Evidence for Gd3+ inhibition of membrane ATP permeability and purinergic signaling

Richard M. Roman1, Andrew P. Feranchak1, Amy K. Davison1, Erik M. Schwiebert2, and J. Gregory Fitz1

1 Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262; and 2 Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Extracellular ATP functions as an important autocrine and paracrine signal that modulates a broad range of cell and organ functions through activation of purinergic receptors in the plasma membrane. Because little is known of the cellular mechanisms involved in ATP release, the purpose of these studies was to evaluate the potential role of the lanthanide Gd3+ as an inhibitor of ATP permeability and to assess the physiological implications of impaired purinergic signaling in liver cells. In rat hepatocytes and HTC hepatoma cells, increases in cell volume stimulate ATP release, and the localized increase in extracellular ATP increases membrane Cl- permeability and stimulates cell volume recovery through activation of P2 receptors. In cells in culture, spontaneous ATP release, as measured by a luciferin-luciferase-based assay, was always detectable under control conditions, and extracellular ATP concentrations increased 2- to 14-fold after increases in cell volume. Gd3+ (200 µM) inhibited volume-sensitive ATP release by >90% (P < 0.001), inhibited cell volume recovery from swelling (P < 0.01), and uncoupled cell volume from increases in membrane Cl- permeability (P < 0.01). Moreover, Gd3+ had similar inhibitory effects on ATP release from other liver and epithelial cell models. Together, these findings support an important physiological role for constitutive release of ATP as a signal coordinating cell volume and membrane ion permeability and suggest that Gd3+ might prove to be an effective inhibitor of ATP-permeable channels once they are identified.

cell volume; liver; chloride channel


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ATP IS PRESENT in low concentrations in many interstitial fluids. By binding to purinergic receptors, it functions as an important autocrine and paracrine signal modulating a broad range of cell and organ processes (4). ATP and its metabolites have well-defined roles in such diverse processes as regulation of cardiac automaticity (18), vasomotor tone (3), hepatic glycogenolysis (12), and epithelial secretion (21, 24). Typically, half-maximal responses occur at nucleotide concentrations between 100 nM and 1 µM, and concentrations are maintained within this range by dynamic interactions involving nucleotide release, degradation, and nucleoside reuptake. Although the primary proteins responsible for extracellular nucleotide reception, degradation (nucleotidases), and nucleoside transport have been recently defined, comparatively little is known regarding the cellular mechanisms involved in ATP release (1, 16, 23, 27, 28).

In many epithelial cells, ATP efflux appears to occur through opening of a conductive pathway consistent with an ATP channel. Increases in membrane ATP permeability result in electrodiffusional movement of charged ATP molecules out of the cell, driven by the ATP concentration gradient and the interior-negative membrane potential difference (19, 20). Coexpression of ATP-binding cassette proteins, including cystic fibrosis transmembrane conductance regulator and multidrug resistance P glycoproteins, increases ATP permeability in some models. However, ATP-binding cassette proteins themselves do not appear to conduct ATP, and the molecular identity of the channel(s) involved is not known (9, 19, 20, 30).

Purinergic signaling pathways modulate many fundamental functions of the liver, including glycogenolysis, cell volume, and bile formation. ATP is present in physiological concentrations in the vascular effluent (hepatic vein) of perfused organs and in mammalian bile (6, 17), and purinergic receptors are expressed by multiple liver cell types (13, 17, 21). In isolated hepatocytes, the rate of ATP release is enhanced by mechanical stimuli and by changes in cell volume, increasing local concentrations to a degree sufficient to signal to neighboring cells (7, 20, 22). However, definition of the mechanisms involved has been difficult in part because of the lack of specific reagents that modulate ATP release.

Recent studies of airway epithelial cells using a luciferin-luciferase assay to measure ATP demonstrate that addition of GdCl3 to the extracellular bath causes a rapid decrease in photon release (25). Because of the prominent role of ATP as a signal modulating liver function and membrane transport, the purpose of these studies was to evaluate the potential role of Gd3+ as an inhibitor of ATP release and to assess the short-term physiological implications of impaired purinergic signaling. The experimental strategy utilized HTC rat hepatoma cells, in which increases in cell volume lead to an adaptive response that involves 1) release of ATP through a conductive pathway, 2) stimulation of P2 receptors by the localized increase in ATP, and 3) solute efflux through opening of membrane K+ and Cl- channels (29). This regulatory cycle plays a key role in recovery of cell volume toward basal values. Accordingly, if Gd3+ functions as an inhibitor of an ATP permeability pathway, then it would be expected to block volume-sensitive ATP release and impair regulatory volume decrease.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and isolation. HTC rat hepatoma cells were used as the primary model for investigation of ATP release. Previous findings indicate that these cells undergo robust regulatory volume decrease in response to cell swelling induced by hypotonic medium or uptake of alanine, express purinergic receptors and volume-sensitive Cl- channels analogous to those in primary rat hepatocytes, and exhibit mechanisms for conductive release of ATP (7, 20, 29). Cells were maintained in culture at 37°C in 5% CO2 in HCO-3-containing modified MEM medium as previously described (7).

