RESUMEN
Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (-/-) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/- mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/- mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/- mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.
Asunto(s)
Acetilcolina/metabolismo , Conducta Exploratoria/fisiología , Proteínas de Transporte de Membrana/fisiología , Actividad Motora/fisiología , Receptores Muscarínicos/metabolismo , Conducta Espacial/fisiología , Animales , Ansiedad/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Colinérgicos/farmacología , Hemicolinio 3/farmacología , Heterocigoto , Aprendizaje por Laberinto/fisiología , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Muscarínicos/efectos de los fármacos , Prueba de Desempeño de Rotación con Aceleración Constante , Escopolamina/farmacologíaRESUMEN
Plasma membrane transporters remove neurotransmitters from the extracellular space and have been postulated to terminate synaptic activity. Their specific roles in synaptic and nonsynaptic neurotransmission at a cellular level, however, remain unclear. We have determined the subcellular location of the dopamine transporter (DAT) by immunoperoxidase and immunogold electron microscopy, using monoclonal antibodies to both the N-terminus and the second extracellular loop. The two DAT epitopes were found on opposite faces of cellular and intracellular membranes, providing confirmation of the predicted molecular topology of DAT. In the striatum, DAT was localized in the plasma membrane of axons and terminals. Double immunocytochemistry demonstrated DAT colocalization with two other markers of nigrostriatal terminals, tyrosine hydroxylase and D2 dopamine receptors. The latter was thus demonstrated to be an autoreceptor. Labeled striatal terminals formed symmetrical synapses with spines, dendrites, and perikarya. DAT was not identified within any synaptic active zones, however, even using serial section analysis. These results suggest that striatal dopamine reuptake may occur outside of synaptic specializations once dopamine diffuses from the synaptic cleft. In the substantia nigra, DAT appears to be specifically transported into dendrites, where it can be found in smooth endoplasmic reticulum, plasma membrane, and pre- and postsynaptic active zones. These localizations suggest that DAT modulates the intracellular and extracellular dopamine levels of nigral dendrites. Within the perikarya of pars compacta neurons, DAT was localized primarily to rough and smooth endoplasmic reticulum, Golgi complex, and multivesicular bodies, identifying probable sites of synthesis, modification, transport, and degradation.
Asunto(s)
Proteínas Portadoras/metabolismo , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Neuropéptidos , Sustancia Negra/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Autorreceptores/metabolismo , Autorreceptores/fisiología , Axones/metabolismo , Proteínas Portadoras/inmunología , Proteínas Portadoras/fisiología , Membrana Celular/metabolismo , Cuerpo Estriado/ultraestructura , Dendritas/metabolismo , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Epítopos/inmunología , Inmunohistoquímica , Masculino , Glicoproteínas de Membrana/metabolismo , Microscopía Inmunoelectrónica , Ratas , Ratas Sprague-Dawley , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D2/fisiología , Sustancia Negra/ultraestructura , Tirosina 3-Monooxigenasa/metabolismo , Proteínas de Transporte Vesicular de Aminas BiógenasRESUMEN
Nine isoforms of the rat NMDAR1 receptor subunit have been previously identified, of which several have an alternatively spliced N-terminal insert believed to be important in proton sensitivity of the receptor. The cloning of the human homologues of NMDAR1-3b (hNMDA1-1) and NMDAR1-4b (hNMDA1-2), both bearing the insert, is reported here. A monoclonal antibody generated against the N-terminal region of these isoforms showed reactivity with at least two distinct human brain proteins of approximately 115 kDa. This antibody was further characterized by using a series of truncated fusion proteins and splice variants of NMDAR1 demonstrating its specific recognition of an epitope within the 21-amino acid N-terminal insert, encoded by exon 5. Western blot and immunocytochemical studies were performed to examine the expression of the exon 5-containing isoforms of the NMDAR1 subunit in both rat and human brain.
