Regulation of mucin secretion from human bronchial epithelial
cells grown in murine hosted xenografts
Jason D.
Conway1,
Tracy
Bartolotta1,
Lubna H.
Abdullah1, and
C. William
Davis1,2
1 Cystic Fibrosis/Pulmonary Research and Treatment
Center and 2 Department of Cell and Molecular
Physiology, University of North Carolina, Chapel Hill, North Carolina
27599-7248
 |
ABSTRACT |
Studies of regulated mucin secretion
from goblet cells in primary cultures of human bronchial epithelial
(HBE) cells have suffered, generally, from poor signal-to-noise ratios,
with reported secretory responses of <100% (less than onefold)
relative to baseline. Using, instead, HBE cells grown as xenografts in
the backs of nude mice, we found that UTP (100 µM) stimulated strong
mucin secretory responses from isolated, luminally perfused
preparations. The peak response (10 min) for 11 control experiments (37 xenografts) was 3.3 ± 0.05-fold relative to baseline, and the
time-integrated response (60 min) was 23.4 ± 0.5-fold. Because
responses to ATP and UTP were approximately equal, an apical membrane
P2Y2-receptor (R) is suggested. Additionally, ADP activated
mucin release from HBE xenografts, whereas UDP and 2-methlythio-ADP did
not, a pattern of response inconsistent with known purinoceptors.
Hence, either a novel receptor to ADP is suggested or there is
significant conversion of ADP to ATP by ecto-adenylate kinase activity.
Adenosine and a nitric oxide donor were without effect. Consistent with
P2Y2-R coupling to phospholipase C, HBE xenografts
responded to ionomycin and PMA; however, they were recalcitrant to
forskolin and chlorophenylthio-cAMP, and to 8-bromo-cGMP. Hence,
human airway goblet cells, like those of other species, appear to be
regulated primarily via phospholipase C pathways, activated
particularly by apical membrane P2Y2-R agonists.
mucus; purinergic regulation; purinoceptor; intracellular
messengers
 |
INTRODUCTION |
IMPROVEMENTS IN
AIRWAY EPITHELIAL cell culture techniques over the past several
years have enabled rigorous studies of regulated mucin secretion from
superficial epithelial goblet cells in a variety of species (see Refs.
12, 30, 53). These efforts have
led to the realization that ATP and UTP acting at apical membrane
P2Y2 receptors provide a principal pathway for the
regulation of mucin secretion from the superficial epithelium in the
airways of all species studied to date; no other G protein-coupled
receptor (GPCR) agonist has been implicated, consistently, in
stimulating mucin secretion (13, 24, 30). Given the
potential of artifact resulting from the use of cell culture models,
however, it is vital that these results be verified in native tissue to
ensure biological and clinical relevancy. On this point, the available data are sparse: the only studies testing the effects of purinergic agonists against goblet cells from native tissues are those from our
laboratory, which employed explants of isolated superficial epithelium
from canine trachea and human nasal turbinates, using video microscopy
to assay for mucin secretory activity (14, 36).
Airway epithelial cells grown in denuded tracheas as xenografts have
been used for many years, originally to study tumor induction (5) and to determine the differentiation potential of
airway epithelial progenitor cells (9, 25). Additionally,
xenografts have also proven useful more recently in providing human
airway epithelia for studies related to cystic fibrosis and gene
therapy (e.g., Refs. 16, 38). In this technique, the
lumens of tracheas denuded of native cells by freeze-thawing are seeded
with airway epithelial cells, and the trachea is implanted
subcutaneously as a tracheal graft in syngeneic rats or mice or as a
tracheal xenograft in an immune-compromised host, commonly a nude
mouse. The grafted trachea is revascularized by the host, and the
epithelial progenitor cells within multiply and develop into a mature
epithelium under the influence of growth and differentiation factors
provided by the host. In this study, we used tracheal xenografts
bearing differentiated human bronchial epithelial cells to test the
mucin secretory responses of human goblet cells to a variety of
purinergic agonists and other secretagogues characteristically active
in the airways. These data may be useful in establishing a surrogate "gold standard," against which results from primary cultures of airway epithelial cells from humans and other species might be compared.
 |
MATERIALS AND METHODS |
Materials.
Purinergic agonists were purchased from Boehringer-Mannheim
(Indianapolis, IN), culture medium was purchased from GIBCO-BRL (Gaithersburg, MD), and the supplements were from Collaborative Research (Bedford, MA). Ionomycin and PMA were purchased from Calbiochem (San Diego, CA). All other chemicals used were purchased from Sigma Chemical (St. Louis, MO) or are specified below.
Cell culture and tracheal xenografts.
SPOC1 cells were grown as described previously (3). Human
bronchial epithelial (HBE) cells were isolated under the auspices of
Institutional Review Board-approved protocols as described in detail
previously (40, 41) from freshly excised human bronchi from an individual with cystic fibrosis. The isolated cells
were grown in primary culture on plastic in bronchial epithelial cell growth medium (BEGM) (20), harvested at 70-80%
confluence, frozen in aliquots, and stored in liquid N2. At
intervals, vials were thawed, passage 1 cells were expanded
on plastic in BEGM and harvested at 70-80% confluence, and
passage 2 cells were seeded into denuded tracheas at
~1 × 106 cells/graft in a volume of 50 µl. The
tracheas used were harvested from 5-day-old chickens and were denuded
of indigenous cells by three freeze-thaw cycles separated by luminal
PBS washes. The tracheas were cannulated at each end with short lengths
of polyethylene (PE) tubing and supported with a stint formed by tying
a length of PE tubing to the cannulas. Batches of prepared tracheas
were frozen until use. After heat sealing the cannulas, we implanted the xenografts subcutaneously into the backs of athymic nu/nu BALB/c
mice (see Ref. 47) and harvested them after a 3-wk
incubation. All animal procedures were performed under Institutional
Animal Care and Use Committee-approved protocols.
At harvest, the xenograft lumens were gently flushed with 1 ml of PBS
and mounted horizontally for perfusion in a bath of DMEM/F-12. The bath
and the affluent tubing floated in a water bath (37°C), and the
xenografts (internal volume ~50 µl) were perfused with DMEM/F-12 at
50 µl/min. The organ and water baths were covered with a clear
plastic sheet, and they, and the perfusion medium, were bubbled with
5% CO2-95% air. After a 2-h equilibration, the perfusates
were collected with a fraction collector, and 5-min fractions were
assessed for mucins.
Mucin assays.
Mucins secreted by SPOC1 cells were detected in microtiter plates by an
SBA ELLA as described previously (3).
Monoclonal antibody production.
A monoclonal antibody (MAb), H6C5, was generated against human mucins
purified from sputum collected from a patient with cystic fibrosis.
