RESUMO
Modulation of neurotrophic factors to protect neurons from damage is proposed as a novel mechanism for the action of antidepressants. However, the effect of antidepressants on modulation of glial cell line-derived neurotrophic factor (GDNF), which has potent and widespread effects, remains unknown. Here, we demonstrated that long-term use of antidepressant treatment significantly increased GDNF mRNA expression and GDNF release in time- and concentration-dependent manners in rat C6 glioblastoma cells. Amitriptyline treatment also increased GDNF mRNA expression in rat astrocytes. GDNF release continued for 24 h following withdrawal of amitriptyline. Furthermore, following treatment with antidepressants belonging to several different classes (amitriptyline, clomipramine, mianserin, fluoxetine and paroxetine) significantly increased GDNF release, but which did not occur after treatment with non-antidepressant psychotropic drugs (haloperidol, diazepam and diphenhydramine). Amitriptyline-induced GDNF release was inhibited by U0126 (10 microM), a mitogen-activated protein kinase (MAPK)-extracellular signal-related kinase (ERK) kinase (MEK) inhibitor, but was not inhibited by H-89 (1 microM), a protein kinase A inhibitor, calphostin C (100 nM), a protein kinase C inhibitor and PD 169316 (10 microM), a p38 mitogen-activated protein kinase inhibitor. These results suggested that amitriptyline-induced GDNF synthesis and release occurred at the transcriptional level, and may be regulated by MEK/MAPK signalling. The enhanced and prolonged induction of GDNF by antidepressants could promote neuronal survival, and protect neurons from the damaging effects of stress. This may contribute to explain therapeutic action of antidepressants and suggest new strategies of pharmacological intervention.
Assuntos
Antidepressivos/farmacologia , Glioblastoma/metabolismo , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Sulfonamidas , Amitriptilina/administração & dosagem , Amitriptilina/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Butadienos/farmacologia , Linhagem Celular , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Imidazóis/farmacologia , Isoquinolinas/farmacologia , Cinética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Naftalenos/farmacologia , Proteínas do Tecido Nervoso/genética , Nitrilas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Psicotrópicos/farmacologia , Ratos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
BACKGROUND: Recent advances in molecular biology, such as polymerase chain reaction (PCR) differential display, have enabled the screening of mRNAs transcriptionally regulated by chronic ethanol treatment. Screening of gene expression after ethanol exposure will be the most needed for new biological insights into alcoholism. METHODS: We used PCR differential display to detect differentially expressed RNAs in N18TG2 cells treated for 4 days with physiologic concentrations of ethanol (25 mM). RESULTS: We succeeded in identifying two differentially expressed RNAs in the ethanol-treated cells. The increase in the expression of the two RNAs was verified by Northern hybridization analysis. Sequence analyses and searches of the sequence databases revealed that one of the RNAs was that of the heat shock cognate protein 73 (HSC73) gene and that the other was the product of a novel gene. The increase in the level of HSC73 mRNA after ethanol administration was consistent with similar reports from other laboratories, and indicated that our assay system would be applicable to the screening of up-regulated gene expression during ethanol treatment. Rapid amplification of cDNA 5'-ends (5'-RACE) allowed us to determine the upstream sequence of the uncharacterized mRNA that would code for a protein of 113 amino acids. A homology search by MPsrch indicated very low homology to the calcium channel L-type alpha I subunit. CONCLUSIONS: The function of this new gene product is presently unknown, but our results indicate that an investigation of the pathophysiological significance of the gene in alcoholism would be worthwhile. Identification of genes that are influenced by chronic ethanol will certainly increase of the molecular mechanisms underlying physiologic dependence.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/efeitos dos fármacos , Reação em Cadeia da Polimerase/métodos , Animais , Proteínas de Choque Térmico/metabolismo , Camundongos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
To improve access to psychiatric consultation for cancer patients as well as non-cancer patients with psychiatric disorders, a psychiatric liaison programme to communicate closely with physicians and ward staff regarding anticipated psychiatric morbidity in patients, was introduced in each ward of a general hospital. The rate of psychiatric consultation referrals for cancer patients was significantly higher after the psychiatric liaison programme was established. The programme had a greater impact on the rate of psychiatric consultation in a unit with cancer patients who were informed of their diagnoses. The greater consultation rates in cancer patients after the liaison programme might be, in part, associated with the physicians' attitude toward the more open disclosure of the cancer diagnosis.
