Zymogen granule - an overview (2023)

Histology and Ultrastructure

The pancreas is a finely nodular, compound gland that bears some resemblance to the salivary glands in outline. The lobules are visible on gross examination and are connected by fibrous septa that contain the ducts, blood vessels, lymphatic vessels, and nerves. The basic subunit of the exocrine part is the acinus, which at its base is called a spherical mass of dark-stained secretory cells.acinar cells(Abb. 55,4). The spherical acinus is followed by a goblet-shaped neck composed of so-called tubular cells.channel cells. The inner lumen of the acinus forms the final portion of the duct.

Caustic casts of the duct system formed by retrograde injection of latex show that there is an extensive network of ducts of increasing size that drain pancreatic secretions from the lumen of the acinus, through intralobular, interlobular, interlobar, and finally into the main duct of the pancreas (Abb. 55,5).²⁰

The duct system of the pancreas is not striated and is lined by columnar epithelium. Goblet cells and occasionally silver-affinity cells are also present. The larger ducts have a thick wall of connective tissue and elastic fibers. The endocrine part consists of the islets of Langerhans, which on H&E staining are pale-staining spherical clusters of cells. A more detailed description of each of the major parenchymal cells of the pancreas reveals remarkable structure-function relationships.

Under light microscopy, acinar cells are tall columnar or pyramidal epithelial cells whose broad bases rest on a basal lamina and whose apices converge in a central lumen. Numerous eosinophils in the resting stateZymogen-GranulatFill in the apical part of the cell. The basal part of the cells contains 1 or 2 centrally located spherical nuclei and basophilic cytoplasm. The Golgi complex lies between the cell nucleus and the zymogen granules and can be seen as a clear area without staining.

The subcellular structure of acinar cells can be visualized by electron microscopy (Abb. 55,6). The most striking feature of the acinar cell is the dense granules of zymogen concentrated at the apical pole. The tight junctions form a belt-like band around the apical end of the cell and result from attachment of the outer membrane leaflets of neighboring cells. These junctions prevent the backflow of substances secreted from the duct into the intercellular space.²¹Gap junctions, which allow the intercellular flow of small molecules, are distributed on the lateral cell membranes and are formed by the attachment of larger, disc-shaped membrane plates.

Mitochondria are elongated cylindrical structures that appear oval in cross section and contain well-developed cristae and matrix granules.Abb. 55,7). They are concentrated in various parts of the acinar cells, including as a belt around the zymogen granules, the nucleus, and the basolateral membrane.²²The gross ER occupies about 20% of the cell volume.²³It occupies most of the basal region of the acinar cells and interdigitates with the zymogen granules in the apical region. This abundance of coarse ER allows the acinar cell to synthesize more protein than any other parenchymal cell in the body.²⁴The Golgi complex consists of flattened, membranous sacs and small vesicles containing scaly, electron-dense material. The Golgi plays an important role in the transport of secretory proteins and the formation of zymogen granules from maturing condensation vacuoles.

50.5.2 Characterization of zymogen granules

because thatZymogen-Granulatit is one of the components of exocytosis and can be easily purified due to its size and density, accumulating considerable information about its protein composition. Originally, this was acquired molecule by molecule, both for content and membrane proteins. Recently, however, proteomic techniques have been applied, in which proteins are identified by high-throughput peptide fragment mass spectrometry.^318–321This led to the identification of more than 100 proteins in the zymogen granule membrane, although not all of them were confirmed by independent methods. Newly identified proteins included several small G proteins, including Rab27B and Rap1; a SNARE protein, SNAP29; and a myosin, MyoVc. Other unexpected proteins were Tm63A (unknown function) and presenilin 2 and other components of the γ-secretase complex together with APP. In one study, the membrane topology of more than 60 zymogen granule membrane proteins was identified by quantitative proteomics.³²⁰Proteins such as ion channels are not fully represented in the proteomic data, probably due to their hydrophobic nature or low copy number.

