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Respiratory Physiology & Neurobiology 166 (2009) 61–67

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology

journa l homepage: www.e lsev ier .com/ locate / resphys io l

egulation of adenosine 5′-triphosphate (ATP)-gated P2X4 receptors onracheal smooth muscle cells

iyuki Nagaokaa, Masayuki Narab,∗, Tsutomu Tamadaa, Hiroaki Kumec, Tetsuya Ogumac,oshiaki Kikuchid, Jamal Zainid, Takuya Moriyae, Masakazu Ichinose f, Gen Tamuraa, Toshio Hattori a

Division of Infectious and Respiratory Diseases, Department of Internal Medicine, Tohoku University School of Medicine, Aoba-ku, Sendai, JapanDivision of Comprehensive Medicine, Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, JapanDivision of Respiratory Medicine, Department of Medicine, Nagoya University School of Medicine, Showa-ku, Nagoya, JapanDivision of Respiratory Medicine, Department of Internal Medicine, Tohoku University School of Medicine, JapanDepartment of Pathology, Kawasaki Medical University, Kurashiki, JapanThe Third Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan

r t i c l e i n f o

rticle history:ccepted 8 February 2009

eywords:urinergic receptorirway smooth muscle

a b s t r a c t

We examined the effects of extracellular adenosine 5′-triphosphate (ATP) on single airway smooth mus-cle (ASM) cells from porcine trachea using a patch-clamp technique. ATP induced a sustained inwardcurrent. Phospholipase C inhibitor U-73122 failed to inhibit the current, suggesting the involvement ofP2X receptor. A specific effecter of P2X4, ivermectin, augmented the current indicating the existence ofP2X4 receptors. Immunohistochemistry and reverse transcription/polymerase chain reaction analysis and

vermectinWestern blot analysis also showed the distribution of the P2X4 receptors. The inward current was reducedby SKF-96365, an inhibitor of both voltage-dependent Ca2+ channels (VDCCs) and voltage-independentCa2+ channels, although a VDCC antagonist, verapamil, did not affect the current. SKF-96365 caused com-plete suppression of both the increase in the intracellular Ca2+ concentration and the contraction of ASMcells induced by ATP. Our results demonstrate that P2X4 receptors exist on ASM and that the receptorsare responsible for Ca2+ influx. These findings suggest that the Ca2+ influx regulated by P2X4 receptors

ASM

plays an important role in

. Introduction

Adenosine 5′-triphosphate (ATP) is an endogenous purineucleotide which acts as a ubiquitous extracellular messenger bind-

ng to purinergic 2 (P2) receptors on the cell surface (Dubyaknd el-Moatassim, 1993). P2 receptors are subclassified into P2Xnd P2Y subtypes on the basis of amino acid sequence homol-gy and distinct signal transduction mechanisms (Abbracchio andurnstock, 1994; North, 2002). P2X receptors are cation-selectivehannels gated by extracellular ATP, initiating an influx of extracel-ular cations into the cell (North, 2002; Khakh and North, 2006).n the other hand, P2Y receptors belong to the superfamily of-protein-coupled receptors (North, 2002). All cloned and func-

ionally expressed P2Y receptors are able to couple through the

nositol 1,4,5-trisphosphate (IP3) pathway involving the activa-ion of phospholipase C (PLC) (von Kugelgen and Wetter, 2000).2 receptors have been found on the cell membranes of manyifferent cell types and their stimulation has a wide range of

∗ Corresponding author. Tel.: +81 22 717 7587; fax: +81 22 717 7508.E-mail address: mnara@mail.tains.tohoku.ac.jp (M. Nara).

569-9048/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.resp.2009.02.002

contraction by a pathway distinct from VDCC.© 2009 Elsevier B.V. All rights reserved.

effects (Dubyak and el-Moatassim, 1993). In airway, ATP mediatesimportant effects. Extracellular ATP is generally thought to interactwith the parasympathetic limb of the autonomic nervous sys-tem resulting in neurogenic bronchoconstriction and neurogenicinflammation, and to modulate the function of various immunecells including mast cells, eosinophils, and dendritic cells, whichin turn induce hypersensitivity responses (Pelleg and Schulman,2002). ATP is also a potent stimulator of airway surface fluid secre-tion through the stimulation of submucosal glands, and epithelialand goblet cells (Lethem et al., 1993; Shimura et al., 1994). Althoughthe effects of extracellular ATP on the airway have been inves-tigated extensively, the direct effect of ATP on airway smoothmuscle (ASM) cells has not been well characterized. Especially,information about the ionic mechanisms of ASM in response toATP is controversial. Bergner and Sanderson (2002) reported thatATP induced a transient contraction and cytosolic Ca2+ oscilla-tion mediated by P2Y purinoceptors in mouse lung. On the other

hand, in rat airways, ATP induced a transient contractile responsedue to extracellular Ca2+ influx. The extracellular Ca2+ influxoccurs through voltage-dependent Ca2+ channels (VDCCs) activatedby external Na+ entry through P2X receptors (Mounkaïla et al.,2005).

