Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells

Dong-Mei Zhao1, Hai-Hui Xue2, Kingo Chida1, Takafumi Suda1, Yutaka Oki1, Miho Kanai1, Chiharu Uchida2, Arata Ichiyama2, and Hirotoshi Nakamura1

1 Second Division, Department of Internal Medicine, and 2 First Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
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DISCUSSION
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ATP induced a biphasic increase in the intracellular Ca2+concentration ([Ca2+]i), an initial spike, and a subsequent plateau in A549 cells. Erythromycin (EM) suppressed the ATP-induced [Ca2+]i spike but only in the presence of extracellular calcium (Ca2+o). It was ineffective against ATP- and UTP-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation and UTP-induced [Ca2+]i spike, implying that EM perturbs Ca2+ influx from the extracellular space rather than Ca2+release from intracellular Ca2+ stores via the G protein-phospholipase C-Ins(1,4,5)P3 pathway. A verapamil-sensitive, KCl-induced increase in [Ca2+]i and the Ca2+ influx activated by Ca2+ store depletion were insensitive to EM. 3'-O-(4-benzoylbenzoyl)-ATP evoked an Ca2+o-dependent [Ca2+]i response even in the presence of verapamil or the absence of extracellular Na+, and this response was almost completely abolished by EM pretreatment. RT-PCR analyses revealed that P2X4 as well as P2Y2, P2Y4, and P2Y6 are coexpressed in this cell line. These results suggest that in A549 cells 1) the coexpressed P2X4 and P2Y2/P2Y4 subtypes contribute to the ATP-induced [Ca2+]i spike and 2) EM selectively inhibits Ca2+ influx through the P2X channel. This action of EM may underlie its clinical efficacy in the treatment of airway inflammation.

P2X receptors; P2Y receptors; calcium release; calcium influx; reverse transcriptase-polymerase chain reaction


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EXTRACELLULAR NUCLEOTIDES regulate a broad range of physiological responses by acting on cell surface P2 purinoceptors (13). These are classified into two families: the intrinsic cation-channel P2X-family receptors and the G protein-coupled P2Y receptors. Activation of both families can lead to an elevation in intracellular free Ca2+ concentration ([Ca2+]i), which is a key process in the regulation of many forms of cellular activity (5). The P2Y2 receptor, formerly named the P2U receptor after its comparable response to ATP and UTP, has been cloned from rat and human lung epithelia (29, 32) and is known to mediate enhancement by ATP of surfactant phospholipid secretion in rat type II pneumocytes (18) and Cl- transport in airway epithelia (23). In such inexcitable cells, the Ca2+ signaling elicited by stimulation of the P2Y2 receptor is a biphasic process consisting of an initial spike representing Ca2+ release from an intracellular Ca2+ store through inositol 1,4,5,-trisphosphate [Ins(1,4,5)P3] receptors and a subsequent plateau representing Ca2+ influx through store-operated Ca2+ channels (SOCs), a process also known as capacitative Ca2+ entry (3, 30). In addition, evidence is accumulating that P2X and P2Y receptors are coexpressed in a variety of cells such as platelets and tissues such as vascular and visceral smooth muscle (19, 25). In this case, the activation of P2X receptors also contributes to the [Ca2+]i spike due to the influx of Ca2+ either through the receptors per se (4), that is, the receptor-operated Ca2+ channels (ROCs), or through the voltage-dependent Ca2+ channels (VDCCs) that open on plasma membrane depolarization caused by the concurrent influx of Na+ with Ca2+ through the P2X receptors (12, 25).

Erythromycin (EM) is a 14-member ring macrolide antibiotic widely used in the treatment of upper and lower respiratory tract infections. In addition to its antimicrobial effect, EM has been shown to improve chronic inflammatory processes including diffuse panbronchiolitis, bronchial asthma, and chronic sinusitis (20), suggesting its direct effects on host cells. Indeed, EM was found to inhibit the secretion of respiratory glycoconjugate from human airways (17) and the transport of Cl- across canine tracheal epithelial cells (39) and to improve ciliary motility in rabbit airway epithelium (38). On the other hand, intracellular Ca2+ is known to be involved in Cl- transport and mucin secretion stimulated by inflammatory mediators such as ATP (21, 23, 36). However, the effect of EM on Ca2+ signaling in the functions of airway cells has not been elucidated. Pharmacological analysis by Clunes and Kemp (7) showed that a functional P2Y2 or P2Y2-like receptor1 is expressed in a human lung epithelial-like carcinoma cell line, A549. The aim of the present study was to clarify how EM exerts its beneficial effects on airway cells from the perspective of signal transduction. To this end, we tried to characterize the signal transducing pathways contributing to the ATP-induced [Ca2+]i spike and plateau in A549 cells and to examine the effect of EM on these processes.


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Materials. Fura 2-AM and N-methyl-D-glucamine (NMDG) were obtained from Wako Pure Chemical Industries (Osaka, Japan), and Cremophor El was from Nacalai Tesque (Kyoto, Japan). EM, ATP, UTP, 3'-O-(4-benzoylbenzoyl)-ATP (BzATP), verapamil, U-73122, U-73343, thapsigargin, and (±)-BAY K 8644 were from Sigma (St. Louis, MO). DMEM was from Life Technologies (Rockville, MD).

EM was dissolved in ethanol, and fura 2-AM, thapsigargin, U-73122, and U-73343 were dissolved in DMSO. These drugs were freshly prepared just before use. Small amounts of ethanol or DMSO (final concentration 0.1%) added to cells as a vehicle did not alter cell morphology, Ins(1,4,5)P3 production or fura 2 fluorescence responsiveness.

Cell culture. The human lung epithelial-like carcinoma cell line A549 was obtained from American Type Culture Collection and maintained in DMEM supplemented with 10% fetal bovine serum and 100 U/ml of penicillin-0.1 mg/ml of streptomycin. The cells were grown in plastic culture flasks at 37°C in a humidified atmosphere of 5% CO2-95% air. The cells were used between passages 6 and 20.

