Cystic Fibrosis Transmembrane Conductance Regulator
Facilitates ATP Release by Stimulating a Separate ATP Release Channel
for Autocrine Control of Cell Volume Regulation*
Gavin M.
Braunstein
,
Richard M.
Roman§,
John P.
Clancy¶
,
Brian A.
Kudlow
¶,
Amanda L.
Taylor¶**,
Vadim Gh.
Shylonsky
,
Biljana
Jovov
,
Krisztina
Peter**,
Tamas
Jilling
,
Iskander I.
Ismailov
,
Dale J.
Benos
¶,
Lisa M.
Schwiebert
¶**,
J. Greg
Fitz§, and
Erik M.
Schwiebert
¶**§§
From the
Department of Physiology and Biophysics,
** Department of Cell Biology, ¶ Gregory Fleming James Cystic
Fibrosis Research Center,
Department of Pediatrics, University
of Alabama at Birmingham, Birmingham, Alabama 35294-0005, the
§ Division of Hepatology, University of Colorado Health
Sciences Center, Denver, Colorado, 80262, and the

Department of Pediatrics, The Evanston
Hospital, Evanston, Illinois 60201
Received for publication, July 5, 2000, and in revised form, November 9, 2000
 |
ABSTRACT |
These studies provide evidence that cystic
fibrosis transmembrane conductance regulator (CFTR) potentiates
and accelerates regulatory volume decrease (RVD) following hypotonic
challenge by an autocrine mechanism involving ATP release and
signaling. In wild-type CFTR-expressing cells, CFTR augments
constitutive ATP release and enhances ATP release stimulated by
hypotonic challenge. CFTR itself does not appear to conduct ATP.
Instead, ATP is released by a separate channel, whose activity is
potentiated by CFTR. Blockade of ATP release by ion channel blocking
drugs, gadolinium chloride (Gd3+) and
4,4'-diisothiocyanatostilbene-2,2'disulfonic acid (DIDS), attenuated the effects of CFTR on acceleration and potentiation of RVD.
These results support a key role for extracellular ATP and autocrine
and paracrine purinergic signaling in the regulation of membrane ion
permeability and suggest that CFTR potentiates ATP release by
stimulating a separate ATP channel to strengthen autocrine control of
cell volume regulation.
 |
INTRODUCTION |
ATP and its metabolites function as potent autocrine and paracrine
agonists that act within tissues to control cell function through
activation of P2 purinergic receptors (1-3) expressed by all cells and
tissues. Purinergic agonists are essential for many specialized
physiological functions (1-10). In cystic fibrosis (CF),1 ATP and a related
triphosphate nucleotide, UTP, stimulate epithelial chloride
(Cl
) channels alternative to CFTR via purinergic
receptors (11-16). Supraphysiological concentrations of ATP also
stimulate CFTR (17). Metabolites of ATP can also act as
Cl
secretagogues (15, 16, 18). Despite the diverse roles
of purinergic signaling, the cellular mechanisms that govern ATP release are not fully defined. CFTR and related ATP-binding cassette (ABC) transporters such as mdr-1 or P-glycoprotein
have been implicated as facilitators of ATP release in some cell models
(14, 19-24), while other laboratories have failed to show evidence of
CFTR-facilitated ATP conduction or release (25-30).
Release of ATP via a conductive pathway has been implicated as an
essential autocrine regulator of cell volume in rat hepatoma cells (5).
Moreover, ABC transporters have been shown to modulate volume-sensitive
Cl
channels and cell volume (31-34). As such, we tested
the hypotheses that CFTR facilitates ATP release under constitutive and
hypotonic conditions for autocrine control of cell volume regulation.
These hypotheses were also based on the fact that airway surface
liquid on CF epithelia is hypertonic with respect to NaCl (35)
and/or reduced in volume (36) or both (37, 38) when compared with non-CF epithelia. These airway surface liquid composition abnormalities may reflect an inability of CF epithelial cells to sense changes in
external mucosal environment and/or an inability of CF cells to
regulate their own cell volume.
To this end, complimentary observations using a variety of techniques
suggest that expression of CFTR enhances ATP release and modulates the
dynamic relationship between cell volume, purinergic signaling, and
membrane ion permeability. Given the cellular challenges related to
changes in solute transport and maintenance of airway surface liquid,
defective cell volume regulation in CF epithelia may underlie the
pathogenesis of CF in the lung and airways.
 |
MATERIALS AND METHODS |
Cell Culture--
The 16HBE14o
non-CF human
bronchial epithelial cell line, the 9HTEo
non-CF human
tracheal epithelial cell line, and the CFBE41o
CF human
bronchial epithelial cell line (homozygous for the
F508-CFTR mutation) were grown as described previously (39-42). COS-7 cells were
grown in similar medium. Parental CFPAC-1 CF human pancreatic epithelial cells and CFPAC-1 cells complemented stably with the wild-type CFTR gene (CFPAC-PLJ-WT clone 20) were grown in
similar medium without and with G418 (1 mg/ml; Cellgro/Mediatech) (43). 3T3-CFTR fibroblasts expressing wild-type CFTR (3T3-WT-H7), expressing
F508-CFTR (3T3-
F508), or mock-transfected (3T3-Mock) were a generous gift from Michael Welsh (University of Iowa, Iowa City, Iowa,
Howard Hughes Medical Institute) (44) and were grown in a
similar medium except that a Dulbecco's minimal essential medium with
high glucose (Life Technologies, Inc.) was used as the basal medium.
