The minor regulated pathway, a rapid component of salivary secretion, may provide docking/fusion sites for granule exocytosis at the apical surface of acinar cells

Anna M. Castle*, Amy Y. Huang* and J. David Castle{ddagger}

Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, VA 22908-0732, USA
* These authors contributed equally to this work

{ddagger} Author for correspondence (e-mail: jdc4r{at}virginia.edu )

Accepted 3 May 2002


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 Materials and Methods
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Recently, we reported that the minor regulated and constitutive-like pathways are the main source of resting secretion by parotid acinar cells. Using tissue lobules biosynthetically labeled with [35S]amino acids, we now show that discharge of the minor regulated pathway precedes granule exocytosis stimulated by isoproterenol (>=1 µM) or carbachol (2 µM). Stimulation of the minor regulated pathway by 40 nM carbachol as well as altering its trafficking, either by adding brefeldin A or by incubating in K+-free medium, cause potentiation of amylase secretion stimulated by isoproterenol, suggesting that the minor regulated pathway contributes to the mechanism of potentiation. Both exocytosis of the minor regulated pathway and the potentiation-inducing treatments induce relocation of immunostained subapical puncta of the SNARE protein syntaxin 3 into the apical plasma membrane. Rab11 and possibly VAMP2 may be concentrated in the same relocating foci. These results suggest that the minor regulated pathway and granule exocytosis are functionally linked and that the minor regulated pathway has a second role beyond contributing to resting secretion — providing surface docking/fusion sites for granule exocytosis. In the current model of salivary protein export, discharge of the minor regulated pathway by either ß-adrenergic or cholinergic stimulation is an obligatory first step. Ensuing granule exocytosis is controlled mainly by ß-adrenergic stimulation whereas cholinergic stimulation mainly regulates the number of surface sites where release occurs.

Key words: Exocytosis, Secretion, Syntaxin 3


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In our previous studies, we have shown that salivary proteins are secreted by at least three distinct pathways from parotid acinar cells. Two pathways, the constitutive-like and minor regulated pathways, export small fractions of total secretory protein while the third and major regulated pathway, granule exocytosis, can export substantial quantities of stored protein. These pathways are used hierarchically with the constitutive-like pathway operating continually, the minor regulated pathway responding to low-level stimulation, and the major regulated pathway requiring stimulation with higher levels of secretagogue. Accordingly, the constitutive-like and minor regulated pathways have been implicated as sources of resting (or basal) salivary secretion (Huang et al., 2001Go), which is driven in situ during waking hours by low frequency parasympathetic stimulation (Emmelin, 1987Go) and is thought to aid in maintaining the physiology of the oral cavity. In contrast, the major regulated pathway is mobilized mainly following more vigorous stimulation and during eating provides large quantities of salivary protein in support of initiating the digestive process (Castle and Castle, 1996Go; Huang et al., 2001Go).

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, 1998Go). 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., 2001Go).

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, 1999Go; Kasai and Takahashi, 1999Go; Ninomiya et al., 1997Go; Campos-Toimil et al., 2000Go). 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.


    Materials and Methods
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 Introduction
 Materials and Methods
 Results
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 References
 
Materials
As described previously (Huang et al., 2001Go), male Sprague-Dawley rats (100-124 g) were obtained from Hilltop (Scottsdale, PA), maintained 7 or 8 days in the vivarium and were used after ad libitum feeding. Expre35S35S used for in vitro biosynthetic labeling was obtained from Perkin Elmer Life Sciences; secretagogues (D,L-isoproterenol (Iso) and carbamyl choline (carbachol; CCh) were from Sigma; brefeldin A (BFA) was from Epicentre Technologies. Immunological reagents included several generous gifts: rabbit antibodies against syntaxin 3 (Pam Tuma and Ann Hubbard, Johns Hopkins Medical School, Baltimore, MD) and Rab3D (Giulia Baldini, Columbia Medical School, New York, NY); mouse monoclonal antibodies against clathrin heavy chain (hybridoma from American Type Culture Collection); dipeptidylpeptidase IV (DPPIV) (Ann Hubbard), syntaxin 6 (Jason Bock and Richard Scheller, Stanford Medical School, CA), and VAMP2 (Reinhard Jahn, Max Planck Institute, Gottingen, Germany). Rabbit anti-Rab11 was obtained from Zymed, Inc. BODIPY-phallacidin and Alexa 488 and 594 conjugated secondary antibodies were from Molecular Probes.

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., 2001Go). 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., 2001Go). 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, 1955Go) and for SDS-PAGE (12.5% gels), Coomassie staining, phosphorimaging, and quantitative analysis of 35S-labeled proteins with Image Quant software (Castle and Castle, 1996Go; Huang et al., 2001Go). 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, 1974Go), 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).


