Functional and Anatomical Identification of a Vesicular Transporter Mediating Neuronal ATP Release

Abstract

ATP is known to be coreleased with glutamate at certain central synapses. However, the nature of its release is controversial. Here, we demonstrate that ATP release from cultured rat hippocampal neurons is sensitive to RNAi-mediated knockdown of the recently identified vesicular nucleotide transporter (VNUT or SLC17A9). In the intact brain, light microscopy showed particularly strong VNUT immunoreactivity in the cerebellar cortex, the olfactory bulb, and the hippocampus. Using immunoelectron microscopy, we found VNUT immunoreactivity colocalized with synaptic vesicles in excitatory and inhibitory terminals in the hippocampal formation. Moreover, VNUT immunolabeling, unlike that of the vesicular glutamate transporter VGLUT1, was enriched in preterminal axons and present in postsynaptic dendritic spines. Immunoisolation of synaptic vesicles indicated presence of VNUT in a subset of VGLUT1-containing vesicles. Thus, we conclude that VNUT mediates transport of ATP into synaptic vesicles of hippocampal neurons, thereby conferring a purinergic phenotype to these cells.

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

“…1 and 2), confirms our early studies and validity of using such fluorescent markers [4]. VNUT and [10][11][12][13][14][15]. In freshly isolated pancreatic acini, secretory/zymogen granule marker Rab3D co-localizes with VNUT ( Fig.…”

“…Soon after, we provided evidence that VNUT is expressed in pancreatic zymogen granules and is engaged in ATP uptake [9]. Since then, expression of VNUT has been demonstrated in several cell types indicating the general importance of VNUT for ATP release [10][11][12][13][14][15].…”

“…The cells were subsequently fixed in 4 % paraformaldehyde in phosphate buffered saline (PBS) for 15 min, treated with 0.2 M TRIS-glycine (pH 7.4) for 15 min, and then rinsed in PBS and permeabilized for 10 min in PBS 0.5 % TritonX-100. The samples were blocked with 10 % bovine serum albumin (BSA) in PBS for 45 min and then incubated with the primary antibodies for VNUT (1:400), which were used in previous studies [8,13], and Rab3D (1:100; Santa Cruz Sc-26559) for 2 h. The slides were then washed in PBS and incubated for 1 h with 1:400 dilution of appropriate secondary antibody conjugated to Alexa 488 or Alexa 568. For nuclear staining, DAPI (4',6-diamidino-2-phenylindole) was used (1:400).…”

Mecanismos purinérgicos no bulbo ventrolateral rostral modulam respostas cardiovasculares e respiratórias promovidas pela ativação dos quimiorreceptores centrais e periféricos.

“…Nossos achados sugerem que a sinalização purinérgica poderia ser co-liberada com glutamato no bulbo ventrolateral rostral durante a ativação de quimiorreceptores periféricos, como já sugerido em outras áreas (LARSSON et al, 2012). Na tentativa de confirmar nossa (SOBRINHO et al, 2014).…”

“…nucleotides are known to be coreleased with glutamate at cer tain central synapses 39 and cNTS projections to the RVLM are known to be glutamatergic, 9 we also assayed for VGLUT2 to determine whether glutamate and nucleotides are colocalized in the same terminals.…”