Exocytosis and movement of zymogen granules observed by
VEC-DIC microscopy in the pancreatic tissue en bloc
Yukio
Ishihara1,
Takashi
Sakurai2,
Taizou
Kimura1, and
Susumu
Terakawa2
1 First Department of Surgery and 2 Photon Medical
Research Center, Hamamatsu University School of Medicine, Hamamatsu
431-3192, Japan
 |
ABSTRACT |
The dynamic
aspects of exocytosis, especially in the normal acinar tissue en bloc,
have remained unclear. We visualized exocytosis directly in the tissue
of the exocrine pancreas of rodents by video-enhanced
contrast-differential interference contrast (VEC-DIC) microscopy to investigate various exocytosis-related rates and the relationship between the movement of granules and exocytotic responses. Stimulation of the tissue with bethanechol or
cholecystokinin caused many of the zymogen granules in the apical pole
to disappear abruptly. The exocytotic transients of individual granules
were completed in 0.48-0.65 s. Granules destined to participate in the exocytotic response moved randomly at velocities of ~0.06 µm/s
or less during stimulation. In the tissue preparation, granules located
far from the apical pole frequently moved back and forth for 1-7
µm without showing exocytosis. Colchicine suppressed this movement
and the late phase of the secretory response. Real-time (VEC-DIC)
observation of granule dynamics revealed that the initial step of
exocytosis was not coupled directly with the microtubule-dependent translocation but with a continuous, slow Brownian fluctuation of granules.
granule movement; pancreas; video microscopy; video-enhanced
contrast-differential interference contrast microscopy
 |
INTRODUCTION |
THE EXOCRINE
PANCREAS SECRETES fluid by channel activity and enzymes by
exocytotic membrane fusion. In contrast to channel dynamics, the
details of fusion dynamics are still unclear because of a lack of the
means to study them in real time. The secretory process of acinar cells
has long been studied morphologically by examining fixed preparations
(12, 26). The exocytosis of secretory granule in the time
domain has been analyzed by capacitance measurements of the plasma
membrane with a patch pipette (19, 24). However, this
technique is rather difficult, is inapplicable to cells surrounded by
an intact tissue, and is complicated by problems caused by membrane
retrieval and other morphological changes of the cell. The secretory
process of catecholamine-containing vesicles has been investigated by
amperometry, that is, by measuring the oxidation current with a thin
carbon-fiber electrode. This has been applied to the exocrine pancreas
charged with exogenous serotonin (36). The spatial
resolution is insufficient to pinpoint the site of exocytosis and to
trace back the origins of the corresponding granules.
McCuskey and Chapman (20) attempted to visualize the
secretory process at the single granule level using a bright-field light microscope combined with a cinephotographic camera. However, it
was not possible to detect exocytosis in living cells without amplification of image intensity. Anderson and McNiven (2) used phase-contrast microscopy to detect exocytosis in pancreatic acinar cells. However, the cells used in their study were derived from
a clone that had lost the acinar structure; therefore, exocytosis was
not convincingly captured, and the spatial aspects of exocytosis in
reference to cellular polarization remained ambiguous and
controversial. Recently, Terakawa and colleagues (14, 27, 28,
32-35) used video-enhanced contrast-differential
interference contrast (VEC-DIC) microscopy for the dynamic analysis of
exocytosis. The DIC lens, with a high numerical aperture, yields an
image of a thin optical section of an intact tissue, and the
enhancement of the contrast with a video system amplifies a discrete
change in the image of the granule. This technique has advantages over
the patch-clamp technique or carbon-fiber amperometry in its ability to
reveal the spatial details of the secretory response in a single cell in normal tissue.
Knowledge of the mechanisms responsible for the release of digestive
enzymes from zymogen granules is important for understanding disorders
of the exocrine pancreas, including pancreatitis. In this study, we
have exploited the advantages of VEC-DIC microscopy, to visualize the
exocytosis of individual granules in pancreatic tissues with the normal
acinar structure, and tracked the movements of secretory granules in
the regions of synthesis and secretion. This allowed us to quantitate
the secretory activities induced by various modes of stimulation and
provided us with an opportunity to analyze explicitly the dynamic
parameters related to exocytosis as well as the relationship between
exocytosis and the movements of secretory granules in exocrine cells.
 |
MATERIALS AND METHODS |
Preparation.
The main body of the pancreas was excised from the male guinea pig
(Hartley strain) or rabbit (Japanese White) anesthetized with
pentobarbital sodium (50 mg/kg ip for guinea pig, 40 mg/kg iv for
rabbit). The tissue was cut immediately into small pieces (~1.0 × 1.0 × 0.5 mm3) with the use of razor blades. A few
pieces of tissue were placed in a chamber, the bottom of which was made
of a coverslip. The tissues were superfused continually at 33°C with
standard medium containing (in mM) 115 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, 1 KH2PO4, 20 NaHCO3 and 16 HEPES (pH
7.4, adjusted with NaOH). The medium was oxygenated with a slow supply
of medicinal gas into a vinyl jacket surrounding silicone tubing for superfusion.
VEC-DIC microscopy.
The instruments used for light microscopic observation were similar to
those described previously (33). Briefly, acinar cells of
the pancreas were observed under an inverted DIC microscope (Axiovert
10, Zeiss, Obercochen, Germany) equipped with a ×100 objective lens
(Plan-Neofluar, numerical aperture = 1.3, oil immersion) and a
×2.5 insertion lens. The DIC microscope produced an image of the
tissue optically sectioned to a slice of about 1 µm thick. The DIC
image was captured with a 0.5-in. charge-coupled device video camera
(TI-23A, NEC, Tokyo, Japan) and then digitized and processed with an
image processor (ARGUS-20, Hamamatsu Photonics, Hamamatsu, Japan) for
real-time enhancement of the contrast (VEC). The processed images were
observed on a video monitor screen (14 in., black and white, Panasonic,
Osaka, Japan) at a final magnification of ×4,400 or ×11,000 and
recorded on videotape with an S-VHS format video cassette recorder
(AG-7500, Panasonic). All pictures and data in this study were
reproduced from these video records. For reproduction, video frames
were digitized and arranged using picture-handling software (Photoshop
3.0J, Adobe, Mountain View, CA). Figures 1-3 and 9-11 were
printed with a digital printer (UP-D7000, Sony, Tokyo, Japan).

