Pepsinogen secretion: coupling of exocytosis visualized by
video microscopy and
[Ca2+]i
in single cells
Chie
Tao1,
Masao
Yamamoto2,
Hiroshi
Mieno1,
Masaki
Inoue1,
Tsutomu
Masujima3, and
Goro
Kajiyama1
1 First Department of Internal
Medicine, 2 Second Department of
Anatomy, and 3 Institute of
Pharmaceutical Sciences, Hiroshima University School of Medicine,
Hiroshima 734-8551, Japan
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ABSTRACT |
Conventional in
vitro studies of pepsinogen secretion have measured secretion into the
bulk medium and have demonstrated the critical role of
Ca2+ in the process. The present
study was undertaken to obtain further details of the process of
secretion and its relation to Ca2+
changes over very short time periods. The relation between
Ca2+ mobilization and exocytosis
in an isolated individual peptic cell of the bullfrog was investigated
by a method to measure both intracellular
Ca2+
([Ca2+]i),
using a fluorescent Ca2+
indicator, fura 2, and exocytosis from single cells using a video microscope analyzing system. Bombesin (3.2 × 10
7 M) and bethanechol (3.2 × 10
4 M) caused a
rapid increase in
[Ca2+]i
(initial peak) and a corresponding high frequency of initial exocytosis. After the initial peak,
[Ca2+]i
was maintained at a somewhat elevated level over the baseline (sustained phase), with a corresponding low frequency of exocytosis. Both the sustained phase of elevated
[Ca2+]i
and the related exocytosis were eliminated by the depletion of
extracellular Ca2+. Low
concentrations of bombesin (3.2 × 10
10 M) and bethanechol
(3.2 × 10
7 M) caused
sustained low-amplitude Ca2+
oscillations with correspondingly low frequencies but also caused sustained exocytosis. These data show that
1) cellular response differs between
high and low concentrations of stimulus,
2) there is a close relation between
[Ca2+]i
and exocytosis, 3) exocytosis
follows elevation of
[Ca2+]i
by 14-45 s (n = 6), and
4) there is a significant positive correlation between the peak
[Ca2+]i
and the number of exocytoses.
bethanechol; bombesin; oscillation; bullfrog
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INTRODUCTION |
ALTHOUGH THE GENERAL relationship between stimulation
and pepsinogen secretion is well established from bulk solution studies over time, i.e., an initial peak of intracellular
Ca2+ concentration
([Ca2+]i)
and a burst of secretion, followed by a sustained lower level of
[Ca2+]i
increase and sustained secretion (1, 7, 8, 21, 30, 31), the
relationship between
[Ca2+]i
in single cells and exocytosis of individual granules in single pepsinogen-secreting cells has not previously been demonstrated. In
this study, using recently available imaging techniques with computer
enhancement (25, 27), we report such data. These data show, in real
time, the relation of granule exocytosis in individual isolated peptic
cells to both the concentration of [Ca2+]i
and to the oscillations in
[Ca2+]i
in response to various doses of cholinergic and peptidergic [bombesin (BB)] stimuli. Before the development of the
video-microscope system, time-resolved patch-clamp capacitance
measurement was the only technique available for the analysis of
exocytosis (11, 12, 17) and its relation to
Ca2+ mobilization (3, 4, 18).
Because the patch-clamp method is technically difficult and causes cell
injury, it was difficult to clarify the real-time relationships in
intact cells. In recent years, much progress has been made in video
microscopy techniques using a small-area charge-coupled device (CCD)
camera and a multiframe digital image processor (25, 27). These
techniques have recently been improved by computer enhancement of
digital images. With this powerful technique it is possible to see a
single zymogen granule in the living state, to study the profile of
secretory activity in isolated intact peptic cells with high
spatiotemporal resolution, and to count individual exocytotic events.
