Raraty, M.G.T.; Neoptolemos, J.P.; Petersen, O.H.; Sutton, R.
April 2003
Gut;Apr2003 Supplement 1, Vol. 52, pA19
Academic Journal
Intracellular activation of both trypsin and cathepsin B is known to occur early in the course of acute pancreatitis. This study aimed to determine the time course and subcellular location of: such activation and its relationship to abnormal Ca[sup 2+] signalling. Isolated mouse pancreatic acinar cells were loaded with 10 µM IPR-CMAC (a fluorescent substrate specific for trypsin) and/or (FR)2-R110 (specific for cathepsin B), together with fura-2 for measurement of cytosolic Ca[sup 2+] ([Ca[sup 2+]],) and were then perifused with stimuli. Enzyme activation was visualised by confocal microscopy, and changes in cellular morphology by electron microscopy. Cells exposed to 10nM CCK or 2 pM thapsigargin showed a rapid rise in [Ca[sup 2+]], followed by a modest sustained elevation. Within 300 seconds of application of: the stimulus, fluorescence appeared within multiple discrete rounded compartments approximately 1 pm in diameter within the granular pole of the cell. These gradually enlarged with continued stimulation and became less distinct. By 60 min, typical vacuoles were apparent on electron microscopy. The fluorescence from both enzyme substrates developed within spatially indistinguishable compartments at the apical pole of the cell but trypsinogen activation preceded cathepsin B activation by a mean of 85 ± 34s. Trypsinogen activation occurred more rapidly and reached a plateau 200 seconds earlier than that from cathepsin B. Attenuation of the abnormal [Ca[sup 2+]][sub i] signals with the Ca[sup 2+] chelating agent BAPTA prevented both trypsinogen activation and vacuolisation. These findings build on our previous findings (PNAS 2000;97:13126-31) and confirm the crucial role of abnormal cytosolic Ca[sup 2+] signals in the initiation of intracellular enzyme activation. Trypsinogen activation occurs within zymogen granules, which then become vacuoles. These results are consistent with the central role trypsinogen activation is thought to play in...


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