Influence of cAMP-effector-agonists on the synthesis of metallothionein in rat primary hepatocytes.
The model of rat primary hepatocytes incubated in DMEM/F12 (Ham) medium was used for studying the influence of the cAMP-effectors epinephrine (100 microM), norepinephrine (100 microM), glucagon (1 microM) and isoproterenol (1-1000 microM) as well as the synthetic cAMP-analogon dibutyryl-cAMP on the metabolism of metallothionein. Liver parenchymal cells isolated by a two-step collagenase perfusion were incubated with DMEM/F12 containing 5% (v/v) fetal calf serum (FCS) and 20 microM zinc in Petri dishes. Experiments were initiated after a 24 h equilibration period by adding the agonists for 18 h. MT in hepatocyte homogenates was quantified by the 109Cd-hemoglobin-binding assay. Cell viability was assessed by the activity of the cytosolic enzyme lactate dehydrogenase (LDH) liberated into the culture medium and by trypan blue exclusion. Isoproterenol and glucagon produced a significant increase of cytosolic MT about 50%. In contrast, incubation with epinephrine and norepinephrine did not lead to any significant effects in the amount of hepatic metallothionein. Simulating the influence of cAMP by dibutyryl-cAMP (500 microM) did not affect the content of hepatic metallothionein. To examine transcriptional and translational regulatory effects supplementation of cycloheximide (0.1-500 microM) and actinomycin D (0.1-100 microM) showed a total inhibition of the agonist induced amounts. Particularly in combination with isoproterenol low LDH activities reflected a high viability of hepatocytes. In conclusion, in primary hepatocyte cultures cAMP-mobilizing-agonists like isoproterenol and glucagon indicate an independent effect on the MT-metabolism. This is possibly due to the de novo synthesis of the protein because suppression by actinomycin D can be observed. However, cAMP-effectors do not seem to be involved in the induction of metallothionein because theophylline and dibutyryl-cAMP did not affect MT-metabolism by suppressing the phosphodiesterase or by stimulating the cAMP-cascade.
Pallauf
J
J
Justus-Liebig-Universität, Institut für Tierernährung und Ernährungsphysiologie, Giessen, Germany.
Fischer
J
J
Lehnert
V
V
D000704:Analysis of Variance; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D003609:Dactinomycin / Q000494:pharmacology; D004837:Epinephrine / Q000494:pharmacology*; D005934:Glucagon / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008668:Metallothionein / Q000494:pharmacology*; D009638:Norepinephrine / Q000494:pharmacology*; D051381:Rats; D013431:Sulfates / Q000494:pharmacology; D013806:Theophylline / Q000494:pharmacology; D017967:Zinc Compounds / Q000494:pharmacology; D019287:Zinc Sulfate
D002087:Butyrates; D013431:Sulfates; D017967:Zinc Compounds; D020148:Butyric Acid; D003609:Dactinomycin; D003994:Bucladesine; D019287:Zinc Sulfate; D005934:Glucagon; D008668:Metallothionein; D013806:Theophylline; D000242:Cyclic AMP; D007545:Isoproterenol; D009638:Norepinephrine; D004837:Epinephrine