Physiology of Cholngiocytes

44.3.1.1

HCO ₃ ⁻ -Rich Aqueous Secretion

The signature secretory function of cholangiocytes is HCO ₃ ⁻ -rich fluid secretion (i.e., ductal bile formation) into the lumen of intrahepatic bile ducts. Physiologically, ductal bile formation is a functional response of the liver to a meal. In the postprandial state, acidic pH stimulates endocrine S cells in the duodenum and jejunum to release a 27-amino-acid peptide, secretin, into the portal venous circulation. In the digestive phase, secretin regulates a number of physiological functions, including cholangiocyte HCO ₃ ⁻ -rich fluid secretion, which determines the alkalinity, hydration, and pH of bile, minimizes passive cholangiocyte absorption of protonated glycine-conjugated bile acids by forming the biliary HCO ₃ ⁻ umbrella, and significantly contributes to the intestinal pool of HCO ₃ ⁻ required for digestion.

In the liver, only large cholangiocytes express heteromeric G protein-coupled secretin receptors (SRs) on the basolateral plasma membrane, which when activated, results in an increase in intracellular cAMP levels followed by the activation of protein kinase A (PKA) that phosphorylates and activates a cAMP-regulated Cl ⁻ channel, the CFTR , located on the apical plasma membrane. The CFTR belongs to a family of adenosine triphosphate (ATP)-binding cassette (ABC) proteins with intracellular N and C termini, and contains two domains spanning the plasma membrane six times each. These two transmembrane domains are separated by an intracellular regulatory domain containing phosphorylation sites for both PKA and protein kinase C (PKC).

Activated CFTR is a Cl ⁻ channel in an open state through which chloride ions pass out of the cell into the ductal lumen. Under basal conditions, the Cl ⁻ permeability of the cholangiocyte apical plasma membrane is low; stimulation with cAMP increases Cl ⁻ transport by 20- to 40-fold.

The current working model of cholangiocyte HCO ₃ ⁻ -rich fluid secretion in humans ( Fig. 44.6 ) proposes that accumulation of Cl ⁻ in the bile duct lumen results in the activation of the apically located Na ⁺ -independent electroneutral Cl ⁻ /HCO ₃ ⁻ exchanger SLC4A2/AE2 , leading to the release of HCO ₃ ⁻ into the bile duct lumen in exchange for Cl ⁻ . HCO ₃ ⁻ secretion is followed by osmotically driven efflux of water into the lumen of intrahepatic bile ducts primarily via water channels or AQPs. The end result is an increase in HCO ₃ ⁻ -rich fluid secretion, which alkalinizes and dilutes ductal bile.

Cholangiocyte HCO ₃ ⁻ secretion in rodents occurs via a Na ⁺ - HCO ₃ ⁻ cotransport (NBC) carrier SLC4A4 that transports one Na ⁺ and three HCO ₃ ⁻ .

Secretin-induced ductal bile secretion might be enhanced by the inhibition of the Na ⁺ /H ⁺ exchanger NHE3/SLC9A3 , which is expressed on the cholangiocyte apical plasma membrane ( Fig. 44.6 ). NHE3 stimulates absorption of fluid from the biliary lumen, thus counterbalancing the fluid secretion in resting bile duct epithelia. The PKA, activated by secretin, inhibits NHE3-mediated fluid absorption, and as a result, increases the net (secretin-induced) fluid secretion.

The secretin signal in cholangiocytes is inactivated by protein phosphatases 1 and/or 2A. These phosphatases dephosphorylate the regulatory domain of CFTR and promote conformational changes causing the occlusion of the Cl ⁻ conductance pathway, thus restoring the basal quiescent state. Thus, the ductal bile secretion stimulated by secretin is regulated at the level of CFTR by a balance between the activities of kinases (inducing activation) and phosphatases (causing inactivation) of this channel.

