Ca2+ and protein kinase C
activation of mucin granule exocytosis in permeabilized SPOC1
cells
C. E.
Scott,
Lubna H.
Abdullah, and
C. William
Davis
Cystic Fibrosis/Pulmonary Research and Treatment Center and the
Department of Physiology, University of North Carolina, Chapel Hill,
North Carolina 27599-7248
 |
ABSTRACT |
Mucin secretion by airway goblet cells is under the control of
apical P2Y2, phospholipase
C-coupled purinergic receptors. In SPOC1 cells, the mobilization of
intracellular Ca2+ by ionomycin or
the activation of protein kinase C (PKC) by phorbol 12-myristate
13-acetate (PMA) stimulates mucin secretion in a fully additive fashion
[L. H. Abdullah, J. D. Conway, J. A. Cohn, and C. W. Davis.
Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17):
L201-L210, 1997]. This apparent independence between PKC and
Ca2+ in the stimulation of mucin
secretion was tested in streptolysin O-permeabilized SPOC1 cells. These
cells were fully competent to secrete mucin when
Ca2+ was elevated from 100 nM to
3.1 µM for 2 min following permeabilization; the
Ca2+
EC50 was 2.29 ± 0.07 µM.
Permeabilized SPOC1 cells were exposed to PMA or 4
-phorbol at
Ca2+ activities ranging from 10 nM
to 10 µM. PMA, but not 4
-phorbol, increased mucin release at all
Ca2+ activities tested: at 10 nM
Ca2+ mucin release was 2.1-fold
greater than control and at 4.7 µM Ca2+ mucin release was maximal
(3.6-fold increase). PMA stimulated 27% more mucin release at 4.7 µM
than at 10 nM Ca2+. Hence, SPOC1
cells possess Ca2+-insensitive,
PKC-dependent, and Ca2+-dependent
PKC-potentiated pathways for mucin granule exocytosis.
lung; airways; mucus; goblet cells; cellular
regulation
 |
INTRODUCTION |
CALCIUM AND protein kinase C (PKC), the active
effectors of the phospholipase C (PLC) signal transduction system, have
been implicated widely in the control of regulated exocytosis. In
contrast to the apparently universal activation of the exocytotic
mechanism by Ca2+, the effects of
PKC vary by cell type: stimulatory, inhibitory, and modulatory effects
on exocytosis have been reported (see Ref. 31). For mucin-secreting
cells of the gastrointestinal tract (18-20) and the airways (1,
16), exocytosis is reported to be activated independently by
Ca2+ and PKC; we have tested this
apparent independence in a permeabilized cell model using SPOC1 cells,
a mucin-secreting cell line from the airways (2, 46).
Native airway goblet cells secrete mucin in response to the interaction
of purinergic agonists [ATP, UTP, adenosine
5'-O-(3-thiotriphosphate) (ATP
S)] with P2Y2
receptors (= P2U) as do primary
cultures of airway epithelial cells and SPOC1 cells (for review, see
Ref. 15). Consistent with the known coupling of this receptor class through heterotrimeric G proteins to PLC (10), primary cultures of
airway epithelial cells comprised predominantly of mucin-secreting cells release inositol phosphates on stimulation (28). Although intracellular Ca2+ levels have yet
to be determined in goblet cells, in SPOC1 cells agents known to
elevate intracellular Ca2+
(ionomycin, thapsigargin) also stimulate mucin secretion (1). Similarly, activation of PKC by phorbol 12-myristate 13-acetate (PMA)
elicits mucin secretion in several cultured airway cell models (24, 27,
35, 52, 53) and in SPOC1 cells (Ref. 1; see also Refs. 15, 16).
Notably, the mucin secretory response to ionomycin and PMA was fully
additive at maximal doses in SPOC1 cells. Downregulation of PKC by
overnight exposure to a half-maximal dose of PMA abolished the ability
of SPOC1 cells to respond to maximal doses of either PMA or UTP, but
they responded maximally to ionomycin (1). These results suggest that
Ca2+ and PKC are independent in
their actions, and, in the experiments reported here, we test whether
PMA is effective in promoting mucin secretion in permeabilized cells
where Ca2+ is controlled by an
exogenous Ca2+ buffer system.
 |
MATERIALS AND METHODS |
Materials.
Culture medium was purchased from GIBCO BRL (Gaithersburg, MD), and the
supplements were from Collaborative Research (Bedford, MA). Nucleotides
were purchased from Boehringer Mannheim (Indianapolis, IN),
streptolysin O (SLO) was from Murex Diagnostics (Norcross, GA), and
TO-PRO was from Molecular Probes (Eugene, OR). All other chemicals were
purchased from Sigma Chemical (St. Louis, MO).
SPOC1 cell culture and mucin secretion.
SPOC1 cells, passages 7-15, were
seeded at densities of 18,000 cells/well in 48-well cluster plates
(Costar, Cambridge, MA) and were grown in a DMEM-F12-based culture
medium described previously (26, 46). Briefly, the medium was
supplemented with 30 mM HEPES, 6.5 mM
L-glutamine, 10 µg/ml insulin,
0.1 µg/ml hydrocortisone, 0.1 µg/ml cholera toxin, 5 µg/ml
transferrin, 50 µM phosphoethanolamine, 80 µM ethanolamine, 25 ng/ml epidermal growth factor, 1% vol/vol bovine pituitary extract, 1 mg/ml bovine serum albumin (essentially globulin-free, Sigma no.
A7638), 50 U/ml penicillin, and 50 µg/ml streptomycin. Except for
cells grown solely for passaging, the medium also contained 10 nM
retinoic acid. Culture media were changed daily, and the cultures were
used for experiments 6-12 days postconfluence.
SPOC1 cell mucin secretion and enzyme-linked lectin assay.
