Influence of heat shock on cell volume regulation: protection from hypertonic challenge in a human monocyte cell line

^a Surgical Research Laboratories, Department of Surgery, University of Vienna, A-1090 Vienna, Austria
^b Department of Human Biological Chemistry and Genetics, University of Texas, Medical Branch at Galveston, Galveston, Texas 77555-0645, USA

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Heat shock response provides cells with higher tolerance against a variety of insults such as heavy metals, reperfusion injury, and endotoxin. In addition, heat treatment is known to affect ion transport mechanisms associated with vital cellular processes, including cell volume regulation. However, there has been no reports to date of a heat shock effect on cellular volume regulation itself. The aim of our study was to investigate whether the heat shock response influences volume regulation of cells. Human promonocytic U937 cells display an increase in volume in response to osmotic shrinkage. This regulatory volume increase (RVI) is mediated mainly by ion antiporters. U937 cells exposed to a temperature of 45°C for 10 min (heat shock) show an enhancement of RVI after hypertonic challenge compared with untreated cells. Also, heat-treated cells display a lower intracellular pH (pH_i) than untreated cells; similar control mechanisms are believed to be involved in regulating both pH_i and RVI. In agreement with this, heat-shocked cells demonstrated increased activity of an HCO₃^--independent/DIDS-sensitive pH_i down-regulator, postulated to be a Cl^-/HCO₃^- exchange. We suggest that heat shock-mediated RVI enhancement is at least partially mediated by an increased Cl^-/HCO₃^- exchange. Our results indicate that heat shock of U937 cells activates a hitherto unknown cytoprotective effect that may help cells to overcome hypertonic challenge.—Oehler, R., Zellner, M., Hefel, B., Weingartmann, G., Spittler, A., Struse, H. M., Roth, E. Influence of heat shock on cell volume regulation: protection from hypertonic challenge in a human monocyte cell line. FASEB J. 12, 553–560 (1998)

Key Words: DIDS • DMA • ion transport • Na⁺/H⁺ exchange • intracellular water space • heat treatment

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
WHEN CONFRONTED with abrupt changes in the local environment, all cells employ a global defense mechanism: the heat shock response. Heat shock response transiently protects both the cell and the parent organism against a variety of insults. Recent studies suggest that the protective effect of the heat shock response is also of clinical importance. Induction of the heat shock response reduces mortality rate and organ damage in sepsis-induced acute lung injury (1) and protects against the lethal effect of endotoxin (2). Overexpression of hsp-70 in a transgenic mouse increases the resistance of the heart to ischemic injury (3). Heat shock is known to influence the ion transport mechanisms associated with vital cellular processes. For example, heat treatment of human epidermoid A431 cells inhibits Na⁺/H⁺ exchange (4). In monkey kidney Vero cells, heat shock decreases the activity of Na⁺-independent Cl^-/HCO₃^- exchange and increases the activity of Na⁺/H⁺ exchange (5).

These ion transport mechanisms are clearly important in the overall functioning of the cell. Especially crucial is the role they play in maintaining intracellular pH (pH_i).² The ion exchangers involved in pH_i regulation have been studied in detail in the human premonocyte cell line U937, the cell line used in the current investigation (6, 7). These cells possess two mechanisms that allow them to recover from intracellular acidification: Na⁺/H⁺ exchange and Na⁺-dependent Cl^-/HCO₃^- exchange. As yet, no system has been described that protects U937 cells from alkalinization.

Another effect mediated by these ion antiporters is maintenance of a constant volume in the face of osmotic perturbation. The processes by which cells subjected to swelling and shrinkage return to a normal volume are called regulatory volume decrease and regulatory volume increase (RVI), respectively. The mechanisms underlying RVI have been studied extensively in lymphoid cells (8). After shrinkage, a joint influx of Na⁺ and Cl^- enters the cell, which, together with water uptake, usually restores the original cell volume. Uptake of Na⁺ is due to increased Na⁺/H⁺ exchange, which appears to be directly stimulated by cell shrinkage. Simultaneous with Na⁺ uptake, extrusion of protons leads to an alkalinization of cytosol, inducing an activation of pH_i down-regulating, Na⁺-independent Cl^-/HCO₃^- exchange.