View larger version (116K):
[in this window]
[in a new window]
|
Fig. 1.
Video-enhanced contrast-differential contrast (VEC-DIC)
microscopic images of an acinus of the guinea pig obtained before
(A) and 30 min after stimulation with 50 µM bethanechol
(BCh; B). After stimulation, acinar lumen was slightly
enlarged and the number of zymogen granules was decreased. Scale
bar = 5 µm.
|
|

View larger version (153K):
[in this window]
[in a new window]
|
Fig. 2.
VEC-DIC microscopic images of the acini of the rabbit.
A: low-magnification view of the tissue. B:
high-magnification view of an acinus. Scale bars = 5 µm.
C: schematic representation of the sites where exocytotic
responses occurred in the initial 20 s of stimulation with 20 µM
ACh in the acinus shown in B. Circular marks were traced
from video images made by processing the original video tape through
the time differential mode. All the changes in images that occurred
during a period as short as 33 ms were captured. Solid curves indicate
the longitudinal axes of the luminal space. Exocytotic responses were
always found in the region near the lumen.
|
|

View larger version (97K):
[in this window]
[in a new window]
|
Fig. 3.
Sequential images of a granule undergoing exocytosis
produced by 50 µM BCh. Interval between each frame was 67 ms.
Frames 4-11 show the process of an exocytotic response.
The granule disappeared in the latter images. Note that there was no
visible change from frame 1 to frame 4. The
diameters of the granule in frames 1-4 ranged from 0.62 to 0.67 µm.
|
|
Stimulation of the pancreas.
The pancreatic tissues were stimulated by adding bethanechol (BCh; 1, 10, 50, and 100 µM) or cholecystokinin octapeptide (CCK-8; 5 nM) to
the standard medium. In some cases, Na+ (65 mM) in the
standard medium was replaced with K+.
Ca2+-chelated medium was prepared by adding EGTA (1 mM) and
an aliquot of NaOH to the Ca2+-omitted standard medium.
Assessment of exocytotic response by light intensity change.
Release of highly condensed substances from the granules was assumed to
give rise to a change in the refractive index, which would appear as a
discrete light intensity change under a DIC microscope. Therefore, we
considered that an abrupt change in light intensity of a granule was an
exocytotic response. To study the dynamic aspects of such an exocytotic
response, we measured the light intensity changes of a single granule
quantitatively. Sequential video images capturing a granule were stored
in multiframe memories of the digital image processor at intervals of
33 or 66 ms for 660-1,320 ms. The light intensities were then
calculated by summing digital values of pixels in a square frame set so
as to circumscribe a single granule. A stepwise change of the light intensity was measured as an indication of the releasing time in an
exocytotic response.
Frequency of exocytosis.
The exocytotic responses in the acinar cells were easily captured by
eye on the monitor screen in real time as well as during replay of the
videotape. Responses of granules appearing in the focal plane (about 1 µm in thickness) were clearly captured, and those located about 1 µm off the focal plane in both sides were also captured, although the
latter images were blurred. The precise numbers of exocytotic responses
every minute were counted to quantitate the secretory activity at the
cellular level. This number would reflect roughly one-half of the total
number of exocytotic events that occur in a spherical region of
4-5 µm in a single acinus.
Movements of zymogen granules.
To examine the movements of zymogen granules, the x-y
coordinates of the centers of a zymogen granule on the monitor screen were measured. A single granule was tracked in every video frame for a
few seconds. The granule movements were measured as the differences of
the coordinates between two frames. The velocities of granule movements
were calculated by dividing the distance by the intervals between
frames. The granules that underwent exocytosis near the lumen (at the
apical membrane) were chosen for analysis for periods of 3.7-5.7 s
immediately before exocytosis. For the purpose of comparison, the
movements of granules chosen randomly from the same sequence of video
records that did not undergo exocytosis and that were located far from
the apical membranes (central region in the cell) were analyzed. The
data in the resting states and the stimulated states were compared. The
resolution for a positional measurement by the VEC-DIC system was about
40 nm in space (7) and 33.3 ms in time.
Amylase activity.
Several pieces of acinar tissue prepared for microscopic observations
were incubated in test tubes (0.5 ml) in the absence or presence of BCh
(10 µM, 100 µM, and 1 mM) for 10 min at 37°C. The amylase
activities in the extracellular medium (~200 µl) in the test tubes
were assayed by Special Reference Laboratory (Tokyo, Japan)
enzymatically by use of
2-chloro-4-nitrophenyl-D-maltoheptaose as a substrate. The
wet weights of the tissues in each tube were measured, and the
activities were normalized to the weights.
Statistical analyses.
Statistical analyses were performed by using the unpaired
t-test. Significance was attributed at P < 0.01. All data were calculated and analyzed with a personal computer
(Power Macintosh 8100/80, Apple Computer, Dallas, TX) using application
software (StatView 4.1, Abacus Concepts, Berkeley, CA). Data are
expressed as means ± SE.
 |
RESULTS |
General view of the acini.
Each acinus of the exocrine pancreas (guinea pig or rabbit), consisting
of 8-10 acinar cells, was clearly visible at a magnification of
×4,000 (Fig. 1). Exocrine acini were
easily distinguished from endocrine islets by several characteristics.