Furthermore, it is also possible to measure
Ca2+ in single cells using a
silicon-intensified targeting (SIT) camera, a fluorescence microscope,
and a multiframe digital image processor controlled by a computer
program (Argus 100CA2). Using these techniques, we studied the relation
between Ca2+ events and granule
exocytosis in isolated individual peptic cells.
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MATERIALS AND METHODS |
Materials.
Bullfrogs (Rana catesbeiana)
(6-7 in.) were obtained from Hiroshima Research Lab Animals
(Hiroshima, Japan). EGTA, HEPES, carbamyl-
-methylcholine chloride
[bethanechol (BCh)], digitonin, albumin, and
poly-L-lysine were purchased
from Sigma (St. Louis, MO). Collagenase (type 1) was obtained from
Worthington Biochemical (Freehold, NJ). Fura 2-acetoxymethyl ester (AM)
and fura 2 were purchased from Wako Chemicals (Osaka, Japan). BB was
obtained from the Peptide Institute (Osaka, Japan). Calcium Calibration Buffer Kit 2 was purchased from Molecular Probes (Eugene, OR). All
other chemicals were of the highest purity
available.
Unless otherwise stated, the standard medium (amphibian Ringer
solution) (ARS) contained (in mM) 89.4 NaCl, 4.0 KCl, 1.8 CaCl2, 0.8 MgSO4, 18 mM
NaHCO3, 11 glucose, 0.1% BSA, and
10 HEPES-NaOH buffer (pH 7.3). The medium was equilibrated with 95%
O2-5%
CO2. The
Ca2+-free medium contained no
CaCl2 or
MgSO4, which were replaced by NaCl
(92.9 mM).
Peptic cell preparation.
The peptic glands were prepared as previously described (14, 15, 21).
The glands were washed with the ARS and allowed to attach naturally to
the bottom of a chamber (50 µl) coated with 0.01%
poly-L-lysine hydrobromide.
Continuous superfusion (1 ml/min) at 20°C of the ARS through the
chamber was maintained while glands were monitored using a ×40
objective lens on a Nikon Diaphot inverted microscope connected to a
Spex Fluorolog spectrofluorimeter system.
Measurement of
[Ca2+]i.
To measure changes in
[Ca2+]i,
glands were incubated in the ARS containing 5 mM fura 2-AM for 30 min
at room temperature and equilibrated with 95%
O2-5%
CO2. After the glands were loaded with fura 2, they were washed three times and allowed to attach naturally to the bottom of a chamber (50 µl) coated with 0.01% poly-L-lysine hydrobromide that
allowed continuous superfusion (1 ml/min) at 20°C. Fluorescent
signals from a single cell were monitored using a ×40 objective
lens on a Nikon Diaphot inverted microscope connected to a Spex
Fluorolog spectrofluorimeter Argus-100 system (Hamamatsu Photonics,
Hamamatsu, Japan) and Ca2+ imaging
software (CA-2; version 3.70) equipped with a SIT video camera
(C2400-08, Hamamatsu Photonics). The emission wavelengths alternated every 2.5 or 5 s from 340 to 380 nm.
[Ca2+]i
was calculated from the fluorescence intensity ratios (340/380 nm) by
fitting the ratios to a calibration curve (13, 16).
Observation of exocytosis in peptic clusters.
Peptic cells were placed in the bottom of the chamber and observed
under a differential interference contrast (DIC) objective lens, using
an inverted Nomarski microscope (Axiovert 135T; Zeiss, Germany). The
DIC images were detected with a CCD camera (SSC-M350; Sony, Tokyo,
Japan). Then the video signals of the camera were contrast-enhanced
with a high-speed digital image processor (Pip4000; ADS, Tokyo, Japan)
controlled by a personal computer (PC980; NEC, Tokyo, Japan) monitored
at a magnification of ×1,960-2,940. The images were recorded
on a videotape using an S-VHS video recorder (AG-7355, Panasonic,
Osaka, Japan). The clusters were placed on the inverted microscope
stage at a temperature of 20-25°C and equilibrated with 95%
O2-5%
CO2 ARS by continuous perfusion.
