Hormone responsiveness of isolated catfish hepatocytes in perifusion system is higher than in flasks incubation

Hepatocytes were isolated from catfish (lctalurus melas) by conventional collagenase digestion. Sensitivities of liver cells isolated from the same fish to the glycogenolytic action of epinephrine, mammalian glucagon, catfish glucagon, catfish glucagon-like peptide, synthetic fragment 19-29 of anglerfish glucagon I, fragment 19-29 of anglerfish glucagon II, and anglerfish glucagon II were compared in two different systems: perifusion in a Bio-Gel P4 column and flask incubation. Both experimental procedures were continued for a total of 100-120 min, while hormones were applied simultaneously to both preparations for 10 min. Effluent fractions from the columns and incubation media from the flasks were collected for glucose determination. The hormonal effects were clearly enhanced in perifused cells compared to those in cells incubated in flasks, the effect being especially evident at physiological concentrations of hormones. The hormonal effects in both systems were dose-dependent. Epinephrine and mammalian glucagon (10 nM), applied separately to the same column, produced two different peaks, glucagon causing more glucose production than epinephrine. In the presence of 0.4 mM glucose in the perifusion system, hormonal effects were diminished, implying that glucose accumulation during incubation of liver cells in flasks might affect hormonal effects. The results obtained in this study indicate that piscine hepatocytes suspended and perifused in a Bio-Gel column are more sensitive to physiological concentrations of glycogenolytic hormones and may represent a new tool for experimental studies of fish liver metabolism and its hormonal regulation.