Department of Cell Biology, University of Virginia Health System, School
of Medicine, Charlottesville, VA 22908-0732, USA
* These authors contributed equally to this work
Author for correspondence (e-mail:
jdc4r{at}virginia.edu
)
Accepted 3 May 2002
![]() |
Summary |
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Key words: Exocytosis, Secretion, Syntaxin 3
![]() |
Introduction |
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The three pathways have been defined and distinguished from each other in
experiments employing biosynthetic (pulse/chase) labeling of parotid tissue
lobules in vitro and analysis of the radiochemical composition of secreted
proteins and the specific radioactivity of amylase, a major acinar protein
that is detected in all pathways. Using this approach, it has been possible to
deduce that all pathways derive from immature secretory granules. The
constitutive-like and minor regulated pathways arise together from immature
granules by vesicular budding that accompanies granule maturation. Their
secretory contents are very similar to each other and are enriched in newly
synthesized and transported proteins. Although the proteins present in these
pathways are mostly the same as those stored in mature granules, the relative
amounts of certain proteins are quite different. These compositional
differences are thought to result from a passive sorting process in which
proteins that efficiently aggregate are stored in granules, whereas those that
either don't aggregate or are less efficiently aggregated become the content
of the budding vesicles (Arvan and Castle,
1998). After exiting the maturing granule, the constitutive-like
and minor regulated pathways diverge from one another to provide the
continually released and stored/stimulatable components of secretion released
by low-level stimulation (Huang et al.,
2001
).
In the present study, we have sought to explore further the significance of
the minor regulated pathway as a second stimulus-dependent and apically
directed vesicular pathway. Previously, it has been noted from measurements of
membrane capacitance and secretion in neural, endocrine and exocrine cells
that regulated secretion exhibits at least two kinetic components, one rapid
and one slower. While the rapid component has been associated mostly with
discharge of small synaptic vesicle-like carriers and the slower component
with granule exocytosis, there are also examples, for example, in pancreatic
ß-cells and acinar cells, of possible kinetic diversity among
granule-like pathways (Kasai,
1999; Kasai and Takahashi,
1999
; Ninomiya et al.,
1997
; Campos-Toimil et al.,
2000
). Thus we have been interested in whether the minor regulated
pathway, which is more sensitive to secretagogue stimulation than granule
exocytosis, is discharged more rapidly than granules in response to strong
exocytotic stimuli. We now show that this is indeed the case. Moreover, we
raise the strong possibility that the minor regulated pathway and granule
exocytosis are linked in a functional sequence in which stimulation of the
minor regulated pathway relocates docking/fusion sites for granule exocytosis
to the apical plasma membrane. Thus discharge of the minor regulated pathway
by Iso or CCh necessarily precedes granule exocytosis and CCh mainly controls
the number of granule release sites while Iso controls granule exocytosis
itself.
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Materials and Methods |
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Tissue incubation and processing
For incubations, parotid glands were dissected from rats sacrificed by
CO2 asphyxiation and cut into lobules in chilled incubation medium
(Dulbecco's modified Eagle's medium equilibrated with 95% O2, 5%
CO2). Preincubations (cys-/met-free medium) and incubations were
performed at 37°C in stoppered Erlenmeyer flasks with periodic
equilibration with the same gas mixture
(Huang et al., 2001). Pulse
labeling was performed for 5 minutes in cys-/met-free medium containing 500
µCi/ml Expre35S35S. In certain experiments,
incubations of labeled lobules were performed in potassium-free medium, which
was prepared by replacing KCl by NaCl in regular medium, as specified.
