Docking of chromaffin granules—A necessary step in exocytosis?

Putative docking of secretory vesicles comprising recognition of and attachment to future fusion sites in the plasma membrane has been investigated in chromaffin cells of the bovine adrenal medulla and in rat phaeochromocytoma (PC 12) cells. Upon permeabilization with digitonin, secretion can be stimulated in both cell types by indreasing the free Ca²⁺-concentration to M levels. Secretory activity can be elicited up to 1 hr after starting permeabilization and despite the loss of soluble cytoplasmic components indicating a stable attachment of granules to the plasma membrane awaiting the trigger for fusion. Docked granules can be observed in the electron microscope in permeabilized PC 12 cells which contain a large proportion of their granules aligned underneath the plasma membrane. The population of putatively docked granules in chromaffin cells cannot be as readily discerned due to the dispersal of granules throughout the cytoplasm. Further experiments comparing PC 12 and chromaffin cells suggest that active docking but not transport of granules can still be performed by permeabilized cells in the presence of Ca²⁺: a short (2 min) pulse of Ca²⁺ in PC 12 cells leads to the secretion of almost all releasable hormone over a 15 min observation period whereas, in chromaffin cells, with only a small proportion of granules docked, withdrawal of Ca²⁺ leads to an immediate halt in secretion. Transport of chromaffin granules from the Golgi to the plasma membrane docking sites seems to depend on a mechanism sensitive to permeabilization. This is shown by the difference in the amount of hormone released from the two permeabilized cell types, reflecting the contrast in the proportion of granules docked to the plasma membrane in PC 12 or chromaffin cells. Neither docking nor the docked state are influenced by cytochalasine B or colchicine. The permeabilized cell system is a valuable technique for thein vitro study of interaction between secretory vesicles and their target membrane.     

Imaging exocytosis of single insulin secretory granules with evanescent wave microscopy: distinct behavior of granule motion in biphasic insulin release.

To study insulin exocytosis by monitoring the single insulin secretory granule motion, evanescent wave microscopy was used to quantitatively analyze the final stage of insulin exocytosis with biphasic release. Green fluorescent protein-tagged insulin transfected in MIN6 beta cells was packed in insulin secretory granules, which appeared to preferentially dock to the plasma membrane. Upon fusion evoked by secretagogues, evanescent wave microscopy revealed that fluorescence of green fluorescent protein-tagged insulin brightened, spread (within 300 ms), and then vanished. Under KCl stimulation, which represents the 1st phase of release, the successive fusion events were seen mostly from previously docked granules for the first minute, followed by the recruitment of new granules to the plasmalemmal docking sites. Stimulation with glucose, in contrast, caused the fusion events from previously docked granules for the first 120 s, thereafter a continuous fusion (2nd phase of release) was observed over 10 min mostly from newly recruited granules that progressively accumulated on the plasma membrane. Thus, our data revealed the distinct behavior of the insulin granule motion during the 1st and 2nd phase of release.     

Docking and fusion of insulin secretory granules in SUR1 knock out mouse beta-cells observed by total internal reflection fluorescence microscopy

To explore how the sulfonylurea receptor (SUR1) is involved in docking and fusion of insulin granules, dynamic motion of single insulin secretory granules near the plasma membrane was examined in SUR1 knock-out (Sur1KO) beta-cells by total internal reflection fluorescence microscopy. Sur1KO beta-cells exhibited a marked reduction in the number of fusion events from previously docked granules. However, the number of docked granules declined during stimulation as a consequence of the release of docked granules into the cytoplasm vs. fusion with the plasma membrane. Thus, the impaired docking and fusion results in decreased insulin exocytosis from Sur1KO beta-cells.     

Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.     

Docking is not a prerequisite but a temporal constraint for fusion of secretory granules

We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic β cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.     

