Expression of Vesicular Nucleotide Transporter in the Mouse Retina

Abstract

Vesicular nucleotide transporter (VNUT) is a membrane protein that is responsible for vesicular storage and subsequent vesicular release of nucleotides, such as ATP, and plays an essential role in purinergic chemical transmission. In the present study, we investigated whether VNUT is present in the rodent retina to define the site(s) of vesicular ATP release. In the mouse retina, reverse transcription polymerase chain reaction (RT-PCR) and immunological analyses using specific anti-VNUT antibodies indicated that VNUT is expressed as a polypeptide with an apparent molecular mass of 59 kDa. VNUT is widely distributed throughout the inner and outer retinal layers, particularly in the outer segment of photoreceptors, outer plexiform layer, inner plexiform layer, and ganglion cell layer. VNUT is colocalized with vesicular glutamate transporter 1 and synaptophysin in photoreceptor cells, while it is colocalized with vesicular γ-aminobutyric acid ( The retina is a photoreceptive organ in which photosignals received by photoreceptor cells are transmitted to the optic nerve mainly through glutamatergic and γ-aminobutyric acid (GABA)ergic chemical transmission. There is, however, evidence that ATP and adenosine are involved as intercellular transmitters and play roles in various functions in the retina. 1-3)The retina has been reported to express various purinoceptors and enzymes involved in ATP hydrolysis.2,3) Photoreceptor cells express P2X2, P2X7, and P2Y1, 2, 4, and 6 receptors.2) P2X7 is localized presynaptically to rod and cone pedicles, and a functional study showed that 3′-O-(4-benzoyl)benzoyl-ATP (BzATP) increases the amplitude of the α-wave, suggesting a neuromodulatory role of purinergic neurotransmission.4) Intravitreal injection of ATP caused rapid loss of photoreceptors, which was reduced by coinjection of the P2X antagonist, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), suggesting the involvement of purinergic chemical transmission in photoreceptor integrity. 5) Bipolar cells and horizontal cells also express P2X3-5 and 7 and P2Y1, 2, 6, and P2X7, respectively.2) Ganglion cells express both P2Xs and P2Y. 6) Stimulation of P2X7, with BzATP, an agonist of P2X7, induced death of the isolated ganglion cells through increases in intracellular Ca 2+ . 6) Loss of ganglion cells in vivo was also observed following injection of BzATP into the retina.7) It has also been reported that A1 and A3 receptors are involved in neuroprotection of ganglion cells. 8-11)Müller cells, retinal glial cells with numerous fine processes, which may make contact with neurons and non-neuronal cells, also express P2X7 and P2Y1, and their stimulation by specific agonists results in increased DNA synthesis and elicitation of the Ca 2+ response, respectively. 12)Extracellular ATP and its degradation product, adenosine, may trigger purinergic responses. In fact, Müller cells were reported to release ATP in a Ca 2+ -dependent manner.9) It was also reported that cultured chick retinal neurons took up radiolabeled ATP then relea...

Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling

“…Endogenous VNUT is colocalized with Lamp1, a lysosomal marker, in these cells [77], [81,92]. For details, see refs [4,81,86,92,93,96,98] but VNUT in astrocytes is not [75,76]. VNUT-mediated Δψ-driven ATP accumulation has been observed in living COS and HEK293 cells [78].…”

“…VNUT-mediated storage of ATP into secretory lysosomes is not characteristic in astrocytes and microglia, but can be observed in cultured cells such as COS1, HEK293T, and C2C12 cells [77]. Endogenous VNUT is colocalized with Lamp1, a lysosomal marker, in these cells [77], [81,92]. For details, see refs [4,81,86,92,93,96,98] but VNUT in astrocytes is not [75,76].…”

“…In the retina, VNUT is present in photoreceptor cells, bipolar cells, amacrine cells, and astrocytes, as well as Mueller cells [76][77][78][79][80][81] (Fig. 4) (Table 1).…”

Neurochemical regulation of the expression and function of glial fibrillary acidic protein in astrocytes

“…Similarly, vesicular nucleotide transporter (VNUT), a membrane protein that is responsible for vesicular storage and subsequent vesicular release of nucleotides including ATP, is present in the photoreceptor cells, bipolar cells, amacrine cells, and astrocytes as well as Muller cells (Moriyama & Hiasa, ). Astrocytes in the SON also express VGAT (Wang, Sun, Hou, & Hamilton, ) that can uptake and release GABA.…”

“…In addition, activation of metabotropic glutamate receptors triggers rapid Ca 2+ ‐and SNARE‐dependent exocytic fusion of astrocytic vesicles and glutamate release (Bezzi et al, ). The release of ATP from VNUT initiates purinergic chemical transmission (Moriyama & Hiasa, ). Thus, astrocytic synaptic vesicles have the functions of extrasynaptic transmission.…”