pathophysiology

The exact mechanism by which predisposing factors such as ethanol and gallstones produce pancreatitis is not fully understood. Most researchers believe that BP is the end result of abnormal activation of pancreatic enzymes in acinar cells. Immunolocalization studies showed that after 15 minutes of pancreatic injury bothZymogen-Granulatand lysosomes are placed inside the acinar cells. The fact that zymogen-lysosome colocalization occurs before amylase elevation, pancreatic edema, and other markers of pancreatitis are evident suggests that colocalization is an early step in the pathophysiologic process and is not a consequence of pancreatitis. Studies also suggest that the lysosomal enzyme cathepsin B activates trypsin in these colocalization organelles. In vitro and in vivo studies have elucidated a complicated model of acinous cell death induced by premature trypsin activation. In this model, once cathepsin B in lysosomes and trypsinogen in zymogen granules are contacted by colocalization induced by pancreatitis-inducing stimuli, activated trypsin induces leakage from colocalized organelles, releasing cathepsin B into the cytosol. It is the cytosolic cathepsin B that then induces apoptosis or necrosis, resulting in the death of the acinar cells. Thus, acinar cell death and, to some extent, the inflammatory response seen in AP can be prevented when acinar cells are pretreated with cathepsin B inhibitors. In vivo studies have also shown that cathepsin B knockout mice show a reduction significant in the severity of pancreatitis.⁴

Activation of intraacinar pancreatic enzyme induces autodigestion of normal pancreatic parenchyma. In response to this initial insult, acinar cells release pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1, IL-2 and IL-6, in addition to anti-inflammatory mediators as IL-. 10 and IL-1 receptor antagonist. These mediators do not induce pancreatic injury, but spread the response locally and systemically. As a result, TNF-α, IL-1 and IL-6, neutrophils and macrophages are recruited into the pancreatic parenchyma and cause the release of more TNF-α, IL-1 and IL-6, reactive oxygen metabolites, prostaglandins, platelets -activating factor and leukotrienes. The local inflammatory response further aggravates pancreatitis because it increases permeability and impairs pancreatic microcirculation. In severe cases, the inflammatory response causes local bleeding and pancreatic necrosis. Furthermore, some of the inflammatory mediators released by neutrophils aggravate pancreatic injury by causing activation of pancreatic enzymes.

The inflammatory cascade is self-limited in approximately 80% to 90% of patients. In other patients, however, there is a vicious circle of recurrent pancreatic injury and local and systemic inflammatory reactions. A small number of patients experience a massive release of inflammatory mediators into the systemic circulation. Active neutrophils mediate acute lung injury and induce adult respiratory distress syndrome, which is commonly seen in patients with severe pancreatitis. The mortality observed in the early stage of pancreatitis is a result of this ongoing inflammatory response. A summary of the inflammatory cascade seen in PA is shown in FIG.Abb. 56,7.

Exocytosis

The SNARE hypothesis for membrane recognition and fusion, which appears to be a generalized mechanism for all cells studied, is particularly relevant for the pancreatic acinar cell, where specific interactions between the zymogen granule membrane and the apical plasma membrane are required for this. exocytosis of the Ensure that enzymes and proenzymes from the digestive tract enter the acinar lumen. Endobrevin-associated membrane/vesicle protein isoform 8 (VAMP-8) appears to function as a key SNARE vesicle (v-SNARE) for zymogen secretion, whereas VAMP-3 does not appear to play a major role. An as yet unidentified t-SNARE and a 25 kDa synaptosome-associated protein (SNAP-25) presumably exist in the apical plasma membrane of the acinar cell.However, the specific interaction of the zymogen granules with the apical plasma membrane is crucial because if the zymogen granules promiscuously fuse with the basolateral plasma membrane, enzymes and proenzymes from the granule contents could enter the interspace and cause injury upon activation. .

