ATP stimulates Ca2+ oscillations and contraction in airway smooth muscle cells of mouse lung slices

Albrecht Bergner and Michael J. Sanderson

Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In airway smooth muscle cells (SMCs) from mouse lung slices, >= 10 µM ATP induced Ca2+ oscillations that were accompanied by airway contraction. After ~1 min, the Ca2+ oscillations subsided and the airway relaxed. By contrast, >= 0.5 µM adenosine 5'-O-(3-thiotriphosphate) (nonhydrolyzable) induced Ca2+ oscillations in the SMCs and an associated airway contraction that persisted for >2 min. Adenosine 5'-O-(3-thiotriphosphate)-induced Ca2+ oscillations occurred in the absence of external Ca2+ but were abolished by the phospholipase C inhibitor U-73122 and the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin. Adenosine, AMP, and alpha ,beta -methylene ATP had no effect on airway caliber, and the magnitude of the contractile response induced by a variety of nucleotides could be ranked in the following order: ATP = UTP > ADP. These results suggest that the SMC response to ATP is impaired by ATP hydrolysis and mediated via P2Y2 or P2Y4 receptors, activating phospholipase C to release Ca2+ via the inositol 1,4,5-trisphosphate receptor. We conclude that ATP can serve as a spasmogen of airway SMCs and that Ca2+ oscillations in SMCs are required to sustain airway contraction.

calcium signaling; confocal microscopy; ATP hydrolysis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ASTHMA IS AN AIRWAY DISEASE characterized by a hyperreactive response of airway smooth muscle cells (SMCs) to spasmogens such as histamine, leukotrienes, and perhaps serotonin (2, 6, 25). A major source of histamine and other agonists is an exocytotic release from inflammatory cells (i.e., mast cells and basophils) within the submucosal tissue, in close proximity to the SMCs. However, mast cell degranulation can simultaneously release another potential spasmogen, ATP, which is packaged in the same exocytotic vesicles (23). In addition, airway epithelial cells have also been identified as a source of extracellular ATP. Although the active release mechanisms of ATP from epithelial cells are not understood (5, 7, 11, 36, 37), epithelial trauma associated with asthma (12, 18) can result in damaged cells that passively release ATP.

In many other tissues, ATP has been found to stimulate P2X and P2Y receptors, both of which have been identified in lung tissue (19, 29, 34, 35). P2X receptors are Ca2+ channels that, on binding of ATP, initiate an influx of extracellular Ca2+ into the cell. P2Y receptors are G protein-linked receptors that, on binding of ATP, activate phospholipase C (PLC), and this leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 binds to specific receptors (IP3R) within the sarcoplasmic reticulum (SR) to release Ca2+ from the SR (for review see Ref. 4). Because increases in the intracellular free Ca2+ concentration ([Ca2+]i) of SMCs usually result in the phosphorylation of myosin light chain and the stimulation of contraction (15, 16), ATP is likely to be a potent spasmogen of airway SMCs.

Surprisingly, the effects of ATP on the [Ca2+]i of airway SMCs or on airway contraction have not been extensively studied, and the few reports that exist are inconsistent. For example, ATP was found to increase in vivo airway resistance in rats (9) or induce contraction in isolated guinea pig tracheae (8). On the other hand, ATP was reported to relax rabbit tracheal smooth muscle strips (1) and isolated mouse trachea (10, 17) that had been previously contracted with acetylcholine (ACh). These tissue preparations were derived from the large airways and consisted of tracheal rings or strips. As a result, these studies were not able to focus on the responses of individual SMCs. In addition, it is not clear whether results obtained from large airways (e.g., trachea) are applicable to the pathophysiology of smaller airways (e.g., lower bronchi and bronchioles). On the other hand, Michoud et al. (22) studied cultured rat tracheal SMCs and reported that ATP induced a Ca2+ transient followed by a lower, but sustained, Ca2+ elevation. However, the contractile responses of these cells were not investigated. By contrast, ATP-induced Ca2+ signaling is better characterized in vascular SMCs (e.g., from the pulmonary artery), where it was found that ATP could induce a series of Ca2+ oscillations (14, 21, 26).

Consequently, the objective of the present study was to characterize the effect of ATP on the Ca2+ signaling and the associated contractility of airway SMCs. To perform these studies, we have refined a technique to cut thin lung slices (~75 µm thick) and have used these slices to simultaneously measure airway contraction and Ca2+ signaling in single airway SMCs with confocal microscopy (3). The major advantages of lung slices are that the in situ organization of the lung and the contractility of the SMCs are maintained for several days.

With this approach, we found that ATP simultaneously induced Ca2+ oscillations in SMCs and contraction of mouse airways. This response is mediated by P2Y receptors acting via PLC and IP3Rs, but not via P2X receptors and Ca2+ influx. These results highlight the potential role for extracellular ATP in regulating airway caliber and are consistent with the hypothesis that sustained airway contraction is maintained by Ca2+ oscillations within SMCs.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Cell culture reagents were obtained from Invitrogen Life Technologies-GIBCO (Carlsbad, CA). Other reagents were obtained from Sigma (St. Louis, MO) unless otherwise stated. In most cases, the Hanks' balanced salt solution was supplemented (sHBSS) with HEPES buffer (25 mM) but lacked phenol red.

