Vesicular Nucleotide Transporter Research Articles

PDE5 inhibitor potentially improves polyuria and bladder storage and voiding dysfunctions in type 2 diabetic rats.

Bladder dysfunction associated with type 2 diabetes mellitus (T2DM) includes urine storage and voiding disorders. We examined pathological conditions of the bladder wall in a rat T2DM model and evaluated the effects of the phosphodiesterase-5 (PDE-5) inhibitor tadalafil. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) rats were used as the T2DM and control groups, respectively. Tadalafil was orally administered for 12 weeks. Micturition behavior was monitored using metabolic cages, and bladder function was evaluated by cystometry. Bladder blood flow was evaluated by laser speckle imaging, and an organ bath bladder distention test was used to measure adenosine triphosphate (ATP) release from the bladder urothelium. The expression levels of vesicular nucleotide transporter (VNUT), hypoxia markers, pro-inflammatory cytokines and growth factors in the bladder wall were measured using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Bladder wall contractions in response to KCl and carbachol were monitored using bladder-strip tests. With aging, OLETF rats had higher micturition frequency and greater urine volume than LETO rats. Although bladder capacity was not significantly different, non-voiding bladder contraction occurred more frequently in OLETF rats than in LETO rats. Bladder blood flow was decreased and ATP release was increased with higher VNUT expression in OLETF rats than in LETO rats. These effects were suppressed by tadalafil administration, with accompanying decreased HIF-1α, 8-OHdG, IL-6, TNF-α, IGF-1, and bFGF expression. The impaired contractile responses of bladder strips to KCl and carbachol in OLETF rats with aging were restored by tadalafil administration. The T2DM rats had polyuria, increased ATP release induced by decreased bladder blood flow and impaired contractile function. PDE5 inhibition improved these changes and may prevent T2DM-associated urinary frequency and bladder storage and voiding dysfunctions.

Deletion of murine astrocytic vesicular nucleotide transporter increases anxiety and depressive-like behavior and attenuates motivation for reward.

Astrocytes are multi-functional glial cells in the central nervous system that play critical roles in modulation of metabolism, extracellular ion and neurotransmitter levels, and synaptic plasticity. Astrocyte-derived signaling molecules mediate many of these modulatory functions of astrocytes, including vesicular release of ATP. In the present study, we used a unique genetic mouse model to investigate the functional significance of astrocytic exocytosis of ATP. Using primary cultured astrocytes, we show that loss of vesicular nucleotide transporter (Vnut), a primary transporter responsible for loading cytosolic ATP into the secretory vesicles, dramatically reduces ATP loading into secretory lysosomes and ATP release, without any change in the molecular machinery of exocytosis or total intracellular ATP content. Deletion of astrocytic Vnut in adult mice leads to increased anxiety, depressive-like behaviors, and decreased motivation for reward, especially in females, without significant impact on food intake, systemic glucose metabolism, cognition, or sociability. These behavioral alterations are associated with significant decreases in the basal extracellular dopamine levels in the nucleus accumbens. Likewise, ex vivo brain slices from these mice show a strong trend toward a reduction in evoked dopamine release in the nucleus accumbens. Mechanistically, the reduced dopamine signaling we observed is likely due to an increased expression of monoamine oxidases. Together, these data demonstrate a key modulatory role of astrocytic exocytosis of ATP in anxiety, depressive-like behavior, and motivation for reward, by regulating the mesolimbic dopamine circuitry.

Hepatic extracellular ATP/adenosine dynamics in zebrafish models of alcoholic and metabolic steatotic liver disease

