RESUMO
The regional and cellular localizations of the previously reported aromatic amino acid binding sites (AABS) in rat brain were investigated using [(3)H]tryptophan as the binding radioligand. An almost 4-fold difference in AABS was observed among the brain regions in the descending order of cerebral cortex ? hippocampus > striatum ? thalamus/hypothalamus ? midbrain ? cerebellum ? brainstem. When rats were injected intracerebroventricularly (i.c.v.) with 5,7-dihydroxytryptamine (0.2 ?mol) after prior administration of desipramine (i.p., 25 mg/kg), [(3)H]tryptophan binding was significantly reduced by 32% in the brainstem but increased by 27% in the midbrain. After injection of 6-hydroxydopamine (i.c.v., 0.2 ?mol), however, binding was significantly reduced in the brainstem by 32%, striatum by 27% and cerebral cortex by 18% but increased by 24% in the midbrain. It is concluded that the AABS are associated with some but not all cells of the three monoaminergic systems in the rat brain. The increases in binding in the midbrain may reflect a localization of the AABS in this region in cells post-synaptic to the degenerating cells.
RESUMO
?(1)-Pyrroline-5-carboxylate dehydrogenase (P5CDH) in rat cerebellum was assayed with a simple spectrophotometric method using high-speed supernatants of whole tissue homogenates. Kinetic analysis showed that the enzyme has K(m) values of 0.83 +/- 0.21 mM for ?(1)-pyrroline-5-carboxylate and 0.47 +/- 0.02 mM for NAD(+). Various cations inhibited P5CDH but only at relatively high concentrations. Several amino acids were strongly inhibitory in the order GABA > glycine > hydroxyproline > cysteine ? proline > glutamine > glutamate > alanine .
RESUMO
[(3)H]Tryptophan binds to a single population of sites in the rat cortical synaptosomal membranes. The binding is reversible and follows the law of mass action. By saturation studies using increasing concentration of [(3)H]tryptophan with decreasing specific radioactivity, the apparent K(d) obtained was approx. 0.8 ?M and the B(max) 110 pmol/mg protein. However, the IC(50) obtained for unlabelled tryptophan in displacing [(3)H]tryptophan binding (3.5 nM) was 0.26 ?M. All six naturally occurring aromatic amino acids studied displaced [(3)H]tryptophan binding with tryptophan and phenylalanine showing higher apparent affinity than histidine, tyrosine, dihydroxyphenylalanine and 5-hydroxytryptophan. These binding sites are proteins in nature as they are sensitive to trypsin and ?-chymotrypsin. It is observed that about 37% of the sites seem to be protected from the proteolytic enzymes by the membrane structure. Furthermore, they are extremely sensitive to phospholipase A(2) presumably because altered membrane phospholipids conduce a conformational change in the binding protein. A considerable degree of stereospecificity was demonstrated with the affinity for l-tryptophan about 90 times higher than that for d-tryptophan. The affinity for l-phenylalanine was 8 times higher than that for d-phenylalanine. Ligand specificity for the aromatic amino acids is remarkable as hydrocinnamic acid, 2-phenylethylamine, 5-hydroxytryptamine, histamine, dopamine, ?-aminobutyric acid, glutamic acid and taurine did not displace [(3)H]tryptophan binding. Therefore, these sites are termed aromatic amino acid binding sites (AABS). Whether or not AABS are involved in neuromodulation at the synapse awaits clarification. If so, the endogenous ligand for the AABS may well be tryptophan.