Before all experiments, SPOC1 cells were removed from the incubator,
washed twice in DMEM-F12, and incubated at 35°C for 30 min; this
procedure was repeated twice for a total equilibration period of 90 min. To study the response of intact cells to nucleotide agonist
challenges, SPOC1 cells were subsequently exposed to UTP or ATP
S
over a wide range of concentrations, each dose in triplicate, during a
single 30-min incubation. Other details of these experiments are given
in RESULTS.
Samples of media removed from the wells of the cluster plate were
assessed for mucin content by enzyme-linked lectin assay (2): 100-µl
samples were bound to 96-well high-binding microtiter plates (Costar
no. 3590) with an overnight or a 2-h incubation at 4 or 37°C,
respectively. The plates were washed (Dynatech MR5000; Chantilly, VA)
with PBS containing 0.05% Tween 20 and 0.02% thimerosal, blocked with
gelatin, and incubated with 1-5 µg/ml horseradish peroxidase-conjugated soybean agglutinin for 1 h at 37°C. After plates were washed and developed (incubation in 0.04% wt/vol of the
substrate, O-phenylenediamine in
0.0175 M citrate-phosphate buffer, pH 5.0, containing 0.01% hydrogen
peroxide), the reaction was stopped by the addition of 4 M sulfuric
acid and optical density was determined at 490 nm (Dynatech model
MR5000 microtiter plate reader). Optical density was converted to
nanograms mucin from a standard curve using known amounts of purified
SPOC1 mucin (2); mucin standards were applied to each microtiter plate
assessed.
Cell permeabilization and
Ca2+ buffering
system.
Differentiated SPOC1 cells were permeabilized, as epithelial sheets in
the bottom of 48-well cluster plates, by SLO (22). SLO was resuspended
at 3 U/ml in intracellular buffer
(Bufi), which had a
final composition (in mM) of 130 potassium glutamate, 20 PIPES, 1.0 MgATP, and 3.0 total EGTA; the pH was adjusted to 6.8, and pCa was
adjusted to a desired activity by the addition of CaEGTA.
Free Ca2+ levels were calculated
with the aid of the computer program Chelator (49); although these
activities were calculated in log units of pCa, for convenience, they
are expressed in nanomolar or micromolar in the
RESULTS and
DISCUSSION. Stock solutions of EGTA
and CaEGTA, nominally 50 mM, were made according to the
Ca2+-buffering system of Gomperts
and Tatham (22). EGTA concentrations were determined by titration (38),
using a Ca2+ solution made from a
freshly opened bottle of
CaCl2 · 2H2O;
aliquots of these stocks were stored at
20°C. Rapid solution
changes during the cell permeabilization procedure were facilitated
with a Finnpipette multistepper pipetter (Needham Heights, MA; using 6 of the 8 tips) and a six-tip vacuum manifold fabricated from Delrin and
disposable, plastic 100-µl pipette tips. With these tools, the medium
in a 48-well cluster plate could be removed and replaced, consistently, in <10 s.
After equilibration (described above), the cells were washed in rapid
succession, twice in PBS (400 µl) and twice in pCa 7.0 Bufi (400 µl). Unless stated
otherwise, the cells were then permeabilized by a 30-s incubation in
pCa 7.0 Bufi containing 1 U/ml SLO
(150 µl), subsequently washed twice in pCa 7.0 Bufi (150 µl), and then incubated in a solution for a time appropriate to the experiment (described in RESULTS). In most
experiments, this medium was then removed and assessed for mucin
content. Last, the degree of cell permeabilization following each
experiment was assessed using the membrane-impermeant, DNA-staining dye
TO-PRO: the cells were washed with PBS before and after a 10-min
incubation in TO-PRO (10 µM in PBS), fixed in 3% formaldehyde in PBS
for 10 min, washed, and covered with 200 µl of PBS-glycerol (1:1).
Nuclear fluorescence was viewed by video microscopy (Hamamatsu C5985
charge-coupled device camera mounted on a Leica IM/DRB microscope),
using a ×5 objective to visualize the central portion of each
well. In experiments characterizing the permeabilization procedure,
fluorescence intensity was quantified by acquiring an image of each
well at constant camera gain and integration period (determined for the
brightest well on the plate) and determining the full-frame integrated
pixel intensity of each image using a MetaMorph image analysis
workstation (Universal Imaging, West Chester, PA). In all other
experiments, each well in a plate was checked by fluorescence
microscopy to confirm that the cells had been permeabilized, as
expected.
 |
RESULTS |
SPOC1 cells grown in 48-well cluster plates.
The spatial variation in mucin secretion and production by intact SPOC1
cells grown on 48-well plates was tested by determining the quantity of
mucin released during 40-min basal and agonist-stimulated periods (100 µM ATP
S) and that in the remaining intracellular pool (lysis in
hypotonic buffer: 1 mM CaCl2, 1 mM
MgCl2, 20 mM TES, pH 7.4). The
mucins released during the basal and stimulated periods and the total
cellular mucin (= basal + stimulated + lysis) are shown for three SPOC1
cell passages in Table 1. Consistent with
previous results (2), the cells responded to ATP
S with a 3.2-fold
increase in mucin secretion; this mucin represented 31.1% of the total
cellular mucin pool. Well-to-well variation in mucin release and
content, determined as the "coefficient of variation," was low
for total mucin production (7.1%), moderately higher for the mucins
secreted during purinergic stimulation (12.1%), and highest for basal
secretion (18%).
The behavior of SPOC1 cells grown in 48-well cluster plates was also
tested by determining the effects of two purinergic agonists on mucin
secretion. Dose-response curves for UTP and ATP
S were constructed
from single plates of SPOC1 cells, with each dose tested in triplicate.
As shown in Fig. 1, these
curves were sigmoidal; the EC50
was 3-4 µM, and the mucin secretory response saturated above 30 µM, consistent with previous results (2). Together, these two studies
show that the cells grown in 48-well cluster plates possess reasonably
uniform well-to-well cellular mucin pools and agonist responses, making
them good candidates for permeabilization experiments.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Purinergic agonist activation of mucin secretion in intact SPOC1 cells.
