RESUMO
Recent findings implicate the calcium-permeable transient receptor potential (TRP) melastatin subtype 2 (TRPM2) and canonical subtype 3 (TRPC3) channels in the pathogenesis of bipolar disorder (BD). As both channels are involved in calcium and oxidative stress signaling, thought to be disrupted in BD, we sought to determine the effects of elevated oxidative stress on their expression and function. Primary rat cortical neurons and astrocytes were treated with oxidative stressors for 1 (acute) and 4 days (chronic). Expression of TRPC3 and TRPM2 were determined by immunoblotting and real-time PCR. Channel functionality was assessed using a TRPC3 activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and live cell, ratiometric fluorometry with the calcium sensitive dye, Fura-2. Neurons treated with rotenone (15-30nM) for 4 days but not 24h showed significant dose-dependent decreases in TRPC3 mRNA (31%, p<0.001) and protein levels (60%, p<0.001). Similar dose-dependent attenuation of TRPC3-mediated calcium fluxes was demonstrated upon chronic rotenone exposure relative to vehicle controls. In contrast, TRPM2 mRNA but not protein levels increased (47%, p=0.017) after acute and chronic rotenone treatment. Chronic exposure of neurons to paraquat (1-2µM), an alternate oxidative stressor, similarly decreased TRPC3 expression (mRNA: 41%; protein: 61%). Unlike neurons, rotenone treatment incurred no changes in astrocyte TRPC3 levels. These findings demonstrate that TRPC3 and TRPM2 channel expression and/or function is sensitive to the redox status of rat primary neurons and that these changes are time dependent. This provides a critical mechanistic link between altered oxidative stress markers, dysfunction of these TRP channels and calcium dyshomeostasis in BD.
Assuntos
Córtex Cerebral/citologia , Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Diglicerídeos/farmacologia , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Regulação da Expressão Gênica/efeitos dos fármacos , Herbicidas/farmacologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Paraquat/farmacologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Rotenona/farmacologia , Canais de Cátion TRPC/genética , Canais de Cátion TRPM/genética , Fatores de TempoRESUMO
Chronic lithium treatment of B-lymphoblast cell lines (BLCLs) from bipolar-I disorder (BD-I) patients and healthy subjects ex vivo attenuates agonist- and thapsigargin-stimulated intracellular calcium (Ca(2+)) responses. As these findings suggest that chronic lithium treatment modifies receptor (ROCE) and/or store-operated Ca(2+) entry (SOCE) mechanisms, we determined whether chronic lithium treatment of BLCLs modified the expression of two members of the transient receptor potential channels (TRPC1 & 3), which participate in ROCE/SOCE. Chronic lithium treatment significantly reduced BLCL TRPC3 immunoreactivity (repeated-measures ANOVA, P=0.00005), with interaction effects of diagnosis (P=0.037) and sex (P=0.040). The lithium-induced decrease was greatest in BLCLs from female BD-I patients compared with those from healthy females (-27%) and with vehicle-treated BLCLs from female BD-I patients (-33%). However, lithium treatment did not affect TRPC1 and 3 mRNA levels, and TRPC1 immunoreactivity. Downregulation of TRPC3 may be an important mechanism by which lithium ameliorates pathophysiological Ca(2+) disturbances as observed in BD.
Assuntos
Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Transtorno Bipolar/sangue , Canais Iônicos/antagonistas & inibidores , Lítio/administração & dosagem , Ativação Linfocitária/efeitos dos fármacos , Adulto , Análise de Variância , Transtorno Bipolar/tratamento farmacológico , Células Cultivadas , Feminino , Humanos , Canais Iônicos/sangue , Canais Iônicos/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/sangue , RNA Mensageiro/genética , Canais de Cátion TRPCRESUMO
BACKGROUND: As altered storage-operated calcium (Ca(2+)) entry (SOCE) may affect Ca(2+) homeostasis in bipolar disorder (BD), we determined whether changes occur in the expression of TRPC7 and SERCA2s, proteins implicated or known to be involved in SOCE, in B lymphoblast cell lines (BLCLs) from BD-I patients and comparison subjects. METHODS: mRNA levels were determined in BLCL lysates from BD-I, BD-II, and major depressive disorder patients, and healthy subjects by comparative reverse transcriptase-polymerase chain reaction, and BLCL basal intracellular Ca(2+) concentration ([Ca(2+)]B) was determined by ratiometric spectrophotometry using Fura-2, in aliquots of the same cell lines, at 13-16 passages in culture. RESULTS: TRPC7 mRNA levels were significantly lower in BLCLs from BD-I patients with high BLCL [Ca(2+)]B compared with those showing normal [Ca(2+)]B (-33%, p =.017) and with BD-II patients (-48%, p =.003), major depressive disorder patients (-47%, p =.049) and healthy subjects (-33%, p =.038). [Ca(2+)]B also correlated inversely with TRPC7 mRNA levels in BLCLs from the BD-I group as a whole (r = -.35, p =.027). CONCLUSIONS: Reduced TRPC7 gene expression may be a trait associated with pathophysiological disturbances of Ca(2+) homeostasis in a subgroup of BD-I patients.