Asunto(s)
Química Encefálica , Clonación Molecular , Exones , Receptores de N-Metil-D-Aspartato/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Western Blotting , Mapeo Epitopo , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , ARN Mensajero/análisis , Ratas , Receptores de N-Metil-D-Aspartato/análisis , Receptores de N-Metil-D-Aspartato/química , Proteínas Recombinantes de Fusión/inmunología , Homología de SecuenciaRESUMEN
Mutations of the presenilin PS1 and PS2 genes are closely linked to aggressive forms of early-onset (< 60 years) familial Alzheimer's disease. A highly specific monoclonal antibody was developed to identify and characterize the native PS1 protein. Western blot analyses revealed a predominant 32-kd immunoreactive polypeptide in a variety of samples, including PC12 cells transfected with human PS1 complementary DNA, brain biopsy specimens from demented patients, and postmortem samples of frontal neocortex from early-onset familial Alzheimer's disease cases (PS1 and PS2), late-onset sporadic Alzheimer's disease cases, and cases of other degenerative disorders. This truncated polypeptide contains the N-terminus of PS1 and appeared unchanged across cases. In 2 early-onset cases linked to missense mutations in the PS1 gene, a PS1 immunoreactive protein (approximately 49 kd) accumulated in the frontal cortex. This protein was similar in size to full-length PS1 protein present in transfected cells overexpressing PS1 complementary DNA, and in lymphocytes from an affected individual with a deletion of exon 9 of the PS1 gene, suggesting that mutations of the PS1 gene peturb the endoproteolytic processing of the protein. Immunohistochemical studies of control brains revealed that PS1 is expressed primarily in neurons, with the protein localized in the soma and dendritic processes. In contrast, PS1 showed striking localization to the neuropathology in early-onset familial Alzheimer's disease and sporadic Alzheimers' disease cases. PS1 immunoreactivity was present in the neuritic component of senile plaques as well as in neurofibrillary tangles. Localization of PS1 immunoreactivity in familial and sporadic Alzheimer's disease suggests that genetically heterogeneous forms of the disease share a common pathophysiology involving PS1 protein.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Proteínas de la Membrana/metabolismo , Adulto , Anciano , Animales , Western Blotting , Encéfalo/metabolismo , Haplorrinos , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Células PC12/metabolismo , Presenilina-1 , Ratas , Distribución Tisular , TransfecciónRESUMEN
The plasma membrane dopamine transporter (DAT) is considered to be a reliable marker of presynaptic dopaminergic terminal loss. Previous in vivo imaging and postmortem binding studies have detected a loss in striatal DAT binding in Parkinson's diseased (PD) brain; however, these techniques have poor spatial resolution and may suffer from nonspecific binding of some ligands. In this study, we use novel highly specific monoclonal antibodies to distinct epitopes of human DAT to quantify and localize the protein. Western blot analysis revealed marked reductions in DAT immunoreactivity in putamen, caudate, and nucleus accumbens of PD brain compared with control cases, and the reductions were significantly correlated to disease duration. Immunohistochemistry revealed DAT-immunoreactive fibers and puncta that were dense throughout the striatum of control brains but that were drastically reduced in putamen of PD brains. Caudate from PD brains showed a significant degree of sparing along the border of the ventricle, and the nucleus accumbens was relatively preserved. An unexpected finding was that discrete islands of DAT immunoreactivity were preserved within the matrix of PD putamen. Thus, immunological analysis of DAT protein provides novel and sensitive means for localizing and quantifying DAT protein in PD and other neurological disorders involving dopaminergic systems.
Asunto(s)
Proteínas Portadoras/análisis , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso/análisis , Enfermedad de Parkinson/patología , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/análisis , Núcleo Caudado/química , Cuerpo Estriado/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Femenino , Humanos , Immunoblotting , Inmunohistoquímica/métodos , Masculino , Persona de Mediana Edad , Putamen/química , Sensibilidad y EspecificidadRESUMEN
Several genes have been implicated in the pathogenesis of early-onset familial Alzheimer's disease. A majority of the autosomal dominant cases are linked to recently identified mutations in the presenilin-1 gene on chromosome 14. The native presenilin-1 protein in primates has not been well characterized, and its precise localization is unknown. We have studied the native presenilin-1 protein in monkey brain and peripheral tissues by using a monoclonal antibody specific for the N-terminal domain of human presenilin-1. Western blots detect polypeptide species of approximately 49 and approximately 32 kDa from COS-7 and PC12 cells transfected with full-length human presenilin-1 cDNA and from in vitro translations of the normal human presenilin-1 mRNA. A 32 kDa polypeptide is detected in monkey peripheral tissues, with the highest expression in testis and lung. In all brain regions the 32 kDa band is the predominant form of presenilin-1, and it is found in particulate subfractions. Light microscopic immunocytochemistry reveals presenilin-1 staining in all brain regions, with the strongest labeling in neurons and neuropil. In addition, weaker immunoreactivity is also present in glia and blood vessels. Neuronal staining shows significant variability, with particularly intense labeling of certain cell types, including large neocortical and hippocampal pyramidal neurons, magnocellular basal forebrain neurons, brainstem motoneurons, and some populations of interneurons. By electron microscopic immunocytochemistry, highly selective presenilin-1 staining is seen on the cytoplasmic surfaces of membranous organelles, which suggest localization to the endoplasmic reticulum-Golgi intermediate compartment, a subdomain of the endoplasmic reticulum, and some coated transport vesicles.