From 9.65 g of sputum solubilized in 6 M
guanidine · HCl (with EGTA and protease
inhibitors), 22 mg of mucins were purified by CsCl density gradient
centrifugation as described previously (3). The material
was relatively free of DNA, as indicated by a negligible absorbance at
260 nm and a complete lack of staining by ethidium bromide, and was
judged to be mucin from its peak density of 1.48 g/ml, an amino acid
composition rich in Gly, Thr, Ser, and Pro (178.3, 158.0, 98.9, and
92.7 residues/1,000, respectively), and high reactivity in a sialic
acid assay (27, 48, 49). Antibodies to this material were
generated in 4- to 6-wk-old mice, which were injected with antigen into
hindfoot pads and the lateral thoracic and inguinal regions on
days 1, 3, 6, 9, and
12. On day 14, lymphocytes were isolated from
dissected lymph nodes (axillary, brachial, inguinal, and poplitea) and
fused with mouse myeloma cells. From the several clones that resulted from this fusion, 11 produced monoclonal antibodies that tested positive in an initial mucin-screening ELISA. Of these, several were
rejected for nonspecific staining patterns. Of the MAbs that bound
mucin with high avidity and exhibited similar staining patterns in
airway tissue sections, we chose one, H6C5, to use as a mucin detection reagent.
H6C5 ELISA.
Samples (100 µl) of perfusate, diluted appropriately to the standard
curve of preliminary assays, and mucin standards were bound to 96-well,
high-binding microtiter plates (Costar no. 3590) overnight at 4°C.
After being washed with PBS containing 0.05% Tween 20 and 0.02%
Thimerosol (PBST), the plates were blocked with 5% dry milk in PBST,
incubated with H6C5 for 1 h at 37°C or overnight at 4°C,
washed in PBST, incubated with horseradish peroxidase
(HRP)-conjugated secondary antibody (1 h, 37°C), washed in PBST, and
developed during a 15-min incubation in 0.04% wt/vol of the substrate
O-phenylenediamine (OPD) in 0.0175 M citrate-phosphate buffer, pH 5.0, containing 0.01% hydrogen peroxide. The reaction was
stopped with the addition of 4 M sulfuric acid, optical density at 490 nm determined in a microtiter plate reader (model MR5000; Dynatech,
Chantilly, VA), and the mucin content (ng) of each well was calculated
with a standard curve for purified human mucins constructed on each
microtiter plate.
Periodic acid, biotin-hydrazide assay.
To validate the results obtained with H6C5, we developed a periodate
staining procedure suitable to small volumes in a microtiter plate
format. Key to the reaction was the substitution of Schiff's reagent,
commonly used in periodic acid-Schiff (PAS) staining, with the
aldehyde-reactive reagent hydrazide. One hundred-microliter samples of
perfusate and mucin standards were bound to 96-well, high-binding
microtiter plates as above. All remaining steps in the procedure were
performed at room temperature. The plates were washed four times with
PBST, oxidized with 1 mM periodic acid (100 µl) for 10 min in the
dark, and then incubated for 45 min with an additional 50 µl
biotin-conjugated hydrazide (0.1 mM) containing 1.0 mM sodium
metabisulfite. After four 5-min washes in PBST, the plates were
incubated with streptavidin-conjugated HRP (1:5,000 in PBST, 100 µl/well) for 20 min and then washed again with four changes of PBST.
Lastly, the plates were developed with OPD substrate solution, the
reaction was stopped with 4 M H2SO4, the
reactions were assessed for optical density (490 nm), and the mucin
content/well was calculated and expressed, as above.
Histology.
Paraffin blocks of human bronchi fixed in 4% paraformaldehyde
(immunostaining) or 10% neutral-buffered formalin [PAS and periodic acid, biotin-hydrazide (PABH) staining] were cut in 8-µm sections and deparaffinized by standard techniques. For immunostaining, sections
were blocked with 5% goat serum in PBS, incubated with or without
(control) H6C5 MAb, washed, incubated with AutoProbe alkaline
phosphatase-conjugated goat anti-mouse IgG secondary antibody
(Biomedia, Foster City, CA), washed, counterstained, and coverslipped.
For PABH staining, sections were oxidized for 30 min in 22 mM periodic
acid, rinsed, incubated in sodium metabisulfite in acetate buffer,
rinsed, blocked with avidin and biotin (Avidin/Biotin Blocking kit;
Vector Laboratories, Burlingame, CA), rinsed, incubated 60 min with
biotin hydrazide (33 mM) plus 1 µl of HRP streptavidin (Vector
Laboratories), rinsed, reduced with sodium borohydrate, and rinsed. The
stain was developed with diaminobenzidine tetrahydrochloride (Sigma),
followed by a rinse, counterstaining, and coverslipping. Sections were
also stained with PAS by standard techniques. All slides were
counterstained with hematoxylin (Bluing Reagent; Richard Allen,
Kalamazoo, MI).
 |
RESULTS |
Airway epithelial cells grown in xenografts.
SPOC1 cells were used to test whether the higher degree of
differentiation achieved by growing the cells in xenografts, versus culture (see Ref. 47), is accompanied by a more robust
mucin secretory response. Figure 1 shows
the time course of mucin release elicited by UTP (100 µM) for three
SPOC1 cell xenografts. For comparison, the figure also shows our
original data derived from UTP-stimulated SPOC1 cells in culture
(3), recalculated to normalize the data to baseline. The
difference between the two response patterns is notable. The
xenograft-grown SPOC1 cells exhibit a mucin secretory response that
rose quickly to a peak and then declined in a manner we have recorded
with native goblet cells of epithelial explants from canine trachea
(13) and from human turbinates (36) and as we
show below for HBE xenografts. The decline in mucin secretion likely
reflects a receptor desensitization phenomenon and/or depletion of
mucin stores. SPOC1 cells grown in culture, in contrast, are stimulated
by UTP in an essentially undiminished pattern of release (with ATP, as
shown in Fig. 7 of Ref. 3, there was
literally no sign of a decline). In addition to an improved waveform,
the magnitude of the xenograft-grown SPOC1 cell mucin secretory
response was greater relative to cells grown in culture: respectively,
the peak responses were 10.6- vs. 3.5-fold relative to baseline
(onefold = 100% above baseline), and the integrated responses (1 h) were 59.3- vs. 34.2-fold. Hence, not only do SPOC1 cells grown in
xenografts have a more robust goblet cell phenotype than those grown in
culture, the time course and magnitude of their mucin secretory
response to purinergic agonists are also superior.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
Mucin secretory response to UTP (100 µM) in xenografts
populated with SPOC1 cells. In this and subsequent mucin secretory time
courses, each point represents the mucins released (means ± SE,
n = 3) at the indicated times for the perfused
xenografts. Mucin secretion for this and most such studies with HBE
xenografts was normalized to the mean of the 30-min baseline values.
Inset: mucin secretory response of SPOC1 cells grown in
culture. Normalized data calculated from the original presented in Ref.