Assuntos
Transtorno Bipolar/genética , Canais de Cálcio/genética , Canais Iônicos , Proteínas de Membrana , Adulto , Linfócitos B , Transtorno Bipolar/classificação , Transtorno Bipolar/diagnóstico , Transtorno Bipolar/psicologia , Cálcio/fisiologia , Linhagem Celular , Transtorno Depressivo Maior/diagnóstico , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/psicologia , Feminino , Expressão Gênica/fisiologia , Homeostase/genética , Homeostase/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPC , Canais de Cátion TRPMRESUMO
The latency in onset of antimanic and mood stabilizing effects of lithium suggest that long-term neuronal adaptations mediated by changes in gene expression may be important to the therapeutic action of lithium treatment. Using differential display-polymerase chain reaction, several novel, hitherto unexpected lithium-regulated genes have been isolated, all of which would not have been predicted with the candidate gene approach. During the process of characterizing one of these novel genes, we have identified a cDNA clone, a homolog of human/mouse transmembrane-4-superfamily (also known as tetraspan) protein, CD151, the expression of which was significantly decreased in rat frontal cortex following chronic (five weeks) lithium treatment. The reduction of CD151 mRNA levels was also observed following chronic administration of carbamazepine and valproate. Conversely, the expression of CD151 was not altered by short-term (one week) lithium treatment and by chronic administration of the tricyclic antidepressant, imipramine, or the typical antipsychotic, haloperidol, further demonstrating time dependence and pharmacological specificity of this effect. Our studies, thus, indicate that CD151 may represent a therapeutically relevant target common to lithium and the anticonvulsant mood stabilizing drugs, carbamazepine and valproate.
Assuntos
Antígenos CD/efeitos dos fármacos , Transtorno Bipolar/tratamento farmacológico , Afeto/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antipsicóticos/farmacologia , Transtorno Bipolar/psicologia , Northern Blotting , Células Cultivadas , Clonagem Molecular , DNA Complementar/genética , Haloperidol/farmacologia , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraspanina 24RESUMO
Reduced inositol monophosphatase (IMPase) activity and elevated basal intracellular calcium levels ([Ca(2+)](B)) have been reported in B lymphoblast cell lines (BLCLs) from bipolar I affective disorder (BD-I) patients, which may reflect cellular endophenotypes of this disorder. As the PI cycle couples to intracellular Ca(2+) mobilization, these two putative endophenotypes may be related. Using an RT-PCR assay, mRNA levels were estimated for IMPA1 and 2 genes encoding human IMPase 1 and 2, respectively, in BLCLs phenotyped on [Ca(2+)](B), from patients with a DSM-IV diagnosis of BD-I (n = 12 per phenotype) and from age- and sex-matched healthy subjects (n = 12). IMPA2 mRNA levels were significantly lower in BLCLs from male BD-I patients with high [Ca(2+)](B) (n = 6) compared with healthy male subjects (n = 5) (-52%, P = 0.013), male BD-I patients with normal BLCL [Ca(2+)](B) (n = 8) (-42%, P = 0.003) and female BD-I patients with high [Ca(2+)](B) (n = 6) (-59%, P = 0.0004). A significant negative correlation was observed between IMPA2 mRNA levels and [Ca(2+)](B) in BLCLs from male (P = 0.046), but not female BD-I patients. Sex-dependent differences were also evident in postmortem temporal cortex IMPA2 mRNA levels which, in contrast to BLCLs, were significantly higher in male BD-I subjects compared with male controls (P = 0.025, n = 4/group). Collectively, these observations suggest a potential sex-dependent link between abnormalities in IMPA2 expression and calcium homeostasis in the pathophysiology of BD.