Parallel studies were also performed using rat hepatocytes isolated by collagenase perfusion and maintained in short- term (<12 h) culture as previously described (15). Although these reliably exhibit ATP release, the approach to isolation and culture was found to produce significant day-to-day variability in constitutive levels of release. Because evaluation of Gd3+ effects required a reproducible model, the principal regulatory paradigms were confirmed in both primary hepatocytes and HTC cells and then explored in detail in HTC cells. In addition, the effects of Gd3+ on basal and volume-sensitive ATP release were studied in biliary epithelial cells [human Mz-ChA-1 cells and normal rat cholangiocytes (NRC)] and human NIH/3T3 fibroblasts.

Solutions. The standard extracellular solution used for all patch-clamp and cell volume studies contained (in mM) 140 NaCl, 4 KCl, 1 KH2PO4, 2 MgCl2, 1 CaCl2, 5 glucose, and 10 HEPES/NaOH (pH 7.4). The osmolarity measured by vapor pressure osmometer averaged ~295 mosmol. In selected studies, osmolality was decreased 10-40% by lowering bath NaCl or by addition of defined amounts of water, depending on the experimental requirements. Exposure to hypotonic buffer did not affect cell viability (propidium iodide staining, data not shown).

ATP bioluminescence assay. ATP release into media was detected by means of a bioluminescence assay (25) as previously described (7). Cells were grown in a 35-mm dish, washed twice with PBS, and incubated with 600 µl OptiMEM-I reduced serum medium plus 2 mg/ml luciferase-luciferin reagent (lyophilized reagent; Calbiochem, La Jolla, CA). NRCs were grown on collagen-coated semipermeable supports (Millicell HA, 12 mm diameter, 1.13 cm2 surface area; Millipore/Fisher, Bedford, MA) and were studied when transepithelial resistance exceeded 1,000 Omega  · cm2 (EVOHM; World Precision Instruments, Sarasota, FL). Semipermeable supports were placed on 35-mm culture dishes in a 200-µl volume of medium to bathe the basolateral side, and 200 µl of medium was then added to the apical chamber. ATP efflux from apical or basolateral NRC membranes was determined selectively by adding luciferin-luciferase reagent to medium on one side of the monolayer only (the side in which ATP is detected), and medium without reagent was added to the other side. Dishes were placed on a platform, lowered directly into the recording chamber of a Turner model TD20/20 luminometer, and studied immediately in real time. Because background luminescence (cells and medium without luciferase-luciferin reagent) is <0.05 arbitrary light unit (ALU), ATP released from cells into the media catalyzes the luciferase-luciferin reaction. Bioluminescence was measured in continuous 15-s photon-collection intervals. To induce cell volume increases, the extracellular buffer was diluted 20-40% by adding water. In control studies performed in parallel, an equal volume of isotonic buffer was added to assess possible ATP release due to mechanical stimulation (9). All solutions added to dishes (media, water, reagents) contained luciferin-luciferase so that the reagent would not be diluted. In both cell models, the small increases in bioluminescence associated with isotonic additions were <20% of those associated with hypotonic additions.

Bioluminescence assay standardization. To approximate ATP released from cells, standard curves of ATP at known concentrations were performed by adding dilutions of an ATP stock (freshly made at the time of measurements; Calbiochem) to OptiMEM-I medium containing 2 mg/ml luciferin-luciferase reagent. In addition, control studies were performed in the absence of cells to test the effects of 1) 20-40% dilution of medium with water, 2) addition of isotonic media, 3) addition of Gd3+ (200 µM), and 4) addition of flufenamic acid (50 µM) on luminescence derived from standard concentrations of ATP to rule out possible nonspecific effects of these maneuvers on the properties of the assay.

Cell volume. Changes in cell volume were measured electronically using a Coulter multisizer (Accucomp 1.19, Coulter Electronics, Hialeah, FL) with an aperture of 100 µm as previously described (20). Measurements of ~20,000 cells in suspension at specified time points after exposure to isotonic or hypotonic buffer were compared with basal values (time 0). Hypotonic buffer contained less NaCl to decrease osmolarity ~30%. Changes in values are expressed as relative volume normalized to the basal period, calculated by dividing experimental values by control values measured in the same cells in standard isotonic buffer.

Volume-sensitive membrane currents. Membrane currents were measured using whole cell patch-clamp techniques as previously described (8, 10). HTC cells were studied at room temperature (22-25°C) 12-24 h after plating. Coverslips were placed in a chamber (volume ~400 µl) and perfused at 2 ml/min with the standard extracellular solution (described in Solutions). Individual cells were viewed through an inverted phase contrast microscope using Hoffman optics at a magnification of ×600 (Olympus IMT-2), and recordings were made using patch pipettes with resistances of 3-6 MOmega and an Axopatch 1C or 1D amplifier (Axon Instruments, Foster City, CA). Currents were filtered at 2-kHz bandwidth using a four-pole low-pass Butterworth filter and were simultaneously recorded on a Gould 2400 chart recorder (Gould, Cleveland, OH) and digitized (5 kHz) for storage on a computer. Currents were analyzed using pCLAMP 6.0 (Axon Instruments). For all studies, the standard pipette (intracellular) solution (pH 7.3) contained (in mM) 130 KCl, 10 NaCl, 2 MgCl2, 10 HEPES/KOH, and free Ca2+ adjusted to 100 nM (0.5 CaCl2, 1 EGTA). Pipette voltages are referred to the bath in which Vp corresponds to the membrane potential; upward and downward deflections of the current trace represent outward and inward membrane current, respectively. Changes in membrane ion permeability were assessed at a test potential of -80 mV (EK) to minimize any contribution of K+ currents, and values were reported as current density (pA/pF) to normalize for differences in cell size as described (20).

