(Received for publication, October 30, 1995; and in revised form, December 11, 1995)
From the
Several lines of evidence suggest that the existence of a
heterogeneous population of inositol 1,4,5-trisphosphate
(Ins(1,4,5)P)-sensitive Ca
stores
underlies the polarized agonist-induced rise in cytosolic
Ca
concentration
([Ca
]
) in pancreatic
acinar cells (Kasai, H., Li, Y. X., and Miyashita, Y.(1993) Cell 74, 669-677; Thorn, P., Lawrie, A. M., Smith, P. M.,
Gallacher, D. V., and Petersen, O. H.(1993) Cell 74,
661-668). To investigate whether the apical pole of acinar cells
contains Ca
stores which are relatively more
sensitive to Ins(1,4,5)P
than those in basolateral areas,
we studied Ca
handling by Ca
stores
in individual streptolysin O (SLO) permeabilized cells using the low
affinity Ca
indicator Magfura-2 and an in situ imaging technique. The uptake of Ca
by
intracellular Ca
stores was ATP-dependent. A
steady-state level was reached within 10 min, and the free
Ca
concentration inside loaded Ca
stores was estimated to be 70 µM. Ins(1,4,5)P
induced Ca
release in a dose-dependent,
``quantal'' fashion. The kinetics of this release were
similar to those reported for suspensions of permeabilized pancreatic
acinar cells. Interestingly, the permeabilized acinar cells showed no
intercellular variation in Ins(1,4,5)P
sensitivity.
Although SLO treatment is known to result in a considerable loss of
cytosolic factors, permeabilization did not result in a redistribution
of zymogen granules, as judged by electron microscope analysis. These
results suggest that Ins(1,4,5)P
-sensitive Ca
stores are unlikely to be redistributed as a result of SLO
treatment. The effects of Ins(1,4,5)P
were therefore
subsequently studied at the subcellular level. Detailed analysis
demonstrated that no regional differences in Ins(1,4,5)P
sensitivity exist in this permeabilized cell system. Therefore,
we propose that additional cytosolic factors and/or the involvement of
ryanodine receptors underlie the polarized pattern of agonist-induced
Ca
signaling in intact pancreatic acinar cells.
In many non-excitable cell types, agonist stimulation results in
repetitive oscillations of the free cytosolic Ca concentration
([Ca
]
) (
)arising largely from Ca
release from
intracellular stores(1) . In the polarized exocrine acinar cell
of exocrine glands, these agonist-induced intracellular Ca
signals are not spatially homogeneous. Thus acetylcholine- or
cholecystokinin-octapeptide-induced
[Ca
]
rises are
initiated in the luminal pole of the cytosol with the Ca
wave subsequently spreading into the basolateral areas of the
cell(2, 3, 4) . In some cases little or no
[Ca
]
increase at all
is observed in basolateral regions(5, 6) . This
spatial pattern of [Ca
]
signaling has been suggested to be important for both
unidirectional fluid secretion (2, 7) and exocytosis (8) .
In permeabilized pancreatic acinar cells, as in many
other permeabilized cell systems, the intracellular messenger D-myo-inositol 1,4,5-trisphosphate
(Ins(1,4,5)P) mobilizes Ca
from
non-mitochondrial intracellular Ca
stores(1, 9) . Recent evidence in both
permeabilized (10, 11, 12) and intact cells (5, 6) favors the existence of a heterogeneous population of Ins(1,4,5)P
-sensitive Ca
stores. In a recent study (12) it was suggested that the
Ca
imaging data obtained by several laboratories (see
above) might be explained by the existence of stores more sensitive to
Ins(1,4,5)P
localized in the apical pole with stores less
sensitive to Ins(1,4,5)P
being located in basolateral
areas.