RESUMO
The effects of death and various treatments that affect the status of nigrostriatal neurones on striatal release of dopamine (DA) and 5-hydroxytryptamine (5-HT) and their metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4-hydroxyphenylethylamine (3-MT), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were studied by in vivo microdialysis. In conscious rats, DA and 5-HT levels were very low compared with their metabolites except 3-MT which was rarely detectable. Upon death by pentobarbitone overdose, there was an immediate surge of the DA level reflecting massive release of the neurotransmitter. This increase was accompanied by a significant increase in 3-MT level but not the other two DA metabolites. Post mortem release of 5-HT was also observed but to a much smaller extent than that of DA. The amounts of amines released appeared to be proportional to the amine stores. When L-dihydroxyphenylalanine (DOPA) was administered to reserpinized rats, the extracellular levels of both DOPAC and HVA increased but not that of DA. However, marked release of DA occurred at death in contrast to reserpinized rats not injected with the precursor. It is evident that exogenous L-DOPA is taken up into the dopaminergic nerve endings and is converted to releasable extravesicular DA, and that this releasable DA is released, at least in part, in accordance with neuronal activities.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Neurônios/metabolismo , Serotonina/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Núcleo Caudado/metabolismo , Cromatografia Líquida de Alta Pressão , Corpo Estriado/efeitos dos fármacos , Ácido Homovanílico/metabolismo , Hidroxidopaminas/farmacologia , Ácido Hidroxi-Indolacético/metabolismo , Ácido Caínico/farmacologia , Levodopa/farmacologia , Masculino , Microdiálise , Neurônios/efeitos dos fármacos , Putamen/metabolismo , Ratos , Ratos Sprague-Dawley , Reserpina/farmacologia , Tiramina/análogos & derivados , Tiramina/metabolismoRESUMO
The effects of some selective dopamine (DA) and 5-hydroxytryptamine (5-HT) antagonists and uptake inhibitors on the outflow of homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the striatum were studied using microdialysis in conscious rats. Eticlopride (D2 antagonist, 2.5 mg/kg) increased HVA but decreased 5-HIAA, while nomifensine (DA uptake inhibitor, 10 mg/kg) decreased both. Mianserin (5-HT2 antagonist, 2 mg/kg) and citalopram (5-HT uptake inhibitor, 11 mg/kg) also decreased both HVA and 5-HIAA. The selectivity of these drugs and the time course of their effects on HVA and 5-HIAA suggested that the dopaminergic drugs may affect the serotonergic systems primarily via changes in extraneuronal DA and conversely, serotonergic drugs affect the dopaminergic systems via changes in extraneuronal 5-HT. The results obtained are consistent with current evidence that DA and 5-HT release from striatal terminals are regulated by D2 and 5-HT1B autoreceptors, respectively. They also support the idea that, in the rat striatum, presynaptic inhibitory heteroreceptors are a major mechanism that contributes to a reciprocal interaction of the dopaminergic and serotonergic systems.
Assuntos
Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Antagonistas de Dopamina/farmacologia , Antagonistas da Serotonina/farmacologia , Animais , Relação Dose-Resposta a Droga , Ácido Homovanílico/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Mianserina/farmacologia , Microdiálise , Nomifensina/farmacologia , Ratos , Salicilamidas/farmacologiaRESUMO
A mouse line was developed by selecting for increased sensitivity to the hypnotic effect of diazepam. These "diazepam sensitive" mice showed a mean duration of loss of righting reflex (LORR) of approximately 150 min at a dose of 20 mg/kg diazepam, this dose failed to induce LORR in the control outbred mice. Rotarod treading times of the diazepam sensitive mice were significantly shorter than that of the control mice over the same dose range indicating that these mice are also more sensitive to the sedative/muscle relaxant effects of diazepam. On the contrary, the ability of diazepam to protect against pentylenetetrazole-induced convulsion was found to be the same in the sensitive and control mice. These observations strongly suggest that the heightened sensitivity to the sedative-hypnotic effects of diazepam in the sensitive mice is unlikely to be due entirely to changes in drug disposition.
Assuntos
Diazepam/farmacologia , Convulsões/tratamento farmacológico , Animais , Diazepam/administração & dosagem , Diazepam/uso terapêutico , Relação Dose-Resposta a Droga , Hipnóticos e Sedativos/farmacologia , Camundongos , Camundongos Endogâmicos , Atividade Motora/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Pentilenotetrazol , Reflexo/efeitos dos fármacos , Convulsões/induzido quimicamente , Especificidade da Espécie , Fatores de TempoRESUMO
Mice treated neonatally with monosodium glutamate (MSG) were found to have learning and memory deficits in performing a non-spatial water escape task. Scopolamine impaired the water-escape performance of the control mice but not that of the MSG-treated mice. It was suggested that the water-escape performance deficit in the MSG-treated mice was a result of impaired central cholinergic mechanisms. As such, scopolamine was unable to further incapacitate an already impaired cholinergic system. This is strongly supported by the decreased affinity of the sodium-dependent high-affinity choline uptake observed in the hippocampus. D-Cycloserine, a partial agonist at the glycine site of the NMDA receptor, did not affect the water-escape performance of the MSG-treated and control mice; nor did it alter the effects of scopolamine. This lack of effect of D-Cycloserine may imply that the NMDA receptors are not involved in non-spatial learning, in contrast to their reported involvement in spatial learning.