To make frequency histograms of exocytoses, we counted the number of
abrupt changes in the brightness of granules in a peptic cell in unit
time on slow playback of the videotape. Furthermore, we used a mode of
image processing by which continuous images were converted into
continuous time-differential images in real time by subtracting the
image obtained at 33 ms from that obtained at 0 ms, the image obtained
at 66 ms from that obtained at 33 ms, and so on, using a multiframe
image processor (Argus-100 system) to reveal the rapid changes in
brightness more clearly.
Freeze-fracture methods.
Tissue specimens of peptic glands were fixed with 1% glutaraldehyde in
a 0.1 M cacodylate buffer (pH 7.3). After immersion in a 40% vol/vol
glycerin solution at room temperature, tissue specimens were placed on
a copper stage and frozen with liquid nitrogen. The specimens were
fractured at
185°C in a JEOL JFD 7000 freeze-fracture
apparatus and shadowed with platinum-carbon without etching and then
digested in filtered commercial bleach. The replicas were washed twice
in distilled water, mounted on grids, and examined in a Hitachi H-7000
electron microscope. Observation was performed on areas in which there
were many large secretory granules in peptic cells.
Data analysis.
Each experiment reported here was performed on preparations from at
least four different frogs. Data are presented as means ± SD.
Statistical analysis was performed using the Mann-Whitney test or
Wilcoxon's signed-rank test. Significant difference was set at
P < 0.05. All data were calculated
and analyzed with a personal computer (Power Macintosh 6100/66; Apple
Computer, Dallas, TX) using application software (Stat View 4.1).
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RESULTS |
Freeze-fracture studies.
Figure 1 shows electron micrographs of typical peptic
cells stimulated for 2 min with 320 nM BB (magnification ×4,500).
The cells had many secretory granules. The peptic cells showed
microvilli projecting into the luminal cavity. Exocytoses were detected
on the apical membrane after the stimulation, although exocytosis was
not observed before stimulation (not shown).

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Fig. 1.
Freeze-fracture study of frog esophageal pepsinogen-secreting cells.
Low-magnification view of exocytosis (arrows) in peptic cells with
apparent acinar formation. Acinar cells have microvilli (small arrows)
projecting into the lumen and secretory granules (G). Microvilli
disappeared in the area of exocytosis. Bar = 1 µm. Arrowheads show
enlarged view of rectangular area. Exocytosis has just finished. Bar = 1 µm.
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Morphological changes of peptic clusters by agonists.
Figure 2 shows DIC microscopic images of typical peptic
clusters (magnification ×1,960). Before the stimulation, peptic
clusters had narrow lumens and abundant secretory granules. Each peptic cell was ~13-18 µm in diameter. The secretory granules
appeared as bright or dark spots 0.2-2.5 µm in diameter (Fig. 2,
A and B). When the agonist was applied to
the perfusion medium, abrupt changes in the brightness of secretory
granules were observed at the apical membrane (Fig. 2,
C-F).
Figure 2G shows time-differential images obtained by subtracting the preceding 33-ms image and by adding
a proper offset value. These brightness changes, representing the
exocytotic events (19, 20, 22, 25-27), were recorded on a
videotape and converted to frequency histograms. A few seconds after
the agonist was applied to the perfusion medium, the lumen was widened
and filled with cloudy material, and the apical membrane became rough
(Fig. 3). Exocytosis seemed to occur on the apical membrane. Rapid movement of granules toward the apical membrane was not
observed before exocytosis. Only some of the many zymogen granules
exocytosed with stimulation. The final step in secretion is that
granules fuse with the apical membrane and discharge their contents.
After apical exocytosis, some of the zymogen granules near the
basolateral membrane transferred to near the apical membrane. They did
not fuse with the apical membrane but remained in the apical cytoplasm.

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Fig. 2.
Differential interference contrast (DIC) images of frog esophageal
pepsinogen-secreting cells dispersed from gland.