After interaction and binding of the zymogen granules to the t-SNARE/SNAP-25 apical plasma membrane, a pre-fusion complex is formed. Four a-helices, two from SNAP-25 and one from the apical v-SNARE granule and t-SNARE, form a compact helix that contracts the granule and apical membranes in one ATP-requiring step. As a result of the juxtaposition, the cytoplasmic surfaces of the granules and the apical membrane fuse, exposing the hydrophobic nuclei of the two membranes.61). This prefusion pore continues to expand until fusion of the two membranes is complete and the contents of the zymogen granules are free to diffuse into the centroacinic lumen.

Acinar cell dysfunction and disease

Acinar cells make up the bulk of the parenchymal mass and are directly or indirectly responsible for most inflammatory diseases of the pancreas, as their products make up the main contribution to ductal content. The main function of the pancreatic acinar cell is to synthesize and secrete pancreatic digestive enzymes (cf.Chapter 56). The process involves synthesis of a series of pancreatic proenzyme proteins (zymogens) in the rough endoplasmic reticulum (RER), transport of the properly folded proteins to the Golgi apparatus for sorting and packaging into zymogen vesicles, vesicular transport of the zymogens to the tip and apical secretion of zymogens into the pancreatic ducts (Abb. 57.3). The process requires large amounts of energy for protein synthesis and the transport of ions, such as B. calcium, from one compartment to another.

Maintaining low calcium concentrations in acinar cells is crucial to protect them from premature trypsinogen activation. Acinar cell calcium may increase due to neurohormonal overstimulation^61,62; high extracellular calcium concentrations⁶³; Bile acid reflux, which opens the apical membrane calcium pathways^64,65; prolonged high-dose alcohol consumption, which lowers the threshold for pacing-induced BP⁶⁶; mitochondrial damage⁶⁷; and other factors that regulate intracellular calcium.⁶⁸Any process that increases acinar cell calcium predisposes to BP through a calcium-dependent trypsinogen activation and stabilization mechanism.⁶⁸

49.6.1 Exocytosis

The SNARE hypothesis of membrane recognition and fusion, which appears to be a generalized mechanism for all cells studied, is particularly relevant for the pancreatic acinar cell, where specific interactions between the zymogen granule membrane and the apical plasma membrane are required for exocytosis of digestive processes. Enzymes and proenzymes enter the acinar lumen.⁶⁵Endobrevin/VAMP8 appears to function as a key V-SNARE for ZG secretion, whereas VAMP3 does not appear to play a major role. As discussed later, VAMP8 is thought to inhibit basolateral exocytosis, except in conditions of alcoholic pancreatitis, where phosphorylation of Munc18c allows VAMP8 to mediate basolateral exocytosis. An as yet unidentified T-SNARE and SNAP25 presumably exist in the apical plasma membrane of the acinar cell. However, the specific interaction of the zymogen granules with the apical plasma membrane is crucial because if the zymogen granules promiscuously fuse with the basolateral plasma membrane, enzymes and proenzymes from the granule contents could enter the interspace and cause injury upon activation. .

After the interaction and binding of the zymogen granules to the T-SNARE/SNAP25 apical plasma membrane, a pre-fusion complex is formed. Four α-helices, two from SNAP25 and one each from the V-SNARE granules and the apical T-SNARE, form a tightly wound coil that pulls the granules and apical membranes together in an ATP-requiring step. As a result of the juxtaposition, the cytoplasmic surfaces of the granules and the apical membrane fuse, exposing the hydrophobic nuclei of the two membranes.⁶⁶This prefusion pore continues to expand until fusion of the two membranes is complete and the contents of the zymogen granules are free to diffuse into the acinar lumen.