CFT-1 cells were grown in LHC-8 medium (Biofluids; Rockville, MD)
without serum. For consistency, all CFTR cell models were grown on
similar substrates and in similar media.
Transient Transfection of COS-7 Cells with LipofectAMINE
PLUS--
COS-7 cells were seeded to 33% confluence 1 day
prior to transfection with LipofectAMINE PLUSTM reagent
(Life Technologies, Inc.) (45-47). To control for transfection efficiency and to correlate relative CFTR protein expression from culture to culture, triple transfections were performed with different CFTR mammalian expression vectors bearing wild-type or mutant CFTR
together with green fluorescence protein- and
luc-bearing mammalian expression vectors. Green fluorescence
protein expression was assessed in each culture as was
luc protein expression to standardize ATP release assays in
the transient transfection cultures.
Transient Infection of COS-7 Cells with Vaccinia
Virus--
These methods have been published previously (48).
Bioluminescent Detection of Released ATP from Heterologous Cell
Cultures and Epithelial Monolayers--
The specifics of this assay
have been published previously (42). Fibroblasts were grown to
or near confluence on collagen-coated dishes. Epithelial cells were
seeded at high density (at least 105 cells per filter) onto
collagen-coated permeable supports (Millicell).
Analysis of Chloride and ATP Channel Activity in a Panel of CFTR
Protein Preparations in Planar Lipid Bilayers--
These methods have
been published previously (49, 50).
Coulter Counter Channelyzer Analysis of Cell Volume--
Mean
cell volume was measured in cell suspensions by electronic cell
sizing (Coulter Multisizer II, AccuComp software version 1.19, Hialeah,
FL) using an 100-µm aperture. Cells in subconfluent culture were
harvested with minimal trypsin (0.05%), suspended in cell culture
medium, centrifuged for 1 min at 1,000 × g,
resuspended in 3 ml of isotonic buffer, and incubated with gentle
agitation for 30-45 min. Aliquots (~500 µl) of cell suspension
were added to 20 ml of isotonic or hypotonic (40% less NaCl) buffer.
Measurements of 20,000 cells on average at specified time points after
exposure to isotonic or hypotonic buffer were compared with basal
values in isotonic buffer (time 0). Changes in values were expressed as
relative volume normalized to the basal period (5).
SPQ Halide Permeability Assay--
SPQ (Molecular Probes) was
solubilized in cell culture medium at a concentration of 2 mg/ml and
was loaded into cell by an overnight incubation. The protocol was 2 min
in NaI Ringer followed by 2-3 min in NaNO3 Ringer and then
several minutes in NaNO3 Ringer containing forskolin or
diluted by 25% with distilled water. Responses were reversed fully in
NaI Ringer. Specific concerning the SPQ assay and system have been
published previously (48).
Immunoprecipitation of CFTR Protein--
Freshly grown
epithelial cells or fibroblasts (a single 35-mm dish or 12-mm diameter
filter confluent and/or tight to fluid) were lysed for 30 min at
4 °C in a buffer containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, and 1% Nonidet P-40 (pH
7.4) supplemented with 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride. These samples were
immunoprecipitated with anti-CFTR antibody as described previously
(47).
Cellular Cyclic AMP Bioassay--
These methods have been
published previously (48).
Data Analysis and Statistics--
Data values were compiled into
Microsoft Excel spreadsheets in which the mean ± S.E. was
calculated for each time point in each set of experimental time
courses. Data were then plotted in SigmaPlot for Windows using the same
arbitrary light unit values. Statistics were performed using SigmaStat
for Windows; paired Student's t test or unpaired ANOVA with
a Bonferroni ad-hoc test were performed as appropriate. A p
value of <0.05 was considered significant.
 |
RESULTS |
Wild-type (WT) CFTR Potentiates and Accelerates Regulatory Volume
Decrease (RVD)--
To evaluate whether CFTR expression plays a role
in cell volume regulation, cell volume recovery from swelling induced
by hypotonic challenge (e.g. RVD) was assessed in cells
stably expressing wild-type CFTR (WT-CFTR) versus
F508-CFTR (Fig. 1A). In all
cells, hypotonic exposure caused an initial increase in cell volume to a similar degree that peaked at 2 min following challenge (Fig. 1A). However, expression of WT-CFTR accelerated the rate of
RVD 2-fold when compared with fibroblasts devoid of CFTR (not shown) or
expressing the
F508-CFTR mutation (Fig. 1, A and
B). When expressed as percent recovery from peak swelling,
expression of WT-CFTR increased the rate of volume recovery 2-fold
(Fig. 1B). Taken together, these results show that wild-type
CFTR accelerates and potentiates cell volume regulation.

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Fig. 1.