3 (n = 6).
|
|
HBE cells seeded into denuded tracheas repopulated the grafts and
differentiated into a mature mucociliary epithelial phenotype over the
3-wk period of incubation in the backs of nude mice (Fig. 2). The epithelium was pseudostratified,
and the columnar cells were predominantly ciliated, but as shown in
Fig. 2, inset, there were also plentiful PAS-positive goblet
cells. Notably, at harvest, the lumens of the HBE xenografts contained
a compact mucous-like plug. After expression by syringe with 1 ml of
PBS and solubilization in 6 M guanidinium hydrochloride, this material
resolved as a single band of low mobility on agarose blots stained with
PAS, which, retrospectively, also stained with mucin-specific
antibodies to MUC5AC and MUC5B (data not shown).

View larger version (123K):
[in this window]
[in a new window]
|
Fig. 2.
Human bronchial epithelial (HBE) xenograft histology.
Larger image is a cross-sectional view of a hematoxylin-eosin-stained,
3-wk-old HBE xenograft, ~3 mm in diameter, after the lumen was
flushed with PBS. Note the uniform pseudostratified epithelium with
ciliated border. Inset: higher-magnification view of the
epithelium stained with periodic acid-Schiff (PAS) to identify goblet
cells.
|
|
H6C5/PABH staining and mucin assays.
During its development, the H6C5 MAb was selected on the basis of its
avid binding of mucin in microtiter plate screening assays. When
applied to paraffin sections of human airways, the MAb intensely
stained goblet cells, including their secretory granules, and the
ciliary border (Fig. 3), and mucous cells
in submucosal glands (data not shown). The heavy staining of cilia by
MAbs generated against intact mucins has been noted previously e.g.,
for 17Q2 (50). In the case of H6C5, the ciliary staining was apparently due to an extracellular epitope, since positive staining
was achieved by the luminal application of the MAb to fresh tissue
before fixation (data not shown).

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 3.
Mucin detection. A-D: staining patterns
of human bronchial tissue for mucin detection reagents. A:
H6C5 (primary MAb) staining of goblet cells and ciliated border. The
image was developed with an alkaline phosphatase-conjugated secondary
Ab. B: control for A, without primary MAb.
C: PAS. D: Periodic acid/biotin-hydrazide (PABH).
The image was developed with horseradish peroxidase-conjugated
streptavidin. For each image, the counterstain was hematoxylin.
E: binding assays. Standard curves from microtiter plate
binding assays generated from purified cystic fibrosis (CF) mucin.
Mucins bound to the plates were detected with H6C5, in an ELISA format,
or with the PABH procedure. The data are presented as means ± SE
of 6 separate assays.
|
|
The procedure for PABH staining is based on the reaction of hydrazide
with the dialdehyde produced in carbohydrates susceptible to periodate
oxidation. In this regard, it is similar to Schiff's reagent; however,
the use of hydrazide has two advantages that makes the PABH assay more
sensitive than PAS. First, each dialdehyde reacts with two molecules of
hydrazide, as opposed to one molecule of Schiff's reagent. Second,
hydrazide can be coupled to many other reagents that might be useful as
markers of periodate oxidation. In the PABH assay, the use of
biotin-hydrazide allowed an enzymatic amplification of the signal with
HRP-conjugated streptavidin. Figure 3, C and D,
compares the staining patterns for PAS and PABH in HBE. Both procedures
stained goblet cells intensely and with good selectivity. The heavily
stained ciliated border in the PABH-treated preparation is the only
readily discernible difference between the two staining patterns and
one that likely reflects signal amplification.
Figure 3E shows the relative sensitivities of the mucin
binding assays developed by PABH staining and the H6C5 ELISA. Both assays were approximately linear over much of their respective ranges,
and both were very sensitive for mucin with lower detection limits in
the low nanogram per milliliter mucin range.
Response of HBE xenografts to UTP and ATP.
After a 2-h equilibration perfusion and a 30-min basal secretion
period, HBE cell xenografts responded to 100 µM UTP added to the
perfusate with a vigorous increase in mucin secretion (Fig. 4). In this experiment, the samples
collected at each 5-min time point were assessed for mucins by both the
H6C5 and PABH assays. As shown, the apparent baseline level of mucin
secretion detected by H6C5 was substantially higher than that detected
by PABH: at 1,526.9 ± 74 ng/fraction the H6C5-detected material
was 40% higher than the 915 ± 36 ng/fraction detected by PABH.
The time courses of the UTP secretory response reported by the two
assays, however, were very similar. In both cases, the mucins released
from the epithelium peaked 10 min after the agonist challenge and then slowly declined to values near baseline over the next 40 min. In fact,
when the mean baseline rates of secreted materials were subtracted from
their respective datasets, the two curves essentially overlaid one
another (Fig. 4, inset). This observation most
likely indicates that H6C5 detects other materials in addition to
polymeric mucins. Because the release of these other materials appears
to be constant, however, they may emanate from sources other than the
regulated secretory pathway, e.g., they may be secreted from constitutive pathways and/or are shed from the luminal surface. Despite
this drawback, we chose to use the H6C5 ELISA for the practical
advantage of a simpler procedure in testing the large number of samples
necessary for the routine monitoring of HBE xenograft mucin secretory
responses.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
Response of HBE goblet cells to UTP (100 µM). HBE
xenografts were perfused at 50 µl/min, and the fractions were
collected at 5-min intervals following a 2-h equilibration. After
collecting fractions during a 30-min baseline period, the perfusate was
switched to one containing UTP (arrow) for a 60-min agonist challenge.
The fractions were assessed for mucin content by H6C5 ELISA and by the
PABH plate assays, and the results are expressed as the means ± SE mucin (ng)/fraction (n = 5). Inset: same
data for both H6C5 and PABH assays, following subtraction from each
point of the mean baseline level of detected mucins secreted (5-30
min).
|
|
Important to our strategy of secretagogue challenges with limited
numbers of preparations was the testing of each HBE xenograft for a
UTP-elicited mucin secretory response as an internal control. To this
end, we examined the ability of HBE xenografts to mount a secretory
response over time with two sets of perfused HBE xenografts. Both sets
received a standard 2-h equilibration perfusion, and fractions were
then collected at 5-min intervals for 3 h. After establishing a
30-min baseline, we gave the first set of xenografts a 100 µM UTP
challenge for 60 min, following which UTP was removed for a 30-min
washout period. The second set was perfused with control medium for
this 120-min period. After collecting fractions to establish a second
baseline, we then challenged both sets of HBE xenografts with 100 µM
UTP for 60 min. As shown in Fig. 5, the
mucin secretory responses elicited by the first UTP challenge to each
set of HBE xenografts were very similar, despite the fact that one had
been perfused for 90 min longer than the other: peak responses were
3.1 ± 0.3 and 3.3 ± 0.3-fold higher than baseline, and the
relative integrated responses were 26.3 ± 2.4- and 21.7 ± 1.4-fold higher than baseline, respectively. This result shows the HBE
xenograft to be a robust preparation capable of mounting a full mucin
secretory response after 4 h of perfusion.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of UTP on mucin secretion from HBE cell
xenografts. After equilibration and collection of fractions during an
initial baseline period, one set of HBE xenografts received a perfusate
containing UTP (100 µM) for 1 h. The UTP was then washed out for
30 min and reintroduced. The other set of xenografts was exposed to UTP
only during the second exposure for the first set. The data are
expressed relative to the mean level of mucin secretion recorded during
the first baseline period (n = 3, each).
|
|
For the HBE xenografts receiving a UTP exposure twice, the 30-min
washout period allowed between the two challenges was apparently too
brief to allow the tissue to recover from the initial challenge. The
weak response to the second challenge could have been due to a receptor
desensitization phenomenon and/or to depletion of mucin granules during
the initial challenge.
