From the Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
Received for publication, January 10, 2001, and in revised form, April 11, 2001
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ABSTRACT |
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Resting secretion of salivary proteins by the
parotid gland is sustained in situ between periods of
eating by parasympathetic stimulation and has been assumed to involve
low level granule exocytosis. By using parotid lobules from ad
libitum fed rats stimulated with low doses of carbachol as an
in vitro analog of resting secretion, we deduce from the
composition of discharged proteins that secretion does not involve
granule exocytosis. Rather, it derives from two other acinar export
routes, the constitutive-like (stimulus-independent) pathway and the
minor regulated pathway, which responds to low doses of cholinergic or
Salivary acinar cells are highly polarized epithelial cells that
are specialized for the secretion of the macromolecular, electrolyte,
and fluid components of saliva. Macromolecular secretion is achieved by
the intracellular transport pathway, culminating in exocytosis of
post-Golgi carriers at the apical plasma membrane. In contrast, export
of electrolytes and fluid is achieved by a transepithelial flux of ions
and passive flow of water mediated by specific membrane-associated
pumps and channels. Secretion is mostly neurally regulated with the
discharge of macromolecules and of electrolytes and fluid being
controlled differentially (1). In the parotid gland, the rate of
macromolecular (mostly protein) secretion is mainly controlled by
sympathetic stimulation through The versatile secretory response of acinar cells enables them to play
two critical physiological roles that are distinguished by the quantity
and quality of secretion. Between periods of eating, an ongoing
"resting" or basal secretion aids in maintaining the homeostasis
(hydration, mineralization, and microbial population) of the oral
cavity, while during eating, an acutely amplified discharge of saliva
facilitates lubrication and initiates digestion of ingested food.
Resting secretion is maintained at least in part by low frequency
parasympathetic stimulation (6). We have been interested in the
pathways and mechanisms of protein secretion used by acinar cells of
the parotid gland as they relate to the differing physiological states
of resting and eating. By using in vitro biosynthetic
labeling of parotid tissue in a pulse-chase protocol coupled with
secretagogue stimulation, we have identified up to four pathways that
export the principal salivary proteins. Two pathways occur without
stimulation, and two others are secretagogue-dependent (1).
The unstimulated pathways are not inhibited by secretory antagonists,
atropine, propranolol, and phentolamine, and thus appear to be truly
secretagogue-independent. Together, they account for 10-15% of the
newly synthesized secretory protein over a 4-6-h time course. The best
characterized unstimulated pathway is the constitutive-like pathway,
which has been observed in various exocrine and endocrine cells and has
been distinguished from the constitutive secretory pathway. It
preferentially exports newly synthesized proteins, beginning soon after
transit through the Golgi, and has been deduced to originate by
vesicular budding that is linked to the maturation of newly formed
secretory granules (11). The secretory composition of the
constitutive-like pathway is enriched in proteins that are
inefficiently sorted by condensation within the cores of maturing
granules (12, 13). A second unstimulated pathway occurring at >3-h
chase times (after the bulk of constitutive-like secretion) exports
proteins having a radiochemical composition resembling the content
stored in mature secretory granules, and it may represent unstimulated
granule exocytosis. Evidently, the magnitude of this type of release,
which has been variable in previous studies (12-14), is of significant
interest with respect to its potential contribution to resting secretion.
Of the two stimulated pathways, the principal one is secretory granule
exocytosis, which we refer to as the major regulated pathway and which
accounts for most secretory protein export. The other is the minor
regulated pathway, which accounts for release of only a few percent of
stored secretory proteins. It is distinguished from granule exocytosis
by preferentially releasing newly synthesized secretory proteins rather
than those that have been in prolonged storage and by its secretory
composition, which closely resembles that of the constitutive-like
pathway. Notably, the minor regulated pathway is much more sensitive to
secretagogue stimulation than is granule exocytosis and is mobilized by
both cholinergic and Since resting secretion in situ is maintained by low level
parasympathetic stimulation, we were interested in whether the minor
regulated pathway serves as its stimulus-responsive component. We now
present evidence that this is the case, and we show that granule
exocytosis contributes to resting secretion only in tissue from fasted
animals where intracellular storage of salivary proteins is near
maximal capacity. In tracing the minor regulated pathway in acinar
cells, we provide experimental support for its common origin with the
constitutive-like pathway by vesicular budding from immature granules
and for its divergence from the constitutive-like pathway at a
brefeldin A-sensitive branch point. Subsequent trafficking of the minor
regulated pathway to the cell surface appears to be direct, albeit
stimulus-controlled, whereas the constitutive-like pathway appears to
be an indirect route involving passage through a distal compartment
postulated to be endosomal.
Materials
Male Sprague-Dawley rats (100-124 g) were obtained from Hilltop
(Scottsdale, PA), maintained 1 week in the vivarium, and were used
either after ad libitum feeding or fasting overnight from 4:00 pm as specified. Expre35S35S used for
biosynthetic labeling of parotid lobules was obtained from PerkinElmer
Life Sciences. Secretagogues, DL-isoproterenol, carbamyl
choline (carbachol; CCh),1
and pilocarpine were obtained from Sigma. Brefeldin A (BFA) was obtained from Epicentre Technologies (Madison, WI), and wortmannin was
obtained from Sigma. Both were dissolved in dimethyl sulfoxide and
stored at Methods
Incubations--
Tissue from one rat was used in each
experiment. Rats were sacrificed by carbon dioxide asphyxiation, and
lobules were dissected from excised parotid glands in chilled
incubation medium (Dulbecco's modified Eagle's medium equilibrated
with 95% O2, 5% CO2). Preincubations (3 times
for 10 min) and incubations including pulse labeling and subsequent
chases (identified below) were performed in stoppered Erlenmeyer flasks
as described previously (14). Although most experiments were conducted
entirely at 37 °C, one set of experiments involved incubation
post-pulse at 19 °C for 2 h to accumulate biosynthetically
labeled proteins in the trans-Golgi network (18) prior to warming to
37 °C for subsequent incubation and experimental analysis (see
"Results"). At designated time intervals, all medium in each sample
(1 ml) was removed and replaced with fresh medium including
secretagogues or perturbants of transport as specified. At the end of
incubation, the tissue in each sample was washed with chilled
phosphate-buffered saline and homogenized. Media and homogenates were
used for assay of amylase activity (19) and for SDS-PAGE (12.5% gels),
Coomassie staining, phosphorimaging, and quantitative analysis of
35S-labeled proteins with ImageQuant software (14, 17).
For biosynthetic labeling, analysis of intracellular transport, and
secretion of cathepsin B (proenzyme and enzyme), incubation with
35S-amino-acids (0.25 mCi/ml) was increased to a 30-min
pulse, and chases were performed for 30-min or hour intervals, as
specified. Immunoprecipitations were performed on aliquots of media and
detergent extracts (1% Nonidet P-40, 0.25% deoxycholate) of tissue
homogenates following addition of protease inhibitors and preclearing
of samples with 1 mg of protein A-Sepharose each. Incubations in a
Triton X-100/deoxycholate/SDS-based RIPA buffer were performed
overnight at 4 °C with anti-cathepsin B immobilized on protein
A-Sepharose. Immunoprecipitates were eluted in Laemmli sample buffer
and were processed for SDS-PAGE and analysis as above.