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It has been established that the smooth muscle contraction isegulated by the intracellular Ca2+ concentration ([Ca2+]i) (Somlyond Somlyo, 1994). An increase in [Ca2+]i by agonists playsn important role in the contraction of airway smooth muscleGerthoffer, 1991; Ito et al., 2001). Reports demonstrated that theontraction induced by receptor agonists is insensitive to inhibitorsf voltage-dependent Ca2+ channels in ASM, indicating that Ca2+

nflux other than from VDCC may be involved in contraction bygonists (Benham and Tsien, 1987; Murray et al., 1993; Gorenne etl., 1998; Ito et al., 2002). To date, however, little is known abouthe Ca2+ influx pathway responsible for the contraction of ASM byTP.

In this study, we investigated the effect of extracellular ATP oningle ASM cells from porcine trachea. The aims of this study wereo determine the purinergic receptor subtype on the ASM and tolarify the relationship between the receptor and the intracellulara2+ mobilization responsible for the contraction of ASM.

. Materials and methods

.1. Cell dissociation

ASM cells were dissociated from porcine tracheae obtained fromlocal abattoir. The smooth muscle layer was isolated from the pos-

erior membranous portion of the trachea by removing surroundingonnective tissue and epithelium, and the isolated smooth mus-le strips were cut into small pieces. Then, these smooth muscleieces were dispersed enzymatically into single cells with collage-ase (0.125 mg/ml), papain (1 mg/ml), anddl-dithiothreitol (2 mM)

or 40 min at 37 ◦C. After incubation, the tissue pieces were gen-ly agitated, and single smooth muscle cells were resuspended inhe extracellular solution. The individual smooth muscle cells wereelaxed and spindle-shaped.

.2. Electrophysiology

A standard patch-clamp recording technique was used as pre-iously described (Nara et al., 1998; Oshiro et al., 2000). Briefly,n EPC9 (HEKA Electronics, Dramstadt, Germany) patch-clampmplifier was used to measure ionic currents. Currents were low-ass filtered at 1 kHz and monitored on both a built-in softwarescilloscope and a thermal pen recorder (RECTI-HORITZ-8K; Nip-ondenki Sanei, Tokyo, Japan) with a bandwidth of 0–300 Hz.he patch electrodes (Drummond Scientific Co., Broomall, PA)ad a tip resistance of 2–5 M�. The junction potential betweenhe patch-pipette and bath solution was nulled by the ampli-er circuitry. After establishing a high-resistance (>2 G�) tighteal, the whole-cell configuration was obtained by rupturing theatch membrane with negative pressure applied to the pipetteip. The capacitance current and series resistance were compen-ated using the amplifier circuitry. The access resistance waslways less than 40 M�, and 75–80% series resistance compen-ation was employed. The solutions used were of the followingomposition (in mM): extracellular (bath) solution, 140 NaCl, 4.7Cl, 1.13 MgCl2, 1.2 CaCl2, 10 glucose, and 10 Hepes; and intra-ellular (pipette) solution, 140 KCl, 1.13 MgCl2, 10 glucose, 10epes, 5 ethyl glycol-bis(�-aminoethyl ether)-N,N,N′,N′-tetraaceticcid (EGTA), 1.72 CaCl2, and 1 Na2-ATP, adjusted to pH 7.2 withaOH (bath solution) or KOH (pipette solution). The free Ca2+ in

he pipette solution was 100 nM (Fabiato, 1988). All experiments

ere carried out at room temperature, 22–24 ◦C. The fluids were

uperfused over the cell by hydrostatic pressure-driven application40–50 cm H2O) through polyethylene tubes (0.5 mm inner diam-ter). The morphology of the smooth muscle cells did not changeven after employment of the whole-cell configuration. In these

& Neurobiology 166 (2009) 61–67

studies, only those cells that contracted in response to ATP wereused.

2.3. Immunohistochemical studies

Specimens of porcine trachea were fixed in periodate–lysine–paraformaldehyde at 4 ◦C for 24 h. After washing in sucrose (10%,15%, and 20%)/phosphate-buffered saline (PBS) for 2 h each, theywere embedded in OCT compound in liquid nitrogen and storedat −80 ◦C until use. The staining was performed with the poly-mer immune complex method. Technical details concerning theprocedures are described by Tamada et al. (2000). Cryostat sec-tions (5 �m) were stained for the rat P2X4 purinergic receptorusing monoclonal rabbit anti-rat P2X4 receptor antibody spe-cific to a 370–388 amino acid residue. Slides were incubatedwith the primary antibody diluted 50-fold in Tris-buffered saline(0.05 M Tris and 0.15 M NaCl, pH 7.6) with 2% NaCl to increase thespecificity of antigen–antibody interactions (Yeung et al., 2004).After overnight incubation at 4 ◦C, the slides were incubated with10% normal goat serum containing PBS and peroxidase blockingreagent to block the endogenous peroxidases. Secondary, slideswere incubated with anti-rabbit Ig goat polyclonal Igs bindingwith peroxidase-labeled dextran for 30 min at room tempera-ture. Slides were then developed by exposure to diaminobenzidine(DAB) for substrate for 5 min at room temperature accordingto the manufacturer’s protocol, and counterstained with hema-toxylin.

