Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules

Rab3D, a member of the Rab3 subfamily of the Rab/ypt GTPases, is expressed on zymogen granules in the pancreas as well as on secretory vesicles in mast cells and in the parotid gland. To shed light on the function of Rab3D, we have generated Rab3D-deficient mice. These mice are viable and have no obvious phenotypic changes. Secretion of mast cells is normal as revealed by capacitance patch clamping. Furthermore, enzyme content and overall morphology are unchanged in pancreatic and parotid acinar cells of knockout mice. Both the exocrine pancreas and the parotid gland show normal release kinetics in response to secretagogue stimulation, suggesting that Rab3D is not involved in exocytosis. However, the size of secretory granules in both the exocrine pancreas and the parotid gland is significantly increased, with the volume being doubled. We conclude that Rab3D exerts its function during granule maturation, possibly by preventing homotypic fusion of secretory granules.     

The regulation of exocytosis in the pancreatic acinar cell.

The pancreatic acinar cell synthesises a variety of digestive enzymes. In transit through the secretory pathway, these enzymes are separated from constitutively secreted proteins and packaged into zymogen granules, which are localised in the apical pole of the cell. Stimulation of the cell by secretagogues such as acetylcholine and cholecystokinin, acting at receptors on the basolateral plasma membrane, causes the generation of an intracellular Ca(2+) signal. This signal, in turn, triggers the fusion of the zymogen granules with the apical plasma membrane, leading to the polarised secretion of the enzymes. This review describes recent advances in our understanding of the control of secretion in the acinar cell. In particular, we discuss the mechanisms underlying the sorting of digestive enzymes into the zymogen granules, the molecular components of the exocytotic "membrane fusion machine," the generation and propagation of the Ca(2+ signal and the development of new techniques for the visualisation of single granule fusion events.     

Exocytosis and movement of zymogen granules observed by VEC-DIC microscopy in the pancreatic tissue en bloc

The dynamicaspects of exocytosis, especially in the normal acinar tissue en bloc,have remained unclear. We visualized exocytosis directly in the tissueof the exocrine pancreas of rodents by video-enhancedcontrast-differential interference contrast (VEC-DIC) microscopy to investigate various exocytosis-related rates and the relationship between the movement of granules and exocytotic responses. Stimulation of the tissue with bethanechol orcholecystokinin caused many of the zymogen granules in the apical poleto disappear abruptly. The exocytotic transients of individual granuleswere completed in 0.48-0.65 s. Granules destined to participate in the exocytotic response moved randomly at velocities of ~0.06 µm/sor less during stimulation. In the tissue preparation, granules locatedfar from the apical pole frequently moved back and forth for 1-7µm without showing exocytosis. Colchicine suppressed this movementand the late phase of the secretory response. Real-time (VEC-DIC)observation of granule dynamics revealed that the initial step ofexocytosis was not coupled directly with the microtubule-dependent translocation but with a continuous, slow Brownian fluctuation of granules.

     

Optical measurement of stimulus-evoked membrane dynamics in single pancreatic acinar cells

Stimulation ofpancreatic acinar cells induces the release of digestive enzymes viathe exocytotic fusion of zymogen granules and activates postfusiongranule membrane retrieval and receptor cycling. In the present study,changes in membrane surface area of rat single pancreatic acinar cellswere monitored by cell membrane capacitance(C_m)measurements and by the membrane fluorescent dye FM1-43. When measuredwith the C_mmethod, agonist treatment evoked a graded, transient increase in acinarcell surface area averaging 3.5%. In contrast, a 13% increase insurface area was estimated using FM1-43, corresponding to the fusion of48 zymogen granules at a rate of 0.5 s¹. After removal of FM1-43from the surface-accessible membrane, a residual fluorescence signalwas shown by confocal microscopy to be localized in endosome-likestructures and confined to the apical regions of acinar cells. Thedevelopment of an optical method for monitoring the membrane turnoverof single acinar cells, in combination with measurements ofC_m changes,reveals coincidence of exocytotic and endocytotic activity in acinarcells after hormonal stimulation.

     

Identification of SNAREs involved in regulated exocytosis in the pancreatic acinar cell.

