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Am J Physiol Lung Cell Mol Physiol 277: L30-L41, 1999;
1040-0605/99 $5.00
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Vol. 277, Issue 1, L30-L41, July 1999

Intracellular calcium oscillations induced by ATP in airway epithelial cells

John H. Evans 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 epithelial cells, extracellular ATP (ATPo) stimulates an initial transient increase in intracellular Ca2+ concentration that is followed by periodic increases in intracellular Ca2+ concentration (Ca2+ oscillations). The characteristics and mechanism of these ATP-induced Ca2+ responses were studied in primary cultures of rabbit tracheal cells with digital video fluorescence microscopy and the Ca2+-indicator dye fura 2. The continual presence of ATPo at concentrations of 0.1-100 µM stimulated Ca2+ oscillations that persisted for 20 min. The frequency of the Ca2+ oscillations was found to be dependent on both ATPo concentration and intrinsic sensitivity of each cell to ATPo. Cells exhibited similar Ca2+ oscillations to extracellular UTP (UTPo), but the oscillations typically occurred at lower UTPo concentrations. The ATP-induced Ca2+ oscillations were abolished by the phospholipase C inhibitor U-73122 and by the endoplasmic reticulum Ca2+-pump inhibitor thapsigargin but were maintained in Ca2+-free medium. These results are consistent with the hypothesis that in airway epithelial cells ATPo and UTPo act via P2U purinoceptors to stimulate Ca2+ oscillations by the continuous production of inositol 1,4,5-trisphosphate and the oscillatory release of Ca2+ from internal stores. ATP-induced Ca2+ oscillations of adjacent individual cells occurred independently of each other. By contrast, a mechanically induced intercellular Ca2+ wave propagated through a field of Ca2+-oscillating cells. Thus Ca2+ oscillations and propagating Ca2+ waves are two fundamental modes of Ca2+ signaling that exist and operate simultaneously in airway epithelial cells.

inositol 1,4,5-trisphosphate; calcium waves; uridine 5'-triphosphate; adenosine 5'-triphosphate; cell signaling


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OSCILLATORY CHANGES in intracellular Ca2+ concentration ([Ca2+]i), or Ca2+ oscillations, occur in a variety of nonexcitable cell types (47). Commonly, Ca2+ oscillations arise after the activation of phospholipase C (PLC) by agonists binding to cell surface receptors (1). This leads to the production of inositol 1,4,5-trisphosphate (IP3) followed by Ca2+ mobilization from internal stores via the IP3 receptor (IP3R), itself a Ca2+ channel, and to an increase in [Ca2+]i. Ca2+ feedback inhibition of the IP3R results in cessation of Ca2+ release (3), and this together with the sequestration and extrusion of Ca2+ from the cytoplasm by pumps into the endoplasmic reticulum or across the plasma membranes, respectively, leads to a decline in [Ca2+]i. Once the [Ca2+]i falls to a permissive level, repetitive cycles of [Ca2+]i increase and decline are maintained by an elevated intracellular IP3 concentration ([IP3]i) (13, 15). Thus Ca2+ oscillations are dependent on the action of both [Ca2+]i and [IP3]i on the IP3R. Experimental data show that the frequency of Ca2+ oscillations are dependent on [IP3]i and, indirectly, agonist concentration (15). Mathematical modeling of Ca2+ oscillations as a function of [IP3]i and [Ca2+]i agrees with the experimental findings (43).

Extracellular ATP (ATPo) serves as an agonist in many organs and tissues (12) and induces an initial transient increase in [Ca2+]i followed by Ca2+ oscillations in a number of cell types including astrocytes (49), smooth muscle cells (24), granulosa luteal cells (44), Madin-Darby canine kidney (MDCK) cells (35), chondrocytes (8), bile duct epithelial cells (29), and megakaryocytes (46). In airway cells, ATPo mobilizes [Ca2+]i via PLC by activating P2U receptors (12). P2U receptors are expressed at the apical pole of rat airway epithelial cells and respond to extracellular UTP (UTPo) as well as to ATPo (19, 26). ATPo and UTPo may serve as important physiological factors in the airway lumen because airway cells are reported to release ATP and UTP in response to stretch (14) and via the cystic fibrosis transmembrane conductance regulator (9). Released ATPo or UTPo may act in an autocrine or paracrine fashion to mediate Cl- secretion (42) and, possibly, ciliary beat frequency (16, 50).

The spatial organization of [Ca2+]i responses to an agonist are also important in coordinating Ca2+-mediated activity at the level of tissues or organs. For example, synchronous Ca2+ oscillations have been reported in tissues such as pancreatic acini (45), islets of Langerhans (2), lung capillary endothelium (51), and liver lobules (30, 33) and in cultures of hepatocytes (48), MDCK cells (35), and chondrocytes (8). The synchrony of Ca2+ oscillations in the above systems invariably relies on gap junction communication between contacting cells, and the synchrony is lost when communication is disrupted. In perfused pancreatic acini (45) and other confluent cell cultures, such as arterial smooth muscle cells (24) and MDCK cells (35), however, no synchrony of Ca2+ oscillations between contacting cells is observed. Thus between cell types and experimental conditions, the spatial coordination of Ca2+ oscillations differs. These differences may then be exploited to investigate different mechanisms of intercellular communication. Because both Ca2+ and IP3 can act as intercellular messengers (36), it remains unclear whether Ca2+ or IP3 is the messenger communicated though the gap junctions to initiate intercellular Ca2+ waves or whether the intercellular messenger employed is cell-type or condition specific. In addition to gap junction communication, cell messengers can be communicated extracellularly (20, 41). In primary cultures of airway epithelial cells, the intercellular diffusion of IP3 is consistent with the observed propagation of intercellular Ca2+ waves under a number of experimental conditions (5).

