Activation of Trypsinogen in Large Endocytic Vacuoles of Pancreatic Acinar Cells
The intracellular activation of trypsinogen, which is both pH- and calcium-dependent, is an important early step in the development of acute pancreatitis. The cellular compartment in which trypsinogen activation occurs currently is unknown. We therefore investigated the site of intracellular trypsin...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2007-03, Vol.104 (13), p.5674-5679 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Sherwood, Mark W. Prior, Ian A. Voronina, Svetlana G. Barrow, Stephanie L. Woodsmith, Jonathan D. Gerasimenko, Oleg V. Petersen, Ole H. Tepikin, Alexei V. |
| description | The intracellular activation of trypsinogen, which is both pH- and calcium-dependent, is an important early step in the development of acute pancreatitis. The cellular compartment in which trypsinogen activation occurs currently is unknown. We therefore investigated the site of intracellular trypsinogen activation by using an established cellular model of acute pancreatitis: supramaximal stimulation of pancreatic acinar cells with cholecystokinin. We used fluorescent dextrans as fluid phase tracers and observed the cholecystokinin-elicited formation and translocation of large endocytic vacuoles. The fluorescent probe rhodamine 110 bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide) dihydrochloride (BZiPAR) was used to detect trypsinogen activation. Fluid phase tracers were colocalized with cleaved BZiPAR, indicating that trypsinogen activation occurred within endocytic vacuoles. The development of BZiPAR fluorescence was inhibited by the trypsin inhibitor benzamidine. Fluorescein dextran and Oregon Green 488 BAPTA-5N were used to measure endosomal pH and calcium, respectively. The pH in endocytic vacuoles was 5.9 ± 0.1, and the calcium ion concentration was 37 ± 11 μM. The caged calcium probe o-nitrophenyl EGTA and UV uncaging were used to increase calcium in endocytic vacuoles. This increase of calcium caused by calcium uncaging was followed by recovery to the prestimulated level within ≈100 s. We propose that the initiation of acute pancreatitis depends on endocytic vacuole formation and trypsinogen activation in this compartment. |
| doi_str_mv | 10.1073/pnas.0700951104 |
| format | Article |
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The cellular compartment in which trypsinogen activation occurs currently is unknown. We therefore investigated the site of intracellular trypsinogen activation by using an established cellular model of acute pancreatitis: supramaximal stimulation of pancreatic acinar cells with cholecystokinin. We used fluorescent dextrans as fluid phase tracers and observed the cholecystokinin-elicited formation and translocation of large endocytic vacuoles. The fluorescent probe rhodamine 110 bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide) dihydrochloride (BZiPAR) was used to detect trypsinogen activation. Fluid phase tracers were colocalized with cleaved BZiPAR, indicating that trypsinogen activation occurred within endocytic vacuoles. The development of BZiPAR fluorescence was inhibited by the trypsin inhibitor benzamidine. Fluorescein dextran and Oregon Green 488 BAPTA-5N were used to measure endosomal pH and calcium, respectively. The pH in endocytic vacuoles was 5.9 ± 0.1, and the calcium ion concentration was 37 ± 11 μM. The caged calcium probe o-nitrophenyl EGTA and UV uncaging were used to increase calcium in endocytic vacuoles. This increase of calcium caused by calcium uncaging was followed by recovery to the prestimulated level within ≈100 s. We propose that the initiation of acute pancreatitis depends on endocytic vacuole formation and trypsinogen activation in this compartment.