Introduction

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Over the last few years, it has become increasingly evident that purine nucleotides and nucleosides such as adenosine 5'-triphosphate (ATP) and adenosine (ADO) not only are involved in cellular metabolism but also act as extracellular mediators (1, 2). Specific purinergic receptors have been found on cell membranes of many different cell types and their stimulation has a wide range of effects, including the modulation of synaptic transmission in the central and peripheral nervous system, mediator release by mastocytes, and the modulation of visceral and vascular smooth-muscle contractility (2).

Purinoceptor stimulation also mediates important effects in the respiratory system. For example, several studies show that purine nucleotides modulate mucociliary clearance. Indeed, ATP and uridine triphosphate (UTP) are potent stimulators of surfactant synthesis by alveolar type II cells and of airway surface fluid secretions through the stimulation of epithelial and goblet cells (3). Chloride ion secretion by epithelial cells and fluid transport by the airway epithelium are also enhanced by P₂-receptor stimulation (9). These properties of the triphosphate nucleotides have led to the investigation of the possible use of aerosolized UTP as a treatment to enhance airway fluid transport in patients with cystic fibrosis (10).

Although the effects of triphosphate nucleotides on respiratory secretory cells have been characterized, their effects on airway smooth-muscle (ASM) cells are not well known. Most reported studies have been carried out in vivo, or in vitro on tracheal preparations, and the published data are often conflicting, some authors observing a contraction and others a relaxation after ATP or UTP administration (11). These discrepancies are most likely attributable to the variety of cells present in in vivo and in vitro preparations and to the various subtypes of purinoceptors present on cell membranes (14, 15).

Recently, we have shown that cultured ASM cells have purinoceptors with the characteristics of P_2Y2 receptors (P_2U receptors), showing equal affinity for ATP and UTP on their surfaces (16). Further, the ATP-induced increase in cytosolic Ca²⁺ was enhanced by preincubation of the cells with ADO, although ADO itself had no measurable effect on the intracellular free-Ca²⁺ concentration ([Ca²⁺]_i) (16).

The interaction between ADO and ATP is of potential interest in the pathophysiology of asthma because airway hypersensitivity to ADO inhalation is one of the hallmarks of asthma (19). It is generally acknowledged that the bronchoconstriction provoked by ADO is due to the mediators released by mast-cell degranulation triggered by the stimulation of P₁ purinoceptors (20). Stimulated mast cells release not only contractile mediators such as histamine but also ATP, which acts as an intercellular messenger, triggering the degranulation of adjacent mast cells (21). Our data suggest that in addition to their action on mast cells, both ATP and ADO could also directly affect ASM cell contractility by modulating the [Ca²⁺]_i. Thus, the aims of this study were (1) to determine whether the enhancing effect of ADO on Ca²⁺ release is specific to P_2Y-receptor stimulation or whether it also applies to other G protein-linked receptors present on ASM, and (2) to determine the mechanisms responsible for the enhancement of the response.

	Materials and Methods

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Cell Cultures

Primary cultures of tracheal smooth-muscle cells were prepared as previously described (16). Briefly, 7- to 9-wk-old Fisher rats (Harlan Sprague Dawley, Walkerville, MD) were injected intraperitoneally with a lethal dose of pentobarbital, and the tracheas were removed and cut longitudinally through the cartilage and placed in Hanks' balanced salt solution (HBSS). Tissue digestion was obtained by incubating the tracheas for 30 min at 37°C in HBSS containing 0.2% collagenase type IV and 0.05% elastase type V. The dissociated cells were then collected by centrifugation, resuspended in culture medium containing 1:1 Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 medium supplemented with 10% fetal bovine serum (FBS), 0.244% NaHCO₃, penicillin (100 U/ml), and streptomycin (100 µg/ml), and plated in 25-cm² culture flasks. Confluent cells were detached with a 0.25% trypsin-0.02% ethylenediaminetetraacetic acid (EDTA) solution and grown on 25-mm glass coverslips for calcium imaging and on 60-mm-diameter wells for the determination of total inositol phosphates. Confluent cells from the first to third passages were used 7 to 12 d after passage. They were identified as smooth-muscle cells by positive immunohistochemical staining for smooth muscle-specific -actin and the absence of cytokeratin (16).