Procedures used for stimulating labeled tissue and collecting and analyzing
incubation media and tissue were the same as previously
(Huang et al., 2001
). At
specified time intervals, all medium in each sample (1 ml) was removed and
replaced with fresh medium with addition of agonists by 1:1000 dilutions from
stocks prepared in H2O. At the end of incubation, lobules were
washed with chilled phosphate-buffered saline and homogenized. Media and
homogenates were used for assay of amylase activity
(Bernfeld, 1955
) and for
SDS-PAGE (12.5% gels), Coomassie staining, phosphorimaging, and quantitative
analysis of 35S-labeled proteins with Image Quant software
(Castle and Castle, 1996
;
Huang et al., 2001
). Specific
radioactivity of amylase was determined by normalizing the intensities of the
amylase bands quantified by phosphorimaging to the amylase enzyme activity in
each sample.
Immunofluorescence microscopy
For immunofluorescence microscopy, incubated parotid lobules were fixed in
periodate/lysine/paraformaldehyde (McLean
and Nakane, 1974), frozen in a 1:1 mixture of 2.3 M sucrose and
50% polyvinylpyrrolidone, and sectioned on a cryomicrotome at -50°C.
Sections were mounted on gelatin-coated slides, blocked in 5% goat serum, PBS
and 0.05% Triton X-100. Primary and secondary antibodies were diluted in 0.5%
goat serum, PBS and 0.05% Triton X-100. Following staining and washing,
sections were mounted in Prolong (Molecular Probes) and examined using either
a Zeiss Axiophot or Axiovert fluorescence microscope. Images were collected as
0.1 µm stacked optical sections using a 63x NA 1.4 objective and
4x magnifier, and the optical sections were digitally deconvolved and
analyzed using Openlab software (Improvision).
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Results |
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Stimulation by CCh over 10 minutes elicited release of modest amounts of
total amylase enzyme activity. At 40 nM CCh, initial output was linear,
reaching 1%, whereas at 200 nM and 2 µM CCh, initial output occurred
as a quick burst and then gradually increased to 3 and 5%, respectively
(Fig. 2A). Examination of the
specific radioactivity of amylase discharged under these conditions showed a
rise to a plateau with 40 nM CCh, while 200 nM and 2 µM CCh showed initial
spikes as with Iso but no secondary gradual rise
(Fig. 2B). Throughout the 10
minute period, the radiochemical composition of the secretion was unchanged at
all levels of CCh (shown by the representative profile in
Fig. 2C) and was identical to
that of the minor regulated pathway. The total protein composition viewed by
Coomassie staining also resembled that of the minor regulated pathway except
at later times of stimulation with 2 µM CCh. Here a faint band of PSP,
indicative of low-level granule exocytosis, was detected
(Fig. 2C, lane 4). These
results argue that CCh stimulates the minor regulated pathway quite
selectively, especially at low doses and that the limited granule exocytosis
that is detected at 2 µM CCh is delayed and restricted to the oldest
unlabeled granules.
|
Possible inter-relationships of the minor regulated pathway and
granule exocytosis
Discharge of the minor regulated pathway preceded granule exocytosis under
all conditions examined (Figs
1,
2). Thus we wondered whether
fusion of the minor regulated carriers might be a prerequisite for granule
exocytosis as might be the case if the minor regulated pathway delivers
components necessary for granule fusion to the apical surface. Because the
minor regulated pathway is stimulated by agonists acting either through
Ca2+- or cAMP-mediated signaling [the same stimuli that regulate
granule exocytosis except at lower concentrations
(Castle and Castle, 1996)], we
have been unable to identify a way of blocking the exocytosis of the minor
regulated pathway selectively. Therefore we have investigated approaches that
are intended to modulate the steady state distribution of minor regulated
carriers within the presumed recycling pathway between the plasma membrane and
apical cytoplasm in order to discern whether the manipulations affect granule
exocytosis.
CCh causes secretory potentiation
If relocation of the minor regulated carriers to the plasma membrane
promotes granule exocytosis, then it might be expected that stimulation with
low doses of CCh, which selectively discharges the minor regulated pathway,
would enhance granule exocytosis stimulated by Iso. Therefore, as a first
approach in testing the inter-relationship between the minor regulated and
granule pathways, we added and removed low doses of CCh during granule
exocytosis stimulated by Iso. Fig.
