RESUMO
Transmission in the "direct" pathway through the basal ganglia, which has an important role in the control of motor movement, is markedly facilitated by the concurrent activation of dopamine D(1) receptors. Consistent with this, Ca(2+)-dependent, depolarization-induced release of [(3)H]-GABA from striatal slices from rats pretreated with reserpine was greatly increased in the presence of 1 microM SKF 38393, a dopamine D(1)-like receptor agonist. The effect of SKF 38393 was mimicked by 1 mM 8-bromo-cyclic AMP (Br-cAMP) and inhibited by the protein kinase A (PKA) inhibitor H-89, mean inhibition 92% +/- 4% with 10 microM H-89 (n = 3). The effects of SKF 38393 and Br-cAMP were not additive. The stimulatory effects of SKF 38393 and Br-cAMP were practically abolished in the presence of the histamine H(3) receptor agonist immepip (1 microM). The depolarization-induced release of [(3)H]-GABA in the presence of SKF 38393 was not significantly inhibited by 5 microM nimodipine, an L-type Ca(2+) channel blocker, or by 0.3 microM omega-conotoxin MVIIA, a selective blocker of N-type channels. However, preincubation of the slices with 0.95 microM omega-agatoxin TK, a P/Q-type channel blocker, followed by washing before changing to a depolarizing medium containing SKF 38393, resulted in a marked inhibition of the stimulated release of [(3)H]-GABA, mean 68% +/- 4% (n = 3). These observations provide evidence that dopamine D(1) agonist facilitation of the depolarization-induced release of GABA from striatal terminals is mediated by the cAMP/PKA pathway and involves mainly P/Q-type Ca(2+) channels.
Assuntos
Canais de Cálcio/metabolismo , Corpo Estriado/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Receptores de Dopamina D1/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio Tipo P/efeitos dos fármacos , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/efeitos dos fármacos , Canais de Cálcio Tipo Q/metabolismo , Corpo Estriado/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Inibidores Enzimáticos/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Receptores de Dopamina D1/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/efeitos dos fármacosRESUMO
The sudden interruption of an intracortical instillation of exogenous gamma-aminobutyric acid (GABA) generates an epileptic focus in mammals. Seizures elicited by GABA withdrawal (GW) last for weeks. A similar withdrawal-induced hyperexcitability is also produced by several GABA(A) receptor agonists. This work reports a quantitative analysis of GW-induced hyperexcitability produced in the hippocampus in vitro. GW produced a left-ward displacement of the input/output (I/O) function, suggesting that the postsynaptic component is predominant to explain the hyperexcitability. A decrease in the inhibitory efficacy of the GABA(A) receptor agonist, muscimol, confirmed that inhibition was impaired. Binding saturation experiments demonstrated a decrease in [(3)H]-muscimol binding after GABA withdrawal showing a close correlation with the development of hyperexcitability. All these modifications coursed without changes in receptor affinity (K(D)) for muscimol or bicuculline as demonstrated by both binding studies and Schild analysis. It is concluded that, in the CA1 region of the hippocampus, it is the number of functional GABA(A) receptors, and not the affinity of the receptor, what is decreased during GW-induced hyperexcitability.
Assuntos
Hipocampo/fisiologia , Receptores de GABA-A/fisiologia , Ácido gama-Aminobutírico/farmacologia , Animais , Bicuculina/farmacologia , Regulação para Baixo , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Cinética , Masculino , Muscimol/farmacologia , Ratos , Ratos Wistar , Receptores de GABA-A/efeitos dos fármacos , Síndrome de Abstinência a SubstânciasRESUMO
The release of glutamate from striatal synaptosomes induced by depolarisation with 4-aminopyridine (4-AP) was studied by a method based on the fluorescent properties of the NAPDH formed by the metabolism of the neurotransmitter by glutamate dehydrogenase.Ca(2+)-dependent, depolarisation-induced glutamate release was inhibited in a concentration-dependent manner by the selective histamine H(3) agonist immepip. Best-fit estimates were: maximum inhibition 60+/-10% and IC(50) 68+/-10 nM. The effect of 300 nM immepip on depolarisation-evoked glutamate release was reversed by the selective H(3) antagonist thioperamide in a concentration-dependent manner (EC(50) 23 nM, K(i) 4 nM). In fura-2-loaded synaptosomes, the increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) evoked by 4-AP-induced depolarisation (resting level 167+/-14 nM; Delta[Ca(2+)](i) 88+/-15 nM) was modestly, but significantly reduced (29+/-5% inhibition) by 300 nM immepip. The action of the H(3) agonist on depolarisation-induced changes in [Ca(2+)](i) was reversed by 100 nM thioperamide. Taken together, our results indicate that histamine modulates the release of glutamate from corticostriatal nerve terminals. Inhibition of depolarisation-induced Ca(2+) entry through voltage-dependent Ca(2+) channels appears to account for the effect of H(3) receptor activation on neurotransmitter release. Modulation of glutamatergic transmission in rat striatum may have important consequences for the function of basal ganglia and therefore for the control of motor behaviour.