Preparation of the cortical reaction: maturation-dependent migration of SNARE proteins, clathrin, and complexin to the porcine oocyte's surface blocks membrane traffic until fertilization

The cortical reaction is a calcium-dependent exocytotic process in which the content of secretory granules is released into the perivitellin space immediately after fertilization, which serves to prevent polyspermic fertilization. In this study, we investigated the involvement and the organization of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins in the docking and fusion of the cortical granule membrane with the oolemma in porcine oocytes. During meiotic maturation, secretory vesicles that were labeled with a granule-specific binding lectin, peanut agglutinin (PNA), migrated toward the oocyte's surface. This surface-orientated redistribution behavior was also observed for the oocyte-specific SNARE proteins SNAP23 and VAMP1 that colocalized with the PNA-labeled structures in the cortex area just under the oolemma and with the exclusive localization area of complexin (a trans-SNARE complex-stabilizing protein). The coming together of these proteins serves to prevent the spontaneous secretion of the docked cortical granules and to prepare the oocyte's surface for the cortical reaction, which should probably be immediately compensated for by a clathrin-mediated endocytosis. In vitro fertilization resulted in the secretion of the cortical granule content and the concomitant release of complexin and clathrin into the oocyte's cytosol, and this is considered to stimulate the observed endocytosis of SNARE-containing membrane vesicles.     

Behavior of cellular cyclic nucleotides after hydrokinetic or ecbolic stimulation of the canine pancreas

The studies deal with the influence of secretin and various ecbolic secretagogues on tissue levels of cAMP and cGMP in vivo and in the isolated perfused canine pancreas. The mutual behaviour of cellular cAMP and cGMP is observed and compared with the time course of the respective secretory events. Synthetic secretin as well as CCK, acetylcholine or Caerulein likewise elevate tissue cAMP and cGMP simultaneously. There exists no difference in the magnitude of increase and in the time course of changes in tissue cyclic nucleotide levels between hydrokinetic and ecbolic stimulation. The rise in cAMP and cGMP coincides with the onset of the respective secretory events and reaches peak values contemporarily to the excretory maxima. The following decrease in tissue cyclic nucleotides approximatively parallels juice or enzyme secretion in the isolated perfused pancreas but differs widely in vivo. Under this condition cAMP and cGMP rapidly fall to basal levels during undiminished excretory function and show a second rise after cessation of the latter. Secretin and various ecbolic secretagogues do not increase tissue content of cyclic nucleotides in the same dose-dependent manner as can be observed with pancreatic secretion. The behaviour of cAMP and cGMP after addition of secretin and CCK or acetylcholine remains widely unchanged during calcium-free perfusion in spite of an extensive excretory inhibition. The corresponding rise in cellular cAMP and cGMP in the sequence of hydrokinetic as well as of ecbolic stimulation points to an analogous intracellular mediation of various secretagogues in different target cells of the exocrine canine pancreas.     

The secretion of adenosin 3',5'-monophosphate after hydrokinetic and ecbolic stimulation in the canine pancreas (author's transl)

The secretion of cAMP is studied in vivo and in the isolated perfused canine pancreas after administration of secretin and CCK or caerulein in comparison with hydrokinetic or ecbolic secretory events as well as with the magnitude and time course of changes in tissue cAMP. 1) The total output of cAMP and pancreatic juice shows a significant and positive correlation after stimulation with secretin. The linear correspondence between cAMP concentration and secretory rates of pancreatic juice beyond 3 ml/5 min and their non-linear, reciprocal correlation at lower rates of fluid secretion point to an active as well as to a passive secretory mechanism for cAMP. 2) CCK and caerulein increase secretion of cAMP too. The output of cAMP however neither corresponds to the time course of protein secretion nor correlates quantitatively with the latter. 3) The behaviour of cAMP secretion and concentration in the pancreatic juice after administration of secretin and CCK or caerulein as well as differs from the changes in tissue cAMP levels. The respective maximum of cAMP output after addition of secretin or ecbolic secretagogues during the greatest decrease in cellular cAMP levels yields on the average about 1% of the estimated reduction in total tissue cAMP content. The results indicate a functional coherence in secretion of pancreatic juice and cAMP but oppose the assumption, that essential amounts of cAMP are released during exocytosis of zymogen granules. The secretion of cAMP may be possibly influenced by cytoplasmatic cAMP levels, but neither reflects the present changes in cellular cAMP nor seems to be of a regulatory importance for the latter.     