Assuntos
Colinérgicos/farmacologia , Reação de Fuga/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Desempenho Psicomotor/efeitos dos fármacos , Glutamato de Sódio/farmacologia , Animais , Animais Recém-Nascidos , Colina/metabolismo , Ciclosserina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Masculino , Camundongos , Tempo de Reação/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Receptores Muscarínicos/metabolismo , Escopolamina/farmacologia , EstereoisomerismoRESUMO
The results of a 6-year review of bulbocavernosus reflex (BCR) latencies in 300 men are presented. The relationship of BCR latency to potency, diabetic status and age was examined. The mean BCR latency in 32 normal men aged 22 to 78 years (x = 44.9 +/- 14.4) was 40.6 +/- 8.9 ms. A significant trend towards increasing BCR latency with age was evident. BCR latency was not found to be significantly associated with potency, the mean measurable BCR latency in impotent men being 43.0 +/- 11.6 ms. Diabetic men were more likely to have a prolonged measurable BCR latency (x = 46.2 +/- 11.2) and impotence, but even in this subset no significant correlation between BCR latency and impotence was detectable. Our results suggest that detectable pudendal neuropathy is unrelated to impotence in non-diabetic and diabetic men and does not usefully distinguish between neurologic and non-neurologic causes of impotence. With the advent of newer objective techniques to assess organic impotence, the value of BCR latency in the routine assessment of impotent subjects is questionable.
Assuntos
Complicações do Diabetes , Disfunção Erétil/fisiopatologia , Pênis/inervação , Reflexo/fisiologia , Adulto , Idoso , Eletromiografia , Disfunção Erétil/etiologia , Resposta Galvânica da Pele/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Ereção Peniana/fisiologia , Tempo de Reação/fisiologia , Estudos RetrospectivosRESUMO
The microbial pattern-recognizing Toll-like receptors (TLRs) are major signal transducers known to shape and influence the postnatal maturation of host intestinal epithelium. Perturbations in this intricate host-microbe cross-talk have been reported to be associated with uncontrolled epithelial cell growth and thus potential cancer development by mechanisms which are largely unknown. We therefore generated transgenic mice carrying a constitutively active TLR4 (CD4-TLR4) linked to an intestinal epithelial cell-specific promoter. Ex vivo analysis of transgenic crypt-villus organoid cultures revealed an increased proliferative capacity and a lowered cyclooxygenase 2 (Cox-2) expression in these organoids compared with wild-type control cultures. Introducing the CD4-TLR4 transgene into APC(Min/+) mice (CD4-TLR4-APC(Min/+)), a model of colorectal carcinoma, resulted in a dramatic drop in tumor load as compared with control APC(Min/+) mice. Intestinal tumors from CD4-TLR4-APC(Min/+) mice displayed reduced Cox-2 protein, elevated interferon ß expression and increased caspase-3 activity, which correlated with increased apoptosis in vivo. Thus, our data reveal that host microbiota-mediated signal transduction via TLR4 in intestinal epithelial cells is far more complex than what is previously reported.
Assuntos
Apoptose , Mucosa Intestinal/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Caspase 3/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ciclo-Oxigenase 2/metabolismo , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Interferon beta/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Organoides/citologia , Organoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 4 Toll-Like/genética , Carga Tumoral/genéticaRESUMO
Evidence that lipocalin 2 (LCN2) is oncogenic has grown in recent years and comes from both animal models and expression analysis from a variety of human cancers. In the intestine, LCN2 is overexpressed in colitis patients and its overexpression is a negative prognostic indicator in colorectal cancer. Functionally, LCN2 has a number of different activities that may contribute to its oncogenic potential, including increasing matrix metalloproteinase activity, control of iron availability and stimulating inflammation. In this report, we examined APCmin intestinal tumorigenesis in an LCN2-deficient background. We found that the loss of LCN2 increased tumor multiplicity specifically in the duodenum, suggesting a potential tumor-suppressive activity. Concurrently, however, LCN2 increased the average small intestinal tumor size particularly in the distal small intestine. We found that this increase was correlated to tumor iron(II) content, suggesting that an iron-scavenging role is important for LCN2 oncogenic activity in the intestine.
