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
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ABSTRACT |
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
13-acetate- 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.
protein kinase C; adenosine 5'-triphosphate; mechanotransduction; purinergic receptor; phospholipase C
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INTRODUCTION |
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 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.
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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 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 inverted-stage Nikon
Diaphot microscope equipped with a ×40 oil-immersion,
1.3-numerical aperture objective with quartz optical elements. The
excitation source was a 100-W mercury lamp. The cells were
alternatively illuminated through 340- or 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
(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.
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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 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).

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Fig. 1.
12-O-tetradecanoylphorbol 13-acetate
(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.
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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.
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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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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
PKC-downregulated cells as evidenced by thapsigargin-induced 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 similar 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
6976-treated 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 mechanical
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).

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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.
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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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Table 1.
Effects of PKC activators and inhibitors on mechanically induced
Ca2+ wave in stimulated, secondary, and tertiary cells
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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 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 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) 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.

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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.
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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 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 occurred
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 TPA-treated cells showed no oscillations. The
reduced response of the TPA-treated cells to ATP addition may be due to a prior activation of a putative PKC-dependent desensitization mechanism (possibly limiting releasable
Ca2+; see
DISCUSSION); however, the parallel
time-dependent decreases in control and TPA-treated cells (Fig.
7D) suggest that they are regulated
similarly on ATP addition.

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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
ATP-induced
[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.
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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 TPA-treated 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 set of cells was 330 ± 46 nM for the control cells and 240 ± 45 nM for the TPA-treated cells (Fig.
8C).

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|
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 TPA- and 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.
|
|
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 thapsigargin-induced [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) 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 |
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
stimulus-dependent
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 inhibitor-induced 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 ATP-induced
[Ca2+]i
oscillations could be due to PKC-dependent 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):
[Ca2+]i
was elevated, but oscillations were not induced. Data presented in
Figs. 6 and 7 are consistent with constant-frequency oscillations that
use PKC feedback inhibition of
Ins(1,4,5)P3
and/or DAG generation.
Relevant to the goals of this work is whether negative feedback on
Ins(1,4,5)P3
production is part of the response of the cells to mechanical
stimulation. The above arguments suggest that airway cells contain the
mechanism for negative feedback and that it is used in the ATP pathway
to generate oscillations. The effect of Gö 6976 on mechanical
stimulation (slowing the rate of spread; Fig. 4) and the lack of an
effect on ATP-induced oscillations suggest that the different stimuli
activate different PKC isoforms. Whether mechanoreceptors use negative
feedback regulation is an open question. It could be argued that, if
they did use negative feedback, mechanical stimulation might generate
[Ca2+]i
oscillations in the directly stimulated cell instead of the apparently
smooth, nonoscillatory
[Ca2+]i
increase that is normally observed. Unfortunately, this issue is
complicated; mechanical stimulation also induces influx of Ca2+ from the medium (7, 8). When
these channels are blocked, mechanically induced oscillations can be
observed (S. Boitano, personal communication). We are
presently testing the effect of PKC activators and inhibitors on the
mechanically induced oscillations that occur in the presence of
Ca2+-channel blockers.
The TPA- or DOG-induced decrease in the rate of
Ca2+ release and the decrease in
the amplitude of the
[Ca2+]i
increase after thapsigargin treatment (see Fig. 8) indicate that PKC
can target proteins associated with intracellular
Ca2+ storage. Ribeiro and Putney
(28) recently obtained a similar result in NIH/3T3 cells. They also
showed that TPA reduced Ca2+
releasable by ionomycin and reduced
45Ca2+
accumulation, suggesting that the decrease in
Ca2+ released with inhibitors of
Ca2+-ATPase was caused by a
PKC-dependent decrease in Ca2+
storage capacity. The shape of the
Ca2+-release curves shown in Fig.
8, A and
B, is consistent with this interpretation. It appears that the absolute amount of releasable Ca2+ is reduced rather than there
being a direct inhibition of transport proteins. A PKC-induced
inhibition of the Ca2+-ATPase or
activation of Ca2+ leakage could
lead to a storage decrease. A decrease in the release of
Ca2+ may be part of the inhibitory
action of PKC on ATP-induced
[Ca2+]i
oscillations and in mechanically induced
[Ca2+]i
increases. A decrease in Ca2+
release would be, by itself, insufficient to limit the extent of the
mechanically induced Ca2+ wave,
which may depend on the diffusion of
Ins(1,4,5)P3 from cell to cell (34, 35). Relevant to this discussion is whether PKC-effective agents influence the rate of return of
[Ca2+]i
to basal levels after mechanical stimulation. TPA-induced PKC activation slowed and both Gö 6976- and BIM-induced PKC
inhibition seemed to hasten recovery of
[Ca2+]i
to prestimulus levels. This is consistent with PKC-dependent inhibition
of Ca2+-ATPase. However, another
PKC activator, DOG, did not slow the [Ca2+]i
recovery. Additional experiments may resolve this conflict.
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 DAG-activated 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.
 |
ACKNOWLEDGEMENTS |
We thank Jennifer Felix for technical support and preparing the
tissue cultures and Andrew Charles for comments on the manuscript.
 |
FOOTNOTES |
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.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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).
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{at}physci.ucla.edu).
Received 25 August 1998; accepted in final form 7 January 1999.
 |
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