Fura 2 loading and measurement of [Ca2+]i. The A549 cells grown to 80-90% confluence on coverslips (35 × 10 mm; MatTek, Ashland, MA), were washed twice with Hanks' balanced salt solution (HBSS) composed of 145 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 1.2 mM CaCl2, 10 mM HEPES, and 1% glucose (pH 7.4) and were then loaded with 5 µM fura 2-AM in HBSS in the dark for 45 min at room temperature. Cremophor El (0.2%) was also included in the incubation medium to improve the uptake of fura 2-AM. Fura 2-AM was removed by a medium change, and then the cells were incubated with HBSS for another 20 min to allow deesterification of fura 2-AM. Next, the cells were washed with HBSS three times and mounted on the stage of an inverted microscope (Diaphot-TMD-300, Nikon, Tokyo, Japan) equipped with an inlet and an outlet for perfusion. At intervals of 5-20 s, a pair of fluorescence intensities emitted (510 nm) by excitation wavelengths of 340 and 380 nm were focused on a silicon-intensified target camera. The signals from the camera were analyzed by a digitized image processor (Argus-50, Hamamatsu Photonics, Hamamatsu, Japan). The ratio of 340- to 380-nm fluorescence intensity was converted to [Ca2+]i with a Ca2+ calibration curve. A fluorescence intensity ratio versus Ca2+ concentration curve was prepared with an Argus-50 internalized calibration program with a Ca2+ calibration buffer kit (Molecular Probes, Eugene, OR). The incubation of cells and measurement of changes in [Ca2+]i were carried out at room temperature (25°C) to reduce the rate of leakage of fura 2 from the cells and to avoid the time-dependent compartmentalization of the probe. Under the conditions used, no vesicular bright spots indicative of the compartmentalization of the probe were observed.

When [Ca2+]i was to be measured in the presence of extracellular Ca2+ (Ca2+o), HBSS containing 1.2 mM CaCl2 was used as the extracellular medium, and the cells were continuously exposed for 3-10 min to a stimulant solution (nucleotides, KCl, or BAY K 8644). For measurement of [Ca2+]i in the absence of Ca2+o, CaCl2 in HBSS was replaced by 1 mM EGTA, and the cells were perfused with the Ca2+-free HBSS for 2-3 min at a flow rate of ~1 ml/min before the agonist addition. In experiments in which the BzATP-induced [Ca2+]i response was measured in the absence of extracellular Na+, NaCl in HBSS was replaced by isotonic NMDG+, and the cells were perfused with the NMDG+-containing HBSS as described above. In some experiments, the cells were exposed to nucleotides only for 45 s to avoid possible receptor desensitization. When the effect of EM on the changes in [Ca2+]i was to be determined, A549 cells were preincubated with the antibiotic or its vehicle for 30 min at room temperature before the addition of stimulants.

Measurement of Ins(1,4,5)P3. A549 cells grown to confluence in 100 × 20-mm dishes (~2 × 106 cells/dish) were washed once with HBSS and incubated in this medium for 20 min at 37°C. EM, U-73122, U-73343, or vehicle (ethanol for EM and DMSO for U-73122 and U-73343) was added, and the incubation was continued at room temperature for 30 (EM and ethanol) or 3 (U-73122, U-73343, and DMSO) min followed by replacement of the incubation medium with 1.5 ml of an agonist solution (ATP or UTP) in HBSS and further incubation at room temperature for given periods. The incubation was finally terminated by adding 0.3 ml of ice-cold 2 M perchloric acid, and the acidified mixture was left on ice for 20 min. The cells were detached from the dishes with a scraper, and the suspension was mixed vigorously and then centrifuged at 4°C for 15 min. One milliliter of the resulting supernatant was neutralized with ice-cold 1.5 M KOH containing 60 mM HEPES to a phenol red end point. After standing at 4°C for 15 min, the samples were centrifuged in the cold to remove potassium perchlorate precipitates. The Ins(1,4,5)P3 content of the supernatant was measured by radioimmunoassay [D-myo-[3H]Ins(1,4,5)P3 assay system, Amersham Pharmacia Biotech, Tokyo, Japan] according to the manufacturer's instructions. The working range of the assay was 0.5-15 pmol of Ins(1,4,5)P3.

Identification of the P2 receptors by RT-PCR, subcloning, and DNA sequencing. Total RNA was prepared from A549 cells grown to confluence with ISOGEN (Wako Pure Chemical Industries), according to the manufacturer's instructions and then treated with DNase I (amplification grade; Life Technologies). Poly(A)+ RNA was isolated from the resulting RNA preparation with Oligotex-dT30 (Roche Diagnostics, Tokyo, Japan), and its concentration was determined spectrophotometrically. First-strand cDNA was synthesized from 500 ng of poly(A)+ RNA by an oligo(dT)-primed reaction (20 µl) with a Superscript preamplification system (Life Technologies). A control reaction was performed in the absence of RT.

For identification of the uridine nucleotide-responsive G protein-coupled P2Y receptors (P2Y2, P2Y4, and P2Y6), inosine (I)-containing degenerate primers were designed based on the amino acids conserved among the three human P2Y subtypes (10, 11, 26, 29; for alignment of amino acid sequences, see Ref. 2). Primer y1 [5'-CGITT(C/T)CTITT(C/T)TA(C/T)(A/G) CIAACCT-3'] (nucleotides in parentheses, wobbles introduced in the primer to obtain complete matches with each template) and primer y4 [5'-AGIA(C/T)IGG(A/G)TCIAG(A/G)CA(A/G)CTGTT-3'] used to amplify first-strand cDNA corresponded to conserved regions in the third and seventh transmembrane domains, respectively. Another set of primers used in the nested PCR, primer y2 [5'-ATCAG(C/T)(G/T)TICAICGIT(A/G)(C/T)CTIGG-3'] and primer y3 [5'-CTGTT(A/G)GC(A/G)CTIGCIA(A/G)IGG(C/T)CG-3'], corresponded to the conserved regions located between those used for the design of primers y1 and y4 (see Fig. 7). Inosine-containing degenerate primers for P2X receptors were also synthesized based on the conserved amino acids among P2X1 to P2X7 receptors (8; for alignment of amino acid sequences, see Ref. 2). Primer x1 [5'-TGGGA(C/T)GTIG(A/C)IGAIT(A/T)(C/T)GT-3'] and primer x4 [5'-GTIGGIATIAT(A/G)TC(A/G)AA(C/T)TTICC-3'] were used to amplify first-strand cDNA, and another set of oligonucleotides, primer x2 [5'-TG(C/T)GA(A/G)(A/G)TIT(C/T)IG(C/G)ITGGTG(C/T)CC-3'] and primer x3 [5'-GC(A/G)AA(C/T)CT(A/G)AA(A/G)TT(A/G)TAICC-3'] were used in the nested PCR. See Fig. 7 for the location of the conserved amino acid regions in the P2X and P2Y receptors.