3A shows that stimulation with 1 µM Iso in combination with 40
nM CCh results in potentiation of amylase secretion. These results are
consistent with many previous findings, which were usually obtained with much
higher doses of cholinergic stimuli (Asking
and Gjorstrup, 1980;
Templeton, 1980
;
Spearman and Butcher, 1981
;
Takemura, 1984
;
Yoshimura et al., 1998
;
Yoshimura et al., 2000
;
Yoshimura and Hiramatsu,
1998
). Fig. 3B
presents a short time course showing amylase-specific radioactivity in
secretion elicited by Iso alone or in combination with either 40 or 200 nM
CCh. Specific radioactivity peaks early, consistent with a rapid release of
the minor regulated pathway. Note that the specific radioactivity of the peak
progressively decreases with increasing CCh, consistent with enhanced
discharge of unlabeled granules (as also reflected in the increased rate of
amylase secretion seen in Fig.
3A). Brief (5 minutes) pretreatment with a low dose of CCh before
addition of 1 µM Iso to stimulate granule exocytosis causes an enhanced
initial release of amylase enzyme with low specific radioactivity compared
with no pretreatment (Fig. 3C).
This observation suggests that activation of the minor regulated pathway
accelerates exocytosis of old (unlabeled) granules.
|
We also noticed that a slight secondary rise in specific radioactivity (after 5 minutes) occurs at about the same rate in the presence and absence of 40 nM CCh but is notably faster in the presence of 200 nM CCh (Fig. 3B), suggesting an accelerated rate of exocytosis of newly formed granules. This possibility is confirmed by comparing the rates of secretion of radiolabeled PSP upon stimulation with Iso alone or in the presence of 40 and 200 nM CCh (Fig. 3D); increased dosage of cholinergic agonist induces the appearance of labeled PSP in the medium at progressively earlier time points. Thus CCh by itself slightly stimulates exocytosis of old granules but together with Iso substantially increases the rates of export of new granules.
Interestingly, if CCh is removed after the initial 10 minutes of stimulation in the extended time course, the potentiation is not sustained and the rate of exocytosis decreases rapidly to that of samples stimulated with 1 µM Iso alone (Fig. 3A). The latter result suggests either rapid inactivation of the potentiation machinery or rapid recycling of minor regulated carriers presumably by endocytosis.
BFA causes secretory potentiation
In the second approach, we tested the effect of BFA on granule exocytosis
stimulated by Iso. We recently showed that BFA blocks the formation of minor
regulated carriers but its secretory contents are redirected to the apical
lumen along the constitutive-like pathway
(Huang et al., 2001).
Consequently, we hypothesized that any membrane components of the minor
regulated carriers would be similarly redirected into the constitutive-like
pathway in the presence of BFA, leading to their increased steady state level
in the apical plasma membrane. If these components are needed for granule
exocytosis, then BFA should increase the rate of granule exocytosis stimulated
by Iso. Fig. 4A shows that
stimulation of the tissue with 1 µM Iso in combination with BFA treatment
results in potentiation of amylase secretion in which the output of the enzyme
activity is greater than the sum of the outputs caused by the secretagogues
individually. BFA alone has a very small effect on amylase release (<1%
over 30-40 minutes), but it increases the Iso-stimulated amount of secreted
amylase by 50% in 60 minutes. The effects of BFA are reversible and, upon its
removal, the rate of amylase secretion reverts to that observed with Iso
alone.
