P2Y2 receptor-stimulated phosphoinositide
hydrolysis and Ca2+ mobilization in tracheal epithelial
cells
Chuen-Mao
Yang,
Wen-Bin
Wu,
Shiow-Lin
Pan,
Yih-Jeng
Tsai,
Chi-Tso
Chiu, and
Chuan-Chwan
Wang
Cellular and Molecular Pharmacology Laboratory, Department of
Pharmacology, College of Medicine, Chang Gung University, Kwei-San,
Tao-Yuan, Taiwan
 |
ABSTRACT |
Extracellular nucleotides have been implicated in the regulation of
secretory function through the activation of P2 receptors in the
epithelial tissues, including tracheal epithelial cells (TECs). In this
study, experiments were conducted to characterize the P2 receptor
subtype on canine TECs responsible for stimulating inositol
phosphate (InsPx) accumulation and
Ca2+ mobilization using a range of nucleotides. The
nucleotides ATP and UTP caused a concentration-dependent
increase in [3H]InsPx
accumulation and Ca2+ mobilization with comparable kinetics
and similar potency. The selective agonists for P1, P2X, and
P2Y1 receptors,
N6-cyclopentyladenosine and AMP,
,
-methylene-ATP and
,
-methylene-ATP, and 2-methylthio-ATP,
respectively, had little effect on these responses. Stimulation of TECs
with maximally effective concentrations of ATP and UTP showed no
additive effect on [3H]InsPx
accumulation. The response of a maximally effective concentration of
either ATP or UTP was additive to the response evoked by bradykinin. Furthermore, ATP and UTP induced a cross-desensitization in
[3H]InsPx accumulation and
Ca2+ mobilization. These results suggest that ATP and UTP
directly stimulate phospholipase C-mediated
[3H]InsPx accumulation and
Ca2+ mobilization in canine TECs. P2Y2
receptors may be predominantly mediating
[3H]InsPx accumulation, and,
subsequently, inositol 1,4,5-trisphosphate-induced Ca2+
mobilization may function as the transducing mechanism for
ATP-modulated secretory function of tracheal epithelium.
canine; inositol phosphates; purinergic receptors; adenosine
5'-triphosphate
 |
INTRODUCTION |
MUCUS
SECRETION PLAYS an important role in defense of the respiratory
tract, and abnormal and excessive mucus secretions are characteristic
features of many chronic inflammatory lung diseases including chronic
obstructive lung disease, asthma, and cystic fibrosis. Mucus
glycoproteins originate from two different secretory cell types:
epithelial goblet cells and submucosal gland cells (17).
The secretion from submucosal glands is under neuronal control based on
anatomic and pharmacological studies. However, the goblet cells are
free of autonomic innervation (35), and their secretion
seems to be induced by chemical irritants and ATP analogs
(18). To date, the signal transduction pathways that link
ATP receptors to secretory function have not been fully established in
canine tracheal epithelial cells (TECs).
ATP is released from neuronal and nonneuronal cells and acts a
well-established physiological role as an extracellular signaling molecule (4, 10). Evidence for the release of
cellular UTP also has been reported (20, 32).
Receptors for extracellular ATP were first subdivided into P2X and P2Y
receptor subtypes on the basis of pharmacological studies with isolated
preparations from a variety of species (5). These receptor
subtypes also differed in their transduction mechanisms: P2X receptors
are transmitter-gated ion channels, whereas P2Y receptors are members
of the G protein-coupled receptor superfamily (1,
11). So far, P2X receptor subtypes have been subclassified
mainly as P2X1 to P2X7 (34).
Pharmacological characterization of purinoceptors relies on agonist
selectivities and potency orders because there is a lack of selective
antagonists for purinoceptors. The stable analog of ATP,
,
-methylene-ATP (
,
-MeATP), is a potent agonist for the
P2X1 receptor (5). P2Y receptors have been
subclassified as P2Y1, P2Y2, P2Y4,
and P2Y6 and a recently identified receptor that has been
given the tentative designation of P2Y11 (15).
