(Received for publication, January 27, 1997, and in revised form, April 4, 1997)
From the Department of Physiology, University of
Texas Southwestern Medical Center, Dallas, Texas 75235, § the Department of Pharmacology, State University of New
York Health Science Center at Syracuse, Syracuse, New York 13210, the
¶ Department of Pathology, Wayne State University School of
Medicine, Detroit, Michigan 48201, and
the Laboratory of
Physiology, University of Leuven, Leuven, Belgium
In polarized epithelial cells
[Ca2+]i waves are initiated in discrete
regions and propagate through the cytosol. The structural basis for
these compartmentalized and coordinated events are not well understood.
In the present study we used a combination of
[Ca2+]i imaging at high temporal resolution,
recording of Ca2+-activated Cl current, and
immunolocalization by confocal microscopy to study the correlation
between initiation and propagation of [Ca2+]i
waves and localization of Ca2+ release channels in
pancreatic acini and submandibular acinar and duct cells. In all cells
Ca2+ waves are initiated in the luminal pole and propagate
through the cell periphery to the basal pole. All three cell types
express the three known inositol 1,4,5-trisphosphate receptors
(IP3Rs). Expression of IP3Rs was confined to
the area just underneath the luminal and lateral membranes, with no
detectable receptors in the basal pole or other regions of the cells.
In pancreatic acini and SMG ducts IP3R3 was also found in
the nuclear envelope. Expression of ryanodine receptor was detected in
submandibular salivary gland cells but not pancreatic acini.
Accordingly, cyclic ADP ribose was very effective in mobilizing
Ca2+ from internal stores of submandibular salivary gland
but not pancreatic acinar cells. Measurement of
[Ca2+]i and localization of IP3Rs in
the same cells suggests that only a small part of IP3Rs
participate in the initiation of the Ca2+ wave, whereas
most receptors in the cell periphery probably facilitate the
propagation of the Ca2+ wave. The combined results together
with our previous studies on this subject lead us to conclude that the
internal Ca2+ pool is highly compartmentalized and that
compartmentalization is achieved in part by polarized expression of
Ca2+ channels.
Ca2+ mobilizing agonists trigger several forms of [Ca2+]i signals, which include [Ca2+]i oscillations and Ca2+ waves (1). It has now been recognized in many cell types that Ca2+ signaling is highly organized and compartmentalized. This issue has been extensively discussed in a series of recent reviews in a special issue of Cell Calcium (2). In the case of pancreatic acini compartmentalization of signaling and intracellular Ca2+ pools was suggested when the quantal nature of Ca2+ release from internal stores (3, 4) was described. Subsequently, a series of elegant papers demonstrated the specialization of the luminal pole (LP)1 of these cells. Ca2+ waves tended to start in this region and spread to the basal pole (BP) (5-8). When the cells are stimulated to trigger [Ca2+]i oscillations, the oscillation tended to occur in and remain confined to the LP (6, 7, 9). Similar LP oscillations could be initiated with infusion of low concentration of IP3 into the cells (7, 9). More recently we showed a higher order of organization of Ca2+ signaling in this pole, in that the initiation site of Ca2+ waves was agonist specific in the same cell type (10).
The structural features responsible for the compartmentalized organization of Ca2+ signaling in acinar and other cell types are not well understood. Several findings suggest that the properties of IP3-mediated Ca2+ release may account for the specialization of the LP. Analysis by reverse transcription polymerase chain reaction and WB showed the presence of all three types of IP3R in the pancreas (11, 12). However, for the most part, their specific cellular expression and localization is not known. In one study clear localization of type 3 IP3R (IP3R3) in the LP has been reported (13). Propagation of the Ca2+ wave from the LP to the BP was suggested to occur through a series of Ca2+-induced Ca2+ release events mediated by the ryanodine receptor (RyR) (5, 14). Accordingly, it was reported that cADPR, an activator of the RyR (15), releases Ca2+ from the IS of pancreatic acini (16). More recently Gerasimenko et al. (17) reported that the secretory granules, which are located in the LP, release Ca2+ in response to IP3 and cADPR.