ATP (100 µM) stimulated mucin secretion from HBE xenografts with a
time course and magnitude similar to that for UTP (data not shown). The
ATP-induced peak response, 4.4 ± 0.4-fold over baseline
(n = 3), occurred 10-15 min following agonist
challenge before declining toward baseline values. UTP, added to the
perfusate with ATP 45 min after the peak response to ATP, had no
additional effect.
Effects of nucleotide diphosphates.
Of the six known P2Y purinoceptors (R), three are specific to
diphosphate nucleotides (Table 1),
P2Y1-R, P2Y6-R, and P2Y12-R. Figure
6 shows that, of the hallmark agonists
for these purinoceptors, only ADP elicited a response from HBE
xenografts. In this case, the peak response was approximately one-half
that elicited by UTP and ATP, and UTP applied subsequently elicited a
second, higher maximal response than did ADP. The response to ADP, with
the lack of response to 2-methylthio (2-MeS)-ADP, is interesting and
may indicate an extracellular adenylate kinase activity or a novel receptor (see DISCUSSION).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of nucleotide diphosphates on mucin secretion
from HBE cell xenografts. In each experiment, after perfusion of the
xenografts for baseline mucin secretion and with 100 µM of the
initial agonist (indicated), the perfusate was switched to one
containing UTP (100 µM).
|
|
Effects of adenosine and nitric oxide.
Adenosine and nitric oxide (NO) are major, local regulators in many
physiological systems, and recent reports have suggested that these
agents are involved in the regulation of airway epithelial cells.
Adenosine, acting through A2B receptors, has been shown recently to be a major regulator of ciliary activity in human nasal
epithelial cells (43) and of CFTR channel activity and Cl
transport in Calu-3 cells (24). NO has
been implicated in many lung functions (e.g., 18), including the
regulation of mucin secretion (4, 17, 52). Neither
adenosine nor an NO donor, however, had detectable effects on mucin
secretion from HBE xenografts (Fig. 7),
whereas subsequent exposures of the same tissues to UTP elicited
typical, peak secretory responses of 4.6- and 3.1-fold over baseline.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 7.
Lack of effects of adenosine (ADO) and a nitric oxide
(NO) donor on mucin secretion from HBE xenografts. After equilibration
and collection of baseline fractions, HBE xenografts were exposed
either to ADO (100 µM; top) or to
S-nitroso-N-acetyl-penicillamine (SNAP, 100 µM), a NO donor. After a 1-h exposure to the putative secretagogue,
UTP was either substituted for (ADO) or added to (SNAP) the perfusate
(n = 3, each).
|
|
Intracellular messengers.
Permeant analogs of intracellular messengers, or agents that
pharmacologically mimic or stimulate cellular messenger production or
release, were tested for their effects on mucin secretion from HBE
xenografts. To test the potential effects of cAMP on mucin secretion,
we used both forskolin and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic
monophosphate (cpt-cAMP), and for cGMP we used 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP). As shown in Fig.
8, none of these reagents elicited a
detectible mucin secretory response from HBE xenografts, whereas
subsequent exposures to UTP elicited normal responses in each case.
Notably, cpt-cAMP used at the same concentration does stimulate ciliary
activity in small explants of HBE cells (43), showing that
this compound does permeate airway epithelial cells as expected.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of cyclic nucleotides on mucin secretion from HBE
xenografts. After equilibration and collection of baseline fractions,
HBE xenografts were exposed either to forskolin (10 µM) or to the
permeant cyclic nucleotide analogs 8-(4-chlorophenylthio)adenosine
3',5'-cyclic monophosphate (cpt-cAMP, 0.5 mM) and 8-bromoguanosine
3',5'-cyclic monophosphate (8-Br-cGMP, 1 mM), for 60 min, as
indicated by the width of the box enclosing the symbol key. After a
30-min washout, UTP (100 µM) was then added to the perfusate for an
additional hour. For clarity, error bars are shown only for the dataset
with the largest SEs (n = 3, each).
|
|
Most P2Y receptors couple to Gq, which activates PLC to
release diacylglycerol (DAG) and inositol 1,4,5-trisphosphate
(45, 51), and activating either limb of this
pathway resulted in a stimulation of mucin secretion (Figs. 9 and 10).
Figure 9, top, shows the time
course of the effects of the Ca2+ ionophore ionomycin (3 µM) on mucin secretion, and the bottom panel shows its
concentration-dependent effects on the integrated response.
Mobilizing intracellular Ca2+ caused approximately
a 2.5-fold increase in peak mucin secretion from human goblet cells at
3 µM, similar to its effects in SPOC1 cells (2). The
ionomycin secretory response was long lasting and was concentration
dependent, saturating above 3 µM. UTP, added at the end of the
ionomycin exposures, stimulated mucin secretion above the plateau level
following ionomycin. The degree of postionomycin UTP stimulation
decreased with increasing ionomycin concentrations.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 9.
Effects of Ca2+ ionophore on mucin secretion
from HBE cell xenografts. Top: time course for ionomycin
effects. Ionomycin (3 µM) was added to the perfusate after
a 30-min baseline period, and UTP (100 µM) was added after the 1-h
ionomycin exposure (n = 3). Bottom:
concentration-effect relationships. Integrated relative mucin secretory
responses are plotted as the means ± SE (n = 3 or
4). For ionomycin, the period integrated was 35-90 min; for
ionomycin + UTP it was 95-120 min.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 10.
Effects of phorbol ester on mucin secretion from HBE
cell xenografts. Top: time course for PMA effects. PMA (300 nM) was added to the perfusate after a 30-min baseline period, and UTP
(100 µM) was added after the 1-h PMA exposure (n = 3). Bottom: integrated concentration-effect relationships.
Integrated relative mucin secretory responses are plotted as means ± SE (n = 3 or 4). For PMA, the period integrated was
35-90 min; for PMA + UTP it was 95-120 min.
|
|
PMA, the DAG mimic, also stimulated mucin secretion from HBE goblet
cells. Figure 10, top, shows
the time course of the concentration having maximal effects (300 µM)
on mucin secretion, and Fig. 10, bottom, shows its
concentration effects on the integrated response. Overall, the effects
of PMA were similar to those of ionomycin: the stimulatory effect was
concentration dependent up to 300 nM and was long lasting, and UTP
added after the 1-h PMA exposure was stimulatory, especially at the
lower PMA concentrations tested. Exposing HBE xenografts to 1 µM PMA,
however, appeared to be deleterious to both phases of the secretory
response, i.e., mucin secretion elicited by PMA and by PMA plus UTP
were substantially reduced over the levels realized by 300 nM PMA.
 |
DISCUSSION |
Mucin secretion from HBE cultures vs. xenografts.