An alternative or additional mechanism through which CFTR contributes to the modification of bile involves Ca ^{2 +} -activated Cl ⁻ channels . According to this model ( Fig. 44.7 ), CFTR regulates the release of ATP from cholangiocytes into the lumen of bile duct by an unknown mechanism, perhaps through ATP-permeable channels and/or exocytosis of ATP-containing vesicles. The ATP then activates apically located P2 receptors resulting in an increase in intracellular Ca ^{2 +} and Ca ^{2 +} -activated Cl ⁻ secretion followed by an increase in HCO ₃ ⁻ and water secretion into the bile duct lumen. A recently identified Ca ^{2 +} -activated Cl ⁻ channel, TMEM16A , on the cholangiocyte apical plasma membrane , favors this model of ductal bile secretion. The activity of TMEM16A in human, rat, and mouse cholangiocytes is increased in response to ATP, and the magnitude of ATP-stimulated TMEM16A-mediated Cl ⁻ currents is threefold greater compared with cAMP-stimulated Cl ⁻ currents mediated by CFTR. Importantly, both the small and large cholangiocytes express TMEM16A and show Ca ^{2 +} -activated Cl ⁻ efflux in response to extracellular nucleotides, whereas only large cholangiocytes show cAMP-stimulated Cl ⁻ currents. These observations suggest that large cholangiocytes may initiate ductal bile formation via activation of both CFTR and TMEM16A, whereas small cholangiocytes may contribute to ductal bile formation via activation of TMEM16A. The contribution of CFTR and TMEM16A to cholangiocyte HCO ₃ ⁻ -rich aqueous secretion may depend on species, but it is likely that TMEM16A provides the predominant mechanisms of ductal bile secretion in human and mouse cholangiocytes.

The cholangiocyte secretion of Cl ⁻ is enhanced in response to activation of a Ca ^{2 +} -activated small conductance (SK2) K ⁺ channel located on the apical plasma membrane. In secretory epithelia, it is thought that activation of K ⁺ channels leads to membrane hyperpolarization by which the electrical driving force for continued Cl ⁻ efflux is maintained.

Thus, the mechanisms of cholangiocyte HCO ₃ ⁻ -rich aqueous secretion are associated with cAMP-activated Cl ⁻ channels, CFTR, Ca ^{2 +} -activated Cl ⁻ channels, TMEM16A, Cl ⁻ /HCO ₃ exchangers, and AQPs. The apically located Na ⁺ /H ⁺ exchanger NHE3, Na ⁺ -HCO ₃ ⁻ cotransport (NBC) carrier SLC4A4, and (SK2) K ⁺ channel play supportive roles in this process.

The apically located mechanisms of cholangiocyte secretion are coordinated with functions of channels, transporters, and exchangers expressed on the basolateral plasma membrane. In particular, a high concentration of intracellular HCO ₃ ⁻ necessary for ductal bile secretion is maintained by coordinated functions of basolaterally located Na ⁺ - H ⁺ exchanger, NHE1/SLC9A1 , Na ⁺ - HCO ₃ ⁻ cotransporter, and Na ⁺ - HCO ⁻ ₃ exchanger . Cytoplasmic carbonic anhydrase, an enzyme that generates HCO ₃ ⁻ from CO ₂ and H ₂ O, also contributes to high level of HCO ₃ ⁻ .

A high concentration of intracellular Cl ⁻ is maintained by an electroneutral, bumetanide-sensitive Na ⁺ - K ⁺ - Cl ⁻ cotransporter that, under physiologic conditions, mediates transport of Na, K, and Cl ions into the cell in an electrically neutral manner with a stoichiometry of 1Na:1K:2Cl, and by Cl ⁻ -HCO ⁻ ₃ exchange that provides active Cl ⁻ uptake. An increase in the apical membrane permeability to Cl ⁻ also results in the activation of two calcium-activated K ⁺ channels, SK2 and intermediate-conductance Ca ^{2 +} -activated K ⁺ channel 1, IK-1. These channels function in a complimentary manner to support the cholangiocyte secretion via recycling of K ⁺ that was brought into the cell by the Na ⁺ -K ⁺ -2Cl ⁻ cotransporter and by Na ⁺ , K ⁺ -ATPase.