2.4. Western blot analysis of P2X4 receptor expression

1 g of porcine tissue (tracheal smooth muscle, tongue, heart,diaphragm) was homogenized in 5 ml ice-cold RIPA buffer, andcentrifuged at 15,000 × g for 30 min at 4 ◦C. The proteins wereseparated on SDS-polyacrylamide gels and electroblotted ontoPVDF membrane. The blots were incubated with a blocking solu-tion containing 5% (v/v) nonfat milk overnight at 4 ◦C and thenwith rabbit polyclonal anti-P2X4 purinergic receptor at 1:200 inBT PBS (0.05% Tween-20, 1% BSA in PBS) for 1 h at 37 ◦C. Fol-lowing washes with PBST, the membranes were incubated withhorseradish peroxidase secondary anti rabbit IgG for 1 h at 37 ◦C(1:5000) and visualized on film using an ECL kit. To compare pro-tein loading, the blots were probed with affinity purified anti-actinantibody. The P2X4 receptor is known to exist as glycosylated(65 kDa) and nonglycosylated (46 kDa) protein (Gonzales et al.,2007).

2.5. Reverse transcription/polymerase chain reaction (RT-PCR)analysis of P2X4 receptor messenger RNA

Freshly isolated porcine tracheae were frozen and storeduntil the experiments to analyze the expression of P2X4 receptormRNA. Total RNA was extracted from tracheal smooth musclecells isolated by capture microdissection (Takara Bio Inc., Shiga,Japan) and was converted to cDNA according to the manufac-turer’s instructions. Since porcine P2X4 receptor gene sequenceswere not found in any databases, we referred to the human,mice and rat P2X4 receptor gene sequences and prospected theporcine P2X4 receptor gene sequence so that its product couldhave high homologies (Takara Bio Inc., Shiga, Japan). As a pos-itive control PCR product, we selected a porcine �-actin genesequence from the NCBI database. Oligonucleotide primers used

in PCR for P2X4 receptor were 5′-CAGGACATGGCCGTCGAGGGA-3′ and 5′-CTCCGGCTTGGCGCTCTAGG-3′ and amplified 311 bpof the P2X4 receptor cDNA segment. Primers for �-actinwere 5′-TCGTCACCAACTGGGACGC-3′ and 5′-GACTACCTC-ATGAAGATCCTGACG-5′ and amplified 359 bp of the �-actin

M. Nagaoka et al. / Respiratory Physiology

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ig. 1. Effect of ATP on airway smooth muscle cells. (A) A representative trace ofTP (100 �M)-induced inward current. The activation of the inward current wasaintained in the continuous presence of ATP. The membrane potential (Mp) was

eld at −40 mV.

DNA segment. The thermal cycling conditions were as follows:rst, denaturation at 94 ◦C for 5 min, 35 cycles of denaturation at4 ◦C for 30 s, annealing at 55 ◦C for 30 s and extension at 72 ◦C for0 s, and finally extension at 72 ◦C for 4 min. The reaction productsere run on a 1.5% agarose gel and visualized after electrophoresis

y ethidium bromide staining.

.6. Isometric force records and measurement of fura-2uorescence

The methods are essentially similar to those described previ-usly (Kume et al., 2001; Ito et al., 2002; Oguma et al., 2007).he smooth muscle layer was isolated from the posterior membra-ous portion of the porcine trachea by removing the surroundingonnective tissue and epithelium. Muscle strips 2 mm long bymm wide were prepared for isometric force recording and [Ca2+]ieasurements. The muscle strips were placed horizontally in the

hamber, and then were treated with 10 �M acetoxymethyl esterf fura-2 (fura-2/AM) for 4 h at room temperature (22–24 ◦C). The

oncytotoxic detergent pluronic F-127 (0.01%wt/vol.) was addedo increase the solubility of fura-2/AM. After loading, the cham-er was perfused with normal bathing solution at 37 ◦C for 50 mino wash out the extracellular fura-2/AM. The normal bathing solu-ion was composed of (in mM): 137 NaCl, 5.9 KHCO3, 2.4 CaCl2,

ig. 2. Effects of PPADS and suramin on ATP-evoked current. Both 100 �M PPADS (A) andotential was −40 mV. n.s., not significant.

& Neurobiology 166 (2009) 61–67 63

1.2 Mg Cl2, and 11.8 glucose, bubbled with a mixture of 99% O2and 1% CO2 (pH 7.4). The bathing solution was infused into theorgan bath at a constant flow of 3 ml/min. The isometric force andfura-2 fluorescence of muscle strips were measured simultane-ously using a displacement transducer and a spectrofluorometer(CAF-110; Japan Spectoscopic, Tokyo, Japan). The intensities of flu-orescence due to excitation at 340 (F340) and 380 (F380) nm weremeasured after background subtraction. The absolute amount of[Ca2+]i was not calculated because the dissociation constant of fura-2 for Ca2+ in smooth muscle may be different from that observed invitro. Therefore, the ratio of F340 to F380 (F340/F380) was used as a rel-ative indicator of [Ca2+]i. Measurements of the tension and F340/F380were made when the response to methacholine (MCh) had attaineda steady state. The temperature of the organ bath was maintainedat 37 ◦C.

2.7. Chemicals

Hepes and Fura-2 were purchased from Dojin Co, Ltd.,Kumamoto, Japan. Collagenase was from Wako Pure Chemicals,Osaka, Japan. U-73122 was purchased from Carbiochem Co., LaJolla, CA. OCT compound was from Miles Laboratories, Naperville,IL. Peroxidase blocking regent, peroxidase-labeled dextran, anddiaminobenzidine were from Dako Co., Carpinteria, CA. Monoclonalrabbit anti human P2X4 receptor antibody was purchased fromAlomone Labs Inc., Jerusalem, Israel. The ECL kit was from Amer-sham Pharmacica Biotec Co. Other chemicals including ATP wereall purchased from Sigma Chemical Co., St. Louis, MO.