Acinar cells were pyramidal or trapezoidal in shape; apicals assembled
together to form a narrow void space presumably an acinar lumen. These cells had nuclei with a very vague outline in the basal side. In
contrast, islet cells showed a rather round shape and a clear nucleus.
Usually, the lumens of most acini in the resting state were shrunken;
occasionally, some wide lumens with a gap width of 1-2 µm were
seen. The zymogen granules in each acinar cell were clearly visible.
They were 0.3-1.5 µm in diameter and were concentrated near the
apical pole of each acinar cell. They fluctuated very slowly within an
area comparable to their diameter in the region crowded with granules.
The granules located outside the crowd and in the perinuclear region
showed a similar fluctuation. However, in addition to the fluctuation,
these granules sometimes moved quite rapidly in a straight manner for a
distance three times their diameter or much longer. Application of 100 µM colchicine suppressed these longer movements significantly, but
not the slower fluctuations (see Movement of zymogen granule below).
Occasionally, after inappropriate preparation procedures, insufficient
oxygenation, or excessive stimulation, large vacuoles formed inside the
acinar cells. We found vacuoles also in cells stimulated with 1 mM BCh
for 30 min. The vacuoles were characterized by a refractive index lower
than the surrounding area, a spherical shape, and an amorphous content.
In these cells, no further physiological responses were observed.
Therefore, care was taken to prevent such morphological disorders.
These preparations were discarded.
Single exocytotic responses.
Stimulation of acinar cells by superfusion with a medium containing BCh
(1, 10, 50, and 100 µM) or CCK-8 (5 nM) caused many zymogen granules
to show abrupt changes in light intensity and to disappear
sequentially. The positions of individual granules did not change
significantly until they disappeared (see Movement of zymogen
granule below). In a single cell, the intervals between such
responses were short at the beginning and then became longer by
1-2 min. The intervals between individual responses seemed to be
quite random throughout. Such responses were always found in the region
near the lumen (Fig. 2). The images of
response were sharper and clearer in the rabbit than in the rat.
In an acinus, each acinar cell showed similar responses to the
stimulants. No responses were observed 1-2 min after removal of
the stimulants from the medium. There was a decrease in the number of
spontaneously responding granules at 3-5 min. The acini were slightly shrunken during the initial few seconds of 100 µM BCh
stimulation. After vigorous releases of substances from many granules,
the outlines of the luminal space became vague.
Time courses of light intensity changes of a single granule during
exocytosis.
The changes in image and light intensity of some zymogen granules in
acinar cells stimulated with BCh were analyzed by using the digital
image processor. Immediately before the occurrence of the changes of
light intensity, the zymogen granules showed no discernible change in
shape and size. For example, the diameter of a granule remained in a
range of 0.62-0.67 µm for more than 100 ms (frames
1-4 in Fig. 3). The changes in
light intensities were assumed to indicate the secretory responses. In
most granules, the initial light intensity was quite stable; the light
intensity then suddenly changed to another level and reached a plateau. When this transition was completed, the granules disappeared. The
dynamics of these secretory responses in many granules were analyzed
(Fig. 4). The dynamics among exocytotic
responses induced by different concentrations of BCh (1, 10, and 100 µM) were compared in histograms (Fig.
5). They were not significantly different (P < 0.01). The average transition times in exocytosis
(releasing time) in the cells stimulated with these concentrations of
BCh ranged from 0.48 to 0.65 s.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.
Time course of the light intensity changes of the granule
during its exocytotic response in the guinea pig. Ordinate represents
the light intensity expressed in percentage of the initial value. Light
intensity changed in 967-1,333 ms. Time required for the light
intensity to change by 95% of the maximum was defined as the release
time (solid line), which was 0.37 s (from point 1 to
point 2).
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
Histograms of the release times for the exocytotic
responses induced by different concentrations of BCh (100 µM in
A, 10 µM in B, and 1 µM in C) and
those found without stimulation (spontaneous release; D) in
the guinea pig. Abscissa represents the time required for the light
intensity to change (release time). Average values of the release times
ranged from 0.48 to 0.65 s. There was no significant concentration
dependence of the release time (P < 0.01).
N, number of responses; m, mean time; n = no. of cells.
|
|
Secretory activity measured from the frequency of exocytosis.
To quantify the secretory activity, frequency histograms were produced
by playing back the videotape and counting the numbers of exocytotic
responses in a single acinus during every minute. When the acini were
stimulated by addition of BCh (1, 10, 50, and 100 µM) to the
superfusion medium, exocytotic responses were observed in almost all
preparations (Fig. 6). A high
concentration of BCh (hyperstimulation; 50 and 100 µM) always induced
the exocytotic responses at a high frequency during an initial period
of a few minutes. During continued application of BCh for about 30 min, the response frequency fell, and the responses tended to disappear. When 1 mM BCh was used, secretory activities were similar to those shown with 100 µM, but large vacuoles appeared inside many acinar cells. Sometimes, after long stimulation (over 30 min) with 100 µM
BCh, similar vacuoles were formed. At lower concentrations of BCh (1 and 10 µM), the frequencies of exocytotic responses were much lower,
and the responses did not display a peak even in the initial phase
(Fig. 6). These responses ceased 1-2 min after acini were returned
to the standard medium.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
Frequency histograms of exocytotic responses in a single
acinus stimulated with BCh in the guinea pig. Different concentrations
of BCh (100 µM in A, 50 µM in B, 10 µM in
C, and 1 µM in D) were applied during the
periods indicated by the horizontal bars. Ordinate represents the
number (N) of exocytotic responses counted every minute. The
secretory activity increased with the concentration of BCh.
|
|
The secretory activity, measured as the frequency of exocytosis in the
dose-response curve, increased with concentrations of BCh from 1 to 100 µM (Fig. 7A). A similar
dose-response relationship was found for the release of amylase from
acinar tissues measured under similar conditions (Fig. 7B).