HCO ⁻ ₃ secreted by cholangiocytes establishes an osmotic gradient that drives water efflux into bile duct lumen via AQPs . The AQPs are organized on the cell membrane as tetramers. The monomeric units of AQPs (26–34 kDa) contain the highly conservative NPA (asparagine-proline-alanine) motifs critical for the formation of the amphipathic pore with the diameter ranging from 1.5 to 3.4 Å, a width sufficient to allow effective passage of water molecules in single files in either direction.

The rat and mouse cholangiocytes express mRNAs for 6 AQPs and for all the 12 known AOPs, respectively. At the protein level, at least four distinct AQPs (i.e., AQP1, AQP4, AQP8, and AQP9) are expressed in mammalian cholangiocytes. Human cholangiocytes express AQP1 and AQP9.

In the basal state, AQP1 is principally expressed in rat cholangiocytes within intracellular vesicles also containing CFTR and SLC4A2/AE2. In the secretin-stimulated state, AQP1, CFTR, and AE2 are translocated to the apical plasma membrane domain facilitating HCO ⁻ ₃ -driven water secretion.

The AQP1-mediated water transport across the cholangiocyte apical plasma membrane is effectively balanced by AQP4 exclusively expressed on the basolateral plasma membrane. In contrast to AQP1, AQP4 functions are not regulated by secretin suggesting that AQP4 is constitutively present in the cholangiocyte basolateral plasma membrane, or alternatively, that its functions are regulated by different signaling mechanisms.

The functional studies on isolated rat enclosed and microperfused intrahepatic bile duct units (IBDUs) in which water transport into the lumen of IBDUs in response to inward (i.e., secretory) osmotic gradients was prevented by an inhibitor of AQPs, mercuric chloride (HgCl ₂ ), as well as by small interfering RNAs to AQP1, strongly supported the importance of AQP1-mediated water transport in biliary epithelia.

The role of AQP1 in ductal bile secretion in mice is controversial. Despite the fact that AQP1 is expressed in mouse small and large intrahepatic bile ducts, this water channel does not appear to be rate limiting for ductal bile formation because this process was not affected by deletion of gene encoding AQP1. Presumably, AQP8 expressed in substantial amounts in large cholangiocytes, is crucial for AQP-mediated water transport in mouse biliary epithelia.

44.3.2.1

Bile Acid Reabsorption

A small amount of bile acids secreted by hepatocytes is reabsorbed from the lumen of intrahepatic bile ducts and returned to hepatocytes instead of being delivered to the intestine. Unconjugated and conjugated bile acids are reabsorbed by two different mechanisms via the chole-hepatic shunt pathway. Unconjugated bile acids secreted by hepatocytes in an anionic form become protonated and passively diffuse into cholangiocytes through their apical plasma membrane. Then they are transported to the basolateral plasma membrane domain, released into the PBP, and returned to hepatocytes. In contrast, conjugated bile acids are transported across the cholangiocyte apical and basolateral plasma membrane domains by specific transporters.

The principal bile acid transporter located on the cholangiocyte apical plasma membrane is the Na ⁺ -dependent bile acid transporter ASBT/SLC10A2, a 48 kDa integral membrane protein identical to the bile acid transporter cloned from rat ileal and renal epithelial cells. ASBT/SLC10A2 is a highly efficient transporter of conjugated bile acid that mediates coupled bile acid—Na ⁺ uptake in a 2:1 ratio. An alternatively spliced form of the Asbt gene generates a truncated, 19 kDa form of ASBR, t-ASBT, located exclusively on the cholangiocyte basolateral plasma membrane presumably providing mechanisms for bile acids efflux from cholangiocytes into the peribiliary plexus. Given the toxic effects associated with intracellular accumulation of bile acids, the mechanisms of bile acids efflux from cholangiocytes via the basolateral plasma membrane are extensive and redundant. In addition to t-ASBT, these mechanisms include the multidrug resistance protein 3 (MRP3/ABCB4), MRP4, the organic anion transporting polypeptide 3 (OATP3), and the organic solute transporter OST alpha-OST beta (OSTα-OSTβ). The physiological relevance of these bile acid transporters remain to be elucidated. In general, the transcellular vectorial transport of bile acids from bile to blood may contribute to the modification of bile via regulation of its chemical composition and properties by removing some amount of bile acids and generating an osmotic pressure that facilitates AQP-mediated water absorption.