Introduction

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In cultured airway epithelial cell monolayers, mechanical stimulation of a single cell initiates an increase in intracellular free calcium concentration ([Ca²⁺]_i) that is radially propagated to adjoining cells (1). This spread of increased [Ca²⁺]_i, termed a "Ca²⁺ wave," functions to communicate a stimulus sensed by one cell to many surrounding cells to coordinate a multicellular response. Although mechanically induced Ca²⁺ waves have been well characterized for airway epithelial cells in vitro, it has not been demonstrated that they occur in vivo. In the present study, we have used confocal microscopy to examine intercellular Ca²⁺ communication in dissected tracheal mucosa, a thick tissue preparation of differentiated cells with a morphology similar to the tracheal epithelium in situ.

In cultured airway epithelial cell monolayers, mechanical stimulation activates phospholipase C (PLC) (2, 3) which hydrolyzes phosphatidylinositol-4,5-bisphosphate to produce inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol. IP₃ appears to mediate the propagation of Ca²⁺ waves by releasing Ca²⁺ from intracellular stores (4), whereas diacylglycerol activates protein kinase C (PKC). In environments of reduced gravitational force, such as in space or in earth-based microgravity simulation chambers, the expression, distribution, and activation of PKC have been modified (5, 6, 7). In addition, microgravity has been shown to alter the actin cytoskeleton and to reduce the number of stress fibers (8), elements that appear to be important in mechanotransduction (9). Altered PKC activation and cytoskeletal modification could inhibit mechanically induced intercellular Ca²⁺ signaling; therefore, we investigated whether the magnitude and propagation of mechanically stimulated Ca²⁺ waves were impaired by maintaining airway epithelium in simulated microgravity.

	Materials and Methods

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Tissue Preparation

Tracheas were dissected from New Zealand White rabbits killed by sodium pentobarbital injection; mucosal layers were microdissected from the cartilaginous backing of tracheas as previously described (10). Tracheal mucosas were rinsed twice in Hanks' balanced salt solution (Gibco BRL, Grand Island, NY), which consists of 1.3 mM CaCl₂, 5.4 mM KCl, 0.3 mM KH₂PO₄, 0.5 mM MgCl₂, 0.4 mM MgSO₄, 140 mM NaCl, 0.3 mM Na₂HPO₄, and 5.6 mM glucose, with 25 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Hepes), pH 7.2, added (referred to as HBSS-Hepes). The mucosas were cut into approximately 0.5-mm² explants. Explants were suspended in culture medium consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 0.37 wt/vol NaHCO₃ (all culture reagents were purchased from Gibco BRL). The suspension of explants was incubated in a humidified 5% CO₂ atmosphere at 37°C for up to 8 d either at unit gravity in untreated Petri dishes (Corning Costar Corp., Oneonta, NY) or under simulated microgravity conditions. Earth-based microgravity was simulated in a rotating, low sheer-stress culture vessel (Synthecon Inc., Houston, TX) which cyclically changes the orientation of the tissue relative to gravity. Because the tissue shed damaged cells during the first 6 h after dissection, experiments were performed on tissue maintained for 1-8 d.

Cells were loaded with fluo-3 acetoxymethyl ester (fluo-3 AM; Molecular Probes, Eugene, OR), a membrane-permeant, calcium-sensitive fluorescent dye, by incubating tracheal mucosal explants in 80 µM fluo-3 AM in HBSS-Hepes with 0.3% Pluronic F-127 (a surfactant to disperse the fluorophore) and 0.5 mM sulfinpyrazone (an inhibitor of organic anion transport) for 2 h at room temperature in the dark. Intracellular esterases cleave the acetoxymethyl group from fluo-3 AM, loading the cells with the membrane-impermeant, fluorescent fluo-3 salt. By monitoring intracellular fluorescence at 2 s intervals over time of incubation in fluo-3 AM, we found that using a high concentration of the fluorophore (80 µM) and inhibiting export of fluo-3 salt were necessary to effectively load the intact epithelium, which normally functions as a barrier to such agents. Explants were allowed to settle for 10 min on coverslips coated with Cell Tak or Matrigel (both from Becton Dickinson Labware, Bedford, MA), then rinsed twice with HBSS-Hepes containing 0.5 mM sulfinpyrazone. Mechanically stimulated Ca²⁺ responses were the same for mucosal explants settled on Matrigel or Cell-Tak.

