RESUMO
Histamine-containing neurons of the tuberomammilary nucleus project to the hippocampal formation to innervate H1 and H2 receptors on both principal and inhibitory interneurons. Here we show that H2 receptor activation negatively modulates outward currents through Kv3.2-containing potassium channels by a mechanism involving PKA phosphorylation in inhibitory interneurons. PKA phosphorylation of Kv3.2 lowered the maximum firing frequency of inhibitory neurons, which in turn negatively modulated high-frequency population oscillations recorded in principal cell layers. All these effects were absent in a Kv3.2 knockout mouse. These data reveal a novel pathway for histamine-dependent regulation of high-frequency oscillations within the hippocampal formation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hipocampo/fisiologia , Interneurônios/fisiologia , Neuropeptídeos/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Células Piramidais/fisiologia , Receptores Histamínicos H2/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Bucladesina/farmacologia , AMP Cíclico/fisiologia , Dimaprit/farmacologia , Técnicas In Vitro , Interneurônios/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Picrotoxina/farmacologia , Canais de Potássio/metabolismo , Células Piramidais/efeitos dos fármacos , Canais de Potássio Shaw , Tetraetilamônio/farmacologiaRESUMO
Cannabis is the most consumed illicit drug worldwide. Its principal psychoactive component, Δ9-tetrahydrocannabinol (THC), affects multiple brain functions, including cognitive performance, by modulating cannabinoid type-1 (CB1) receptors. These receptors are strongly enriched in presynaptic terminals, where they modulate neurotransmitter release. We analyzed, through a proteomic screening of hippocampal synaptosomal fractions, those proteins and pathways modulated 3â¯h after a single administration of an amnesic dose of THC (10â¯mg/kg, i.p.). Using an isobaric labeling approach, we identified 2040 proteins, 1911 of them previously reported in synaptic proteomes, confirming the synaptic content enrichment of the samples. Initial analysis revealed a significant alteration of 122 proteins, where 42 increased and 80 decreased their expression. Gene set enrichment analysis indicated an over-representation of mitochondrial associated functions and cellular metabolic processes. A second analysis focusing on extreme changes revealed 28 proteins with altered expression after THC treatment, 15 of them up-regulated and 13 down-regulated. Using a network topology-based scoring algorithm we identified those proteins in the mouse proteome with the greatest association to the 28 modulated proteins. This analysis pinpointed a significant alteration of the proteasome function, since top scoring proteins were related to the proteasome system (PS), a protein complex involved in ATP-dependent protein degradation. In this regard, we observed that THC decreases 20S proteasome chymotrypsin-like protease activity in the hippocampus. Our data describe for the first time the modulation of the PS in the hippocampus following THC administration under amnesic conditions that may contribute to an aberrant plasticity at synapses.
Assuntos
Dronabinol/farmacologia , Hipocampo/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Hipocampo/enzimologia , Hipocampo/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteoma/efeitos dos fármacos , Proteômica , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
Kv3.1 and Kv3.2 K(+) channel proteins form similar voltage-gated K(+) channels with unusual properties, including fast activation at voltages positive to -10 mV and very fast deactivation rates. These properties are thought to facilitate sustained high-frequency firing. Kv3.1 subunits are specifically found in fast-spiking, parvalbumin (PV)-containing cortical interneurons, and recent studies have provided support for a crucial role in the generation of the fast-spiking phenotype. Kv3.2 mRNAs are also found in a small subset of neocortical neurons, although the distribution of these neurons is different. We raised antibodies directed against Kv3.2 proteins and used dual-labeling methods to identify the neocortical neurons expressing Kv3.2 proteins and to determine their subcellular localization. Kv3.2 proteins are prominently expressed in patches in somatic and proximal dendritic membrane as well as in axons and presynaptic terminals of GABAergic interneurons. Kv3.2 subunits are found in all PV-containing neurons in deep cortical layers where they probably form heteromultimeric channels with Kv3.1 subunits. In contrast, in superficial layer PV-positive neurons Kv3.2 immunoreactivity is low, but Kv3.1 is still prominently expressed. Because Kv3.1 and Kv3.2 channels are differentially modulated by protein kinases, these results raise the possibility that the fast-spiking properties of superficial- and deep-layer PV neurons are differentially regulated by neuromodulators. Interestingly, Kv3. 2 but not Kv3.1 proteins are also prominent in a subset of seemingly non-fast-spiking, somatostatin- and calbindin-containing interneurons, suggesting that the Kv3.1-Kv3.2 current type can have functions other than facilitating high-frequency firing.
