MATERIALS AND METHODS

Animals. Adult male CD-1 outbred mice (25-30 g)(Charles River Breeding Laboratory, Raleigh, NC) were acclimated for at least 1 wk before use. Animals were maintained on a 12: 12-h light-dark cycle and allowed free access to standard rodent chow and tap water. These studies were performed in adherence to the Guide for the Care and Use of Laboratory Animals [DHEW Publication No.(NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRRNIH, Bethesda, MD 208921 and were approved by the Institutional Animal Care and Use Committee.

ChemicaZs and reagents. Porcine pancreatic cx-amylase was obtained from Boehringer Mannheim (Indianapolis, IN). Caerulein, DEM, and LPS (Escherichia coZi serotype Olll: B4) were from Sigma Chemical (St. Louis, MO). All other chemicals and solvents were obtained from either Sigma Chemical or Fisher Scientific (Chicago, IL). Experimental protocols. Acute pancreatitis was induced in CD-l mice, as described previously (18). Specifically, mice were injected intraperitoneally with 50 pg caeruleinkg body wt for a total of seven hourly injections over a 6-h period. Control or sham-treated mice either received no injections or were injected intraperitoneally with an equal volume of saline on the same injection schedule. All the experiments were initiated between 8: 30 AM and 9: 30 AM. Animals (3 mice/group) were killed at different time points after the initiation of caerulein injections. Blood was collected by heart puncture and allowed to clot, and serum cc-amylase was assayed using the Procion yellow starch method, as reported previously (18). The pancreas was removed and quickly trimmed free of fat in cold phosphate-buffered saline (PBS). A small piece of tissue was weighed and immediately homogenized in 10 vol of 10% trichloroacetic acid (TCA). The supernatant was recovered by centrifugation, and total pancreatic GSH [GSH plus oxidized GSH (GSSG)] content was measured, as previously described (18). For protein extraction and analysis, one piece of pancreas was frozen in liquid nitrogen and stored in-80 C (see below). Another piece was minced and fixed in Bouin’s fixative for histological evaluation and immunohistochemistry or fixed in a standard fixative (4% paraformaldehyde, 1.6% glutaraldehyde in PBS) for electron microscopy. The remainder of the gland was processed immediately for RNA extraction (see below).