PKC role in mechanically induced Ca2⫹ waves
and ATP-induced Ca2⫹ oscillations in airway epithelial cells
MICHAEL L. WOODRUFF, VICTOR V. CHABAN, CHRISTOPHER M. WORLEY,
AND ELLEN R. DIRKSEN
Department of Neurobiology, University of California, Los Angeles School of Medicine,
Los Angeles, California 90095-1763
protein kinase C; adenosine 58-triphosphate; mechanotransduction; purinergic receptor; phospholipase C
MECHANICAL STIMULATION of a single airway epithelial
cell causes an increase in the intracellular free Ca2⫹
concentration ([Ca2⫹ ]i ) among a group of cells both in
intact epithelia (13) and in monolayer cultures (32, 33).
The increased [Ca2⫹]i, referred to as a ‘‘Ca2⫹ wave,’’
spreads radially from the stimulated cell to an average
of 20 neighboring cells in the intact epithelium and to
over 50 cells in culture. Mechanical stimulation generates inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] (14) and
the phospholipase C inhibitor U-73122 blocks the spread
of the Ca2⫹ wave (18), suggesting that the physical
stimulus activates phospholipase C, which hydrolyzes
phosphatidylinositol 4,5-bisphosphate to form Ins(1,4,5)P3.
Ins(1,4,5)P3 diffuses in the cytoplasm to release Ca2⫹
from intracellular stores in the stimulated cell and
probably also diffuses through gap junctions to release
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Ca2⫹ in neighboring cells (for a model, see Refs. 34, 35;
for reviews, see Refs. 10, 31).
Hydrolysis of phosphatidylinositol 4,5-bisphosphate
also forms diacylglycerol (DAG), an activator of protein
kinase (PK) C, in the plasma membrane of the stimulated cell, and it is possible that PKC may modulate
Ins(1,4,5)P3-dependent Ca2⫹ signaling. Stretch has been
shown to activate PKC in endothelial cells (29). Exogenous activation of PKC has been shown to decrease
astroglial gap junction permeability to lucifer yellow
dye and to limit mechanically induced Ca2⫹ waves in
the glial cells (12), a result consistent with most
observations on the effect of PKC-dependent phosphorylation on junctional permeability (15, 20). However,
activation of PKC increased total gap junctional conductance in cardiomyocytes (21), an effect that could
increase mechanically induced Ca2⫹-wave communication. The principal goal of this report is to examine the
effect of PKC activators and inhibitors on mechanically
induced Ca2⫹ waves in airway epithelial cells.
The airway epithelial cells in culture also produce
Ins(1,4,5)P3-dependent [Ca2⫹]i increases when the purinerigic-receptor activator ATP is added (17), and we
tested the effect of PKC activators and inhibitors on the
ATP response. ATP-dependent increases in [Ca2⫹]i appear to occur independently in the cells; that is, there is
no evidence for gap junction-mediated signaling influencing the responses in the individual cells (17). If PKC
agents affect the ATP-induced [Ca2⫹]i increases, it will
provide evidence of PKC modulation of Ca2⫹ signaling
in the airway cells that would be independent of a
putative effect of PKC on gap junctional communication. We also examined the effect of PKC agents on Ca2⫹
release from internal stores that occurs after treatment
of airway epithelial cells with thapsigargin, an inhibitor of endoplasmic reticulum Ca2⫹-ATPase.
MATERIALS AND METHODS
Cell culture. Primary cultures of rabbit tracheal airway
epithelial cells were prepared as previously described (11).
Tracheal mucosal layers from New Zealand White rabbits
were cut into small pieces, placed onto collagen-coated coverslips, and incubated for 8–20 days at 37°C under a humidified
5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum, 100 U/ml of
penicillin, 100 µg/ml of streptomycin, 0.25 µg/ml of amphotericin B, and 0.37% (wt/vol) NaHCO3. All culture reagents were
purchased from GIBCO BRL (Grand Island, NY) .
Mechanical stimulation. Borosilicate glass capillaries
(1B150-4, World Precision Instruments, Sarasota, FL) were
pulled with a Narishige puller (Tokyo, Japan) and heat
polished to produce 1-µm-diameter tips. Microprobes were
mounted in a piezoelectric device driven by a Grass SD9
1040-0605/99 $5.00 Copyright r 1999 the American Physiological Society
L669
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Woodruff, Michael L., Victor V. Chaban, Christopher
M. Worley, and Ellen R. Dirksen. PKC role in mechanically
induced Ca2⫹ waves and ATP-induced Ca2⫹ oscillations in
airway epithelial cells. Am. J. Physiol. 276 (Lung Cell. Mol.
Physiol. 20): L669–L678, 1999.—Mechanical stimulation of
airway epithelial cells generates the Ca2⫹ mobilization messenger inositol 1,4,5-trisphosphate and the protein kinase
(PK) C activator diacylglycerol. Inositol 1,4,5-trisphosphate
diffuses through gap junctions to mediate intercellular communication of the mechanical stimulus (a ‘‘Ca2⫹ wave’’); the
role that diacylglycerol-activated PKC might play in the
response is unknown. Using primary cultures of rabbit tracheal cells, we show that 12-O-tetradecanoylphorbol 13acetate- or 1,2-dioctanyl-sn-glycerol-induced activation of PKC
slows the Ca2⫹ wave, decreases the amplitude of induced
intracellular free Ca2⫹ concentration ([Ca2⫹]i ) increases, and
decreases the number of affected cells. The PKC inhibitors
bisindolylmaleimide and Gö 6976 slowed the spread of the
wave but did not change the number of affected cells. We show
that ATP-induced [Ca2⫹]i increases and oscillations, responses
independent of intercellular communication, were inhibited
by PKC activators. Bisindolylmaleimide decreased the amplitude of ATP-induced [Ca2⫹]i increases and blocked oscillations, suggesting that PKC has an initial positive effect on
Ca2⫹ mobilization and then mediates feedback inhibition.