A: a peptic cluster with apparent
acinar formation reproduced from sequential video frames. Cells contain
many secretory granules. Bar = 10 µm.
B: illustration of a peptic cluster.
N, nucleus; A, apical membrane; B, basolateral membrane; S, secretory
granule; L, lumen. C-F: sequential
video images showing abrupt changes of brightness induced by bombesin
(BB) in a single granule (arrows). Bar = 10 µm.
G: sequence of video frames processed
by time differentiation. Time-differential images were obtained by
subtracting frame 2 from
frame 1,
frame 3 from
frame 2, and so on, every 33 ms, using a
multiframe image processor. This calculation method clearly reveals the
exocytotic event (arrow).
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Fig. 3.
Morphological changes of pepsinogen-secreting cells reproduced from
sequential video frames (DIC images). Arrow indicates border of apical
membrane. A: pepsinogen-secreting
cluster obtained 1 s before application of bethanechol (BCh).
B: 1 min after application of BCh (32 µM). Lumen has become slightly wider.
C: 8 min after application of BCh.
Lumen has become wider and still contains cloudy material. Apical
membrane has become rough. D: 15 min
after application of BCh. Changes in lumen and apical membrane are more
pronounced than in C. Bar = 10 µm.
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Frequency histograms and intracellular
Ca2+
mobilization.
In prior studies of dose-related pepsinogen secretion in isolated acini
of frog pepsinogen cells, maximal effective concentrations of BCh and
BB were found to be 32 µM and 320 nM, respectively (5). Figure
4 compares the exocytotic responses and
Ca2+ mobilization. Figure
4A shows BCh- and BB-mediated
Ca2+ mobilization in a medium
containing Ca2+ (ARS), with an
initial rapid increase followed by a plateau elevation. There were
significant statistical differences between basal
[Ca2+]i
and peak
[Ca2+]i
(Table 3) and between basal
[Ca2+]i
and sustained
[Ca2+]i
(data not shown) stimulated by both BCh and BB. Figure
4B shows BCh-mediated and Fig.
4C shows similar BB-mediated
exocytotic histograms in a medium containing
Ca2+ (ARS). The pattern of
exocytosis closely followed the
Ca2+ mobilization pattern seen in
Fig. 4A. Exocytosis was always
observed 10-20 s after the application of agonists. There were
significant statistical differences in lag time from the onset of
stimuli (both 32 µM BCh and 320 nM BB) to the peak
[Ca2+]i
and the peak frequency of exocytosis (Table 1). The lag
times between the peak
[Ca2+]i
and the peak frequency of exocytosis were 15.5 ± 4.8 s
(n = 5) (stimulated by BCh) and 13.6 ± 5.1 s (n = 5) (stimulated by BB). Although there was probable variation in the shape of the histograms, the application of agonists caused a large peak of secretion during an initial period of <2 min and a small sustained secretion after the peak. Sustained secretion continued for >30 min
(exocytosis had not ended during the 40 min of the observation). Figure
5 shows the same agonists used with a
Ca2+-free medium. The sustained
phase of Ca2+ mobilization was not
observed (Fig. 5A), and there was no
sustained phase of exocytotic events. However, the initial large peaks
of exocytosis occurred within about the same time frame after the application of the agonists (Fig. 5, B
and C; cf. Fig. 4).

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Fig. 4.
Time course of BCh (32 µM)- and BB (320 nM )-stimulated increase of
intracellular Ca2+ concentration
([Ca2+]i)
and frequency of exocytotic events in
Ca2+-containing solution
[amphibian Ringer solution (ARS)].
A: BCh and BB caused a rapid initial
increase and subsequent sustained elevation in
[Ca2+]i.
