From the Cystic Fibrosis Research and Treatment Center, Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7248
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cystic fibrosis (CF)
transmembrane regulator (CFTR) is a cyclic AMP-dependent
Cl channel that is defective in CF cells. It has
been hypothesized that CFTR exhibits an ATP release function that
controls the airway surface ATP concentrations. In airway epithelial
cells, CFTR-independent Ca2+-activated Cl
conductance is regulated by the P2Y2 receptor. Thus, ATP
may function as an autocrine signaling factor promoting
Cl
secretion in normal but not CF epithelia if ATP
release is defective. We have tested for CFTR-dependent ATP
release using four independent detection systems. First, a luciferase
assay detected no differences in ATP concentrations in the medium from
control versus cyclic AMP-stimulated primary normal human
nasal epithelial (HNE) cells. A marked accumulation of extracellular
ATP resulted from mechanical stimulation effected by a medium
displacement. Second, high pressure liquid chromatography analysis of
3H-labeled species released from
[3H]adenine-loaded HNE cells revealed no differences
between basal and cyclic AMP-stimulated cells. Mechanical stimulation
of HNE cells again resulted in enhanced accumulation of extracellular [3H]ATP and [3H]ADP. Third, when measuring
ATP concentrations via nucleoside diphosphokinase-catalyzed
phosphorylation of [
-33P]dADP, equivalent formation of
[33P]dATP was observed in the media of control and cyclic
AMP-stimulated HNE cells and nasal epithelial cells from wild-type and
CF mice. Mechanically stimulated [33P]dATP formation was
similar in both cell types. Fourth, 1321N1 cells stably expressing the
human P2Y2 receptor were used as a reporter system for
detection of ATP via P2Y2 receptor-promoted formation of
[3H]inositol phosphates. Basal [3H]inositol
phosphate accumulation was of the same magnitude in control and
CFTR-transduced cells, and no change was observed following addition of
forskolin and isoproterenol. In both cell types, mechanical stimulation
resulted in hexokinase-attenuable [3H]inositol phosphate
formation. In summary, our data suggest that ATP release may be
triggered by mechanical stimulation of cell surfaces. No evidence was
found supporting a role for CFTR in the release of ATP.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The wide distribution of cell surface P2 receptors (1, 2) and the presence in most tissues of ectoenzymes that rapidly degrade extracellular nucleotides (3) support the notion that regulated process(es) for the cellular release of ATP may exist. Indeed, ATP has been found in an extracellular location in many tissues (4-10), and ATP secretion from intracellular granules during platelet activation as well as nerve transmission are well described events where physiological release of nucleotides occurs (4). However, in most tissues where significant ATP concentrations in the extracellular space have been detected, the mechanism(s) of ATP release have not been identified.
In the airways, the volume and composition of the liquid secretions may
be regulated by extracellular nucleotides (11-14). In cystic fibrosis
(CF)1 airway epithelia, the
P2Y2 receptor is linked to a
Ca2+-dependent chloride channel that provides
an alternative Cl secretory pathway to the CF
transmembrane regulator (CFTR) Cl
channel (11). This
alternative chloride channel (Cla) has been identified as a
potential target for therapy of CF lung disease. The localization of
P2Y2 receptors on the apical surface of airway epithelia
suggests the possibility that these receptors are regulated endogenously by the release of ATP onto the lumen.
