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The Initiating Mechanism of Premature Trypsin Activation in Pancreatitis Kai Yang

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The Initiating Mechanism of Premature Trypsin Activation in Pancreatitis Kai Yang Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2004 The Initiating Mechanism of Premature Trypsin Activation in Pancreatitis Kai Yang Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES THE INITIATING MECHANISM OF PREMATURE TRYPSIN ACTIVATION IN PANCREATITIS By KAI YANG A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science Degree Awarded: Summer Semester, 2004 The members of the Committee approve the Thesis of Kai Yang defended on 21 April, 2004. Wei-Chun Chin Professor Co - Directing Thesis George Bates Professor Co - Directing Thesis Thomas Keller Committee Member Laura Keller Committee Member Approved: Timothy S.Moerland, Chair, Department of Biological Science The Office of Graduate Studies has verified and approved the above named committee members. ii ACKNOWLEDGEMENTS I would like to express my thanks and appreciation to my advisor, Dr. Wei- Chun Chin for his continuous encouragement and kind help in my research and study. When I was in trouble, he gave me a chance to continue my study. I am also very grateful to Dr. George Bates, my co-major professor for his help. Without their support, I never would have finished my master degree in the department of biological science. I also wish to thank Dr. Thomas Keller and Dr. Laura Keller for their contribution as my committee members. I also would like to express my love and gratitude to my family for their support, patient, kindness, inspiration and love. Without them, I cannot recover from the trouble. iii TABLE OF CONTENTS List of Figures .................................................................................... v Abstract .......................................................................................... vi INTRODUCTION.................................................................................... 1 MATERIALS AND METHODS................................................................ 6 RESULTS .................................................................................... 11 DISCUSSION .................................................................................... 16 REFERENCES .................................................................................... 34 BIOGRAPHICAL SKETCH .................................................................... 40 iv LIST OF FIGURES Figure 1: Illustration of the validation of the deconvolution program ..... .........21 Figure 2: Effect of incubation time on trypsin activity in zymogen granules.....22 2+ 2+ Figure 3: Effect of increasing [Ca ] in the intracellular solution on [Ca ]G , [pH]G and trypsin activity in zymogen granules ...................................... .........23 Figure 4: Effect of increasing [Ca2+] in the intracellular solution on the [K+] in isolated mouse pancreatic zymogen granules ...................................... .........24 Figure 5: Ca2+/K+ ion exchange in zymogen granules ........................... .........25 Figure 6: H+/K+ ion exchange in zymogen granules .............................. .........26 2+ Figure 7: The effect of [Ca ]G alone on trypsinogen autoactivation in zymogen granules .................................................................................... .........27 Figure 8: The effect of [pH]G alone on trypsinogen autoactivation in zymogen granules .................................................................................... .........28 2+ Figure 9: The effect of both increased [Ca ]G and decreased [pH]G on trypsinogen autoactivation in zymogen granules ................................... .........29 2+ Figure 10: The effect of TEA (20 mM and 10 mM) on [Ca ]G, [pH]G and trypsin activity in zymogen granules .................................................................. .........30 2+ Figure 11: The effect of apamin (100 nM) on [Ca ]G , [pH]G and trypsin activity in zymogen granules ............................................................................. .........31 2+ Figure 12: The effect of charybdotoxin (10 nM) on [Ca ]G , [pH]G and trypsin activity in zymogen granules .................................................................. .........32 Figure 13: Model of the dynamics of H+ and Ca2+ inside zymogen granules. ..33 v ABSTRACT Under normal physiological conditions, trypsin remains inactive as trypsinogen inside the pancreas. Upon entering the small intestine, trypsinogen is converted to active trypsin. Acute pancreatitis is caused by premature activation of trypsinogen and the digestion of the pancreas. Up to now the exact initiating mechanism of this premature activation