The BCh-induced responses were blocked almost completely in the
presence of 1 µM atropine (Fig.
8A). Elevation of the
K+ concentration (to 65 mM) in the medium did not induce an
exocytotic response. However, in the same preparation, stimulation with
CCK-8 (5 nM) 10 min after reduction of the K+ concentration
to the normal level induced a large burst of exocytotic responses (Fig.
8B). When acini were stimulated with BCh-containing medium
supplemented with 1 mM EGTA (no addition of Ca2+), the
secretory activity was strongly suppressed, and only a few exocytotic
responses were detected (Fig. 8C). The BCh-induced exocytotic responses were also suppressed strongly and reversibly when
lanthanum chloride (10 µM) was added to the medium (Fig. 8D).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 7.
Dose-response curves for secretory activity measured by
counting the frequency of exocytotic events (A) or by
assaying the enzymatic activity (B). In A, the
numbers of exocytotic events were counted for an initial period of 5 min in the tissues stimulated at 3 different concentrations of BCh (1, 10, and 100 µM). Numbers of preparations measured were 4, 6, and 5, respectively. In B, dose-response curves are for amylase
released from several pieces of tissue to the medium, measured in test
tubes. Tissues were stimulated with BCh at 10, 100, and 1,000 µM.
Activities were normalized for the wet weight of acinar tissues and
expressed in IU/mg. The numbers of samples were 3, 4, 5, and 3, respectively, for each of the points shown.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of various reagents on the secretory activity
(guinea pig). A: atropine (1 µM; Atr). B: high
concentration of K+ (65 mM) and cholecystokinin octapeptide
(CCK-8; 5 nM). C: Ca2+-free medium (1 mM EGTA).
D: lanthanum (La; 10 µM). Ordinate represents the number
of exocytotic events counted every minute. A-D represent
data obtained from different preparations.
|
|
Movement of zymogen granules.
To study the movement of a granule immediately before its exocytosis,
the traveling path (trajectory) of a single granule that underwent
exocytosis was traced retrospectively by playing back the videotape.
For a period of ~5 s immediately before the exocytosis, the average
velocity of granule movement was 0.06 µm/s (Table
1). During this period, the average
maximal distance of granule movement was 0.16 µm. In contrast, the
average velocity of granules that did not undergo exocytosis, because
they were located in the central region of the cell, was 0.11 µm/s.
The maximal distance of movement of these granules was 0.29 µm. To compare these data with those of granules in the resting state, granules of the same acinus were also measured before stimulation. Groups of granules were chosen from the area near the lumen and from
the central region in the cell. The average velocity and the maximal
distance of granule movement were, respectively, 0.08 µm/s and 0.17 µm near the lumen and 0.10 µm/s and 0.21 µm in the central
region. These values were not significantly different (P < 0.01) from those obtained in the stimulated
state. Actually, these movements appeared on the monitor screen as very
slow fluctuations as described above. A typical example of the movement
of a granule that underwent exocytosis is shown in Fig.
9. Many granules underwent exocytosis
without any spatially directed drift in the apical region, similarly to
this example.

View larger version (84K):
[in this window]
[in a new window]
|
Fig. 9.
A: DIC image of an acinus of the guinea pig.
The origin of the coordinates (0,0) was set at the center
of the lumen. The ordinate was set in the direction of the crossing of
the central point of the basal membrane of a cell. The abscissa was set
at a right angle to the ordinate. Scale bar = 5 µm.
B: sequential images of a granule observed immediately
before exocytosis. Numbers correspond to those in C and
D. The granule in B1 and B2 appeared
in almost the same position; the granule then abruptly disappeared in
B3. Scale bar = 2 µm. C and D:
movement of a single granule (arrow in B along the
coordinates x and y) was traced from sequential
video images. This particular granule moved within a range of 0.17 µm
without showing a significant drift in the direction toward the center
of the lumen. Average velocity was only 0.08 µm/s during the 10.7-s
period immediately before exocytosis. All these measurements were
performed in the same preparation 3 min after the onset of 100 µM BCh
stimulation.
|
|
Occasionally, granules in the central region showed a linear, long
movement (>1 µm). Granules frequently glided 5-10 µm in 3 s at a stretch. Some moved from the nuclear region to the back row of the granule accumulation in the apical pole (Fig.
10). Others moved in a reverse
direction along a similar track. Such long movements (traveling or
gliding) were not observed in the luminal region. These movements were
thought to be of different nature and thus were excluded from the
analyses of the central region, displayed in Table 1, and were analyzed
separately. A typical case of the linear and long-distance movement of
a granule is shown in Fig. 11. To
ascertain the nature of the long movements of granules, the effects of
colchicine and BCh on the numbers of granules that moved over 1 µm
were examined. The numbers of traveling granules were decreased by 100 µM colchicine and were increased by 20 µM BCh (Fig.
12). Neither colchicine nor BCh
affected the smaller fluctuating movements. Such long-distance
traveling of granules never led directly to exocytosis in the central
region. The presence of 100 µM colchicine (for 10 min before BCh
application) failed to suppress the secretory activity, as measured by
the frequency of exocytosis (Fig. 12A). The presence of 100 µM colchicine (for 20 min before BCh application) slightly suppressed
the number of exocytotic responses (Fig. 12B). This
suppression was stronger in the later phase of the secretory response
than in the initial phase (compare with Fig. 12A).

View larger version (164K):
[in this window]
[in a new window]
|
Fig. 10.
