MATERIALS AND METHODS isoprostanes resulting from lipid peroxidation in
Materials. [ 3 H]PGE 2 (120 Ci/mmol), [ 3 H]PGF 2a (168 Ci/mmol), and hepatocytes. F 2 -isoprostane release was detected by im-[ 3 H]L-leucine (5 Ci/mmol) were purchased from New England Numunoassay. Conventional cultures of rat hepatocytes clear (Boston, MA). Collagenase type D was purchased from Boehcontain Kupffer and endothelial cells, which can syntheringer-Mannheim Biochemicals (Indianapolis, IN). Percoll solution size significant amounts of cyclooxygenase products. was obtained from Pharmacia LKB (Uppsala, Sweden). Prostaglan- Highlypurified hepatocytes do not produce cyclooxygendins and 8-iso-PGF 2a were obtained from Cayman Chemical (Ann ase products, even with a maximal stimulus to lipid per-Arbor, MI). Primaria plates were purchased from Falcon (Becton oxidation. CCl 4 causes the release of F 2 -isoprostanes Dickinson, Lincoln Park, NJ). Most other chemicals were purchased from Sigma (St. Louis, MO) except as indicated. from hepatocytes with or without observable cell injury, Animal Experiments. Adult male Sprague-Dawley rats (range, as detected by Trypan blue exclusion or lactate dehydro-200-250 g) were obtained from Taconic Farms, Inc. (Germantown, genase (LDH) release. (HEPATOLOGY 1996;24:677-684.) NY) or in later experiments from Harlan (Indianapolis, IN). All animal research protocols were approved by the Animal Research Com-Cyclooxygenase products have been implicated in the conmittees of New England Medical Center and of the University of trol of liver carbohydrate metabolism, 1 in liver regeneration, 2
New Mexico, observing guidelines set forth in the National Institutes and in protection against liver injury. 3,4 Liver endothelial of Health's Guide for the Care and Use of Laboratory Animals. and Kupffer cells are known to produce large amounts of Liver Cell Isolations. Collagenase perfusion was performed as previously described. 6,11,12 The resulting cell suspension was washed with iced Dulbecco's phosphate-buffered saline (without calcium and Abbreviations: PGD2, prostaglandin D2; PGE2, prostaglandin E2; PGF2a, prostaglandin magnesium) supplemented with 10 mmol/L of glucose, 18 mmol/L of F2a; HDM, hormonally defined medium; RPMI, Roswell Park Memorial Institute; LDH, HEPES, and 2 g/L of bovine serum albumin. Hepatocytes were isolactate dehydrogenase; TBARS, thiobarbituric reactive substances; EIA, enzyme immunoaslated by three successive centrifugations at 50g for 2 minutes in say; TLC, thin-layer chromatography.