Asunto(s)
Encéfalo/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Animales , Encéfalo/ultraestructura , Humanos , Inmunohistoquímica , Macaca , Microscopía Electrónica , Presenilina-1RESUMEN
The Huntington disease (HD) phenotype is associated with expansion of a trinucleotide repeat in the IT15 gene, which is predicted to encode a 348-kDa protein named huntington. We used polyclonal and monoclonal anti-fusion protein antibodies to identify native huntingtin in rat, monkey, and human. Western blots revealed a protein with the expected molecular weight which is present in the soluble fraction of rat and monkey brain tissues and lymphoblastoid cells from control cases. In lymphoblastoid cell lines from juvenile-onset heterozygote HD cases, both normal and mutant huntingtin are expressed, and increasing repeat expansion leads to lower levels of the mutant protein. Immunocytochemistry indicates that huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. In the human striatum, huntingtin is enriched in a patch-like distribution, potentially corresponding to the first areas affected in HD. Subcellular localization of huntingtin is consistent with a cytosolic protein primarily found in somatodendritic regions. Huntingtin appears to particularly associate with microtubules, although some is also associated with synaptic vesicles. On the basis of the localization of huntingtin in association with microtubules, we speculate that the mutation impairs the cytoskeletal anchoring or transport of mitochondria, vesicles, or other organelles or molecules.
Asunto(s)
Química Encefálica , Células Madre Hematopoyéticas/química , Enfermedad de Huntington , Linfocitos/química , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas Nucleares/aislamiento & purificación , Animales , Encéfalo/citología , Compartimento Celular , Línea Celular , Citosol/química , Haplorrinos , Humanos , Proteína Huntingtina , Inmunohistoquímica , Microscopía Inmunoelectrónica , Proteínas del Tejido Nervioso/inmunología , Neuronas/química , Proteínas Nucleares/inmunología , Ratas , Proteínas Recombinantes de Fusión/inmunología , Distribución TisularRESUMEN
The precise localization of D1 and D2 dopamine receptors within striatal neurons and circuits is crucial information for further understanding dopamine pharmacology. We have used subtype specific polyclonal and monoclonal antibodies against D1 and D2 dopamine receptors to determine their cellular and subcellular distributions, their colocalization, and their differential connectivity with motor cortical afferents labeled either by lesion-induced degeneration or by anterograde transport of biotinylated dextrans. D1 and D2 are primarily expressed in medium-sized neurons and spiny dendrites. Axon terminals containing D1 were rare whereas D2-immunoreactive axon terminals forming symmetrical synapses with dendrites and spines were common. In 2 microns sections, D1 was localized to 53% of neurons, and D2 to 48% of neurons, while mixing D1 and D2 antibodies labeled 78%. By electron microscopy, D1 was localized to 43% of dendrites and 38% of spines while D2 was localized to 38% of dendrites and 48% of spines. Combining D1 and D2 antibodies resulted in the labeling of 88.5% of dendrites and 92.6% of spines. Using different chromogens for D1 and D2, colocalization was not observed. Ipsilateral motor corticostriatal afferents were primarily axospinous and significantly more synapsed with D1 than D2-positive spines (65% vs 47%). Contralateral motor corticostriatal afferents were frequently axodendritic and no difference in their frequency of synapses with D1 and D2 dendrites and spines was observed. These findings demonstrate differential patterns of expression of D1 and D2 receptors in striatal neurons and axon terminals and their differential involvement in motor corticostriatal circuits.