Calcium Measurements

As previously described, cells were incubated for 20 to 30 min at 37°C with Hanks' buffer (in mM: NaCl 137, NaHCO₃ 4.2, glucose 10, Na₂HPO₄ 3, KCl 5.4, KH₂PO₄ 0.4, CaCl₂ 1.3, MgCl₂ 0.5, MgSO₄ 0.8, N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid [Hepes] 5) containing 6 µM Fura-2-acetoxymethylester (Fura-2AM) and 0.02% pluronic F-127. The loaded cells were then washed and the coverslips placed in a Leiden chamber (Medical Systems Corp., Greenville, NY) containing 500 µl of Hanks' buffer on the stage of an inverted microscope equipped for epifluorescence with a ×20 objective (Nikon, Montréal, PQ, Canada). The temperature in the chamber was maintained at 35 ± 0.5°C using a temperature controller (model TC-102; Medical Systems Corp.). Fluorescence measurements of fields containing about 20 cells were made at 345/380 excitatory wavelengths and 510 emission wavelength using a PTI D401 microphotometer (Photon Technology International Inc., Princeton, NJ). Background fluorescence and autofluorescence were automatically subtracted.

All test drugs were diluted in Hanks' buffer from frozen stock solutions. They were prewarmed to 35°C before being added in the appropriate concentrations in a 250-µl volume after an equivalent volume of Hanks' buffer had been withdrawn. This volume was used to ensure rapid diffusion of the test drug and thus avoid asynchronous responses because ratio measurements were obtained in cell populations rather than single cells. Control experiments have shown that adding and withdrawing 250 µl of Hanks' buffer has no significant effect on the resting ratio (16). [Ca²⁺]_i was measured for 30 s before and for 5 min after the addition of the drugs. Only one measurement per slide was performed.

The [Ca²⁺]_i was calculated using a calculated dissociation constant of Ca²⁺ to Fura-2 of 224 nM (22). Maximum ratio was determined in cells exposed to 10⁵ M ionomycin in the presence of 1.3 mM CaCl₂ and minimum ratio in Ca²⁺-free Hanks' buffer to which ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) 10³ M and ionomycin 10⁵ M had been added.

Measurement of Total Inositol Phosphates

Confluent cells were incubated with 1 Ci/ml ³H-myo-inositol (Amersham Life Science, Oakville, ON, Canada) in inositol-free DMEM supplemented with 2% FBS. After 48 h of radiolabeling, the cells were washed and placed in 1 ml Krebs-Henseleit buffer containing 10 mM LiCl. Appropriate concentrations of agonists were then added and the cells challenged for 10 min. The reactions were stopped by adding 200 µl of 3 M trichloroacetic acid and the dishes immediately placed on ice for 30 min. The cells were then scraped off the dishes and the resulting cell suspensions were first sonicated and then centrifuged at 4,000 × g for 20 min. A total of 250 µl of 10 mM EDTA (pH 7.0) and 1 ml of fresh trichloro-trifluoroethane/tri-n-octylamine (50% vol/vol) were added to 1 ml of the supernatant. After vigorous vortexing, the tubes were centrifuged at 12,000 × g for 4 min and the first milliliter of the top layer was removed and neutralized with 200 µl of 60 mM NaHCO₃. Total [³H]inositol phosphates were separated from free [³H]myo-inositol by anion exchange chromatography. The samples were loaded onto columns containing a 1-ml bed of formate-charged Dowex resin (Bio-Rad Laboratories, Mississauga, ON, Canada); [³H]myo-inositol was eluted with 12 ml of 60 mM ammonium formate plus 5 mM sodium tetraborate, whereas the [³H]inositol phosphates were eluted with 4 ml of 1.2 M ammonium formate plus 100 mM formic acid. The aliquots were then diluted 1:1 with H₂O and total [³H]inositol phosphates were measured by liquid scintillation counting (23).

