From the Departments of Physiology and Biophysics,
** Cell Biology, and
Medicine and
the § Gregory Fleming James Cystic Fibrosis Research Center,
University of Alabama, Birmingham, Alabama 35294-0005 and the
¶ Department of Pathophysiology, Semmelweis University,
Budapest 1085, Hungary
Received for publication, December 3, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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Purinergic receptor stimulation has potential
therapeutic effects for cystic fibrosis (CF). Thus, we explored roles
for P2Y and P2X receptors in stably increasing
[Ca2+]i in human CF (IB3-1) and non-CF
(16HBE14o In cystic fibrosis
(CF),1 cyclic AMP- and
protein kinase A-dependent transepithelial Cl It is widely accepted that CFTR plays a crucial role in ATP release
from cells (8-10). The same is true for mdr ABC
transporters in hepatocytes and heterologous cells (11, 12). Once ATP
is released into the extracellular space, it can bind to purinoceptors regulating a variety of functions in different epithelia (13-15). ATP
and other agonists of purinoceptors are known to increase intracellular
Ca2+ concentration ([Ca2+]i) potently
in airway epithelial cells which, in turn, leads to stimulation of
Cl Purinoceptors are divided into two classes: P1 or adenosine receptors,
and P2, which recognize primarily extracellular ATP, ADP, UTP, and UDP.
The P2 receptors are further subdivided into two subclasses. P2X
receptors are extracellular ATP-gated calcium-permeable non-selective
cation channels that are modulated by extracellular Ca2+,
Mg2+, H+, and metal ions such as
Zn2+ and/or Cu2+ (25). P2Y receptors couple to
heterotrimeric G proteins and phospholipases (primarily phospholipase
C In this study, we used both CF (IB3-1) (30) and non-CF
(16HBE14o Cell Cultures--
IB3-1 cells derive from airway epithelia of a
CF patient carrying two different mutations of the CFTR gene, the most
common trafficking mutation ( Fura-2 Imaging of Intracellular Ca2+--
Cytosolic
Ca2+ concentration was measured with dual excitation
wavelength fluorescence microscopy (Deltascan, Photon Technologies, Princeton, NJ) after cells were loaded with the permeant form of the
fluorescence dye Fura-2/acetoxymethyl ester (Fura-2/AM; Teflabs,
Austin, TX). Fura-2 fluorescence was measured at an emission wavelength
of 510 nm in response to the excitation wavelength of 340 and 380 nm,
alternated at a rate of 50 Hz by a computer-controlled chopper
assembly. Ratios (340/380 nm) were calculated at a rate of 5 points/s
using PTI software. Cells were incubated in Dulbecco's phosphate-buffered saline containing 2 mM CaCl2
and 1 mM MgCl2 in the presence of 5 µM Fura-2/AM and 1 mg/ml Pluronic F-127 dissolved in
Me2SO for 120 min to allow loading of the dye into
the cells. After loading, coverslips were rinsed at least for 10 min in
Dulbecco's phosphate-buffered saline to remove extracellular Fura-2/AM
and the surfactant and were positioned in the cuvette at a 45° angle from the excitation light. Two glass capillary tubes were inserted into
the top of the cuvette out of the patch of the excitation light. One
tube was extended to the bottom of the cuvette and connected by way of
polyethylene tubing to an infusion pump. The other capillary tube was
positioned at the top of the cuvette and served to remove fluid from
the cuvette. The volume of the cuvette was ~1.5 ml, and the flow rate
was ~5 ml/min. It is important to note that switch in perfusion
solutions is removed in time and space for the cuvette, such that a
10-15-s time lag exists before agonist is exposed to the cells.
Experiments were performed at room temperature. Fluorescence
intensities at both wavelengths were assessed, and only those
preparations in which there were >200,000 counts/s for both
wavelengths were used for experiments. At the beginning of each
experiment, cells were perfused with solution A (see below), and the
fluorescence ratio was monitored for at least for 100 s to
establish a stable base-line value. Agonists and antagonists were then
added to the appropriate solutions (see later). The 340/380 nm ratios
(R) were converted into [Ca2+]i values
using the equations of Grynkiewicz et al. (32) as follows:
[Ca2+]i = Kd × ((R Fura-2 Quenching Experiments--
Cells were loaded and washed
as described for intracellular [Ca2+] measurement.
Fluorescence signal was measured at 359 nm (isosbestic wavelength) in
the presence of MnCl2 (500 µM) to detect
Ca2+-independent changes in Fura-2 fluorescence (33).
Immunoblotting with P2X Receptor Channel Isoform-specific
Antibodies--
Cells were lysed in a buffer containing 10 mM Tris, 0.5 mM NaCl, 0.5% Triton X-100, 50 µg/ml aprotinin (Sigma), 100 µg/ml leupeptin (Sigma), and 100 µg/ml pepstatin A (Sigma) adjusted to pH 7.2-7.4. Twenty micrograms
of protein were run per lane and separated on an 8% SDS-polyacrylamide
gel and then transferred to a polyvinylidene difluoride membrane
(Osmonics, Westborough, MA). Immunoblotting was performed with a rabbit
polyclonal antibody to P2X4 (Alomone Laboratories,
Jerusalem, Israel) at a dilution of 1:500. P2X1,
P2X2, and P2X7 antibodies were also obtained
from Alomone Laboratories and were tested in a similar manner.
Reactivity was detected by horseradish peroxidase-labeled goat
anti-rabbit secondary antibody (1:3,000 dilution, New England Biolabs,
Beverly, MA). Enhanced chemiluminescence was used to visualize the
secondary antibody.
Biotinylation of Plasma Membrane P2X Receptor
Channels--
Cells were seeded on Vitrogen-coated (collagen types I
and IV diluted 1:15 in Dulbecco's phosphate-buffered saline) 12-mm filters and grown as polarized monolayers with a transepithelial resistance that exceeded 400 ohms/cm2. Cells were placed on
ice and washed 3 times with cold PBS supplemented with 0.1 mM CaCl2 and 1.0 mM
MgCl2. Cells were then incubated in 1.0 mg/ml
poly(ethylene)oxid maleimide (Pierce) or sulfo-NHS-LC biotin
(Pierce) in cold supplemented PBS for 25 min at 4 °C. Cells were
washed 4 times with cold supplemented PBS, and the biotin was quenched
with 0.1% bovine serum albumin (Sigma). Cells were then washed 3 times
with cold supplemented PBS. Alternatively, cells could be biotinylated
with biocytin hydrazide. Filters were first incubated in 300 µl of a
stock solution containing 30 mM NaIO4 and 600 µl of a stock solution containing 100 mM sodium acetate
and 0.02% sodium azide, pH 5.5, for 30 min at room temperature in the
dark. Filters were washed and subsequently incubated with 1.0 mg/ml
biocytin hydrazide (Pierce) for 1 h at 4 °C. The reaction was
quenched with 0.1 M Tris, pH 7.5. Cell lysates were
collected as described above in immunoblotting procedures. Immobilized
streptavidin beads (Pierce) were added to the lysates at a 1:10
dilution and rocked overnight at 4 °C. Beads were washed 3 times
with lysis buffer and incubated in sample buffer for 5 min at 95 °C.
The mixture was centrifuged, and the supernatant was loaded onto an SDS-PAGE gel. The immunoblotting procedure then continued as described above.
Solutions--
Buffers for [Ca2+]i
measurement contained (mmol/liter) the following: for solution A: NaCl
140, KCl 3, KH2PO4 1.3, Na2HPO4 8, MgCl2 1, CaCl2 2; for solution B: NaCl 140, KCl 3, KH2PO4 1.3, Na2HPO4 8, MgCl2 1, Na-EGTA 1; for solution C: NMDG-Cl 140, KCl 4.5, Hepes 10, MgCl2 1, CaCl2 2; and for solution D: NMDG-Cl 100, KCl 40, Hepes 10, MgCl2 1, CaCl2
2. The solutions are at pH 7.3 unless indicated otherwise. In Fura-2
quenching experiments MnCl2 (500 µM) was
added to Ca2+- and EGTA-free solutions.