Under Ca²⁺-free conditions the epithelium was rinsed with Ca²⁺-free HBSS-Hepes, which consisted of 5.4 mM KCl, 0.3 mM KH₂PO₄, 2.8 mM MgCl₂, 0.4 mM MgSO₄, 140 mM NaCl, 0.3 mM Na₂HPO₄, 5.6 mM glucose, 25 mM Hepes (pH 7.2), and 1.0 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), three times over 10 min to wash extracellular Ca²⁺ from the thick tissue and substrate. Mechanical stimuli were applied 10-40 min after changing to Ca²⁺-free HBSS-Hepes. Experiments were performed on cells at room temperature.

Fluorescence Measurements

Fluorescence was measured with a Zeiss 410 inverted confocal laser scanning microscope (Zeiss, Thornwood, NY). Two infinity-corrected, long-working distance objectives were used: a Zeiss Plan-Neofluar 40x/0.75 N.A. dry and an Olympus LUMPlanFl 40x/0.8 N.A. water-immersion objective. Fluo-3 was excited by an argon-krypton laser with output at 488 nm. The laser intensity was reduced to 20% power and attenuated by one-third with a neutral density filter to minimize photobleaching. Emitted fluorescent light was screened with a 515-nm long-pass filter and detected by a photomultiplier tube. The confocal pinhole was adjusted to take a 2.3-3.1-µm-thick optical slice. The Zeiss microscope contrast and brightness settings were fixed to aid in comparisons between experiments.

The fluorescent images of cells on the upper surface of the thick tissue explants were faint due to absorption of excitation light and light scattering through 100-200 µm of connective tissue. To improve image quality, we viewed image planes midway through the tissue in a folded-down area near the explant margin, which provided a cross-sectional image of the epithelial cells (Figure 1b). Digital images were recorded at 0.5 s intervals to the hard drive as Tagged-Image File Format (TIFF) files with a 0-255 relative grayscale intensity value for each pixel. Fluo-3 fluorescence values represent the average grayscale intensity, after background subtraction, for an area extending over most of the cell. To eliminate background fluorescence from subsequent analysis, we used a macro to subtract an initial resting image from each image in an experiment series. Overall resting fluo-3 fluorescence declined in Ca²⁺-free HBSS-Hepes. The lower resting fluorescence was normalized to the experiments in standard HBSS-Hepes by background subtraction. Relative grayscale values were normalized to the maximum grayscale intensity of the stimulated cell set equal to 1.0. By measuring the fluorescence of fluo-3 salt solutions with known Ca²⁺ concentrations (from 0 to 1 mM), we confirmed that changes in intracellular fluo-3 fluorescence were proportional to changes in [Ca²⁺]_i. A time-averaged (8×, 8.06-s scan) fluorescence image (Figure 1c) was taken to show cell borders. The cell borders of basal cells were not evident; because basal cells do not extend to the lumen, they did not load appreciably with fluo-3.

View larger version (41K): [in this window] [in a new window]   Figure 1.   (a) The non-confocal brightfield image shows a glass pipette (P) in position to stimulate an epithelial cell of a tracheal mucosal explant. The ciliated (Ci) apical surface of the epithelium and connective tissue (CT) are indicated. (b) The three-dimensional diagram shows, in cutaway view, the position of the 2.3-3.1 µm-thick confocal image plane. (c) A time-averaged confocal fluorescence image of the fluo-3-loaded tissue shown in (a) reveals approximate cell borders and the Texas Red-filled pipette tip. The confocal microscope collects emitted fluorescent light only from the plane of focus and provides a cross-sectional view of the epithelial cells.

Mechanical Stimulation

Micropipettes were pulled to produce a ~ 1-µm diameter tip. In some experiments, micropipettes were filled with 0.1 mM Texas Red to clearly identify the location of the pipette relative to the cells. Changes in fluo-3 fluorescence were not different for cells stimulated with empty or Texas Red-filled micropipettes. The tip of the pipette was positioned about 10 µm away from the apical membrane of a ciliated cell, in the same optical plane as the cells being imaged. A ciliated cell was mechanically stimulated by advancing and retracting the micropipette manually using a Narishige hydraulic micromanipulator (Tokyo, Japan). The contact between the micropipette and the cell was transient, less than 0.5 sec.