Chemicals

Elastase, collagenase, trichloro-trifluoroethane/tri-n-octylamine, ATP, ADO, serotonin (5-hydroxytryptamine [5-HT], creatinine sulfate complex), and nifedipine were purchased from Sigma (St. Louis, MO). Fura-2-AM and pluronic F-127 were from Molecular Probes (Eugene, OR). DMEM, FBS, penicillin, and streptomycin were from GIBCO Canada (Mississauga, ON, Canada); Ham's F-12 was from ICN (Mississauga, ON, Canada); and ionomycin and EGTA were from Calbiochem (San Diego, CA). 1-Deoxy-1-(6-{[(3-iodophenyl)methyl]amino}-9H-purin-9-yl)-N-methyl--D-ribofuranuronamide (IB-MECA) was obtained from RBI Research Biochemicals (Natick, MA). AACOCF3 was from Biomolecular Research (Plymouth Meeting, PA).

Statistical Analysis

The data are expressed as means ± standard error of the mean (SEM). Throughout, n refers to the number of coverslips studied. Differences between means were compared by Student's t test. Log transformation of the data was performed before testing when distributions were log normal. Analysis of variance was not performed because of the significant difference between the variances of the different treatment groups.

	Results

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Effect of ADO on ATP

The mean resting level of [Ca²⁺]_i in rat tracheal smooth-muscle cells was 70 ± 3 nM (n = 121) and did not differ significantly among the various experimental groups. Section A in Figure 1 shows the peak [Ca²⁺]_i release after the addition of ADO (10⁵ M), ATP (10⁴ M), and ADO (10⁵ M) + ATP (10⁴ M). Incubation for 60 s with ADO 10⁵ M had no significant effect on the resting levels of [Ca²⁺]_i (70 ± 8 versus 75 ± 6 nM [n = 21]) but pretreatment with ADO 10⁵ M significantly increased the peak response to ATP (10⁴ M) from 585 ± 70 to 955 ± 211 nM [Ca²⁺]_i (n = 22 and 21, respectively; P = 0.05) (16). Section B in Figure 1 shows the effect of 60 s incubation with the A₃ receptor agonist IB-MECA. Baseline [Ca²⁺]_i was 79 ± 10 before and 93 ± 14 after the addition of the drug (P = NS). After the addition of ATP (10⁴ M), peak [Ca²⁺]_i was 2,062 ± 630 nM (n = 8; P < 0.000), a value significantly higher than that obtained for ATP alone.

View larger version (13K): [in this window] [in a new window]   Figure 1.   Effects of ATP and ADO + ATP (A) and of IB-MECA and IB-MECA + ATP (B) on peak Ca2+ release. Peak Ca2+ release induced by ADO + ATP and by IB-MECA + ATP was significantly greater than by ATP alone. Data presented are means ± SEM.

Effect of ADO on 5-HT

We wished to know whether ADO augmented only ATP-induced Ca²⁺ changes, so we tested the interactions of ADO with 5-HT. Figure 2 shows the peak [Ca²⁺]_i response to 5-HT with and without preincubation with ADO (10⁵ M) for 60 s. Baseline [Ca²⁺]_i was 69 ± 9 nM for the 5-HT (10⁶ M) group and 72 ± 5 nM for the ADO (10⁵ M) + 5-HT (10⁶ M) group. After the addition of ADO, baseline [Ca²⁺]_i was 88 ± 14 nM (P = NS). Peak [Ca²⁺]_i was 559 ± 48 nM after the addition of 5-HT (10⁶ M) and 854 ± 126 nM after the addition of ADO (10⁵ M) + 5-HT (10⁶ M) (n = 12, P < 0.025).