Asunto(s)
Cuerpo Estriado/fisiología , Corteza Motora/fisiología , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Sinapsis/fisiología , Animales , Anticuerpos Monoclonales , Cuerpo Estriado/citología , Cuerpo Estriado/ultraestructura , Inmunohistoquímica , Masculino , Microscopía Electrónica , Corteza Motora/citología , Neuronas Aferentes/fisiología , Ratas , Ratas Sprague-Dawley , Sinapsis/ultraestructura , Distribución TisularRESUMEN
A family of muscarinic ACh receptor genes are expressed in hippocampus, but little is known about the localization of the encoded proteins and their regulation by cholinergic innervation. Subtype-specific antibodies were used to localize m1-m4 proteins in the hippocampal formation by immunocytochemistry and to determine the alterations in the subtypes following deafferentation. Each of the receptors is differentially localized in Ammon's horn and dentate gyrus, with highly complementary distributions. m1 is widely expressed in somata and dendrites of pyramidal neurons and granule cells in dentate gyrus. m2 immunoreactivity is expressed mostly in nonpyramidal neurons, and in several discrete bands of fibers and puncta surrounding pyramidal neurons and other layers. m3 is enriched in pyramidal neurons, the neuropil in stratum lacunosum-moleculare and the outer third of the molecular layer of dentate gyrus. m4 is enriched in nonpyramidal neurons, in fiber pathways (alveus, fimbria, and hippocampal commissure), and in the inner third of the molecular layer. Fimbria-fornix lesions decreased ipsilateral m2- and m4-immunoreactive axons in the fimbria, with no apparent changes in the distribution of any of the receptors in hippocampus. 192-IgG immunotoxin lesions of the cholinergic septohippocampal projections, which spare noncholinergic projections, produced a small decrease in m2-immunoreactive fibers in the fimbria with no other major changes in the distribution of subtypes. Immunoprecipitation studies at 3-28 d following fimbria-fornix lesions revealed a 25% loss of m2 at 3 d in hippocampus, and upregulation of both m1 (20-29% at 7-14 d) and m4 (44% at 28 d). Thus, the vast majority of muscarinic receptor subtypes are intrinsic to the hippocampal formation and/or nonseptal hippocampal afferents. A subset of m2 and m4 are presynaptically localized, with m2 in cholinergic axons and m2 and m4 possibly in noncholinergic axons that comprise the septohippocampal pathway. The unique laminar and regional distributions of m1-m4 in the hippocampus reflect differential cellular and subcellular distributions of the subtypes and/or selective association of receptor subtypes with certain afferent and intrinsic connections. These results indicate that each subtype likely has a different role in cholinergic modulation of excitatory and inhibitory hippocampal circuits.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Expresión Génica , Hipocampo/metabolismo , Receptores Muscarínicos/biosíntesis , Vías Aferentes/citología , Vías Aferentes/metabolismo , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Inmunoglobulina G , Inmunohistoquímica , Masculino , N-Metilescopolamina , Neuronas/citología , Neuronas/fisiología , Especificidad de Órganos , Conejos/inmunología , Ratas , Ratas Sprague-Dawley , Receptores Muscarínicos/análisis , Derivados de Escopolamina/metabolismoRESUMEN
The m2 muscarinic acetylcholine receptor gene is expressed at high levels in basal forebrain, but the paucity of information about localization of the encoded receptor protein has limited the understanding of cellular and subcellular mechanisms involved in cholinergic actions in this region. The present study sought to determine the cellular localization of m2 protein, its relationship to cholinergic neurons, and its pre- and postsynaptic distribution in the rat medial septum-diagonal band complex using immunocytochemistry with polyclonal rabbit antibodies and a newly developed rat monoclonal antibody specific to the m2 receptor. Light microscopic colocalization studies demonstrated that m2 was present in a subset of choline acetyltransferase immunoreactive neurons, in choline acetyltransferase-negative neurons, and in more neuropil elements than was choline acetyltransferase. Intraventricular injections of 192 IgG-saporin, an immunotoxin directed to the low-affinity nerve growth factor receptor, resulted in depletion of choline acetyltransferase-immunoreactive neurons in the medial septum-diagonal band complex, whereas m2 immunoreactivity in neurons and in the neuropil was unchanged. By electron microscopy, m2 receptor in medial septum-diagonal band complex was localized to the plasmalemma of a small population of small to medium-sized neurons, and it was also found in dendrites, axons, and axon terminals in the neuropil. Neurons expressing m2 immunoreactivity received synaptic contacts from unlabelled axon terminals. A small distinct subpopulation of large neurons, unlabelled by m2 immunoreactivity, received synaptic contacts from m2-immunoreactive terminals. Thus, m2 receptor is situated to mediate the local effects of acetylcholine on basal forebrain cholinergic and noncholinergic neurons and, also, at both pre- and postsynaptic sites.
