1 Department of Anatomy, School of Medicine, Kitasato University, 1-15-1
Kitasato, Sagamihara, Kanagawa 228-8555, Japan
2 Department of Pharmacology, School of Medicine, Sapporo Medical University,
South 1, West 17, Sappro 060-8556
* Author for correspondence (e-mail: segawa{at}kitasato-u.ac.jp )
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Summary |
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Key words: Calcium signalling, Salivary gland, Gap junction, Ins(1,4,5)P3 receptor, Epithelia
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Introduction |
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Cellular activities in virtually all cell types are, despite tremendous
diversities in their expression, regulated by common intracellular signalling
systems, and calcium is one important signalling molecule involved in the
regulation of diverse cell functions
(Berridge et al., 2000). In
response to adequate stimuli, [Ca2+]i increases, oscillates and
decreases, leading to the activation, modulation and termination of cell
function. It is likely that the integration of multicellular activity in the
organ is achieved through the regulation of Ca2+ signalling.
Although extensive studies have elucidated subcellular mechanisms responsible
for Ca2+ signalling, little is known of its regulation at the
supracellular level.
The salivary gland is an exocrine organ capable of secreting fluid and
macromolecules under the autonomic nerve regulation
(Garrett, 1987). It is composed
of tubular epithelia that can be divided into two major domains. The distal
end is the secretory unit, the acini, where the primary saliva is produced. At
the proximal part are the ducts, which modify the primary saliva by absorbing
or secreting certain ions, such as Na+, Cl- and
HCO3- (Young et al.,
1987
). By using high-speed confocal microscopy, we previously
studied Ca2+ signalling in the rat salivary ducts
(Yamamoto-Hino et al., 1998
)
and acini (Takemura et al.,
1999
). Millisecond analyses demonstrated that [Ca2+]i
responses in the ducts started in some `pioneer cells' and then spread to the
neighboring cells, showing asynchrony and heterogeneity
(Yamamoto-Hino et al., 1998
),
whereas those in the acini occurred synchronously
(Takemura et al., 1999
). These
data prompted us to study whether the Ca2+ signalling system in
salivary glands is constructed according to the tissue architecture.
In the present study, we analysed details of calcium signalling in the rat
parotid gland. We dissociated the glands with collagenase to obtain
morphologically and functionally intact preparation of acini and ducts
(Segawa et al., 1985). These
specimens were loaded with fluo-3 AM and stimulated with calcium mobilising
agents carbachol (CCh) and ATP in the presence or absence of extracellular
Ca2+. Changes in [Ca2+]i of acini and ducts were
analysed by high-speed confocal microscopy both at the regional level and at
the single cell level. The possible role of intercellular communication
through the gap junction was studied functionally by octanol pretreatment and
morphologically by connexin 32 immunohistochemistry. The distribution of
inositol (1,4,5)-trisphosphate receptor type 2
[Ins(1,4,5)P3R2], a receptor molecule involved in the
release of Ca2+ from intracellular stores, was also examined.
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Materials and Methods |
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High-speed confocal microscopy
The fluo-3-loaded dissociated tissues were placed on the coverslip coated
with Cell Tak (Collaborative Biomedical Products, Bedford, MA)
(Segawa, 1999). They were
viewed with a inverted light microscope (Nikon Diaphot 300, Nikon, Tokyo,
Japan) equipped with a high-speed confocal microscope (Oz with InterVision
software, Noran Instruments Inc., Middleton, WI). For stimulation of the
dissociated tissues, 10 µM carbachol (Wako Pure Chemical Industries, Osaka,
Japan) or 100 µM ATP (adenosine 5'-triphosphate: Sigma Chemical, St
Louis, MO) were added directly onto the tissues. When necessary, pretreatment
with octanol (Wako Pure Chemical Industries) was performed at 3 mM for 3
minutes before addition of CCh. The specimens were observed with the plan apo
objective lenses x20, x40, x60 with NA 0.75, 1.00 and 1.20,
respectively. The plane of approximately 20-50 µm depth from the coverslip
was observed. Confocal images were taken every 8-33 milliseconds. Excitation
was performed with an Ar/Kr laser at 488 nm and the emission signals were
collected through a 515 nm barrier filter. Pseudo-ratio imaging was performed
by dividing raw fluorescence images by an image immediately before addition of
CCh or ATP, representing the resting distribution of fluo-3 fluorescence in
the specimens. The pseudo-ratio images were used for the quantitative
measurement.
