Extracellular nucleotides stimulate Cl- currents in biliary epithelia through receptor-mediated IP3 and Ca2+ release
ABSTRACT Extracellular ATP regulates bile formation by binding to P2 receptors on cholangiocytes and stimulating transepithelial Cl(-) secretion. However, the specific signaling pathways linking receptor binding to Cl(-) channel activation are not known. Consequently, the aim of these studies in human Mz-Cha-1 biliary cells and normal rat cholangiocyte monolayers was to assess the intracellular pathways responsible for ATP-stimulated increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and membrane Cl(-) permeability. Exposure of cells to ATP resulted in a rapid increase in [Ca(2+)](i) and activation of membrane Cl(-) currents; both responses were abolished by prior depletion of intracellular Ca(2+). ATP-stimulated Cl(-) currents demonstrated mild outward rectification, reversal at E(Cl(-)), and a single-channel conductance of approximately 17 pS, where E is the equilibrium potential. The conductance response to ATP was inhibited by the Cl(-) channel inhibitors NPPB and DIDS but not the CFTR inhibitor CFTR(inh)-172. Both ATP-stimulated increases in [Ca(2+)](i) and Cl(-) channel activity were inhibited by the P2Y receptor antagonist suramin. The PLC inhibitor U73122 and the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB both blocked the ATP-stimulated increase in [Ca(2+)](i) and membrane Cl(-) currents. Intracellular dialysis with purified IP3 activated Cl(-) currents with identical properties to those activated by ATP. Exposure of normal rat cholangiocyte monolayers to ATP increased short-circuit currents (I(sc)), reflecting transepithelial secretion. The I(sc) was unaffected by CFTR(inh)-172 but was significantly inhibited by U73122 or 2-APB. In summary, these findings indicate that the apical P2Y-IP3 receptor signaling complex is a dominant pathway mediating biliary epithelial Cl(-) transport and, therefore, may represent a potential target for increasing secretion in the treatment of cholestatic liver disease.
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ABSTRACT: Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca2+) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca2+ signaling is unknown. Ca2+ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca2+ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca2+ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity.PLoS ONE 12/2014; 9(12):e114043. DOI:10.1371/journal.pone.0114043 · 3.53 Impact Factor
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ABSTRACT: Purinergic signalling plays major roles in the physiology and pathophysiology of digestive organs. Adenosine 5'-triphosphate (ATP), together with nitric oxide and vasoactive intestinal peptide, is a cotransmitter in non-adrenergic, non-cholinergic inhibitory neuromuscular transmission. P2X and P2Y receptors are widely expressed in myenteric and submucous enteric plexuses and participate in sympathetic transmission and neuromodulation involved in enteric reflex activities, as well as influencing gastric and intestinal epithelial secretion and vascular activities. Involvement of purinergic signalling has been identified in a variety of diseases, including inflammatory bowel disease, ischaemia, diabetes and cancer. Purinergic mechanosensory transduction forms the basis of enteric nociception, where ATP released from mucosal epithelial cells by distension activates nociceptive subepithelial primary afferent sensory fibres expressing P2X3 receptors to send messages to the pain centres in the central nervous system via interneurons in the spinal cord. Purinergic signalling is also involved in salivary gland and bile duct secretion.Purinergic Signalling 12/2013; 10(1). DOI:10.1007/s11302-013-9397-9 · 2.64 Impact Factor
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ABSTRACT: Background & Aims Both hepatocytes and cholangiocytes release ATP into bile, where it acts as a potent autocrine/paracrine stimulus that activates biliary secretory mechanisms. ATP is known to be metabolized into multiple breakdown products, ultimately yielding adenosine. However, the elements implicated in the adenosine-dependent purinergic regulation of cholangiocytes are not known. Methods Normal rat cholangiocytes (NRC) were used to study the expression of adenosine receptors and transporters and their functional interactions at the apical and basolateral membrane domains of polarized cholangiocytes. Results We found that: (1) cholangiocytes exclusively express two concentrative nucleoside transporters (CNT) known to be efficient adenosine carriers: CNT3 locating at the apical membrane, and CNT2 locating at the apical and basolateral domains; (2) in both domains NRC also express the high affinity adenosine receptor A2A, which modulates the activity of apical CNT3 in a domain-specific manner; (3) the regulation exerted by A2A on CNT3 is dependent upon the cAMP/PKA/ERK/CREB axis, intracellular trafficking mechanisms and AMPK phosphorylation; (4) secretin increases the activity of the apically-located CNT3, and promotes an additional basolateral CNT3-related activity; and (5) extracellular ATP (a precursor of adenosine) is able to exert an inhibitory effect on the apical activity of both CNT3 and CNT2. Conclusions This study uncovers the functional expression of nucleoside transporters in cholangiocytes and provides evidence for direct crosstalks between adenosine transporters and the receptors for adenosine and for its natural extracellular precursor, ATP. Our data anticipate the possibility of adenosine playing a major role in the physiopathology of the biliary epithelia.Journal of Hepatology 12/2014; DOI:10.1016/j.jhep.2014.06.036 · 10.40 Impact Factor