Although Ca2+-sensitive cell functions are often mediated by oscillatory rather than prolonged sustained increases in [Ca2+]i (32), only a few brief reports have shown Ca2+ oscillations in ciliated airway epithelial cells (21, 31, 37), goblet cells (31), or airway gland cells (25). Using digital microscopy techniques and the Ca2+ indicator fura 2, we investigated the temporal aspects of the [Ca2+]i response to ATPo and UTPo in airway epithelial cells and describe here the mechanism by which Ca2+ oscillations are initiated and maintained. We found that ATP-induced Ca2+ oscillations in airway epithelial cells were dependent on the ATPo concentration ([ATP]o), were mediated by G protein-coupled receptors involving an IP3 signaling pathway, and were not communicated to adjacent cells. Thus airway epithelial cells exhibit two fundamental modes of Ca2+ signaling, intracellular Ca2+ oscillations and intercellular Ca2+ waves, which may occur simultaneously within a cell.


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

The techniques for culturing airway epithelial cells, mechanically stimulating individual cells, and the measurement of [Ca2+]i by fluorescent video microscopy have been described in detail elsewhere (7, 23, 38-40) and will be only briefly reviewed here.

Cell culture. Primary cultures of airway epithelial cells were prepared from the tracheae of New Zealand White rabbits as previously described (11) except that the collagen on the coverslips was fixed with formaldehyde instead of glutaraldehyde. After dissection of the epithelial mucosa from the trachea, the tissue was cut into ~0.5-mm squares, placed onto collagen-coated coverslips, and cultured in DMEM supplemented with 10% fetal bovine serum, 10 mM HEPES, and antibiotics-antimycotics at 37°C in 10% CO2 for 5-9 days.

Measurement of [Ca2+]i. Cells were loaded with fura 2 by incubation at 37°C for 1 h in 5 µM fura 2-AM (Calbiochem) in Ca2+ (1.3 mM)-containing Hanks' buffered salt solution without phenol red (HBSS; GIBCO BRL) and additionally buffered with 25 mM HEPES (HHBSS, pH 7.4). The cells were washed and allowed to incubate at room temperature for 30 min to allow for complete deesterification of the fura 2-AM. In Ca2+-free experiments, Ca2+/Mg2+-free Dulbecco's phosphate-buffered saline (DPBS; GIBCO BRL) was used in place of HHBSS. Cells were visualized with a Nikon inverted microscope equipped with fluorescence optics and a ×40 objective lens. Fluorescence was detected with a silicon-intensified target camera, recorded with an optical memory disk recorder, and digitized by computer (7, 38). Images of [Ca2+]i were calculated by single-wavelength recordings referenced to ratiometric measurements (23). Initial [Ca2+]i reference images were based on 10 frames recorded at 340 and 380 nm. Changes in [Ca2+]i were recorded by monitoring changes in fluorescence with an illumination wavelength of 380 nm. Additional reference images were taken at 340 nm every 30 s. [Ca2+]i was calculated from the change in fluorescence intensity at 380 nm (7, 23). All images were subjected to background subtraction and correction for shading and bleaching. For plots of [Ca2+]i versus time, single points encompassing an area of 2.1 × 2.3 µm were selected from the cells of interest, and [Ca2+]i was calculated only at those points. Time-lapse recordings were made at 2 images/s (each frame recorded at 30 frames/s).

Drug application. ATP and UTP (Sigma) were dissolved in distilled H2O at 10 mM and stored in aliquots at -20°C. Desired final concentrations were made by dilution of the stock in HHBSS or Ca2+/Mg2+-free DPBS. Thapsigargin, U-73122, and U-73343 (BIOMOL) were dissolved in dimethyl sulfoxide (DMSO; 1, 5, and 7.5 mM, respectively), divided into aliquots, and stored at -20°C. Final concentrations were made by dilution in HHBSS. Controls for thapsigargin experiments were performed in 0.1% DMSO. Two hundred microliters of the required experimental solution were exchanged for the 200 µl of HHBSS in the cell chamber for each experiment. Between trials, the cells were washed with >3 ml (>15 volumes) of HHBSS. In all experiments, the cells were allowed to recover for >15 min between trials or between control and experimental conditions.

Mechanical stimulation. Mechanical stimulation of a single cell was performed by brief displacement of the apical surface of the cell membrane with a glass micropipette (~1-µm tip diameter) for 100 ms. The magnitude and duration of the membrane displacement was controlled by applying a voltage pulse with a Grass stimulator to a piezoelectric crystal to which the micropipette was attached (40).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Response to ATPo. The Ca2+ response of airway epithelial cells to ATPo was quantified with digital video microscopy in confluent primary cell cultures (5-9 days old) loaded with the Ca2+ indicator fura 2. The application of ATPo (0.1-100 µM) induced an initial Ca2+ transient in >90% of cells in the field of view. However, the magnitude of this [Ca2+]i increase varied greatly from cell to cell, but in general, it increased with increasing [ATP]o. After the initial [Ca2+]i increase induced by ATPo, 7-36% of all the cells, depending on the [ATP]o, displayed periodic Ca2+ oscillations (Fig. 1). These Ca2+ oscillations ceased immediately on ATPo washout (data not shown). The greatest number of cells displayed Ca2+ oscillations in response to 5 µM ATP (n = 386-503 cells, 3-4 experiments; Fig. 2A). These Ca2+ oscillations were initiated 15 s to several minutes after the initial [Ca2+]i transient and consisted of a sharp increase in [Ca2+]i followed by a slower [Ca2+]i decline; however, the [Ca2+]i increases of the Ca2+ oscillations were smaller than that of the initial Ca2+ transient. In many experiments, the initial two to three Ca2+ oscillations were characterized by a higher frequency and were initiated from a higher baseline [Ca2+]i than subsequent oscillations.