Scanning electron microscopy
Dissociated tissues were fixed with 2% glutaraldehyde in 0.1 M cacodylate
buffer, pH 7.2, for 1 hour at 4°C, and post-fixed with 1% OsO4
for 1 hour at 4°C. They were dehydrated through a graded series of
ethanol, dried in a critical point dryer and coated with Au/Pd at 4 nm by a
sputter coater. The specimens were observed by a Hitachi S-4500 field emission
scanning electron microscope (Hitachi Co. Ltd., Hitachi, Japan) operated at 5
kV.
Immunohistochemistry
Pieces of parotid glands were fixed with 10% formalin for 1 hour at
4°C, and cut in a cyrostat at 20 µm. The sections were pretreated with
1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) at 4°C
for 1 hour. They were then treated with mouse monoclonal antibodies against
Ins(1,4,5)P3R2 (IgGIIa, KM1083)
(Sugiyama et al., 1994) and
connexin 32 (IgG1, Zymed Laboratories, Inc., CA) at 4°C for overnight.
Control specimens were incubated solely with BSA-PBS. After washing with PBS,
the sections were treated with RITC-labelled anti-mouse IgGIIa and
FITC-labelled anti-mouse IgG1, mounted with anti-fader reagent Fluoro-Guard
(Bio-Rad Lab., CA) and observed under the conventional confocal microscope
(Bio-Rad MRC 1024, Nippon Bio-Rad Lab., Tokyo, Japan). Serial confocal images
were taken along the Z-axis at a distance of 1.5 µm and then reconstructed
into the `through focus' image by summating each image.
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Results |
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Chanes in [Ca2+]i in acini and ducts in response to
CCh
Following application of CCh to the tissues, both acinar and ductal
segments exhibited a rise in fluo-3 fluorescence intensity, indicating a rise
in [Ca2+]i. Fluo-3 loading was efficiently applied in the outer
layers of cell clumps so that the response of acini was defined in those
located in the periphery of the specimen.
Fig. 2 shows the typical
response. Acini were always the first to respond (0.32-2.5 seconds after CCh;
mean=0.76 seconds, n=28), and the ducts followed (0.87-3.1 seconds
after CCh, mean=1.70 seconds, n=12) with a time lag of 0.38-2.0
seconds (mean=1.0 seconds, n=12). We did not observe any propagation
of Ca2+ waves from acini to the ducts. Instead, within the ducts,
the rise in [Ca2+]i started in some `pioneer cells' and then spread
to the neighboring cells (Fig.
2C), as described previously in the ducts of submandibular glands
(Yamamoto-Hino et al.,
1998).
[Ca2+]i response to CCh and ATP in the acini and ducts:
analyses at the regional level
The response of acini and ducts to CCh and ATP was analysed by the
consecutive application of each reagent in the presence or absence of
extracellular Ca2+. Acini and ducts exhibited a distinct response
pattern. A typical example is shown in Fig.
3 (n=14).
|
When CCh was applied to the specimens in the absence of extracellular Ca2+ (Fig. 3C), [Ca2+]i increased in the acinar area but not in the ductal area. However, addition of Ca2+ to the perfusion medium resulted in a rise in [Ca2+]i in the ductal area (Fig. 3D). After washing the specimen thoroughly with Ca2+-free KRH, and adding ATP in the absence of extracellular Ca2+, the acinar response was weak or negligible (Fig. 3E). By contrast, the ductal area exhibited a rise in [Ca2+]i, although the response was transient and [Ca2+]i soon declined. When Ca2+ was added to the perfusion medium under these conditions, the ductal area exhibited a rise in [Ca2+]i and showed a sustained plateau (Fig. 3F).
When the application protocol was reversed and ATP was applied prior to CCh, the acinar and ductal responses were similar to those described above (not shown). Thus the sequence of application of ATP and CCh did not seem to alter the pattern of acinar and ductal response.