Statistical Analysis

Data are presented as mean ± SEM with n equal to the number of experiments. Differences were considered significant when P < 0.05.

	Results

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Mechanical Stimulation Initiates an Intercellular Ca²⁺ Wave in Intact Tracheal Epithelium

A brief (~ 0.5 s) mechanical stimulus applied to the apical membrane of a single cell caused an increase in [Ca²⁺]_i that originated at the site of mechanical stimulation. After a brief delay, a "wave" of increased [Ca²⁺]_i spread to adjacent cells (Figures 2a and 2b). In neighboring cells, increases in [Ca²⁺]_i spread across cells from the lateral membrane nearest the stimulated cell through the cytoplasm to the opposite lateral membrane.

View larger version (93K): [in this window] [in a new window]   View larger version (27K): [in this window] [in a new window]   Figure 2.   (a) Mechanical stimulation increased [Ca2+]i of cells in the intact tracheal epithelium. Approximate cell borders and the position of the glass micropipette are outlined in white. Relative [Ca2+]i levels are represented in pseudocolor (see color bar for scale). Increased [Ca2+]i spread from the mechanically stimulated cell to the fourth neighboring cells to the left and right by 10 s after stimulation. (b) Changes in fluo-3 fluorescence over time for the stimulated cell and cells to the left shown in (a). After mechanical stimulation (at arrow), [Ca2+]i rose immediately in the stimulated cell, then increased in surrounding cells sequentially with distance from the stimulated cell.

In the cross-sectional views of the cells used in these experiments (Figure 1), we observed linear propagation of the Ca²⁺ wave in both directions away from the stimulated cell (Figures 2a, 5a, and 5c). The propagation was roughly equal in both directions in cross-sectional planes taken at a wide variety of angles in the tissue; therefore, it seems reasonable that increased [Ca²⁺]_i also spread to adjoining cells above and below the optical plane of focus, and that the Ca²⁺ wave spread radially, as was observed in monolayer cultures (1). The mechanically induced [Ca²⁺]_i increases spread through the stimulated cell and approximately two surrounding tiers of cells (2.1 ± 0.2, n = 28). Propagation through a radius of 2.6 cells corresponds to a Ca²⁺ wave in which 20 cells are affected by mechanical stimulation of a single cell. The number of cells involved in a mechanically stimulated Ca²⁺ wave did not vary significantly over the 8 d of tissue maintenance.

The peak increase in fluo-3 fluorescence intensity was greatest in the stimulated cell and declined as a function of the distance from this cell (Figure 3). The cells immediately adjacent to the stimulated cell ("second" cells) showed an increase above background fluorescence only half as large (55 ± 7%) as that of the stimulated cell. The third and fourth cells from the stimulated cell showed increases 30 ± 6% and 14 ± 4% of the stimulated cell, respectively. In Figure 4, the lag time for the onset of the [Ca²⁺]_i elevation following mechanical stimulation is plotted as a function of distance from the stimulated cell. Fluorescence in the stimulated cell increased 1.2 ± 0.3 s after mechanical stimulation and returned to near resting level after 29.9 ± 3.1 s. By comparison, fluorescence in the second row of cells increased 3.0 ± 0.4 s after mechanical stimulation and returned to near resting level after 14.2 ± 1.2 s (n = 14; tissue from five different rabbits). The average speed of cell-to-cell propagation of the [Ca²⁺]_i increases was approximately 6 µm/s. Because there were pauses in the spread of elevated [Ca²⁺]_i at the cell borders, the actual speed of the Ca²⁺ wave through the cytoplasm was greater than 6 µm/s.

View larger version (33K): [in this window] [in a new window]   Figure 3.   The peak fluo-3 fluorescence was smaller in cells more distant from the mechanically stimulated cell. The amplitudes of the mechanically induced [Ca2+]i increases were larger in normal HBSS-Hepes (1.3 mM Ca2+) (dark gray and cross-hatched bars) than in Ca2+-free medium (light gray and striped bars). The amplitudes of the changes in fluorescence were not significantly different for tissue maintained at unit gravity (solid bars) in comparison with tissue maintained in simulated microgravity under the same extracellular Ca2+ condition (patterned bars to the immediate right).