Morphological Studies--
Immunofluorescent staining of
cryosectioned parotid lobules was performed essentially as described
before (20). Lobules were fixed by the
periodate/lysine/paraformaldehyde procedure (21), frozen in 2.3 M sucrose, 50% polyvinylpyrrolidone (1:1), sectioned (~1
µm) at
Lobules of control and stimulated tissue for morphological analysis by
bright field microscopy and electron microscopy were fixed in
phosphate-buffered 2% formaldehyde, 2% glutaraldehyde, post-fixed in
1% osmium tetroxide, stained en block with 0.5% magnesium uranyl
acetate, dehydrated in acetone, and embedded in Epon resin according to
standard procedures. Thick (0.5 µm) sections were stained with
toluidine blue, and thin sections for EM were mounted on copper grids
and stained with uranyl acetate and lead citrate.
Cannulation of Parotid Ducts and Collection of Stimulated
Secretion in Situ--
Rats were anesthetized with urethane, and
parotid ducts, one per animal, were surgically exposed and cannulated
on the surface of the masseter muscle. Secretagogues freshly dissolved
in sterile PBS were administered by local interstitial injections in
glandular parenchyma, 100 µl at each of two sites per gland.
Stimulations were performed in the order 1 µM carbachol,
5 µM carbachol, 10 µM isoproterenol. The
resulting secretions were collected manually from the cannula into
microcentrifuge tubes, and they were assayed for amylase and analyzed
by SDS-PAGE, Coomassie staining, and densitometry.
Major Secretory Products in the Parotid Are Expressed in All Acinar
Cells--
In analyzing the secretory pathways that are present in
parotid acinar cells, we have focused our attention on some of the major secretory products. In particular, we have examined the intracellular transport and discharge of amylase, the most abundant acinar protein in rat, and two other proteins, an ~38-kDa
proline-rich protein (PRP) and 25-kDa leucine-rich parotid secretory
protein (PSP). As can be seen in the immunofluorescence micrographs
shown in Fig. 1, all three proteins are
expressed in substantial amounts throughout the entire acinar cell
population. Furthermore, none of the proteins accumulates in the acinar
extracellular space under the unstimulated or carbachol-stimulated
conditions used in this
study.2 Consequently,
compositional differences involving these three proteins among
different secretory pathways are almost certainly the consequence of
intracellular sorting events occurring generally in acinar cells and
also do not reflect different cellular origins for distinct secretory
pathways.
"Resting Secretion" by Parotid Lobules in Vitro and by Tissue
in Situ Occurs Almost Entirely by the Constitutive-like and Minor
Regulated Pathways--
More than 20 years ago, it was established in
parotid acinar cells that secretory granules appear to be discharged in
order of their synthesis with older granules being released first (22). Previously, we used biosynthetic labeling and analysis of radioactive secretory products to identify the constitutive-like and minor regulated pathways in parotid and to distinguish them from granule exocytosis as routes that preferentially export newly synthesized proteins (12, 14). Furthermore, we found that the two non-granule pathways exported amylase but almost no PSP, whereas both proteins were
abundant in secretion released by granule exocytosis. (PRP was not
analyzed because it contains no methionine or cysteine and thus was not
labeled by the 35S-amino-acid mixture.) Examples of these
differences in radiochemical composition are illustrated in Fig.
2A. As noted previously (14), the compositional differences are independent of the chase interval selected for observation. Whereas the biosynthetic labeling strategy enabled us to detect and characterize the minor pathways over any
secretion resulting from exocytosis of unlabeled granules, we did not
previously address their relative contributions as putative sources of
resting secretion. Consequently, we have now examined the composition
of total secretory proteins released from parotid lobules in the
absence of stimulation and in the presence of low dose carbachol
stimulation and compared it with the composition released by high dose
isoproterenol stimulation and with the content of purified secretory
granules. As shown in Fig. 2B, the Coomassie staining
profiles for unstimulated secretion and carbachol-stimulated secretion
are highly similar to each other but differ from those of
isoproterenol-induced secretion and granule content, which are
identical to each other. Just as observed for the radiolabeled
secretory profiles (Fig. 2A), PSP is nearly absent from the
protein staining profiles of unstimulated and carbachol-stimulated
secretions, although it is prominent in granule content and
isoproterenol-stimulated
secretion.3 In addition, PRP
(which is not labeled by 35S in Fig. 2A) is
poorly represented in unstimulated and carbachol-stimulated secretions
even though it is prominent in stored and released granule contents
(Fig. 2B). Thus the composition of resting secretion is
distinct from that of secretory granule content, indicating that
unstimulated exocytosis of granules contributes negligibly to the
output of salivary proteins by lobules of ad libitum fed animals.
To document that the secretion observed in vitro reflects
what occurs in situ, we injected low concentrations of
carbachol and subsequently a high concentration of isoproterenol into
the glandular interstitium of an anesthetized rat and collected
secretion from the cannulated parotid duct. Local application of a
cholinergic agonist avoids significant activation of adrenal
catecholamine secretion and consequent adrenergic stimulation of
parotid tissue as can occur following systemic administration (3). As
can be seen in Fig. 2C, the composition of secreted proteins
following low doses of carbachol closely resembles that obtained
in vitro with unstimulated and carbachol-stimulated lobules,
implying that resting secretion is derived from the constitutive-like
and minor regulated pathways and that the pathways are apically directed.
Unstimulated Exocytosis of Secretory Granules Contributes to
Resting Secretion by Parotid Acinar Cells of Fasted Animals but Is Not
a Dominant Pathway--
Rats are nocturnal feeders, and the total
amylase content in parotid typically decreases by ~40% overnight as
a result of secretion (23). In initiating morning experiments using
parotid tissue from ad libitum fed animals, we are examining
the secretory pathways in acinar cells that have a complement of
storage granules that is below capacity. Although our experimental
conditions satisfactorily approximate the physiological state, we were
interested to examine whether overnight fasting, which should maximize
the cellular storage pool of granules, affects the composition of
resting secretion and the pathways that contribute to it. Thus, with
tissue from fasted animals, we carried out biosynthetic labeling using
a pulse-chase protocol, and we analyzed the radiochemical and total
protein compositions of unstimulated secretion and secretion stimulated by 40 nM carbachol and by 10 µM
isoproterenol. As shown in Fig. 2D, the radiochemical
composition released by the constitutive-like and minor regulated
pathways is similar to that observed with tissue from ad
libitum fed animals. Secretion of labeled PSP is underrepresented
as compared with the level observed upon stimulating granule exocytosis
with isoproterenol. However, the spectrum of total proteins secreted,
as revealed by Coomassie staining, now includes increased amounts of
PRP and PSP, suggesting participation of granule exocytosis (Fig.
2E). To distinguish whether the increased amount of PSP
represents newly synthesized or older protein, we compared the specific
radioactivities of amylase and PSP (normalized to Coomassie staining)
in secretions following 40 nM carbachol and 10 µM isoproterenol addition. Whereas the specific
radioactivity of amylase was 6-fold higher after carbachol than after
isoproterenol, the specific radioactivity of PSP was lower (by
1.2-fold) after carbachol than after isoproterenol. Thus basal
exocytosis of older (unlabeled) granules becomes a contributing pathway
to resting secretion along with the other two pathways in acinar cells
that are replete with granules.
Fig. 2F summarizes the output of newly synthesized and total
amylase in the absence and presence of stimulation in a typical experiment. The data illustrate that the pathways contributing to
resting secretion account for only small fractions of newly synthesized
and stored amylase, whereas stimulated granule exocytosis releases much
larger fractions. It is also apparent that the fractional output of
amylase in resting secretion is increased during fasting.
Stimulation of the Minor Regulated Pathway Correlates with
Expansion of Apical Luminal Profiles Occurring Rapidly in a Large
Fraction of Acinar Cells--
In an attempt to visualize morphological
changes in response to low doses of carbachol and to assess whether the
minor regulated pathway is activated broadly among acinar cells, we
incubated lobules for brief times in the presence and absence of 40 nM carbachol and then chilled, fixed, and processed the
samples for light and electron microscopy. Fig.