Data Analysis--
Data are expressed as mean ± S.D. An
unpaired Student's t test was used to compare the data in
different experimental groups. Results were considered significant if
p < 0.05. For original Fura-2 traces shown in the
figures, data are graphed with calibrated cytosolic free calcium on the
y axis, because data from an individual preparation of cells
was accumulated for all of the experiments in that figure where a
calibration was also performed. Because not all data were generated
from cells of the same passage or where a calibration was not performed
for every preparation, the data in tables are shown as ratiometric data.
Purinergic Agonists Trigger a Transient Increase in
[Ca2+]i in the Presence of Extracellular
Na+ in IB3-1 Cells--
To test for the presence of
purinergic receptors in IB3-1 cells, we measured the cytosolic free
Ca2+ concentration after stimulation with different
agonists to both P2Y and P2X receptors in physiologic bath solution
(solution A) containing Na+. Superfusion of cells with
solution containing ATP (100 µM) caused a rapid increase
in the ratio (340/380 nm) of Fura-2 fluorescence (rbasal = 0.89 ± 0.09 to
rpeak = 1.19 ± 0.13; n = 15). However, the response was transient, and the
[Ca2+]i returned close to basal value within
200 s after stimulation, even in the continuous presence of
agonist (r = 0.92 ± 0.09; n = 15)
(Fig. 1A). Furthermore, when
cells were exposed to ATP for the second time, only a small and even
more transient change was detected in Fura-2 fluorescence (Fig.
1A). Administration of 10 µM ATP caused a
comparable but smaller change in [Ca2+]i (Table
I). The effect of ATP was completely
inhibited by the application of suramin (100 µM) (Table
I). ADP, 2MeSATP (100 µM each), and ADP Purinergic Agonists Trigger a Transient Increase in
[Ca2+]i in the Absence of Extracellular
Ca2+--
Activation of P2Y1 receptors leads
to G protein-coupled phospholipase C- and inositol
1,4,5-trisphosphate-dependent release of Ca2+
from intracellular stores. As such, P2Y agonists should increase cytosolic Ca2+ even in the absence of extracellular
Ca2+. Therefore, we repeated the experiments with ATP (100 µM) (Fig. 2A)
and ADP (100 µM) superfusing IB3-1 cells with solutions
containing EGTA (1 mM) instead of CaCl2
(solution B). Similar to control conditions, both agonists increased
[Ca2+]i transiently, indicating that their
effects, at least partially, were independent from extracellular
Ca2+ (Table I). Nonetheless, the absence of extracellular
Ca2+ reduced the agonist-induced peak increase in
[Ca2+]i (Table I). Again, under these conditions,
the Ca2+ transients decayed fully back to base line within
200 s. Interestingly, these data did suggest that, besides
P2Y1 receptor activation, purinergic agonists may also
trigger Ca2+ influx from extracellular stores, which
contributes to the peak increase in [Ca2+]i.
Nevertheless, under these ionic conditions, Ca2+ influx was
not sufficient to support a sustained elevation of [Ca2+]i, the goal of this study. Experiments
described below lend clarification to these early data.
P2X Receptor-selective Agonists Fail to Trigger an Increase in
[Ca2+]i in the Presence of Extracellular
Na+ and Ca2+--
Multiple subtypes of P2X
receptors have already been described in human, rabbit, and rodent
airway epithelial cells (27, 34, 35). Thus, we speculated that the
higher peak in [Ca2+]i in the presence of
extracellular Ca2+ and the loss of the full response in
Ca2+-free extracellular solution could be explained by the
concomitant activation of P2X receptors activated by ATP. To test this
hypothesis, we superfused IB3-1 cells with "solution A" containing
either ATP and BzBzATP Trigger an Increase in
[Ca2+]i with Transient and Sustained Components
in the Absence of Extracellular Na+--
Despite the
negative data above with regard to P2X-selective agonists, we
maintained the hypothesis that P2X receptors were involved in the full
Ca2+ response induced by ATP in the presence of
extracellular Ca2+. Rationale for this hypothesis is given
by the fact that, in human and mouse lymphocytes, Na+ might
compete with Ca2+ for entry through P2X receptors from
extracellular stores (36-38) as well as other families of
Ca2+ entry channels like the transient receptor potential
channels (TRPs) or the store-operated Ca2+ channels (SOCs)
(39, 40). Thus, we speculated that extracellular Na+ might
suppress the Ca2+ permeability of P2X receptor channels in
IB3-1 cells. To verify this hypothesis, we substituted extracellular
Na+ by N-methyl-D-glucamine (NMDG)
(solution C) and tested the effects of a non-discriminant P2Y and P2X
agonist (ATP), P2X-specific agonists (BzBzATP and
Following removal of extracellular Na+ and changes in
[Ca2+]i, we applied ATP (100 µM).
Under these conditions, ATP induced a further increase in
[Ca2+]i displaying a biphasic Ca2+
response consisting of an initial transient peak
and a sustained component (Fig.
2B and Tables II and
III). In addition, as shown in Fig.
2B, a second application of ATP elicited a smaller increase in the [Ca2+]i peak; however, the sustained
Ca2+ plateau was comparable with that observed after the
first stimulation by ATP. When [Ca2+]i reached a
stable value after withdrawal of extracellular Na+, we also
added either BzBzATP (100 µM) (Fig. 3B) or
P2X4 Receptor Channel Protein
Biochemistry--
Due to the lack of other specific agonists or
inhibitors, our functional studies did not distinguish further agonists
among the P2XR subtypes. However, biochemical evidence suggests that IB3-1 cells express the P2X4 receptor channel robustly.
Membrane protein lysates from IB3-1 cells were prepared and were
subjected to immunoblotting with a P2X4-specific polyclonal
antibody. Fig. 4A shows the
positive results for P2X4 receptor channel protein in total
membrane protein lysates from IB3-1 cells grown on collagen-coated plastic as confluent monolayers. Inconsistent signals or a lack of
signal was observed for P2X1, P2X2, and
P2X7 using specific antibodies to those subtypes (data not
shown). The P2X4 signal displayed a similar biochemical
phenotype compared with human vascular endothelial cells and human
polycystic kidney disease renal epithelial cells performed in our
laboratory (13, 41) as well as a recent study of P2X4
receptor biochemistry in cardiac tissue and myocytes (42). An
unglycosylated band was detected at ~46 kDa (the predicted molecular
mass for P2X4) and a larger and broader glycosylated band
at 60-65 kDa. These immunoblotting data show that P2X4 is
the most abundant P2X subtype expressed in IB3-1 cells. However, these
data do not rule out less abundant expression of other P2X subtypes
that is below the limit of detection with these antibodies. Further
chemical modification of the extracellular solution also supports the
abundant expression of P2X4 receptor channels as the major
P2X receptor subtype mediating Ca2+ entry (see below).
Fig. 4, B-D, shows additional data in
16HBE14o The Extracellular ATP-gated P2X4 Receptor Channel Is
the Major Ca2+ Entry Channel Stimulated by ATP in IB3-1 and
16HBE14o The P2X4-mediated Ca2+ Entry Is Sustained,
Long Lived, Reversible, and Re-acquired upon Re-addition of
Agonist--
For any therapeutic approach to be effective, especially
one that targets an endogenous receptor, stimulation should be
sustained and long lived. Even more desirable, the effect should be
reversible to control the response. Ultimately, it is ideal if this
endogenous receptor target did not desensitize or inactivate, as is
apparent in this study for P2Y-mediated transient Ca2+
signal. Fig. 7 shows experiments designed
to determine whether P2X4-mediated Ca2+ entry
was sustained and long lived in IB3-1 cells. In the first protocol, ATP
(100 µM) was added in Na+-free solution that
has pH 7.9. A transient increase in [Ca2+]i
mediated by P2Y receptors was followed by a sustained plateau that
persisted for over 60 min, until ATP was removed (Fig. 7A).