Lung slices. Lung slices were cut as previously described (3). Briefly, male BALB/C inbred mice (Charles River Breeding Labs, Needham, MA; 42-77 days old) were killed by intraperitoneal injection of pentobarbital sodium (Nembutal), and the chest wall was removed. The trachea was cannulated using an intravenous catheter system (20G Intima, Becton Dickinson, Sandy, UT), and the lungs were inflated with 2% agarose-sHBSS at 37°C (~1 ml). Subsequently, 0.1-0.2 ml of air was injected to flush the agarose-sHBSS out of the airways. To stiffen the soft lung tissue for sectioning, the agarose was gelled at 4°C. The lungs were removed, and ~75-µm-thick slices were cut with a tissue slicer (model EMS-4000, Electron Microscopy Sciences, Fort Washington, PA). The slices were maintained by flotation in DMEM supplemented with 10% fetal bovine serum and antibiotics and antimycotics at 37°C in 10% CO2 for up to 5 days. Immunohistochemistry was performed using monoclonal mouse anti-alpha -actin antibodies and FITC-conjugated anti-mouse IgG antibodies (for details see Ref. 3).

Measurement of airway contraction. Experiments were performed on airways with a lumen that was free of agarose and lined by epithelial cells showing ciliary activity. For all experiments, the slices (~7,000-8,000 µm long × 3,000-4,000 µm wide × 75 µm thick) and were bathed in 150 µl of sHBSS. The cross-sectional area of the airways was 29,989 ± 1,885 (SE) µm2 (n = 104), and there were no significant differences between groups of airways used for different experiments. The slices were placed on cover glasses within a custom-made Plexiglas chamber and held in position by a piece of nylon mesh (CMN-300-B, Small Parts, Miami Lakes, FL). Phase-contrast images were recorded using a digital charge-coupled device camera (model TM-6710, Pulnix America, Sunnyvale, CA), a digital camera interface (Road Runner, BitFlow, Woburn, MA), and image acquisition software (Video Savant, IO Industries, London, ON, Canada). Frames were captured in time lapse (1 frame/s), and the cross-sectional area of the airway was measured with respect to time by pixel summing using the image analysis software Scion (Scion, Frederick, MD; download available at no cost at www.scioncorp.com).

Measurements of [Ca2+]i. Slices were loaded with the Ca2+ indicator dye Oregon green (20 µM; Molecular Probes, Eugene, OR) for 1 h (for details see Ref. 3), and confocal microscopy was performed using a custom-built microscope (31). Images were recorded in time lapse (2 frames/s). Regions of interest of 8.4 × 8.4 µm were defined in single SMCs, and the average fluorescence intensities of each region of interest were obtained. Bleach correction was calculated from the bleaching rate 15-30 s before the addition of drugs. Data were corrected with the bleach correction before a rolling average of three frames was calculated. Final fluorescence values are expressed as a fluorescence ratio (F/F0) normalized to the initial fluorescence (F0). Simultaneous airway contraction was analyzed by measuring the area of the part of the airway lumen that was visible in the confocal images.

Drug application. ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S), UTP, ADP, AMP, alpha ,beta -methylene ATP, and adenosine were dissolved in sHBSS. A 10× concentrated solution was added to the volume of the bath solution to be diluted to the final working concentration. Xestospongin (Calbiochem, La Jolla, CA), U-73122, U-73433, and indomethacin were dissolved in dimethyl sulfoxide and diluted in sHBSS to the final concentrations used. Final dimethyl sulfoxide concentrations were 0.1% for indomethacin, 0.05% for xestospongin, and 2.5% for U-73122 and U-73433.

Statistics. Statistical analysis was performed using the t-test and the Kruskal-Wallis one-way analysis of variance on ranks; for all pairwise multiple comparisons, the Student-Newman-Keuls method was used. Values are means ± SE. P < 0.05 was considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ATP-induced airway contraction. The addition of 10-100 µM ATP initiated airway contraction (Figs. 1 and 2). This airway contraction was followed by relaxation of the airway in the continued presence of ATP [relaxation began 61 ± 23 (SE) s after addition of 100 µM ATP, n = 6; Fig. 2A]. To distinguish whether the hydrolysis of ATP, presumably by ectonucleotidases, or receptor accommodation was responsible for the brevity of the response to ATP, we tested the effect of the nonhydrolyzable ATP analog ATPgamma S on airway contraction. At >= 500 nM, ATPgamma S induced contraction (Fig. 2B) that was maintained for a significantly longer time than that induced by ATP (relaxation began 164 ± 5 s after addition of 100 µM ATPgamma S, n = 6, P < 0.002). Airway contraction was dependent on ATP concentration in the range 10-100 µM (Fig. 2C). A decrease in lumen size of 10.9 ± 1.4% (n = 6) of the initial cross-sectional area occurred in response to 100 µM ATP. A similar concentration dependence of airway contraction was induced by 0.5-100 µM ATPgamma S (Fig. 2C). However, the airways responded more strongly to ATPgamma S than to ATP, and a decrease of ~10% in the lumen area was induced by ~1 µM ATPgamma S compared with 100 µM ATP. This greater sensitivity to ATPgamma S resulted in a leftward shift of the concentration-dependence curve compared with ATP. A near-maximal reduction in lumen area of 14.6 ± 5.1% (n = 6) was induced by 100 µM ATPgamma S.


