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A review of the recent findings related to the morphological heterogeneity of …
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A number of in vivo models (e.g., BDL, acute admini-stration of CCl4, partial hepatectomy, chronic feeding of ANIT or bile salts)[47-52] demonstrated that the intrahepatic biliary epithelium is functionally heterogeneous, with specific sized bile ducts (i.e., small and large) differentially responding to liver injury/toxins with changes in proliferative, apoptotic and secretory activities[2,3,12,47-52,54,62,71-73]. A number of in vitro experimental models (i.e., small and large cholangiocytes and IBDU and small and large immortalized normal murine cholangiocytes) (Figure 1)[12,13,47,48,50,51,75] have allowed us to suggest that the intrahepatic biliary epithelium is morphologically and functionally heterogeneous[2, 3,12,47-52,54,62,71-73]. The very first approach that was employed and that significantly contributed to lay down the basis of this field of research was the purification of small and large cholangiocytes from rat liver by counterflow elutriation[12,54,76]. Coupling such a technique to immunoaffinity separation[12,18,54], it was possible to isolate two distinct subpopulations of small (approximately 8 mm in diameter, obtained at the centripetal flow rate of 25 ml/min) and large (approximately 14 mm in diameter, collected at the flow rate of 55 mL/min) cholangiocytes (Figure 1)[12,54]. The two subpopulations of small and large cholangiocytes are further purified by immunoaffinity separation[18] using an antibody against an unidentified antigen (expressed by all intrahepatic cholangiocytes)[18] and characterized morphologically (by computerized image analysis) (Figure 1)[12,54], phenotypically (expression of g-glutamyltransferase and cytokeratin-19 genes)[12,54] and functionally (by measurement of gene expression of secretin receptor, CFTR and Cl-/HCO3- exchanger and basal and secretin-stimulated cAMP levels, Cl- efflux and Cl-/HCO3- exchanger activity)[12,54].
In addition, we have developed a technique for isolating small (diameter smaller than 15 mm) and large (diameter greater than 15 mm) IBDU from small and large bile ducts, respectively (Figure 1)[13]. This important tool allowed us to directly evaluate the differential secretory responses of different portions of the biliary epithelium to selected gastrointestinal hormones/peptides[13,25,65,77]. As shown in Figure 1, the small duct was pruned off from the large duct by a brief exposure of a laser focused on the junction between large and small ducts (arrow) leading to separation of small from large ducts[13]. Small and large IBDU were characterized by morphometric analysis, gene expression for secretin receptor, CFTR and Cl-/HCO3- exchanger, secretin-induced cAMP levels, and secretion by change in luminal size in response to agonists including secretin, insulin, the a1-adrenergic receptor agonist, the a2-adrenergic receptor agonist, UK14,304 and the D2 dopaminergic receptor agonist, quinelorane[13,25,65,66,77].
Most recently. we have immortalized, from normal mice (BALB/c), small and large cholangiocytes by the introduction of the SV40 large T antigen gene, that allowed, after cloning, to establish small and large cholangiocyte cell lines[75]. The characteristics of the two subpopulations were evaluated by electron microscopy (EM) and measurement of trans-epithelial electrical resistance (TER), and secretin-stimulated cAMP levels[75]. EM, TER and differential cAMP response to secretin are consistent with the concept that small and large immortalized cholangiocytes originate from small and large ducts, respectively[75]. Microarray successfully displayed characteristic differential cDNA expression between small and large cholangiocytes[75]. Using the above described methods individually or in tandem, has allowed us to clearly demonstrate heterogeneity of the intrahepatic biliary epithelium and to dissect the differential physiological responses of these distinct subpopulations of cholangiocytes to endogenous stimuli.
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