In a second approach, a 15-min stimulation was performed with ATP and
then was reversed with washout. Following re-addition of ATP, a similar
sustained calcium plateau was acquired that persisted for 40 min. A
third washout and stimulation was performed at the end of the protocol
(Fig. 7B), showing lack of desensitization of the
P2X4 receptors or inactivation of their channel function. In contrast, the transient spike observed in the first application of
ATP was lost. These data show, these data show that the
P2X4-mediated Ca2+ entry is sustained, long
lived, reversible, and re-acquirable upon washout and re-addition of
agonist.
Neither the Reverse Operation Mode of the
Na+/Ca2+ Exchanger Nor
Voltage-dependent Ca2+ Channels or
Store-operated Ca2+ Channels Are Involved in ATP-induced
Ca2+ Entry in IB3-1 Cells--
Theoretically, both the
initial increase in [Ca2+]i after removal of
extracellular Na+ and the sustained Ca2+
plateau induced by administration of ATP could be due to the activation
of the Na+/Ca2+ exchanger in its reverse
operation mode and/or other classes of Ca2+ entry channels.
Thus, we removed extracellular Na+ and added ATP in the
presence of KB-R7943 (30 µM), a specific inhibitor of
reverse operation mode of the Na+/Ca2+
exchanger (43). Since KB-R7943 had no effect under these experimental conditions, we excluded the presence of this exchanger at the plasma
membrane (Fig. 8A and Tables
II and III). Although airway epithelial cells are non-excitable cells
and should not express voltage-dependent Ca2+
channels, we asked the question whether cell membrane depolarization stimulated or inhibited the Ca2+ response induced by ATP.
Therefore, we exposed the cells to high extracellular KCl concentration
(40 mM) in Na+-free medium (solution D), and
then we added ATP. As shown in Fig. 8B and Tables II and
III, membrane depolarization inhibited the peak increase of
[Ca2+]i, and the sustained Ca2+
plateau was completely abolished, indicating that IB3-1 cells do not
express voltage-dependent Ca2+ channels.
SOCs or TRPs represent other pathways by which Ca2+ can
enter non-excitable cells besides the ATP-gated P2X receptor channels. Theoretically, both SOCs and TRPs could be responsible for the sustained Ca2+ influx induced by ATP in
Na+-free medium. Therefore, we tested whether SOCs are
present in IB3-1 cells. We treated the cells with thapsigargin (100 nM), an inhibitor of Ca2+ pump in the ER
membrane, in the presence of extracellular Ca2+.
This maneuver induced a large initial increase in Fura-2 fluorescence ratio (rbasal = 1.00 ± 0.05 to
rpeak = 2.92 ± 0.17; n = 3) followed by a sustained Ca2+ plateau
(rsustained = 1.58 ± 0.29;
n = 3). In the absence of extracellular
Ca2+, stimulation with thapsigargin resulted in a small
transient increase in [Ca2+]i due to the
depletion of intracellular Ca2+ stores, and the re-addition
of extracellular Ca2+ elicited a large
[Ca2+]i increase (Fig.
9). These data indicate that IB3-1 cells
possess SOCs, which are activated by a decrease in
[Ca2+]ER. Next, we have asked whether SOCs or
store-independent TRP-like channels contribute to the sustained
Ca2+ increase after P2Y1 receptor stimulation
in Na+-free medium. To address this question, we used
2APB, which has recently been reported to inhibit SOCs (44, 45),
and SKF-96365, which is a blocker of the store-independent TRPs (46).
Neither 2APB (75 µM) nor SKF-56365 (50 µM) abolished the ATP-induced sustained increase in
[Ca2+]i in the absence of extracellular
Na+ (Table III). Interestingly, the sustained
Ca2+ plateau was further augmented by the SKF-96365
compound (Table III). These data indicate that, in IB3-1 cells, SOCs
and/or TRPs do not play a role in regulating
[Ca2+]i following purinergic receptor
stimulation.
Stimulation of purinergic receptors exerts biological effects,
which are mediated in part through elevation of intracellular Ca2+ concentration (47-52). In the present study, we show
evidence that IB3-1 cells express P2Y1 and P2X4
receptors abundantly. P2Y1 receptors have been found
recently in airway epithelia of P2Y2 receptor-knockout mice
(54), in rat lung (55), and in Calu-3 human airway epithelial cells
(56). ADP Nevertheless, in addition to the beneficial targeting of
P2Y receptors for CF therapy, we argue here for the beneficial
targeting of P2X receptors as well. Activation of these receptors would also have the added benefit of eliciting a sustained increase in
[Ca2+]i, an effect not observed with P2Y-specific
agonists. The transient nature of the Ca2+ signal induced
by purinergic agonists accounts presumably for transient
Cl However, our data could conceivably be explained in the following ways:
1) opening of extracellular ATP-gated P2X receptor channels; 2)
activation of Na+/Ca2+ exchanger in reverse
operation mode due to Na+ removal; 3) opening of
voltage-dependent Ca2+ channels following
membrane depolarization; and 4) activation of SOCs or TRPs after
depletion of intracellular Ca2+ stores. All lines of
evidence indicate that activation of ATP-gated P2X4
receptor channels led to augmentation of Ca2+ signal and
the sustained Ca2+ plateau. First, in IB3-1 cells, BzBzATP,
a P2X receptor-specific agonist, increases
[Ca2+]i only in Na+-free medium.
Second, the ATP-induced Ca2+ plateau was enhanced by
alkaline extracellular pH and inhibited by acidic extracellular pH.
Third, ATP-induced Mn2+ entry caused quenching of Fura-2 in
a pH-dependent manner exhibiting significant increase in
Mn2+ permeability at alkaline pH. Fourth, application of
Zn2+ further enhanced the effects of ATP. Fifth, a
P2Y1 receptor-specific agonist, ADP Although BzBzATP is primarily known to be an agonist of
P2X7 and antibodies used in this study were raised against
rat P2X receptors, stimulation by Zn2+ and inhibition by
H+ are most consistent with activation of the
P2X4 receptors and inconsistent with other P2X receptor
subtypes (25). For instance, stimulatory effects by Zn2+
rule out a role for P2X7, because Zn2+ is a
P2X7 antagonist (25). Inhibition of Ca2+ entry
by acidic pH rules out P2X2 receptors, which are stimulated by acidic pH (25). The only phenotype that is not completely explained
by P2X4 alone is the alkaline pH stimulation.
Heterologously expressed P2X4 is only mildly stimulated by
alkaline pH (64). As such, we cannot rule out that additional P2X
receptor subtypes (perhaps P2X5 (65), P2X6
(66), or splice variants of P2X4, P2X5, and
P2X6 (67)) may be conferring these pH effects in a P2XR
heteromultimer. Interestingly, in 16HBE14o In a past study (27), our laboratory showed that a P2X-selective
agonist, BzBzATP, stimulated transepithelial chloride secretion in
Ussing chamber experiments on airway epithelia that had both transient
and sustained components and in nasal potential difference assays on
mouse nasal mucosa that were transient stimulations that averaged 1-2
mV. These stimulations occurred in Na+-rich solutions (27).
Despite this knowledge, we did not perform experiments designed to
examine P2XR-mediated signaling in this study (27). Because
Na+ is in great excess to Ca2+ in physiological
saline, the contribution of Ca2+-permeable non-selective
cation channels to a Ca2+ entry phenotype is often masked.
