Cholangiocytes exhibit dynamic, actin-dependent apical membrane turnover

Calcyon is called a "cross-talk accessory protein" because the mechanism by which it enables the typically G s -linked D1 dopamine receptor to stimulate intracellular calcium release depends on a priming step involving heterologous G q -linked G-protein-coupled receptor activation. The details of how priming facilitates the D1R calcium response have yet to be precisely elucidated. The present work shows that calcyon is constitutively localized both in vesicular and plasma membrane compartments within HEK293 cells. In addition, surface biotinylation and luminescence assays revealed that priming stimulates a 2-fold increase in the levels of calcyon expressed on the cell surface and that subsequent D1R activation produces further accumulation of the protein in the plasma membrane. The effects of priming and D1R agonists were blocked by nocodazole implicating microtubules in the delivery of calcyoncontaining vesicles to the cell surface. Accumulation of calcyon in the plasma membrane correlated well with increased intracellular calcium levels as thapsigargin mimicked, and 2-aminoethoxydiphenylborane abrogated, the effects of priming. KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII) also blocked the effects of priming and D1R agonists. Furthermore, expression of constitutively active forms of the kinase bypassed the requirement for priming indicating that CaMKII is a key effector in the Ca 2؉ and microtubule-dependent delivery of calcyon to the cell surface.The D1-like class of dopamine (DA) 1 receptors (D1R and D5R subtypes) belong to the superfamily of seven transmembrane domain, G-protein-coupled receptors (GPCRs), which regulate various biological functions, including movement, endocrine function, and memory processes by G-protein-coupled intracellular signaling pathways (1). In addition to the heterotrimeric G-proteins, signaling via D1Rs appears to be regulated by direct association with other proteins called dopamine receptor interacting proteins (DRIPs). Currently identified DRIPs for the D1R include adenosine (2) and NMDA (3) receptors, as well as a group of non-receptor proteins like calcyon (4), DriP78 (5), and neurofilament-M (6). Both DriP78 and neurofilament-M regulate the levels of D1Rs expressed on the cell surface, but by different mechanisms. For example, neurofilament-M hinders agonist promoted internalization of D1Rs, whereas DriP78 regulates the processing of newly synthesized D1Rs in the endoplasmic reticulum (ER).Calcyon is a predicted single transmembrane protein that is widely distributed in brain (7) and is abundant in D1R-positive neurons and dendrites in the prefrontal cortex and hippocampus (4). Interaction with calcyon has been shown to enhance the signaling capabilities of D1Rs. Specifically, calcyon enables typically G s -linked D1Rs to elicit robust intracellular calcium ([Ca 2ϩ ] i ) release by a mechanism that requires that heterologous G q -linked GPCRs have first been activated in a step called priming (4). Recent evidence from cells in primary cult...

Section: Discussionsupporting

confidence: 93%

Elevated Intracellular Calcium Triggers Recruitment of the Receptor Cross-talk Accessory Protein Calcyon to the Plasma Membrane

Ali

Bergson

2003

“…6, B and C show that DMSO also increased FM1-43 fluorescence. However, these changes were about 10 -20% in 10 min, which correspond to the rates of exocytosis of 1-2% per min that have been previously found in HTC and Mz-Cha-1 cells and result from constitutive exocytosis under basal conditions (27,39). Fig.…”

Section: Nppb-dependent Atp Release-supporting

confidence: 66%

5-Nitro-2-(3-phenylpropylamino)benzoic Acid (NPPB) Stimulates Cellular ATP Release through Exocytosis of ATP-enriched Vesicles