PCR amplification of first-strand cDNA was carried out with the hot-start method with AmpliWax PCR Gem 100 (Perkin-Elmer, Foster City, CA). Briefly, up to 100 pmol of degenerate primers ( primers y1 and y4 for P2Y subtypes and primers x1 and x4 for P2X subtypes) were mixed with 10 nmol of each deoxynucleotide triphosphate to give a lower mixture of 20 µl. One pilule of wax Gem was added to each reaction, and the mixture was incubated at 75°C for 7 min followed by further incubation at 25°C for 3 min. Then, 30 µl of the upper mixture containing 1 µl of first-strand cDNA and 2.5 U of Taq plus Pwo polymerase (Roche Diagnostics) was layered on the solidified wax phase. PCR was started by incubation at 94°C for 90 s followed by 10 (94°C for 45 s, 42°C for 45 s, and 72°C for 90 s) and 25 (94°C for 45 s, 45°C for 45 s, and 72°C for 90 s) cycles of amplification, and a further extension at 72°C for 7 min. The product (1 µl) was then subjected to nested PCR amplification with primers y2 and y3 for P2Y subtypes and primers x2 and x3 for P2X subtypes. The conditions for the nested PCR were the same as above except that the annealing temperatures were 45 and 50°C for the 10- and 25-cycle amplifications, respectively.

The PCR products were resolved by electrophoresis on 5% polyacrylamide gels and visualized with an ultraviolet transilluminator. PCR products from the nested PCR were extracted from the gels and then cloned into pGEM-T Easy vector with a TA cloning system (Promega, Madison, WI). Two microliters of the ligation mixture were transformed into Escherichia coli. DH5-competent cells and positive colonies were selected based on increases in size of plasmids. Plasmid DNA was isolated with Wizard Plus SV minipreps DNA purification systems (Promega) and then sequenced with an automated fluorescent sequencing system (ABI Prism BigDye terminator cycle sequencing kit and ABI Prism 310 genetic analyzer, Perkin-Elmer). The sequencing was performed from both sides of the inserted PCR fragments with T7 and SP6 primers.

Selective primers for P2X4 and those for P2X7 were designed according to the published sequences (15; GenBank accession no. for human P2X7 is Y09561): 5'-GGAGATACGTTGTGCTCAACG-3' and 5'-ATGCCATAGGCCTTGATGAGC-3' for P2X4 and 5'-AGGAGATCGTGGAGAATGGAG-3' and 5'-AAGGACACGTTGGTGGTCTTG-3' for P2X7. These primers were used to amplify first-strand cDNA from A549 cells. The PCR mixture (50 µl) contained 25 pmol of each primer, 2.5 U of Taq and Pwo polymerase, 10 pmol of each deoxynucleotide triphosphate, 1 µl of cDNA, and 5 µl of 10× buffer provided with the polymerase. PCR conditions were 95°C for 3 min followed by 30 cycles (95°C for 1 min, 45°C for 1 min, and 72°C for 2 min) of amplification, and a further 7-min extension at 72°C.

Data analysis. In experiments involving the measurement of changes in [Ca2+]i, 4-10 images were recorded before administration of the agonist to calculate the basal [Ca2+]i level. The increase in [Ca2+]i (Delta [Ca2+]i) was calculated by subtraction of the basal [Ca2+]i. The maximum [Ca2+]i response observed within 30 s after agonist administration was regarded as the [Ca2+]i spike. In each experiment, changes in [Ca2+]i in 7-14 individual cells were measured, and the experiment was repeated at least three times. Delta [Ca2+]i is expressed as means ± SD, and differences were analyzed by Student's t-test. A probability of P < 0.05 was considered to be significant.


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Effect of EM on the ATP-induced [Ca2+]i spike in A549 cells. In standard extracellular medium containing 1.2 mM CaCl2, [Ca2+]i in A549 cells showed a rapid increase in response to ATP in a dose-dependent manner, in agreement with Clunes and Kemp (7). The increase in [Ca2+]i was biphasic, with an initial spike followed by a subsequent sustained phase (plateau) (Fig. 1A). In this experiment, we used a high dose (100 µM) of ATP to fully activate its receptors. In the experiment shown in Fig. 1A, the cells were continuously exposed to 100 µM ATP for 10 min. When the cells were exposed to ATP only for 45 s, the [Ca2+]i spike was essentially the same as that observed under the continuous stimulation, although the sustained phase was shorter.


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Fig. 1.   Effect of erythromycin (EM) on ATP-induced intracellular Ca2+ concentration ([Ca2+]i) spike. Fura 2-loaded A549 cells were treated with either vehicle (0.1% ethanol) or EM (0.1 mM in A and C and indicated concentrations in B) at room temperature for 30 min. After perfusion with standard extracellular medium [Hanks' balanced salt solution (HBSS) containing 1.2 mM CaCl2] or Ca2+-free medium (HBSS containing 1 mM EGTA in place of CaCl2) for 2-3 min, cells were exposed to 100 µM ATP for 10 min (A) or 45 s to 10 min (B and C). Fluorescent signals were recorded at 10-s intervals, and increase in (Delta ) [Ca2+]i and [Ca2+]i spike were calculated from fluorescence ratio (340- to 380-nm fluorescence intensity) as described in METHODS. A: time course of ATP-induced [Ca2+]i response in presence of extracellular Ca2+ (Ca2+o) after pretreatment with EM or vehicle. Experiments were carried out >10 times with similar results. Representative recordings, each from a single cell, are shown. B: dose-dependent inhibition of [Ca2+]i spike by EM in presence of Ca2+o. C: effect of EM on [Ca2+]i spike in presence (+) and absence (-) of Ca2+o. Values are means ± SD of 4-8 experiments; nos. in parentheses, no. of cells observed. ** P < 0.05 vs. control value without EM pretreatment.