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.0700951104</identifier><identifier>PMID: 17363470</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>Acinar cells ; Alcoholic pancreatitis ; Animals ; Benzamidines ; Biological Sciences ; Body fluids ; Calcium ; Calcium - metabolism ; Cell membranes ; Cells ; Cells, Cultured ; Dextrans ; Dextrans - chemistry ; Endocytosis ; Endosomes ; Enzyme Activation ; Excretory system ; Fluorescence ; Fluorescent Dyes - pharmacology ; Hydrogen-Ion Concentration ; Medical disorders ; Mice ; Pancreas ; Pancreas - cytology ; Pancreatitis - metabolism ; Protein Transport ; Trypsin - chemistry ; Trypsinogen - metabolism ; Vacuoles ; Vacuoles - metabolism</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2007-03, Vol.104 (13), p.5674-5679</ispartof><rights>Copyright 2007 The National Academy of Sciences of the United States of America</rights><rights>Copyright National Academy of Sciences Mar 27, 2007</rights><rights>2007 by The National Academy of Sciences of the USA 2007</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c594t-9a364489d62bdd408f35f592b4450006a6f0b90bee22b2d5c8e9edebdcef575d3</citedby><cites>FETCH-LOGICAL-c594t-9a364489d62bdd408f35f592b4450006a6f0b90bee22b2d5c8e9edebdcef575d3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/104/13.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/25427254$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/25427254$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,803,885,27924,27925,53791,53793,58017,58250</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17363470$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sherwood, Mark W.</creatorcontrib><creatorcontrib>Prior, Ian A.</creatorcontrib><creatorcontrib>Voronina, Svetlana G.</creatorcontrib><creatorcontrib>Barrow, Stephanie L.</creatorcontrib><creatorcontrib>Woodsmith, Jonathan D.</creatorcontrib><creatorcontrib>Gerasimenko, Oleg V.</creatorcontrib><creatorcontrib>Petersen, Ole H.</creatorcontrib><creatorcontrib>Tepikin, Alexei V.</creatorcontrib><title>Activation of Trypsinogen in Large Endocytic Vacuoles of Pancreatic Acinar Cells</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>The intracellular activation of trypsinogen, which is both pH- and calcium-dependent, is an important early step in the development of acute pancreatitis. The cellular compartment in which trypsinogen activation occurs currently is unknown. We therefore investigated the site of intracellular trypsinogen activation by using an established cellular model of acute pancreatitis: supramaximal stimulation of pancreatic acinar cells with cholecystokinin. We used fluorescent dextrans as fluid phase tracers and observed the cholecystokinin-elicited formation and translocation of large endocytic vacuoles. The fluorescent probe rhodamine 110 bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide) dihydrochloride (BZiPAR) was used to detect trypsinogen activation. Fluid phase tracers were colocalized with cleaved BZiPAR, indicating that trypsinogen activation occurred within endocytic vacuoles. The development of BZiPAR fluorescence was inhibited by the trypsin inhibitor benzamidine. Fluorescein dextran and Oregon Green 488 BAPTA-5N were used to measure endosomal pH and calcium, respectively. The pH in endocytic vacuoles was 5.9 ± 0.1, and the calcium ion concentration was 37 ± 11 μM. The caged calcium probe o-nitrophenyl EGTA and UV uncaging were used to increase calcium in endocytic vacuoles. This increase of calcium caused by calcium uncaging was followed by recovery to the prestimulated level within ≈100 s. We propose that the initiation of acute pancreatitis depends on endocytic vacuole formation and trypsinogen activation in this compartment.