The GTPase RalA Regulates Different Steps of the Secretory Process in Pancreatic β-Cells

Background

RalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic β-cells and analyzed the impact on different steps of the insulin-secretory process.

Methodology/Principal Findings

We found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3–5 min and reaches a plateau after 10–15 min. The activation of the GTPase is triggered by increases in intracellular Ca²⁺ and cAMP and is prevented by the L-type voltage-gated Ca²⁺ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane.

Conclusions/Significance

Taken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca²⁺ and cAMP controls hormone release from pancreatic β-cell by coordinating the execution of different events in the secretory pathway.     

Site of docking and fusion of insulin secretory granules in live MIN6 beta cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy

To determine the site of insulin exocytosis in the pancreatic beta cell plasma membrane, we analyzed the interaction between the docking/fusion of green fluorescent protein-tagged insulin granules and syntaxin 1 labeled by TAT-conjugated Cy3-labeled antibody (Ab) using total internal reflection fluorescence microscopy (TIRFM). Monoclonal Ab against syntaxin 1 was labeled with Cy3 then conjugated with the protein transduction domain of HIV-1 TAT. TAT-conjugated Cy3-labeled anti-syntaxin 1 Ab was transduced rapidly into the subplasmalemmal region in live MIN6 beta cells, which enabled us to observe the spatial organization and distribution of endogenous syntaxin 1. TIRFM imaging revealed that syntaxin 1 is distributed in numerous separate clusters in the intact plasma membrane, where insulin secretory granules were docked preferentially to the sites of syntaxin 1 clusters, colocalizing with synaptosomal-associated protein of 25 kDa (SNAP-25) clusters. TIRFM imaging analysis of the motion of single insulin granules demonstrated that the fusion of insulin secretory granules stimulated by 50 mm KCl occurred exclusively at the sites of the syntaxin 1 clusters. Cholesterol depletion by methyl-beta-cyclodextrin treatment, in which the syntaxin 1 clusters were disintegrated, decreased the number of docked insulin granules, and, eventually the number of fusion events was significantly reduced. Our results indicate that 1) insulin exocytosis occurs at the site of syntaxin 1 clusters; 2) syntaxin 1 clusters are essential for the docking and fusion of insulin granules in MIN6 beta cells; and 3) the sites of syntaxin 1 clusters are distinct from flotillin-1 lipid rafts.     

Phospholipase A2 activation by melittin causes amylase release from exocrine pancreas

Phospholipase A2-induced deacylation of membrane phospholipids is associated with changes in membrane fluidity. The importance of this reaction in the pancreatic amylase secretory process was tested using melittin, a phospholipase A2 stimulating peptide. Phospholipase A2 activity (using [3H]arachidonic acid release as an index) and amylase secretion were both increased in a time- and concentration-dependent manner by melittin. Phospholipids prelabelled with [3H]oleic acid or [14C]linoleic acid also released radioactive free fatty acids in response to melittin. Prostaglandin synthesis was not involved in the melittin response, since inhibitors of arachidonic acid oxidation (indomethacin, 5,8,11,14-eicosatetraynoic acid) did not alter the ability of melittin to release [3H]arachidonic acid or amylase. When melittin was co-applied with carbachol, cholecystokinin octapeptide, or vasoactive intestinal peptide, amylase secretion was additive. The effect of melittin on both fatty acid and amylase release was dependent on extracellular calcium, though melittin's effects were not dependent on the intracellular accumulation of second messengers such as calcium or cAMP. The data suggest that activation of phospholipase A2 by melittin results in the triggering of the secretory process in exocrine pancreas by a different mechanism than that for other pancreatic secretagogues.     