To address this question we have now imaged the
Ca concentration within Ca
stores
in pancreatic acinar cells using the in situ imaging technique
originally described by Hofer and Machen(13) . The low affinity
Ca
indicator Magfura-2 was loaded into intracellular
stores and the properties of these stores were studied in streptolysin
O-permeabilized cells. ATP-driven Ca
uptake and
Ins(1,4,5)P
-dependent Ca
release could be
clearly demonstrated within individual permeabilized pancreatic acinar
cells. However, we were unable to detect any subcellular regional
differences in Ins(1,4,5)P
-sensitivity. This may indicate
that cytosolic modulators of Ins(1,4,5)P
-operated
Ca
channels and/or the involvement of ryanodine
receptors, underlie the polarized nature of
[Ca
]
signaling
patterns in acinar cells.
Since a
considerable amount of the total accumulated Magfura-2 was present in
the cytosolic compartment, the permeabilization process could be
followed on-line (as loss of cytosolic dye) by using the imaging
system. At the start of the permeabilization procedure, loaded cells
were excited at the isosbestic wavelength for Magfura-2, i.e. 360 nm. Permeabilization was achieved within 10 min and, as a
consequence, a significant drop in fluorescence was observed as
cytosolic Magfura-2 was lost into the incubation medium (results not
shown). Perfusion of the permeabilized cells was subsequently continued
with the Ca uptake medium devoid of SLO, as described
above.
Figure 5:
Effect of stepwise increases in
Ins(1,4,5)P concentration on loaded intracellular
Ca
stores. Intracellular stores of permeabilized
pancreatic acinar cells loaded Ca
in an ATP-dependent
fashion for 15 min. The experiment was started and image pairs were
collected at 15 s intervals. After 1.5 min 0.1 µM Ins(1,4,5)P
was included for 5 min in the medium and
its concentration was increased at 5-min intervals to 0.3, 1.0, and 10
µM. Finally, permeabilized cells were reperfused with
control medium until the end of the experiment. A, individual
responses of seven acinar cells plotted in a similar format to that of Fig. 2. B and C show, respectively, a
brightfield image and a fluorescence image (340 nm excitation). Six
ratio images are presented in D. The ratio values were
converted to pseudocolor to clarify the ratio levels measured. The
pseudocolor scale used is shown at the side. The images shown are taken
at time points indicated with the corresponding numbers in A.
The calibration squares in B and C, and the
sizes of the boxes used to number the images in D,
represent 25 µm. The results shown are representative for three
independent experiments.
Figure 1: Electron micrographs on intact and permeabilized pancreatic acinar cells. Intact pancreatic acinar cells or cells permeabilized by steptolysin O treatment were fixed and processed as described under ``Experimental Procedures.'' A and B show the morphology of a triplet of intact cells and two coupled permeabilized cells, respectively. The magnification was in both cases 3000 times and the calibration bar represents 5 µM.
Figure 2:
Effect of ATP and Ca on
the Ca
content of intracellular Ca
stores in permeabilized pancreatic acinar cells. Magfura-2-loaded
pancreatic acinar cells were permeabilized by SLO treatment, and the
Magfura-2 remaining inside the intracellular compartments was used to
monitor store lumen Ca
changes. A field of nine
permeabilized cells was excited alternately at 340 and 380 nm and the
emitted fluorescence captured by a digital camera as described under
``Experimental Procedures.'' Pairs of images were acquired at
1 min intervals. The off-line calculated 340/380 nm ratio of each
individual cell is depicted. After assessment of permeabilization,
cells were initially perfused with Ca
uptake medium
in the absence of Ca
or ATP, but with a free
Mg
concentration of 0.9 mM. At the beginning
of the experiment, ATP was added to this medium. After 19 min,
Ca
was introduced to the Mg
- and
ATP-containing medium at a free concentration of 0.2 µM.
The observation is representative of two independent
experiments.