Steatotic liver disease (SLD) is a burgeoning health problem predominantly associated with excessive alcohol consumption, which causes alcohol-related liver disease (ALD), and high caloric intake, which results in metabolic dysfunction-associated SLD (MASLD). The pathogenesis of ALD and MASLD, which can progress from steatohepatitis to more severe conditions such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, is complicated by several factors. Recently, extracellular ATP and adenosine (Ado), as damage-associated molecular patterns, were reported to promote inflammation and liver fibrosis, contributing to SLD pathogenesis. Here, we explored the in vivo dynamics of hepatic extracellular ATP and Ado during the progression of steatohepatitis using a genetically encoded GPCR-activation-based sensor (GRAB) in zebrafish models. We established hepatocyte-specific GRABATP and GRABAdo in zebrafish and investigated the changes in in vivo hepatic extracellular ATP and Ado levels under ALD or MASLD conditions. Disease-specific changes in hepatocyte extracellular ATP and Ado levels were observed, clearly indicating a correlation between hepatocyte extracellular ATP/Ado dynamics and disease progression. Furthermore, clodronate, a vesicular nucleotide transporter inhibitor, alleviated the MASLD phenotype by reducing the hepatic extracellular ATP and Ado content. These findings provide deep insights into extracellular ATP/Ado dynamics in disease progression, suggesting therapeutic potential for ALD and MASLD.

Immunohistochemical localization of P2Y12 purinoceptors in the rat carotid body

The present study investigated the localization of the adenosine 5′-diphosphate (ADP)-selective P2Y12 purinoceptors in the rat carotid body using multilabeling immunofluorescence. Punctate immunoreactive products for P2Y12 were distributed in chemoreceptive type I cells immunoreactive to vesicular nucleotide transporter (VNUT) or dopamine beta-hydroxylase, but not in S100B-immunoreactive glial-like type II cells. P2Y12 immunoreactivity was localized in cell clusters containing VNUT-immunoreactive type I cells surrounded by the perinuclear cytoplasm and cytoplasmic processes of type II cells immunoreactive for ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and NTPDase3, which hydrolyze extracellular nucleotide tri- and/or di-phosphates. In ATP bioluminescence assays using carotid bodies, the degradation of extracellular ATP was attenuated in the presence of the selective NTPDases inhibitor ARL67156, suggesting ATP-degrading activity by NTPDases in the tissue. These results suggest that ATP released from type I cells is degraded into ADP and adenosine 5′-monophosphate by NTPDases expressed in type II cells, and that ADP modulates type I cells via P2Y12 purinoceptors.

Lipopolysaccharide-mediated ATP signaling regulates interleukin-6 mRNA expression via the P2-purinoceptor in human dental pulp cells.

Dental pulp cells play a crucial role in maintaining the balance of the pulp tissue. They actively respond to bacterial inflammation by producing proinflammatory cytokines, particularly interleukin-6 (IL-6). While many cell types release adenosine triphosphate (ATP) in response to various stimuli, the mechanisms and significance of ATP release in dental pulp cells under inflammatory conditions are not well understood. This study aimed to investigate ATP release and its relationship with IL-6 during the inflammatory response in immortalized human dental pulp stem cells (hDPSC-K4DT) following lipopolysaccharide (LPS) stimulation. We found that hDPSC-K4DT cells released ATP extracellularly when exposed to LPS concentrations above 10 μg/mL. ATP release was exclusively attenuated by N-ethylmaleimide, whereas other inhibitors, including clodronic acid (a vesicular nucleotide transporter inhibitor), probenecid (a selective pannexin-1 channel inhibitor), meclofenamic acid (a selective connexin 43 inhibitor), suramin (a nonspecific P2 receptor inhibitor), and KN-62 (a specific P2X7 antagonist), did not exhibit any effect. Additionally, LPS increased IL-6 mRNA expression, which was mitigated by the ATPase apyrase enzyme, N-ethylmaleimide, and suramin, but not by KN-62. Moreover, exogenous ATP induced IL-6 mRNA expression, whereas ATPase apyrase, N-ethylmaleimide, and suramin, but not KN-62, diminished ATP-induced IL-6 mRNA expression. Overall, our findings suggest that LPS-induced ATP release stimulates the IL-6 pathway through P2-purinoceptor, indicating that ATP may function as an anti-inflammatory signal, contributing to the maintenance of dental pulp homeostasis.

ADP-mediated Modulation of Intracellular Calcium Responses in Chromaffin Cells: The Role of Ectonucleoside Triphosphate Diphosphohydrolase 2 on Rat Adrenal Medulla Function.

