Foetal‐rat liver hepatocytes were cultured in primary monolayer culture on a collagen‐coated substratum and the viability of the hepatocytes was monitored using biochemical markers. The cellular activity of glutamate dehydrogenase, glucose‐6‐phosphate dehydrogenase and acid phosphatase increased markedly during the first 32 h of culture in medium contafoetal calf serum. The ATP concentration of the collagen‐attached hepatocytes was 625 pmol/μg DNA and did not change significantly during culture. The activity of cytosolic phosphoenolpyruvate carboxykinase could be increased during the first 10 h of culture by the addition of glucagon, epinephrine or N6,O2′‐dibutyryladenosine 3′:5′‐monophosphate (Bt2cAMP). This response to hormones or Bt2cAMP was retained in hepatocytes during three days of culture. Treatment of cells with glucagon or epinephrine caused an increase in enzyme activity of between 4–7‐fold higher than in untreated cells. Enzyme induction by hormones was shown to be dose‐dependent: significant responses were obtained with 1 nM glucagon or 10 nM epinephrine. The induction was evident within 2 h and was maximal 4 h after the addition of optimal hormone doses. Application of either the α‐adrenergic agonist phenylephrine (100 μM) or the β agonist isoproterenol (10 μM) produced an effect equivalent to 1 μM epinephrine. The effects of epinephrine or phenylephrine could be completely abolished by the prior addition of the β antagonist propranolol (100 μM) however the same concentration of the α antagonist phentolamine did not inhibit the induction due to epinephrine or isoproterenol. The induction by Bt2cAMP was almost completely prevented (90%) by the simultaneous addition of actinomycin D (0.2 μg/ml) suggesting that the nucleotide acts on transcription to increase phosphoenolpyruvate carboxykinase synthesis. These results suggest that cultured foetal rat liver hepatocytes provide a suitable system for the study of the role of hormones in the initiation of specific enzyme synthesis. The demonstration of a direct effect in cultured foetal hepatocytes gives further support for a role for epinephrine and glucagon as the primary stimuli for the appearance of hepatic phosphoenolpyruvate carboxykinase in the newborn rat.
|Number of pages||8|
|Journal||European Journal of Biochemistry|
|Publication status||Published - Dec 1979|