(Received for publication, December 4, 1995; and in revised form, January 9, 1996)
From the
We have investigated the localization of Ca extrusion sites in mouse pancreatic acinar cells. Employing a new
technique, in which high resolution localization of cellular
Ca
exit is achieved by confocal microscopy and a
Ca
-sensitive fluorescent probe coupled to heavy
dextran to slow down diffusion of extracellular Ca
,
it is shown directly that the secretory pole (secretory granule area)
is the major site for Ca
extrusion following agonist
stimulation. This Ca
extrusion appears not to be a
consequence of exocytosis, as assessment of secretion under our
experimental conditions (low external Ca
concentration, room temperature) using the technique of
monitoring quinacrine fluorescence shows little loss of secretory
granules in spite of sustained Ca
exit. We conclude
that Ca
is primarily extruded by Ca
pumps from the secretory pole and propose that this process is
useful for maintaining a high Ca
concentration in the
acinar lumen, which is necessary for promotion of endocytosis.
Hormone or neurotransmitter-evoked intracellular Ca release is rapidly followed by activation of plasma membrane
Ca
pumps extruding a considerable fraction of the
Ca
liberated from the
stores(1, 2, 3, 4) . Pancreatic
acinar cells are highly polarized with an apical secretory pole that is
particularly sensitive to Ca
mobilizing messengers (5, 6, 7) . Recent indirect evidence suggests
that continuous maximal agonist stimulation evoking a sustained global
rise in the cytosolic Ca
concentration
([Ca
]
) (
)results in activation of plasma membrane Ca
pumps in all regions of these cells but that the basolaterally
located pumps are partly switched off after only a few
seconds(8) .
We have now devised a new method for direct
visualization of the regional Ca extrusion sites from
single cells. To detect Ca
extruded from the
stimulated cells as well as to slow down diffusion of Ca
in the external milieu, we use confocal microscopy and a
Ca
-sensitive fluorescent probe linked to heavy
dextran in the extracellular solution. We show directly that the
secretory pole is the major Ca
extrusion site in
pancreatic acinar cells following agonist stimulation. This arrangement
would appear to have substantial physiological advantages.
Ca
extrusion across the luminal membrane would help
to confine physiological Ca
signals to the apical
part of the cell. Furthermore, endocytosis, which must follow
Ca
-activated exocytosis, requires extracellular
Ca
(9, 10) , and the Ca
pumping into the acinar lumen would secure this.
Fragments of mouse pancreas were digested by pure collagenase
to obtain single acinar cells or small clusters as described previously (11) . In some experiments, cells were loaded with the
fluorescent indicator fura-red acetoxymethyl ester for 30 min at room
temperature as described previously for fura-2
loading(6, 11) . A group of fura-red containing single
cells/cell clusters or non-loaded cells/cell clusters were centrifuged
twice in Ca-free solution and then placed in a small
experimental chamber (approximately 200 µl) containing nominally
calcium-free solution and 30-100 µM Calcium Green 1
bound to dextran (M
500,000) (Molecular Probes).
The free calcium concentration in the extracellular solution under such
conditions was around 0.2 µM. The substantial difference
between the emission spectra of Calcium Green 1 dextran and fura-red
allowed us to monitor simultaneously intracellular and extracellular
calcium levels. The fluorescent signals from the intracellular and
extracellular dyes were recorded using a Noran Odyssey confocal
microscope (Noran Instruments) with the excitation wavelength of 488 nm
and emission wavelengths of 530 and 650 nm for Calcium Green 1 dextran
and fura-red, respectively. Several boxes were placed in different
areas of the field, and the average fluorescence levels from these
boxes were acquired during the course of experiments. Measurements of
Ca
extrusion were performed on both loaded and
unloaded cells. Images were analyzed and processed by two-dimensional
Intervision analysis software (Noran Instruments). The experimental
data are presented as fluorescence intensity rather than free calcium
concentration.
Quinacrine was loaded into the cells by incubation in
Ca-containing solution (1 mM) with 1
µM of the dye for 7-10 min at 37 °C. After
loading, cells were centrifuged and resuspended twice in the nominally
calcium-free solution (except for experiments of the type shown in Fig. 5C).
Figure 5:
Short lasting ionophoretic ACh application
does not deplete the cells of secretory granules. A,
transmitted light and quinacrine fluorescence images of three-cell
cluster. The scale bar is 10 µm. Boxes 1 and 2 show the areas from which quinacrine fluorescence was
recorded in B. Elevation in fluorescence intensity is
presented as a change in color from blue to red. B, changes in quinacrine fluorescence due to ACh iontophoretic
application to the cluster that is shown in A. The onset of
the ACh application (20 s; 25 nA) is indicated by the arrow.