The amylase-secreting cells of the stomach of the scallop, Pecten maximus: ultrastructural, immunohistochemical and immunocytochemical characterizations

(1) alpha-amylase was extracted and purified from the stomach/digestive gland complex of the scallop Pecten maximus and an anti-serum was induced against the purified amylase by rabbit immunization. (2) The anti scallop amylase was used to localize the amylase-secreting cells in the stomach of Pecten maximus by immunofluorescence and immunogold labelling. The amylase-secreting cells are glandular cells particularly numerous in the main sorting area of the stomach. Their secretory granules were found strongly positive for anti-amylase. Three types of glandular cells were observed, actually corresponding to the three stages of the glandular-cell activity, synthesis, secretion and excretion. (3) The synthesizing cell shows the characteristic features of a protein-synthesizing cell: a conspicuous nucleolus and abundant granular endoplasmic reticulum. In the secretory cell, the secretory granules are formed by the Golgi apparatus and accumulate in the apical part of the cell. The secretory cell is filled with two types of secretory granules which are released in the stomach lumen by apocrine excretion. (4) The present study brings the first demonstration of the synthesis and extracellular release of amylase by glandular cells of the stomach epithelium of a bivalve.     

Effect of dopamine on amylase secretion from guinea pig pancreatic acinar cells in vitro

Dopamine has been shown to effect pancreatic flow, protein output and amylase secretion in a variety of species. However, there is conflicting evidence regarding the role of dopamine on amylase release in vitro. Specific studies were conducted to evaluate the effect of dopamine and to compare its effects with other substances on basal- and secretagogue-stimulated amylase secretion in a guinea pig dispersed pancreatic acinar cells preparation. Dopamine (10(-6) M) induced a small, but significant (P less than 0.05) increase of amylase secretion. Established secretagogues (10(-6) M) including bombesin, cholecystokinin-octapeptide (CCK-8) and carbachol as anticipated induced significantly larger responses. Other substances tested (10(-6) M) including thyrotropin-releasing hormone (TRH) and muscimol were without effect. Complete dose-response studies (10(-11)-10(-3) M) in the presence of bombesin, CCK-8 and carbachol revealed that dopamine does not affect amylase release in response to these secretagogues. These findings suggest that dopamine is a weak stimulant of amylase secretion in vitro, and that it may therefore play a minor role in regulation of pancreatic enzyme secretion. Several factors including vascular, hormonal and neural have been implicated in regulation of pancreatic exocrine secretion. In particular, autonomic nervous system activity, notably cholinergic, has been shown to affect the secretory status of the pancreatic acinar cell. In addition, several biologically active peptides including bombesin, cholecystokinin (CCK), secretin, vasoactive intestinal peptide (VIP), substance P, gastrin and stimulation of cholinergic (muscarinic) receptors with carbachol have been shown to stimulate pancreatic enzyme secretion both in vivo and in vitro. Certain controversy regarding the role of the sympathetic nervous system in regulation of pancreatic exocrine secretion does exist. For example, several studies with agonists and antagonists of noradrenergic and dopaminergic receptor subtypes suggest a stimulatory effect on pancreatic fluid, electrolyte and enzyme secretion. However, these responses are species-specific and variations inherent to the model have been described. Dopamine administration has been shown to stimulate pancreatic bicarbonate and enzyme secretion in a variety of species including mice, dogs, and man. Radioligand binding studies with 3H-dopamine have revealed the presence of high- and low-affinity dopamine binding sites in dog pancreatic acinar cells. Stimulation of these receptors has been correlated with dose-dependent increases in intracellular cAMP levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transduction of MIN6 beta cells with TAT-syntaxin SNARE motif inhibits insulin exocytosis in biphasic insulin release in a distinct mechanism analyzed by evanescent wave microscopy

To investigate the in vivo interaction of syntaxin-mediated soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) assembly and insulin exocytosis in biphasic release, we examined the dynamics of insulin granule motion such as docking and fusion with the plasma membrane when the syntaxin SNARE motif (H3 domain) was transduced into living MIN6 beta cells. TAT-H3, produced by fusion of the protein transduction domain of human immunodeficiency virus-1 TAT to the syntaxin-H3 domain, was rapidly transduced into the subplasmalemmal region in living MIN6 cells. Immunoblotting analysis followed by immunoprecipitation on TAT-H3-treated MIN6 cells showed that TAT-H3 binds SNAP-25 and VAMP-2 in vivo. Transduction of MIN6 cells with TAT-H3 caused a decrease in both the first and second phase of insulin release. We therefore quantitatively analyzed approaching, docking, and fusing of green fluorescent protein-labeled single insulin granules in TAT-H3-transduced MIN6 cells by evanescent wave microscopy. Under high glucose stimulation, TAT-H3 treatment not only reduced the fusion events from previously docked granules for the first 120 s (first phase of release) but also strongly inhibited the docking and fusion from newly recruited insulin granules after this point (second phase of release). During the second phase of release we observed a marked reduction in the accumulation of newly docked insulin granules; subsequently, fusion events were significantly decreased. TAT-H3 treatment by itself, however, did not alter the number of previously docked granules without stimulation. We conclude that introduction of the H3 domain into MIN6 cells inhibits biphasic insulin release by two mechanisms. 1) In the first phase of insulin release, the H3 domain interferes with previously docked granules to be fused, and 2) in the second phase of insulin release reduced fusion events result from a marked decline of newly docked granules. Thus, syntaxin-mediated SNARE assembly modulates insulin exocytosis in biphasic insulin release in a distinct way.     