Wild-type, but not
F508-CFTR, accelerates and potentiates RVD
dynamics: dependence of RVD on CFTR, ATP release, and extracellular ATP
signaling. A, raw cell volume data illustrating the
difference in RVD dynamics between wild-type CFTR- and
F508-CFTR-expressing fibroblasts (n = 3 for each
cell type for this representative data set; n = 6 overall). Using a confidence interval of 0.99 for a linear regression,
the slope of the line fit to the cell volume values measured from the
peak of hypotonicity-induced cell swelling to the end of the RVD time
course was measured to estimate the rate (* reflects p < 0.05 significance in slope of RVD dynamics by ANOVA). The slope of
the wild-type CFTR RVD time course was 4.77 × 10 3, while the F508-CFTR RVD slope was
2.93 × 10 3. B, percent RVD
from the peak of swelling (2 min) to the 30-min time point following
RVD is plotted (* reflects p < 0.05 significance by
ANOVA). C, apyrase (1 unit/ml) blocks RVD in wild-type
CFTR-expressing fibroblasts; this effect is rescued fully by ATP S
(25 µM) (n = 3 for each fibroblast
clone). Note different expanded scale on the graph in C
versus A.
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WT-CFTR-facilitated RVD Depends upon Extracellular ATP
Signaling--
To assess whether the effects of CFTR expression on
cell volume regulation are related to release of ATP, the effects of
the ATP scavenger apyrase were determined. In fibroblasts expressing WT-CFTR, depletion of extracellular ATP by apyrase blocked RVD completely (Fig. 1C). Addition of ATP
S, a poorly
hydrolyzable ATP analog resistant to apyrase, reversed this inhibition
(Fig. 1C). These results suggest that extracellular ATP is
required for RVD and for maintenance of cell volume.
WT-CFTR Potentiates ATP Release under Basal Conditions--
To
determine whether these effects of CFTR on volume regulation are
related to ATP release, additional studies were performed using a
luciferin-luciferase assay to quantify ATP release into the medium
(Fig. 2A). Under basal
conditions, WT-CFTR-expressing fibroblasts release 3-fold greater ATP
than mock-transfected fibroblasts and 2-fold greater ATP than
F508-CFTR-expressing fibroblasts (Fig. 2A).
Interestingly, ATP release was always detectable (above background),
even in the absence of CFTR or in the presence of
F508-CFTR,
suggesting that the ATP release mechanism is an entity separate from
the ABC transporter itself.

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Fig. 2.
ABC transporters augment ATP release under
basal or isotonic conditions. A, luminescence values in
arbitrary light units (ALU) from mock or WT-CFTR- or
F508-CFTR-expressing fibroblasts. Data from COS-7 cells lacking or
transiently expressing WT-CFTR or F508-CFTR is provided in the text.
Experimental number (n) is shown in each bar in
A. Data are expressed as mean ± S.E. (* reflects
p < 0.01 value for ATP release significantly
greater than control, ** reflects p < 0.05 value
for ATP release significantly greater than control, ANOVA and a
Bonferroni ad-hoc test). B, immunoprecipitations of WT and
F508-CFTR protein in fibroblasts lacking or stably expressing
wild-type CFTR or F508-CFTR and in COS-7 cells lacking or
transiently expressing wild-type or mutant CFTR proteins
(representative of three independent experiments in each heterologous
system).
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Additional studies were performed to assess the effects of transient
transfection of CFTR on ATP release using COS-7 cells. Mock-transfected
cells had no detectable CFTR. WT-CFTR-transfected cells expressed both
partially (band B) and fully glycosylated (band C) forms of CFTR, while
F508-CFTR-transfected cells had only the partially glycosylated band
B form of CFTR (47, 51). To more carefully monitor the amount of
transduction of a given heterologous cell with CFTR, COS-7 cells were
transfected transiently with two different WT-CFTR constructs or a
F508-CFTR constructs (Fig. 2B). Similar results were also
found in the fibroblast clones stably expressing WT-CFTR and
F508-CFTR (Fig. 2B). As in the fibroblast clones studied
above, wild-type CFTR potentiated ATP release 2.9 ± 0.5-fold when cultures were studied 48 h post-transfection, while
F508-CFTR failed to potentiate ATP release (1.2 ± 0.5-fold; mock-transfected and parental data normalized to 1-fold;
n = 6-9). Taken together, these data show that
wild-type CFTR potentiates ATP release from cells under basal conditions.
WT-CFTR Potentiates and Sensitizes Hypotonicity-induced ATP
Release--
Because WT-CFTR potentiates RVD and ATP release, we
tested the hypothesis that CFTR may also potentiate ATP release
triggered by an increase in cell volume after a hypotonic challenge.
Hypotonic challenge to increase cell volume triggered an immediate and
robust increase in ATP release from non-CF epithelia expressing WT-CFTR (Fig. 3A). As little as 4%
dilution triggered a change in luminescence of
+10.9 ALU from non-CF
epithelia (Fig. 3A). In sharp contrast, 33% dilution was
required to stimulate any ATP release (only a
+0.7 ALU) from
CFBE41o
monolayers. These results were confirmed by
another model of CF. In CFPAC-1 monolayers lacking CFTR, a large
dilution of 24% was again required to elicit a small change (only a
+1.6 ALU) in ATP release (Fig. 3A). When CFPAC-1
monolayers are stably complemented with WT-CFTR, as little as 4%
dilution is enough to trigger ATP release (
+6.8 ALU) (Fig.