Assuntos
Corpo Estriado/metabolismo , Regulação para Baixo/fisiologia , Antagonistas de Aminoácidos Excitatórios/metabolismo , Ácido Glutâmico/metabolismo , Receptores Histamínicos H3/metabolismo , Sinaptossomos/metabolismo , Animais , Corpo Estriado/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Imidazóis/farmacologia , Masculino , Piperidinas/farmacologia , Ratos , Ratos Wistar , Receptores Histamínicos H3/fisiologia , Sinaptossomos/efeitos dos fármacosRESUMO
Glial glutamate receptors are likely to play a role in plasticity, learning, and memory and in a number of neuropathologies. An enhanced glutamate-dependent tyrosine phosphorylation has been detected in such processes. Using primary cultures of chick Bergmann glia cells and chick cerebellar slices, we addressed whether glial glutamate receptors can activate the nonreceptor tyrosine kinase pp125 focal adhesion kinase (pp125(FAK)). A dose- and time-dependent tyrosine phosphorylation of pp125(FAK) was found in both preparations upon glutamate treatment. This effect was mediated through alpha-amino-3-hydroxy-5-methyl-4-isoaxazolepropionate (AMPA)/kainate (KA) receptors, as shown by its inhibition by the specific antagonists 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7- sulfonamide (NBQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) and the lack of effect of metabotropic agonists. FAK tyrosine phosphorylation was dependent on phosphatidylinositol 3-kinase activity. As expected, an increase in pp125(FAK) catalytic activity was found upon glutamate treatment. Immunprecipitation experiments demonstrated that FAK associates with ionotropic glutamate receptors. Taken together, these results suggest a role for glial glutamate receptors in cytoskeletal rearrengments and focal adhesion contact formation and provide new insight into the signaling transactions elicited by this neurotransmitter in glial cells.
Assuntos
Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Cerebelo/metabolismo , Ácido Glutâmico/metabolismo , Neuroglia/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de AMPA/metabolismo , Animais , Anticorpos/farmacologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/embriologia , Embrião de Galinha , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Proteína-Tirosina Quinases de Adesão Focal , Ácido Glutâmico/farmacologia , Immunoblotting , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/efeitos dos fármacos , Receptores de AMPA/efeitos dos fármacos , Receptores de Ácido Caínico/efeitos dos fármacos , Receptores de Ácido Caínico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tirosina/metabolismoRESUMO
BACKGROUND: Histamine stimulates cell proliferation in some tumor cell lines through the activation of H(1) receptors coupled to phosphoinositide hydrolysis. We therefore set out to study the presence of H(1) receptors in the prostate cancer cell line DU-145 and the effect of their stimulation on cell growth. METHODS: The presence of histamine receptors was studied by radioligand binding. Phosphoinositide hydrolysis was assessed by measuring [(1)H]-inositol phosphate ([(1)H]-IPs) accumulation and changes in the intracellular concentration of free Ca(2+) ([Ca(2+)](i)). Proliferation was assessed by cell counting and by [(1)H]-thymidine incorporation. RESULTS: DU-145 cells express H(1) receptors (110+/-14 fmol/mg of protein) whose stimulation results in [(1)H]-IPs accumulation (602+/-23% of basal, EC(50) 2.2+/-0.4 microM) and calcium mobilization (resting level 96+/-5 nM, Delta[Ca(2+)](i) 517+/-32 nM, EC(50) 6.2+/-0.1 microM). Incubation with histamine (100 microM, 24 hr) resulted in a decrease in both cell number and [(1)H]-thymidine incorporation, blocked by the H(1) antagonist mepyramine (1 microM). CONCLUSIONS: Histamine inhibits the proliferation of DU-145 cells through the activation of H(1) receptors coupled to phosphoinositide hydrolysis.