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  		Fig. 1.   Response of individual airway epithelial cells to extracellular ATP (ATPo). Representative traces of intracellular Ca2+ concentration ([Ca2+]i) changes with respect to time of 3 different cells exposed to increasing ATPo concentration ([ATP]o; 0.1-100 µM) are shown. Sensitivities of cells were determined by minimum [ATP]o value required to elicit multiple Ca2+ oscillations. A: high ATP-sensitive cells displayed multiple Ca2+ oscillations at ~0.5 µM ATP. B: intermediate ATP-sensitive cells displayed Ca2+ oscillations at ~5 µM ATP. C: low ATP-sensitive cells displayed Ca2+ oscillations at ~50 µM ATP. Characteristics of Ca2+ oscillation responses to ATPo were similar between each ATP sensitivity category; frequency of Ca2+ oscillations increased with increased [ATP]o until, at high relative [ATP]o values, Ca2+ oscillations ceased and [Ca2+]i remained elevated. Traces are representative of data sets of 265 cells analyzed from 5 independent experiments.



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  		Fig. 2.   Summary of Ca2+ response of airway epithelial cells to ATPo. A: airway epithelial cells displayed greatest Ca2+ oscillatory behavior at 5 µM ATP. [ATP], ATP concentration. B: in a single culture, average number of Ca2+ oscillations/cell (defined as average number of [Ca2+]i increases/cell during a 5-min recording period after initial [Ca2+]i increase) increased with increasing [ATP]o values. However, [ATP]o value generating the greatest number of Ca2+ oscillations/cell was dependent on ATPo sensitivity of cell. C: no. of cells in each ATP sensitivity category in a single cell culture (same culture as in B). Intermed, intermediate. Data in A are from 388-503 cells from 3-5 experiments analyzed at each [ATP]o value. Data in B and C are from a single experiment with 63 cells analyzed.

Frequently, each Ca2+ oscillation occurred as an intracellular Ca2+ wave that spread across the individual cell. The region from which the Ca2+ wave initiated and the direction of the wave propagation was constant for each intracellular Ca2+ oscillation and was unique to each individual cell. The relationship of the Ca2+ wave initiation site to a specific cell organelle or structure was not determined but may result from the heterogeneity in the distribution and sensitivity of the Ca2+ pools or IP3Rs. This relationship will be the subject of a future study. Simultaneous phase-contrast imaging also demonstrated that the ciliary beat frequency of the cells increased and decreased in concert with the [Ca2+]i changes of each Ca2+ oscillation, and studies correlating the [Ca2+]i and ciliary beat frequency changes also need to be completed.

Dose-response experiments revealed that individual cells within a population exhibited differing sensitivities to ATPo and that this determined the characteristics of the Ca2+-oscillatory behavior of each cell. Cells could be categorized as having a high, intermediate, or low sensitivity to ATPo. Within each category, four basic responses to ATPo were well identified (Fig. 1). These consisted of 1) a single Ca2+ transient after exposure to ATPo; 2) the initiation of a few irregular Ca2+ oscillations; 3) the initiation of well-defined, multiple Ca2+ oscillations; and 4) a prolonged elevation of the baseline [Ca2+]i after the initial spike. For each cell, this range of responses required a 5- to 10-fold increase in ATPo. For example, a cell with high sensitivity to ATPo (Fig. 1A) responded to 0.5 or 1 µM ATP with regular, periodic Ca2+ oscillations. However, the same cell responded to 5 µM ATP with an initial [Ca2+]i transient followed by a prolonged elevated [Ca2+]i without any subsequent Ca2+ oscillations. By contrast, a cell with an intermediate sensitivity to ATPo (Fig. 1B) responded to 5 µM ATP with well-defined Ca2+ oscillations and required 50 µM ATP to induce an elevated baseline of [Ca2+]i without Ca2+ oscillations. On the other hand, a cell with low sensitivity to ATPo required 50 µM ATP to induce regular Ca2+ oscillations (Fig. 1C).

These differing Ca2+-oscillatory responses to ATPo are summarized in Fig. 2. The greatest number of cells (46% of the culture) was classified as having an intermediate sensitivity to ATPo, and these cells displayed the greatest number of Ca2+ oscillations in response to 10 µM ATP. Cells classified as having either a high (32% of the culture) or low sensitivity (22% of the culture) to ATPo displayed the greatest number of Ca2+ oscillations in response to 1 or 100 µM ATP, respectively. Therefore, in a single culture, individual cells displayed similar [Ca2+]i responses to ATPo stimulation but over a 100-fold concentration range.

Response to UTPo. In an earlier study by Hansen et al. (16), the [Ca2+]i response of tracheal epithelial cells to ATPo was blocked by the antagonist suramin, suggesting P2 purinergic-receptor involvement. Because P2U receptors exhibit a greater or equal selectivity for UTPo over ATPo (12), we determined the [Ca2+]i response of cultured epithelial cells to UTPo. The [Ca2+]i response of individual cells to 0.1 µM UTPo or ATPo is shown in Fig. 3. In one cell (Fig. 3A), UTPo elicited an initial [Ca2+]i transient, whereas ATPo had no effect on the [Ca2+]i. In another cell (Fig. 3B), the initial [Ca2+]i transient in response to UTPo was followed by a single Ca2+ oscillation, whereas ATPo elicited only an initial [Ca2+]i increase. However, the initial [Ca2+]i transients were virtually identical for both agonists. In a third cell (Fig. 3C), UTPo elicited a higher frequency of Ca2+ oscillations compared with that elicited by ATPo.


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  		Fig. 3.   Differential response of individual cells to extracellular UTP (UTPo) and ATPo. Cultured cells were sequentially exposed to 0.1 µM ATP and 0.1 µM UTP or vice versa. Traces of [Ca2+]i change over time are representative of individual cells in which UTPo but not ATPo elicited an initial [Ca2+]i increase (A) or initiated a Ca2+ oscillation subsequent to the initial [Ca2+]i increase (B). C: trace is representative of cells in which UTPo initiated a higher frequency of Ca2+ oscillations compared with that initiated by ATPo. Traces are representative of 229 cells analyzed from 4 experiments.

