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Wasting of lean tissue as a consequence of metabolic acidosis is a serious problem in patients with chronic renal failure. A possible contributor is inhibition by low pH of the System A (SNAT2) transporter, which carries the amino acid L-glutamine (L-Gln) into muscle cells. The aim of this study was to determine the effect of selective SNAT2 inhibition on intracellular amino acid profiles and amino acid-dependent signaling through mammalian target of rapamycin in L6 skeletal muscle cells. Inhibition of SNAT2 with the selective competitive substrate methylaminoisobutyrate, metabolic acidosis (pH 7.1), or silencing SNAT2 expression with small interfering RNA all depleted intracellular L-Gln. SNAT2 inhibition also indirectly depleted other amino acids whose intracellular concentrations are maintained by the L-Gln gradient across the plasma membrane, notably the anabolic amino acid L-leucine. Consequently, SNAT2 inhibition strongly impaired signaling through mammalian target of rapamycin to ribosomal protein S6 kinase, ribosomal protein S6, and 4E-BP1, leading to impairment of protein synthesis comparable with that induced by rapamycin. It is concluded that even though SNAT2 is only one of several L-Gln transporters in muscle, it may determine intracellular anabolic amino acid levels, regulating the amino acid signaling that affects protein mass, nucleotide/nucleic acid metabolism, and cell growth.

Presynaptic P2X(7) receptors are thought to play a role in the modulation of transmitter release and have been localised to terminals with the location and morphology typical of excitatory boutons. To test the hypothesis that this receptor is preferentially associated with excitatory terminals we combined immunohistochemistry for the P2X(7) receptor subunit (P2X(7)R) with that for two vesicular glutamate transporters (VGLUT1 and VGLUT2) in the rat CNS. This confirmed that P2X(7)R immunoreactivity (IR) is present in glutamatergic terminals; however, whether it was co-localised with VGLUT1-IR or VGLUT2-IR depended on the CNS region examined. In the spinal cord, P2X(7)R-IR co-localised with VGLUT2-IR. In the brainstem, co-localisation of P2X(7)R-IR with VGLUT2-IR was widespread, but co-localisation with VGLUT1-IR was seen only in the external cuneate nucleus and spinocerebellar tract region of the ventral medulla. In the cerebellum, P2X(7)R-IR co-localised with both VGLUT1 and VGLUT2-IR in the granular layer. In the hippocampus it was co-localised only with VGLUT1-IR, including in the polymorphic layer of the dentate gyrus and the substantia radiatum of the CA3 region. In other forebrain areas, P2X(7)R-IR co-localised with VGLUT1-IR throughout the amygdala, caudate putamen, striatum, reticular thalamic nucleus and cortex and with VGLUT2-IR in the dorsal lateral geniculate nucleus, amygdala and hypothalamus. Dual labelling studies performed using markers for cholinergic, monoaminergic, GABAergic and glycinergic terminals indicated that in certain brainstem and spinal cord nuclei the P2X(7)R is also expressed by subpopulations of cholinergic and GABAergic/glycinergic terminals. These data support our previous hypothesis that the P2X(7)R may play a role in modulating glutamate release in functionally different systems throughout the CNS but further suggest a role in modulating release of inhibitory transmitters in some regions.

Glutamate transport into synaptic vesicles is a prerequisite for its regulated neurosecretion. Here we functionally identify a second isoform of the vesicular glutamate transporter (VGLUT2) that was previously identified as a plasma membrane Na+-dependent inorganic phosphate transporter (differentiation-associated Na+/P(I) transporter). Studies using intracellular vesicles from transiently transfected PC12 cells indicate that uptake by VGLUT2 is highly selective for glutamate, is H+ dependent, and requires Cl- ion. Both the vesicular membrane potential (Deltapsi) and the proton gradient (DeltapH) are important driving forces for vesicular glutamate accumulation under physiological Cl- concentrations. Using an antibody specific for VGLUT2, we also find that this protein is enriched on synaptic vesicles and selective for a distinct class of glutamatergic nerve terminals. The pathway-specific, complementary expression of two different vesicular glutamate transporters suggests functional diversity in the regulation of vesicular release at excitatory synapses. Together, the two isoforms may account for the uptake of glutamate by synaptic vesicles from all central glutamatergic neurons.

