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Am J Physiol Lung Cell Mol Physiol 276: L669-L678, 1999;
1040-0605/99 $5.00
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Vol. 276, Issue 4, L669-L678, April 1999

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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

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, 4alpha -phorbol-12,13-didecanoate (4alpha -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 (approx 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, 4alpha -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; 4alpha -phorbol, 4alpha -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.

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

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.

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.

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 4alpha -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 4alpha -phorbol and the PKC inhibitors (BIM and Gö 6976) did not significantly influence the thapsigargin-induced release.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 276(4):L669-L678
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