This was true for our CF cell model. In IB3-1 cells, removal of
extracellular Na+ was required to observe any increase in
[Ca2+]i with BzBzATP and a sustained
Ca2+ signal with ATP. Nonetheless, in
16HBE14o Interestingly, although controversial, recent data indicate that airway
surface liquid (ASL) in non-CF subjects is hypotonic and low in
Na+ with respect to the plasma (68). In contrast, other
studies (69) have concluded that non-CF and CF ASL are isotonic.
Nevertheless, it is noteworthy that, in Na+-replete medium,
extracellular ATP stimulation of ciliary beat is attenuated, whereas in
Na+-free medium, ATP induction of ciliary beat was
profound, suggesting a role for P2X receptors on cilia (35). Because
cilia reside and need to function optimally in the ASL environment, we
postulate that normal ASL may be hypotonic and, in particular, low in
Na+, allowing P2X receptor agonists to stimulate sustained
signaling that may impact ion transport and ciliary beat. These
specialized chemical and ionic conditions may also be critical in the
delivery of agonists for CF therapy. This is tenable, because the
vehicle for delivery during nebulization, aerosolization, or
instillation would merely need to be modified to suit these optimal conditions.
Taken together, these findings are profound with regard to therapy in
CF, because they suggest that endogenously expressed P2X receptors do
not desensitize or inactivate, and under appropriate conditions, their
activation leads to a prolonged Ca2+ signal that could
translate into a sustained Cl) airway epithelial cells. Cytosolic
Ca2+ was measured by fluorospectrometry using the
fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes
was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP
and other P2Y agonists caused only a transient increase in
[Ca2+]i derived from intracellular stores in a
Na+-rich environment. In contrast, ATP induced an increase
in [Ca2+]i that had transient and sustained
components in a Na+-free medium; the sustained plateau was
potentiated by zinc or increasing extracellular pH.
Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased
[Ca2+]i only in Na+-free medium,
suggesting competition between Na+ and Ca2+
through P2XRs. Biochemical evidence showed that the P2X4
receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca2+ channels,
voltage-dependent Ca2+ channels, or
Na+/Ca2+ exchanger in the ATP-induced sustained
Ca2+ signal was ruled out. In conclusion, these data show
that epithelial P2X4 receptors serve as ATP-gated calcium
entry channels that induce a sustained increase in
[Ca2+]i. In airway epithelia, a P2XR-mediated
Ca2+ signal may have therapeutic benefit for CF.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
transport is impaired because of mutations in the CF gene that encodes
for the protein, the "cystic fibrosis transmembrane conductance regulator" or CFTR (1). Originally, CFTR was thought to function exclusively as a low conductance Cl
channel (2, 3). More
recently, it has become clear that CFTR also regulates a series of
other transporters and ion channels, such as the
Cl
/HCO
transport is shared as a key disease
phenotype by CF epithelia from all affected tissues and that this
pathway is lost in CF. Therefore, activation of a cAMP-independent
Cl
secretory pathway through exploitation of a naturally
expressed epithelial protein could be of interest for CF therapy. In
certain cases, stimulation of Ca2+-dependent
Cl
channels can correct the impaired
HCO
secretion (14-17) and inhibition of Na+
absorption (18-22). In fact, earlier studies have proposed the use of
UTP and non-hydrolyzable UTP analogs as therapeutic agonists targeted
to the P2Y2 receptors in the treatment of CF lung disease (23, 24).
) to raise intracellular free calcium concentration (26). In CF
epithelial cells from multiple tissues, expression of P2X and P2Y
receptors appears unaffected, offering the possibility to increase
[Ca2+]i through targeting a naturally expressed
receptor in the apical or basolateral membrane domains (27, 28).
Nonetheless, in different CF epithelial cell models, the
desensitization of P2Y receptors and the transient nature of the
Ca2+ response upon chronic and repeated delivery of a
P2Y-specific agonist have made it difficult to generate stable stimuli
for ion secretion (7, 29).
) (31) human airway epithelial cell models, to
dissect out P2X-specific and P2Y-specific mechanisms of triggering an
increase in [Ca2+]i. We characterized a broad
range of P2Y-selective, P2X-selective, and non-discriminant P2Y and P2X
agonists under different chemical and ionic conditions to explore
possible strategies to elicit an increase in
[Ca2+]i that is sustained and prolonged. Results
described herein, using Fura-2/AM-based imaging, show that activation
of P2Y and P2X receptors increases [Ca2+]i by
completely distinct mechanisms. P2Y receptors elicit a transient
increase in [Ca2+]i derived from intracellular
endoplasmic reticulum (ER) stores, whereas P2X receptors trigger a
sustained rise in [Ca2+]i, allowing
Ca2+ influx from the extracellular space. In addition,
biochemical evidence shows that the P2X4 receptor is the
major epithelial subtype present in both cell lines. Thus, we conclude
that epithelial P2X receptors function as ATP-gated Ca2+
entry channels in the plasma membrane and have profound potential as a
target for CF pharmacotherapy.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Phe-508) and a premature stop
codon mutation (W1282X) (30). 16HBE14o
cells
are non-CF or normal airway epithelial cells, which express CFTR at the
plasma membrane. The cells were grown on Vitrogen 100-coated
tissue-culture flasks in 5% CO2 incubator at 37 °C. IB3-1 cells were cultured in LHC-8 (Biofluids, Rockville, MD) medium
supplemented with 5% fetal bovine serum (Invitrogen), 100 units/ml
penicillin/streptomycin (Invitrogen), 1× L-glutamine (Invitrogen), and 1.25 µg/ml Fungizone (Invitrogen).
16HBE14o
cells were cultured in minimum Eagle's medium
(Invitrogen) supplemented with 10% fetal bovine serum and 100 units/ml
penicillin/streptomycin. When cells reached confluency, they were
washed twice with Ca2+/Mg2+-free PBS. The cells
were then suspended using trypsin/EDTA solution and plated on diluted
Vitrogen-coated (collagen types I and IV diluted 1:15 in Dulbecco's
phosphate-buffered saline) glass coverslips. For
[Ca2+]i measurements, cells were used 48-72 h
after plating.
Rmin)/(Rmax
R)) × (Sf380/Sb380) where Kd is the dissociation constant of Fura-2 for
Ca2+, Rmax and
Rmin are R values under saturating
and Ca2+-free conditions, respectively, and
Sf380 and
Sb380 are the fluorescent signals
(S) emitted by Ca2+-free (f) and
Ca2+-bound (b) forms of Fura-2 at a wavelength
of 380 nm. In situ cell calibrations were accomplished after
the cells were permeabilized with ionomycin (2 µM) under
Ca2+-free (10 mM EGTA) and saturating
Ca2+ (3 mM CaCl2) conditions. The
Kd was assumed to be 224 nM (32).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S (10 and 100 µM) also caused an increase in cytosolic Ca2+
concentration, showing similar characteristics described for ATP (Fig.
1, B-D, and Table I). Because 2MeSATP and ADP
S increased [Ca2+]i in a similar manner to ATP and ADP, these
data argue strongly for activation of P2Y1 receptors over
other P2Y subtypes. In contrast, neither UTP (100 µM)
(Fig. 1A) nor UDP (100 µM) had any effect on
Ca2+ concentration (Table I). To explore whether
degradation of ATP or ADP plays role in elevation of
[Ca2+]i, we tested the effects of adenosine (100 µM). Because adenosine did not increase
[Ca2+]i, we did not pursue the participation of
P1 receptors in increasing [Ca2+]i (Table I).
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Fig. 1.