There are statistical differences between basal and peak
[Ca2+]i
stimulated by both BCh and BB (see Table 3). There are also statistical
differences between basal and sustained
[Ca2+]i
stimulated by both BCh and BB (data not shown). Trace represents means ± SD of 6 cells in 1 cluster. B
and C: in the same way, in solution
containing Ca2+ the frequencies of
exocytotic events were enhanced many times by BCh and BB during the
first few minutes and continued through a sustained phase with a lower
grade of frequency. Lag times from the beginning of stimuli (both BCh
and BB) to peak
[Ca2+]i
and peak frequency of exocytosis are statistically different (the
Mann-Whitney test) (see Table 1). Data are representative of
experiments with 5 different cell preparations.
[Ca2+]o,
extracellular Ca2+
concentration.
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Fig. 5.
Time course of BCh (32 µM)- and BB (320 nM)-stimulated increase of
[Ca2+]i
and frequency of exocytotic events in
Ca2+-free medium with 1 mM EGTA.
A: BCh and BB caused a rapid initial
increase but no sustained elevation in
[Ca2+]i.
Differences between basal and peak
[Ca2+]i
stimulated by both BCh and BB are statistically significant (data not
shown). Trace represents means ± SD of 6 cells in 1 cluster.
B and
C: in
Ca2+-free medium BB and BCh caused
numerous exocytoses, but only for 45-70 s. Lag times from the
beginning of the stimulation (both BCh and BB) to peak
[Ca2+]i
and peak frequency of exocytosis differ significantly (data not shown).
Data are representative of experiments with 5 different cell
preparations.
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Repeated stimulation with BCh and BB.
To determine whether BB and BCh release
Ca2+ from the same pools,
sequential stimulation by BB and BCh was applied, and the order was
then reversed. Figure
6A shows
[Ca2+]i
mobilization from the pools by sequential stimulation with BB and BCh
in Ca2+-free medium containing 1 mM EGTA. Figure 6B shows the
exocytotic events under the same conditions.
[Ca2+]i
mobilization and exocytosis were only detected during the period of BB
stimulation. BCh did not induce an increase in
[Ca2+]i
or exocytosis after the BB stimulation. When the order was reversed and
BCh was applied before BB (Fig.
7A) in a
Ca2+-free solution containing 1 mM
EGTA, both agents elicited peaks of
[Ca2+]i
and exocytotic events (Fig. 7) that were limited to 30-60 s.

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Fig. 6.
Repeated stimulation of peptic glands by BB (320 nM; first stimulation)
and BCh (32 µM; second stimulation) in
Ca2+-free medium containing 1 mM
EGTA. After BB, BCh did not induce an increase in either
[Ca2+]i
(A) or in exocytotic events
(B). Trace in
A represents means ± SD of 6 cells
in 1 cluster, and data in B are
representative of experiments with 5 different cell preparations.
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Fig. 7.
Repeated stimulation of the peptic glands by BCh (32 µM; first
stimulation) and BB (320 nM; second stimulation) in
Ca2+-free medium containing 1 mM
EGTA. Despite prior BCh stimulation, BB induced an initial rapid
increase in
[Ca2+]i
(A) and exocytotic events
(B). Trace in
A represents means ± SD
of 6 cells in 1 cluster, and data in B
are representative of experiments with 5 different cell preparations.
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The data suggest that the larger BB-sensitive
Ca2+ pool incorporates the smaller
BCh-sensitive Ca2+ pool. Initial
spikes of Ca2+ caused by either
stimulus produced exocytosis.
Relation between
Ca2+ oscillation
and exocytoses.