3, A and B, shows
light micrographs of toluidine blue-stained sections that have been
printed in reverse contrast to enhance visualization and comparison of
extracellular spaces bordering acinar cells, and particularly the
apical lumina. In controls, the lumina appear mostly as small dark
spots bordered by the light adjoined acinar cells. Following 1 min and
especially 3 min of stimulation, a substantial fraction of the luminal
spaces within individual lobules has expanded, implicating structural reorganization at the apical surface and possible secretion.
Stereological measurements of the surface area of >1500 luminal
profiles from multiple acini of unstimulated specimens and >2400
profiles from stimulated specimens made using the Openlab program
(Improvision Inc.) indicated that the mean luminal diameter increased
1.5-2-fold by 3 min of stimulation with at least a 25% shift in the
distribution of total profiles to larger dimensions. Not only is the
response observed broadly in the acinar tissue, but also it occurs
quite rapidly and correlates well with the rapid export of secretory products observed in our earlier study (Fig. 2 in Ref. 14). Examination
by EM (Fig. 3, C and D) indicated that luminal
expansion decreases the incidence of microvilli protruding into the
lumen and brings the apical surface into closer proximity to the
stored granule population. These observations suggest that the
actin-rich terminal web may be reorganized in a way that is conducive
to granule exocytosis, although there is no evidence suggesting that low dose carbachol treatment causes granule exocytosis.
The Minor Regulated and Constitutive-like Pathways Form in
Parallel, but Their Outputs Have Different Specific
Radioactivities--
We showed previously that the minor regulated
pathway could be stimulated to export pulse-labeled salivary proteins
of high specific radioactivity at prolonged chase times, well after
most of the constitutive-like secretion of the labeled proteins had occurred (14). This distinguished the minor regulated pathway as a
separate secretagogue-responsive storage compartment and discounted the
possibility that stimulation was accelerating the constitutive-like
pathway. Although the two pathways are distinct, they draw on the same
pool of secretory proteins during formation as judged by comparing the
radiochemical compositions at equal specific radioactivity of amylase
(Fig. 2). Thus we have been interested in defining the divergence of
the pathways and clarifying their separate routes to the cell surface.
To begin, we have compared the formation of the two types of carriers
by following the time dependence of specific radioactivity of amylase
in unstimulated secretion and in secretion elicited by 40 nM carbachol in a pulse-chase experiment. Fig.
4A shows a sample time course
of specific radioactivity for amylase released without stimulation or
with single intervals of carbachol stimulation. Fig. 4B
compares the specific radioactivity of amylase secreted within single
intervals of stimulation when carbachol is administered at different
times of chase with that secreted in the absence of stimulation. We
performed these studies on parotid lobules from both ad
libitum-fed and fasted rats and obtained the same results with
both types of tissue. As can be seen, the specific radioactivity of
amylase rises and falls during the chase concomitantly in both
unstimulated and stimulated secretions, demonstrating that carriers of
both pathways are continually formed in parallel. Strikingly, however,
the specific radioactivity of amylase released by the minor regulated
pathway is Brefeldin A Inhibits Formation of the Minor Regulated Pathway and
Amplifies the Export of Secretory Proteins in the Constitutive-like
Pathway--
Formation of many kinds of vesicular carriers including
several involved in post-Golgi transport is inhibited by BFA
(e.g. Refs. 18, 24, and 25), which blocks GDP/GTP exchange
on certain ADP-ribosylation factor family GTPases (26). We were interested to compare the sensitivities of the constitutive-like and
minor regulated pathways to BFA. Accordingly, we used our standard
pulse labeling protocol and added BFA during the chase for time
intervals beginning at 80 min or later. Earlier times of addition were
not used in initial experiments in order to minimize the well known
effects of BFA's inhibition of more proximal (endoplasmic reticulum-Golgi) intracellular transport steps. When 10 µg/ml BFA was
added at 80 min, it inhibited the subsequent output of radiolabeled
proteins in response to 40 nM carbachol by ~80%. In
contrast, when BFA was added at 160 min (after most carriers containing
labeled proteins had formed) and followed by carbachol stimulation,
secretion was decreased by only ~40% (Fig.
5). Although BFA blocked the output of
labeled protein, it caused at most a small inhibition of
carbachol-stimulated output of amylase enzyme activity (data not
shown). Thus the results indicate that BFA inhibits formation of
carriers of the minor regulated pathway but not their discharge.
Although BFA inhibited formation of the minor regulated pathway, it
enhanced unstimulated secretion as reflected in the discharge of
amylase detected enzymatically (Fig. 6A) and radiochemically.
Notably, the specific radioactivity of amylase appearing in
unstimulated secretion increased ~3-fold following BFA addition at 80 min of chase (Fig. 6B). The radiochemical composition of the
enhanced secretion immediately following BFA was not noticeably changed
from before drug addition (Fig. 6C). Two alternative
explanations for this result come to mind. First, inhibition of
formation of minor regulated carriers leads to a compensatory amplified
export of the newly synthesized proteins in the constitutive-like
pathway. As these proteins have a high specific radioactivity, they
would increase the specific radioactivity of constitutive-like
secretion, which is much lower (Fig. 4). Second, BFA may itself cause
exocytosis of minor regulated secretory carriers thereby adding a
separate high specific radioactivity component to "unstimulated"
output. To distinguish between these possibilities, we examined the
effect of BFA addition at increasing chase times beyond 80 min.
According to the first alternative, the increment of specific
radioactivity of amylase in the medium following BFA addition at
increasing times should decrease in parallel with the normal decrease
in specific radioactivity observed in constitutive-like secretion. In
contrast, the second alternative predicts that the BFA-induced increase
in specific radioactivity of amylase in the medium at increasing times
of drug addition should decrease only slightly (
The observation that BFA enhanced output in the constitutive-like
pathway was quite interesting on two accounts. First, it seemed to
contrast with previous observations suggesting that formation of
constitutive-like secretory carriers from immature granules is blocked
by BFA (e.g. Ref. 24). Second, it appeared potentially
analogous to the recently reported enhanced constitutive-like secretion
of procathepsin B in pancreatic Wortmannin Has No Effect on the Minor Regulated Pathway but Extends
the Duration of Constitutive-like Secretion of Newly Synthesized
Proteins Over the Same Period--
The phosphatidylinositol
3-kinase inhibitor wortmannin at concentrations The Constitutive-like and Minor Regulated Pathways Are Not
Significant Export Routes for Lysosomal Hydrolases or Their
Precursors--
Lysosomal hydrolase precursors are useful markers for
monitoring traffic from immature granules to endosomes (18, 31-33). By
having established that the constitutive-like and minor regulated pathways are distinct yet collaborate to form resting secretion, we
were interested to evaluate whether the trafficking of a prohydrolase under normal conditions or upon addition of wortmannin might aid in
further understanding the interrelationship of the two pathways. Also,
regulated exocytosis of mature lysosomal hydrolases has been reported
in various cell types (34, 35), raising the possibility that there
might be a relationship of this process to the operation of the minor
regulated pathway. In order to address these issues, we have used
extended (30 min) pulse labeling and immunoprecipitation of
(pro)cathepsin B from the media and detergent extracts of homogenized
lobules. Initially, we confirmed that procathepsin B enters and exits
immature granules (32) by showing that 10 µM
isoproterenol added between 0-1 and 1-2 h of chase, respectively,
stimulated secretion of about half and about a quarter of the total
labeled pro- and mature cathepsin B (Fig.