The present study investigated the localization and the adenosine 5'-triphosphate (ATP)-degrading function of the plasma membrane-bound ecto-nucleotidase, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), in the rat adrenal medulla. The effect of ATP degradation product, adenosine 5'-diphosphate (ADP), on carbachol (CCh)-induced intracellular Ca2+ ([Ca2+]i) responses in adrenal chromaffin cells was examined using calcium imaging. NTPDase2-immunoreactive cells were distributed between chromaffin cells. NTPDase2-immunoreactive cells were immunoreactive for glial fibrillary acidic protein and S100B, suggesting that they were sustentacular cells. NTPDase2-immunoreactive cells surrounded chromaffin cells immunoreactive for vesicular nucleotide transporter and P2Y12 ADP-selective purinoceptors. In ATP bioluminescence assays using adrenal medullary slices, ATP was rapidly degraded and its degradation was attenuated by the NTPDase inhibitors sodium polyoxotungstate (POM-1) and 6-N, N-diethyl-d-β,γ-dibromomethylene ATP (ARL67156). ADP inhibited CCh-induced [Ca2+]i increases of chromaffin cells in adrenal medullary slices. The inhibition of CCh-induced [Ca2+]i increases by ADP was blocked by the P2Y12 purinoceptor antagonist AZD1283. CCh-induced [Ca2+]i increases were also inhibited by the P2Y1, P2Y12, and P2Y13 purinoceptor agonist 2-methylthioadenosine diphosphate trisodium (2MeSADP), in combination with the P2Y1 purinoceptor antagonist MRS2179. These results suggest that sustentacular cells express NTPDase2 to degrade ATP released from adrenal chromaffin cells, and ADP modulates the excitability of chromaffin cells via P2Y12 purinoceptors to regulate catecholamine release during preganglionic sympathetic stimuli. (J Histochem Cytochem 72: 41-60, 2024).

Intrathecal administration of botulinum toxin type a antagonizes neuropathic pain by countering increased vesicular nucleotide transporter expression in the spinal cord of chronic constriction injury of the sciatic nerve rats

Botulinum toxin type A (BoNT/A) induces direct analgesic effects in neuropathic pain by inhibiting the release of substance P, calcitonin gene-related peptide (CGRP) and glutamate. Vesicular nucleotide transporter (VNUT) was responsible for the storage and release of ATP in vivo, and one of the mechanisms underlying neuropathic pain is VNUT-dependent release of extracellular ATP from dorsal horn neurons. However, the analgesic effect of BoNT/A by affecting the expression of VNUT remained largely unknown. Thus, in this study, we aimed to elucidate the antinociceptive potency and analgesic mechanism of BoNT/A in chronic constriction injury of the sciatic nerve (CCI) induced neuropathic pain. Our results showed that a single intrathecal injection of 0.1 U BoNT/A seven days after CCI surgery produced significant analgesic activity and decreased the expression of VNUT in the spinal cord of CCI rats. Similarly, BoNT/A inhibited the CCI-induced increase in ATP content in the rat spinal cord. Overexpression of VNUT in the spinal cord of CCI-induced rats markedly reversed the antinociceptive effect of BoNT/A. Furthermore, 33 U/mL BoNT/A dramatically reduced the expression of VNUT in pheochromocytoma (PC12) cells but overexpressing SNAP-25 increased VNUT expression in PC12 cells. Our current study is the first to demonstrate that BoNT/A is involved in neuropathic pain by regulating the expression of VNUT in the spinal cord in rats.

MP74-03 LONG-TERM ADMINISTRATION OF A PDE-5 INHIBITOR PREVENTS THE DEVELOPMENT OF BLADDER EPITHELIAL ATP INCREASE AND DECREASED DETRUSOR CONTRACTILITY IN TYPE 2 DIABETIC RATS