|
K+ depletion causes secretory potentiation
If endocytosis of minor regulated carriers is responsible for removing
plasma membrane components and hence decreasing the rate of granule
exocytosis, then blocking recycling might also be expected to result in an
enhanced rate of granule exocytosis. Therefore, in the third approach, we
sought to inhibit endocytosis by depleting intracellular potassium and
examined the effect on Iso-stimulated secretion of amylase. Previously, it has
been shown that incubation of cells in K+-depleted medium results
in lowering of intracellular potassium and rapid disruption of endocytosis in
fibroblasts (Larkin et al.,
1983; Altankov and Grinnell,
1993
). Before testing out this possibility, we used pulse-chase
labeled parotid lobules to evaluate the output of amylase and radiolabeled
proteins in K+-depleted medium in the absence of stimulation. After
10 minutes, the time used to affect endocytosis elsewhere
(Altankov and Grinnell, 1993
),
there was no detectable effect on either parameter. At more extended times
(>30 minutes), we noticed a small increase in secretion above basal level,
but the amount of amylase (<0.4% of total) and its specific radioactivity
(approximately twice that of unstimulated secretion) were modest. The
radiochemical composition was identical to that of the constitutive-like and
minor regulated pathways. However, we were unable to distinguish whether the
small release reflected inhibition of endocytosis coupled to constitutive-like
secretion (Huang et al., 2001
)
or slight stimulation of the minor regulated pathway. When granule exocytosis
was induced by 1 µM Iso in K+-depleted medium, we observed a
very robust potentiation in which the rate of amylase secretion nearly doubled
(Fig. 4B), while the
radiochemical composition of biosynthetically labeled proteins and the time
course of specific radioactivity changes were the same as shown in
Fig. 1B,C. The potent effect is
consistent with accumulation of granule fusion sites at the cell surface by
preventing endocytosis of minor regulated carriers in K+-depleted
medium.
Discharge of the minor regulated pathway correlates with relocation
of syntaxin 3
In our previous analysis of resting secretion, we noted that stimulation of
the minor regulated pathway was accompanied by expansion of luminal spaces,
suggesting reorganization of acinar apical surfaces
(Huang et al., 2001). At least
part of the change may reflect addition of the membranes of minor regulated
carriers following their exocytosis. Consequently, we sought to identify
candidate markers of the carriers that might enable us to study the operation
and regulation of the pathway more directly. Parotid lobules were subjected to
in vitro incubation, stimulated (or not) for brief time periods, rapidly
chilled, and then fixed, processed and cryosectioned. The sections were
immunostained with antibodies specific for various membrane proteins that have
been implicated to function in exocrine secretion and counterstained with
either BODIPY-phalloidin, which readily identifies the actin-rich terminal web
beneath the apical plasma membrane, or with anti-DPPIV, which marks the apical
plasma membrane. Fig. 5
presents sample fluorescence micrographs of sections from unstimulated tissue
stained with anti-syntaxin 3. Syntaxin 3 is a secretory granule membrane
component in acinar cells (Gaisano et al.,
1996a
) (A.M.C., A.Y.H. and J.D.C., unpublished) and mast cells
(Guo et al., 1998
), and
quantitative analysis following subcellular fractionation in parotid indicates
that
80% of this SNARE protein is associated with secretory granules in
parotid (A.M.C. and J.D.C., unpublished). Immunostaining is observed
throughout the stored population of granules, consistent with presence of the
protein in granule membranes. In addition, many of the acinar cells exhibit
bright punctate staining, which is often located close to the apical surface
just beneath (on the cytoplasmic side of) the terminal web. Notably, the
syntaxin 3 foci are distinct from DPPIV staining
(Fig. 5D). Following
stimulation with 40 nM CCh for 1 or 3 minutes, the granule-like staining
remained unaffected but the positioning of the bright puncta changed
significantly. Apical luminal spaces were expanded as signified by increased
separation of the terminal webs in neighboring cells, and many of the syntaxin
3 foci were clearly located on the luminal side of the terminal webs
(Fig. 6A). Under these
conditions, syntaxin 3 staining was aligned with DPPIV staining; however, the
syntaxin 3 staining remained focal (Fig.