Structure and dynamics of the fusion pore in live cells.

Atomic force microscopy reveal pit-like structures typically containing three or four, approximately 150 nm in diameter depressions at the apical plasma membrane in live pancreatic acinar cells. Stimulation of secretion causes these depressions to dilate and return to their resting size following completion of the process. Exposure of acinar cells to cytochalasin B results in decreased depression size and a loss in stimulable secretion. It is hypothesized that depressions are the fusion pores, where membrane-bound secretory vesicles dock and fuse to release vesicular contents. Zymogen granules, the membrane-bound secretory vesicles in exocrine pancreas, contain the starch digesting enzyme, amylase. Using amylase-specific immunogold labeling, localization of amylase at depressions following stimulation of secretion is demonstrated. This study confirms depressions to be the fusion pores in pancreatic acinar cells. High-resolution images of the fusion pore in live pancreatic acinar cells reveal the structure in much greater detail than has previously been observed.     

Insight in the exocytotic process in chromaffin cells: regulation by trimeric and monomeric G proteins

Catecholamine secretion from chromaffin cells has been used for a long time as a general model to study exocytosis of large dense core secretory granules. Permeabilization and microinjection techniques have brought the possibility to dissect at the molecular level the multi-protein machinery involved in this complex physiological process. Regulated exocytosis comprises distinct and sequential steps including the priming of secretory granules, the formation of a docking complex between granules and the plasma membrane and the subsequent fusion of the granule with the plasma membrane. Key proteins involved in the exocytotic machinery have been identified. For instance, SNAREs which participate in the docking events in most intracellular transport steps along the secretory pathway, play a role in exocytosis in both neuronal and endocrine cells. However, in contrast to intracellular transport processes for which the highest fusion efficiency is required after correct targeting of the vesicles, the number of exocytotic events in activated secretory cells needs to be tightly controlled. We describe here the multistep control exerted by heterotrimeric and monomeric G proteins on the progression of secretory granules from docking to fusion and the molecular nature of some of their downstream effectors in neuroendocrine chromaffin cells.     

Rab3D redistribution and function in rat parotid acini

The original article to which this Erratum refers was published in J. Cell. Physiol. (2003) 197(3) 400–408 . Rab3D is a low molecular weight GTP‐binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cytosol as a complex with Rab escort protein. In the present study, we examined the redistribution of membrane‐associated Rab3D during secretion in permeabilized parotid acini. When permeabilized acini were stimulated with calcium and cAMP, amylase release increased greater than twofold over basal. Quantitative immunoblotting of subcellular fractions revealed that Rab3D did not dissociate from parotid membranes during secretion. Immunohistochemical staining demonstrated that Rab3D co‐localizes with amylase containing granules that are found in the apical pole of the cell. Upon stimulation with calcium and cAMP, Rab3D and amylase immunostaining of granules appeared to be more dispersed. However, Rab3D immunostaining was not observed on the plasma membrane and appeared to reside in the apical cytoplasm. To examine the role of Rab3D in amylase release, cytosolic extracts containing myc‐tagged Rab3D and Rab3DQ81L, a GTP‐binding mutant, were prepared and incubated with streptolysin O‐permeabilized acini. Rab3D, but not Rab3DQ81L, bound to parotid membranes suggesting that Rab3D‐binding to parotid membranes is guanine nucleotide‐dependent. Moreover, wild‐type and mutant Rab3D inhibited agonist‐induced amylase release from permeabilized parotid acini. These observations indicate that in parotid acini, Rab3D does not dissociate from parotid membranes or redistribute to the plasma membrane during secretion, and may play an inhibitory role in regulated secretion. The fact that both wild‐type Rab3D and the GTP‐binding mutant inhibit amylase release suggests that binding of Rab3D to the membrane is not essential for secretory inhibition. J. Cell. Physiol. 199: 316, 2004© 2004 Wiley‐Liss, Inc.     