PKC activators also reduced the [Ca2⫹]i increase that followed
thapsigargin treatment, indicating a PKC effect associated
with the Ca2⫹ release mechanism.
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PKC ROLE IN CA2⫹ WAVES AND OSCILLATIONS IN EPITHELIA
Fig. 1. 12-O-tetradecanoylphorbol 13acetate (TPA) inhibited spread of Ca2⫹
waves induced by mechanical stimulation. Pseudocolor images of intracellular free Ca2⫹ concentration ([Ca2⫹]i )
were calculated from fura 2 fluorescence as described in MATERIALS AND
METHODS. Top: under control conditions,
most cells in image field increased
[Ca2⫹]i within 15 s of mechanical stimulation of cell indicated by arrow. Middle:
a typical response 10 min after addition
of TPA. Only ⬃9 cells show [Ca2⫹]i
increases. Mechanical stimulation occurred at border between 2 cells. Bottom: a typical response 40 min after
addition of TPA. Approximately 19 cells
show [Ca2⫹]i increases.
(St. Louis, MO). 1,2-Dioctanyl-sn-glycerol (DOG), bisindolylmaleimide (BIM), Gö 6976, 4␣-phorbol-12,13-didecanoate (4␣phorbol), and calphostin C were purchased from Calbiochem
(Irvine, CA). The PKC activators and inhibitors were used at
concentrations 20 times the published EC50 values. These
concentrations were used to nearly fully affect the enzyme
while still preserving specificity.
Presentation of data. A field of cells (60–80 cells) was used
to determine responding cells in each experiment. Not all
cells in a field were analyzable because of focusing and
dye-loading considerations; the number of data-generating
cells in each field was between 30 and 75. The averages of
changes in [Ca2⫹ ]i between experiments were used to obtain
SDs, with n equal to the number of experiments. All errors are
SEs. The experimental means were considered significant at
P ⬍ 0.05. Plots of [Ca2⫹]i as a function of time were calculated
from an area of the cell covering 6 ⫻ 6 pixels (⬇5 µm2 ), with
data collected at 1 Hz, except in Fig. 4C where the averages
were calculated every 0.033 s. The individual points plotted in
the graphs are averages of data from video frames taken at 4
frames/s or from single frames.
RESULTS
TPA suppresses mechanically induced intercellular
Ca2⫹ waves. When a single cell in a monolayer culture
was mechanically stimulated by touching it with a
glass microprobe, an average of 50.1 ⫾ 4.8 (SE) cells
(n ⫽ 11) showed a [Ca2⫹]i increase. Figure 1, top, shows
a typical response. [Ca2⫹]i increased first in the cell
directly stimulated (Fig. 1, top, arrow), and then the
[Ca2⫹]i increase spread radially to adjacent cells, presumably as the Ca2⫹ mobilization messenger
Ins(1,4,5)P3 diffused from the stimulated cell to adjacent cells through gap junctions (6). TPA treatment
restricted this intercellular communication to only a
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stimulator (Grass Instruments, West Warwick, RI) and were
positioned near the apical membrane of the cells with a
Narishige hydraulic micromanipulator. The pipette was deflected downward for ⬃150 ms to distort the cell membrane.
Stimulator initiation sends an electrical pulse to the image
recording system so that the precise time of mechanical
stimulation was obtained in each experiment.
Fluorescence measurements of [Ca2⫹]i. Fluorescence image
analysis was performed as previously described (32). The
cells were incubated in 5 µM fura 2-AM (Molecular Probes,
Eugene, OR) for 1 h at 37°C in modified phenol red-free
Hanks’ balanced salt solution consisting of (in mM) 1.3 CaCl2,
5.0 KCl, 0.3 KH2PO4, 0.5 MgCl2, 0.4 MgSO4, 138 NaCl, 0.3
Na2HPO4, and 0.1% glucose (GIBCO BRL) buffered with 25
mM HEPES (pH 7.2). Thereafter, the cells were washed twice
in Hanks’ balanced salt solution-HEPES and allowed to
incubate for an additional 30 min before use. All experiments
were done at room temperature.
Coverslips were mounted in a chamber over an invertedstage Nikon Diaphot microscope equipped with a ⫻40 oilimmersion, 1.3-numerical aperture objective with quartz
optical elements. The excitation source was a 100-W mercury
lamp. The cells were alternatively illuminated through 340or 380-nm filters (Omega Optical, Brattleboro, VT). A 405-nm
dichroic mirror separated excitation and emission signals,
and emitted light was passed through a 510-nm long-pass
filter into a silicon-intensified target camera (Cohu, San
Diego, CA). Images were recorded with an optical-memory
disk recorder (Panasonic TQ2026F) and computer-processed
with a frame grabber and image processor boards (Data
Translation, Marlborough, MA). The signals were calculated
by a ratiometric method (16) to estimate [Ca2⫹ ]i. Data
processing and ratio value conversions to [Ca2⫹ ]i were carried
out with software designed by Michael Sanderson (see Ref.