To demonstrate that the Magfura signal was not saturated in
loaded Ca stores under these conditions, the
Ca
ionophore 4-Br-A23187 (2 µM) was
included and the ambient free Ca
concentration in the
medium was increased to 1 mM (for details of the medium used,
see ``Experimental Procedures''). This treatment resulted in
a further increase in the ratio to 3.43 (S.E. 0.02; n =
3 cell preparations and 19 individual cells analyzed; results not
shown, but see also Fig. 3). This indicates that the free
Ca
concentration of steady-state loaded intracellular
Ca
stores of pancreatic acinar cells is not in the
millimolar range. A tentative calibration was made by perfusing
permeabilized cells with Ca
ionophore and a medium
containing different ambient free Ca
concentrations. Fig. 3shows the result of this calibration. The average Magfura
ratio in stores loaded at steady-state was 1.44 (see above), and
therefore the free Ca
concentration in loaded
intracellular Ca
stores is estimated to be 70
µM. The minimum ratio, obtained with permeabilized cells
in a Ca
free medium, including ionophore, was 0.37
(S.E. = 0.02; n = 4 cell preparations and 37
individual cells analyzed). The average ratio in unloaded stores
without ionophore in the same set of experiments was 0.47 (S.E.
= 0.03; n = 4 cell preparations and 37 cells
analyzed). This shows that, before initiation of Ca
uptake, the Ca
stores in the permeabilized
cells were virtually depleted of their Ca
content.
Figure 3:
Calibration of the free Ca levels inside intracellular Ca
stores of
permeabilized pancreatic acinar cells. Permeabilized pancreatic acinar
cells were allowed to load Ca
in the presence of ATP.
After 15 min, Mg
and ATP were removed from the medium
and various Ca
concentrations were imposed by using
the non-fluorescent Ca
ionophore 4-Br-A23187 (2
µM). Image pairs were acquired every 3.5 min. Details of
the various Ca
conditions are described under
``Experimental Procedures.'' The observation is
representative of two independent
experiments.
Figure 4:
Effects of Ins(1,4,5)P on
loaded intracellular Ca
stores in permeabilized
pancreatic acinar cells. Permeabilized pancreatic acinar cells were
perfused during a 15-min period, allowing intracellular Ca
stores to accumulate Ca
to a steady-state
level. The experiment was started after the loading period and a pair
of images was acquired each 15 s as described under ``Experimental
Procedures'' and the legend of Fig. 2. After 1.5 min,
Ins(1,4,5)P
was added with a concentration of 0.3 or 1.0
µM in A and B, respectively. After a
period of 12 min, the Ins(1,4,5)P
concentration was
increased to 10 µM until the experiment was terminated.
For reasons of clarity, the averaged responses of five and seven cells
are presented in A and B, respectively. For each
situation, all permeabilized cells responded in a similar manner. The
results represent a single observation; the effects of 0.3 and 1.0
µM Ins(1,4,5)P
applied for a period of 5 min
were tested in four independent experiments and gave results similar to
those presented.
In
permeabilized gastric epithelial cells, mitochondrial uptake of
Ca contributed substantially to the ATP- and
Ca
-dependent increase in the signal from
compartmentalized Magfura-2(21) . Thus a considerable part of
the ATP-dependent Ca
pool in this cell type was
Ins(1,4,5)P
-insensitive but sensitive to mitochondrial
Ca
uptake inhibitors. In pancreatic acinar cells,
however, a maximal dose of Ins(1,4,5)P
released virtually
all the Ca
that had been taken up in an ATP-dependent
manner, indicating that mitochondria were not active under the
conditions used (see above). In addition, when Ca
uptake was performed in the presence of the mitochondrial
inhibitors antimycin and oligomycin, no change in uptake
characteristics was observed (results not shown). These observations
confirm previous studies with radiotracer techniques on permeabilized
pancreatic acinar cells, in which it was shown that mitochondrial
Ca
uptake was inactive at an ambient free
Ca
concentration identical to that used in the
present study(16) .