The P2Y1 receptor is activated by adenine nucleotides and
not by uridine nucleotides. ADP is the most potent natural agonist for
this receptor, and whether ATP is an agonist for this receptor remains
uncertain (21). The P2Y2 receptor is activated
equipotently by ATP and UTP but is activated, at most only poorly, by
diphosphate nucleotides (23, 26). The
P2Y4 receptor is activated by UTP and weakly, if at all, by
ATP, UDP, or ADP (7, 26). The
P2Y6 receptor is UDP sensitive and is activated weakly, if
at all, by UTP, ATP, or ADP (8, 26). The
P2Y11 receptor has been shown to be activated by ATP and
ADP but not by UTP or UDP (6). In several cell types, P2Y
receptors are G protein-coupled receptors that are commonly associated with phospholipase (PL) C activation, with a
subsequent increase in inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3]
formation and intracellular Ca2+ release,
diacylglycerol production, and activation of protein kinase C
(2, 9, 14, 40). A
detailed pharmacological characterization of these receptors in canine
TECs is still lacking.
Although the potency order for some nucleotides has been studied
(2, 9, 14, 40), the
exact P2Y receptor subtype that mediates the hydrolysis of
phosphoinositide (PI) and Ca2+ mobilization in canine TECs
has not been fully understood. Therefore, the purpose of this study was
to identify which subtype of P2Y receptor on canine TECs mediates
[3H]inositol phosphate (InsPx)
accumulation and Ca2+ mobilization induced by ATP and UTP.
Nucleotide agonist potency, pharmacological additivity, and
cross-desensitization were examined to determine whether ATP and UTP
acted on the same putative extracellular receptors. The data
demonstrate that in canine TECs, ATP might activate PLC through
the P2Y2 receptors, leading to generation of
Ins(1,4,5)P3,
and subsequent Ca2+ release from
Ins(1,4,5)P3-sensitive
internal stores may function as a transducing mechanism for regulation
of tracheal secretory function.
 |
METHODS |
Materials.
Dulbecco's modified Eagle's medium (DMEM)-Ham's nutrient
mixture F-12 medium and fetal bovine serum (FBS) were purchased from GIBCO BRL (Life Technologies, Gaithersburg, MD).
myo-[2-3H]inositol (18 Ci/mmol) was from
Amersham. Fura 2-AM was from Molecular Probes (Eugene, OR). ATP and
analogs were obtained from RBI (Natick, MA). Enzymes and other
chemicals were from Sigma (St. Louis, MO).
Animals.
Mongrel dogs, 10-20 kg, both male and female, were purchased from
a local supplier. Dogs were housed indoors in the animal facility under
automatically controlled temperature and light-cycle conditions and fed
standard laboratory chow and tap water ad libitum. Dogs were
anesthetized with ketamine (20 mg/kg intramuscularly) and pentobarbital
sodium (30 mg/kg intravenously). The tracheae were surgically removed.
Isolation and culture of TECs.
Cells were isolated essentially as described by Wu et al.
(38). The trachea was cut longitudinally through the
cartilage rings, and strip epithelium was pulled off the submucosa,
rinsed with phosphate-buffered saline (PBS) containing 5 mM
dithiothreitol, and digested with 0.05% protease XIV in PBS at 4°C
for 24 h; after vigorous shaking of the strips at room
temperature, 5 ml of FBS were added to terminate the digestion. The
released cells were collected and washed twice with 50% DMEM-50%
Ham's nutrient F-12 medium that contained 5% FBS, 1× nonessential
amino acids, 100 U/ml of penicillin, 100 µg/ml of
streptomycin, 50 µg/ml of gentamicin, and 2.5 µg/ml of Fungizone.