View larger version (10K): [in this window] [in a new window]   Figure 2.   Effects of 5-HT and ADO + 5-HT on peak Ca2+ release. Data are means ± SEM.

Mechanisms of ADO Enhancement of ATP-Induced [Ca²⁺]_i Increase

First we tested the effects of ADO on ATP-induced increases in inositol phosphates. Figure 3A shows the increase in total inositol phosphates produced by increasing concentrations of ATP (mean ± SEM, n = 7). At a concentration of 10⁴ M, ATP induced an increase in total inositol phosphates of 211 ± 63% of control; this increased further to 325 ± 34% of control with ATP 10³ M (P < 0.001). Figure 3B shows that pretreatment of the cells with ADO at two different concentrations had no effect on the increase in total inositol phosphates produced by ATP 10⁴ M, 218 ± 30% (n = 6); ADO (10⁶ M) + ATP (10⁴ M), 200 ± 19% (n = 6); and ADO (10⁵ M) + ATP (10⁴ M), 190 ± 19% (n = 6).

View larger version (13K): [in this window] [in a new window]   Figure 3.   Production of inositol phosphates following stimulation with ATP (A) and with ADO + ATP (B). Data are expressed in percentages of the control value. Each point represents the mean ± SEM.

The role of extracellular Ca²⁺ in ADO-induced augmentation of ATP was then evaluated. In Figure 4, the effects on the peak [Ca²⁺]_i response of extracellular Ca²⁺ removal and of the nifedipine (10⁶ M), a Ca²⁺ channel blocker, are shown. The peak response to ATP (10⁴ M) alone was not significantly affected by either treatment: [Ca²⁺]_i rose to 432 ± 77 nM in Ca²⁺-free medium (n = 10) and 572 ± 92 nM in the presence of nifedipine (10⁶ M; n = 18) compared with 585 ± 70 nM [Ca²⁺]_i after ATP (10⁴ M). However, the ADO-enhanced response to ATP was abolished: Peak [Ca²⁺]_i was 474 ± 90 nM following the addition of ADO (10⁵ M) + ATP (10⁴ M) in Ca²⁺-free medium (n = 14) and 614 ± 80 nM in the presence of nifedipine (10⁶ M; n = 18) versus 955 ± 211 nM in normal Hanks' buffer.

View larger version (16K): [in this window] [in a new window]   Figure 4.   Effects of extracellular calcium removal and of nifedipine on peak Ca2+ release by ATP and by ADO + ATP. Data presented are means ± SEM. Numbers in parentheses indicate the number of experiments. *P < 0.03.

Thus the data presented in Figures 3 and 4 establish that the enhanced response to ATP induced by ADO is not due to a synergistic increase in inositol triphosphate (IP₃) but to Ca²⁺ entry from the extracellular fluid through Ca²⁺ channels. Because it has been reported that the permeability of voltage-dependent Ca²⁺ channels can be modulated by increased arachidonic acid (AA) production after P₁-purinoceptor stimulation (24), the effect of the cystolic phospholipase A₂ (cPLA₂) inhibitor AACOCF3 (10 min incubation, 5 × 10⁵ M) was measured on the responses to ATP and ADO + ATP; these results are shown in Section A of Figure 5. It can be seen that the peak response to ATP 10⁴ M decreased from 585 ± 70 to 281 ± 40 nM [Ca²⁺]_i (P < 0.01) and the peak response to ADO 10⁵ M + ATP 10⁴ M from 955 ± 211 to 389 ± 79 nM [Ca²⁺]_i (P < 0.02). Figure 5B shows that preincubation with AACOCF3 had no significant effect on 5-HT-induced peak Ca²⁺ release (559 ± 48 versus 462 ± 48 nM) but significantly abolished the ADO enhancement effect on 5-HT (854 ± 126 versus 415 ± 60 nM, P  0.02).