Original traces showing the effects of
ATP and UTP (100 µM each)
(A), ADP (100 µM)
(B), 2MeSATP (100 µM) (C), and
ADP S (100 µM) (D) on
[Ca2+]i. IB3-1 cells were superfused with
Na+-containing medium (solution A). A, please
note that the second application of ATP was without effect. In these
traces and in all others below, please note that there is a time lag of
10-15 s before agonist-containing perfusate enters the cuvette. As all
of these experiments were performed on coverslips prepared on the same
day, a calibration was used on the same cell preparation to allow
conversion and plotting of the data as cytosolic calcium.
Maximum changes in Fura-2 fluorescence in IB3-1 cells in
Na+-containing medium
ratios (340/380 nm) are maximum changes in Fura-2 fluorescence in
response to purinergic agonists versus basal fluorescence.
Values for % are percent changes in fluorescence versus ATP
(100 µM), except ADP (100 µM) +Ca2+
free media whose value for % is versus ADP (100 µM). Values are means ± S.D.; n = number of experiments.
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Fig. 2.
Original traces showing the effects of ATP
(100 µM) on
[Ca2+]i in IB3-1 cells exposed to nominally
Ca2+-free, Na+-containing solution
(A) and in cells exposed to
Ca2+-containing Na+-free solution
(B) as indicated. B, please note the
slight sustained increase in [Ca2+]i upon
substitution of Na+ by NMDG. This sustained plateau was the
first hint that in Na+-free medium Ca2+ entry
channels could also be involved in the ATP-induced sustained
Ca2+ response.
,
-methylene ATP (
,
-MeATP, 100 µM) or
benzoyl-benzoyl-ATP (BzBzATP, 100 µM) (Fig.
3A), selective agonists for
different P2X receptor subtypes. Under these conditions, P2X-selective
purinergic agonists failed to change [Ca2+]i
(Table I). However, we were aware of the fact that
,
-MeATP and
BzBzATP, although potent agonists at P2X1,
P2X3, and P2X7 receptors, have little or no
effect at other P2XR subtypes. Thus, we hypothesized that changing the
ionic composition of the superfusion medium might reveal activation of
a Ca2+ entry mechanism by these agonists (see below).
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Fig. 3.
A includes an original trace
showing the lack of an effect of BzBzATP (100 µM) on
[Ca2+]i in IB3-1 cells in
Na+-containing medium. B shows an original trace
where cells exposed to Na+-free medium are responsive to
BzBzATP (100 µM) with a rise in
[Ca2+]i in IB3-1 cells. Note that a second trace
(in magenta) is shown to illustrate the lack of effect of
BzBzATP (100 µM) in a Ca2+-free and
Na+-free solution.
,
-MeATP), and a
P2Y1-specific agonist (ADP
S). As shown in Fig.
2B (and in Fig. 5B and Fig. 7, A and B), substitution of extracellular Na+ by NMDG
itself caused a small but sustained increase in
[Ca2+]i (rbasal = 0.89 ± 0.04 to rNMDG = 0.94 ± 0.04;
n = 31; p < 0.05) which was completely
absent when extracellular Ca2+ was also omitted from the
superfusion medium. These observations suggest the presence of a
mechanism that allows sustained Ca2+ entry, even in
non-stimulated cells.
,
-MeATP (100 µM). BzBzATP, but not
,
-MeATP,
induced a small increase in [Ca2+]i (Fig.
3B and Tables II and III). This increase was completely
dependent on the presence of extracellular Ca2+, indicating
a role for P2X receptors in Ca2+ influx (Fig. 3B
and Table II). In Na+-free media, P2Y1-specific
agonist, ADP
S (100 µM), augmented the peak increase in
[Ca2+]i (Table II) but failed to elicit a
sustained Ca2+ plateau (Table III). Taken together, these
data argue for a role for P2X receptors as Ca2+ entry
channels in IB3-1 cells.
Maximum changes in Fura-2 fluorescence in IB3-1 cells in
Na+-free medium
ratios (340/380 nm) are maximum changes in Fura-2 fluorescence in
response to purinergic agonists versus basal fluorescence.
All values for % are percent changes in fluorescence versus
ATP (100 µM). Values are means ± S.D.;
n = number of experiments.
Changes in Fura-2 fluorescence in IB3-1 cells 5 min after the peak
stimulation
ratios (340/380 nm) are changes in Fura-2 fluorescence 5 min after
the peak stimulation versus unstimulated conditions. All
values for % are percent changes in fluorescence versus ATP
(100 µM) in Na+-containing medium. Values are
means ± S.D.; n = number of experiments.
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Fig. 4.
A, immunoblot analysis of IB3-1 cells
grown as non-polarized monolayers in flasks using rabbit polyclonal
antibodies against P2X4 receptors. A smaller band of the
predicted molecular mass for P2X4 (46 kDa) was detected, as
was a larger, broader, glycosylated band at 60-70 kDa. The positions
of molecular mass markers are shown on the left (in kDa).
This is representative of 3 such experiments. B, immunoblot
analysis of 16HBE14o cells and CFPAC-1 cells grown as
polarized cell monolayers (CFPAC-1 cells were screened as another
CF-relevant cell line and were grown in a similar manner than
16HBE14o
cells except that Iscove's modified essential
medium was used for the basal medium with all other additives kept
similar). Note the stronger expression in polarized cell monolayers and
the presence of a 40-50-kDa band (unglycosylated
predicted molecular mass), a 60-80-kDa band (glycosylated form),
and an even larger form at ~100 kDa (glycosylated form). This is
representative of 6 such experiments. C, tunicamycin (10 µM), an inhibitor of glycosylation, added to the culture
medium in an overnight 24-h incubation of confluent cell monolayers
grown in flasks abolished the 100-kDa form and inhibited the expression
of the 60-80-kDa band, yielding more of the 40-50-kDa unglycosylated
form. This is representative of 2 such experiments. D, three
water-soluble forms of biotin reagents were used to biotinylate apical
membrane P2X4. The data reveal that
poly(ethylene)oxid-maleimide biotin, a reagent that reagents
with primary amines primarily on lysine residues, detected only the
glycosylated forms in the apical plasma membrane of
16HBE14o
epithelial cell monolayers. Biocytin hydrazide
failed to work in this experiment, likely because our conditions for
oxidizing the carbohydrate residues were not optimal.
Sulfo-NHS-LC-biotin detected all of the forms, indicating that it may
have detected apical P2X4; however, it may have gained
access to the cell interior to find the unglycosylated form as well.
This is representative of 2 such experiments. Note pertaining to all
panels: no secondary antibody controls were performed for all of the
above experiments, as were peptide immunogen blocking experiments that
effectively blocked the signal. Peptide immunogens for
P2X1, P2X2, and P2X7 did not block
the P2X4 signaling, revealing additional specificity. In
addition to data from our laboratory in human ADPKD kidney epithelial
cells (41) and human vascular endothelial cells (13), this is the first
documentation of biochemical detection of native airway epithelial
P2X4 receptor protein.
non-CF airway epithelial cells. Immunoblotting
of non-polarized cells grown in flasks (Fig. 4, B and
C) as well as biotinylation (Fig. 4D) of
polarized monolayers grown on permeable supports revealed robust and
apical membrane-localized expression of P2X4. In these
lysates, a third band of ~100 kDa was also found. Biotinylation was
performed on the apical and basolateral surface of these monolayers.
Only the apical signal is shown in Fig. 4D, although a
detectable signal was also observed in basolateral biotinylated
material (data not shown). Secondary antibody controls and blocking of
antibody binding with the peptide immunogen, provided with the primary
antibody in all biochemical assays, verified the specificity of
P2X4 receptor expression (data not shown). These data
suggest that P2X4 receptors are expressed abundantly by
human airway epithelial cells grown under non-polarized and polarized conditions.