It has been recently proposed that CFTR itself modulates the
composition of airway surface liquids by acting as a channel for ATP,
regulating Cla pathways via activation of P2Y2
receptors (15). In CF with defective CFTR, an implication of this
hypothesis is that resting levels of ATP would be reduced, resulting in
reduced Cla as well as CFTR activation. The notion that
CFTR mediates the release of ATP evolved from studies showing that
protein kinase A stimulated a single channel (CFTR) current in
CFTR-expressing cells when 100 mM ATP was present in the
intracellular compartment (15, 16). In addition, CFTR was shown to
regulate the activity of a second chloride channel in excised patches,
an activity thought to reflect ATP release and activation of outwardly
rectifying Cl channels by P2Y2 receptors
(15). However, other studies found either no evidence for CFTR-mediated
ATP conductance (17-19) or that some but not all CFTR Cl
channels could be associated with an ATP permeability (20). Measurements of ATP released from cells have also produced results that
either support (15, 21) or do not support (22, 23) a role for CFTR in
the regulation of ATP release. The current debate (24-26) reflects the
uncertainty that prevails on this issue (reviewed in Ref. 27). In this
study, we tested the function of CFTR as a pathway for ATP release
using both biochemical methods for directly measuring ATP release in
intact cells and an assay utilizing the human P2Y2 receptor
as a biological reporter for ATP release within the relevant cellular
biophase (receptor domain).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture--
Primary cultures of human nasal epithelial
(HNE) cells and immortalized nasal epithelial cells from either normal
or CF mice (28) were grown as polarized epithelia on 12.5-mm
porous Transwell Col filters (Costar) as reported previously
(29). Assays with HNE cells were carried out 7-10 days after seeding,
a time coincident with the development of the maximal ion transport
activity (29). T84 human colonic carcinoma cells, a cell line
expressing high levels of endogenous CFTR (30), were grown as a
polarized epithelium onto cross-linked collagen supports as described
previously (30). Control and CFTR-transduced NIH-3T3 fibroblasts were
grown on 12-well plastic plates as described (31). HP2U-1321N1 cells, a
clonal cell line derived from human astrocytoma cells stably expressing
the human P2Y2 receptor (32), were grown on 12-well plastic
plates (inositol phosphate assay) or on 6-well plates (36Cl efflux measurement and Western
blotting) in DMEM-H containing 5% fetal bovine serum and antibiotics
as described previously (9).
Luciferin/Luciferase Assay-- HNE cells were preincubated for 1 h in a Krebs-Ringer solution (2.4 mM K2HPO4, 0.4 mM KH2PO4, 115 mM NaCl, 1.2 mM MgCl2, 1.2 mM CaCl2, 25 mM NaH2CO3, and 5 mM glucose) at 37 °C and 5% CO2. The cells were exposed for the indicated times to the designated drug. The mucosal medium (0.3 ml) was collected, centrifuged to remove potentially detached cells, and boiled for 1 min. A 100-µl sample aliquot was diluted with 200 µl of H2O prior to measurements. The luciferin/luciferase mixture (300 µM luciferin, 5 µg/ml luciferase, 25 mM HEPES (pH 7.8), 6.25 mM MgCl2, 0.63 mM EDTA, 75 µM dithiothreitol, and 1 mg/ml bovine serum albumin) was added to samples via an LB953 AutoLumat luminometer (Berthold GmbH, Wildbad, Germany), and the sample luminescence was compared with an ATP standard curve performed for each individual experiment. To assess intracellular ATP content, cells were lysed with 5% trichloroacetic acid followed by ethyl ether extraction and neutralization.
Release of [3H]Adenine-labeled Nucleotides-- The cells were labeled for 3 h with 10 µCi/ml [3H]adenine as described previously (33). Labeled cells were exposed to the indicated drug without washing away the label to avoid unnecessary agitation of the cells. The mucosal medium was collected, and 3H-labeled species were resolved by high pressure liquid chromatography (HPLC).
Nucleoside Diphosphokinase (NDPK)-catalyzed Formation of
[-33P]dATP--
NDPK catalyzes the phosphorylation of
nucleoside diphosphates utilizing ATP as the
-phosphate donor
molecule (34). We used [
-33P]dADP, obtained as
described previously (29), as the acceptor molecule to quantitatively
determine the formation of [
-33P]dATP as a function of
ATP concentration. HNE cells and nasal epithelial cells from either
wild-type or CF mice were preincubated for 1 h in 0.3 ml of
(mucosal) HEPES (pH 7.4)-buffered DMEM-H (HEPES/DMEM). Incubations were
in the presence of 0.5 units/ml NDPK and 10 nM
[
-33P]dADP (0.2 µCi) added to the mucosal bath.
33P-Labeled species were resolved by HPLC.