Assuntos
Neocórtex/fisiologia , Neurônios/fisiologia , Neuropeptídeos/fisiologia , Parvalbuminas/análise , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Somatostatina/análise , Sequência de Aminoácidos , Animais , Anticorpos , Axônios/fisiologia , Axônios/ultraestrutura , Encéfalo/fisiologia , Linhagem Celular , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Dendritos/fisiologia , Dendritos/ultraestrutura , Humanos , Interneurônios/citologia , Interneurônios/fisiologia , Camundongos , Dados de Sequência Molecular , Neocórtex/citologia , Neurônios/classificação , Neurônios/citologia , Neuropeptídeos/análise , Neuropeptídeos/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Canais de Potássio/análise , Canais de Potássio/genética , Coelhos , Ratos , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Canais de Potássio Shaw , Córtex Somatossensorial/citologia , Córtex Somatossensorial/fisiologia , TransfecçãoRESUMO
Voltage-gated K(+) channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to -10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 -/- mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing.
Assuntos
Córtex Cerebral/fisiopatologia , Predisposição Genética para Doença , Inibição Neural , Neuropeptídeos/deficiência , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/deficiência , Convulsões/fisiopatologia , Potenciais de Ação/genética , Animais , Comportamento Animal , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Clonagem Molecular , Eletroencefalografia , Feminino , Marcação de Genes , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Técnicas de Patch-Clamp , Fenótipo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Convulsões/genética , Canais de Potássio Shaw , Células-Tronco , Tetraetilamônio/farmacologiaRESUMO
BACKGROUND: Platelet imidazoline receptors have been shown to be up-regulated in patients with unipolar major depression. This study examines the status of imidazoline receptor proteins in platelets of euthymic bipolar patients and in brains of lithium-treated rats. METHODS: Platelets were collected from 12 bipolar patients (lithium-treated or drug-free) and brains from chronic lithium-treated rats. Imidazoline receptors were quantitated by immunoblotting, using a specific antiserum, and/or radioligand binding. RESULTS: No changes in platelet imidazoline receptors (35-kDa and 45-kDa proteins) were found. Lithium treatment did not alter brain imidazoline receptors (29/30-kDa, 45-kDa, and 66-kDa proteins or density/affinity of [3H]-idazoxan binding sites). CONCLUSIONS: Imidazoline receptor proteins are not altered in platelets of euthymic patients with bipolar affective disorder.
Assuntos
Antimaníacos/farmacologia , Transtorno Bipolar/sangue , Plaquetas/metabolismo , Química Encefálica/efeitos dos fármacos , Imidazóis/sangue , Lítio/farmacologia , Receptores de Droga/sangue , Timo/fisiologia , Adulto , Animais , Transtorno Bipolar/psicologia , Western Blotting , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Feminino , Proteínas de Ligação ao GTP/metabolismo , Humanos , Receptores de Imidazolinas , Técnicas In Vitro , Masculino , Ensaio Radioligante , Ratos , Ratos Sprague-DawleyRESUMO
1. The densities of various imidazoline receptor proteins (with apparent molecular masses of approximately 29/30-45- and 66-kDa) were quantitated by immunoblotting in the rat cerebral cortex after various drug treatments. The modulation of these imidazoline receptor proteins was then compared with the changes in the density of non-adrenoceptor [3H]-idazoxan binding sites (I2-sites) induced by the same drug treatments. 2. Chronic treatment (7 days) with the I2-selective imidazol(in)e drugs idazoxan (10 mg kg-1), cirazoline (1 mg kg-1) and LSL 60101 (10 mg kg-1) differentially increased the immunoreactivity of imidazoline receptor proteins. The levels of the 29/30-kDa protein were increased by idazoxan and LSL 60101 (23%), the levels of the 45-kDa protein only by cirazoline (44%) and those of the 66-kDa protein only by idazoxan (50%). These drug treatments also increased the density of I2-sites (32-42%). 3. Chronic treatment (7 days) with efaroxan (10 mg kg-1), RX821002 (10 mg kg-1) and yohimbine (10 mg kg-1), which possess very low affinity for I2-imidazoline receptors, did not alter either the immunoreactivity of imidazoline receptor proteins or the density of I2-sites. 4. Chronic treatment (7 days) with the monoamine oxidase (MAO) inhibitors clorgyline (10 mg kg-1) and phenelzine (10 mg kg-1) decreased the immunoreactivity of the 29/30-kDa (17-24%), 45-kDa (19%) and 66-kDa (23-31%) imidazoline receptor proteins. The alkylating agent N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (1.6 mg kg-1, 6 h) also decreased the levels of the three imidazoline receptor proteins (20-47%). These drug treatments consistently decreased the density of I2-sites (31-57%). 5. Significant correlations were found when the mean percentage changes in immunoreactivity of imidazoline receptor proteins were related to the mean percentage changes in the density of I2-sites after the various drug treatments (r = 0.92 for the 29/30-kDa protein, r = 0.69 for the 45-kDa protein and r = 0.75 for the 66-kDa protein). 6. In the rat cerebral cortex the I2-imidazoline receptor labelled by [3H]-idazoxan is heterogeneous in nature and the related imidazoline receptor proteins (29/30-, 45- and 66-kDa) detected by immunoblotting contribute differentially to the modulation of I2-sites after drug treatment.