Assuntos
Adenocarcinoma/patologia , Divisão Celular , Neoplasias da Próstata/patologia , Receptores Histamínicos H1/fisiologia , Cálcio/metabolismo , Sinalização do Cálcio , Humanos , Hidrólise , Masculino , Fosfatidilinositóis/farmacologia , Células Tumorais CultivadasRESUMO
1. A study was made of the regulation of [(3)H]-gamma-aminobutyric acid ([(3)H]-GABA) release from slices of rat striatum by endogenous dopamine and exogenous histamine and a histamine H(3)-agonist. Depolarization-induced release of [(3)H]-GABA was Ca(2+)-dependent and was increased in the presence of the dopamine D(2) receptor family antagonist, sulpiride (10 microM). The sulpiride-potentiated release of [(3)H]-GABA was strongly inhibited by the dopamine D(1) receptor family antagonist, SCH 23390 (1 microM). Neither antagonist altered basal release. 2. The 15 mM K(+)-induced release of [(3)H]-GABA in the presence of sulpiride was inhibited by 100 microM histamine (mean inhibition 78+/-3%) and by the histamine H(3) receptor-selective agonist, immepip, 1 microM (mean inhibition 81+/-5%). The IC(50) values for histamine and immepip were 1.3+/-0.2 microM and 16+/-2 nM, respectively. The inhibitory effects of histamine and immepip were reversed by the H(3) receptor antagonist, thioperamide, 1 microM. 3. The inhibition of 15 mM K(+)-induced [(3)H]-GABA release by immepip was reversed by the H(3) receptor antagonist, clobenpropit, K(d) 0.11+/-0.04 nM. Clobenpropit alone had no effect on basal or stimulated release of [(3)H]-GABA. 4. Elevated K(+) caused little release of [(3)H]-GABA from striatal slices from reserpinized rats, unless the D(1) partial agonist, R(+)-SKF 38393, 1 microM, was also present. The stimulated release in the presence of SKF 38393 was reduced by 1 microM immepip to the level obtained in the absence of SKF 38393. 5. These observations demonstrate that histamine H(3) receptor activation strongly inhibits the dopamine D(1) receptor-dependent release of [(3)H]-GABA from rat striatum; primarily through an interaction at the terminals of GABA neurones.
Assuntos
Neostriado/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores Histamínicos H3/metabolismo , Tioureia/análogos & derivados , Ácido gama-Aminobutírico/metabolismo , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Animais , Cálcio/farmacologia , Dopamina/metabolismo , Antagonistas dos Receptores de Dopamina D2 , Histamina/farmacologia , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Imidazóis/antagonistas & inibidores , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Neostriado/efeitos dos fármacos , Piperidinas/antagonistas & inibidores , Piperidinas/farmacologia , Potássio/farmacologia , Ratos , Ratos Wistar , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D2/metabolismo , Reserpina/farmacologia , Sulpirida/antagonistas & inibidores , Sulpirida/farmacologia , Tioureia/farmacologiaRESUMO
In human astrocytoma U373 MG cells that express histamine H1 receptors (180 +/- 6 fmol/mg protein) but not H2 or H3 receptors, histamine stimulated mitogenesis as assessed by [3H]-thymidine incorporation (173 +/- 2% of basal; EC50, 2.5 +/- 0.4 microM). The effect of 100 microM histamine was fully blocked by the selective H1 antagonist mepyramine (1 microM) and was markedly reduced (93 +/- 4% inhibition) by the phospholipase C inhibitor U73122 (10 microM). The activator of protein kinase C (PKC) phorbol 12-tetradecanoyl-13-acetate (TPA, 100nM) stimulated [3H]-thymidine incorporation (270 +/- 8% of basal), and this response was not additive with that to 100 microM histamine. The incorporation of [3H]-thymidine induced by 100 microM histamine was partially reduced by the PKC inhibitor Ro 31-8220 (57 +/- 7% inhibition at 300 nM) and by the compound PD 098,059 (30 microM, 62 +/- 14% inhibition), an inhibitor of the mitogen-activated kinase (MAPK) kinases MEK1/MEK2. These results show that histamine H1 receptor activation stimulates the proliferation of human astrocytoma U373 MG cells. The action of histamine appears to be partially mediated by PKC stimulation and MAPK activation.