Purinergic stimulation of mucin secretion from HBE-passaged primary
cultures has been reported previously (11, 37, 54). Although statistically relevant, the mucin secretory responses reported
for these cultures lacked rigor, exhibiting increases relative to
baseline of only 0.6- to 0.75-fold over a period of 2 h.1 In our own hands with
highly differentiated HBE cultures (e.g., Refs. 40,
41), UTP-induced mucin secretion has been equally poor;
mucin secretion has been plentiful during these experiments, but the
increases in secretion obtained during UTP challenges have been trivial
to undetectable (data not shown). By way of comparison, ATP and UTP
induce mucin secretion from SPOC1 cells grown in culture or in
xenografts with peak responses ~3- to 10-fold, relative to baseline,
and 1-h integrated responses of ~30- to 60-fold (Fig. 1). An
additional concern with HBE cultures is that they appear to be
refractory to PMA (37), an agent that has been shown to
stimulate mucin secretion in every other airway goblet cell model so
far tested (2, 13, 28, 30, 33). At this point, it is not
clear whether the apparently universal problem with HBE cultures is
caused by poor handling during experiments, inadequate mucin assays,
and/or inappropriate culture conditions. In any case, the weak
responses reported for HBE cultures to date make it difficult to
understand even the basics of agonist-induced mucin secretion from
human airways, let alone the more complicated phenomena associated with
inflammatory agents. Hence, we sought a more robust experimental model
that might be used to resolve these issues and to this end turned to
xenograft cultures incubated in the backs of nude mice. In this
situation, not only do the cells differentiate under the influence of
growth and differentiation factors offered by a mammalian host, but the
precannulated xenografts proved optimal for a gentle dissection from
the host and preparation for perfusion. As shown in Fig. 1, SPOC1 cells
grown in xenografts where they achieve a more robust goblet cell
phenotype (see Ref. 47) also responded in a more
physiological manner to agonist than do cells grown in culture: the
magnitude of the response was greater, and the time course typified
that expected for an agonist-induced response.
The HBE xenografts proved remarkably robust during experiments: grafts
perfused for a total of 4 h responded equally well to UTP as
grafts perfused for 2.5 h before the UTP challenge (Fig. 5).
Furthermore, the mucin secretory responses mounted by HBE xenografts to
UTP were substantial. In the control experiments (Figs. 4 and 5) and
all the agonist challenge experiments in which there was no response to
the primary secretagogue (e.g., Fig. 6, middle and
bottom), the peak response to 100 µM UTP was 3.3 ± 0.05-fold and the integrated response over 60 min was 23.4 ± 0.5-fold relative to baseline (n = 11, taking the mean
data from each experiment, over a total of 37 xenografts). These data
indicate a preparation more suitable for a pharmacological
characterization of agonist signaling in human goblet cells than
previously reported models.
Responses of HBE goblet cells to purinergic agonists.
Of the nucleotide triphosphate agonists tested, goblet cells in the HBE
xenografts were responsive to both UTP (Figs. 4 and 5) and ATP
(RESULTS). These results are in accordance with our previous findings with video microscopy showing that goblet cells in
isolated epithelial explants of human turbinate epithelium were
stimulated to degranulate by these same agonists (36). For
the three nucleotide triphosphate purinoceptors (P2Y2-R,
P2Y4-R, and P2Y11-R), the approximate equality
of the mucin secretory responses by HBE xenografts to ATP and UTP are
most consistent with a P2Y2-R-mediated response to ATP and
UTP (see Table 1). These nucleotide triphosphates are full agonists at
P2Y2-R, whereas P2Y4-R is activated by UTP and
antagonized by ATP, and P2Y11-R is activated by ATP but not
UTP (45, 51). Hence, on pharmacological grounds there is
no need to hypothesize more than the expression of P2Y2-R
in HBE goblet cells.
Of the nucleotide diphosphate agonists tested, human goblet cells were
responsive to ADP and unresponsive to UDP and 2-MeS-ADP (Fig. 6). ADP
activates P2Y1-R and P2Y12-R; however, both of
these purinoceptors are activated to an even greater degree by
2-MeS-ADP, an agonist to which the HBE xenografts were recalcitrant
(Table 1). Interestingly, canine tracheal goblet cells observed by
video microscopy underwent a partial degranulation in response to ADP but did not respond to 2-MeS-ATP, another full agonist at
P2Y1-R (14). One explanation is that ADP may
act as a partial agonist at a P2Y11, an ATP-selective
purinoceptor that couples to both Gq and Gs
(45, 51). Favoring this possibility is the rather low-grade response to ADP and the fact that UTP applied subsequently to
HBE xenografts elicited an additional, sizable mucin secretory response
(compare Figs. 5 and 6). Against the notion of P2Y11-R involvement in the ADP response, however, is its apparent basolateral localization in epithelial cells (44, 51). Another
possibility is that the effects ascribed to ADP were in fact due to ATP
generated from ADP plus phosphate by extracellular
nucleotide/nucleoside metabolism. Support for this notion is
offered by the recent finding of significant adenylate kinase activity
in airway gland secretions and on epithelial cell surfaces
(15). Hence, the mucin secretory response to ADP by both
human and canine goblet cells is curious and needs to be examined
further to distinguish between these two possibilities and that of a
novel purinoceptor.
Given the lack of response by goblet cells of HBE xenografts (Fig. 7)
and canine trachea (14) to adenosine, the cells appear to
lack apical membrane adenosine receptors. As discussed below, these
results are interesting in light of the strong response to adenosine we
reported recently for ciliated cells (43). Adenosine and
its analogs thus appear to be the only known purinergic agonists that
stimulate ciliary activity (43) and fluid secretion into the airway lumen (24) without stimulating mucin secretion.
This result therefore raises the possibility of an adenosine
receptor-based therapy to stimulate mucociliary clearance in airway
obstructive diseases. Unfortunately, it also has the limiting caveats
that inappropriately high levels of adenosine can elicit airway
bronchospasm and inflammatory responses, including goblet cell
metaplasia (6, 22, 42).
Intracellular messenger systems in HBE goblet cells.
The lack of response by HBE xenografts to permeant cyclic nucleotide
analogs and forskolin (Fig. 8) suggests that mucin secretion from human
goblet cells is not regulated by agonists or other factors whose
effects are mediated by these cellular messenger systems.
These results are consistent with the negative results obtained with adenosine and
S-nitroso-N-acetyl-penicillamine, the NO donor
(Fig. 7), since adenosine effects are generally mediated by adenylate
cyclase and cAMP and since NO effects are mediated by soluble guanylate
cyclase and cGMP. Additionally, the results are consistent with the
lack of response by goblet cells in human turbinates and by SPOC1 cells
to cyclic nucleotides (2).