2.8. Statistical analysis

mean comparison, the two-tailed paired or unpaired Student’st-test was used. A value of P < 0.05 was considered statisticallysignificant. n denotes the number of experiments in differentcells.

100 �M suramin (B) failed to inhibit the ATP-evoked inward current. The holding

64 M. Nagaoka et al. / Respiratory Physiology

Fig. 3. Effect of ivermectin (IVM) on ATP-evoked current. (A) Representative recordsof 100 �M ATP-evoked current before (upper) and after (lower) the addition of IVM(3 �M). IVM potentiates the amplitude of the ATP-evoked current. The holdingpotential was −40 mV. (B) Summary of data from 9 cells showing that IVM poten-tiates ATP-evoked currents. The amplitude of the currents increased by 360% in thepresence of IVM. * represents P < 0.01.

Fig. 4. (A) Immunolocalization of the P2X4 receptor in porcine ASM. Polymer immune cP2X4-R. The section was with mAb against P2X4-R, and was counterstained weakly withsignificant label was detected in porcine airway smooth muscle cells in the case of negatanalysis. The abundance of P2X4 receptor protein (65/46 kDa) in the porcine tracheal smtongue, heart, and diaphragm. Expression of �-actin is shown as a marker for protein load

& Neurobiology 166 (2009) 61–67

3. Results

3.1. Electrophysiological effects of ATP on ASM

Fig. 1 shows the membrane currents recorded in the whole-cellconfiguration of the patch-clamp technique in response to 100 �Mextracellular ATP at a holding potential of −40 mV. ATP elicitedan inward current. The current amplitudes were 78.8 ± 26.6 pA(n = 17 cells from 10 animals). The activation of the inward currentwas maintained in the continuous presence of ATP. ATP was thenwashed away and the current returned to its base line within a fewseconds.

3.2. P2X4 receptor-mediated currents

We first used the PLC inhibitor U-73122 in order to examinewhether the P2Y signaling pathway is involved because ATP-mediated activation of P2Y receptors has been shown to activatePLC with the subsequent generation of IP3 (von Kugelgen andWetter, 2000). Exposure of ASM to U-73122 (10 �M) for 30 min didnot alter the induction of the inward current by ATP (74.2 ± 17.4 pAfor ATP alone and 70.8 ± 30.1 pA for ATP in the presence of U-73122;P = 0.83; n = 6 cells from 4 animals), suggesting that not P2Y recep-tors but P2X receptors are involved in the ATP-induced current.We then applied the P2X receptor antagonists PPADS and suramin.

As shown in Fig. 2, however, these antagonists failed to inhibitthe current (81.4 ± 27.2 pA for ATP alone and 73.8 ± 33.1 pA for ATPplus PPADS; P = 0.30; n = 7 cells from 3 animals, 84.2 ± 40.0 pA forATP alone and 80.0 ± 36.0 pA for ATP plus suramin; P = 0.38; n = 6cells from 5 animals, respectively). As far as P2X receptors are

omplex stain of porcine airway smooth muscles using rabbit mAb against humanhematoxylin. The positive brown stain was detected in smooth muscle cells. No

ive control Igs (bar: 100 �m). (B) P2X4 receptor expression in ASM by Western blotooth muscle was determined. Controls included extracts prepared from porcine

ing.

iology & Neurobiology 166 (2009) 61–67 65

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Fig. 6. Effect of SKF-96365 and verapamil on ATP-evoked current. (A) Representa-tive records of 100 �M ATP-evoked current before (upper) and after the addition(lower) of SKF-96365 (100 �M). SKF-96365 reduced the ATP-evoked current. Theholding potential was −40 mV. (B) Summary of data from 9 cells showing that SKF-

M. Nagaoka et al. / Respiratory Phys

oncerned, most of the antagonists available are highly limited inerms of receptor-affinity, subtype-selectivity (Lambrecht, 2000).PADS and suramin are known to be ineffective as antagonists to2X4 receptors although they are specific for other P2X receptorsSoto et al., 1996; Khakh et al., 1999; Khakh and North, 2006).hen, we applied ivermectin (IVM), a specific positive allostericffecter of P2X4 (Khakh et al., 1999; Priel and Silberberg, 2004;ilberberg et al., 2007), to ASM cells. As shown in Fig. 3A, in theresence of 3 �M IVM in the extracellular solution, the current inesponse to ATP increased. IVM significantly increased the currenty 360% (72.2 ± 10.6 pA for ATP alone and 261.1 ± 53.8 pA for ATPlus IVM; P < 0.01; n = 9 cells from 4 animals) (Fig. 3B). We alsomployed immunohistochemistry to confirm the presence of P2X4eceptors, supporting the identification of the receptors carriedut by pharmacological methods as described above. As shown inig. 4A, immunofluorescence for P2X4 receptors was evident in ASMells. P2X4 receptor expression was also confirmed by Western blotnalysis (Fig. 4B). In addition, the P2X4 expression of the smoothuscles was demonstrated by RT-PCR (Fig. 5). The intactness of

NA was confirmed by amplification of �-actin.