Assuntos
Ligação Competitiva , Encéfalo/efeitos dos fármacos , Idazoxano/farmacologia , Receptores de Droga/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Receptores de Imidazolinas , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
1. I2-Imidazoline sites ([3H]-idazoxan binding) have been identified on monoamine oxidase (MAO) and proposed to modulate the activity of the enzyme through an allosteric inhibitory mechanism (Tesson et al., 1995). The main aim of this study was to assess the inhibitory effects and nature of the inhibition of imidazol(ine)/guanidine drugs on rat liver MAO-A and MAO-B isoforms and to compare their inhibitory potencies with their affinities for the sites labelled by [3H]-clonidine in the same tissue. 2. Competition for [3H]-clonidine binding in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the pharmacological profile of the interaction (2-styryl-2-imidazoline, LSL 61112 > idazoxan > 2-benzofuranyl-2-imidazoline, 2-BFI = cirazoline > guanabenz > oxymetazoline > > clonidine) was typical of that for I2-sites. 3. Clonidine inhibited rat liver MAO-A and MAO-B activities with very low potency (IC50S: 700 microM and 6 mM, respectively) and displayed the typical pattern of competitive enzyme inhibition (lineweaver-Burk plots: increased K(m) and unchanged Vmax values). Other imidazol(ine)/guanidine drugs also were weak MAO inhibitors with the exception of guanabenz, 2-BFI and cirazoline on MAO-A (IC50S: 4-11 microM) and 2-benzofuranyl-2-imidazol (LSL 60101) on MAO-B (IC50: 16 microM). Idazoxan was a full inhibitor although with rather low potency, on both MAO-A and MAO-B isoenzymes (IC50S: 280 microM and 624 microM, respectively). Kinetic analyses of MAO-A inhibition by these drugs revealed that the interactions were competitive. For the same drugs acting on MAO-B the interactions were of the mixed type inhibition (increased K(m) and decreased Vmax values), although the greater inhibitory effects on the apparent value of Vmax/K(m) than on the Vmax value indicated that the competitive element of the MAO-B inhibition predominated. 4. Competition for [3H]-Ro 41-1049 binding to MAO-A or [3H]-Ro 19-6327 binding to MAO-B in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the drug inhibition constants (Ki values) were similar to the IC50 values displayed for the inhibition of MAO-A or MAO-B activities In fact, very good correlations were obtained when the affinities of drugs at MAO-A or MAO-B catalytic sites were correlated with their potencies in inhibiting MAO-A (r = 0.92) or MAO-B (r = 0.99) activity. This further suggested a direct drug interaction with the catalytic sites of MAO-A and MAO-B isoforms. 5. No significant correlations were found when the potencies of imidazol(ine)/guanidine drugs at the high affinity site (pKiH, nanomolar range) or the low-affinity site (pKiL, micromolar range) of I2-imidazoline receptors labelled with [3H]-clonidine were correlated with the pIC50 values of the same drugs for inhibition of MAO-A or MAO-B activity. These discrepancies indicated that I2-imidazoline receptors are not directly related to the site of action of these drugs on MAO activity in rat liver mitochondrial fractions. 6. Although these studies cannot exclude the presence of additional binding sites on MAO that do not affect the activity of the enzyme, they would suggest that I2-imidazoline receptors represent molecular species that are distinct from MAO.
