RESUMO
Markers of identified neuronal populations have previously suggested selective degeneration of projection neurons in Huntington's disease (HD) striatum. Interpretations are, however, limited by effects of compensatory regulation and atrophy. Studies of the vesicular monoamine transporter type-2 (VMAT2) and of the vesicular acetylcholine transporter (VAChT) in experimental animals indicate that they are robust markers of presynaptic integrity and are not subject to regulation. We measured dopamine and acetylcholine vesicular transporters to characterize the selectivity of degeneration in HD striatum. Brains were obtained at autopsy from four HD patients and five controls. Autoradiography was used to quantify radioligand binding to VMAT2, VAChT, the dopamine plasmalemmal transporter (DAT), benzodiazepine (BZ) binding sites, and D2-type dopamine receptors. The activity of choline acetyltransferase (ChAT) was determined as an additional marker of cholinergic neurons. Autoradiograms were analyzed by video-assisted densitometry and assessment of atrophy was made from regional structural areas in the coronal projection. Striatal VMAT2, DAT, and VAChT concentrations were unchanged or increased, while D2 and BZ binding and ChAT activity were decreased in HD. After atrophy correction, all striatal binding sites were decreased. However, the decrease in ChAT activity was 3-fold greater than that of VAChT binding. In addition to degeneration of striatal projection neurons, there are losses of extrinsic nigrostriatal projections and of striatal cholinergic interneurons in HD on the basis of vesicular transporter measures. There is also markedly reduced expression of ChAT by surviving cholinergic striatal interneurons.
Assuntos
Proteínas de Transporte/análise , Doença de Huntington/metabolismo , Glicoproteínas de Membrana/análise , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Neuropeptídeos , Sinapses/química , Tetrabenazina/análogos & derivados , Proteínas de Transporte Vesicular , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Autorradiografia , Proteínas de Transporte/metabolismo , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/metabolismo , Fibras Colinérgicas/química , Fibras Colinérgicas/enzimologia , Corpo Estriado/química , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Feminino , Flumazenil/metabolismo , Flumazenil/farmacologia , Moduladores GABAérgicos/metabolismo , Moduladores GABAérgicos/farmacologia , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Mudanças Depois da Morte , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/análise , Receptores de Dopamina D2/metabolismo , Receptores de GABA-A/análise , Receptores de GABA-A/metabolismo , Substância Negra/química , Substância Negra/metabolismo , Sinapses/metabolismo , Trítio , Proteínas Vesiculares de Transporte de Acetilcolina , Proteínas Vesiculares de Transporte de Aminas Biogênicas , Proteínas Vesiculares de Transporte de MonoaminaRESUMO
A series of 31 compounds based on the piperidyl or pyrrolidyl benzilate scaffold were prepared from methyl benzilate and 4-piperidinol, (R)-(+)-3-piperidinol, or (R)-(+)-3-pyrrolidinol. Amine substituents included alkyl and aralkyl groups. In vitro K(i) values ranged from 0.05 nM to >100 nM. (R)-N-(2-Fluoroethyl)-3-piperidyl benzilate (3-FEPB, 22, K(i) = 12.1 nM) and N-(2-fluoroethyl)-4-piperidyl benzilate (4-FEPB, 8, K(i) = 1. 83 nM) were selected for radiolabeling with fluorine-18. Using alkylation with 2-[(18)F]fluoroethyl triflate, 3-[(18)F]FEPB (42) and 4-[(18)F]FEPB (43) were produced in 7-9% radiochemical yield and >97% radiochemical purity. For in vivo studies, retention was moderate in mouse brain for 42; however, blocking with scopolamine showed that uptake was not muscarinic cholinergic receptor-mediated. Conversely, 43 exhibited high, receptor-mediated retention in mouse brain, with significant clearance after 1 h. These results suggest that 43 could have applications as an in vivo probe for measuring endogenous acetylcholine levels.
Assuntos
Benzilatos/síntese química , Compostos de Benzil/síntese química , Piperidinas/síntese química , Pirrolidinas/síntese química , Receptores Muscarínicos/metabolismo , Acetilcolina/metabolismo , Animais , Benzilatos/química , Benzilatos/metabolismo , Benzilatos/farmacologia , Compostos de Benzil/química , Compostos de Benzil/metabolismo , Compostos de Benzil/farmacologia , Encéfalo/metabolismo , Feminino , Radioisótopos de Flúor , Marcação por Isótopo , Ligantes , Camundongos , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/farmacologia , Pirrolidinas/química , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Ensaio Radioligante , Ratos , Solubilidade , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
The properties as well as the distribution of high specific activity alpha-[O-methyl-3H]methyoxytetrabenazine binding to the synaptic vesicular monoamine transporter were studied autoradiographically in rat brain sections. Saturation analysis revealed [3H]methoxytetrabenazine interaction with a homogeneous population of striatal sites (Hill coefficient 1.00 +/- 0.05), with an apparent equilibrium dissociation binding constant of 3.9 +/- 0.4 nM and a maximal binding capacity of 1.2 +/- 0.1 fmol/micrograms protein. Highest levels of [3H]methoxytetrabenazine binding sites were observed in regions richly innervated by the monoamine systems. In the presence of 1 microM concentrations of a variety of competing drugs, only reserpine significantly inhibited [3H]methoxytetrabenazine binding. The presynaptic nigrostriatal location of [3H]methoxytetrabenazine binding was demonstrated by unilateral lesion of the median forebrain bundle with 6-hydroxydopamine. The resulting decrease of striatal [3H]methoxytetrabenazine binding showed an excellent correlation with tyrosine hydroxylase-positive neuron density in the substantia nigra pars compacta (r2 = 0.96; P < 0.001). The present studies demonstrate that in vitro [3H]methoxytetrabenazine binding is a reliable, quantitative marker of the synaptic vesicular monoamine transporter. Further, it is indicated that [3H]methoxytetrabenazine binding provides an accurate assessment of monoamine neuronal losses and may thus be of great value in future studies of neurodegenerative diseases.
