Departments of 1 Pharmacology
and 2 Surgery, Alveolar type II epithelial (ATII) cells
repopulate the alveolus after acute lung injury. We hypothesized that
injury would initiate signals in nearby survivors. When rat ATII
monolayers were wounded, elevations in intracellular free
Ca2+ concentration
([Ca2+]i)
began at the edge of the wound and propagated outward as a wave for at
least 300 µm. The
[Ca2+]i
wave was due to both influx of extracellular
Ca2+ and release of intracellular
Ca2+ stores. Reducing
Ca2+ influx with brief treatments
of ethylene glycol-bis(
alveolar epithelial cells; acute lung injury; mechanical injury; intercellular signaling; intracellular calcium stores
SURVIVAL AFTER ACUTE lung injury is dependent on the
restoration of the integrity of the alveolar-capillary interface.
Preservation of lung function requires the reestablishment of an intact
epithelial barrier (24). Cells that are primarily responsible for the
reestablishment of this barrier are alveolar type II (ATII) cells. ATII
cells repopulate the alveolus after lung injury, restoring barrier
function, alveolar fluid absorption, and the synthesis of pulmonary
surfactant and surfactant proteins (9, 18). The ability of the ATII cells to respond quickly to injury is critical to the prevention of
later development of fibrosis (26). For ATII cells to initiate processes such as proliferation to restore the epithelial lining, they
must first detect the onset of alveolar injury. The mode of
transmission of such a signal and the type of signal (positive or
negative) is currently unknown. To determine which cellular signaling
systems might underlie rapid detection of nearby injury, we have
investigated some of the earliest signaling responses of ATII cells to
nearby injury. These early signals may begin the cascade of events that
regulate the healing response after injury.
Mechanical injury produces immediate
Ca2+ signals in several cell
types. Enomoto et al. (8) and Sammak et al. (27) have found that cell
rupture releases factors that can stimulate elevations in intracellular
free Ca2+ concentrations
([Ca2+]i)
in endothelial and epithelial cells. It has also been shown that gentle
stimulation with microneedles (30, 34) or shear stress (33) elevates
[Ca2+]i
in single cells. Gentle mechanical stretching, without membrane rupture, leads to propagation of
[Ca2+]i
waves in monolayers of endothelial cells as well as tracheal, mammary,
lens, and ATII epithelial cells (6, 8, 25, 27, 30, 38). Therefore, we
hypothesize that elevations in
[Ca2+]i
might be involved in the ATII response to injury.
If
[Ca2+]i
were elevated during injury, it might regulate cell behavior such as
motility, proliferation, and alveolar fluid absorption and secretion.
It is known that elevations in
[Ca2+]i
lead to secretion of pulmonary surfactant and surfactant proteins (5).
Although the signals that cause ATII cell motility, proliferation, and
differentiation into type I cells are still unknown, it has been shown
in other cell types that
[Ca2+]i
influences motile speed (2, 11, 12, 19, 20; P. J. Sammak, P. O. T. Tran, L. E. Hinman, Q.-H. P. Tran, G. M. Unger, and R. L. Bellrichard, unpublished observations), proliferation (1,
3, 19, 21, 22, 36, 38), and differentiation (13, 15).
Because we could not directly observe the alveolus in situ, we wounded
primary cultures of rat ATII cells. Confluent monolayers of ATII cells
were wounded by making a scratch in the monolayer with an 18-gauge
needle held in a micromanipulator. This model system allowed us to look
for
[Ca2+]i
signals that originated during and immediately after wounding and how
these signals might be propagated throughout the alveolus. This model
also allows us to look at signals that are generated after injury that
results in immediate cell rupture.
The property of intercellular communication is fundamental to any
multicellular system, including the lung. Intercellular communication
could be used to coordinate a response to many stimuli, including
wounding.
[Ca2+]i-dependent
intercellular communication has been studied in respiratory tract cilia
where it coordinates ciliary beat frequency (17) and in endothelial
cells where it stimulates movement of endothelial cell monolayers
during wound closure (Sammak et al., unpublished observations).