View larger version (25K): [in this window] [in a new window]   Figure 5.   Effects of the cPLA2 inhibitor AACOCF3 (hatched columns) on peak Ca2+ release by ATP and ADO + ATP (A) and by 5-HT and ADO + 5-HT (B). Data represented are means ± SEM. Open columns, controls.

	Discussion

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The importance of extracellular ATP as a mediator of cellular function has become apparent over the last few years, with the discovery that specific receptors, the P₂ receptors, are present on the cell membrane of many different types of cells and the realization that sources of extracellular ATP are numerous: for example, it is a neural transmitter present in both the central and the autonomic nervous system and is packaged with catecholamines and enkephalins in adrenal chromaffin granules and with serotonin in platelet granules (2, 25). In addition, several different types of cells (including smooth-muscle and epithelial cells) can secrete up to 60% of their ATP content in the absence of cytolysis (2, 25). Mast-cell degranulation has also been shown to produce the release of ATP that acts as an intercellular messenger, triggering the degranulation of adjacent mast cells (21).

Our data confirm our previous observations that stimulation of ASM cells with ATP produces an increase in [Ca²⁺]_i which is enhanced in the presence of ADO. An enhancement of the ATP-induced increase in [Ca²⁺]_i is also observed following incubation with IB-MECA, a specific agonist for the A₃ purinoceptor subtype. This latter observation is consistent with other published data showing that in ASM cells, the enhancement by ADO of the ATP- induced Ca²⁺ release is not due to the stimulation of the A₁ or A₂ purinoceptor subtypes (16). Indeed, in this previous work, the effects of ADO on ATP were enhanced rather than abolished in presence of xanthine amine congener, a blocker of A₁ and A₂ purinoceptor subtypes.

Considering that [Ca²⁺]_i is a primary determinant of the contractile response of smooth muscle, this enhancing effect of ADO, particularly if it is not specific for P₂-receptor stimulation, could be an important determinant of ASM responsiveness to contractile agonists. The results we obtained, showing that 5-HT-induced Ca²⁺ release is also enhanced in presence of ADO, confirm this hypothesis.

An enhancement of agonist response through P₁-purinoceptor stimulation was first reported in the DDT₁ MF-2 smooth-muscle cell line by Gerwins and Fredholm (26, 27). These authors showed that ADO potentiated the effects of ATP and bradykinin and that this effect was mediated by the activation of the A₁ subtype of P₁ purinoceptors. In their experiments, the formation of inositol 1,4,5-triphosphate [(1,4,5)IP₃] was observed following the stimulation of either A₁- or P₂-purinoceptor stimulation; when both receptors were stimulated simultaneously, the subsequent increase in (1,4,5)IP₃ was not additive but synergistic. However, in various types of tissues there are also reports that ADO can potentiate the effects of receptors coupled to the activation of phospholipase C, including histamine H₁ receptors, ₁ adrenoceptors, and muscarinic and nucleotide receptors, without having an effect per se on phospholipase C activation (24, 28). This also appears to be the case in our experiments because the increase in total inositol phosphates produced by incubation of the cells with ADO and ATP was comparable to that measured in the presence of ATP alone. Numerous studies on signal transduction have shown that elaborate cross-talk mechanisms exist for the regulation of [Ca²⁺]_i when more than one stimulus acts on a target cell (34). One such mechanism is the phosphorylation by cyclic adenosine monophosphate (cAMP)-dependent protein kinase of the (1,4,5)IP₃ receptor (35). The outcome of this phosphorylation appears to vary in different cell types: In the brain, it decreases the potency of (1,4,5)IP₃ in releasing Ca²⁺ whereas in liver cells the opposite occurs and the dose-response curve for (1,4,5)IP₃-mediated Ca²⁺ release is shifted to the left (35, 36). Because P₁-receptor activation regulates adenylyl cyclase activity, the role of such a mechanism on the enhancement of the ATP response by ADO was assessed in our system by measuring the peak Ca²⁺ release by ATP and by ADO + ATP in Ca²⁺-free medium. Peak Ca²⁺ release by ATP in the absence of extracellular Ca²⁺ was comparable to that obtained in Ca²⁺-rich medium; and also, the difference in peak [Ca²⁺]_i produced by ATP and by ADO + ATP was abolished in Ca²⁺-free medium. These data confirm that ATP-induced Ca²⁺ release comes from intracellular stores (16). In addition, the fact that the peak [Ca²⁺]_i release by ATP and by ADO + ATP did not differ in Ca²⁺-free medium is consistent with the measurements of total inositol phosphates and also indicates that the potentiation effect of ADO is not due to an increased sensitivity of the (1,4,5)IP₃ receptors to (1,4,5)IP₃ but is due rather to extracellular Ca²⁺ entry. Administration of nifedipine, a voltage-dependent Ca²⁺ channel blocker, confirmed this hypothesis because the enhancing effect of ADO on the ATP-induced response was abolished in the presence of this compound.