3A). When challenged with more robust dilutions of the
medium osmolality, 51% hypotonicity triggered a robust ATP release in
WT-CFTR-expressing non-CF epithelia (
+48.7 ALU) and in
WT-CFTR-complemented CFPAC-1 monolayers (
+33.0 ALU) (Fig.
3A). In contrast, 51% dilution with distilled water only stimulated small increases in luminescence in CFBE41o
monolayers (
+3.0 ALU) and in CFPAC-1 monolayers (
+3.9 ALU)) (Fig.
3A). Taken together, these results show that WT-CFTR
potentiates ATP release into the apical medium and heightens the
sensitivity of the apical membrane of the epithelium to hypotonic
challenge and subsequent increases in cell volume.

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Fig. 3.
CFTR augments the sensitivity to hypotonic
challenge and the magnitude of hypotonicity-induced ATP release in
epithelial cell monolayers across the apical membrane.
A, hypotonicity-induced ATP release into the apical medium
(graphed as luminescence in ALU) for non-CF airway epithelial
(16HBE14o ) monolayers, CF airway epithelial
(CFBE41o ) monolayers, CF pancreatic epithelial (CFPAC-1)
monolayers, and WT-CFTR-complemented CFPAC-1 epithelial monolayers.
Apical-directed ATP release triggered by hypotonicity was
significantly greater in non-CF and WT-CFTR-complemented monolayers
versus CF monolayers expressing the F508-CFTR mutation
(ANOVA, p < 0.05 or lower at all dilutions;
n = 6-8). Data shown for all cells is graphed on the
same scale to show the lack of responsiveness of the CF cell models.
B, immunoprecipitation of WT-CFTR band C protein from
non-CF, but not the CF airway epithelial cell monolayers
(representative of three independent experiments).
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To better define CFTR protein expression in these non-CF and CF airway
epithelial cell lines, immunoprecipitation of CFTR protein was
performed in parallel. Expression of the mature band C form of CFTR
protein was demonstrated in the non-CF 16HBE14o
cell line
with a trace amount of the band B form (Fig. 3B). In sharp
contrast, the CF human airway epithelial cell line
(CFBE41o
) lacked any detectable CFTR protein (Fig.
3B). Characterization of the parental CFPAC-1 cells and the
stably complemented CFPAC-1 clone have been published previously
(43). Thus, these dynamic changes in ATP release correlate with
expression of CFTR protein.
WT-CFTR Potentiates Volume-sensitive ATP Release: Further
Potentiation by Cyclic AMP in WT-CFTR-Expressing
Fibroblasts--
Because the cyclic AMP-dependent protein
kinase signaling cascade stimulates CFTR chloride channel (51) and
because cyclic AMP stimulates ATP release in WT-CFTR-expressing cells
(14, 23), we tested the hypothesis that protein kinase A activity modulates hypotonicity-induced ATP release. A typical time course of
the response to forskolin (2 µM) and to 33% dilution of
the medium osmolality is shown in Fig.
4A. Forskolin stimulated ATP release transiently and only in fibroblasts expressing WT-CFTR (Fig.
4A). Hypotonicity stimulated ATP release in all three
fibroblast clones, but most robustly in WT-CFTR-expressing fibroblasts
(Fig. 4B). More intriguingly, pretreatment with forskolin
potentiated volume-sensitive ATP release, but, again, only in
WT-CFTR-expressing fibroblasts (Fig. 4B), where values
increased 3-fold above those in the absence of cyclic AMP (Fig.
4B). No such enhancement was observed in mock or
F508-CFTR-expressing fibroblasts (Fig. 4B). These results
suggest that cyclic AMP and hypotonicity function in a synergistic
manner to stimulate ATP release, but only in cells expressing
WT-CFTR.

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Fig. 4.
CFTR augments hypotonicity-induced ATP
release in two different heterologous cell culture systems:
hypotonicity-induced ATP release is potentiated by cyclic AMP, but only
in wild-type CFTR-expressing cells. A, typical time
course of forskolin (2 µM)- and hypotonicity-induced ATP
release in wild-type CFTR-expressing fibroblasts versus mock
and F508-expressing controls. Forskolin potentiated ATP release
(p < 0.01 in the first minute following stimulation by
paired Student's t test, n = 6), but only
in wild-type CFTR-expressing cells. B, summary of data shown
in A. Hypotonicity stimulated ATP release that was
significantly elevated throughout stimulation in all clones; forskolin
potentiated that release, but only in wild-type CFTR-expressing cells
(ANOVA, p < 0.01 in the first minute following
stimulation, n = 6).
