SPECIAL COMMUNICATION
Cell swelling-induced ATP release and gadolinium-sensitive channels

Francis Boudreault and Ryszard Grygorczyk

Centre hospitalier de l'Université de Montréal and Department of Medicine, Université de Montréal, Montréal, Québec, Canada H2W 1T8


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Gadolinium (Gd3+) inhibits luciferase bioluminescence. A: luciferase bioluminescence is rapidly enhanced by reducing salt concentration in the sample (arrow marked "30% hypo"), while 50 µM Gd3+ causes rapid inhibition by ~50%. In this cell-free experiment, a test tube contained 500 µl of physiological extracellular saline and 0.1 mg/ml of commercially available EDTA-containing luciferase reagent (see METHODS). To reduce salt concentration in the sample, an appropriate volume of distilled water containing the luciferase reagent was added so that only salts and ATP were diluted by 30%. The addition of isotonic saline containing luciferase had little effect on luminescence, demonstrating that enhancement of bioluminescence is not due to added fresh luciferase reagent (arrow marked "iso"). The traces shown here were recorded ~10 min after the addition of 100 nM ATP ("high" ATP) or 10 nM ATP ("low" ATP), when bioluminescence had already declined to ~50% of the initial value and reached a relatively steady level (time 0). The relative light signal from the luminometer was acquired at the 5-Hz sampling rate and was interrupted for ~10 s during each addition (breaks on the time axis). Data are representative of 3 experiments. B: dose-dependent inhibition of bioluminescence observed with EDTA-containing luciferase reagent. The reagent concentration was 1 mg/ml. Luminescence was recorded ~10 min after the addition of 10 nM ATP, as described in A. Gd3+ was added to the test tube (arrow) at final concentrations of 100, 200, or 500 µM. The addition of water served as a control. The figure shows that inhibition of bioluminescence required a relatively high concentration of Gd3+ (>= 100 µM) when an EDTA-containing luciferase reagent was used. Data are representative of 4 experiments. C: inhibition of bioluminescence by Gd3+ was time dependent. Gd3+ was added at different concentrations to 10 nM Mg-ATP samples. At different time points, 50-µl aliquots were taken, and luciferase bioluminescence was measured after mixing with 50 µl of luciferase reagent. The luminescence observed in the absence of Gd3+ was set to 100%. Note the significant time-dependent shift of the dose-inhibition curve. Hypo, hypotonic; iso, isotonic.

The extent of bioluminescence inhibition by Gd3+ may vary with time and, to some extent, it also depends on the experimental protocol used. For example, somewhat different inhibition was observed when Gd3+ was first incubated with luciferase and bioluminescence was recorded upon the subsequent addition of ATP, compared with the protocol in which Gd3+ was preincubated with ATP before addition of the luciferase reagent. This latter case is illustrated in Fig. 1C, where the dose-inhibition curves of luciferase luminescence were determined at different time points after GdCl3 was mixed with ATP. It shows that the GdCl3 concentration required to produce 50% inhibition of bioluminescence was ~300 µM at 0.5 min but decreased significantly to 50 µM at 150 min. Longer incubation of ATP with Gd3+ for up to 24 h did not significantly change this inhibition (not shown). Time-dependent inhibition may be relevant in ATP efflux assays when aliquots of extracellular media are taken at different time points, but their ATP content is measured later, after all the samples are collected.

Several commercially available luciferase reagents contain EDTA. In the experiments shown in Fig. 1, we used glycine-buffered firefly extract with added luciferin (Sigma, catalog no. F3641), which contains ~0.5 mM EDTA when dissolved at a concentration of 1 mg/ml. Because of strong binding of Gd3+ to EDTA (14), the actual concentration of free Gd3+ responsible for the observed inhibition of the luciferase reaction could be significantly lower. Indeed, this was confirmed in the experiment illustrated in Fig. 2. When nominally EDTA-free luciferase-luciferin reagent was used, 10 µM Gd3+ was sufficient to inhibit luciferase activity by >50% (Fig. 2A). Addition of 250 µM EDTA abolished the inhibition by 10 µM Gd3+ (Fig. 2A, trace labeled + EDTA) and shifted the dose-response inhibition curve to the right, consistent with the chelation of Gd3+ by added EDTA (Fig. 2B).