Rab3D redistribution and function in rat parotid acini

Rab3D is a low molecular weight GTP-binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cytosol as a complex with Rab escort protein. In the present study, we examined the redistribution of membrane-associated Rab3D during secretion in permeabilized parotid acini. When permeabilized acini were stimulated with calcium and cAMP, amylase release increased greater than twofold over basal. Quantitative immunoblotting of subcellular fractions revealed that Rab3D did not dissociate from parotid membranes during secretion. Immunohistochemical staining demonstrated that Rab3D co-localizes with amylase containing granules that are found in the apical pole of the cell. Upon stimulation with calcium and cAMP, Rab3D and amylase immunostaining of granules appeared to be more dispersed. However, Rab3D immunostaining was not observed on the plasma membrane and appeared to reside in the apical cytoplasm. To examine the role of Rab3D in amylase release, cytosolic extracts containing myc-tagged Rab3D and Rab3DQ81L, a GTP-binding mutant, were prepared and incubated with streptolysin O-permeabilized acini. Rab3D, but not Rab3DQ81L, bound to parotid membranes suggesting that Rab3D-binding to parotid membranes is guanine nucleotide-dependent. Moreover, wild-type and mutant Rab3D inhibited agonist-induced amylase release from permeabilized parotid acini. These observations indicate that in parotid acini, Rab3D does not dissociate from parotid membranes or redistribute to the plasma membrane during secretion, and may play an inhibitory role in regulated secretion. The fact that both wild-type Rab3D and the GTP-binding mutant inhibit amylase release suggests that binding of Rab3D to the membrane is not essential for secretory inhibition.     

Unstimulated amylase secretion is proteoglycan-dependent in rat parotid acinar cells

It is well-known that amylase is secreted in response to extracellular stimulation from the acinar cells. However, amylase is also secreted without stimulation. We distinguished vesicular amylase as a newly synthesized amylase from the accumulated amylase in secretory granules by short time pulse and chased with ³⁵S-amino acid. The newly synthesized amylase was secreted without stimulation from secretory vesicles in rat parotid acinar cells. The secretion process did not include microtubules, but was related to microfilaments. p-Nitrophenyl β-xyloside, an inhibitor of proteoglycan synthesis, inhibited the newly synthesized amylase secretion. This indicated that the newly synthesized amylase was secreted from secretory vesicles, not via the constitutive-like secretory route, which includes the immature secretory granules, and that proteoglycan synthesis was required for secretory vesicle formation.     

High extracellular glucose inhibits exocytosis through disruption of syntaxin 1A-containing lipid rafts

Diabetes is characterized by high blood glucose which eventually impairs the secretion of insulin. Glucose directly affects cholesterol biosynthesis and may in turn affect cellular structures that depend on the sterol, including lipid rafts that help organize the secretory apparatus. Here, we investigated the long-term effects of glucose upon lipid rafts and secretory granule dynamics in pancreatic β-cells. Raft fractions, identified by the presence of GM1 and flotillin, contained characteristically high levels of cholesterol and syntaxin 1A, the t-SNARE which tethers granules to the plasma membrane. Seventy-two hours exposure to 28 mM glucose resulted in ∼30% reduction in membrane cholesterol, with consequent redistribution of raft markers and syntaxin 1A throughout the plasma membrane. Live cell imaging indicated loss of syntaxin 1A from granule docking sites, and fewer docked granules. In conclusion, glucose-mediated inhibition of cholesterol biosynthesis perturbs lipid raft stability, resulting in a loss of syntaxin 1A from granule docking sites and inhibition of insulin secretion.