Exocytotic Release of ATP from Cultured Astrocytes

Pangršič, Tina; Potokar, Maja; Stenovec, Matjaž; Kreft, Marko; Fabbretti, Elsa; Nistri, Andrea; Pryazhnikov, Evgeny; Khiroug, Leonard; Giniatullin, Rashid; Zorec, Robert

doi:10.1074/jbc.m700290200

Cited by 218 publications

(227 citation statements)

References 54 publications

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“…Live human osteosarcoma U2OS cells maintained in control conditions and stained with the ATP-sensitive fluorochrome quinacrine, which is usually employed for the detection of intracellular ATP-containing vesicles, 36 exhibit a green fluorescence that can readily be monitored by cytofluorometry. This signal is significantly reduced when U2OS cells are co-treated with 2-deoxyglucose (an inhibitor of glycolysis) and antimycin A (which blocks mitochondrial respiration at the level of complex III; Figure 1a), reflecting a significant depletion of intracellular ATP stores.…”

Section: Resultsmentioning

confidence: 99%

Molecular mechanisms of ATP secretion during immunogenic cell death

et al. 2013

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The immunogenic demise of cancer cells can be induced by various chemotherapeutics, such as anthracyclines and oxaliplatin, and provokes an immune response against tumor-associated antigens. Thus, immunogenic cell death (ICD)-inducing antineoplastic agents stimulate a tumor-specific immune response that determines the long-term success of therapy. The release of ATP from dying cells constitutes one of the three major hallmarks of ICD and occurs independently of the two others, namely, the pre-apoptotic exposure of calreticulin on the cell surface and the postmortem release of high-mobility group box 1 (HMBG1) into the extracellular space. Pre-mortem autophagy is known to be required for the ICD-associated secretion of ATP, implying that autophagy-deficient cancer cells fail to elicit therapy-relevant immune responses in vivo. However, the precise molecular mechanisms whereby ATP is actively secreted in the course of ICD remain elusive. Using a combination of pharmacological screens, silencing experiments and techniques to monitor the subcellular localization of ATP, we show here that, in response to ICD inducers, ATP redistributes from lysosomes to autolysosomes and is secreted by a mechanism that requires the lysosomal protein LAMP1, which translocates to the plasma membrane in a strictly caspase-dependent manner. The secretion of ATP additionally involves the caspase-dependent activation of Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)-mediated, myosin II-dependent cellular blebbing, as well as the opening of pannexin 1 (PANX1) channels, which is also triggered by caspases. Of note, although autophagy and LAMP1 fail to influence PANX1 channel opening, PANX1 is required for the ICD-associated translocation of LAMP1 to the plasma membrane. Altogether, these findings suggest that caspase-and PANX1-dependent lysosomal exocytosis has an essential role in ATP release as triggered by immunogenic chemotherapy.

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Section: Resultsmentioning

confidence: 99%

Molecular mechanisms of ATP secretion during immunogenic cell death

et al. 2013

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“…ATP is released from cells through various mechanisms, including exocytosis [34], maxi-anion channels [35], volumesensitive outwardly rectifying chloride channels [36], and gap-junction hemichannels [37]. Although the mechanism of release of ATP from cancer cells has not been clearly established, it is possible that the ability to release ATP is correlated with malignancy or motility of cancer cells.…”

Section: Discussionmentioning

confidence: 99%

Autocrine signaling via release of ATP and activation of P2X7 receptor influences motile activity of human lung cancer cells

Takai

Tsukimoto

Hishida

et al. 2014

Purinergic Signalling

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Extracellular nucleotides, such as ATP, are released from cells and play roles in various physiological and pathological processes through activation of P2 receptors. Here, we show that autocrine signaling through release of ATP and activation of P2X7 receptor influences migration of human lung cancer cells. Release of ATP was induced by stimulation with TGF-β1, which is a potent inducer of cell migration, in human lung cancer H292 cells, but not in noncancerous BEAS-2B cells. Treatment of H292 cells with a specific antagonist of P2X7 receptor resulted in suppression of TGF-β1-induced migration. PC-9 human lung cancer cells released a large amount of ATP under standard cell culture conditions, and P2X7 receptor-dependent dye uptake was observed even in the absence of exogenous ligand, suggesting constitutive activation of P2X7 receptor in this cell line. PC-9 cells showed high motile activity, which was inhibited by treatment with ecto-nucleotidase and P2X7 receptor antagonists, whereas a P2X7 receptor agonist enhanced migration. PC-9 cells also harbor a constitutively active mutation in epidermal growth factor receptor (EGFR). Treatment with EGFR tyrosine kinase inhibitor AG1478 suppressed both cell migration and P2X7 receptor expression in PC-9 cells. Compared to control PC-9 cells, cells treated with P2X7 antagonist exhibited broadened lamellipodia around the cell periphery, while AG1478-treated cells lacked lamellipodia. These results indicate that P2X7-mediated signaling and EGFR signaling may regulate migration of PC-9 cells through distinct mechanisms. We propose that autocrine ATP-P2X7 signaling is involved in migration of human lung cancer cells through regulation of actin cytoskeleton rearrangement.

