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In in vitro and in vivo systems, understanding localization and the functional role of ATP is essential, but effective methods to monitor ATP in cells and tissues are limited. Although quinacrine dihydrochloride is a well-known fluorescent dye used to detect ATP, it is limited in its use because it shows non-specific nuclear staining both in vitro and in vivo. A commercial luciferin-luciferase bioluminescence assay has also been used to detect ATP, but it can not be easily used in vivo. Thus, to effectively monitor ATP in vivo, we employed a novel two-photon ATP fluorescent probe, acedan-based Zn(DPA). Using the acedan-based Zn(DPA) probe, we show that this probe produces high quality images of ATP in lung, spleen, liver and spinal cord tissues.

After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The adenosine triphosphate (ATP) plays important roles under physiological and pathological conditions such as traumatic brain injury, neuroinflammation and neuropathic pain. In the present study, we set out to study the role of lysosomal vesicles on ATP release from the dorsal root ganglion neurons. We found that the lysosomal vesicles, which contain the quinacrine-positive fluorescence and express the vesicular nucleotide transporter (VNUT), were localized within the soma and growth cone of the cultured dorsal root ganglion neurons. In addition, the number of the quinacrine staining was decreased by application of lysosomal exocytosis activators, and this decrease was suppressed by the metformin and vacuolin-1, which suppressed lysosomal exocytosis. Thus, these findings suggest that ATP release via the lysosomal exocytosis may be one of the pathways for ATP release in response to stimulation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The present study demonstrates that adenosine triphosphate (ATP) is released from Schwann cells through lysosomal exocytosis during Wallerian degeneration and in response to stimulation. In primary Schwann cell cultures, ATP was stored in lysosomal vesicles. ATP could then induce Ca(2+)-dependent lysosomal exocytosis. Among three stimulants of lysosomal exocytosis (glutamate, NH(4)Cl and zymosan), only NH(4)Cl was sufficient to induce ATP release from ex vivo sciatic nerve explants at 3 days in vitro. Lysosomal exocytosis inhibitors (metformin, chlorpromazine and vacuolin-1) reversed the effect of NH(4)Cl-enhanced ATP release, replicating the state of explants treated with NH(4)Cl in the absence of lysosomal exocytosis inhibitors. Furthermore, we observed ATP release through lysosomal exocytosis during Wallerian degeneration in sciatic explant cultures using the recently identified vesicular nucleotide transporter (VNUT). From these experiments, we conclude that the exocytosis of lysosomes in Schwann cells during Wallerian degeneration is Ca(2+)-dependent, and that it induces ATP release from Schwann cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)