H antiport activity are important components of this pathway.
In separate experiments, we determined the effect of phorbol ester
treatment on HPAEC viability following a metabolic insult (Fig.
2). The rationale for this experiment was
that phorbol ester treatment is known to upregulate Na/H antiport
activity and expression. Therefore, we investigated the effect of this
intervention on cell death as a way to extend the findings with the
Na/H ionophore monensin. Treatment of monolayers with PMA (1 µM) for
2 h, followed by a metabolic inhibition with Az-IAA (as in Fig.
1C) 18 h after the phorbol ester treatment, potentiated the
loss of viability compared with that in monolayers subjected to
metabolic inhibition alone in medium at pH 7.4. Incubation of the
monolayers in acidic medium (pH 6.4) or with a specific Na/H antiport
inhibitor protected the PMA-treated monolayers from cell death
following metabolic inhibition. PMA had no effect on viability in
control monolayers not subjected to metabolic inhibition (data not
shown). Finally, a similar treatment protocol using an inactive phorbol
ester (-PMA) did not potentiate cell death but led to a loss of
viability, which was identical to treatment with metabolic inhibitors
in medium at pH 7.4 alone (data not shown).
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Effect of metabolic inhibition on pHi. The viability experiments suggested that the changes in pHi and Na/H antiport activity following a metabolic insult are critical factors linked to cell death. Therefore, we determined the pHi response during the first 60 min following a metabolic insult under the same conditions in which viability was monitored. These experiments were performed using antimycin (20 µg/ml)-2-deoxyglucose (10 mM) because the magnitude of cell death with this regimen was smaller than the loss of viability observed over 6 h with the other models (~30-50% with A-D vs. >80% with the other models, data not shown). Therefore, pHi could be monitored with minimal interference secondary to loss of probe (BCECF) from the intracellular compartment as a result of altered membrane permeability.
Metabolic inhibition with A-D produced a biphasic pattern of
pHi response over the 60-min monitoring period (Fig.
3). A metabolic insult was consistently
associated with a rapid, acute drop in pHi
(~0.25-0.30 pH units) over the first 10-15 min, which was followed by a rise in pHi slowly back toward baseline
during the rest of the experiment. In several instances,
pHi returned to a level that was significantly higher than
the baseline pHi value in a given monolayer preparation as
illustrated in Fig. 3. This experiment was repeated several times
(n = 10) with similar results. This biphasic response was
not observed in control monolayers incubated in MEM, pH 7.4, without
metabolic inhibitors. Control monolayers demonstrated no significant
change in pHi over the same time period. Altering the doses
of antimycin (5 and 10 µM) produced a modification of the biphasic
response, leading to a smaller acute drop in pHi, which was
again followed by a rebound back toward baseline pHi (data
not shown), suggesting that the biphasic response was dose related and
varied with the level of metabolic inhibition.
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In contrast, the biphasic response pattern observed following a metabolic insult in MEM, pH 7.4, was altered by the conditions that modified cell viability (Fig. 1). The maneuvers that preserved cell viability were associated with specific changes in the biphasic response compared with the maneuvers that potentiated cell death. Metabolic inhibition in MEM, pH 6.2, (extracellular acidosis) induced the same initial drop in pHi, which was not followed by a subsequent rise in pHi back to baseline (Fig. 3). Metabolic inhibition in MEM, pH 7.4, in the presence of a specific Na/H antiport inhibitor or Na+-free medium produced a response that was identical to the effect of extracellular acidosis (data not shown). This inhibition of the second phase of the biphasic pHi response, consisting of the increase in pHi back toward baseline, with maneuvers that inhibit Na/H antiport activity (extracellular acidosis, Na+-free medium, or a specific Na/H antiport inhibitor) suggested that this component of the response is mediated by active Na/H exchange. Finally, the typical biphasic response following a metabolic insult was altered in a different way by the maneuver that potentiated cell death. In the presence of the Na/H ionophore monensin, there was no acute drop in pHi (Fig. 3). Instead, there was a rapid rise in pHi above baseline, and pHi remained above the baseline level during the entire experiment. The results of the experiments with acidosis, Na/H antiport inhibitors, Na+-free medium, and monensin were repeated several times (n = 4-6) with similar results.
