Centre hospitalier de l'Université de Montréal and Department of Medicine, Université de Montréal, Montréal, Québec, Canada H2W 1T8
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ABSTRACT |
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ATP release induced by hypotonic swelling
is an ubiquitous phenomenon in eukaryotic cells, but its underlying
mechanisms are poorly defined. A mechanosensitive (MS) ATP channel has
been implicated because gadolinium (Gd3+), an inhibitor of
stretch-activated channels, suppressed ATP efflux monitored by
luciferase bioluminescence. We examined the effect of Gd3+
on luciferase bioluminescence and on ATP efflux from hypotonically swollen cells. We found that luciferase was inhibited by 10 µM Gd3+, and this may have contributed to the previously
reported inhibition of ATP release. In ATP efflux experiments,
luciferase inhibition could be prevented by chelating Gd3+
with EGTA before luminometric ATP determinations. Using this approach,
we found that 10-100 µM Gd3+, i.e., concentrations
typically used to block MS channels, actually stimulated hypotonically
induced ATP release from fibroblasts. Inhibition of ATP release
required at least 500, 200, or 100 µM Gd3+ for
fibroblasts, A549 cells, and 16HBE14o
cells,
respectively. Such biphasic and cell-specific effects of
Gd3+ are most consistent with its action on membrane lipids
and membrane-dependent processes such as exocytosis.
mechanosensitive adenosine 5'-triphosphate release; luciferase bioluminescence
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INTRODUCTION |
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ATP RELEASE induced by mechanical stimuli, such as hypotonic swelling, shear stress, or mechanical strain, has been observed in all eukaryotic cells examined (4, 16, 20-22, 27, 29, 31). Extracellular ATP and other nucleotides then interact with purinergic P2Y and P2X receptors to regulate a broad range of physiological responses, including vascular tone, muscle contraction, cell proliferation, mucociliary clearance, synaptic transmission, and platelet aggregation (5, 7, 12). The mechanism of mechanosensitive (MS) ATP release is not known, and several mechanisms have been proposed, including the fusion of ATP-enriched protein transport vesicles, ATP transporters, and MS ATP channels (6, 20, 23, 28, 29). The latter hypothesis was based on the observation that gadolinium (Gd3+), an inhibitor of stretch-activated cation channels, inhibited swelling-induced ATP efflux when it was monitored by luciferase bioluminescence. In particular, for epithelial cells, it was proposed that the cystic fibrosis transmembrane conductance regulator (CFTR) facilitates swelling-induced ATP release through a separate ATP-permeable channel and contributes to cell volume regulation. An MS channel was implicated because ATP release was blocked by Gd3+ (6).
ATP-dependent luciferase bioluminescence allows the detection of femtomoles of ATP; however, light output from the reaction depends on a number of factors, including ionic strength, pH, temperature, and the concentration of divalent cations (25), all of which may vary during the experiment. For example, experimental manipulations aimed at modulating ATP efflux from cells, such as application of hypotonic media or putative inhibitors, may directly interfere with the bioluminescence reaction and hinder accurate ATP detection. The trivalent lanthanide Gd3+ blocks mechanogated channels and has diverse, often nonspecific effects that include blockage of other channel types, induction of liposome fusion, and pore formation in erythrocytes (2, 9, 14, 18, 19, 32). Many of these actions may originate from binding of Gd3+ to phospholipids and alteration of physical properties of the cell membranes (13). In addition to direct and indirect inhibition of ion channels, lanthanides have been reported to inhibit several enzymes, including calcium ATPases, proteinases, and kinases (14, 15, 33). Use of Gd3+ is further complicated by its strong binding to certain anions that are often present in physiological and cell culture solutions such as phosphate, carbonate, sulfate, EDTA, albumin, and ATP (8, 14). Thus the concentration of free Gd3+ in the presence of these anions could be significantly overestimated, leading to erroneous conclusions (8).
In this study, we sought to examine the effect of Gd3+ on
ATP detection by luciferase-luciferin bioluminescence and on ATP efflux from hypotonically swollen cells. We found that ATP-dependent luciferase bioluminescence was directly inhibited by 10 µM free Gd3+ and may have contributed to the previously reported
apparent inhibition of ATP efflux from several cell types. When
luciferase inhibition was prevented by chelating any Gd3+
before luminometric ATP determinations, we found that Gd3+
at 10-100 µM stimulated ATP efflux from hypotonically swollen 3T3 fibroblasts. Inhibition of ATP efflux from fibroblasts and lung
epithelial A549 cells by Gd3+ was observed at 200-500
µM, i.e., concentrations significantly higher than those used
typically to directly block MS ion channels. Our results do not support
the view that Gd3+-sensitive channels are involved in ATP
release and suggest that indirect and nonspecific effects of
Gd3+ on lipid membrane and membrane-dependent processes
should be considered.