The effects of potential blockers of nonselective cation channels were assessed using whole cell techniques and in excised membrane patches as previously described (14). Nonselective cation channels are abundant in liver cells, have nearly equal permeability to Na+ and K+, and are opened by increases in cytosolic Ca2+ concentration ([Ca2+]) (half-maximal effects ~660 nM). The standard NaCl solution was used in both the bath and pipette, except that the [Ca2+] in the solution bathing the cytosolic surface of the patch was lowered to 1 µM, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (10 µM) and CsCl (2 mM) were added to the pipette solution to minimize opening of Cl- and K+ channels, respectively. Under these conditions, nonselective cation channels open immediately after patch excision and are readily identified by a linear conductance of ~28 pS and reversal near 0 mV (14). Because all patches contained multiple channels, mean open probability was estimated using all-points amplitude histograms (pCLAMP 6.0). Briefly, in a patch with N active channels, each level representing the simultaneous opening of n channels was fitted with a Gaussian curve. The probability of n channels being open simultaneously (Pn) was calculated from the area under the peak corresponding to the level n divided by the total area (14).

Statistics. Results are presented as means ± SE, with n representing the number of cells for patch-clamp studies and the number of culture plates or repetitions for other assays. Student's paired or unpaired t-test was used to assess statistical significance as indicated, and P values <0.05 were considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Gd3+ inhibits volume-sensitive ATP release. Cells in subconfluent culture were washed with standard isotonic buffer, covered with isotonic OptiMEM-I buffer (volume 600 µl), and then allowed to equilibrate in the luminometer for ~10 min. Under these conditions, luminescence achieves a stable basal value that is proportional to the amount of ATP released into the extracellular buffer (7, 25). There was significant constitutive release of ATP in all plates tested, and luminescence returned to zero after addition of apyrase (2 U/ml) to hydrolyze extracellular ATP (Fig. 1). Mechanical stimulation caused by addition of 200 µl isotonic buffer (total volume 800 µl) and gentle swirling of the culture dish to ensure mixing caused a small but consistent increase in luminescence that did not reach statistical significance. In contrast, in the same cells, subsequent addition of 200 µl distilled water (total volume 1,000 µl, 20% decrease in osmolarity) in the same manner to increase cell volume increased luminescence ~ 2- to 14-fold, depending on the cell type studied. The addition of Gd3+ (final concentration 200 µM) rapidly and significantly inhibited this response.


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Fig. 1.   Effect of cell volume and Gd3+ on ATP bioluminescence. A: HTC cells were incubated in isotonic OptiMEM-I medium containing luciferin-luciferase for ~10 min. Under these conditions, bioluminescence achieves a stable basal value that is proportional to amount of ATP released into extracellular medium. Mechanical stimulation caused by addition of 200 µl isotonic media (Iso) and gentle swirling of culture dish caused a small but consistent increase in bioluminescence (all studies). In contrast, in same cells, subsequent addition of 200 µl distilled water [Hypotonic (Hypo), 20% decrease in osmolarity] in same manner to increase cell volume increased bioluminescence in every study. In contrast, addition of a similar volume of media at same time point instead of water had little effect (Isotonic + Gd3+). Addition of Gd3+ (final concentration 200 µM) rapidly and significantly inhibited both response to hypotonic challenge (Hypotonic + Gd3+) and basal luminescence (Isotonic + Gd3+). Figures shown represent results from single study days, in which each point represents >= 5 studies. B: percent change in bioluminescence for each condition compared with unperturbed controls (0) at 8 min are shown. Bioluminescence for cells exposed to repeated isotonic additions did not significantly vary from controls (Iso, n = 8), but Gd3+ inhibited basal ATP release ~63% (Iso Gd3+, n = 7). An ~3-fold increase in bioluminescence observed during 20% medium dilution (Hypo, n = 11) was not affected by addition of flufenamic acid (50 µM, Hypo FFA, n = 6). However, exposure to Gd3+ (200 µM, Hypo Gd3+, n = 12) decreased bioluminescence ~90%. * P < 0.01.

In the example in Fig. 1, the relative responses of HTC cells to isotonic and hypotonic exposures are shown. Repeated addition of isotonic buffer had little effect on ATP release, but buffer dilution (hypotonic exposure to increase cell volume) caused an approximately threefold increase in luminescence. These effects of cell volume were reversible in that return of swollen cells to isotonic media causes a decrease of luminescence toward basal values (25). In the presence of hypotonic buffer, subsequent exposure to Gd3+ caused a rapid decrease in luminescence. From standard curves, the estimated peak amount of ATP in the extracellular buffer is ~60 nM (Fig. 2A). Together, these findings confirm previous evidence for volume-sensitive ATP release in HTC cells and suggest that cellular ATP release is inhibited by Gd3+.