Assuntos
Guanidinas/farmacologia , Imidazóis/farmacologia , Isoenzimas/antagonistas & inibidores , Fígado/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Receptores de Droga/efeitos dos fármacos , Animais , Clonidina/metabolismo , Clonidina/farmacologia , Receptores de Imidazolinas , Cinética , Fígado/efeitos dos fármacos , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Ácidos Picolínicos/farmacologia , Ratos , Ratos Sprague-Dawley , Tiazóis/farmacologiaRESUMO
Imidazoli(di)nes bind to molecular entities different from alpha 2-adrenoceptors: the so-called imidazoline receptors (IRs). Two main types of IRs have been described, the clonidine- and the idazoxan-preferring types, as well as other IRs whose pharmacological properties do not fit either type, but little is known about the molecular features of these receptors. In this study, IR proteins have been solubilized from the rat brain, using the zwitterionic detergent CHAPS, and analyzed by pharmacological and immunological means two of the four peak discriminated by gel filtration chromatography using [3H]idazoxan binding and a specific antibody. The IR eluted in the first peak accounted for 80% of the specific binding of [3H]idazoxan to solubilized brain membranes, and its pharmacological features corresponded to the non-adrenoceptor component of [3H]idazoxan binding in rat brain native membranes. The elution volume of this peak corresponded to a 130-140-kDa protein, but immunoblot analysis with a specific anti-IR antiserum showed the presence of a approximate 45-kDa IR protein, suggesting that this receptor is either an oligomeric protein complex or that it is associated with other proteins. This result was in agreement with the isolation and immunodetection of a 45-kDa peptide by affinity chromatography, which supported the relationship between this protein and a rat brain imidazoline binding site. The second peak, accounting for 15% of the specific binding of [3H]idazoxan to solubilized membranes, had a Mr of approximately 65-70,000, as determined by gel filtration chromatography and immunoblotting.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Encéfalo/metabolismo , Proteínas/química , Receptores de Droga/metabolismo , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Ligação Competitiva , Clonidina/farmacologia , Dioxanos/farmacologia , Relação Dose-Resposta a Droga , Eletroforese , Idazoxano , Imidazóis/farmacologia , Receptores de Imidazolinas , Immunoblotting , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Biochemical and pharmacologic evidence supports the heterogeneous nature of imidazoline receptors (IRs). However, only monoamine oxidase (MAO) (55- and 61-kD) isozymes have been identified as imidazoline binding site-containing proteins. Idazoxan-binding proteins of approximately 70- and approximately 45-kD of unknown amino acid sequences have been isolated from chromaffin cells and rat brain, respectively. Other proteins of approximately 27-30 to > 80 kD have been visualized by immunologic and photoaffinity labeling techniques in different tissues and species. The specific antiserum that recognizes the approximately 70-, approximately 45-, and approximately 29-kD IR proteins, but not MAO, was used to quantitate these proteins in the rat brain cortex. Treatments (7 days) with the I2-selective imidazoline drugs idazoxan (10 mg/kg), cirazoline (1 mg/kg), and LSL 60101 ([2-(2-benzofuranyl) imidazole; 10 mg/kg]) induced differential changes in these proteins: levels of the approximately 29-kD IR were increased by idazoxan and LSL 60101 (23%), levels of the approximately 45-kD protein only by cirazoline (44%), and those of the approximately 66-kD protein only by idazoxan (50%). These treatments also increased the densities of [3H]-idazoxan (I2) binding sites (32-42%). Chronic treatment with efaroxan, RX821002, and yohimbine (10 mg/kg), which possess very low affinity for I2-IRs, did not alter either their immunoreactivities or the density of I2 sites. Chronic treatment with MAO inhibitors clorgyline and phenelzine (10 mg/kg) and acute treatment with EEDQ (1.6 mg/kg, 6 h) induced decreases in the levels of these IR proteins (17-47%) and I2 sites (31-57%). Significant correlations were found when the mean percentage changes in immunoreactivity of IR proteins were related to the mean percentage changes in the density of I2 sites after treatment with the foregoing drug (r = 0.92, r = 0.69, and r = 0.75 for the approximately 29-, approximately 45-, and approximately 66-kD proteins, respectively). These results indicate that in the rat cerebral cortex, the I2 sites labeled by [3H]idazoxan are heterogeneous and that the related immunoreactive IR proteins contribute differently to the modulation of I2 sites after drug treatment.