Asunto(s)
N-Glicosil Hidrolasas , Prosencéfalo/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Anticuerpos Monoclonales , Axones/enzimología , Axones/ultraestructura , Colina O-Acetiltransferasa/metabolismo , Glicósido Hidrolasas/toxicidad , Hibridomas/metabolismo , Inmunoglobulina G/toxicidad , Inmunohistoquímica , Inmunotoxinas/toxicidad , Masculino , Microscopía Electrónica , Terminaciones Nerviosas/enzimología , Terminaciones Nerviosas/ultraestructura , Neuronas/enzimología , Neuronas/ultraestructura , Proteínas de Plantas/toxicidad , Prosencéfalo/ultraestructura , Ratas , Ratas Sprague-Dawley , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saporinas , Sinapsis/enzimología , Sinapsis/ultraestructuraRESUMEN
A family of receptor subtypes, defined either by molecular (m1-m5) or pharmacological (M1-M4) analysis, mediates muscarinic cholinergic neurotransmission in brain. The distribution and functions of the m3 receptor protein in brain and its relation to M3 ligand binding sites are poorly understood. To better characterize the native brain receptors, subtype-specific antibodies reactive with the putative third inner loops were used: (i) to measure the abundance of m3 protein and its regional distribution in rat brain by immunoprecipitation; (ii) to determine the cellular and subcellular distribution of m3 protein by light microscopic immunocytochemistry; and (iii) to compare the distribution of m3 immunoreactivity with the autoradiographic distribution of M3 binding sites labeled by [3H]4-diphenylacetoxy-N-methyl piperidine methioxide in the presence of antagonists selective for the other receptor binding sites. The m3 protein, measured by immunoprecipitation, accounted for 5-10% of total solubilized receptors in all brain regions studied. Immunocytochemistry also revealed a widespread distribution of m3-like immunoreactivity, and localized the subtype to discrete neuronal populations and distinct subcellular compartments. The distribution of m3 protein was consistent with the messenger RNA expression, and like M3 binding sites, the protein was enriched in limbic cortical regions, striatum, hippocampus, anterior thalamic nuclei, superior colliculus and pontine nuclei. However, m3 immunoreactivity and M3 binding were differentially localized in regions and lamina of cortex and hippocampus. The results confirm the presence of m3 protein in brain, its low abundance compared to other muscarinic receptor subtypes, and provide the first immunocytochemical map of its precise localization. The distribution of m3 suggests that it mediates a wide variety of cholinergic processes in brain, including possible roles in learning and memory, motor function and behavioral state control. However, since the distribution of the molecularly-defined receptor protein is distinct from the pharmacologically-defined M3 binding site, investigations of the functions of m3 in brain must await development of more selective ligands or use of non-pharmacological approaches.
Asunto(s)
Encéfalo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Autorradiografía , Inmunohistoquímica , Masculino , N-Metilescopolamina , Parasimpatolíticos/metabolismo , Prosencéfalo/anatomía & histología , Prosencéfalo/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M3 , Derivados de Escopolamina/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Muscarinic ACh receptors mediate complex and clinically important effects in the striatum. To better understand the roles of the different muscarinic receptor subtypes (m1-m4), we have determined the cellular and subcellular distribution of the m1-m4 receptor proteins in the rat neostriatum using subtype-specific antibodies and avidin-biotin-peroxidase immunocytochemistry for light and electron microscopy. m1 receptor protein is expressed in 78% of neurons and is enriched in spiny dendrites and at postsynaptic densities. A small number of m1-immunoreactive axon terminals were observed, all forming asymmetrical synapses. About 2.5% of striatal neurons express m2 receptor protein with reaction product evident, by light microscopy in scattered large oval neurons with enfolded nuclei and long aspiny dendrites. By electron microscopy, m2 immunocytochemistry labeled somata, aspiny dendrites, and many axon terminals. Most axon terminals containing m2 make symmetrical synapses with somata, and dendritic shafts and spines. In addition, many m2-immunoreactive axon terminals formed asymmetrical synapses with spines or dendrites. m3 receptor protein was not evident in somata by light microscopy but was present in a distinct population of small-caliber spiny dendrites as well as in axon terminals forming asymmetrical synapses with spines. m4 receptor protein was heterogeneously distributed in the neostriatum and localized to 44% of striatal cells. m4-positive neurons had the ultrastructural features of medium spiny neurons with reaction product particularly concentrated in spines, often at postsynaptic densities. Axon terminals containing m4 form asymmetrical synapses, primarily with spines. These findings indicate that the muscarinic receptor proteins occur in distinct populations of striatal neurons; that the receptor proteins concentrate postsynaptically at synapses, including many considered to be noncholinergic; that m2 is the predominant muscarinic autoreceptor in the striatum; and that each receptor subtype may be a presynaptic heteroceptor in the striatum modulating extrinsic striatal afferents.