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Fig. 2.   Assay properties in cell-free conditions. Sensitivity of bioluminescence assay to ATP concentrations and salt concentrations was measured under cell-free conditions as described in METHODS. A: with use of standard solutions, bioluminescence increased in a linear manner (r2 = 0.995) with increasing concentrations of ATP; n = 7 for each concentration. B: At a fixed ATP concentration of 125 nM, addition of isotonic media (Iso) and lowering NaCl concentrations by 20% or 40% had no consistent effect on bioluminescence compared with controls (Ctl). Similarly, neither Gd3+ (200 µM, Gd3+) nor flufenamic acid (50 µM, FFA) altered bioluminescence; n = 8 for each experiment.

Gd3+ effects on ATP in solution. The inhibitory effect of Gd3+ on bioluminescence could reflect direct binding of Gd3+ to ATP4- or MgATP2- in solution, effects of changing salt concentrations on binding parameters, or other nonspecific chemical effects. To assess these possibilities, additional studies were performed in the luminometer under cell-free conditions in which ATP (final concentration 125 nM) was added to medium (Fig. 2B). Addition of Gd3+ (final concentration 200 µM) did not significantly alter bioluminescence (values of 54.1 ± 7.1 to 52.3 ± 6.4 ALU in the absence vs. presence of Gd3+; n = 8, not significant). Similarly, Gd3+ had no apparent effect on the rate of spontaneous ATP hydrolysis, as indicated by the change in bioluminescence over time. Finally, addition of isotonic media or medium dilution by 20% or 40% in the absence of cells had no consistent effect on bioluminescence (Fig. 2B). Thus the inhibition of ATP-related bioluminescence by Gd3+ is not detectable in cell-free systems.

Effects of cation channel inhibition on ATP release. Gd3+ inhibits the opening of mechanosensitive, nonselective cation channels in some but not all cell types (5). Nonselective cation channels with a conductance of ~28 pS are abundant in HTC and liver cells (14, 15). However, they are not likely to be involved in this process because they are closed under basal conditions (NPo < 0.01) and open in response to cell volume decreases rather than increases (31). To assess more directly whether the inhibition of ATP release by Gd3+ could be due to unanticipated effects on cation channels, the effects of a structurally unrelated cation channel blocker on ATP release were tested. For these studies, putative blockers were assessed in excised membrane patches as described previously (14). In patches containing open channels, flufenamic acid proved to be a potent blocker, with half-maximal inhibition of NPo at concentrations of 5-10 µM and nearly complete inhibition at 50 µM (Fig. 3A). Flufenamic acid also inhibited cation currents when applied from the extracellular aspect of the membrane (Fig. 3B). In the whole cell configuration, Ca2+ mobilization leads to transient activation of inward currents due to opening of the nonselective cation conductance (8). Ionomycin-stimulated currents (1 µM) decreased from -80.1 ± 15.7 pA/pF (-80 mV) under control conditions (n = 3) to -8.1 ± 5.2 pA/pF in the presence of flufenamic acid (50 µM; n = 4, P < 0.01). This inhibitory effect of flufenamic acid is similar to results in cortical collecting duct cells (26).




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Fig. 3.   Effect of flufenamic acid (FFA) on ATP release. To assess whether inhibitory effects of Gd3+ were due to blockade of nonselective cation channels, effects of a structurally unrelated channel blocker were assessed. A: currents carried by nonselective cation channels were measured in an excised membrane patch as described in METHODS at a potential of +40 mV (pipette referenced to bath). Addition of flufenamic acid (bar) was followed by channel closure. B: compared with controls (n = 3), extracellular application of flufenamic acid to bath solution (50 µM, n = 4) markedly inhibited whole cell nonselective cation currents activated by exposure to ionomycin (1 µM). Values represent means ± SE currents measured at -80 mV. * P < 0.01. C: bioluminescence was determined by similar methods to those in Fig. 1A. Same concentration of flufenamic acid had no effect on volume-activated ATP bioluminescence (Hypo + FFA; n = 6). In the same study, Gd3+ (200 µM, Hypo + Gd3+; n = 6) significantly decreased volume-activated bioluminescence to values near isotonic controls (Iso control; n = 5).

If the effects of Gd3+ on ATP release are due to inhibition of nonselective cation channels, then flufenamic acid would also be expected to inhibit ATP release. This proved not to be the case, as illustrated in Fig. 3C. In the study shown, basal and volume-stimulated bioluminescence was unaffected by addition of flufenamic acid (50 µM). Thus the inhibitory effect of Gd3+ on ATP release is not likely to be related to indirect effects on these nonselective cation channels.

