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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha ,beta -methylene-ATP and beta ,gamma -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha ,beta -methylene-ATP (alpha ,beta -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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) (ATPgamma S; 100 µM) elicited a substantial [3H]InsPx response, whereas 2-methylthio-ATP (2-MeS-ATP; 100 µM), ADP (100 µM), alpha ,beta -MeATP (100 µM), beta ,gamma -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 ATPgamma 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 ATPgamma S increased at a slower rate to a maximum at ~7 min (Fig. 1).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   [3H]InsPx accumulation stimulated by P1 and P2 receptor agonists in TECs



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Time course of [3H]inositol phosphate (InsPx) accumulation after stimulation with ATP, UTP and adenosine 5'-O-(3-thiotriphosphate) (ATPgamma 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 ATPgamma 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 alpha ,beta -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).


View larger version (19K):
[in this window]
[in a new window]
 
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, alpha ,beta -methylene-ATP (alpha ,beta -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 alpha ,beta -MeATP nor beta ,gamma -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.


View larger version (14K):
[in this window]
[in a new window]
 
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).



View larger version (15K):
[in this window]
[in a new window]
 
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.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Additivity of agonist effects on [3H]InsPx accumulation in TECs

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).


View larger version (20K):
[in this window]
[in a new window]
 
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).

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Cross-desensitization by ATP- and UTP-stimulated [Ca2+]i changes in TECs


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha ,beta -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 alpha ,beta -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, alpha ,beta -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 ATPgamma 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abbracchio, MP, and Burnstock G. Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol Ther 64: 445-475, 1994[ISI][Medline].

2.   Boarder, MR, Weisman GA, Turner JT, and Wilkinson GF. G protein-coupled P2 purinoceptors: from molecular biology to functional responses. Trends Pharmacol Sci 16: 133-139, 1995[ISI][Medline].

3.   Brown, HA, Lazarowski ER, Boucher RC, and Harden TK. Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5'-nucleotide receptor in human airway epithelial cell. Mol Pharmacol 40: 648-655, 1991[Abstract].

4.   Burnstock, G. The past, present and future of purine nucleotides as signalling molecules. Neuropharmacology 36: 1127-1139, 1997[ISI][Medline].

5.   Burnstock, G, and Kennedy C. Is there a basis for distinguishing two types of P2-purinoceptors? Gen Pharmacol 16: 433-440, 1985[Medline].

6.   Communi, D, Goverts C, Parmentier M, and Boeynaems J-M. Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase. J Biol Chem 272: 31969-31973, 1997[Abstract/Free Full Text].

7.   Communi, D, Motte S, Boeynaems J-M, and Pirotton S. Pharmacological characterization of the human P2Y4 receptor. Eur J Pharmacol 317: 383-389, 1996[ISI][Medline].

8.   Communi, D, Parmentier M, and Boeynaems J-M. Cloning, functional expression and tissue distribution of the human P2Y6 receptor. Biochem Biophys Res Commun 222: 303-308, 1996[ISI][Medline].

9.   Dalzeil, HH, and Westfall DP. Receptors for adenine nucleotides and nucleosides: subclassification, distribution, and molecular characterization. Pharmacol Rev 46: 449-466, 1994[ISI][Medline].

10.   Dubyak, GR, and El-Moatassim C. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol Cell Physiol 265: C577-C606, 1993[Abstract/Free Full Text].

11.   Fredholm, BB, Abbracchio MP, Burnstock G, Daly JW, Harden TK, Jacobson KA, Leff P, and Williams M. Nomenclature and classification of purinoceptors. Pharmacol Rev 46: 143-156, 1994[ISI][Medline].

12.   Gerwins, P, and Fredholm BB. ATP and its metabolite adenosine act synergistically to mobilize intracellular calcium via the formation of inositol 1,4,5-trisphosphate in a smooth muscle cell line. J Biol Chem 267: 16081-16087, 1992[Abstract/Free Full Text].

13.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

14.   Harden, TK, Boyer JL, and Nicholas RA. P2-purinergic receptors: subtype-associated signaling responses and structure. Annu Rev Pharmacol Toxicol 35: 541-579, 1995[ISI][Medline].

15.   Hourani, SMO, and Hall DA. Receptors for ADP on human blood platelets. Trends Pharmacol Sci 15: 103-108, 1994[ISI][Medline].

16.   Humphrey, PPA, Buell G, Kennedy I, Khakh BS, Michel AD, Surprenant A, and Derek TJ. New insight on P2X purinoceptors. Naunyn Schmiedebergs Arch Pharmacol 352: 585-596, 1995[ISI][Medline].

17.   Jeffery, PK. Morphologic features of airway surface epithelial cells and glands. Am Rev Respir Dis 128: S14-S20, 1983[ISI][Medline].

18.   Kai, H, Yoshitake K, Isohama Y, Hamaura 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].

19.   Kondo, M, Kanoh S, Tamaoki J, Shirakawa H, Miyazaki S, and Nagai A. Erythromycin inhibits ATP-induced intracellular calcium responses in bovine tracheal epithelial cells. Am J Respir Cell Mol Biol 19: 799-804, 1998[Abstract/Free Full Text].

20.   Lazarowski, ER, Homolya L, Boucher RC, and Harden TK. Direct demonstration of mechanically induced release of cellular UTP and its implication for uridine nucleotide receptor activation. J Biol Chem 272: 24348-24354, 1997[Abstract/Free Full Text].