[Ca2+]i response to CCh and ATP in the acini and ducts:
analyses at the individual cell level
Analyses of the individual cell response
(Fig. 4) were carried out using
the same preparation as that shown in Fig.
3. The pattern of acinar cell response was similar to that
measured at the regional level. As described below, each acinar cell exhibited
an almost homogenous pattern of response. By contrast, ductal cells exhibited
an heterogeneous pattern of response. Although the gross measurement of the
specimen failed to detect the ductal response to CCh in the absence of
extracellular Ca2+ (Fig.
3D), a few duct cells were found to respond under this condition
(Fig. 4B). Addition of
Ca2+ to the perfusion medium caused [Ca2+]i levels to
rise in most duct cells (Fig.
4C) but several cells showed a weak or negligible response; ATP
stimulation caused [Ca2+]i levels to rise in these duct cells
(Fig. 4D). In the absence of
extracellular Ca2+, the response of each cell was transient, but in
the presence of extracellular Ca2+ it exhibited a sustained plateau
(Fig. 4E). The magnitude of the
response and the latency period differed from one cell to another.
|
Effect of octanol on the synchronised [Ca2+]i response in
the acini
10 µM CCh treatments often caused an oscillation in the
[Ca2+]i response in acinar cells
(Fig. 5A,C). The oscillatory
response occurred synchronously among the cells within an acinus. Gap
junctions have been shown to be involved in the synchronised
[Ca2+]i response in several cell systems
(Stauffer et al., 1993;
Guerineau et al., 1998
). We
thus analysed the effects of octanol, a gap junction inhibitor, on the acinar
cell response. Following pretreatment of the specimen with octanol for 3
minutes, the response became asynchronous
(Fig. 5B,D).
|
Distribution of connexin 32 and Ins(1,4,5)P3R2 in
the acini and ducts
The involvement of gap junctions in the observed [Ca2+]i
response was further analysed by immunohistochemistry using connexin antibody.
As shown in Fig. 6A, positive
immunofluorescence was found predominantly in the acinar area, whereas the
ductal area was mostly devoid of positive signals. An exceptional area was the
distal end of the intercalated ducts, in which a positive reaction was
sometimes detected. In an attempt to discover the intracellular molecule
responsible for the supracellular regulation of Ca2+ signalling, we
also observed the distribution of Ins(1,4,5)P3R2.
Ins(1,4,5)P3R2 immunofluorescence was detected along the
apico-lateral area of acinar cells (Fig.
6B). This area corresponds to the intercellular canaliculi, a
specialised form of the lumen in the rat parotid acini
(Takemura et al., 1999). In
the ducts, strong immunofluorescence was observed along the apical area; many
cells showed positive signals, but some cells were found to exhibit weak or
negative fluorescence. Thus, in contrast to the acini, the distribution of
Ins(1,4,5)P3R2 in the ducts exhibits heterogeneity. The
merged image (Fig. 6C) clearly
showed the different immunolocalisation of connexin 32 and
Ins(1,4,5)P3R2 in the acini and ducts. Control specimens
treated without primary antibodies did not reveal any specific staining.
|
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Discussion |
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Many investigators have observed the elevation of [Ca2+]i in the
acini and ducts of salivary glands in response to CCh and ATP stimulation
(Valdez and Turner, 1991;
Gromada et al., 1993
;
Dinudom et al., 1993
;
Hurley et al., 1993
; Soltof et
al., 1993; Hurley et al.,
1994
; Jorgensen et al.,
1995
; Xu et al.,
1996
; Lee et al.,
1997a
; Lee et al.,
1997b
; Tojyo et al.,
1997a
; Tojyo et al.,
1997b
; Bird et al.,
1998
). As discussed previously, the time course of
[Ca2+]i elevation proceeds rapidly, making it difficult to clarify
the spatiotemporal details of Ca2+ signalling dynamics in the
tissue (Takemura et al.,
1999
). Highspeed confocal microscopy of dissociated glands allowed
analysis of the changes in [Ca2+]i of the order of a millisecond,
with precise measurement of acinar and ductal responses from the supracellular
to the subcellular level.