Pretreatment of A549 cells with EM caused a concentration-dependent inhibition of the ATP-induced [Ca2+]i spike (Fig. 1, A and B), and the concentrations of EM used were clinically achievable (24). In preliminary experiments, EM alone did not cause any significant change in [Ca2+]i during the 30-min preincubation, and the 30-min pretreatment with EM gave a maximum inhibition of the ATP-induced [Ca2+]i spike, although the inhibition by EM was only partial (~40%) even when higher concentrations (up to 10-3 M) were used.

In the absence of Ca2+o, on the other hand, the ATP-induced [Ca2+]i spike was lower than that in the presence of Ca2+o and was not appreciably inhibited by pretreatment with EM (Fig. 1C). These results suggest that a Ca2+ entry process also contributes to the ATP-induced [Ca2+]i spike in the presence of Ca2+o and that this process is the target of EM.

Effects of EM and U-73122 on ATP- and UTP-stimulated Ins(1,4,5)P3 formation. The existence of a functional P2Y2 or P2Y2-like receptor in A549 cells has been suggested from a pharmacological analysis (7) showing that both ATP and UTP induced a [Ca2+]i response with similar potency. The P2Y2 or P2Y2-like receptor is a G protein-coupled receptor linked to the phospholipase effector enzymes in a variety of cells (13). In the present study, both ATP and UTP caused a prompt increase in Ins(1,4,5)P3 formation in A549 cells in a dose-dependent manner, with the peak response 5 s after the addition of ATP or UTP (Fig. 2, A and B). Pretreatment of cells for 3 min with 10 µM U-73122, an uncoupler of G protein-mediated phospholipase C activation (34), almost completely abolished ATP- and UTP-induced Ins(1,4,5)P3 formation, whereas pretreatment with U-73343, an inactive analog of U-73122, was without effect (Fig. 2C). In addition, the ATP-induced [Ca2+]i spike in the absence of Ca2+o was completely abolished by U-73122 (compare with Fig. 4D), suggesting that the [Ca2+]i spike induced by activation of the P2Y2 or P2Y2-like receptor in A549 cells is principally mediated by G proteins and phospholipase C. On the other hand, pretreatment of A549 cells with 0.1 mM EM for 30 min did not affect ATP- or UTP-stimulated Ins(1,4,5)P3 formation (Fig. 2C). Together with the ineffectiveness of EM in inhibiting the [Ca2+]i spike in the absence of Ca2+o, these results suggest that EM does not affect the Ins(1,4,5)P3-mediated intracellular signaling events that lead to Ca2+mobilization on activation of the P2Y2 or P2Y2-like receptor.


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Fig. 2.   Changes in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3; IP3] in response to extracellular nucleotides. A: time course of Ins(1,4,5)P3 accumulation in response to ATP. A549 cells were incubated with 100 µM ATP at room temperature for indicated periods. B: Ins(1,4,5)P3 accumulation in response to various concentrations of ATP and UTP. A549 cells were exposed to indicated concentrations of ATP or UTP for 5 s. C: effect of EM, its vehicle [ethanol (EtOH)], U-73122, and U-73343 on ATP- and UTP-induced Ins(1,4,5)P3 accumulation. Cells were subjected to pretreatment with 0.1 mM EM, 0.1% EtOH, 10 µM U-73122, or 10 µM U-73343 as described in METHODS and then incubated with 100 µM ATP or UTP for 5 s. Ins(1,4,5)P3 in each sample was determined in duplicate. Values are means ± SD of 4 separate experiments.

Effects of EM and verapamil on the [Ca2+]i response induced by KCl or BAY K 8644. Ca2+ influx through VDCCs is a candidate for the Ca2+ entry processes that may contribute to the ATP-induced [Ca2+]i spike, although the T-type VDCC was reported to be absent in A549 cells (33). To examine the possibility that other subtypes of VDCCs are expressed in this cell line, we used a high concentration of KCl (55 mM) to directly activate the putative Ca2+ channel. On the addition of KCl, the elevation in [Ca2+]i was first observed right underneath the membrane (Fig. 3A), implying an immediate Ca2+ influx through a VDCC. Then the [Ca2+]i increased further and spread through whole cells, probably due to a Ca2+-induced Ca2+ release (CICR) mechanism. The Ca2+ channel responsible was identified to be the L-type VDCC because the [Ca2+]i response was mimicked by an L-type-specific agonist, BAY K 8644 (10 µM), and was abrogated by an L-type-selective antagonist, verapamil (20 µM; Fig. 3, B and C). The [Ca2+]i response induced by either KCl or BAY K 8644 was insensitive to pretreatment with EM (Fig. 3, B and C).


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Fig. 3.   KCl- and BAY K 8644-induced [Ca2+]i response. A: pseudocolor images of [Ca2+]i response to addition of KCl. KCl (55 mM) was applied to fura 2-loaded A549 cells, and changes in [Ca2+]i were followed at 5-s intervals. Images were taken before (i) and 5 (ii) and 10 (iii) s after KCl application. Images were processed with an Argus-50 digitized image processor as described in METHODS. Increases in [Ca2+]i are color coded: blue and pink correspond to low and high [Ca2+]i levels, respectively (right). B: effects of verapamil and EM on KCl-induced [Ca2+]i response. A549 cells were treated with 20 µM verapamil for 3 min or 0.1 mM EM for 30 min before KCl application. C: effects of verapamil and EM on BAY K 8644-induced [Ca2+]i response. Cells were treated with verapamil or EM as above before BAY K 8644 application. Fluorescent signals were recorded at 5-s intervals, and Delta [Ca2+]i was calculated as described in METHODS. Experiments were carried out at least 5 times, with similar results. Representative recordings, each from a single cell, are shown.