32) for an AT computer (Gateway, North Sioux City, SD).
Drugs. 12-O-tetradecanoylphorbol 13-acetate (TPA), ATP,
HEPES, EGTA, and thapsigargin were purchased from Sigma
PKC ROLE IN CA2⫹ WAVES AND OSCILLATIONS IN EPITHELIA
1 A third inhibitor, calphostin C, completely suppressed the wave.
Further analysis with calphostin C indicated that its principal effect
in restricting the Ca2⫹ wave may not be due to inhibition of PKC but
to a nonspecific action, depletion of internal Ca2⫹ stores. A large, slow
increase in [Ca2⫹]i in all cells in the field occurred after a short delay
of introducing calphostin C to the bath. This increase occurred with
and without extracellular Ca2⫹ present and so probably represents
release of Ca2⫹ from intracellular stores. Treatment with thapsigargin (1 µM), which would normally result in the release of Ca2⫹ from
internal stores and a large increase in [Ca2⫹]i (see Fig. 8 for example),
produced no [Ca2⫹]i increase after calphostin C. PKC inhibitors BIM
and Gö 6976 had no effect on basal [Ca2⫹]i, and unlike calphostin C,
they do not obviate the thapsigargin-induced release of Ca2⫹ from
internal stores (see Fig. 8C).
Fig. 2. TPA-induced inhibition of Ca2⫹ wave was fairly slow in onset
and showed recovery from inhibition during 40 min of exposure. No.
of cells affected by mechanical stimulation was determined by
counting all cells in microscopic field that met criterion of showing a
sustained increase in [Ca2⫹]i ⬎ 30 nM within 30 s of stimulation.
Data are from 11 control, 4 5-min, 5 10-min, 5 20-min, and 3 4-min
stimulations. Inset: effect of TPA at several concentrations ([TPA]), all
after 10-min exposure.
lar to values previously published (32). The first 10 s of
the responses (i.e., Fig. 4A, area outlined by dashed
lines) are shown in Fig. 4B along with the responses of
TPA-treated cells (10 min, 160 nM) and Gö 6976treated cells (10 min, 32 nM). TPA-induced PKC activation and Gö 6976-induced inhibition added an ⬃1-s
delay to the transfer of information to the secondary
cell and 2–3 s of delay to the transfer of the increase to
the tertiary cell. Estimating delays to cells further than
tertiary cells with the PKC activator was not feasible
because few distant cells were influenced by mechani-
Fig. 3. Extent of mechanically induced Ca2⫹ wave is reduced by
protein kinase (PK) C activators but not by PKC inhibitors. DOG,
1,2-dioctanyl-sn-glycerol; 4␣-phorbol, 4␣-phorbol-12,13-didecanoate;
BIM, bisindolylmaleimide. Control cells for each set of data were
from the same cell population obtained immediately before addition
of PKC-effective agent. Values are means ⫾ SE from 12 or more
determinations. Control bar indicates error in control determinations.
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few adjacent cells. Figure 1, middle, shows a response
to mechanical stimulation after a 10-min exposure to
TPA (160 nM). Including all cells that showed an
increase in [Ca2⫹]i ⬎ 30 nM above the basal concentration within 30 s of the stimulus, only nine cells participated in the response to mechanical stimulation. Figure 1, bottom, shows that by 40 min there was some
recovery from the TPA-induced inhibition. In this experiment, ⬃19 cells participated in the response to mechanical stimulation. A time course of the TPA-induced
suppression of the Ca2⫹ wave is shown in Fig. 2. The
maximum inhibition of the wave occurred at 10 min of
TPA treatment, and by 20 and 40 min, there was some
recovery. Figure 2, inset, shows the dose-response curve
for TPA. A 50% effective dose is ⬃5 nM. A phorbol ester
ineffective in activating PKC, 4␣-phorbol (160 nM),
does not inhibit the Ca2⫹ wave (Fig. 3). A highly specific
PKC activator, DOG (32 µM), restricted the Ca2⫹ wave
to the same extent as TPA (Fig. 3).
The data in Fig. 3 also show that two PKC inhibitors,
BIM and Gö 6976, had no significant effect on the
number of cells participating in the mechanically induced Ca2⫹ wave.1 When cultures were treated overnight with 160 nM TPA to downregulate PKC, the
number of cells participating in mechanically induced
intercellular Ca2⫹ waves was dramatically reduced
(Fig. 3). Only an average of nine cells was affected by
stimulation of a single cell, and in no experiment did
stimulation affect cells more than two cells removed
from the stimulated cell. Treatment with TPA did not
further suppress the waves, consistent with PKC absence. The internal stores were intact in the PKCdownregulated cells as evidenced by thapsigargininduced release (data not shown).
Both the speed of the cell-to-cell spread of the Ca2⫹
waves (Fig. 4) and the magnitude of the [Ca2⫹]i increases that occur in the cells that participated in the
Ca2⫹ waves (Fig. 5) were reduced by TPA (also see Table
1). In Fig. 4, the responses of the stimulated cells, the
cells immediately adjacent to the stimulated cells (‘‘secondary cells’’) and the cells two cells distant from the
stimulated cells (‘‘tertiary cells’’) were superimposed at
the point of mechanical stimulation and then averaged
to show the mean delay time between cells as the Ca2⫹
waves spread outward from the point of stimulation.
