Polyamine release and vesicular polyamine transporter expression in megakaryoblastic cells and platelets.

Polyamines not only play essential roles in cell growth and function of living organisms but are also released into the extracellular space and function as regulators of chemical transduction, although the cells from which they are released and their mode of release are not well understood. The vesicular polyamine transporter (VPAT), encoded by the SLC18B1 is responsible for the vesicular storage of spermine and spermidine, followed by their vesicular release from secretory cells. Focusing on VPAT will help identify polyamine-secreting cells and new polyamine functions. In this study, we investigated the possible involvement of VPAT in vesicular release of polyamines in MEG-01 clonal megakaryoblastic cells and platelets. RT-PCR, western blotting, and immunohistochemistry revealed VPAT expression in MEG-01 cells. MEG-01 cells secreted polyamines upon A23187 stimulation in the presence of Ca2+, which is temperature-dependent and sensitive to bafilomycin A1. A23187-induced polyamine secretion from MEG-01 cells was reduced by treatment with reserpine, VPAT inhibitors, or VPAT RNA interference. Platelets also expressed VPAT, displaying a punctate distribution, and released spermidine upon A23187 and thrombin stimulation. These findings have demonstrated VPAT-mediated vesicular polyamine release from MEG-01 cells, suggesting the presence of similar vesicular polyamine release mechanisms in platelets.

Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors.

S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate the expression of differentiated phenotype of chondrocytes. We also found that the cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2, and chondroitin sulfate biosynthetic enzymes. The blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, Alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

Vesicular ATP release from hepatocytes plays a role in the progression of nonalcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is becoming a growing public health problem along with the increase of metabolic syndrome worldwide. Extracellular nucleotides are known to serve as a danger signal by initiating purinergic signaling in many inflammatory disorders, although the role of purinergic signaling in the progression of NASH remains to be clarified. Vesicular nucleotide transporter (VNUT) is a key molecule responsible for vesicular ATP release to initiate purinergic signaling. Here, we studied the role of VNUT in the progression of nonalcoholic steatohepatitis. VNUT was expressed in mouse hepatocytes and associated, at least in part, with apolipoprotein B (apoB)-containing vesicles. High glucose stimulation evoked release of appreciable amount of ATP from hepatocytes, which disappeared in hepatocytes of Vnut knockout (Vnut-/-) mice. Glucose treatment also stimulated triglyceride secretion from hepatocytes, which was inhibited by PPADS and MRS211, antagonists of P2Y receptors, and clodronate, a VNUT inhibitor, and was significantly reduced in Vnut-/- mice. In vivo, postprandial secretion of triglyceride from hepatocytes was observed, while the serum triglyceride level was significantly reduced in Vnut-/- mice. On a high-fat diet, the liver of wild type mice exhibited severe inflammation, fibrosis, and macrophage infiltration, which is similar to NASH in humans, while this NASH pathology was not observed in Vnut-/- mice. These results suggest that VNUT-mediated vesicular ATP release regulates triglyceride secretion and involves in chronic inflammation in hepatocytes. Since blockade of vesicular ATP release protects against progression of steatohepatitis, VNUT may be a pharmacological target for NASH.

Functional characterization and tissue localization of the facilitative glucose transporter GLUT12.

Facilitative glucose transporters (GLUTs) play crucial roles in glucose utilization and homeostasis. GLUT12 was initially isolated as a novel GLUT4-like transporter involved in insulin-dependent glucose transport. However, tissue distribution and biochemical properties of GLUT12 are not well understood. In this study, we investigated the basic kinetic properties and tissue distribution of GLUT12. Human GLUT12 and GLUT1 were overexpressed and purified using Ni-NTA column chromatography. Reconstituted proteoliposomes showed time-dependent d-glucose transport activity, which was inhibited by phloretin and dehydroascorbate. Dose dependence of glucose transport revealed a KM and Vmax values of 6.4 mM and 1.2 µmol/mg/min, respectively, indicating that GLUT12 is a high-affinity type GLUT. Glucose transport by GLUT12 was inhibited by ATP and glucose-1-phosphate, glucose-6-phosphate and disaccharides (properties similar to those of GLUT1). Indirect immunohistochemistry revealed the distribution of mouse GLUT12 in the apical region of distal tubules and collecting ducts in the kidney and epithelial cells of the jejunum. In addition to these cells, GLUT12 was present in chromaffin cells in the adrenal medulla, the anterior pituitary lobe, as well as the thyroid and pyloric glands. These tissue distributions suggest a unique function of GLUT12, besides that of an insulin-dependent glucose transport.