Asunto(s)
Cuerpo Estriado/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Cuerpo Estriado/ultraestructura , Immunoblotting , Inmunohistoquímica , Masculino , Microscopía Electrónica , Ratas , Ratas Sprague-Dawley , Receptores Muscarínicos/inmunología , Distribución TisularRESUMEN
The physiological responses to activation of the m5 muscarinic acetylcholine receptor were compared with those of m3 and m4 in transformed Chinese hamster ovary cells, using patch-clamp electrophysiological and biochemical techniques. Stimulation of the m5 receptor induced increases in both a calcium-dependent potassium conductance and phosphoinositide (PI) metabolism of similar magnitude to those activated by m3. Raising of intracellular calcium or injection of inositol-1,4,5-trisphosphate mimicked the activation of the calcium-dependent potassium conductance by both of these receptors. Although similar regarding these responses, the m3 and m5 receptors induced different cAMP responses. Stimulation of m5 receptors induced a 2-fold increase in cAMP levels, whereas m3 induced a 20-fold increase. These cAMP responses required greater than 100-fold more agonist than the PI responses, and both PI and cAMP responses were insensitive to pertussis toxin. Stimulation of m4 receptors caused little increase in PI metabolism and no electrophysiological effects. Stimulation of m4 receptors with low concentrations of agonist decreased cAMP levels, but at high agonist concentrations cAMP levels were elevated. After treatment with pertussis toxin, the decrease in cAMP levels induced by m4 was blocked and a marked increase in cAMP levels, comparable to those observed for m3 receptors, was uncovered at higher doses. The data indicate that each of the receptors has distinct functional properties.
Asunto(s)
Receptores Muscarínicos/fisiología , Acetilcolina/farmacología , Animales , Línea Celular Transformada , Células Cultivadas , AMP Cíclico/análisis , Cobayas , Inositol 1,4,5-Trifosfato/farmacología , Potenciales de la Membrana/efectos de los fármacos , Fosfatidilinositoles/metabolismo , Receptores Muscarínicos/efectos de los fármacos , Canales de Sodio/fisiologíaRESUMEN
Glycoprotein D (gD) is a virion envelope component of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) which plays an important role in viral infection and pathogenesis. Previously, anti-gD monoclonal antibodies (MAbs) were arranged into groups which recognize distinct type-common and type-specific sites on HSV-1 gD (gD-1) and HSV-2 gD (gD-2). Several groups recognize discontinuous epitopes which are dependent on tertiary structure. Three groups, VII, II, and V, recognize continuous epitopes present in both native and denatured gD. Previously, group II consisted of a single MAb, DL6, whose epitope was localized between amino acids 268 and 287. In the study reported here, we extended our analysis of the antigenic structure of gD, concentrating on continuous epitopes. The DL6 epitope was localized with greater precision to residues 272 to 279. Four additional MAbs including BD78 were identified, each of which recognizes an epitope within residues 264 to 275. BD78 and DL6 blocked each other in binding to gD. In addition, a mutant form of gD was constructed in which the proline at 273 was replaced by serine. This change removes a predicted beta turn in gD. Neither antibody reacted with this mutant, indicating that the BD78 and DL6 epitopes overlap and constitute an antigenic site (site II) within residues 264 to 279. A separate antigenic site (site XI) was recognized by MAb BD66 (residues 284 to 301). This site was only six amino acids downstream of site II, but was distinct as demonstrated by blocking studies. Synthetic peptides mimicking these and other regions of gD were screened with polyclonal antisera to native gD-1 or gD-2. The results indicate that sites II, V, VII, and XI, as well as the carboxy terminus, are the major continuous antigenic determinants on gD. In addition, the results show that the region from residues 264 through 369, except the transmembrane anchor, contains a series of continuous epitopes.