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METHODS |
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Cell culture.
Adult human lung carcinoma A549 cells and NIH/3T3 fibroblasts (American
Type Culture Collection) were grown in DMEM supplemented with 10%
fetal bovine serum, 20 mM L-glutamine, 60 µg/ml
penicillin G, and 100 µg/ml streptomycin. Human bronchial epithelial
16HBE14o cells, a generous gift from Dr. D. Gruenert,
were cultured as described (10). All constituents of the
culture media were from GIBCO BRL (Burlington, ON). Cell monolayers
were grown to near confluency on culture-treated plastic in 12-well
plates with an area of 3.14 cm2/well.
ATP efflux experiments. Before the experiments, culture media was removed from the wells, and the cell monolayers were washed two times with physiological NaCl solution. Because of massive ATP release during the wash, the cells were allowed to equilibrate for ~1 h at 37°C and to reduce ATP to its basal level (16). In addition, repeated gentle replacement of one-half of the extracellular solution allows the extracellular ATP concentration to be quickly reduced close to the basal level. ATP efflux experiments were performed at 37°C on a rotating platform (30 rpm) to ensure mixing and to minimize the unstirred layer above the cells. Each well with cells contained 500 µl of solution, and hypotonic shock was applied by adding the appropriate volume of distilled water or solution containing only divalent cations (see below). Samples (25 or 50 µl) of the medium covering the cells were taken at various time points, and ATP content was determined immediately by luciferin/luciferase bioluminescence assay.
Luciferin/luciferase luminometric assay. To measure ATP content in the samples, we used glycine-buffered firefly luciferase with added luciferin (Sigma, catalog no. L3641) at a concentration of 1 mg/ml of the total reagent. In some experiments, we employed a custom-made mix of EDTA-free luciferin and luciferase. It contained 150 µM luciferin (catalog no. L6882), ~2 µM luciferase (catalog no. L9506), 50 mM glycine, 0.1 mM Tris, 10 mM MgSO4, and 1 mg/ml BSA, all purchased from Sigma. During the assay, 50 µl of the medium was mixed in the cuvette with 50 µl of luciferin/luciferase reagent, and the average light signal was measured for 10 s with a TD20/20 luminometer (Turner Design, Sunnyvale, CA). ATP content was determined using [ATP] vs. luminescence standard curves, which were made for each experimental solution and each luciferase reagent preparation. In experiments in which Gd3+ was used to block ATP efflux from the cells, samples of extracellular media were first treated with EGTA plus MgSO4 to chelate any Gd3+ before luminometric measurements, as described in more detail in RESULTS. For experiments requiring prolonged luminescence measurement, the luminometer was connected to a computer, and the luminescence signal was recorded and displayed in real time with software provided by the luminometer manufacturer and Microsoft Excel.
Cell viability test. The uptake of ethidium bromide, a nuclei marker, was monitored in selected experiments to evaluate cell viability and the possible contribution of cell lysis to ATP release. Cell monolayers were incubated in the presence of 0.15-0.3 µg/ml of ethidium bromide, and red-stained dead cells were counted by epifluorescence microscopy.
Solutions and chemicals. Physiological NaCl solution contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose, pH 7.4, adjusted with NaOH. For hypotonic cell swelling, tonicity of the NaCl solution was reduced by adding an appropriate volume of distilled water or, in the majority of experiments, a solution that contained 1 mM MgCl2 and 1 mM CaCl2 to preserve the concentration of divalent cations during hypotonic shock. GdCl3 hexahydrate (Sigma) was prepared as a 100 mM stock solution in distilled water.
Statistical analysis. Statistical significance was assessed at P < 0.05 by ANOVA.
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RESULTS |
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Gd3+ directly inhibits luciferase
bioluminescence.
Hypotonicity-induced ATP release experiments are often performed by
adding luciferase reagent directly to the cell's extracellular media
and measuring ATP-dependent bioluminescence in real time. However,
change in salt concentration during hypotonic shock as well as the
addition of putative ATP-release blockers could have a direct effect on
the luciferase bioluminescence reaction. This is illustrated in Fig.