Quantification of Nucleotides by HPLC-- Nucleotides were separated by HPLC (Shimadzu) via a strong anion-exchange column (Rainin Instrument Co. Inc.) with a mobile phase developed from 0.45 M NH4COOH (pH 4.8) to 0.5 M Na2H2PO4 (pH 2.7) over a 30-min period (9). Radioactivity was measured on line with a Radiomatic 500TR analyzer (Packard Instrument Co.). Species were identified and quantified as described previously (9).
Release of 51Cr from HNE Cells-- Confluent polarized HNE cells were incubated for 3 h in HEPES/DMEM containing 10 µCi of [51Cr]Na2CrO3 added to the mucosal bath. The cells were washed (four times) and preincubated for 1 h in HEPES/DMEM. The mucosal solution (0.5 ml) was removed at the times indicated, and the radioactivity was quantified with a Cobra Autogamma counter (Packard Instrument Co.).
Expression of CFTR in HP2U-1321N1 Astrocytoma Cells-- Subconfluent HP2U-1321N1 cells grown on 12-well plastic plates were infected with adenoviral vector (5 × 109 particles/well) containing DNA encoding either the CFTR gene (AdCFTR) or LacZ gene (AdLacZ) as described (31). Cells were assayed for CFTR expression 48 h after infection. For Western blot analysis, CFTR was localized with an antiserum raised against a carboxyl-terminal peptide of CFTR (35). [36Cl]Chloride efflux was quantified as described previously (31).
Measurement of [3H]Inositol Phosphates-- HP2U-1321N1 cells were labeled overnight in 0.5 ml of inositol-free DMEM-H containing 4 µCi/ml myo-[3H]inositol. At the time of assay, 10 mM LiCl was added to the cells for 15 min, followed by a further 15-min incubation in the presence of the indicated drugs. Incubations were terminated by the addition of 5% trichloroacetic acid, and the resulting [3H]inositol phosphates were separated and quantified by chromatography on Dowex columns as described previously (9).
Reagents--
ATP and dATP were purchased from Pharmacia
(Uppsala, Sweden). Hexokinase, NDPK, forskolin, and dADP were from
Boehringer Mannheim. Luciferin, luciferase, and isoproterenol were
obtained from Sigma. [3H]Adenine (17 Ci/mmol) and
myo-[3H]inositol (20 Ci/mmol) were from
American Radiolabeled Chemicals (St. Louis, MO).
[-33P]dATP (3000 Ci/mmol),
[51Cr]Na2CrO3 (300-500
mCi/mg cromium), and [36Cl]NaCl (3 mCi/g chloride) were
from Amersham Pharmacia Biotech.
Expression of the Results-- Except where stated otherwise, pooled data are expressed as means ± S.E. and are representative of at least three independent experiments performed with duplicate or triplicate samples. For statistical comparisons, unpaired t test was used, and p < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Release of ATP from HNE Cells--
ATP was quantified in the
mucosal medium bathing HNE cells utilizing the luciferin/luciferase
method. Under resting conditions, i.e. the cells were kept
undisturbed for 1 h prior to the assay, accumulation of ATP was
18.5 ± 3.4 pmol/106 cells (3.3 ± 0.6 nM in 0.3 ml of medium; n = 9). Cell
monolayers incubated with forskolin and isoproterenol (20 µM each, 10 min), a drug combination that is maximally
effective in initiating cyclic AMP-dependent
Cl secretion via CFTR (33), exhibited a concentration of
(mucosal) ATP of 4.2 ± 0.7 nM (25.1 ± 4.1 pmol/106 cells; n = 9), a value not
significantly different from control incubations. In contrast,
perturbing the cell surface by a medium change (data not shown) or by
gently pipetting the mucosal medium up and down twice resulted in a
marked accumulation of mucosal ATP (49.5 ± 8 nM,
297 ± 61 pmol/106 cells). Mechanically released ATP
by a medium displacement represented 0.9-1.6% of the cellular ATP
content, and it was unaffected by exposing the cells to the cyclic AMP
mixture (51 ± 12 nM, 306 ± 79 pmol/106 cells). The possibility that cell lysis occurred
during mechanical stimulation was investigated with
51Cr-loaded HNE cells. No changes in 51Cr
base-line levels were observed during a 10-min period following a
medium displacement (Table I). Moreover,
base-line radioactivity remained unchanged after five repetitive medium
displacements (data not shown). Thus, although we cannot rule out the
possibility that a small number of damaged cells contributed to
51Cr base-line levels, our results suggest that ATP was
released from intact (not damaged) cells. Consistent with this, we have recently shown that no cell lysis occurred during mechanically promoted
ATP release from 1321N1 cells (36).