</description><subject>Acinar cells</subject><subject>Alcoholic pancreatitis</subject><subject>Animals</subject><subject>Benzamidines</subject><subject>Biological Sciences</subject><subject>Body fluids</subject><subject>Calcium</subject><subject>Calcium - metabolism</subject><subject>Cell membranes</subject><subject>Cells</subject><subject>Cells, Cultured</subject><subject>Dextrans</subject><subject>Dextrans - chemistry</subject><subject>Endocytosis</subject><subject>Endosomes</subject><subject>Enzyme Activation</subject><subject>Excretory system</subject><subject>Fluorescence</subject><subject>Fluorescent Dyes - pharmacology</subject><subject>Hydrogen-Ion Concentration</subject><subject>Medical disorders</subject><subject>Mice</subject><subject>Pancreas</subject><subject>Pancreas - cytology</subject><subject>Pancreatitis - metabolism</subject><subject>Protein Transport</subject><subject>Trypsin - chemistry</subject><subject>Trypsinogen - metabolism</subject><subject>Vacuoles</subject><subject>Vacuoles - metabolism</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0b9vEzEUB3ALgWgozEzAiaFiSfv8-7xUiqIWkCLRobBaPp8vOLrYwb6ryH-PT4kaYIDFlp4_7yvbD6HXGC4xSHq1CyZfggRQHGNgT9AMg8JzwRQ8RTMAIuc1I-wMvch5AxOr4Tk6w5IKyiTM0N3CDv7BDD6GKnbVfdrvsg9x7ULlQ7Uyae2qm9BGux-8rb4ZO8be5YnemWCTM1N5YX0wqVq6vs8v0bPO9Nm9Ou7n6Ovtzf3y03z15ePn5WI1t1yxYa4MFYzVqhWkaVsGdUd5xxVpGOPlnsKIDhoFjXOENKTltnbKta5preu45C09R9eH3N3YbF0phyGZXu-S35q019F4_edJ8N_1Oj5oXNOa1aIEXBwDUvwxujzorc-2PMEEF8esJVBMVS3_C7ESXFBMCnz_F9zEMYXyC5pAyWKSQ0FXB2RTzDm57vHKGPQ0Uz3NVJ9mWjre_v7Skz8OsYAPRzB1nuKYxlRzIZnuxr4f3M-h0Hf_pkW8OYhNHmJ6JIQzIstCfwHPO794</recordid><startdate>20070327</startdate><enddate>20070327</enddate><creator>Sherwood, Mark W.</creator><creator>Prior, Ian A.</creator><creator>Voronina, Svetlana G.</creator><creator>Barrow, Stephanie L.</creator><creator>Woodsmith, Jonathan D.</creator><creator>Gerasimenko, Oleg V.</creator><creator>Petersen, Ole H.</creator><creator>Tepikin, Alexei V.</creator><general>National Academy of Sciences</general><general>National Acad Sciences</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20070327</creationdate><title>Activation of Trypsinogen in Large Endocytic Vacuoles of Pancreatic Acinar Cells</title><author>Sherwood, Mark W. ; 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The cellular compartment in which trypsinogen activation occurs currently is unknown. We therefore investigated the site of intracellular trypsinogen activation by using an established cellular model of acute pancreatitis: supramaximal stimulation of pancreatic acinar cells with cholecystokinin. We used fluorescent dextrans as fluid phase tracers and observed the cholecystokinin-elicited formation and translocation of large endocytic vacuoles. The fluorescent probe rhodamine 110 bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide) dihydrochloride (BZiPAR) was used to detect trypsinogen activation. Fluid phase tracers were colocalized with cleaved BZiPAR, indicating that trypsinogen activation occurred within endocytic vacuoles. The development of BZiPAR fluorescence was inhibited by the trypsin inhibitor benzamidine. Fluorescein dextran and Oregon Green 488 BAPTA-5N were used to measure endosomal pH and calcium, respectively. The pH in endocytic vacuoles was 5.9 ± 0.1, and the calcium ion concentration was 37 ± 11 μM. The caged calcium probe o-nitrophenyl EGTA and UV uncaging were used to increase calcium in endocytic vacuoles. This increase of calcium caused by calcium uncaging was followed by recovery to the prestimulated level within ≈100 s. We propose that the initiation of acute pancreatitis depends on endocytic vacuole formation and trypsinogen activation in this compartment.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>17363470</pmid><doi>10.1073/pnas.0700951104</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Acinar cells Alcoholic pancreatitis Animals Benzamidines Biological Sciences Body fluids Calcium Calcium - metabolism Cell membranes Cells Cells, Cultured Dextrans Dextrans - chemistry Endocytosis Endosomes Enzyme Activation Excretory system Fluorescence Fluorescent Dyes - pharmacology Hydrogen-Ion Concentration Medical disorders Mice Pancreas Pancreas - cytology Pancreatitis - metabolism Protein Transport Trypsin - chemistry Trypsinogen - metabolism Vacuoles Vacuoles - metabolism |
| title | Activation of Trypsinogen in Large Endocytic Vacuoles of Pancreatic Acinar Cells |
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