Decay of the BCECF signal while measuring the pHi response was similar in control vs. A-D-treated monolayers (data not shown), suggesting little change in membrane integrity and permeability during the experiment. These findings correspond with the results of the viability experiments (Fig. 1), which demonstrate that loss of viability generally began ~120 min after initiation of the metabolic insult. A similar biphasic pHi response pattern was observed following metabolic inhibition with Mx (10 µM)-IAA (0.5 mM) in medium at pH 7.4 (data not shown), as observed with A-D. Similarly, the pHi response following metabolic inhibition with Mx-IAA in acidotic medium, pH 6.2, and in the presence of a specific Na/H antiport inhibitor was also identical to the findings with A-D (data not shown).
Effect of metabolic inhibition on Na/H antiport activity. In
separate experiments, we measured Na/H antiport activity at different time points following metabolic inhibition with A-D. These experiments were designed to confirm the results in Fig. 3, which suggested that
activation of Na/H exchange mediates the return of pHi to baseline during the second phase of the biphasic pHi
response following metabolic inhibition. Na/H antiport activity,
measured as the rate of acid recovery, was compared between control
monolayers vs. monolayers subjected to metabolic inhibition in medium
at pH 7.4. Antiport activity was reduced at both 15 and 60 min
following a metabolic insult (Table 1). The
rate of acid recovery at the 15- and 60-min time points was reduced 40 and 45%, respectively, compared with the recovery rate of control
monolayers. In both the control monolayers and monolayers subjected to
a metabolic insult for 60 min, addition of a specific antiport
inhibitor during acid recovery (MIA, 10 µM) markedly inhibited the
recovery (n = 4, data not shown), indicating that acid recovery
under these conditions is mediated by Na/H exchange.
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In separate experiments, monolayers were pretreated with PMA, as in
Fig. 2, prior to measuring the rate of acid recovery to determine the
effect of this treatment on Na/H antiport activity in either the
presence or absence of metabolic inhibitors. The results demonstrate
that phorbol ester pretreatment increased the rate of acid recovery in
both the presence and absence of metabolic inhibitors (Table
2), consistent with an increase in Na/H
exchange in monolayers pretreated with the phorbol ester.
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Effect of metabolic inhibition on cell ATP content. Table 1 also illustrates the results of experiments in which the ATP content of monolayers was determined under the same conditions in which Na/H antiport activity was determined in the acid recovery experiments following metabolic inhibition with A-D. These results demonstrate that metabolic inhibition produced a 58 and 87% decrease in cell ATP content at the 15- and 60-min time points, respectively, compared with that in control monolayers incubated under identical conditions but not subjected to a metabolic insult.
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DISCUSSION |
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The present results are the first detailed characterization of the role of pHi and the Na/H antiport in pulmonary endothelial cell injury, demonstrating the presence of a pH-dependent cell death pathway following a metabolic insult. Cell death is attenuated by maneuvers that decrease pHi and inhibit Na/H exchange, such as altering the transmembrane pH gradient (extracellular acidosis) or pharmacological inhibition of the Na/H antiport. In contrast, treatments that increase pHi or stimulate Na/H exchange, such as incubation with the Na/H ionophore monensin or protein kinase C-mediated activation of the Na/H antiport, potentiate the loss of viability. Similar results with different models of metabolic inhibition support the conclusion that loss of endothelial cell viability under these conditions is a pH-dependent event involving changes in pHi and Na/H antiport activity. This conclusion is reinforced by the findings related to the temporal pattern of the pHi response following a metabolic insult. Maneuvers that protect HPAEC following metabolic inhibition (extracellular acidosis, Na/H antiport inhibition) are associated with a prolonged depression of pHi. This response is distinctly different from the response observed following a metabolic insult in medium at pH 7.4 alone, in which pHi decreases but returns to baseline within 30-60 min via Na/H exchange. In contrast, maneuvers that potentiate cell death (monensin, PMA) initially elevate pHi and/or stimulate Na/H exchange. These findings suggest that changes in pHi mediated via Na/H exchange are early events that initiate the pH-dependent cell death pathway, leading to loss of viability at later time points.
Recent findings suggest that alterations of membrane function, ion transport, and intracellular ion homeostasis play a critical role in cellular injury in a variety of cell types (38), including lung cells. For example, recent studies involving the alveolar epithelium have highlighted the role of altered ion homeostasis and ion transport in response to hyperoxia (10, 34). In contrast, prior work on the mechanisms of endothelial cell injury has generally focused on the external factors that modify cell viability (4). Little information is available concerning the sequence of changes in cell membrane function or the key intracellular parameters linked to cell death in endothelial cells. Specifically, the role of membrane ion transport sites in the loss of endothelial cell viability under conditions associated with lung injury has not been systematically investigated (16, 30).
