Cholangiocytes along the biliary tree are morphologically heterogeneous [8]. The small ducts are lined by 4–5 cholangiocytes, termed small cholangiocytes, which exhibit a cuboidal or flattened shape and possess a basement membrane on their basolateral domain. On their apical domain, microvilli and primary cilia face the bile duct lumen. Large bile ducts are lined by columnar-shaped cells known as large cholangiocytes that also express both microvilli and cilia on their apical domain [8]. When compared to small cholangiocytes, large cholangiocytes have a smaller nuclear to cytoplasmic ratio with a higher content of rough endoplasmic reticulum [1]. This feature implies that large cholangiocytes are more differentiated and have less plasticity relative to small cholangiocytes [8].

Cholangiocytes are connected to each other via tight junctions that maintain cholangiocyte polarity through cell-to-cell adhesion [8]. The apical plasma membrane domain faces the ductal lumen, which functions as the secretory pole for ductal bile formation; the basolateral plasma membrane domain faces the extracellular matrix and underlying connective tissue [10], Fig. 7.3.

The main function of intrahepatic cholangiocytes is to modify bile via a series of secretory/absorptive events. These are regulated by several gastrointestinal peptides/hormones including gastrin, endothelin-1, somatostatin, TGR-5, and secretin, which display inhibitory and stimulatory effects on water and bicarbonate (HCO₃ ⁻) secretion. These modifications ultimately influence the bile volume, content, tonicity, and alkalinity [8, 14]. While there is considerable species variation, intrahepatic cholangiocytes directly generate up to 40 % of daily bile secretion [2]. Secretory functions are performed mainly by large intrahepatic cholangiocytes via a mechanism dependent on cAMP activation. Large cholangiocytes abundantly express the appropriate ion transport systems and hormone receptors for these functions. For example, cholangiocytes in the large ducts are the major functional anatomic sites for expression of secretin and somatostatin receptors necessary for bile modification and secretion. In contrast, cholangiocytes lining small bile ducts, including the finest branches of the biliary system, do not express the secretin and somatostatin receptors exerting secretory activities independent of cAMP activation [19, 20]. For instance, during injury of large bile ducts, small cholangiocytes, which lack the cystic fibrosis transmembrane conductance regulator (CFTR), activate an alternative pathway for water and electrolyte secretion dependent of Ca⁺ signaling [21].

Small cholangiocytes, which are mitotically quiescent, proliferate via activation of Ca⁺² signaling in response to liver injury and toxins [19, 20]. For instance, small cholangiocytes may replicate upon stimulation with histamine or secretin or injury by α-naphthylisothiocyanate or acute carbon tetrachloride. Le Sage et al. demonstrated that acute administration of carbon tetrachloride to rats induces apoptosis of large cholangiocytes and proliferation of small cholangiocytes. Furthermore, small cholangiocytes acquired de novo expression of secretin receptors. Stimulation of small cholangiocytes with secretin-induced activation of cAMP suggests that upon injury small cholangiocyte may acquire secretory features of large cholangiocytes. This suggested that small cholangiocytes compensated for the functions of the injured large cholangiocytes [22]. Also, after partial hepatectomy, rat small cholangiocytes function as a niche for hepatobiliary progenitor cells. Studies performed in human cholestatic livers and in human-regenerating liver after alcohol-induced injury suggest that human cholangiocytes behave in a similar manner [23]. Thiese et al. reported that acetaminophen-induced hepatic massive necrosis stimulates a niche of stem cells containing small cells positive for the cholangiocyte marker cytokeratin 19 (CK19) within the Canals of Hering [24]. Thus, the main biological properties of small cholangiocytes are their ability to proliferate, to acquire features of large cholangiocytes, to differentiate into hepatocytes, and to be a cell reservoir upon injury [1, 25].

Moreover, cAMP and/or Ca²⁺-sensitive basolateral potassium (K⁺) channels, expressed in cholangiocytes, mediate K⁺ release which leads to membrane hyperpolarization to maintain the electrical driving force for continued apical Cl⁻ secretion [30]. Under basal conditions, the permeability of the apical membrane is low but can be increased several fold following cAMP stimulation [31, 32]. Furthermore, Cl⁻ secretion and subsequent reuptake is required for HCO₃ ⁻ secretion by the Cl⁻/HCO₃ ⁻ anion exchanger 2 (AE2). Cl⁻ uptake is mediated by the sodium (Na⁺)/K⁺/Cl⁻ cotransporter NKCC1, which is localized in the basolateral membrane of rat cholangiocytes. In an electrically neutral manner with stoichiometry of 1Na⁺:1 K⁺:2Cl⁻, a gradient is established, which maintains a high concentration of intracellular Cl⁻ [33]. This is important as HCO₃ ⁻ is secreted in exchange for luminal Cl⁻. The movement of ions across the cholangiocyte apical and basolateral membranes promotes osmotic-driven bile secretion [34].