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Fig. 1.   Appearance of airways and location of smooth muscle cells (SMCs) in mouse lung slices. A and B: phase-contrast micrographs showing an airway in a lung slice immediately before (A) and 30 s after (B) addition of 100 µM ATP. AL, airway lumen; EC, epithelial cells. Scale bar, 50 µm. C: lung slices loaded with Ca2+ indicator dye Oregon green and observed using confocal microscopy. SMCs (arrows) in airway wall are separated from the airway lumen by a single column of epithelial cells (star ). Scale bar, 5 µm. D: lung slices stained using anti-alpha -actin antibodies and observed with confocal microscopy. Brightly stained SMCs surround the airway lumen. Scale bar, 5 µm.



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Fig. 2.   Airway contraction induced by ATP or adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S). Cross-sectional area of the airway lumen, recorded with phase-contrast microscopy at 1 frame/s, was calculated and plotted against time. Decrease in cross-sectional area is airway contraction; increase in cross-sectional area is airway relaxation. A: 100 µM ATP induced airway contraction; after ~1 min, airway relaxed again. B: nonhydrolyzable ATP analog ATPgamma S (100 µM) induced contraction that lasted >= 2 min. Traces in A and B are representative of 6 experiments in 6 different slices from >= 2 different mice. C: after addition of agonists, cross-sectional area of the airway lumen was monitored, and maximum reduction in area attained within 3 min in each experiment was plotted against agonist concentration. ATP and ATPgamma S showed a concentration-dependent increase in airway contraction (equivalent to a decrease in cross-sectional area). Response to ATPgamma S was more pronounced over concentrations used and resulted in a leftward shift of the response curve relative to that of ATP. Each point represents mean ± SE of 6 experiments in 6 different slices from >= 2 different mice. Mean cross-sectional area of airways was 30,633 ± 2,716 µm2 (n = 54).

In response to ATP and ATPgamma S, brief transient contractions followed by rapid relaxation or "twitches" of airways were sometimes observed. These twitches occurred when the airway was contracted as well as when the airway was relaxing (Fig. 3, arrows). The twitches could involve the whole airway circumference with a marked change in cross-sectional area, or the twitches could be localized to only parts of the airway wall, reflecting presumably the activity of single SMCs, without a significant narrowing of the airway lumen. Confocal microscopy revealed that the twitches occurred simultaneously with Ca2+ transients in the associated SMCs (data not shown). Although this is an interesting response, it is relatively minor compared with the major contractile response to ATP and is included primarily for completeness.


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Fig. 3.   Transient airway contractions. Cross-sectional area of the airway lumen was calculated and plotted against time. After addition of 10 µM ATPgamma S, the airway contracted, and transient contractions were followed by rapid relaxations or "twitches" (arrows). Twitches occurred while the airway was relaxing; in other experiments, twitches occurred while the airway was still contracted.

The prolonged and more sensitive contraction in response to ATPgamma S than to ATP suggests that ATP is hydrolyzed after addition to the lung slice. Consequently, we investigated the influence of the ATP hydrolysis products ADP, AMP, and adenosine on airway cross-sectional area. Neither adenosine (100 µM, n = 6; Fig. 4A) nor AMP (100 µM, n = 6; Fig. 4B) had any effect on airway caliber. ADP (100 µM) had only a small effect and stimulated a reduction of airway cross-sectional area of 2.95 ± 1.0% (n = 6; Fig. 4B).


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Fig. 4.   Airway contraction induced by nucleotide receptor agonists. A: addition of 100 µM UTP induced airway contraction; addition of 100 µM adenosine had no effect on airway cross-sectional area. B: ADP induced slight airway contraction; AMP had no effect on airway cross-sectional area. Each trace is representative of 6 experiments in 6 different slices from >= 2 different mice. Arrows, agonist addition. C: maximum reduction in cross-sectional area induced within 3 min after addition of different agonists (100 µM). alpha ,beta -Methylene ATP (alpha ,beta -Me-ATP) had only a marginal effect on airway caliber; ADP contracted the airways slightly. Contraction induced by UTP was similar to that induced by ATP. *P < 0.05 vs. alpha ,beta -methylene ATP and ADP. Values are means ± SE of 6 experiments for each agonist using 6 airways in 6 different slices from >= 2 different mice. Mean cross-sectional area of the airways was 31,720 ± 3,157 µm2 (n = 36).

To investigate the classification of the purinergic receptor(s) involved in ATP-induced airway contraction, the airway responses to UTP and alpha ,beta -methylene ATP (an agonist primarily of P2X receptors) were also examined. Although 100 µM alpha ,beta -methylene ATP had only a marginal effect on airway caliber (0.24 ± 0.2%, n = 6), the reduction in airway caliber induced by 100 µM UTP (9.1 ± 2.0%, n = 6) was similar to that induced by 100 µM ATP (10.9 ± 1.4%, n = 6; Fig. 4).

Previous studies (1, 10, 17) reported that ATP had a relaxing effect on tracheal SMCs that had been stimulated to contract with 10 µM ACh. This response was thought to be mediated by prostaglandins released by the epithelial cells and was attributed to a regulatory role of epithelial cells in SMC contraction. To test for a relaxing effect of ATP in lung slices, the airways were contracted with ACh as previously described (3) and subsequently exposed to ATP. The airways showed a large reduction in cross-sectional area in response to 10 µM ACh, but this contraction was not influenced by the addition of 100 µM ATP (n = 4; Fig. 5A). However, if airway contraction was induced with 100 nM ACh, a concentration previously shown to contract airways to ~50% of their maximum contraction (3), a slight relaxation was observed in response to 100 µM ATP (n = 5; Fig. 5A). This relaxation was small and had little effect on the cross-sectional area.