View larger version (38K): [in this window] [in a new window]   Figure 4.   The mean lag time for cells to respond with elevated [Ca2+]i following mechanical stimulation increased as a function of distance from the stimulated cell. The spread of the Ca2+ wave was faster in the presence of extracellular Ca2+ (dark gray and cross-hatched bars) than in Ca2+-free medium (light gray and striped bars). The lag time for the onset of an increase in [Ca2+]i in tissue maintained at unit gravity (solid bars) was not significantly different from tissue maintained at microgravity under the same extracellular Ca2+ condition (patterned bars to the immediate right).

The cells did not appear to be seriously injured by mechanical stimulation. Both stimulated and adjoining cells were able to return their elevated [Ca²⁺]_i to near resting level within 1 min after mechanical stimulation. A second mechanical stimulus, applied to the same cell 1 min after the first stimulus, caused a second, similar Ca²⁺ wave. Additionally, the stimulated cell responded like a neighboring cell when a second mechanical stimulus was applied to a neighboring cell. The mechanically stimulated cell appeared wounded when fluorescent dye was lost immediately after mechanical stimulation, [Ca²⁺]_i remained at a high level minutes after mechanical stimulation, the membrane blebbed, and/or ciliary beating slowed or ceased. In six of 107 experiments cells showed signs of mechanical injury, and those experiments were excluded from the analysis.

Using brightfield optics, we monitored ciliary movement by eye. Ciliary beating increased robustly in response to mechanical stimulation. Ciliary beat frequency increased in the stimulated cell first, then the increase spread to adjoining cells.

Removal of Extracellular Ca²⁺ Attenuates, but Does Not Eliminate, the Ca²⁺ Wave

To determine whether an influx of Ca²⁺ from the extracellular medium contributed to the elevation of cytosolic [Ca²⁺], tissues were incubated for 10-40 min in Ca²⁺-free HBSS-Hepes. In the absence of extracellular Ca²⁺, mechanical stimulation caused cell-to-cell Ca²⁺ signaling (Figure 5a) and a wave of increased ciliary beat frequency.

View larger version (90K): [in this window] [in a new window]   Figure 5.   The pseudocolor images show relative [Ca2+]i (see color bar) before and after mechanical stimulation of a cell in (a) tissue in Ca2+-free medium, (b) tissue in Ca2+-containing medium with 10 µM thapsigargin, and (c) tissue maintained in simulated microgravity in Ca2+-containing medium. Images shown are from single experiments and are representative of the responses observed in 14, 14, and 13 experiments, respectively.

Under Ca²⁺-free conditions, the magnitudes of the fluorescence increases in response to mechanical stimulation were reduced (Figure 3). The fluorescence increases in the mechanically stimulated, second, third, and fourth cells were reduced by approximately 50, 30, 60, and 70%, respectively. The lag times for the onset of [Ca²⁺]_i elevations in response to mechanical stimulation were significantly greater in Ca²⁺-free medium (Figure 4). The average lag time of the stimulated cell was twice as long in Ca²⁺-free than in Ca²⁺-containing medium. In Ca²⁺-free HBSS-Hepes, the second and third cells exhibited an increase in [Ca²⁺]_i after 30 and 50% longer lag times, respectively, compared with the results obtained in HBSS-Hepes (with 1.3 mM Ca²⁺). Without Ca²⁺ in the extracellular medium, the mechanically induced Ca²⁺ wave spread through a shorter radius of 1.8 ± 0.3 cells (n = 14) in comparison with 2.6 ± 0.2 (n = 14) cells in Ca²⁺-containing medium (Figure 6).

View larger version (37K): [in this window] [in a new window]   Figure 6.   The mean radius of spread of the mechanically induced [Ca2+]i increases is compared for tissue maintained at unit gravity (gray bars) or in simulated microgravity (white bars), in 1.3 mM Ca2+-containing or Ca2+-free medium, with or without thapsigargin.