A: DIC image of an acinus of the rabbit. L,
luminal space. Scale bar = 5 µm. B: schematic
representation of the directions and loci of granules, showing
long-distance movements (>1 µm) of the acinus shown in A.
Some moved from the nuclear region to the back row of the granule
accumulation region in the apical pole. Others turned backward.
C-E: sequential images of a granule showing a basoapical
gliding followed by a reverse gliding movement. Each position of
granule marked by the arrow corresponds to c, d,
and e in B. Scale bar = 3 µm.
|
|

View larger version (89K):
[in this window]
[in a new window]
|
Fig. 11.
A: DIC image of a whole acinus observed in
the guinea pig. The coordinates were set as described in Fig. 9. Scale
bar = 2 µm. B: sequential images of a granule gliding
in the central region of the acinus. The numbers correspond to those in
C and D. Occasionally, the granule (such as the
one indicated by arrows) moved linearly over 1 µm from the nuclear
region to the back row of granule accumulation region in the apical
pole. However, none of those granules showed exocytotic responses.
Scale bar = 2 µm. C and D: movements of
the single granule (shown by arrows in B) were traced along
the coordinates x and y by measuring the
positions of the granule in its center. The average velocity was 0.65 µm/s, and the maximal distance of movement was 3.38 µm for this
granule.
|
|

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 12.
Effects of colchicine (100 µM) and BCh (20 µM) on
the long-distance movement (>1 µm) of granules and the frequency
histogram of exocytotic responses in the rabbit. Left ordinate
represents the numbers of exocytotic responses counted every minute
(solid columns). Right ordinate represents the numbers of granules that
moved over 1 µm of an acinus in 1 min ( ).
A: mean numbers of such granules were increased by BCh to
twice that of the resting state. Colchicine decreased the mean number.
The presence of 100 µM colchicine (for 10 min before BCh application)
failed to suppress the secretory responses. No. of subjects = 4. B: presence of 100 µM colchicine (for 20 min before BCh
application) suppressed the long-distance movements completely but not
the secretory response.
|
|
 |
DISCUSSION |
In this study, exocytosis of zymogen granules in the pancreatic
tissue was visualized directly in real time by VEC-DIC microscopy. The
fine movements of zymogen granules in individual cells with normal
acinar structure could be analyzed in both the absence and presence of
exocytosis. Thus granule movements in the resting secretory states were
compared. In the resting state, secretory granules showed frequent
bidirectional gliding movements between the perinuclear region and the
subapical region, but granules near the apical pole showed only very
slow movements. Stimulation induced exocytosis at the apical membrane
and produced a slight decrease or no change in the fluctuation
movements and a slight increase in the gliding movements of granules in
the perinuclear region. Therefore, we conclude that the result of the
directed movements in the middle part of the cell was to cause
secretory granules to accumulate in the apical pole and that the
granules were driven to the final docking site only by Brownian movement.
Exocytosis visualized by VEC-DIC microscopy.
The exocytotic response of a single granule was clearly visualized by
the present technique as an abrupt change in light intensity followed
by the disappearance of the granule. This made it possible to
quantitate and analyze the secretory process dynamically at a
subcellular level. The abrupt light intensity changes of a granule under a VEC-DIC microscope were generally considered to be exocytotic responses for the following reasons: 1) the responses of
granules were induced only when cells were stimulated with specific
agonists; 2) the responses were always followed by the
disappearance of the granule; 3) the responses could be
suppressed by addition of a specific antagonist or by removal of
Ca2+ from the medium; and 4) similar optical
changes of vesicles in chromaffin cells were associated with the
release of substances oxidizable with a carbon-fiber electrode at +500
mV (34). In the present experiments, the exocytotic
responses appeared exclusively when the tissue was stimulated with BCh
(10-100 µM) (Fig. 6) and only in the vicinity of the lumen (Fig.
2C). In accordance with the similar responses in other
secretory cells, the granules in pancreatic acinar cells also
disappeared after such responses (Fig. 3). In addition, the dose
dependence of the secretory activity, measured as the frequency of
exocytotic events, was similar to that measured for the release of
amylase activity (Fig. 7). Therefore, it was concluded that the optical
responses we observed were due to exocytosis.
The responses were completely suppressed by 1 µM atropine (Fig. 8),
suggesting that muscarinic receptors are involved in triggering the
response (6). The BCh-induced exocytotic responses were significantly suppressed by removal of Ca2+ from the
external medium (Fig. 8). The exocytotic responses were also strongly
suppressed when lanthanum, known to block Ca2+ channels on
the cytoplasmic membrane, was added to the medium. These results are in
full accordance with the properties of the intracellular
Ca2+ response and amylase secretion measured earlier
(37). Furthermore, these results are compatible with other
studies of exocytosis performed by VEC-DIC microscopy (14, 27,
28, 32-35).
In pancreatic acini prepared by other methods, the dose-response curves
for amylase release induced by muscarinic agonists showed some
inhibitory effects at concentrations in the range of 50-100 µM
(10). These preparations showed receptor desensitization (38) as well as a rapid decay of both the Ca2+
transients (21) and amylase releases (36) at
high doses. In salivary glands, water secretion, which provides a
vehicle for amylase, also shows reduced efficiency during muscarinic
stimulation at high doses, whereas the energy consumption is enhanced
at the same doses (22, 23). One of the unique aspects of
our preparation was that the basal release observed without stimulation
was extremely low. Another was that the response induced by 100 µM
BCh did not decay much during a 30-min period of stimulation (Fig. 6),
and it was larger than that induced by 10 µM (Fig. 7). The absence of
enzymatic treatment of our en bloc tissue preparations might better
preserve the natural characteristics of the receptors and the
intracellular signaling pathways. Alternatively, the tight intercellular space might hamper the rapid access of stimulants to receptors.