The normal (control), averaged responses to mechanical stimulation are shown at full scale in Fig. 4A. The
relative amplitudes and delays between cells are simi-
L671
L672
PKC ROLE IN CA2⫹ WAVES AND OSCILLATIONS IN EPITHELIA
cal stimulation after TPA was added. The PKC activator DOG also added an ⬃3-s delay to the response
initiation of the tertiary cells; however, delay in the
secondary cell response was not significantly different
in these experiments (see Table 1). The delay induced
by prior inhibition of PKC with Gö 6976 was surprising
in that the inhibitor seemed to have no effect on the
extent of the Ca2⫹ wave. The effect is probably real
because it appears in both the secondary and tertiary
cells and occurs as well with the PKC inhibitor BIM
(Table 1).
Some of the delay in the transfer of information could
arise in agent-induced delays in transduction within
the stimulated cell, delays not resolvable in the above
experiments where the data were acquired at 1 point/s.
To increase the time resolution, data were obtained at
video rate (30 data points/s) for control and TPA-,
DOG-, and BIM-treated cells. The means for 12 cells
each are shown in Fig. 4C. Activation of PKC did
appear to induce a small delay in the [Ca2⫹]i increase in
the stimulated cell, but BIM-induced inhibition of PKC
did not. The time between the stimulus and the first
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Fig. 4. TPA decreased rate of spread of mechanically induced Ca2⫹
waves. A: control responses in stimulated, secondary, and tertiary
cells. Area in box was replotted in B where it was compared with data
after addition of TPA or Gö 6976. C: data for only stimulated cells
under control, TPA-treated, DOG-treated, and BIM-treated conditions taken at high resolution to show delay within stimulated cell
induced by PKC activators. In A and B, data used in this analysis
were from control and 5-, 10-, and 20-min TPA-treated cells in Fig. 2.
Responses were superimposed at point of mechanical stimulation
and then averaged to show relative delays. In C, 12 cells were
stimulated under control conditions and 12 or more cells were
stimulated after 10–16 min of agent treatment. Data were acquired
at 30 Hz for 8–10 s at 380-nm excitation and calculated assuming
minimal bleaching.
calculated [Ca2⫹]i value to exceed two SDs of the mean
basal [Ca2⫹]i (determined for the 1-s period before
stimulation) was defined as the ‘‘delay time.’’ This time
was determined for each of the 24 cells, and these
values were also averaged. This delay was 60 ⫾ 7 (SE)
ms (n ⫽ 12) for the control cells and 88 ⫾ 9 ms (n ⫽ 12)
for the TPA-treated cells. The delay was slightly longer
than this in the DOG-treated cells.
To show the effect of PKC activation and inhibition on
the approximate magnitude of the mechanically induced [Ca2⫹]i increases in stimulated, secondary, and
tertiary cells, the responses of each cell that showed an
increase in [Ca2⫹]i ⬎ 30 nM were superimposed at the
point of increase in [Ca2⫹]i and then averaged. Figure 5,
top, shows the results of this analysis for control and
TPA- and Gö 6976-treated cells. The average peak
increases of the control cells in the stimulated, secondary, and tertiary cells were 781 ⫾ 73 (SE; n ⫽ 10), 543 ⫾
26 (n ⫽ 55), and 482 ⫾ 19 (n ⫽ 69) nM, respectively.
There was no significant difference in the amplitude of
the [Ca2⫹]i increases in the stimulated cells with either
TPA or DOG; however, the stimulated cell amplitude
was increased by both PKC inhibitors, BIM and Gö
6976 (Table 1). The average values shown for the
inhibitors (for the stimulated cells) in Table 1 are lower
than the actual values because the magnitude of the
[Ca2⫹]i increases saturated the fura 2 dye in many of
the determinations.
A different result was obtained in the neighboring
cells in that the inhibitors had little effect on amplitude, but the activators of PKC reduced the mechanically induced [Ca2⫹]i increases (Table 1). Only the
nearest neighbors to the stimulated cells showed an
increase with one of the inhibitors, Gö 6976. BIM had
no effect on secondary cells, and neither BIM nor Gö
6976 had a significant effect on tertiary cells. TPA
significantly reduced the response amplitudes in secondary and tertiary cells, and DOG reduced the response in
tertiary cells. For secondary and tertiary cells, dye
saturation was not a problem, and the values given for
these cells in Table 1 are reliable.
Figure 5, bottom, shows the control, TPA, and Gö
6976 responses from Fig. 5, top, normalized for each
condition to aid in comparing the kinetics of the responses. Within the resolution of these experiments,
the rates of increase in [Ca2⫹]i appear to be approximately equal in the control and the PKC-activated and
PKC-inhibited cells; the delays to peak [Ca2⫹]i (by 1–3 s
in most cases) suggest that there may be some slowing
of the responses for both activation and inhibition.
The recovery of [Ca2⫹]i to the basal concentration in
stimulated, secondary, and tertiary cells was significantly delayed by TPA. The PKC inhibitors BIM and Gö
6976 did not significantly affect the rate of [Ca2⫹]i
recovery in the stimulated cells but increased the rate
of recovery in both the secondary and tertiary cells. The
results with the PKC activator DOG were less clear.