The action of
Ins(1,4,5)P was studied in more detail by analyzing the
ratio images of the experiment. Fig. 5, B and C show a brightfield and a fluorescence image respectively of the
selected field of cells. The brightfield image shows again that
permeabilized cells organized in triplets clearly maintained their
polarized morphology. The first ratio image (Fig. 5D, image
1) shows Ca
stores in permeabilized cells loaded
to a steady-state level. The ratio intensity throughout the different
regions of the cells was not homogeneous, although virtually all
regions had a ratio value of about 1 or higher (i.e. the free
[Ca
] was 40 µM or higher).
Stimulation with a low dose of Ins(1,4,5)P
(0.3
µM) resulted in a simultaneous decrease of Ca
levels in all regions of the permeabilized cells (Fig. 5D, images 2 and 3). Elevation of the
dose to 1.0 µM (Fig. 5D, image 4) and then
to 10.0 µM (Fig. 5D, image 5) resulted
again in a simultaneous reaction in all subcellular regions in all
permeabilized acinar cells. Subsequent removal of Ins(1,4,5)P
resulted in Ca
reuptake in all regions of the
permeabilized cells (Fig. 5D, image 6). (The regional
analysis, as presented in Fig. 5D, was performed in
four additional experiments; in two experiments sequential
Ins(1,4,5)P
additions were made as presented in Fig. 5, and in the other two experiments, 0.3 µM Ins(1,4,5)P
added to loaded stores of permeabilized
cells. All experiments analyzed gave similar results to those presented
in Fig. 5). To further demonstrate the uniform Ins(1,4,5)P
sensitivity, the kinetics of Ins(1,4,5)P
-induced
Ca
release were compared between selected areas of
interest in apical and basolateral regions in the same field of cells. Fig. 6A shows the selected areas and Fig. 6B shows the averaged and normalized kinetics of Ca
release induced by Ins(1,4,5)P
in apical and
basolateral regions. Again, the results demonstrate that both regions
were equally sensitive to Ins(1,4,5)P
. Taken together, the
results demonstrate that Ca
stores in
SLO-permeabilized acinar cells display neither regional nor
intercellular differences in their sensitivity toward
Ins(1,4,5)P
.
Figure 6:
Comparison of Ins(1,4,5)P sensitivity in apical and basolateral regions of permeabilized
pancreatic acinar cells. A, in the same field of seven
selected cells shown in Fig. 5, a region of interest was
selected in the apical (black square symbols) and basolateral
part of each cell (white square symbols). The selected regions
of interest are shown on the corresponding brightfield image. B shows the effect of stepwise increases in Ins(1,4,5)P
on the averaged and normalized size of the
Ins(1,4,5)P
-sensitive store in the apical regions (solid line) and in the basolateral (dashed line)
regions. Maximal store size was defined as the difference in ratio
between unstimulated and maximally Ins((1,4,5)P
-stimulated
Ca
stores and the averaged results were normalized to
this value; this is directly analogous to the definition of
``Ins(1,4,5)P
-sensitive Ca
pools'' commonly employed in radiotracer experiments. The
results presented are from the same experiment as shown in Fig. 5and are typical for three independent
experiments.
Figure 7:
Effect of stepwise increases in
Ins(1,4,5)P concentration on loaded intracellular
Ca
stores in the absence of intracellular
Ca
-ATPase activity. Intracellular Ca
stores of permeabilized pancreatic acinar cells were loaded with
Ca
to a steady-state level for 15 min as described
under ``Experimental Procedures.'' At the start of the
experiment, Ca
pumps of the loaded intracellular
stores were inhibited by including 1 µM thapsigargin in
the medium. After 5 min the Ins(1,4,5)P
concentration was
increased from 0.1 to 0.3 µM and finally to 1.0 µM at 5-min intervals. The Magfura ratio was determined each 15 s and
the average value obtained from the eight cells in the field is shown.
All permeabilized cells responded to the applications in a similar
fashion and the result shown is representative of three independent
experiments.
