Isolation and primary culture of bovine hepatocytes: albumin synthesis and adrenergic activation of glycogenolysis.
We describe a technique for isolation and primary culture of bovine hepatocytes, and their metabolic characterization. Hepatocytes were isolated from the caudate lobe of bovine liver by perfusion with 0.25 mM ethylene-glycol tetraacetic acid and 0.05% collagenase. The viability and yield of the cells were 70-92% and 0.1-3.6 x 10(7) cells/g liver, respectively. When the isolated hepatocytes were cultured in Williams' medium E, they began to spread in 3 hr and formed monolayers in 24 hr. These monolayers were retained for at least 6 days. To monitor the metabolic activities specific to liver, synthesis and secretion of albumin were measured by labeling with [35S]-methionine and immunoprecipitation. This activity was low in isolated hepatocytes, but increased after culturing 1-3 days, and decreased again after 6 days. Glycogenolytic activity was also assessed by measuring glucose release to the medium by stimulation with epinephrine. The glycogenolytic response to epinephrine was also enhanced by culturing the hepatocytes 1-3 days, but was decreased after 6 days. Since the isolated bovine hepatocytes retained the liver-specific activities of albumin synthesis and glycogenolysis for several days in culture, these cells are useful for cellular and molecular studies on the functions of bovine liver.
Nakagawa-Tosa
N
N
Department of Biochemistry, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Morimatsu
M
M
Mominoki
K
K
Nakatsuji
H
H
Syuto
B
B
Saito
M
M
D000818:Animals; D002417:Cattle; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D017364:Collagenases; D046508:Culture Techniques / Q000379:methods; D004533:Egtazic Acid; D004837:Epinephrine / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D008112:Liver Glycogen / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D010477:Perfusion / Q000295:instrumentation / Q000379:methods; D012709:Serum Albumin / Q000096:biosynthesis*; D013997:Time Factors
D008112:Liver Glycogen; D012709:Serum Albumin; D004533:Egtazic Acid; D008715:Methionine; D017364:Collagenases; D004837:Epinephrine