is still not clear. In these experiments, pH fluctuations, Ca2+ concentration changes and trypsin activity inside pancreatic zymogen granules were monitored. The effects of possible pharmacological inhibitors were also assessed. The results show that a sustained increase of Ca2+ in the cytosol can trigger K+ influx into 2+ + + zymogen granules (ZGs) via a Ca -activated K channel (ASKCa). This influx of K then mobilizes bound Ca2+ by Ca2+/K+ ion-exchange to increase free Ca2+ concentration in the ZGs and also mobilizes bound H+ by H+/K+ ion-exchange to decrease the pH in the ZGs. Both the increase of free Ca2+ concentration and the decrease of pH in the ZGs will facilitate trypsinogen autoactivation and stabilize active trypsin. Moreover these investigations show that the ASKCa in the membrane of ZGs may be a small 2+ + conductance Ca -activated K channel (SKCa channel), because it can be activated by 300 nM [Ca2+] and inactivated by apamin (100 nM) and TEA (20 mM). vi INTRODUCTION Pancreatitis is an inflammation of the pancreas associated with pancreas damage (Mergener & Baillie, 1998). There are an estimated 50,000 to 80,000 cases in the United States each year. Nearly 20% of them develop life-threatening complications, such as renal or respiratory failure. The overall mortality is 5-10% and may increase to 35% with complications (Mergener & Baillie, 1998; NIH, 2001). Moreover pancreatic inflammation is a risk factor for pancreatic cancer, which is the fourth leading cause of cancer death in US with extremely poor prognosis (Farrow & Evers, 2002). People with alcohol abuse, the human immunodeficiency virus (HIV) infection or hereditary genetics defects are at high risk to develop acute pancreatitis (Steinberg & Tenner, 1994; NIH, 2001). Pancreatitis includes chronic pancreatitis and acute pancreatitis. In acute pancreatitis the duration of inflammation that can result in the damage of the pancreas is usually short and reversible, while in chronic pancreatitis the inflammation is mild but it lasts for a long time. Most cases of pancreatitis are caused either by alcohol abuse or by gallstones. Investigations find that alcohol can sensitize the receptors of agonists in the membrane of the pancreatic acinar cells. Alcohol stimulates the zymogen activation process induced by agonists, but it has no effect alone (Lu et al, 2002). It has long been hypothesized that in gallstone pancreatitis, gallstones block the bile ducts and bile flows back into the pancreatic ductal system. This hypothesis has been challenged by some investigators who favor pancreatic ductal hypertension as the main reason for gallstone pancreatitis (Steer et al, 1988). In this review, gallstones can block pancreatic ducts and result in ductal hypertension, so small ducts are ruptured ΁ and digestive enzymes will be released, leading to acute pancreatitis. Other less common triggering factors include nicotine (Chowdhury et al, 2002) and hereditary genetics defects (Whitcomb et al, 1996). The development of acute pancreatitis consists of three phases: an initiating phase, an intra-acinar cell phase such as cell injury, and an extra-acinar cell phase including local and systemic inflammation such as pulmonary and renal failure. The earliest events in pancreatitis include the loss of secretory cell polarity and the appearance of intracellular vacuoles. These phenomena are all associated with the abnormal activation of digestive enzymes (Steer, 1999; Raraty et al., 1999). The pancreatic acinar cell can synthesize many digestive enzymes such as trypsinogen, proelastase and procarboxypeptidase. These proteins are produced in the rough endoplasmic reticulum (RER) and modified by the Golgi complex. Digestive enzymes existing primarily as inactive zymogens are packaged in condensing vesicles at the trans side of the Golgi and carried towards the plasma membrane. At the plasma membrane, these vesicles will form the mature ZGs. When stimulated, those ZGs fuse with the plasma membrane and digestive enzymes are released. Many pancreatic digestive enzymes exist as inactive zymogens in the pancreatic acinar cells. When entering the small intestine, trypsinogen is converted to trypsin by the brush border hydrolase enterokinase, which is normally only found in the small intestine (Gorelick et al., 1992). Under normal physiological conditions, trypsinogen has only a small amount of enzymatic ΂ activity. But in pancreatitis, trypsinogen is prematurely activated in pancreas. The activated trypsin then activates the other pancreatic zymogens. It also can activate acinar cells, duct cells and imflammatory cells via the trypsin receptor, namely protease activated receptor isoform 2 (PAR-2) (Hirota et al., 2003), and result in the cascade reactions and damage
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