The major aim of the present study was to characterize the
spatial organization of intracellular Ca stores in
pancreatic acinar cells. To address this question we imaged
Ca
stores in individual permeabilized pancreatic
acinar cells using a Ca
-sensitive dye
compartmentalized in organelles. Our main finding is that
Ins(1,4,5)P
-sensitive Ca
stores are
located throughout the acinar cell cytoplasm and that no regional
differences in Ins(1,4,5)P
sensitivity exist, at least in
the absence of cytosolic modulatory factors.
During the cell
permeabilization process, cytosolic factors are lost via the pores
created by SLO in the plasma membrane(23) . One of our concerns
was that SLO treatment affected intracellular structures. Both
conventional and electron microscopy demonstrated that SLO
permeabilized pancreatic acinar cells had a more swollen appearance.
However, the cell architecture remained polarized, since the
localization of zymogen granules remained restricted to the apical pole
of the cells. Other studies on SLO-permeabilized pancreatic acinar
cells have also shown that they retain their polarity and remain
functionally active, both in terms of agonist- or
Ins(1,4,5)P-stimulated Ca
release from
intracellular stores and in terms of agonist- or
Ins(1,4,5)P
- or Ca
-stimulated enzyme
secretion(24, 25) . Electron microscopy revealed that
the endoplasmic reticulum was less strictly arranged compared with
intact cells, an effect that was most likely caused by the swelling. We
therefore cannot rule out the possibility that some rearrangement of
the endoplasmic reticulum Ca
stores may have
occurred. However, our hypothesis is that the relative position of the
components of the Ca
stores is most likely not
altered given that the permeabilized cells clearly retained their
polarized morphology.
The characteristics of store loading, and the
ratio values in unloaded and loaded stores of permeabilized pancreatic
acinar cells, were similar to those observed in permeabilized gastric
epithelial cells by Hofer and Machen(13) . Our estimate of the
free intra-organellar Ca concentration in
steady-state loaded intracellular Ca
stores as 70
µM is also in broad agreement with the value of 127
µM found in gastric epithelial cells. It might be argued
that Mg
interferes to some extent with the
Ca
signals reported by Magfura-2. However, this seems
unlikely, since (i) Mg
is not transported in an
ATP-dependent manner and (ii) the resting Magfura ratio in unloaded
stores was very low, i.e. nearly equivalent to the minimum
ratio for the dye in the total absence of all divalent cations. In
addition, if Mg
was present inside stores its free
concentration would need to exceed a value of 150 µM to
give a significant contribution to the Magfura-2 signal, since the
apparent affinity of the dye for Mg
is very low, i.e. 1.5 mM(26) . The Magfura-2 signal in
organelles was clearly not saturated with Ca
under
normal conditions, since the ratio was increased markedly by exposure
of Ca
stores to the Ca
ionophore
4-Br-A23187 in the presence of 1 mM ambient
Ca
.
Recent developments have allowed measurements
of Ca levels inside the endoplasmic reticulum by
targeting the Ca
-sensitive bioluminescent protein
aequorin to this organelle(27, 28, 29) . In
the first work of this type, Kendall et al. (27, 28) have reported that in COS-7 cells free
Ca
inside the endoplasmic reticulum was around
1-5 µM, approximately 5-20 times the free
cytosolic Ca
concentration. Very recently, however,
it has been reported by Montero et al.(29) that
Ca
concentrations inside the endoplasmic reticulum of
HeLa cells exceeded 100 µM. By using the Ca
surrogate Sr
, these workers concluded that even millimolar free concentrations of divalent cations could occur
within the endoplasmic reticulum and they argued from this that
Ca
levels might reach similar values. Taken at face
value, however, the widely differing estimates reported using the
aequorin technique suggest that a ubiquitous conclusion about
Ca
levels inside Ca
stores cannot
be reached. Although, as discussed above, we cannot exclude the
possibility that properties of the endoplasmic reticulum are altered
during permeabilization, we suggest that our estimate of a free
Ca
concentration within the endoplasmic reticulum of
70 µM might well be applicable to loaded Ca
stores in intact pancreatic acinar cells.