1A, where
we simulated a typical hypotonicity-induced ATP efflux
experiment in the absence of cells. The bioluminescence reaction was
initiated in a test tube containing physiological extracellular saline
with the luciferase reagent, by adding 10 or 100 nM ATP. Approximately
10 min later, when ATP-dependent luciferase bioluminescence reached a
relatively stable level, the NaCl was diluted by 30% with distilled
water containing the luciferase reagent to mimic the hypotonic shock
applied to cells without diluting the luciferase reagent. This resulted
in a steplike enhancement of light output despite concurrent dilution
of ATP. Subsequent application of 50 µM GdCl3 caused
~50% inhibition of bioluminescence. These results demonstrate that
Gd3+ and changes in NaCl concentration have a direct effect
on luciferase bioluminescence and may contribute to an apparent
modulation of ATP efflux observed with cells. It is noteworthy that the
enhancement and inhibition of bioluminescence were smaller when 10 nM
ATP was used instead of 100 nM (Fig. 1A, traces labeled
"low" ATP and "high" ATP, respectively). These effects,
therefore, may go unnoticed in experiments when ATP concentration in
the media is low. The direct inhibitory effect of Gd3+ on
luciferase bioluminescence was dose dependent and, under our experimental conditions, required relatively high concentrations similar to those reported previously to inhibit ATP efflux from cells,
typically 100 µM (Fig. 1B).
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Chelation of Gd3+ by EGTA prevents
inhibition of luciferase in ATP efflux experiments.
Strong binding of Gd3+ by EDTA or EGTA may be used to
remove Gd3+ from aliquots of extracellular media before
determining ATP content by luminometry. This could allow studies of
Gd3+ effects on ATP efflux from cells while avoiding its
inhibitory action on luciferase bioluminescence. Figure
3 shows that incubation of
Gd3+-containing media with 25 mM EGTA plus 5 mM
MgSO4 for 30 min chelated any Gd3+ and almost
completely removed inhibition of bioluminescence. We used this method
in subsequent studies of swelling-induced ATP efflux from cells.
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Reexamination of the effect of Gd3+
on ATP release from hypotonically swollen cells.
Figure 4 shows the
accumulation of ATP in extracellular media bathing NIH/3T3 fibroblasts,
human lung carcinoma A549 cells, and human bronchial epithelial
16HBE14o cells, determined 25 min after hypotonic shock.
Hypotonic shock was applied in the absence or presence of increasing
concentrations of Gd3+, from 0 to 500 µM. After
Gd3+ was chelated with EGTA plus MgSO4, ATP
content in aliquots of extracellular media was determined by
luminometry. Figure 4A demonstrates that in response to 50%
hypotonic shock, the fibroblasts released approximately fivefold more
ATP in the presence of 10-200 µM Gd3+ compared with
the hypotonic shock applied in the absence of Gd3+. At 500 µM Gd3+, the amount of released ATP declined and was
similar to that observed in the absence of Gd3+. Similar
behavior was observed with A549 cells (Fig. 4B). The amount
of ATP released in the presence of 30 µM Gd3+ was
reproducibly, although statistically not significantly, increased and
it was inhibited ~80% by 200 µM Gd3+. For
16HBE14o
cells, slight inhibition of hypotonically
induced ATP release could be noticed already with 30 µM
Gd3+, but statistically significant inhibition required
100 µM (Fig. 4C). Hypotonic shock and Gd3+
did not cause any detectable cell lysis during these experiments, as
evaluated by ethidium bromide uptake and fluorescence microscopy (see
METHODS). Under control conditions, typically 3-10
cells per 1,000 were found to be stained. The number of dead cells, however, did not increase during a 25-min exposure to 50% hypotonic shock.
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DISCUSSION |
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The mechanisms underlying cell swelling-induced ATP release remain poorly defined. In this study, we have tested the hypothesis that Gd3+-inhibitable channels are involved by evaluating the direct effect of Gd3+ on luciferase bioluminescence and on ATP efflux from cells. We also have taken into account the tight binding of Gd3+ to ATP and to other anions, such as EDTA, that are often present in luciferase reagent.