DOG delayed the recovery in the stimulated cells but
increased the rate of recovery in the secondary and
tertiary cells, a result opposite to the effect of the PKC
activator TPA (see Table 1).
PKC ROLE IN CA2⫹ WAVES AND OSCILLATIONS IN EPITHELIA
L673
PKC activation also inhibits ATP-induced Ca2⫹ mobilization. ATP causes the release of Ca2⫹ from internal
stores in airway epithelial cells through a purinergic
receptor- and/or Ins(1,4,5)P3-dependent Ca2⫹ mobilization mechanism (17). When ATP was added, [Ca2⫹]i
oscillations were initiated in individual cells (Fig. 6A)
Table 1. Effects of PKC activators and inhibitors
on mechanically induced Ca2⫹ wave in stimulated,
secondary, and tertiary cells
Initial [Ca2⫹ ]i
Increase
Amplitude of [Ca2⫹ ]i
Increase, nM
Rate of [Ca2⫹ ]i
Recovery
Stimulated cells
Control
TPA
DOG
BIM
Gö 6976
Delayed ⬇30 ms
Delayed ⬇30 ms
Same
ND
781 ⫾ 73 (10)
726 ⫾ 58 (12)
830 ⫾ 88 (7)
947 ⫾ 29* (15)
998 ⫾ 2* (13)
Slower
Slower
Same
Same
Secondary cells
Control
TPA
DOG
BIM
Gö 6976
Delayed 1 s
Same
Delayed 1 s
Delayed 1 s
543 ⫾ 26 (55)
385 ⫾ 42* (31)
442 ⫾ 51 (24)
525 ⫾ 27 (66)
690 ⫾ 51* (26)
Slower
Faster
Faster
Faster
Tertiary cells
Control
TPA
DOG
BIM
Gö 6976
Delayed 3 s
Delayed 3 s
Delayed 1 s
Delayed 2 s
482 ⫾ 19 (69)
274 ⫾ 41* (18)
374 ⫾ 35* (23)
495 ⫾ 26 (82)
458 ⫾ 44 (27)
Slower
Faster
Faster
Faster
Values are means ⫾ SE calculated with peak value for each cell;
nos. in parentheses, no. of determinations. PKC, protein kinase C;
TPA, 12-O-tetradecanoylphorbol 13-acetate; DOG, 1,2-dioctanoyl-snglycerol; BIM, bisindolylmaleimide; [Ca2⫹ ]i , intracellular free Ca2⫹
concentration; ND, not determined. Initial [Ca2⫹ ]i increase and rate
of [Ca2⫹ ]i recovery are relative to control. * Significantly different
from control average at 0.05 level.
that had a maximum frequency of 4 oscillations/min (in
the first minute of exposure). There was cell-to-cell
heterogeneity in the ATP response even in the same
microscopic field such that, for example, although some
cells in the field approached the maximum oscillation
frequency, others showed no response or only one or two
oscillations during the sampling period (3 min). Figure
6B shows a normalized histogram distribution of the
number of oscillations within 3 min of the addition of
different concentrations of ATP. There was a concentration dependence such that as ATP concentration increased, a higher proportion of the cells approached the
maximum frequency. The maximum proportion of cells
showing high-frequency responses was obtained as
ATP concentration increased to only 2 µM (Fig. 6, A and
B). (At 0.1 µM ATP, no cells oscillated at ‘‘high’’ frequency.) The average delay to the first Ca2⫹ oscillation
(Fig. 6C) was also concentration dependent and became
minimal between 2 and 4 µM ATP. Although ATP is
continuously present in the bath, the ATP-induced
oscillations in any given cell become smaller in amplitude (Fig. 6A) and less frequent with time (Fig. 6, A and
D). The decrease in response may be partially due to a
decreased availability of releasable Ca2⫹. The airway
epithelial cells in culture do not appear to have a robust
capacitative Ca2⫹ entry that might otherwise assist in
replenishing internal stores after evoked Ca2⫹ release.
When 2 mM Ca2⫹ is added to fura 2-loaded cells 10 min
after they have been treated with thapsigargin in
‘‘Ca2⫹-free’’ medium, only a modest, very slow increase
in [Ca2⫹]i is observed (data not shown), a response not
typical of cells that have activated store-operated Ca2⫹
channels.
When ATP was added after activation or inhibition of
PKC, the number and shape of ATP-induced [Ca2⫹]i
oscillations were changed. Figure 7A shows single-cell
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Fig. 5. TPA decreased amplitude and
slowed rate of recovery of responses to
mechanical stimulation, whereas PKC
inhibitors had no effect on amplitude
but increased rate of recovery. Data are
the same as those used in Fig. 4. Here,
individual responses are superimposed
at rising phase of [Ca2⫹]i increases so
that response amplitudes and [Ca2⫹]i
increase and decrease kinetics in stimulated, secondary, and tertiary cells could
be more accurately determined. Top:
averages of [Ca2⫹]i data. Bottom: same
data normalized (arrows). Averages
were normalized by subtracting lowest
concentration of Ca2⫹ from each data
point in average and then dividing these
values by highest concentration of Ca2⫹.
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PKC ROLE IN CA2⫹ WAVES AND OSCILLATIONS IN EPITHELIA
responses to 1 µM ATP for control, PKC-activated
(TPA), and PKC-inhibited (BIM) conditions, and Fig.