Gd3+ inhibition of regulatory volume decrease. During regulatory volume decrease, ATP functions as a signaling molecule coupling increases in HTC cell volume to increases in membrane Cl- permeability (20). To assess whether the inhibition of ATP release is physiologically relevant, the effects of Gd3+ on regulatory volume decrease (using a Coulter counter; Fig. 4) and volume-sensitive Cl- currents (whole cell patch-clamp studies; Fig. 5) were assessed. In each case, cells were exposed to hypotonic buffer (NaCl lowered to decrease osmolarity 15 or 30% for patch-clamp and Coulter studies, respectively) in the absence or presence of Gd3+ (200 µM). In the presence of Gd3+, there was essentially complete inhibition of cell-volume recovery from swelling (Fig. 4A) and activation of volume-sensitive Cl- currents (Fig. 5). For the latter studies, current density was measured at a test potential of -80 mV (EK) to eliminate any contribution of volume-sensitive K+ currents. With the standard intra- and extracellular solutions, basal current density averaged -2.0 ± 0.5 pA/pF. Under control conditions, hypotonic exposure to increase cell volume increased current density -75.4 ± 20.5 pA/pF (n = 7, P < 0.001). In the presence of Gd3+, the response to hypotonic exposure was markedly decreased to -10.0 ± 3.2 pA/pF (n = 12, P < 0.001). Examination of the representative recording in Fig. 5 indicates that the effects of Gd3+ are rapid in onset and fully reversible on return to Gd3+-free solutions. Moreover, exposure to exogenous ATP in the presence of Gd3+ resulted in activation of inward currents (n = 4). Together these findings are consistent with an inhibitory role for Gd3+ in ATP release but not in P2 receptor binding or Cl- channel opening.


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Fig. 4.   Effect of Gd3+ on cell volume regulation. Mean cell volume was measured in HTC cells in suspension, and changes are presented as relative volume by normalizing to basal values in isotonic buffer. A: under control conditions (control, n = 6), exposure to hypotonic buffer (30% decrease in osmolarity) is followed by a rapid initial increase in relative volume followed by gradual recovery toward basal values. In the presence of Gd3+ (Gd3+, 200 µM, n = 6), regulatory volume decrease is inhibited. B: in isotonic buffer, exposure to increasing concentrations of Gd3+ results in a gradual increase in cell volume (n = 5 for each). C: increase in cell volume caused by Gd3+ (Gd3+, 200 µM, n = 6) was completely inhibited by coincubation of cells with the nonhydrolyzable ATP analog ATPgamma S (Gd3+ + ATPgamma S, 20 µM, n = 6).



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Fig. 5.   Effect of Gd3+ on volume-sensitive current activation. In this representative recording, whole cell currents were measured using patch-clamp techniques at a holding potential of -40 mV and test potentials of 0 mV (upward deflection of current) and -80 mV (downward deflection of current). In presence of Gd3+, cell volume increases produced by hypotonic exposure failed to activate currents. In absence of Gd3+, same maneuver was followed by reversible activation of volume-sensitive currents. In same cell, Gd3+ had no effect on activation of currents by P2 receptor agonist ATP (10 µM). In this example, amplitude of volume-sensitive currents exceeded range of chart recorder. Inset: average Cl- current density (measured at -80 mV) measured in isotonic (basal) solution (n = 15), control hypotonic solution (n = 8), and hypotonic solution in presence of Gd3+ (n = 12). Values in the absence vs. presence of Gd3+ are significantly different (P < 0.001).

Concentration-dependent effects of Gd3+ on cell volume. To evaluate the concentration dependence of these effects, cell volume was measured in the presence of increasing concentrations of Gd3+. These studies were performed in cells maintained in standard isotonic buffer (in the absence of hypotonic challenge) and take advantage of the stable release of ATP observed under these conditions. If constitutive release of ATP contributes to maintenance of basal volume, then inhibition of ATP release would be anticipated to increase cell volume. In the absence of Gd3+, the volume of cells suspended in the standard isotonic buffer was stable or decreased slightly over 60 min. Addition of Gd3+ resulted in significant increases in cell volume, and both the rate and magnitude of the response increased in a concentration-dependent manner (Fig. 4). Maximal effects on volume were observed at Gd3+ concentrations >300 µM, and half-maximal effects (measured at 10 min) were observed at Gd3+ concentrations of ~80 µM. Induction of cell swelling by Gd3+ (200 µM) was completely prevented by coincubation with ATPgamma S (20 µM). Thus inhibition of ATP release in isotonic media leads to an increase in cell volume, suggesting that constitutive purinergic signaling contributes to cell volume homeostasis under basal conditions as well as during regulatory volume decrease.

Gd3+ inhibits ATP permeability in multiple epithelial models. Additional studies were performed to establish the role of Gd3+ as a regulator of ATP release in freshly isolated rat hepatocytes and other epithelial cell types (summarized in Table 1). As shown in Fig. 6A, exposure of hepatocytes to hypotonic stress (20% medium dilution) increased ATP permeability ~14-fold; this represents an increase in extracellular ATP from ~50 to >400 nM, values within the physiological range for P2 receptor activation. Subsequent exposure to Gd3+ (200 µM) rapidly decreased bioluminescence; both basal and volume-dependent ATP release were significantly inhibited. In a similar fashion, increases in cell volume enhanced ATP release two- to fourfold from cultured human Mz-ChA-1 cholangiocarcinoma cells, from both apical and basolateral membranes of polarized normal rat cholangiocytes (NRC), and from human NIH/3T3 fibroblasts. In all models, exposure to Gd3+ markedly decreased both volume-sensitive and basal ATP release (Fig. 6B). These findings indicate that results in HTC cells are applicable to primary hepatocytes and suggest that Gd3+-sensitive ATP permeability pathways are broadly expressed in epithelial tissues.