The molecular basis of exocytotic membrane fusion in the pancreatic acinar cell was investigated using an in vitro assay that measures both zymogen granule-plasma membrane fusion and granule-granule fusion. These two fusion events were differentially sensitive to Ca(2+), suggesting that they are controlled by different Ca(2+)-sensing mechanisms. Botulinum neurotoxin C (BoNT/C) treatment of the plasma membranes caused cleavage of syntaxin 2, the apical isoform of this Q-SNARE, but did not affect syntaxin 4, the basolateral isoform. BoNT/C also cleaved syntaxin 3, the zymogen granule isoform. BoNT/C treatment of plasma membranes abolished granule-plasma membrane fusion, whereas toxin treatment of the granules reduced granule-plasma membrane fusion and abolished granule-granule fusion. Tetanus toxin cleaved granule-associated synaptobrevin 2 but caused only a small reduction in both granule-plasma membrane fusion and granule-granule fusion. Our results indicate that syntaxin 2 is the isoform that mediates fusion between zymogen granules and the apical plasma membrane of the acinar cell. Syntaxin 3 mediates granule-granule fusion, which might be involved in compound exocytosis. In contrast, the major R-SNARE on the zymogen granule remains to be identified.     

Regulation of zymogen granule exocytosis by Ca2+, cAMP, and PKC in pancreatic acinar cells

The effect of cAMP and PKC on zymogen granule exocytosis was investigated by simultaneously measuring cytosolic Ca2+ concentration ([Ca2+]c) and individual zymogen granule exocytosis in isolated mouse pancreatic acini. When acinar cells were stimulated with acetylcholine (ACh, 10 microM), exocytic events were detected through granule-attached apical membranes with [Ca2+]c rise. Application of secretin, forskolin (an adenylate cyclase activator), or PMA (a PKC activator) alone did not elicit any [Ca2+]c rise or zymogen granule exocytosis, but co-stimulation with ACh led to exocytosis in that the total number of secreted granules increased markedly without a significant difference in [Ca2+]c rises. When we evoked exocytosis by [Ca2+]c ramps, pretreatment with forskolin or PMA elicited exocytosis at lower [Ca2+]c levels. These results indicate that PKC or cAMP alone could not directly elicit zymogen granule exocytosis, but that they increase the total releasable pool by rendering zymogen granules more sensitive to Ca2+.     

Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

Exocytosis is evoked by intracellular signals, including Ca²⁺ and protein kinases. We determined how such signals interact to promote exocytosis in exocrine pancreatic duct epithelial cells (PDECs). Exocytosis, detected using carbon-fiber microamperometry, was stimulated by [Ca²⁺]_i increases induced either through Ca²⁺ influx using ionomycin or by activation of P2Y2 or protease-activated receptor 2 receptors. In each case, the exocytosis was strongly potentiated when cyclic AMP (cAMP) was elevated either by activating adenylyl cyclase with forskolin or by activating the endogenous vasoactive intestinal peptide receptor. This potentiation was completely inhibited by H-89 and partially blocked by Rp-8-Br-cAMPS, inhibitors of protein kinase A. Optical monitoring of fluorescently labeled secretory granules showed slow migration toward the plasma membrane during Ca²⁺ elevations. Neither this Ca²⁺-dependent granule movement nor the number of granules found near the plasma membrane were detectably changed by raising cAMP, suggesting that cAMP potentiates Ca²⁺-dependent exocytosis at a later stage. A kinetic model was made of the exocytosis stimulated by UTP, trypsin, and Ca²⁺ ionophores with and without cAMP increase. In the model, without a cAMP rise, receptor activation stimulates exocytosis both by Ca²⁺ elevation and by the action of another messenger(s). With cAMP elevation the docking/priming step for secretory granules was accelerated, augmenting the releasable granule pool size, and the Ca²⁺ sensitivity of the final fusion step was increased, augmenting the rate of exocytosis. Presumably both cAMP actions require cAMP-dependent phosphorylation of target proteins. cAMP-dependent potentiation of Ca²⁺-induced exocytosis has physiological implications for mucin secretion and, possibly, for membrane protein insertion in the pancreatic duct. In addition, mechanisms underlying this potentiation of slow exocytosis may also exist in other cell systems.     