7B shows averaged data for control, activated (DOG),
and inhibited (BIM) conditions. With PKC activation
(either TPA or DOG; both gave the same results), the
magnitude of the [Ca2⫹]i increases and the number of
oscillations were dramatically reduced. The histogram
distributions of the number of oscillations that oc-
Fig. 7. TPA suppressed ATP-induced
[Ca2⫹]i increases and oscillations, whereas
BIM allowed ATP-induced [Ca2⫹]i increases
while eliminating oscillations. A: data from
an individual cell in a field of cells showing
a [Ca2⫹]i oscillation pattern induced by 1
µM ATP under control, TPA, and BIM
conditions. B: data averaged after 1st oscillations in individual cells were superimposed at rising phase of [Ca2⫹]i increase.
This allowed comparison of effect of PKC
activation and inhibition on ATP-induced
[Ca2⫹]i increases. DOG and BIM were
added ⬃10 min before ATP addition. C:
histogram distribution of no. of ATPinduced [Ca2⫹]i oscillations before and after TPA addition. In control cells, most
cells responded with 3 or more [Ca2⫹]i
oscillations. After TPA, most cells responded with 1 or no oscillations. BIM
data are not shown because all cells gave 1
[Ca2⫹]i increase. D: no. of oscillations that
occurred between 0 and 50, 51 and 100,
and 101 and 150 s after ATP addition were
counted for control and TPA-treated cells
to show decrease in oscillation frequency.
curred within 160 s of the addition of 1 µM ATP with
and without TPA treatment for all of the cells analyzed
in the TPA experiments are shown in Fig. 7C. In the 135
cells followed under control conditions, 50 showed 4 or
more oscillations. For the 118 TPA-treated cells, only 1
cell showed as many as 4 oscillations; 55 of the TPAtreated cells showed no oscillations. The reduced response of the TPA-treated cells to ATP addition may be
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Fig. 6. ATP induces [Ca2⫹]i oscillations
in airway epithelial cells. A: representative traces from individual cells. Arrow,
addition of indicated [ATP]. B: histogram distribution of no. of oscillations
within 3 min of adding ATP. Distribution was normalized to directly compare distributions. No. of cells counted
were 185, 140, 69, 518, 204, and 245 for
0.1–4 µM, respectively. C: delay to 1st
ATP-induced oscillation became shorter
at higher concentrations. Notice that
for high frequency-responding cells (5
oscillations/3 min), delay to 1st oscillation was fairly concentration independent. D: for any given frequency of
response, oscillations slowed down.
Here, data are from addition of 4 µM
ATP. Time shown for 1 oscillation is
time from ATP addition to 1st peak;
time shown for 2nd oscillation is time
from 1st to 2nd peak, etc.
PKC ROLE IN CA2⫹ WAVES AND OSCILLATIONS IN EPITHELIA
Fig. 8. Thapsigargin-induced release of Ca2⫹ from internal stores
was reduced after TPA treatment. Both rate of [Ca2⫹]i increase and
peak amplitude of [Ca2⫹]i increase when thapsigargin was added
were decreased after 10 min of TPA. A: average [Ca2⫹]i increases in a
field of cells (⬃40 cells) from a control culture and a field of cells (⬃40
cells) from a TPA-treated culture. B: average [Ca2⫹]i increases from
control and TPA-treated cultures after removal of extracellular Ca2⫹
to eliminate capacitative Ca2⫹ influx. C: average rates of [Ca2⫹]i
increase and average peak [Ca2⫹]i increases from control and TPAand DOG-treated cultures, each under normal extracellular free
Ca2⫹ concentration ([Ca2⫹]o ) or Ca2⫹-free medium conditions and
from 4␣-phorbol-, BIM-, and Gö 6976-treated cultures. Rate of
increase for each culture was determined by finding maximum slope
in rising phase of averaged [Ca2⫹]i from each field of cells tested.
Values are means ⫾ SE. Each condition was repeated 6 times.
* Significantly different from parallel control, P ⫽ 0.05 by Student’s
t-test.
set of cells was 330 ⫾ 46 nM for the control cells and
240 ⫾ 45 nM for the TPA-treated cells (Fig. 8C).
The rate of [Ca2⫹]i increase and the [Ca2⫹]i peak
shown in Fig. 8A probably reflect the release of Ca2⫹
from internal stores; however, some of the [Ca2⫹]i signal
could be due to activation of store-operated Ca2⫹ channels in the plasma membrane (19) and thus to Ca2⫹
influx, although, as suggested above, capacitative Ca2⫹
entry in these cells may be very low. The thapsigargininduced [Ca2⫹]i increases obtained in the extracellular
solution without added Ca2⫹ and with 1 mM EGTA
(Ca2⫹-free medium; Fig. 8B) indicate that Ca2⫹ influx
from the extracellular medium does not play a significant role in the [Ca2⫹]i increases. The averaged (⫾SE)
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due to a prior activation of a putative PKC-dependent
desensitization mechanism (possibly limiting releasable Ca2⫹; see DISCUSSION ); however, the parallel timedependent decreases in control and TPA-treated cells
(Fig. 7D) suggest that they are regulated similarly on
ATP addition.