The cell number was counted, and the cells were diluted with
DMEM-Ham's F-12 medium to 2 × 106 cells/ml. The
cells were plated onto 12-well (1 ml/well) and 6-well (2 ml/well)
culture plates containing glass coverslips coated with collagen
for [3H]InsPx accumulation and
Ca2+ measurement, respectively. The culture medium
was changed after 24 h and then changed every 2 days.
To characterize the isolated and cultured TECs, an indirect
immunofluorescent staining was performed as described by O'Guin et al.
(28) with AE1 and AE3 mouse monoclonal antibodies and fluorescein isothiocyanate-labeled goat anti-mouse IgG.
Accumulation of InsPx.
The effect of ATP on the hydrolysis of PI was assayed by
monitoring the accumulation of
[3H]InsPx as described by Yang et
al. (39). Cultured TECs were incubated with 5 µCi/ml of
myo-[2-3H]inositol at 37°C for 24 h.
TECs were washed two times with and incubated in Krebs-Henseleit buffer
(pH 7.4) containing (in mM) 117 NaCl, 4.7 KCl, 1.1 MgSO4, 1.2 KH2PO4, 20 NaHCO3, 2.4 CaCl2, 1 glucose, 20 HEPES,
and 10 LiCl at 37°C for 30 min. After 1 mM ATP was added, the
incubation continued for another 60 min or for the times indicated in
Figs. 1-5. Reactions were terminated by addition of 5% perchloric
acid followed by sonication and centrifugation at 3,000 g
for 15 min.
The perchloric acid-soluble supernatants were extracted four times with
ether, neutralized with potassium hydroxide, and applied to a column of
AG1-X8 (formate form, 100- to 200-µm mesh; Bio-Rad). The
resin was washed successively with 5 ml of water and 5 ml of 60 mM
ammonium formate-5 mM sodium tetraborate to eliminate free
[3H]inositol and glycerophosphoinositol, respectively.
Sequential washes with 5 ml of 0.2 M ammonium formate-0.1 M formic
acid, 0.4 M ammonium formate-0.1 M formic acid, and 1 M ammonium
formate-0.1 M formic acid were used to elute inositol 1-monophosphate
[Ins(1)P], inositol 4,5-bisphosphate
[Ins(4,5)P2], and
Ins(1,4,5)P3, respectively. Total
[3H]InsPx was eluted with 5 ml of
1 M ammonium formate-0.1 M formic acid. The amount of
[3H]InsPx was determined in a
radiospectrometer (model LS5000TA, Beckman, Fullerton, CA).
Measurement of intracellular Ca2+
level.
Intracellular Ca2+ concentration
([Ca2+]i) was measured in confluent
monolayers with the Ca2+-sensitive dye fura 2-AM as
described by Grynkiewicz et al. (13). On confluence, the
cells were cultured in DMEM-Ham's F-12 medium with 1% FBS 1 day
before measurements were made. The monolayers were covered with 1 ml of
DMEM-Ham's F-12 medium with 1% FBS containing 5 µM fura 2-AM and
were incubated at 37°C for 45 min. At the end of the period, the
coverslips were washed twice with a physiological buffer solution
containing (in mM) 125 NaCl, 5 KCl, 1.8 CaCl2, 2 MgCl2, 0.5 NaH2PO4, 5 NaHCO3, 10 HEPES, and 10 glucose, pH 7.4. The cells were
incubated in PBS for 30 min more to complete dye deesterification. The
coverslip was inserted into a quartz cuvette at an angle of ~45° to
the excitation beam and placed in the temperature-controlled holder of
a Hitachi F-4500 spectrofluorometer (Tokyo, Japan). Continuous
stirring was achieved with a magnetic stirrer. The ratio of the
fluorescence at the two wavelengths was computed and used to calculate
changes in [Ca2+]i. The ratios of maximum and
minimum fluorescence of fura-2 were determined by adding ionomycin (10 µM) in the presence of PBS containing 5 mM Ca2+ and by
adding 5 mM EGTA at pH 8 in Ca2+-free PBS, respectively.