In the current study, we show that wounding ATII cells produces an
[Ca2+]i
wave that is initiated at the site of injury and propagates throughout
the monolayer for several hundred micrometers. In addition, we find
that the signal for the
[Ca2+]i
wave is carried, at least in part, via diffusion through the extracellular media. The source of the
Ca2+ for the
[Ca2+]i
wave was from both intracellular and extracellular
Ca2+ pools; however, the
[Ca2+]i
wave was totally dependent on the filling of intracellular Ca2+ stores. These findings
suggest that ATII cells can detect wounding in nearby cells as
monitored by increases in
[Ca2+]i.
It is plausible that these changes in
[Ca2+]i
may have an important role in the determination of the response of ATII
cells after wounding.
Cell culture.
ATII cells were harvested in a manner similar to that described by
Dobbs et al. (7). Male Sprague-Dawley rats weighing 250-275 g
(Harlan Sprague Dawley, Indianapolis, IN) were used. Cells were plated
on fibronectin-coated glass coverslips at a concentration of 1 × 106 cells/coverslip and were grown
for 2 days in 10% fetal calf serum (GIBCO BRL, Gaithersburg, MD) in
5% CO2 at 37°C. The viability of cells by trypan blue exclusion was always >90%. Cells had >90% ATII morphology by light microscopy. Morphology was confirmed in
representative samples by electron microscopy.
Ca2+
imaging.
Cells were loaded for 1 h at room temperature with 2 µM acetoxymethyl
ester of fura 2 (Molecular Probes, Eugene, OR) and 0.03% pluronic acid
F-127 (Molecular Probes). Both loading and imaging were
done in Hanks' balanced salt solution (HBSS) with 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and without bicarbonate or phenol red.
[Ca2+]i
measurements were made by fluorescence ratio imaging of confluent monolayers at room temperature in HBSS. The imaging system consisted of
a Diaphot 300 inverted microscope (Nikon, Melville, NY), a cooled
charge-coupled device camera (PXL KAF 1400; Photometrics, Tucson, AZ),
and a filter wheel (Mac 2000; Ludl Electronics, Hawthorne, NY), which
were all controlled by IP Lab Spectrum (Signal Analytics, Vienna, VA)
on an Apple Power Macintosh computer. Excitation light was provided by
a 150-W xenon arc lamp (Opti-Quip, Highland Mills, NY). Excitation
light was selected with 340- and 385-nm band-pass filters (Chroma
Technology, Battleboro, VT). Cells were observed with a Nikon CF fluor
×20 objective with a numerical aperture of 0.75.
Calibrations.
Ratios of 340- to 385-nm intensity (R) were calibrated to
[Ca2+]i
by an in vitro approach similar to Grynkiewiez et al. (10). Briefly, 5 µM fura 2 in 10 mM Ca2+ HBSS was
imaged to record the ratio of fluorescence intensity at saturating
Ca2+
(Rmax). Five micromolar fura
2-free acid in Ca2+-free HBSS and
10 mM ethylene glycol-bis(
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid or Gd3+ reduced both the
amplitude and the apparent speed. Draining intracellular Ca2+ stores by pretreatment with
cyclopiazonic acid eliminated the [Ca2+]i
wave. Therefore, the
[Ca2+]i
wave depended critically on intracellular
Ca2+ stores.
[Ca2+]i
elevations propagated over a break in the monolayer, suggesting that
extracellular pathways were involved. Furthermore, extracellular factors from injured cells elevated
[Ca2+]i
in uninjured cultures. We conclude that wounding produces a [Ca2+]i
wave in surviving cells and part of this response is mediated by
soluble factors released into the extracellular space during injury.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA) were imaged to record the ratio of fluorescence intensity at zero Ca2+
(Rmin). The following formula
was then used to generate a calibration curve
where
(1)
is the ratio of 385-nm intensities at zero
Ca2+ over maximal
Ca2+ and
Kd is the
dissociation constant at 224 nM (10).