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CFTR Is Closely Associated with an ATP Release Channel That Appears
Separate from the CFTR Channel Protein--
To further define whether
CFTR conducts ATP or whether CFTR is closely associated with a separate
ATP release channel, protein preparations containing or lacking WT-CFTR
were analyzed for both Cl
and ATP
single
channel conductance in planar lipid bilayers. The protein preparation
used in these bilayer studies represents an epithelial cell protein
extract from membrane vesicles derived from epithelial cells (49, 50).
Consistent with the studies of Kopito and co-workers and of Bear and
colleagues (28, 30), CFTR immunoprecipitated and highly purified from
this epithelial vesicle protein and fused with planar lipid bilayers
formed channels that conducted Cl
in 18 out of 18 preparations (Cl
conductance = 11 ± 2 picosiemens) in which channels were observed. DPC (200 µM) blocked CFTR Cl
channel activity, while
DIDS or gadolinium chloride (200 µM each) had no effect
on CFTR Cl
channel activity (data not shown). Conversely,
an ATP conductance was observed in only 1 out of 18 preparations (ATP
conductance = 22 picosiemens). We hypothesized that this one
example of ATP channel activity may represent the fusion and activity
of a contaminating protein that conducts ATP. In support of this latter
hypothesis, less pure vesicle protein preparations containing CFTR and
other proteins were also examined (Fig.
5A). In this preparation, CFTR Cl
channels, outwardly rectifying Cl
channels (ORCCs), and ATP channels were observed frequently and in
similar incidence (see example in Fig. 5A). In these
recordings, ATP channel activity was recorded first in symmetrical 100 mM NaATP, so as not to have possible contamination by
Cl
channel activity or by trace amounts of
Cl
in the recording chamber. Subsequently, ORCC and CFTR
Cl
channel activity were observed when ATP-containing
solutions were replaced with Cl
-containing solutions
(Fig. 5A). Addition of DIDS (300 µM) inhibited ORCCs, while DPC blocked residual CFTR Cl
channel
activity (Fig. 5A). These results show that two different Cl
conductances and an ATP conductance are present in
this bovine tracheal epithelial vesicle protein material that contains
CFTR. However, because highly purified CFTR does not appear to reliably conduct ATP, this ATP conductance is likely not conferred by CFTR, but
may be conferred by another anion channel protein.

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Fig. 5.
Chloride and ATP conductances in less
purified CFTR-containing and CFTR-immunodepleted protein preparations:
highly purified CFTR cannot conduct ATP. A, lipid
bilayer recording of less purified bovine tracheal epithelial vesicle
protein preparation in which an ATP channel was observed initially in
symmetrical Na2ATP solutions, followed by recording of both
ORCC and then CFTR Cl channel activity (after DIDS was
applied to block the ORCC) in 100 mM CsCl
(n = 5). B, ATP channel activity in a
unpurified protein preparation immunodepleted of CFTR. ATP channel
Po was increased by a pressure gradient, while
GdCl3 decreased Po to virtually 0.00 (n = 6; see text for Po
data).
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We next asked the question: what if CFTR is removed from this material?
Are CFTR Cl
channels and ATP channels lost or can this
activity be dissociated? To answer these questions, CFTR was
immunodepleted out of this material, and this preparation was fused
with planar lipid bilayers. Importantly, an ATP conductance was still
observed, even in the nominal absence of CFTR protein (Fig.
5B). In these experiments, a pressure gradient applied to
the membrane increased the open probability (Po)
of the ATP channel from 0.50 ± 0.10 to 0.80 ± 0.10, while
GdCl3, a mechanosensitive ion channel blocker, decreased markedly the Po of ATP channels to 0.13 ± 0.05 (Fig. 5B; n = 6). DPC and DIDS, broad
specificity Cl
channel blockers, also inhibit this ATP
conductance (data not shown). Taken together, the simplest
interpretation of these data is that an anion channel distinct from
CFTR plays a primary role in conferring the ATP conductance measured in
this assay. However, the ATP conductance appears to be closely
associated with CFTR. Because this channel is inhibited by gadolinium
chloride and DPC and activated by a pressure gradient, this
ATP-permeable channel may belong to the family of mechanosensitive
anion channels. Although these data argue that CFTR itself is not
likely to conduct ATP, the possibility cannot be ruled out that a
regulatory cofactor required to induce CFTR to conduct ATP may be
purified away from CFTR in these experiments.
CFTR Expression, ATP Transport, Extracellular ATP Signaling, and
Cell Volume Regulation Are Linked--
To provide a causal link
between each of the entities described above, we tested the possibility
that a mechanosensitive anion channel may be involved in conductive ATP
transport out of cells and autocrine control of RVD. Focusing again on
WT-CFTR-expressing fibroblasts, Fig. 6
shows the effect of ion channel-blocking drugs on basal and
hypotonicity-induced ATP release and on RVD dynamics. DIDS, a broad
specificity Cl
channel and transporter blocking drug, was
used in lieu of DPC, because DPC has inhibitory effects on the
luciferase detection reagent while DIDS and GdCl3 do not
(see Fig. 6 legend for data). The presence of DIDS blocked fully basal
and hypotonicity-induced ATP release (Fig. 6A, left
panel) and inhibited RVD markedly (Fig. 6A, right
panel). To confirm that DIDS was inhibiting RVD through effects on
ATP release, ATP
S was added in the presence of DIDS. ATP
S rescued
the effects of DIDS fully (Fig. 6A, right panel), consistent with an effect of DIDS as an inhibitor of autocrine ATP
control of cell volume regulation. Similar experiments were performed
with the mechanosensitive ion channel blocker, GdCl3. GdCl3 inhibited basal and hypotonicity-induced ATP release
markedly (Fig. 6B, left panel). GdCl3
also blocked RVD and caused cells to swell markedly (Fig.