Asunto(s)
Antígenos Virales/inmunología , Simplexvirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Cricetinae , Análisis Mutacional de ADN , Epítopos , Peso Molecular , Oligopéptidos/síntesis química , Oligopéptidos/inmunología , Conformación ProteicaRESUMEN
The abilities of whole herpes simplex virus type 1 (HSV-1) antigen (HSV-ag) and purified HSV-1 native and recombinant envelope proteins to stimulate in vitro T-lymphocyte responses were compared in patients with recurrent herpes labialis. Immunochemically purified preparations of native glycoproteins B, C, and D (ngB, ngC, ngD) from cultured HSV-1 as well as expressed recombinant plasmid preparations of gD (rgD-1t, rgD-45K) elicited lymphocyte proliferation (LT) and production of gamma interferon (IFN-gamma) and interleukin-2 (IL-2) only in seropositive individuals. The IFN-gamma induced by rgD-1t correlated with the time to the next herpetic lesion in 19 volunteers followed to recurrence (r = 0.69, P less than 0.008), although the magnitude and frequency of LT and IFN-gamma responses were lower with either recombinant or native purified antigens than with the whole-virus antigen. Combinations of ngB plus ngD or ngB plus ngC plus ngD stimulated more IFN-gamma, equivalent to whole-virus-antigen responses. Recombinant-derived human IL-2 also specifically increased LT and IFN-gamma responses in antigen-driven cultures. ngD stimulated IL-2 and LT responses similar to those of whole-virus antigen and higher than those of ngC. HSV-ag and ngB induced significantly higher titers of total IFN than could be accounted for by IFN-gamma; this was not seen for the other antigens, which induced only IFN-gamma. HSV-ag-driven Leu 2a-, plastic-nonadherent blood cells, unlike whole peripheral blood mononuclear cells, showed evidence of an increase and then a decline in the frequency of HSV-responsive cells after a lesion recurrence. These studies suggest that HSV-1 envelope proteins are capable of stimulating an immune T-helper-cell response which is associated with the prevention of human herpes simplex lesion recurrence. Although the whole virus probably contains additional important antigens, increasing concentrations or combinations of certain purified glycoproteins or the addition of nonspecific enhancers of T-lymphocyte function can drive in vitro immune responses to the same level as the complete set of viral antigens.
Asunto(s)
Simplexvirus/inmunología , Linfocitos T/inmunología , Proteínas del Envoltorio Viral/inmunología , Antígenos Virales/inmunología , Células Cultivadas , Humanos , Interferón gamma/biosíntesis , Interleucina-2/farmacología , Activación de Linfocitos , Proteínas Recombinantes/inmunologíaRESUMEN
This single-dose, double-blind, randomized, placebo-controlled study assessed the efficacy and safety of 50 mg of flurbiprofen (Ansaid, Upjohn) in the relief of postoperative pain following cesarean section, as well as vaginal or abdominal hysterectomies. Results show that both 50 mg of oral flurbiprofen and 10 mg of intramuscular morphine sulfate were significantly superior to placebo in 161 patients with respect to pain intensity after medication, pain relief scores, need for additional analgesia, and overall clinical evaluation of pain relief. By two hours after treatment, there were no significant differences between morphine sulfate and flurbiprofen in terms of pain intensity or degree of pain relief. According to investigators' global evaluations of efficacy, both active treatments were statistically superior to placebo. The only adverse reaction occurred in the morphine treatment group. Flurbiprofen administered orally for the relief of moderate to severe pain following major gynecologic surgery appears to be equal to morphine sulfate and superior to placebo in efficacy and safety. Unlike morphine, flurbiprofen is a nonparenteral, uncontrolled substance, and thus patient acceptance is improved while nursing time is decreased.