Classical views of regulated exocytosis include complete fusion of the zymogen granules with the apical plasma membrane, followed by a collapse of the granule membrane into the apical plasma membrane, temporarily enlarging the apical plasma membrane. About an hour after withdrawal of the secretagogue, this excess membrane is somehow internalized back into the cell, possibly into the Golgi complex or another compartment for reuse or destruction. The net result is the return of the apical luminal dimensions back to their resting dimension. This type of secretion, also known as "total fusion", is believed to facilitate the initial rapid release of about 5 seconds, followed by the opening of the granules into the acinar lumen, which lasts for 5 minutes or more. This prolonged exposure of the granule lumen can facilitate solubilization and mixing of the highly concentrated zymogen granule contents for slower delivery to the duodenum as needed for digestion of the meal.

An alternative view of pancreatic acinar cell exocytosis is thought to involve a transient fusion of the granule membrane with the apical plasma membrane, allowing limited amounts of secretory proteins to be released into the acinar lumen without the granule membrane collapsing entirely into the apical plasma membrane. This mechanism is also known as "kiss and run", in which the granule membrane is removed more or less intact and refilled for another round of exocytosis. This mechanism has already been extensively demonstrated, mainly in neuronal cells, where there is extremely rapid release of neurotransmitters and the vesicle membranes can be rapidly internalized and refilled, in addition to the complete collapse of neurotransmitter vesicles in the presynaptic membrane, followed by rescue and refilling. ( recycling). ). Applying the kiss-and-run mechanism to the pancreatic acinar cell is problematic because atomic force microscopy images were likely seen from the basolateral membrane and zymogen granules subjected to classical exocytosis do not contain newly synthesized proteins, so there is a problem with the way recovered kiss and run granules absorb newly synthesized proteins, apparently bypassing the known secretory pathway.

A final observed mechanism is compound exocytosis, in which secretory granules fuse before interacting with the apical plasma membrane and fusing.⁶⁷Although the physiological significance of compound exocytosis is not currently understood, it may facilitate more rapid release of zymogen granule contents because the acinar lumen represents a small fraction of the total surface area, limiting the number of granules that can fuse with the membrane. apex at once. What triggers grain-to-grain fusion prior to contact with the apical plasma membrane is not known, but it may involve pH changes, acquisition of various SNARE proteins, etc. included and requires further research.

39.6.1 Exocytosis

the release ofZymogen-GranulatContents in the lumen of the acinus require fusion of the vesicle membrane with the apical plasma membrane. Four main steps are probably involved in this process: approximation of the secretion granules in the apical region of the acinar cell, close to the plasmatic membrane; Binding of secretion granules to the plasmatic membrane; docking and priming, which involves SNARE proteins; and finally the calcium-dependent melting event.⁹⁷

The SNARE hypothesis of membrane recognition and fusion, which appears to be a generalized mechanism for all cells studied, is particularly relevant for the pancreatic acinar cell, where specific interactions between the zymogen granule membrane and the apical plasma membrane are required for exocytosis of digestive processes. Enzymes and proenzymes enter the acinar lumen.¹⁴¹Endobrevin/VAMP8 appears to function as a key V-SNARE for ZG secretion, whereas VAMP3 does not appear to play a major role. As discussed later, VAMP8 is thought to inhibit basolateral exocytosis, except in conditions of alcoholic pancreatitis, where phosphorylation of Munc18c allows VAMP8 to mediate basolateral exocytosis.¹⁴²An as yet unidentified T-SNARE and SNAP25 presumably exist in the apical plasma membrane of the acinar cell. However, the specific interaction of the zymogen granules with the apical plasma membrane is critical because, if the zymogen granules were promiscuously fused with the basolateral plasma membrane, enzymes and proenzymes from the granule contents could enter the interstitial space and cause injury after the activation.