Of cells displaying an [Ca2+]i increase in response to 0.1 µM UTP, 91% of these cells exhibited an [Ca2+]i increase in response to 0.1 µM ATP (n = 229 cells, 4 experiments; Fig. 4A). Less than 2% of cells exhibited an [Ca2+]i increase in response to ATPo but not to UTPo. Also, 0.1 µM UTP initiated Ca2+ oscillations in 59% of cells compared with 40% with 0.1 µM ATP (Fig. 4A), and the cells stimulated with UTPo showed a greater number of oscillations than those stimulated with ATPo (Fig. 4B). These responses to ATPo or UTPo were independent of the order of agonist challenge.


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  		Fig. 4.   Summary of differential responses to ATPo and UTPo. A: of the cells which exhibited at least 1 [Ca2+]i increase in response to 0.1 µM UTP, 91% also responded to 0.1 µM ATP. Only 4 of 229 cells from 4 experiments that responded to ATPo failed to respond to UTPo. Of the responding cells, 59% of cells exhibited oscillations in UTPo, whereas only 40% exhibited oscillations in ATPo. B: average no. of oscillations/cell induced by UTPo was higher than that induced by ATPo in the 5-min recording period.

Ca2+ oscillations and PLC activity. The stimulation of [Ca2+]i increase by ATPo and UTPo via P2U receptors is believed to involve the release of Ca2+ from IP3-sensitive Ca2+ stores after the activation of PLC and the production of IP3 (12). In support of this hypothesis, Hansen et al. (16) found that neither ryanodine nor caffeine increases [Ca2+]i in airway epithelial cells and suggested that the internal Ca2+ release does not appear to involve a ryanodine receptor-mediated Ca2+-induced Ca2+ release. The involvement of PLC in the response of epithelial cells to ATPo is supported by the finding that an increase in [Ca2+]i was abolished by the aminosteroid U-73122, a PLC inhibitor, whereas its inactive analog U-73343 had no effect (17). To determine whether PLC activation was part of the mechanism of ATP-induced Ca2+ oscillations, we investigated the effect of U-73122 on ATP-induced Ca2+ oscillations.

Incubation of cells for 20 min in 10 µM U-73122 before application of 5 µM ATP completely abolished the initial [Ca2+]i transient as well as the subsequent Ca2+ oscillations in the cells (data not shown). A 20-min incubation in 10 µM U-73343 had no effect on the initial [Ca2+]i response or subsequent Ca2+ oscillations (data not shown). Because it is possible that Ca2+ oscillations require an initial PLC-dependent [Ca2+]i increase but do not require continual activity of PLC, we added 10 µM U-73122 to cells exhibiting ongoing ATP-induced Ca2+ oscillations. ATP-induced Ca2+ oscillations quickly ceased on addition of U-73122 (Fig. 5A). The addition of U-73343 had no effect on ongoing Ca2+ oscillations (n = 83 cells, 3 experiments; Fig. 5B).


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  		Fig. 5.   Effect of phospholipase C inhibitor U-73122 and an inactive analog, U-73343, on [Ca2+]i changes induced by ATPo. A: U-73122 was added to cells 2 min after exposure to 5 µM ATP. Trace shows that ATPo induced a characteristic Ca2+ oscillation pattern and that Ca2+ oscillations ceased after addition of U-73122. B: similar experiment shows that U-73343 had no effect on Ca2+ oscillations (n = 83 cells from 3 experiments).

Ca2+ oscillations and the release of internal Ca2+ stores. IP3-mediated Ca2+ oscillations typically are dependent on Ca2+ release from internal stores and are independent of the extracellular Ca2+ concentration ([Ca2+]o). To investigate the dependence of ATP-induced Ca2+ oscillations on [Ca2+]o, cells were stimulated with 5 µM ATP in Ca2+/Mg2+-free DPBS. Because extracellular unbound EGTA has been shown to interfere with Ca2+ release from intracellular stores in airway epithelial cells (18) and because the Ca2+ response to histamine in airway cells was essentially the same in nominally Ca2+-free medium, medium containing 1 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, and medium with an [Ca2+]o of ~7 nM (18), we decided to use a nominally Ca2+-free medium for these experiments. Ca2+ oscillations induced by ATPo occurred in the absence of extracellular Ca2+ (n = 33 cells, 3 experiments; Fig. 6A). Treatment with Ca2+/Mg2+-free DPBS alone failed to induce Ca2+ oscillations (Fig. 6B).


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  		Fig. 6.   Effect of low extracellular Ca2+ concentration on [Ca2+]i changes induced by ATPo. A: cells were washed once in Ca2+/Mg2+-free Dulbecco's PBS (DPBS) and then stimulated with 5 µM ATP in Ca2+/Mg2+-free DPBS. Trace shows that ATPo induced a characteristic Ca2+ oscillation pattern in absence of extracellular Ca2+. B: similar experiment shows that in the same cell Ca2+/Mg2+-free DPBS alone failed to induce Ca2+ oscillations (n = 33 cells from 3 experiments).

To investigate further the dependence of Ca2+ oscillations on internal Ca2+ stores, we used the Ca2+-ATPase inhibitor thapsigargin. Prolonged incubation (20 min) of cells with 1 µM thapsigargin elevated the baseline [Ca2+]i in the cells and abolished the Ca2+ response of the cells to 5 µM ATP (data not shown). Again, Ca2+ oscillations may be dependent on an initial Ca2+ increase but independent of internal Ca2+ pools. To investigate this possibility, cells were exposed for a short time (1 min) to thapsigargin before stimulation with ATPo. This short-term exposure to thapsigargin stimulated a small rise in [Ca2+]i but allowed a large ATP-induced [Ca2+]i increase (n = 91 cells, 4 experiments; Fig. 7A). Baseline [Ca2+]i levels remained elevated in these cells, and the cells failed to exhibit Ca2+ oscillations. An identical treatment of cells with DMSO, the vehicle for thapsigargin, resulted in a normal Ca2+ response to ATPo treatment (Fig. 7B). To determine whether internal release of Ca2+ is the sole mechanism driving ATP-induced Ca2+ oscillations, we treated cells displaying ATP-induced Ca2+ oscillations with thapsigargin. After the addition of thapsigargin to Ca2+-oscillating cells, the amplitude of the Ca2+ oscillations decreased and the baseline [Ca2+]i increased with each successive oscillation until oscillations ceased (n = 154 cells, 5 experiments; Fig. 7, C and D).