Asunto(s)
Flurbiprofeno/uso terapéutico , Morfina/uso terapéutico , Dolor Postoperatorio/tratamiento farmacológico , Propionatos/uso terapéutico , Adulto , Anciano , Cesárea , Ensayos Clínicos como Asunto , Método Doble Ciego , Evaluación de Medicamentos , Femenino , Humanos , Histerectomía , Persona de Mediana Edad , Placebos , Distribución AleatoriaRESUMEN
Glycoprotein D (gD) of herpes simplex virus (HSV) protects mice from a lethal challenge by either HSV type 1 (HSV-1; oral) or HSV-2 (genital). We evaluated whether synthetic peptides representing residues 1 through 23 of gD (mature protein) can be used as a potential synthetic herpesvirus vaccine. The immunogenicity of the peptides was demonstrated by the biological reactivity of antipeptide sera in immunoprecipitation and neutralization assays. All sera which immunoprecipitated gD had neutralizing against both HSV-1 and HSV-2. The highest titers were found in animals immunized with the longest peptides. The region of residues 1 through 23 was immunogenic regardless of whether the type 1 or type 2 sequence was presented to the animal. Immunization of mice with gD or synthetic peptides conferred solid protection against a footpad challenge with HSV-2. However, the peptides were not as effective as gD in protection against an intraperitoneal challenge. The results suggested that synthetic vaccines based on gD show promise and should be more rigorously tested in a variety of animal models.
Asunto(s)
Simplexvirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Inmunización , Fragmentos de Péptidos/inmunología , Proteínas del Envoltorio Viral/síntesis química , Proteínas Virales/síntesis química , Proteínas Virales/inmunologíaRESUMEN
Herpes simplex virus type 1 (HSV-1) glycoprotein gC was purified by affinity chromatography with an immunosorbent column containing monoclonal antibody to HSV-1 gC, and its reactivity with rabbit antisera was measured by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of radioimmunoprecipitates. Positive reactions were detected between HSV-1 gC and rabbit hyperimmune antisera to both HSV-1 and HSV-2. Electrophoretic analysis also revealed reactivity between the rabbit antisera and peptides of HSV-1 gC generated by partial digestion with trypsin. These findings indicate that HSV-1 gC has one or more cross-reactive or type-common determinants that are readily detected, and therefore, the presence of antibodies reacting with HSV-1 gC in sera may not necessarily be indicative of an earlier infection with HSV-1.
Asunto(s)
Anticuerpos Antivirales/análisis , Sueros Inmunes/análisis , Simplexvirus/inmunología , Proteínas del Envoltorio Viral , Proteínas Virales/inmunología , Animales , Anticuerpos Monoclonales/análisis , Cromatografía de Afinidad , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas/inmunología , Glicoproteínas/aislamiento & purificación , Inmunización , Masculino , Péptidos/análisis , Conejos , Proteínas Virales/aislamiento & purificaciónRESUMEN
The 104-S monoclonal antibody immunoprecipitated from herpes simplex virus type 2 (HSV-2)-infected cell extracts the 75,000-molecular-weight glycoprotein gF and its 65,000-molecular-weight precursor (pgF). The precursor pgF was sensitive to endoglycosidase H digestion, indicating the presence of high mannose-type oligosaccharides, whereas the stable gF product was sensitive to neuraminidase digestion, indicating the presence of sialic acid residues. The 104-S antibody also weakly precipitated the 130,000-molecular-weight herpes simplex virus type 1 (HSV-1) glycoprotein gC from both infected cell extracts and purified preparations obtained through the use of monoclonal antibody-containing immunoadsorbent columns. Immunofluorescence tests demonstrated that the 104-S antibody reacted with antigen present in cells infected with HSV-2 strain 333 and HSV-1 strain 14012 but not with antigen present in cells infected with HSV-1 strain MP, a strain deficient in HSV-1 gC production. These findings indicate that HSV-1 gC and HSV-2 gF have antigenic determinants that are related.
Asunto(s)
Epítopos/análisis , Simplexvirus/genética , Proteínas del Envoltorio Viral , Proteínas Virales/inmunología , Proteínas Virales/aislamiento & purificación , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , RiñónRESUMEN
Intracellular p40 is a class of protein ranging in molecular weight from 39,000 to 45,000 that is immunoprecipitated from herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell extracts by mouse monoclonal antibodies or guinea pig antisera against HSV-1 and HSV-2 nucleocapsid p40. Analysis by a two-dimensional gel system showed that HSV-1 and HSV-2 intracellular p40 each consisted of three major components. However, these HSV-1 and HSV-2 proteins differed in charge and size. Analysis of Staphylococcus aureus V8 protease partial digests by two-dimensional gel electrophoresis indicated that none of the peptides of HSV-1 and HSV-2 intracellular p40 were identical. Immunoprecipitation of the partial digest products of intracellular p40-1 and p40-2 with homologous and heterologous guinea pig antisera resulted in the precipitation of various combinations of peptides indicating the presence of either type-specific or cross-reactive antigenic determinants.