Vesicular transporters are responsible for the loading of neurotransmitters into specialized secretory organelles in neurons and neuroendocrine cells to make them available for regulated neurosecretion. The exocytotic release of neurotransmitter therefore depends on the functional activity of the vesicular transporters and their efficient sorting to these secretory organelles. Molecular analysis of vesicular transport proteins has revealed important information regarding structural domains responsible for their functional properties, including substrate specificity and trafficking to various classes of secretory vesicles. These studies have established the existence of an important functional relationship between transporter activity and presynaptic quantal neurosecretion.

A cDNA clone encoding a plasma membrane alanine-preferring transporter (SAT2) has been isolated from glutamatergic neurons in culture and represents the second member of the system A family of neutral amino acid transporters. SAT2 displays a widespread distribution and is expressed in most tissues, including heart, adrenal gland, skeletal muscle, stomach, fat, brain, spinal cord, colon, and lung, with lower levels detected in spleen. No signal is detected in liver or testis. In the central nervous system, SAT2 is expressed in neurons. SAT2 is significantly up-regulated during differentiation of cerebellar granule cells and is absent from astrocytes in primary culture. The functional properties of SAT2, examined using transfected fibroblasts and in cRNA-injected voltage-clamped Xenopus oocytes, show that small aliphatic neutral amino acids are preferred substrates and that transport is voltage- and Na(+)-dependent (1:1 stoichiometry), pH-sensitive, and inhibited by alpha-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of system A. Kinetic analyses of alanine and MeAIB uptake by SAT2 are saturable, with Michaelis constants (K(m)) of 200-500 microm. In addition to its ubiquitous role as a substrate for oxidative metabolism and a major vehicle of nitrogen transport, SAT2 may provide alanine to function as the amino group donor to alpha-ketoglutarate to provide an alternative source for neurotransmitter synthesis in glutamatergic neurons.

Glutamine is the preferred precursor for the neurotransmitter pool of glutamate, the major excitatory transmitter in the mammalian central nervous system. We have isolated a complementary DNA clone (designated GlnT) encoding a plasma membrane glutamine transporter from glutamatergic neurons in culture, and its properties have been examined using the T7 vaccinia system in fibroblasts. When GlnT is transfected into CV-1 cells, L-glutamine is the preferred substrate. Transport is Na(+)-dependent and inhibited by alpha-methylaminoisobutyric acid, a specific inhibitor of neutral amino acid transport system A. Kinetic analysis of glutamine uptake by GlnT is saturable, with a Michaelis constant (K(m)) of 489 +/- 88 microM at pH 7.4. Glutamine uptake mediated by GlnT is pH-sensitive with a 5-fold greater efficiency of uptake at pH 8.2 than at pH 6.6. Only the maximal velocity of transport increases without a significant change in K(m). The distribution of GlnT mRNA and protein in the central nervous system is widespread and is expressed on neurons that use glutamate as their neurotransmitter. In cultured cerebellar granule cells, GlnT is expressed only on neurons and is absent from astrocytes. GlnT expression increases concomitantly with the morphologic and functional differentiation of these cells in vitro, consistent with its role of supplying glutamatergic neurons with their neurotransmitter precursor. GlnT is the first member of the system A family of neutral amino acid transporters with 11 putative membrane-spanning domains and is a potential target to modulate presynaptic glutamatergic function.