Although the effect of heat shock upon the ion exchangers regulating pH_i is well documented, if not completely understood, there have been no reports of a heat shock effect on cellular volume regulation. The two effects may potentially be interactive. In the present work, the effect of heat shock on the antiporters involved in volume and pH_i regulation of U937 cells is investigated.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Chemicals and solutions
2',7'-bis (carboxyethyl) carboxyfluorescein acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes (Eugene, Oreg.). [³H]Inulin and [¹⁴C]urea were from Amersham (Buckinghamshire, U.K.). 5-(N,N-dimethyl)amiloride (DMA) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) were from Sigma (St. Louis, Mo.). All other chemicals were from Merck (Darmstadt, Germany). Nominally bicarbonate-free phosphate-buffered saline (PBS) was 10 mM Na₂HPO₄/NaH₂PO₄ (pH=7.4), 135 mM NaCl, 2.7 mM KCl. In Na⁺-free PBS, NaCl was substituted by equimolar concentrations of choline chloride and K⁺-phosphate was used instead of Na⁺-phosphate.

Cell culture
Human promonocytic U937 cells (American Type Culture Collection, Rockville, Md.) were grown in RPMI-1640 cell culture medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 0.1% penicillin, and 0.1% streptomycin at 37°C/5% CO₂. Cells from passages 15–25 were used for experiments. Cell viability was determined in two different ways: trypan blue and propidium iodide exclusion (9, 10). For measuring the percentage of apoptotic cells, an annexin V apoptosis detection kit (Genzyme, Cambridge, Mass.) was used. Cells stained with annexin V but not with propidium iodide were identified as apoptotic.

pHⁱ measurements
To determine the pH_i, we used the acetoxymethylester of the pH-sensitive fluorochrome BCECF (BCECF-AM). BCECF-AM permeates through the cell membrane and becomes cleaved by cytoplasmatic esterases. The product BCECF is charged and therefore trapped in the cytoplasm. Accordingly, the fluorescence of BCECF-loaded cells reflects the pH in the cytosol.

Cells were incubated in PBS (1x10⁶ cells/ml) supplemented with 2 µg/ml BCECF-AM for 30 min at 37°C. At the end of the incubation period, cells were washed twice with PBS, resuspended to 1.2 x 10⁶ cells/ml in PBS, and transferred to a cuvette maintained at 37°C. The BCECF fluorescence was measured with a spectrofluorometer (FP777 JASCO, Tokyo, Japan) as the ratio of emission at 525 for dual excitation at 490 and 450 nm (slit with 4 nm). BCECF leaks out of U937 cells at a rate of 0.4%/min. The fluorescence signal was calibrated using the nigericin/KCl method, as described previously (11).

Determinations of intracellular water space (ICS)
ICS was measured with an isotopic inulin–urea assay as described previously (12). Briefly, cells were washed in PBS, resuspended to 10⁷ cells/ml in PBS supplemented with [¹⁴C]urea (0.5 µCi/ml) and [³H]inulin (1 µCi/ml), and incubated for at least 5 min at 37°C. Urea, which can diffuse through the cell membrane, is a marker for total water space; inulin, which does not permeate the cell membrane, is a marker for extracellular water space. Cell suspensions were then incubated in PBS solutions with different osmolarities in 96-well microtiter plates, for 5 min at 37°C and 5% CO₂, and then pelleted by gentle centrifugation. The [¹⁴C]urea and [³H]inulin contents of the cell pellet and of the supernatant were determined by scintillation counting. The protein content of the pellet was measured by Bradford assay. From these data, the intracellular water space per total cell protein content (in µl/mg) was calculated.

Statistical analysis
All data are expressed as median ± standard deviation. Student's t test was used for comparison of groups. Linear least-squares regression to the data was used to calculate the slope, which was used as a measure of initial pH_i recovery rate or initial acidification rate. The Boltzmann equation was used to fit a sigmoidal curve to the data in Fig. 2and Fig. 3.

