Parotid Secretory Granules: Crossroads of Secretory Pathways and Protein Storage

Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins—including amylase, proline-rich proteins, and parotid secretory protein (PSP)—to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols aimed at targeting therapeutic proteins either to the oral cavity or to circulation. To be successful, such protocols must be based on a solid understanding of protein trafficking in salivary gland cells. In this paper, model systems available to study the secretion of salivary proteins are reviewed. Parotid secretory proteins are stored in large dense-core secretory granules that undergo stimulated secretion in response to extracellular stimulation. Secretory proteins that are not stored in large secretory granules are secreted by either the minor regulated secretory pathway, constitutive secretory pathways (apical or basolateral), or the constitutive-like secretory pathway. It is proposed that the maturing secretory granules act as a distribution center for secretory proteins in salivary acinar cells. Protein distribution or sorting is thought to involve their selective retention during secretory granule maturation. Unlike regulated secretory proteins in other cell types, salivary proteins do not exhibit calcium-induced aggregation. Instead, sulfated proteoglycans play a role in the storage of secretory proteins in parotid acinar cells. This work suggests that unique sorting and retention mechanisms are responsible for the distribution of secretory proteins to different secretory pathways from the maturing secretory granules in parotid acinar cells.

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Immunohistochemical localization of pancreatic exocrine enzymes in normal and neoplastic pancreatic acinar epithelium of rat.

Journal of Histochemistry & Cytochemistry ◽

10.1177/29.2.6166657 ◽

1981 ◽

Vol 29 (2) ◽

pp. 309-313 ◽

Cited By ~ 8

Author(s):

L J Hansen ◽

M Mangkornkanok/Mark ◽

J K Reddy

Keyword(s):

Secretory Granules ◽

Acinar Cells ◽

Immunohistochemical Localization ◽

Model Systems ◽

Secretory Process ◽

Secretory Proteins ◽

Variable Extent ◽

Immunofluorescent Technique ◽

Pancreatic Exocrine ◽

Indirect Immunofluorescent

The transplantable pancreatic acinar carcinomas of rat established recently provide useful model systems to examine the composition of secretory proteins as well as the secretory process in transformed pancreatic exocrine epithelium. The neoplastic acinar cells exhibit considerable variation in the extent of cytodifferentiation. In the present study the enzymatic profile of this heterogeneous tumor cell population has been investigated by the indirect immunofluorescent technique using antibodies against six pancreatic enzymes. By immunofluorescence, all neoplastic cells stained positively for the six enzymes tested: amylase, lipase, carboxypeptidase A, chymotrypsinogen, trypsinogen, and ribonuclease. Some variability in the intensity of immunofluorescence was noted, suggesting possible quantitative differences in the content of a given enzyme among tumor cells. These observations suggest that neoplastic acinar cells with or without secretory granules contain secretory proteins, but to a variable extent.

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Molecular chaperones in pancreatic tissue: the presence of cpn10, cpn60 and hsp70 in distinct compartments along the secretory pathway of the acinar cells

Journal of Cell Science ◽

10.1242/jcs.107.3.539 ◽

1994 ◽

Vol 107 (3) ◽

pp. 539-549 ◽

Cited By ~ 1

Author(s):

C.S. Velez-Granell ◽

A.E. Arias ◽

J.A. Torres-Ruiz ◽

M. Bendayan

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Secretory Pathway ◽

Secretory Granules ◽

Acinar Cells ◽

Pancreatic Tissue ◽

Secretory Proteins ◽

Trans Golgi Network ◽

Hsp70 Protein ◽

Golgi Network

Three chaperones, the chaperonins cpn10 and cpn60, and the hsp70 protein, were revealed by immunochemistry and cytochemistry in pancreatic rat acinar cells. Western immunoblotting analysis of rat pancreas homogenates has shown that antibodies against cpn10, cpn60 and hsp70 protein recognize single protein bands of 25 kDa, 60 kDa and 70 kDa, respectively. Single bands for the cpn10 and cpn60 were also detected in pancreatic juice. Immunofluorescence studies on rat pancreatic tissue revealed a strong positive signal in the apical region of the acinar cells for cpn10 and cpn60, while an immunoreaction was detected at the juxtanuclear Golgi region with the anti-hsp70 antibody. Immunocytochemical gold labeling confirmed the presence of these three chaperones in distinct cell compartments of pancreatic acinar cells. Chaperonin 10 and cpn60 were located in the endoplasmic reticulum, Golgi apparatus, condensing vacuoles and secretory granules. Interestingly, the labeling for both cpn10 and cpn60 followed the increasing concentration gradient of secretory proteins along the RER-Golgi-granule secretory pathway. On the contrary, the labeling for hsp70 was mainly concentrated in the endoplasmic reticulum and the Golgi apparatus. In the latter, the hsp70 was found to be primary located in the trans-most cisternae and to colocalize with acid phosphatase in the trans-Golgi network. The three chaperones were also present in mitochondria. In view of the role played by the chaperones in the proper folding, sorting and aggregation of proteins, we postulate that hsp70 assists the adequate sorting and packaging of proteins from the ER to the trans-Golgi network while cpn10 and cpn60 play key roles in the proper packaging and aggregation of secretory proteins as well as, most probably, in the prevention of early enzyme activation in secretory granules.