Assuntos
Transtorno Bipolar/genética , Transtorno Bipolar/metabolismo , Cálcio/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Adulto , Linfócitos B/metabolismo , Transtorno Bipolar/tratamento farmacológico , Feminino , Expressão Gênica , Homeostase/genética , Humanos , Lítio/uso terapêutico , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Transdução de Sinais/genética , Lobo Temporal/metabolismoRESUMO
The mechanisms underlying the therapeutic effects of lithium are largely unknown but may involve progressive adaptive alterations at the level of gene expression. Using differential display PCR, we identify a novel cDNA fragment, the expression of which was increased in the rat frontal cortex after 5 weeks of lithium administration. A full-length cDNA (2954-nt) was cloned by arrayed cDNA library screening, and sequencing of the clone revealed an open reading frame of 537-bp encoding a 179-residue protein. Amino acid sequence comparisons revealed that our clone is a member of the Nudix hydrolase family, with the highest percentage of homology (95%) being with a subtype of human diphosphoinositol polyphosphate phosphohydrolase, hDIPP2. Northern blot analysis revealed that chronic lithium treatment significantly increased rDIPP2 mRNA levels in frontal cortex, but not in hippocampus, midbrain, and cerebellum. The effect of lithium on rDIPP2 mRNA expression was not shared by two other anticonvulsant mood stabilizers, carbamazepine and valproate. Time-course studies showed that 1-week of lithium had no effect on rDIPP2 mRNA abundance in the frontal cortex. Our results suggest that DIPP2 may represent a biologically relevant target of lithium therapy, further supporting the notion that abnormalities in inositol phosphate metabolism may be significant in the pathophysiology and pharmacotherapy of bipolar disorder.
Assuntos
Hidrolases Anidrido Ácido/efeitos dos fármacos , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/isolamento & purificação , Encéfalo/efeitos dos fármacos , Fosfatos de Inositol/metabolismo , Lítio/farmacologia , Neurônios/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Hidrolases Anidrido Ácido/química , Sequência de Aminoácidos , Animais , Anticonvulsivantes/farmacologia , Sequência de Bases , Encéfalo/enzimologia , Carbamazepina/farmacologia , Clonagem Molecular/métodos , DNA Complementar/análise , Masculino , Dados de Sequência Molecular , Neurônios/enzimologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Psicotrópicos/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Ácido Valproico/farmacologiaRESUMO
BACKGROUND: Evidence of extensive cross-talk between calcium (Ca(2+))- and cAMP-mediated signaling systems suggests that previously reported abnormalities in Ca(2+) homeostasis in bipolar I (BP-I) patients may be linked to disturbances in the function of G proteins that mediate cAMP signaling. METHODS: To test this hypothesis, the beta-adrenergic agonist, isoproterenol, and the G protein activator, sodium fluoride (NaF), were used to stimulate cAMP production in B lymphoblasts from healthy and BP-I subjects phenotyped on basal intracellular calcium concentration ([Ca(2+)](B)). cAMP was measured by radioimmunoassay and [Ca(2+)](B) by ratiometric fluorometry with fura-2. RESULTS: Isoproterenol- (10 microM) stimulated cAMP formation was lower in intact B lymphoblasts from BP-I patients with high [Ca(2+)](B) (>/= 2 SD above the mean concentration of healthy subjects) compared with patients having normal B lymphoblast [Ca(2+)](B) and with healthy subjects. Although basal and NaF-stimulated cAMP production was greater in B lymphoblast membranes from male BP-I patients with high versus normal [Ca(2+)](B), there were no differences in the percent stimulation. This suggests the differences in NaF response resulted from higher basal adenylyl cyclase activity. CONCLUSIONS: These findings suggest that trait-dependent disturbances in processes regulating beta-adrenergic receptor sensitivity and G protein-mediated cAMP signaling occur in conjunction with altered Ca(2+) homeostasis in those BP-I patients with high B lymphoblast [Ca(2+)](B).
