TI - Discussion . AB - Our study demonstrates that prolactin regulates the growth of female cholangiocytes presumably by an autocrine mechanism . We first demonstrated in liver sections that cholangiocytes from normal and BDL female and male rats express prolactin receptors . By real time PCR : (i) normal female cholangiocytes expressed both the short and long form of prolactin receptor mRNA ; and ( ii ) following BDL , the expression of the short and long form of prolactin receptors increased in female cholangiocytes . Our data on cholangiocyte prolactin receptor expression are slightly different to those of previous studies in albino mongrel rats [23] showing that : (i) normal isolated intrahepatic bile duct units ( IBDU ) predominantly express the message for the long form of the prolactin receptor , whereas the expression of the short form of the prolactin receptor is very low or absent [23] ; and ( ii ) following BDL , the genetic expression of the long form markedly increases in IBDU whereas the short form of prolactin receptor slightly increased in IBDU [23] . The slight difference between these data is presumably due to the different strain of rats used in our studies ( female 344 Fischer ) and the other studies ( albino mongrel ) [23] . Prolactin receptors are expressed by rat hepatocytes in the sinusoidal domain of cellular membranes and in perinuclear areas [32] . Prolactin receptors are also expressed by human hepatocytes of patients with obstructive jaundice of different etiology , but prolactin receptor expression is lower in hepatocytes compared to human cholangiocytes [21] . Although these previous studies have shown that cholangiocytes express prolactin receptors [21,23] , no information exists on the role of prolactin in the regulation of cholangiocyte hyperplasia . We next performed in vivo studies and demonstrated that the administration of : (i) prolactin to normal female rats induces cholangiocyte hyperplasia devoid of portal inflammation and hepatic damage ; and ( ii ) anti-prolactin antibody to BDL female rats decreases cholangiocyte proliferation and ameliorates portal inflammation and hepatic damage . The most likely explanation why prolactin increased cholangiocyte growth but not portal inflammation and hepatic damage in normal rats is that prolactin induces cholangiocyte hyperplasia as a direct effect and not as a consequence of obstructive cholestasis ( ie , BDL ) , a pathological condition associated with increased portal inflammation [33,34] . In support of our findings , a number of studies have shown that certain bile acids , vascular endothelial growth factor and forskolin induce cholangiocyte hyperplasia devoid of apoptosis , necrosis , hepatic damage or portal inflammation [31,35,36] . However , in BDL , which is accompanied by an inflammatory response along with cholestasis [33,37,38] , the blocking of prolactin with an antibody reduces hepatic damage and cholangiocyte proliferation along with suppression of some inflammatory responses . Concomitant with enhanced ductal hyperplasia , there was increased prolactin serum levels in normal female rats treated with prolactin compared to NaCl treated rats . In BDL female rats , the serum levels of prolactin increased approximately 15-fold as compared to the levels of normal female rats . Moreover , the administration of anti-prolactin antibody to BDL rats reduces not only cholangiocyte proliferation but also prolactin serum levels . We suggest that changes in prolactin serum levels may be important in the regulation of cholangiocyte growth in chronic cholestatic liver diseases . In different cell types , prolactin effects are mediated by an increase in Ca2+ levels and PKC activation [11, 39] . Thus , we evaluated the role of the Ca2+/PKC signaling pathway in prolactin regulation of cholangiocyte hyperplasia . Our results show that [Ca2+] i levels are increased in normal female cholangiocytes after in vitro prolactin stimulation as compared to cholangiocytes stimulated with BSA . From our previous experience with purified cholangiocytes and cholangiocarcinoma cell lines we do not expect a traditional calcium spike [24,26,29,40,41] . In our previous studies [24,26,29,40,41] , we demonstrated that the Ca2+ dynamics of cholangiocytes are in general slower and not characterized by a Ca2+ spike [24,26,29,40,41] . The method that we currently employ results in Ca2+ measurements [29,31] , which are an average signal of 400,000 cells rather than typical single cell measurements [24,26,40] . This approach gives us similar data [29,31] to that obtained with measurements , which were made in single cells loaded with Fluo-3AM [24,26,40] . Since the measurements are taken in a large number of cells the cellular response to prolactin is not synchronized in the studies . Thus , any peaks present will be muted and spread out over time , which is a factor contributing to the slow drift observed . In support of our finding , Ducret et al . demonstrated a similar slow response calcium wave due to prolactin in glia cells [42] . We next evaluated if prolactin stimulation of cholangiocyte proliferation was coupled with phosphorylaTION/dePHOSphorylation of specific Ca2+ -dependent PKC isoforms . Our finding that prolactin stimulation of normal female cholangiocyte proliferation is associated with concomitant increased ( PKCbeta-I ) and decreased ( PKCalpha ) PKC PHOSphorylation suggests that a counterbalancing system between PCKbeta isoform and PKCalpha may regulate cholangiocyte proliferation following prolactin stimulation . In support of this concept , while enhanced phosphorylaTION of PKCbeta-II mediates the activation of secretin-stimulated ductal secretion [29] ( a functional marker of cholangiocyte growth ) [1,2,4,6,25,43] of BDL rats , increased PHOSphorylation of PKCalpha ( which is inversely related to cholangiocyte growth ) is associated with reduced cholangiocyte growth [24,25,40] . In agreement with the view , in hematopoietic [44] and glioma [45] cells , the activation of PKCbeta-I and beta II isoforms leads to an increase in cell proliferation . Furthermore , in intestinal cell lines the overexpression and activation of PKCalpha decreases cell growth and tumorigenicity [46] . Taken together , our studies show that the Ca2+/PKCbeta-I and alpha signaling pathway is one of the players involved in prolactin regulation of cholangiocyte proliferation , but did not evaluate if other pathways ( eg , JAK/STAT and 17-hydroxy-steroid dehydrogenase ) modulate prolactin effects on cholangiocyte growth . Also , our studies do not establish which isoform ( short or long ) of the prolactin receptor mediates the effects of prolactin on cholangiocyte growth . However , based upon previous studies showing that the long form of prolactin receptor mediates increases in [Ca2+] i in other cells [18,19,47] , we propose that the long form of the prolactin receptor may be the major player in prolactin modulation of cholangiocyte growth . Next , we demonstrated that normal and BDL female cholangiocytes express the message and protein for prolactin and secrete prolactin in primary cultures . The reason why prolactin secretion is similar in normal and BDL female cholangiocytes ( although prolactin message expression increases in BDL cholangiocytes ) may be due to post-transcriptional events ( eg , message stability/degradation ) affecting the translation of the prolactin message . On the basis of these findings , although our studies do not provide direct evidence for the following concept , we propose that prolactin may regulate cholangiocyte proliferation by an autocrine mechanism ( in addition to a paracrine pathway ) . In agreement with the latter concept , a number of cells including mammary epithelial cells , fibroblasts and cancer cell lines [12, 15] secrete prolactin , thus regulating their functions . Furthermore , in support of the concept that prolactin regulates cholangiocyte proliferation by an autocrine mechanism , we have previously shown that cholangiocytes express/secrete neurotrophins [33] , vascular endothelial growth factor [31] and serotonin [48] , thus regulating intrahepatic ductal mass by an autocrine mechanism [31,33,48] .