Fluorescence intensity was recorded from boxes 1 and 2 in A. C, the effect of 10 µM ACh at
37 °C on a quinacrine-loaded cluster in a Ca (1
mM)-containing solution. Duration of ACh administration is
shown by the bar. Curve represents fluorescence
intensity from the secretory pole.
ACh applications were made either by a small droplet addition of a concentrated ACh stock solution to achieve a concentration of about 10 µM in the experimental chamber or by iontophoresis from a microelectrode filled with 20 mM ACh chloride (a second electrode was in this case placed in the experimental chamber). The iontophoretic currents used were from 10 to 100 nA and lasted from 1 to 20 s.
The extracellular solution
contained (in mM): NaCl 140, MgCl 0.66-1.13,
KCl 4.7, glucose 10, Hepes 10, pH 7.2, adjusted with NaOH. Experiments
were carried out at room temperature with the exception of one series
specifically mentioned in the text.
Fig. 1shows simultaneous measurements of
Ca-sensitive fluorescence of fura-red loaded into a
pancreatic acinar cell and Calcium Green 1 dextran present in the
external solution. It can be seen that the rapid rise in
[Ca
]
evoked by ACh,
causing a decrease in the intensity of fura red fluorescence, is
followed by an increase in the fluorescence of Calcium Green 1 dextran
signifying extrusion of Ca
from the cell to the
surrounding medium. Stimulation of this type (with 10 µM ACh) always evokes a [Ca
]
signal that is initiated in the secretory pole but then
rapidly (within a few seconds) spreads to the whole of the acinar
cell(11, 12) . It is theoretically possible that there
could be an uneven intracellular distribution of fura-red, which might
influence the time courses of Ca
extrusion in
different parts of the cell, and for that reason the majority of the
experiments described in what follows were done on cells that had not
been loaded with any fluorescent indicators. In these experiments only
extracellular indicator was present monitoring Ca
extrusion.
Figure 1:
Simultaneous measurement of
Ca extrusion and changes in the cytosolic free
Ca
concentration
([Ca
]
) in response to
stimulation with 10 µM ACh. The upper trace shows
the increase in fluorescence intensity (percent) from the extracellular
Calcium Green 1 dextran representing an increase in the external
Ca
concentration (Ca
extrusion)
whereas the lower trace shows the decrease in fluorescence
intensity from the intracellular fura-red representing an increase in
[Ca
]
.
Fig. 2shows the result from an experiment
on a cluster of acinar cells bathed in a solution containing Calcium
Green 1 dextran, where the confocal microscope was used to locate the
sites of Ca exit from stimulated cells. Fig. 2A shows the transmitted light picture of the
cluster demonstrating the central localization of the secretory
granules. Fig. 2B shows a fluorescence image with the
optical slice going through part of the cluster consisting of 4 cells
surrounding a pseudolumen. The three boxes shown represent the
sites from which the Ca
-sensitive fluorescence was
recorded. A brief but vigorous period of ionophoretic ACh stimulation
causes rises of the extracellular Ca
concentration in
all of the three areas in which fluorescence was monitored, but as seen
in Fig. 2C by far the steepest rise occurs in box 1 placed close to the center of the cluster near the pseudolumen.
Figure 2:
Localization of Ca extrusion from an acinar cell cluster in response to ionophoretic
ACh stimulation. A, transmitted light image of acinar cell
cluster. The central localization of the secretory granules is seen.
The scale bar represents 10 µm. B, fluorescence
intensity image using optical slice through part of cluster consisting
of four cells surrounding a pseudolumen. The average fluorescence
intensities in boxes 1-3 are displayed in C as
a function of time. C, time course of fluorescence intensity
(%) from the boxes (1-3) shown in B.
The base lines in traces 1-3 have been deliberately
off-set so that the three traces can be seen more
clearly.
It could be argued that the results shown in Fig. 2might be
explained by restrictions to the diffusion of Ca away
from the lumen. We therefore decided to do experiments in which the
optical slice did not go through the cell cluster itself. In the
following experiments the fluorescent light was collected from a layer
just above the upper surface of the cluster.
Fig. 3shows the
result from such an experiment on a cluster of four acinar cells bathed
in a solution containing Calcium Green 1 dextran. Fig. 3a shows the transmitted light picture of the cluster demonstrating
the central localization of the secretory granules. All other pictures
in this set show pseudocolor representations of the fluorescence
intensity of Calcium Green 1 dextran in the extracellular solution. The
outline of the cell cluster taken from the transmitted light picture
has for convenience been placed on all the fluorescence images. Fig. 3, b-h, represents images taken at 10-s
intervals immediately before b and following ACh stimulation (c-h), whereas Fig. 3i shows the
situation more than 100 s after the maximal external Ca concentration had been reached. It is seen in Fig. 3that
the maximum fluorescence intensity is close to the secretory granule
area. The rise in extracellular Ca
-sensitive
fluorescence is transient, lasting only about 200 s, in spite of
continuous ACh stimulation. This is expected as the external
Ca
concentration in our experiments is kept
sufficiently low to prevent Ca
entry into the cell so
that the cell runs out of mobilizable Ca
as shown
previously(1, 2) . Fourteen experiments of the type
shown in Fig. 3were carried out with similar results.