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  		Fig. 7.   Effect of thapsigargin on [Ca2+]i changes induced by ATPo. A: cells were exposed to 5 µM ATP after a 60-s incubation with thapsigargin. Trace shows that after a transient increase, [Ca2+]i remained elevated. C: cells exposed to 5 µM ATP were treated with thapsigargin at 60 s. Trace shows that thapsigargin induced a diminution of Ca2+ oscillation amplitude, with a concomitant elevation in baseline [Ca2+]i followed by a cessation of oscillations at an elevated baseline [Ca2+]i. In similar experiments where 0.1% DMSO was added 60 s before (B) or after (D) exposure to 5 µM ATP, characteristic ATP-induced Ca2+ oscillations were observed. Traces in A and B are representative of 108 cells analyzed from 3 independent experiments and in C and D are representative of 154 cells analyzed from 5 independent experiments.

Spatial characteristics of Ca2+ oscillations. To ascertain the ability of cells exhibiting Ca2+ oscillations to influence the Ca2+ activity of neighboring cells, we analyzed the temporal and spatial [Ca2+]i responses of cells to ATPo in confluent cultures. Pseudocolor images of the [Ca2+]i change in airway epithelial cells exposed to 5 µM ATP (Fig. 8) showed that after an initial [Ca2+]i increase stimulated by the addition of ATPo (Fig. 8A), many cells displayed repetitive increases in [Ca2+]i of differing frequency and amplitude (Fig. 8, B-I). The asynchronous nature of the Ca2+ oscillations is exemplified by five adjacent cells (Fig. 8, star ) that exhibited Ca2+ oscillations independently from one another.


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  		Fig. 8.   Pseudocolor images of [Ca2+]i change in cells exposed to ATPo. Airway epithelial cells in a confluent culture were treated with 5 µM ATP and responded with an initial increase in [Ca2+]i (A). After recovery from initial [Ca2+]i increase (B and C), several cells initiated Ca2+ oscillations with frequencies and amplitudes that were intrinsic to each cell (D-I). Individual nature of Ca2+ oscillations is highlighted by asynchrony of Ca2+ oscillations in 5 adjacent cells (star ). White lines, approximate cell outlines. Times are relative to ATPo addition.

Graphic representations of the [Ca2+]i changes in four adjacent cells (Fig. 9) show that all cells responded to the addition of 5 µM ATP with a nearly simultaneous [Ca2+]i increase. However, the patterns of Ca2+ oscillations differed greatly between cells. For example, the first Ca2+ oscillation in cell B (central cell) occurred at ~40 s (Fig. 9, line 1) and failed to propagate to cell C. This Ca2+ oscillation preceded the first Ca2+ oscillation of cell A by ~50 s and followed the first Ca2+ oscillation of cell D by ~8 s. Similarly, the second Ca2+ oscillation in cell B (Fig. 9, line 2) preceded the first Ca2+ oscillation in cell A by ~6 s and followed the second Ca2+ oscillation in cell D by ~20 s. By 150 s, cells A-D have experienced 1, 3, 0, and 4 Ca2+ oscillations, respectively.


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  		Fig. 9.   Temporal and spatial organization of ATP-induced Ca2+ oscillations. Outlines (top) and Ca2+ traces (bottom) of 4 cells treated with 5 µM ATP are shown. In response to ATPo (line A), cell B exhibited characteristic Ca2+ oscillations and was bordered by cells that exhibited Ca2+ oscillations of lower (cell A) and higher (cell D) frequencies and by a cell that exhibited no oscillations (cell C). Ca2+ oscillations in cell B (lines 1-3) were temporally distinct from Ca2+ oscillations in neighboring cells.

Intercellular Ca2+ signaling. In a previous study (6), it has been demonstrated that in response to mechanical stimulation, airway epithelial cells propagated increases in [Ca2+]i similar to those associated with Ca2+ oscillations to adjacent cells as intercellular Ca2+ waves. The mechanism responsible for the intercellular waves was proposed to be the diffusive spread of IP3 rather than of Ca2+ between adjacent cells via gap junctions. Consequently, the asynchronous nature and failure of ATP-induced Ca2+ oscillations to influence the Ca2+ activity of adjacent cells may result either from an inherent but independent regulation of cell function or, alternatively, from a lack of gap junction communication between cells. To explore this hypothesis and to estimate the extent of cell-cell communication, the ability of cells displaying ATP-induced Ca2+ oscillations to propagate intercellular Ca2+ waves was tested.

Figure 10 shows pseudocolor images of the change in [Ca2+]i in confluent cells that have been exposed to 5 µM ATP and have been subsequently mechanically stimulated. In response to ATPo, many cells exhibit Ca2+ oscillations (Fig. 10, A-C, star ). Because of the variability in sensitivity of individual cells to ATPo, the single dose of ATPo evoked differing Ca2+ responses in adjacent cells. After mechanical stimulation of a single cell (Fig. 10D, arrow), an intercellular Ca2+ wave spread through Ca2+-oscillating and nonoscillating cells (Fig. 10, E and F). After passage of the intercellular Ca2+ wave (Fig. 10, G-I), many cells (star ) resumed Ca2+ oscillations.