The dissociation constant of fura 2 for Ca2+ was assumed to
be 224 nM (13).
Analysis of data.
Concentration-effect curves were fitted, and EC50
values were estimated by Prism Program (GraphPad, San
Diego, CA). The data are expressed as means ± SE of the
experiments, with statistical comparisons based on a two-tailed
Student's t-test at a P < 0.01 level of significance.
 |
RESULTS |
Characteristics of [3H]InsPx formation.
[3H]inositol-labeled TECs were stimulated in the presence
of 10 mM LiCl, and total
[3H]InsPx was separated and
counted. The results obtained from a series of experiments with a
variety of agonists are shown in Table 1.
ATP (1 mM), UTP (1 mM), and adenosine
5'-O-(3-thiotriphosphate) (ATP
S; 100 µM) elicited
a substantial [3H]InsPx response,
whereas 2-methylthio-ATP (2-MeS-ATP; 100 µM), ADP (100 µM),
,
-MeATP (100 µM),
,
-MeATP (100 µM),
N6-cyclopentyladenosine (CPA; 200 µM), and
AMP (1 mM) did not. These results show that the effect of ATP was not
due to its breakdown products such as ADP or AMP. Furthermore, the
responses obtained were apparently not due to the activation of P1
receptors because AMP and CPA, both P1 receptor agonists, induced
little response. In contrast to P1 receptor agonists, ATP (1 mM),
UTP (1 mM), and ATP
S (100 µM) induced a rapid
accumulation of
[3H]Ins(1)P,
[3H]Ins(4,5)P2,
and
[3H]Ins(1,4,5)P3,
respectively, in a time-dependent manner (Fig. 1). In the presence of these
three agonists, the formation of [3H]Ins(1,4,5)P3
appeared first, reached maximum by 5 min of stimulation, and then
slightly declined, whereas the
[3H]Ins(1)P and
[3H]Ins(4,5)P2
responses to ATP, UTP, and ATP
S increased at a slower rate to a
maximum at ~7 min (Fig. 1).

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Fig. 1.
Time course of [3H]inositol phosphate
(InsPx) accumulation after stimulation with ATP,
UTP and adenosine 5'-O-(3-thiotriphosphate) (ATP S) in
tracheal epithelial cells (TECs). A: inositol
1-monophosphate. B: inositol 4,5-bisphosphate. C:
inositol 1,4,5-trisphosphate. [3H]inositol-labeled cells
were washed and incubated in Krebs-Henseleit buffer containing 10 mM
LiCl at 37°C for 30 min and then exposed to 1 mM ATP, 1 mM UTP, or
100 µM ATP S for the various times. Data are means ± SE from
3 separate experiments determined in triplicate.
|
|
Analysis of concentration-effect curves when TECs were exposed to
agonists for 10 min (Fig. 2) indicated
that the EC50 values were 10 ± 4 (UTP) and 40 ± 15 (ATP) µM, respectively. The maximum responses to 2-MeS-ATP and
,
-MeATP (P2Y1 and P2X receptor agonists, respectively) were less than those of UTP and ATP, and thus the EC50 values were not calculated. The EC50
values for the late responses (exposure to agonists for 60 min) were
similar to those of the rapid responses (data not shown).

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Fig. 2.
Concentration-dependent stimulation of
[3H]InsPx accumulation by P2
receptor agonists in TECs. The [3H]inositol-labeled cells
were washed and incubated in Krebs-Henseleit buffer containing 10 mM
LiCl at 37°C for 30 min and then exposed to increasing concentrations
([Drug]) of UTP, ATP, , -methylene-ATP ( , -MeATP), or
2-methylthio-ATP (2-MeS-ATP) for another 10 min. Data were normalized
to the basal levels of InsPx accumulation
(10,350 ± 1,100 dpm/well) from 3 separate experiments determined
in triplicate.
|
|
Agonist specificity for the Ca2+ transient in TECs.
