Evidence of Intracellular Activation of Serine Proteases in Acute Cerulein-Induced Pancreatitis in Rats

Abstract

It is believed that activation of zymogen proteases occurs in the early development of acute pancreatitis. This hypothesis was proved on subcellular fractions of rat pancreas after induction of pancreatitis by infusion of high doses of cerulein for 2 h. Secretory enzyme activities were measured spectrophotometrically in subcellular fractions obtained by differential ultracentrifugation. Additionally, trypsin and chymotrypsin activities were detected by enzyme blots after isoelectric focusing. Finally immunoblotting (Western-blot analysis) for amylase, lipase, trypsin/ogen, and chymotrypsin/ogen was carried out on fractions separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In cerulein pancreatitis, subcellular fractions of secretory granules and vacuoles showed significant amounts of free trypsin and chymotrypsin activities compared with controls. The presence of free activities of serine proteases was paralleled by the appearance of numerous low molecular weight peptides detected by 2-dimensional electrophoresis and SDS-PAGE, which in part represented proteolytically cleaved secretory proteins. It is concluded that the intracellular activation of serine proteases that occurs in cerulein pancreatitis could contribute to further acinar cell destruction.

Key Words:

• Acute pancreatitis
• enzyme blotting
• serine proteases
• two-dimensional electrophoresis
• Western blot