The inhibitory effect of TPA- and DOG-induced PKC
activation is consistent with negative feedback by PKC
on the ATP transduction mechanism. Under control
conditions, ATP-dependent activation of PKC would
turn off ATP-induced generation of Ins(1,4,5)P3 and
DAG, [Ca2⫹]i would return toward basal levels, and
PKC would be turned off. In the presence of ATP still
bound to the purinergic receptor, oscillatory [Ca2⫹]i
increases would be generated. Inhibition of PKC before
ATP addition should reduce the feedback effects of
PKC, and [Ca2⫹]i should show a sustained increase in
response to ATP. After BIM treatment, each cell responded to 1 µM ATP with a single, sharp [Ca2⫹]i
increase followed by a relatively slow decline (Fig. 7, A
and B). The cellular increases were asynchronous, but
the shape of the response in each cell was fairly
consistent. The averaged traces in Fig. 7B were obtained only from cells that showed a [Ca2⫹]i increase
after ATP addition and only after all the responses were
superimposed at the rising phase of the initial [Ca2⫹]i
increase so that the amplitude and kinetics of the
responses could be compared. We expected the response
amplitude for BIM-treated cells to be greater than that
for control cells because, according to our model, feedback inhibition would be suppressed; however, the
control cells consistently gave larger responses. Parallel analysis of the data with 2 µM ATP generated
similar results; control amplitudes were greater than
BIM-treated amplitudes. Interestingly, the PKC inhibitor Gö 6976, which has a narrower specificity than
BIM, did not affect the ATP-induced [Ca2⫹]i increases
(see DISCUSSION ).
When PKC downregulation was induced by overnight TPA treatment, the cells became fairly unresponsive to ATP. Fewer cells responded to ATP with [Ca2⫹]i
increases, and those that did showed either no oscillations (similar to the BIM result in Fig. 7, A and B) or
very shallow oscillations.
TPA reduces the rate of release of Ca2⫹ from internal
stores induced by thapsigargin. To assess whether TPA
influences Ca2⫹ storage or Ca2⫹ release from internal
stores, we used thapsigargin-induced release as an
assay. Thapsigargin inhibits endoplasmic reticulum
Ca2⫹-ATPase (22) and causes depletion of Ca2⫹ from
intracellular stores in airway epithelial cells (6). Figure
8A shows the typical effects of 1 µM thapsigargin on
[Ca2⫹]i in both control and TPA-treated (160 nM, 10
min) cells. [Ca2⫹]i increased under both conditions, but
the rate of [Ca2⫹]i increase was slower for the TPAtreated cells and the peak of [Ca2⫹]i increase was
reduced. The rate of [Ca2⫹]i increase in the control cells
was 12.5 ⫾ 1.4 (SE) nM/s (n ⫽ 6), and the rate of
increase in the TPA-treated cells was 4.5 ⫾ 1.2 nM/s
(n ⫽ 6; Fig. 8C). The average [Ca2⫹]i peak for the same
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PKC ROLE IN CA2⫹ WAVES AND OSCILLATIONS IN EPITHELIA
data for six determinations each with and without TPA
in Ca2⫹-free medium are plotted alongside the data for
the medium with a physiological extracellular Ca2⫹
concentration in Fig. 8C. There was no significant
difference with and without Ca2⫹ outside; the effect of
TPA was intact under both conditions.
The PKC activator DOG generated similar results
(Fig. 8C), whereas inactive 4␣-phorbol and the PKC
inhibitors (BIM and Gö 6976) did not significantly
influence the thapsigargin-induced release.
DISCUSSION
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We have shown that PKC activation suppresses
mechanically induced intercellular Ca2⫹ waves in airway epithelial cells, a result similar to the inhibition of
mechanically induced Ca2⫹ waves in cultured astroglial
cells (12). The inhibition of the Ca2⫹ wave in the airway
cells manifests itself as 1) a decrease in the rate of
spread of the wave, 2) decreases in the amplitude of the
average [Ca2⫹]i increases in stimulus-affected cells, and
3) a decrease in the number of cells participating in
stimulus-induced [Ca2⫹]i increases. In addition, PKC
activation suppresses the occurrence of ATP-induced
[Ca2⫹]i oscillations and decreases the Ca2⫹ release
induced by thapsigargin. These results suggest that
PKC activation must, in addition to possibly reducing
gap junctional communication (12, 15, 20), regulate
several aspects of stimulus-dependent Ca2⫹ mobilization and/or Ca2⫹ wave events, including stimulusdependent Ins(1,4,5)P3 generation and Ca2⫹ transport
across membranes of internal storage organelles.
Inhibition of PKC before mechanical stimulation
decreases the rate of spread of the Ca2⫹ wave; however,
the amplitude of the average [Ca2⫹]i increase in the
participating cells is unaffected (or is slightly greater;
see Fig. 5), and the extent of the Ca2⫹ wave is not
significantly different (see Fig. 3). The inhibitorinduced delay in the transfer of stimulus information to
the neighboring cells (1–2 s; see Fig. 4) and the slight
decrease in the rate of [Ca2⫹]i increase (see Fig. 5)
suggest that PKC may play an initial positive role in
stimulus-dependent mobilization of Ca2⫹. This putative
positive effect precedes temporally the negative effects
of PKC suggested above. An initial positive effect of
stimulus-dependent PKC activation is suggested, too,
in the ATP experiments. In control ATP additions, the
initial increase in [Ca2⫹]i is sharper and of greater
amplitude than when PKC is inhibited or activated (see
Fig. 7B). When PKC is agent inhibited, the positive
effect is missing so that the [Ca2⫹]i increase is less and
negative feedback is missing so that the [Ca2⫹]i increase is prolonged. When PKC is agent activated, the
negative feedback effects predominate, and the [Ca2⫹]i
increase is eliminated or reduced to one or two small
oscillations. Possible molecular mechanisms for this
positive effect might include PKC-dependent protein
phosphorylations that increase Ca2⫹ influx across the
plasma membrane. Boitano and colleagues (7, 8) previously presented evidence suggesting that mechanical
stimulation activates a plasma membrane Ca2⫹ channel. Consistent with this idea, mechanically induced
Ca2⫹ waves are slower in Ca2⫹-free extracellular solutions; however, they are roughly equal in extent (32).