Classical views of regulated exocytosis include the complete fusion of the zymogen granules with the apical plasma membrane, followed by the collapse of the granule membrane into the apical plasma membrane, temporarily enlarging the apical plasma membrane. About an hour after withdrawal of the secretagogue, this excess membrane is internalized back into the cell, possibly into the Golgi complex or another compartment for reuse or destruction.¹⁴³The net result is the return of the apical luminal dimensions back to their resting dimension. This type of secretion, also known as "full fusion", is believed to facilitate the initial rapid discharge of about 5 seconds. followed by opening the granules into the acinar lumen lasting approximately 5 minutes or more. This prolonged exposure of the granule lumen may facilitate solubilization of its contents by fluids and electrolytes from the acinar and ductal cells necessary for delivery of the contents to the duodenum as needed for digestion of the meal.

An alternative view of pancreatic acinar cell exocytosis is thought to involve a transient fusion of the granule membrane with the apical plasma membrane, allowing limited amounts of secretory proteins to be released into the acinar lumen without the granule membrane collapsing entirely into the apical plasma membrane. This mechanism is also known as "kiss and run", whereby the granule membrane is recovered more or less intact and recharged for another round of exocytosis. This mechanism has been extensively demonstrated, particularly in neuronal cells, where extremely rapid release of neurotransmitters occurs and vesicle membranes can be rapidly internalized and recharged, in addition to complete collapse of neurotransmitter vesicles at the presynaptic membrane, followed by rescue and refilling ( recycling). Applying the kiss-and-run mechanism to the pancreatic acinar cell is problematic because atomic force microscopy images were likely seen from the basolateral membrane and zymogen granules subjected to classical exocytosis do not contain newly synthesized protein, so it is problematic how The retrieved kiss-and-run granules absorb newly synthesized proteins, apparently bypassing the known secretory pathway.

A final observed mechanism is that of compound exocytosis, in which secretory granules fuse together (compound exocytosis) before interacting with the apical plasma membrane and fusing.^144.145Although the physiological significance of compound exocytosis is not currently understood, it may facilitate more rapid and efficient release of the contents of zymogen granules, since the acinar lumen represents a small fraction of the total surface area, limiting the number of granules that can be released. may fuse with the apical membrane at any time. What triggers grain-to-grain fusion prior to contact with the apical plasma membrane is not known, but it may involve pH changes, acquisition of various SNARE proteins, etc. included and requires further research.⁹⁷

FURTHER STUDIES

attendance of documentsZymogen-Granulator the production of exocrine enzymes by tumor cells establishes the diagnosis of acinar cell carcinoma. If sufficient, periodic acid-Schiff (PAS) staining with and without diastatic digestion highlights red-stained intracytoplasmic granules. Electron microscopy can also be used to detect granules. Although not widely used, butyrate esterase staining detects lipase activity about 75% of the time, and 75 to 95% of cases stain with antibodies to exocrine enzymes, including trypsin, chymotrypsin, lipase, and elastase. Low and high molecular weight cytokeratins (CAM 5.2 and AE1, respectively) and cytokeratins 8 and 18 stain carcinoma cells, but cytokeratins 7, 19 and 20 are typically negative. As acinar cell carcinomas may contain low numbers of endocrine cells (1–2%), positive immunocytochemical staining with the endocrine markers chromogranin and synaptophysin should not be misinterpreted.

serum lipase

Serum lipase is derivedZymogen-Granulatof pancreatic acinar cells, gastric mucosal cells, hepatocytes, adipocytes and myocytes. Most non-pancreatic lipase activity in serum is produced by hepatocytes and mucosal cells. Normally, an increase in serum lipase three to four times above the reference range indicates damage to the pancreas. In dogs, acute injury produces an increase in lipase activity within 24 hours, peaking after 2 to 5 days. Elevated lipase activity tends to be more sensitive to prolonged longitudinal injury than amylase. Because lipase concentrations in zymogen granules are approximately 4.5 times greater than amylase concentrations, recurrent lesions are more likely to be detected by leakage of lipase into the circulation. Lipase is less frequently elevated than amylase by hepatobiliary and intestinal injury or renal failure and is considered more specific for mild localized exocrine injury in dogs.