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  		Fig. 10.   Pseudocolor image of [Ca2+]i change in cells exposed to ATPo and after mechanical stimulation of a single cell (arrows). Cells in a confluent culture exposed to 5 µM ATP exhibited Ca2+ oscillations (A-C, star ). Mechanical stimulation of a single cell resulted in passage of an intercellular Ca2+ wave throughout field of Ca2+-oscillating and nonoscillating cells (D-F). After passage of Ca2+ wave, cells continued to exhibit Ca2+ oscillations (G-I). White lines, approximate cell outlines. Times are relative to mechanical stimulation.

The traces in Fig. 11 show typical [Ca2+]i responses of several adjacent cells after application of ATPo; the variability of the Ca2+ response is clearly demonstrated in the traces. Cell B shows a response typical for a cell with a high sensitivity to ATPo, whereas cells A, C, and D show responses typical for cells with an intermediate sensitivity. Although the Ca2+ response of cells A, C, and D are fundamentally similar, these adjacent cells clearly display Ca2+ oscillations of different frequencies and magnitudes, and this emphasizes the asynchronous nature of adjacent cell Ca2+ oscillations. At 120 s, mechanical stimulation (Fig. 11, line S) of a single cell (cell A) initiated the propagation of an intercellular Ca2+ wave that spread throughout the cell culture, passing through both Ca2+-oscillating and nonoscillating cells. The mechanical stimulation of cell A resulted in an almost immediate and sustained [Ca2+]i increase in that cell. With a very short time delay (~1 s), the [Ca2+]i in cell B rapidly increased and subsequently decreased to a higher baseline [Ca2+]i. In cell C, the [Ca2+]i increased after a ~3-s delay to a higher amplitude than the preceding oscillatory [Ca2+]i increases. Interestingly, the next two Ca2+ oscillations initiated after a shorter period and at a higher baseline [Ca2+]i than did the Ca2+ oscillations preceding the intracellular wave. In cell D, after a ~5-s delay, the [Ca2+]i increase in the intercellular Ca2+ wave was higher than the initial [Ca2+]i increase; however, it failed to reinitiate Ca2+ oscillations.


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  		Fig. 11.   Initiation of an intercellular Ca2+ wave through cells experiencing ATP-induced Ca2+ oscillations. Outlines (top) and Ca2+ traces (bottom) of 4 cells treated with 5 µM ATP are shown. ATPo was applied to cells (line A) and elicited characteristic Ca2+ oscillations in the 4 cells. After 120 s, cell A was mechanically stimulated with a micropipette (line S), which resulted in an immediate increase in [Ca2+]i of cell A and generation of an intercellular Ca2+ wave. Sequential increases in [Ca2+]i associated with intercellular Ca2+ wave were observed in cells B-D, with an increasing time delay. [Ca2+]i increase in cell B was transient and resulted in a slightly elevated baseline [Ca2+]i. In cell C, [Ca2+]i increase was followed first by a single Ca2+ oscillation that had a shorter period and initiated at a higher [Ca2+]i than the previous Ca2+ oscillations and then by resumption of Ca2+ oscillations similar to those occurring before Ca2+ wave. In cell D, [Ca2+]i increase was transient and failed to reinitiate Ca2+ oscillations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated by digital imaging fluorescence microscopy that airway epithelial cells exhibit Ca2+ oscillations in response to ATPo. Although Ca2+ oscillations have been briefly reported in airway epithelial cells from four species (21, 25, 31, 37), no in-depth characterization has been made of the phenomenon. Here, we analyzed the mechanism underlying the generation of the ATP-induced Ca2+ oscillations, the intercellular heterogeneity of ATPo responsiveness, and the intercellular signaling associated with ATP-induced Ca2+ oscillations.

Signaling pathway involved in the generation of Ca2+ oscillations. Airway epithelial cells exhibit a suramin-sensitive [Ca2+]i response to ATPo (16), which suggests that P2 purinogenic receptors are involved in the [Ca2+]i response observed in these studies. Airway epithelial cells have been reported to express several ATP-sensitive purinoceptors, including P2U, P2T, P2Y (19), and P2X (22). The P2X receptor/channel tentatively identified in rabbit ciliated epithelium (22) required low (150 µM) [Ca2+]o for activity, was not active at high (1.5 mM) [Ca2+]o, and was not activated by UTPo (22). Because our experiments were conducted in ~1.3 mM [Ca2+]o, we believe that P2X-receptor activity did not contribute to the [Ca2+]i increases observed. UTPo is ineffective in stimulating P2T and P2X receptors and is much less potent than ATPo in stimulating P2Y receptors but is of equal or greater potency to ATPo in stimulating P2U receptors (12). Consequently, the fact that UTPo elicited a more robust [Ca2+]i response from cells (greater number of cells displaying Ca2+ oscillations and a greater number of Ca2+ oscillations per cell) than ATPo at equimolar concentrations suggests the involvement of P2U receptors. Cl- secretion in rat airway epithelial cells, attributed to apically located P2U receptors, displayed a similar enhancement by UTPo (19). In addition, ATP released from the cystic fibrosis transmembrane conductance regulator is thought to be involved in the autocrine regulation of Ca2+-dependent Cl- channels via apically located P2U receptors in human airway epithelium cells (10, 42).

The aminosteroid U-73122, a PLC antagonist, prevented an initial [Ca2+]i increase in response to ATPo, consistent with an earlier report suggesting that the Ca2+ response to ATPo was mediated by a PLC/IP3 pathway (17). U-73122, when added to cells experiencing ATP-induced Ca2+ oscillations, resulted in the immediate cessation of Ca2+ oscillations. This indicates that both the initial Ca2+ increase and the subsequent Ca2+ oscillations utilize common signaling elements and that Ca2+ oscillations require continuous PLC activity for their maintenance. In addition, when ATPo is removed from cells displaying ATP-induced Ca2+ oscillations, the oscillations immediately cease, suggesting that continuous receptor signaling is also required for the maintenance of Ca2+ oscillations. These experiments, however, cannot distinguish between a cyclic increase in [IP3]i produced by the positive feedback of Ca2+ on Ca2+-sensitive PLC as proposed by the cross-coupling model of Ca2+ oscillations (27) and the steady-state increase in [IP3]i that drives Ca2+ oscillations as described in the Ca2+-induced Ca2+ release model (10). However, the propagation of intercellular waves in airway epithelial cells does not appear to involve the regenerative increase in IP3, which would be expected to occur if Ca2+-sensitive PLC isoforms were present in the cell. Hence we believe that the activation of PLC by ATPo binding to its receptor results in the steady-state increase in [IP3]i.