Cells were grown on two 48-well cluster plates and after equilibration
were exposed to the indicated doses of agonist for 40 min, with each
dose tested in triplicate. Quantity of mucin released into the medium
was determined by enzyme-linked lectin assay; mean values for each dose
are presented.
|
|
SLO permeabilization.
In extensive experiments not shown, various SLO permeabilization
protocols were tested on SPOC1 cells. These attempts included a SLO
exposure and wash at 4°C, followed by the cells being rewarmed such
that the permeabilization step occurred after unbound SLO and potential
contaminants in the material provided by the manufacturer were removed
from the medium (34). The only procedure attempted that produced a
consistent activation of mucin release by high Ca2+, however, was a brief
exposure to SLO in pCa 7.0 Bufi
(100 nM Ca2+) at 35°C
followed by an immediate wash. Figure 2
depicts the 35°C dose-permeabilization effects of SLO on SPOC1
cells, including videomicrographs of TO-PRO-stained cells at selected
doses. The low molecular weight (MW) dye, TO-PRO (MW 645), was chosen
as a marker of permeabilization because it is similar to EGTA (MW 380)
in size and its fluorescence excitation and emission spectra are
similar to fluorescein. As shown in Fig. 2, SLO effectively permeabilized the cells to TO-PRO at doses above 0.1 U/ml. The clustering pattern of nuclear fluorescence that is observed at 0.3 and
1 U/ml SLO is consistent with the pattern of SPOC1 cell differentiation
that occurs in culture (2, 17, 46). That is, the cells grow as
extensive patches of multilayered cells, with cells in the outermost
layers containing mucin secretory granules. The more uniform staining
observed at 3 U/ml reflects the permeabilization of cells in areas of
the well occupied by those cells growing as a single layer in contact
with the plastic substratum. These cells, and their adjacent
counterparts in the multilayered areas, resemble the basal cells of
pseudostratified airway epithelia.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 2.
Streptolysin O (SLO) permeabilization of SPOC1 cells. Cells were
exposed to indicated doses of SLO in pCa 7.0 intracellular buffer
(Bufi; 100 nM) at 35°C for 10 min and then stained with TO-PRO. Fluorescence from the central portion
of each well in 48-well cluster plates was imaged by a ×5
objective, acquired by a cooled charge-coupled device video camera, and
quantified as the full-frame, integrated pixel intensity.
Insets: sample images for selected SLO
doses. Each dose was tested in triplicate on each plate, and data are
presented as means ± SE (n = 3 SPOC1 cell passages).
|
|
The time courses of SLO permeabilization to TO-PRO at 1 and 3 U/ml are
shown in Fig. 3. TO-PRO fluorescence
increased rapidly with increasing SLO exposure time at both doses to a
pseudo-plateau between 30 and 60 s at 70-80% of maximal. Longer
exposures resulted in only moderately higher levels of TO-PRO
fluorescence.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Time course of SLO permeabilization of SPOC1 cells. Cells were
incubated at 1 or 3 U/ml SLO in pCa 7.0 Bufi (100 nM), in triplicate, at
35°C for time periods indicated and then washed and incubated for
10 min in TO-PRO in the same buffer. After fixation, cellular
fluorescence was quantified as described in Fig. 2. Data are presented,
normalized to the maximal fluorescence on each plate, as means ± SE
(n = 3 SPOC1 cell passages).
|
|
Mucin release in permeabilized SPOC1 cells.
In preliminary experiments,
Ca2+-dependent mucin release was
maximal following a 30-s exposure of SPOC1 cells to 1 U/ml SLO; a
higher SLO dose (3 U/ml) or a longer exposure time (60 s) resulted in a
reduced amount of mucin released to the bath (data not shown). Figure
4 shows the results of experiments designed
to establish two important parameters related to permeabilization with
a 30-s, 1 U/ml SLO protocol, namely, the postpermeabilization period of time necessary to achieve maximal mucin release and recovery and the
period of time SPOC1 cells were capable of supporting
Ca2+-dependent mucin release
following permeabilization. The data show first that a 10- to 15-min
incubation in high Ca2+ (3.2 µM)
was necessary to achieve a maximal release of mucin to, and recovery
from, the medium (Fig. 4A). Second,
the permeabilized cells were fully competent to secrete mucin in
response to a high Ca2+ stimulus
for a minimum of 2 min. When the cells were held at 100 nM
Ca2+ for longer periods, they
exhibited a reduced response to the subsequent high
Ca2+ stimulus such that after 10 min the cells released ~50% of the amount of mucin released by cells
stimulated by high Ca2+
immediately following permeabilization. This 2-min window of full
secretory competency for permeabilized SPOC1 cells is narrower than
that observed in other permeabilized cell models (see
DISCUSSION); however, it was
sufficiently broad for the purposes of the experiments reported herein.
That the cells were capable of maximal secretion for 2 min following
permeabilization, and yet 10-15 min were required to achieve a
maximal recovery of mucin after stimulation by high Ca2+, suggests that the relatively
longer time requirement for mucin recovery resulted from a need for
secreted mucins to hydrate, temper, and then solubilize into the medium
(see Refs. 12, 51).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Temporal parameters for
Ca2+-dependent mucin release in
permeabilized SPOC1 cells. In one-half of each plate
(A), immediately following the pCa
7.0 (100 nM) postpermeabilization wash,
Ca2+ was elevated to pCa 5.5 (3.2 µM); cells were incubated for the indicated time intervals, and then
the media were harvested for mucin determinations. Cells in the other
half of each plate (B) were washed
after permeabilization and held at pCa 7.0 (100 nM) for the indicated
time intervals, and then pCa was elevated to 5.5 (3.2 µM) for a
constant 10-min incubation. Data were normalized to the maximal amount
of mucin released on each plate and are presented as means ± SE
(n = 3 SPOC1 cell passages).