Membrane interactions between secretion granules and plasmalemma in three exocrine glands

Three types of membrane interactions were studied in three exocrine systems (the acinar cells of the rat parotid, rat lacrimal gland, and guinea pig pancrease) by freeze- fracture and thin-section electron microscopy: exocytosis, induced in vivo by specific pharmacological stimulations; the mutual apposition of secretory granule membranes in the intact cell; membrane appositions induced in vitro by centrifugation of the isolated granules. In all three glandular cells, the distribution of intramembrane particles (IMP) on the fracture faces of the luminal plasmagranule membrane particles (IMP) on the fracture faces of the lumenal plasmalemma appeared random before stimulation. However, after injection of secretagogues, IMP were rapidly clearly from the areas of granule- plasmalemma apposition in the parotid cells and, especially, in lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid they were less pronounced. Exocytotic openings were usually large and the fracture faces of their rims were covered with IMP. In contrast, in stimulated pancreatic acinar cells, the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrown necks, and no images which might correspond to early stages of membrane fusion were revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat, circular areas, also devoid of IMP. In thin sections, the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures. Isolated secretion granules were forced together in vitro by centrifugation. Under these conditions, increasing the centrifugal force from 1,600 to 50,000 g for 10 min resulted in a progressive, statistically significant increase of the frequency of IMP-free flat appositions between parotid granules. In contrast, no such areas were seen between freeze-fractured pancreatic granules, although some focal pentalaminar appositions appeared in section after centrifugation at 50 and 100,000 g for 10 min. On the basis of the observation that, in secretory cells, IMP clearing always develops in deformed membrane areas (bulges, depressions, flat areas), it is suggested that it might result from the forced mechanical apposition of the interacting membranes. This might be a preliminary process not sufficient to initiate fusion. In the pancreas, IMP clearing could occur over surface areas too small to be detected. In stimulated parotid and lacrimal glands they were exceptional. These structures were either attached at the sites of continuity between granule and plasma membranes, or free in the acinar lumen, with a preferential location within exocytotic pockets or in their proximity. Experiments designed to investigate the nature of these blisters and vesicles revealed that they probably arise artifactually during glutaraldehyde fixation. In fact, (a) they were large and numerous in poorly fixed samples but were never observed in thin sections of specimens fixed in one step with glutaraldehyde and OsO(4); and (b) no increase in concentration of phospholipids was observed in the parotid saliva and pancreatic juice after stimulation of protein discharge, as was to be expected if release of membrane material were occurring after exocytosis.     

Exocytosis in living salivary glands: direct visualization by video-enhanced microscopy and confocal laser microscopy

Although exocytosis is widely believed to involve granule movement, membrane fusion and the emptying of granule content, direct study of these processes has been difficult in living cells because of the limited resolution of conventional light microscopy. Using video-enhanced microscopy and confocal laser microscopy, we have now studied these processes in living rat parotid and submandibular gland acinar cells. Under a differential interference contrast (DIC) microscope equipped with a CCD camera and a high speed image processor, secretory granules were in general stationary even after secretory stimulation with isoproterenol (IPR). Following IPR stimulation, however, there were abrupt changes in light intensity of secretory granules, and many granules disappeared. Confocal microscopy was then performed to confirm whether the observed changes in granules were related to membrane fusion and content release. For this, cells were perfused with the fluid-phase tracer Lucifer Yellow; confocal images thus obtained clearly demonstrated the appearance of fluorescence in omega-shaped invaginations of the apical plasma membrane which corresponded to the sites at which changes were observed in DIC images. The time sequence analyses of confocal images showed that there was a repetitive appearance and disappearance of omega-shaped fluorescent foci at the apical plasma membrane until most of the granules were depleted. During this time, there did not appear to be any significant expansion of the apical plasma membrane and if endocytic uptake of the tracer occurred, it was below the limit of detection. These observations provide new insights into the exocytotic process in salivary glands and are at variance in some respects with previous interpretations made from electron microscopy.     

