Role of AE2 for pHi regulation in biliary epithelial cells

Abstract

The Cl(-)/HCO(-) 3anion exchanger 2 (AE2) is known to be involved in intracellular pH (pHi) regulation and transepithelial acid-base transport. Early studies showed that AE2 gene expression is reduced in liver biopsies and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic non-suppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. Microfluorimetric analysis of the Cl(-)/HCO(-) 3 anion exchange (AE) in isolated cholangiocytes showed that the cAMP-stimulated AE activity is diminished in PBC compared to both healthy and diseased controls. More recently, it was found that miR-506 is upregulated in cholangiocytes of PBC patients and that AE2 may be a target of miR-506. Additional evidence for a pathogenic role of AE2 dysregulation in PBC was obtained with Ae2 (-/-) a,b mice, which develop biochemical, histological, and immunologic alterations that resemble PBC (including development of serum AMA). Analysis of HCO(-) 3 transport systems and pHi regulation in cholangiocytes from normal and Ae2 (-/-) a,b mice confirmed that AE2 is the transporter responsible for the Cl(-)/HCO(-) 3exchange in these cells. On the other hand, both Ae2 (+/+) a,b and Ae2 (-/-) a,b mouse cholangiocytes exhibited a Cl(-)-independent bicarbonate transport system, essentially a Na(+)-bicarbonate cotransport (NBC) system, which could contribute to pHi regulation in the absence of AE2.

Keywords: Cl−/HCO−3 anion exchange; bile flow; biliary HCO−3 secretion; cholangiocytes; primary biliary cirrhosis.

Figure 1

Major ion carriers involved in pH_i regulation in cholangiocytes. (A) Acid extruders or HCO⁻₃ loaders: Cells are loaded with HCO⁻₃ via CO₂ hydration catalyzed by carbonic anhydrases [CO_2(g)+H₂O_(l) ↔ HCO_3−(aq) + H⁺_(aq)] and subsequent H⁺ extrusion through Na⁺/H⁺ exchange, mainly mediated by the basolateral amiloride-sensitive NHE1, that is recognized as a potent acid extruder. Amiloride-insensitive NHE2 and amiloride-sensitive NHE3 may also participate, though these apical exchangers seems to play a major role for NaCl and fluid absorption from the bile duct lumen (Strazzabosco, ; Spirlì et al., ; Mennone et al., 2001). Additionally, Na⁺: HCO⁻₃ cotransporters (NBC) with a stoichiometry of 1:2 or a Na⁺-dependent Cl⁻/HCO⁻₃ exchanger (NDCBE) may load HCO⁻₃ (in rodent or human cholangiocytes, respectively). (B) Acid loaders: The Na⁺-independent Cl⁻/HCO⁻₃ exchanger AE2 is the major acid loader in cholangiocytes. Physiologically, it extrudes HCO⁻₃ in exchange with Cl⁻ once a high outside to inside gradient has been established following stimulation of a variety of apical Cl⁻ channels (the cAMP-activated CFTR and the Ca²⁺-dependent TMEM16A—illustrated in Figure 2—among other channels pending a complete characterization). Characteristically, mouse cholangiocytes possess an additional capability for acid loading through Na⁺: HCO⁻₃ cotransport (putatively with a stoichiometry of 1:3) (Uriarte et al., 2010). The biliary epithelial cells have other ion carriers like those for Cl⁻, Na⁺, and K⁺ (not shown) which may contribute, at least indirectly, to pH_i regulation and/or HCO⁻₃ secretion. Asterisks are used to indicate that locations for NBC(s) remain to be definitely determined.

Figure 2

Main mechanisms involved in biliary HCO⁻₃ secretion in cholangiocytes. Lower left: illustrates that the hormone secretin induces trafficking of vesicles with the chloride channel CFTR, the anion exchanger AE2/SLC4A2, and the water channel AQP1. Vesicle exocytosis at the apical membrane allows for bicarbonate-rich hydrocholeresis. Lower right: cholinergic stimulation of basolateral M3 muscarinic receptors increases InsP3 and leads to Ca²⁺ release. Activation of the apical Ca²⁺-dependent Cl⁻ channel TMEM16A results in efflux of Cl⁻ which is then exchanged with HCO⁻₃ via AE2. Moreover, CFTR activation that follows secretin stimulation may induce apical release of ATP with further stimulation of apical P2Y receptors, increases in InsP3 and Ca²⁺, activation of apical Ca²⁺-dependent Cl⁻ channel TMEM16A, Cl⁻ efflux and final AE2-mediated Cl⁻/HCO⁻₃ exchanger for apical HCO⁻₃ secretion. Upper right: release of ATP that follows PKC-dependent exocytosis of ATP-enriched intracellular vesicles upon increases in cell volume. Further stimulation of apical P2Y receptors may end up with apical HCO⁻₃ secretion as described for the CFTR-dependent release of ATP.