J. Pallauf et al., INFLUENCE OF CAMP-EFFECTOR-AGONISTS ON THE SYNTHESIS OF METALLOTHIONEIN IN RAT PRIMARY HEPATOCYTES, Zeitschrift fur Ernahrungswissenschaft, 34(4), 1995, pp. 293-300
The model of rat primary hepatocytes incubated in DMEM/F12 (Ham) mediu
m was used for studying the influence of the cAMP-effectors epinephrin
e (100 mu M), norepinephrine (100 mu M), glucagon (1 mu M) and isoprot
erenol (1-1000 mu M) as well as the synthetic cAMP-analogon dibutyryl-
cAMP on the metabolism of metallothionein. Liver parenchymal cells iso
lated by a two-step collagenase perfusion were incubated with DMEM/F12
containing 5 % (v/v) fetal calf serum (FCS) and 20 mu M 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 quan
tified by the Cd-109-hemoglobin-binding assay. Cell viability was asse
ssed by the activity of the cytosolic enzyme lactate dehydrogenase (LD
H) liberated into the culture medium and by trypan blue exclusion. Iso
proterenol and glucagon produced a significant increase of cytosolic M
T about 50 %. In contrast, incubation with epinephrine and norepinephr
ine did not lead to any significant effects in the amount of hepatic m
etallothionein. Simulating the influence of cAMP by dibutyryl-cAMP (50
0 mu M) did not affect the content of hepatic metallothionein. To exam
ine transcriptional and translational regulatory effects supplementati
on of cycloheximide (0.1-500 mu M) and actinomycin D (0.1-100 mu M) sh
owed a total inhibition of the agonist induced amounts. Particularly i
n combination with isoproterenol low LDH activities reflected a high v
iability 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 d
e novo synthesis of the protein because suppression by actinomycin D c
an be observed. However, cAMP-effectors do not seem to be involved in
the induction of metallothionein because theophylline and dibutyryl-cA
MP did not affect MT-metabolism by suppressing the phosphodiesterase o
r by stimulating the cAMP-cascade.