It was essential to maintain the tissue under good conditions to obtain
the exocytotic responses. For example, formation of vacuoles inside
acinar cells is one of the characteristics of an early phase of acute
pancreatitis (8). These vacuoles are known to be formed
also by supramaximal stimulation with cerulein or CCK-8
(25). In fact, we occasionally observed vacuoles in our
preparations under inappropriate conditions and found that they never
showed exocytosis on chemical stimulation. For physiological studies,
therefore, the absence of vacuole was one of criteria for a good preparation.
Dynamics of exocytosis.
Before exocytosis, no increases in granule diameter of more than 50 nm
were observed (Fig. 3). This absence of significant enlargements of the
granules observed in the present preparation seems to exclude the
possibility that one granule fused with another before exocytosis.
However, one granule may fuse with another when one had fused with the
apical membrane. This occurs frequently in colonic goblet cells,
resulting in a large invagination or cavity in the apical pole of the
cells after prolonged stimulation (35). In pancreatic
acinar cells, formation of the apical invagination was rare, indicating
that the membrane retrieval after an exocytotic response was
sufficiently fast (~1 s).
The discrete light intensity changes of a single zymogen granule
reflected the release of highly condensed substances into the
extracellular space. The time required for this optical change reflected the process from the initial phase of fusion to the complete
release of its contents (releasing time). According to our
measurements, the average times required for BCh-induced exocytosis ranged from 0.48 to 0.65 s (Fig. 5). In the case of vesicles
containing catecholamines, the estimates of the time required for the
contents to diffuse out (releasing time) from measurements of the
oxidation current by means of a carbon-fiber electrode ranged from 1 to 100 ms (3, 5, 39, 42). It is reasonable to assume that the
larger the granule size, the slower the dynamics. Our video microscopic
measurement of zymogen granules gave a release time compatible for
larger granules (35).
Other factors that affect the dynamics of the exocytotic process might
include the nature of the granule membrane, solubility of granule
contents, density of substances in the acinar lumen, kinds of
stimulants, and so on. It is also possible that water secretion,
depending on the concentration of BCh, alters the luminal environment.
However, the dynamics in the presence of different concentrations of
BCh (1, 10, and 100 µM) were very similar. This suggests that the
release time depends more on the granule itself than on the luminal environment.
Movement of a single granule before exocytosis.
Agonist-induced exocytosis has been assumed to involve the following
sequential steps (9, 12, 26): 1) movement of
zymogen granules toward the apical pole, 2) docking of the
granule to an appropriate membrane site, 3) fusion of the
granule membrane with the cytoplasmic membrane, and 4)
release of granule contents into the luminal space. These processes
were assumed to occur mainly as a result of studies of stationary
images obtained by electron microscopy. Although the movements of
granules in several types of secretory cells were studied (1, 11,
15, 16, 30), the relationship between the movement and
exocytosis remains unclear because the properties of both processes
were not observed simultaneously.
Anderson and McNiven (2) claimed that they detected
exocytosis in pancreatic acinar cells under a phase-contrast
microscope. However, the clonal cells they used had lost the acinar
structure; therefore, neither exocytosis nor cellular polarization was
convincingly determined. Because it was difficult to quantitate
secretory activity at a single granule level, the movement of granules
they observed could be irrelevant to exocytosis. In fact, no
directed shift of granules toward the cell membrane has been reported
in mast cells (43), goblet cells (35), and
chromaffin cells (33) stimulated with agonists. Therefore,
a question still remained as to when and how granules translocated from
their site of synthesis to the site of exocytosis.
The normal basoapical polarities of the acinar cells were maintained
intact in the present study. The side views indicated that
long-distance movements of granules occurred frequently in the central
region of the cells; these were found not to be directly related to
exocytosis (Fig. 11). The suppression of these movements by colchicine
(Fig. 12) suggests that granules are transported along the microtubule
track (4, 29, 40). Thus granules synthesized in the
perinuclear region are transported rapidly toward the apical pole by
the microtubule-mediated gliding mechanism. After the transport, most
granules stay in the region of granule accumulation, but some leave by
the retrograde transport mechanism. The probability of granule
transport to the region of granule accumulation is always higher than
that of the retrograde transport away from it. Because of these
dynamics, granules accumulate near the apical pole even in the
unstimulated state. This view is in disagreement with the findings
described in a recent report in which granules flocked after
stimulation (2). However, the increase in overall
transport during stimulation (Fig. 12) is in accordance with a report
in which the association of kinesin with zymogen granules increased
during stimulation (18).
The finding that application of colchicine suppressed exocytosis more
strongly in the later phase of the secretory response than during the
initial 1-min period (Fig. 11B) supports the view that the
microtubule-mediated supply of granules to the apical pole is one of
the limiting processes for the secretion only during the persistent
phase. Our direct observation of granule transport provides an
explanation of why colchicine inhibited the amylase release by only
25-30% (12). In anterior pituitary cells, colchicine also suppressed exocytosis by a similar degree sometime after inhibition of granule trafficking (our unpublished observation).
Zymogen granules moved <0.16 µm in the 5-s period immediately before
exocytosis. The average velocity of the granule movement was 0.06 µm/s. The slowness of this Brownian movement near the apical membrane
was probably due to restriction by cytoskeletal network (particles of a
similar size show much faster Brownian motion in water). In fact, the
actin cytoskeleton in the vicinity of the apical membrane in the
pancreatic acini of the rat is dense (17). However,
granules seem to move through the network, since individual actin
filaments can bend and swing quite readily (42). Although
granule movements were very small, they clearly continued to move until
they underwent exocytosis. This suggests that granules are docked to
the plasma membrane in a form of a dynamic equilibrium that allows some reversibility.
