Characterization of cytosolic Ca2+ signaling in rat bile duct epithelia

Abstract

Bile duct epithelia play an important role in the formation and conditioning of bile. However, hormonal responses in this epithelial tissue are incompletely understood. Secretin increases ductular secretion through the intracellular messenger adenosine 3',5'-cyclic monophosphate (cAMP), but whether hormones increase cytosolic Ca2+ (Ca2+(i)) in these cells and whether Ca2+(i) regulates duct secretion is unknown. To address these questions, we examined Ca2+(i) signaling in isolated rat bile duct units using ratio microspectrofluorometry and confocal microscopy. We also used videomicroscopy to examine secretion and cell volume in isolated bile duct cells and duct units. Acetylcholine (ACh) and ATP both increased Ca2+(i) in bile duct units and elicited patterns of Ca2+(i) increases and oscillations that were distinct and dose dependent. In contrast, Ca2+(i) was not increased by the hepatocyte Ca2+(i) agonists vasopressin, angiotensin, and phenylephrine or by the exocrine pancreas agonists cholecystokinin (CCK) and bombesin. In addition, secretin did not increase Ca2+(i) in the isolated bile duct units, whereas ACh did not increase Ca2+(i) in isolated hepatocytes. Mobilization of internal, thapsigargin-sensitive Ca2+ stores contributed more than influx of extracellular Ca2+ to the Ca2+(i) increases induced in the duct units, and ATP-induced increases in Ca2+(i) could be blocked by microinjection of heparin but not de-N-sulfated heparin. ACh transiently decreased bile flow in the isolated perfused rat liver, although neither ACh nor ATP altered secretion in isolated ducts or changed the volume of single isolated bile duct cells. These findings demonstrate that bile duct epithelial cells possess both muscarinic and purinergic receptors that activate Ca2+(i) signaling pathways similar to those seen in other types of epithelia, but that the two types of receptors elicit distinct patterns of Ca2+(i) signals. Increases in Ca2+(i) have minimal direct effects on bile duct secretion, although it remains to be determined whether such signals selectively modulate other aspects of bile duct epithelial cell function.

Cyclic AMP Regulates Bicarbonate Secretion in Cholangiocytes Through Release of ATP Into Bile

“…The cAMP/CFTR pathway is thought to be of primary importance in maintaining ductular bile flow, and indeed cystic fibrosis can result in chronic cholestasis and secondary biliary cirrhosis (6). The second secretory pathway in cholangiocytes is stimulated by neurotransmitters such as acetylcholine (ACh) and autocrine agents such as ATP, and is mediated by cytosolic Ca 2+ (7). The downstream effect of Ca 2+ is to activate Ca 2+ -dependent chloride channels on the apical plasma membrane, which in turn release chloride that is exchanged for bicarbonate via a chloride-bicarbonate exchanger (3)(4)(5).…”

The Anti-apoptotic Protein Mcl-1 Inhibits Mitochondrial Ca2+ Signals

“…As an additional control, mitochondrial Ca 2ϩ signals in all transfected cells were compared with signals observed in co-cultured non-transfected cells on the same coverslips. Cells were stimulated with ATP (10 and 100 M), which is known to increase Ca 2ϩ via P2Y receptor-mediated InsP 3 formation in this cell type (21,29). Mitochondrial Ca 2ϩ increased just as often in cells transfected with GFP alone as in matched non-transfected controls (p Ͼ 0.1, Fig.…”

Physiology of Cholangiocytes

“…For example, lower concentrations of ATP predominantly induce sustained Ca 2+ oscillations, whereas higher concentrations predominantly induce single, brief Ca 2+ increases. In contrast, ACh does not display clear dose dependence, and the spikes it induces tend to be irregular in duration (although typically longer as compared to ATP induced) and frequency (161,200,210). This variability may enable different stimuli to elicit nuanced and distinct physiological effects even though they all act through modulation of intracholangiocyte Ca 2+ signaling (203).…”

“…Acetylcholine-The ACh-induced potentiation of secretin-stimulated HCO 3 − secretion results from activation of muscarinic M 1 and M 3 receptors that are expressed on the cholangiocyte basolateral plasma membrane followed by an increase of the intracellular Ca 2+ concentration (15,74,75,210). In cholangiocytes, ACh does not act via a Ca 2+ -activated PKC, but presumably by activation of Ca 2+ -sensitive AC isoforms, whereby it effectuates a twofold increase in cAMP (15).…”

“…Activation of apically located P2Y receptors in rat cholangiocytes causes a rapid and substantial increase in membrane Cl − permeability, favoring efflux of Cl − from the cell into the ductal lumen, which presumably occurs through Ca 2+ -activated Cl − channels (82,231,236). In rat IBDUs, both basolateral and apical membrane P2Y receptor activation results in an increase in intracellular Ca 2+ concentration and biliary HCO 3 − secretion, thus suggesting the importance of this mechanism in ductal bile modification (71,210). Activation of basolateral P2Y receptors by ATP and other nucleotides is less effective, however, in increasing intracellular Ca 2+ concentration compared to apical P2Y receptor activation (71).…”