6B, right panel). Again, exposure to ATP
S partially reversed the effect on cell volume (Fig. 6B,
right panel).

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Fig. 6.
Ion channel blocking drugs inhibit
hypotonicity-induced ATP release and RVD. WT-CFTR-expressing
fibroblasts were assessed with both assays. A: right panel,
representative ATP release assay under isotonic and hypotonic
conditions; DIDS inhibited both basal and hypotonicity-induced ATP
release fully (representative of six independent experiments).
A: right panel, RVD measurements in the absence
of either drug, in the presence of DIDS, and in the presence of DIDS
plus ATP S (n = 3 each). DIDS blocked RVD; ATP S
rescued DIDS inhibition. B: left panel,
representative ATP release assay under isotonic and hypotonic
conditions; GdCl3 inhibited both basal and
hypotonicity-induced ATP release markedly (representative of nine
independent experiments). B: right panel, RVD
measurements in the absence of either drug, in the presence of
GdCl3, and in the presence of GdCl3 plus
ATP S (n = 3 for each fibroblast clone).
GdCl3 blocked RVD and caused cells to swell to a greater
degree than DIDS; ATP S blocked partially GdCl3-induced
swelling, but failed to fully rescue RVD (n = 3 each).
With respect to effects on luciferase activity, bioluminescence
detection of known amounts of ATP in the presence and absence of
GdCl3 and DIDS (200 µM) was assessed:
10 7 M ATP: control, 189 ± 4 ALU; DIDS, 178 ± 6 ALU; GdCl3, 190 ± 5 ALU;
10 6 M ATP: control, 1378 ± 111 ALU; DIDS, 1466 ± 55; GdCl3, 1257 ± 85 (n = 3 each). In contrast, DPC (200 µM)
inhibited luciferase activity 100-fold (data not shown).
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WT-CFTR and Extracellular ATP Signaling Are Required for
Volume-sensitive Cl
Permeability--
RVD depends upon
activation of a volume-sensitive anion conductance in most cells. To
measure hypotonicity-induced Cl
channel activity in
multiple cells within a culture, SPQ halide fluorophore imaging assays
were performed on cells that lacked or expressed WT or mutant CFTR.
COS-7 cells infected with T7 vaccinia virus controls or virus bearing
WT-CFTR or
F508-CFTR were compared for either forskolin- or
hypotonicity-induced SPQ fluorescence. While T7 controls failed to
respond significantly to forskolin (permeability coefficient or pI = 0.29), a small increase in SPQ fluorescence was observed with a 25%
hypotonic challenge (pI = 0.71; Fig.
7C). Overexpression of
WT-CFTR-expressing cultures responded robustly and reversibly to both
stimuli (Fig. 7A), with permeability coefficients that were
10-fold greater for forskolin (pI = 3.86) and 3.5-fold greater for
hypotonicity (pI = 2.37) versus T7 controls.
F508-CFTR-expressing cultures had an intermediate response to both
stimuli (forskolin pI = 0.47 and hypotonicity pI = 1.33; Fig.
7B), consistent with the modest effects of
F508-CFTR on
ATP release. Because overexpression of CFTR may overestimate CFTR
function, CFT-1 CF epithelial cells homozygous for the
F508-CFTR mutation were transiently transfected with WT-CFTR plasmid constructs (Fig. 7D). Although levels of expression are lower than in
vaccinia-driven systems, WT-CFTR also potentiated hypotonicity-induced
Cl
efflux (pI = 18.77 versus pI of
"mock" control = 7.03; Fig. 7D). In
9HTEo
non-CF airway epithelial cells expressing WT-CFTR,
hypotonicity-induced Cl
efflux was assessed in the
absence and presence of hexokinase or apyrase, two different ATP
scavengers. As with RVD assays shown above, the ATP scavenger blunted
hypotonicity-induced Cl
efflux (hypotonicity pI = 5.29; hypotonicity + hexokinase pI = 0.59; hypotonicity + apyrase
pI =
0.03; Fig. 7E), suggesting that extracellular
autocrine ATP signaling is required for triggering Cl
efflux during RVD. Hypotonicity fails to increase cyclic AMP levels,
suggesting that stimulation of CFTR Cl
channel activity
is not contributing to hypotonicity-induced Cl
efflux
(Fig. 7F). Taken together, these data indicate that both CFTR and extracellular ATP signaling are required for volume-sensitive Cl
efflux during RVD.

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Fig. 7.