Figure 3:
Localization of Ca extrusion from a small acinar cell cluster in response to ACh
stimulation (10 µM). a, transmitted light image
of acinar cell cluster. The central localization of the secretory
granules is seen. The scale bar represents 10 µm. b-i, pseudocolor images of
Ca
-sensitive fluorescence intensity taken just before (b) and then at 10-s intervals following and during ACh
stimulation except i, which was obtained 95 s after h. The outline of the acinar cell cluster is shown in red on each of the images. The color map to the right shows the relationship between fluorescence intensity and color.
The optical slice from which the fluorescent light was collected did
not go through the cells but was right above the cell
cluster.
In
order to improve the resolution of Ca exit sites we
carried out a series of experiments on isolated single cells. In this
case the single acinar cells were stimulated with 10-15-s pulses
of ionophoretically applied ACh. In experiments with fura-red loaded
cells it was established that this protocol always gave rise to a
global elevation of
[Ca
]
(12) . One
of the experiments designed to visualize the major Ca
extrusion site from a single cell after this type of ACh
stimulation is shown in Fig. 4. The polarization of the single
cell is clearly seen in Fig. 4A, a. The confocal system
collected the fluorescent light from a layer that went through the
cell, which is shown as a black spot in each of the
fluorescence images (Fig. 4A, b-i) that were
obtained at 3-s intervals following the start of ACh stimulation. The
intensity of the fluorescence of the extracellular Calcium Green 1
dextran increased much more near the secretory pole than near the basal
pole, and this is also illustrated graphically in Fig. 4B. In every one of the six experiments of this
type, the fluorescence intensity grew more markedly near the secretory
pole than the basal pole, and in no case was the peak intensity at the
basal pole more than 50% of that seen near the secretory pole,
irrespective of the exact position of the stimulating ACh pipette. As
seen in Fig. 4B there was a steep increase in the
Ca
-sensitive fluorescence also at the basal pole, and
we tried to assess whether this might be due to diffusion of
Ca
primarily extruded from the apical (secretory)
pole or whether it was more likely to arise from extrusion through the
basal membrane. We therefore compared the rise in
Ca
-sensitive fluorescence from three boxes placed
equidistantly on a straight line with the box in the middle near the
secretory pole and the two other boxes at the basal pole or in the
exact opposite direction from the secretory pole, respectively. In such
cases the rise near the basal pole occurred earlier and was always
steeper and larger (at least 2 times) than in the box on the opposite
side of the secretory pole, indicating that the rise in Ca
concentration at the basal pole is mostly due to Ca
extrusion through the basal pole rather than diffusion of
Ca
extruded at the secretory pole.
Figure 4: Calcium extrusion from a single isolated pancreatic acinar cell following ionophoretic ACh application. A, a, transmitted light image of the cell. Boxes denote the places from which the average values of fluorescence intensity are presented as graphs in B. The scale bar represents 10 µm. b-i are fluorescence intensity images taken from the beginning of the ACh application at 3-s intervals. The optical slice went through the cell, and the shape of the cell is shown in black on each of the fluorescent images. B, the average fluorescence intensity in boxes 1-3 that are depicted in A (off-set in the vertical direction to improve the clarity of separation of the three traces during the experiment).
Can the
Ca extrusion observed (Fig. 2Fig. 3Fig. 4) be explained simply as a
result of exocytotic Ca
release? From experiments
with the droplet technique, in which the total amount of Ca
extruded from maximally stimulated pancreatic acinar cells was
measured, it is known that the whole of the mobilizable Ca
pool, corresponding to about 0.7 mM of total cellular
calcium concentration(1, 13) , is exported in about
200 s(1) . Within that time frame the pancreatic acinar cell is
very far from being depleted of secretory granules. We could not
observe any significant loss of quinacrine fluorescence from secretory
granules (quinacrine is accumulated in the acidic granules, and loss of
quinacrine fluorescence is used as a measure of secretion(14) )
after our standard 10-15-s ACh pulse under the conditions of our
experiments (room temperature, Ca
-free solution) (n = 6) (Fig. 5, A and B). In
the presence of 1 mM external Ca
, sustained
ACh stimulation (10 µM) at 37 °C did evoke a
measurable decrease in quinacrine fluorescence, indicating secretion (Fig. 5C).
The novel technique described here, in which high resolution
localization of cellular Ca exit is achieved by
confocal microscopy and a Ca
-sensitive fluorescent
probe coupled to heavy dextran to slow down diffusion of extracellular
Ca
, has enabled us to show directly that the
secretory pole in pancreatic acinar cells is the major site of
Ca
extrusion following agonist stimulation.