Immunocytochemistry for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) was used to examine the expression of these linked cholinergic markers in human basal forebrain, including cases with early stages of Alzheimer's disease (AD). Previous neurochemical studies have measured decreased ChAT activity in terminal fields, but little change or even increased levels of VAChT. To determine total cholinergic neuron numbers in the nucleus basalis of Meynert (nbM), stereologic methods were applied to tissue derived from three groups of individuals with varying levels of cognition: no cognitive impairment (NCI), mild cognitive impairment (MCI), and early-stage Alzheimer's disease (AD). Both markers were expressed robustly in nucleus basalis neurons and across all three groups. On average, there was no significant difference between the number of ChAT- (210,000) and VAChT- (174, 000) immunopositive neurons in the nbM per hemisphere in NCI cases for which the biological variation was calculated to be 17%. There was approximately a 15% nonsignificant reduction in the number of cholinergic neurons in the nbM in the AD cases with no decline in MCI cases. The number of ChAT- and VAChT-immunopositive neurons was shown to correlate significantly with the severity of dementia determined by scores on the Mini-Mental State Examination, but showed no relationship to apolipoprotein E allele status, age, gender, education, or postmortem interval when all clinical groups were combined or evaluated separately. These data suggest that cholinergic neurons, and the coexpression of ChAT and VAChT, are relatively preserved in early stages of AD., (Copyright 1999 Wiley-Liss, Inc.)

The human homologue of the vesicular acetylcholine transporter (hVAChT) and the neuronal isoform of the vesicular monoamine transporter (hVMAT2) are differentially targeted to two populations of regulated secretory organelles when expressed in PC12 cells. Western blot analysis of subcellular fractions from sucrose equilibrium density gradients and glycerol velocity gradients of homogenates from stably transfected cells revealed hVAChT immunoreactivity in fractions that contain synaptophysin, a marker of synaptic vesicles, while hVMAT2 immunoreactivity was confined to heavy fractions containing chromogranin B, a marker of large dense core vesicles. In cells treated with nerve growth factor, hVAChT immunoreactivity alone co-localized with synaptophysin and was abundantly expressed on synaptic vesicle clusters. Chimeras between hVMAT2 and hVAChT were utilized to identify the domain of hVAChT required for its expression on synaptic vesicles and which would shift the expression of hVMAT2 from large dense core vesicles to synaptic vesicles. Biochemical, immunocytochemical, and electron microscopic analyses revealed that a chimera in which the cytoplasmic tail of hVMAT2 was replaced with hVAChT sequences was now preferentially targeted to synaptic vesicles. In addition, hVAChT expression on synaptic vesicles was nearly abolished when the hVMAT2 cytoplasmic tail was present. Thus, structural information resides within the terminal cytoplasmic domain of VAChT, which specifically targets it to synaptic vesicles.

Chimeras between the human vesicular acetylcholine transporter (hVAChT) and the neuronal isoform of the human vesicular monoamine transporter (hVMAT2) have been constructed and stably expressed in a rat pheochromocytoma cell line (PC12) in an effort to identify cholinergic-specific domains of VAChT. Examination of the transport properties of a chimera in which the N-terminal portion (up to putative transmembrane domain II and including the lumenal glycosylated loop) of hVAChT was replaced with hVMAT2 sequences (2/V@NheI) revealed that its apparent affinity for acetylcholine (ACh) was reduced approximately seven-fold compared to wild-type. However, the affinity of this chimera for vesamicol did not significantly differ from hVAChT. Similarly, the 2/V@NheI chimera retained its preferential targeting to the small synaptic-like vesicles found in PC12 cells in agreement with our recently reported observations that the synaptic vesicle targeting domain resides in the cytoplasmic tail of VAChT.

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.