Secretion of gamma-glutamyltranspeptidase by the pancreas: evidence for a membrane shedding process during exocytosis

Gamma Glutamyltranspeptidase (GGT) is a membrane-bound enzyme involved in glutathione metabolism. It is present in rat exocrine pancreas at a level which is only exceeded by the kidney. It has been previously shown that most of the enzyme activity is located in the apical area of the acinar cell, more precisely at the level of zymogen granules and plasma membrane. The aim of the present study was to examine the secretory behavior of that enzyme. Under resting conditions, in vivo, high levels of GGT were found in pancreatic juice and its level was not related to protein concentration. Under secretin infusion, a relatively constant level of GGT was released, and again, there was no correlation between enzyme activity and protein concentration. Following a bolus injection of caerulein, an analog of cholecystokinin, marked and concomitant rises in protein and GGT levels were observed. Ultracentrifugation, as well as gel filtration on Sepharose 4B, demonstrated that the enzyme was not released in a soluble form. This observation is in agreement with in vitro determinations on isolated zymogen granules showing that GGT is totally associated with the ZG membrane and undetect-able in the content of these organelles. The present data show that 1 degree GGT is released from the rat pancreas acinar cells in a particulate form; 2 degree GGT release is elicited by hormonal stimulation coinciding with the exocytotic release of secretory proteins. Our observations lead us to propose that in rat pancreas, ZG membrane fragments are released along with secretory proteins during exocytosis.     

Stabilization of exocytosis by dynamic F-actin coating of zymogen granules in pancreatic acini

Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca(2+)-dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini. Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a caged-Ca(2+) compound. Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho. Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis. Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation. Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating. Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis.     

Involvement of phospholipase D in the cAMP-regulated exocytosis of rat parotid acinar cells

The activation of beta-adrenergic receptors in rat parotid acinar cells causes intracellular cAMP elevation and appreciably stimulates the exocytotic release of amylase into saliva. The activation of Ca(2+)-mobilizing receptors also induces some exocytosis. We investigated the role of phospholipase D (PLD) in regulated exocytosis in rat parotid acinar cells. A transphosphatidylation assay detected GTPgammaS (a nonhydrolyzable analogue of GTP)-dependent PLD activity in lysates of rat parotid acinar cells, suggesting that PLD is activated by small molecular mass GTP-binding proteins. The PLD inhibitor, neomycin, suppressed cAMP-dependent exocytosis in saponin-permeabilized cells. Signaling downstream of PLD was disrupted by 1-butanol due to conversion of the PLD reaction product (phosphatidic acid) to phosphatidylbutanol. The stimulation of exocytosis by isoproterenol as well as by a Ca(2+)-mobilizing agonist (methacholine) was inhibited by 1-butanol. These results suggest that PLD is important for regulated exocytosis in rat parotid acinar cells.     

Regulated apical secretion of zymogens in rat pancreas. Involvement of the glycosylphosphatidylinositol-anchored glycoprotein GP-2, the lectin ZG16p, and cholesterol-glycosphingolipid-enriched microdomains

We examined the role of glycosphingolipid- and cholesterol-enriched microdomains, or rafts, in the sorting of digestive enzymes into zymogen granules destined for apical secretion and in granule formation. Isolated membranes of zymogen granules from pancreatic acinar cells showed an enrichment in cholesterol and sphingomyelin and formed detergent-insoluble glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose density gradients and contained the glycosylphosphatidylinositol (GPI)-anchored glycoprotein GP-2, the lectin ZG16p, and sulfated matrix proteoglycans. Morphological and pulse-chase studies with isolated pancreatic lobules revealed that after inhibition of GPI-anchor biosynthesis by mannosamine or the fungal metabolite YW 3548, granule formation was impaired leading to an accumulation of newly synthesized proteins in the Golgi apparatus and the rough endoplasmic reticulum. Furthermore, the membrane attachment of matrix proteoglycans was diminished. After cholesterol depletion or inhibition of glycosphingolipid synthesis by fumonisin B1, the formation of zymogen granules as well as the formation of detergent-insoluble complexes was reduced. In addition, cholesterol depletion led to constitutive secretion of newly synthesized proteins, e.g. amylase, indicating that zymogens were missorted. Together, these data provide first evidence that in polarized acinar cells of the exocrine pancreas GPI-anchored proteins, e.g. GP-2, and cholesterol-sphingolipid-enriched microdomains are required for granule formation as well as for regulated secretion of zymogens and may function as sorting platforms for secretory proteins destined for apical delivery.     

