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.     

Identification of a region from the human cholinergic gene locus that targets expression of the vesicular acetylcholine transporter to a subset of neurons in the medial habenular nucleus in transgenic mice

We use a transgenic mouse model system to elucidate the regulatory regions within the human cholinergic gene locus responsible for vesicular acetylcholine transporter gene expression in vivo. In this report we characterized two transgenes for their ability to confer cholinergic-specific expression of the encoded vesicular acetylcholine transporter. An 11.2 kb transgene (named hV11.2) that spanned from about 5 kb upstream of the start of vesicular acetylcholine transporter translation down to the first choline acetyltransferase coding exon gave expression in the somatomotor neurons and a subpopulation of cholinergic neurons in the medial habenular nucleus. The second transgene (named hV6.7), a 5-prime truncated version of hV11.2 that was devoid of 4.5 kb of gene-regulatory sequences completely lacked vesicular acetylcholine transporter expression in vivo. Our data indicate that vesicular acetylcholine transporter expression in somatomotor neurons and in the medial habenular nucleus is uniquely specified within the cholinergic gene locus, and separable from cholinergic expression elsewhere. The identification of these two subdivisions of the cholinergic nervous system suggests that other cholinergic neurons in the CNS and PNS are similarly regulated by additional discrete domains within the cholinergic gene locus.     

A shared vesicular carrier allows synaptic corelease of GABA and glycine

The type of vesicular transporter expressed by a neuron is thought to determine its neurotransmitter phenotype. We show that inactivation of the vesicular inhibitory amino acid transporter (Viaat, VGAT) leads to embryonic lethality, an abdominal defect known as omphalocele, and a cleft palate. Loss of Viaat causes a drastic reduction of neurotransmitter release in both GABAergic and glycinergic neurons, indicating that glycinergic neurons do not express a separate vesicular glycine transporter. This loss of GABAergic and glycinergic synaptic transmission does not impair the development of inhibitory synapses or the expression of KCC2, the K+ -Cl- cotransporter known to be essential for the establishment of inhibitory neurotransmission. In the absence of Viaat, GABA-synthesizing enzymes are partially lost from presynaptic terminals. Since GABA and glycine compete for vesicular uptake, these data point to a close association of Viaat with GABA-synthesizing enzymes as a key factor in specifying GABAergic neuronal phenotypes.     

Differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI) is a vesicular glutamate transporter in endocrine glutamatergic systems

Vesicular glutamate transporter is present in neuronal synaptic vesicles and endocrine synaptic-like microvesicles and is responsible for vesicular storage of L-glutamate. A brain-specific Na(+)-dependent inorganic phosphate transporter (BNPI) functions as a vesicular glutamate transporter in synaptic vesicles, and the expression of this BNPI defines the glutamatergic phenotype in the central nervous system (Bellocchio, E. E., Reimer, R. J., Fremeau, R. T., Jr., and Edwards, R. H. (2000) Science 289, 957-960; Takamori, S., Rhee, J. S., Rosenmund, C., and Jahn, R. (2000) Nature 407, 189-194). However, since not all glutamatergic neurons contain BNPI, an additional transporter(s) responsible for vesicular glutamate uptake has been postulated. Here we report that differentiation-associated Na(+)-dependent inorganic phosphate cotransporter (DNPI), an isoform of BNPI (Aihara, Y., Mashima, H., Onda, H., Hisano, S., Kasuya, H., Hori, T., Yamada, S., Tomura, H., Yamada, Y., Inoue, I., Kojima, I., and Takeda, J. (2000) J. Neurochem. 74, 2622-2625), also transports L-glutamate at the expense of an electrochemical gradient of protons established by the vacuolar proton pump when expressed in COS7 cells. Molecular, biological, and immunohistochemical studies have indicated that besides its presence in neuronal cells DNPI is preferentially expressed in mammalian pinealocytes, alphaTC6 cells, clonal pancreatic alpha cells, and alpha cells of Langerhans islets, these cells being proven to secrete L-glutamate through Ca(2+)-dependent regulated exocytosis followed by its vesicular storage. Pancreatic polypeptide-secreting F cells of Langerhans islets also expressed DNPI. These results constitute evidence that DNPI functions as another vesicular transporter in glutamatergic endocrine cells as well as in neurons.     