Assuntos
Transtorno Bipolar/fisiopatologia , Cálcio/fisiologia , AMP Cíclico/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Homeostase/fisiologia , Transdução de Sinais/fisiologia , Adulto , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Feminino , Homeostase/efeitos dos fármacos , Humanos , Isoproterenol/farmacologia , Masculino , Transdução de Sinais/efeitos dos fármacos , Fluoreto de Sódio/farmacologiaRESUMO
BACKGROUND: Substantial evidence indicates that lithium may exert its therapeutic effects through progressive adaptive changes at the level of gene expression; however, the study of lithium-regulated genes has been primarily undertaken with the "candidate gene" approach based on a specific testable hypothesis. The aim of our study was to identify lithium-regulated genes that would not be predicted a priori by the candidate gene approach. METHODS: Differential display polymerase chain reaction was used to isolate and identify messenger RNAs (mRNAs) that are differentially expressed in the frontal cortex of rats given lithium for 5 weeks to achieve plasma lithium concentrations of 0.6 to 0.9 mmol/L. RESULTS: A putative lithium-regulated complementary DNA fragment (LRG1) was identified. Northern blot analysis revealed that 5 weeks of lithium treatment, but not 1 week, significantly reduced LRG1 mRNA levels. LRG1 mRNA levels were similarly reduced by 5 weeks of carbamazepine, but not valproate administration. Sequence analysis and search of the GenBank database revealed that LRG1 is analogous to the sequence of the gene for rat aldolase A. CONCLUSIONS: These results demonstrate that chronic administration of lithium, but not short-term administration, down regulates the levels of aldolase A mRNA, suggesting this effect may play a role in mediating the therapeutic action of this agent.
Assuntos
Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Lítio/farmacologia , Animais , Sequência de Bases , Northern Blotting , Relação Dose-Resposta a Droga , Regulação para Baixo , Frutose-Bifosfato Aldolase/efeitos dos fármacos , Frutose-Bifosfato Aldolase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Lítio/administração & dosagem , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Ratos WistarRESUMO
Although the nucleus accumbens is assumed to be a critical brain "pleasure center," its function in humans is unknown. As animal data suggest that a unique feature of this small brain area is its high sensitivity to down-regulation of an inhibitory G protein by drugs of abuse, we compared G protein levels in postmortem nucleus accumbens with those in seven other brain regions of chronic users of cocaine, methamphetamine, and heroin, and of matched controls. Biochemical changes were restricted to the nucleus accumbens in which concentrations of G(alpha)1 and/or G(alpha)2 were reduced by 32-49% in the methamphetamine and heroin users. This selective responsiveness to these abused drugs implies a special role for the human nucleus accumbens in mechanisms of drug reinforcement and suggests that some features of the drug-dependent state (e.g., tolerance) might be related to inhibition of G(alpha)1-linked receptor activity.
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Heroína/farmacologia , Metanfetamina/farmacologia , Entorpecentes/farmacologia , Núcleo Accumbens/metabolismo , Adulto , Encéfalo/metabolismo , Cadáver , Cocaína/farmacologia , Regulação para Baixo , Feminino , Proteínas de Ligação ao GTP/metabolismo , Humanos , Masculino , Valores de Referência , Transtornos Relacionados ao Uso de Substâncias/metabolismoRESUMO
Previous observations of reduced [3H]cyclic AMP binding in postmortem brain regions from bipolar affective disorder subjects imply cyclic AMP-dependent protein kinase function may be altered in this illness. To test this hypothesis, basal and stimulated cyclic AMP-dependent protein kinase activity was determined in cytosolic and particulate fractions of postmortem brain from bipolar disorder patients and matched controls. Maximal enzyme activity was significantly higher (104%) in temporal cortex cytosolic fractions from bipolar disorder brain compared with matched controls. In temporal cortex particulate fractions and in the cytosolic and particulate fractions of other brain regions, smaller but statistically nonsignificant increments in maximal enzyme activity were detected. Basal cyclic AMP-dependent protein kinase activity was also significantly higher (40%) in temporal cortex cytosolic fractions of bipolar disorder brain compared with controls. Estimated EC50 values for cyclic AMP activation of this kinase were significantly lower (70 and 58%, respectively) in both cytosolic and particulate fractions of temporal cortex from bipolar disorder subjects compared with controls. These findings suggest that higher cyclic AMP-dependent protein kinase activity in bipolar disorder brain may be associated with a reduction of regulatory subunits of this enzyme, reflecting a possible adaptive response of this transducing enzyme to increased cyclic AMP signaling in this disorder.