|
|
As expected from the presence of
P2Y2 receptors on SPOC1 cell
apical membranes, interpretation of the data on mucin release from
SLO-permeabilized cells was complicated by the millimolar levels of ATP
contained in Bufi. Figure
5 shows the results of an experiment in
which SPOC1 cells were exposed to 0.1-3 U/ml SLO followed by a
Bufi incubation with
Ca2+ ranging from 100 nM to 3.2 µM. Note that mucin release was highest in the cells exposed to the
lowest dose of SLO; because the cells were mostly intact at 0.1 U/ml
SLO (Fig. 2), mucin release at this dose was due to the activation of
P2Y2 receptors by ATP (Fig. 1;
Ref. 2), with the subsequent activation of PLC, release of
Ca2+ from internal stores, and the
production of diacylglycerol (DAG). At 0.3 U/ml SLO, at which an
intermediate degree of permeabilization occurs (Fig. 2), the mucins
released at all Ca2+ activities
were reduced relative to the 0.1 U/ml dose of SLO. Mucin release with
0.3 U/ml SLO was the lowest at the lower
Ca2+ activities (100 and 316 nM),
thereby indicating some degree of Ca2+ specificity in the overall
secretory response. At the full levels of permeabilization produced by
1 and 3 U/ml SLO, SPOC1 cells exhibited a clear
Ca2+-dependent mucin-secretory
response: at low Ca2+ (100 and 316 nM) mucin release was greatly diminished, and at high levels of
Ca2+ it was stimulated.
Presumably, the high degree of permeabilization achieved with these
doses of SLO allowed efficient intracellular buffering of
Ca2+, such that the cells
responded to the free Ca2+
controlled by the EGTA-buffer system rather than to ATP activation of
P2Y2 receptors. Last, note in Fig.
5 that SPOC1 cells permeabilized with 1 U/ml SLO achieved a release of
mucin at 3.2 µM Ca2+, which was
82.9% of that secreted by the agonist-stimulated cells (0.1 U/ml SLO),
showing that the permeabilization procedure had relatively minor
effects on the ability of the cells to secrete.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Mucin release by SPOC1 cells permeabilized with different doses of SLO.
After a 30-s permeabilization at pCa 7.0 (100 nM) with indicated doses
of SLO, cells were washed and incubated at the indicated
Ca2+ activities for 15 min; mucins
released to the bath were then quantified. Data were normalized to the
maximal amount of mucin on each plate and are presented as means ± SE (n = 3 SPOC1 cell passages). Note
that mucin release at all Ca2+
activities was highest at the lowest doses of SLO (see text for
explanation).
|
|
Ca2+-dependent
and PKC-dependent mucin release.
The effects of Ca2+ on mucin
release in permeabilized SPOC1 cells was investigated in a fine-grain,
Ca2+ dose-response study on cells
grown in single 48-well cluster plates over four separate passages.
Figure 6, depicting an individual result,
shows that relative to control of 100 nM
Ca2+ mucin release by
permeabilized SPOC1 cells incubated at 10 nM Ca2+ was moderately inhibited (36 ± 12%, n = 4). This apparent
reduction in mucin release at lower than normal
Ca2+ activities, however, was not
always observed. In other experiments, there was no difference between
the mucins released at 10 and 100 nM
Ca2+ (e.g., see Figs. 5 and 7).
Mucin release in these studies was slightly increased at
Ca2+ activities between 100 nM and
1 µM Ca2+, strongly stimulated
above 1 µM Ca2+, and saturated
above 10 µM Ca2+, with a maximal
2.49 ± 0.55-fold increase over control. The
Ca2+
EC50 for the mucin secretory
response was 2.29 ± 0.07 µM.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6.
Ca2+-dependent mucin release in
permeabilized SPOC1 cells. Result is shown of an individual experiment
in which SPOC1 cells were permeabilized with 1 U/ml SLO (30 s), washed,
and incubated for 15 min in Bufi
containing the indicated Ca2+
activities. Data are presented as mean values of triplicate wells,
normalized to the amount of mucins released at pCa 7.0 (100 nM).
Ca2+-dependent waveform depicted
is typical of 4 experiments; summary data are given in
RESULTS.
|
|
To test whether PKC activation of mucin granule exocytosis is dependent
on or independent of Ca2+, SPOC1
cells grown in pairs of 48-well plates were permeabilized and incubated
at Ca2+ activities ranging from 10 nM to 10 µM. One-half of the cells on one plate was exposed to a
maximal dose of PMA (300 nM; Ref. 1), whereas one-half of the cells on
the other plate was exposed to the same concentration of 4
-phorbol,
an inactive phorbol ester. PMA stimulated mucin release in
permeabilized SPOC1 cells over the entire range of
Ca2+ activities (Fig.
7). At 10 nM
Ca2+, which is approximately
one-tenth of basal intracellular
Ca2+ levels in most cells, mucin
release was stimulated 2.1-fold over the 100 nM
Ca2+ control. This stimulation by
PMA, in fact, was as strong as the maximal
Ca2+-dependent response, a
2.1-fold stimulation at 10 µM
Ca2+. As
Ca2+ stimulated mucin granule
exocytosis at activities >1 µM, the PMA response was potentiated.
At 4.7 µM Ca2+, PMA-stimulated
cells released 27% more mucin relative to their paired controls than
at 10 nM Ca2+
(P < 0.05, paired
t-test); the
Ca2+
EC50 for cells exposed to PMA was
1.0 µM compared with 2.2 µM for the paired control. The PMA
response at 2.1 µM Ca2+ appeared
to be diminished relative to the maximal levels recorded at 4.7 and 10 µM, but it was still significantly higher than its paired control.