IP3-mediated Ca2+ oscillations usually rely on the repetitive release of Ca2+ from internal Ca2+ pools, and when airway epithelial cells were exposed to ATPo in nominally Ca2+-free medium, the cells displayed characteristic Ca2+ oscillations for a number of minutes. These results indicate a dependence on internal Ca2+ stores for the oscillations. Refilling of the internal Ca2+ stores is mediated by the action of Ca2+-ATPases, which can be inhibited by thapsigargin. Airway epithelial cells treated for 60 s with thapsigargin showed a slight increase in the [Ca2+]i during thapsigargin treatment; however, when ATPo was applied, the cells responded with a large initial [Ca2+]i increase that remained elevated and failed to exhibit Ca2+ oscillations. Thus no ATP-induced Ca2+ oscillations were observed after discharge of the internal pools when the pool was not allowed to refill. Furthermore, by applying thapsigargin 60 s after the addition of ATP and the onset of Ca2+ oscillations, the amplitude of the Ca2+ oscillations was progressively diminished and the baseline [Ca2+]i was increased until the Ca2+ oscillations ceased at an elevated [Ca2+]i. A dependence on thapsigargin-sensitive internal Ca2+ pools is a similar requirement for the ATP-induced Ca2+ oscillations of chondrocytes (8) and bile duct epithelial cells (29).

Characteristics of Ca2+ oscillations. Four basic responses to ATPo were identified in this study. These were 1) a single Ca2+ increase after ATPo stimulation; 2) the initiation of a few irregular Ca2+ oscillations; 3) the initiation of regular, periodic Ca2+ oscillations; and 4) a sustained elevation in the baseline [Ca2+]i after an initial Ca2+ increase. The heterogeneity of the Ca2+ response in airway epithelial cells was very similar to the heterogeneous Ca2+ responses to ATPo reported in glial cells (49), bile duct cells (29), megakaryocytes (46), and chondrocytes (8). In airway epithelial cells, however, these four Ca2+ responses were reproduced in individual cells exposed to different [ATP]o values. Therefore, the heterogeneity of the Ca2+ response was due in part to a 100-fold difference in the sensitivity of individual cells to ATPo and allowed us to characterize individual cells as having low, intermediate, and high sensitivities to ATPo. The basis of this differential sensitivity was not determined but may arise from variations in the expression, density, or sensitivity of the ATP receptor (P2U), IP3Rs, or other components of the signaling pathway. Although these differences may result in different resting [Ca2+]i values, a correlation between ATP sensitivity and resting [Ca2+]i was not found. This suggests that the ATP sensitivity may only be manifested after stimulation.

Ca2+-signaling differences may also arise from species-specific differences. Although Ca2+ oscillations have been observed in airway epithelial cells from rabbit, cow (21), mouse (Evans, unpublished observations), and sheep (37), there has been no report of Ca2+ oscillations in human airway cells except for serous gland cells (25). In view of the widespread occurrence of Ca2+ oscillations, it would be surprising to find that human cells fail to demonstrate Ca2+ oscillations.

Differences in the signaling components and the Ca2+ response of cells can also arise from culturing conditions such as variations in the composition of the matrix and medium, variations in the Ca2+-signaling phenotype between normal and transformed cell lines, and the applied agonist concentrations. In addition, [Ca2+]i measurements from populations of cells rather than from individual cells would fail to reveal heterogeneous Ca2+ oscillations such as those reported here. These reasons may also account for why the observation of Ca2+ oscillations in airway epithelial cells has varied among investigators.

If the steady-state [IP3]i is controlled by the degree of receptor activation, as it is in other cell types (4), then the four basic Ca2+ responses observed in airway epithelial cells reflect the changes in [IP3]i, a hypothesis that is consistent with the mathematical modeling of Ca2+ oscillations (43). When the [ATP]o was relatively low (relative to the intrinsic sensitivity of the cell to ATPo), reflecting relatively low receptor activation and a low steady-state [IP3]i, the cell was unable to support Ca2+ oscillations and responded to ATPo with a single [Ca2+]i increase. In the [ATP]o range that yielded Ca2+ oscillations, the Ca2+ oscillation frequency increased with increased [ATP]o, an observation common to many cell types stimulated by a wide variety of agonists such as vasopressin (33), phenylephrine (34), acetylcholine (29), and ATP (8) and possibly reflected increased [IP3]i. In some experiments, the first few Ca2+ oscillations had shorter periods and initiated at a higher [Ca2+]i than subsequent Ca2+ oscillations, which may be due to an initial [IP3]i spike that declines to a steady-state [IP3]i. Exhibition of preceding higher-frequency Ca2+ oscillations initiating at higher [Ca2+]i values is a feature common in cells to many agonists including ATP (8, 29, 46). At relatively high [ATP]o values, when the steady-state [IP3]i would be high, the cells responded with a sustained increase in [Ca2+]i and no Ca2+ oscillations.