                              
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Table 1.   Gd 3+ decreases epithelial ATP permeability



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Fig. 6.   Gd3+ inhibits ATP release in multiple epithelial cells. A: bioluminescence was used to characterize ATP release from freshly isolated rat hepatocytes. As shown, increasing cell volume by diluting medium by 20% (Hypotonic, n = 6) markedly increased ATP release (~14-fold) compared with isotonic medium addition (Isotonic, n = 5). Addition of Gd3+ (200 µM) rapidly decreased bioluminescence. B: effects of Gd3+ on basal and volume-sensitive ATP release from rat hepatocytes (RH), human cholangiocarcinoma cells (Mz-ChA-1), human fibroblasts (3T3), and apical (NRC-A) and basolateral membranes (NRC-BL) of polarized rat cholangioctyes are summarized. Percent change bioluminescence was calculated as in Fig. 1B. Significant increases in bioluminescence during a 20% hypotonic stress were apparent in all cell types (Hypo Control). In each study, Gd3+ (200 µM) significantly decreased extracellular ATP levels during both isotonic (Iso + Gd3+) and hypotonic (Hypo + Gd3+) stress; n > 6 for each data point.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Extracellular ATP represents an important signaling molecule that modulates a broad range of cell and organ functions. Recently, cell volume has been shown to be a potent stimulus for ATP release in several epithelial cell types (20, 25). The principal findings of these studies of liver cells are that ATP release is detectable under basal conditions, is stimulated by increases in cell volume, and is inhibited by Gd3+. Moreover, inhibition of release has significant physiological effects on membrane transport and cell volume homeostasis. Together, these findings support an important physiological role for constitutive release of ATP and suggest that Gd3+ might prove to be an effective inhibitor of ATP-permeable channels once they are identified.

Previous studies of liver cells and cell lines demonstrate that 5'-ectonucleotidases and nucleoside transporters are abundant, and by degradation of extracellular ATP and recycling of adenosine they function to modulate local concentrations of purinergic agonists. The origin of extracellular ATP, however, has been more difficult to define. Several observations indicate that hepatocytes themselves are capable of regulated ATP release. First, mechanical stimulation of hepatocytes leads to release of a soluble factor that binds to target cells and stimulates mobilization of intracellular Ca2+. The response is inhibited by the ATPase apyrase and by purinergic receptor blockade (22). Second, with the use of whole cell patch-clamp techniques, increases in HTC cell volume result in parallel increases in currents carried by anionic ATP (20, 29). These findings are consistent with opening of volume-sensitive channels permeable to anionic ATP as detected in other cell types (19, 25).

In these studies, ATP release was measured in cells in subconfluent culture by a sensitive and specific luminometric assay (25) and in single cells by monitoring volume-sensitive Cl- currents as a bioassay for purinergic stimulation (20). The whole cell approach takes advantage of the fact that volume-sensitive Cl- currents in HTC cells are strictly dependent on local ATP release. Under control conditions, ATP-dependent bioluminescence was always detectable, and increases in cell volume (stimulated by hypotonic exposure) caused a rapid increase in extracellular ATP and activation of whole cell Cl- currents. These responses were fully reversible by return to isosmotic media and were rapidly inhibited by Gd3+ (200 µM), resulting in marked impairment of cell volume homeostasis.

These findings suggest that Gd3+ is an effective inhibitor of cellular ATP release, but several alternative interpretations were considered. Under cell-free conditions, Gd3+ failed to inhibit bioluminescence in standard ATP-containing solutions and had no effect on spontaneous hydrolysis of ATP. Similarly, salt concentrations in the range tested had no consistent effect on standard ATP solutions, indicating that Gd3+ does not alter the properties of the assay under cell-free conditions. Potential blocking effects of Gd3+ on nonselective cation channels (5) also appear to be unlikely because these channels are essentially always closed under the conditions of these studies and are not known to regulate ATP permeability. Moreover, flufenamic acid in concentrations sufficient to inhibit channel opening had no effect on volume-sensitive ATP release. It is important to emphasize that these studies do not rule out an effect of Gd3+ on other mechanosensitive channels too small to be detected by single-channel recordings or nonspecific effects on cytosolic Ca2+ or other potential mediators.

In the absence of other explanations, the most direct interpretation of these findings is that Gd3+ inhibits ATP release through direct and reversible interactions with an ATP-permeable channel. Although biophysical evidence for such channels continues to accumulate, there is no specific information regarding their molecular identity. Thus more direct evidence for functional interactions between Gd3+ and ATP channels depends on definitive identification of the protein(s) involved.