Assuntos
Receptores de Droga/análise , Receptores de Droga/metabolismo , Animais , Sítios de Ligação , Encéfalo/metabolismo , Humanos , Idazoxano/metabolismo , Imidazóis/metabolismo , Receptores de Imidazolinas , Imunoensaio , Especificidade de Órgãos , Ratos , Receptores de Droga/imunologiaRESUMO
K+ channel principal subunits are by far the largest and most diverse of the ion channels. This diversity originates partly from the large number of genes coding for K+ channel principal subunits, but also from other processes such as alternative splicing, generating multiple mRNA transcripts from a single gene, heteromeric assembly of different principal subunits, as well as possible RNA editing and posttranslational modifications. In this chapter, we attempt to give an overview (mostly in tabular format) of the different genes coding for K+ channel principal and accessory subunits and their genealogical relationships. We discuss the possible correlation of different principal subunits with native K+ channels, the biophysical and pharmacological properties of channels formed when principal subunits are expressed in heterologous expression systems, and their patterns of tissue expression. In addition, we devote a section to describing how diversity of K+ channels can be conferred by heteromultimer formation, accessory subunits, alternative splicing, RNA editing and posttranslational modifications. We trust that this collection of facts will be of use to those attempting to compare the properties of new subunits to the properties of others already known or to those interested in a comparison between native channels and cloned candidates.
Assuntos
Canais de Potássio/genética , Processamento Alternativo , Animais , Clonagem Molecular , Regulação da Expressão Gênica/genética , Humanos , Ativação do Canal Iônico , Filogenia , Canais de Potássio/classificação , Conformação Proteica , Processamento de Proteína Pós-Traducional/genética , Edição de RNA , RNA Mensageiro/genética , XenopusRESUMO
Four mammalian Kv3 genes have been identified, each of which generates, by alternative splicing, multiple protein products differing in their C-terminal sequence. Products of the Kv3.1 and Kv3.2 genes express similar delayed-rectifier type currents in heterologous expression systems, while Kv3.3 and Kv3.4 proteins express A-type currents. All Kv3 currents activate relatively fast at voltages more positive than -10 mV, and deactivate very fast. The distribution of Kv3 mRNAs in the rodent CNS was studied by in situ hybridization, and the localization of Kv3.1 and Kv3.2 proteins has been studied by immunohistochemistry. Most Kv3.2 mRNAs (approximately 90%) are present in thalamic-relay neurons throughout the dorsal thalamus. The protein is expressed mainly in the axons and terminals of these neurons. Kv3.2 channels are thought to be important for thalamocortical signal transmission. Kv3.1 and Kv3.2 proteins are coexpressed in some neuronal populations such as in fast-spiking interneurons of the cortex and hippocampus, and neurons in the globus pallidus. Coprecipitation studies suggest that in these cells the two types of protein form heteromeric channels. Kv3 proteins appear to mediate, in native neurons, similar currents to those seen in heterologous expression systems. The activation voltage and fast deactivation rates are believed to allow these channels to help repolarize action potentials fast without affecting the threshold for action potential generation. The fast deactivating current generates a quickly recovering after hyperpolarization, thus maximizing the rate of recovery of Na+ channel inactivation without contributing to an increase in the duration of the refractory period. These properties are believed to contribute to the ability of neurons to fire at high frequencies and to help regulate the fidelity of synaptic transmission. Experimental evidence has now become available showing that Kv3.1-Kv3.2 channels play critical roles in the generation of fast-spiking properties in cortical GABAergic interneurons.
