The objective is the elucidation of the molecular mechanisms by which the functional organization of the adult liver acinus is attained. It is our hypothesis that the functional heterogeneity of hepatocytes is necessary to accomplish the ultimate function of the liver acinus, the regulation of solute concentration in the terminal hepatic venule and in bile. We propose that regulation of gene expression is one of the mechanisms by which the functional heterogeneity of hepatocytes develops. Consequently, of particular interest are: a. the changes in gene expression occuring during the transition from fetal to adult liver, and b. the modulation of gene expression within the adult liver acinus. Phenobarbital (PB)-induction of liver cytochromes P-450 b and e (two major BP-inducible forms) will be used as a model since after PB administration, cytochrome P-450 proteins and mRNAs are induced predominantly in hepatocytes of the distal half of the acinus (zones 3 and 2). Specifically, we propose to answer these questions: Is induction of cytochromes P-450 b and e mRNAS occuring in all or in some hepatocytes? Secondly, is the heterogeneous PB-induction of cytochrome P-450 proteins within the liver acinus controlled predominantly at the level of gene transcription? And thirdly, when is this manifestation of hepatocyte heterogeneity established? It is imprinted in fetal life, or does it develop post-natally, and by which molecular mechanisms? In situ hybridization techniques will be used to determine the zonal localization of hepatocytes responding to PB with induction of cytochrome P-450 b and e mRNAs in the adult liver. It will also be used to assess the time of liver development at which the heterogeneous pattern of expression of the PB-inducible cytochrome P-450 genes is attained. In vitro transcription assays using nuclei isolated from hepatocytes of different zones of the adult liver acinus will be used to assess whether differences in transcription rate are the main mechanisms responsible for the heterogenous expression of the cytochromes P-450 b and e genes after PB. By probing the molecular mechanisms involved in the establishment and regulation of cytochrome P-450 b and e gene expression within the hepatic acinus, these experiments should provide insight into regulatory steps which are necessary for the liver to attain the adult organization of the liver acinus.
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