View larger version (16K): [in this window] [in a new window]   Figure 2. Recovery of pHi after acid load in heat-treated and untreated cells. For heat treatment, cells were exposed to 45°C for 10 min and then incubated at 37°C/5% CO2 for 90 min in RPMI-1640 medium. Heat-treated and untreated cells were washed in PBS containing BCECF-AM and incubated for another 30 min at 37°C/5% CO2. Cells were then loaded with NH4Cl (75 mM) for 15 min, placed in Na+-free PBS for 2 min, resuspended in Na+-containing PBS, and pHi was measured. The apparent initial recovery was determined by linear regression of pHi values measured in the first 2.5 min (r=0.94 hs+, r=0.92 hs-). The fitting curve was calculated using the Boltzmann equation.

View larger version (19K): [in this window] [in a new window]   Figure 3. Intracellular acidification after withdrawal of bicarbonate. For heat treatment, cells were exposed to 45°C for 10 min and then incubated at 37°C/5% CO2 for 90 min in RPMI-1640 medium. Heat-treated and untreated cells were washed in PBS with 25 mM HCO3- containing BCECF-AM and incubated for another 30 min at 37°C/5% CO2. Bicarbonate was then removed (by washing cells in nominally bicarbonate-free PBS) and pHi was measured. The apparent initial acidification was determined by linear regression of pHi values measured in the first 6 min (r=0.98 hs+, r=0.96 hs-). The fitting curve was calculated using the Boltzmann equation.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Heat shock effect on pH_i of U937 cells
The resting pH_i of U937 cells in nominally bicarbonate-free PBS is 7.08 ±0.031. Cells subjected to heat shock (experimentally defined as a temperature of 45°C for 10 min) and then placed at 37°C for 2 h to recover have a pH_i of 6.99 ±0.031 ( Fig. 1).

View larger version (14K): [in this window] [in a new window]   Figure 1. Intracellular pH after heat treatment in the presence and absence of bicarbonate. For heat treatment, cells were exposed to 45°C for 10 min and incubated at 37°C/5% CO2 for 90 min in RPMI-1640 medium. Heat-treated and untreated cells were washed in PBS with and without 25 mM HCO3- containing BCECF-AM and incubated for another 30 min at 37°C/5% CO2; pHi was then measured (n=4–8). The results are given as median ± standard deviation (**P<0.005 vs. hs-).

Heat shock effect on cell death
Heat-induced intracellular acidification is not related to cell death, as ascertained via apoptosis and cell viability assays at different times after heat shock (0, 1, 2, 3, 24 h). Percentage of numbers of viable cells was not different in heat-treated vs. control cells. All cases contained less than 6% apoptotic cells.

Heat shock effect on Na⁺-dependent Cl^-/HCO₃^- exchange
When suspended in PBS with bicarbonate (25 mM HCO₃^-), the pH_i of U937 cells increases within a few minutes by 0.2 pH units ( Fig. 1). This indicates the existence of an HCO₃^--dependent, acid extruding mechanism in U937 cells, believed to be Na⁺-dependent Cl^-/HCO₃^- exchange. To determine whether the heat shock-mediated acidification seen under nominally bicarbonate-free conditions also occurs in the presence of bicarbonate, cells were heat treated and then incubated at 37°C for 2 h in the presence and absence of bicarbonate; pH_i was then measured. As seen in Fig. 1, the presence of bicarbonate prevents heat shock-mediated acidification.

Heat shock effect on Na⁺/H⁺ exchange
The second pH_i up-regulating ion transport mechanism in U937 cells is Na⁺/H⁺ exchange. Cells suspended in Na⁺-free PBS and acid loaded (40 mM NH₄Cl for 15 min) display a pH_i reduction by about 0.9 pH units (to a pH_i of 6.23 ±0.12). When cells are placed in Na⁺-containing PBS, pH_i returns to 7.09 ±0.016 within 4 min ( Fig. 2). This recovery from acid load is Na⁺ dependent and is inhibited by DMA (data not shown). These results seem to indicate that Na⁺/H⁺ exchange is the primary process responsible for recovery from an acid load in a bicarbonate-free system. Hence, if hyperthermia affects Na⁺/H⁺ exchange, then the recovery rate from acid loading might be impaired in heated cells. To address this issue, heat-shocked cells were acid loaded after recovery from heat treatment. Although heated cells recover to a lower pH_i than untreated cells, in accordance with the data presented previously, the rate of recovery does not differ between the two groups ( Fig. 2).