Treatment with Thapsigargin to Deplete Intracellular Ca²⁺ Stores Inhibits the Mechanically Induced Ca²⁺ Wave

Since the [Ca²⁺]_i increases observed without a source of external Ca²⁺ must be due to release of Ca²⁺ from intracellular stores, we treated the tissue with thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase (11, 12), in Ca²⁺-containing and Ca²⁺-free medium. Exposing the tissue to 1 µM thapsigargin caused a slow increase in [Ca²⁺]_i over ~ 2 min. During exposure to thapsigargin for 1 h, cells continued to display ciliary beating. Mechanically induced [Ca²⁺]_i increases were significantly inhibited by thapsigargin treatment in comparison with matched control responses in the same explant before the addition of thapsigargin (Figure 5b). We observed a variety of attenuated Ca²⁺ responses in thapsigargin-treated tissue. Of 14 experiments in Ca²⁺-containing medium, thapsigargin treatment inhibited the spread of increased [Ca²⁺]_i to three cells (once), to two cells (five times), to only the stimulated cell (five times), and to zero cells (three times). In contrast, mechanical stimulation caused [Ca²⁺]_i elevations in two or more cells in 13 of 14 matched control experiments. In 10 experiments in Ca²⁺-free medium, thapsigargin restricted the spread of increased [Ca²⁺]_i to three cells (once), to only the stimulated cell (three times), and to zero cells (six times).

Mechanical Stimulation Initiates Ca²⁺ Waves in Tissue Maintained in Simulated Microgravity Similar to the Responses in Tissue Maintained at Unit Gravity

Observation by light microscopy and measurements of cell height and width showed no apparent difference in the morphology of cells in tissue maintained at unit gravity versus simulated microgravity (data not shown). Cells retained a columnar-to-cuboidal morphology, averaging a height of 16 ± 1 µm (n = 20) from 2-8 d in incubation, both at unit gravity and in simulated microgravity.

A transient mechanical stimulus evoked a pattern of intercellular Ca²⁺ signaling in tissue kept in simulated microgravity not significantly different from tissue kept at unit gravity. Figure 5c shows a typical [Ca²⁺]_i response to mechanical stimulation in tissue maintained in simulated microgravity. The magnitude of the mechanically induced increases in fluorescence in stimulated, second, third, and fourth cells were not significantly affected by keeping explants in simulated microgravity (Figure 3). The lag times for the onset of [Ca²⁺]_i elevations in the mechanically stimulated cell and sequential neighboring cells were not influenced by maintaining the tissue in simulated microgravity (Figure 4). The mechanically induced [Ca²⁺]_i wave spread through a radius of 2.6 ± 0.2 (n = 28) cells in tissue kept at unit gravity, not significantly different from the propagation through 2.4 ± 0.2 cells (n = 13) in tissue kept at simulated microgravity (Figure 6). As in control tissues maintained at unit gravity, the absence of extracellular Ca²⁺ reduced the magnitude (Figure 3) and slowed the cell-to-cell spread of the [Ca²⁺]_i increases (Figure 4), but did not eliminate intercellular Ca²⁺ signaling. In Ca²⁺-free medium, the spread of the mechanically induced Ca²⁺ wave was through a smaller radius (1.8 ± 0.2 cells; n = 18) compared with the spread in Ca²⁺-containing medium in tissues kept in simulated microgravity. The attenuation of mechanically induced Ca²⁺ waves in the absence of extracellular Ca²⁺ in tissue maintained in simulated microgravity was not significantly different from the results obtained in tissue maintained at unit gravity (Figure 6).

	Discussion

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In previous reports we have characterized the mechanically stimulated intercellular Ca²⁺ waves which occur in cultured monolayers of tracheal epithelial cells (for review: 13, 14). Sneyd and colleagues (15) developed a model of Ca²⁺ wave propagation based on these and similar studies in cultured glial cell monolayers. According to this model, mechanical stimulation of a cell activates both Ca²⁺-conducting channels, which allow the influx of extracellular Ca²⁺, and PLC, which generates IP₃ in the stimulated cell. IP₃ diffuses through the stimulated cell and through gap junctions to adjoining cells where it binds IP₃-receptors to release Ca²⁺ from intracellular stores.