Wounding. Cells were wounded with an 18-gauge needle held in a micromanipulator (Narishige). The needle was held at an ~45° angle to the coverslip. The needle was placed so that it touched the coverslip but did not deflect the coverslip. Deflection was monitored by a loss of focus. The needle was then smoothly moved with the micromanipulator for a distance of 2-3 mm at a rate of 2 mm/s, which removed a path of cells that was 4-5 cells wide and 4-6 mm long.
Solutions. Ca2+-EGTA solutions were prepared by adding EGTA (Sigma Chemical, St. Louis, MO) to Ca2+-free HBSS (GIBCO) to a final concentration of 100 µM or 10 mM. Cyclopiazonic acid (CPA; Calbiochem, La Jolla, CA) solutions were prepared in HBSS to a final concentration of 10 µM. Gd3+ solutions were freshly prepared by adding enough gadolinium(III) chloride hexahydrate (Aldrich, Milwaukee, WI) to phosphate-free HBSS (chloride in place of phosphate to avoid precipitation with Gd3+) to obtain a final concentration of 100 µM.
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RESULTS |
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Wounding caused an elevation in [Ca2+]i. To assess if wounding elicited a [Ca2+]i response, confluent monolayers of ATII cells were loaded with fura 2 and were wounded with an 18-gauge needle. The [Ca2+]i was then measured by fluorescence ratio imaging at 5-s intervals. Before wounding, the monolayer had an average basal [Ca2+]i of 0.23 ± 0.05 (SD) µM (n = 31 monolayers). Immediately after the cells were wounded, [Ca2+]i was elevated in the cells at the wound edge (Fig. 1A2). After 15 s, the cells farthest from the wound edge also had elevated [Ca2+]i (Fig. 1A3). Twenty-five seconds postwounding, cells up to 250 µm from the wound had elevated [Ca2+]i (Fig. 1A4). Thirty-five seconds postwounding, cells had begun to return to baseline (Fig. 1A5). After 2 min, nearly all cells in the field of view had returned to baseline. The magnitude of the elevation was greatest at the wounded edge (>2.5 µM; Fig. 1A3) and dropped off with increasing distance from the wound edge.
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|
The [Ca2+]i wave required intracellular Ca2+ stores. To determine if the source for the rise in [Ca2+]i was from intracellular Ca2+ stores or extracellular media, the contribution of each pathway was individually blocked. To block influx of extracellular Ca2+, cells were wounded in Ca2+-free HBSS with 100 µM EGTA to chelate any Ca2+ in the extracellular media. To limit the exposure to EGTA and a Ca2+-free environment, imaging was started in HBSS, and then solutions were switched to 100 µM EGTA 1 min before wounding. When extracellular Ca2+ was chelated, the wave was still present, but the amplitude, speed, and range were all reduced (compare Fig. 1, A1-A6 with B1-B6). A representative trace of the [Ca2+]i elevations at three distances from the wound is shown in Fig. 2B. In 100 µM EGTA, the peak was reduced from saturating levels in HBSS to 1.37 ± 0.65 (SD) µM (n = 4; Fig. 2, A vs. B), and the range was reduced to 194 ± 112 (SD) µm (n = 4; Fig. 1B4). The apparent wave speed was reduced by 45% to 7.18 ± 1.31 (SD) µm/s (n = 4). However, the rate of deceleration of the wave speed became highly variable and was no longer linear. This suggests that the wave speed and the mechanism of deceleration were highly dependent on an influx of extracellular Ca2+. These data demonstrate that the kinetic properties of the [Ca2+]i wave depend on the concentration of extracellular Ca2+, but cells continue to elevate [Ca2+]i in the absence of extracellular Ca2+.