Anti-Depressant Fluoxetine Reveals its Therapeutic Effect Via Astrocytes.

Although psychotropic drugs act on neurons and glial cells, how glia respond, and whether glial responses are involved in therapeutic effects are poorly understood. Here, we show that fluoxetine (FLX), an anti-depressant, mediates its anti-depressive effect by increasing the gliotransmission of ATP. FLX increased ATP exocytosis via vesicular nucleotide transporter (VNUT). FLX-induced anti-depressive behavior was decreased in astrocyte-selective VNUT-knockout mice or when VNUT was deleted in mice, but it was increased when astrocyte-selective VNUT was overexpressed in mice. This suggests that VNUT-dependent astrocytic ATP exocytosis has a critical role in the therapeutic effect of FLX. Released ATP and its metabolite adenosine act on P2Y11 and adenosine A2b receptors expressed by astrocytes, causing an increase in brain-derived neurotrophic factor in astrocytes. These findings suggest that in addition to neurons, FLX acts on astrocytes and mediates its therapeutic effects by increasing ATP gliotransmission.

Vesicular nucleotide transporter mediates ATP release and migration in neutrophils.

Neutrophils migrate to sites infected by pathogenic microorganisms. This migration is regulated by neutrophil-secreted ATP, which stimulates neutrophils in an autocrine manner through purinergic receptors on the plasma membrane. Although previous studies have shown that ATP is released through channels at the plasma membrane of the neutrophil, it remains unknown whether it is also released through alternate secretory systems involving vesicular mechanisms. In this study, we investigated the possible involvement of vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and nucleotide release, in ATP secretion from neutrophils. RT-PCR and Western blotting analysis indicated that VNUT is expressed in mouse neutrophils. Immunohistochemical analysis indicated that VNUT mainly colocalized with matrix metalloproteinase-9 (MMP-9), a marker of tertiary granules, which are secretory organelles. In mouse neutrophils, ATP release was inhibited by clodronate, which is a potent VNUT inhibitor. Furthermore, neutrophils from VNUT-/- mice did not release ATP and exhibited significantly reduced migration in vitro and in vivo These findings suggest that tertiary granule-localized VNUT is responsible for vesicular ATP release and subsequent neutrophil migration. Thus, these findings suggest an additional mechanism through which ATP is released by neutrophils.

Identification of a vesicular ATP release inhibitor for the treatment of neuropathic and inflammatory pain.

Despite the high incidence of neuropathic and inflammatory pain worldwide, effective drugs with few side effects are currently unavailable for the treatment of chronic pain. Recently, researchers have proposed that inhibitors of purinergic chemical transmission, which plays a key role in the pathological pain response, may allow for targeted treatment of pathological neuropathic and inflammatory pain. However, such therapeutic analgesic agents have yet to be developed. In the present study, we demonstrated that clodronate, a first-generation bisphosphonate with comparatively fewer side effects than traditional treatments, significantly attenuates neuropathic and inflammatory pain unrelated to bone abnormalities via inhibition of vesicular nucleotide transporter (VNUT), a key molecule for the initiation of purinergic chemical transmission. In vitro analyses indicated that clodronate inhibits VNUT at a half-maximal inhibitory concentration of 15.6 nM without affecting other vesicular neurotransmitter transporters, acting as an allosteric modulator through competition with Cl- A low concentration of clodronate impaired vesicular ATP release from neurons, microglia, and immune cells. In vivo analyses revealed that clodronate is more effective than other therapeutic agents in attenuating neuropathic and inflammatory pain, as well as the accompanying inflammation, in wild-type but not VNUT -/- mice, without affecting basal nociception. These findings indicate that clodronate may represent a unique treatment strategy for chronic neuropathic and inflammatory pain via inhibition of vesicular ATP release.