Heat shock effect on pH_i down-regulating systems
As described above, removing bicarbonate from the solution leads to an inactivation of Na⁺-dependent Cl^-/HCO₃^- exchange, resulting in a decrease in pH_i. This acidification takes place within 8 min, with an initial acidification rate of 0.061 ±0.007 pH_i/min ( Fig. 3). To determine whether this drop in pH_i is mediated by an Cl^-/HCO₃^- exchange mechanism, cells were incubated at 37°C for 30 min in PBS containing bicarbonate and 1 mM DIDS (a potent inhibitor of Cl^-/HCO₃^- exchange). After withdrawal of bicarbonate from the solution, U937 cells showed a reduced initial acidification rate of 0.027 ±0.011 pH_i/min (n=8). This clearly demonstrates the existence of an HCO₃^--independent, DIDS-sensitive, pH_i down-regulating mechanism in U937 cells.

Our results suggest that if heat shock has an effect on this mechanism, the initial rate of acidification after bicarbonate removal should change in heat-shocked cells. Indeed, heated cells allowed to recover at 37°C for 2 h in bicarbonate containing PBS and then subjected to removal of bicarbonate show a drop in pH_i to levels lower than those seen in control cells ( Fig. 3). Further, the initial rate of acidification in heat-treated cells (0.080±0.007 pH_i/min) is significantly (P=0.003) higher than in untreated cells (0.061±0.007 pH_i/min).

Heat shock effect on cell volume under hypertonic conditions
Because it was known that in other leukocytes Cl^-/HCO₃^- exchange is involved in both pH_i and volume regulation, playing a major role in RVI, the results of the above pH_i experiments encouraged us to investigate any potential effects of heat shock on RVI that might be mediated by this cellular mechanism. The resting volume of U937 cells at 37°C in isotonic PBS (305 mosmol/l) is 1.09 ±0.01 pl, as measured by Coulter counter analysis. To determine cell volume changes, we used an inulin–urea assay, which measures the intracellular water space per cellular protein content (ICS) (12). In U937 cells, the resting volume corresponds to an ICS value of 4.01 ±0.17 µl/mg. Heat-shocked U937 cells exhibit a decrease in their volume by 17 ±4% (n=4, see Fig. 4).

View larger version (14K): [in this window] [in a new window]   Figure 4. Intracellular water space (ICS) of heat-treated and untreated cells under isotonic and hypertonic conditions. For heat treatment, cells were exposed to 45°C for 10 min and then incubated at 37°C/5% CO2 for 120 min in RPMI-1640 medium. Heat-treated and untreated cells were washed in PBS with and without 25 mM HCO3-, incubated for 30 min at 37°C/5% CO2, and ICS was determined. The results are given as median ± standard deviation (**P<0.005 vs. hs-, ***P<0.001 vs. hs-, n=8).

To ascertain the influence of heat shock on RVI, we measured the ICS of heat-treated and untreated U937 cells exposed to hypertonic conditions (455 mosmol/l) for 5 min. Untreated cells in hypertonic solution had a 41% lower ICS than those in isotonic conditions ( Fig. 4). Heat-treated cells, in contrast, displayed only a 20% lower ICS in hypertonic conditions than in isotonic conditions. This highly significant difference (P=0.0023) indicates that heat shock influences cellular behavior in hypertonic solutions.

To determine whether the higher ICS of heat-treated cells in a hypertonic solution correlates with the RVI, experiments were performed to further characterize the RVI in U937 cells.

Characterization of RVI in U937 cells
To determine the RVI process in U937 cells, we measured cell volume changes after exposing cells to a hypertonic solution (455 mosmol/l). As depicted in Fig. 5, cells display an initial reduction in volume that is quite massive, followed by a modest recovery of volume. These data demonstrate that RVI is activated after osmotic shrinkage.

View larger version (15K): [in this window] [in a new window]   Figure 5. Changes in intracellular water space (ICS) after exposure to hypertonic conditions. Cells were incubated at 37°C/5% CO2 in hypertonic PBS (455 mosmol/l) and ICS was measured at different times. The results are given as the percentage of isotonic control (median ± standard deviation, n=4).