.3. Ca2+ entry through the ATP-activated inward current

It is known that ATP acting through P2X purinoceptors can acti-ate a cation current permeant to Ca2+ ions in several types ofells (Benham, 1989; Nakazawa et al., 1991; Evans et al., 1996;apiod, 1998; Lundy et al., 2002). A recent report suggested thathe extra cellular Ca2+ influx occurred through VDCC activated byxternal Na+ entry through P2X receptors (Mounkaïla et al., 2005).n the first instance, we applied SKF-96365, an inhibitor of bothDCC and voltage-independent Ca2+ channel (VICC) (Merrit et al.,990), to ASM cells. Fig. 6A shows the effect of SKF-96365 on thenward current evoked by ATP. The current was reduced by 47% inhe presence of 100 �M SKF-96365 (78.3 ± 7.5 pA for ATP alone and1.7 ± 7.9 pA for ATP in the presence of 100 �M SKF-96365; P < 0.01;= 9 cells from 5 animals) (Fig. 6B). On the other hand, the current

as unaffected by the VDCC antagonist verapamil (n = 5 cells fromanimals) (Fig. 6C), suggesting that VDCC is not involved in the

urrent. These data indicate that the ATP-induced inward currentnvolves the influx of Ca2+, and that the Ca2+ influx in response toTP is mediated not by VDCC but by the P2X4 receptor.

ig. 5. cDNA segments of P2X4 receptor (left panel) and �-actin (right panel) ampli-ed by RT-PCR from porcine trachea. Tracheal smooth muscle cells selectivelyxtracted from porcine tracheae by laser capture microdissection showed a clearand of P2X4 receptor cDNA. The 311 bp and 359 bp DNA segments were expectedo be amplified from the P2X4 receptor cDNA and �-actin cDNA, respectively.

96365 significantly reduced the ATP-evoked currents. The amplitude of the currentsreduced by 47% in the presence of SKF-96365. * represents P < 0.01. (C) A representa-tive trace of 100 �M ATP-evoked current before and after the addition of verapamil.Verapamil did not affect the current.

3.4. Effect of SKF-96365 on contraction and Ca2+ mobilizationinduced by ATP

We examined the extent to which Ca2+ activated by ATP wouldcontribute to the contraction using fura-2 fluorescence methodsand by measuring the isometric force. As shown in Fig. 7A, the appli-cation of 100 �M ATP caused tonic contraction and a rapid increasein F340/F380. The increase in F340/F380 was sustained without a tran-sient increase in the continuous presence of ATP. When the tensionand F340/F380 in response to 1 �M methacholine were taken as100%, these values by 100 �M ATP were 86.8 ± 6.6, and 90.6 ± 4.8%,respectively (n = 6). When 100 �M SKF-96365 was applied to thetissue pre-contracted by 100 �M ATP, SKF-96365 caused roughlya complete suppression of both the tension and the increase inF340/F380 induced by ATP. As shown in Fig. 7B, the applicationof SKF-96365 resulted in a concentration-dependent inhibition ofthe tension and F340/F380 induced by 100 �M ATP. When 10 �MSKF-96365 was applied in the same way, the values of percentcontraction and percent F340/F380 were 65.1 ± 9.8, and 72.8 ± 12.1%,respectively (n = 6). When the concentration of SKF-96365 wasincreased to 100 �M, those values were decreased to 3.8 ± 4.0,and 2.1 ± 1.6%, respectively (n = 6). Together with the effect of SKF-96365 on the inward current (Fig. 6), these data suggest that asignificant portion of the inward current following P2X4 receptoractivation, that is Ca2+ influx, contributes to the smooth musclecontraction.

4. Discussion

The present study provides evidence for an ATP-induced inwardcurrent after stimulation of P2X4 receptors in porcine ASM cells. Itfurther demonstrates the physiological relevance of this current toCa2+ mobilization in these cells.

Purinoceptors are a diverse group of purine- and pyrimidine-sensitive receptors that have been divided into two major families,

classified as P2X and P2Y (Dubyak and el-Moatassim, 1993). P2Xreceptors are ligand-gated, cation-selective channels, whereas P2Yreceptors are G-protein-coupled receptors (North, 2002). All clonedand functionally expressed P2Y receptors are able to couple through

66 M. Nagaoka et al. / Respiratory Physiology & Neurobiology 166 (2009) 61–67

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ig. 7. Effects of SKF-96365 on the contraction and Ca2+ mobilization induced byupper trace) and F340/F380 (lower trace). (B) The values of percent contraction and pe< 0.05 and P < 0.01, respectively.