Because 4
-phorbol had no discernible effect at any
Ca2+ activity, the PMA effects
appeared to be specific and were presumably due to PKC activation.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of phorbol 12-myristate 13-acetate (PMA) on mucin release in
permeabilized SPOC1 cells. After permeabilization, one-half of the
SPOC1 cells on each 48-well cluster plate was treated with 300 nM
4 -phorbol ( ) or PMA ( ) and the other half served as control
(open symbols). Cells were exposed to the indicated range of
Ca2+ activities, in triplicate,
for a 15-min incubation. Mucin released in each well was normalized to
that released under control conditions (pCa 7.0 or 100 nM), and data
are presented as means ± SE (n = 4 SPOC1 cell passages). For clarity, SE bars not shown for the
4 -phorbol control data. PMA values are all different from control,
P < 0.05, paired
t-test. Dotted line indicates a 2-fold
stimulation over the pCa 7.0 (100 nM) controls.
|
|
 |
DISCUSSION |
Regulated exocytosis by neurons and secretory cells has been the focus
of intense investigation over the past 20 years (recent reviews in
Refs. 4, 41, 57). These efforts were aided substantially by the
development of permeabilization techniques pioneered by Baker et al.
(7, 30) and Gomperts et al. (8, 21, 25), which allowed access to and
control of the intracellular milieu (see also Refs. 3, 9, 39). Many
permeabilized secretory cell models have been subsequently described
for study of the control of exocytosis by intracellular
Ca2+, PKC, nucleotides, and
specific proteins (e.g., see Refs. 11, 22, 23, 34). Pancreatic acini,
however, represent the only other permeabilized epithelial cell model
so developed, and this study with SPOC1 cells represents the first
model developed for an epithelium studied in a polarized configuration.
The bacterial toxin SLO was chosen as the permeabilization agent for
these studies to ensure efficient intracellular
Ca2+ buffering by EGTA. This toxin
binds plasma membrane cholesterol and then polymerizes to form
ring-shaped 24- to >30-nm pores that render the plasma membrane
permeable to organic molecules as large as lactate dehydrogenase (MW
140,000; Refs. 3, 50). Use of ionophores to control intracellular
Ca2+ is impractical because of the
difficulties in controlling the intracellular quantities of permeant
EGTA or related buffers. Use of
-toxin for this purpose is also
questionable because of restricted EGTA diffusivity through its 2- to
3-nm pores. In
-toxin-permeabilized gonadotropes, for instance, 30 mM CaEGTA buffers, or a 20-min preequilibration with 10 mM CaEGTA
buffers at 0°C, were required to effectively demonstrate
Ca2+-activated luteinizing hormone
release (6). Because efficient Ca2+ buffering was required in
these studies with SPOC1 cells to test the effects of PKC activation at
very low levels of intracellular Ca2+, we chose to use SLO.
Permeabilization was most efficient with short luminal SLO exposures of
SPOC1 cells at 35°C, followed by a rapid wash in
Bufi. These cells were fully
competent to secrete mucin in response to an elevation in
Ca2+ for 2 min following
permeabilization (Fig. 4). In other SLO-permeabilized cells, the period
of secretory competency is tens of minutes in duration (e.g., see Ref.
29, 55), and secretory activity in rundown cells can be restored
through the addition to the medium of cytosol (14, 47) or purified
proteins (37, 43, 44). Under optimal conditions (30-s exposure to 1 U/ml SLO; Figs. 3 and 5), and following a maximal activation by
Ca2+, permeabilized SPOC1 cells
released a quantity of mucin only ~20% less than that secreted by
intact cells following purinergic stimulation (Fig. 5). Thus, although
brief, the window of secretory competency for permeabilized SPOC1 cells
was sufficiently broad and the secretory response was sufficiently
robust to allow the experiments necessary to test for
Ca2+- and PKC-activated exocytotic
release of mucin.
Permeabilized SPOC1 cells exhibited a graded mucin secretory response
to increases in Ca2+ activity
(Figs. 5-7), with the initial responses occurring above 320 nM. In
preliminary measurements with intact SPOC1 cells, we have determined
basal Ca2+ activities to be
80-100 nM (data not shown). Consequently, these cells are similar
to virtually every other secretory cell studied in possessing a
secretory pathway activated by
Ca2+ at suprabasal levels. The
Ca2+
EC50 of this response in SPOC1
cells, 2.29 ± 0.07 µM, was in the same low micromolar range
described for most other permeabilized cells (e.g., see Refs. 13, 29,
33, 36, 40, 48). These data are consistent with the positive effects of
ionomycin and thapsigargin on intact SPOC1 cells (1), and together they
lend strong support for a role of
Ca2+ in mediating agonist
responses in airways mucin secreting cells (see also Ref. 16; cf. Ref.
32).
The effects of PKC activation in secretory cells are more varied than
the effects of Ca2+. At
Ca2+ activities below the nominal
100 nM basal intracellular Ca2+
levels, some cells are not affected by PMA or other PKC-activating reagents (i.e., pancreatic acini, Ref. 29; chromaffin cells, Ref. 30);
however, most secretory cells exhibit some degree of PKC-activated
exocytosis (e.g., gonadotropes, Ref. 36; PC-12 cells, Ref. 48). At 10 nM Ca2+, SPOC1 cells were
powerfully stimulated by PMA; the amount of mucin released in response
to PMA under these conditions was the same as that released maximally
by micromolar levels of Ca2+ (Fig.
7). Indeed, in the robustness of this PMA response at subbasal Ca2+ levels, SPOC1 cells stand out
from all other secretory cells studied.
In most other secretory cells, PMA has been shown to potentiate
Ca2+-dependent responses (e.g.,
PC-12 cells, Ref. 48; mast cells, Ref. 40). In SPOC1 cells, PMA had
slight synergism with Ca2+, with
the PMA-related increase in
Ca2+-dependent secretion being
27% greater than the effects of PMA at subbasal
Ca2+ (Fig. 7). Given the apparent
additivity of ionomycin and PMA in intact SPOC1 cells (1), this minor
degree of synergism between PKC and
Ca2+ is not surprising.