Coexpression of glutamate vesicular transporter (VGLUT1) and choline acetyltransferase (ChAT) proteins in fetal rat hippocampal neurons in culture

A very small population of choline acetyltransferase (ChAT) immunoreactive cells is observed in all layers of the adult hippocampus. This is the intrinsic source of the hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection. This study aimed at quantifying and identifying the origin of this small population of ChAT-immunoreactive cells in the hippocampus at early developmental stages, by culturing the fetal hippocampal neurons in serum-free culture and on a patternable, synthetic silane substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine. Using this method, a large proportion of glutamatergic (glutamate vesicular transporter, VGLUT1-immunoreactive) neurons, a small fraction of GABAergic (GABA-immunoreactive) neurons, and a large proportion of cholinergic (ChAT-immunoreactive) neurons were observed in the culture. Interestingly, most of the glutamatergic neurons that expressed glutamate vesicular transporter (VGLUT1) also co-expressed ChAT proteins. On the contrary, when the cultures were double-stained with GABA and ChAT, colocalization was not observed. Neonatal and adult rat hippocampal neurons were also cultured to verify whether these more mature neurons also co-express VGLUT1 and ChAT proteins in culture. Colocalization of VGLUT1 and ChAT in these relatively more mature neurons was not observed. One possible explanation for this observation is that the neurons have the ability to synthesize multiple neurotransmitters at a very early stage of development and then with time follows a complex, combinatorial strategy of electrochemical coding to determine their final fate.     

Localization and osmotic regulation of vesicular glutamate transporter-2 in magnocellular neurons of the rat hypothalamus

In this report we present immunocytochemical and in situ hybridization evidence that magnocellular vasopressin and oxytocin neurons in the hypothalamic supraoptic and paraventricular nuclei express type-2 vesicular glutamate transporter, a marker for their glutamatergic neuronal phenotype. To address the issue of whether an increase in magnocellular neuron activity coincides with the altered synthesis of the endogenous glutamate marker, we have introduced a new dual-label in situ hybridization method which combines fluorescent and autoradiographic signal detection components for vasopressin and vesicular glutamate transporter-2 mRNAs, respectively. Application of this technique provided evidence that 2% sodium chloride in the drinking water for 7 days produced a robust and significant increase of vesicular glutamate transporter-2 mRNA in vasopressin neurons of the supraoptic nucleus. The immunocytochemical labeling of pituitary sections, followed by the densitometric analysis of vesicular glutamate transporter-2 immunoreactivity in the posterior pituitary, revealed a concomitant increase in vesicular glutamate transporter-2 protein levels at the major termination site of the magnocellular axons. These data demonstrate that magnocellular oxytocin as well as vasopressin cells contain the glutamatergic marker vesicular glutamate transporter-2, similarly to most of the parvicellular neurosecretory neurons examined so far. The robust increase in vesicular glutamate transporter-2 mRNA and immunoreactivity after salt loading suggests that the cellular levels of vesicular glutamate transporter-2 in vasopressin neurons are regulated by alterations in water–electrolyte balance. In addition to the known synaptic actions of excitatory amino acids in magnocellular nuclei, the new observations suggest novel mechanisms whereby glutamate of endogenous sources can regulate magnocellular neuronal functions.     

A putative vesicular transporter expressed in Drosophila mushroom bodies that mediates sexual behavior may define a neurotransmitter system

Vesicular transporters are required for the storage of?all classical and amino acid neurotransmitters in synaptic vesicles. Some neurons lack known vesicular transporters, suggesting additional neurotransmitter systems remain unidentified. Insect mushroom bodies (MBs) are critical for several behaviors, including learning, but the neurotransmitters released by the intrinsic Kenyon cells (KCs) remain unknown. Likewise, KCs do not express a known vesicular transporter. We report the identification of a novel Drosophila gene portabella (prt) that is structurally similar to known vesicular transporters. Both larval and adult brains express PRT in the KCs of the MBs. Additional PRT cells project to the central complex and optic ganglia. prt mutation causes an olfactory learning deficit and an unusual defect in the male's position during copulation that is rescued by expression in KCs. Because prt is expressed in neurons that lack other known vesicular transporters or neurotransmitters, it may define a previously unknown neurotransmitter system responsible for sexual behavior and a component of olfactory learning.     