To further characterize the P2 receptor subtype-mediated
[Ca2+]i response, the ability of various
agonists to mobilize Ca2+ was assessed in TECs. Figure
3 illustrates a
typical response elicited by 1 mM ATP showing that
[Ca2+]i increased rapidly [from a resting
level of 115 ± 13 nM (n = 4 experiments) to a peak at 409 ± 9 nM (n = 4 experiments)] within ~10 s and subsequently declined to basal
levels within 1 min. There was no evidence of a sustained elevation in
[Ca2+]i. Similar results were obtained in
TECs stimulated with 1 mM UTP (Fig. 3). ADP (100 µM) and 2-MeS-ATP
(100 µM) had a smaller response than ATP and UTP. In contrast, P1
receptor agonists CPA (200 µM) and AMP (1 mM) induced a slight
increase in [Ca2+]i, indicating that the
responses observed were not mediated through the activation of P1
receptors. Moreover, neither
,
-MeATP nor
,
-MeATP elicited a
rise in [Ca2+]i (data not shown), ruling out
the involvement of P2X receptors in this response. In addition,
application of ATP and UTP was found to evoke a concentration-dependent
increase in [Ca2+]i (Fig.
4). This effect was maximal at 1 mM ATP
or UTP and concentrations < 10 nM failed to evoke any response.
The EC50 values for ATP and UTP were 10 ± 3 and
7 ± 3 µM, respectively (n = 6 experiments), close to those of [3H]InsPx
accumulation induced by these agonists. These data suggest that the
predominant receptors implicated in the Ca2+ response are
the P2Y2 receptors.

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Fig. 3.
P1 and P2 receptor agonist-stimulated intracellular
Ca2+ concentration ([Ca2+]i)
changes in TECs. A: cells grown on glass coverslips were
loaded with 5 µM fura 2-AM, and fluorescence measurement of
[Ca2+]i was carried out in a dual-excitation
wavelength spectrophotometer with excitation at 340 and 380 nm after
addition of N6-cyclopentyladenosine (CPA; 200 µM), AMP (1 mM), ADP (100 µM), 2-MeS-ATP (100 µM), ATP (1 mM), or
UTP (1 mM). The responses of ATP and UTP showed an initial transient
increase of [Ca2+]i, but the sustained
plateau in [Ca2+]i was not obviously seen in
TECs. B: summary of increased
[Ca2+]i induced by these agonists. Data were
derived from 4 separate experiments. Results are means ± SE of
the increase above the resting level (115 ± 13 nM).
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Fig. 4.
Dependence of the rise in
[Ca2+]i on ATP and UTP concentration. Cells
grown on glass coverslips were loaded with 5 µM fura 2-AM, and
fluorescent measurement of [Ca2+]i was
carried out in a dual-excitation wavelength spectrophotometer with
excitation at 340 and 380 nm. The log concentration-effect curves of
ATP- and UTP-induced rise in [Ca2+]i were
derived from 6 separate experiments. Results are means ± SE of
the increase above the resting level.
|
|
Evidence that ATP and UTP act on the same receptor.
The additivity of the effect of ATP and UTP was investigated to
determine whether they acted on the same receptor. As shown in Table
2,
[3H]InsPx production in
response to the combination of maximally effective
concentrations of ATP and UTP was not greater than that observed with
each agonist alone. In contrast, the
InsPx response induced by either
ATP or UTP was additive in combination with bradykinin.
These results indicated that ATP and UTP shared a common
receptor.
The effects of preincubation with UTP and ATP on the subsequent
[3H]InsPx responses to ATP and UTP
as a function of concentration were similar. As shown in Fig.