PKC has been shown in recent years to be a family of at least 11 isoforms that may be categorized into 3 or 4 subfamilies (for review,
see Ref. 42). Pertinent to this discussion are those isoforms activated
by phosphatidylserine and DAG or PMA, that is, the conventional or cPKC
isoforms (which are
Ca2+-dependent) and the novel or
nPKC isoforms (which are
Ca2+-insensitive) (PMA does not
activate the atypical isoforms or PKCµ). Because secretion in SPOC1
and other cells is activated by PMA at subbasal
Ca2+ levels, a likely possibility
is that nPKC isoforms will prove to be responsible for this effect.
Recent data in fact support this notion: nPKC isoforms have been
implicated in the agonist regulation of secretion for colonic cell
lines (24), pancreatic acini (45), and lachrymal glands (56), and
overexpression of nPKC
in GH4 cells leads to a selective increase in
basal prolactin secretion rates (5). In other secretory cells, however,
cPKC isoforms have been implicated in modulating secretion (e.g., RBL cells, Ref. 11). Hence, the PKC isoforms active in activating and/or modulating secretion are likely cell-type dependent.
In conclusion, the purinergic regulation of mucin secretion in SPOC1
cells appears to possess
Ca2+-independent, PKC-activated,
and PKC-potentiated Ca2+-dependent
pathways; in this regard, they are similar to many other nonepithelial
secretory cells but not to pancreatic acini. The identities of the PKC
isoforms responsible for
Ca2+-independent mucin secretion
and the degree of independence between these two pathways at the
molecular level require further investigation. For the
latter topic, a major question is whether multiple exocytotic mechanisms exist in a given secretory cell type or, alternately, whether the apparent independence between
Ca2+ and PKC lies with one or more
rate-limiting steps (e.g., cortical microfilaments; Ref. 54) situated
proximal to exocytotic docking sites.
 |
ACKNOWLEDGEMENTS |
We gratefully thank Drs. Peter Tatham and Bastien Gomperts for critical
advice and encouragement during these studies.
 |
FOOTNOTES |
This work was supported by a research grant from Glaxo Wellcome.
Address for reprint requests: C. W. Davis, 6009 Thurston-Bowles, CB
7248, Univ. of North Carolina, Chapel Hill, NC 27599.
Received 15 September 1997; accepted in final form 6 April 1998.
 |
REFERENCES |
1.
Abdullah, L. H.,
J. D. Conway,
J. A. Cohn,
and
C. W. Davis.
Protein kinase C and Ca2+ activation of mucin secretion in airway goblet cells.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L201-L210,
1997[Abstract/Free Full Text].
2.
Abdullah, L. H.,
S. W. Davis,
L. Burch,
M. Yamauchi,
S. H. Randell,
P. Nettesheim,
and
C. W. Davis.
P2u purinoceptor regulation of mucin secretion in SPOC1 cells, a goblet cell line from the airways.
Biochem. J.
316:
943-951,
1996[Medline].
3.
Ahnert-Hilger, G.,
W. Mach,
K. J. Fohr,
and
M. Gratzl.
Poration by alpha-toxin and streptolysin O: an approach to analyze intracellular processes.
Methods Cell Biol.
31:
63-90,
1989[Medline].
4.
Ahnert-Hilger, G.,
and
B. Wiedenmann.
Requirements for exocytosis in permeabilized neuroendocrine cells. Possible involvement of heterotrimeric G proteins associated with secretory vesicles.
Ann. NY Acad. Sci.
733:
298-305,
1994[Abstract].
5.
Akita, Y.,
S. Ohno,
Y. Yajima,
Y. Konno,
T. C. Saido,
K. Mizuno,
K. Chida,
S. Osada,
T. Kuroki,
S. Kawashima,
and
K. Suzuki.
Overproduction of a Ca(2+)-independent protein kinase C isozyme, nPKC epsilon, increases the secretion of prolactin from thyrotropin-releasing hormone-stimulated rat pituitary GH4C1 cells.
J. Biol. Chem.
269:
4653-4660,
1994[Abstract/Free Full Text].
6.
Augustine, G. J.,
M. E. Burns,
W. M. DeBello,
D. L. Pettit,
and
F. E. Schweizer.
Exocytosis: proteins and perturbations.
Annu. Rev. Pharmacol. Toxicol.
36:
659-701,
1996[Medline].
7.
Baker, P. F.,
and
D. E. Knight.
Calcium control of exocytosis and endocytosis in bovine adrenal medullary cells.
Philos. Trans. R. Soc. Lond. B Biol. Sci.
296:
83-103,
1981[Medline].
8.
Bennett, J. P.,
S. Cockcroft,
and
B. D. Gomperts.
Rat mast cells permeabilized with ATP secrete histamine in response to calcium ions buffered in the micromolar range.
J. Physiol. (Lond.)
317:
335-345,
1981[Abstract].
9.
Bhakdi, S.,
U. Weller,
I. Walev,
E. Martin,
D. Jonas,
and
M. Palmer.
A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes.
Med. Microbiol. Immunol. (Berl.)
182:
167-175,
1993[Medline].
10.
Brown, H. A.,
E. R. Lazarowski,
R. C. Boucher,
and
T. K. Harden.
Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5'-nucleotide receptor in human airway epithelial cells.
Mol. Pharmacol.
40:
648-655,
1991[Abstract].
11.
Buccione, R.,
G. Di Tullio,
M. Caretta,
M. R. Marinetti,
C. Bizzarri,
S. Francavilla,
A. Luini,
and
M. A. De Matteis.
Analysis of protein kinase C requirement for exocytosis in permeabilized rat basophilic leukaemia RBL-2H3 cells: a GTP-binding protein(s) as a potential target for protein kinase C.
Biochem. J.
298:
149-156,
1994[Medline].
12.
Carlstedt, I.,
J. K. Sheehan,
A. P. Corfield,
and
J. T. Gallagher.
Mucous glycoproteins: a gel of a problem.
Essays Biochem.
20:
40-76,
1985[Medline].
13.