Cells--
Like other subtypes of the P2X
receptor channel family, the P2X4 receptors are also
regulated by different cations, such as H+ or
Zn2+ (25). Thus, if it is true that in IB3-1 cells the
prolonged Ca2+ response in Na+-free medium was
due to activation of P2X4 receptors, then extracellular pH
and Zn2+ should modify the ATP-induced Ca2+
signal. To test this hypothesis, we measured
[Ca2+]i after changing extracellular pH or in the
presence of Zn2+ in both IB3-1 and 16HBE14o
cells. We exposed IB3-1 cells to ATP after changing the pH of the superfusion solution. As shown in Table III, increasing
extracellular pH potentiated the ATP-induced sustained increase in
[Ca2+]i only in Na+-free medium.
Furthermore, in a Na+-free environment, acidic pH
significantly reduced the ATP-induced peak increase in
[Ca2+]i (Table II). To demonstrate directly the
effect of ATP on Ca2+ influx from extracellular sources via
another approach, we measured quenching of Fura-2 at 359 nm in the
presence of MnCl2 (500 µM). Mn2+
is known to permeate the same entry channels as Ca2+ and
quenches Fura-2 fluorescence when it enters the cells. As shown in Fig.
5A, in Na+-free
medium, acidic extracellular pH (6.4) inhibited Mn2+ entry,
whereas alkaline extracellular pH (7.9) potentiated markedly Mn2+ entry and quenching of the dye. To further support the
involvement of P2X4 receptor channels, we tested the effect
of the P2X receptor co-agonist, Zn2+, on ATP-induced
Ca2+ entry mechanisms. Inclusion of ZnCl2 (20 µM) further augmented the sustained increase in
[Ca2+]i induced by ATP in Na+-free
medium (Fig. 5B and Table III) but had no effect in
Na+-containing medium (Table III). Since our biochemical
data (see above) indicated that P2X4 receptors are also
present in 16HBE14o
non-CF airway epithelial cells, we
tested whether increasing extracellular pH or addition of
Zn2+ augmented the ATP-induced sustained Ca2+
entry in Na+-free medium in 16HBE14o
cells.
As shown in Fig. 6A, ATP
elicited extracellular pH-dependent quenching of Fura-2,
suggesting that ATP-stimulated Ca2+ influx is facilitated
by alkaline pH. In addition, similar to results obtained with IB3-1
cells, both inclusion of Zn2+ and increasing pH potentiated
the effects of ATP on sustained Ca2+ signal (Fig.
6B). Taken together, these data argue for a prominent role
for the P2X4 receptor as a Ca2+ entry channel
in human airway epithelial cells and argue against a functional role
for other P2X receptor subtypes.
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Fig. 5.
Representative traces showing the pH
dependence of ATP-induced Mn2+ entry in IB3-1 cells.
A, quenching of Fura-2 was measured at the isosbestic
wavelength of Fura-2 (359 nm). Cells were exposed to MnCl2
(500 µM) in Na+- and Ca2+-free
medium at pH 7.3. After 200 s, ATP (100 µM) was
added to the superfusion medium having three different pH values, as
indicated. At least 3 experiments have been done in each group with
similar results. A representative trace shows the effects of ATP (100 µM) in presence of ZnCl2 (20 µM) in cells exposed to Na+-free medium
(B) as indicated. Please note the augmentation of the
sustained plateau of increased [Ca2+]i in IB3-1
cells by inclusion of ZnCl2 (compare with original trace in
Fig. 2B).
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Fig. 6.
Representative traces on the left
showing the pH dependence of ATP-induced Mn2+ entry
in 16HBE14o cells. A, experiments were
performed in a similar manner to those in Fig. 5A. Changes
in cytosolic Ca2+ concentration 5 min after the peak
stimulation versus the basal [Ca2+]i
in 16HBE14o
cells are shown in B. Effects on
the sustained plateau of increased [Ca2+]i in
16HBE14o
cells are shown illustrating the potentiating
effect of ZnCl2 and of alkaline pH. All experiments have
been done in Na+-free medium. *, p < 0.05
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Fig. 7.
Representative traces showing the duration of
the sustained plateau in [Ca2+]i in IB3-1 CF
cells induced by ATP under Na+-free conditions (pH 7.9)
(A) and the reversibility, long lived, and
reproducible nature of the sustained plateau induced by ATP and
mediated by P2X4 (B). Each trace for
each protocol is typical of 3 such experiments.
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Fig. 8.
Representative traces showing the effects of
KB-R7943 (30 µM)
(A) and high [KCl]e (40 mM)
(B) on ATP-induced Ca2+ signal.
Experiments were done in a Na+-free environment.
B, note that substitution of Na+ by NMDG causes
a slight increase in [Ca2+]i, an effect that was
inhibited in high KCl-containing solution.
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Fig. 9.
Representative trace shows the effect of
thapsigargin (100 nM) on [Ca2+]i in
the absence and in the presence of extracellular Ca2+ as
indicated. This maneuver reveals the presence of SOCs involved in
Ca2+ entry, albeit induced by emptying ER stores
completely.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S, a specific agonist of P2Y1 receptors,
increased [Ca2+]i to a similar extent as ATP,
ADP, and 2MeSATP, suggesting the presence of P2Y1
receptors. Although recent data (57) indicate that 2MeSATP and,
possibly, ADP
S at a concentration of 100 µM may
activate P2Y11 receptors, we believe it is very unlikely
that the increase in [Ca2+]i observed in this
study was due to the activation of P2Y11 receptors. This
conclusion derives from the fact that P2Y11 receptors are
poorly stimulated by ADP (26), whereas our data show that ADP is at
least as potent an agonist as ATP. In addition, ADP
S also elicited a
significant increase in [Ca2+]i at a
concentration of 10 µM. In other airway epithelial cell
models, the presence of P2Y2 has already been demonstrated (16, 58, 59). Furthermore, in vivo studies demonstrate that aerosolized UTP has beneficial effects in treatment of CF lung disease,
confirming the presence of P2Y2 and/or P2Y4 on
the apical membrane of airway epithelium (23, 48). Interestingly,
neither UTP nor UDP increased [Ca2+]i in IB3-1
cells; however, both agonists do rescue impaired cell volume regulation
in IB3-1 cells.2 These
differences may reveal additional signal transduction pathways triggered by P2Y receptors that are independent of cytosolic calcium.
and fluid secretion observed in different CF
epithelial cell models (7, 29). Activation of P2X receptor channels
under appropriate conditions would lead to Ca2+ influx from
the extracellular space. Furthermore, this Ca2+ response is
sustained for at least 1 h, is reversible, and is re-acquired to
the same sustained level upon re-addition of agonists under conditions
designed to stimulate P2X4.
S, did not cause a
sustained increase in [Ca2+]i. Sixth, neither
2APB, an inhibitor of SOCs, nor SKF-56365, a blocker of
store-independent TRP-like channels, abolished the sustained increase
in [Ca2+]i induced by ATP. Seventh, recent data
(60, 61) indicate that Zn2+ inhibits SOCs. Eighth,
biochemical evidence showed abundant expression of P2X4.
Roles for the reverse mode of the Na+/Ca2+
exchanger and/or voltage-dependent Ca2+
channels were ruled out with a variety of different cell biological maneuvers and/or pharmacological inhibitors. It is noteworthy that
Vennekens et al. (62) have recently reported that epithelial Ca2+ channels are regulated by extracellular pH. However,
these channels are mainly expressed in kidney and intestinal epithelia
and inhibited by metal ions at low micromolar concentration (63).
cells,
ATP-driven Mn2+ entry was also enhanced by alkaline pH, and
Zn2+ potentiated the ATP-induced sustained increase in
[Ca2+]i. Taken together, these data indicate that
P2X4 receptors function as ATP-gated Ca2+ entry
channels in both CF and non-CF airway epithelial cells.
non-CF cells, extracellular Na+ (140 mM) prevented neither the BzBzATP-dependent
Ca2+ response nor the ATP-induced Ca2+
plateau3; however, responses
to both BzBzATP and ATP were much more profound under
Na+-free conditions. Thus, we speculate that P2XR agonists
might be useful in CF therapy regardless of extracellular
Na+ concentration, although modification of the
extracellular environment (Na+ removal, among other
maneuvers) may strengthen their efficacy and was required to optimally
study Ca2+ entry mechanisms in Fura-2 spectrofluorometry.