CCh stimulation caused a rise in [Ca2+]i in the acini before the
ducts (Fig. 2). We did not
observe any Ca2+ waves from acini to ducts; thus direct propagation
of Ca2+ signals from acini to ducts seems unlikely. 3D propagation
of Ca2+ signals out of and into the plane of focus might be another
explanation. Since CCh triggers fluid secretion from the acini, it is also
possible that acinar fluid elicited the ductal response from the luminal side
(Xu et al., 1996;
Lee et al., 1997a
;
Lee et al., 1997b
). However,
it was noted that the presence of acini was not obligatory to initiate the
ductal response (Fig. 3). This
indicates that the ductal system has its own intrinsic control system for
[Ca2+]i mobilisation. Based on the fact that the Ca2+
response started in certain cells from which the Ca2+ wave then
propagated to neighboring cells (Fig.
2), it is likely that these cells act as `pioneer' or `pacemaker'
cells to control the regional ductal response
(Yamamoto-Hino et al.,
1998
).
The pattern of Ca2+ response to CCh and ATP in the presence or absence of extracellular Ca2+ was considerably different in acini and ducts (Fig. 3). The acinar domain responded well to CCh but not to ATP, and the responsiveness to CCh did not require the presence of extracellular Ca2+. This indicates that the acinar domain raises [Ca2+]i by the release of Ca2+ from the intracellular pool upon muscarinic receptor stimulation. Purinergic receptor stimulation seems to have little effect on both the intra- and extracellular mechanisms of Ca2+ mobilisation in the acinar domain. By contrast, the ductal domain responded both to CCh and ATP, but in the absence of extracellular Ca2+ the responsiveness to CCh was abolished. This suggests that the observed ductal response to CCh occurs mostly through the entry of Ca2+ from the extracellular fluid. Alternatively, the release of Ca2+ from the intracellular store might require the presence of extracellular Ca2+ in the case of CCh stimulation. In the same duct, ATP triggered both the release of Ca2+ from the intracellular pool (Fig. 3E) and the Ca2+ entry from the extracellular fluid (Fig. 3F). However, it should be pointed out that the response of individual duct cells varied from one to the other (Fig. 4). This indicates that the intracellular signalling cascade downstream of receptor activation is diverse among the duct cells.
Extensive studies have elucidated that Ca2+ signalling involves
a molecular cascade of reactions, including phosphoinositide turnover and the
activation of receptors/channels present on the plasma membrane and the
intracellular stores, leading to the elevation of [Ca2+]i either by
entry of Ca2+ from the extracellular fluid or release of
Ca2+ from the intracellular stores
(Berridge et al., 2000;
Meldolesi and Pozzan, 1998
;
Michikawa et al., 1996
). Our
findings suggest that such subcellular mechanisms are not equally activated in
the cells of duct domain. Since salivary ducts are known to be composed of
heterogeneous cell populations (Sato and
Miyoshi, 1988
; Sato and
Miyoshi, 1998
), it is possible that the heterogeneity of
Ca2+ signalling reflects the heterogeneity of cell types. Whether
this involves the heterogeneity of ER Ca2+ stores
(Meldolesi and Pozzan, 1998
)
remains to be elucidated. Further studies are needed to clarify the biological
significance of diverse Ca2+ signalling occurring in the duct
domain.
In contrast to the ducts, acini exhibited a synchronous [Ca2+]i
response (Fig. 5). Gap
junctional communication has been implicated in synchronised intercellular
Ca2+ signalling (Stauffer et
al., 1993; Guerineau et al.,
1998
). In mammalian salivary glands, the presence of gap junctions
between the acinar cells has been shown by electron microscopy
(Dewery and Barr, 1964
;
Hand, 1972
; Nagato and Tandler,
1986) and connexin immunohistochemistry
(Hirono et al., 1995
;
Lee et al., 1998
;
Shimono et al., 2000
).