To confirm that the effect of EGTA was limited to chelating extracellular Ca2+ and did not predrain intracellular Ca2+ stores, we repeated the experiment using Gd3+ instead of EGTA. Gd3+ is an effective and reliable Ca2+ channel blocker that has been used with success in other cell lines (Ref. 23 and Sammak et al., unpublished observations). When cells were wounded in phosphate-free HBSS with 1.3 mM Ca2+ and 100 µM Gd3+, the wave was still present, but the peak elevation, speed, and range were all reduced (compare Fig. 1, A1-A6 with C1-C6). A representative trace of the [Ca2+]i elevations at three distances from the wound is shown in Fig. 2C. In 100 µM Gd3+, the peak elevation was reduced from saturating levels in HBSS to 1.42 ± 0.69 (SD) µM (n = 6; Fig. 2, A vs. C). The range was reduced to 74.6 ± 22.3 (SD) µm (n = 3; Fig. 1, A5 vs. C5), and the apparent wave speed was reduced by 75% to 3.38 ± 0.39 (SD) µm/s (n = 3). The rate of deceleration in wave speed could not be measured because of limits in the rate that data could be acquired. In agreement with the EGTA experiments, the Gd3+ experiments suggest that, whereas extracellular Ca2+ is a significant source of Ca2+ for the [Ca2+]i wave, it is not required for the phenomenon. To test if intracellular Ca2+ stores were required for the [Ca2+]i wave, we emptied intracellular Ca2+ stores by pretreatment with CPA, an endoplasmic reticulum Ca2+-adenosinetriphosphatase (SERCA) inhibitor. By inhibiting the SERCA pump, intracellular stores can no longer be filled and the Ca2+ leaks out into the cytoplasm, eventually emptying the stores. After a 1-h treatment with CPA, [Ca2+]i had returned to levels seen in untreated cells. Treated cultures were then wounded, and [Ca2+]i was recorded (Fig. 1, D1-D6). A representative trace of the [Ca2+]i elevations at three distances from the wound is shown in Fig. 2D. After CPA treatment, the peak elevation in the second row of cells was reduced from saturating levels in HBSS to 0.35 ± 0.06 (SD) µM (n = 3; Fig. 2, A vs. D). Even more dramatic was the abolishment of wave propagation beyond the second row of cells (Fig. 1, A2-A6 vs. Fig. 2, D2-D6). To ensure that CPA did not permanently damage the cells, CPA was subsequently washed out, and the cells were allowed to refill intracellular Ca2+ stores in HBSS for 35 min. Cultures were wounded again, and [Ca2+]i elevations were observed that were qualitatively similar to untreated cultures (compare Fig. 1, A1-A6 with D1-D6, and compare Fig. 2, A with E). The reversibility of the effects of CPA suggested that CPA was specific to the SERCA and that it was nontoxic. These data suggested that the release of Ca2+ from intracellular stores was necessary for the [Ca2+]i wave to occur. Therefore, wound-induced [Ca2+]i signaling depends critically on intracellular stores regardless of the extracellular Ca2+ concentration.An extracellular signal was sufficient to cause an [Ca2+]i wave. Previously, we have found [Ca2+]i waves in endothelial cells that are totally dependent on extracellular communication (27). We examined whether this [Ca2+]i wave had the same dependencies. To test this, a confluent monolayer of cells was wounded to provide a break in the monolayer that was devoid of cells (Fig. 1F1). One hour after wounding, a second wound parallel to the old wound was made. We found that the new wound produced elevations in [Ca2+]i (Fig. 1F2). A representative trace of the [Ca2+]i elevations at two distances from the wound is shown in Fig. 2F; the 100-µm distance was omitted due to the fact that it was in the middle of the old wound. Interestingly, cells on the far side of the old wound also elevated [Ca2+]i (Fig. 1, F3-F5). The elevation on the far side of the old wound was similar in amplitude and timing to that observed for a confluent monolayer at the same distance from the wound (Fig. 1, A3-A5 vs. F3-F5, and Fig. 2, A vs. F), although not all cells responded. Because the [Ca2+]i wave was able to jump over a break in the monolayer, we conclude that some signal for the [Ca2+]i can be transmitted without direct cell-cell contact.