Again, like human turbinate goblet cells and SPOC1 cells
(2), HBE xenografts responded robustly to challenges with
a Ca2+ ionophore (ionomycin; Fig. 9) and a PKC activator
(PMA; Fig. 10). Although the number of HBE xenografts available for
these studies was limited, we were able to demonstrate a concentration dependency in the goblet cell secretory response for both ionomycin and
PMA. Hence, these results suggest that mucin secretion in human goblet
cells is regulated by cellular messengers generated by PLC.
The results with PMA deserve special comment in light of those recently
derived from SPOC1 cells (1). This study showed that PMA
activated PKC maximally at 30 nM, whereas mucin secretion was
stimulated at levels up to 300 nM, suggesting a PKC-independent effect
of PMA. PMA has been suggested to activate other C1 domain proteins in
cells (29), a prime candidate for which is MUNC13, an
obligate accessory protein to the exocytotic complex (10) that we found was expressed in SPOC1 cells (1).
Interestingly, HBE xenografts were stimulated nearly threefold more at
300 nM PMA than at 100 nM, the concentration chosen to ensure that PKC was activated maximally (Fig. 10). Hence, this result suggests that
PMA, at high concentrations, acts via a PKC-independent mechanism to
stimulate mucin secretion from human goblet cells, similar to its
effects in SPOC1 cells.
A variety of studies have implicated NO and/or cGMP in stimulating
mucin secretion from primary cultures of guinea pig tracheal (4,
17, 52) and HBE cells (37). These results are at variance, however, with those presented herein with HBE xenografts (Figs. 7 and 8) and with our previous results from SPOC1 cells (2), both of which showed NO- and/or cGMP-active agents to be without effect. Mucin secretion from submucosal glands is also either unaffected or inhibited by NO (8). One general
problem with the studies claiming NO/cGMP responsiveness is that the
cultures used exhibited relatively low secretory responses (less than
onefold relative to baseline; see above), which can easily
hinder data analysis. A more specific problem was apparent in the
studies with HBE cultures (37) in which PMA (100 nM) and 8 Br-cGMP (1 mM) elicited a small, 2-h, integrated mucin secretory
response (of ~1.1-fold) only when used in combination. When used
separately, neither reagent elicited mucin release. In the present
study with HBE xenografts, by contrast, 100 nM PMA alone caused a
large, 1-h, integrated mucin secretory response of 5.3 ± 1.9-fold
(n = 4) relative to baseline, and the response
increased to 14.4 ± 4.2-fold (n = 5) at 300 nM
PMA (Fig. 10). Hence, activation of PKC by PMA, alone, appears
sufficient to stimulate mucin secretion from human goblet cells. This
result is consistent with the effects of PMA in eliciting mucin release
from SPOC1 cells (2) from primary cultures of hamster
(28) and feline (33) tracheal epithelial
cells and from a variety of other mucin-secreting cells (e.g., 19, 23, 32). In light of these considerations, the roles of NO and cGMP in the
mucin secretory responses of HBE and other airway cultures may need to
be reconsidered.
Goblet cells and purinergic signaling in the airways.
Two important questions raised by the original study indicate the
importance of purinergic agonists in airway signaling
(39): what is the source of ATP in the airway lumen, and
how are the different cell types regulated differentially from one
another? After some 12 years, it is now clear that cells in and out of the nervous system release ATP and UTP (7, 21) and
metabolize it in the extracellular space (55), including
airway epithelia (15, 35, 46). Outside of the nervous
system, these agonists and their active metabolites generally work
through P2Y purinoceptors and have local actions. In the lumen of the
airways, they appear to regulate mucociliary clearance
(31). P2Y2-R appears to be a principal
receptor on both ciliated and goblet cells, at which ATP and UTP
stimulate Cl
and fluid secretion (26, 39),
ciliary activity (43), and mucin secretion from goblet
cells (Ref. 36; Fig. 4). More pertinent to the discussion
is the role of nonnucleotide triphosphate receptors. UDP
(P2Y6) and adenosine (A2BAR) both stimulate
increases in Cl
and fluid secretion from ciliated cells
(34), as well as ciliary activity (43), but
these agonists have no effect on goblet cells (Figs. 6 and 7). For
goblet cells, ADP may be a mucin secretagogue (Fig. 6), perhaps acting
through a novel purinoceptor or in conjunction with an ecto-adenylate
kinase, but the agonist has no apparent direct effects on ciliary cell
function (43). Hence, the luminal metabolites of UTP and
ATP may regulate ciliary and goblet cell function independently. These
actions are likely to be controlled by the local rates of ATP and UTP
secretion into the lumen, depth of airway surface liquid, rates of free
and surface-active extracellular nucleotide metabolism and uptake
mechanisms, and the availability of appropriate purinoceptors in the
vicinity of agonists. Thus mucociliary clearance and its local
regulation represent dynamic phenomena, for which continued
investigations into specific signaling mechanisms and therapies promise
to yield interesting academic and clinical possibilities.
 |
ACKNOWLEDGEMENTS |
The authors thank Dr. Bruce Caterson and colleagues for services in
generating the H6C5 MAb.
 |
FOOTNOTES |
These studies were supported by the North American Cystic Fibrosis
Foundation and National Heart, Lung, and Blood Institute Grant
HL-63756. Additionally, J. D. Conway was partially supported by
medical student research fellowships or traineeships from the Southern
Medical Association, the Cystic Fibrosis Foundation, and the University
of North Carolina Holderness Foundation.
1
The study of Yerxa et al. (54)
reported mucin secretion as raw data (OD units). Without a standard
curve, the multiplicity of the mucin secretory response in this study
could not be calculated.
Address for reprint requests and other correspondence:
C. W. Davis, 6009 Thurston-Bowles, CB 7248, Univ. of North
Carolina, Chapel Hill, NC 27599 (E-mail:
cwdavis{at}med.unc.edu).
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.
First published January 17, 2003;10.1152/ajplung.00410.2002
Received 2 December 2002; accepted in final form 13 January 2003.
 |
REFERENCES |
1.
Abdullah LH, Bundy JT, Ehre C, and Davis CW. Differential effects
of purinergic agonist and PMA on protein kinase C and mucin secretion
from SPOC1 cells. Am J Physiol Lung Cell Mol Physiol.
In press.
2.
Abdullah, LH,
Conway JD,
Cohn JA,
and
Davis CW.
Protein kinase C and Ca2+ activation of mucin secretion in airway goblet cells.
Am J Physiol Lung Cell Mol Physiol
273:
L201-L210,
1997[Abstract/Free Full Text].
3.
Abdullah, LH,
Davis SW,
Burch L,
Yamauchi M,
Randell SH,
Nettesheim P,
and
Davis CW.
P2u purinoceptor regulation of mucin secretion in SPOC1 cells, a goblet cell line from the airways.
Biochem J
316:
943-951,
1996[ISI][Medline].
4.