Spatial characteristics of Ca2+ oscillations. The pattern of ATP-induced Ca2+ oscillations was intrinsic to each cell, and Ca2+ oscillations within one cell failed to influence the [Ca2+]i activity of adjoining cells in a manner similar to the asynchronous ATP-induced Ca2+ oscillations that have been observed in bile duct cells (29) and MDCK cells (35) and are consistent with the behavior of spontaneous Ca2+ oscillations in airway epithelial cells (6). After a nearly synchronous increase in [Ca2+]i in the cells in response to the addition of ATPo, each cell displayed a unique Ca2+ response. In contrast, ATP-induced Ca2+ oscillations initiate intercellular Ca2+ waves in cultures of chondrocytes (8), and a variety of agonists generate Ca2+ oscillations that spread as intercellular Ca2+ waves in liver tissue (28, 33) and hepatocytes (48). Interestingly, in MDCK cells, bradykinin and thrombin, but not ATPo, stimulate synchronized Ca2+ oscillations, suggesting that the synchrony of Ca2+ oscillations in adjoining cells may be a function of the agonist (35). The synchrony of Ca2+ oscillations in MDCK cells and the spreading of intercellular Ca2+ waves from Ca2+ oscillations in hepatocytes and salivary glands, however, are dependent on gap junction communication. Disruption of communication by octanol (35) or alpha -glycyrrhetinic acid (48) leads to asynchronous Ca2+ oscillations or the failure of Ca2+ oscillations to initiate intercellular Ca2+ waves without affecting Ca2+ oscillations themselves, suggesting the gap junction communication of a signaling molecule between cells to maintain synchrony.

Intercellular communication during Ca2+ oscillations. In airway epithelial cells, mechanical stimulation of one cell results in propagated increases in [Ca2+]i to neighboring cells or an intercellular Ca2+ wave. Passage of an intercellular Ca2+ wave in airway epithelial cells is mediated by the gap junction diffusion of IP3 generated in the stimulated cell (6). Consequently, the passage of an intercellular Ca2+ wave through a field of ATP-stimulated cells, some of which were displaying Ca2+ oscillations, suggests that the inability of the Ca2+-oscillatory behavior of one cell to influence a neighboring cell is not due to the disruption of gap junction communication between cells. This inability instead demonstrates that Ca2+ alone is insufficient as an intercellular messenger to communicate Ca2+ changes to neighboring airway epithelial cells even when the IP3Rs of the neighboring cells are sensitized to IP3, although Ca2+ may act as the messenger in other cell types (28, 33, 48). Indeed, the magnitude of the Ca2+ increase associated with the Ca2+ oscillations in the cells is not different from the magnitude of the [Ca2+]i change associated with the passage of the intercellular Ca2+ wave, thus demonstrating that the [Ca2+]i increase associated with the wave cannot account for Ca2+ wave transmission.

Although it has been reported that mechanical stimulation of airway epithelial cells results in the release of ATP (14), we do not believe that the intercellular propagation of Ca2+ waves described above relies on this mechanism. We repeatedly failed to observe Ca2+ oscillations in cells after the passage of a mechanically stimulated intercellular Ca2+ wave, which we would expect if Ca2+ waves were dependent on ATPo. Also, although the Ca2+ response in airway epithelial cells is blocked by suramin, a P2-receptor antagonist, the propagation of mechanically stimulated intercellular Ca2+ waves is not (17). Moreover, fluid flow over cells during mechanical stimulation fails to bias the direction of spread of the subsequent intercellular Ca2+ waves (17).

Associated with the passage of the intercellular Ca2+ wave through ATP-induced Ca2+-oscillating cells was a transient increase in the oscillation frequency and the initiation of Ca2+ oscillations at a higher [Ca2+]i. As discussed above, this observation is consistent with a transient increase in [IP3]i over the steady-state [IP3]i, which would be expected because IP3 diffuses outward from the stimulated cell ahead of the Ca2+ wave and then is metabolized and diffused into the surrounding cells. Although IP3 can apparently traverse the gap junctions between neighboring cells stimulated with ATPo, we suggest that no IP3 gradient is established between ATP-stimulated cells, so no intercellular Ca2+ waves are initiated from any cell.

In summary, two separate Ca2+ signals can occur in airway epithelial cells: Ca2+ oscillations that are intrinsic and confined to an individual cell and intercellular Ca2+ waves that are generated at a distance and encompass a number of cells. We show that the two Ca2+ signals can occur simultaneously within cells and suggest that Ca2+ oscillations may serve to regulate individual cell functions, whereas intercellular Ca2+ waves coordinate cooperative cell activity.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Grant HL-49288 (to M. J. Sanderson).


    FOOTNOTES

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

Address for reprint requests and other correspondence: J. H. Evans, Dept. of Physiology, S4-315, Univ. of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655 (E-mail: John.Evans{at}ummed.edu).

Received 5 November 1998; accepted in final form 8 March 1999.


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DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 277(1):L30-L41
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ATP- and UTP-activated P2Y receptors differently regulate proliferation of human lung epithelial tumor cells
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Expression of nucleotide-regulated Cl- currents in CF and normal mouse tracheal epithelial cell lines
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J. A. Felix, E. R. Dirksen, and M. L. Woodruff
Physiology of a Microgravity Environment: Selected Contribution: PKC activation inhibits Ca2+ signaling in tracheal epithelial cells kept in simulated microgravity
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Endothelin-Mediated Calcium Signaling in Preglomerular Smooth Muscle Cells
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A. J. Ratner, R. Bryan, A. Weber, S. Nguyen, D. Barnes, A. Pitt, S. Gelber, A. Cheung, and A. Prince
Cystic Fibrosis Pathogens Activate Ca2+-dependent Mitogen-activated Protein Kinase Signaling Pathways in Airway Epithelial Cells
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J. H. Evans, D. M. Spencer, A. Zweifach, and C. C. Leslie
Intracellular Calcium Signals Regulating Cytosolic Phospholipase A2 Translocation to Internal Membranes
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Adenosine A3 receptor-mediated potentiation of mucociliary transport and epithelial ciliary motility
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cGMP abolishes agonist-induced [Ca2+]i oscillations in human bladder epithelial cells
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