Assuntos
Neoplasias/psicologia , Encaminhamento e Consulta , Idoso , Feminino , Humanos , Relações Interprofissionais , Masculino , Pessoa de Meia-Idade , Neoplasias/complicações , Defesa do Paciente , Psiquiatria , Revelação da VerdadeRESUMO
Although there are several reports on the alteration of intracellular signal transduction during heat shock in somatic cells, the long term effects of heat shock on neuronal cells remain unknown. In this report, we investigated cyclic AMP (cAMP) accumulation and the expression of heat shock proteins following heat shock in mouse neuroblastoma N18TG2 cells. Basal cAMP accumulation, or that stimulated by serotonin (10 microM), cholera toxin (1 microg/ml), and forskolin (1 microM) was suppressed at 0, 3, and 6 h following heat shock (45 degrees C for 30 min). The cAMP levels were restored at 15 and 24 h after heat shock, corresponding with the expression of stress-induced heat shock protein 72 (HSP72). Quercetin, an inhibitor of HSP expression, decreased the expression of HSP72 and inhibited the recovery of cAMP levels 24 h after heat shock. Quercetin also decreased the basal expression of the constitutive heat shock cognate protein 70 (HSC70) and suppressed cAMP accumulation in non-heat shocked cells. These results suggest that stress-induced HSP72 restores cAMP accumulation to control levels following heat shock and that constitutive HSC70 is related to cAMP levels in non-stress conditions.
Assuntos
AMP Cíclico/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Animais , Western Blotting , Proteínas de Transporte/biossíntese , Proteínas de Transporte/metabolismo , Toxina da Cólera/farmacologia , Colforsina/farmacologia , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP72 , Proteínas de Choque Térmico/análise , Proteínas de Choque Térmico/biossíntese , Camundongos , Neuroblastoma , Quercetina/farmacologia , Serotonina/farmacologia , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Our previous study has demonstrated that astrocytes derived from the rat frontal cortex contain 5-hydroxytryptamine (5-HT)7 receptors positively coupled to adenylyl cyclase. In this study, we observed a desensitization of 5-HT7 receptors induced by a treatment with agonists (0.1, 1, and 10 muM, 0.5 to 3.5 h). Maximum responses, but not the EC50 values, in the concentration-response curve of 5-HT-induced cyclic AMP formation were decreased after pretreatment with 5-HT. Pretreatment with 5-carboxamidotryptamine (5-CT) elicited a potent desensitization of 5-HT-induced cyclic AMP formation. Neither 2-methyl-5-HT nor alpha-methyl-5-HT caused the desensitization. When the astrocytes were treated with isoproterenol, N-ethylcarboxamidoadenosine, and dibutyryl cyclic AMP (all of which increase intracellular cyclic AMP levels), 5-HT-induced cyclic AMP responses were not affected. Conversely, adenylyl cyclase activity mediated by either isoproterenol or N-ethylcarboxamidoadenosine was attenuated by pretreatment with each of these agonists, but not 5-HT. In addition, our study showed that the administration of 5-HT, 5-CT, and 8-hydroxy-2-(di-n-propylamino)tetralin to the astrocytes stimulated cyclic AMP formation both in the presence and absence of forskolin, whereas in neuron-rich cultures of the frontal cortex, these agonists did not change basal cyclic AMP levels and decreased forskolin-stimulated cyclic AMP formation. Neurons may predominantly contain 5-HT1A receptors that are negatively coupled to adenylyl cyclase. These results suggest that 5-HT7 receptors are highly expressed in astrocytes but not in neuronal cells, and that pretreatment with their agonists results in a homologous desensitization of the receptors.