You have accessJournal of UrologyCME1 Apr 2023MP74-03 LONG-TERM ADMINISTRATION OF A PDE-5 INHIBITOR PREVENTS THE DEVELOPMENT OF BLADDER EPITHELIAL ATP INCREASE AND DECREASED DETRUSOR CONTRACTILITY IN TYPE 2 DIABETIC RATS Kabuto Takafumi, So Inamura, Hisato Kobayashi, Xinmin Zha, Keiko Nagase, Minekatsu Taga, Masaya Seki, Nobuki Tanaka, Yoshinaga Okumura, Osamu Yokoyama, and Naoki Tearada Kabuto TakafumiKabuto Takafumi More articles by this author , So InamuraSo Inamura More articles by this author , Hisato KobayashiHisato Kobayashi More articles by this author , Xinmin ZhaXinmin Zha More articles by this author , Keiko NagaseKeiko Nagase More articles by this author , Minekatsu TagaMinekatsu Taga More articles by this author , Masaya SekiMasaya Seki More articles by this author , Nobuki TanakaNobuki Tanaka More articles by this author , Yoshinaga OkumuraYoshinaga Okumura More articles by this author , Osamu YokoyamaOsamu Yokoyama More articles by this author , and Naoki TearadaNaoki Tearada More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003348.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We examined a series of pathological conditions of bladder wall in a type 2 diabetes mellitus (T2DM) model (Otsuka Long-Evans Tokushima Fatty [OLEFT] rats) and evaluated the effects of a long-term administration of the PDE-5 inhibitor tadalafil (TA) on these conditions. METHODS: Male OLETF and Long-Evans Tokushima Otsuka (LETO) rats were used as T2DM and control groups, respectively. TA was orally administered for 12 weeks (wks) from the age of 36 wks. Bladder blood flow and ATP, prostaglandin E2 (PGE2), and nerve growth factor (NGF) released from the bladder epithelium were measured at 36 and 48 wks of age. Vesicular nucleotide transporter (VNUT), hypoxia-inducible factor-1 alpha (HIF-1α), 8-hydroxy-2′-deoxyguanosine (HIF-1α), the oxidative stress marker 8-OHdG, and the mRNA and protein of pro-inflammatory cytokines and growth factors in the bladder wall were measured by RT-PCRs and ELISAs. Bladder strip contractions to KCl and carbachol were monitored in the OLETF rat bladder. RESULTS: The bladder blood flow in the OLETF rats was decreased at 48 wks compared to 36 wks. TA increased the bladder blood flow. Epithelial ATP was significantly increased in OLETF compared to LETO and decreased by TA in both LETO and OLETF. The bladder VNUT expression was significantly elevated in OLETF versus LETO at 36 wks; it was further enhanced at 48 wks, but a significant decrease occurred with TA in both rat strains. HIF-1α and 8-OHdG of OLETF were upregulated at 48 wks and decreased by TA. In OLETF, the expressions of IL-6, IL-8, bFGF, TNF-α, TGF-β, IGF-1, and IGF1R were increased at 48 wks versus 36 wks. TA decreased IL-6, TNF-α, IGF-1 and IGF1R. In OLETF, the contractile response of bladder strips to KCl and carbachol decreased at 48 wks versus 36 wks, but it recovered after TA administration. CONCLUSIONS: These data suggest that impaired bladder blood flow resulted in increased expressions of HIF-1α, 8-OhdG, and various pro-inflammatory cytokines and growth factors In T2DM. As a result, the bladder epithelial ATP release was enhanced, possibly via VNUT upregulation. A long-term administration of a PDE-5 inhibitor could prevent the development of storage and voiding dysfunction associated with T2DM. This study is the first to suggest that epithelium-derived ATP may be involved in urine storage dysfunction in T2DM and tadalafil inhibits ATP release and improves bladder contraction. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1069 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kabuto Takafumi More articles by this author So Inamura More articles by this author Hisato Kobayashi More articles by this author Xinmin Zha More articles by this author Keiko Nagase More articles by this author Minekatsu Taga More articles by this author Masaya Seki More articles by this author Nobuki Tanaka More articles by this author Yoshinaga Okumura More articles by this author Osamu Yokoyama More articles by this author Naoki Tearada More articles by this author Expand All Advertisement PDF downloadLoading ...

Hindbrain Adenosine 5-Triphosphate (ATP)-Purinergic Signaling Triggers LH Surge and Ovulation via Activation of AVPV Kisspeptin Neurons in Rats.