7). About 70% of total amylase is
discharged concomitantly from each sample. The bulk (87%) of
intracellular procathepsin B is processed to the mature ~30-kDa form
by 60 min of chase (data not shown), signifying transport to late
endosomes/lysosomes. Very little precursor and no mature enzyme were
detected in the medium of unstimulated samples during either chase
interval, and <0.3% of total antigen was detected in the medium
following stimulation of the minor regulated pathway with 40 nM carbachol between 1.5 and 2 h (Fig. 7). Thus the
constitutive-like and minor regulated pathways do not normally serve as
significant export routes for lysosomal hydrolases in parotid acinar
cells.
Inclusion of 100 nM wortmannin in chase media immediately
post-pulse did not result in increased unstimulated secretion of procathepsin B or in significant detection of procathepsin B in the
minor regulated pathway. Also, wortmannin did not perceptibly increase
the level of procathepsin B released by granule exocytosis in response
to isoproterenol (data not shown). In these latter respects, our
results differ from those reported for pancreatic Minimal Contribution of Granule Exocytosis to Resting
Secretion--
In the present studies, we have made a compelling case
through examination of the total composition of parotid secretory
proteins that secretion under resting (basal) conditions and under
conditions of acute strong stimulation is supported by different
intracellular pathways. Surprisingly, we have been able to deduce that
granule exocytosis, which customarily has been equated with most forms of regulated exocrine secretion of proteins, is largely reserved for
the latter response where massive quantities of protein are needed to
efficiently process ingested food. In contrast, the minor regulated
secretory pathway, which is a derivative of the maturing secretory
granule, normally handles the need for regulated secretion under
resting conditions. Secretory granule exocytosis makes a contribution
to resting secretion only when acinar cells approach full-capacity
storage (during fasting), and even then the contribution is modest
(Fig. 2). The selective mobilization of a smaller capacity and more
sensitive-responding pathway to support resting secretion represents a
clever and economic specialization.
Previously, we deduced that the minor regulated pathway was capable of
releasing as much as 10% of newly synthesized amylase and thus
concluded that the pathway is broadly derived from the acinar cell
population (14). In the present study, we have shown that low dose
carbachol stimulation visibly affects the organization and dimensions
of the apical surfaces of a sizable fraction of the acinar cell
population (Fig. 3) and that these changes correlate with production of
an apically directed secretion in situ (Fig. 2C).
Although a morphological response to stimulation is not evident in all
cells, we suspect that the breadth of the effect may be limited by the
dose of stimulant. Indeed, our previous results show that the magnitude
of the minor regulated secretory response increases with secretagogue
dose (Fig. 2 in Ref. 14), suggesting that acinar cells may be
differentially sensitive to stimulation. Physiological resting
secretion may normally involve only a fraction of cells responding to
low dose parasympathetic stimulation at any given time. Differential
sensitivity to secretory stimulation within populations of regulated
secretory cells appears to be a general property of exocrine and
endocrine glands (36-39). Notably, whereas only a portion of
total acinar cells may supply resting secretion at any point in time,
the formation of new carriers probably proceeds continuously in all
cells since filling and discharge of the minor regulated pathway seem
to be indifferent to the fasting/feeding state of the animal (Figs. 2
and 4).
Intracellular Organization of Minor Regulated and Constitutive-like
Pathways--
Our findings regarding the intracellular relationships
of the minor regulated and constitutive-like pathways have led to the model shown in Fig. 8. Both pathways
originate by a common step of vesicular budding that is linked to
maturation of secretory granules and is shown to derive from both
condensing vacuoles and immature granules. The two pathways overlap at
this level with the route followed by lysosomal prohydrolases. They
diverge subsequently by formation of minor regulated carrier vesicles, which are maintained as a storage pool until they are induced to
undergo exocytosis at the apical surface. After the branch point,
constitutive-like carriers proceed to a junction with endosomes. Here
the lysosomal prohydrolase pathway diverges, and the proteins destined
for constitutive-like secretion are delivered to the cell surface via
recycling endosomes. Notably, the separation from the prohydrolase
pathway appears especially efficient in the acinar cell as
compared with the pancreatic
Formation of the two pathways of resting secretion together is
supported by the observations that the relative radiochemical compositions of the major salivary proteins in both pathways are the
same (Fig. 2) and that initial detection of radiolabeled proteins in
unstimulated and carbachol-stimulated secretions occurs concurrently (Fig. 4). Divergence of the two pathways distal to the common origin is
supported by at least three findings. First, we showed previously that
the minor regulated pathway alone behaves as a storage compartment from
which biosynthetically labeled proteins could be discharged long after
the bulk of unstimulated (constitutive-like) secretion of labeled
proteins had occurred (14). Second, we have observed a striking
difference in the specific radioactivity of amylase released by the two
pathways, with that of the minor regulated pathway being substantially
higher than that of the constitutive-like pathway (Fig. 4). Third, the
two pathways are distinguished by their differential sensitivity to
BFA, particularly when it is applied at chase times that implicate an
effect distal to immature granule exit (Figs. 5 and 6).
Whereas passage of the constitutive-like pathway through endosomes has
been analyzed previously by following trafficking of lysosomal
prohydrolases in pancreatic
The use of BFA has identified two levels where the pathways comprising
resting secretion are perturbed (Fig. 8) and has been key in placing
the branch point of the constitutive-like and minor regulated pathways
in the overall transport process (Figs. 5 and 6). We were able to block
production of the characteristic constitutive-like secretion,
presumably by inhibiting vesicular budding from condensing vacuoles and
immature granules, if we added BFA while biosynthetically labeled
proteins were accumulated in the TGN. Under these conditions, the
radiochemical composition of the ensuing secretion (Fig. 6G) suggested that sorting had been preempted, whereas the reduced specific
radioactivity of amylase (Fig. 6F) probably reflected extracellular dilution by older unlabeled enzyme, whose export is
facilitated by the later effect of BFA (Fig. 6, A and
E). Blockade of constitutive-like secretion at its origin
agrees with previous observations (24, 25), and the secretion of newly
synthesized proteins that occurs under these conditions is likely to
derive proximally, possibly from components of the TGN vesiculated by BFA (41, 42). If we added BFA later during chase, either with or
without prior accumulation of labeled secretory proteins in the TGN, we
found that the drug had the opposite effects of increasing the specific
radioactivity of exported amylase and preserving the composition
characteristic of constitutive-like secretion. Moreover, these effects
were linked in a compensatory relationship to inhibition of formation
of the minor regulated pathway. Simultaneously, the results identified
the second level of action of BFA on resting secretion and signified
the presence of the branch point of its two pathways. Interestingly,
this compensatory relationship where constitutive-like secretion is
increased when the minor pathway is inhibited is the reverse of one
noted previously where discharge of the minor regulated pathway
transiently reduced constitutive-like secretion of labeled amylase
during replenishment of the minor regulated carriers (14).
In contrast to BFA, wortmannin did not have major effects on the
constitutive-like and minor regulated pathways over the same time
course. The minor effects observed late during chase appear to be
secondary to the formation of both pathways and may be related to well
known perturbations within the endosomal system (15). The lack of
perturbation of protein exit from immature granules by wortmannin in
salivary acinar cells appears distinct from the effect observed in
insulin-secreting cells (18). However, we presently cannot fully rule
out the possibility that decreased drug access to the transport
machinery in acinar cells of parotid lobules might contribute to the difference.