Odor suppression of voltage-gated currents contributes to the odor-induced response in olfactory neurons

Olfactorychemotransduction involves a signaling cascade. In addition totriggering transduction, odors suppress ion conductances. Bystimulating with brief odorant pulses, we observed a current associatedwith odor-induced suppression of voltage-gated conductances and studiedits time dependence. We characterized this suppression current inisolated Caudiverbera caudiverberaolfactory neurons. All four voltage-gated currents are suppressed byodor pulses in almost every neuron, and suppression is caused by odorsinducing excitation and by those inducing inhibition, indicating anonselective phenomenon, in contrast to transduction. Suppression has a10-fold shorter latency than transduction. Suppression was morepronounced when odors were applied to the soma than to the cilia,opposite to transduction. Suppression was also present in rat olfactory neurons. Furthermore, we could induce it inDrosophila photoreceptor cells,demonstrating its independence from the chemotransduction cascade. Weshow that odor concentrations causing suppression are similar to thosetriggering chemotransduction and that both suppression and transductioncontribute to the odor response in isolated olfactory neurons.Furthermore, suppression affects spiking, implying a possiblephysiological role in olfaction.

     

Vesicular neurotransmitter transporters

Neurotransmission depends on the regulated release of chemical transmitter molecules. This requires the packaging of these substances into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific vesicular transporters. The family of genes encoding the vesicular transporters for biogenic amines and acetylcholine have recently been cloned. Direct comparison of their transport characteristics and pharmacology provides information about vesicular transport bioenergetics, substrate feature recognition by each transporter, and the role of vesicular amine storage in the mechanism of action of psychopharmacologic and neurotoxic agents. Regulation of vesicular transport activity may affect levels of neurotransmitter available for neurosecretion and be an important site for the regulation of synaptic function. Gene knockout studies have determined vesicular transport function is critical for survival and have enabled further evaluation of the role of vesicular neurotransmitter transporters in behavior and neurotoxicity. Molecular analysis is beginning to reveal the sites involved in vesicular transporter function and the sites that determine substrate specificity. In addition, the molecular basis for the selective targeting of these transporters to specific vesicle populations and the biogenesis of monoaminergic and cholinergic synaptic vesicles are areas of research that are currently being explored. This information provides new insights into the pharmacology and physiology of biogenic amine and acetylcholine vesicular storage in cardiovascular, endocrine, and central nervous system function and has important implications for neurodegenerative disease.     

Features of Egg Cells of Living Representatives of Ancient Families of Ferns

Examination of two species of osmundaceous ferns, two speciesof marattialean, and one dipterid, all representative of fernswith a long fossil history, has shown that the egg cells possessnumerous large amyloplasts. The nuclei of these egg cells alsoproduce no vesicular or sheet-like protrusions during maturation.In these respects the egg cells of the ferns examined differsharply from those of more recent ferns such as Pteridium andDryopteris. The significance of these findings is discussed. Egg cells, amyloplast, Macroglossum, Todea, Osmunda, Dipteris     

The abts and sulp families of anion transporters from Caenorhabditis elegans

The slc4 and slc26 gene families encode two distinct groups of gene products that transport HCO₃^– and other anions in mammalian cells. The SLC4 and SLC26 proteins are important contributors to transepithelial movement of fluids and electrolytes and to cellular pH and volume regulation. Herein we describe the cDNA cloning from the nematode Caenorhabditis elegans of four anion bicarbonate transporter (abts) homologs of slc4 cDNA and eight sulfate permease (sulp) homologs of slc26 cDNA. Analysis of transgenic nematode strains carrying promoter::GFP fusions suggests relatively restricted expression patterns for many of these genes. At least three genes are expressed primarily in the intestine, three are expressed primarily in the excretory cell, and one is expressed in both of these polarized cell types. One of the genes is also expressed exclusively in the myoepithelium-like cells of the pharynx. Many of the sulp gene products localize to the basolateral membrane rather than to the apical membrane. Several ABTS and SULP proteins exhibited anion transport function in Xenopus oocytes. The strongest Cl^– transporter among these also mediated Cl^–/HCO₃^– exchange. These findings encourage exploitation of the genetic strengths of the nematode model system in the study of the physiological roles of anion transport by the proteins of these two highly conserved gene families.     