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Discharge of the minor regulated pathway precedes granule exocytosis
In our earlier studies, we demonstrated the selective discharge of the minor regulated pathway under low-level stimulation conditions by either cholinergic or ß-adrenergic agonists, and we noted its rapid kinetics (Castle and Castle, 1996Go; Huang et al., 2001Go). Thus we were curious whether upon more robust stimulation, discharge of the minor regulated pathway preceded granule exocytosis. Using parotid lobules that were pulse-labeled and chase-incubated >=150 minutes to insure labeling of minor regulated carriers and mature secretory granules with newly synthesized proteins, we applied agonists and then replaced incubation media at time intervals that were short yet separated enough to enable collection of sufficient secretory protein for compositional analysis. Stimulation by Iso alone (1 or 10 µM) caused near linear amylase release, which typically reaches 10-15% (1 µM) and 20-25% (10 µM) by 15 minutes. As seen in Fig. 1A, during the first three minutes of discharge, the protein composition detected by Coomassie staining resembles that elicited by low doses of CCh, which is poor in the 25 kDa protein PSP (Huang et al., 2001Go). Subsequently, the protein profile switches to that of granule exocytosis, which has abundant PSP. The radiochemical profile mimics the Coomassie profile (Fig. 1B) and, notably, the specific radioactivity of amylase exhibits an initial rapid spike and subsequently begins a slow rise as the protein composition changes towards the granule profile (Fig. 1C). Based on the protein composition and high specific radioactivity, the initial secretion represents export by the minor regulated pathway (Huang et al., 2001Go). The spike in specific radioactivity signifies that cellular release and not merely ductal clearance of previously secreted protein is responsible for the immediate secretory response. The ensuing drop in specific radioactivity and compositional shift indicate that granule exocytosis has begun subsequently with the older unlabeled granules being released before newer labeled granules (Sharoni et al., 1976Go).



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Fig. 1. Discharge of the minor regulated pathway precedes granule exocytosis. (A) Coomassie staining profiles for parotid secretory proteins released at early time points after pulse-chase biosynthetic labeling with [35S]amino acids upon stimulation of lobules by 10 µM isoproterenol. At each time point, the medium was removed and replaced with fresh medium containing freshly added stimulant. Amylase (Amy.), proline-rich protein (PRP), and parotid secretory protein (PSP) are identified. The composition of secretion elicited by 40 nM carbachol (CCh) and of the content of isolated secretory granules (Gr) is also shown. (B) Radiochemical composition of protein profiles illustrated in panel A. PRP contains no methionine or cysteine and is unlabeled. (C) Specific radioactivity of amylase for the samples shown in panels A and B obtained by normalizing the intensities of the amylase bands in B (quantified by phosphorimaging) to the amylase enzyme activity in each sample.

 

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.



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Fig. 2. Characterization of secretion stimulated by different doses of carbachol. Parotid lobules were pulse labeled and chased for 180 minutes before stimulation and, at each timepoint, the medium was completely removed and replaced. (A) Time course of secretion of amylase enzyme upon stimulation with 40 nM CCh, 200 nM CCh and 2 µM CCh. (B) Specific radioactivity of amylase was calculated as in Fig. 1. (C) Left panel, radiochemical composition of secretion elicited by 2 µM CCh after 1 minute of stimulation. Right panel, coomassie-stained profiles for samples of secretion collected following stimulation with 40 nM (lanes 1,2) and 2 µM (lanes 3,4) CCh. Lanes 1 and 3 show profiles for the first 3 minutes of stimulation, lanes 2 and 4 show profiles from the 10-15 minute interval. For comparison, lane 5 shows a sample collected after 15 minutes of stimulation with 1 µM Iso. Data are from one of three experiments with the same outcome.

 

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, 1996Go)], 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, 1980Go; Templeton, 1980Go; Spearman and Butcher, 1981Go; Takemura, 1984Go; Yoshimura et al., 1998Go; Yoshimura et al., 2000Go; Yoshimura and Hiramatsu, 1998Go). 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.