Potentially Related Pathways and Functions in Other Cell
Types--
The preferential export of newly synthesized secretory
proteins and the enhanced secretagogue sensitivity characterizing the minor regulated pathway of parotid acinar cells are reminiscent of
various earlier studies in endocrine cells, for example pancreatic
Although we have no information about the function of the minor
regulated pathway in the trafficking of membrane components, we believe
that there are several potentially interesting avenues to be explored
here. Exocrine granule maturation entails progressive reduction in the
surface density of intramembranous particles as viewed by freeze
fracture (47), suggesting that vesicles emanating from maturing
granules may engage in selective membrane protein removal. SNARE and
associated proteins have been implicated in nucleating membrane protein
sorting during vesicle formation (48-50), and one or more SNAREs that
eventually support exocytosis of vesicles derived from immature
granules may also function in forming the vesicles. A recent study
identified a BFA-sensitive remodeling process involving
vesicle-associated membrane protein-4 and synaptotagmin-4 removal from
maturing neuroendocrine granules that appears to enhance stimulus
responsiveness of the granules (51). Although removal of a negative
regulator of granule exocytosis, synaptotagmin-4, was emphasized, there
is likely to be an accompanying role of the exiting vesicles as
secretory carriers. We are intrigued by the possibility that the
vesicles are related to constitutive-like and/or minor regulated
carriers in acinar cells.
We anticipate that other membrane proteins may be concentrated in minor
regulated carriers as well. In particular, the membrane machinery
regulating fluid and electrolyte secretion may be included in the
vesicles, especially as the minor regulated pathway is stimulated by
the same secretagogues that cause fluid and electrolyte secretion and
is mobilized by levels of stimulation that are just sufficient to
initiate salivary flow. As acinar cells are epithelial, it is possible
that the minor regulated pathway may be related to apical
carriers/endosomes, which undergo increased export to the apical
surface in response to cell signaling (52-57). The identification of
markers and the isolation of minor regulated carriers should greatly
enhance our ability to explore these relationships and to address
possible general secretory and transport functions.
-adrenergic agonists (Castle, J. D., and Castle, A. M. (1996) J. Cell Sci. 109, 2591-2599). The protein
composition collected in vitro mimics that collected from
cannulated ducts of glands given low level stimulation in situ. Analysis of secretory trafficking along the two pathways of
resting secretion has indicated that the constitutive-like pathway may
pass through endosomes after diverging from the minor regulated pathway
at a brefeldin A-sensitive branch point. The branch point is deduced to
be distal to a common vesicular budding event by which both pathways
originate from immature granules. Detectable perturbation of neither
pathway in lobules was observed by wortmannin addition, and neither
serves as a significant export route for lysosomal procathepsin B. These findings show that parotid acinar cells use low capacity, high
sensitivity secretory pathways for resting secretion and reserve
granule exocytosis, a high capacity, low sensitivity pathway, for
massive salivary protein export during meals. An analogous strategy may
be employed in other secretory cell types.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic receptors. Rates of
fluid and electrolyte secretion are mainly controlled by
parasympathetic stimulation through muscarinic acetylcholine receptors
and, to a lesser extent, sympathetic stimulation through
-adrenergic
receptors (see Refs. 2-4 and reviewed in Refs. 5 and 6). Although
these are the primary divisions in secretory signaling, they are not
absolute. Indeed, the dilute secretion elicited by parasympathetic
stimulation in situ (or muscarinic cholinergic agonists
in vitro) contains a protein content, whereas the
protein-rich, low volume secretion produced by
-adrenergic agonists
contains fluid and electrolytes. Furthermore, parasympathetically and
sympathetically controlled signaling pathways are synergistic in
enhancing the rates of protein export and fluid and electrolyte
transport (4, 7-10). Thus alone and in combination, the signaling
pathways enable acinar cells to vary the amount and composition of
saliva over a wide range.
-adrenergic stimuli (14).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. The activity of the specific batch of wortmannin used was confirmed by demonstrating its ability to elicit endosomal vesiculation in HeLa cells (15). Antibodies against amylase and
proline-rich protein used in the present studies have been described
previously (16, 17). A rabbit antibody against parotid secretory
protein was generously provided by Dr. William Ball (Howard
University). Rabbit antibody against rat procathepsin B, which
recognizes both precursor and mature forms of the enzyme, was from
Upstate Biotechnologies, Inc. (Lake Placid, NY).
50 °C, and mounted on gelatin-coated slides. The sections
were blocked in 5% goat serum, 0.05% Triton X-100, and
phosphate-buffered saline; suitable dilutions of primary and secondary
(Alexa 594-conjugated) antibodies (Molecular Probes, Inc., Eugene, OR)
were made in 0.5% goat serum, 0.05% Triton X-100, and
phosphate-buffered saline. Stained sections were mounted and examined
by fluorescence microscopy.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Immunofluorescence micrographs of amylase,
PRP, and PSP. Cryosections of fixed rat parotid were stained with
antibodies against amylase, PRP, and PSP followed by Alexa-594
conjugated secondary antibody. Corresponding differential interference
contrast images are shown in bottom panel, and ducts, where
the acinar proteins not produced, are labeled D. Bar, 20 µm.
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Fig. 2.
Composition of parotid secretory proteins
collected under different conditions. A and
B, proteins released in vitro by parotid lobules
of ad libitum fed rats that have been incubated without
stimulation (Ctl), with 40 nM carbachol
(CCh), or with 10 µM isoproterenol
(Iso). Amylase (Amy), proline-rich protein
(PRP), and parotid secretory protein (PSP) are
identified in A and B. A, radiochemical profile
following pulse labeling with Expre35S35S and
chase incubation. The samples have been normalized to load equal
radioactivity (band density) of amylase in order to emphasize the
relative compositional differences of other proteins, particularly PSP,
in the different secretions. B, composition of total
secreted protein as viewed by Coomassie staining. The composition of
the content of isolated secretory granules (Gran) is also
shown. C, Coomassie staining of total salivary proteins
collected in situ by cannulation of the parotid duct.
Stimulation was by sequential interstitial injection of 1 and 5 µM carbachol and 10 µM isoproterenol. The
results shown are representative of three separate cannulation
experiments. D and E, radiochemical and Coomassie
staining profiles for unstimulated, carbachol-stimulated, and
isoproterenol-stimulated secretions released in vitro by
parotid lobules from rats fasted overnight. The Coomassie profile for
granules purified from the glands of fasted animals is shown in
E for comparison. The 22-kDa protein observed at
variable levels in A-E is common salivary protein, a
product mainly of intercalated duct cells (58). It has not been
considered in the present analysis of acinar products. F,
summary of secretion of newly synthesized amylase
(35S) and amylase enzyme (Enz.
Act.) during a 30-min interval expressed as fractions of total
(tissue + all media). Ctl values for 35S-amylase
are taken from the peak (90-120 min) of unstimulated secretion (14);
CCh values reflect stimulated release between 150 and 180 min;
isoproterenol values reflect stimulated release between 240 and 270 min.
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Fig. 3.
Widespread rapid morphological changes at the
apical surfaces of parotid acinar cells induced by low dose stimulation
with carbachol. Lobules incubated at 37 °C in vitro
with or without brief stimulation using 40 nM carbachol
were chilled, fixed in aldehydes, and processed for embedding and
sectioning for light and electron microscopy. A and
B, light micrographs of unstimulated and 3-min
carbachol-stimulated tissue, respectively. Sections were stained with
toluidine blue, photographed, and imaged in reverse contrast to
highlight extracellular, particularly luminal, spaces, which appear as
darkened foci between acinar cells (arrows). C
and D, electron micrographs illustrating examples of luminal
profiles from unstimulated and stimulated (3 min) tissue showing that
the expansion of the luminal space upon stimulation reflects
reorganization of the apical surface involving reduction of microvilli
and closer proximity of the surface to secretory granules. The lumen
(L) and an example of a secretory granule (G) are
identified.
