RESUMO
We studied the distribution and cellular localization of Na(+)-coupled neutral amino acid transporter 2, a member of the system A family of amino acid transporters, in the rat and human cerebral cortex using immunocytochemical methods. Na(+)-coupled neutral amino acid transporter 2-positive neurons were pyramidal and non-pyramidal, and Na(+)-coupled neutral amino acid transporter 2/GABA double-labeling studies revealed that Na(+)-coupled neutral amino acid transporter 2 was highly expressed by GABAergic neurons. Double-labeling studies with the synaptophysin indicated that rare axon terminals express Na(+)-coupled neutral amino acid transporter 2. Na(+)-coupled neutral amino acid transporter 2-immunoreactivity was also found in astrocytes, leptomeninges, ependymal cells and choroid plexus. Electron microscopy showed robust Na(+)-coupled neutral amino acid transporter 2-immunoreactivity in the somato-dendritic compartment of neurons and in glial processes, but, as in the case of double-labeling studies, failed to reveal Na(+)-coupled neutral amino acid transporter 2-immunoreactivity in terminals. To rule out the possibility that the absence of Na(+)-coupled neutral amino acid transporter 1- and Na(+)-coupled neutral amino acid transporter 2-positive terminals was due to insufficient antigen detection, we evaluated Na(+)-coupled neutral amino acid transporter 1/synaptophysin and Na(+)-coupled neutral amino acid transporter 2/synaptophysin coexpression using non-standard immunocytochemical procedures and found that Na(+)-coupled neutral amino acid transporter 1 and Na(+)-coupled neutral amino acid transporter 2+ terminals were rare in all conditions. These findings indicate that Na(+)-coupled neutral amino acid transporter 1 and Na(+)-coupled neutral amino acid transporter 2 are virtually absent in cortical terminals, and suggest that they do not contribute significantly to replenishing the Glu and GABA transmitter pools through the glutamate-glutamine cycle. The strong expression of Na(+)-coupled neutral amino acid transporter 2 in the somato-dendritic compartment and in non-neuronal elements that are integral parts of the blood-brain and brain-cerebrospinal fluid barrier suggests that Na(+)-coupled neutral amino acid transporter 2 plays a role in regulating the levels of Gln and other amino acids in the metabolic compartment of cortical neurons.
Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Córtex Cerebral/citologia , Neurônios/metabolismo , Animais , Western Blotting/métodos , Proteínas de Ligação ao Cálcio/metabolismo , Córtex Cerebral/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica/métodos , Masculino , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Pessoa de Meia-Idade , Proteínas Musculares/metabolismo , Neurônios/ultraestrutura , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/metabolismoRESUMO
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.
Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/análise , Proteínas de Membrana Transportadoras , Receptores Purinérgicos P2/análise , Medula Espinal/metabolismo , Proteínas de Transporte Vesicular , Animais , Química Encefálica/fisiologia , Proteínas de Transporte/biossíntese , Terminações Pré-Sinápticas , Ratos , Ratos Wistar , Receptores Purinérgicos P2/biossíntese , Receptores Purinérgicos P2X7 , Medula Espinal/química , Proteína Vesicular 1 de Transporte de Glutamato , Proteína Vesicular 2 de Transporte de GlutamatoRESUMO
Cholinergic neurotransmission depends upon the regulated release of acetylcholine. This requires the loading of acetylcholine into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Here, we identify point mutants in Caenorhabditis elegans that map to highly conserved regions of the VAChT gene of Caenorhabditis elegans (CeVAChT) (unc-17) and exhibit behavioral phenotypes consistent with a reduction in vesicular transport activity and neurosecretion. Several of these mutants express normal amounts of VAChT protein and exhibit appropriate targeting of VAChT to synaptic vesicles. By site-directed mutagenesis, we have replaced the conserved amino acid residues found in human VAChT with the mutated residue in CeVAChT and stably expressed these cDNAs in PC-12 cells. These mutants display selective defects in initial acetylcholine transport velocity (K(m)), with values ranging from 2- to 8-fold lower than that of the wild-type. One of these mutants has lost its specific interaction with vesamicol, a selective inhibitor of VAChT, and displays vesamicol-insensitive uptake of acetylcholine. The relative order of behavioral severity of the CeVAChT point mutants is identical to the order of reduced affinity of VAChT for acetylcholine in vitro. This indicates that specific structural changes in VAChT translate into specific alterations in the intrinsic parameters of transport and in the storage and synaptic release of acetylcholine in vivo.
Assuntos
Acetilcolina/metabolismo , Receptores Colinérgicos/química , Vesículas Sinápticas/química , Sequência de Aminoácidos , Animais , Transporte Biológico , Caenorhabditis elegans , Dados de Sequência Molecular , Células PC12 , Piperidinas/metabolismo , Mutação Puntual , Ratos , Receptores Colinérgicos/fisiologiaRESUMO
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.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Exocitose , Proteínas de Membrana Transportadoras , Neuropeptídeos , Organelas/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Aminas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Transporte Proteico , Prótons , Proteínas Vesiculares de Transporte de Acetilcolina , Proteínas Vesiculares de Transporte de Aminas BiogênicasRESUMO
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.