Ovulation disorders are a serious problem for humans and livestock. In female rodents, kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) are responsible for generating a luteinizing hormone (LH) surge and consequent ovulation. Here, we report that adenosine 5-triphosphate (ATP), a purinergic receptor ligand, is a possible neurotransmitter that stimulates AVPV kisspeptin neurons to induce an LH surge and consequent ovulation in rodents. Administration of an ATP receptor antagonist (PPADS) into the AVPV blocked the LH surge in ovariectomized (OVX) rats treated with a proestrous level of estrogen (OVX + high E2) and significantly reduced the ovulation rate in proestrous ovary-intact rats. AVPV ATP administration induced a surge-like LH increase in OVX + high E2 rats in the morning. Importantly, AVPV ATP administration could not induce the LH increase in Kiss1 KO rats. Furthermore, ATP significantly increased intracellular Ca2+ levels in immortalized kisspeptin neuronal cell line, and coadministration of PPADS blocked the ATP-induced Ca2+ increase. Histologic analysis revealed that the proestrous level of estrogen significantly increased the number of P2X2 receptor (an ATP receptor)-immunopositive AVPV kisspeptin neurons visualized by tdTomato in Kiss1-tdTomato rats. The proestrous level of estrogen significantly increased varicosity-like vesicular nucleotide transporter (a purinergic marker)-immunopositive fibers projecting to the vicinity of AVPV kisspeptin neurons. Furthermore, we found that some hindbrain vesicular nucleotide transporter-positive neurons projected to the AVPV and expressed estrogen receptor α, and the neurons were activated by the high E2 treatment. These results suggest that hindbrain ATP-purinergic signaling triggers ovulation via activation of AVPV kisspeptin neurons.SIGNIFICANCE STATEMENT Ovulation disorders, which cause infertility and low pregnancy rates, are a serious problem for humans and livestock. The present study provides evidence that adenosine 5-triphosphate, acting as a neurotransmitter in the brain, stimulates kisspeptin neurons in the anteroventral periventricular nucleus, known as the gonadotropin-releasing hormone surge generator, via purinergic receptors to induce the gonadotropin-releasing hormone/luteinizing hormone surge and ovulation in rats. In addition, histologic analyses indicate that adenosine 5-triphosphate is likely to be originated from the purinergic neurons in the A1 and A2 of the hindbrain. These findings may contribute to new therapeutic controls for hypothalamic ovulation disorders in humans and livestock.

Phylogenetic and localisation analyses of mammalian VNUT reveal a unique ER-retention sequence replaces vesicular targeting motifs in the C-terminus.

Adenosine 5′-triphosphate (ATP) acts as a neurotransmitter in central and peripheral nerves, and the Vesicular Nucleotide Transporter (VNUT) is thought to load synaptic vesicles (SVs) with ATP. Related neurotransmitter transporters use C-terminal dileucine and tyrosine motifs to target them to SVs. We previously found that (1) rat, mouse and human VNUT weakly localise to synaptic vesicle pools, and (2) that their C-terminals contain a KDEL-like endoplasmic reticulum retention motif rather than a SV-targeting motif. Here we report a phylogenetic analysis of VNUT's C-terminal KDEL-like motif and study whether it promotes endoplasmic reticulum/Golgi retention of VNUT.

Exploring the transport cycle of DgoT, a bacterial homolog of human vesicular glutamate transporters.

The solute carrier 17 (SLC17) family belongs to the major facilitator superfamily (MFS) and encompasses vesicular glutamate transporters (VGLUTs), inorganic phosphate transporters, the lysosomal H+/sialic acid cotransporter sialin, and the vesicular nucleotide transporter (VNUT). SLC17 members mediate symport or exchange of various substrates with H+ at distinct stoichiometries. A bacterial D-galactonate transporter (DgoT) from E. coli shows high sequence similarity with mammalian VGLUTs and sialin. We used a combination of experimental and computational approaches to explore DgoT transport mechanism. In solid-supported membrane (SSM)-based electrophysiology, rapid galactonate application triggers a transient current generated by substrate binding and subsequent conformational changes, followed by a slower electrogenic transport. Transport current at different pH values demonstrates inhibition of H+ release and binding with apparent pK values of 6.7 and 9.2, respectively. We demonstrated that DgoT functions as galactonate/H+ symporter with 1:2 stoichiometry. There are four charged residues present in the membrane spanning region of DgoT, of which three (R47, R126 and E133) are conserved among SLC17 family. Mutations D46N, E133Q, R47Q and R126Q lead to loss of transport activity, while selective binding of galactonate remains intact. Analysis of pre-steady currents indicates that D46 and E133 are involved in proton translocation, while R47 and R126 are necessary for conformational changes. In all-atom molecular dynamics simulations, protonation of D46 and E133 leads to rearrangement of transmembrane (TM) helices TM1 and TM7 that gate access to the substrate-binding site from the extracellular side. Substrate binding then induces the closure of the periplasmic gate and transition to the inward-open conformation, where substrate is released, in agreement with the alternating access mechanism used by other MFS transporters. Based on our experimental and computational results, we propose a detailed transport model for galactonate/H+ cotransport in DgoT.