7-fold higher than that released by the constitutive-like
pathway throughout the time course. This substantial difference
suggests that the newly sorted proteins in the constitutive-like
pathway undergo a dilution into a pool of unlabeled proteins en route
to discharge, whereas the newly sorted proteins of the minor regulated
pathway do not. Notably, any secretion of unlabeled amylase, for
example by low level granule exocytosis in tissue from fasted animals, has only a minor effect on the specific radioactivities and does not
alter the relative specific radioactivities for stimulated and
unstimulated pathways. Thus the difference in specific radioactivity between the constitutive-like and minor regulated pathways is created
intracellularly.
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Fig. 4.
The minor regulated and constitutive-like
secretory pathways form in parallel, but secretions released by the two
pathways have different specific radioactivities. Parotid lobules
from ad libitum fed or fasted animals were pulse-labeled
with 35S-amino-acids and divided into separate samples for
chase incubation with medium changes at 30-min intervals. At specified
time points, individual samples were stimulated for 30 min with 40 nM carbachol to elicit secretion by the minor regulated
pathway. Secretions from all samples and all time points were assayed
for amylase enzyme activity and subjected to SDS-PAGE, and radioactive
amylase was quantitated by phosphorimaging. A, examples of
specific radioactivity profiles of amylase for unstimulated lobules
(open circles) and lobules stimulated for a single 30-min
interval beginning at 90 (open triangles) and 210 min
(open inverted triangles). B, plots summarizing
the results of experiments conducted as in A from ad
libitum fed (squares) and fasted (circles)
animals. The open symbols show the specific radioactivity of
amylase in unstimulated secretion. The filled symbols
correspond to the net specific radioactivity of amylase in
CCh-stimulated secretion. Each data point derives from a separate
sample where carbachol was applied at a single time interval as shown.
Net specific radioactivity of amylase was calculated after correcting
the radioactivity and enzyme activity of amylase for the contributions
from unstimulated secretion. The data are representative of at least
six experiments using tissue from ad libitum fed animals and
three experiments using tissue from fasted animals.
30%; Fig. 4)
reflecting secretion from the high specific radioactivity compartment.
As can be seen in Fig. 6D, the increase in specific
radioactivity of secreted amylase following BFA addition progressively
declines and is more than 10-fold lower by 320 min. Thus the first
alternative applies, and BFA blockade appears to divert newly
synthesized protein to the constitutive-like pathway.
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Fig. 5.
Inhibition of formation of the minor
regulated pathway by BFA. BFA was added to pulse-labeled tissue at
either 80 or 160 min chase at 37 °C and maintained until 240 min at
which time the minor regulated pathway was stimulated with 40 nM carbachol. The results are normalized to a control in
which BFA was not added but stimulation was performed at 240 min. The
results shown are from three experiments (mean ± S.E.) for BFA
added at 80 min and two experiments (mean; vertical line is
range) for BFA added at 160 min.
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Fig. 6.
The effects of BFA on unstimulated
secretion. A-C show results of incubation entirely at
37 °C. Parotid lobules were pulse-labeled and chased, and media were
replaced and analyzed every 40 min. BFA (open circles) or
Me2SO (closed circles) was added at 80 min post-pulse and maintained throughout. A, amylase
activity was assayed, and unstimulated secretion of amylase was plotted
as a function of time. B, specific radioactivity of secreted
amylase with time evaluated as described under "Experimental
Procedures." C, profiles of radiolabeled proteins secreted
before (40 and 80 min) and after BFA addition (120 min). The results in
A-C are representative of three separate experiments.
D, effect of BFA addition at increasing chase times on the
specific radioactivity of amylase in the ensuing 40-min interval of
unstimulated secretion. Results are normalized to the increment in
specific radioactivity observed in the interval beginning at 80 min.
E-G show results where lobules were incubated for 2 h
at 19 °C following pulse labeling and subsequently warmed to
37 °C for the rest of incubation. Me2SO (filled
circles) or BFA was added at the time of warming (120 min,
filled triangles) or at 25 min after warming (145 min, open circles) and maintained throughout. Media were
replaced every 30 min. E, amylase enzyme activity;
F, amylase-specific radioactivity. Data from two identical
experiments were normalized to the maximal values obtained with BFA
added 25 min post warm-up, and means are plotted. Vertical
bars identify ranges that differ by more than the width of the
symbol. G, profiles of radiolabeled secretory proteins
detected in secretions at 180 min for untreated lobules
(Ctl), lobules where BFA was added at 120 min
(warm-up), and lobules where BFA was added at 145 min
(after warm-up). For untreated cells, the profile is quite
similar to the control in C except that the level of labeled
PSP is slightly increased as a consequence of imposing the 19 °C
block. Positions of amylase (Amy) and PSP are
indicated.
-cells in response to BFA action at
a site distal to exit from the immature granule (18). Consequently, we
wondered whether there might be two sites of BFA blockade, one at exit
and one beyond exit from immature granules, and that drug addition at
80 min post-pulse might have missed the first one. Thus in an effort to
evaluate how BFA action on early post-Golgi events might affect the
pathways of resting secretion, we incubated parotid lobules post-pulse
for 2 h at 19 °C to accumulate labeled proteins in the TGN
(18). In parallel samples, we either made no addition or added BFA
either at the same time or 25 min after raising incubation temperature
to 37 °C. The results are shown in Fig. 6,
E-G.4 Warming the
tissue promptly increased the release of amylase enzyme activity to
~1% of total per 30 min in the control (the level observed in our
other experiments), and BFA at both times of addition elevated
unstimulated release above control (Fig. 6E). The effects of
BFA on the specific radioactivity of amylase and on the radiochemical
composition of secreted proteins, however, depended on when the drug
was added (Fig. 6, F and G). When added at the
time of warm-up, BFA inhibited the rise in amylase-specific radioactivity. Although the output of radioactive proteins was reduced,
the composition of the secretion also changed such that it was enriched
in labeled PSP along with other acinar proteins as if imposing early
blockade had resulted in low level discharge of unsorted proteins. When
BFA was added after warm-up, the specific radioactivity of secreted
amylase immediately increased (as in the experiments conducted entirely
at 37 °C), and the radiochemical composition of the amplified
secretion was comparable to that typically observed in the
constitutive-like and minor regulated pathways of untreated tissue.
Quantitation of the PSP/amylase band density ratio of the 180-min
samples from two separate experiments yielded a ratio of 0.01-0.03
(range) for control and for BFA added 25 min after warm-up and
0.10-0.15 (range) for BFA added at warm-up. These outcomes document
decreased sorting as an indirect consequence of inhibiting vesicular
budding by early BFA addition. Taken together, the observations
indicate that BFA imposes two blocks, an early one that impairs the
constitutive-like pathway and a later one that inhibits formation of
minor regulated carriers distal to exit from immature granules where
sorting occurs.
100 nM
perturbs a variety of post-Golgi trafficking pathways with little or no
effects on constitutive and regulated secretion (15, 18, 27-30).
Because wortmannin has been reported to interfere with lysosomal
prohydrolase trafficking from immature secretory granules to endosomes
in endocrine cells (18), we were interested to test whether it affected
the exocrine constitutive-like and minor regulated secretory pathways.
We applied it immediately after pulse labeling parotid lobules and
monitored its effects on unstimulated and carbachol-stimulated
secretion during subsequent chase incubation. In three separate
experiments, we observed that wortmannin did not affect delivery to
either the constitutive-like pathway or minor regulated pathway, the
latter tested by stimulation at 80 and 160 min. Newly synthesized
amylase appeared in the medium from both pathways of drug-treated
tissue at the same rates as in control tissue, and the radiochemical
composition of the secretions was not altered (data not shown). At
prolonged times (>160 min chase), however, wortmannin had two minor
effects. It maintained the unstimulated release of labeled protein
above control levels without increasing the overall level of
unstimulated (amylase enzyme) secretion, and it subtly reduced
carbachol-stimulated discharge of amylase activity without altering
secretory composition. Isoproterenol-induced discharge of labeled
proteins by granule exocytosis was not altered by wortmannin (data not shown).