In cell-free experiments, we found that the luciferase bioluminescent reaction is directly blocked by <10 µM Gd3+ and enhanced in hypotonic solution. Reduction of the luciferase assay sensitivity by Gd3+ and its increase in hypotonic solution also were noticed in a recent study on MS ATP release from Xenopus oocytes (23). Block of luciferase bioluminescence by Gd3+ may be responsible, at least in part, for the reported inhibition of ATP efflux from cells. Lanthanides have been previously reported to inhibit several enzymes, including proteinase, phosphoglycerate kinase, hexokinase, pyruvate kinase, calcium ATPase, and malic enzyme (14, 15, 33). It has been suggested that the mechanism of inhibition of several kinases involves competitive replacement of Mg2+ by lanthanides in the biologically active Mg-ATP complex (14, 15). A similar mechanism may be responsible for the inhibition of luciferase bioluminescence observed in our study. Negatively charged phosphates on ATP bind more tightly to Gd3+ than to Mg2+ or Ca2+. The logarithm of the association constant (pKa) for the ATP-metal ion complex is 7.1 for Gd3+ at pH 8 (14), compared with 5.8 for Mg2+ and 3.8 for Ca2+ (24). This favors competitive displacement of Mg2+ by Gd3+ in the Mg-ATP complex. Inhibition of luciferase activity by the formation of biologically inactive Gd-ATP complex rather than direct binding of Gd3+ to the luciferase enzyme agrees with our observation that after inhibition of bioluminescence by Gd3+, the addition of fresh Mg-ATP to the reaction mixture produced a strong luminescence signal (data not shown).
Inhibition of bioluminescence required high concentrations of Gd3+ (100-200 µM) when the luciferase reagent contained EDTA. However, when a nominally EDTA-free luciferase reagent was used, we observed a 50% block of bioluminescence by only 10-17 µM Gd3+ (Fig. 2, A and B, respectively). This is because Gd3+ strongly binds to EDTA and EGTA, with affinities even higher than for ATP (pKa of 17.4 and 17.5, respectively) (24). The presence of these molecules or other anions of high affinity for Gd3+ in the luciferase reaction solution will affect the concentration of free Gd3+ available to form the Gd-ATP complex and shift the threshold of inhibition to apparently higher GdCl3 concentrations. This is demonstrated in Fig. 2B where the addition of 250 µM EDTA shifted the dose-inhibition curve to the right, a result consistent with ~1:1 binding of EDTA and Gd3+. Chelation of Gd3+ may explain why inhibition of luciferase bioluminescence was not detected in cell-free tests in some studies. Furthermore, because GdCl3 is typically >1,000-fold in excess of ATP (10 µM vs. 10 nM), it could be estimated that even when 95% of Gd3+ is chelated by anions, essentially 100% of ATP will be bound in a Gd-ATP complex, leading to complete inhibition of the luciferase reaction. This may account for the very steep dose dependence of inhibition seen in Fig. 2B. As a consequence, minor differences in Gd3+ binding capacities of solutions used in experiments with and without cells may lead to entirely different results, depending on whether any Gd3+ was available to form the Gd-ATP complex. In some studies, 200-500 µM Gd3+ was required to produce noticeable inhibition of bioluminescence, while in others it was blocked by 10-30 µM Gd3+, the concentrations typically used to block mechanogated channels and luciferase bioluminescence in our experiments (20, 28-30). These differences may reflect the varying composition of experimental solutions and their different capacity to bind Gd3+. Although we have observed luciferase inhibition by as low as 10 µM GdCl3, the concentration of free Gd3+ in these experiments was certainly lower than that of total added GdCl3 due to binding by other anions present in this luciferase reagent, such as sulfate, Tris, albumin, glycine (see METHODs), and traces of EDTA, which were left from the luciferase purification step (~100 nM according to the manufacturer).
To examine the effect of Gd3+ on ATP efflux from cells while avoiding its inhibitory influence on the luciferase reaction, the samples of extracellular media containing Gd3+ in this study were treated with EGTA plus MgSO4 to chelate any Gd3+ before the luminometry determinations of ATP content. Usually, to properly determine ATP content in extracellular solutions of different compositions, it is sufficient to make luminescence-ATP calibration curves for each experimental solution tested. This approach, however, will not work reliably for solutions containing Gd3+ because it binds directly to ATP and often leads to complete loss of the bioluminescence signal. This is further complicated by the fact that inhibition is time dependent (Fig. 1C). This time dependence may be due to slow equilibration/redistribution of Gd3+ between ATP and other Gd-binding anions such as phosphate, sulfate, carbonate, or EDTA, often present in the reaction solution.
The present study revealed that the effect of Gd3+ on cell
swelling-induced ATP release was cell-type specific and showed biphasic dependence for some cells. ATP released by fibroblasts was enhanced by
low concentrations of Gd3+ but was inhibited by higher
concentrations, although differently for different cell types. Our
results vary from those of earlier studies, which did not detect
stimulation of ATP release, but reported 50-70% inhibition by
~200 µM Gd3+, regardless of the cell type tested,
including fibroblasts, human bronchial epithelial
16HBE14o cells, rat hepatocytes, and cholangiocytes
(28, 30). We believe that these differences could be
attributed to direct inhibition of luciferase bioluminescence combined
with partial chelation of the Gd3+ added in those studies.