Compared with pancreatic acini, there is little information on Ca2+ signaling by SMG acinar and duct cells. Several studies reported the response of the two cell types to Ca2+-mobilizing agonists including carbachol, epinephrine, substance P, and isoproterenol (18-20). Studying Ca2+ signaling in SMG cells has several advantages. First, it allowed us to determine the generality of polarized arrangement of Ca2+ signaling and expression of Ca2+ transporters in secretory epithelial cells. Second, SMG cells were shown to express mRNA for type 1 RyR (21), which allows a more clear evaluation of the role of RyR in Ca2+ signaling in polarized cells. Third and most important for this study, is that the SMG duct is a single layer epithelium with very well defined LP and BP, which allows an unequivocal localization of Ca2+ transporters, in particular the Ca2+ pumps described in the accompanying manuscript (33).
In the present study we used [Ca2+]i imaging at high temporal resolution to show that in all three cell types Ca2+ waves start in the LP and spread through the cell periphery to the BP. This pattern correlated well with the polarized expression of IP3Rs in all cells and RyR in SMG cells. The results also highlight the problem of the mechanism of Ca2+ wave propagation in the face of the paucity of Ca2+ release channels from the cell interior.
cADPR was purchased from Calbiochem or Sigma. Preparation and characterization of the pAb raised against IP3R1-3 are described in Ref. 12. IgG2a Clone 2 mAb against IP3R3 was from Transduction Laboratories (Lexington, KT). Two anti-ryanodine receptor Abs were used, mAb Clone 34-C from Affinity Bioreagents (22) and mAb anti-RyR from Calbiochem (23).
Preparation of Tissue Slices, Acini, and Duct FragmentsThe
pancreas and SMG of 100-150-g rats were removed immediately after
sacrifice of animals, immersed in the O.C.T. compound (Miles, Elkhard,
IN) and snap frozen in liquid nitrogen. Frozen glands were used to cut
4-µm sections. The sections were plated on polylysine-coated slides,
dried, and kept at 20 °C until use. A mixture of acini and duct
fragments from all glands were prepared by published procedures (4, 10,
20). The cells were kept suspended in cold PSA until use. The
composition of PSA was (in mM) NaCl 140, KCl 5, MgCl2 1, CaCl2 1, Hepes 10 (pH 7.4 with NaOH), glucose 10, pyruvate 10, bovine serum albumin 0.1%, and soybean trypsin inhibitor 0.02%.
After removal of the glands and fine mincing, the minced pancreas was treated with trypsin/EDTA and then collagenase to liberate single pancreatic acinar cells exactly as described before (24). The minced SMG was washed once with 20 ml of PSA by 10 s of centrifugation at 120 × g. The pellet was resuspended in 5 ml of PSA containing 0.8 mg/ml collagenase type CLS4, 254 units/mg (Worthington) and was digested for 20 min at 37 °C. The tissue was washed twice with a PBS solution resuspended in 5 ml of PBS containing 0.05% trypsin/0.02% EDTA and incubated for 8 min at 37 °C. The trypsin-treated tissue was washed twice with 30 ml of PSA, resuspended in 6 ml of PSA containing 0.53 mg/ml collagenase CLS4, and incubated for 20 min at 37 °C. The cells were washed twice with 20 ml of PSA by a 2-min centrifugation at 150 × g and kept on ice until use. Single duct and acinar cells were distinguishable by microscopy based on their different size, and their identity was verified during electrophysiological recording by measurement of membrane capacitance that averaged 19.74 ± 0.55 picofarad (n = 12) in acinar and 4.33 ± 0.06 picofarad (n = 6) in duct cells.
[Ca2+]i ImagingLoading cells with
Fura 2 and image acquisition and analysis was as detailed before (10).
Acini and ducts with well defined basal and apical borders were
selected for experimentation. To maximize the temporal resolution,
fluorescence was measured at a single excitation wavelength of 380 nm,
averaging four consecutive images for each time point. Under these
conditions using a frame size of 256 × 240 pixels allowed
recording the fluorescence of 4-5 acinar cells at a resolution of up
to 7.7 Hz. During perfusion with the control solution and just before
the first stimulation, the image of the resting cells was acquired.
This was taken as the fluorescence at time 0 (F0). Pixel values of all subsequent images were
divided by this image, and the traces are the calculated F0/Ft, where
Ft is the fluorescence at time t. For the
experiments in Figs. 7 and 8 at the end of fluorescence recording, the
perfusion medium was aspirated, and the bright field image at high
(experimental) and low magnification was recorded. Then the bottom of
the coverslip was tagged with a marker before fixation and extraction
of the cells with cold methanol. The extracted cells were processed for
IC staining with IP3Rs Ab as described below. At the end of
Ab staining, coordinates were labeled around the tag, the tag was
cleared to allow imaging by confocal microscopy, and the coordinates
together with the low magnification bright field image were used to
identify the cell cluster from which [Ca2+]i was
recorded.
