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Matthew Voas, Nishika Muddasani, Yoshi Moriyama, Salvatore Stella; Characterization of the vesicular nucleotide transporter (VNUT) in mammalian retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2507.
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© ARVO (1962-2015); The Authors (2016-present)
The recent discovery of a novel vesicular nucleotide transporter (VNUT) has established that one of the major mechanisms for release of ATP occurs through a classical Ca2+-dependent vesicular process. The source of purines in the retina has been unclear, however, using highly specific antibodies against VNUT, potential release sites can now be mapped in the mammalian retina. The aim of the present study was to use anatomical and cell biological approaches to investigate the vesicular localization and putative release sites in the mammalian retina.
In order to investigate VNUT expression in the outer retina, mouse retinas were evaluated with immunohistochemistry and Western blots using antibody markers of various cell types and synaptic structures using newly developed antibodies targeted to VNUT. All images were collected on a confocal microscope and analyzed for fluorescence.
VNUT expression was localized to horizontal cells in the outer retina, and amacrine cells, Müller cells and ganglion cells in the inner retina. The western blot indicates a band with a relative mobility of ~60-65 kDa. Preadsorption of the VNUT antibodies to both human and mouse VNUT protein completely abolished VNUT immunoreactivity in retinas. VNUT immunoreactivity co-localized with calbindin and the vesicular GABA transporter (VGAT) in horizontal cells of mouse retina. In addition, VNUT immunoreactivity labeled structures including the somata, processes and tips of horizontal cells that were co-labeled with calbindin D-28K. VNUT also labeled subset of amacrine cells and Muller cells in the inner retina which co-localized with markers to each respectively. Ganglion cells and their processes were strongly labeled with VNUT and co-localized with Brn3a and SMI-32.
These findings provide evidence for VNUT expression in the retina and strongly support the hypothesis that ATP is stored and released from horizontal cells, amacrine cells, Muller cells, and ganglion cells in the mammalian retina. The presence of VNUT in both the inner and outer retina argues strongly that vesicular release of ATP and the balance of purines have the potential to significantly impact visual processing in the retina. Taken together, this study provides the first evidence for a significant purine source in the outer retina that can contribute to both ATP and adenosine levels in retina.
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