Figure 8A shows
dose-related
[Ca2+]i
data from a single cell of frog pepsinogen acini induced by high (32 µM) and low (320 nM) concentrations of BCh. Oscillations were most
pronounced with the middle concentrations of 320 nM and 3.2 µM BCh
but not at lower (32 nM) or higher concentrations (320 µM) (data not
shown). Figure 8B shows exocytotic
events induced by high (32 µM) and low (320 nM) concentrations of
BCh, which produced different patterns of
[Ca2+]i
and corresponding patterns of secretion. Figure 9 shows
dose-related [Ca2+]i
data and exocytotic events induced by high (320 nM) and low (320 pM)
BB. Oscillations were most pronounced with the lower concentrations of
320 pM and 32 pM (data not shown). At the concentration of stimulus
showing the greatest oscillation, there was neither an initial peak of
[Ca2+]i
nor an initial spike of secretion, but low frequency of exocytosis continued for >30 min (Figs. 10 and 11) (exocytosis
had not ended by the time the 40-min observation was over). There are
clearly large differences in the pattern and magnitude of
[Ca2+]i
responses at various concentrations of both BB and BCh, which are
reflected in the pattern of the exocytosis of pepsinogen. The
mechanisms responsible for these differences are not yet clear. Nor is
it clear which of these responses represents physiological conditions.

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Fig. 8.
A: pattern of
[Ca2+]i
stimulated by BCh at two different concentrations (32 µM and 320 nM).
Data are means ± SD of 6 cells (32 µM BCh) and are representative
of 5 experiments (320 nM BCh). B:
pattern of exocytosis at low (320 nM) and high (32 µM) concentrations
of BCh shows the lack of an initial spike of secretion at 320 nM. Data
are representative of experiments with 5 different cell preparations.
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Fig. 9.
A: pattern of
[Ca2+]i
stimulated by BB at 2 different concentrations (320 nM and 320 pM).
Data are means ± SD of 6 cells (320 nM BB) and are representative
of 5 experiments (320 pM BB). B:
pattern of exocytosis at high (320 nM) and low (320 pM) concentrations
of BB shows the lack of an initial spike of secretion at 320 pM. Data
are representative of experiments with 5 different cell preparations.
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Fig. 10.
A low concentration of BCh (320 nM) produces an oscillatory pattern of
[Ca2+]i
with oscillations of a similar magnitude
(A). Moreover, the exocytosis
pattern and the frequency of oscillations
(B) were also very similar. Data are
representative of experiments with 5 different cell preparations each.
The lag times from the beginning of stimulation to the elevation of
[Ca2+]i
and the onset of exocytosis were significantly different (Mann-Whitney
test) (see Table 2).
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Fig. 11.
At low concentrations, BB (320 pM) produces an oscillatory pattern of
[Ca2+]i
with oscillations of similar magnitude
(A). Moreover, exocytosis pattern
and frequency (B) are also very similar. Data are
representative of experiments with 5 different cell preparations each.
The lag times from the beginning of stimulation to the elevation of
[Ca2+]i
and the onset of exocytosis are significantly different (Mann-Whitney
test) (see Table 2).
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There were significant statistical differences in lag times from the
beginning of stimulation (32 µM BCh and 320 nM BB) to the elevation
of
[Ca2+]i
and the onset of exocytosis (Table 2). The lag times
between the elevation of
[Ca2+]i
and the onset of exocytosis are 43.9 ± 20.3 s
(n = 5) (stimulated by BCh) and 28.6 ± 19.2 s (n = 5)
(stimulated by BB). The time from the beginning of stimulation to the
elevation of
[Ca2+]i
and the onset of exocytosis diminishes with higher concentrations of
stimuli (Table 1).
Relationship between peak
[Ca2+]i
and number of exocytoses.
Table 3 and Fig. 12 show the relation between the peak
[Ca2+]i
and the number of exocytoses stimulated by five different
concentrations of BCh and six different concentrations of BB. Since the
major exocytotic events appeared in the initial 5-min period of
stimulation, the number of them found in a region of ~400
µm2 (about one cell) was counted
in this period for relative quantification. There was a significant
positive correlation between the peak [Ca2+]i
and the number of exocytoses stimulated by both BCh and BB (Fig. 12,
A and
B). The relation between
[Ca2+]i
and secretion is apparently independent of the type of
stimulus (Fig. 12C).

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Fig. 12.