Measurement of Ca2+ Release in Permeable Cells
Ca2+ release from SLO-permeabilized cells was measured exactly as described previously (25). The SLO permeabilization medium was composed of a Chelex-treated solution containing 145 mM KCl and 10 mM Hepes (pH 7.2 with NaOH) and supplemented with 0.02% soybean trypsin inhibitor, 3 mM ATP, 5 mM MgCl2, 10 mM creatine phosphate, 5 units/ml creatine phosphokinase, 10 µM antimycin A, 10 µM oligomycin, 1 µM Fluo 3, and 3 mg/ml SLO (Difco). Fluo 3 fluorescence was recorded and calibrated as detailed previously (25).
Whole Cell Current RecordingThe tight seal whole cell
current recording of the patch clamp technique (26) was used for
measurement of Ca2+-activated Cl current,
which directly correlates with changes in [Ca2+]i
(27). The experiments were performed with single acinar or duct cells
perfused with solution A. The standard pipette solution contained (in
mM): KCl 140, MgCl2 1, EGTA 0.5, Na2ATP 5, and Hepes 10 (pH 7.3 with KOH) as described in
previous studies (25). In some experiments this solution also contained
1-100 µM cADPR and 1.5 mg/ml heparin. Seals of 6-10
gigaohms were produced on the cell membrane, and the whole cell
configuration was obtained by gentle suction or voltage pulses of 0.5 V
for 0.3-1 ms. The patch clamp output (Axopatch-1B, Axon Instruments)
was filtered at 20 Hz. Recording was performed with patch clamp 6 and a
Digi-Data 1200 interface (Axon Instruments). In all experiments the
Cl
and cation equilibrium potentials were about 0 mV. All
the traces shown were at a holding potential of
40 mV.
Packed, purified pancreatic acini, and SMG ducts and acini were extracted with a buffer containing 62.5 mM Tris (pH 6.8 with HCl), 8 M urea, 2% SDS, and 100 mM dithiothreitol in a ratio of 1:2. 10-µl samples containing about 75 µg of protein were separated by an SDS-polyacrylamide gel electrophoresis using a 7.5% polyacrylamide gel. The separated proteins were transferred to 0.2-µm polyvinylidene difluoride membranes, and the membranes were blocked by a 1-h incubation at room temperature in 5% nonfat dry milk in a solution containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20 (TTBS). The IP3Rs were detected by a 1-2-h incubation of individual membranes with the respective Ab diluted in TTBS.
ImmunocytochemistryTissue slices and the cells attached to
glass coverslips used for [Ca2+]i measurements
were fixed and permeabilized with 0.5 ml of cold methanol for 10 min at
20 °C. After removal of methanol, the cells were washed with PBS
and incubated in 0.5 ml of PBS containing 50 mM glycine for
10 min at room temperature. This buffer was aspirated and the
nonspecific sites were blocked by a 1 h incubation at room
temperature with 0.25 ml of a PBS solution containing 5% goat serum,
1% bovine serum albumin, and 0.1% gelatin (blocking medium). The
medium was aspirated and replaced with 50 µl of blocking medium
containing control serum or a 1:50 dilution of pAb against
IP3R1 and IP3R2, 1:25 dilution of pAb and
1:2000 dilution of mAb against IP3R3, and 1:250 dilution of
mAb against the RyR. In several control experiments, the primary pAbs
against the different IP3Rs were preincubated with the
respective peptides used to raise them (see Ref. 12). Such
preincubation completely blocked detection of the respective
IP3Rs by WB or IC. After incubation with the primary Ab for
1.5 h at room temperature and three washes with blocking medium,
the Ab were detected with goat IgG tagged with fluorescein. Images were
collected with a Bio-Rad MRC 1000 confocal microscope.
In a previous study we showed that in maximally stimulated
pancreatic acini, Ca2+ waves tend to initiate in the apical
pole and spread through the cell periphery to the basal pole (10). Fig.
1 and Table I show that this is not a
unique phenomenon to pancreatic cells, because it can be demonstrated
in SMG acinar and duct cells. With all agonists the Ca2+
wave tended to initiate in the LP or the lateral region adjacent to the
LP. However, differences among agonists were noted in terms of wave
speed and the time required to cause maximal increase in
[Ca2+]i. Epinephrine induced the fastest wave in
duct cells; the [Ca2+]i wave initiated by
isoproterenol in duct cells was significantly slower than the others as
well as the time required to achieve peak Ca2+ (Table I).
