Prolactin participates in the regulation of liver function. However, prolactin receptor (PrlR) expression and its regulation have been described only for hepatocytes. In this study, we investigated the expression and regulation of PrlR isoforms in the other important intrahepatic cellular compartment: the biliary epithelial cells, or cholangiocytes. Our aim was to determine whether prolactin should be considered as a potential regulator of cholangiocyte function under normal and pathological conditions. Cholangiocytes and hepatocytes were differentially isolated from rat liver. PrlR expression was analysed at the mRNA level by isoform-specific semiquantitative PCR, and at the protein level by immunostaining of liver sections. Hormonal regulation of PrlR expression was evaluated by comparing intact rats with gonadectomized, pituitary-grafted or bromocriptine-treated animals. Common bile-duct ligation was used as the experimental model of cholestasis. Our results demonstrate that the expression pattern and regulation of PrlR isoforms is totally different in cholangiocytes compared with hepatocytes: (1) mature rat cholangiocytes express low levels of PrlR, while it is very high in hepatocytes, (2) only the long isoform is detected in cholangiocytes, while the short isoform predominates in hepatocytes and (3) PrlR levels in cholangiocytes are induced by obstructive cholestasis, but not by sex hormones or prolactin, while it is the opposite in hepatocytes. From these data, the actions of prolactin on liver are anticipated to exhibit strong cell-type specificity in both normal and pathological conditions.
Immunoperoxidase method combined with cytofluorimetry showed that in contrast to hepatocytes, enhanced expression of prolactin receptors on rat cholangiocytes induced by common bile duct ligation cannot be suppressed by the prolactin secretion inhibitor bromocriptine. Key Words: prolactin receptors; rat liver cells; common bile duct ligation; bromocriptine; immunohistochemistryExpression of prolactin receptors (PRL-R) in hepatocytes is regulated by a multihormonal assembly, that includes prolactin (PRL), the major positive regulator of PRL-R, sex steroids, insulin, thyroid hormones, and glucocorticoids [2]. Hepatotropic effects of PRL, in particular, induction of ornithine decarboxylase [7], phosphoenolpyruvate carboxykinase [12], and protein kinase C [5], expression of the growth hormone genes [7], stimulation of DNA synthesis [6], and DNA hypomethylation [9] are studied in detail.Unlike hepatocytes, cholangiocytes express little or no PRL-R in mature rats, but intensely express these receptors during active proliferation (in prepubertal animals [1]) or after common bile duct ligation (CBDL) [3]. The physiological role of increased sensitivity of cholangiocytes to PRL during the prepubertal period remains unclear. Another peculiarity is that the expression of PRL-R in cholangiocytes does not depend on sex steroids [3]. These data suggest different mechanisms of regulation of PRL-R expression in hepatocytes and cholangiocytes. To verify this assumption we studied the effect of PRL on PRL-R expression in rat cholan- MATERIALS AND METHODSExperiments were carried out o11 random-bred albino rats of both sexes weighing 200-220 g. The common bile duct was ligated by a standard method described previously [4].On days 5-13 postoperation, 5 males and 8 females were subcutaneously injected with 10 nag/ kg bromocriptine dissolved in 50% ethanol (2 nag/ 0.5 ml/rat). Control animals (4 males and 8 females) received 0.5 lnl 50% ethanol every days. The animals were decapitated on day 14 postoperation. The liver was fixed in 4% paraform for 18-20 h at 4~ and embedded in paraplast; PRL-R were visualized by indirect immunoperoxidase method [11]. To this end, 3-gm-thick sections pretreated with 10 mM sodium periodate and 0.01% sodium borohydrite were incubated with mouse monoclonal antibodies clone U6 (0.1 mg/ml IgG fraction from ascitic fluid, kindly provided by Dr. P. A. Kelly, France) in 0.05
The presence of prolactin receptor and peculiarities of its isoform expression in bile duct cells (cholangiocytes) differentially isolated from rat liver under different conditions were investigated in the present study. Normal cholangiocytes express prolactin receptor at relatively low level comparable to those of some prolactin-dependent tissues. Long receptor isoform is predominant in cholangiocytes but not in hepatocytes. The prolactin receptor level increases significantly under obstructive cholestasis due to evaluation of long and appearance of short isoforms. In rat cholangiocytes, unlike other tissues, the main positive regulators of prolactin receptor expression are cholestasis-induced factors instead of sex hormone and prolactin levels. Long isoform is predominant and induced primarily by cholestasis-induced factors.
In rats with ligated common bile duct, the inhibitor of prolactin secretion bromocriptine inhibited proliferation of bile ducts in males, reduced Na § concentration in bile in females, and elevated blood bilirubin. In males with pituitary transplants, proliferation of bile ducts increased. It was concluded that the effects of prolactin on cholangiocyte proliferation and bile ion content after ligation of the common bile duct are sex-dependent. Key Words: prolactin; rat liver cells; common bile duct ligation; bromocriptineSome liver pathologies, in particular diseases associated with bile duct obstruction are accompanied by proliferation of ductal structures. In experimental animals this reaction can be induced by common bile duct ligation (CBDL) [9].Cholangiocyte hyperplasia induced by CBDL is accompanied by intense expression of prolactin receptors (PRL-R) [2]. This study was aimed at investigation of the effect of prolactin (PRL) on liver cells under conditions of cholestasis induced by CBDL. Blood concentration of PRL was reduced by administration of the PRL secretion inhibitor bromocriptine (Be) or elevated by transplantation of pituitary grafts beneath the renal capsule. These grafts produced large amount of PRL due to the absence of the hypothalamic inhibitory control [8].We studied the effects of PRL on bile duct proliferation and bile ion content. Functional activity of liver cells in CBDL and after administration of BC was assessed by serum bilirubin concentration. MATERIALS AND METHODSExperiments were carried out on outbred and Wistar rats of both sexes weighing 150-220 g. CBDL was reproduced as described previously [4].
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