6B), suggesting the SNARE remained concentrated rather than
diffusing after entry into the apical surface. For comparison,
Fig. 6C shows the relative
positions of actin and DPPIV in CCh-stimulated tissue.
|
|
As an additional test of the correlation of relocation of syntaxin 3 with stimulation of the minor regulated pathway, we examined tissue following a 3 minute stimulation with 1 µM Iso. Here also syntaxin 3 was relocated into the apical surface (Fig. 6D), in advance of granule exocytosis. For both CCh and Iso stimulation, we quantitated the number of relocated syntaxin 3 foci per length of apical surface as defined by underlying actin staining. The results obtained from 24 luminal profiles indicated a frequency of relocation of 1±0.1 per µm. Thus our findings point to a tight association of syntaxin 3 relocation and mobilization of the minor regulated pathway.
Evaluation of additional prospective markers of the minor regulated
pathway
We also examined other membrane proteins for possible relocation in
response to stimulation by 40 nM CCh. The SNARE protein VAMP2 and the small
GTPase Rab11 are components of apical secretory and recycling compartments in
epithelial cells (Hansen et al.,
1999; Gaisano et al.,
1994
; Fujita-Yoshigaki et al.,
1996
; Hori et al.,
1996
; Calhoun et al.,
1998
). VAMP2 exhibited extensive granule-like staining and
occasionally subapical staining that appeared similar to the foci that were
brightly stained with anti-syntaxin 3, while Rab11 showed significant
concentration in foci that resembled those containing syntaxin 3
(Fig. 7A,C). Stimulation by 40
nM CCh appeared to cause some relocation of both proteins to the luminal side
of the actin-rich terminal web (Fig.
7B,D); however, the extent of relocation was not as impressive as
with syntaxin 3. We also examined Rab3D which, like VAMP2, exhibits extensive
granule staining (Valentijn et al.,
1996
; Ohnishi et al.,
1996
); however, unlike Rab11, it was not highly concentrated in
foci that were just beneath the apical plasma membrane (data not shown).
Syntaxin 6 is concentrated in immature but not mature secretory granules in
exocrine cells (Klumpermann et al.,
1998
) and is thought to function in exocytosis in certain cell
types (Martin-Martin et al.,
2000
). These features suggested a possible association with the
minor regulated pathway. However, we found that syntaxin 6 was almost entirely
localized in the central cytoplasm well away from apical lumina and did not
redistribute detectably upon CCh stimulation (not shown), making it unlikely
as a marker for the minor regulated pathway. Thus this limited search
identified VAMP2 and Rab11 as prospective markers along with syntaxin 3 for
the minor regulated carriers.
|
Both BFA and K+ depletion induce relocation of syntaxin
3
To further test the correlation between relocation of syntaxin 3 and the
prospective role of the minor regulated pathway in promoting granule
exocytosis, we examined whether BFA treatment and K+ depletion
affected the localization of syntaxin 3. As seen in
Fig. 8A, lobules treated for 15
minutes with BFA in the absence of Iso exhibited an increased relocation of
syntaxin 3 across the terminal web and into the apical surface. Similarly,
incubation of tissue for 15 minutes in K+-depleted medium with no
Iso stimulation produced images of expanded apical lumina with relocation of
syntaxin 3, although the incidence is lower than observed following brief CCh
stimulation (Fig. 8B). While
these observations strengthen the correlation being tested, the latter result
implies that K+-depletion is not acting solely to block recycling
of minor regulated carriers because agonist stimulation has been omitted. In
view of the small stimulatory effect on secretion by K+ depletion
noted above and the expanded lumina noted in
Fig. 8B, we suspect that
K+ depletion acts to discharge the minor regulated pathway at a low
level as well as efficiently blocking endocytosis. The latter action would
then be the major cause of potentiation of Iso-stimulated amylase
secretion.