Norepinephrine transporter expression in cholinergic sympathetic neurons: differential regulation of membrane and vesicular transporters

Sympathetic neurons that undergo a noradrenergic to cholinergic change in phenotype provide a useful model system to examine the developmental regulation of proteins required to synthesize, store, or remove a particular neurotransmitter. This type of change occurs in the sympathetic sweat gland innervation during development and can be induced in cultured sympathetic neurons by extracts of sweat gland-containing footpads or by leukemia inhibitory factor. Sympathetic neurons initially produce norepinephrine (NE) and contain the vesicular monoamine transporter 2 (VMAT2), which packages NE into vesicles, and the norepinephrine transporter (NET), which removes NE from the synaptic cleft to terminate signaling. We have used a variety of biochemical and molecular techniques to test whether VMAT2 and NET levels decrease in sympathetic neurons which stop producing NE and make acetylcholine. In cultured sympathetic neurons, NET protein and mRNA decreased during the switch to a cholinergic phenotype but VMAT2 mRNA and protein did not decline. NET immunoreactivity disappeared from the developing sweat gland innervation in vivo as it acquired cholinergic properties. Surprisingly, NET simultaneously appeared in sweat gland myoepithelial cells. The presence of NET in myoepithelial cells did not require sympathetic innervation. VMAT2 levels did not decrease as the sweat gland innervation became cholinergic, indicating that NE synthesis and vesicular packaging are not coupled in this system. Thus, production of NE and the transporters required for noradrenergic transmission are not coordinately regulated during cholinergic development.     

Molecular and functional analysis of a novel neuronal vesicular glutamate transporter.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Packaging and storage of glutamate into glutamatergic neuronal vesicles requires ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. VGLUT1, the first identified vesicular glutamate transporter, is only expressed in a subset of glutamatergic neurons. We report here the molecular cloning and functional characterization of a novel glutamate transporter, VGLUT2, from mouse brain. VGLUT2 has all major functional characteristics of a synaptic vesicle glutamate transporter, including ATP dependence, chloride stimulation, substrate specificity, and substrate affinity. It has 75 and 79% amino acid identity with human and rat VGLUT1, respectively. However, expression patterns of VGLUT2 in brain are different from that of VGLUT1. In addition, VGLUT2 activity is dependent on both membrane potential and pH gradient of the electrochemical proton gradient, whereas VGLUT1 is primarily dependent on only membrane potential. The presence of VGLUT2 in brain regions lacking VGLUT1 suggests that the two isoforms together play an important role in vesicular glutamate transport in glutamatergic neurons.     

Cholinergic interneurons mediate fast VGluT3-dependent glutamatergic transmission in the striatum

The neurotransmitter glutamate is released by excitatory projection neurons throughout the brain. However, non-glutamatergic cells, including cholinergic and monoaminergic neurons, express markers that suggest that they are also capable of vesicular glutamate release. Striatal cholinergic interneurons (CINs) express the Type-3 vesicular glutamate transporter (VGluT3), although whether they form functional glutamatergic synapses is unclear. To examine this possibility, we utilized mice expressing Cre-recombinase under control of the endogenous choline acetyltransferase locus and conditionally expressed light-activated Channelrhodopsin2 in CINs. Optical stimulation evoked action potentials in CINs and produced postsynaptic responses in medium spiny neurons that were blocked by glutamate receptor antagonists. CIN-mediated glutamatergic responses exhibited a large contribution of NMDA-type glutamate receptors, distinguishing them from corticostriatal inputs. CIN-mediated glutamatergic responses were insensitive to antagonists of acetylcholine receptors and were not seen in mice lacking VGluT3. Our results indicate that CINs are capable of mediating fast glutamatergic transmission, suggesting a new role for these cells in regulating striatal activity.     

From glutamate co-release to vesicular synergy: vesicular glutamate transporters

Recent data indicate that 'classical' neurotransmitters can also act as co-transmitters. This notion has been strengthened by the demonstration that three vesicular glutamate transporters (vesicular glutamate transporter 1 (VGLUT1), VGLUT2 and VGLUT3) are present in central monoamine, acetylcholine and GABA neurons, as well as in primarily glutamatergic neurons. Thus, intriguing questions are raised about the morphological and functional organization of neuronal systems endowed with such a dual signalling capacity. In addition to glutamate co-release, vesicular synergy - a process leading to enhanced packaging of the 'primary' transmitter - is increasingly recognized as a major property of the glutamatergic co-phenotype. The behavioural relevance of this co-phenotype is presently the focus of considerable interest.     