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Secretory proteins without a transport signal are retained in secretory granules during maturation in rat parotid acinar cells

Archives of Oral Biology ◽

10.1016/j.archoralbio.2015.01.006 ◽

2015 ◽

Vol 60 (4) ◽

pp. 642-649 ◽

Cited By ~ 2

Author(s):

Osamu Katsumata-Kato ◽

Megumi Yokoyama ◽

Miwako Matsuki-Fukushima ◽

Takanori Narita ◽

Hiroshi Sugiya ◽

...

Keyword(s):

Secretory Granules ◽

Acinar Cells ◽

Secretory Proteins ◽

Parotid Acinar Cells ◽

Rat Parotid

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Regulated and constitutive secretion of distinct molecular forms of acetylcholinesterase from PC12 cells

Journal of Cell Science ◽

10.1242/jcs.106.3.731 ◽

1993 ◽

Vol 106 (3) ◽

pp. 731-740 ◽

Cited By ~ 2

Author(s):

E.S. Schweitzer

Keyword(s):

Pc12 Cells ◽

Secretory Pathway ◽

Intracellular Localization ◽

Synaptic Function ◽

Molecular Forms ◽

Secretory Proteins ◽

Secretory Pathways ◽

Regulated Secretion ◽

Constitutive Secretion ◽

A Cell

PC12 cells secrete the enzyme acetylcholinesterase (AChE) while at rest, and increase the overall rate of this secretion 2-fold upon depolarization. This behavior is different from the release of other markers by the constitutive or regulated secretory pathways in PC12 cells. Both the resting and stimulated release of AChE are unchanged after treatment with a membrane-impermeable esterase inhibitor, demonstrating that it represents true secretion and not shedding from the cell surface. The stimulation release of AChE is Ca(2+)-dependent, while the unstimulated release is not. Analysis of the molecular forms of AChE secreted by PC12 cells indicates that the release of AChE actually involves two concurrent but independent secretory processes, and that the G4 form of the enzyme is secreted constitutively, while both the G2 and G4 forms are secreted in a regulated manner, presumably from regulated secretory vesicles. Compared with other regulated secretory proteins, a much smaller fraction of cellular AChE is secreted, and the intracellular localization of this enzyme differs from that of other regulated secretory proteins. The demonstration that a cell line that exhibits regulated secretion of acetylcholine (ACh) is also capable of regulated secretion of AChE provides additional evidence for the existence of multiple regulated secretory pathways within a single cell. Moreover, there appears to be a selective packaging of different molecular forms of AChE into the regulated versus the constitutive secretory pathway. Both the specificity of sorting of AChE and the regulation of its secretion suggest that AChE may play a more dynamic role in synaptic function than has been recognized previously.

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Sorting and storage during secretory granule biogenesis: looking backward and looking forward

Biochemical Journal ◽

10.1042/bj3320593 ◽

1998 ◽

Vol 332 (3) ◽

pp. 593-610 ◽

Cited By ~ 363

Author(s):

Peter ARVAN ◽

David CASTLE

Keyword(s):

Secretory Pathway ◽

Molecular Mechanisms ◽

Secretory Granules ◽

Secretory Proteins ◽

Membrane Composition ◽

Granule Formation ◽

Granule Membrane ◽

Regulated Secretory Pathway ◽

Secretory Products

Secretory granules are specialized intracellular organelles that serve as a storage pool for selected secretory products. The exocytosis of secretory granules is markedly amplified under physiologically stimulated conditions. While granules have been recognized as post-Golgi carriers for almost 40 years, the molecular mechanisms involved in their formation from the trans-Golgi network are only beginning to be defined. This review summarizes and evaluates current information about how secretory proteins are thought to be sorted for the regulated secretory pathway and how these activities are positioned with respect to other post-Golgi sorting events that must occur in parallel. In the first half of the review, the emerging role of immature secretory granules in protein sorting is highlighted. The second half of the review summarizes what is known about the composition of granule membranes. The numerous similarities and relatively limited differences identified between granule membranes and other vesicular carriers that convey products to and from the plasmalemma, serve as a basis for examining how granule membrane composition might be established and how its unique functions interface with general post-Golgi membrane traffic. Studies of granule formation in vitro offer additional new insights, but also important challenges for future efforts to understand how regulated secretory pathways are constructed and maintained.