In this experiment, the [Ca2+]i response induced by 10 µM BAY K 8644 was lower than that induced by KCl. This probably arises from the use of (±)-BAY K 8644, which is composed of two optical isomers. It has been reported that only the (-)-enantiomer of BAY K 8644 has strongly Ca2+ agonistic properties and that the (+)-enantiomer is a weak antagonist (14).

Effect of EM on the cation-channel P2X receptor(s). Because EM does not affect Ca2+ influx through the L-type VDCC, other Ca2+ channels and/or regulatory events located upstream from VDCC activation may be the target of EM. The P2X-family receptor can be considered a candidate Ca2+ channel because it functions as a ligand-gated nonselective cation channel. On activation, P2X receptors function as ROCs and provide a "brief high-intensity burst" of [Ca2+]i (5). In addition, stimulation of P2X receptors has been shown to be accompanied by activation of the L-type VDCC in cardiac myocytes (12) and smooth muscle (25), probably through the influx of Na+. Thus we examined the possibility that the P2X receptor(s) is coexpressed with the P2Y2 or P2Y2-like receptor in A549 cells and that its function is blocked by EM.

BzATP has been repeatedly shown to be least effective in the induction of Ins(1,4,5)P3 in a variety of cells such as submandibular ductal cells (1) and HL-60 cells (35) and has been used as a selective agonist for P2X receptors (13, 40, 41). In our preliminary experiments (n = 2), a 5-s incubation of A549 cells with 100 µM BzATP caused only 15 and 7% increases (vs. control value) in Ins(1,4,5)P3, confirming the reported ineffectiveness or least effectiveness of BzATP in the induction of Ins(1,4,5)P3. As shown in Fig. 4A, BzATP induced a dose-dependent [Ca2+]i response in A549 cells. The [Ca2+]i response to BzATP was totally dependent on Ca2+o (Fig. 4Aiii), validating the absence of P2Y-mediated Ca2+ mobilization. Pretreatment of the cells with EM abolished the BzATP-induced [Ca2+]i response in the presence of Ca2+o (Fig. 4, A and B).


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Fig. 4.   Influx of Ca2+ through putative P2X receptor(s). A: effect of EM on 3'-O-(4-benzoylbenzoyl)-ATP (BzATP)-induced [Ca2+]i response. A549 cells were perfused with standard extracellular (i, ii, and iv) or Ca2+-free (iii) medium for 2-3 min, and then 30 (i) or 100 (ii-iv) µM BzATP in respective medium was applied to cells. In experiment iv, A549 cells were treated for 30 min with 0.1 mM EM. Fluorescent signals were recorded at 5-s intervals, and Delta [Ca2+]i was calculated as described in METHODS. Experiments were carried out 3-5 times with similar results. Representative recordings, each from a single cell are shown. B: summary of experiments ii-iv in A. C: effect of EM on UTP-induced [Ca2+]i spike in presence and absence of Ca2+o. Experiments were carried out as in A except that 100 µM UTP was applied to cells as agonist for 45 s. D: effects of U-73122 and EM on ATP-induced [Ca2+]i spike in presence and absence of Ca2+o. A549 cells were treated with either 10 µM U-73122 for 3 min, 0.1 mM EM for 30 min, or both U-73122 and EM (for 27 min with EM and then for 3 min with both U-73122 and EM). Cells were then perfused with standard extracellular or Ca2+-free medium followed by exposure to 100 µM ATP for 45 s. U-73122 (10 µM) was also included in ATP solution in experiment with U-73122-treated cells. Cells treated with 10 µM U-73343, an inactive analog of U-73122, showed the same response as that of untreated cells (data not shown). Values are means ± SD from 3-6 experiments; nos. in parentheses, no. of cells observed. ** P < 0.05 vs. corresponding control value.

UTP is a selective agonist for P2Y2 and P2Y4 (27) and does not act on P2X-family receptors. In agreement with a previous report (7), UTP as well as ATP induced a biphasic [Ca2+]i response in A549 cells in the presence of Ca2+o. However, the UTP-induced spike was lower than the ATP-induced one, although it was slightly higher than that induced by ATP in the absence of Ca2+o. In addition, neither the removal of extracellular Ca2+ nor the pretreatment of cells with EM further lowered the UTP-induced [Ca2+]i spike (Fig. 4C). These results suggest that the [Ca2+]i spike elicited by UTP can be ascribed to the Ins(1,4,5)P3-mediated Ca2+ mobilization caused by activation of the P2Y2 or P2Y2-like receptor and that this process is not affected by EM.

A combination of the removal of Ca2+o and the treatment of cells with U-73122 led to an almost complete inhibition of the ATP-induced [Ca2+]i spike (Fig. 4D). In contrast, when U-73122-treated cells were stimulated by ATP in the presence of Ca2+o, a compromised [Ca2+]i response was observed, and this response was again abolished by pretreatment of the cells with EM. These results further corroborated that an EM-sensitive Ca2+influx is activated by ATP independently of activation of the P2Y2 or P2Y2-like receptor.

As for the inhibitory mechanism of EM, two possibilities can be considered. One is direct inhibition of the P2X-receptor cation-channel activity, and the other is interference with the events between P2X activation and opening of the VDCC. As mentioned above, the VDCC can be secondarily activated by membrane depolarization caused by the influx of Na+ through the P2X channels (12, 25). To narrow down the possibilities, we measured the BzATP-induced [Ca2+]i response 1) in the presence of verapamil to block VDCC or 2) with NMDG+ in place of NaCl in HBSS to prevent Na+ influx and the ensuing membrane depolarization. As shown in Fig. 5, neither verapamil nor the replacement of Na+ with NMDG+ had a significant effect on the BzATP-induced [Ca2+]i response, suggesting that the BzATP-induced [Ca2+]i response is entirely or largely mediated by P2X and independent of activation of the VDCC. In addition, the BzATP-induced [Ca2+]i response in the absence of Na+ was completely abolished in EM-pretreated cells (Fig. 5B), implying direct inhibition by EM of Ca2+influx through the P2X channels.