Inhibition of PKC does not appear to affect the thapsigargin-induced release of Ca2⫹ from internal stores,
suggesting that constitutive PKC activity does not play
a role in basal Ca2⫹ release and uptake.
Enkvist and McCarthy (12) used astroglial cultures,
which stain positively for connexin (Cx) 43, to demonstrate a TPA-dependent decrease in mechanically induced intercellular Ca2⫹ waves. Positive immunostaining for Cx43 has been obtained in airway epithelial
cell-smooth muscle cell cocultures (24); however, it is
unclear whether Cx43 mediates the Ca2⫹ waves stimulated in these cocultures (24) or in our monolayer
cultures of airway epithelia. Lucifer yellow transfer
occurs between astroglial cells in culture but does not
occur between cells in airway cell cultures (32), suggesting that the gap junction proteins in airway epithelial
cells may not be Cx43 or, if they are, show different
regulation. Antibodies to Cx32 have been shown to 1)
block Ca2⫹ waves in airway epithelial monolayer cultures, 2) recognize substrates in immunohistochemical
staining of airway epithelial sections, and 3) stain
Western blots of the epithelial proteins (5). Note that
both Cx43 and Cx32 are phosphorylated by PKC (3, 9,
25, 27, 30) and that the effect of phosphorylation is
reduced permeability (23, 25).
Overnight treatment with TPA to downregulate PKC
restricts the Ca2⫹ wave to only a few cells (see Fig. 3). In
many cell types, the principal effect of long-term TPA
exposure is a decrease in the number of gap junctions
and permanent intercellular communication loss (e.g.,
Refs. 1, 2, 9, 37). Downregulation of PKC in astroglial
cells (by chronic TPA treatment) decreased but did not
eliminate mechanically induced Ca2⫹ wave propagation (12). Reduction of gap junction proteins may play a
role in communication loss; however, it should also be
pointed out that [Ca2⫹]i increases induced by ATP
binding were also reduced after long-term TPA treatment.
Gap junction proteins are not directly involved in the
cellular response to ATP; therefore, PKC-dependent
inhibition of gap junctional permeability could not
cause the observed decrease in ATP-induced [Ca2⫹]i
oscillations. The TPA and DOG inhibition of ATPinduced [Ca2⫹]i oscillations could be due to PKCdependent inhibition of airway epithelial cell
Ins(1,4,5)P3 and/or DAG generation. Bird et al. (4) have
shown that PKC-dependent negative feedback on ligand-induced Ins(1,4,5)P3 and/or DAG production in
mouse lacrimal acinar cells is important in generating
constant-frequency [Ca2⫹]i oscillations (for a review, see
Ref. 36). Similar results and conclusions were obtained
for ATP-induced [Ca2⫹]i oscillations in chicken granulosa cells (26). For the lacrimal acinar cells (4), Ca2⫹
release mechanisms were not implicated in generating
the oscillations because injection of Ins(1,4,5)P3 directly into the cytoplasm of the lacrimal cells increased
[Ca2⫹]i but did not generate oscillations. A similar
result was obtained when Ins(1,4,5)P3 was injected into
airway epithelial cells in monolayer cultures (32):
PKC ROLE IN CA2⫹ WAVES AND OSCILLATIONS IN EPITHELIA
In summary, from our data on mechanically induced
Ca2⫹ waves and ATP-induced oscillations, we suggest
that stimulus-dependent and/or Ins(1,4,5)P3-dependent Ca2⫹ mobilization can be influenced by DAGactivated PKC by four mechanisms in airway epithelial
cells. First, PKC promotes Ca2⫹ influx, which can
positively affect the Ca2⫹ mobilization. Second, PKC
negatively influences generation of the mobilization
messenger Ins(1,4,5)P3 and the PKC activator DAG.
Third, PKC inhibits storage membrane Ca2⫹-ATPase
(or promotes Ca2⫹ leak), and fourth, PKC may inhibit
gap junctional-mediated intercellular communication.
We thank Jennifer Felix for technical support and preparing the
tissue cultures and Andrew Charles for comments on the manuscript.
This work was supported by a grant from the National Aeronautics
and Space Administration Microgravity Research and from a grant
from the State of California Tobacco-Related Disease Research
Program of the University of California.
Address for reprint requests and other correspondence and present address of M. L. Woodruff: Dept. of Physiological Sciences, PO
Box 951527, UCLA, Los Angeles, CA 90095-1527 (E-mail:
michaelw@physci.ucla.edu).
Received 25 August 1998; accepted in final form 7 January 1999.
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TPA-induced PKC activation slowed and both Gö 6976and BIM-induced PKC inhibition seemed to hasten
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However, another PKC activator, DOG, did not slow the
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this conflict.
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