|
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Discussion |
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Contributions to secretory potentiation by the minor regulated
pathway
In efforts to explore ways that discharge of the minor regulated pathway
and granule exocytosis might be functionally linked, we have been unable to
dissociate their discharge except by lowering the dose of their common
stimuli. Consequently, we have employed experimental strategies designed to
manipulate the pathway and alter the steady state distribution of its cargo
and have assessed whether the treatments have enhanced or inhibited granule
exocytosis. Both BFA and K+ depletion acted synergistically in
accelerating isoproterenol-stimulated amylase secretion
(Fig. 4). These treatments
qualify as new ways to achieve secretory potentiation, and they point to a
vesicular mechanism being involved in the potentiation process. Further, as 40
nM CCh selectively stimulates the minor regulated pathway, our data showing
gain and loss of potentiation, respectively, upon adding and removing CCh
(Fig. 3A) are also consistent
with a vesicular mechanism of potentiation involving this pathway. Taken
together, these findings led us to propose that potentiation in acinar cells
results at least in part from increasing the number of granule docking/fusion
sites in the cell surface via the minor regulated pathway, which are used in
turn for granule exocytosis. Interestingly, this type of mechanism involving
an increase in the number of functional release sites might be analogous to
neurons, where potentiation has been interpreted to reflect an increase in the
fraction of releasable synaptic vesicles that undergo exocytosis per stimulus
(Stevens and Wesseling,
1999).
Association of exocytotic machinery with the minor regulated
pathway
Our studies have also led to the identification of prospective markers of
the minor regulated pathway, namely syntaxin 3, Rab11 and VAMP2. This was made
possible by initially showing that 40 nM CCh discharges the minor regulated
pathway exclusively (Fig. 2),
and then correlating stimulation with relocation of these proteins across the
terminal web and into the apical membrane (Figs
5,6,7,8).
All three proteins are candidate exocytotic machinery
(Galli et al., 1998;
Hori et al., 1996
;
Calhoun et al., 1998
;
Fujita-Yoshigaki et al., 1996
;
Gaisano et al., 1996b
).
Relocation of syntaxin 3 is particularly impressive with concentrated foci
being observed in the apical surface wherever luminal profiles are expanded.
In contrast, relocation of VAMP2 and Rab11, while readily observed, is not as
striking.
The putative association of exocytotic machinery with vesicles of the minor
regulated pathway is interesting on several accounts. First, while part of
this machinery [e.g. the v-SNARE (R-SNARE) VAMP2] may function in fusing minor
regulated carrier vesicles to the cell surface, another part, particularly
syntaxin 3, may be cargo that is destined to serve as a t-SNARE (Q-SNARE) for
granule exocytosis after delivery. Second, the realization that syntaxin 3
might be the t-SNARE for granule exocytosis is relevant to earlier deductions
regarding the exocrine granule fusion machinery. Previously, syntaxin 2 was
regarded as the likely t-SNARE for granule release in exocrine pancreas and
parotid based on its apical localization and its sensitivity to botulinum
neurotoxin C (Gaisano et al.,
1996a; Hansen et al.,
1999
). However, syntaxin 3, which is also sensitive to the same
toxin (Hansen et al., 1999
),
can now be considered a likely candidate. It may be the case that syntaxin 2
functions as a t-SNARE in exocytosis of the minor regulated pathway, while
syntaxin 3 serves this role for granules. Application of isoform-specific
syntaxin perturbants will be required to address these possibilities.
A third point of interest stems from the appearance of syntaxin 3 as
concentrated foci both before and after delivery to the apical surface (Figs
5,
6,
8). This observation suggests
that syntaxin 3 may be delivered in prefabricated docking/fusion sites.
Potentially these sites may be related to the fusion hot spots that have been
implicated recently for constitutive secretory vesicles in fibroblasts and for
secretory granules in pituitary lactotrophs
(Keller et al., 2001;
Schmoranzer et al., 2000
;
Cochilla et al., 2000
).
Moreover, if the intracellular foci mark minor regulated carriers as
suspected, then assembly of the docking/fusion sites may have occurred as part
of a sorting process when minor regulated carrier vesicles bud from immature
granules. Syntaxin 3 in foci appears more highly concentrated than in granule
membranes, and the selection process may be analogous to sorting events for
SNARE and related proteins at other vesicular budding sites during
intracellular transport (Springer and
Schekman, 1998
; Salem et al.,
1998
; Haucke and De Camilli,
1999
).