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Fig. 5.   Direct inhibition by EM of Ca2+-channel activity of P2X receptor(s). A: effects of verapamil and EM on BzATP-induced [Ca2+]i response in presence of extracellular Na+. B: effect of EM on BzATP-induced [Ca2+]i response in absence of extracellular Na+. Fura 2-loaded A549 cells were perfused for 2-3 min with HBSS (A) or N-methyl-D-glucamine (NMDG+)-containing HBSS (B) and then exposed to BzATP. Where indicated, cells were pretreated with 0.1 mM EM for 30 min or 20 µM verapamil for 3 min. Fluorescent signals were recorded at 5-s intervals, and Delta [Ca2+]i was calculated as described in METHODS. Experiments were carried out 3 times, and representative recordings, each from a single cell, are shown.

Effects of EM and Ni2+ on Ca2+ influx through SOCs. In EM-pretreated cells, the [Ca2+]i plateau after the ATP-induced [Ca2+]i spike was also lower (compare with Fig. 1A). In nonexcitable cells, the plateau phase is known to primarily result from Ca2+ influx through SOCs, which is indispensable for replenishment of intracellular Ca2+ stores (30). The influx of Ca2+ through SOCs can be activated in the absence of measurable phosphoinositide hydrolysis by passive depletion of the Ca2+ stores with thapsigargin, a selective inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (28). To examine whether the lowered Ca2+ plateau resulted from direct inhibition of SOCs by EM, we applied thapsigargin (0.1 µM) to A549 cells in the absence of Ca2+o. On addition of thapsigargin, release of Ca2+ from the intracellular stores occurred (Fig. 6A), and after the released Ca2+ was pumped out, readdition of Ca2+o gave rise to an elevation in [Ca2+]i, indicating activation of Ca2+ influx through SOCs. The influx was abrogated by 10 mM Ni2+ (Fig. 6B), a nonspecific Ca2+-channel blocker, but not by verapamil up to 50 µM (data not shown), confirming that Ca2+ entry evoked by store depletion was unrelated to the L-type VDCC in A549 cells. In EM-pretreated cells, neither the content of the intracellular stores nor the Ca2+ influx through SOCs was affected (Fig. 6C).


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Fig. 6.   Influx of Ca2+ through store-operated Ca2+ channels (SOCs). Thapsigargin (Tg; 0.1 µM) was added to untreated (A and B) or EM-treated (C) fura 2-loaded A549 cells in absence of Ca2+o to deplete Tg-sensitive intracellular Ca2+ stores. After [Ca2+]i returned to basal level, Tg was removed by perfusion and standard extracellular medium containing 1.2 mM CaCl2 was added to cells. In B, cells were incubated with 10 mM Ni2+ (final concentration) for 3 min before addition of extracellular Ca2+. In C, cells were pretreated with EM (0.1 mM) for 30 min before addition of Tg. Fluorescent signals were recorded at 20-s intervals, and Delta [Ca2+]i was calculated as described in METHODS. Experiments were carried out at least 3 times with similar results. Representative recordings, each from a single cell, are shown.

Identification of P2X and P2Y subtypes. To ascertain, on a molecular basis, the coexpression of P2X and P2Y receptors in A549 cells, we amplified their mRNAs by RT-PCR using degenerate primers. In the case of P2Y receptors, the degenerate primers were designed based on the best-conserved amino acids among P2Y2, P2Y4, and P2Y6 receptors (Fig. 7B), which share a common feature of the response to uridine nucleotides. Primers y1 and y4 were used in low-stringency PCR on DNase I-treated mRNA from A549 cells and generated a major product of ~580 bp, the size expected for these P2Y subtypes. This product was further amplified with the nested primers y2 and y3 to enhance the specificity, resulting in an ~510-bp fragment (Fig. 7D). This fragment was extracted from the gel, ligated into a vector, subcloned in E. coli DH5, and sequenced after screening. Of the 29 positive colonies, 17 contained a sequence identical to that of P2Y6, and 7 and 5 colonies had sequences identical to those of P2Y2 and P2Y4, respectively. These results indicate that the three P2Y subtypes are coexpressed in A549 cells.


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Fig. 7.   Identification of P2X and P2Y subtypes. A and B: conserved amino acid regions in P2X and P2Y receptors, respectively, used for design of degenerate primers. P2Xs are composed of 2 transmembrane domains (TM1 and TM2) separated by a long extracellular loop, relatively short NH2 and COOH termini in cytoplasm, and a highly conserved hydrophobic segment (H5) that precedes TM2. Primers x1, x2, and x3 correspond to conserved regions in extracellular loop, and primer x4 corresponds to an extracellular region between H5 and TM2 (2). P2Ys constitute a subfamily of G protein-coupled receptors and are characterized by 7 transmembrane domains (TM1 to TM7). Primers y1 and y2 correspond to a conserved region in TM3 and that immediately downstream of TM3, respectively. Primers y3 and y4 correspond to conserved regions in TM7 that partially overlap each other. Italic type, overlapped amino acids; boldface type, well-conserved amino acids among respective subtypes. Note that length of the lines does not represent actual size of primers or cDNAs. C and D: RT-PCR amplification of P2X and P2Y mRNAs, respectively, with degenerate primers. Experimental details are described in METHODS. Primers used are given above each lane. E: RT-PCR with 2 sets of primers each specific to P2X4 and P2X7, respectively. A fragment of the expected size (650 bp) was produced in RT-PCR with P2X4-specific primers, but no product was detected with P2X7-specific primers. If P2X7 mRNA was expressed, a product of 642 bp was expected. Hinf I digest of pBR322 was used as a size marker. Nos. at left, bp.

The same strategy was used to detect P2X subtypes. Primers x1 and x4 amplified an ~770-bp fragment from the mRNA preparation, and this product was amplified in the nested PCR with primers x2 and x3, generating an ~440-bp fragment (Fig. 7, A and C). This fragment was sequenced as described above, and the sequence of the 24 positive colonies was identical to that of P2X4, indicating that P2X4 is the most abundant, if not the exclusive, subtype in this cell line.