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Fig. 5.   Effect of ATP on precontracted and indomethacin-treated airways. A: airways were contracted with ACh and subsequently exposed to 100 µM ATP. After contraction with 10 µM ACh, addition of ATP had no effect on airway cross-sectional area. After contraction with 100 nM ACh, addition of ATP resulted in a slight relaxation of the airway. Traces are representative of 4 experiments in 4 different airways in 4 different lung slices. B: after airway contraction with 100 nM ACh, addition of 100 µM nonhydrolyzable ATPgamma S also induced a slight relaxation. C: lung slices were incubated for 30 min with 100 µM indomethacin and subsequently contracted with 100 nM ACh. No relaxation was observed on addition of 100 µM ATP. D: no difference was observed in contraction of airways induced by ATP (10 or 100 µM) with or without incubation with 100 µM indomethacin for 30 min. Values are means ± SE (n = 6). E: after the airways had been contracted with 10 µM ACh, addition of 1 µM prostaglandin E2 (PGE2) led to a small relaxation that was reversed by addition of 10 µM ATPgamma S. Trace is representative of 3 experiments in 3 different airways in 3 different lung slices. Mean cross-sectional area of the airways was 28,700 ± 3,172 µm2 (n = 43).

To test whether the hydrolysis products of ATP such as adenosine, rather than ATP itself, caused this relaxation, airways contracted with 100 nM ACh were exposed to 100 µM ATPgamma S, instead of ATP. The airways showed a similar response, i.e., a slight relaxation, in response to ATPgamma S (n = 4; Fig. 5B). To test whether cyclooxygenase products such as prostaglandins, released by epithelial cells, were involved in the relaxation, lung slices were incubated for 30 min with 100 µM indomethacin, a cyclooxygenase inhibitor, before exposure to 100 nM ACh. With this indomethacin pretreatment, a relaxation of the ACh-induced airway contraction in response to 100 µM ATP was not observed (n = 4; Fig. 5C).

To investigate the possibility that ATP-induced prostaglandin release may also influence ATP-induced airway contraction, lung slices were incubated for 30 min with 100 µM indomethacin before the addition of ATP. We found no difference between the indomethacin-treated and control lung slices with respect to the reduction in airway cross-sectional area induced by 10 or 100 µM ATP: 2.44 ± 1.0 and 2.10 ± 1.0% area reduction with and without indomethacin, respectively, for 10 µM ATP and 10.3 ± 2.2 and 10.9 ± 1.4% area reduction with and without indomethacin, respectively, for 100 µM ATP (n = 6; Fig. 5D). Similarly, ATPgamma S-induced airway contraction appeared to be unaltered by preincubation with indomethacin (n = 6; data not shown). The addition of indomethacin alone had no effect on airway caliber (n = 4; data not shown). To test whether the airways in lung slices were, in fact, sensitive to prostaglandins, airways were contracted with 10 µM ACh and subsequently exposed to 1 µM prostaglandin E2 (PGE2). PGE2 had a small relaxing effect on the airways, but this effect was reversed by the addition of 10 µM ATPgamma S (n = 3; Fig. 5E).

ATP-induced Ca2+ signaling in airway SMCs. The Ca2+ response of the SMCs to ATP consisted of a brief burst of Ca2+ oscillations (Fig. 6A). Because of the small number of oscillations that occurred, their frequency was difficult to accurately calculate. However, the number of oscillations was dependent on the ATP concentration: 1.25 ± 0.25 and 10.9 ± 1.4 oscillations in response to 10 and 100 µM ATP, respectively (n = 6). The initial [Ca2+]i increase associated with the ATP-induced oscillations, defined as the percent change in the F/F0 measured immediately before and at the peak of the initial Ca2+ oscillation, also increased with concentration (Fig. 6D).


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Fig. 6.   ATP- and ATPgamma S-induced Ca2+ signaling in airway SMCs. Ca2+ changes in SMCs were recorded using confocal microscopy and expressed as fluorescence ratio (F/F0). A: Ca2+ oscillations in airway SMCs were induced by ATP but subsided after ~1 min. Only a limited number (3-6) of oscillations were observed. B: ATPgamma S induced a prolonged series of Ca2+ oscillations with an initial decline in magnitude that stabilized after 3-5 oscillations. Traces are representative of 6 experiments from 6 different airways in 6 different slices from 2 different mice. C: frequency of Ca2+ oscillations induced by 0.1-100 µM ATPgamma S was concentration dependent. No oscillation frequency was calculated for ATP, because ATP induced only a small number of Ca2+ oscillations. D: initial increase in F/F0 (percent change in F/F0 measured immediately before and at the peak of the initial Ca2+ oscillation) was concentration dependent for ATP and ATPgamma S. Values in C and D are means ± SE of 6 experiments of 6 different airways in 6 different slices from >= 2 different mice.