To date, several attempts to isolate a population of minor regulated carrier vesicles based on their presumed content of syntaxin 3 and high specific radioacitivity secretory proteins have proven unsuccessful. Thus other approaches may be necessary in order to move the association of syntaxin 3 with the minor regulated pathway beyond the level of correlation.
Fitting the minor regulated pathway and compound exocytosis into a
model of protein secretion by salivary acinar cells
In attempting to define the role of the minor regulated pathway through its
selective stimulation by low concentrations of CCh, we also uncovered what
appears to be an important distinction in the granule exocytotic pathways that
occur in response to ß-adrenergic and cholinergic stimulation. While Iso
readily induced granule exocytosis, progressively involving newly synthesized
granules as defined by pulse-chase labeling
(Fig. 1), CCh induced almost no
granule exocytosis except at a concentration of 2 µM, where discharge was
restricted to old unlabeled granules (Fig.
2). We believe that elevated CCh is stimulating granule fusion
only to the cell surface, where the oldest granules are accumulated. In
contrast, Iso is able to stimulate granule-to-granule fusion and thus involve
newer labeled granules that are located deeper in the apical cytoplasm.
Indeed, micromolar (or smaller) concentrations of CCh generate spatially
restricted calcium signals that are largely confined to the apical cytoplasm
of acinar cells (Straub et al.,
2000; Thorn et al.,
1993
) and might therefore activate exocytosis locally (especially
if promoted by exocytosis of the minor regulated pathway). Further, Iso, which
mainly acts through cAMP-mediated signaling, is well known to stimulate
compound exocytosis extending deep into the acinar cell cytoplasm
(Amsterdam et al., 1969
).
Accordingly, it now seems likely that regulation of granule release in parotid
acinar cells resembles the mechanisms controlling granule release in pituitary
lactotrophs. Here Ca2+ signaling increases the number of plasma
membrane sites involved in granule exocytosis and cAMP contributes (along with
PKC) to amplifying granule-to-granule fusion
(Cochilla et al., 2000
). An
endocrine equivalent of the minor regulated pathway has not been identified,
however.
Taking together the insights that we have gained about the activation of
the minor regulated pathway and the organization of ensuing granule
exocytosis, we have developed a model for regulation of protein secretion in
the acinar cell (Fig. 9). At
low levels of stimulation, either cholinergic
(Fig. 9A) or ß-adrenergic,
the minor regulated pathway is activated rapidly and selectively
(Castle and Castle, 1996) (this
study). This response occurs widely among acinar cells
(Huang et al., 2001
), and in
addition to releasing modest amounts of secretory protein, it delivers granule
docking/fusion sites to the apical cell surface. The minor regulated pathway
may be equivalent to or a substitute for the ready release pool of secretory
granules/vesicles that have been implicated in the immediate secretory
response in acinar cells and other cell types (e.g.
Gillis et al., 1996
;
Stevens and Sullivan, 1998
;
Yoshimura et al., 1998
). At
higher levels of stimulation, the intracellular calcium signal induces
multiple exocytotic events of minor regulated carriers, which in turn
facilitates local granule exocytotic events involving the oldest granules that
are closest to the newly created release sites. This is what occurs at 2 µM
CCh in our experimental system (Fig.
9B) and at higher concentrations of cholinergic agonists used by
others. While low-level ß-adrenergic stimulation mobilizes some of the
granule docking/fusion sites via the minor regulated pathway, higher level
stimulation mainly triggers compound exocytosis from these sites via
cAMP-based signaling and leads to release of newly matured granules
(Fig. 9C). When combined with
low-level cholinergic stimulation, we suggest that the outcome is compound
exocytosis from a larger number of fusion sites and that this represents the
vesicle-mediated component of secretory potentiation
(Fig. 9D).
|
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Acknowledgments |
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