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Fig. 7.
The constitutive-like and minor regulated
pathways are not significant export routes for lysosomal hydrolase
precursors (or enzymes). Lobules pulse-labeled 30 min were chased
and stimulated (or not) with 10 µM isoproterenol
(Iso) between 0-1 and 1-2 h or with 40 nM
carbachol (CCh) between 1.5 and 2 h of chase. Samples
of solubilized cell lysates and medium were immunoprecipitated with
anticathepsin B antibody under conditions that quantitatively
immunoprecipitate procathepsin B/cathepsin B. The fluorographs of cell
samples identify the mobilities of procathepsin B (ProB) and
mature cathepsin B (B) taken from samples at 0 and 2.5 h of chase, respectively. For secretions, isoproterenol stimulates
export of 48% of total labeled procathepsin B/cathepsin B during the
first time interval and 22% during the second time interval. The *
identifies a secretory contaminant migrating slightly slower than
procathepsin B in unstimulated secretion. CCh stimulated export of only
a small amount (<0.3% of total) of procathepsin B. Lanes showing
samples with and without CCh stimulation were immunoprecipitated from
three times the amount of medium used for the isoproterenol
sample.
-cells (18). We
did not conduct analogous studies to explore prospective perturbations
of procathepsin B trafficking by BFA because we felt that the
inhibitory effect of BFA on formation of the minor regulated pathway
(Fig. 5) would preclude gaining further insight about how the pathways
are interrelated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell (Fig. 7, (18)), possibly
reflecting enhanced membrane (receptor) association of procathepsin B
at the elevated pH characterizing apical endosomes (40).
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Fig. 8.
Current model of the organization of pathways
comprising parotid resting secretion showing common exit from immature
secretory granules (IG) and condensing vacuoles
(CV) of the constitutive-like (CL)
and minor regulated (MR) pathways, divergence at a
distal site, and junction of the constitutive-like pathway with the
endosomal system. Endocytosis from the apical lumen is thought to
be responsible for dilution of newly synthesized proteins within the
constitutive-like pathway. A putative pathway leading from endosomes
(E) to lysosomes (L) is also shown. Deduced
BFA-sensitive sites are identified. The model is discussed further in
the text.
-cells (18, 32, 33), we have been
motivated to propose this route in acinar cells to explain the
difference in specific radioactivities of secretory proteins released
by the constitutive-like and minor regulated pathways. Accordingly, we
view endosomes as the probable site distal to branching of the minor
regulated pathway where proteins traveling in the constitutive-like
pathway are diluted into a pool of unlabeled (or less radioactive)
proteins. It seems quite likely that the steady state level of
secretory protein present within acinar endosomes is significant as a
consequence of continual internalization by endocytosis from the apical
lumen. Apical luminal spaces of exocrine tissues, which have a small
caliber and are tortuous, may be uniquely capable of accumulating
discharged secretory proteins as prospective cargo for endocytosis if
passive drainage of the ductal system is slow, as it clearly is when
there is little or no stimulation. In contrast to the constitutive-like
carriers, minor regulated carriers, once formed, are stored until
discharged directly by exocytosis. The peak and subsequent decline in
specific radioactivity of amylase in the minor regulated pathway
(Fig. 4) suggests that there is continual turnover within the storage pool.
-cells where preferential secretion of newly synthesized insulin in
response to moderate glucose elevation was reported (43). Furthermore,
a potentially related pathway involving rapid "piecemeal" secretion
of stored secretory products has been described in hematopoietic cells
(44). Consequently, there is good reason to believe that an analog of
the exocrine minor regulated pathway might exist in most if not all
types of regulated secretory cells and possibly even other cell types
(45). In the case of endocrine cells, it may be particularly intriguing
to consider in future studies whether a minor regulated pathway analog
functions in pulsatile secretion that widely characterizes resting
secretion of hormones (46). Whether the minor regulated pathway and its
prospective analogs carry any unique secretory cargo also remains a
significant issue. Our data show that the content of the parotid minor
regulated pathway is essentially identical to that of the
constitutive-like pathway and mainly contains proteins that are less
efficiently retained in maturing granules (Fig. 2). Nevertheless, we do
not discount the possibility that the pathway might be used selectively to export minor components that are required to maintain oral physiology during periods between ingestion of food. At present, however, it seems clear that the minor regulated pathway does not
function in stimulus-evoked export of active lysosomal enzymes (Fig.
7), a process characterized elsewhere (34, 35).
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Sam Green and Jim Casanova and the members of their laboratories for comments on the manuscript and insightful discussions during the course of this work. We thank Dr. William Ball (Howard University) for the generous gift of anti-PSP antibody and Hans Stukenbrok (Pfizer Inc.) for advice on cryomicrotomy.
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FOOTNOTES |
---|
* This work was supported by Grant DE08941 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, VA 22908. Tel.: 804-924-1786; Fax: 804-982-3912; E-mail: jdc4r@virginia.edu.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M100211200
2 A. Y. Huang, A. M. Castle, B. T. Hinton, and J. D. Castle, unpublished data.
3 Two observations indicate that the relative lack of PSP in unstimulated and carbachol-stimulated secretions is not due to its selective retention in the glandular duct system following secretion, particularly when the overall level of discharged protein is low. First, the radiochemical compositions of the secretions remain distinct in the presence of a background of unstimulated granule exocytosis by lobules from fasted animals (Fig. 2, D and E). Second, in experiments presented in Fig. 6, secretion from parotid lobules following a 19 °C temperature block and brefeldin A treatment is low, yet the relative level of labeled PSP is increased (due to missorting), suggesting unrestricted secretion.
4 We also tested the addition of BFA 15 min before warming, but we have not shown the results because they resemble the findings for BFA added at warm-up and do not provide further insight.
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ABBREVIATIONS |
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The abbreviations used are: CCh, carbamylcholine; BFA, brefeldin A; PRP, proline-rich protein; PSP, parotid secretory protein; TGN, trans-Golgi network; SNARE, SNAP receptor where SNAP is soluble NSF attachment protein; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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1. | Castle, D., and Castle, A. (1998) Crit. Rev. Oral. Biol. Med. 9, 4-22[Abstract] |
2. | Asking, B. (1985) Acta Physiol. Scand. 124, 535-542[Medline] [Order article via Infotrieve] |
3. | Asking, B., and Gjorstrup, P. (1980) Acta Physiol. Scand. 109, 407-413[Medline] [Order article via Infotrieve] |
4. | Asking, B., and Gjorstrup, P. (1980) Acta Physiol. Scand. 109, 415-420[Medline] [Order article via Infotrieve] |
5. | Baum, B. J. (1987) J. Dent. Res. 66, 628-632[Medline] [Order article via Infotrieve] |
6. | Emmelin, N. (1987) J. Dent. Res. 66, 509-517[Abstract] |
7. | Templeton, D. (1980) Pfluegers Arch. 384, 287-289[Medline] [Order article via Infotrieve] |
8. | Takemura, H. (1984) Jpn. J. Pharmacol. 36, 107-109[Medline] [Order article via Infotrieve] |
9. | Larsson, O., and Olgart, L. (1989) Acta Physiol. Scand. 137, 231-236[Medline] [Order article via Infotrieve] |
10. | Hirono, C., Sugita, M., Furuya, K., Yamagishi, S., and Shiba, Y. (1998) J. Membr. Biol. 164, 197-203[CrossRef][Medline] [Order article via Infotrieve] |
11. | Arvan, P., and Castle, D. (1998) Biochem. J. 332, 593-610[Medline] [Order article via Infotrieve] |
12. | von Zastrow, M., and Castle, J. D. (1987) J. Cell Biol. 105, 2675-2684[Abstract] |
13. |
von Zastrow, M.,
Castle, A. M.,
and Castle, J. D.