None of the granules that underwent exocytosis moved faster in the
stimulated state than in the resting state (Table 1). The granules
actually tended to slow down their movement. These findings suggest
that granules in front of the region of accumulation may bind to and
unbind from the apical membrane in a form of dynamic equilibrium
irrespective of the presence or absence of stimulation. The decrease of
movement would reflect a slight shift of the equilibrium from the
unbound to the bound state. Stimulation triggers the chemical step
necessary to induce exocytosis only for those granules that happen to
be in the bound state so that the fusion of granules with the apical
membrane proceeds.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Charles Edwards for critical reading of the manuscript.
 |
FOOTNOTES |
The video sequences of exocytosis and movement of zymogen granules are
accessible at http://www.hama-med.ac.jp/w3a/photon.
Address for reprint requests and other correspondence: S. Terakawa, Photon Medical Research Center, Hamamatsu Univ. School of
Medicine, 3600 Handa, Hamamatsu, 431-3192 Japan (E-mail:
terakawa{at}hama-med.ac.jp).
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. Section 1734 solely to indicate this fact.
Received 10 July 1998; accepted in final form 10 May 2000.
 |
REFERENCES |
1.
Allen, RD,
Allen NS,
and
Travis JL.
Video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: a new method capable of analyzing microtubule-related motility in the reticulopodial network of Allogromia laticollaris.
Cell Motil Cytoskeleton
1:
291-302,
1981[ISI].
2.
Anderson, KL,
and
McNiven MA.
Vesicle dynamics during regulated secretion in a novel pancreatic acinar cell in vitro model.
Eur J Cell Biol
66:
25-38,
1995[ISI][Medline].
3.
Bruns, D,
and
Jahn R.
Real-time measurement of transmitter release from single synaptic vesicles.
Nature
377:
62-65,
1995[ISI][Medline].
4.
Busson-Mabillot, S,
Chambaut-Guérin-M A,
Ovtracht L,
Muller P,
and
Rossignol B.
Microtubules and protein secretion in rat lacrimal glands: localization of short-term effects of colchicine on the secretory process.
J Cell Biol
95:
105-117,
1982[Abstract].
5.
Chow, RH,
Klingauf J,
and
Neher E.
Time course of Ca2+ concentration triggering exocytosis in neuroendocrine cells.
Proc Natl Acad Sci USA
91:
12765-12769,
1994[Abstract/Free Full Text].
6.
Dehaye, J,
Winand J,
Poloczek P,
and
Christopher J.
Characterization of muscarinic cholinergic receptors on rat pancreatic acini by N-[3H]methyl-scopolamine binding.
J Biol Chem
259:
294-300,
1984[Abstract/Free Full Text].
7.
Gelles, J,
Schnapp BJ,
and
Sheetz MP.
Tracking kinesin-driven movements with nanometre-scale precision.
Nature
331:
450-453,
1988[ISI][Medline].
8.
Goodale, RL,
Manivel JC,
Borner JW,
Liu S,
Judge J,
Li C,
and
Tanaka T.
Organophosphate sensitizes the human pancreas to acinar cell injury: an ultrastructural study.
Pancreas
8:
171-175,
1993[ISI][Medline].
9.
Gorelick, FS,
and
Jamieson JD.
The pancreatic acinar cell.
In: Physiology of the Gastrointestinal Tract (3rd ed.). New York: Raven, 1994, vol. 2, p. 1353-1376.
10.
Habara, Y,
and
Kanno T.
Dual effects of chlorobutanol on secretory response and intracellular Ca2+ dynamics in isolated pancreatic acini of the rat.
Br J Pharmacol
109:
685-692,
1993[Abstract].
11.
Herman, B,
and
Albertini DF.
A time-lapse video image intensification analysis of cytoplasmic organelle movements during endosome translocation.
J Cell Biol
98:
565-576,
1984[Abstract].
12.
Jamieson, JD.
Transport and discharge of exportable protein in pancreatic exocrine cells: in vitro studies.
Curr Top Membr Transp
3:
273-338,
1972.
13.
Jamieson, JD,
and
Palade GE.
Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells.
J Cell Biol
50:
135-158,
1971[Abstract/Free Full Text].
14.
Kamijo, A,
Terakawa S,
and
Hisamatsu K.
Neurotransmitter-induced exocytosis in goblet and acinar cells of rat nasal mucosa studied by video microscopy.
Am J Physiol Lung Cell Mol Physiol
265:
L200-L209,
1993[Abstract/Free Full Text].
15.
Karnaky, KJ, Jr,
Garretson LT,
and
O'Neil RG.
Video-enhanced microscopy of organelle movement in an intact epithelium.
J Morphol
213:
21-31,
1992[ISI][Medline].
16.
Kreis, TE,
Matteoni R,
Hollinshead M,
and
Tooze J.
Secretory granule and endosomes show saltatory movement biased to the anterograde and retrograde directions, respectively, along microtubules in AtT20 cells.
Eur J Cell Biol
49:
128-139,
1989[ISI][Medline].
17.
Kurihara, H,
and
Uchida K.
Distribution of microtubules and microfilaments in exocrine (ventral prostatic epithelial cells and pancreatic exocrine cells) and endocrine cells (cells of the adenohypophysis and islets of Langerhans).
Histochemistry
87:
223-227,
1987[ISI][Medline].
18.
Marlowe, KJ,
Farshori P,
Torgerson RR,
Anderson KL,
Miller LJ,
and
McNiven MA.
Changes in kinesin distribution and phosphorylation occur during regulated secretion in pancreatic acinar cells.
Eur J Cell Biol
75:
140-152,
1998[ISI][Medline].
19.
Maruyama, Y.
Ca2+-induced excess capacitance fluctuation studied by phase-sensitive detection method in exocrine pancreatic acinar cells.