Assuming that these findings are relevant to cells in vivo, several points merit additional emphasis. First, the effects of Gd3+ on cell volume provide evidence that constitutive release of ATP is physiologically important. In cells in isotonic buffer, exposure to Gd3+ caused a concentration-dependent increase in volume, with half-maximal effects near 80-100 µM. The actual concentration of ATP necessary for signaling remains unclear because the bioluminescence assay detects ATP activity in bulk solution and not at the cell surface. However, that amount of ATP release involves only a fraction of total cellular ATP stores because total cellular ATP content remains constant under the conditions of these studies (unpublished observations). Recently, alternative approaches to assess surface-associated luminescence have been introduced and may permit more accurate quantitation of pericellular nucleotide release and metabolism (2).

Second, the findings in isolated cells provide further support for the notion that ATP serves as a signal coupling changes in cell volume to membrane Cl- permeability. However, the overall significance of these findings to cells in vivo is difficult to assess. More physiological stimuli, including insulin and uptake of amino acids, have been shown to significantly increase liver cell volume (11). Given the wide distribution of purinergic receptors among many different liver cell types, it is attractive to speculate that ATP may serve as a more general signal, coordinating the response to volume changes at the organ level as well.

In aggregate, these findings support a role for Gd3+ as a potent and reversible inhibitor of cellular ATP permeability, uncoupling changes in cell volume from ATP release (Fig. 7). Further definition of the mechanisms involved will require identification of the specific channel proteins that contribute to membrane ATP permeability. Given the diverse roles of extracellular ATP as a signaling molecule, Gd3+ may serve as a useful probe for characterization of the mechanisms involved in regulation of local ATP concentrations and constitutive purinergic signaling pathways.


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Fig. 7.   Proposed model for inhibition of purinergic signaling by Gd3+. In liver epithelial cells, constitutive and volume-sensitive ATP release regulates cell volume homeostasis by enhancing P2 receptor (P2R)-coupled membrane Cl- permeability. ATP is rapidly degraded by membrane ectoATPases. Inhibition of ATP release (Gd3+), dephosphorylation of extracellular ATP (apyrase, hexokinase), and blockade of P2 receptors (suramin, reactive blue 2) disrupt this cascade, decreasing Cl- efflux, which leads to an increase in cell volume and impaired recovery from swelling.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. M. Roman, Campus Box B158, Rm. 6412, Univ. of Colorado Health Sciences Center, Denver CO 80262 (E-mail: rick.roman{at}uchsc.edu).

Received 25 January 1999; accepted in final form 13 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arias, I. M., M. Che, Z. Gatmaitan, C. Leveille, T. Nishida, and M. St. Pierre. The biology of the bile canaliculus. Hepatology 17: 318-329, 1993[Medline].

2.   Beigi, R., E. Kobatake, M. Aizawa, and G. R. Dubyak. Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase. Am. J. Physiol. 276 (Cell Physiol. 45): C267-C278, 1999[Abstract/Free Full Text].

3.   Burnstock, G. Vascular control by purines with emphasis on the coronary system. Eur. Heart J. 10: 15-21, 1989[Medline].

4.   Burnstock, G. P2 purinoceptors: historical perspective and classification. In: P2 Purinoceptors: Localization, Function and Transduction Mechanisms, edited by D. J. Chadwick, and J. A. Goode. Chichester, UK: John Wiley & Sons, 1996, p. 1-34.

5.   Caldwell, R. A., H. F. Clemo, and C. M. Baumgarten. Using gadolinium to identify stretch-activated channels: technical considerations. Am. J. Physiol. 275 (Cell Physiol. 44): C619-C621, 1998[Abstract/Free Full Text].

6.   Chari, R. S., S. M. Schutz, J. A. Haebig, G. H. Shimokura, P. B. Cotton, J. G. Fitz, and W. C. Meyers. Adenosine nucleotides in bile. Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33): G246-G252, 1996[Abstract/Free Full Text].

7.   Feranchak, A. P., R. M. Roman, E. M. Schwiebert, and J. G. Fitz. Phosphatidyl inositol 3-kinase represents a novel signal regulating cell volume through effects on ATP release. J. Biol. Chem. 273: 14906-14911, 1998[Abstract/Free Full Text].

8.   Fitz, J. G., and A. Sostman. Nucleotide receptors activate cation, potassium, and chloride currents in a liver cell line. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G544-G553, 1994[Abstract/Free Full Text].

9.   Grygorczyk, R., and J. W. Hanrahan. CFTR-independent ATP release from epithelial cells triggered by mechanical stimuli. Am. J. Physiol. 272 (Cell Physiol. 41): C1058-C1066, 1997[Abstract/Free Full Text].

10.   Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. Improved patch clamp techniques for high resolution recording from cells and cell-free membrane patches. Pflügers Arch. 391: 85-100, 1981[Medline].

11.   Haussinger, D. Regulation and functional significance of liver cell volume. In: Progress in Liver Disease, edited by J. L. Boyer, and R. K. Ockner. Philadelphia, PA: W. B. Saunders, 1996, p. 29-53.

12.   Keppens, S., and H. De Wulf. Characterization of the biological effects of 2-methylthio-ATP on rat hepatocytes: clear-cut differences with ATP. Br. J. Pharmacol. 104: 301-304, 1991[Abstract].