Although there are debates as to whether gap junctions are present between the
duct cells (Dewery and Barr,
1964
; Hirono et al.,
1995
; Lee et al.,
1998
), our findings indicate that the ductal domain is mostly
devoid of gap junctions and, if present, they are located at the distal end of
the intercalated ducts close to the acini
(Fig. 6). The
immunofluorescence spots observed in the intercalated duct area presumably
correspond to the gap junctions between myoepithelial cells
(Lee et al., 1998
) or
intercalated duct cells themselves (Dewery
and Barr, 1964
; Hirono et al.,
1995
). Whatever the explanations are, previous data and our
studies indicate that the acinar domain is the principal site where gap
junctions are present in salivary glands. The abolishment by octanol of the
synchronised Ca2+ response in the acini
(Fig. 5) strongly suggests that
the acinar domain acts as the functional syncitium through the gap
junction.
Immunohistochemical distribution of Ins(1,4,5)P3R2 was
homogeneous in the acini but heterogeneous in the ducts
(Fig. 6). The distribution of
Ins(1,4,5)P3R2 in the intercellular canaliculi area of
salivary acini has been observed by us
(Yamamoto-Hino et al., 1998;
Takemura et al., 1999
) and by
others (Lee et al., 1997a
).
Heterogeneous expression of Ins(1,4,5)P3Rs in salivary
ducts has also been noted by our previous study
(Yamamoto-Hino et al., 1998
).
Heterogeneous expression of Ins(1,4,5)P3Rs has also been
observed in airway epithelia (Sugiyama et
al., 1996
), kidney collecting ducts
(Monkawa et al., 1998
) and
gastrointestinal epithelia (Matovick et
al., 1996
), and thus is considered as the common feature in
tubular epithelia. Since Ins(1,4,5)P3Rs play key roles in
the release of Ca2+ from the intracellular stores
(Berridge et al., 2000
;
Meldolesi and Pozzan, 1998
;
Michikawa et al., 1996
), the
particular tissue distribution of Ins(1,4,5)P3R2 is likely
to determine the type of Ca2+ signalling: either acinar
(synchronised) or ductal (non-synchronised).
Our observations indicate that acini and ducts create distinct local
Ca2+ signalling communities. Since acini and ducts constitute the
elementary units of exocrine organs, the organised Ca2+ signalling
might have some impact on the mechanisms underlying the harmonised organ
function. Coordination of Ca2+ signalling according to the tissue
unit has been observed in the liver
(Nathanson et al., 1995;
Robb-Gaspers and Thomas,
1995
), and suggested to facilitate diverse cells to perform the
integrative, organ-level response. Distinct Ca2+ signalling among
different domains (i.e. the acini and ducts), may help to develop the
versatility needed for the organ to perform its complex functions.
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Acknowledgments |
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Footnotes |
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References |
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Berridge, M. J., Lipp, P. and Bootman, M. D. (2000). The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell. Biol. 1, 11-21.[Medline]
Bird, G. S., Lounzao, M. C., Ribeiro, C. M. P. and Putney, J. W., Jr (1998). Calcium signaling in exocrine glands. Eur. J. Morph. 36,153 -156.
Dewery, M. M. and Barr, L. (1964). A study of
the structure and distribution of the nexus. J. Cell
Biol. 23,553
-585.
Dinudom, A., Poronnik, P., Allen, D. G. and Cook, D. I. (1993). Control of intracellular Ca2+ by adrenergic and muscarinic agonists in mouse mandibular duct and endpieces. Cell Calcium 14,631 -638.[Medline]
Garrett, J. R. (1987). The proper role of nerves in salivary secretion: a review. J. Dent. Res. 66,387 -397.[Abstract]
Gromada, J., Jorgensen, T. D., Triststaris, K., Nauntofte, B. and Dissing, S. (1993). Ca2+ signalling in exocrine acinar cells: the diffusional properties of cellular inositol 1,4,5-triphosphate and its role in the release of Ca2+. Cell Calcium 14,711 -723.[Medline]
Guerineau, N. C., Bonnefont, X., Stoeckel, L. and Mollard,
P. (1998). Synchronized spontaneous Ca2+
transients in acute anterior pituitary slices. J. Biol.
Chem. 273,10389
-10395.