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“…6). Our immunohistochemical studies with anti-mSLC17A9 antibodies indicated that mSLC17A9 protein is expressed in a population of astrocytes, suggesting involvement of this transporter in storage of ATP in ATP-secreting astrocytes (25)(26)(27). Thus, identification of SLC17A9 protein as a VNUT may reveal the molecular mechanisms behind how ATP is secreted from purinergic cells and provide a novel molecular target for the pathophysiology of purinergic signaling and its therapeutic potential (9)(10)(11).…”

Section: Slc17a9 Protein As a Vnutmentioning

confidence: 99%

Identification of a vesicular nucleotide transporter

Sawada

Echigo

Juge

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

374

457

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ATP is a major chemical transmitter in purinergic signal transmission. Before secretion, ATP is stored in secretory vesicles found in purinergic cells. Although the presence of active transport mechanisms for ATP has been postulated for a long time, the proteins responsible for its vesicular accumulation remains unknown. The transporter encoded by the human and mouse SLC17A9 gene, a novel member of an anion transporter family, was predominantly expressed in the brain and adrenal gland. The mouse and bovine counterparts were associated with adrenal chromaffin granules. Proteoliposomes containing purified transporter actively took up ATP, ADP, and GTP by using membrane potential as the driving force. The uptake properties of the reconstituted transporter were similar to that of the ATP uptake by synaptic vesicles and chromaffin granules. Suppression of endogenous SLC17A9 expression in PC12 cells decreased exocytosis of ATP. These findings strongly suggest that SLC17A9 protein is a vesicular nucleotide transporter and should lead to the elucidation of the molecular mechanism of ATP secretion in purinergic signal transmission.ATP ͉ chromaffin granule ͉ purinergic signaling ͉ storage and exocytosis ͉ synaptic vesicle V esicular storage and subsequent exocytosis of neurotransmitters is essential for chemical transmission in neurons and endocrine cells. Thus far four distinct classes of transporters are known to participate in the uptake of neurotransmitters into neuronal synaptic vesicles and secretory granules in endocrine cells. These are vesicular monoamine transporters, vesicular acetylcholine transporters, vesicular inhibitory amino acid transporters, and vesicular glutamate transporters (VGLUTs) (1-7). These vesicular transporters mediate the active accumulation of their respective neurotransmitters through an electrochemical gradient of protons across the membrane generated by vacuolar proton-ATPase. ATP is stored in secretory vesicles and subsequently exocytosed, which leads to the various purinergic responses, such as central control of autonomic functions, pain and mechanosensory transduction, neural-glial interactions, control of vessel tone and angiogenesis, and platelet aggregation through purinoceptors, thus establishing its role as a chemical transmitter (8)(9)(10)(11)(12). Because the concentration of nucleotides in the vesicles was maintained at Ϸ0.1-1 M, an active transport mechanisms to accumulate nucleotides has been postulated (8-18). Although evidence increasingly supports the presence of a vesicular ATP transporters in secretory vesicles such as synaptic vesicles and adrenal chromaffin granules (13-19), the protein responsible for the ATP accumulation has not yet been identified.Here, we report the expression and function of human and mouse SLC17A9, an isoform of the SLC17 phosphate transporter family. We present evidence that the SLC17A9 protein acts as a vesicular nucleotide transporter (VNUT) and that it plays an essential role in vesicular storage of ATP in the ATP-secreting cells. Results a...

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Exocytotic Release of ATP from Cultured Astrocytes

Cited by 218 publications

References 54 publications

Molecular mechanisms of ATP secretion during immunogenic cell death

Molecular mechanisms of ATP secretion during immunogenic cell death

Autocrine signaling via release of ATP and activation of P2X7 receptor influences motile activity of human lung cancer cells

Identification of a vesicular nucleotide transporter

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