5, after the cells were desensitized by
preincubation with either 1 mM UTP or 1 mM ATP for 4 h, the maximal response to ATP or UTP for 10 min was greatly reduced. However,
the EC50 values for the induction of
[3H]InsPx accumulation in ATP- and
UTP-pretreated cells evoked by ATP and UTP were 80 ± 20 and
73 ± 27 µM and 83 ± 24 and 72 ± 19 µM,
respectively (n = 3 experiments), close to the values
in control cells (40 ± 15 and 21 ± 7 µM, respectively).

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Fig. 5.
Concentration-effect curves for ATP- and UTP-stimulated
[3H]InsPx accumulation in ATP- and
UTP-desensitized TECs. [3H]inositol-labeled cells
were washed twice with Krebs-Henseleit buffer and then preincubated
with vehicle (control), 1 mM UTP, or 1 mM ATP in this buffer for 3 h. The cells were then rapidly washed 3 times with Krebs-Henseleit
buffer, incubated in this buffer containing 10 mM LiCl for 30 min, and
then exposed to various concentrations of ATP ([ATP]; A)
and UTP ([UTP]; B) for another 10 min. Data were
normalized to the basal levels of
[3H]InsPx accumulation from 4 separate experiments determined in triplicate. The basal level of
[3H]InsPx accumulation in
nonpretreated cells was 11,300 ± 1,400 dpm/well.
|
|
To further determine whether ATP and UTP were acting via the same
receptors to change [Ca2+]i in TECs, the
cells were preincubated with either 1 mM ATP or 1 mM UTP for 3 h
and then challenged with a maximally effective concentration of 1 mM
ATP or 1 mM UTP (Table 3). Pretreatment with ATP reduced the subsequent exposure to ATP and UTP to 18.0 ± 2.9 and 18.0 ± 4.7%, respectively (n = 3 experiments) of the responses seen in control cells (no pretreatment).
Similar response patterns were seen when the cells were pretreated with
UTP and attenuated the subsequent response to ATP and UTP to 27.1 ± 4.6 and 30.9 ± 3.6%, respectively, of the control values
(n = 3).
 |
DISCUSSION |
Extracellular ATP has been well established as a regulatory
agonist of a large variety of cellular functions (1).
Several studies (1, 11, 14,
16) have shown that the effects of ATP are mediated
through the stimulation of specific P2Y receptor subtypes present on
the cell surface that activate PI-specific PLC and lead to generation
of [3H]InsPx and release of
Ca2+ from internal stores. However, the effects of ATP on
canine TECs are not well established. The aim of this work was to
establish whether ATP had a direct effect on canine TECs and
whether this effect was mediated by P2Y receptor subtypes. The results
presented here show that P2Y receptors are activated by ATP and UTP.
This is shown by the formation of
[3H]InsPx and the increase in
[Ca2+]i on stimulation of canine TECs with
ATP and UTP. Stimulation of TECs with the ATP analogs CPA and AMP,
,
-MeATP, and 2-MeS-ATP, assumed to interact with P1, P2X, and
P2Y1 receptors, respectively, had little effect on these
responses, suggesting that these receptor subtypes were not responsible
for the [3H]InsPx and
Ca2+ responses to ATP.