Assuntos
Adenilil Ciclases/metabolismo , Astrócitos/enzimologia , Córtex Pré-Frontal/enzimologia , Receptores de Serotonina/efeitos dos fármacos , Agonistas do Receptor de Serotonina/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/efeitos dos fármacos , Inibidores de Proteínas Quinases , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Astrocytes derived from rat frontal cortex contain 5-hydroxytryptamine7 (5-HT)7 receptors positively coupled to adenylyl cyclase. In the present study, we investigated the effects of glucocorticoids on adenylyl cyclase activity and 5-HT7 receptor gene expression in astrocytes. Addition of dexamethasone (0.01-100 nM, 12-72 h) to the culture medium decreased cyclic AMP formation induced by 5-HT in a time- and concentration-dependent manner. Dexamethasone treatment (10 nM, 48 h) reduced maximum responses of cyclic AMP formation induced by 5-HT and 5-carboxamidotryptamine without alterations in the EC50 value. In contrast, treatment with the mineralocorticoid aldosterone (48 h) had no significant effects on 5-HT-induced cyclic AMP formation with concentrations up to 10 nM. It was observed that dexamethasone treatment (1-100 nM, 3-72 h) also decreased the expression of 5-HT7 receptor mRNA, using reverse transcription and polymerase chain reaction analysis. A significant reduction in expression of 5-HT7 mRNA appeared at 3 h of dexamethasone treatment and reached a maximum at 6 h of treatment. On the other hand, dexamethasone treatment (10 nM, 48 h) did not affect basal levels of cyclic AMP and cyclic AMP synthesis stimulated by isoproterenol, N-ethylcarboxamidoadenosine, cholera toxin, and forskolin. These results suggest that dexamethasone decreases the expression of the 5-HT7 receptor gene and, consequently, 5-HT7 receptor-mediated signal transduction in frontocortical astrocytes.
Assuntos
Astrócitos/química , Dexametasona/farmacologia , Regulação para Baixo/efeitos dos fármacos , Glucocorticoides/farmacologia , Receptores de Serotonina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida) , Agonistas Adrenérgicos beta/farmacologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Células Cultivadas , Toxina da Cólera/farmacologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Lobo Frontal/citologia , Expressão Gênica/efeitos dos fármacos , Isoproterenol/farmacologia , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/genética , Serotonina/farmacologia , Vasodilatadores/farmacologiaRESUMO
We have investigated the effects of interleukin (IL)-1 beta and lipopolysaccharide (LPS) on endothelin (ET)-induced intracellular Ca2+ rise in C6 rat glioma cells in order to study the mechanisms of their effects on Ca2+ signaling systems. Pretreatment with IL-1 beta (10(3) U/mL) and LPS (1 microgram/mL) for 24 h significantly inhibited 100 nM ET-1-induced increase in intracellular Ca2+ either in the presence or absence of external Ca2+. Their inhibitory effects were in dosedependent (IL-1 beta; 50-1000 U/mL, LPS; 10-1000 ng/mL) and time-dependent (12-24 h) manners. A tyrosine kinase antagonist genistein (50 microM) but not a protein kinase C inhibitor H7 (30 microM) prevented the inhibition of the ET response by IL-1 beta and LPS. These results suggest that activation of tyrosine kinase may be essential for the inhibition of the ET receptor-mediated Ca2+ signaling systems by IL-1 beta and LPS.