Dynamic regulation of the large exocytotic fusion pore in pancreatic acinar cells

Loss of granule content during exocytosis requires the opening of a fusion pore between the secretory granule and plasma membrane. In a variety of secretory cells, this fusion pore has now been shown to subsequently close. However, it is still unclear how pore closure is physiologically regulated and contentious as to how closure relates to granule content loss. Here, we examine the behavior of the fusion pore during zymogen granule exocytosis in pancreatic acinar cells. By using entry of high-molecular-weight dyes from the extracellular solution into the granule lumen, we show that the fusion pore has a diameter of 29-55 nm. We further show that by 5 min after granule fusion, many granules have a closed fusion pore with evidence indicating that pore closure is a prelude to endocytosis and that in granules with a closed fusion pore the chymotrypsinogen content is low. Finally, we show that latrunculin B treatment promotes pore closure, suggesting F-actin affects pore dynamics. Together, our data do not support the classical view in acinar cells that exocytosis ends with granule collapse. Instead, for many granules the fusion pore closes, probably as a transition to endocytosis, and likely involving an F-actin-dependent mechanism.     

Fine structure of the silk gland in the mite Ornithocheyletia sp. (Prostigmata,Cheyletidae)

Silk spinning is widely-spread in trombidiform mites, yet scarse information is available on the morphology of their silk glands. Thus this study describes the fine structure of the prosomal silk glands in a small parasitic mite, Ornithocheyletia sp. (Cheyletidae). These are paired acinous glands incorporated into the podocephalic system, as typical of the order. Combined secretion of the coxal and silk glands is released at the tip of the gnathosoma. Data obtained show Ornithocheyletia silk gland belonging to the class 3 arthropod exocrine gland. Each gland is composed of seven pyramidal secretory cells and one ring-folded intercalary cell, rich in microtubules. The fine structure of the secretory cells points to intensive protein synthesis resulted in the presence of abundant uniform secretory granules. Fibrous content of the granules is always subdivided into several zones of two electron densities. The granules periodically discharge into the acinar cavity by means of exocytosis. The intercalary cell extends from the base of the excretory duct and contributes the wall of the acinar cavity encircling the apical margins of the secretory cells. The distal apical surface of the intercalary cell is covered with a thin cuticle resembling that of the corresponding cells in some acarine and myriapod glands. Axon endings form regular synaptic structures on the body of the intercalary cell implying nerve regulation of the gland activity.     

A role of VAMP8/endobrevin in regulated exocytosis of pancreatic acinar cells

Despite our general understanding that members of the SNARE superfamily participate in diverse intracellular docking/fusion events, the physiological role of the majority of SNAREs in the intact organism remains elusive. In this study, through targeted gene knockout in mice, we establish that VAMP8/endobrevin is a major player in regulated exocytosis of the exocrine pancreas. VAMP8 is enriched on the membrane of zymogen granules and exists in a complex with syntaxin 4 and SNAP-23. VAMP8-/- mice developed normally but showed severe defects in the pancreas. VAMP8 null acinar cells contained three times more zymogen granules than control acinar cells. Furthermore, secretagogue-stimulated secretion was abolished in pancreatic fragments derived from VAMP8-/- mice. In addition, VAMP8-/- mice were partially resistant to supramaximal caerulein-induced pancreatitis. These results suggest a major physiological role of VAMP8 in regulated exocytosis of pancreatic acinar cells by serving as a v-SNARE of zymogen granules.     

Maintenance of quantal size and immediately releasable granules in rat chromaffin cells by glucocorticoid

Glucocorticoid is reported to regulate catecholamine synthesis and storage. However, it is not clear whether the actual amount of catecholamine released from individual granules (quantal size, Q) in mature chromaffin cells is affected by glucocorticoid. Using carbon fiber amperometry, we found that dexamethasone did not affect mean cellular Q or the proportional release from different populations of granules in rat chromaffin cells cultured for 1 day in a serum-free defined medium. After two extra days of culture in the defined medium, there was a rundown in mean cellular Q, and it was associated with a shift in the proportional release from the different granule populations. This phenomenon could not be rescued by serum supplementation but could be prevented by dexamethasone via an action that was independent of changes in voltage-gated Ca²⁺ channel (VGCC) density. Using simultaneous measurements of membrane capacitance and cytosolic Ca²⁺ concentration, we found that for cells cultured in defined medium dexamethasone enhanced the exocytotic response triggered by a brief depolarization (50 ms) without affecting the VGCC density or the fast exocytotic response triggered via flash photolysis of caged Ca²⁺. Thus glucocorticoid may regulate the number of immediately releasable granules that are in close proximity to a subset of VGCC. Because chromaffin cells in vivo are exposed to high concentrations of glucocorticoid, our findings suggest that the paracrine actions of glucocorticoid maintain the mean catecholamine content in chromaffin cell granules as well as the colocalization of releasable granules with VGCCs. catecholamines; paracrine action; exocytosis; calcium channels     