The traditional view is that acidosis is not beneficial for optimal cell function under normal conditions (7) and, by extension, under conditions associated with cell injury. Recent work has challenged this notion. Changes in pHi and/or the activity of the ion transport systems that regulate this parameter modify viability in different cell types. Extracellular acidosis or inhibition of the ion transport sites that regulate pHi may actually protect cells under conditions associated with cell injury (13, 19, 23, 24). The protective effects of extracellular acidosis against anoxic cell damage in cardiac muscle, renal tubule cells, and hepatocytes are well known (3, 13, 25, 33), but this recent work has extended these earlier findings to cellular models of ischemic injury. In this scheme, acidosis or inhibition of the return of pHi to normal following a hypoxic or ischemic insult attenuates the changes directly linked to cell death.
The basic features of pH-dependent cell death in HPAEC are similar to what has been described in hepatocytes and myocytes (19, 23). One notable difference is that HPAEC appear to be more resistant to the loss of viability following a metabolic insult than other cell types. Hepatocyte and myocyte cell death occurred more rapidly (40-60 min) (15, 19, 24) than the time course of altered viability in HPAEC (Fig. 1). Protection of HPAEC with maneuvers that inhibit the return of pHi to normal suggests that changes in pHi play a key role in initiating the loss of cell viability. The present findings extend the concept of pH-dependent cell injury to a cell type (endothelial cells) relevant to ischemic lung injury. These findings at the cellular level also extend work at the organ-system level, demonstrating that Na/H antiport inhibition attenuated the ischemia-induced changes in microvascular endothelial cell permeability in the whole lung (31).
Metabolic inhibition in cultured cells has been widely utilized to investigate questions related to the cellular response to an ischemic insult. This work has provided valuable insights into several aspects of altered cell function, including microfilament assembly, altered membrane function and intracellular ion homeostasis, and oxidant stress following an ischemic insult (5, 15, 17, 29, 43). Metabolic inhibition is associated with an acute drop in pHi, related to proton accumulation following ATP hydrolysis (19, 38, 40). In addition, the ATP depletion following metabolic inhibition is associated with inhibition of Na/H exchange in several cell types (8, 20, 26), providing another explanation for the decrease in pHi following a metabolic insult. Thus the prevailing view is that the Na/H antiport is an ATP-dependent ion transport site. Less well appreciated, but relevant to the current findings, is the observation that the effect of ATP depletion on Na/H exchange may vary among cell types. Human A431 cells and foreskin fibroblasts do not demonstrate inhibition of Na/H exchange following a metabolic insult (12). Renal epithelial cells demonstrate an ~80% recovery of antiport-mediated Na+ uptake 30 min into recovery from metabolic inhibition when cell ATP content is still only 14% of baseline values (6). The present findings demonstrate that Na/H antiport activity is reduced, but not abolished, in HPAEC in the first hour (15-60 min) following a metabolic insult (Table 1). In addition, Na/H exchange contributes to the return of pHi to normal under these conditions. The reason for the variability in the ATP dependence of Na/H exchange among cell types is unknown despite advances in understanding the molecular features of the ATP dependence, which resides in the cytoplasmic domain of the antiport protein (8, 20).
The temporal pattern of the pHi response and the level of activity of the Na/H antiport beyond the first 15 min following a metabolic insult have not been defined in most cell types. HPAEC demonstrate a biphasic pHi response over 60 min following a metabolic insult. Inhibition of the second half of this response, the return of pHi to normal, with maneuvers that inhibit Na/H exchange (Fig. 3) indicates that Na/H exchange is activated following a metabolic insult. This conclusion is supported by direct evidence of active Na/H exchange during this part of the biphasic response (Table 1). HPAEC contain only one antiport isoform (NHE1) located on the basolateral cell surface (14). Therefore, the present findings demonstrate that activation of NHE1 is a key component of the pH-dependent cell death pathway in HPAEC. These results suggest that changes in pHi and NHE1 activity initiate the events leading to cell death at later time points.