One possible mechanism of the enhanced ATP accumulation that we observed in the presence of Gd3+ could be inhibition of ATP hydrolysis by ectonucleotidases. This mechanism, however, is unlikely to contribute significantly, since ATP hydrolysis by cell ectonucleotidases is relatively slow at the ATP concentrations observed in our experiments (16). Thus an increased rate of ATP release in the presence of Gd3+ is likely responsible for the enhanced ATP accumulation. This phenomenon may be related to the reported fusogenic effects of low concentrations of lanthanides (2), which could facilitate fusion of ATP-containing vesicles with the plasma membrane. Such a mechanism would, however, require extracellularly applied Gd3+ to have access to the cytosolic side of the membrane. Although cell membranes are considered to be impermeable to lanthanides (14), a recent study suggests that La3+, and to some extent Gd3+, could be rapidly taken up into the cytosol of Chinese hamster ovary cells (17).
ATP release is clearly inhibited at higher Gd3+ concentrations of 200-500 µM. Although 10 µM Gd3+ is often sufficient to block mechanogated channels (19), the effects of higher Gd3+ concentrations may be explained in terms of Gd3+ interaction with the outer surface of cell membranes. Lanthanides have a high affinity for cellular membrane phospholipids, with Kd values in the micromolar range (14). Binding of lanthanides to the outer surface of cell membranes not only decreases its negative surface charge but also produces structural changes in the membrane/water interface, lipid packing, and phase equilibrium (13). Membrane stiffening caused by Gd3+ could decrease a cell's osmotic sensitivity. For example, lanthanides have been reported to decrease osmotic fragility of erythrocytes, thus protecting them from hypotonic hemolysis (14).
The present data do not entirely exclude the possibility that
stretch-activated channels may contribute to MS ATP release from some
cell types. Although Gd3+ and other lanthanides were often
found to have biphasic stimulator/inhibitory effects on exocytotic
release of various neurotransmitters (1, 14), it should be
noted that small stimulation of MS channel activity by low
concentrations and inhibition by higher concentrations of
Gd3+ were reported in Escherichia coli
protoplasts (11). However, in Xenopus oocytes,
which also possess MS and Gd3+-sensitive channels,
electrophysiological studies have demonstrated that mechanical stimuli
much stronger than that required to cause ATP release failed to
activate an increase in membrane conductance, ruling out the
involvement of such channels in these cells (23, 27, 34).
Further investigations are required to verify whether similar responses
could be seen in cell lines tested in this study. Besides
Gd3+, certain anion channel blockers, such as
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and DIDS, were also
reported to inhibit ATP release (6, 20, 26, 29). However,
our preliminary experiments showed that 100 µM NPPB caused ~20%
inhibition of the luciferase bioluminescence (data not shown).
Furthermore, DIDS was also recently reported to directly interfere with
luciferase activity (3). It may be desirable, therefore,
to systematically reexamine the effect of various Cl
channel blockers on luciferase bioluminescence before any firm conclusions can be drawn regarding their block of ATP release.
In summary, our data showing cell-specific and, for some cells, biphasic effects of Gd3+ on swelling-induced ATP release do not support the view that channels directly inhibitable by Gd3+ are involved. Rather, they are consistent with indirect effects of Gd3+ on lipid membranes. Therefore, other mechanisms should also be considered, such as the recently proposed exocytotic release due to fusion of ATP-enriched vesicles involved in transporting proteins from the Golgi complex to the cell surface (23).
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ACKNOWLEDGEMENTS |
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The authors thank Drs. J. Hanrahan, A. Guyot, Y. Berthiaume, A. Dagenais, and A. Kubalski for comments on this manuscript.
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FOOTNOTES |
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This study was supported by the Canadian Cystic Fibrosis Foundation (CCFF) and funds from the Canada Foundation for Innovation.
R. Grygorczyk is a CCFF Scholar. F. Boudreault is the recipient of a CCFF studentship.
Address for reprint requests and other correspondence: R. Grygorczyk, Centre hospitalier de l'Université de Montréal - Hôtel-Dieu, 3850 Saint-Urbain, Montréal, Québec, Canada H2W 1T8 (E-mail: ryszard.grygorczyk{at}umontreal.ca).
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. Section 1734 solely to indicate this fact.
First published September 5, 2001; 10.1152/ajpcell.00317.2001
Received 12 July 2001; accepted in final form 10 September 2001.
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