Relation between peak
[Ca2+]i
and number of exocytoses stimulated by BCh
(A) and BB
(B) (data from Table 3). There is a
significant positive correlation between peak
[Ca2+]i
and number of exocytoses stimulated by both BCh and BB (Student's
t-test). The threshold
[Ca2+]i
values for stimulation of exocytosis are >128 nM for BCh and >158
nM for BB. C: overlap data from BCh
( ) and BB ( ) stimulation of peptic cells (from
A and
B). Values are means ± SD of 3-6
experiments.
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DISCUSSION |
Previous studies have indicated that cholinergic agonists such as BCh
and peptidergic agonists such as BB bind their receptors, cause the
breakdown of phosphoinositides by activating phospholipase C, mobilize
cellular Ca2+ (1), and finally
induce pepsinogen secretion. These prior studies suggested that initial
pepsinogen secretion by these agonists is triggered by mobilization of
Ca2+ from internal
Ca2+ pools, and sustained
secretion is dependent on the influx of Ca2+ (1).
Furthermore, pepsinogen secretion is believed to be mediated by
exocytosis, a fusion of secretory granules with the plasma membrane
that depends on processes that bind phospholipids in the presence of
Ca2+, e.g., via annexins (2),
followed by an extrusion of their contents to the extracellular space
(6). To further define the process of exocytosis and to elucidate the
direct relationship between pepsinogen secretion and
Ca2+ mobilization, we used a video
microscope system. This system has high spatial resolution of at least
160 nm, compared with resolutions of 150 µm obtained using
conventional microscope systems. Additionally, this system has a high
time resolution (i.e., images can be taken every 33 ms). These
capabilities provide powerful tools for dynamic studies of the
secretory process at the level of a single granule in a single cell.
This system allowed us to observe "popping" responses (abrupt
changes in appearance and in light intensity) of the granules, and we
concluded that these popping responses are exocytosis in the peptic
cells for the following reasons: 1)
These popping responses were induced only when peptic cells were
stimulated and were never observed without stimulation. 2) After such responses, granules
were not recovered by readjustment of the microscope focus.
3) The popping responses were
similar to exocytotic responses found in other secretory cells, namely, chromaffin cells (22), colonic goblet cells (21), salivary acinar cells
(20), nasal epithelial goblet cells (9), pancreatic
-cells (19), and
neutrophils (22). These images are supported by the freeze-fracture
studies (Fig. 1).
The present studies provide a much more detailed mechanistic and
temporal picture of the secretion processes of the peptic cell than
studies of secretion into bulk solutions over long time periods (5, 8,
21) or even studies such as continuous perfusion of isolated glands, in
which observations are made at intervals of 10 s or more (14). In bulk
solution studies (5, 8, 21, 31) the temporal relation between cell
[Ca2+]i
and secretion cannot be directly resolved.
In this study we have been able to define secretion temporally and
quantitatively, i.e., by timing and counting exocytosis of secretory
granules. These data have been related to measurements of
[Ca2+]i
in peptic cells from the same preparations studied under identical experimental conditions. There were significant statistical differences in lag times from the onset of stimuli (both BCh and BB) to the peak
[Ca2+]i
and the peak frequency of exocytosis. This is the first time data have
been proved showing the time lag between Ca2+ signaling and
exocytosis. About 15 s are required for secretory granules to initiate
contact and fuse with the apical membrane when a submaximal dose of
stimulation is used, and about 40 s are required when a low-dose
stimulation is used. A low concentration of
Ca2+ may need more time (about 20 s) to initiate the first exocytosis. The present data show that in
every instance the initial Ca2+
peak derived from intracellular
Ca2+ pools was followed, within
seconds, by the onset of a large number of exocytoses. Such a peak
elevation of
[Ca2+]i
could be induced by sequential stimulation, e.g., BCh followed by BB;
BCh and BB appear to act on the same intracellular
Ca2+ pool (23). In addition to the