Assuntos
Cálcio/metabolismo , Endotelina-1/farmacologia , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Inibidores Enzimáticos/farmacologia , Genisteína , Glioma , Isoflavonas/farmacologia , Cinética , Proteínas Tirosina Quinases/antagonistas & inibidores , Ratos , Células Tumorais CultivadasRESUMO
In rat frontal cortical astrocytes, 5-hydroxytryptamine (5-HT), 5-carboxamidotryptamine and 5-methoxytryptamine elicited a concentration-dependent increase of cyclic AMP accumulation, with EC50 values of 137 +/- 7 nM (5-HT), 10.4 +/- 3.9 nM (5-carboxamidotryptamine) and 57.8 +/- 13.6 nM (5-methoxytryptamine). Accumulation of cyclic AMP stimulated by 5-HT (10 microM) was inhibited by methiothepin (IC50 = 21 +/- 13 nM) but not by pindolol, SCH 23390, ICS 205-930 or GR 113808. These pharmacological effects on cyclic AMP formation in astrocytes are consistent with those observed in 5-HT7 receptor binding studies. Indeed, reverse transcription-polymerase chain reaction analysis revealed the presence of 5-HT7 mRNA in astrocytes. Chronic exposure of astrocytes to mianserin (0.05-4 microM) or amitriptyline (1-8 microM) for 3 days produced a concentration-dependent enhancement of 5-HT-stimulated cyclic AMP accumulation, with EC50 values of 0.47 +/- 0.04 microM (mianserin) and 2.5 +/- 0.3 microM (amitriptyline). The enhancement of cyclic AMP accumulation was time dependent, reaching maximal levels within 2 days. Mianserin exposure (2 microM, for 3 days) resulted in a comparable enhancement of cyclic AMP accumulation mediated by 5-carboxamidotryptamine and 5-methoxytryptamine, whereas this treatment had no effect on cyclic AMP production induced by G protein-specific stimulators and catalytic subunit-selective stimulators. The mianserin-induced enhancement of 5-HT-stimulated cyclic AMP accumulation was decreased by methiothepin (IC50 = 15 +/- 8 nM) and significantly attenuated by pretreatment with 5-HT7 receptor antisense oligonucleotides, suggesting that chronic mianserin exposure produces an increase in 5-HT7 receptor activity. Chronic exposure to maprotiline, setiptiline or clomipramine (5 microM, for 3 days) mimicked the effect of mianserin. The enhancement of 5-HT7 receptor activity after chronic antidepressant exposure may be a mechanism underlying the therapeutic effects.
Assuntos
Antidepressivos/farmacologia , Astrócitos/metabolismo , AMP Cíclico/metabolismo , Lobo Frontal/metabolismo , Receptores de Serotonina/fisiologia , Adenilil Ciclases/metabolismo , Amitriptilina/administração & dosagem , Amitriptilina/farmacologia , Animais , Antidepressivos/administração & dosagem , Astrócitos/enzimologia , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Lobo Frontal/citologia , Lobo Frontal/enzimologia , Metiotepina/farmacologia , Mianserina/administração & dosagem , Mianserina/farmacologia , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Serotonina/genética , Antagonistas da Serotonina/farmacologia , Transdução de SinaisRESUMO
In the present study, we investigated the effects of chronic in vitro administration of amitriptyline, a tricyclic antidepressant, on cyclic GMP formation stimulated by 5-hydroxytryptamine (5-HT) in the neuroblastoma x glioma hybrid cell line, NG 108-15, 5-HT (0.01-100 microM)-stimulated cyclic GMP formation was concentration-dependent and was sensitive to ICS 205-930, a 5-HT3 receptor antagonist. Exposure of NG 108-15 cells to 5 microM amitriptyline for 3 days significantly reduced 5-HT-stimulated cyclic GMP formation. Acute treatment with amitriptyline had no effect on 5-HT-stimulated cyclic GMP formation. The reduction by chronic amitriptyline exposure of 10 microM 5-HT-stimulated cyclic GMP formation was concentration-dependent over the concentration range examined (0.5 to 10 microM). The IC50 of amitriptyline was 1.9 microM. In contrast, amitriptyline exposure, even at a concentration of 8 microM, failed to modify cyclic GMP formation stimulated by bradykinin, sodium nitroprusside, or atrial natriuretic peptide. Increases in intracellular Ca2+ concentration ([Ca2+]i) evoked by 10 microM 5-HT were attenuated in amitriptyline-exposed cells, while 100 nM bradykinin-induced [Ca2+]i increases were not affected. In addition, chronic exposure to 5 microM amitriptyline caused a decrease in affinity (Kd) of [3H]zacopride specific binding to 5-HT3 recognition sites. The Bmax for the labelled ligand remained unchanged. These results suggest that chronic amitriptyline exposure reduces 5-HT-stimulated cyclic GMP formation and [Ca2+]i increases, and this may reflect the functional changes of 5-HT3 receptors.