Cazalis, M.,
G. Dayanithi,
and
J. J. Nordmann.
Requirements for hormone release from permeabilized nerve endings isolated from the rat neurohypophysis.
J. Physiol. (Lond.)
390:
71-91,
1987[Abstract].
14.
Cockcroft, S.,
J. P. Bennett,
and
B. D. Gomperts.
f-MetLeuPhe-induced phosphatidylinositol turnover in rabbit neutrophils is dependent on extracellular calcium.
FEBS Lett.
110:
115-118,
1980[Medline].
15.
Davis, C. W.
Goblet cells: physiology and pharmacology.
In: Airway Mucus: Basic Mechanisms and Clinical Perspectives, edited by D. F. Rogers,
and M. I. Lethem. Basel: Berkhauser, 1996.
16.
Davis, C. W.,
L. H. Abdullah,
and
R. C. Boucher.
Cellular basis for the purinergic regulation of mucin secretion in the airways.
In: Cilia, Mucus, and Mucociliary Interactions, edited by G. L. Baum. New York: Marcel Dekker, 1997, p. 153-166.
17.
Doherty, M. M.,
J. Liu,
S. H. Randell,
C. A. Carter,
C. W. Davis,
P. Nettesheim,
and
P. C. Ferriola.
Phenotype and differentiation potential of a novel rat tracheal epithelial cell line.
Am. J. Respir. Cell Mol. Biol.
12:
385-395,
1995[Abstract].
18.
Forstner, G.
Signal transduction, packaging and secretion of mucins.
Annu. Rev. Physiol.
57:
585-605,
1995[Medline].
19.
Forstner, G.,
Y. Zhang,
D. McCool,
and
J. Forstner.
Mucin secretion by T84 cells: stimulation by PKC, Ca2+, and a protein kinase activated by Ca2+ ionophore.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G1096-G1102,
1993[Abstract/Free Full Text].
20.
Forstner, G.,
Y. Zhang,
D. McCool,
and
J. Forstner.
Regulation of mucin secretion in T84 adenocarcinoma cells by forskolin: relationship to Ca2+ and PKC.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G606-G612,
1994[Abstract/Free Full Text].
21.
Gomperts, B. D.,
J. M. Baldwin,
and
K. J. Micklem.
Rat mast cells permeabilized with Sendai virus secrete histamine in response to Ca2+ buffered in the micromolar range.
Biochem. J.
210:
737-745,
1983[Medline].
22.
Gomperts, B. D.,
and
P. E. Tatham.
Regulated exocytotic secretion from permeabilized cells.
Methods Enzymol.
219:
178-189,
1992[Medline].
23.
Holz, R. W.,
M. A. Bittner,
and
R. A. Senter.
Regulated exocytotic fusion I: chromaffin cells and PC12 cells.
Methods Enzymol.
219:
165-178,
1992[Medline].
24.
Hong, D. H.,
J. F. Forstner,
and
G. G. Forstner.
Protein kinase C-
is the likely mediator of mucin exocytosis in human colonic cell lines.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G31-G37,
1997[Abstract/Free Full Text].
25.
Howell, T. W.,
and
B. D. Gomperts.
Rat mast cells permeabilised with streptolysin O secrete histamine in response to Ca2+ at concentrations buffered in the micromolar range.
Biochim. Biophys. Acta
927:
177-183,
1987[Medline].
26.
Kaartinen, L.,
P. Nettesheim,
K. B. Adler,
and
S. H. Randell.
Rat tracheal epithelial cell differentiation in vitro.
In Vitro Cell. Dev. Biol. Anim.
29A:
481-492,
1993.
27.
Kai, H.,
K. Yoshitake,
Y. Isohama,
I. Hamamura,
K. Takahama,
and
T. Miyata.
Involvement of protein kinase C in mucus secretion by hamster tracheal epithelial cells in culture.
Am. J. Physiol.
267 (Lung Cell. Mol. Physiol. 11):
L526-L530,
1994[Abstract/Free Full Text].
28.
Kim, K. C.,
Q. X. Zheng,
and
I. Van-Seuningen.
Involvement of a signal transduction mechanism in ATP-induced mucin release from cultured airway goblet cells.
Am. J. Respir. Cell Mol. Biol.
8:
121-125,
1993[Medline].
29.
Kitagawa, M.,
J. A. Williams,
and
R. C. De Lisle.
Amylase release from streptolysin O-permeabilized pancreatic acini.
Am. J. Physiol.
259 (Gastrointest. Liver Physiol. 22):
G157-G164,
1990[Abstract/Free Full Text].
30.
Knight, D. E.,
and
P. F. Baker.
Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields.
J. Membr. Biol.
68:
107-140,
1982[Medline].
31.
Knight, D. E.,
H. von Grafenstein,
and
C. M. Athayde.
Calcium-dependent and calcium-independent exocytosis.
Trends Neurosci.
12:
451-458,
1989[Medline].
32.
Ko, K. H.,
M. Jo,
K. McCracken,
and
K. C. Kim.
ATP-induced mucin release from cultured airway goblet cells involves, in part, activation of protein kinase C.
Am. J. Respir. Cell Mol. Biol.
16:
194-198,
1997[Abstract].
33.
Koopmann, W. R., Jr.,
and
R. C. Jackson.
Calcium- and guanine-nucleotide-dependent exocytosis in permeabilized rat mast cells. Modulation by protein kinase C.
Biochem. J.
265:
365-373,
1990[Medline].
34.
Larbi, K. Y.,
and
B. D. Gomperts.
Practical considerations regarding the use of streptolysin-O as a permeabilising agent for cells in the investigation of exocytosis.
Biosci. Rep.
16:
11-21,
1996[Medline].
35.
Larivee, P.,
S. J. Levine,
A. Martinez,
T. Wu,
C. Logun,
and
J. H. Shelhamer.
Platelet-activating factor induces airway mucin release via activation of protein kinase C: evidence for translocation of protein kinase C to membranes.