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

Vesicular storage of ATP is one of the processes initiating purinergic chemical transmission. Although an active transport mechanism was postulated to be involved in the processes, a transporter(s) responsible for the vesicular storage of ATP remained unidentified for some time. In 2008, SLC17A9, the last identified member of the solute carrier 17 type I inorganic phosphate transporter family, was found to encode the vesicular nucleotide transporter (VNUT) that is responsible for the vesicular storage of ATP. VNUT transports various nucleotides in a membrane potential-dependent fashion and is expressed in the various ATP-secreting cells. Mice with knockout of the VNUT gene lose vesicular storage and release of ATP from neurons and neuroendocrine cells, resulting in blockage of the initiation of purinergic chemical transmission. Thus, VNUT plays an essential role in the vesicular storage and release of ATP. The VNUT knockout mice exhibit resistance for neuropathic pain and a therapeutic effect against diabetes by way of increased insulin sensitivity. Thus, VNUT inhibitors and suppression of VNUT gene expression may be used for therapeutic purposes through suppression of purinergic chemical transmission. This review summarizes the studies to date on VNUT and discusses what we have learned about the relevance of vesicular ATP release as a potential drug target.

Function and expression of a splicing variant of vesicular glutamate transporter 1.

Vesicular glutamate transporter (VGLUT) is an active transporter responsible for vesicular storage of glutamate in synaptic vesicles and plays an essential role in glutamatergic neurotransmission. VGLUT consists of three isoforms, VGLUT1, VGLUT2, and VGLUT3. The VGLUT1 variant, VGLUT1v, with an additional 75-base pair sequence derived from a second intron between exons 2 and 3, which corresponds to 25 amino acid residues in the 1st loop of VGLUT1, is the only splicing variant among VGLUTs, although whether VGLUT1v protein is actually translated at the protein level remains unknown. In the present study, VGLUT1v was expressed in insect cells, solubilized, purified to near homogeneity, and its transport activity was examined. Proteoliposomes containing purified VGLUT1v were shown to accumulate glutamate upon imposition of an inside-positive membrane potential (Δψ). The Δψ-driven glutamate uptake activity requires Cl- and its pharmacological profile and kinetics are comparable to those of other VGLUTs. The retinal membrane contained two VGLUT1 moieties with apparent molecular masses of 65 and 57kDa. VGLUT1v-specific antibodies against an inserted 25-amino acid residue sequence identified a 65-kDa immunoreactive polypeptide. Immunohistochemical analysis indicated that VGLUT1v immunoreactivity is present in photoreceptor cells and is associated with synaptic vesicles. VGLUT1v immunoreactivity is also present in pinealocytes, but not in other areas, including the brain. These results indicated that VGLUT1v exists in a functional state in rat photosensitive cells and is involved in glutamatergic chemical transmission.

Vesicular Polyamine Transporter Mediates Vesicular Storage and Release of Polyamine from Mast Cells.

Mast cells are secretory cells that play an important role in host defense by discharging various intragranular contents, such as histamine and serotonin, upon stimulation of Fc receptors. The granules also contain spermine and spermidine, which can act as modulators of mast cell function, although the mechanism underlying vesicular storage remains unknown. Vesicular polyamine transporter (VPAT), the fourth member of the SLC18 transporter family, is an active transporter responsible for vesicular storage of spermine and spermidine in neurons. In the present study, we investigated whether VPAT functions in mast cells. RT-PCR and Western blotting indicated VPAT expression in murine bone marrow-derived mast cells (BMMCs). Immunohistochemical analysis indicated that VPAT is colocalized with VAMP3 but not with histamine, serotonin, cathepsin D, VAMP2, or VAMP7. Membrane vesicles from BMMCs accumulated spermidine upon the addition of ATP in a reserpine- and bafilomycin A1-sensitive manner. BMMCs secreted spermine and spermidine upon the addition of either antigen or A23187 in the presence of Ca2+, and the antigen-mediated release, which was shown to be temperature-dependent and sensitive to bafilomycin A1 and tetanus toxin, was significantly suppressed by VPAT gene RNA interference. Under these conditions, expression of vesicular monoamine transporter 2 was unaffected, but antigen-dependent histamine release was significantly suppressed, which was recovered by the addition of 1 mm spermine. These results strongly suggest that VPAT is expressed and is responsible for vesicular storage of spermine and spermidine in novel secretory granules that differ from histamine- and serotonin-containing granules and is involved in vesicular release of these polyamines from mast cells.

Urothelial ATP exocytosis: regulation of bladder compliance in the urine storage phase.