In contrast to ATP, ATPgamma S induced a prolonged series of Ca2+ oscillations (Fig. 6B). The frequency of the ATPgamma S-induced Ca2+ oscillations was concentration dependent, increasing from ~4/min at 0.1 µM to ~8/min at 100 µM (Fig. 6C). Similarly, the ATPgamma S-induced [Ca2+]i increase in the initial Ca2+ oscillation was concentration dependent and was more sensitive than the response to ATP (Fig. 6D). Although a [Ca2+]i increase of ~24% was stimulated by 100 µM ATP, the same [Ca2+]i increase was stimulated by ~0.5 µM ATPgamma S. After the first Ca2+ oscillation, the next three to five Ca2+ oscillations declined in peak magnitude, but subsequent oscillations persisted, with a near-constant magnitude for >= 2 min (Fig. 6B).

To determine whether ATP-induced Ca2+ oscillations were dependent on a Ca2+ influx from the extracellular space, airways were exposed to 10 µM ATPgamma S in a Ca2+-free solution containing 5 mM EGTA. Under these conditions of zero external Ca2+, ATPgamma S still induced Ca2+ oscillations (n = 6; Fig. 7A) with an initial Ca2+ increase (31.5 ± 5.1 and 33.7 ± 3.7% increase in F/F0 without and with external Ca2+, respectively) and a Ca2+ oscillation frequency (4.3 ± 0.5 and 4.7 ± 0.9/min without and with external Ca2+, respectively) that was not different from the Ca2+ oscillations stimulated in the presence of external Ca2+.


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Fig. 7.   Mechanisms of ATPgamma S-induced Ca2+ signaling. A: 10 µM ATPgamma S induced a prolonged series of Ca2+ oscillations in SMCs of lung slices exposed to a Ca2+-free solution containing 5 mM EGTA. B: after incubation with the phospholipase C inhibitor U-73122 (50 µM) for 30 min, no Ca2+ oscillations in response to 10 µM ATPgamma S were observed. C: after incubation with the inositol 1,4,5-trisphosphate receptor antagonist xestospongin (10 µM) for 45 min, Ca2+ signaling in response to 10 µM ATPgamma S was not observed. Traces are representative of 4-6 experiments from 4-6 different airways in 4-6 different slices from >= 2 different mice.

To test the involvement of PLC in the signaling pathway leading to Ca2+ oscillations, lung slices were exposed to the PLC inhibitor U-73122 (50 µM) for 30 min. On addition of 10 µM ATPgamma S, no Ca2+ changes were observed (n = 6; Fig. 7B). To serve as a control experiment, lung slices were exposed to the inactive analog U-73433 (50 µM) for 30 min. In this case, 10 µM ATPgamma S still induced Ca2+ oscillations (n = 6; data not shown). To confirm that the intracellular Ca2+ signaling involved the IP3R, lung slices were incubated with the IP3R antagonist xestospongin (10 µM) for 45 min. This treatment abolished the Ca2+ signaling in response to 10 µM ATPgamma S (n = 4; Fig. 7C). These treatments with U-73122 and xestospongin prevented ATP-induced airway contraction.

Correlation between ATP-induced airway contraction and Ca2+ signaling. The Ca2+ signaling in the SMCs was correlated with airway contraction by analysis of the changes that occurred in the cross-sectional area of the airway lumen that were visible in the confocal images simultaneously with the changes in SMC fluorescence. The change in lumen size is given in absolute units, rather than as a percentage of the starting lumen area. This data representation is necessary because, at the magnifications required to observe the changes in [Ca2+]i of single SMCs, only a part of the area of the airway lumen is visible in the confocal images. Furthermore, because the size of this area can vary depending on the tissue morphology and alignment, reporting the change in area as a percentage of the starting area may misrepresent the size of the airway contraction.

As described earlier, 100 µM ATP induced a small number of Ca2+ oscillations that terminated after ~1 min (Fig. 8A). The initial Ca2+ oscillation was associated with the initiation of airway contraction. The following two to three Ca2+ oscillations correlated with the maintenance of contraction. However, with the subsequent decline in magnitude and cessation of the ATP-induced Ca2+ oscillations, a simultaneous relaxation of the airway was observed. By contrast, ATPgamma S induced sustained Ca2+ oscillations, and the airway rapidly contracted in synchrony with the first Ca2+ oscillation and remained contracted during the following Ca2+ oscillations (Fig. 8B).


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Fig. 8.   Correlation of ATP- and ATPgamma S-induced Ca2+ signaling and airway contraction. Ca2+ changes in SMCs were recorded using confocal microscopy and expressed as fluorescence ratio. Simultaneous change in cross-sectional area of the airway lumen that was visible in the confocal images was measured and expressed as an absolute change in area, rather than percent change of the starting area as for data acquired from phase-contrast images. The reason for this is that, because of the higher magnification used, only part of the cross-sectional area of the lumen is visible in the confocal images. Consequently, the amount of the lumen visualized can vary, and similar contractions of the airway can result in different percent contractions (i.e., a larger visible area results in a smaller percentage). A: ATP induced a small number of Ca2+ oscillations, and the initial oscillation was associated with initiation of airway contraction. When Ca2+ oscillations stopped, the airway relaxed (visible area before contraction = 359 µm2). B: ATPgamma S also induced Ca2+ oscillations, which persisted during maintenance of airway contraction and for the duration of the experiment (visible area before contraction = 466 µm2). Traces are representative of 8 experiments of 8 different airways in 8 different slices from >= 2 different mice.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We present a versatile airway preparation, the lung slice, which we have used in conjunction with confocal microscopy to simultaneously record Ca2+ signaling in SMCs and the associated airway contraction, a measure of the integrated activity of airway SMCs. We have found that ATP and ATPgamma S induced Ca2+ oscillations in airway SMCs and that these Ca2+ oscillations correlated with the initiation and maintenance of airway contraction. Conversely, when the Ca2+ oscillations subsided, the airways relaxed.