13.   Keppens, S., A. Vandekerchove, H. Moshage, S. H. Yap, R. Aerts, and H. De Wulf. Regulation of glycogen phosphorylase activity in isolated human hepatocytes. Hepatology 17: 610-614, 1993[Medline].

14.   Lidofsky, S. D., A. Sostman, and J. G. Fitz. Regulation of cation-selective channels in liver cells. J. Membr. Biol. 157: 231-236, 1997[Medline].

15.   Lidofsky, S. D., M. H. Xie, A. Sostman, B. F. Scharschmidt, and J. G. Fitz. Vasopressin increases cytosolic sodium concentration in hepatocytes and activates calcium influx through cation-selective channels. J. Biol. Chem. 268: 14632-14636, 1993[Abstract/Free Full Text].

16.   Misumi, Y., S. Ogata, S. Hirose, and Y. Ikehara. Primary structure of rat liver 5'-nucleotidase deduced from the cDNA. J. Biol. Chem. 265: 2178-2183, 1990[Abstract/Free Full Text].

17.   Nukina, S., T. Fusaoka, and R. G. Thurman. Gylcogenolytic effect of adenosine involves ATP from hepatocytes and eicosanoids from Kupffer cells. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G99-G105, 1994[Abstract/Free Full Text].

18.   Olah, M. E., and G. L. Stiles. Adenosine receptor subtypes: characterization and therapeutic regulation. Annu. Rev. Pharmacol. Toxicol. 35: 581-606, 1995[Medline].

19.   Pasyk, E. A., and J. K. Foskett. Cystic fibrosis transmembrane conductance regulator-associated ATP and adenosine 3'-phosphate 5'-phosphosulfate channels in endoplasmic reticulum and plasma membranes. J. Biol. Chem. 272: 7746-7751, 1997[Abstract/Free Full Text].

20.   Roman, R. M., Y. Wang, S. D. Lidofsky, A. P. Feranchak, N. Lomri, B. F. Scharschmidt, and J. G. Fitz. Hepatocellular ATP-binding cassette protein expression enhances ATP release and autocrine regulation of cell volume. J. Biol. Chem. 272: 21970-21976, 1997[Abstract/Free Full Text].

21.   Schlenker, T., J. M.-J. Romac, A. Sharara, R. M. Roman, S. Kim, N. LaRusso, R. Liddle, and J. G. Fitz. Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G1108-G1117, 1997[Abstract/Free Full Text].

22.   Schlosser, S. F., A. D. Burgstahler, and M. H. Nathanson. Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides. Proc. Natl. Acad. Sci. USA 93: 9948-9953, 1996[Abstract/Free Full Text].

23.   Smith, T. M., and T. L. Kirley. Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the ecto-ATPase and CD39 ecto-apyrases. Biochim. Biophys. Acta 1386: 65-78, 1998[Medline].

24.   Stutts, M. J., E. R. Lazarowski, A. M. Paradiso, and R. C. Boucher. Activation of CFTR Cl- conductance in polarized T84 cells by luminal extracellular ATP. Am. J. Physiol. 268 (Cell Physiol. 37): C425-C433, 1995[Abstract/Free Full Text].

25.   Taylor, A. L., B. A. Kudlow, K. L. Marrs, D. Gruenert, W. B. Guggino, and E. M. Schwiebert. Bioluminescence detection of ATP release mechanisms in epithelia. Am. J. Physiol. 275 (Cell Physiol. 44): C1391-C1406, 1998[Abstract/Free Full Text].

26.   Volk, T., E. Fromter, and C. Korbmacher. Hypertonicity activates nonselective cation channels in mouse cortical collecting duct cells. Proc. Natl. Acad. Sci. USA 92: 8478-8482, 1995[Abstract].

27.   Wang, J., M. E. Schaner, S. Thomassen, S.-F. Su, M. Piquette-Miller, and K. M. Giacomini. Functional and molecular characteristics of Na+-dependent nucleoside transporters. Pharmacol. Res. 14: 1524-1532, 1997.

28.   Wang, T. F., P. A. Rosenberg, and G. Guidotti. Characterization of a brain ecto-apyrase: evidence for only one ecto-apyrase (CD39) gene. Mol. Brain Res. 47: 295-302, 1997[Medline].

29.   Wang, Y., R. M. Roman, S. D. Lidofsky, and J. G. Fitz. Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation. Proc. Natl. Acad. Sci. USA 93: 12020-12025, 1996[Abstract/Free Full Text].

30.   Watt, W. C., E. R. Lazarowski, and R. C. Boucher. Cystic fibrosis transmembrane regulator-independent release of ATP. Its implications for the regulation of P2Y2 receptors in airway epithelia. J. Biol. Chem. 273: 14053-14058, 1998[Abstract/Free Full Text].

31.   Wehner, F., and H. Tinel. Role of Na+ conductance, Na+-H+ exchange and Na+-K+-2Cl- symport in the regulatory volume increase of rat hepatocytes. J. Physiol. (Lond.) 506: 127-142, 1998[Abstract/Free Full Text].


Am J Physiol Gastroint Liver Physiol 277(6):G1222-G1230
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