Pflügers Arch
407:
561-563,
1986[ISI][Medline].
20.
McCuskey, RS,
and
Chapman TM.
Microscopy of the living pancreas in situ.
Am J Anat
126:
395-408,
196[ISI][Medline].
21.
Muallem, S,
Pandol SJ,
and
Beeker TG.
Modulation of agonist-activated calcium influx by extracellular pH in rat pancreatic acini.
Am J Physiol Gastrointest Liver Physiol
257:
G917-G924,
1989[Abstract/Free Full Text].
22.
Murakami, M,
Seo Y,
and
Watari H.
Dissociation of fluid secretion and energy supply in rat mandibular gland by high dose of ACh.
Am J Physiol Gastrointest Liver Physiol
254:
G781-G787,
1988[Abstract/Free Full Text].
23.
Murakami, M,
Seo Y,
and
Watari H.
Effects of acetylcholine on salivary secretion and energy metabolism: measured by NMR spectroscopy.
Biomed Res
8, Suppl:
83-90,
1987.
24.
Neher, E,
and
Marty A.
Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.
Proc Natl Acad Sci USA
79:
6712-6716,
1982[Abstract].
25.
Niederau, C,
and
Grendell JH.
Intracellular vacuoles in experimental acute pancreatitis in rats and mice are an acidified compartment.
J Clin Invest
81:
229-236,
1988[ISI][Medline].
26.
Palade, GE.
Subcellular particles.
In: Functional Changes in the Structure of Cell Components. New York: Ronald, 1959, p. 64-83.
27.
Sakurai, T,
and
Terakawa S.
Insulin secretion from pancreatic
-cells directly visualized as exocytosis by video microscopy.
Bioimages
3:
85-92,
1995.
28.
Segawa, A,
Terakawa S,
Yamashina S,
and
Hopkins CR.
Exocytosis in living salivary glands: direct visualization by video-enhanced microscopy and confocal laser microscopy.
Eur J Cell Biol
54:
322-330,
1991[ISI][Medline].
29.
Seybold, J,
Bierger W,
and
Kern HF.
Studies on intracellular transport of secretory proteins in the rat exocrine pancreas.
Virchows Arch
368:
309-327,
1975.
30.
Somers, G,
Blondel B,
Orci L,
and
Malaisse W.
Motile events in pancreatic endocrine cells.
Endocrinology
104:
255-264,
1979[Abstract].
31.
Tao, C,
Yamamoto M,
Mieno H,
Inoue M,
Masujima T,
and
Kajiyama G.
Pepsinogen secretion: coupling of exocytosis visualized by video microscopy and [Ca2+] in single cells.
Am J Physiol Gastrointest Liver Physiol
274:
G1166-G1177,
1998[Abstract/Free Full Text].
32.
Terakawa, S.
Optical studies on the intracellular processes for secretion.
Gunma Symp Endocrinol
26:
137-144,
1989.
33.
Terakawa, S,
Fan JH,
Kumakura K,
and
Ohara-Imaizumi M.
Quantitative analysis of exocytosis directly visualized in living chromaffin cells.
Neurosci Lett
123:
82-86,
1991[ISI][Medline].
34.
Terakawa, S,
Kumakura K,
and
Duchen MR.
Spatio-temporal analysis of quantal secretory events from single bovine adrenal chromaffin cells in culture.
J Physiol (Lond)
487:
59-60,
1995.
35.
Terakawa, S,
and
Suzuki Y.
Exocytosis in colonic goblet cells visualized by video-enhanced light microscopy.
Biochem Biophys Res Commun
176:
466-472,
1991[ISI][Medline].
36.
Tomita, Y,
Inooka G,
Shimada H,
and
Maruyama Y.
Ca2+-dependent unidirectional vesicular release detected with a carbon-fibre electrode in rat pancreatic acinar cell triplets.
Pflügers Arch
428:
69-75,
1994[ISI][Medline].
37.
Tsunoda, Y,
Stuenkel EL,
and
Williams JA.
Characterization of sustained [Ca2+]i increase in pancreatic acinar cells and its relation to amylase secretion.
Am J Physiol Gastrointest Liver Physiol
259:
G792-G801,
1990[Abstract/Free Full Text].
38.
Vinayek, R,
Murakami M,
Sharp CM,
Jensen RT,
and
Gardner JD.
Carbachol desensitizes pancreatic enzyme secretion by downregulation of receptors.
Am J Physiol Gastrointest Liver Physiol
258:
G107-G121,
1990[Abstract/Free Full Text].
39.
Wightman, RM,
Jankowski JA,
Kennedy RT,
Kawagoe KT,
Schroeder TJ,
Leszczyszyn DJ,
Near JA,
Diliberto EJ, Jr,
and
Viveros OH.
Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells.
Proc Natl Acad Sci USA
88:
10754-10758,
1991[Abstract].
40.
Williams, JA,
and
Lee M.
Microtubule and pancreatic amylase release by mouse pancreas in vitro.
J Cell Biol
71:
795-806,
1976[Abstract].
41.
Yanagida, T,
Nakase M,
Nishiyama K,
and
Oosawa F.
Direct observation of motion of single F-actin filaments in the presence of myosin.
Nature
307:
58-60,
1984[ISI][Medline].
42.
Zhou, Z,
and
Misler S.
Amperometric detection of quantal secretion from patch-clamped rat pancreatic
-cells.
J Biol Chem
270:
270-277,
1996.
43.
Zimmerberg, J,
Curran M,
Cohen FS,
and
Brodwick M.
Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cell.
Proc Natl Acad Sci USA
84:
1585-1589,
1987[Abstract].
Am J Physiol Cell Physiol 279(4):C1177-C1188
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society