In various cell types, stimulation of G protein-coupled receptors can result in PLA₂ activation and the release of AA from membrane lipids. Moreover, it has been shown that in some cells, such as macrophages, the phospholipase C (PLC) and PLA₂ pathways can be activated simultaneously through different subunits of a given G protein (37). AA itself, and/or its metabolites derived through the cyclooxygenase or the lipoxygenase pathways, have a variety of biologic effects, one of them being the modulation of plasma membrane ion channels (38, 39). In the DDT₁ MF-2 cells for example, histamine H₁-receptor stimulation resulted in the activation of both PLC and PLA₂ with the subsequent formation of (1,4,5)IP₃ and AA. Van der Zee and colleagues (40) showed that the released AA acts as a second messenger, activating Ca²⁺ influx from extracellular sources through the opening of Ca²⁺ channels. This effect was specific for AA because blockers of the cyclooxygenase or the lipoxygenase pathways did not affect Ca²⁺ entry but lanthanum, a Ca²⁺ channel blocker, did. Moreover, other studies indicate that AA not only activates Ca²⁺ entry but also induces the release of Ca²⁺ from intracellular stores (41). PLA₂ activation has been reported following both P₁- and P₂-receptor stimulation (42, 43). In a thyroid cell line, the stimulation of P₁ receptors had no effect per se on AA release but produced a synergistic increase of the AA release by P₂-purinoceptor stimulation (24).

Thus, we hypothesized that Ca²⁺ entry in cells was due to an increase in AA release produced by P₁-purinoceptor stimulation. The data we obtained confirmed this hypothesis by showing that indeed, the cPLA₂ inhibitor AACOCF3 abolishes the enhancing effect of ADO on both ATP- induced and 5-HT-induced Ca²⁺ release. Interestingly, incubation with AACOCF3 had no effect on 5-HT-induced Ca²⁺ release but reduced the peak Ca²⁺ response to ATP. Thus, these data show that in ASM cells, the peak Ca²⁺ response to ATP is modulated not only by (1,4,5)IP₃- induced Ca²⁺ release, but also by AA-induced Ca²⁺ release from intracellular stores (41).

In conclusion, we have shown that the enhancing effect of ADO on receptor stimulation is not specific for P₂ purinoceptor but is also present when other agonists, such as the contractile agonist 5-HT, are used. This effect of ADO is due to cPLA₂ activation and presumably reflects the action of AA liberated by cPLA₂. AA, in turn, regulates [Ca²⁺]_i by modulating both Ca²⁺ release from intracellular stores and Ca²⁺ entry through Ca²⁺ channels. These data suggest that ADO may enhance ASM response to inflammatory mediators. Thus, in addition to its well-known effect on mast-cell degranulation, ADO might contribute to airway hyperresponsiveness in asthmatic and cystic fibrosis patients by enhancing the smooth-muscle response to the mediator released.