The second messenger
Ins(1,4,5)P released Ca
from
intracellular Ca
stores in permeabilized pancreatic
acinar cells. The characteristics of this release were similar to the
fluxes observed in suspensions of permeabilized acinar cells using the
radioactive tracer
Ca
(10, 12) .
Ca
release was of a quantal nature, apparently due to
the compensatory action of the organelle Ca
pump
during suboptimal stimulation. Thus, suboptimal concentrations of
Ins(1,4,5)P
were much more efficient in releasing
Ca
in the absence of Ca
pumping
activity. An interesting observation was that all permeabilized cells
showed similar sensitivities to Ins(1,4,5)P
. This
observation rules out the often raised possibility that intercellular
differences in Ins(1,4,5)P
sensitivity determine the
quantal nature of Ins(1,4,5)P
-induced Ca
release.
Compartmentalized dye techniques similar to those
applied here have been employed in a number of cell types, including
hepatocytes, gastric epithelial cells, AR4-2J pancreatoma cells,
and smooth muscle cells (13, 21, 30, 31, 32) . In
both hepatocytes (31) and DDTMF-2 smooth muscle
cells (32) the intracellular Ca
stores
function as a single homogeneous pool, and electron microscopy has
shown that the endoplasmic reticulum, which presumably acts as the
intracellular Ca
storage compartment, is a continuous
compartment. However, the properties of Ins(1,4,5)P
-induced
Ca
release from intracellular Ca
stores clearly differed between the two cell types. In
permeabilized hepatocytes attached to coverslips, the
Ins(1,4,5)P
-induced Ca
release was
non-quantal, in that, while the kinetics of Ca
release depended on the concentration of Ins(1,4,5)P
used, all doses of Ins(1,4,5)P
eventually induced
total depletion of the Ca
stores. In smooth muscle
cells, in contrast, Ca
release induced by
Ins(1,4,5)P
was of a quantal nature, similar to what we
have observed for pancreatic acinar cells.
Agonist stimulation of
intact acinar cells initiates a rise of cytosolic Ca in the apical pole of the cell, with the increase in
[Ca
]
subsequently spreading
into basolateral areas of the
cell(2, 3, 4, 5, 6, 7) .
It is notable, however, that some reports indicate that
[Ca
]
rises uniformly throughout
acinar cells upon agonist stimulation(33, 34) . In
recent studies on intact acinar cells, employing the combination of
imaging techniques and patch-clamp recording, evidence was obtained for
a heterogeneous distribution of Ca
stores(5, 6) . By infusing acinar cells with a
low dose of Ins(1,4,5)P
, or its non-metabolizable analogue
inositol 1,4,5-trisphosphorothioate, it was shown that Ca
spikes could be generated exclusively in the apical pole. These
results therefore strongly support the idea that a heterogeneous
population and distribution of Ins(1,4,5)P
-sensitive
Ca
pools do exist in individual pancreatic acinar
cells. Biochemical evidence suggested that this store heterogeneity
might be explained by differences in numbers of
Ins(1,4,5)P
-operated Ca
channels and/or
by differences in sensitivity to Ins(1,4,5)P
(12) .