It is important to note that acinar cells are about 2.5 times bigger
than duct cells, which indicates that the wave travels about 2-3 times
faster in SMG acinar compared with duct cells.
|
To study whether and how the
expression of different IP3Rs in specific parts of the
cells correlate with the [Ca2+]i wave, we
analyzed the expression of type 1-3 IP3R by WB and
determined their localization by IC in pancreatic and SMG cells. Fig.
2 shows WB analysis of the different IP3R in
pancreatic acini and SMG duct and acinar cells. For this we took
advantage of our technique for separation between SMG acinar and duct
cells, which yield better than 95% pure duct and acinar preparations (20). IP3Rs in all cells were sensitive to proteolysis.
Including 100 mM of the potent protease inhibitor
benzamidine and 0.3% soybean trypsin inhibitor reduced the intensity
of minor bands and increased the level of intact receptors. However,
even under these conditions proteolysis could not be completely
prevented. Because of this problem it is not possible to reliably
determine the relative abundance of the different types of
IP3R. In the case of IP3R3 we used two separate
Ab to verify the expression of this receptor because the two Ab gave
somewhat different results in IC (see below). Both Abs showed that SMG
acinar cells express the highest level of IP3R3. The pAb,
but not the mAb, detected expression of IP3R3 in SMG ducts.
The relative intensity of labeling of SMG duct and acini by the pAb
(about 1:3) excludes contamination of the duct preparation with acini
as the source of labeling.
Immunolocalization of IP3R
Previously only the
localization of IP3R3 in the LP of pancreatic acini has
been reported (13). Fig. 3 demonstrates the localization
of all IP3Rs in the LP of pancreatic acini and SMG acinar
and duct cells. Remarkably all three epithelial cell types expressed
high levels of IP3R1 and IP3R2 only close to
the luminal and lateral membranes. In additional experiments when up to
2-3 times higher concentrations of Ab were used, still no
IP3R expression could be observed in any region of the
cells beyond that shown in Fig. 3.
Cell-specific expression and localization was observed with IP3R3 using two different Ab, a pAb raised against a 13-amino acid sequence of the C-terminal of IP3R3 (12) and a mAb raised against amino acids 22-230 of IP3R3 (28). In pancreatic acini both Ab detected expression of IP3R3 in the LP and next to the lateral membrane, which closely resembled that of IP3R1 and IP3R2. However, only the pAb detected expression around the nuclear envelope. The ability of the pAb but not the mAb to detect the IP3R3 in the nuclear envelope is further highlighted when the pattern of expression of IP3R3 in the SMG is examined. In SMG acini, expression of IP3R3 was confined to the LP and lateral membrane. With the pAb IP3R3 was detected only around the nuclear envelope of duct cells. The mAb did not stain the SMG duct. Unlike both types of acini, the SM duct did not express any IP3R3 in the LP or lateral membrane. After submission of the present study similar localization of IP3Rs in pancreatic acini using the same pAb was reported (29).
RyR in SM and Pancreatic CellsThe confinement of
IP3R expression to just underneath the luminal and lateral
membranes in the face of the propagated Ca2+ waves raised
the possibility that RyR may mediate the propagation of the waves. We
expected all cell types to express the RyR because it was reported that
cADPR releases Ca2+ from IS (16) and isolated zymogen
granules (17) of pancreatic acini and the SMG express type 1 RyR (21).
The use of mAb that recognize all isoforms of known RyR and functional
characterization of Ca2+ release showed that this may not
be the case. Fig. 4 shows that we could not detect any
RyR in pancreatic acini by IC. On the other hand both SMG acinar and
duct cells stained by the same Ab. Also in this case expression of RyR
was confined to the LP and lateral membrane.
Considering the reported effect of cADPR on Ca2+ release in
pancreatic acini (16, 17), we examined the effect of this compound on
Ca2+ release from IS of pancreatic acini and SM cells using
two different experimental protocols. In Fig.
5A it is shown that 10 µM cADPR infused through a patch pipette into pancreatic acinar cells caused Ca2+ release from IS. However, with 10 µM
cADPR Ca2+ release was observed in only four of nine cells,
was much smaller than that caused by agonist (Fig. 5A), and
was blocked by heparin (Fig. 5B) to the extent that in five
of five experiments with heparin no Ca2+ release from IS
was observed. These findings confirm all aspects of the data reported
by Thorn et al. (16). Unlike the case in pancreatic acini,
10 µM cADPR potently released Ca2+ from IS of
SMG acinar and duct cells (Fig. 5, C and E), the
release was observed in eight of nine acinar and four of four duct
cells, and it was not blocked by heparin (Fig. 5, D and
F) in three of three acinar and three of three duct
cells.