Assuntos
Neurônios/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Química Encefálica , Linhagem Celular , Evolução Molecular , Regulação da Expressão Gênica , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Canais de Potássio/análise , Proteínas Quinases/metabolismo , RNA/análise , Ratos , Alinhamento de Sequência , Canais de Potássio ShawRESUMO
An antibody against a sequence from the divergent third intracellular loop of the human alpha2A-adrenoceptor, amino acids 262-276, was produced. The antiserum was tested, along with the preimmune serum, by means of ELISA and dot blot assays which demonstrated that the alpha2A-peptide used for the antibody production was recognized by the immune serum. The antibody also recognized the alpha2A-adrenoceptor protein in the human brain (immunoblot analysis). In cortical membranes a major immunoreactive peptide of approximately 70 kDa (mature glycosylated receptor) was detected and after treatment with N-glycosidase F only a approximately 50 kDa peptide (non-glycosylated receptor) was immunodetected. This antibody was used to demonstrate that a chemical modification of the alpha2A-adrenoceptor induced by the alkylating agent EEDQ facilitates the protease-mediated receptor degradation. Thus in vitro, normal receptor degradation (24-44% at 2-4 h) was enhanced by EEDQ (10(-6) M) (38-71% at 2-4 h) but in the presence of protease inhibitors this effect was almost abolished.
Assuntos
Alquilantes/farmacologia , Encéfalo/metabolismo , Endopeptidases/metabolismo , Quinolinas/farmacologia , Receptores Adrenérgicos alfa 2/metabolismo , Antagonistas Adrenérgicos alfa/farmacologia , Sequência de Aminoácidos , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Glicosilação , Humanos , Imunoglobulina G , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Receptores Adrenérgicos alfa 2/química , Receptores Adrenérgicos alfa 2/efeitos dos fármacosRESUMO
Cannabis derivatives have become the most widely used illicit substances in developed countries, and constitute a major health concern. The psychoactive compounds contained in cannabis induce their pharmacological effects by the activation of at least two different receptors, CB1 and CB2 cannabinoid receptors. Multiple studies have demonstrated the specific involvement of CB1 cannabinoid receptors in the addictive properties of cannabinoids. Several neurotransmitter systems involved in the addictive effects of other prototypical drugs of abuse, such as the dopaminergic and the opioid system are also involved in cannabis addiction. The participation of other neurochemical systems in behavioural responses of cannabinoids related to their addictive effects has also been reported. This review describes the experimental methods now available to study the pharmacological responses of cannabinoids related to their addictive effects and how these methods have contributed to advance the knowledge of the specific contribution of different neurochemical systems in cannabis addiction.
Assuntos
Química Encefálica/fisiologia , Canabinoides/farmacologia , Abuso de Maconha/metabolismo , Receptores de Canabinoides/metabolismo , Animais , Agonistas de Receptores de Canabinoides , Canabinoides/química , Modelos Animais de Doenças , Humanos , Abuso de Maconha/fisiopatologiaAssuntos
Encéfalo/metabolismo , Receptores de Droga/metabolismo , Reserpina/farmacologia , Animais , Monoaminas Biogênicas/metabolismo , Encéfalo/efeitos dos fármacos , Córtex Cerebral/metabolismo , Receptores de Imidazolinas , Immunoblotting , Masculino , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/isolamento & purificação , Regulação para CimaAssuntos
Adenoma/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Monoaminoxidase/metabolismo , Neoplasias Hipofisárias/metabolismo , Receptores de Droga/metabolismo , Acromegalia/etiologia , Benzofuranos/farmacocinética , Feminino , Lobo Frontal/metabolismo , Humanos , Imidazóis/farmacocinética , Receptores de Imidazolinas , Isoenzimas/metabolismo , Cinética , Masculino , Pessoa de Meia-Idade , Hipófise/metabolismo , Ensaio Radioligante , Valores de Referência , TrítioRESUMO
Voltage-gated K(+) channels containing pore-forming subunits of the Kv3 subfamily have specific roles in the fast repolarization of action potentials and enable neurons to fire repetitively at high frequencies. Each of the four known Kv3 genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K(+) channel subunits differing only in their C-terminal sequence. The alternative splicing does not affect the electrophysiological properties of the channels, and its physiological role is unknown. It has been proposed that one of the functions of the alternative splicing of Kv3 genes is to produce subunit isoforms with differential subcellular membrane localizations in neurons and differential modulation by signaling pathways. We investigated the role of the alternative splicing of Kv3 subunits in subcellular localization by examining the brain distribution of the two alternatively spliced versions of the Kv3.1 gene (Kv3.1a and Kv3.1b) with antibodies specific for the alternative spliced C-termini. Kv3.1b proteins were prominently expressed in the somatic and proximal dendritic membrane of specific neuronal populations in the mouse brain. The axons of most of these neurons also expressed Kv3.1b protein. In contrast, Kv3.1a proteins were prominently expressed in the axons of some of the same neuronal populations, but there was little to no Kv3.1a protein expression in somatodendritic membrane. Exceptions to this pattern were seen in two neuronal populations with unusual targeting of axonal proteins, mitral cells of the olfactory bulb, and mesencephalic trigeminal neurons, which expressed Kv3.1a protein in dendritic and somatic membrane, respectively. The results support the hypothesis that the alternative spliced C-termini of Kv3 subunits regulate their subcellular targeting in neurons.