Am. J. Respir. Cell Mol. Biol.
11:
199-205,
1994[Abstract].
36.
Macrae, M. B.,
J. S. Davidson,
R. P. Millar,
and
P. A. van der Merwe.
Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium.
Biochem. J.
271:
635-639,
1990[Medline].
37.
Martin, T. F.,
and
J. H. Walent.
A new method for cell permeabilization reveals a cytosolic protein requirement for Ca2+-activated secretion in GH3 pituitary cells.
J. Biol. Chem.
264:
10299-10308,
1989[Abstract/Free Full Text].
38.
Miller, D. J.,
and
G. L. Smith.
EGTA purity and the buffering of calcium ions in physiological solutions.
Am. J. Physiol.
246 (Cell Physiol. 15):
C160-C166,
1984[Abstract/Free Full Text].
39.
Miller, S. G.,
and
H. P. Moore.
Movement from trans-Golgi network to cell surface in semiintact cells.
Methods Enzymol.
219:
235-248,
1992.
40.
Moller, K.,
D. Benz,
D. Perrin,
and
H. D. Soling.
The role of protein kinase C in carbachol-induced and of cAMP-dependent protein kinase in isoproterenol-induced secretion in primary cultured guinea pig parotid acinar cells.
Biochem. J.
314:
181-187,
1996[Medline].
41.
Morgan, A.
Exocytosis.
Essays Biochem.
30:
77-95,
1995[Medline].
42.
Newton, A. C.
Regulation of protein kinase C.
Curr. Opin. Cell Biol.
9:
161-167,
1997[Medline].
43.
O'Sullivan, A. J.,
A. M. Brown,
H. N. Freeman,
and
B. D. Gomperts.
Purification and identification of FOAD-II, a cytosolic protein that regulates secretion in streptolysin-O permeabilized mast cells, as a rac/rhoGDI complex.
Mol. Biol. Cell
7:
397-408,
1996[Abstract].
44.
Ozawa, K.,
Z. Szallasi,
M. G. Kazanietz,
P. M. Blumberg,
H. Mischak,
J. F. Mushinski,
and
M. A. Beaven.
Ca2+-dependent and Ca2+-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells.
J. Biol. Chem.
268:
1749-1756,
1993[Abstract/Free Full Text].
45.
Pollo, D. A.,
J. J. Baldassare,
T. Honda,
P. A. Henderson,
V. D. Talkad,
and
J. D. Gardner.
Effects of cholecystokinin (CCK) and other secretagogues on isoforms of protein kinase C (PKC) in pancreatic acini.
Biochim. Biophys. Acta
1224:
127-138,
1994[Medline].
46.
Randell, S. H.,
J. Y. Liu,
P. C. Ferriola,
L. Kaartinen,
M. M. Doherty,
C. W. Davis,
and
P. Nettesheim.
Mucin production by SPOC1 cells
an immortalized rat tracheal epithelial cell line.
Am. J. Respir. Cell Mol. Biol.
14:
146-154,
1996[Abstract].
47.
Sarafian, T.,
D. Aunis,
and
M. F. Bader.
Loss of proteins from digitonin-permeabilized adrenal chromaffin cells essential for exocytosis.
J. Biol. Chem.
262:
16671-16676,
1987[Abstract/Free Full Text].
48.
Schafer, T.,
U. O. Karli,
E. K. Gratwohl,
F. E. Schweizer,
and
M. M. Burger.
Digitonin-permeabilized cells are exocytosis competent.
J. Neurochem.
49:
1697-1707,
1987[Medline].
49.
Schoenmakers, T. J.,
G. J. Visser,
G. Flik,
and
A. P. Theuvenet.
CHELATOR: an improved method for computing metal ion concentrations in physiological solutions.
Biotechniques
12:
870-879,
1992[Medline].
50.
Sekiya, K.,
H. Danbara,
K. Yase,
and
Y. Futaesaku.
Electron microscopic evaluation of a two-step theory of pore formation by streptolysin O.
J. Bacteriol.
178:
6998-7002,
1996[Abstract].
51.
Sheehan, J. K.,
D. J. Thornton,
M. Somerville,
and
I. Carlstedt.
Mucin structure. The structure and heterogeneity of respiratory mucus glycoproteins.
Am. Rev. Respir. Dis.
144:
S4-S9,
1991[Medline].
52.
Shimura, S.,
H. Ishihara,
M. Nagaki,
H. Sasaki,
and
T. Takishima.
A stimulatory role of protein kinase C in feline tracheal submucosal gland secretion.
Respir. Physiol.
93:
239-247,
1993[Medline].
53.
Steel, D. M.,
and
J. W. Hanrahan.
Muscarinic-induced mucin secretion and intracellular signaling by hamster tracheal goblet cells.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L230-L237,
1997[Abstract/Free Full Text].
54.
Trifaro, J. M.,
M. L. Vitale,
and
A. Rodriguez Del Castillo.
Cytoskeleton and molecular mechanisms in neurotransmitter release by neurosecretory cells.
Eur. J. Pharmacol.
225:
83-104,
1992[Medline].
55.
Yamamoto, T.,
Y. Furuki,
S. Guild,
and
J. W. Kebabian.
Adenosine 3',5'-cyclic monophosphate stimulates secretion of alpha-melanocyte-stimulating hormone from permeabilized cells of the intermediate lobe of the rat pituitary gland.
Biochem. Biophys. Res. Commun.
143:
1076-1084,
1987[Medline].
56.
Zoukhri, D.,
R. R. Hodges,
C. Sergheraert,
A. Toker,
and
D. A. Dartt.
Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion.
Am. J. Physiol.
272 (Cell Physiol. 41):
C263-C269,
1997[Abstract/Free Full Text].
57.
Zucker, R. S.
Exocytosis: a molecular and physiological perspective.
Neuron
17:
1049-1055,
1996[Medline].
Am J Physiol Cell Physiol 275(1):C285-C292
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society