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.

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 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.

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.

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.

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 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.

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.

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.

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.

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.

Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules.

Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca(2+)-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

Functional and anatomical identification of a vesicular transporter mediating neuronal ATP release.

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.

Type 1 sodium-dependent phosphate transporter (SLC17A1 Protein) is a Cl(-)-dependent urate exporter.

SLC17A1 protein (NPT1) is the first identified member of the SLC17 phosphate transporter family and mediates the transmembrane cotransport of Na(+)/P(i) in oocytes. Although this protein is believed to be a renal polyspecific anion exporter, its transport properties are not well characterized. Here, we show that proteoliposomes containing purified SLC17A1 transport various organic anions such as p-aminohippuric acid and acetylsalicylic acid (aspirin) in an inside positive membrane potential (Deltapsi)-dependent manner. We found that NPT1 also transported urate. The uptake characteristics were similar to that of SLC17 members in its Cl(-) dependence and inhibitor sensitivity. When arginine 138, an essential amino acid residue for members of the SLC17 family such as the vesicular glutamate transporter, was specifically mutated to alanine, the resulting mutant protein was inactive in Deltapsi-dependent anion transport. Heterologously expressed and purified human NPT1 carrying the single nucleotide polymorphism mutation that is associated with increased risk of gout in humans exhibited 32% lower urate transport activity compared with the wild type protein. These results strongly suggested that NPT1 is a Cl(-)-dependent polyspecific anion exporter involved in urate excretion under physiological conditions.

Identification of a vesicular aspartate transporter.

Aspartate is an excitatory amino acid that is costored with glutamate in synaptic vesicles of hippocampal neurons and synaptic-like microvesicles (SLMVs) of pinealocytes and is exocytosed and stimulates neighboring cells by binding to specific cell receptors. Although evidence increasingly supports the occurrence of aspartergic neurotransmission, this process is still debated because the mechanism for the vesicular storage of aspartate is unknown. Here, we show that sialin, a lysosomal H(+)/sialic acid cotransporter, is present in hippocampal synaptic vesicles and pineal SLMVs. RNA interference of sialin expression decreased exocytosis of aspartate and glutamate in pinealocytes. Proteoliposomes containing purified sialin actively accumulated aspartate and glutamate to a similar extent when inside positive membrane potential is imposed as the driving force. Sialin carrying a mutation found in people suffering from Salla disease (R39C) was completely devoid of aspartate and glutamate transport activity, although it retained appreciable H(+)/sialic acid cotransport activity. These results strongly suggest that sialin possesses dual physiological functions and acts as a vesicular aspartate/glutamate transporter. It is possible that people with Salla disease lose aspartergic (and also the associated glutamatergic) neurotransmission, and this could provide an explanation for why Salla disease causes severe neurological defects.

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.

Vesicular inhibitory amino acid transporter is a Cl-/gamma-aminobutyrate Co-transporter.

The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of gamma-aminobutyrate (GABA) and glycine which plays an essential role in GABAergic and glycinergic neurotransmission. The transport mechanism of VIAAT remains largely unknown. Here, we show that proteoliposomes containing purified VIAAT actively took up GABA upon formation of membrane potential (Deltapsi) (positive inside) but not DeltapH. VIAAT-mediated GABA uptake had an absolute requirement for Cl(-) and actually accompanied Cl(-) movement. Kinetic analysis indicated that one GABA molecule and two Cl(-) equivalents were transported during one transport cycle. VIAAT in which Glu(213) was specifically mutated to alanine completely lost the ability to take up both GABA and Cl(-). Essentially the same results were obtained with glycine, another substrate of VIAAT. These results demonstrated that VIAAT is a vesicular Cl(-) transporter that co-transports Cl(-) with GABA or glycine in a Deltapsi dependent manner. It is concluded that Cl(-) plays an essential role in vesicular storage of GABA and glycine.

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.

Identification of the vesicular nucleotide transporter (VNUT) in taste cells.

Taste cells are chemosensory epithelial cells that sense distinct taste qualities. It is the type II taste cell that express G-protein coupled receptors to sense either umami, sweet, or bitter compounds. Whereas several reports have suggested involvement of ATP in taste signal transduction, there is a paucity of molecular information about how ATP is stored and being released. The recent discovery of a novel vesicular nucleotide transporter (VNUT) led us to examine whether VNUT exist in the taste tissue where ATP is to be released for taste signal transmission. Here, we report that VNUT is selectively expressed in type II cell but not in type III taste cell. In addition, we show that during taste bud development VNUT expression is always accompanied by the expression of type II taste cell markers. Our results, together with previous studies, strongly suggest that VNUT plays a role in type II taste cell.