Lipase passes the glomerular filtration barrier and is inactivated by the kidney in the proximal tubules in a manner similar to amylase, and like amylase, hyperlipasemia can occur in severe kidney disease. How does pancreatic lipase require Ca²⁺, colipase and bile salts as cofactors for digestion, increased lipase with simultaneous decrease in serum Ca²⁺may indicate concomitant peripancreatic injury and abdominal steatitis. Lipase concentrations are usually measured as serum lipase activity; however, measurement of pancreatic lipase immunoreactivity (PLI) offers the advantage of directly measuring lipase protein concentrations, and PLI has been reported to be more specific for canine exocrine injury than serum lipase activity.

Histopathology

Differentiation of serous acinar cells with cytoplasmZymogen-Granulatidentifies these tumors. Neoplastic serous cells are generally polygonal with slightly basophilic cytoplasm and regular, round, eccentric nuclei. The cytoplasm varies from distinctly granular to finely reticular or foamy.Abb. 12-25). The cells are PAS positive, resistant to diastasis. They do not react or react only weakly with the Mucicarmin stain. Serous cells vary from leaves, often with an organoid pattern, to randomly scattered foci (seeAbb. 12-25). In most tumors, they represent a minority of tumor cells and are sometimes rare.

Other cell types are characterized as intercalated, vacuolated, clear, nonspecific ductal glands. Cubic, eosinophilic to amphophilic cells, similar to intercalated ducts, are smaller than serous cells; have a large nucleus to cytoplasm ratio; and surround luminal spaces ranging from small canal lumen sizes to large cystic spaces.Abb. 12-26). They are found in most tumors.

Variously large clear cytoplasmic vacuoles characterize vacuolated cells. The cytoplasmic membranes of some cells appear to be dilated by vacuoles (Abb. 12-27). Vacuolated cells are present in a minority of tumors but are characteristic.

In contrast to vacuolated cells, clear cells have unstained cytoplasmic compartments. They appear in small aggregates or extended sheets in only about 6% of tumors. They rarely dominate. Clear cells are PAS negative.

Nonspecific glandular cells are amphophilic to eosinophilic, polygonal cells with ill-defined cell borders that occur in syncytial sheets (cf.Abb. 12-25). They do not react with the PAS stain. They occur in most acinar cell adenocarcinomas.

Tumor cell growth patterns in acinar cell adenocarcinoma are identified as solid, microcystic, papillary-cystic and follicular. Many tumors show a mixture of these patterns, although one pattern usually predominates.

In the fixed pattern, closely spaced cells are arranged in layers, nodules, and aggregates (cf.Abb. 12-25). In the microcystic pattern, numerous small spaces of varying size are surrounded by tumor cells.Abb. 12-28). The papillary-cystic pattern shows epithelial proliferations, some of which fill relatively large cystic spaces (cf.Abb. 12-27). One to several cells line papillae and luminal surfaces, which sometimes bulge into the lumen and produce the appearance of a "tombstone row" (cf.Abb. 12-27). Interspersed duct-like and vacuolated cells usually predominate. The follicular pattern resembles that of large thyroid follicles and is characterized by multiple cysts lined by cells similar to intercalated ducts and containing eosinophilic proteinaceous material (cf.Abb. 12-26). The luminal material reacts with the PAS stain. The follicular pattern is the least common architectural pattern.

Rare acinar cell adenocarcinomas have areas of undifferentiated carcinoma and are often referred to as "undifferentiated" acinar cell adenocarcinomas.⁸¹The biological potential and treatment of these tumors are determined by the undifferentiated carcinoma component.

Many acinar cell adenocarcinomas are associated with prominent lymphoid infiltrates of their stroma (cf.Abb. 12-25).⁴³The stroma ranges from delicate fibrovascular tissue to dense collagen. Hemorrhage and hemosiderin are prominent in some tumors, and tumor cells sometimes contain cytoplasmic hemosiderin granules. Most infiltrate adjacent normal tissue.