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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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).
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 · 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 M 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).
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.
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RESULTS |
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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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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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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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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 ATPS (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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DISCUSSION |
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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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FOOTNOTES |
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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.
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