Zonal down-regulation and redistribution of the multidrug resistance protein 2 during bile duct ligation in rat liver.

We have studied regulation of the multidrug resistance protein 2 (mrp2) during bile duct ligation (BDL) in the rat. In hepatocytes isolated after 16, 48, and 72 hours of BDL, mrp2-mediated dinitrophenyl-glutathione (DNP-GS) transport was decreased to 65%, 33%, and 33% of control values, respectively. The impaired mrp2-mediated transport coincided with strongly decreased mrp2 protein levels, without any significant changes in mrp2 RNA levels. Restoration of bile flow after a 48-hour BDL period resulted in a slow recovery of mrp2-mediated transport and protein levels. Immunohistochemical detection of the protein in livers of rats undergoing BDL showed strongly reduced mrp2 staining after 48 hours, which was initiated in the periportal areas of the liver lobule and progressed toward the pericentral areas after 96 hours. Immunofluorescent detection of mrp2 in livers of rats undergoing 48 hours of BDL revealed decreased staining accompanied by intracellular localization of the protein in pericanalicular vesicular structures. Within this intracellular compartment, mrp2 colocalized with the bile salt transporter (bsep) and was still active as shown by vesicular accumulation of the fluorescent organic anion glutathione-bimane (GS-B). We conclude that down-regulation of mrp2 during BDL-induced obstructive cholestasis is mainly posttranscriptionally regulated. We propose that this down-regulation is caused by endocytosis of apical transporters followed up by increased breakdown of mrp2, probably in lysosomes. This breakdown of mrp2 is more severe in the periportal areas of the liver lobule.

Increased C/EBP in fetal rat small intestine precedes initiation of differentiation marker mRNA synthesis.

Morphogenesis, initiation of differentiation marker gene expression, and their correlation with CCAT/enhancer binding protein (C/EBP) expression were analyzed in the developing fetal rat small intestine. Expressions of mRNAs for lactase-phlorizin hydrolase (LPH), intestinal alkaline phosphatase (IALP), carbamoyl-phosphate synthetase (CPS), and three isoforms of C/EBP were simultaneously determined by Northern blot analysis from 15 to 19 days of gestation. At 17 days of gestation, prior to villus formation as demonstrated by light and electron microscopy, only CPS and C/EBPalpha, -beta, and -delta expression could clearly be detected. Both LPH and IALP mRNA were definitely detectable in proximal and middle intestine on day 18, as soon as the stratified epithelium of the early intestine had been transformed into a single layer of columnar epithelium lining villi. This distribution was confirmed by in situ hybridization for LPH mRNA. During the period of transformation when the columnar epithelium and villi were forming, no LPH or IALP mRNA was detectable in the immature distal one-third of the fetal intestine. Preceding villus morphogenesis, immunostaining demonstrated nuclear localization of C/EBPalpha protein in intestinal epithelial cells, with continued expression in all enterocytes through 19 days of gestation. Enhanced expression of C/EBPalpha mRNA and protein began 24 h prior to the initiation of the differentiation markers, suggesting that it may play a role in regulation of fetal intestinal differentiation.

Coordinate expression of lactase-phlorizin hydrolase mRNA and enzyme levels in rat intestine during development.

The development of rat intestinal lactase-specific activity displays a well-known post-weaning decline. In contrast, total lactase activity increases to reach maximal levels around weaning, and remains high subsequently. In order to elucidate the molecular basis for these patterns, a rat lactase cDNA was isolated and characterized, and used in the quantification of lactase mRNA during development. This lactase cDNA uniquely hybridized to a 6.8-kilobase mRNA in the small intestine. To assess the amount of lactase mRNA encoding for lactase enzyme activity in the small intestine, total intestinal RNA was isolated and analyzed by Northern and dot-blot hybridization. The pattern of total lactase mRNA during development followed that of total lactase activity, suggesting that over this time span the level of lactase activity is primarily controlled at the transcriptional level. However, the magnitude of increase of total lactase activity during lactation compared to that of total lactase mRNA suggests that additional mechanisms are involved in regulating lactase levels. Analysis of the regional distribution of lactase mRNA along the small intestine at 14 days revealed that mRNA was high in the proximal three regions, but was dramatically lower in the distal regions. Total lactase activity, in contrast, displayed maximum activity in the mid-intestine with decreased levels both proximally and distally. Thus, lactase activity in the intestine appears to be regulated during development predominantly by transcriptional mechanisms, while alterations during lactation, and along the proximal to distal gradient, are the result of other control mechanisms.