Assuntos
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Histamina/farmacologia , Receptores Histamínicos H1/metabolismo , Células Tumorais Cultivadas/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Divisão Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Humanos , Fosfatos de Inositol/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Pirilamina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Timidina/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
1. The effects of chronic pharmacological modulation of L-type Ca2+ channel activity on the cell surface expression of Na+ channels were examined in GH3 cells. 2. Prolonged inhibition (4-5 days) of L-channels with nimodipine caused a 50-60 % decrease in the peak amplitude of whole-cell Na+ currents recorded with the patch-clamp technique. On the contrary, prolonged exposure to the L-channel agonist Bay K 8644 induced an approximately 2.5-fold increase in peak Na+ current. In both cases, there were only minor changes in cell capacitance and no significant changes in Na+ channel gating properties. 3. Measurements of the specific binding of radiolabelled saxitoxin to intact cells showed that nimodipine treatment reduced the number of cell surface Na+ channels, whereas treatment with Bay K 8664 produced the opposite effect. The dual regulation of Na+ channel abundance explained the mentioned changes in Na+ current amplitude. 4. Plasma membrane Na+ channels had a half-life of approximately 17 h both in control cells and in cells treated with Bay K 8644, as estimated from the rate of decay of peak Na+ current after inhibition of protein synthesis with cycloheximide. Actinomycin D, an inhibitor of gene transcription, and also cycloheximide, occluded the stimulatory effect of Bay K 8644 on Na+ current density when measured over a 24 h period. 5. These findings indicate that the entry of Ca2+ through L-type channels influences in a positive way the number of functional Na+ channels in GH3 cells, and suggest that Ca2+ influx stimulates either Na+ channel gene expression or the expression of a regulatory protein that promotes translocation of pre-assembled Na+ channels into the plasma membrane.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Hipófise/metabolismo , Canais de Sódio/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Eletrofisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Nimodipina/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Hipófise/citologia , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Saxitoxina/metabolismo , Canais de Sódio/fisiologia , Transcrição Gênica/efeitos dos fármacosRESUMO
In synaptosomes from rat substantia nigra pars reticulata, [3H] gamma-aminobutyric acid (GABA) uptake was inhibited by GABA, (+/-)-nipecotic acid, beta-alanine and SKF 89976-A. Inhibition was concentration-dependent and monophasic, with IC50 values that agree with those reported for the cloned rat GABA transporter GAT-1. [3H]GABA uptake was modestly, but significantly, reduced (21 +/- 3% inhibition) by 100 nM phorbol 12-tetradecanoyl-13-acetate (TPA), an activator of protein kinase C (PKC). The inhibitory action of TPA was reversed by the PKC inhibitor staurosporine (100 nM). Saturation analysis revealed that TPA reduced the maximum capacity of transport with no change in the affinity for GABA. [3H]GABA uptake was unaffected by either forskolin (10 microM) or 8-bromo-cAMP (500 microM). These results indicate that SNr GABAergic afferents express the GAT-1 transporter whose activity can be regulated by a PKC-mediated mechanism.
Assuntos
Carcinógenos/farmacologia , Prolina/análogos & derivados , Substância Negra/metabolismo , Sinaptossomos/enzimologia , Acetato de Tetradecanoilforbol/farmacologia , Ácido gama-Aminobutírico/farmacocinética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Gânglios da Base/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Colforsina/farmacologia , Inibidores Enzimáticos/farmacologia , GABAérgicos/farmacologia , Ácidos Nipecóticos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Estaurosporina/farmacologia , Sinaptossomos/efeitos dos fármacos , Trítio , beta-Alanina/farmacologiaRESUMO
We report herein the synthesis of a novel DNA delivery system and in vitro evidence of its ability to transfect cell lines by binding to the high-affinity neurotensin receptor and subsequent internalization of ligand-receptor complexes. The targeting vehicle consisted of neurotensin crosslinked with poly-L-lysine via N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). The SPDP-derivatives with either neurotensin or poly-L-lysine were purified by gel filtration. The conjugate resulting of the reaction of neurotensin-SPDP with HS-SPDP-poly-L-lysine was purified through Biogel A 1.5. The neurotensin-SPDP-poly-L-lysine conjugate was able to bind plasmidic DNAs (pSV2cat and pGreen Lantern-1) at optimal molar ratios of 1:5 and 1:6 (DNA: conjugate), respectively. The conjugate internalized those plasmids in the cell lines (N1E-115 and HT-29) bearing the high-affinity neurotensin receptor. Expression of the plasmid products, chloramphenicol acetyltransferase and green fluorescent protein, was observed in such cell lines. Both internalization and expression of the plasmids transferred by the neurotensin-SPDP-poly-L-lysine conjugate were prevented by neurotensin (1 microM) and SR-48692 (100 nM), a specific antagonist of the high-affinity neurotensin receptor. The neurotensin-SPDP-poly-L-lysine conjugate was unable to transfect cell lines lacking the neurotensin receptor (COS-7 and L-929). In rat brain, the high-affinity neurotensin receptor is expressed by specific neurons such as those of the nigrostriatal and mesolimbic dopaminergic systems. Therefore, the neurotensin-SPDP-poly-L-lysine conjugate could be a useful tool for gene delivery to those neuronal systems.