The pathologic activation of inactive zymogens to mature enzymes within the pancreatic acinar cell very likely plays a role in initiating pancreatitis. This proteolytic activation can be detected by a sensitive immunoblot assay developed to detect by the zymogen precursor and mature enzyme forms (JCI 87:363, '91). Multiple zymogens undergo processing to mature enzyme forms after hyper stimulation (10-100 fold concentrations greater than those which cause maximum secretion) with the secretagogue cholecystokinin (CCK). Active forms generated by CCK hyper-stimulation remain in the acinar cell. Autoactivation o of trypsinogen is most likely the event that initiates intracellular zymogen processing. Ethanol dramatically sensitizes the cell to the effects of CCK (AJP 270: G171, '96). Zymogen activation depends on a low pH compartment, a serine protease, and an increase in intracellular Ca2+ and cGMP. Bombesin hyper- stimulation activates zymogens, but unlike CCK, the active enzymes are released from the cell thus separating activation from inhibition of secretion. CCK may cause retention of active enzymes by disrupting the apical actin cytoskeleton, This proposal will examine the role of zymogen activation in causing cell injury, define the intracellular site of zymogen activation, and explore the role of compartmental acidification in this pathologic processing. Preliminary studies demonstrates that CCK caused acinar cell injury in a time and concentration-dependent manner, whereas bombesin does not cause injury. Thus, both zymogen activation and retention within the acinar cell are required to cause injury. To detect the intracellular site of zymogen activation, a highly specific antibody has been generated to the trypsinogen activation peptide (TAP) that does not recognize trypsinogen. In an in vivo hyper-stimulation model of pancreatitis, the antibody detects TAP generation in distinct acinar cell compartments in a time-dependent manner: in small vesicles (100 nM) after 30 minutes of hyper-stimulation and predominantly in large vacuoles (1-3 mu m) by 60 minutes. The proposed studies will explore the proteases involved in generating cellular injury and examine the role of the actin- based cytoskeleton in causing the retention of active enzymes. The effects of selectively disrupting the apical actin cytoskeleton with chimeric form of C3 botulinum toxin on acinar cell cytoskeleton, secretion and injury will be examined. The identify of the compartment containing TAP immunoreactivity will be determined using double-labeling light-level and electron microscopy techniques. Finally, the importance of compartmental acidification and vacuolar ATPases in this pathologic processing will be tested.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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General Medicine A Subcommittee 2 (GMA)
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Serrano, Jose
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Yale University
Internal Medicine/Medicine
Schools of Medicine
New Haven
United States
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Gorelick, Fred S; Lerch, Markus M (2017) Do Animal Models of Acute Pancreatitis Reproduce Human Disease? Cell Mol Gastroenterol Hepatol 4:251-262
Gorelick, Fred S (2016) Advances in pancreatology: 2016. Curr Opin Gastroenterol :
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