21.   Leon, C, Hechler B, Vial C, Leray C, Cazenave J-P, and Gachet C. The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells. FEBS Lett 403: 26-30, 1997[ISI][Medline].

22.   Luo, SF, Pan SL, Wu WB, Wang CC, Chiu CT, Tsai YJ, and Yang CM. Bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal epithelial cells. Br J Pharmacol 126: 1341-1350, 1999[Abstract/Free Full Text].

23.   Lustig, KD, Shiau AK, Brake AJ, and Julius D. Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc Natl Acad Sci USA 90: 5113-5117, 1993[Abstract].

24.   Murrin, RJ, and Boarder MR. Neuronal `nucleotide' receptor linked to phospholipase C and phospholipase D? Stimulation of PC12 cells by ATP analogues and UTP. Mol Pharmacol 41: 561-568, 1992[Abstract].

25.   Nakanishi, M, Kawasaki M, Ogino H, Yoshida M, and Yagawa K. Extracellular ATP regulates the proliferation of alveolar macrophages. Am J Respir Cell Mol Biol 10: 560-564, 1994[Abstract].

26.   Nicholas, RA, Watt WC, Lazarowski ER, Li Q, and Harden TK. Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: identification of a UDP-selective, a UTP-selective, and an ATP- and UTP-specific receptor. Mol Pharmacol 50: 224-229, 1996[Abstract].

27.   O'Connor, SE, Dainty IA, and Leff P. Further subclassification of ATP receptors based on agonist studies. Trends Pharmacol Sci 12: 137-141, 1991[ISI][Medline].

28.   O'Guin, WM, Schermer A, and Sun TT. Immunofluorescence staining of keratin filaments in cultured epithelial cells. J Tissue Cult Methods 9: 123-128, 1985.

29.   Paradiso, AM, Mason SJ, Lazarowski ER, and Boucher RC. Membrane-restricted regulation of Ca2+ release and influx in polarized epithelia. Nature 377: 643-646, 1995[ISI][Medline].

30.   Parr, CE, Sullivan DM, Paradiso AM, Lazarowski ER, Burch LH, Olsen JC, Erb L, Weisman GA, Boucher RC, and Turner JT. Cloning and expression of a human P2U nucleotide receptor, a target for cystic fibrosis pharmacotherapy. Proc Natl Acad Sci USA 91: 3275-3279, 1994[Abstract].

31.   Pfeilschifter, J. Comparison of extracellular ATP and UTP signalling in rat renal mesangial cells. Biochem J 272: 469-472, 1990[ISI][Medline].

32.   Saiag, B, Bodin P, Shacoori V, Catheline M, Rault B, and Burnstock G. Uptake and flow-induced release of uridine nucleotides from isolated vascular endothelial cells. Endothelium 2: 279-285, 1995.

33.   Stutchfield, J, and Cockcroft S. Undifferentiated HL-60 cells respond to extracellular ATP and UTP by stimulating phospholipase C activation and exocytosis. FEBS Lett 262: 256-258, 1990[ISI][Medline].

34.   Surprenant, A, Rassendren F, Kawashima E, North RA, and Buell G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272: 735-738, 1996[Abstract].

35.   Tokuyama, K, Kuo HP, Rohde JAL, Barnes PJ, and Rogers DF. Neural control of goblet cell secretion in guinea pig airway. Am J Physiol Lung Cell Mol Physiol 259: L108-L115, 1990[Abstract/Free Full Text].

36.   Von Kugelgen, I, and Starke K. Evidence for the separate vasoconstriction-mediating nucleotide receptors, both distinct from the P2X-receptor, in rabbit basilar artery: a receptor for pyrimidine nucleotides and a receptor for purine nucleotides. Naunyn Schmiedebergs Arch Pharmacol 341: 538-546, 1990[ISI][Medline].

37.   Wilson, SM, Law VWY, Pediani JD, Allen EA, Wilson G, Khan ZE, and Ko H. Nucleotide-evoked calcium signals and anion secretion in equine cultured epithelia that express apical P2Y2 receptors and pyrimidine nucleotide receptors. Br J Pharmacol 124: 832-838, 1998[Abstract].

38.   Wu, R, Yankaskas J, Cheng E, Knowles MR, and Boucher JR. Growth and differentiation of human nasal epithelial cells in culture: serum-free, hormone-supplemented medium and proteoglycan synthesis. Am Rev Respir Dis 132: 311-320, 1985[ISI][Medline].

39.   Yang, C-M, Hsia H-C, Chou S-P, Ong R, Hsieh J-T, and Luo S-F. Bradykinin-stimulated phosphoinositide metabolism in cultured canine tracheal smooth muscle cells. Br J Pharmacol 111: 21-28, 1994[Abstract].

40.   Zegarra-Moran, O, Romeo G, and Galietta JV. Regulation of transepithelial ion transport by two different purinoceptors in the apical membrane of canine kidney (MDCK) cells. Br J Pharmacol 114: 1052-1056, 1995[Abstract].


Am J Physiol Lung Cell Mol Physiol 279(2):L235-L241
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Google Scholar
Articles by Yang, C.-M.
Articles by Wang, C.-C.
Articles citing this Article
PubMed
PubMed Citation
Articles by Yang, C.-M.
Articles by Wang, C.-C.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online