Nanomolar clodronate induces adenosine accumulation in the perfused rat mesenteric bed and mesentery-derived endothelial cells.

The vesicular nucleotide transporter (VNUT) is critical for sympathetic co-transmission and purinergic transmission maintenance. To examine this proposal, we assessed whether the bisphosphonate clodronate, claimed as a potent in vitro VNUT blocker, modified spontaneous and/or the electrically evoked overflow of ATP/metabolites and NA from mesentery sympathetic perivascular nerve terminals. Additionally, in primary endothelial cell cultures derived from this tissue, we also evaluated whether clodronate interfered with ATP/metabolite cell outflow and metabolism of N6-etheno adenosine 5'-triphosphate (eATP), N6-etheno adenosine (eADO), and adenosine deaminase enzyme activity. Rat mesenteries were perfused in the absence or presence of .01-1,000nM clodronate, 1-1,000nM Evans blue (EB), and 1-10µM DIDS; tissue perfusates were collected to determine ATP/metabolites and NA before, during, and after perivascular electrical nerve terminal depolarization. An amount of 1-1,000nM clodronate did not modify the time course of ATP or NA overflow elicited by nerve terminal depolarization, and only 10nM clodronate significantly augmented perfusate adenosine. Electrical nerve terminal stimulation increased tissue perfusion pressure that was significantly reduced only by 10nM clodronate [90.0 ± 18.6 (n = 8) to 35.0 ± 10.4 (n = 7), p = .0277]. As controls, EB, DIDS, or reserpine treatment reduced the overflow of ATP/metabolites and NA in a concentration-dependent manner elicited by nerve terminal depolarization. Moreover, mechanical stimulation of primary endothelial cell cultures from the rat mesentery added with 10 or 100nM clodronate increased adenosine in the cell media. eATP was metabolized by endothelial cells to the same extent with and without 1-1,000nM clodronate, suggesting the bisphosphonate did not interfere with nucleotide ectoenzyme metabolism. In contrast, extracellular eADO remained intact, indicating that this nucleoside is neither metabolized nor transported intracellularly. Furthermore, only 10nM clodronate inhibited (15.5%) adenosine metabolism to inosine in endothelial cells as well as in a commercial crude adenosine deaminase enzyme preparation (12.7%), and both effects proved the significance (p < .05). Altogether, present data allow inferring that clodronate inhibits adenosine deaminase activity in isolated endothelial cells as in a crude extract preparation, a finding that may account for adenosine accumulation following clodronate mesentery perfusion.

A promising drug for neuropathic pain: identification of vesicular nucleotide transporter as a novel target of eicosapentaenoic acid.

A promising drug for neuropathic pain: identification of vesicular nucleotide transporter as a novel target of eicosapentaenoic acid.

María Teresa Miras Portugal: a pioneer for vesicular nucleotide storage.

Chromaffin granules are secretory granules present in adrenal medulla chromaffin cells. They contain high contents of catecholamines and nucleotides and have been regarded as a model system for the study of vesicular transmitter uptake and release. In 1988, Dr. María Teresa Miras Portugal, when studying catecholamine biosynthesis, detected a novel group of nucleotides, the diadenosine polyphosphates, in the adrenal chromaffin granules. Based on this finding, she unraveled the existence of diadenosine polyphosphate-mediated chemical transmission, leading to a paradigm shift in the field of purinergic signaling. She is also a pioneer in the studies on vesicular nucleotide storage. First, María Teresa and her group characterized nucleotide transport in chromaffin granules and synaptic vesicles using fluorescent nucleotide derivatives such as 1, N6-ethenoadenosine triphosphates. Then, they revealed the presence of a hypothetical vesicular nucleotide transporter with unique properties in terms of substrate specificity. In this article, we will describe her contributions to vesicular nucleotide storage and the foundations she laid for future studies.