Assuntos
Sistema A de Transporte de Aminoácidos , Aminoácidos/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Sódio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cerebelo/metabolismo , Clonagem Molecular , Primers do DNA , DNA Complementar , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Nitrogênio/metabolismo , Ratos , Ratos Sprague-Dawley , XenopusRESUMO
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.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos , Proteínas de Transporte/genética , Ácidos Aminoisobutíricos/farmacologia , Animais , Células COS , Proteínas de Transporte/química , Células Cultivadas , Cerebelo/metabolismo , Clonagem Molecular , Imunofluorescência , Concentração de Íons de Hidrogênio , Hibridização In Situ , Cinética , Proteínas de Membrana/química , Dados de Sequência Molecular , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
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.
Assuntos
Proteínas de Transporte/análise , Colina O-Acetiltransferase/análise , Transtornos Cognitivos/enzimologia , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso/análise , Neurônios/enzimologia , Substância Inominada/enzimologia , Proteínas de Transporte Vesicular , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Biomarcadores , Transtornos Cognitivos/patologia , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Masculino , Substância Inominada/citologia , Proteínas Vesiculares de Transporte de AcetilcolinaAssuntos
Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Neuropeptídeos , Proteínas de Transporte Vesicular , Acetilcolina/metabolismo , Animais , Transporte Biológico Ativo , Proteínas de Transporte/análise , Técnicas de Cultura de Células/métodos , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Imuno-Histoquímica/métodos , Cinética , Glicoproteínas de Membrana/análise , Neurotransmissores/metabolismo , Ensaio Radioligante/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Serotonina/metabolismo , Transfecção/métodos , Trítio , Proteínas Vesiculares de Transporte de Acetilcolina , Proteínas Vesiculares de Transporte de Aminas BiogênicasRESUMO
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.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Neuropeptídeos , Piperidinas/farmacocinética , Vesículas Sinápticas/fisiologia , Proteínas de Transporte Vesicular , Acetilcolina/metabolismo , Neoplasias das Glândulas Suprarrenais , Animais , Sítios de Ligação , Transporte Biológico , Humanos , Cinética , Fármacos Neuromusculares Despolarizantes/farmacocinética , Células PC12 , Feocromocitoma , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas Vesiculares de Transporte de Acetilcolina , Proteínas Vesiculares de Transporte de Aminas Biogênicas , Proteínas Vesiculares de Transporte de MonoaminaRESUMO
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.
Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Transporte/química , Proteínas de Membrana Transportadoras , Neurônios/metabolismo , Organelas/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Acetilcolina/metabolismo , Animais , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Citoplasma/metabolismo , Humanos , Microscopia Imunoeletrônica , Organelas/ultraestrutura , Células PC12 , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Transdução de Sinais , Vesículas Sinápticas/ultraestrutura , Transfecção , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
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.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurotransmissores/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Bovinos , Regulação da Expressão Gênica , Proteínas de Helminto/genética , Proteínas de Helminto/fisiologia , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Degeneração Neural/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/genética , Conformação Proteica , Ratos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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.
Assuntos
Acetilcolina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras , Proteínas de Transporte Vesicular , Neoplasias das Glândulas Suprarrenais , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Proteínas de Transporte/biossíntese , Citosol/metabolismo , Humanos , Cinética , Fármacos Neuromusculares Despolarizantes/metabolismo , Fármacos Neuromusculares Despolarizantes/farmacologia , Células PC12 , Feocromocitoma , Piperidinas/metabolismo , Piperidinas/farmacologia , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tetrabenazina/metabolismo , Tetrabenazina/farmacologia , Transfecção , Proteínas Vesiculares de Transporte de AcetilcolinaAssuntos
Proteínas de Transporte/biossíntese , Proteínas de Membrana Transportadoras , Proteínas de Transporte Vesicular , Acetilcolina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Clonagem Molecular , Glicosilação , Humanos , Mamíferos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Torpedo , Transfecção , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
The gene encoding the vesicular acetylcholine transporter (VAChT) has recently been localized within the first intron of the gene encoding choline acetyltransferase (ChAT) and is in the same transcriptional orientation. These two genes, whose products are required for the expression of the cholinergic phenotype, could therefore be coregulated. We thus tested the effects on VAChT gene expression of the cholinergic differentiation factor/leukemia inhibitory factor and retinoic acid, both of which induce ChAT activity and increase ChAT mRNA levels in cultured sympathetic neurons. These factors increased both the number of binding sites for vesamicol, a specific ligand of VAChT, and VAChT immunoreactivity. This increase in the number of VAChT molecules resulted from an increase in the amount of VAChT mRNA, as assessed by reverse transcription-PCR and which paralleled that of ChAT mRNAs. These data suggest a functional role for ChAT and VAChT gene organization and are consistent with the existence of a coregulatory mechanism for the embedded ChAT and VAChT genes.