Activity of medullary respiratory neurons during ventilator-induced apnea in sleep and wakefulness

Mechanical ventilation of cats in sleep andwakefulness causes apnea, often within two to three cycles of theventilator. We recorded 137 medullary respiratory neurons in four adultcats during eupnea and during apnea caused by mechanical ventilation. We hypothesized that the residual activity of respiratory neurons during apnea might reveal its cause(s). The results showed that residual activity depended on 1) theamount of nonrespiratory inputs to the cell (cells with morenonrespiratory inputs had greater amounts of residual activity);2) the cell type (expiratory cellshad more residual activity than inspiratory cells); and 3) the state of consciousness (moreresidual activity in wakefulness and rapid-eye-movement sleep than innon-rapid-eye-movement sleep). None of the cells showed an activationduring ventilation that could explain the apnea. Residual activity ofapproximately one-half of the cells was modulated in phase with theventilator. The strength of this modulation was quantified by using aneffect-size statistic and was found to be weak. The patterns ofmodulation did not support the idea that mechanoreceptors excite somerespiratory cells that, in turn, inhibit others. Indeed, most cells,inspiratory and expiratory, discharged during the deflation-inflationtransition of ventilation. Residual activity failed to reveal the causeof apnea but showed that during apnea respiratory neurons act as ifthey were disinhibited and disfacilitated.

     

Methamphetamine rapidly decreases vesicular dopamine uptake

Vesicular sequestration is important in the regulation of cytoplasmic concentrations of monoamines such as dopamine. Moreover, recent evidence suggests that increases in cytoplasmic dopamine levels, perhaps attributable to changes in vesicular monoamine transporter function, contribute to methamphetamine-induced dopaminergic deficits. Hence, we examined whether striatal vesicular uptake is altered following methamphetamine treatment. Multiple administrations of methamphetamine rapidly (within 1 h) decreased vesicular dopamine uptake and dihydrotetrabenazine binding, an effect that (a) persisted at least 24 h, (b) was associated with dopamine and not serotonin neurons, and (c) was unrelated to residual drug introduced by the original methamphetamine treatment. These data suggest that methamphetamine rapidly decreases vesicular monoamine transporter function in dopaminergic neurons, a phenomenon that may be associated with the long-term damage caused by this stimulant.     

Identification of maize silicon influx transporters

Maize (Zea mays L.) shows a high accumulation of silicon (Si),but transporters involved in the uptake and distribution havenot been identified. In the present study, we isolated two genes(ZmLsi1 and ZmLsi6), which are homologous to rice influx Sitransporter OsLsi1. Heterologous expression in Xenopus laevisoocytes showed that both ZmLsi1 and ZmLsi6 are permeable tosilicic acid. ZmLsi1 was mainly expressed in the roots. By contrast,ZmLsi6 was expressed more in the leaf sheaths and blades. Differentfrom OsLsi1, the expression level of both ZmLsi1 and ZmLsi6was unaffected by Si supply. Immunostaining showed that ZmLsi1was localized on the plasma membrane of the distal side of rootepidermal and hypodermal cells in the seminal and crown roots,and also in cortex cells in lateral roots. In the shoots, ZmLsi6was found in the xylem parenchyma cells that are adjacent tothe vessels in both leaf sheaths and leaf blades. ZmLsi6 inthe leaf sheaths and blades also exhibited polar localizationon the side facing towards the vessel. Taken together, it canbe concluded that ZmLsi1 is an influx transporter of Si, whichis responsible for the transport of Si from the external solutionto the root cells and that ZmLsi6 mainly functions as a Si transporterfor xylem unloading.     

Mice with selective elimination of striatal acetylcholine release are lean,show altered energy homeostasis and changed sleep/wake cycle

Cholinergic neurons are known to regulate striatal circuits; however, striatal‐dependent physiological outcomes influenced by acetylcholine (ACh) are still poorly under;?>stood. Here, we used vesicular acetylcholine transporter (VAChT)^{D2‐Cre‐flox/flox} mice, in which we selectively ablated the vesicular acetylcholine transporter in the striatum to dissect the specific roles of striatal ACh in metabolic homeostasis. We report that VAChT^{D2‐Cre‐flox/flox} mice are lean at a young age and maintain this lean phenotype with time. The reduced body weight observed in these mutant mice is not attributable to reduced food intake or to a decrease in growth rate. In addition, changed activity could not completely explain the lean phenotype, as only young VAChT^{D2‐Cre‐flox/flox} mice showed increased physical activity. Interestingly, VAChT^{D2‐Cre‐flox/flox} mice show several metabolic changes, including increased plasma levels of insulin and leptin. They also show increased periods of wakefulness when compared with littermate controls. Taken together, our data suggest that striatal ACh has an important role in the modulation of metabolism and highlight the importance of striatum cholinergic tone in the regulation of energy expenditure. These new insights on how cholinergic neurons influence homeostasis open new avenues for the search of drug targets to treat obesity.