A different protocol comparing the effect of cADPR in the two cell
types is shown in Fig. 6. In SLO permeabilized cells
cADPR up to 100 µM failed to release Ca2+
from IS of pancreatic acini (Fig. 6, trace a). On the other
hand 10, 100 (not shown) and 25 µM cADPR released
Ca2+ from IS of SMG cells (Fig. 6, trace b), and
this release was not blocked by heparin (Fig. 6, trace c).
Relatively high concentrations of cADPR were needed, probably because
of the presence of an ATP regeneration system and high concentration of
Mg2+ in the uptake medium. Additional evidence for the
differences between SMG and pancreatic cells was obtained with
caffeine. At 2 and 5 mM, caffeine failed to increase
[Ca2+]i in pancreatic acinar cells, but it
inhibited the effect of agonist stimulation, as reported before (30).
On the other hand 5 mM caffeine increased
[Ca2+]i in SMG acinar and duct cells by about
220 ± 36 nM (n = 4 for each cell
type) (results not shown).
Correlation between IP3R Localization and [Ca2+]i Initiation Sites
Considering the quantal behavior of Ca2+ release (3, 4), it was of interest to determine whether initiation sites of Ca2+ release correlated with the entire or part of the IP3R pool. To achieve that we recorded [Ca2+]i at high temporal resolution from small acinar clusters, fixed the cells, and stained them with Ab raised against IP3Rs. An example for each IP3R using SMG acinar cells is shown in Fig. 7. Despite the low efficiency of labeling with IP3R1 in isolated SM acinar cells (Fig. 7A, c), it is clear that area 1 did not include the entire IP3R1 pool in the LP of cell 1. This is even more clear for IP3R2 (Fig. 7B) and IP3R3 (Fig. 7C). In each case IP3R is apparent in the entire LP and most of the lateral membrane, but the initial [Ca2+]i increase occurred in a limited region of the cells.
A better resolution of the initiation sites and their correlation with IP3R expression was obtained in three cells clusters of pancreatic acini (Fig. 8). In this case we also attempted to correlate the [Ca2+]i initiation sites with the special distribution of IP3Rs. High background precluded such studies with IP3R1 Ab in isolated pancreatic acini. In the cases of IP3R2 and IP3R3, the sharp [Ca2+]i initiation sites correlated with only part of the IP3R pool. Interestingly, it appears that the [Ca2+]i wave initiation sites correlated best with expression of IP3Rs in specific parts of the cells. For example the initiation sites correlated best with expression of IP3R2 in the bottom part of cells 1 and 2. Similar correlations were observed in several experiments with each IP3R. Whether this indicates the spacial location of the initiation site or it represents cell fixation artifact remains to be determined.
Ca2+ signaling is governed by the action of Ca2+ pumps and Ca2+ channels (31). The pumps remove Ca2+, whereas the channels release Ca2+ into the cytosol. The coordinated activation and inactivation of these transporters yield [Ca2+]i signals in the form of Ca2+ oscillations and/or [Ca2+]i waves. The localization and participation of the Ca2+ pumps in the [Ca2+]i wave initiated by agonists is described in the accompanying manuscript (33). In the present studies we show the polarized expression of Ca2+ channels in three secretory epithelial cells and provide evidence for their compartmentalization to initiate and propagate the Ca2+ waves.
WB analysis and IC revealed that all three cell types express the three known IP3Rs. In agreement with the reported high levels of type 1 RyR mRNA in SMG tissue (21), IC showed the presence of RyR in SMG cells. This is further supported by the ability of cADPR to release Ca2+ from IS of SMG cells. As was shown for other cells (15, 32), the effect of cADPR in SMG cells was not inhibited by a high concentration of heparin. cADPR was particularly effective in SMG cells because stimulation with maximal concentration of agonist released only a small amount of Ca2+ from IS of cells infused with 10 µM cADPR. Finally, caffeine caused a small Ca2+ release form IS of SMG acinar and duct cells. Hence, we believe that SMG acinar and duct cells indeed express an active RyR.