The [3H]InsPx and Ca2+
responses elicited by ATP were mimicked by UTP with the following rank
order of potency: UTP = ATP
2-MeS-ATP =
,
-MeATP. In
TECs, neither AMP nor CPA elicited any significant [3H]InsPx accumulation and
Ca2+ mobilization, indicating that the receptor involvement
in these responses does not belong to the P1 receptors. Furthermore, it is unlikely that the actions of ATP are mediated via one of its breakdown products because ADP was much less potent and AMP was ineffective in these responses. Therefore, the subtype of purinoceptors coupling to [3H]InsPx accumulation
and Ca2+ mobilization seems to be P2 receptors. Because P2X
and P2Y receptors have been suggested to be present in several lung
cell types (25), we further examined whether the receptor
mediating the accumulation of
[3H]InsPx and increase in
[Ca2+]i by ATP belonged to one of
these receptor subtypes. In present study,
,
-MeATP (the highly
potent P2X receptor agonist) was able to elicit only a very small
accumulation of
[3H]InsPx and increase
in [Ca2+]i, suggesting that P2X receptors are
unlikely to be involved in the responses to ATP. In addition, 2-MeS-ATP
(the highly potent P2Y1 receptor agonist) was the least
potent of the purinoceptor agonists examined, indicating that the
responses of cultured TECs to ATP are not mediated via P2Y1
receptor subtype. Indeed, of the ATP analogs investigated, only ATP
S
was able to cause InsPx accumulation
and Ca2+ mobilization with similar potency and efficacy as
ATP. Moreover, the [3H]InsPx
accumulation and Ca2+ mobilization evoked by ATP and UTP
were similar in both their maximum effect and potency, consistent with
those of P2Y2 purinoceptors (19,
23, 27, 29, 30,
37). The structural differences between the pyrimidine
(UTP) and purine (ATP) bases raise the question of whether these two
agonists act via the same nucleotide receptor, and previous studies
(33, 36) have suggested the existence of
separate pyrimidine and purine receptors, although other workers
(3, 24, 31) have suggested that
a single receptor recognizes both types of agonists. It should be noted that there was no evidence of a sustained elevation in
[Ca2+]i induced by ATP and UTP in canine TECs
similar to that of the responses to bradykinin in these cells
(22). These responses are different from those of human
airway epithelial cell lines (3, 29) and
primary culture of TECs and nasal epithelial cells (19,
37). This may reflect a species-specific difference between canine and human airway epithelial cells. The results obtained
from the present study in TECs suggest that UTP- and ATP-induced
[3H]InsPx accumulation and
Ca2+ mobilization are mediated through the activation of
the same receptor population. These findings demonstrate that the
pharmacological properties of P2Y receptors coupled to the signal
transduction pathways in canine TECs were consistent with those of
P2Y2 receptors (3, 4,
11, 12, 19, 27,
29, 37).
The formation of [3H]InsPx
in TECs stimulated with ATP and UTP showed a similar time course, and
corresponding [3H]InsPx levels
were reached. Besides the similarity in time course of
InsPx accumulation and increase in
[Ca2+]i, the
[3H]InsPx accumulation induced by
optimal concentrations of ATP and UTP was not additive. Furthermore,
ATP- and UTP-induced Ca2+ mobilization showed
cross-desensitization, whereas cross-desensitization was absent in TECs
stimulated with one of these nucleotides and bradykinin. Consequently,
these observations further strongly support that the
[3H]InsPx accumulation and changes
in [Ca2+]i elicited by ATP and UTP in TECs
are mediated by a common receptor, identified as a P2Y2 receptor.
In conclusion, these results provide evidence for the existence of the
P2Y2 receptor subtype in canine cultured TECs. This receptor is linked to
Ins(1,4,5)P3
production and subsequent Ca2+ mobilization. It is
activated by both ATP and UTP with similar potencies and efficacies and
resembles the receptors previously described in human airway epithelial
cells (3) and PC12 cells (24). These data,
added to that of many other studies showing that P2 receptors
are present on several lung cell types including epithelial and goblet
cells (3, 29), submucosal glands (37), alveolar type II cells (14), and
lung macrophages (25), suggest that extracellular ATP
might play an important role in the physiological functions of
respiratory system.
 |
ACKNOWLEDGEMENTS |
This work was supported by Chang Gung Medical Research Foundation
Grant CMRP-680 and National Science Council, Taiwan, Grant NSC86-2314-B182-107.
 |
FOOTNOTES |
Address for reprint requests and other
correspondence: C.-M. Yang, Dept. of Pharmacology, College of Medicine,
Chang Gung Univ., 259 Wen-Hwa 1 Rd., Kwei-San, Tao-Yuan, Taiwan
(E-mail: Chuenmao{at}mail.cgu.edu.tw).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 23 March 1999; accepted in final form 15 March 2000.
 |
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