|
Release of [3H]Adenine-labeled Nucleotides--
We
loaded the intracellular pool of ATP with [3H]adenine,
and the release of 3H-labeled species was quantified by
HPLC. Because the relatively large release of [3H]ATP
during medium changes (data not shown and Ref. 9) could mask a
potential contribution by CFTR, a protocol was utilized that avoided
cell washes. HPLC analysis of (mucosal) medium bathing resting
[3H]adenine-labeled HNE cells demonstrated a small
peak of [3H]ADP, barely distinguishable from background
levels (Fig. 1A) (non-incorporated [3H]adenine eluted at 5-8 min). No
increase in [3H]ATP or [3H]ADP accumulation
was observed in forskolin/isoproterenol-stimulated HNE cells (Fig.
1B). Incubation of cells with ionomycin to induce elevation
of intracellular Ca2+ resulted in a small accumulation of
extracellular [3H]ATP and greater accumulation of
[3H]ADP (Fig. 1C). A marked accumulation of
[3H]ATP and [3H]ADP was observed after a
medium displacement (Fig. 1D). This protocol was repeated
with T84 cells and with NIH-3T3 cells stably expressing either normal
CFTR or the -subunit of the interleukin-2 receptor as a control
(31). In all cases, addition of forskolin/isoproterenol (2-10 min)
promoted no release of [3H]adenine-labeled species, but a
large accumulation of 3H-labeled nucleotides was observed
after a medium displacement irrespective of the presence of CFTR (data
not shown).
|
ATP-dependent Conversion of [-33P]dADP
to [
-33P]dATP--
The possibility that CFTR mediates
the release of ATP from a nucleotide pool not accessed by
[3H]adenine (37) was investigated by trapping released
ATP with NDPK and [
-33P]dADP. The NDPK-catalyzed
conversion of [
-33P]dADP to
[
-33P]dATP was measured by HPLC as a function of ATP
concentration (Fig. 2). Moreover, since
exogenous NDPK activity greatly exceeds endogenous ecto-ATPase
activity, the assay resulted in a system that effectively locks the
-phosphate of ATP onto the [
-33P]dADP molecule upon
release. Fig. 3 illustrates the
conversion of [
-33P]dADP to
[
-33P]dATP in the mucosal HNE cell baths under various
conditions. A basal conversion (16 ± 3%) of
[
-33P]dADP to [
-33P]dATP was observed
with cells that had not been treated with agonists (Fig.
2A). Addition of forskolin and isoproterenol (20 µM each) did not result in increased conversion (14 ± 4%) of [
-33P]dADP to [
-33P]dATP,
indicating that elevation of cellular cyclic AMP did not promote the
release of ATP (Fig. 2B). However, an ~2-fold greater conversion (26 ± 3%) of [
-33P]dADP to
[
-33P]dATP was observed following addition of 1 µM ionomycin, consistent with a Ca2+-promoted
ATP release (Fig. 2C). A 4-fold increased formation (71 ± 6%) of [
-33P]dATP occurred following mechanical
stimulation of the cells (Fig. 2D). The calculated ATP
concentrations were 6 ± 1, 5 ± 1, 17 ± 3, and 97 ± 11 nM for control cells, cAMP-stimulated cells, ionomycin-treated cells, and mechanically stimulated cells,
respectively.
