Pavlov and the zymogen granule

Radioautographs of pancreatic acinar cells were prepared in rats and mice sacrificed at various times after injection of leucine-, glycine-, or methionine-H3. Measurements of radioactivity concentration (number of silver grains per unit area) and relative protein concentration (by microspectrophotometry of Millon-treated sections) yielded the mean specific activity of proteins in various regions of the acinar cells. The 2 to 5 minute radioautographs as well as the specific activity time curves demonstrate protein synthesis in ergastoplasm. From there, most newly synthesized proteins migrate to and accumulate in the Golgi zone. Then they spread to the whole zymogen region and, finally, enter the excretory ducts. An attempt at estimating turnover times indicated that two classes of proteins are synthesized in the ergastoplasm: "sedentary" with a slow turnover (62.5 hours) and "exportable" with rapid turnover (4.7 minutes). It is estimated that the exportable proteins spend approximately 11.7 minutes in the Golgi zone where they are built up into zymogen granules, and thereafter 36.0 minutes as fully formed zymogen granules, before they are released outside the acinar cell as pancreatic secretion. The mean life span of a zymogen granule in the cell is estimated to be 47.7 minutes.

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Functional role of J domain of cysteine string protein in Ca2+-dependent secretion from acinar cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.90592.2008 ◽

2009 ◽

Vol 296 (5) ◽

pp. G1030-G1039 ◽

Cited By ~ 19

Author(s):

Ning Weng ◽

Megan D. Baumler ◽

Diana D. H. Thomas ◽

Michelle A. Falkowski ◽

Leigh Anne Swayne ◽

...

Keyword(s):

Membrane Protein ◽

Secretory Pathway ◽

Digestive Enzyme ◽

Family Members ◽

Protective Role ◽

Amylase Secretion ◽

Zymogen Granule ◽

Hsp70 Family ◽

Cysteine String Protein ◽

J Domain

The heat shock protein 70 family members Hsc70 and Hsp70 are known to play a protective role against the onset of experimental pancreatitis, yet their molecular function in acini is unclear. Cysteine string protein (CSP-α) is a zymogen granule (ZG) membrane protein characterized by an NH2-terminal “J domain” and a central palmitoylated string of cysteine residues. The J domain functions as a cochaperone by modulating the activity of Hsc70/Hsp70 family members. A role for CSP-α in regulating digestive enzyme exocytosis from pancreas was investigated by introducing CSP-α truncations into isolated acini following their permeabilization with Perfringolysin O. Incubation of acini with CSP-α1-82, containing the J domain, significantly augmented Ca2+-stimulated amylase secretion. Effects of CSP-α1-82 were concentration dependent, with a maximum 80% increase occurring at 200 μg/ml of protein. Although CSP-α1-82 had no effects on basal secretion measured in the presence of ≤10 nM free Ca2+, it did significantly augment GTP-γS-induced secretion under basal Ca2+ conditions by ∼25%. Mutation of the J domain to abolish its cochaperone activity failed to augment Ca2+-stimulated secretion, implicating the CSP-α/Hsc70 cochaperone system as a regulatory component of the secretory pathway. CSP-α physically associates with vesicle-associated membrane protein 8 (VAMP 8) on ZGs, and the CSP-α-VAMP 8 interaction was dependent on amino acids 83-112 of CSP-α. Immunofluorescence analysis of acinar lobules or purified ZGs confirmed the CSP-α colocalization with VAMP 8. These data establish a role for CSP-α in regulating digestive enzyme secretion and suggest that CSP-α and Hsc70 modulate specific soluble N-ethylmaleimide-sensitive attachment receptor interactions necessary for exocytosis.

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Immunocytochemical assays of amylase and chymotrypsinogen in rat pancreas secretory granules. Efficacy of using immunogold-labeled ultrathin cryosections to estimate relative protein concentrations.

Journal of Histochemistry & Cytochemistry ◽

10.1177/32.10.6207220 ◽

1984 ◽

Vol 32 (10) ◽

pp. 1028-1034 ◽

Cited By ~ 27

Author(s):

G Posthuma ◽

J W Slot ◽

H J Geuze

Keyword(s):

Secretory Granules ◽

Relative Concentration ◽

Data Sets ◽

Zymogen Granule ◽

Protein Levels ◽

Pancreatic Cells ◽

Intracellular Proteins ◽

Ultrathin Cryosections ◽

Respective Protein ◽

Normal Laboratory

An exploration was conducted as to whether the relative concentration of two intracellular proteins could be evaluated quantitatively from their labeling densities in ultrathin cryosections labeled with the immunogold technique. As a model rat pancreatic cells were used in which the content of amylase (Am) and chymotrypsinogen (Ch) was experimentally altered. Rats were fed either normal laboratory chow or food containing soybean trypsin inhibitor (STI), which affects the Am/Ch ratio in the tissues. The changes in Am and Ch protein levels and enzyme activities were measured biochemically in cell suspension homogenates or in zymogen granule fractions. Within 5 days a maximal change in the Am/Ch was observed as a result of adaptation to the STI diet. The Am/Ch ratio determined biochemically was compared with that from counts of gold particles bound to the respective protein in immunogold-labeled cryosections. The two data sets matched fairly well, indicating that the intensity of the immunoreaction is a reliable reflection of antigen concentration in this system.

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Role of sulfated O-linked glycoproteins in zymogen granule formation

Journal of Cell Science ◽

10.1242/jcs.115.14.2941 ◽

2002 ◽

Vol 115 (14) ◽

pp. 2941-2952 ◽

Cited By ~ 2

Author(s):

Robert C. De Lisle

Keyword(s):

Acinar Cell ◽

Secretory Granules ◽

Sodium Chlorate ◽

Pancreatic Acinar Cell ◽

Secretory Proteins ◽

Zymogen Granule ◽

Acidic Ph ◽

Zymogen Granules ◽

Granule Formation

Packaging of proteins into regulated secretory granules is mediated by the mildly acidic pH of the trans Golgi network and immature secretory granules. This need for an acidic pH indicates that ionic interactions are important. The mouse pancreatic acinar cell contains four major sulfated glycoproteins,including the zymogen granule structural component Muclin. I tested the hypothesis that sulfation and the O-linked glycosylation to which the sulfates are attached are required for normal formation of zymogen granules in the exocrine pancreas. Post-translational processing was perturbed with two chemicals: sodium chlorate was used to inhibit sulfation and benzyl-N-acetyl-α-galactosaminide was used to inhibit O-linked oligosaccharide elongation. Both chemicals resulted in the accumulation in the Golgi region of the cell of large vacuoles that appear to be immature secretory granules, and the effect was much more extensive with benzyl-N-acetyl-α-galactosaminide than chlorate. Both chemical treatments inhibited basal secretion at prolonged chase times, and again benzyl-N-acetyl-α-galactosaminide had a greater effect than chlorate. In addition, benzyl-N-acetyl-α-galactosaminide, but not chlorate, totally inhibited stimulated secretion of newly synthesized proteins. These data provide evidence for a role of sulfated O-linked glycoproteins in protein condensation and maturation of zymogen granules. Under maximal inhibition of O-linked oligosaccharide biosynthesis, anterograde post-Golgi traffic in the regulated pathway is almost totally shut down, demonstrating the importance of these post-translational modifications in progression of secretory proteins through the regulated pathway and normal granule formation in the pancreatic acinar cell.

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