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Fig. 3. Potentiation of isoproterenol-stimulated amylase secretion by CCh. Parotid lobules were pulse-labeled and chased as in Fig. 1. (A) Secretion of amylase enzyme from samples stimulated with 40 nM CCh alone (CCh), 1 µM Iso (Iso), 40 nM CCh and 1 µM Iso in combination (CCh + Iso). A parallel sample was treated with 40 nM CCh and 1 µM Iso for 10 minutes and subsequently with 1 µM Iso alone (CCh take-away). At each timepoint the medium was removed in entirely and replaced with medium to which additives had been freshly added. (B) Specific radioactivity of amylase in samples stimulated with 1 µM Iso, 1 µM Iso and 40 nM CCh, or 1 µM Iso and 200 nM CCh during the first 10 minutes of stimulation. At each time point the medium was completely removed and replaced. (C) Effect of pre-stimulation with 40 nM CCh on initial secretion and specific radioactivity of amylase. Lobules were pulsed and chased as above. Amylase enzyme (left panel) and specific radioactivity of amylase (right panel) were determined in secretion from the first 1 minute of stimulation in samples treated with 1 µM Iso (open bar), 1 µM Iso and 40 nM CCh added simultaneously (hashed bar), and a sample pre-treated with 40 nM CCh 5 minutes prior to addition of 1 µM Iso plus 40 nM CCh (filled bar). Medium was completely replaced at the end of the pretreatment. (D) Time course of secretion of radiolabeled PSP as a marker of newly formed granules. The gels shown were those from the experiment shown in B.

 

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., 2001Go). 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.



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Fig. 4. Potentiation of isoproterenol-stimulated amylase secretion by BFA and by K+ depletion. Parotid lobules were pulse-labeled and chased as in Fig. 1. (A) Effects of BFA treatment on amylase secretion. Lobules were pretreated with 10 µg/ml BFA or carrier Me2SO for 30 minutes. Subsequently samples were treated with 10 µg/ml BFA alone (BFA), 1 µM Iso and carrier Me2SO (Iso) or 10 µg/ml BFA (Iso + BFA) and incubation was continued for 60 minutes with complete changes of medium every 10 minutes. A parallel sample was treated with 1 µM Iso and 10 µg/ml BFA and subsequently with 1 µM Iso alone (BFA take-away). The results shown are representative of four separate experiments. (B) Potentiation of isoproterenol-induced amylase secretion in K+-depleted medium. Samples were incubated in DMEM or K+-free DMEM (Na+ replacing K+) for 30 minutes before adding 1 µM isoproterenol. Subsequently, incubation was continued with complete changes of medium every 10 minutes. The results shown are representative of four separate experiments.

 

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., 1983Go; Altankov and Grinnell, 1993Go). 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, 1993Go), 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., 2001Go) 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., 2001Go). 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., 1996aGo) (A.M.C., A.Y.H. and J.D.C., unpublished) and mast cells (Guo et al., 1998Go), 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.



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Fig. 5. Localization of syntaxin 3 in unstimulated parotid tissue. (A,C,D) Immunofluorescence of cryosections of parotid tissue have been stained with anti-syntaxin 3 (red) and either BODIPY-phallacidin to mark the actin-rich terminal web or anti-DPPIV to mark the apical plasma membrane (green). All other fluorescent images are of a single 0.1 µM layer following digital deconvolution. Closed arrowheads identify some of the syntaxin 3 foci that are concentrated beneath (on the cytoplasmic side of) the actin band or the apical plasma membrane and are found where little or no luminal space is visible. Open arrrowheads identify some of the few syntaxin 3 foci that have relocated to the luminal side of the actin band or into the DPPIV band signifying association with the apical plasma membrane. Relocated foci are generally observed where the luminal space is evident. (B) Differential interference contrast (DIC) image paired with the image in A. Bar, 10 µm.

 


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Fig. 6. Relocation of syntaxin 3 in response to stimulation of minor regulated pathway. Parotid tissue was processed for immunofluorescence following a 3 minute treatment with 40 nM CCh or 1 µM Iso. (A,B,D) Immunofluorescent specimens were stained with syntaxin 3 (red) and counterstained with BODIPY-phallacidin (green) or DPPIV (green). (C) Comparison of staining of DPPIV (red) and BODIPY-phallacidin (green). Open arrowheads identify examples of syntaxin 3 foci that have relocated to the luminal side of the actin band or into the DPPIV band. Bar, 10 µm.

 

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., 1999Go; Gaisano et al., 1994Go; Fujita-Yoshigaki et al., 1996Go; Hori et al., 1996Go; Calhoun et al., 1998Go). 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., 1996Go; Ohnishi et al., 1996Go); 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., 1998Go) and is thought to function in exocytosis in certain cell types (Martin-Martin et al., 2000Go). 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.