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An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

The Journal of Cell Biology ◽

10.1083/jcb.109.1.17 ◽

1989 ◽

Vol 109 (1) ◽

pp. 17-34 ◽

Cited By ~ 73

Author(s):

P Rosa ◽

U Weiss ◽

R Pepperkok ◽

W Ansorge ◽

C Niehrs ◽

...

Keyword(s):

Secretory Pathway ◽

Secretory Granules ◽

Intracellular Localization ◽

Secretory Protein ◽

Secretory Proteins ◽

Chromogranin B ◽

Double Labeling ◽

Protein Protein Interaction ◽

And Function ◽

Secretogranin I

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.

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PKA inhibitor, H-89, affects the intracellular transit of regulated secretory proteins in rat lacrimal glands

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.1.c262 ◽

1998 ◽

Vol 274 (1) ◽

pp. C262-C271 ◽

Cited By ~ 22

Author(s):

P. Robin ◽

B. Rossignol ◽

M. N. Raymond

Keyword(s):

Protein Kinase ◽

Secretory Pathway ◽

Kinase Inhibitors ◽

Secretory Granules ◽

Protein Kinase Inhibitors ◽

Secretory Proteins ◽

Lacrimal Glands ◽

Calphostin C ◽

A 23187 ◽

Golgi Network

We tested the effect of H-89, a protein kinase A (PKA) inhibitor, on the intracellular transit of the regulated secretory proteins in rat lacrimal glands. We show that H-89, by itself, induces the secretion of newly synthesized proteins trafficking in its presence but not of proteins already stored in the mature secretory granules. This secretion does not depend on the presence of extracellular Ca2+. The proteins released are identical to those secreted after cholinergic stimulation or under the action of the ionophore A-23187, but the secretion level is ∼40% lower. The effect of H-89 seems to be due to PKA inhibition because other protein kinase inhibitors (calphostin C, chelerythrine, H-85) do not induce secretion. We further show that H-89 does not modify the rate of glycoprotein galactosylation but induces the secretion of newly galactosylated glycoproteins. Finally, we used a “20°C block” procedure to show that H-89 affects a trans-Golgi network (TGN) or post-TGN step of the secretory pathway. Our results demonstrate that, in lacrimal cells, H-89 affects the intracellular trafficking of secretory proteins, suggesting a role for PKA in this process.

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Regulation of renin processing and secretion: chemiosmotic control and novel secretory pathway

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.2.c305 ◽

1993 ◽

Vol 265 (2) ◽

pp. C305-C320 ◽

Cited By ~ 35

Author(s):

J. A. King ◽

D. J. Lush ◽

J. C. Fray

Keyword(s):

Secretory Pathway ◽

Secretory Granules ◽

Surface Membrane ◽

Water Flux ◽

Cellular Level ◽

Macula Densa ◽

Secretory Proteins ◽

Juxtaglomerular Cells ◽

Cl Channels ◽

Health And Disease

The renin-angiotensin-aldosterone system (RAAS) plays an important role in cardiovascular and electrolyte regulation in health and disease. Juxtaglomerular cells in the kidney regulate endocrine RAAS by physiologically controlling conversion of prorenin and secretion of renin. The classical baroceptor, neurogenic, and macula densa mechanisms regulate renin expression at the cellular level by Ca2+, adenosine 3',5'-cyclic monophosphate (cAMP), and chemiosmotic forces (K+, Cl-, and water flux coupled to H+ movement). The baroceptor mechanism (through Ca2+) activates K+ and Cl- channels in the surface membrane and deactivates a KCl-H+ exchange chemiosmotic transporter in the secretory granular membrane. The neurogenic mechanism (through cAMP) promotes prorenin processing to renin. The macula densa mechanism (through K+ and Cl-) involves the processing of prorenin to renin. Ca2+, by inhibiting the KCl-H+ exchange transporter, prevents secretory granules from engaging in chemiosmotically mediated exocytosis. cAMP, on the other hand, by stimulating H+ influx, provides the acidic granular environment for prorenin processing to renin. It is concluded that, in the presence of a favorable chemiosmotic environment, prorenin is processed to renin, which may then be secreted by regulative degranulation or divergence translocation, a novel secretory pathway used by several secretory proteins, including renin.

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In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules.

The Journal of Cell Biology ◽

10.1083/jcb.105.2.659 ◽

1987 ◽

Vol 105 (2) ◽

pp. 659-668 ◽

Cited By ~ 37

Author(s):

T L Burgess ◽

C S Craik ◽

L Matsuuchi ◽

R B Kelly

Keyword(s):

Signal Peptide ◽

Secretory Pathway ◽

Protein Targeting ◽

Secretory Granules ◽

Tumor Cell Line ◽

Signal Peptidase ◽

Secretory Proteins ◽

In Vitro Mutagenesis ◽

Regulated Secretory Pathway

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.

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