(1989)
J. Biol. Chem.
264,
6566-6571 |
14. |
Castle, J. D.,
and Castle, A. M.
(1996)
J. Cell Sci.
109,
2591-2599 |
15. |
Martys, J.,
Wjasow, C.,
Gangi, D.,
Kielian, M.,
McGraw, T.,
and Backer, J.
(1996)
J. Biol. Chem.
271,
10953-10962 |
16. |
Castle, A. M.,
Stahl, L. E.,
and Castle, J. D.
(1992)
J. Biol. Chem.
267,
13093-13100 |
17. |
Castle, A. M.,
Huang, A. Y.,
and Castle, J. D.
(1997)
J. Cell Biol.
138,
45-54 |
18. |
Turner, M. D.,
and Arvan, P.
(2000)
J. Biol. Chem.
275,
14025-14030 |
19. | Bernfeld, P. (1955) Methods Enzymol. 1, 149-158 |
20. | Cameron, R. S., Cameron, P. L., and Castle, J. D. (1986) J. Cell Biol. 103, 1299-1313[Abstract] |
21. | McLean, W., and Nakane, P. F. (1974) J. Histochem. Cytochem. 22, 1077-1083[Medline] [Order article via Infotrieve] |
22. | Sharoni, Y., Eimerl, S., and Schramm, M. (1976) J. Cell Biol. 71, 107-122[Abstract] |
23. | Sreebny, L. M., and Johnson, D. A. (1969) Arch. Oral Biol. 14, 397-405[Medline] [Order article via Infotrieve] |
24. |
Fernandez, C. J.,
Haugwitz, M.,
Eaton, B.,
and Moore, H. H.
(1997)
Mol. Biol. Cell
8,
2171-2185 |
25. | De Lisle, R. C., and Bansal, R. (1996) Eur. J. Cell Biol. 71, 62-71[Medline] [Order article via Infotrieve] |
26. | Jackson, C. L., and Casanova, J. E. (2000) Trends Cell Biol. 10, 60-67[CrossRef][Medline] [Order article via Infotrieve] |
27. | Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H., and Balch, W. E. (1995) J. Cell Biol. 130, 781-796[Abstract] |
28. | Davidson, H. W. (1995) J. Cell Biol. 130, 797-805[Abstract] |
29. | Nakajima, Y., and Pfeffer, S. R. (1997) Mol. Biol. Cell 8, 577-582[Abstract] |
30. |
Kundra, R.,
and Kornfeld, S.
(1998)
J. Biol. Chem.
273,
3848-3853 |
31. | Kuliawat, R., and Arvan, P. (1994) J. Cell Biol. 126, 77-86[Abstract] |
32. |
Kuliawat, R.,
Klumperman, J.,
Ludwig, T.,
and Arvan, P.
(1997)
J. Cell Biol.
137,
595-608 |
33. |
Klumpermann, J.,
Kuliawat, R.,
Griffith, J. M.,
Geuze, H. J.,
and Arvan, P.
(1998)
J. Cell Biol.
141,
359-371 |
34. |
Rodriguez, A.,
Webster, P.,
Ortego, J.,
and Andrews, N. W.
(1997)
J. Cell Biol.
137,
93-104 |
35. |
Martinez, I.,
Chakrabarti, S.,
Hellevik, T.,
Morehead, J.,
Fowler, K.,
and Andrews, N. W.
(2000)
J. Cell Biol.
148,
1141-1149 |
36. | Phaneuf, S., Grondin, M., LeBel, D., Roberge, M., Lord, A., and Beaudoin, A. (1984) Cell Tissue Res. 235, 699-701[Medline] [Order article via Infotrieve] |
37. | Phaneuf, S., Grondin, G., Lord, A., and Beaudoin, A. R. (1985) Cell Tissue Res. 239, 105-109[Medline] [Order article via Infotrieve] |
38. | Walker, A. M., and Farquhar, M. G. (1980) Endocrinology 107, 1095-1104[Abstract] |
39. |
Leong, D. A.,
and Thorner, M. O.
(1991)
J. Biol. Chem.
266,
9016-9022 |
40. | Wang, E. S., Brown, P. S., Aroeti, B., Chapin, S. J., Mostov, K. E., and Dunn, K. W. (2000) Traffic 1, 480-493[CrossRef][Medline] [Order article via Infotrieve] |
41. | Hendricks, L. C., McClanahan, S. L., Palade, G. E., and Farquhar, M. G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7242-7246[Abstract] |
42. | Komhoff, M., Hollinshead, M., Tooze, J., and Kern, H. F. (1994) Eur. J. Cell Biol. 63, 192-207[Medline] [Order article via Infotrieve] |
43. | Halban, P. A. (1982) Endocrinology 110, 1183-1188[Medline] [Order article via Infotrieve] |
44. | Dvorak, A., Furitsu, T., Letourneau, L., Ishizaka, T., and Ackerman, S. J. (1991) Am. J. Pathol. 138, 69-82[Abstract] |
45. | Chavez, R. A., Miller, S. G., and Moore, H.-P. (1996) J. Cell Biol. 133, 1177-1191[Abstract] |
46. | Veldhuis, J. D. (2000) Novartis Found. Symp. 227, 1-4[Medline] [Order article via Infotrieve] |
47. | Sesso, A., Assis, J. E., Kuwajima, V. Y., and Kachar, B. (1980) Acta Anat. 108, 521-539[Medline] [Order article via Infotrieve] |
48. |
Springer, S.,
and Schekman, R.
(1998)
Science
281,
698-700 |
49. |
Haucke, V.,
and De Camilli, P.
(1999)
Science
285,
1268-1271 |
50. | Salem, N., Faundez, V., Horng, J. T., and Kelly, R. B. (1998) Nat. Neurosci. 1, 551-556[CrossRef][Medline] [Order article via Infotrieve] |
51. |
Eaton, B. A.,
Haugwitz, M.,
Lau, D.,
and Moore, H. P.
(2000)
J. Neurosci.
20,
7334-7344 |
52. | Barroso, M., and Sztul, E. S. (1994) J. Cell Biol. 124, 83-100[Abstract] |
53. |
Brewer, C. B.,
and Roth, M. G.
(1995)
J. Cell Sci.
108,
789-796 |
54. |
Brignoni, M.,
Pignataro, O. P.,
Rodriguez, M. L.,
Alvarez, A.,
Vega-Salas, D. E.,
Rodriguez-Boulan, E.,
and Salas, P. J. I.
(1995)
J. Cell Sci.
108,
1931-1943 |
55. | Hansen, S. H., and Casanova, J. E. (1994) J. Cell Biol. 126, 677-687[Abstract] |
56. | Christensen, B. M., Zelenina, M., Aperia, A., and Nielsen, S. (2000) Am. J. Physiol. 278, F29-F42 |
57. |
Calhoun, B. C.,
Lapierre, L. A.,
Chew, C. S.,
and Goldenring, J. R.
(1998)
Am. J. Physiol.
275,
C163-C170 |
58. |
Girard, L. R.,
Castle, A. M.,
Hand, A. R.,
Castle, J. D.,
and Mirels, L.
(1993)
J. Biol. Chem.
268,
26592-26601 |