Hypotonicity-induced Cl
permeability is augmented by WT-CFTR and attenuated by ATP
scavengers. A-C, comparison of forskolin and 25%
hypotonicity-induced Cl permeability (measured by
fluorescence of the halide fluorophore, SPQ) in COS-7 cells infected
with T7 control virus (C) or vaccinia virus bearing WT-CFTR
(A) or F508-CFTR (B) (number of cells screened
shown in parentheses; result was repeated in three
independent infections; pI = permeability coefficient following
stimulation with forskolin or hypotonicity). D, CFT-1 CF
epithelial cells transiently transfected with WT-CFTR when compared
with mock-transfected cells (number of cells screened was 25; result
representative of three independent transfections). E,
comparison in 9HTEo non-CF airway epithelial cells of
hypotonicity-induced Cl permeability in the absence and
presence of two different ATP scavengers, apyrase and hexokinase (2 units/ml each) (number of cells screened was 25; result representative
of three independent sets of experiments). HYPO, hypotonic.
F, cellular cyclic AMP levels in response to forskolin
(FORSK) or 25% hypotonic (HYPO) challenge and
companion controls using a bioassay (n = 3-6;
forskolin-induced cAMP levels were p < 0.05 greater than all other samples by ANOVA). PAP,
papaverine.
|
|
 |
DISCUSSION |
CFTR-dependent ATP Release and
Signaling--
Potential interactions between ABC transporters and ATP
signaling pathways have been difficult to define. The primary
observations of these studies are that CFTR expression 1) enhances cell
volume recovery from swelling or RVD, 2) stimulates both constitutive and volume-sensitive ATP release, and 3) facilitates autocrine control
of cell volume regulation via purinergic receptors. While the mechanism
of ATP release is not fully defined, these effects appear to be due to
CFTR-dependent modulation of a separate ATP channel. These
results are in agreement with volume-sensitive ATP release and ABC
transporter potentiation of that release in hepatocytes and biliary
epithelial cells (5,52). These studies also synergize with evidence for
CFTR-dependent ATP release in red blood cells (53) and
defective cell volume regulation in CF (
/
) intestinal crypts (54).
These results also correlate with work implicating the closest relative
of CFTR, the ABC transporter mdr-1 or P-glycoprotein,
initially as a volume-activated Cl
channel and later as a
regulator of volume-activated Cl
channels (31-33).
Indeed, mdr-1 was also shown recently to accelerate RVD in
heterologous cells (34). Thus, CFTR may function as a versatile and
sensitive transducer that modulated volume-sensitive ATP release and
membrane ion permeability.
Based on these observations, our working hypothesis is that cell volume
is sensed and transduced in an ABC transporter-dependent manner, leading to potentiation of ATP release through stimulation of a
separate, yet tightly associated, ATP channel. In lipid bilayers, this
ATP release pathway is stimulated by a transmembrane pressure gradient
and is inhibited by the mechanosensitive ion channel blockers, the
lanthanides (GdCl3 and LaCl3), and the broad
specificity Cl
channel blockers, DIDS and DPC (Fig.
8). This working model is bolstered by
the data that DIDS and GdCl3 do not inhibit CFTR Cl
channel activity. The finding that cyclic AMP
potentiates volume-sensitive ATP release only in wild-type
CFTR-expressing cells underscores a key regulatory role for CFTR in
this process. Because the sensitivity as well as the magnitude of
extracellular ATP release is lost in CF epithelia or in heterologous
cells that lack wild-type CFTR, it is attractive to speculate that the
sensor for hypotonicity may be related to CFTR itself. The
possibility, that CFTR as well as other ABC transporters may be
"sensors" or receptors, has been suggested in a previous report
(23).

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Fig. 8.
Working model of CFTR and purinergic
signaling in normal versus CF epithelia: loss of CFTR
and purinergic signaling leads to defects in cell volume regulation and
may lead to abnormal airway surface liquid composition and/or
volume. Shown is a working model illustrating non-CF epithelial
cell's ability to sense hypotonicity (as reduced or diluted external
Cl ions) in a CFTR-dependent manner, intact
purinergic signaling from a non-CF cells that is antagonized by ATP
scavengers and ATP release channel inhibitors, and ATP- and
hypotonicity-induced Cl efflux and RVD. In a CF cell, all
is attenuated due to a lack of CFTR in the apical membrane, defects in
epithelial cell volume regulation, and dysregulation of other anion and
cation conductances.
|
|
ATP Release Channel Can Be Dissociated from CFTR--
The specific
cellular mechanisms whereby CFTR facilitates the release of ATP are not
known and are limited by the lack of a molecular candidate other than
CFTR (14, 19-30). Observations herein are consistent with the view
that CFTR itself does not conduct ATP but, instead, is associated
closely with a separate ATP-permeant channel that is expressed in both
epithelial and heterologous cells. First, an ATP channel conductance
was not reliably detected in a highly purified preparation of CFTR in lipid bilayers, consistent with other published work (28, 30). Second,
in less purified systems containing many proteins, both Cl
and ATP channels are observed. Third, ATP channels are
still detectable after rigorous immunodepletion of CFTR. The fact that ATP channels are active independently of CFTR is intriguing and suggests that, in the bilayer, the cofactor regulatory machinery is no
longer present. These findings are consistent with other studies that
demonstrate that ATP release from intact cells is diminished but still
detectable in the absence of CFTR. It is important to emphasize,
however, that CFTR, as a member of the ABC transporter family, may
transport ATP itself, albeit at rates that are nonconductive and more
consistent with a transporter. Moreover, it is also possible that CFTR
may transport ATP at conductive rates, in that purification of CFTR may
have eliminated a regulatory protein that is essential for helping CFTR
conduct ATP. These uncertainties underscore the need for identification
of new molecular candidates for the ATP release pathway.
