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Regulation of purinergic signaling in biliary epithelial cells by exocytosis of SLC17A9-dependent ATP-enriched vesicles.

ABSTRACT: ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. Dynamic, multiscale, live cell imaging (confocal and total internal reflection fluorescence microscopy and a luminescence detection system with a high sensitivity charge-coupled device camera) was utilized to detect vesicular ATP release from cell populations, single cells, and the submembrane space of a single cell. In response to increases in cell volume, BECs release ATP, which was dependent on intact microtubules and vesicular trafficking pathways. ATP release occurred as stochastic point source bursts of luminescence consistent with exocytic events. Parallel studies identified ATP-enriched vesicles ranging in size from 0.4 to 1 ?m that underwent fusion and release in response to increases in cell volume in a protein kinase C-dependent manner. Present in all models, SLC17A9 contributed to ATP vesicle formation and regulated ATP release. The findings are consistent with the existence of an SLC17A9-dependent ATP-enriched vesicular pool in biliary epithelium that undergoes regulated exocytosis to initiate purinergic signaling.

SUBMITTER: Sathe MN

PROVIDER: S-EPMC3137107 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Regulation of purinergic signaling in biliary epithelial cells by exocytosis of SLC17A9-dependent ATP-enriched vesicles.

Sathe Meghana N MN Woo Kangmee K Kresge Charles C Bugde Abhijit A Luby-Phelps Kate K Lewis Matthew A MA Feranchak Andrew P AP

The Journal of biological chemistry 20110525 28

ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. ...[more]

PMID: 21613220

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Project description:Cryptosporidiosis is a zoonotic intestinal disease that affects humans, wildlife, and neonatal cattle, caused by Cryptosporidium parvum. Neutrophil extracellular traps (NETs), also known as suicidal NETosis, are a powerful and ancient innate effector mechanism by which polymorphonuclear neutrophils (PMN) battle parasitic organisms like protozoa and helminths. Here, C. parvum oocysts and live sporozoites were utilized to examine suicidal NETosis in exposed bovine PMN under both 5% O2 (physiological conditions within small intestinal tract) and 21% O2 (normal hyperoxic conditions in research facilities). Both sporozoites and oocysts induced suicidal NETosis in exposed PMN under physioxia (5% O2) and hyperoxia (21% O2). Besides, C. parvum-induced suicidal NETosis was affirmed by total break of PMN, co-localization of extracellular DNA decorated with pan-histones (H1A, H2A/H2B, H3, H4) and neutrophil elastase (NE) by means of confocal- and immunofluorescence microscopy investigations. C. parvum-triggered NETs entrapped sporozoites and impeded sporozoite egress from oocysts covered by released NETs, according to scanning electron microscopy (SEM) examination. Live cell 3D-holotomographic microscopy analysis visualized early parasite-induced PMN morphological changes, such as the formation of membrane protrusions towards C. parvum while undergoing NETosis. Significant reduction of C. parvum-induced suicidal NETosis was measured after PMN treatments with purinergic receptor P2X1 inhibitor NF449, under both oxygen circumstances, this receptor was found to play a critical role in the induction of NETs, indicating its importance. Similarly, inhibition of PMN glycolysis via 2-deoxy glucose treatments resulted in a reduction of C. parvum-triggered suicidal NETosis but not significantly. Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) were not increased in C. parvum-exposed cells, according to measurements of PMN energetic state. Treatments with inhibitors of plasma membrane monocarboxylate transporters (MCTs) of lactate failed to significantly reduce C. parvum-mediated NET extrusion. Concerning Notch signaling, no significant reduction was detected after PMN treatments with two specific Notch inhibitors, i.e., DAPT and compound E. Overall, we here describe for the first time the pivotal role of ATP purinergic receptor P2X1 in C. parvum-mediated suicidal NETosis under physioxia (5% O2) and its anti-cryptosporidial properties.

Dataset Information

Regulation of purinergic signaling in biliary epithelial cells by exocytosis of SLC17A9-dependent ATP-enriched vesicles.

Publications

Regulation of purinergic signaling in biliary epithelial cells by exocytosis of SLC17A9-dependent ATP-enriched vesicles.

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