It had previously been stated that the primary event during RVI in leukocytes, at least with respect to ion transport, is the activation of Na⁺/H⁺ exchange (8). This results in a volume increase that is DMA sensitive and in a concomitant alkalinization of the cytosol. To assess whether RVI in U937 cells also depends on Na⁺/H⁺ exchange, we used the specific Na⁺/H⁺ exchange inhibitor DMA. Figure 6 shows that DMA reduces the ICS of both heat-shocked and control cells exposed to hypertonic conditions for 5 min. However, the ICS difference between heat-treated and untreated cells in the presence of DMA is similar to that in the absence of DMA. These data suggest that RVI of U937 cells depends at least partially on Na⁺/H⁺ exchange and that the heat shock-mediated ICS increase under hypertonic conditions cannot be inhibited by DMA.

View larger version (14K): [in this window] [in a new window]   Figure 6. Influence of DMA on volume of heat-treated and untreated cells under isotonic and hypertonic conditions. For heat treatment, cells were exposed to 45°C for 10 min and then incubated at 37°C/5% CO2 for 120 min in RPMI-1640 medium. Heat-treated and untreated cells were washed in PBS with and without 100 µM DMA and incubated for 30 min at 37°C/5% CO2. Cells were then washed in hypertonic PBS (455 mosmol/l, with and without 100 µM DMA), incubated for 5 min 37°C/5% CO2, and ICS was measured (n=4–8). The results are given as the percentage of untreated isotonic control (median±standard deviation, **P<0.005, ***P<0.001 vs. hs-).

For further characterization of RVI in U937 cells, the influence of hypertonicity on pH_i was determined. As stated above, the resting pH_i of U937 cells in nominally bicarbonate-free PBS is 7.08 ±0.03. After undergoing hypertonic challenge (455 mosmol/l for 3 min), U937 cells showed an increase in pH_i of 0.08 (pH_i=7.16 ±0.03), indicating an osmotic activation of Na⁺/H⁺ exchange. Together, these data show that after osmotic shrinkage, U937 cells display a volume recovery, accompanied by an activation of Na⁺/H⁺ exchange.

In view of these results, it is hypothesized that the ICS increase measured 5 min after the onset of hypertonic challenge is already the result of ongoing RVI. These data strongly suggest that the heat shock-mediated ICS increase under hypertonic conditions is directly attributable to an accelerated volume recovery in heated cells.

Influence of heat shock on RVI under different hypertonic conditions
To examine whether heat shock enhances RVI at different hypertonic conditions, heat-treated and untreated cells were incubated in PBS solutions with different osmolarities for 5 min, and ICS was then measured. Figure 7 shows that under isotonic conditions, heat-treated U937 cells have a lower cell volume than do untreated cells. The ICS difference is nearly the same (18±5%) as in the previous experiment (17±4%; see Fig. 4). The ICS of untreated cells decreased in a linear manner with increasing osmolarity. In contrast, heat-treated cells had nearly the same volume at low hypertonic conditions (405 mosmol/l) as at normotonic conditions. At moderate hypertonicity (455 mosmol/l), the ICS of heat-treated cells decreases but is still higher than that of untreated cells. At high hypertonicity (555 mosmol/l), heat-treated cells show an ICS similar to that of untreated cells.

View larger version (18K): [in this window] [in a new window]   Figure 7. Intracellular water space (ICS) of heat-treated and untreated cells under different hypertonic conditions. For heat treatment, cells were exposed to 45°C for 10 min and then incubated at 37°C/5% CO2 for 120 min in RPMI-1640 medium. Heat-treated and untreated cells were washed in PBS with different osmolarities and incubated for 5 min 37°C/5% CO2. ICS was then measured (n=8). The results are given as the percentage of untreated isotonic control (median ±standard deviation, ***P<0.001 vs. hs-).