In this light, several anion channels beyond CFTR are candidates for
ATP release channels. For example, because DIDS inhibits ORCCs, because
ORCC Cl
channels are present in the less purified protein
material derived from bovine tracheal epithelial membrane vesicles, and
because ORCCs are stimulated by membrane stretch (55), it is possible that ORCCs may function as a conduction pathway for ATP.
Voltage-sensitive organic anion channels are blocked in a
voltage-dependent manner by 10 mM ATP as an
open channel blocker (56); as such, voltage-sensitive organic anion
channels may also be permeable to ATP. Thinnes and colleagues
(57) have identified plasma membrane-expressed forms of the
voltage-dependent anion channel (PL-VDAC) or "porin,"
which, in inner mitochondrial membrane, functions to conduct newly
synthesized ATP in and out of the mitochondrion. Plasmalemmal forms of
VDAC may also conduct ATP (57). These or other ATP-permeable channels may also be present in vesicles (as borne out by the bilayer studies) and may be inserted into the plasma membrane as ATP-filled vesicles fuse to release ATP by an exocytic mechanism (9).
CFTR as a Cell Volume Regulator--
These considerations suggest
a working model for ATP release and signaling that places CFTR in a
different context (Fig. 8). In non-CF cells, this signaling system
would be active under basal conditions, and volume-sensitive ATP
release would be augmented by hypotonicity and would culminate
in normal RVD in a manner governed by CFTR at the apical plasma
membrane (Fig. 8). However, in CF cells with the
F508 mutation, the
lack of functional CFTR leads to impaired ATP release and a depletion
of this autocrine signaling system. The result is that cell volume
regulation would be desensitized or down-regulated under both basal and
hypotonic conditions (Fig. 8). Theoretically, in the CF epithelium,
this could lead to an impaired ability to maintain normal cell volume under basal conditions and a decreased ability to RVD under hypotonic conditions. Indeed, abnormalities in ionic and osmotic strength and/or
volume of CF airway surface liquid have been measured in multiple
studies (35-38). The fact that airway surface liquid appears regulated
in its depth and in its composition implies a regulatory role of the
airway surface epithelium itself and an involvement of cell volume
regulatory processes. Thus, dysfunction of RVD mechanisms, cell volume
regulation, or both due to defective CFTR and a lack of extracellular
autocrine ATP signaling may contribute to the pathogenesis of CF lung dysfunction.
 |
ACKNOWLEDGEMENTS |
All authors contributed much to this work in
a team effort! We thank Dieter Gruenert for the non-CF and CF airway
epithelial cell models; Monique Mansoura and Melissa Ashlock at the
NHGRI for the gift of the CFT-1 cells as well as their YAC
clones; and Pary Weber, Michael Welsh, and the Howard Hughes Medical
Institute for the generous gift of the 3T3 fibroblasts
expressing wild-type and
F508 CFTR.
 |
FOOTNOTES |
*
This work was supported by a CF New Investigator Grant from
the Cystic Fibrosis Foundation (CFF) (to E. M. S.), by
National Institutes of Health Grant R01 DK/HL 54367 (to E. M. S.), by a CF New Investigator Grant and Research Grant from the CFF (to L. M. S.), by an National Institutes of Health Grant DK-48764 (to D. J. B.), by a CFF Fellowship F981 (to B. J.), and
by a Leroy Matthews award from the CFF (to J. P. C.).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.
§§
To whom correspondence should be addressed: Assistant Professor
of Physiology and Biophysics, Assistant Professor of Cell Biology, and
Research Scientist in the Gregory Fleming James CF Research Center,
University of Alabama at Birmingham, BHSB 740, 1918 University Blvd.,
Birmingham, AL 35294-0005. Tel.: 205-934-6234; Fax: 205-934-1445;
E-mail: eschwiebert@physiology.uab.edu.
Published, JBC Papers in Press, November 10, 2000, DOI 10.1074/jbc.M005893200
 |
ABBREVIATIONS |
The abbreviations used are:
CF, cystic
fibrosis;
CFTR, cystic fibrosis transmembrane conductance regulator;
ABC, ATP-binding cassette;
SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium, inner salt;
WT, wild-type;
RVD, regulatory volume decrease;
ATP
S, adenosine
5'-O-(thiotriphosphate);
ALU, arbitrary light unit(s);
DPC, diphenylamine carboxylic acid;
DIDS, 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid;
ORCC, outwardly
rectifying Cl
channel;
ANOVA, analysis of variance.
 |
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