he IP3 pathway involving the activation of PLC (von Kugelgen andetter, 2000). Our data indicate that the ATP-activated inward

urrent is associated with P2X receptors because U-73122, a PLCnhibitor, failed to inhibit the current. To date, seven clones of P2Xeceptors expressed in various cells have been identified. These P2Xeceptors show differences in their sensitivity to antagonists andn their kinetics of desensitization (Evans and Surprenant, 1996;orth, 2002). In the present study, the ATP-induced current showed

ittle desensitization (Fig. 1) and, moreover, this current was notlocked by the conventional antagonists of P2X receptors, PPADSnd suramin (Fig. 2). Among the P2X receptors, the P2X4 recep-or is unusual in its insensitivity to blockade by these antagonistsSoto et al., 1996; North, 2002). ATP-induced current through P2X4eceptors is also reported to show non-desensitization (Evans andurprenant, 1996). In addition, the most useful distinguishing fea-ure of ATP-evoked currents by P2X4 receptors is their potentiationy IVM (Khakh et al., 1999; Priel and Silberberg, 2004; Silberbergt al., 2007). IVM is a specific allosteric effector of P2X4. It canncrease the gating of the P2X4 channel. The P2X4 channel is sensi-ive to IVM, whereas other subtypes of P2X receptor are insensiveo the compound (Khakh et al., 1999; Jelínkova et al., 2008). Collec-ively, our observations clearly indicate the functional expressionf P2X4 receptors on ASM cells. The existence of the receptors waslso evident by immunostaining, RT-PCR and Western blot analysisFigs. 4 and 5). However, as we do not have a specific P2X4 recep-or antagonist, further investigation may be required to confirmbsolutely the function of the P2X4 receptor.

It has been established that the smooth muscle contractions regulated by [Ca2+]i (Somlyo and Somlyo, 1994). An increasen [Ca2+]i by agonists plays an important role in the contrac-ion of airway smooth muscle (Gerthoffer, 1991; Ito et al., 2001).

t is generally accepted that voltage-dependent Ca2+ channelsnd voltage-independent Ca2+ channels (VICCs), which include aeceptor-operated Ca2+ permeable channel, a store-operated Ca2+

hannel and a transmitter-gated cation channel, are involved in Ca2+

nflux and the induced contraction (Worley and Kotlikoff, 1990;

) A representative trace showing the effects of SKF-96365 on the change in forceF340/F380 for 100 �M ATP with SKF-96365 (10–100 �M) inhibition. * and ** represent

Large, 2002; Egan and Khakh, 2004). P2X receptors are known astransmitter (ATP)-gated cation channels (Egan and Khakh, 2004;Abbracchio and Burnstock, 1994; North, 2002). Fig. 6 shows that asignificant proportion (47%) of the inward current following P2X4receptor activation in physiological solution is carried by calcium.P2X receptors are reported to have a relatively high calcium per-meability in several types of cells including smooth muscle cells(Benham, 1989; Evans et al., 1996; Capiod, 1998; Lundy et al., 2002).Several laboratories have quantified Ca2+ movement through thepores of P2X channels (Abbracchio and Burnstock, 1994; Egan andKhakh, 2004). For example, Egan and Khakh (2004) estimated thatthe Ca2+ flux ranged from ∼3% to 15% of the total current throughrecombinant P2X2 receptors. Rogers and Dani (1995) have shownthat 6.5% of the total charge is carried by Ca2+ through the P2Xreceptor in sympathetic neurons. These studies used different cells,different P2X receptors, and different extracellular and intracellu-lar concentrations of ions. Therefore, it is not possible to directlycompare the values among such studies. We estimated the Ca2+

flux roughly by using SKF-96365. It is possible that SKF-96365inhibits cations other than Ca2+ (Zhang and Hancox, 2003). Thus,we may have overestimated the Ca2+ influx through P2X4 recep-tors in ASM. However, Figs. 6 and 7 support the role of this inwardcurrent in producing airway contraction that is dependent on Ca2+

influx. The inward current was resistant to the VDCC antagonistverapamil (Fig. 6). Mounkaïla et al. described that ATP induced acontractile response due to extracellular Ca2+ influx and that theextracellular Ca2+ influx occurs through VDCC activated by exter-nal Na+ entry through P2X receptors. In our study, however, thecontribution of external Na+ was small because verapamil, a VDCCantagonist, did not work (Fig. 6). Direct Ca2+ entry through P2X4receptors caused the contractile response in our study. The differ-

ence between the observations by Mounkaïla et al. and our seemsto depend on the species or P2X subtypes. In our study (Fig. 7), ATPelicited a sustained increase in F340/F380, that is [Ca2+]i, without atransient increase. P2Y receptors are known to couple to G-proteinand PLC to raise free Ca2+ transiently (Ralevic and Burnstock, 1998;

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G

I

I

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Yeung, D., Kharidia, R., Brown, S.C., Górecki, D.C., 2004. Enhanced expression of the

M. Nagaoka et al. / Respiratory Phys

on Kugelgen and Wetter, 2000). Therefore, this lack of a transientncrease in [Ca2+]i together with the effect of U-73122 suggests that2Y receptors were not involved in this Ca2+ mobilization.

ATP is known to be released from storage compartments inerve terminals, chromaffin cells, circulating platelets, and mastells (Dubyak and el-Moatassim, 1993; Pelleg and Schulman, 2002).ast cells stimulated with antigen release not only inflammatoryediators such as histamine but also ATP, which acts as a cell-to-cellessenger, triggering the degranulation of neighbouring mast cells

Osipchuk and Cahalan, 1992). Our results suggest that, in additiono its effect on mast cells, ATP directly affects ASM cell contractil-ty through P2X4 receptors. Considering these results, ATP may alsoave a direct role in bronchial asthma via its direct action on ASMells.