Assuntos
Proteínas de Transporte/genética , Colina O-Acetiltransferase/genética , Regulação da Expressão Gênica , Interleucina-6 , Proteínas de Membrana Transportadoras , Proteínas de Transporte Vesicular , Animais , Sequência de Bases , Western Blotting , Proteínas de Transporte/metabolismo , Células Cultivadas , Colina O-Acetiltransferase/metabolismo , Gânglios Simpáticos/citologia , Gânglios Simpáticos/metabolismo , Inibidores do Crescimento/farmacologia , Fator Inibidor de Leucemia , Linfocinas/farmacologia , Sondas Moleculares/genética , Dados de Sequência Molecular , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos , Tretinoína/farmacologia , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
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.
Assuntos
Acetilcolina/metabolismo , Proteínas de Transporte/genética , Fibras Colinérgicas/metabolismo , Proteínas de Membrana Transportadoras , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Células Cultivadas , Colina O-Acetiltransferase/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 10 , DNA Complementar , Humanos , Dados de Sequência Molecular , Células PC12 , Conformação Proteica , RNA Mensageiro/metabolismo , Ratos , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/química , Proteínas de Transporte/química , Proteínas de Helminto/química , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Neuropeptídeos , Piperidinas/metabolismo , Receptores Colinérgicos/genética , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Clonagem Molecular , Glicoproteínas/química , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Colinérgicos/química , Receptores Colinérgicos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Torpedo/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina , Proteínas Vesiculares de Transporte de Aminas Biogênicas , Proteínas Vesiculares de Transporte de MonoaminaRESUMO
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.
Assuntos
Química Encefálica , Proteínas de Transporte/análise , Gânglios/química , Proteínas de Membrana Transportadoras , Medula Espinal/química , Proteínas de Transporte Vesicular , Animais , Biomarcadores , Proteínas de Transporte/genética , Masculino , RNA Mensageiro/análise , Ratos , Ratos Wistar , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
Gangliosides were isolated from four subcellular fractions of the electric organ of Torpedo marmorata: synaptosomes, presynaptic membranes, postsynaptic membranes, and synaptic vesicle membranes. This exploited a principal advantage offered by this tissue: facile separation of pre-and postsynaptic elements. Total ganglioside concentration in presynaptic membranes was approximately twice that of synaptosomes and 15 times that of postsynaptic membranes (47.7, 24.4, and 3.21 micrograms of lipid sialic acid per mg protein, respectively). Synaptic vesicle membranes had the highest overall concentration (78.9) relative to protein, but a concentration approximately comparable to that of presynaptic membranes when expressed relative to phospholipid. The thin-layer patterns of these two fractions were similar, both in terms of total pattern and the specific pattern of gangliotetraose structures as revealed by overlay with cholera toxin B subunit; these were notable for the paucity of monosialo structures and the virtual absence of GM1. Postsynaptic membranes, on the other hand, had a significantly higher content of monosialogangliosides including the presence of GM1. The synaptosomal pattern resembled that of the presynaptic membranes and synaptic vesicles. Thus, a clear difference in ganglioside pattern could be discerned between the pre- and postsynaptic elements of the electric organ.
Assuntos
Órgão Elétrico/ultraestrutura , Gangliosídeos/análise , Frações Subcelulares/química , Membranas Sinápticas/química , Torpedo , Animais , Cromatografia em Camada Fina , Gangliosídeo G(M1)/análise , Gangliosídeo G(M3)/análise , Ácido N-Acetilneuramínico , Ácidos Siálicos/análise , Vesículas Sinápticas/química , Sinaptossomos/químicaRESUMO
Using isolated cholinergic synaptosomes prepared from Torpedo electric organ, we studied the effects of N,N'-dicyclohexylcarbodiimide (DCCD) on acetylcholine (ACh) synthesis, compartmentation, and release after stimulation. Whereas ACh synthesis was unchanged, ACh compartmentation inside synaptosomes was affected by the presence of DCCD. In resting conditions, the uptake into the synaptic vesicle pool of newly synthesized ACh (i.e., [14C]ACh synthesized in the presence of the drug) was progressively and markedly inhibited as the duration of DCCD preincubation was increased, whereas compartmentation of endogenous ACh was unchanged in the presence of DCCD. After stimulation, the release of endogenous ACh from DCCD-treated synaptosomes was similar to that of control, in contrast to the release of [14C]ACh, which was markedly inhibited. This inhibition was observed whatever the conditions of stimulation used (gramicidin D, calcium ionophore A23187, or KCl depolarization). The study of the compartmentation of [14C]ACh during stimulation revealed a transfer of highly labeled ACh from the free to the bound ACh compartment in the presence of DCCD, suggesting the existence of several ACh subcompartments within the free and bound ACh pools. The present results are discussed in comparison with the previously reported effects of vesamicol (AH5183) on ACh compartmentation and release.