Using confocal microscopy we have extended our previous studies on tracheal epithelial monolayer cultures by examining intercellular Ca²⁺ signaling in intact tracheal mucosal explants, a thick tissue preparation in which the epithelial cells have retained much of their in vivo cell-cell and cell-connective tissue associations and structure. In intact epithelium, mechanical stimulation evoked intercellularly propagated increases in [Ca²⁺]_i. The magnitude of the [Ca²⁺]_i elevations became significantly smaller with distance from the stimulated cell until the Ca²⁺ signal died out, consistent with generation of an intercellular messenger in the stimulated cell and propagation via diffusion to adjoining cells. The magnitude and propagation of the Ca²⁺ wave was markedly inhibited by treatment with thapsigargin and slightly attenuated by Ca²⁺-free conditions, suggesting that the release of Ca²⁺ from intracellular stores supplied the bulk of the observed [Ca²⁺]_i increases with, perhaps, a small contribution from Ca²⁺ influx. Thus, the results that we have obtained in the intact tissue are consistent with the model outlined above.

Furthermore, the results from the intact tissue are similar to the monolayer results. Both respond to mechanical stimulation with increased [Ca²⁺]_i and communicate the [Ca²⁺]_i increases to neighboring cells. Both exhibit intercellular [Ca²⁺]_i communication in the absence of extracellular Ca²⁺ and show dramatically decreased Ca²⁺ signaling when intracellular Ca²⁺ stores are emptied by thapsigargin treatment. However, the results in the intact tissue differ from the results obtained in the monolayer cultures in several important respects. First, in the intact tissue, fewer cells participated in the mechanically induced increases in [Ca²⁺]_i. Second, under Ca²⁺-free conditions, the stimulated cell in the intact tissue showed a significant increase in [Ca²⁺]_i; whereas in the monolayer cells, the stimulated cell showed no change or a slight decrease in [Ca²⁺]_i in Ca²⁺-free medium (1). Note that the stimulated cell in monolayer cultures does not increase [Ca²⁺]_i, but the adjacent cells show a fairly normal Ca²⁺ wave response in Ca²⁺-free medium following mechanical stimulation. Third, the thapsigargin-induced inhibition of the spread of the [Ca²⁺]_i increases was less effective in the intact tissue than in the monolayer cultures.

Mechanically induced cell-to-cell propagation of increased [Ca²⁺]_i spread to an average of 20 adjacent cells in the intact epithelium, in comparison with 50-75 cells in monolayer cultures (1). If the Ca²⁺ wave is propagated by diffusion of IP₃ through gap junctions, then this disparity could originate from differences in IP₃ production or gap-junction protein expression. If the relevant sensory transduction mechanisms, including releasable IP₃, are located in the apical membrane, then intact cells would be expected to release less IP₃ based on surface area. The cells of monolayer cultures have an approximately 6-fold larger apical membrane surface area (without a significant change in cell volume) than cells of the intact tissue. If the density of the lipids remains the same, about 6-fold more transmitter could be released in stimulated cells of monolayer cultures. Also, the cells of monolayer cultures could express a higher density of gap-junction proteins or gap junctions with less resistance to the diffusion of IP₃ than the more differentiated cells of the intact tissue. Recently, Brissette and coworkers (16) showed that the level and type of gap-junction protein changes as mouse primary keratinocytes terminally differentiate; the intercellular transfer of the small dyes Lucifer yellow and neurobiotin was significantly reduced in the differentiated cells. Also, Widdicombe and coworkers (17) reported that gap junctions were larger and more numerous in cultured canine tracheal epithelial monolayers than in the intact epithelium.