. Conclusion

In conclusion, our study demonstrates that P2X receptors existn ASM cells, and the evidence presented here suggests that theseeceptors are of the P2X4 subtype. P2X4 receptors play an importantole in Ca2+-dependent events, that is contraction, by a pathwayistinct from VDCC.

cknowledgement

We thank Mr. B. Bell for reading the manuscript.

eferences

bbracchio, M.P., Burnstock, G., 1994. Purinoceptors: are there families of P2X andP2Y purinoceptors? Pharmacol. Ther. 64, 445–475.

enham, C.D., 1989. ATP-activated channels gate calcium entry in single smoothmuscle cells dissociated from rabbit ear artery. J. Physiol. 419, 689–701.

enham, C.D., Tsien, R.W., 1987. A novel receptor-operated Ca2+-permeable channelactivated by ATP in smooth muscle. Nature 328, 275–278.

ergner, A., Sanderson, M.J., 2002. ATP stimulates Ca2+ oscillations and contractionin airway smooth muscle cells of mouse lung slices. Am. J. Physiol. Lung Cell.Mol. Physiol. 283, L1271–L1279.

apiod, T., 1998. ATP-activated cation currents in single guinea-pig hepatocytes. J.Physiol. 507, 795–805.

ubyak, G.R., el-Moatassim, C., 1993. Signal transduction via P2-purinergic recep-tors for extracellular ATP and other nucleotides. Am. J. Physiol. Cell Physiol. 265,C577–C606.

gan, T.M., Khakh, B.S., 2004. Contribution of calcium ions to P2X channel responses.J. Neurosci. 24, 3413–3420.

vans, R.J., Lewis, C., Virginio, C., Lundstrom, K., Buell, G., Surprenant, A., North, R.A.,1996. Ionic permeability of, and divalent cation effects on, two ATP-gated cationchannels (P2X receptors) expressed in mammalian cells. J. Physiol. 497, 413–422.

vans, R.J., Surprenant, A., 1996. P2X receptors in autonomic and sensory neurons.Semin. Neurosci. 8, 217–223.

abiato, A., 1988. Computer programs for calculating total from specified free orfree from specified total ionic concentrations in aqueous solutions containingmultiple metals and ligands. Methods Enzymol. 157, 378–417.

erthoffer, W.T., 1991. Regulation of the contractile element of airway smooth mus-cle. Am. J. Physiol. Lung Cell. Mol. Physiol. 261, L15–L28.

onzales, E., Prigent, S., About-Lovergne, A., Boucherie, S., Tordjmann, T., Jacquemin,E., Combettes, L., 2007. Rat hepatocytes express functional P2X receptors. FEBSLett. 581, 3260–3266.

orenne, I., Labat, C., Gascard, J.P., Norel, X., Nashashibi, N., Brink, C., 1998.Leukotriene D4 contractions in human airways are blocked by SK&F 96365,an inhibitor of receptor-mediated calcium entry. J. Pharmacol. Exp. Ther. 284,549–552.

to, S., Kume, H., Honjo, H., Katoh, H., Kodama, I., Yamaki, K., Hayashi, H., 2001. Possibleinvolvement of Rho kinase in Ca2+ sensitization and mobilization by MCh intracheal smooth muscle. Am. J. Physiol. Lung Cell. Mol. Physiol. 280, L1218–L1224.

to, S., Kume, H., Yamaki, K., Katoh, H., Honjo, H., Kodama, I., Hayashi, H., 2002.Regulation of capacitative and noncapacitative receptor-operated Ca2+ entry

by Rho-kinase in tracheal smooth muscle. Am. J. Respir. Cell Mol. Biol. 26,491–498.

elínkova, I., Vávra, V., Jindrichova, M., Obsil, T., Zemkova, H.W., Zemkova, H., Sto-jilkovic, S.S., 2008. Identification of P2X4 receptor transmembrane residuescontributing to channel gating and interaction with ivermectin. Pflügers Arch.456, 939–950.

& Neurobiology 166 (2009) 61–67 67

Khakh, B.S., North, A., 2006. P2X receptors as cell-surface ATP sensor in health anddisease. Nature 442, 527–532.

Khakh, B.S., Proctor, W.R., Dunwiddie, T.V., Labarca, C., Lester, H.A., 1999. Allostericcontrol of gating and kinetics at P2X4 receptor channels. J. Neurosci. 19,7289–7299.

Kume, H., Ito, S., Ito, Y., Yamaki, K., 2001. Role of lysophosphatidylcholine in the desen-sitization of �-adrenergic receptors by Ca2+ sensitization in tracheal smoothmuscle. Am. J. Respir. Cell Mol. Biol. 25, 291–298.

Lambrecht, G., 2000. Agonists and antagonists acting at P2X receptors: selectivityprofiles and functional implications. Naunyn Schmiedebergs Arch. Pharmacol.362, 340–350.

Large, W.A., 2002. Receptor-operated Ca2+-permeable nonselective cation channelsin vascular smooth muscle: a physiologic perspective. J. Cardiovasc. Electrophys-iol. 13, 493–501.

Lethem, M.I., Dowell, M.L., Scott, M.V., Yankaskas, J.R., Egan, T., Boucher, R.C., Davis,C.W., 1993. Nucleotide regulation of goblet cells in human airway epithelialexplants: normal exocytosis in cystic fibrosis. Am. J. Respir. Cell Mol. Biol. 9,315–322.