relation between initial
[Ca2+]i
spikes and exocytosis, the data also show a subsequent sustained but
lower rate of exocytosis (secretion) that is related to a lower
sustained elevation of
[Ca2+]i,
which is dependent on the influx of
Ca2+ from the extracellular medium
(8, 30, 31). Whereas higher concentrations of stimuli produce spikes of
[Ca2+]i
followed by lower sustained plateaus, low concentrations of BCh (3.2 × 10
7 M) and BB (3.2 × 10
10 M) induced
Ca2+ oscillations that continued
for almost 30 min, but without an initial
[Ca2+]i
peak. These modest oscillatory elevations of
[Ca2+]i
were associated with a continuous low frequency of exocytosis, but
without an initial spike of
[Ca2+]i;
thus there was also no initial burst of exocytosis. Tsunoda (28) felt
that although there was insufficient experimental evidence to define
any precise mehanism for the biological effect of
Ca2+ oscillation, it was likely
that small repetitive signals are as effective as large sustained
signals. How the oscillations are generated intracellularly and how
they function in Ca2+ signaling
remain to be elucidated (29). It has been suggested that the
Ca2+ oscillation may be a form of
frequency-modulated signaling that enhances signal recognition and
avoids desensitization and Ca2+
toxicity (10). We found almost identical
Ca2+ oscillation and exocytosis
patterns with two different agonists, BCh and BB (Fig. 10), suggesting
that such patterns may represent physiological conditions.
Although granule movement relative to the plasma membrane is required
for exocytosis and is likely to be an integral part of
stimulus-secretion coupling (20), in previous video microscopic studies, granule movement toward the captured substance was observed in
neutrophils during phagosome formation (22), and granule movement
toward the apical membrane was not detectable in chromaffin cells (25,
26), pancreatic
-cells (19), nasal acinar cells, nasal epithelial
goblet cells (9), colonic goblet cells (27), or salivary gland cells
(20). These observations and our present observations seem to support
the concept that in both endocrine and exocrine glands the granules of
unstimulated and stimulated cells maintain relatively fixed positions
in the apical cytoplasm. Consequently,
Ca2+ may not play an important
role in translocation of the secretory granule to the apical membrane.
In contrast, with the depletion of all mucin granules in colonic cells
in 30-40 min (27), only the apical zymogen granules of the peptic
cell are secreted in the 40 min of observation. This may be explained
by the more prolonged maturation time of peptic granules or by rapid
replacement of secreted granules, since synthesis is stimulated by
secretion and peptic cells are not depleted of pepsinogen even after
prolonged secretion.
There was a significant positive correlation between the peak
[Ca2+]i
and the number of exocytoses stimulated by both BCh and BB. The
relation between
[Ca2+]i
and exocytosis appears to be independent of the stimulus, with a
threshold
[Ca2+]i
of 120-160 nM and a possible plateau of ~2,000 nM.
In conclusion, our studies demonstrate that
1) initial exocytosis is related to
the mobilization of Ca2+ from
intracellular pools, 2)
Ca2+ influx from the extracellular
medium is responsible for continuing exocytosis in frog
pepsinogen-secreting cells, 3)
exocytosis follows elevation of
[Ca2+]i
by 15-40 s, 4) there is a
significant positive correlation between peak
[Ca2+]i
and the number of exocytoses, independent of the nature of the
stimulus, and 5) continued
exocytosis at lower levels is related to
Ca2+ oscillation, which may be the
physiological state.
These conclusions are based on visualization of granule exocytosis in
individual cells and related to
[Ca2+]i
changes after stimulation in real time.
 |
ACKNOWLEDGEMENTS |
We are grateful to B. I. Hirschowitz for reading an earlier draft
of the manuscript. We also thank S. Terakawa for helpful advice, and
Dr. E. Suzaki and E. Kawai for technical assistance with microvideo
analysis.
 |
FOOTNOTES |
A portion of these results has been published previously in abstract
form (Gastroenterology 106: A193,
1994).
Address for reprint requests: C. Tao, Kasumi 1-2-3 Minami ku, Hiroshima
734-8551, Japan.
Received 19 August 1997; accepted in final form 25 February 1998.
 |
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