ACh-induced Ca2+ oscillations in airway SMCs have been reported by several groups (20, 27, 28, 30, 32, 33), and Pabelick et al. (24) assumed that ACh-induced Ca2+ oscillations may function to regulate global [Ca2+]i in airway SMCs and, thereby, regulate airway contraction. In our previous study reporting ACh-induced Ca2+ oscillations in airway SMCs in lung slices (3), we demonstrated that the Ca2+ oscillations occurred during the maintenance of airway contraction and that whenever the Ca2+ oscillations were abolished by a variety of approaches, the airways relaxed. As a result, we hypothesized that ACh-induced Ca2+ oscillations maintain airway contraction. Our findings with ATP in the present study support and extend this hypothesis by demonstrating that this principle is not limited to a single agonist (e.g., ACh) but also applies to ATP. As a result, the maintenance of contraction by Ca2+ oscillations may be a general principle of airway SMCs in response to agonists.

The maximal frequencies of ACh-induced Ca2+ oscillations (~20/min) (3) were higher than the frequencies of the ATPgamma S-induced Ca2+ oscillations (~8/min), and the corresponding maximal contraction induced was also higher (~20 and ~14% of starting area for ACh and ATPgamma S, respectively). This suggests that higher frequencies are required to maintain stronger airway contraction. This relationship between Ca2+ oscillation frequency and airway contraction supports the idea that airway contraction is generated or regulated in a frequency-modulated manner.

Although ATP induced airway contraction, the airways relaxed after ~1 min. Extracellular ATP is commonly subject to hydrolysis by ectonucleotidases (for review see Ref. 38), and Gerwins and Fredholm (13) found that, in a cultured SMC line, hydrolysis could reduce ATP concentrations to <50% of starting value within a few minutes. Lung slices contain a variety of cell types that are in close proximity to the SMCs, making it likely that ectonucleotidase activity is present. Consistent with this idea is our finding that lower concentrations of the nonhydrolyzable ATP analog ATPgamma S induced airway contraction that was sustained for longer periods than that induced by ATP. Consequently, we believe that the relaxation of the airway after the addition of ATP results primarily from a degradation of ATP. This proposed hydrolysis of ATP would be expected to release adenosine, AMP, and ADP, but none of these had any relaxing effect on airway size. This is consistent with the idea that airway relaxation resulted from a reduction of ATP concentration. Alternatively, it is possible that a complex mixture of ATP and ATP metabolites might have a relaxant effect on airways and that this contributed to the brevity of the ATP-induced contraction. Another explanation for the airway relaxation is receptor desensitization. Although it was found that ATPgamma S induced contraction for a longer period than ATP, the contractile response to ATPgamma S subsided after ~10 min. If it is assumed that the ATPgamma S is still present, relaxation could be explained by the fact that the P2Y receptors or another intracellular site became desensitized (29). This would also infer that the relaxation of airways after ATP exposure resulted from ATP degradation and receptor accommodation.

The nucleotide receptor agonist alpha ,beta -methylene ATP is selective for P2X over P2Y receptors (29) but had only marginally contractile effects on airways. In addition, P2X receptors are Ca2+ channels leading to Ca2+ influx on binding of ATP (19, 29), but in Ca2+-free solution, ATPgamma S still induced Ca2+ oscillations with a magnitude and frequency comparable to that in the presence of external Ca2+. The effects of ATP on airways are therefore likely to be mediated by P2Y, rather than P2X, receptors. Given the order of potency for inducing airway contraction (ATP = UTP > ADP), the receptor subtypes P2Y2 and P2Y4 would be predicted. In addition, because ADP induced a slight contraction, P2Y1 receptors may also be present, but a small contractile response could also be mediated via P2Y2 or P2Y4 receptors. P2Y receptors are coupled to G proteins and increase intracellular IP3 levels via activation of PLC (29, 35). This is consistent with our data, which indicate a need for PLC activity and IP3Rs for the initiation of Ca2+ oscillations. Therefore, we propose that ATP-induced Ca2+ signaling requires an activation of PLC by G protein-coupled P2Y2 or P2Y4 receptors, leading to increased formation of IP3 and release of Ca2+ from the SR via IP3Rs.

ATP, acting via prostaglandins released from epithelial cells, has been previously reported to extensively relax perfused tracheae that were precontracted with 10 µM ACh (1, 10, 17). However, in this study, airways fully contracted with 10 µM ACh or airways partly contracted with 100 nM ACh [~50% of their maximal contraction (3)] showed no response or very little response to 100 µM ATP or ATPgamma S. ATP probably did not induce further contraction, because ACh and ATP utilize similar signal transduction pathways, and the stimulation by ACh exceeded the stimulation induced by ATP. The small relaxing effect of ATP could be abolished with indomethacin, and this is consistent with the hypothesis that cyclooxygenase products may be released from epithelial cells. We also found that PGE2 had a relaxing effect on precontracted airways, but this effect was reversible by ATPgamma S, a result indicating that the contraction induced by ATPgamma S was sufficient to counter the relaxing effect of PGE2. Because there was no difference in ATP-induced airway contraction in the presence or absence of indomethacin, we believe that neither ATP nor its metabolites release sufficient quantities of prostaglandins to influence the ATP-induced airway contraction. We therefore conclude that ATP primarily functions to contract airways in mouse lung slices.