Adler, KB,
Fischer BM,
Li H,
Choe NH,
and
Wright DT.
Hypersecretion of mucin in response to inflammatory mediators by guinea pig tracheal epithelial cells in vitro is blocked by inhibition of nitric oxide synthase.
Am J Respir Cell Mol Biol
13:
526-530,
1995[Abstract].
5.
Bankert, RB,
Hess SD,
and
Egilmez NK.
SCID mouse models to study human cancer pathogenesis and approaches to therapy: potential, limitations, and future directions.
Front Biosci
7:
c44-c62,
2002.
6.
Blackburn, MR,
Volmer JB,
Thrasher JL,
Zhong H,
Crosby JR,
Lee JJ,
and
Kellems RE.
Metabolic consequences of adenosine deaminase deficiency in mice are associated with defects in alveogenesis, pulmonary inflammation, and airway obstruction.
J Exp Med
192:
159-170,
2000[Abstract/Free Full Text].
7.
Bodin, P,
and
Burnstock G.
Purinergic signalling: ATP release.
Neurochem Res
26:
959-969,
2001[ISI][Medline].
8.
Bredenbroker, D,
Dyarmand D,
Meingast U,
Fehmann HC,
Staats P,
von Wichert P,
and
Wagner U.
Effects of the nitric oxide/cGMP system compared with the cAMP system on airway mucus secretion in the rat.
Eur J Pharmacol
411:
319-325,
2001[ISI][Medline].
9.
Brody, AR,
Hook GE,
Cameron GS,
Jetten AM,
Butterick CJ,
and
Nettesheim P.
The differentiation capacity of Clara cells isolated from the lungs of rabbits.
Lab Invest
57:
219-229,
1987[ISI][Medline].
10.
Brose, N,
Rosenmund C,
and
Rettig J.
Regulation of transmitter release by Unc-13 and its homologues.
Curr Opin Neurobiol
10:
303-311,
2000[ISI][Medline].
11.
Chen, Y,
Zhao YH,
and
Wu R.
Differential regulation of airway mucin gene expression and mucin secretion by extracellular nucleotide triphosphates.
Am J Respir Cell Mol Biol
25:
409-417,
2001[Abstract/Free Full Text].
12.
Davis, CW,
and
Abdullah LH.
In vitro models for airways mucin secretion.
Pulm Pharmacol Ther
10:
145-155,
1997[ISI][Medline].
13.
Davis, CW,
Abdullah LH,
and
Boucher RC.
Cellular basis for the purinergic regulation of mucin secretion in the airways.
In: Cilia, Mucus, and Mucociliary Interactions, edited by Baum GL.. New York: Dekker, 1998, p. 153-166.
14.
Davis, CW,
Dowell ML,
Lethem M,
and
Van Scott M.
Goblet cell degranulation in isolated canine tracheal epithelium: response to exogenous ATP, ADP, and adenosine.
Am J Physiol Cell Physiol
262:
C1313-C1323,
1992[Abstract/Free Full Text].
15.
Donaldson, SH,
Picher M,
and
Boucher RC.
Secreted and cell-associated adenylate kinase and nucleoside diphosphokinase contribute to extracellular nucleotide metabolism on human airway surfaces.
Am J Respir Cell Mol Biol
26:
209-215,
2002[Abstract/Free Full Text].
16.
Filali, M,
Zhang Y,
Ritchie TC,
and
Engelhardt JF.
Xenograft model of the CF airway.
Methods Mol Med
70:
537-550,
2002[Medline].
17.
Fischer, BM,
Rochelle LG,
Voynow JA,
Akley NJ,
and
Adler KB.
Tumor necrosis factor-alpha stimulates mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro.
Am J Respir Cell Mol Biol
20:
413-422,
1999[Abstract/Free Full Text].
18.
Folkerts, G,
Kloek J,
Muijsers RB,
and
Nijkamp FP.
Reactive nitrogen and oxygen species in airway inflammation.
Eur J Pharmacol
429:
251-262,
2001[ISI][Medline].
19.
Forstner, G,
Zhang Y,
McCool D,
and
Forstner J.
Mucin secretion by T84 cells: stimulation by PKC, Ca2+, and a protein kinase activated by Ca2+ ionophore.
Am J Physiol Gastrointest Liver Physiol
264:
G1096-G1102,
1993[Abstract/Free Full Text].
20.
Gray, TE,
Guzman K,
Davis CW,
Abdullah LH,
and
Nettesheim P.
Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells.
Am J Respir Cell Mol Biol
14:
104-112,
1996[Abstract].
21.
Harden, TK,
and
Lazarowski ER.
Release of ATP and UTP from astrocytoma cells.
Prog Brain Res
120:
135-143,
1999[ISI][Medline].
22.
Holgate, ST.
Adenosine: a key effector molecule of asthma or just another mediator?
Am J Physiol Lung Cell Mol Physiol
282:
L167-L168,
2002[Free Full Text].
23.
Hong, DH,
Forstner JF,
and
Forstner G.
Protein kinase C-
is the likely mediator of mucin exocytosis in human colonic cell lines.
Am J Physiol Gastrointest Liver Physiol
272:
G31-G37,
1997[Abstract/Free Full Text].
24.
Huang, P,
Lazarowski ER,
Tarran R,
Milgram SL,
Boucher RC,
and
Stutts MJ.
Compartmentalized autocrine signaling to cystic fibrosis transmembrane conductance regulator at the apical membrane of airway epithelial cells.
Proc Natl Acad Sci USA
98:
14120-14125,
2001[Abstract/Free Full Text].
25.
Inayama, Y,
Hook GE,
Brody AR,
Cameron GS,
Jetten AM,
Gilmore LB,
Gray T,
and
Nettesheim P.
The differentiation potential of tracheal basal cells.
Lab Invest
58:
706-717,
1988[ISI][Medline].
26.
Jiang, C,
Finkbeiner WE,
Widdicombe JH,
McCray PB, Jr,
and
Miller SS.
Altered fluid transport across airway epithelium in cystic fibrosis.
Science
262:
424-427,
1993[ISI][Medline].
27.
Jourdian, GW,
Dean L,
and
Roseman S.
The sialic acids. XI. A periodate-resorcinol method for the quantitative estimation of free sialic acids and their glycosides.
J Biol Chem
246:
430-435,
1971[Abstract/Free Full Text].
28.
Kai, H,
Yoshitake K,
Isohama Y,
Hamamura I,
Takahama K,
and
Miyata T.
Involvement of protein kinase C in mucus secretion by hamster tracheal epithelial cells in culture.
Am J Physiol Lung Cell Mol Physiol
267:
L526-L530,
1994[Abstract/Free Full Text].
29.
Kazanietz, MG.
Novel "nonkinase" phorbol ester receptors: the C1 domain connection.
Mol Pharmacol
61:
759-767,
2002[Abstract/Free Full Text].
30.
Kim, KC,
McCracken K,
Lee BC,
Shin CY,
Jo MJ,
Lee CJ,
and
Ko KH.