Assuntos
Monoaminas Biogênicas/fisiologia , Neurônios/efeitos dos fármacos , Tetrabenazina/análogos & derivados , Animais , Autorradiografia , Ligação Competitiva/efeitos dos fármacos , Cinética , Ligantes , Feixe Prosencefálico Mediano/citologia , Feixe Prosencefálico Mediano/enzimologia , Feixe Prosencefálico Mediano/fisiologia , Oxidopamina/toxicidade , Ratos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
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.
Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Muscarínicos/metabolismo , Animais , Autorradiografia , Imuno-Histoquímica , Masculino , N-Metilescopolamina , Parassimpatolíticos/metabolismo , Prosencéfalo/anatomia & histologia , Prosencéfalo/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M3 , Derivados da Escopolamina/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
Plasma membranes were purified from deciduoma of pseudopregnant rats, rat liver and intestine, and calf uterus. Steroid binding evaluated with deciduoma plasma membranes showed competitive progestin binding, in contrast with estradiol binding which was nondisplaceable as measured by competition binding assay. When the photosensitive steroid [3H]-R5020 was photocrosslinked to plasma membrane, binding was reduced competitively by either progesterone or R5020. These results indicate that the decidual cell plasma membrane contains specific sites for interactions with progestins.
Assuntos
Decídua/metabolismo , Endométrio/metabolismo , Estradiol/metabolismo , Norpregnadienos/metabolismo , Progesterona/metabolismo , Promegestona/metabolismo , Animais , Ligação Competitiva , Fracionamento Celular , Membrana Celular/metabolismo , Endométrio/ultraestrutura , Feminino , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Fotoquímica , Pseudogravidez , Ratos , Útero/metabolismoRESUMO
Inclusion of sodium tetradecyl sulfate in the Laemmli polyacrylamide gel electrophoresis system produces resolution of the kidney Na,K-ATPase catalytic subunit into a doublet and sharpens demarcation of catalytic subunit isoforms in NA,K-ATPase from brain.
Assuntos
Álcoois Graxos , Isoenzimas/metabolismo , Rim/enzimologia , Tetradecilsulfato de Sódio , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Encéfalo/enzimologia , Calibragem , Catálise , Bovinos , Eletroforese em Gel de Poliacrilamida/normas , Camundongos , Dodecilsulfato de Sódio , ATPase Trocadora de Sódio-Potássio/normasRESUMO
The myelin-deficient Shiverer (Shi/Shi) mutant mouse may be a useful model in assessing the dependence of brain (Na+ + K+)-ATPase concentration and composition on myelin membrane formation. Brain microsomal membranes from age-matched control (+/+) and Shiverer (Shi/Shi) mice were fractionated by differential centrifugation and sucrose gradient sedimentation. No reduction in (Na+ + K+)-ATPase specific activity was measured in whole homogenates, high- and low-speed fractions or gradient fractions from brains of Shi/Shi mice as compared to those of +/+ mice. In addition, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with antisera specific for mouse brain (Na+ + K+)-ATPase revealed no significant difference in catalytic subunit composition between fractions of +/+ and Shi/Shi brains. The similar results obtained for both +/+ and myelin-deficient Shi/Shi mice suggest that myelin contributes little to total brain (Na+ + K+)-ATPase.
Assuntos
Encéfalo/enzimologia , Camundongos Mutantes Neurológicos/metabolismo , ATPase Trocadora de Sódio-Potássio/análise , Animais , Masculino , Camundongos , Microssomos/análise , Frações SubcelularesRESUMO
The content and distribution of the membrane-bound enzyme (Na+ + K+)-ATPase in a rat cerebral C6 glioma was determined by immunocytochemistry, immunoblots and enzyme assay. In the C6 glioma cell culture (Na+ + K+)-ATPase activity was about 20% of (Mg2+ + Na+ + K+)-ATPase activity. However, (Mg2+ + Na+ + K+)-ATPase activity in the cerebral C6 gliomas was very close to Mg2+ baseline and not significantly increased by Na+ and K+. As shown by immunoblotting, (Na+ + K+)-ATPase catalytic subunit was detected in excised samples of control cerebrum and as a trace in the intracerebral portions of C6 glioma but not at all in the extracranial portions of C6 glioma or in C6 glioma cell culture. (Na+ + K+)-ATPase was not detected immunocytochemically in paraffin sections of the extracranial or intracerebral portions of rat cerebral C6 glioma. The absence of staining for (Na+ + K+)-ATPase clearly demarcated projections of glioma within normal brain. These results suggest that C6 glioma has little if any expression of (Na+ + K+)-ATPase in vitro or in vivo. The small amount of enzyme epitope in the intracerebral portions represents contamination by normal cerebrum in the extracts.