Am J Physiol Lung Cell Mol Physiol
279: L235–L241, 2000.
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
Received 23 March 1999; accepted in final form 15 March 2000
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
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:
[email protected]).
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.
canine; inositol phosphates; purinergic receptors; adenosine
5⬘-triphosphate
MUCUS SECRETION PLAYS
http://www.ajplung.org
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society
L235
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Yang, Chuen-Mao, Wen-Bin Wu, Shiow-Lin Pan, YihJeng Tsai, Chi-Tso Chiu, and Chuan-Chwan Wang.
P2Y2 receptor-stimulated phosphoinositide hydrolysis and
Ca2⫹ mobilization in tracheal epithelial cells. Am J Physiol
Lung Cell Mol Physiol 279: L235–L241, 2000.—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.
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ATP AND SIGNAL TRANSDUCTION
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 phosphatebuffered 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 ␮g/ml of streptomycin, 50 ␮g/ml of gentamicin, and 2.5
␮g/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 antimouse 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 ␮Ci/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-␮m 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 ␮M 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
Table 1. [3H]InsPx accumulation stimulated by P1
and P2 receptor agonists in TECs
Treatment
Basal
ATP
UTP
ATP␥S
2-MeS-ATP
ADP
␣,␤-MeATP
␤,␥-MeATP
CPA
AMP
[3H]InsPx, dpm ⫻ 104/well
1.39 ⫾ 0.03
3.47 ⫾ 0.12
3.68 ⫾ 0.22
4.85 ⫾ 0.47
1.63 ⫾ 0.10
2.05 ⫾ 0.11
1.82 ⫾ 0.05
1.59 ⫾ 0.36
1.34 ⫾ 0.12
1.40 ⫾ 0.13
Values are means ⫾ SE from 3 separate experiments determined
in triplicate. [3H]inositol-labeled tracheal epithelial cells (TECs)
were washed and incubated in Krebs-Henseleit buffer containing 10
mM LiCl at 37°C for 30 min and then exposed to the agonists [1 mM
ATP, UTP, and AMP; 100 ␮M ADP, adenosine 5⬘-O-(3-thiotriphosphate) (ATP␥S), 2-methylthio-ATP (2-MeS-ATP), ␣,␤-methyleneATP (␣,␤-MeATP), and ␤,␥-MeATP; and 200 ␮M N6-cyclopentyladenosine (CPA)] for another 60 min.
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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.
ATP AND SIGNAL TRANSDUCTION
L237
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 ␮M) 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 twotailed Student’s t-test at a P ⬍ 0.01 level of significance.
RESULTS
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.
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 1,4,5trisphosphate. [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 ␮M ATP␥S for the
various times. Data are means ⫾ SE from 3 separate experiments
determined in triplicate.
a variety of agonists are shown in Table 1. ATP (1 mM),
UTP (1 mM), and adenosine 5⬘-O-(3-thiotriphosphate)
(ATP␥S; 100 ␮M) elicited a substantial [3H]InsPx response, whereas 2-methylthio-ATP (2-MeS-ATP; 100
␮M), ADP (100 ␮M), ␣,␤-MeATP (100 ␮M), ␤,␥-MeATP
(100 ␮M), N6-cyclopentyladenosine (CPA; 200 ␮M), 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 ␮M) 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).
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) ␮M, 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).
Agonist specificity for the Ca2⫹ transient in TECs. To
further characterize the P2 receptor subtype-mediated
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Characteristics of [3H]InsPx formation. [3H]inositollabeled 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
L238
ATP AND SIGNAL TRANSDUCTION
[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 ␮M) and 2-MeS-ATP
(100 ␮M) had a smaller response than ATP and UTP.
In contrast, P1 receptor agonists CPA (200 ␮M) 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 concentrationdependent 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 ␮M, 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.
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
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 ␮M 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 ␮M), AMP (1 mM),
ADP (100 ␮M), 2-MeS-ATP (100 ␮M), 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 ␮M
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.
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ATP AND SIGNAL TRANSDUCTION
Table 2. Additivity of agonist effects on [3H]InsPx
accumulation in TECs
Treatment
[3H]InsPx, dpm ⫻ 104/well
Basal
ATP
UTP
BK
ATP ⫹ UTP
ATP ⫹ BK
UTP ⫹ BK
ATP ⫹ UTP ⫹ BK
1.16 ⫾ 0.38
4.47 ⫾ 0.37
3.75 ⫾ 0.58
3.28 ⫾ 0.10
4.64 ⫾ 0.38
6.37 ⫾ 0.40*
5.71 ⫾ 0.30*
5.84 ⫾ 0.52
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 UTPpretreated cells evoked by ATP and UTP were 80 ⫾ 20
and 73 ⫾ 27 ␮M and 83 ⫾ 24 and 72 ⫾ 19 ␮M,
respectively (n ⫽ 3 experiments), close to the values in
control cells (40 ⫾ 15 and 21 ⫾ 7 ␮M, respectively).
Increase in [Ca2⫹]i, nM
Pretreatment
Control
ATP
UTP
ATP
UTP
529 ⫾ 46
95 ⫾ 17*
143 ⫾ 21*
482 ⫾ 73
143 ⫾ 29*
148 ⫾ 25*
Values are means ⫾ SE from 3 separate experiments determined
in triplicate. The cells were preincubated without (control) and with
1 mM ATP or 1 mM UTP in the medium for 1.5 h. The cells were then
loaded with fura 2-AM and incubated with buffer containing 1 mM
ATP or 1 mM UTP for a further 1.5 h. When ATP or UTP was added,
fluorescent measurement of intracellular Ca2⫹ concentration
([Ca2⫹]i) was carried out in a dual-excitation wavelength spectrophotometer, with excitation at 340 and 380 nm. * P ⬍ 0.01 compared
with control.
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 func-
Fig. 5. Concentration-effect curves for ATPand 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 KrebsHenseleit 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.
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Values are means ⫾ SE from 3 independent experiments determined in triplicate. [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, and 10 ␮M
bradykinin (BK) either alone or in combination for another 60 min.
* P ⬍ 0.05 compared with [3H]InsPx accumulation induced by ATP,
UTP, or BK alone.
Table 3. Cross-desensitization by ATP- and
UTP-stimulated [Ca2⫹]i changes in TECs
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ATP AND SIGNAL TRANSDUCTION
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, ATPand UTP-induced Ca2⫹ mobilization showed crossdesensitization, 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.
This work was supported by Chang Gung Medical Research Foundation Grant CMRP-680 and National Science Council, Taiwan,
Grant NSC86-2314-B182-107.
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tions (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
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