The bladder urothelium is more than just a barrier. When the bladder is distended, the urothelium functions as a sensor to initiate the voiding reflex, during which it releases ATP via multiple mechanisms. However, the mechanisms underlying this ATP release in response to the various stretch stimuli caused by bladder filling remain largely unknown. Therefore, the aim of this study was to elucidate these mechanisms. By comparing vesicular nucleotide transporter (VNUT)-deficient and wild-type male mice, we showed that ATP has a crucial role in urine storage through exocytosis via a VNUT-dependent mechanism. VNUT was abundantly expressed in the bladder urothelium, and when the urothelium was weakly stimulated (i.e. in the early filling stages), it released ATP by exocytosis. VNUT-deficient mice showed reduced bladder compliance from the early storage phase and displayed frequent urination in inappropriate places without a change in voiding function. We conclude that urothelial, VNUT-dependent ATP exocytosis is involved in urine storage mechanisms that promote the relaxation of the bladder during the early stages of filling.

Expression of Vesicular Nucleotide Transporter in the Mouse Retina.

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 (GABA) transporter in amacrine cells and bipolar cells. VNUT is also present in astrocytes and Müller cells. The retina from VNUT knockout (VNUT(-/-)) mice showed the loss of VNUT immunoreactivity. The retinal membrane fraction took up radiolabeled ATP in diisothiocyanate stilbene disulfonic acid (DIDS)-, an inhibitor of VNUT, and bafilomycin A1-, a vacuolar adenosine triphosphatase (ATPase) inhibitor, in a sensitive manner, while membranes from VNUT(-/-) mice showed the loss of DIDS-sensitive ATP uptake. Taken together, these results indicate that functional VNUT is expressed in the rodent retina and suggest that ATP is released from photoreceptor cells, bipolar cells, amacrine cells, and astrocytes as well as Müller cells to initiate purinergic chemical transmission.

Involvement of multiple CCN family members in platelets that support regeneration of joint tissues.

OBJECTIVES: Platelet-rich plasma (PRP) has been widely used to enhance the regeneration of damaged joint tissues, such as osteoarthritic and rheumatoid arthritic cartilage. The aim of this study is to clarify the involvement of all of the CCN family proteins that are crucially associated with joint tissue regeneration. METHODS: Cyr61-CTGF-NOV (CCN) family proteins in human platelets and megakaryocytic cells were comprehensively analyzed by Western blotting analysis. Production of CCN family proteins in megakaryocytes in vivo was confirmed by immunofluorescence analysis of mouse bone marrow cells. Effects of CCN family proteins found in platelets on chondrocytes were evaluated by using human chondrocytic HCS-2/8 cells. RESULTS: Inclusion of CCN2, a mesenchymal tissue regenerator, was confirmed. Of note, CCN3, which counteracts CCN2, was newly found to be encapsulated in platelets. Interestingly, these two family members were not detectable in megakaryocytic cells, but their external origins were suggested. Furthermore, we found for the first time CCN5 and CCN1 that inhibits ADAMTS4 in both platelets and megakaryocytes. Finally, application of a CCN family cocktail mimicking platelets onto HCS-2/8 cells enhanced their chondrocytic phenotype. CONCLUSIONS: Multiple inclusion of CCN1, 2 and 3 in platelets was clarified, which supports the harmonized regenerative potential of PRP in joint therapeutics.

Structure, Function, and Drug Interactions of Neurotransmitter Transporters in the Postgenomic Era.

Vesicular neurotransmitter transporters are responsible for the accumulation of neurotransmitters in secretory vesicles and play essential roles in chemical transmission. The SLC17 family contributes to sequestration of anionic neurotransmitters such as glutamate, aspartate, and nucleotides. Identification and subsequent cellular and molecular biological studies of SLC17 transporters unveiled the principles underlying the actions of these transporters. Recent progress in reconstitution methods in combination with postgenomic approaches has advanced studies on neurotransmitter transporters. This review summarizes the molecular properties of SLC17-type transporters and recent findings regarding the novel SLC18 transporter.

Wide expression of type I Na+-phosphate cotransporter 3 (NPT3/SLC17A2), a membrane potential-driven organic anion transporter.