The increase in [Ca²⁺]_i in the mechanically stimulated cell of intact epithelium in Ca²⁺-free medium was unexpected because the stimulated cell of monolayer cultures shows no change or a decrease in [Ca²⁺]_i (1). Based on imaging data and membrane potential measurements obtained in the monolayer cultures, we have suggested that cultured ciliated epithelial cells contain a voltage-dependent, Ca²⁺-conducting channel activated indirectly by mechanical stimulation (18, 19). The activation of this putative channel in Ca²⁺-free medium could lower [Ca²⁺]_i and thereby compromise IP₃-dependent release of Ca²⁺ from intracellular stores, which has been shown to be less effective at low Ca²⁺ concentrations (20, 21). It is possible that the Ca²⁺ channels are only expressed, or are over-expressed, by the epithelial cells in the monolayer culture. Another possibility is that channel activation in the cultured monolayer cells results in significant lowering of cytoplasmic [Ca²⁺] (by Ca²⁺ efflux along the concentration gradient to lower extracellular [Ca²⁺]), but the same activation in the intact tissue does not decrease [Ca²⁺]_i because of differences in cell architecture. In monolayer cultures, cells are flattened, ~ 2 µm high and 20-40 µm wide. A relatively high surface-area-to-volume ratio could permit a more rapid Ca²⁺ efflux, and a more efficient lowering of [Ca²⁺]_i. In intact epithelium, cells are cuboidal to columnar, ~ 16 µm high and ~ 12 µm wide. The lower surface-area-to-volume ratio might reduce the impact of Ca²⁺ efflux on cytoplasmic [Ca²⁺]. Recent reports have indicated that cell attachments to the extracellular matrix influence Ca²⁺ signaling (22, 23) and that changes in cell shape alone, with all other factors held constant, can regulate cellular responses (24). By using intact tissue, we have minimized the distinct changes in cell shape and extracellular matrix introduced in the culturing of epithelial cell monolayers.

In the absence of extracellular Ca²⁺, mechanical stimulation initiated intercellular Ca²⁺ signaling in intact epithelium; however, the [Ca²⁺]_i increases had a smaller amplitude and spread to fewer cells. At the present time, we do not know whether the lessening of the Ca²⁺ waves was due to a lack of contribution from Ca²⁺ influx or to a reduction of intracellular Ca²⁺ stores during exposure to Ca²⁺-free medium.

In monolayer cultures, thapsigargin treatment completely blocked the spread of the Ca²⁺ wave from the stimulated cell to adjacent cells (4). In the intact tissue, although the [Ca²⁺]_i increase was greatly diminished after thapsigargin treatment, [Ca²⁺]_i did increase in one or two adjacent cells in six of 14 experiments. The force of mechanical stimulation may affect more cells in the intact epithelium because these cells are more tightly packed than cells of monolayer cultures. The increase in [Ca²⁺]_i in the adjacent cells in the intact epithelium could be due to mechanically induced Ca²⁺ influx. Alternatively, thapsigargin may not be as effective in emptying intracellular stores of Ca²⁺ in the intact tissue as it is in the monolayer cells. The [Ca²⁺]_i increases obtained in Ca²⁺-free medium after thapsigargin treatment are hard to explain otherwise. Many cells show thapsigargin-insensitive internal Ca²⁺ stores and the airway epithelial cells in situ may show this characteristic.

The capability of assaying mechanically induced Ca²⁺ communication in explant tissues also allowed us to examine tissue that had been maintained in a rotating wall vessel which simulates the microgravity conditions experienced in space travel. Several studies (5, 6, 7) have suggested that microgravity can change the expression and distribution of PKC, an enzyme activated by the IP₃ co-product, diacylglycerol. PKC-dependent protein phosphorylations could modify the signal transduction pathway of intercellular Ca²⁺ communication. We have recently obtained evidence that phorbol esters, which activate PKC directly, inhibit mechanically stimulated intercellular Ca²⁺ communication (unpublished data).

The results in this report suggest that the mechanosensitive Ca²⁺ response of the intact epithelium was not significantly modified by simulated microgravity. We found that mechanical stimulation of a cell in tissues maintained for 3-8 d in simulated microgravity exhibited mechanically induced Ca²⁺ waves not significantly different in magnitude, speed, or number of cells affected, compared with tissue kept at unit gravity, indicating that mechanically induced Ca²⁺ signaling was not compromised by the simulated microgravity environment.

Although mechanically stimulated intercellular Ca²⁺ signaling has been demonstrated in many types of cultured cells (25), evidence for mechanically induced Ca²⁺ signaling in intact tissue has been demonstrated only in glial cells of the intact retina (31). In the present study, we demonstrate that mechanical stimulation of a single cell evokes a propagated wave of increased [Ca²⁺]_i and accelerated ciliary beating in several neighboring cells of intact tracheal epithelium. Ca²⁺ is involved in regulating ciliary activity, the function of which is to aid mucociliary clearance in the airways (1, 32). We postulate that in intact tracheal epithelium a local mechanical stimulus initiates a Ca²⁺ wave and, in turn, a coordinated wave of increased ciliary beating in an effort to propel airborne particles from the airways.