ATP depletion alone is an unlikely cause for the loss of viability for several reasons. Endothelial cells demonstrate tolerance to short-term ATP depletion (43), whereas maneuvers, such as extracellular acidosis, that protect cells from injury are still associated with profound ATP depletion (24, 28). Nevertheless, the precise role of ATP depletion as an initiating event in cell death remains controversial and may differ among cell types (38). Other events involving alterations of membrane integrity, intracellular ion homeostasis, or organelle function have been proposed as critical components of pH-dependent cell death. Activation of Na/H exchange may lead to a cytotoxic elevation of cytosolic sodium concentration (9) or initiate Na/Ca exchange, leading to cytotoxic elevations in cytosolic calcium (2, 18). Additional mechanisms initiated by increases in pHi and Na/H exchange, including altered mitochondrial membrane permeability (28, 36), increased intracellular oxidant production (15), or phospholipase activation leading to loss of membrane integrity (42), may also contribute to altered viability. In addition, there are differences among cell types in the components of this cell death pathway (37). The precise contribution of each of these mechanisms in the pH-dependent cell death pathway in endothelial cells will require further investigation.
Recent findings indicate that necrotic cell death is not a passive process but an active signal transduction cascade linked to phosphorylation pathways (35). The findings that phorbol ester treatment potentiated cell death in HPAEC (Fig. 2), suggesting that phosphorylation events are part of the cell death pathway, are compatible with this new concept. Protein kinase C-mediated activation of NHE1 is a well-documented pathway for activation of Na/H exchange (32). A similar phorbol ester treatment as used in this study upregulated NHE1 expression and activity in renal cells (1). The present results indicate that phorbol ester treatment significantly increased NHE1 activity in normal cells and in cells following a metabolic insult. Potentiation of cell death following phorbol ester treatment and inhibition of this effect with NHE1 inhibition suggest that protein kinase C-mediated phosphorylation and NHE1 activation are components of the HPAEC death pathway. These findings also suggest that protein kinase C-mediated events occur upstream to NHE1 activation. Additional work will be required to define these phosphorylation events at the molecular level.
Important questions remain unanswered. A limitation of the present
results is that the experiments were conducted in
HCO3-free medium, and thus conclusive
proof of the in vivo relevance of these findings will require use of
HCO
3/CO2-containing medium
to evaluate the role of Na/H exchange in the presence of other
functioning systems that regulate pHi. The present results demonstrate that incubation in acidotic medium was the most effective protective strategy for preserving HPAEC viability, which is in contrast to the prevailing view that acidosis disrupts cell function and leads to diminished viability (7). The protective effect of
acidosis has been noted in more than one cell type, but the mechanisms
are poorly defined (25). Acidosis is not universally or equally
protective against a metabolic insult in all cell types or organ
systems (23, 37). Our results demonstrate that this protective effect
involves modification of the pHi response and Na/H exchange
following a metabolic insult and probably modification of the changes
in other key intracellular parameters triggered by the initial changes
in pHi and Na/H exchange. Differences in the degree of
protection provided by acidosis and antiport inhibition among cell
types may involve differences in the signal transduction pathways
activated following an ischemic insult. Therefore, the optimal design
of strategies for preservation of cell viability in different organ
systems requires a precise determination of the mechanisms involved in
loss of viability in specific cell types.
Our results are relevant to lung injury and organ transplantation for several reasons. Preservation of organ function is highly dependent on endothelial cell viability. The unique location of endothelial cells at the blood-organ interface makes these cells a key target for cytotoxic stimuli. Second, conditions associated with cell injury in many organ systems include alterations of ion transport and intracellular ion homeostasis (38, 41). Third, a defect in mitochondrial respiration is a critical element of the cellular dysfunction, which precedes loss of viability under conditions associated with lung injury, such as ischemia-reperfusion (38, 39, 41). Finally, modification of the activity of ion transport systems, including the Na/H antiport, is associated with preservation of organ function (21, 22, 27, 31). Therefore, a better understanding of the role of specific ion transport systems in cell injury may lead to new approaches for preservation of cell viability in vivo.
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ACKNOWLEDGEMENTS |
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This study was supported by the Veterans Affairs Merit Review Program (grants to M. Cutaia and S. Rounds) and the University of Pennsylvania, Pulmonary, Allergy, and Critical Care Division.
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FOOTNOTES |
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A preliminary report of this work was presented at the American Thoracic Society Meeting, San Francisco, CA, in May 1998, and published in abstract form. (Am. J. Respir. Crit. Care Med. 159: A350, 1999).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Cutaia, VA Medical Center, Research Service, 3900 University and Woodland Aves., Philadelphia, PA 19104-9019.
Received 14 May 1999; accepted in final form 11 October 1999.
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