BzATP has been recognized as the most potent agonist for P2X7 but has not been tested for P2X4. We noticed that the amino acids corresponding to primer x1 [WDV(A,S)(D,E)(F,Y)V] are highly conserved among the P2X1 to P2X6 subtypes, but the conservation is less in P2X7 [FDTADYT] (boldface type denotes well-conserved amino acids among P2X subtypes). To find out whether P2X7 is also coexpressed with P2X4 in this cell line, two sets of primers specific for P2X4 and P2X7 were synthesized and used to amplify first-strand cDNA. As shown in Fig. 7E, the expression of P2X4 was confirmed, but that of P2X7 was not detectable.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we provided pharmacological and molecular evidence that P2X and P2Y receptors are coexpressed in A549 cells. We also showed that both the Ca2+ mobilization through Ins(1,4,5)P3 receptors and the Ca2+ influx through the P2X receptor are involved in the spike phase of the [Ca2+]i response when cells are exposed to ATP. The presence of a functional P2Y2 or P2Y2-like receptor in A549 cells had been shown by Clunes and Kemp (7), and this was confirmed in our experiments as shown by both ATP and UTP, at a concentration of 100 µM, inducing a similar [Ca2+]i spike in the absence of Ca2+o. In addition, we found that both ATP and UTP stimulate similar amounts of Ins(1,4,5)P3 accumulation in a dose-dependent manner (Fig. 2B). These findings are compatible with the results of RT-PCR analyses, which showed that P2Y2, P2Y4, and P2Y6 are coexpressed in this cell line. It has been shown that the P2Y2 subtype responds to ATP and UTP equipotently, whereas the P2Y4 subtype responds preferentially to UTP (9). A study by Nicholas et al. (27) in which purity of the nucleotides was carefully controlled further showed that both ATP and UTP are full agonists for P2Y4, although the affinity of this receptor for ATP [concentration of agonist causing half-maximal effect (K0.5) = 3.9 ± 0.7 µM] is 50-fold less than that for UTP (K0.5 = 0.8 ± 0.2 µM). In our experiments, ATP and UTP were used at concentrations of 10-100 µM, and under these conditions, both P2Y2 and P2Y4 can be fully activated, resulting in an apparently similar response such as Ins(1,4,5)P3 accumulation and the [Ca2+]i response in the absence of Ca2+o. P2Y6 is a UDP-selective receptor and thus not involved in the ATP- and UTP-induced intracellular response. The physiological significance of the expression of various P2Y subtypes including P2Y6 remains to be elucidated.

P2Y2 and P2Y4 are coupled to G proteins to stimulate phosphoinositide metabolism and Ca2+ mobilization (9, 13). In A549 cells, both the ATP- and UTP-induced Ins(1,4,5)P3 formation (Fig. 2C) and the increase in [Ca2+]i in the absence of Ca2+o (Fig. 4D) were almost completely suppressed by pretreatment with U-73122, an uncoupler of the G protein-mediated phospholipase C activation. These results suggest that the [Ca2+]i response induced by activation of P2Y2 and P2Y4 is accounted for by the phospholipase C-mediated Ca2+ release from intracellular stores. However, the ATP-induced [Ca2+]i spike in the absence of Ca2+o was obviously lower than that in the presence of Ca2+o (Fig. 1C), suggesting that Ca2+ influx is also involved in the [Ca2+]i response in the presence of Ca2+o. A P2X-family receptor was considered to be responsible for the Ca2+ influx based on the following observations. 1) BzATP, known as a selective agonist for some subtypes of P2X (1, 41), elicited a Ca2+o-dependent increase in [Ca2+]i (Fig. 4, A and B) without stimulating the formation of Ins(1,4,5)P3. 2) The ATP-induced [Ca2+]i spike in the absence of Ca2+o was completely abolished by U-73122, whereas in the presence of Ca2+o, a portion of the increase in [Ca2+]i was preserved (Fig. 4D). 3) UTP, a selective agonist for P2Y2 and P2Y4, induced a [Ca2+]i response similar to that induced by ATP in the absence of Ca2+o, whereas ATP, but not UTP, caused an additional increase in [Ca2+]i in the presence of Ca2+o. Indeed, the expression of P2X4 in A549 cells was confirmed by RT-PCR. This subtype of the P2X-family receptors appears to be most abundant, if not exclusive, and may be the responsible channel that permits Ca2+ entry in response to ATP. These results are in contrast to those described by Clunes and Kemp (7) in that they measured a similar [Ca2+]i spike in response to ATP whether Ca2+o was present or not. We believe that Ca2+entry through the P2X receptor participates in the spike phase of the ATP-induced [Ca2+]i response because we were able to detect P2X4 mRNA and a Ca2+o-dependent rapid increase in [Ca2+]i in response to a P2X-selective agonist BzATP.

Unexpectedly, a functional VDCC was found in this cell line and was probably the L type as judged by both the induction by BAY K 8644 and the inhibition by verapamil (Fig. 3). Ca2+ entry via a VDCC is also a rapid process, and opening of the VDCC by ATP occurs subsequent to activation of P2X in cardiac myocytes and smooth muscle (12, 25). In A549 cells, however, the BzATP-induced [Ca2+]i response was not affected by either pretreatment with verapamil or isotonic replacement of Na+ with NMDG+ in the extracellular medium, suggesting that Ca2+ influx through the P2X receptor per se evoked the [Ca2+]i response independent of opening of the VDCC.

Low concentrations of BzATP (30 µM or lower) were found to elicit an increase in [Ca2+]i after a latency (Fig. 4A). A similar precedent has been provided by Sun et al. (37). In their study, human platelet-derived P2X1 expressed in a different cell line showed a slow-onset [Ca2+]i response to low concentrations of ADP. The latency may represent a period that is necessary for a local increase in [Ca2+]i to propagate to a global response via CICR. Underlying CICR is the release of Ca2+ from intracellular stores through Ins(1,4,5)P3 and/or ryanodine receptors. These receptors, both forming homotetrameric Ca2+ channels, show bell-shaped open probability curves versus the free Ca2+ concentrations to which they were exposed (6), although their kinetic constants are different. Therefore, when the Ca2+ influx is small in response to low concentrations of agonist for P2X channels, a lag may arise before the regenerative release of Ca2+ through CICR excites the whole cell. When the Ca2+ influx is triggered by high concentrations of KCl or BzATP, as shown in Figs. 3A and 4A, CICR provides a rapid [Ca2+]i response. In addition to the role of the ryanodine receptor in CICR (ryanodine receptor type I was found to be expressed in A549 cells; Xue H-H, Zhao D-M, Suda T, Uchida C, Oda T, Chida K, Ichiyama A, and Nakamura H, unpublished observations), the opening of Ins(1,4,5)P3 receptors is proposed to be under the dual control of Ins(1,4,5)P3 and Ca2+ [reviewed by Berridge (5)], and this has been clearly established in Xenopus oocytes by showing that cytosolic Ca2+ potentiates the ability of Ins(1,4,5)P3 to liberate Ca2+ from intracellular stores (42). These observations and considerations raised a possibility that Ca2+ entering the cells through ROCs and/or VDCCs can coordinate with Ins(1,4,5)P3 to enhance Ca2+ mobilization in addition to directly participating in the [Ca2+]i spike by itself, resulting in an elevated [Ca2+]i spike. Thus P2X and P2Y receptors may cooperate, when they are coexpressed, to participate in shaping the [Ca2+]i spike in response to extracellular nucleotides.