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Fig. 7. Evaluation of other vesicular trafficking proteins as possible marker of carriers of the minor regulated pathway. Parotid tissue was processed for immunofluorescence without (Ctl) or with (Cch) a 3 minute treatment with 40 nM CCh. Immunofluorescent specimens have been counterstained with BODIPY-phallacidin (green) for actin in each case. (A) VAMP2 (red) staining of control tissue; (B) VAMP2 (red) staining of stimulated tissue; (C) Rab11 (red) staining of control tissue; (D) Rab11 (red) staining of stimulated tissue. Solid arrowheads identify prospective subapical foci of VAMP2 and Rab 11, and open arrowheads identify some of the foci that have relocated to the luminal side of the actin band. Bar, 10 µm.

 

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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Fig. 8. Effect of BFA treatment or K+-depletion on localization of syntaxin 3. Parotid lobules were incubated for 15 minutes with 10 µg/ml BFA (A) or in K+-free medium (B) prior to fixation. Tissue sections were stained with syntaxin 3 (red) and counterstained with BODIPY-phallacidin (green). Open arrowheads point to examples of fluorescent foci that have relocated to the luminal aspect of the actin band. Bar, 10 µm.

 


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The minor regulated pathway is the earliest secretory response
Our previous studies showed that the minor regulated pathway can be selectively discharged by low-level cholinergic and ß-adrenergic stimulation (Castle and Castle, 1996Go; Huang et al., 2001Go). The present results have demonstrated clearly that its discharge precedes secretory granule exocytosis in response to elevated levels of Iso or CCh (Fig. 1). Thus this pathway qualifies as both a rapid and a sensitive response pathway that apparently is always mobilized at the outset of stimulation. We believe that it may be an analog of rapid, stimulus-evoked exocytotic events that have been reported in several other recent studies of regulated secretory cells and other cell types. In particular, fusion of the equivalent of minor regulated carriers may be the source of the `small-step' increases in membrane capacitance observed in electrophysiological recordings (e.g. Giovannucci et al., 1998Go; Kasai, 1999Go). Also, the minor regulated pathway is likely to contribute significantly to the rapid and acute peak of amylase secreted by dissociated parotid acini in response to various stimuli in perifusion experiments (Yoshimura et al., 1998Go; Yoshimura et al., 2000Go; Yoshimura and Hiramatsu, 1998Go). Unfortunately, the time resolution in our diagnosis is limited by the necessity to collect secretory protein in sufficient quantities for compositional and specific radioactivity analyses and thus is insufficient to relate the minor regulated pathway unambiguously to these other exocytotic events, which have been followed over much shorter intervals. Nevertheless, our approach has had the key advantage of linking the exocytotic event to at least one specific function — exporting secretory protein and very likely to novel roles in facilitating granule exocytosis and contributing to secretory potentiation.

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, 1999Go).

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., 1998Go; Hori et al., 1996Go; Calhoun et al., 1998Go; Fujita-Yoshigaki et al., 1996Go; Gaisano et al., 1996bGo). 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., 1996aGo; Hansen et al., 1999Go). However, syntaxin 3, which is also sensitive to the same toxin (Hansen et al., 1999Go), 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., 2001Go; Schmoranzer et al., 2000Go; Cochilla et al., 2000Go). 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, 1998Go; Salem et al., 1998Go; Haucke and De Camilli, 1999Go).

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., 2000Go; Thorn et al., 1993Go) 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., 1969Go). 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., 2000Go). 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, 1996Go) (this study). This response occurs widely among acinar cells (Huang et al., 2001Go), 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., 1996Go; Stevens and Sullivan, 1998Go; Yoshimura et al., 1998Go). 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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Fig. 9. Current model for the regulation of granule exocytosis in parotid acinar cells in which the minor regulated pathway provides granule docking/fusion sites. Abbreviations: CL, constitutive-like pathway; CV, condensing vacuole; IG, immature granule; MR, minor regulated pathway; SG, secretory granule. (A) Selective stimulation of the minor regulated pathway (red vesicles) by 40 nM CCh. Analogous selective stimulation occurs in response to <100 nM Iso (Castle and Castle, 1996Go). Discharge creates docking/fusion sites for secretory granules. (B) Sequential exocytosis of the minor regulated pathway and oldest secretion granules accumulated nearest to the apical surface in response to 2 µM CCh. (C) Sequential exocytosis of the minor regulated pathway and compound exocytosis leading to release of newly synthesized granules deep in the storage pool in response to 1 µM isoproterenol (Iso). (D) Secretory potentiation in response to 40 nM CCh and 1 µM isoproterenol resulting from multiple pathways of compound exocytosis at multiple docking/fusion sites created by the minor regulated pathway.

 


    Acknowledgments
 
We are very grateful to Pam Tuma, Ann Hubbard, Giulia Baldini, Reinhard Jahn, Jason Bock and Richard Scheller for their gifts of antibodies. These studies were support by a grant (DE08941) from the National Institutes of Health.


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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