Hand, A. R. (1972). Adrenergic and cholinergic terminals in the rat parotid gland. Electron microscopic observations on permanganate-fixed glands. Anat. Rec. 173,131 -140.[Medline]
Hirono, C., Shiba, Y. and Kanno, Y. (1995). Different localizations of 21 and 27 kDa gap-junction proteins in rat salivary glands. Histochem. Cell Biol. 103, 39-46.[Medline]
Hurley, T. W., Shoemaker, D. D. and Ryan, M. P. (1993). Extracellular ATP prevents the release of stored Ca2+ by autonomic agonists in rat submandibular gland acini. Am. J. Physiol. 34,C1472 -C1478.
Hurley, T. W., Ryan, M. P. and Shoemaker, D. D. (1994). Mobilization of Ca2+ influx, but not of stored Ca2+, by extracellular ATP in rat submandibular gland acini. Arch. Oral Biol. 39,205 -212.[Medline]
Jorgensen, T. D., Gromada, J., Tritsaris, K., Nauntofte, B. and Dissing, S. (1995). Activation of P2z purinoceptors diminishes the muscarinic cholinergic-induced release of inositol 1,4,5-triphosphate and stored calcium in rat parotid acini. Biochem. J. 312,457 -464.[Medline]
Lee, M. G., Xu, X., Zeng, W., Diaz, J., Wojcikiewicz, J. H.,
Kuo, T. H., Wuytack, F. Racymaekers, L. and Muallem, S.
(1997a). Polarized expression of Ca2+ channels in
pancreatic and salivary gland cells. Correlation with initiation and
propagation of [Ca2+]i waves. J. Biol.
Chem. 272,15765
-15770.
Lee, M. G., Zeng, W. and Muallem, S. (1997b).
Characterization and localization of P2 receptors in rat submandibular gland
acinar and duct cells. J. Biol. Chem.
272,32951
-32955.
Lee, C. Y., Muramatsu, T. and Shimono, M. (1998). Localization of connexin 26, 32 and 43 in rat parotid gland. Acta Histochem. Cytochem. 31,211 -216.
Matovick, L. M., Maranto, A. R., Soroka, C. J., Gorelick, F. S.,
Smith, J. and Goldenring, J. R. (1996). Co-distribution of
calmodulin-dependent protein kinase II and inositol triphosphate receptors in
an apical domain of gastrointestinal mucosal cells. J. Histochem.
Cytochem. 44,1243
-1250.
Meldolesi, J. and Pozzan, T. (1998). The
heterogeneity of ER Ca2+ stores has a key role in nonmuscle cell
signaling and function. J. Cell Biol.
142,1395
-1398.
Michikawa, T., Miyawaki, A., Furuichi, T. and Mikoshiba, K. (1996). Inositol 1,4,5-triphosphate receptors and calcium signaling. Crit. Rev. Neurobiol. 10, 39-55.[Medline]
Monkawa, T., Hayashi, M., Miyawaki, A., Sugiyama, T., Yamamoto-Hino, M., Hasegawa, M., Furuichi, T., Mikoshiba, K. and Saruta, T. (1998). Localization of inositol 1,4,5-triphosphate receptors in the rat kidney. Kidney Int. 53,296 -301.[Medline]
Nagatao, T. and Tandler, B. (1986). Gap junctions in rat sublingual gland. Anat. Rec. 214, 71-75.[Medline]
Nathanson, M. H., Burgstahler, A. D., Mennone, A., Fallon, M.
B., Gonzalez, C. B. and Saez, J. C. (1995). Ca2+
waves are organized among the hepatocytes in the intact organ. Am.
J. Physiol. 269,G167
-G171.
Pinkstaff, C. A. (1980). The cytology of salivary glands. Int. Rev. Cytol. 63,141 -261.
Robb-Gaspers, L. D. and Thomas, A. P. (1995).
Coordination of Ca2+ signaling by intercellular propagation of
Ca2+ waves in the intact liver. J. Biol.
Chem. 270,8102
-8107.
Sato, A. and Miyoshi, S. (1988). Ultrastructure of the main excretory ducts epithelia of the parotid and submandibular glands with a review of the literature. Anat. Rec. 220,239 -251.[Medline]
Sato, A. and Miyoshi, S. (1998). Cells in the duct system of the rat submandibular gland. Eur. J. Morph. 36 Suppl.,61 -66.