Because cell-cell contact was not required, we attempted to ascertain if a signal in the extracellular space was sufficient to cause an elevation in [Ca2+]i. To test this possibility, cells from one coverslip were crushed with a rubber policeman in 100 µl of HBSS and were then immediately applied to a reporter coverslip of fura 2-loaded cells. In the reporter coverslip, [Ca2+]i levels rose to 0.52 ± 0.14 (SD) µM (n = 3). The elevation produced by conditioned media was less then the elevation seen during wounding; however, it was statistically different from baseline (unpaired t-test,
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DISCUSSION |
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To participate in the recovery process after acute lung injury, ATII cells must be able to react to injury-generated stimuli with the appropriate cellular responses. In primary ATII cells, we found that, immediately after a wound was made in the monolayer, [Ca2+]i began to elevate in the surviving cells at the wound edge. The [Ca2+]i elevation was transient and traveled for a limited distance in the monolayer before falling to baseline levels. Peak [Ca2+]i levels were similar to those we find after wounding in endothelial cells (27). The source of the [Ca2+]i wave was from both intracellular and extracellular stores of Ca2+. However, the [Ca2+]i wave was critically dependent on intracellular Ca2+ stores. Blocking the influx of extracellular Ca2+ with Gd3+ or chelating extracellular Ca2+ with EGTA reduced the range, speed, and magnitude of the wave but did not abolish it. However, draining the intracellular Ca2+ stores with CPA stopped propagation of the wave beyond the second row of cells and dramatically reduced the magnitude of the elevation. This suggests that the [Ca2+]i elevation was dependent on the presence of Ca2+ in the intracellular stores.
To better understand this phenomenon, we examined how the signal for the [Ca2+]i elevations was propagated from cell to cell. This propagation could be accomplished by several mechanisms, including 1) the diffusion of [Ca2+]i-elevating molecules through gap junctions, 2) stretch-activated Ca2+ influx and stores release, 3) electrically operated Ca2+ channels, or 4) diffusion of [Ca2+]i-elevating molecules through the extracellular space.
In airway epithelial cells, it has been shown that [Ca2+]i waves generated by mechanical stimulation are propagated by diffusion of D-myo-inositol 1,4,5-trisphosphate through gap junctions (29). If diffusion of any signals through gap junctions was responsible for the [Ca2+]i elevation, we would have expected the [Ca2+]i wave to stop at a break in the monolayer. However, in our experiments the [Ca2+]i elevation did cross the break in the monolayer as can be seen in Fig. 1, F1-F6. This experiment did not test for the presence of gap-junctional contributions, but it did demonstrate that they were not necessary for the [Ca2+]i wave. Furthermore, the wave had a similar speed and amplitude after crossing the barrier as when it traveled through a confluent monolayer (compare Fig. 1, A5 with F5). This experiment suggested that if gap junction transmission was present, then it was not critical to the timing and amplitude of the [Ca2+]i wave.
The second possibility for propagation of the [Ca2+]i wave was through stretch or electrical coupling. This model has been observed in both airway epithelial cells (16) and endothelial cells (23). We performed our experiments in a manner that would minimize the contributions of these pathways. During our experiments, we prevented flexing of the substrate by controlling the amount of pressure that was applied to the coverslip. We also tested to see if movement of the needle through an area devoid of cells right next to reporter cells could cause enough fluid flow to elevate [Ca2+]i, which it did not. It was still possible that the passage of the needle through the monolayer directly stretched the cells in the plane of the monolayer during wounding. However, if this lateral stretch were the cause of the [Ca2+]i wave, we would have expected the wave to stop at a break in the monolayer because there would be no mechanism for transfer of the stretch stimulus across the break. Therefore, stretch-activated channels were insufficient to account for ability of the [Ca2+]i wave to jump over breaks in the monolayer. A similar argument can be made for the third possibility, electrically coupled propagation. Electrical propagation would also have required direct cell-cell contacts for propagation. These experiments did not directly test for the presence of stretch or electrical coupling. It is quite likely that one or both of these mechanisms was present, but neither mechanism alone could explain the jumping of the wave over the old wound.