ATP-induced changes of [Ca2+]i have been investigated in several types of SMCs, and ATP-induced Ca2+ oscillations have been observed, for example, in vascular SMCs (14, 21, 26). In contrast, little is known about ATP-induced Ca2+ signaling in airway SMCs. Michoud et al. (22) reported a Ca2+ transient followed by a Ca2+ plateau in response to ATP in cultured rat tracheal SMCs. Besides differences in species and tissue preparation, the study averaged the fluorescence from ~20 cells, and this probably prevented the observation of Ca2+ oscillations.

In summary, we have demonstrated that ATP induces airway contraction and Ca2+ oscillations in mouse airway SMCs. We believe that these responses are mediated via P2Y receptors to stimulate Ca2+ oscillations that rely on PLC activity and IP3Rs, but not on Ca2+ influx. The strength of the response appears to be attenuated by hydrolysis of ATP. Also, in view of the fact that the airways remained contracted whenever Ca2+ oscillations were occurring in the SMCs, we provide further evidence that airway contraction is maintained by Ca2+ oscillations. These studies suggest that ATP is a potential spasmogen of airway SMCs and that it may play a role in the regulation of airway caliber and in the pathogenesis of airway disease.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-49288.


    FOOTNOTES

Address for reprint requests and other correspondence: M. J. Sanderson, Dept. of Physiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655 (E-mail: michael.sanderson{at}umassmed.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

August 2, 2002;10.1152/ajplung.00139.2002

Received 7 May 2002; accepted in final form 31 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aksoy, MO, and Kelsen SG. Relaxation of rabbit tracheal smooth muscle by adenine nucleotides: mediation by P2 purinoceptors. Am J Respir Cell Mol Biol 10: 230-236, 1994[Abstract].

2.   Barnes, PJ. Histamine and serotonin. Pulm Pharmacol Ther 14: 329-339, 2001[ISI][Medline].

3.   Bergner, A, and Sanderson MJ. Acetylcholine-induced calcium signaling and contraction of airway smooth muscle cells in lung slices. J Gen Physiol 119: 187-198, 2002[Abstract/Free Full Text].

4.   Bootman, MD, Collins TJ, Peppiatt CM, Prothero LS, MacKenzie L, De Smet P, Travers M, Tovey SC, Seo JT, Berridge MJ, Ciccolini F, and Lipp P. Calcium signalling---an overview. Semin Cell Dev Biol 12: 3-10, 2001[ISI][Medline].

5.   Braunstein, GM, Roman RM, Clancy JP, Kudlow BA, Taylor AL, Shylonsky VG, Jovov B, Peter K, Jilling T, Ismailov I, Benos DJ, Schwiebert LM, Fitz JG, and Schwiebert EM. Cystic fibrosis transmembrane conductance regulator facilitates ATP release by stimulating a separate ATP release channel for autocrine control of cell volume regulation. J Biol Chem 276: 6621-6630, 2001[Abstract/Free Full Text].

6.   Cazzola, I, and Matera MG. 5-HT modifiers as a potential treatment of asthma. Trends Pharmacol Sci 21: 13-16, 2000[ISI][Medline].

7.   Donaldson, SH, Lazarowski ER, Picher M, Knowles MR, Stutts MJ, and Boucher RC. Basal nucleotide levels, release, and metabolism in normal and cystic fibrosis airways. Mol Med 6: 969-982, 2000[ISI][Medline].

8.   Fedan, JS, Belt JJ, Yuan LX, and Frazer DG. Contractile effects of nucleotides in guinea pig isolated, perfused trachea: involvement of respiratory epithelium, prostanoids and Na+ and Cl- channels. J Pharmacol Exp Ther 264: 210-216, 1993[Abstract].

9.   Flezar, M, Olivenstein R, Cantin A, and Heisler S. Extracellular ATP stimulates elastase secretion from human neutrophils and increases lung resistance and secretion from normal rat airways after intratracheal instillation. Can J Physiol Pharmacol 70: 1065-1068, 1992[ISI][Medline].

10.   Fortner, CN, Breyer RM, and Paul RJ. EP2 receptors mediate airway relaxation to substance P, ATP, and PGE2. Am J Physiol Lung Cell Mol Physiol 281: L469-L474, 2001[Abstract/Free Full Text].

11.   Foskett, JK. ClC and CFTR chloride channel gating. Annu Rev Physiol 60: 689-717, 1998[ISI][Medline].

12.   Gaillard, D, Jouet JB, Egreteau L, Plotkowski L, Zahm JM, Benali R, Pierrot D, and Puchelle E. Airway epithelial damage and inflammation in children with recurrent bronchitis. Am J Respir Crit Care Med 150: 810-817, 1994[Abstract].

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

14.   Guibert, C, Pacaud P, Loirand G, Marthan R, and Savineau JP. Effect of extracellular ATP on cytosolic Ca2+ concentration in rat pulmonary artery myocytes. Am J Physiol Lung Cell Mol Physiol 271: L450-L458, 1996[Abstract/Free Full Text].