Assuntos
Proteínas de Fase Aguda/biossíntese , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Lipocalinas/biossíntese , Proteínas Oncogênicas/biossíntese , Proteínas de Fase Aguda/deficiência , Proteínas de Fase Aguda/genética , Animais , Apoptose/fisiologia , Progressão da Doença , Feminino , Genes APC , Neoplasias Intestinais/genética , Lipocalina-2 , Lipocalinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genéticaRESUMO
Specific [3H]phenytoin binding in rat cortical membranes is associated with a micromolar affinity site through which diazepam can exert an enhancing effect. In contrast to diazepam, (+)bicuculline, isoguvacine and pentobarbital inhibited phenytoin binding, while GABA and (-)baclofen had no effect. Decreased phenytoin binding and a loss of the diazepam effect were observed in phospholipase A2-treated membranes. Binding in the absence and presence of diazepam demonstrated differential postnatal development. Furthermore, the degree to which binding was enhanced by diazepam varied from one brain region to another. The regional variations of phenytoin binding, both in the presence and absence of diazepam, did not correlate positively with the regional distribution of either the central benzodiazepine receptors or the peripheral type binding sites in the brain.
Assuntos
Química Encefálica , Encéfalo/crescimento & desenvolvimento , Receptores de Droga/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Diazepam/farmacologia , Masculino , Membranas/metabolismo , Neurotransmissores/farmacologia , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Ratos , Ratos Endogâmicos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Phenytoin was observed to inhibit competitively the sodium dependent high affinity synaptosomal transport of both glutamate (Glu) and gamma-aminobutyric acid (GABA) with Ki values of 66 +/- 10 and 185 +/- 65 microM, respectively. This contrasted with a previous report that the uptakes of Glu and GABA were enhanced by phenytoin. The degree of inhibition is dependent on the concentrations of the competing drug and substrate present. Taking the therapeutic levels of phenytoin and the overall brain Glu and GABA contents, the degrees of inhibition obtainable appear to be negligible. However, as most of the high levels of Glu and GABA in the brain are intracellular, Glu, and GABA concentrations in the microenvironment of the uptake sites may be sufficiently small so that the ability of phenytoin to inhibit Glu and GABA transport may contribute significantly to the anticonvulsant property of this drug.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Glutamatos/metabolismo , Inibidores da Captação de Neurotransmissores , Fenitoína/farmacologia , Sinaptossomos/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Córtex Cerebral/metabolismo , Técnicas In Vitro , Ratos , Sinaptossomos/metabolismoRESUMO
[3H]Phenytoin binding to rat cortical membrane was significantly enhanced in the presence of diazepam. This binding is saturable, reversible and displacable by unlabelled phenytoin. Analyses of the binding data either by the Scatchard plot or by the displacement curve revealed a high and a low affinity sites with Kd values of 32 +/- 5 nM and 8.5 +/- 1.1 microM, respectively. Similar enhancement of [3H]phenytoin binding was observed when diazepam was replaced by Ro 5-4864 (4'-chlorodiazepam) which is selective for the 'peripheral' type benzodiazepine binding sites. In contrast, neither the 'central' type receptor selective agonist clonazepam nor the antagonist Ro 15-1788 enhanced [3H]phenytoin binding. Therefore, it seems that these phenytoin binding sites in rat cerebral cortex are associated with a benzodiazepine site similar to the 'peripheral' type binding site for its selective affinity for Ro 5-4864. However, judging from the micromolar concentrations required for the enhancement of [3H]phenytoin binding, they appear unlikely to be the same 'peripheral' type binding sites as measured by [3H]Ro 5-4864 binding (Kd approx. 1 nM). The micromolar affinity benzodiazepine recognition sites are a possibility, if they indeed exist.