The absorption of ions, BAs, amino acids, and glucose are additional processes that contribute to ductal bile modification [8]. Glucose is removed from bile in a Na⁺-dependent manner by the Na⁺-glucose cotransporter, SGLT1, localized in the apical plasma membrane of the bile ducts. Conjugated BAs enter cholangiocytes through the apical Na⁺ -dependent bile salt uptake transporter (ASBT) [35]. This is a 48 kDa integral membrane protein, localized on the cholangiocyte apical membrane. A truncated form of this transporter (t-ASBT), responsible for the final reabsorption of bile salts from the bile into the blood, is found on the basolateral membrane [36, 37]. To prevent the cytotoxic effects of intracellular BA accumulation, basolateral extrusion of bile salts is mediated by MRP3, a member of the multidrug resistance protein (MRP) subfamily of transporters. MRP3 substrates include the organic anions estradiol-17-glucuronide, bilirubin glucuronide, monovalent bile salts taurocholate and glycocholate, as well as divalent sulfated bile salts [8, 38].

Hepatocytes secrete glutathione into the bile. After glutathione in bile is hydrolyzed, the amino acids, glutamate, cysteine, and glycine are produced and then absorbed by cholangiocytes for the resynthesis of glutathione, which mediates bile salt-independent secretion of canalicular bile. Additionally, taurine and glycine play a key role in the formation of conjugated BAs, preventing the reabsorption of the conjugated BAs as they traffick through the biliary tract [28].

Cholangiocytes also express the G protein-coupled bile acid receptor, TGR5 (GPBAR-1, M-Bar, or GPR131). TGR5 is a transmembrane receptor linked to cAMP signaling expressed in a variety of human and rodent tissues that is encoded by a gene located on chromosome 1C3 in mouse and 2q35 in humans [60]. In cholangiocytes, TGR5 is found in multiple intracellular locations, including primary cilia on the apical domain, on the non-ciliary portion of the apical membrane, and on the inner and outer membrane of the cholangiocyte nucleus [61]. TGR5 is a major receptor for bile acid signaling in cholangiocytes, and its activation affects intracellular cAMP via coupling to Gα_s or Gα_i proteins subsequently triggering downstream signaling events [62]. A role for TGR5 in the development of gallstones was proposed by Keitel et al. In vitro experiments from the same study also showed that TGR5 stimulates the CFTR-dependent release of biliary Cl⁻ [63]. As mentioned above, primary cilia are key organelles involved in intracellular signaling and, as such, influence the response to TGR5 cholangiocyte activation. For example, in cholangiocytes, experimentally devoid of primary cilia, stimulation by TGR5 agonists enhanced cAMP activation via Gα_i, partially inhibiting ERK signaling, which results in reduced cholangiocyte proliferation [61]. Interestingly, the reverse outcomes were noted when ciliated cholangiocytes were challenged with the same TGR5 agonists [61]. Masyuk et al. demonstrated that primary cilia act as mechanosensors, responding to luminal fluid flow by alterations in intracellular Ca²⁺ and cAMP. The ciliary proteins involved in this transduction of mechanical stimuli include polycystin-1, a cell surface receptor, and polycystin-2, a Ca²⁺ channel [14]. Primary cilia also express the transient receptor potential 4 (TRPV4) protein, a Ca²⁺ permeable, nonselective cation channel, through which they can detect changes in osmolarity [15]. Gradilone et al. demonstrated that hypotonicity induces a rise in intracellular Ca²⁺ via TRPV4 activation in rat cholangiocytes. Furthermore, in vivo stimulation of cholangiocyte TRPV4 by intrabiliary saline increased adenosine triphosphate (ATP) production, HCO₃ ⁻ release, and thereby bile movement [15]. The role of cholangiocyte primary cilia as chemosensors has also been demonstrated. Primary cilia on rat cholangiocytes express the purinergic receptors, P2Y₁₂ and P2Y₁₃, that respond to changes in cAPM induced by adenosine diphosphate (ADP) and ATP-γS (nonhydrolyzed analog of ATP), two known agonists of P2Y₁₂ receptors. Moreover, suramin, an inhibitor of P2Y receptors, can prevent the ADP-dependent decrease of cAMP [16].