The characteristics of ATP-dependent transport of acetylcholine (ACh) in homogenates of pheochromocytoma (PC-12) cells stably transfected with the human vesicular acetylcholine transporter (VAChT) cDNA are described. The human VAChT protein was abundantly expressed in this line and appeared as a diffuse band with a molecular mass of approximately 75 kDa on Western blots. Vesicular [3H]ACh accumulation increased approximately 20 times over levels attained by the endogenous rat VAChT, expressed at low levels in control PC-12 cells. The transport of [3H]ACh by human VAChT was dependent upon the addition of exogenous ATP at 37 degrees C. Uptake was abolished by low temperature (4 degrees C), the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (2.5 microM) and bafilomycin A1 (1 microM), a specific inhibitor of the vesicular H+-ATPase. The kinetics of [3H]ACh uptake by human VAChT were saturable, exhibiting an apparent Km of 0.97 +/- 0.1 mM and Vmax of 0.58 +/- 0.04 nmol/min/mg. Maximal steady-state levels of vesicular [3H]ACh accumulation were directly proportional to the concentration of substrate present in the medium with saturation occurring at approximately 4 mM. Uptake was stereospecifically inhibited by L-vesamicol with an IC50 of 14.7 +/- 1.5 nM. The apparent affinity (Kd) of [3H]vesamicol for human VAChT was 4.1 +/- 0.5 nM, and the Bmax was 8.9 +/- 0.6 pmol/mg. The turnover (Vmax/Bmax) of the human VAChT was approximately 65/min. This expression system should prove useful for the structure/function analysis of VAChT.

Immunohistochemical visualization of the rat vesicular acetylcholine transporter (VAChT) in cholinergic neurons and nerve terminals has been compared to that for choline acetyltransferase (ChAT), heretofore the most specific marker for cholinergic neurons. VAChT-positive cell bodies were visualized in cerebral cortex, basal forebrain, medial habenula, striatum, brain stem, and spinal cord by using a polyclonal anti-VAChT antiserum. VAChT-immuno-reactive fibers and terminals were also visualized in these regions and in hippocampus, at neuromuscular junctions within skeletal muscle, and in sympathetic and parasympathetic autonomic ganglia and target tissues. Cholinergic nerve terminals contain more VAChT than ChAT immunoreactivity after routine fixation, consistent with a concentration of VAChT within terminal neuronal arborizations in which secretory vesicles are clustered. These include VAChT-positive terminals of the median eminence or the hypothalamus, not observed with ChAT antiserum after routine fixation. Subcellular localization of VAChT in specific organelles in neuronal cells was examined by immunoelectron microscopy in a rat neuronal cell line (PC 12-c4) expressing VAChT as well as the endocrine and neuronal forms of the vesicular monoamine transporters (VMAT1 and VMAT2). VAChT is targeted to small synaptic vesicles, while VMAT1 is found mainly but not exclusively on large dense-core vesicles. VMAT2 is found on large dense-core vesicles but not on the small synaptic vesicles that contain VAChT in PC12-c4 cells, despite the presence of VMAT2 immunoreactivity in central and peripheral nerve terminals known to contain monoamines in small synaptic vesicles. Thus, VAChT and VMAT2 may be specific markers for "cholinergic" and "adrenergic" small synaptic vesicles, with the latter not expressed in nonstimulated neuronally differentiated PC12-c4 cells.

The cholinergic synapse has long been a model for biochemical studies of neurotransmission. The molecules that are responsible for synaptic transmission are being identified rapidly. The vesicular transporter for ACh, which is responsible for the concentration of ACh within synaptic vesicles, has been characterized recently, both at the molecular and functional level. Definitive identification of the cloned gene involved genetics of Caenorhabditis elegans, the specialized Torpedo electromotor system, and expression in mammalian tissue culture. Comparison of the vesicular transporter for ACh with the vesicular transporters for monoamines demonstrates a new gene family. Gene mapping has demonstrated a unique relationship between the genes for the vesicular ACh transporter and for choline acetyltransferase.