Heat shock-mediated RVI enhancement at different times after heat treatment
In all the experiments described above, heat-treated cells were allowed to recover at 37°C for 2 h. To investigate whether this time delay between heat shock and ICS measurement is necessary in order to observe heat-mediated RVI enhancement, time course experiments were performed. Heat-treated cells were incubated at 37°C for up to 4 h in nominally bicarbonate-free PBS. After exposing cells to hypertonic conditions (455 mosmol/l) for 5 min, the ICS was measured. As seen in Fig. 8, ICS immediately after heat-shock does not differ between heated and control cells. The strongest effect of heat treatment on volume recovery was detected 1 to 2 h after heat shock; the heat shock-mediated RVI enhancement is seen to decline after 4 h.

View larger version (17K): [in this window] [in a new window]   Figure 8. Intracellular water space (ICS) of heat-treated and untreated cells under hypertonic conditions at different times after heat shock. For heat treatment, cells were exposed to 45°C for 10 min and then incubated at 37°C/5% CO2 for up to 4 h in PBS. Heat-treated and untreated cells were washed in hypertonic PBS (455 mosmol/l) and incubated for 5 min 37°C/5% CO2; ICS was then measured (n=4–8). The results are given as a percentage of untreated isotonic control (median±standard deviation, **P<0.005, ***P<0.001 vs. hs-).

Influence of different heat treatments on RVI
For heat treatment in all of the experiments described above, cells were exposed to 45°C for 10 min. To investigate whether heat shock-mediated RVI enhancement also occurs after moderate heating of cells, we exposed U937 cells to 42°C for 1 h. In parallel, cells were exposed to 45°C for 10 min, followed by 50 min at 37°C. After cells were exposed to hypertonic conditions (455 mosmol/l) for 5 min, ICS was measured. Figure 9 shows that after both heat treatments, cells exhibit a higher ICS than untreated cells.

View larger version (17K): [in this window] [in a new window]   Figure 9. Intracellular water space (ICS) under hypertonic conditions after different heat treatments. For heat treatment, cells were exposed to 42°C for 60 min or to 45°C for 10 min, followed by 50 min at 37°C in PBS. Heat-treated and untreated cells were washed in hypertonic PBS (455 mosmol/l) and incubated for 5 min at 37°C/5% CO2; ICS was then measured (n=8). The results are given as the percentage of untreated isotonic control (median±standard deviation, ***P<0.001 vs. hs-).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
We report that heat shock of U937 cells affects pH_i and cell volume regulation, presumably via its effects on ion transport mechanisms. This is the first report of such an effect. Heat shock stimulates a hitherto unknown Cl^-/HCO₃^- exchange mechanism in these cells that appears to mediate both pH_i and cell volume regulation. Further work is needed to delineate the nature of this potentially novel mechanism.

U973 cells display an acidification of the cytosol after heat treatment in the absence of bicarbonate. Heat-mediated intracellular acidification has been described in various cell types (4, 5, 13, 14). In contrast to effects reported for A431 cells and Vero cells (4, 5), U937 cells seem not to involve Na⁺/H⁺ exchange in heat-mediated acidification of cytosol. The activity of this exchange mechanism remains unaffected after heat treatment. We report, however, a heat-induced increase of a potentially novel HCO₃^--independent, DIDS-sensitive, pH_i down-regulating mechanism. This mechanism is presumed to be a Cl^-/HCO₃^- antiport that exchanges extracellular Cl^- for intracellular HCO₃^-. Support for this notion comes from the observation that another premonocytic cell line, HL-60 (which shares many features in common with the U937 cell line), uses a Na⁺-independent Cl^-/HCO₃^- exchange as the major pH_i down-regulating system (15). Additional corroboration is provided by the reports of Ladoux et al. (6), who found that a bicarbonate-independent pH_i recovery is seen in U937 cells after alkali loading.

In further agreement with our observations, Ludt and co-workers (5) reported a heat-mediated alteration of Na⁺-independent Cl^-/HCO₃^- exchange in Vero cells. In addition, Amonio and Fox (16) found a hyperthermia induced increase of intracellular Cl^- in CHO cells that could be blocked by furosemide, an inhibitor of Cl^-/HCO₃^- exchange (16). Our findings, as well as those of other workers, demonstrate that acidification of cytosol in heat-treated U937 cells is mediated, at least in part, by an increased Cl^-/HCO₃^- exchange.