Is the vesicular nucleotide transporter a molecular target of eicosapentaenoic acid?

Vesicular nucleotide transporter (VNUT), an active transporter for nucleotides in secretory vesicles, is responsible for the vesicular storage of ATP and plays an essential role in purinergic chemical transmission. Inhibition of VNUT decreases the concentration of ATP in the luminal space of secretory vesicles, followed by decreased vesicular ATP release, resulting in the blockade of purinergic chemical transmission. Very recently, Miyaji and colleagues reported that eicosapentaenoic acid (EPA) is a potent VNUT inhibitor and effective in treating neuropathic and inflammatory pain and insulin resistance through inhibition of vesicular storage and release of ATP. However, our validation study indicated that, in bovine adrenal chromaffin granule membrane vesicles, EPA inhibited the formation of an electrochemical gradient of protons across the membrane with the concentration of 50% inhibition (IC50) being 1.0μM without affecting concanamycin B-sensitive ATPase activity. Essentially, similar results were obtained with proteoliposomes containing purified vacuolar H+-ATPase. Consistent with these observations, EPA inhibited the ATP-dependent uptakes of ATP and dopamine by chromaffin granule membrane vesicles, with ID50 being 1.2 and 1.0μM, respectively. Furthermore, EPA inhibited ATP-dependent uptake of L-glutamate by mouse brain synaptic vesicles with ID50 being 0.35μM. These results indicate that EPA at sub-μM acts as a proton conductor and increases proton permeability across the membrane, regardless of the presence or absence of VNUT, thereby inhibiting non-specifically the vesicular storage of neurotransmitters. Thus, EPA may affect a broader range of chemical transmission than proposed.

Immunohistochemical localization of vesicular nucleotide transporter in small intensely fluorescent (SIF) cells of the rat superior cervical ganglion

Our previous study reported that a part of small intensely fluorescent (SIF) cells in the rat superior cervical ganglion were innervated by P2X3 purinoceptor-expressing glossopharyngeal sensory nerve endings, suggesting the occurrence of adenosine 5′-triphosphate (ATP)-mediated transmission between them. The present study investigated the immunolocalization of vesicular nucleotide transporter (VNUT) in SIF cells of the superior cervical ganglion in male Wistar rats. VNUT was immunohistochemically localized in tyrosine hydroxylase-immunoreactive SIF cells and sympathetic postganglionic neurons, but not in a few SIF cells with immunoreactivity for dopamine beta-hydroxylase. P2X3-immunoreactive ramified nerve endings formed flat leaf-like or spherical terminal parts to surround some VNUT-immunoreactive SIF cells, but not other VNUT-immunoreactive SIF cells attached to ganglionic neurons. VNUT-immunoreactive SIF cells contained bassoon-immunoreactive products at the contacting surface of P2X3-immunoreactive nerve endings. Immunoreactivity for ectonucleoside triphosphate diphosphohydrolase 2, which hydrolyzes extracellular ATP, was observed in S100B-immunoreactive satellite glial cells surrounding VNUT-immunoreactive SIF cells, but not in the attachment surfaces between SIF cells and nerve endings with P2X3 immunoreactivity. The present results suggest that SIF cells release ATP by exocytosis to modulate the excitability of sensory nerve endings and postganglionic neurons in the superior cervical ganglion.

Non-Nitrogen-Containing Bisphosphonates Prevent Pyrophosphorylation of Exocytosis Proteins.

Clodronate, a non-nitrogen-containing bisphosphonate (non-NBP), is intracellularly converted into non-hydrolyzable ATP analogs. Clodronate and its analogs impair normal cell functions, including the exocytosis process. However, how this occurs in mast cells is still not well characterized. To summarize the possible mechanisms of clodronate-mediated exocytosis inhibition in mast cells. Non-NBPs display several possible mechanisms of exocytosis inhibition in various cell types, including vesicular nucleotide transporter (VNUT) and purinergic receptor inhibition. Inhibition of purinergic receptors has been shown in mast cells, but VNUT inhibition remains to be confirmed. Inhibition of protein prenylation by non-NBPs has also been shown; however, direct evidence of non-NBPs in prenylated exocytosis proteins is still contradictory. Finally, non-NBPs may inhibit mast cell exocytosis via impairment of protein pyrophosphorylation. This mechanism is less studied, and direct evidence of the involvement of pyrophosphorylated proteins in exocytosis is still lacking. Non-NBPs may affect mast cell exocytosis by interacting with purinergic receptors or VNUT or by preventing post-translational modifications of exocytosis protein(s), i.e., prenylation and pyrophosphorylation. The latter needs further investigation to provide direct evidence of a role for non- NBPs.