The fourth possibility for propagation of the wave was the release of [Ca2+ ]i-elevating molecules from the wound into the extracellular fluid. This mechanism has been shown to be responsible for [Ca2+]i waves in endothelial (27) and some epithelial (8) cells. If [Ca2+]i-elevating molecules were released into the extracellular fluid, we would expect the [Ca2+]i wave to jump over a break in the monolayer. Not only did the wave jump over the break in the monolayer, but it maintained a similar magnitude and speed compared with an intact monolayer. This is in agreement with the release of [Ca2+]i-elevating molecules from the wound into the extracellular fluid. Therefore, the release of [Ca2+]i-elevating molecules from the wound into the extracellular fluid is most consistent with our data. It is possible that the other signaling mechanisms have a role in mobilizing Ca2+, but under our conditions they are insufficient to explain our results. If release of [Ca2+]i-elevating molecules from the wound into the extracellular fluid were responsible for the wave, we would expect the wave to exhibit kinetics consistent with diffusion. For simple one-dimensional diffusion, the distance that the wave front travels should be proportional to the square root of time (4), resulting in a time-dependent decrease in velocity. When performed in the presence of extracellular Ca2+, our experimental data fit this general model. In the absence of extracellular Ca2+, the wave speed became highly variable. One possible interpretation is that wave speed is dependent on the influx of Ca2+ as well as on signaling molecule concentration such that without an influx of Ca2+ the extracellular signaling molecules cannot elevate [Ca2+]i in a reproducible way (27).
The release of [Ca2+]i-elevating molecules from the wound into the extracellular fluid would also explain why crushing cells and adding them back to an unwounded reporter monolayer elevated [Ca2+]i. When crushed cells were added back to reporter monolayers, the [Ca2+]i elevation peaked at 0.52 ± 0.14 µM. This elevation is modest compared with the elevation during wounding. However, this value should not be compared with wounding because the concentration of [Ca2+]i-elevating molecules that was applied was indeterminable. Last, it should be noted that the crushed cells were capable of producing an elevation in [Ca2+]i that was comparable to 10 µM ATP, which is known to cause secretion of surfactant at these concentrations (5).
There are a number of functional implications for the role of Ca2+ in signaling in the alveolus. The range of propagation of the Ca2+ wave is comparable to alveolar size (diameter of ~100 µm) and thus is appropriate for intercellular communication. Ca2+ elevations have been demonstrated in ATII cells in a number of model systems of surfactant production with secretagogues (14, 31, 32, 37). It is plausible that elevation of Ca2+ through receptor-mediated (35) or other mechanisms is one of the first intracellular steps to increased surfactant secretion. Although the role of [Ca2+]i in ATII cell proliferation and differentiation is currently unknown, changes in intracellular Ca2+ are associated with increased proliferation in other cell types (1, 3, 19, 21, 36, 38) and differentiation in other cell types (13, 15). This places the [Ca2+]i wave at the right place and possibly the right time to regulate both surfactant secretion and proliferation.
In summary, the present study demonstrates that wounding confluent monolayers of ATII cells produced an [Ca2+]i wave. This [Ca2+]i wave involved the influx of Ca2+ from the extracellular medium but was critically dependent on the release of Ca2+ from internal stores. The signal for the wave was partly carried in the extracellular medium, but other pathways might be involved in the [Ca2+]i response. The role for the [Ca2+]i wave has yet to be determined, but it is poised at a position both temporally and spatially to coordinate the healing response to acute lung injury.
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ACKNOWLEDGEMENTS |
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This work as supported by National Science Foundation Grant MCB-9596838 and American Lung Association Grant RG-175-N (to P. J. Sammak) and The North Trauma Institute and National Institutes of Health Grant BRSGS07RR06007-01 (to G. J. Beilman).
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FOOTNOTES |
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Address for reprint requests: P. J. Sammak, 3-249 Millard Hall, 435 Delaware St. SE, Dept. of Pharmacology, Univ. of Minnesota, Minneapolis, MN 55455.
Received 27 May 1997; accepted in final form 5 September 1997.
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