In particular, it was suggested that, during suboptimal stimulation,
the most sensitive stores were completely depleted, whereas less
sensitive stores remained partially filled due to a compensatory
pumping mechanism. This model (12) could explain a number of
observations in intact acinar cells in which the apical pole
Ca
stores display higher apparent Ins(1,4,5)P
and Ca
sensitivity(5, 6) . In
the present study we have examined directly whether Ca
stores were heterogeneously distributed in individual
permeabilized cells. However, no subcellular differences in
Ins(1,4,5)P
sensitivity could be detected. One possible
interpretation of this result is that subcellular regional differences
in Ins(1,4,5)P
sensitivity depend critically on some
aspects of cellular or cytoskeletal or endoplasmic reticulum
architecture, which is disrupted by permeabilization. However, as
discussed above, we feel it is unlikely that cell permeabilization
results in a major redistribution of Ca
stores. If
our results can be extrapolated to the intact acinar cell, an
alternative explanation of why Ca
starts to rise in
the apical region on agonist stimulation may be the selective presence
of an additional Ca
release mechanisms in this area
of the cell(5, 35) . Several studies suggest the
existence of cyclic ADP-ribose-induced Ca
release
which might be mediated by ryanodine receptors (35, 36) . The experimental evidence in those studies
has been interpreted to suggest that the combined activation of
Ins(1,4,5)P
-sensitive and cyclic ADP-ribose-sensitive
mechanisms is required to explain polarized Ca
spike
generation. However, 5 µM cyclic ADP-ribose failed to
change the Magfura signal in permeabilized pancreatic acinar cells,
whereas in the same cells Ins(1,4,5)P
induced a normal
response. (
)It is possible that cytosolic factors, which are
lost during permeabilization and the subsequent extensive perfusion,
may be required for the cyclic ADP-ribose response.
Interestingly,
other lines of evidence argue against our finding that no regional
differences in Ins(1,4,5)P sensitivity exist. Several
isoforms of the Ins(1,4,5)P
receptor are known to be
expressed in pancreatic acinar cells(37, 38) .
Therefore, multiple isoforms are likely to be translated into
functionally operating receptors in pancreatic acinar cells, possibly
with non-homogeneous distributions within the cell. So far,
immunocytochemistry with antibodies directed against Ins(1,4,5)P
receptors has shown that these receptors are present in the
apical pole of pancreatic and airway gland acinar
cells(24, 39) . In pancreatic acinar cells only type 3
Ins(1,4,5)P
receptors were detected, with no evidence being
found for the presence of type 1 Ins(1,4,5)P
receptors(24) . This observation is surprisingly for two
reasons: (i) Ins(1,4,5)P
sensitivity in basolateral areas
has been demonstrated many times in intact cells (5, 6) and in permeabilized cells (this study) and
(ii) more than half of the Ins(1,4,5)P
receptor mRNA
expressed in the whole pancreas was mRNA of type 1
receptors(37, 38) .
The present study is, however,
consistent with Ins(1,4,5)P binding studies in a pancreatic
microsomal fraction, which revealed the presence of a single class of
binding sites(20) . In addition, the dose-response curve for
Ins(1,4,5)P
-induced Ca
release gave no
indications of the presence of multiple binding sites (e.g.(19) and (20) ). Multiple Ins(1,4,5)P
binding sites and a broad dose-response relationship for
Ins(1,4,5)P
-induced Ca
release would be
expected if the intrinsic properties of Ins(1,4,5)P
receptor subtypes included different Ins(1,4,5)P
sensitivities. If the spatial pattern of Ca
signaling observed in pancreatic acinar cells is not a result of
the intrinsic properties of Ins(1,4,5)P
receptors, it must
involve additional cytosolic factors regulating the opening of these
receptor-operated ion channels. Multiple kinases and cytosolic factors
like Ca
are known to be involved in the complex
regulation of Ins(1,4,5)P
receptors(1, 40, 41) . In peripherial
tissues, cytosolic Ca
levels and/or the
phosphorylation status of Ins(1,4,5)P
receptors have been
shown to play an important role in controlling Ca
release mechanisms(42, 43) .
In conclusion,
imaging of Ca within intracellular stores revealed
that Ins(1,4,5)P
-sensitive Ca
stores are
found in all regions of the polarized pancreatic acinar cell.
Furthermore, the stores did not display a heterogeneous sensitivity
toward Ins(1,4,5)P
. The polarized Ca
signaling observed in intact acinar cells is therefore
likely to be controlled by additional cytosolic factors and/or
ryanodine receptors possibly present in the apical pole of acinar
cells.