Airway goblet cell mucin: its structure and regulation of secretion.
Eur Respir J
10:
2644-2649,
1997[Abstract/Free Full Text].
31.
Knowles, MR,
and
Boucher RC.
Mucus clearance as a primary innate defense mechanism for mammalian airways.
J Clin Invest
109:
571-577,
2002[Free Full Text].
32.
Koh, DS,
Moody MW,
Nguyen TD,
and
Hille B.
Regulation of exocytosis by protein kinases and Ca(2+) in pancreatic duct epithelial cells.
J Gen Physiol
116:
507-520,
2000[Abstract/Free Full Text].
33.
Larivee, P,
Levine SJ,
Martinez A,
Wu T,
Logun C,
and
Shelhamer JH.
Platelet-activating factor induces airway mucin release via activation of protein kinase C: evidence for translocation of protein kinase C to membranes.
Am J Respir Cell Mol Biol
11:
199-205,
1994[Abstract].
34.
Lazarowski, ER,
and
Boucher RC.
UTP as an extracellular signaling molecule.
News Physiol Sci
16:
1-5,
2001[Abstract/Free Full Text].
35.
Lazarowski, ER,
Boucher RC,
and
Harden TK.
Constitutive release of ATP and evidence for major contribution of ecto-nucleotide pyrophosphatase and nucleoside diphosphokinase to extracellular nucleotide concentrations.
J Biol Chem
275:
31061-31068,
2000[Abstract/Free Full Text].
36.
Lethem, MI,
Dowell ML,
Van Scott M,
Yankaskas JR,
Egan T,
Boucher RC,
and
Davis CW.
Nucleotide regulation of goblet cells in human airway epithelial explants: normal exocytosis in cystic fibrosis.
Am J Respir Cell Mol Biol
9:
315-322,
1993[ISI][Medline].
37.
Li, Y,
Martin LD,
Spizz G,
and
Adler KB.
MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.
J Biol Chem
276:
40982-40990,
2001[Abstract/Free Full Text].
38.
Liu, X,
Jiang Q,
Mansfield SG,
Puttaraju M,
Zhang Y,
Zhou W,
Cohn JA,
Garcia-Blanco MA,
Mitchell LG,
and
Engelhardt JF.
Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing.
Nat Biotechnol
20:
47-52,
2002[ISI][Medline].
39.
Mason, SJ,
Paradiso AM,
and
Boucher RC.
Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium.
Br J Pharmacol
103:
1649-1656,
1991[Abstract].
40.
Matsui, H,
Grubb BR,
Tarran R,
Randell SH,
Gatzy JT,
Davis CW,
and
Boucher RC.
Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease.
Cell
95:
1005-1015,
1998[ISI][Medline].
41.
Matsui, H,
Randell SH,
Peretti SW,
Davis CW,
and
Boucher RC.
Coordinated clearance of periciliary liquid and mucus from airway surfaces.
J Clin Invest
102:
1125-1131,
1998[Abstract/Free Full Text].
42.
Meade, CJ,
Dumont I,
and
Worrall L.
Why do asthmatic subjects respond so strongly to inhaled adenosine?
Life Sci
69:
1225-1240,
2001[ISI][Medline].
43.
Morse, DM,
Smullen JL,
and
Davis CW.
Differential effects of UTP, ATP, and adenosine on ciliary activity of human nasal epithelial cells.
Am J Physiol Cell Physiol
280:
C1485-C1497,
2001[Abstract/Free Full Text].
44.
Nguyen, TD,
Meichle S,
Kim US,
Wong T,
and
Moody MW.
P2Y(11), a purinergic receptor acting via cAMP, mediates secretion by pancreatic duct epithelial cells.
Am J Physiol Gastrointest Liver Physiol
280:
G795-G804,
2001[Abstract/Free Full Text].
45.
Nicholas, RA.
Identification of the P2Y(12) receptor: a novel member of the P2Y family of receptors activated by extracellular nucleotides.
Mol Pharmacol
60:
416-420,
2001[Free Full Text].
46.
Picher, M,
and
Boucher RC.
Metabolism of extracellular nucleotides in human airways by a multi-enzyme system.
Drug Dev Res
52:
66-75,
2000[ISI].
47.
Randell, SH,
Liu JY,
Ferriola PC,
Kaartinen L,
Doherty MM,
Davis CW,
and
Nettesheim P.
Mucin production by SPOC1 cells-an immortalized rat tracheal epithelial cell line.
Am J Respir Cell Mol Biol
14:
146-154,
1996[Abstract].
48.
Rose, MC.
Mucins: structure, function, and role in pulmonary diseases.
Am J Physiol Lung Cell Mol Physiol
263:
L413-L429,
1992[Abstract/Free Full Text].
49.
Sheehan, JK,
Thornton DJ,
Somerville M,
and
Carlstedt
Mucin structure. I. The structure and heterogeneity of respiratory mucus glycoproteins.
Am Rev Respir Dis
144:
S4-S9,
1991[ISI][Medline].
50.
St. George, JA,
Cranz DL,
Zicker SC,
Etchison JR,
Dungworth DL,
and
Plopper CG.
An immunohistochemical characterization of rhesus monkey respiratory secretions using monoclonal antibodies.
Am Rev Respir Dis
132:
556-563,
1985[ISI][Medline].
51.
Von Kugelgen, I,
and
Wetter A.
Molecular pharmacology of P2Y-receptors.
Naunyn Schmiedebergs Arch Pharmacol
362:
310-323,
2000[ISI][Medline].
52.
Wright, DT,
Fischer BM,
Li C,
Rochelle LG,
Akley NJ,
and
Adler KB.
Oxidant stress stimulates mucin secretion and PLC in airway epithelium via a nitric oxide-dependent mechanism.
Am J Physiol Lung Cell Mol Physiol
271:
L854-L861,
1996[Abstract/Free Full Text].
53.
Wu, R,
Zhao YH,
and
Chang MM.
Growth and differentiation of conducting airway epithelial cells in culture.
Eur Respir J
10:
2398-2403,
1997[Abstract/Free Full Text].
54.
Yerxa, BR,
Sabater JR,
Davis CW,
Stutts MJ,
Lang-Furr M,
Picher M,
Jones AC,
Cowlen M,
Dougherty R,
Boyer J,
Abraham WM,
and
Boucher RC.
Pharmacology of INS37217 [P(1)-(uridine 5')-P(4)- (2'-deoxycytidine 5')tetraphosphate, tetrasodium salt], a next-generation P2Y(2) receptor agonist for the treatment of cystic fibrosis.
J Pharmacol Exp Ther
302:
871-880,
2002[Abstract/Free Full Text].
55.
Zimmermann, H.
Extracellular metabolism of ATP and other nucleotides.
Naunyn Schmiedebergs Arch Pharmacol
362:
299-309,
2000[ISI][Medline].
Am J Physiol Lung Cell Mol Physiol 284(6):L945-L954
1040-0605/03 $5.00
Copyright © 2003 the American Physiological Society