Segawa, A. (1999). Measurement of secretion in confocal microscopy. Methods Enzymol. 307,328 -340.[Medline]
Segawa, A., Sahara, N., Suzuki, K. and Yamashina, S. (1985). Acinar structure and membrane regionalization as a prerequisite for exocrine secretion in the rat submandibular gland. J. Cell Sci. 78,67 -85.[Abstract]
Shimono, M., Lee, C. Y., Matsuzaki, H., Ishikawa, H., Inoue, T., Hashimoto, S. and Muramatsu, T. (2000). Connexins in salivary glands. Eur. J. Morph. 38,257 -261.
Soltoff, S. P., McMillian, M. K., Talamo, B. R. and Cantley, L. C. (1993). Blockade of ATP binding site of P2 purinoceptors in rat parotid acinar cells by isothiocyanate compounds. Biochem. Pharmacol. 45,1936 -1940.[Medline]
Stauffer, P. L., Zhao, H., Luby-Phelps, K., Moss, R. L., Star,
R. A. and Muallem, S. (1993). Gap junction communication
modulates [Ca2+]i oscillations and enzyme secretion in pancreatic
acini. J. Biol. Chem.
268,19769
-19775.
Sugiyama, T., Furuya, A., Monkawa, T., Yamamoto-Hino, M., Satoh, S., Ohmori, K., Miyawaki, A., Hanai, N., Mikoshiba, K. and Hasegawa, M. (1994). Monoclonal antibodies distinctively recognizing the subtypes of inositol 1,4,5-triphosphate receptor: application to the studies on inflammatory cells. FEBS Lett. 354,149 -154.[Medline]
Sugiyama, T., Yamamoto-Hino, M., Wasano, K., Mikoshiba, K. and
Hasegawa, M. (1996). Subtype-specific expression patterns of
inositol 1,4,5-triphosphate receptors in rat airway epithelial cells.
J. Histochem. Cytochem.
44,1237
-1242.
Takemura, H., Yamashina, S. and Segawa, A. (1999). Millisecond analyses of Ca2+ initiation sites evoked by muscarinic receptor stimulation in exocrine acinar cells. Biochem. Biophys. Res. Commun. 259,656 -660.[Medline]
Tojyo, Y., Tanimura, A., Matsui, S. and Matsumoto, Y. (1997a). Effects of extracellular ATP on cytosolic Ca2+ concentration and secretory responses in rat parotid acinar cells. Arch. Oral Biol. 42,393 -399.[Medline]
Tojyo, Y., Tanimura, A. and Matsumoto, Y. (1997b). Imaging of intracellular Ca2+ waves induced by muscarinic receptor stimulation in rat parotid acinar cells. Cell Calcium 22,455 -462.[Medline]
Valdez, I. H. and Turner, R. J. (1991). Effects
of secretagogues on cytosolic Ca2+ levels in rat submandibular
granular ducts and acini. Am. J. Physiol.
261,G359
-G363.
Xu, X., Diaz, J., Zhao, H. and Muallem, S. (1996). Characterization, localization and axial distribution of Ca2+ signalling receptors in the rat submandibular salivary gland ducts. J. Physiol. 491,647 -662.[Abstract]
Yamamoto-Hino, M., Miyawaki, A., Segawa, A., Adachi, E.,
Yamashina, S., Fujimoto, T., Sugiyama, T., Furuichi, T., Hasegawa, M. and
Mikoshiba, K. (1998). Apical vesicles bearing inositol
1,4,5-triphosphate receptors in the Ca2+ initiation site of ductal
epithelium of submandibular gland. J. Cell Biol.
141,135
-142.
Young, J. A. and van Lennep, E. W. (1978). The Morphology of Salivary Glands. London, New York, San Francisco: Academic Press.
Young, J. A., Cook, D. I., van Lennep, E. W. and Roberts, M. L. (1987). Secretion by the major salivary glands. In Physiology of the Gastrointestinal Tract (2nd edn) (ed. L. Johnson, J. Christensen, M. Jackson, E. Jacobson and J. Waksh), pp.773 -815. New York: Raven Press Ltd.