Vesicular nucleotide transporter is a molecular target of eicosapentaenoic acid for neuropathic and inflammatory pain treatment

Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC50) of 67 nM, acting as an allosteric modulator through competition with Cl-. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, VNUT-/- mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in VNUT-/- mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the VNUT-/- mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.

Nitrergic and Purinergic Nerves in the Small Intestinal Myenteric Plexus and Circular Muscle of Mice and Guinea Pigs.

ATP is an excitatory and inhibitory neurotransmitter, while nitric oxide (NO) is an inhibitory neurotransmitter in the enteric nervous system (ENS). We used a vesicular nucleotide transporter (SLC17A9, VNUT) antibody and a nitric oxide synthase (NOS) antibody to identify purinergic and nitrergic nerves in mouse and guinea ileum. Mouse: VNUT-immunoreactivity (ir) was detected in nerve fibers in myenteric ganglia and circular muscle. VNUT-ir fibers surrounded choline acetyltransferase (ChAT), nitric oxide synthase (nNOS), and calretinin-ir neurons. VNUT-ir nerve cell bodies were not detected. Tyrosine hydroxylase (TH)-ir nerves were detected in myenteric ganglia and the tertiary plexus. Guinea pig: VNUT-ir was detected in neurons and nerves fibers and did not overlap with NOS-ir nerve fibers. VNUT-ir was detected in nerve fibers in ganglia but not nerve cell bodies. VNUT-ir nerve fibers surrounded NOS-ir and NOS- neurons. NOS-ir and VNUT-ir nerve fibers did not overlap in myenteric ganglia or circular muscle. VNUT-ir nerves surrounded some ChAT-ir neurons. VNUT-ir and ChAT-ir were detected in separate nerves in the CM. VNUT-ir nerve fibers surrounded calretinin-ir neurons.Conclusions: VNUT-ir neurons likely mediate purinergic signaling in small intestinal myenteric ganglia and circular muscle. ATP and NO are likely released from different inhibitory motorneurons. VNUT-ir and ChAT-ir interneurons mediate cholinergic and purinergic synaptic transmission in the myenteric plexus.

Single-nucleotide polymorphisms of the SLC17A9 and P2RY12 genes are significantly associated with phantom tooth pain.

Phantom tooth pain (PTP) is a rare and specific neuropathic pain that occurs after pulpectomy and tooth extraction, but its cause is not understood. We hypothesized that there is a genetic contribution to PTP. We focused on solute carrier family 17 member 9 (SLC17A9)/vesicular nucleotide transporter (VNUT) and purinergic receptor P2Y12 (P2RY12), both of which have been associated with neuropathic pain and pain transduction signaling in the trigeminal ganglion in rodents. We sought to corroborate these associations in humans. We investigated gene polymorphisms that contribute to PTP. We statistically examined the association between genetic polymorphisms and PTP vulnerability in 150 patients with orofacial pain, including PTP, and 500 healthy subjects. We found that the rs735055 polymorphism of the SLC17A9 gene and rs3732759 polymorphism of the P2RY12 gene were associated with the development of PTP. Carriers of the minor allele of rs735055 and individuals who were homozygous for the major allele of rs3732759 had a higher rate of PTP. Carriers of the minor allele of rs735055 reportedly had high SLC17A9 mRNA expression in the spinal cord, which may increase the storage and release of adenosine triphosphate. Individuals who were homozygous for the major allele of rs3732759 may have higher P2RY12 expression that is more active in microglia. Therefore, these carriers may be more susceptible to PTP. These results suggest that specific genetic polymorphisms of the SLC17A9 and P2RY12 genes are involved in PTP. This is the first report on genes that are associated with PTP in humans.