A secretion granule membrane protein (GRAMP 92) is found in non-granule membranes including those of the endocytic pathway.

GRAMP 92, a secretion granule-associated membrane protein, has been identified in exocrine and endocrine storage granule membranes using a monoclonal antibody against rat parotid secretion granule membranes. This integral membrane glycoprotein has a M(r) of 92,000 in pancreatic zymogen granule membranes, and is slightly smaller in endocrine granule membranes. In both cases, deglycosylation produces core proteins of M(r) 52,000, that have identical peptide fingerprints. Unlike the slightly smaller zymogen granule membrane glycoprotein GP-2, GRAMP 92 does not appear to be bound to the membrane by a glycophosphatidyl inositol anchor, is not found on the plasma membrane and is not released into the secretion. Within acinar cells, low levels of antigen are observed immunocytochemically over the membranes of most granules. Antigen is highly concentrated on small vesicles that are closely apposed to (and possibly interact with) granules. As well, antigen is localized to organelles in the Golgi and basolateral regions that are part of the endocytic pathway. In hepatocytes a glycoprotein similar if not identical to GRAMP 92 marks the endocytic pathway including lysosomes. These findings indicate that GRAMP 92 is a widely distributed endocytic component and suggest that cells specialized for regulated secretion may adapt such components for storage granule function. Granule-associated GRAMP 92-rich membranes may link the exocytotic and endocytic pathways.     

Ca-regulated secretory granule exocytosis in pancreatic and parotid acinar cells

Protein secretion from acinar cells of the pancreas and parotid glands is controlled by G-protein coupled receptor activation and generation of the cellular messengers Ca²⁺, diacylglycerol and cAMP. Secretory granule (SG) exocytosis shares some common characteristics with nerve, neuroendocrine and endocrine cells which are regulated mainly by elevated cell Ca²⁺. However, in addition to diverse signaling pathways, acinar cells have large ∼1 μm diameter SGs (∼30 fold larger diameter than synaptic vesicles), respond to stimulation at slower rates (seconds versus milliseconds), demonstrate significant constitutive secretion, and in isolated acini, undergo sequential compound SG–SG exocytosis at the apical membrane. Exocytosis proceeds as an initial rapid phase that peaks and declines over 3 min followed by a prolonged phase that decays to near basal levels over 20–30 min. Studies indicate the early phase is triggered by Ca²⁺ and involves the SG proteins VAMP2 (vesicle associated membrane protein2), Ca²⁺-sensing protein synatotagmin 1 (syt1) and the accessory protein complexin 2. The molecular details for regulation of VAMP8-mediated SG exocytosis and the prolonged phase of secretion are still emerging. Here we review the known regulatory molecules that impact the sequential exocytic process of SG tethering, docking, priming and fusion in acinar cells.     

Intracellular transport of sulfated macromolecules in parotid acinar cells

Intracellular transport of sulfated macromolecules in parotid acinar cells was investigated by electron microscopic radioautography after injection of 35S-sulfate. Ten minutes after injection radiosulfate was concentrated in the Golgi region. By 1 hr, much of the radioactive material had been transported to condensing vacuoles. These vacuoles were subsequently transformed into zymogen granules which contained almost 70% of the radioactivity 4 hrs after injection. These results indicate that, in addition to its packaging function, the Golgi apparatus in parotid acinar cells is capable of utilizing inorganic sulfate for the production of sulfated macromolecules. These molecules, following an intracellular route similar to that taken by digestive enzymes, become an integral component of zymogen granules. The possibility that sulfated macromolecules play a role in exocrine secretion by aiding in the packaging of exportable proteins is discussed.