Membrane potential (Δψ)-driven and Cl(-)-dependent organic anion transport is a primary function of the solute carrier family 17 (SLC17) transporter family. Although the transport substrates and physiological relevance of the major members are well understood, SLC17A2 protein known to be Na(+)-phosphate cotransporter 3 (NPT3) is far less well characterized. In the present study, we investigated the transport properties and expression patterns of mouse SLC17A2 protein (mNPT3). Proteoliposomes containing the purified mNPT3 protein took up radiolabeled p-aminohippuric acid (PAH) in a Δψ- and Cl(-)-dependent manner. The mNPT3-mediated PAH uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDs) and Evans blue, common inhibitors of SLC17 family members. The PAH uptake was also inhibited by various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs (NSAIDs) and urate. Consistent with these observations, the proteoliposome took up radiolabeled urate in a Δψ- and Cl(-)-dependent manner. Immunohistochemistry with specific antibodies against mNPT3 combined with RT-PCR revealed that mNPT3 is present in various tissues, including the hepatic bile duct, luminal membranes of the renal urinary tubules, maternal side of syncytiotrophoblast in the placenta, apical membrane of follicle cells in the thyroid, bronchiole epithelial cells in the lungs, and astrocytes around blood vessels in the cerebrum. These results suggested that mNPT3 is a polyspecific organic anion transporter that is involved in circulation of urate throughout the body.

Identification of a mammalian vesicular polyamine transporter.

Spermine and spermidine act as neuromodulators upon binding to the extracellular site(s) of various ionotropic receptors, such as N-methyl-d-aspartate receptors. To gain access to the receptors, polyamines synthesized in neurons and astrocytes are stored in secretory vesicles and released upon depolarization. Although vesicular storage is mediated in an ATP-dependent, reserpine-sensitive fashion, the transporter responsible for this process remains unknown. SLC18B1 is the fourth member of the SLC18 transporter family, which includes vesicular monoamine transporters and vesicular acetylcholine transporter. Proteoliposomes containing purified human SLC18B1 protein actively transport spermine and spermidine by exchange of H(+). SLC18B1 protein is predominantly expressed in the hippocampus and is associated with vesicles in astrocytes. SLC18B1 gene knockdown decreased both SLC18B1 protein and spermine/spermidine contents in astrocytes. These results indicated that SLC18B1 encodes a vesicular polyamine transporter (VPAT).

Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity.

Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut(-/-)) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut(-/-) mice. Glucose-responsive ATP release was also absent in pancreatic ß-cells in Vnut(-/-) mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut(-/-) mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis.

Immunological identification of vesicular nucleotide transporter in intestinal L cells.

It is well established that vesicular nucleotide transporter (VNUT) is responsible for vesicular storage of nucleotides such as ATP, and that VNUT-expressing cells can secrete nucleotides upon exocytosis, playing an important role in purinergic chemical transmission. In the present study, we show that VNUT is expressed in intestinal L cells. Immunohistochemical evidence indicated that VNUT is present in glucagon-like peptide 1 (GLP-1) containing cells in rat intestine. VNUT immunoreactivity is not co-localized with GLP-1, a marker for secretory granules, and synaptophysin, a marker for synaptic-like microvesicles (SLMVs). Essentially the same results were obtained for GLUTag clonal L cells. Sucrose density gradient analysis confirmed that VNUT is present the light fraction, unlike secretory granules. These results demonstrate that intestinal L cells express VNUT in either the unidentified organelles at light density other than secretory granules and SLMVs or a subpopulation of SLMVs, and suggest that L cells are purinergic in nature and secrete nucleotides independent of GLP-1 secretion.

Essential role of vesicular nucleotide transporter in vesicular storage and release of nucleotides in platelets.

Nucleotides are stored in the dense granules of platelets. The release of nucleotides triggers one of the first steps in a series of cascades responsible for blood coagulation. However, the mechanism of how the nucleotides are accumulated in the granules is still far less understood. The transporter protein responsible for storage of nucleotides in the neuroendocrine cells has been identified and characterized. We hypothesized that the vesicular nucleotide transporter (VNUT) is also involved in the vesicular storage of nucleotides in platelets. In this article, we present three lines of evidence that VNUT is responsible for the vesicular storage of nucleotides in platelets and that vesicular ATP transport is crucial for platelet function, detection and characterization of VNUT activity in platelets isolated from healthy humans and MEG-01 cells, RNA interference experiments on MEG-01 cells, and studies on nucleotide transport and release with a selective inhibitor.

Vesicular nucleotide transport: a brief history and the vesicular nucleotide transporter as a target for drug development.

Neurons and neuroendocrine cells store nucleotides in vesicles and release them upon stimulation, leading to intercellular purinergic signaling. The molecular machinery responsible for the vesicular storage of nucleotides was a long standing enigma, however, recently the transporter involving in the process was identified. This article summarizes the history of vesicular storage of nucleotides and the identification of the vesicular nucleotide transporter (VNUT) responsible for the process. The significance of VNUT as a drug target to control purinergic chemical transmission is also discussed.