There is less certainty as to the expression of RyR in pancreatic acini. Thus, Ryr was not detected by IC, cADPR was ineffective in releasing Ca2+ from IS of permeabilized cells, and caffeine did not increase [Ca2+]i in intact pancreatic acinar cells. The only exception was that infusion of 10 µM cADPR through a patch pipette into pancreatic acini causes a small Ca2+ release from internal stores. However, this observation is weakened by the finding that the effect of cADPR was inhibited by heparin (see also Ref. 16), an inhibitor of the IP3 channel (4, 25). A possible explanation for all the findings is that pancreatic acini express very low levels of RyR, below the detection threshold of the Ab, which is not sufficient to observe Ca2+ release in permeable cells. This may be inferred from the observation that 10 µM cADPR induced a much smaller Ca2+ release in pancreatic compared with SMG cells. The inhibition by heparin may suggest functional interaction between the IP3R and RyR. In SMG cells the high abundance of RyR allowed Ca2+ release even when IP3R were inhibited by heparin. Obviously further work is needed to completely resolve the question of RyR in pancreatic acinar cells.
With the exception of IP3R3 all Ca2+ channels were confined to the LP and lateral membrane. The pAb raised against IP3R3 detected clear expression of this receptor in the nuclear region of pancreatic acinar and SMG duct cells. On the other hand, the mAb against the same receptor failed to detect any nuclear staining, although they detected the IP3R3 in the LP of pancreatic and SM acinar cells. Because the mAb and pAb recognize different sequences of IP3R3, WB analysis with the pAb clearly shows the expression of IP3R3 in duct cells, and the only staining of IP3R3 in duct cells is found in the nuclear region, we can conclude that IP3R3 is indeed expressed in the nuclear envelope. The differential reactivity with the mAb and pAb may indicate expression of two different isoforms of IP3R3 in the cell periphery and the nuclear envelope.
In all three cell types and with all Ab we could not detect any Ca2+ channels in the granular region. We paid particular attention to this region because it was reported that both IP3 and cADPR released Ca2+ from single isolated zymogen granules (17). After submission of the present work, IP3Rs localization in pancreatic acini and analysis of isolated granules also failed to detect IP3Rs associated with the granular region (29). However, it is possible that the level of IP3Rs in the granules and other parts of the cells are too low to be detected by IC. A more interesting possibility is that only subset of the granules respond to IP3 and may have originated from the so called "fusion competent" pool next to the luminar membrane, the site of high levels of IP3Rs.
The most popular model for propagation of Ca2+ waves is sequential Ca2+-induced Ca2+ release events mediated by the effect of Ca2+ on IP3R and/or RyR (1, 27). This and all other models assume and require expression of Ca2+ release channels throughout the cytoplasm. This was not found to be the case for all the cells studied. The Ca2+ release channels were concentrated next to the luminal and lateral membranes. Such a localization can account well for the finding that in most pancreatic acinar (see Ref. 10) and SMG cells (Fig. 1 and Table I) the Ca2+ waves were initiated in the LP and tended to propagate along the cell periphery. However, in all cells [Ca2+]i eventually increased in all parts of the cytoplasm. How the Ca2+ propagated from sites of high Ca2+ release channel expression to the center of the cells is not clear. One possibility is that peripheral to central propagation occurs by diffusion. This is not very likely because, as shown in the following manuscript (33), wave propagation is a controlled process dependent on the activity of sarco/endoplasmic reticulum Ca2+ ATPase pumps. Another possibility is that Ca2+ release events in the cell periphery are sufficiently large to reach the nuclear membrane and activate IP3R3. In the space between these two sites of high levels of Ca2+ release channels, expression of the channels may be below the detection level of all Ab used but sufficient to allow Ca2+ release when activated by IP3 and/or the Ca2+ released in the initial event.
Polarized expression of Ca2+ release channels and propagation of the Ca2+ wave through the cell periphery highlight the compartmentalized arrangement of Ca2+ signaling complexes. In previous studies we (4, 10, 25) and others (5-9) provided multiple functional evidence for compartmentalization of Ca2+ signaling. The results in Figs. 7 and 8 provide the first correlation between pattern of IP3R expression and initiation sites of the Ca2+ wave. Multiple and agonist specific initiation sites (10) would suggest that specificity of initiation sites is determined by the agonist but that initiation sites always reside at a region of high expression level of IP3Rs. Other Ca2+ transporters that control the initiation sites and propagation of Ca2+ waves are the Ca2+ pumps. The role of the pumps in these events are described in the accompanying manuscript (33).
We thank Mary Vaughn for superb administrative support and Su Ge Luo for technical assistance.