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Fig. 2.   Chelation of Gd3+ by EDTA shifts the luminescence dose-inhibition curve. A: when a nominally EDTA-free luciferin/luciferase mixture was used in an experiment similar to that shown in Fig. 1B, 50% inhibition of bioluminescence was observed with 10 µM Gd3+. This inhibition could be prevented by adding 250 µM EDTA. Data are representative of 3 experiments. B: comparison of bioluminescence dose-inhibition curves obtained in the absence and presence of 250 µM EDTA. The observed shift of the dose-inhibition curve is consistent with ~1:1 binding of Gd3+ to added EDTA. To avoid the time dependence shown in Fig. 1C, the protocol used here was slightly modified. In the experiment in Fig. 1C, Gd3+ and ATP were first mixed together and incubated for different time periods before being added to the test tube containing the luciferase reagent. In this experiment, Gd3+ was preincubated with the luciferase reagent for 5 min, and then peak luminescence was recorded immediately upon the addition of 10 nM ATP. Luminescence was normalized to that observed in the absence of Gd3+.

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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Fig. 3.   Chelation of Gd3+ by EGTA fully restores luciferase bioluminescence. Luminescence was observed with 100 nM Mg-ATP in samples of physiological solution containing Gd3+ at a final concentration of 0, 200, or 500 µM (open bars). Gd3+ was incubated with Mg-ATP for 15 min before peak luminescence was determined using Sigma F3641 luciferase reagent. Note the complete inhibition of luciferase bioluminescence by 500 µM Gd3+. This inhibition could be reversed if the samples were mixed at a 1:1 ratio with a solution containing 50 mM EGTA and 10 mM MgSO4. After an additional 15-min incubation to chelate Gd3+, bioluminescence was almost completely restored to the control level observed with the Gd3+-free, EGTA-treated sample (filled bars). Note that the 2 sets of control samples, one treated with EGTA and the other not treated, were set to 100%, although the EGTA-treated sample showed ~10% reduction in absolute luminescence due to the combined effect of a twofold dilution of ATP, the salt content in the sample, and the presence of EGTA. Data are means ± SD of 6 independent experiments.

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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Fig. 4.   Effect of Gd3+ on ATP accumulation in the extracellular media of hypotonically swollen cells. ATP efflux was stimulated by applying 50% hypotonic shock to NIH/3T3 fibroblasts (A), lung carcinoma A549 cells (B), and bronchial epithelial 16HBE14o- cells (C) in the absence or presence of different GdCl3 concentrations. This was achieved by diluting extracellular salts with hypotonic solution (see METHODS), which contained GdCl3 at final concentrations as indicated. Twenty-five minutes after hypotonic shock, 25-µl aliquots of extracellular media were taken from control (no Gd3+) and Gd3+-treated cells and then mixed with 25 µl of solution containing EGTA + MgSO4 to chelate Gd3+. After a 15-min incubation, 50 µl of the luciferase reagent was added to each sample, and peak luminescence was recorded. ATP that accumulated in the extracellular media was expressed in % of control after subtraction of basal ATP content observed before hypotonic shock. The control values of ATP released by hypotonic shock, i.e., in the absence of Gd3+, were 1.5 ± 0.6, 17 ± 4, and 11 ± 5 pmol/105 cells for 3T3 fibroblasts, A549 cells, and 16HBE14o- cells, respectively. Data are the average ± SE of 6 cell monolayers from 2-3 independent experiments. Data points show statistically significant differences (*) compared with the control (ANOVA and Fisher's protected least significant differences test, P < 0.05). For 3T3 fibroblasts, the accumulated ATP was significantly enhanced by 10-200 µM Gd3+ (P <=  0.01), but it was not different from control for 500 µM Gd3+. For A549 cells, the accumulated ATP was statistically not different from the control for Gd3+ concentrations of up to 100 µM but showed significant inhibition (P < 0.0001) at 200 and 500 µM. For 16HBE14o- cells, all tested concentrations of Gd3+ appear to inhibit ATP accumulation, although the effect was only statistically significant (P < 0.001) at concentrations of 100 µM and higher.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    ACKNOWLEDGEMENTS

The authors thank Drs. J. Hanrahan, A. Guyot, Y. Berthiaume, A. Dagenais, and A. Kubalski for comments on this manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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