Assuntos
Processamento Alternativo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Canais de Potássio/metabolismo , Frações Subcelulares/metabolismo , Sequência de Aminoácidos/genética , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Linhagem Celular , Humanos , Masculino , Membranas/metabolismo , Membranas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Subunidades Proteicas , Canais de Potássio Shaw , Distribuição TecidualRESUMO
The activation of T-lymphocytes is dependent upon, and accompanied by, an increase in voltage-gated K+ conductance. Kv1.3, a Shaker family K+ channel protein, appears to play an essential role in the activation of peripheral human T cells. Although Kv1.3-mediated K+ currents increase markedly during the activation process in mice, and to a lesser degree in humans, Kv1.3 mRNA levels in these organisms do not, indicating post-transcriptional regulation. In other tissues Shaker K+ channel proteins physically associate with cytoplasmic beta-subunits (Kvbeta1-3). Recently it has been shown that Kvbeta1 and Kvbeta2 are expressed in mouse T cells and that they are up-regulated during mitogen-stimulated activation. In this study, we show that the human Kvbeta subunits substantially increase K+ current amplitudes when coexpressed with their Kv1.3 counterpart, and that unlike in mouse, protein levels of human Kvbeta2 remain constant upon activation. Differences in Kvbeta2 expression between mice and humans may explain the differential K+ conductance increases which accompany T-cell proliferation in these organisms.
Assuntos
Peptídeos/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Canal de Potássio Kv1.3 , Camundongos , Canais de Potássio/química , Canais de Potássio/genética , RNA Complementar/genética , RNA Complementar/metabolismo , Superfamília Shaker de Canais de Potássio , Xenopus laevisRESUMO
Aminopeptidase activities, assayed as arylamidase activities, were investigated in selected tissues of 1, 6, 12 and 24-month-old rats. The enzyme activities were found to have a heterogeneous distribution and age-related changes were observed. The highest levels of soluble arginyl-aminopeptidase activity were detected in brain homogenate at all the studied ages, whereas membrane-bound activity presented the highest levels in brain and kidney in the four ages tested. Aspartyl-aminopeptidase activity was detected mainly in the particulate fraction of kidney at all four ages. In 1, 6 and 12-month-old animals, soluble aspartyl-aminopeptidase activity was also higher in the kidney than in the rest of the tissues, whereas in the group of 2-year-old rats, the highest levels were found in both kidney and liver. Age-related changes were observed in all the studied tissues and for all the assayed enzymatic activities. In general, the maximal levels were detected in both the youngest and the oldest animals, and the minimal ones in 6 and 12-month-old rats. However, in the adrenals, the soluble and membrane-bound arginyl-aminopeptidase activity was higher in 6-month and 2-year-old rats than in 1-month and 12-month-old rats. These changes may reflect the functional status of the susceptible endogenous substrates of aminopeptidases.
Assuntos
Envelhecimento/metabolismo , Aminopeptidases/metabolismo , Animais , Glutamil Aminopeptidase , Ratos , Ratos Sprague-DawleyRESUMO
The levels of specific soluble aspartyl aminopeptidase activity were assayed in retina, occipital cortex, anterior pituitary, posterior pituitary, pineal gland and serum of adult male rats, using Asp-2-naphthylamide as substrate, in a 12:12 h light:dark cycle (7-19 h light). Significant diurnal variations appeared in retina, pineal gland, occipital cortex and serum. In addition, different patterns of diurnal variation of the enzymatic activity were observed in the tissues analyzed. A regular increase of the activity was noticed at the end of the dark period in all the tissues as a common feature, except in serum, in which the enzymatic activity reached a peak in the middle of the light period, decreasing progressively during the dark hours. After the last hours of darkness, the pattern of variation in the activity differed in each tissue. These diurnal variations in aspartyl aminopeptidase activity could reflect the functional status of its putative endogenous substrates, such as angiotensin II, and it may also suggest the existence of differential regulatory mechanisms associated with each location.