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.
- [Show abstract] [Hide abstract]
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; · 2.64 Impact Factor
- [Show abstract] [Hide abstract]
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 01/2014; 9(12):e114043. · 3.53 Impact Factor
Article: Purinergic signaling[Show abstract] [Hide abstract]
ABSTRACT: The concept of purinergic neurotransmission was proposed in 1972, after it was shown that adenosine 5′-triphosphate (ATP) was a transmitter in non-adrenergic, non-cholinergic inhibitory nerves in the guinea pig taenia coli. Subsequently, ATP was identified as a cotransmitter in sympathetic and parasympathetic nerves, and it is now recognized that ATP acts as a cotransmitter in most nerves in both the peripheral nervous system and central nervous system (CNS). ATP acts as a fast excitatory neurotransmitter or neuromodulator and has potent long-term (trophic) roles in cell proliferation, differentiation, and death in development and regeneration, as well as in disease. Three subclasses of receptors to purines and pyrimidines have been identified, P1 adenosine receptors (with four subtypes), P2X ionotropic nucleotide receptors (seven subtypes), and P2Y metabotropic nucleotide receptors (eight subtypes). ATP is released physiologically by many different cell types by mechanical deformation, and after release ATP undergoes rapid enzymatic degradation by ectonucleotidases. Purinergic receptors appeared early in evolution and have a widespread distribution on many different non-neuronal cell types as well as neurons. There is evidence for the involvement of purinergic signaling in embryonic development and in the activities of stem cells. There is a rapidly growing literature about the pathophysiology of purinergic signaling, and there are therapeutic developments for a variety of diseases, including stroke and thrombosis, osteoporosis, kidney failure, bladder incontinence, cystic fibrosis, dry eye, cancer, and disorders of the CNS, such as Alzheimer's, Parkinson's, and Huntington's disease, multiple sclerosis, epilepsy, migraine, and neuropsychiatric disorders. WIREs Membr Transp Signal 2012, 1:116–125. doi: 10.1002/wmts.14 For further resources related to this article, please visit the WIREs website.WIREs Membr Transp Signal. 03/2012; 1(1):116-125.