|
|
|
The P2Y2 Receptor as a Biosensor for ATP--
Finally,
in an attempt to assay for CFTR-dependent ATP release in
the relevant physiological environment, i.e. the liquid layer associated with the cell surface, we used the P2Y2
receptor as a biosensor for ATP to investigate the role of CFTR in the activation of P2Y2 receptors in intact cells. As such, we
took advantage of a cell line (1321N1 human astrocytoma cells) that is
null for expression of P2 receptors (32). First, we achieved a high
expression level of P2Y2 receptors by retroviral infection of 1321N1 cells with the cDNA encoding the human P2Y2
receptor (32). 1321N1 cells stably expressing the human
P2Y2 receptor (HP2U-1321N1 cells) exhibit a marked
ATP-stimulated formation of inositol phosphates, whereas control cells
are unresponsive (9). Overexpression of the P2Y2 receptor
in 1321N1 cells resulted in large receptor reserve, and consequently,
ATP potency in HP2U-1321N1 cells (EC50 = 180 nM
(9)) is increased 30-fold relative to ATP potency observed with the
P2Y2 receptor natively expressed in airway epithelial cells
(38). More important, significant accumulation of inositol phosphates
in HP2U-1321N1 cells was detectable at concentrations of ATP as low as
10 nM (see Fig. 6 and Ref. 9). Second, we infected the
HP2U-1321N1 cells with an adenoviral vector containing the CFTR
cDNA (AdCFTR) or with adenoviral vectors encoding the
LacZ gene (AdLacZ) as a control and assayed for the effect
of CFTR expression on the accumulation of inositol phosphates. Fig.
5A shows a Western blot for
CFTR of lysates from AdLacZ- or AdCFTR-infected cells. A band (~180
kDa), revealed by an anti-CFTR antiserum (antiserum 858 (35)), was
present in AdCFTR-infected cells, but not in cells infected with the
LacZ vector. A functional cyclic AMP-activated
Cl permeability was demonstrated in AdCFTR-infected
HP2U-1321N1 cells, but not in AdLacZ-infected cells, as indicated by
forskolin- and isoproterenol-stimulated 36Cl
efflux (Fig. 5B).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study demonstrates the presence of ATP in the mucosal surface liquid of nonstimulated airway epithelial cells and that accumulation of extracellular ATP increases substantially when cells are subjected to mechanical stimuli. Unlike previous studies in which CFTR was reported to act as an ATP channel (15, 16, 21), we found no evidence involving CFTR in the release of ATP from intact epithelial and non-epithelial cells. The cause of this discrepancy may reside, at least in part, in the methodological approaches.
Previously, studies implicating CFTR as an ATP channel have employed permeabilized cells, membrane patches, and/or repetitive changes of cell media and in general have subjected the cells to stresses not consistent with physiological conditions. We have used both classical methods for extracellular detection of ATP, e.g. the luciferase assay, and HPLC separation of 3H-labeled nucleotides released from [3H]adenine-preloaded cells as well as newly developed approaches for trapping ATP with NDPK and have adopted conditions in which sampling for ATP release was carefully controlled. Although we were able to detect ATP accumulation in the liquids bathing the surface of resting normal HNE cells, we observed no differences after incubating the cells with agents that promoted elevation of intracellular cyclic AMP. Moreover, no differences were found in extracellular ATP accumulation with nasal epithelial cells from normal or CF mice under basal conditions or as a function of cyclic AMP pathway stimulation. We have shown here that a marked release of ATP occurred after mechanical stimulation of airway epithelial cell surfaces by a medium displacement. However, mechanically stimulated release of ATP was of the same magnitude in normal or CF airway epithelial cells as well as in control or CFTR-expressing non-epithelial cells. A CFTR-independent mechanically stimulated ATP release was also recently reported with colonic and human airway epithelial cell lines (23).