Assuntos
Córtex Cerebral/metabolismo , Diazepam/farmacologia , Fenitoína/metabolismo , Animais , Benzodiazepinonas/farmacologia , Sítios de Ligação , Membrana Celular/metabolismo , Córtex Cerebral/efeitos dos fármacos , Clonazepam/farmacologia , Flumazenil/farmacologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
Twenty percent of the Swiss albino mice administered with a single dose (60 mg/kg) of oxazepam dissolved in dimethyl sulphoxide (DMSO) died within 7 days. Similarly high mortality rate (37%) was observed in diazepam sensitive mice derived from a Swiss albino stock. In contrast, zero mortality was observed in Swiss albino mice administered with DMSO or diazepam (35 mg/kg). Mortality at 60 mg/kg diazepam was only 10%. The high mortality caused by oxazepam in Swiss albino mice seemed to be strain-related as only 7% mortality was observed with identically treated BALB/c mice. Since DMSO is the only convenient vehicle for the administration of oxazepam by injection, it is suggested that a suitable strain must be selected for experimentation in order to avoid unnecessary loss of animals.
Assuntos
Mortalidade , Oxazepam/toxicidade , Animais , Dimetil Sulfóxido/administração & dosagem , Feminino , Injeções Intraperitoneais/veterinária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxazepam/administração & dosagem , SolventesRESUMO
We studied the early events of de novo formation of adventitious shoot meristems in stem segments of Brassica oleracea. A regeneration system was used that is efficient, rapid, highly responsive to cytokinins and does not involve callus formation, thus allowing studies on a direct developmental switch of cells in the stem segment to form adventitious shoot meristem cells. Shoot meristem cells and dividing cells were marked from very early stages using in situ hybridization studies with Brostm, a Brassica homologue of the Arabidopsis SHOOTMERISTEMLESS (STM) gene, and a cyclin box-derived probe, Brocyc, respectively. We show that the process of developmental switching starts before any cell division occurs in the stem explants. This switching occurs synchronously both longitudinally and transversely in the explant, in groups of 5-7 phloem parenchyma cells subtending vascular bundles in the explant. Brostm is induced specifically in response to a cytokinin, benzyladenine, within 4 h of treatment and the transcripts persist during cell proliferation leading to shoot differentiation. We also show that during adventitious shoot formation, cells expressing Brostm are distinct from those expressing Brocyc. Lastly, our data suggest that, although developmental switching is initiated synchronously within 4 h of treatment, it requires 8 h of treatment for the establishment of organogenic determinance. The latter process is aynchronous, implying that additional factors formed later than Brostm are required to achieve maximal levels of determined cell populations to form adventitious shoots in vitro.
Assuntos
Adenina/análogos & derivados , Proteínas de Arabidopsis , Brassica/genética , Proteínas de Homeodomínio/genética , Meristema/genética , Proteínas de Plantas , Adenina/farmacologia , Sequência de Aminoácidos , Compostos de Benzil , Northern Blotting , Southern Blotting , Brassica/citologia , Brassica/crescimento & desenvolvimento , DNA Complementar/química , DNA Complementar/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hibridização In Situ , Cinetina , Meristema/efeitos dos fármacos , Meristema/crescimento & desenvolvimento , Dados de Sequência Molecular , Caules de Planta/efeitos dos fármacos , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Purinas , RNA de Plantas/genética , RNA de Plantas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição TecidualRESUMO
[3H]PK 11195 binding to the peripheral benzodiazepine binding site was investigated in the brain and liver of mice treated with ethanol (4 g/kg, p.o.) daily for 5 days. In the brain stem, Bmax was decreased by 78% in the ethanol-treated group with unaltered Kd (2 nM). The ethanol-withdrawn group did not differ from the control group in both parameters. In the cerebellum, Bmax was decreased by 74% but the binding affinity increased 5-fold as the Kd decreased from 10 to 2 nM. The ethanol-withdrawn group did not differ significantly from the ethanol-treated group. No changes were observed in the cerebrum and liver. These results further support the idea that [3H]PK 11195 binding may be a useful marker for ethanol consumption. The observed changes in these binding sites may represent a functional adaptive response to the ethanol insult and/or a role in the mediation of the effects of ethanol.