The
relatively high rate of Ca extrusion at the secretory
pole, uncovered in our experiments (Fig. 4), could be due to a
relatively high density of Ca
pumps in this region
and/or be explained by the Ca
pumps in this part of
the cell having different characteristics from those in the basal
membrane. A number of isoforms of the plasma membrane
Ca
-ATPase have been described(15) , and it is
known that the Ca
affinity of the pump can be
controlled by alternative splicing via changes in the affinity for
calmodulin(16) . It is also possible that agonist stimulation
could differentially regulate Ca
pumps in the luminal
and basolateral membranes.
The results shown in Fig. 5, A and B, should not necessarily be taken as an indication
that there was no secretion in our experiments, although secretion
would be expected to be reduced because of the low external calcium
concentration(17) . The quinacrine technique employed may not
be sufficiently sensitive, but the crucial point is that the majority
of secretory granules was still in the cell after the period of
stimulation resulting in the Ca extrusion shown in Fig. 2Fig. 3Fig. 4. The total calcium
concentration in isolated zymogen granules appears to be about 15
mM(18, 19) . Taking into account the known
magnitude of the acutely hormone-mobilizable Ca
pool (1, 13) and the ratio of zymogen granule volume to
cell volume(20) , it can be estimated that the zymogen granules
apparently contain about the same amount of calcium as the acutely
hormone-mobilizable pool. Since the whole of the acutely
hormone-mobilizable Ca
pool is extruded within 200 s (1) it means that if this extrusion were to be accounted for
solely in terms of exocytotic release, all the zymogen granules should
have been lost within this period. This is obviously very far from
being the case (Fig. 5)(1) . The maximal rate of enzyme
secretion, under optimal conditions, is such that only about 7% of the
cellular enzyme content has been released after a half hour of
continuous maximal stimulation(21) .
Let us, for the sake of
argument, suppose that most of the Ca is extruded
homogeneously from the entire cell membrane but that calcium secretion
occurs only in the secretory region. Such an arrangement would lead to
inhomogeneity of calcium release. But under the above mentioned
assumptions, this inhomogeneity would not appear to explain the whole
difference observed with regard to calcium release between basal parts
of the cells and their secretory regions. In all our experiments the
difference in values of fluorescent signals between these parts of the
extracellular milieu was not less than a factor of 2. It means that
there is at least a 2-fold difference in calcium flux, leaving a unit
area of cell membrane in these two regions. The part of the cell
perimeter associated with the secretory granule area where calcium
efflux was at least 2 times higher than in the basolateral part was, in
our experiments, about one-fifth of the whole cell perimeter. In this
case, calcium secretion should be 10% of the total calcium efflux in
order to account for the difference between the basal parts of the
cells and their secretory regions, i.e. on the basis of
previous estimations of the total calcium releasable pool and total
calcium granule content, 10% of all granules should be released during
ACh application at room temperature and low external Ca
concentration in less than 4 min. This is unlikely since only 7%
of the granules could be released during a half hour of ACh stimulation
at 37 °C with a high external Ca
concentration.
We therefore conclude that the Ca
extrusion observed
in our experiments is unlikely to be accounted for by exocytotic
release of Ca
, although there may well be a small
contribution from this process, but is most likely due to plasma
membrane Ca
-activated ATPases, since
Na
-Ca
exchange is virtually absent
in pancreatic acinar cells(1, 13) .
The marked
Ca extrusion through the secretory pole would appear
to be advantageous for the overall function of the acinar cells.
Ca
signals evoked by physiological agonist
concentrations are essentially confined to the secretory granule
area(5, 6, 7) , and selective Ca
pumping across the luminal membrane would help to prevent
spreading of the signal into the basolateral regions. This may be
important to avoid Ca
-induced Ca
release in the nucleus via the inositol trisphosphate and
ryanodine receptors localized in the inner nuclear
membrane(22, 23) . While Ca
extruded
across the basolateral membrane would appear to have no special
function, Ca
pumping into the acinar lumen would
maintain a high Ca
concentration in this
extracellular compartment. This is important as it is known that
endocytosis in the pancreas cannot proceed in the absence of external
Ca
(9, 10) . We can now envisage an
interesting Ca
cycle in the pancreas where agonist
stimulation primarily releases Ca
from stores in the
secretory granule area(24) , most likely from the granules
themselves(19, 25) . The Ca
released
into the cytosol is then mainly transported across the luminal membrane
into the acinar lumen where it promotes
endocytosis(9, 10) , following the stimulant-evoked
exocytosis, and may also in part be taken back into the cell via the
endocytotic process or through a Ca
entry pathway, in
this way being recycled into new secretory granules.