Lundy, P.M., Hamilton, M.G., Mei, L., Gong, W., Vair, C., Sawyer, T.W., Frew, R.,2002. Stimulation of Ca2+ influx through ATP receptors on rat brain synapto-somes: identification of functional P2X7 receptor subtypes. Br. J. Pharmacol. 135,1616–1626.

Merrit, J.E., Armstrong, W.P., Benham, C.D., Hallam, T.J., Jacob, R., Jaxa-Chamiec,A., Leigh, B.K., McCarthy, S.A., Moores, K.E., Rink, T.J., 1990. SK&F 96365, novelinhibitor of receptor-mediated calcium entry. Biochem. J. 271, 515–528.

Mounkaïla, B., Marthan, R., Roux, E., 2005. Biphasic effect of extra cellular ATP onhuman and rat airways is due to multiple P2 purinoceptor activation. Respir.Res. 6, 143–158.

Murray, R.K., Fleischmann, B.K., Kotlikoff, M.I., 1993. Receptor-activated Ca influx inhuman airway smooth muscle: use of Ca imaging and perforated patch-clamptechniques. Am. J. Physiol. Cell Physiol. 264, C485–C490.

Nakazawa, K., Fujimori, K., Tanaka, A., Inoue, K., 1991. Comparison of adenosinetriphosphate- and nicotine-activated inward currents in rat phaeochromocy-toma cells. J. Physiol. 434, 647–660.

Nara, M., Sasaki, T., Shimura, S., Oshiro, T., Irokawa, T., Kakuta, Y., Shirato, K., 1998.Effects of histamine and endothelin-1 on membrane potentials and ion currentsin bovine tracheal smooth-muscle cells. Am. J. Respir. Cell Mol. Biol. 19, 805–811.

North, R.A., 2002. Molecular physiology of P2X receptors. Physiol. Rev. 82, 1013–1067.Oguma, T., Ito, S., Kondo, M., Makino, Y., Shimokata, K., Honjo, H., Kamiya, K., Kume,

H., 2007. Roles of P2X receptors and Ca2+ sensitization in extracellular adenosinetriphosphate-induced hyperresponsiveness in airway smooth muscle. Clin. Exp.Allergy 37, 893–900.

Oshiro, T., Sasaki, T., Nara, M., Tamada, T., Shimura, S., Maruyama, Y., Shirato, K.,2000. Suppression of maxi-K channel and membrane depolarization by syn-thetic polycations in single tracheal myocytes. Am. J. Respir. Cell Mol. Biol. 22,528–534.

Osipchuk, Y., Cahalan, M., 1992. Cell-to-cell spread of calcium signals mediated byATP receptors in mast cells. Nature 359, 241–244.

Pelleg, A., Schulman, E.S., 2002. Adenosine 5′-triphosphate axis in obstructive airwaydiseases. Am. J. Ther. 9, 454–464.

Priel, A., Silberberg, S.D., 2004. Mechanism of ivermectin facilitation of human P2X4

receptor channels. J. Gen. Physiol. 123, 281–293.Ralevic, V., Burnstock, G., 1998. Receptors for purines and pyrimidines. Pharmacol.

Rev. 50, 413–492.Rogers, M., Dani, J.A., 1995. Comparison of quantitative calcium flux through NMDA,

ATP, and ACh receptor channels. Biophys. J. 68, 501–506.Shimura, S., Sasaki, T., Nagaki, M., Takishima, T., Shirato, K., 1994. Extracellular ATP

regulation of feline tracheal submucosal gland secretion. Am. J. Physiol. LungCell. Mol. Physiol. 267, L159–L164.

Silberberg, S.D., Li, M., Swartz, K.J., 2007. Ivermectin interaction with transmembranehelices reveals widespread rearrangements during opening of P2X receptorchannels. Neuron 54, 263–274.

Somlyo, A.P., Somlyo, A.V., 1994. Signal transduction and regulation in smooth mus-cle. Nature 372, 231–236.

Soto, F., Garcia-Guzman, M., Gomez-Hernandez, J.M., Hollmann, M., Karschin, C.,Stühmer, W., 1996. P2X4: an ATP-activated ionotropic receptor cloned from ratbrain. Proc. Natl. Acad. Sci. U.S.A. 93, 3684–3688.

Tamada, T., Sasaki, T., Saitoh, H., Ohkawara, Y., Irokawa, T., Sasamori, K., Oshiro, T.,Tamura, G., Shimura, S., Shirato, K., 2000. A novel function of thyrotropin as apotentiator of electrolyte secretion from the tracheal gland. Am. J. Respir. CellMol. Biol. 22, 566–573.

von Kugelgen, I., Wetter, A., 2000. Molecular pharmacology of P2Y-receptors. NaunynSchmiedebergs Arch. Pharmacol. 362, 310–323.

Worley, J.F., Kotlikoff, M.I., 1990. Dihydropyridine-sensitive calcium channels in air-way smooth muscle cell. Am. J. Physiol. Lung Cell. Mol. Physiol. 259, L468–L480.

P2X4 receptor in Duchenne muscular dystrophy correlates with macrophageinvasion. Neurobiol. Dis. 15, 212–220.

Zhang, Y.H., Hancox, J.C., 2003. A novel, voltage-dependent nonselective cation cur-rent activated by insulin in guinea pig isolated ventricular myocytes. Circ. Res.92, 765–768.