Vesicular expression and release of ATP from dopaminergic neurons of the mouse retina and midbrain.

Tracy Ho,Andrew I Jobling,Kirstan A Vessey,Trinette Chuang,Archana Ramesh,Erica L Fletcher,Ursula Greferath

doi:10.3389/fncel.2015.00389

Abstract

Vesicular nucleotide transporter (VNUT) is required for active accumulation of adenosine tri-phosphate (ATP) into vesicles for purinergic neurotransmission, however, the cell types that express VNUT in the central nervous system remain unknown. This study characterized VNUT expression within the mammalian retina and brain and assessed a possible functional role in purinergic signaling. Two native isoforms of VNUT were detected in mouse retina and brain based on RNA transcript and protein analysis. Using immunohistochemistry, VNUT was found to co-localize with tyrosine hydroxylase (TH) positive, dopaminergic (DA) neurons of the substantia nigra and ventral tegmental area, however, VNUT expression in extranigral non-DA neurons was also observed. In the retina, VNUT labeling was found to co-localize solely with TH-positive DA-cells. In the outer retina, VNUT-positive interplexiform cell processes were in close contact with horizontal cells and cone photoreceptor terminals, which are known to express P2 purinergic-receptors. In order to assess function, dissociated retinal neurons were loaded with fluorescent ATP markers (Quinacrine or Mant-ATP) and the DA marker FFN102, co-labeled with a VNUT antibody and imaged in real time. Fluorescent ATP markers and FFN102 puncta were found to co-localize in VNUT positive neurons and upon stimulation with high potassium, ATP marker fluorescence at the cell membrane was reduced. This response was blocked in the presence of cadmium. These data suggest DA neurons co-release ATP via calcium dependent exocytosis and in the retina this may modulate the visual response by activating purine receptors on closely associated neurons.

Highlights

Extracellular adenosine 3′-triphosphate acts as a potent signaling molecule in various tissues, mediating physiological and pathological responses on binding to cell surface purinergic receptors (Burnstock, 2007; Abbracchio et al, 2009)
Multiple Isoforms of the Slc17a9 Gene are Expressed in the Mouse Retina and Brain In addition to full length Slc17a9 sequence, an additional three potential protein coding isoforms have been predicted (X1, X2, and X3)
For the first time, that Vesicular nucleotide transporter (VNUT) expression is apparent in DA-cells of the rodent retina and midbrain suggesting that adenosine tri-phosphate (ATP) is stored and released by way of exocytosis from DA cells across the CNS

Summary

Introduction

Extracellular adenosine 3′-triphosphate (eATP) acts as a potent signaling molecule in various tissues, mediating physiological and pathological responses on binding to cell surface purinergic receptors (Burnstock, 2007; Abbracchio et al, 2009). Vesicular nucleotide transporter (VNUT) protein, encoded by the solute carrier 17, member 9 (SLC17A9) gene, was found to be a novel member of an anion transporter family It was identified as a mediator of active accumulation of nucleotides (i.e., ATP, ADP, and UTP) into secretory vesicles and was found to be required for exocytosis of ATP from various tissue types (Sawada et al, 2008; Iwatsuki et al, 2009; Tokunaga et al, 2010; Sathe et al, 2011; Larsson et al, 2012; Geisler et al, 2013; Oya et al, 2013; Sesma et al, 2013; Haanes et al, 2014; Harada and Hiasa, 2014)

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