Dolovcak

Waldrop

Fitz

et al. 2009

Self Cite

Extracellular ATP acts as a potent signaling molecule in many different tissues including the immune system, neurons, endothelial cells, and secretory epithelia by activation of purinergic receptors in the plasma membrane (1). Cells release ATP in response to physiologic stimuli such as shear stress, stretch, osmotic swelling, and hypoxia (2-5). One mechanism for ATP release involves movement of ATP through transporters or channel proteins in the plasma membrane. There is evidence for ATP release through ATP-binding cassette (ABC) transporters, connexin and pannexin hemichannels, P2X7-pannexin1 receptorchannel complex, and multiple Cl Ϫ channels (6 -11). In addition, there is evidence for exocytic vesicular release of ATP (4, 12). Under basal conditions, the concentration of ATP in extracellular medium is in the low nanomolar range. Vesicles store ATP in the millimolar range, and exocytosis of these ATP-enriched vesicles increases local ATP concentrations. It has been difficult to study the contribution of exocytosis in ATP release because many cells are capable of releasing ATP through more than one mechanism. For example, ATP release from astrocytes is mediated by both vesicular exocytosis and transport proteins (8,(13)(14)(15). Thus, the role of vesicular exocytosis in ATP release is still poorly understood.5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) 2 is widely used as an inhibitor of many different Cl Ϫ channels and has been reported to inhibit ATP release mediated by Cl Ϫ channels (16,17). In some cells, NPPB also inhibits ATP release mediated by mechanosensitive and pannexin1 channels, and other channels that have not been defined (2,10,18,19). Thus, NPPB is an inhibitor of channel-mediated ATP release.The purpose of these studies was to assess the role of vesicular exocytosis in cellular ATP release. Using FM1-43 fluorescence to measure exocytosis and bioluminescence assay to measure ATP release in real time, we found that exposure to NPPB under basal conditions potently stimulates ATP release. These previously unknown effects of NPPB appear to be mediated through stimulation of exocytosis of a pool of ATP-enriched vesicles. EXPERIMENTAL PROCEDURESCell Models-Studies of ATP release were performed in HTC and Mz-Cha-1 cells. Both cell lines have been utilized as models for cellular ATP release, degradation, and purinergic signaling in secretory epithelia (7,20). HTC cells are derived from rat hepatoma, and Mz-Cha-1 cells are derived from human adenocarcinoma of the gall bladder. The procedures for culturing these cells have been previously described (21,22). Cells were used within 48 h after plating.Measurement of ATP Release-Cellular release of ATP was measured using the luciferin-luciferase assay as previously described (23,24). All cells were grown to confluence in 35-mm Petri dishes. Prior to study, cells were gently washed twice with 1 ml of OptiMEM (Invitrogen) and then 800 l of OptiMEM containing 2 mg/ml firefly luciferin-luciferase (Sigma cat. num.

“…Biliary cells, for example, possess a dense population of vesicles ϳ140 nm in diameter in the subapical space (44), and increases in cell volume increase the rate of exocytosis ϳ10-fold to values sufficient to replace 15-30% of plasma membrane surfaceareawithinminutesthroughaphosphoinositide3-kinasedependent mechanism (16). Both the rate of exocytosis and the amount of ATP released are potentiated by prior activation of PKC, suggesting that PKC is involved in the recruitment of these vesicles to a readily releasable state (16).…”

Section: Discussionmentioning

confidence: 99%

Initiation of Purinergic Signaling by Exocytosis of ATP-containing Vesicles in Liver Epithelium

Feranchak

Lewis

Kresge

et al. 2010

Self Cite

Extracellular ATP represents an important autocrine/paracrine signaling molecule within the liver. The mechanisms responsible for ATP release are unknown, and alternative pathways have been proposed, including either conductive ATP movement through channels or exocytosis of ATP-enriched vesicles, although direct evidence from liver cells has been lacking. Utilizing dynamic imaging modalities (confocal and total internal reflection fluorescence microscopy and luminescence detection utilizing a high sensitivity CCD camera) at different scales, including confluent cell populations, single cells, and the intracellular submembrane space, we have demonstrated in a model liver cell line that (i) ATP release is not uniform but reflects point source release by a defined subset of cells; (ii) ATP within cells is localized to discrete zones of high intensity that are ϳ1 m in diameter, suggesting a vesicular localization; (iii) these vesicles originate from a bafilomycin A 1 -sensitive pool, are depleted by hypotonic exposure, and are not rapidly replenished from recycling of endocytic vesicles; and (iv) exocytosis of vesicles in response to cell volume changes depends upon a complex series of signaling events that requires intact microtubules as well as phosphoinositide 3-kinase and protein kinase C. Collectively, these findings are most consistent with an essential role for exocytosis in regulated release of ATP and initiation of purinergic signaling in liver cells.Extracellular ATP functions within liver as a key autocrine/ paracrine signaling molecule. The purinergic signaling cascade is initiated by release of ATP from intracellular stores, but the mechanisms involved are poorly understood. However, P2 receptors are expressed on all liver cell types, and once outside of the cell ATP has multiple effects, including (i) coordination within the liver lobule of cell-to-cell [Ca 2ϩ ] i signaling (1), (ii) maintenance of cell volume within a narrow physiological range (2), and (iii) coupling of the separate hepatocyte and cholangiocyte contributions to bile formation and stimulation of biliary secretion (3). Specifically, cellular ATP release leads to increased concentrations of ATP in bile sufficient to activate P2 receptors in the apical membrane of targeted cholangiocytes, resulting in a robust secretory response through activation of Cl Ϫ channels in the apical membrane. Moreover, multiple signals including intracellular calcium, cAMP and bile acids appear to coordinate ATP release, which has been recognized recently as a final common pathway responsible for biliary secretion (3-5). Accordingly, definition of the mechanisms involved in ATP release represents a key focus for efforts to modulate liver function and the volume and composition of bile.Previous studies indicate that increases in cell volume serve as a potent stimulus for physiologic ATP release in many epithelia and in liver cells increase extracellular nucleotide concentrations 5-10-fold (6). Two broad models for ATP release by nonexcitatory cells have been...