To directly test the potential regulation of P2Y2 receptors by CFTR, we have used a functional assay in which the P2Y2 receptor acted as a biosensor for releasable ATP. We were able to couple the advantage of the high expression level of recombinant P2Y2 receptors attainable by retroviral infection of 1321N1 cells with adenovirus-mediated expression of CFTR in these cells to test the effect of CFTR activation on P2Y2 receptor-promoted inositol phosphate formation. Our hypothesis was that local accumulation of CFTR-releasable ATP in the unstirred and consequently difficult to sample liquid layer on the cell surface would be detected by coexpression of these two recombinant proteins on the surface of 1321N1 cells. Consistent with our previous reports showing mechanically promoted release of ATP from 1321N1 cells (9, 36), a sustained accumulation of inositol phosphates was observed following a medium displacement (Fig. 6). More important, inositol phosphate accumulation in 1321N1 cells was of the same magnitude regardless of CFTR expression, indicating that CFTR was not involved in mechanically induced ATP release. Although HP2U-1321N1 cells were sensitive to a 10 nM concentration of exogenously added ATP, no effect on inositol phosphates was observed following addition of forskolin and isoproterenol to CFTR-transduced HP2U-1321N1 cells as compared with control cells. Thus, activation of CFTR in 1321N1 cells did not result in accumulation of extracellular ATP in concentrations high enough to promote P2Y2 receptor activation. In summary, by functionally expressing both CFTR and P2Y2 receptors in a null cell line, we were able to directly test the hypothesis that CFTR regulates the activity of P2Y2 receptors. We have found no evidence to support this hypothesis.
The presence of P2Y2 receptors in the airway epithelia (32,
38) and their coupling to a Cl secretory pathway on the
mucosal surface of HNE cells (11) suggest that extracellular
accumulation of nucleotides might be important for the regulation of
ion permeabilities. The recent identification of an ectonucleotidase
activity on HNE cells (29) suggests one mechanism for regulating
extracellular accumulation of nucleotides. Our present study indicates
that ~20 pmol of ATP/106 cells accumulate at steady state
on the mucosal surface of resting normal HNE cells. This closely
resembles the ATP concentration observed in the lumen of normal human
airways in vivo (39). Furthermore, ATP transiently
accumulates following mechanical stimulation of cells in concentrations
capable of activating P2Y2 receptors. Previously,
mechanically induced non-lytic release of ATP had been described in a
variety of tissues. For example, changes in blood fluxes were shown to
cause the release of ATP from endothelial cells (10, 40); shear forces
promoted ATP release from mouse fibroblasts (6); and ATP release
secondary to mechanical stimulation was also described with rat
basophilic cells (7), 1321N1 human astrocytoma cells (9, 36), and rat
hepatocytes (8). Stretch-promoted ATP release was reported with rabbit
urinary bladder epithelial cells (41) and human lung adenocarcinoma
Calu-3 cells (40), and ATP release triggered by bath turbulence was
observed with T84 cells and with immortalized human tracheal epithelial
cell lines (23, 26). Our current study extends these observations to
primary human airway epithelial cells. It is not clear whether
mechanical stimulation of airway epithelial cells and release across
the epithelial surface occurs in vivo and what its
functional significance might be. Mechanical forces applied on the
airways by air fluxes may represent a primary mechanism for autocrine
and paracrine regulation of electrolyte homeostasis in airway
epithelia.
In conclusion, the techniques used in this study for detecting extracellular ATP are thought to closely approximate physiological conditions, creating a system in which pertinent and reliable information is generated. We found that ATP is released from each of the studied cell types in response to mechanical stimulation and/or secondary to elevation of intracellular calcium levels. No evidence was found indicating that CFTR was involved in ATP release in airway epithelia from two species. This study suggests that CFTR itself does not directly effect ATP or regulate other possible ATP release pathways in the cells we have studied, but our data do not rule out possible CFTR-dependent regulation of ATP release pathways in other cell types. The occurrence of ATP release independent of activation of CFTR raises new possibilities for treatment of CF. For potential therapeutic targeting, it may important to identify, at the molecular level, the mechanism involved in nucleotide release from airway epithelia.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. R. Yankaskas for assistance in obtaining tissues and L. Brown for editorial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL34322 and HL42384 and by Cystic Fibrosis Foundation Grant R026. W. C. W. and E. R. L. contributed equally to this work.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: Dept. of Medicine, CB
7248, School of Medicine, University of North Carolina, Chapel Hill, NC
27599-7248. Tel.: 919-966-7046: Fax: 919-966-7524; E-mail:
edu{at}med.unc.edu.
1 The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane regulator; Cla, alternative chloride channel; HNE, human nasal epithelial; DMEM-H, Dulbecco's modified high glucose Eagle's medium; HPLC, high pressure liquid chromatography; NDPK, nucleoside diphosphokinase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|