It has been known for some time that, in other cell lines, regulation of cell volume is closely coupled to that of pH_i (8). Considering that several ion antiport mechanisms, such as those involved in Na⁺/H⁺ and Cl^-/HCO₃^- exchanges, are involved in both processes, it seems reasonable to postulate that a heat-mediated alteration of Cl^-/HCO₃^- exchange should affect cell volume regulation. Indeed, heat-treated U937 cells show an enhancement of volume gain in response to osmotic shrinkage compared with untreated cells. This effect is not seen directly after heat treatment, which would preclude a direct effect of heat on RVI. After reaching a maximum value at 1 h after heat shock, the effect diminishes. Since heat shock proteins first begin to be expressed 2 h after heat shock (17), it is clear they cannot be involved in this process. In addition, because heat shock-mediated enhancement of RVI cannot be abolished by DMA, Na⁺/H⁺ exchange is not involved.

In RVI, the joint operations of cation and anion exchange produce a net gain of NaCl, which together with the uptake of osmotically obliged water restores the original cell volume after hypertonic challenge. Accordingly, the rate of Cl^-/HCO₃^- exchange directly affects this process. Therefore, we conclude that heat shock-mediated RVI enhancement in U937 cells is at least partially mediated by an increased Cl^-/HCO₃^- exchange. Any consequences of heat treatment on target sensitivity, protein turnover, proliferation capacity, as well as response to growth factors, hormones, and drugs and the potential involvement of increased Cl^-/HCO₃^- exchange in these cellular functions, must be addressed in future studies.

Our results show that heat shock generates a response in U937 cells that helps cells overcome volume loss. This cytoprotective effect can also be induced by hyperthermic conditions such as those observed in patients with high fever. Therefore, it is possible that high body temperature could lead to an enhancement of cellular RVI in these patients. Maintenance of a constant volume is crucial for cellular function. Cell volume is a dynamic parameter, which changes within minutes in response to a large number of different factors (18). Cell volume is also reduced by a variety of disease states that are associated with substantial elevation of plasma osmolarity, such as diabetes mellitus, dehydration, and renal failure (19). Cell shrinkage affects several important cellular functions, including protein turnover (20) and respiratory burst (21). A decrease in cell volume in liver and skeletal muscle triggers the protein catabolic states that accompany various diseases such as burns, polytrauma, and acute necrotizing pancreatitis (22). These and other findings illustrate the relation between cell volume homeostasis and pathophysiology. Consequently, the cytoprotective effect of heat shock response—the enhancement of RVI—could be of great clinical relevance.

	ACKNOWLEDGMENTS

 
We are grateful to Susanne Oehler and the late Manuel Simon for helpful discussions.

	FOOTNOTES

 
¹ Correspondence: Surgical Research Laboratories, Allgemeines Krankenhaus (8G9.05), Waehringer-Guertel 18–20, A-1090 Vienna, Austria. E-mail: rudolf.oehler{at}akh-wien.ac.at

² Abbreviations: RVI, regulatory volume increase; pH_i, intracellular pH; ICS, intracellular water space, PBS, phosphate-buffered saline; DMA, 5-(N,N-dimethyl)amiloride; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonate; BCECF-AM, 2',7'-bis (carboxyethyl) carboxyfluorescein acetoxymethyl ester.

Received for publication October 15, 1997. Accepted for publication January 5, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 