Nevertheless, further studies are required to determine whether the
presence of extracellular Na+ inhibits P2XR-mediated rescue
of Cl
secretion in CF therapy.
secretion in CF and non-CF epithelia.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant R01 HL63934 (to E. M. S.), OTKA Grant T037524, and ETT Grant 226/2000 (to A. Z.).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 may be addressed: Dept. of Physiology
and Biophysics and the Gregory Fleming James Cystic Fibrosis Research
Center, University of Alabama, MCLM 740, 1918 University Blvd.,
Birmingham, AL 35294-0005. Tel.: 205-934-6235; Fax: 205-934-1445; E-mail: zsembery@physiology.uab.edu.
§§ To whom correspondence may be addressed: Dept. of Physiology and Biophysics and Dept. of Cell Biology and the Gregory Fleming James CF Research Center, University of Alabama, MCLM 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, February 3, 2003, DOI 10.1074/jbc.M212277200
2 G. M. Braunstein and E. M. Schwiebert, unpublished observations.
3 Á. Zsembery and E. M. Schwiebert, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CF, cystic fibrosis;
ASL, airway surface liquid;
CFTR, cystic fibrosis transmembrane
conductance regulator;
ER, endoplasmic reticulum;
NMDG, N-methyl-D-glucamine;
P2XR, P2X purinergic
receptor channel;
SOC, store-operated calcium channel;
TRP, transient
receptor potential channel;
PBS, phosphate-buffered saline;
ADPS, adenosine 5'-[
-thio]diphosphate;
2MeSATP, 2-methylthio ATP;
,
-meATP, methylene ATP;
BzBzATP, benzoyl-benzoyl-ATP;
2APB, 2-amino-ethoxyliphenyl borate.
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REFERENCES |
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---|
1. | Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J. L., Drumm, M. L., Iannuzzi, M. C., Collins, F. S., and Tsui, L. C. (1989) Science 245, 1066-1073[Medline] [Order article via Infotrieve] |
2. | Gregory, R. J., Cheng, S. H., Rich, D. P., Marshall, J., Paul, S., Hehir, K., Ostedgaard, L., Klinger, K. W., Welsh, M. J., and Smith, A. E. (1990) Nature 27, 382-386 |
3. | Berger, H. A., Anderson, M. P., Gregory, R. J., Thompson, S., Howard, P. W., Maurer, R. A., Mulligan, R., Smith, A. E., and Welsh, M. J. (1991) J. Clin. Invest. 88, 1422-1431[Medline] [Order article via Infotrieve] |
4. | Schreiber, R., Greger, R., Nitschke, R., and Kunzelmann, K. (1997) Pfluegers Arch. 434, 841-847[CrossRef][Medline] [Order article via Infotrieve] |
5. | Schwiebert, E. M., Benos, D. J., Egan, M. E., Stutts, M. J., and Guggino, W. B. (1999) Physiol. Rev. 79, S145-S166[Medline] [Order article via Infotrieve] |
6. |
Zsembery, Á.,
Strazzabosco, M.,
and Graf, J.
(2000)
FASEB J.
14,
2345-2356 |
7. | Zsembery, Á., Jessner, W., Sitter, G., Spirli, C., Strazzabosco, M., and Graf, J. (2002) Hepatology 35, 95-104[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Braunstein, G. M.,
Roman, R. M.,
Clancy, J. P.,
Kudlow, B. A.,
Taylor, A. L.,
Shylonsky, V. G.,
Jovov, B.,
Peter, K.,
Jilling, T.,
Ismailov, I. I.,
Benos, D. J.,
Schwiebert, L. M.,
Fitz, J. G.,
and Schwiebert, E. M.
(2001)
J. Biol. Chem.
276,
6621-6630 |
9. | Schwiebert, E. M., Egan, M. E., Hwang, T. H., Fulmer, S. B., Allen, S. S., Cutting, G. R., and Guggino, W. B. (1995) Cell 81, 1063-1073[Medline] [Order article via Infotrieve] |
10. | Schwiebert, E. M. (1999) Am. J. Physiol. 276, C1-C8[Medline] [Order article via Infotrieve] |
11. | Lomri, N., Fitz, J. G., and Scharschmidt, B. F. (1996) Semin. Liver Dis. 16, 201-210[Medline] [Order article via Infotrieve] |
12. |
Roman, R. M.,
Wang, Y.,
Lidofsky, S. D.,
Feranchak, A. P.,
Lomri, N.,
Scharschmidt, B. F.,
and Fitz, J. G.
(1997)
J. Biol. Chem.
272,
21970-21976 |
13. |
Schwiebert, L. M.,
Rice, W. C.,
Kudlow, B. A.,
Taylor, A. L.,
and Schwiebert, E. M.
(2002)
Am. J. Physiol.
282,
C289-C301 |
14. |
Stutts, M. J.,
Lazarowski, E. R.,
Paradiso, A. M.,
and Boucher, R. C.
(1995)
Am. J. Physiol.
268,
C425-C433 |
15. |
Hwang, T. H.,
Schwiebert, E. M.,
and Guggino, W. B.
(1996)
Am. J. Physiol.
270,
C1611-C1623 |
16. |
Watt, W. C.,
Lazarowski, E. R.,
and Boucher, R. C.
(1998)
J. Biol. Chem.
273,
14053-14058 |
17. |
Huang, P.,
Lazarowski, E. R.,
Tarran, R.,
Milgram, S. L.,
Boucher, R. C.,
and Stutts, M. J.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
14120-14125 |
18. | Devor, D. C., and Pilewski, J. M. (1999) Am. J. Physiol. 276, C827-C837[Medline] [Order article via Infotrieve] |
19. | Kunzelmann, K., Schreiber, R., Nitschke, R., and Mall, M. (2000) Pfluegers Arch. 440, 193-201[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Mall, M.,
Wissner, A.,
Gonska, T.,
Calenborn, D.,
Kuehr, J.,
Brandis, M.,
and Kunzelmann, K.
(2000)
Am. J. Respir. Cell Mol. Biol.
23,
755-761 |
21. | Inglis, S. K., Collett, A., McAlroy, H. L., Wilson, S. M., and Olver, R. E. (1999) Pfluegers Arch. 438, 621-627[CrossRef][Medline] [Order article via Infotrieve] |
22. | Iwase, N., Sasaki, T., Shimura, S., Yamamoto, M., Suzuki, S., and Shirato, K. (1997) Respir. Physiol. 107, 173-180[CrossRef][Medline] [Order article via Infotrieve] |
23. | Knowles, M. R., Olivier, K., Noone, P., and Boucher, R. C. (1995) Am. J. Respir. Crit. Care Med. 151, S65-S69[Medline] [Order article via Infotrieve] |
24. | Bennett, W. D., Olivier, K. N., Zeman, K. L., Hohneker, K. W., Boucher, R. C., and Knowles, M. R. (1996) Am. J. Respir. Crit. Care Med. 153, 1796-1801[Abstract] |
25. | North, R. A., and Surprenant, A. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 563-580[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Ralevic, V.,
and Burnstock, G.
(1998)
Pharmacol. Rev.
50,
413-492 |
27. |
Taylor, A. L.,
Schwiebert, L. M.,
Smith, J. J.,
King, C.,
Jones, J. R.,
Sorscher, E. J.,
and Schwiebert, E. M.