15.   Gunst, SJ, and Tang DD. The contractile apparatus and mechanical properties of airway smooth muscle. Eur Respir J 15: 600-616, 2000[Abstract/Free Full Text].

16.   Hall, IP. Second messengers, ion channels and pharmacology of airway smooth muscle. Eur Respir J 15: 1120-1127, 2000[Abstract/Free Full Text].

17.   Kao, J, Fortner CN, Liu LH, Shull GE, and Paul RJ. Ablation of the SERCA3 gene alters epithelium-dependent relaxation in mouse tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol 277: L264-L270, 1999[Abstract/Free Full Text].

18.   Laitinen, LA, Heino M, Laitinen A, Kava T, and Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 131: 599-606, 1985[ISI][Medline].

19.   Lambrecht, G. Agonists and antagonists acting at P2X receptors: selectivity profiles and functional implications. Naunyn Schmiedebergs Arch Pharmacol 362: 340-350, 2000[ISI][Medline].

20.   Liu, X, and Farley JM. Acetylcholine-induced chloride current oscillations in swine tracheal smooth muscle cells. J Pharmacol Exp Ther 276: 178-186, 1996[Abstract].

21.   Mahoney, MG, Randall CJ, Linderman JJ, Gross DJ, and Slakey LL. Independent pathways regulate the cytosolic [Ca2+]i initial transient and subsequent oscillations in individual cultured arterial smooth muscle cells responding to extracellular ATP. Mol Biol Cell 3: 493-505, 1992[Abstract].

22.   Michoud, MC, Tolloczko B, and Martin JG. Effects of purine nucleotides and nucleosides on cytosolic calcium levels in rat tracheal smooth muscle cells. Am J Respir Cell Mol Biol 16: 199-205, 1997[Abstract].

23.   Osipchuk, Y, and Cahalan M. Cell-to-cell spread of calcium signals mediated by ATP receptors in mast cells. Nature 359: 241-244, 1992[ISI][Medline].

24.   Pabelick, CM, Sieck GC, and Prakash YS. Significance of spatial and temporal heterogeneity of calcium transients in smooth muscle. J Appl Physiol 91: 488-496, 2001[Abstract/Free Full Text].

25.   Page, S, Ammit AJ, Black JL, and Armour CL. Human mast cell and airway smooth muscle cell interactions: implications for asthma. Am J Physiol Lung Cell Mol Physiol 281: L1313-L1323, 2001[Abstract/Free Full Text].

26.   Pauvert, O, Marthan R, and Savineau J. NO-induced modulation of calcium oscillations in pulmonary vascular smooth muscle. Cell Calcium 27: 329-338, 2000[ISI][Medline].

27.   Prakash, YS, Kannan MS, and Sieck GC. Regulation of intracellular calcium oscillations in porcine tracheal smooth muscle cells. Am J Physiol Cell Physiol 272: C966-C975, 1997[Abstract/Free Full Text].

28.   Prakash, YS, Pabelick CM, Kannan MS, and Sieck GC. Spatial and temporal aspects of ACh-induced [Ca2+]i oscillations in porcine tracheal smooth muscle. Cell Calcium 27: 153-162, 2000[ISI][Medline].

29.   Ralevic, V, and Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413-492, 1998[Abstract/Free Full Text].

30.   Roux, E, Guibert C, Savineau JP, and Marthan R. [Ca2+]i oscillations induced by muscarinic stimulation in airway smooth muscle cells: receptor subtypes and correlation with the mechanical activity. Br J Pharmacol 120: 1294-1301, 1997[Abstract].

31.  Sanderson MJ and Parker I. Video-rate confocal microscopy. In: Methods in Enzymology, edited by Marriott G and Parker I. In press.

32.   Sieck, GC, Kannan MS, and Prakash YS. Heterogeneity in dynamic regulation of intracellular calcium in airway smooth muscle cells. Can J Physiol Pharmacol 75: 878-888, 1997[ISI][Medline].

33.   Sims, SM, Jiao Y, and Zheng ZG. Intracellular calcium stores in isolated tracheal smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 271: L300-L309, 1996[Abstract/Free Full Text].

34.   Suarez-Huerta, N, Pouillon V, Boeynaems J, and Robaye B. Molecular cloning and characterization of the mouse P2Y4 nucleotide receptor. Eur J Pharmacol 416: 197-202, 2001[ISI][Medline].

35.   Von Kugelgen, I, and Wetter A. Molecular pharmacology of P2Y receptors. Naunyn Schmiedebergs Arch Pharmacol 362: 310-323, 2000[ISI][Medline].

36.   Walsh, DE, Harvey BJ, and Urbach V. CFTR regulation of intracellular calcium in normal and cystic fibrosis human airway epithelia. J Membr Biol 177: 209-219, 2000[ISI][Medline].

37.   Watt, WC, Lazarowski ER, and Boucher RC. Cystic fibrosis transmembrane regulator-independent release of ATP: its implications for the regulation of P2Y2 receptors in airway epithelia. J Biol Chem 273: 14053-14058, 1998[Abstract/Free Full Text].

38.   Zimmermann, H. Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedebergs Arch Pharmacol 362: 299-309, 2000[ISI][Medline].


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