1. Villar, J., Ribeiro, S. P., Mullen, J. B., Kuliszewski, M., Post, M., and Slutsky, A. S. (1994) Induction of the heat shock response reduces mortality rate and organ damage in a sepsis-induced acute lung injury model [see comments]. Crit. Care Med. 22, 914–921[Medline]
2. Hotchkiss, R., Nunnally, I., Lindquist, S., Taulien, J., Perdrizet, G., and Karl, I. (1993) Hyperthermia protects mice against the lethal effects of endotoxin. Am. J. Physiol 265, R1447–R1457[Abstract/Free Full Text]
3. Marber, M. S., Mestril, R., Chi, S. H., Sayen, R., Yellon, D. M., and Dillmann, W. H. (1995) Overexpression of the rat inducible 70-kD stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J. Clin. Invest. 95, 1446–1456
4. Kiang, J. G., McKinney, L. C., and Gallin, E. K. (1990) Heat induces intracellular acidification in human A-431 cells: role of Na(+)-H+ exchange and metabolism. Am. J. Physiol. 259, C727–C737[Abstract/Free Full Text]
5. Ludt, J., Sandvig, K., and Olsnes, S. (1993) Rapid increase in pH set-point of the Na(⁺)-independent chloride/bicarbonate antiporter in Vero cells exposed to heat shock. J. Membr. Biol. 134, 143–153[Medline]
6. Ladoux, A., Krawice, I., Cragoe, E. J., Jr., Abita, J. P., and Frelin, C. (1987) Properties of the Na⁺-dependent Cl^-/HCO₃^- exchange system in U937 human leukemic cells.. Eur. J. Biochem. 170, 43–49[Medline]
7. Ladoux, A., Damais, C., Krawice, I., Abita, J. P., and Frelin, C. (1988) An increase in intracellular pH is a general response of promonocytic cells to differentiating agents. FEBS Lett. 234, 353–356[Medline]
8. Grinstein, S., and Foskett, J. K. (1990) Ionic mechanisms of cell volume regulation in leukocytes. Annu. Rev. Physiol. 52, 399–414[Medline]
9. Kaltenbach, H. T., Kaltenbach, M. H., and Lyons, W. B. (1958) Nigrosin as a dye for differentiating live and dead ascites cells. Exp. Cell. Res. 15, 112–117[Medline]
10. Holden, H. T., Lichter, W., and Sigel, M. M. (1973) Quantitative methods for measuring cell growth and death. In Tissue Culture Methods and Applications (Kruse, P. F., and Patterson, K. M., eds) pp. 408–412, Academic Press, New York
11. Thomas, J. A., Buchsbaum, R. N., Zimniak, A., and Racker, E. (1979) Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18, 2210–2218[Medline]
12. Oehler, R., Hefel, B., and Roth, E. (1996) Determination of cell volume changes by an inulin urea assay in 96 well plates: a comparison with Coulter counter analysis. Analyt. Biochem. 241, 269–271
13. Yi, P. N., Chang, C. S., Tallen, M., Bayer, W., and Ball, S. (1983) Hyperthermia-induced intracellular ionic level changes in tumor cells. Radiat. Res. 93, 534–544[Medline]
14. Eisner, D. A., Nichols, C. G., O'Neill, S. C., Smith, G. L., and Valdeolmillos, M. (1989) The effects of metabolic inhibition on intracellular calcium and pH in isolated rat ventricular cells. J. Physiol. (London) 411, 393–418[Abstract/Free Full Text]
15. Restrepo, D., Kozody, D. J., Spinelli, L. J., and Knauf, P. A. (1988) pH homeostasis in promyelocytic leukemic Hl60 cells. J. Gen. Physiol. 92, 489–507[Abstract/Free Full Text]
16. Amorino, G. P., and Fox, M. H. (1996) Effects of hyperthermia on intracellular chloride. J. Membr. Biol. 152, 217–222
17. Ding, X. Z., Smallridge, R. C., Galloway, R. J., and Kiang, J. G. (1996) Increases in HSF1 translocation and synthesis in human epidermoid A-431 cells: role of protein kinase C and [Ca²⁺]_i. J. Invest. Med. 44, 144–153[Medline]
18. Häussinger, D., and Schliess, F. (1995) Cell volume and hepatocellular function. J. Hepatol. 22, 94–100[Medline]
19. McManus, M. L., Churchwell, K. B., and Strange, K. (1995) Regulation of cell volume in health and disease. N. Engl. J. Med. 333, 1260–1266[Free Full Text]
20. Häussinger, D., and Lang, F. (1991) The mutual interaction between cell volume and cell function: a new principle of metabolic regulation. Biochem. Cell. Biol. 69, 1–4[Medline]
21. Miyahara, M., Watanabe, Y., Edashige, K., and Yagyu, K. (1993) Swelling-induced O₂^- generation in guinea-pig neutrophils. Biochim. Biophys. Acta 1177, 61–70[Medline]
22. Häussinger, D., Roth, E., Lang, F., and Gerok, W. (1993) Cellular hydration state: an important determinant of protein catabolism in health and disease. Lancet 341, 1330– 1332[Medline]