(1999)
J. Clin. Invest.
104,
875-884 |
28. | Kunzelmann, K., and Mall, M. (2001) Clin. Exp. Pharmacol. Physiol. 28, 857-867[CrossRef][Medline] [Order article via Infotrieve] |
29. | Warth, R., and Greger, R. (1993) Cell. Physiol. Biochem. 3, 2-16 |
30. | Zeitlin, P. L., Lu, L., Rhim, J., Cutting, G., Stetten, G., Kieffer, K. A., Craig, R., and Guggino, W. B. (1991) Am. J. Physiol. 4, L313-L319 |
31. | Gruenert, D. C., Basbaum, C. B., and Widdicombe, J. H. (1990) In Vitro Cell. Dev. Biol. 26, 411-418[Medline] [Order article via Infotrieve] |
32. | Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract] |
33. |
Merritt, J. E.,
Jacob, R.,
and Hallam, T. J.
(1989)
J. Biol. Chem.
264,
1522-1527 |
34. |
Korngreen, A.,
Ma, W.,
Priel, Z.,
and Silberberg, S. D.
(1998)
J. Physiol. (Lond.)
508,
703-720 |
35. | Ma, W., Korngreen, A., Uzlaner, N., Priel, Z., and Silberberg, S. D. (1999) Nature 400, 894-897[CrossRef][Medline] [Order article via Infotrieve] |
36. | Wiley, J. S., and Dubyak, G. R. (1989) Blood 73, 1316-1323[Abstract] |
37. | Pizzo, P., Zanovello, P., Bronte, V., and Di Virgilio, F. (1991) Biochem. J. 274, 139-144[Medline] [Order article via Infotrieve] |
38. |
Baricordi, O. R.,
Ferrari, D.,
Melchiorri, L.,
Chiozzi, P.,
Hanau, S.,
Chiari, E.,
Rubini, M.,
and Di Virgilio, F.
(1996)
Blood
87,
682-690 |
39. | Balzer, M., Lintschinger, B., and Groschner, K. (1999) Cardiovasc. Res. 42, 543-549[CrossRef][Medline] [Order article via Infotrieve] |
40. | Arnon, A., Hamlyn, J. M., and Blaustein, M. P. (2000) Am. J. Physiol. 278, C163-C173 |
41. | Schwiebert, E. M., Wallace, D. P., Braunstein, G. M., King, S. R., Peti-Peterdi, J., Hanaoka, K., Guggino, W. B., Guay-Woodford, L. M., Bell, P. D., Sullivan, L. P., Grantham, J. J., and Taylor, A. L. (2002) Am. J. Physiol. 282, F763-F775[Medline] [Order article via Infotrieve] |
42. |
Hu, B.,
Mei, Q. B.,
Yao, X. J.,
Smith, E.,
Barry, W. H.,
and Liang, B. T.
(2001)
FASEB J.
15,
2739-2741 |
43. | Wang, X., Kim, S. U., van Breemen, C., and McLarnon, J. G. (2000) Cell Calcium 27, 205-212[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Dobrydneva, Y.,
and Blackmore, P.
(2001)
Mol. Pharmacol.
60,
541-552 |
45. | Gregory, R. B., Rychkov, G., and Barritt, G. J. (2001) Biochem. J. 354, 285-290[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Bennett, B. D.,
Alvarez, U.,
and Hruska, K. A.
(2001)
Endocrinology
142,
1968-1974 |
47. |
Rugolo, M.,
Mastrocola, T.,
Whorle, C.,
Rasola, A.,
Gruenert, D. C.,
Romeo, G.,
and Galietta, L. J.
(1993)
J. Biol. Chem.
268,
24779-24784 |
48. |
Lazarowski, E. R.,
Boucher, R. C.,
and Harden, T. K.
(1994)
Am. J. Physiol.
266,
C406-C415 |
49. |
Van Scott, M. R.,
Chinet, T. C.,
Burnette, A. D.,
and Paradiso, A. M.
(1995)
Am. J. Physiol.
269,
L30-L37 |
50. |
Furukawa, M.,
Ikeda, K.,
Yamaya, M.,
Oshima, T.,
Sasaki, H.,
and Takasaka, T.
(1997)
Am. J. Physiol.
272,
C827-C836 |
51. | Zsembery, Á., Spirli, C., Granato, A., LaRusso, N. F., Okolicsanyi, L., Crepaldi, G., and Strazzabosco, M. (1998) Hepatology 28, 914-920[Medline] [Order article via Infotrieve] |
52. |
Okada, Y.,
Maeno, E.,
Shimizu, T.,
Dezaki, K.,
Wang, J.,
and Morishima, S.
(2001)
J. Physiol. (Lond.)
532,
3-16 |
53. | Lazarowski, E. R., Watt, W. C., Stutts, M. J., Boucher, R. C., and Harden, T. K. (1995) Br. J. Pharmacol. 116, 1619-1627[Abstract] |
54. |
Homolya, L.,
Watt, W. C.,
Lazarowski, E. R.,
Koller, B. H.,
and Boucher, R. C.
(1999)
J. Biol. Chem.
274,
26454-26460 |
55. | Laubinger, W., and Reiser, G. (1998) Biochem. Pharmacol. 55, 687-695[CrossRef][Medline] [Order article via Infotrieve] |
56. |
Communi, D.,
Paindavoine, P.,
Place, G. A.,
Parmentier, M.,
and Boeynaems, J. M.
(1999)
Br. J. Pharmacol.
127,
562-568 |
57. | Sak, K., and Webb, T. E. (2002) Arch. Biochem. Biophys. 397, 131-136[CrossRef][Medline] [Order article via Infotrieve] |
58. | Donaldson, S. H., Lazarowski, E. R., Picher, M., Knowles, M. R., Stutts, M. J., and Boucher, R. C. (2000) Mol. Med. 6, 969-982[Medline] [Order article via Infotrieve] |
59. |
Homolya, L.,
Steinberg, T. H.,
and Boucher, R. C.
(2000)
J. Cell Biol.
150,
1349-1360 |
60. | Uehara, A., Yasukochi, M., Imanaga, I., Nishi, M., and Takeshima, H. (2002) Cell Calcium 31, 89-96[CrossRef][Medline] [Order article via Infotrieve] |
61. |
Jung, S.,
Pfeiffer, F.,
and Deitmer, J. W.
(2000)
J. Physiol. (Lond.)
527,
549-561 |
62. | Vennekens, R., Prenen, J., Hoenderop, J. G., Bindels, R. J., Droogmans, G., and Nilius, B. (2001) Pfluegers Arch. 442, 237-242[CrossRef][Medline] [Order article via Infotrieve] |
63. |
Nilius, B.,
Prenen, J.,
Vennekens, R.,
Hoenderop, J. G.,
Bindels, R. J.,
and Droogman, G.
(2001)
Br. J. Pharmacol.
134,
453-462 |
64. |
Wildman, S. S.,
King, B. F.,
and Burnstock, G.
(1999)
Br. J. Pharmacol.
126,
762-768 |
65. | Collo, G., North, R. A., Kawashima, E., Merlo-Rich, E., Neidhart, S., Surprenant, A., and Buell, G. (1996) J. Neurosci. 16, 2495-2507[Abstract] |
66. | Le, K. T., Babinski, K., and Seguela, P. (1998) J. Neurosci. 18, 7152-7159[Abstract] |
67. | Dhulipala, P. D., Wang, Y. X., and Kotlikoff, M. I. (1998) Gene (Amst.) 207, 259-266[CrossRef][Medline] [Order article via Infotrieve] |
68. | Smith, J. J., Travis, S. M., Greenberg, E. P., and Welsh, M. J. (1996) Cell 85, 229-236[Medline] [Order article via Infotrieve] |
69. | Matsui, H., Grubb, B. R., Tarran, R., Randell, S. H., Gatzy, J. T., Davis, C. W., and Boucher, R. C. (1998) Cell 95, 1005-1015[Medline] [Order article via Infotrieve] |