The predicted C-terminal dodecapeptide of the human vesicular acetylcholine transporter (VAChT), deduced from the unique open reading frame of the recently cloned human VAChT cDNA, was conjugated through an N-terminal cysteine to keyhole limpet hemocyanin and used as an immunogen to generate polyclonal antihuman VAChT antibodies in rabbits. The distribution of the VAChT antigen in representative regions of the cholinergic nervous system was examined and compared to that of the acetylcholine biosynthetic enzyme choline acetyltransferase (ChAT), a specific marker for cholinergic neurons. VAChT immunoreactivity was localized in cell bodies of neurons in the basal forebrain and ventral horn of the spinal cord, regions in which major cholinergic projection systems to the cerebral cortex and to skeletal muscle, respectively, originate. The primate caudate nucleus contained numerous VAChT-positive interneurons. VAChT immunoreactivity was visualized in both cell bodies and extensive terminals in striatal interneurons, in contrast to formalin-fixed, deparaffinized sections stained for ChAT, in which cell bodies and fibers were stained but nerve terminals were less well visualized than with the VAChT antiserum. VAChT-positive nerve fibers were visualized in routinely immersion-fixed, paraffin-embedded human cerebral cortex, comparable to the density of fibers observed in perfusion-fixed Bouin's-postfixed monkey cerebral cortex. Extensive investment of virtually all principal ganglion cells of thoracic sympathetic ganglia of monkey and human with VAChT-positive nerve terminals was observed. VAChT-positive cell bodies, presumably corresponding to cholinergic sympathetic sudomotor neurons, were a significant fraction of the total principal cell population in monkey and human thoracic sympathetic ganglia.

The vesicular acetylcholine transporter (VAChT) has been identified and characterized based on the acquisition of high affinity vesamicol binding and proton-dependent, vesamicol-sensitive acetylcholine accumulation by a fibroblast cell line transfected with a clone from a rat pheochromocytoma cDNA library encoding this protein. The distribution of VAChT mRNA coincides with that reported for choline acetyltransferase (ChAT), the enzyme required for acetylcholine biosynthesis, in the peripheral and central cholinergic nervous systems. A human VAChT cDNA was used to localize the VAChT gene to chromosome 10q11.2, which is also the location of the ChAT gene. The entire sequence of the human VAChT cDNA is contained uninterrupted within the first intron of the ChAT gene locus. Transcription of VAChT and ChAT mRNA from the same or contiguous promoters within a single regulatory locus provides a previously undescribed genetic mechanism for coordinate regulation of two proteins whose expression is required to establish a mammalian neuronal phenotype.

Complementary DNA clones corresponding to a messenger RNA encoding a 56 kDa polypeptide have been obtained from Torpedo marmorata and Torpedo ocellata electric lobe libraries, by homology screening with a probe obtained from the putative acetylcholine transporter from the nematode Caenorhabditis elegans. The Torpedo proteins display approximately 50% overall identity to the C. elegans unc-17 protein and 43% identity to the two vesicle monoamine transporters (VMAT1 and VMAT2). This family of proteins is highly conserved within 12 domains which potentially span the vesicle membrane, with little similarity within the putative intraluminal glycosylated loop and at the N- and C-termini. The approximately 3.0 kb mRNA species is specifically expressed in the brain and highly enriched in the electric lobe of Torpedo. The Torpedo protein, expressed in CV-1 fibroblast cells, possesses a high-affinity binding site for vesamicol (Kd = 6 nM), a drug which blocks in vitro and in vivo acetylcholine accumulation in cholinergic vesicles.

Expression of the acetylcholine biosynthetic enzyme choline acetyltransferase (ChAT), the vesicular acetylcholine transporter (VAChT), and the high-affinity plasma membrane choline transporter uniquely defines the cholinergic phenotype in the mammalian central (CNS) and peripheral (PNS) nervous systems. The distribution of cells expressing the messenger RNA encoding the recently cloned VAChT in the rat CNS and PNS is described here. The pattern of expression of VAChT mRNA is consistent with anatomical, pharmacological, and histochemical information on the distribution of functional cholinergic neurons in the brain and peripheral tissues of the rat. VAChT mRNA-containing cells are present in brain areas, including neocortex and hypothalamus, in which the existence of cholinergic neurons has been the subject of debate. The demonstration that VAChT is a completely adequate marker for cholinergic neurons should allow the systematic delineation of cholinergic synapses in the rat nervous system when antibodies directed to this protein are available.