One of our major goals has been to elucidate how EM affects the signal Ca2+ in host cells. In this study, the following observations suggest that EM suppresses the ATP-induced [Ca2+]i spike by selectively inhibiting the cation-channel activity of the P2X receptor. First, neither the [Ca2+]i spike elicited by UTP (Fig. 4C) or by ATP in the absence of Ca2+o (Fig. 1C) nor Ins(1,4,5)P3 formation by both ATP (Fig. 2B) and UTP was affected by pretreatment of the cells with EM, indicating that the P2Y2- and P2Y4-initiated intracellular signaling leading to Ca2+ mobilization is independent of EM inhibition. Second, the Ca2+ influx evoked on activation of the P2X receptor, stimulated selectively by either BzATP (Fig. 4, A and B) or ATP in the presence of U-73122 to block the P2Y2 and P2Y4 pathways (Fig. 4D), was effectively inhibited by EM. Third, Ca2+ influx through the L-type VDCC can be completely blocked by verapamil but not by EM (Fig. 3). Furthermore, the BzATP-induced [Ca2+]i response in the absence of extracellular Na+ was also abolished by EM pretreatment, suggesting a direct effect of EM on the channel activity of the P2X receptor rather than the processes downstream of the Na+ influx. The most abundant subtype of P2X receptors expressed in A549 cells was identified to be P2X4, and studies on stably expressed recombinant P2X4 are under way.

We noticed that the [Ca2+]i plateau after the ATP-induced [Ca2+]i spike was also lower in EM-pretreated A549 cells. It seems unlikely that this phenomenon is attributable to the direct inhibition of SOCs by EM because Ca2+ entry activated by store depletion with thapsigargin was independent of EM inhibition (Fig. 6). Ca2+ influx through SOCs is known to be important in refilling the discharged intracellular Ca2+ stores (30). Although the signal that couples the store content to SOCs remains to be identified, the quantity of Ca2+ entering the cytoplasm is thought to be in tune with that mobilized from the stores (28). Considering these quantal properties of Ca2+ entry, we assume that the inhibition by EM of Ca2+ influx through the P2X receptor compromises intracellular Ca2+ mobilization, and thus there is less depletion of Ca2+ stores, which, in turn, leads to a reduced Ca2+ influx through SOCs, resulting in the lowered [Ca2+]i plateau in EM-treated A549 cells. Recently, Kondo et al. (22) showed that EM inhibits the sustained [Ca2+]i response rather than ATP-induced [Ca2+]i spike in bovine airway epithelial cells. The discrepancy needs to be elucidated in future studies.

The finding presented in this study is of some clinical importance because the concentrations of EM employed were clinically reachable. One of the most conspicuous effects of EM is the suppression of fluid secretion from bronchial epithelial cells in the treatment of bronchitis (17, 41). Mucin exocytosis and Cl- secretion have been shown to be Ca2+-regulated processes in airway-derived cells (21, 23). A recent study by Taylor et al. (40) provided strong evidence that multiple P2X isoforms are expressed on both the apical and basolateral membranes of airway epithelia and that the P2X receptors in both membranes stimulate secretory Cl- transport. Our results that EM blocks the P2X-induced Ca2+ influx may represent one mechanism by which EM exerts its antisecretory effect in the treatment of chronic respiratory tract infections. The inhibition by EM requires a 30-min preincubation with cells for the maximum effect, probably reflecting the reported necessity of its accumulation in the cells (16).

In conclusion, we showed that P2X4 as well as three P2Y subtypes are coexpressed in A549 cells and that Ca2+ influx through both P2X4 and the P2Y2/P2Y4-mediated Ca2+mobilization can be simultaneously activated by ATP, contributing to the spike phase of the ATP-induced [Ca2+]i response. We also found that Ca2+ influx through the P2X channel, but not that through the L-type VDCC and SOCs, is inhibited by EM. Inhibition of the P2X-mediated Ca2+ influx by EM may represent a mechanism underlying its effectiveness in improving chronic inflammatory processes through suppression of the Ca2+-dependent airway secretion.


    ACKNOWLEDGEMENTS

We thank Prof. Atsuo Miyagawa (Third Division) and Prof. Susumu Terakawa (Second Division, Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu, Japan) and Dr. Hiroshi Sato (Third Division, Department of Internal Medicine, Hamamatsu University School of Medicine) for useful discussions and technical instructions in measuring intracellular calcium concentration.


    FOOTNOTES

This work was supported in part by a scholarship awarded to D.-M. Zhao from the College Women's Association of Japan. H.-H. Xue is a recipient of a Japanese Government (Ministry of Education) Scholarship.

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.

1 Because P2Y4 is also expressed in A549 cells, as shown in this study, and a nomenclature, P2Y2-like receptor, has been proposed by Ralevic and Burnstock (31) to describe native P2Y receptors, which are activated by both ATP and UTP but are not simply equated with recombinant P2Y2, the P2U receptor found by Clunes and Kemp (7) in A549 cells is called "P2Y2 or P2Y2-like receptor" in this paper.

Address for reprint requests and other correspondence: K. Chida, Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan (E-mail: chidak11{at}tm.hama-med.ac.jp).

Received 6 July 1999; accepted in final form 30 November 1999.


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