Assuntos
Transtorno Bipolar/enzimologia , Encéfalo/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Autopsia , Transtorno Bipolar/tratamento farmacológico , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Citosol/enzimologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Cinética , Lítio/farmacocinética , Lítio/uso terapêutico , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peptídeos/farmacologia , Valores de Referência , Lobo Temporal/enzimologiaRESUMO
ADP-ribosylation of the stimulatory G protein alpha subunit, alpha(s), has been demonstrated in a number of different mammalian tissues. However, little is known about the occurrence and role of this process in modifying alpha(s) levels/function in human brain. In the present study, endogenous and cholera toxin (CTX)-catalyzed [32P]ADP-ribosylated products were characterized in postmortem human temporal cortex by (1) immunoprecipitation with alpha(s) antisera (RM/1), (2) comparisons of immunoblots and autoradiograms of the [32P]ADP-ribosylated products, and (3) limited protease digestion. Of the three major endogenous [32P]ADP-ribosylated products (48, 45, and 39 kDa) in postmortem brain, the 48-kDa and 45-kDa bands were clearly identified as alpha(s-L) (long isoform) and alpha(s-S) (short isoform), respectively. RM/1 immunoprecipitated the 39-kDa [32P]ADP-ribosylated protein, and overlays of immunoblots and autoradiograms showed that this product corresponded to an alpha(s)-like-immunoreactive protein. Furthermore, limited protease digestion of the 39-kDa endogenous [32P]ADP-ribosylated band generated peptide fragments similar to both endogenous and CTX-catalyzed [32P]ADP-ribosylated alpha(s-S). Two major CTX-catalyzed [32P]ADP-ribosylated products were also identified as alpha(s-L) (52 kDa) and alpha(s-S) (45 kDa). These findings clearly demonstrate that alpha(s) is a substrate for endogenous and CTX-catalyzed [32P]ADP-ribosylation in postmortem human brain. Furthermore, a lower molecular weight alpha(s)-like immunoreactive protein is also expressed in human brain and is a substrate for endogenous but not CTX-catalyzed [32P]ADP-ribosylation.
Assuntos
Encéfalo/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , NAD/metabolismo , Adenosina Difosfato Ribose/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Autopsia , Autorradiografia , Toxina da Cólera/metabolismo , Ditiotreitol/farmacologia , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Mapeamento de Peptídeos , Radioisótopos de Fósforo , Lobo Temporal/metabolismoRESUMO
We recently reported that the activity of a calcium-independent subtype of phospholipase A2 is increased in blood of patients with schizophrenia. The present investigation examined whether similar changes take place in brain of patients with this disorder, and for comparison, in patients with bipolar disorder. The activity of two classes of PLA2, calcium-stimulated and independent, were assayed in autopsied temporal, prefrontal and occipital cortices, putamen, hippocampus and thalamus of 10 patients with schizophrenia, 8 patients with bipolar disorder and 12 matched control subjects. Calcium-independent PLA2 activity was increased by 45% in the temporal cortex of patients with schizophrenia as compared with the controls but was not significantly altered in other brain areas. In contrast, calcium-stimulated PLA2 activity was decreased by 27-42% in the temporal and prefrontal cortices and putamen, with no significant alterations in other brain regions. Brain PLA2 activity was normal in patients with bipolar disorder. Calcium-stimulated PLA2 activity was normal in cortex, cerebellum and striatum of rats treated acutely or chronically with haloperidol, whereas calcium-independent PLA2 activity was decreased in striatum of chronically treated animals, indicating that altered PLA2 activity in patients with schizophrenia is unlikely to be a direct effect of medication. Studies of the cellular role played by PLA2 suggest that decreased calcium-stimulated PLA2 activity, as also occurs in striatum of chronic human cocaine users, may be due, in part, to increased dopaminergic activity in the disorder, whereas increased calcium-independent PLA2 activity may be related to abnormal fatty acid metabolism and oxidative stress in schizophrenia.
Assuntos
Encéfalo/enzimologia , Fosfolipases A/metabolismo , Esquizofrenia/enzimologia , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Adulto , Idoso , Animais , Transtorno Bipolar/enzimologia , Cálcio/metabolismo , Feminino , Humanos , Lisofosfolipase/metabolismo , Masculino , Pessoa de Meia-Idade , Fosfolipases A2 , Fosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The phosphodiesterase type IV (PDEIV) family of enzymes contributes to the metabolism of cAMP formed by the stimulation of beta-adrenergic, A2-adenosine, and H2-histamine receptors in the brain. Disturbances in cAMP-mediated signaling have been implicated in several neuropsychiatric disorders, and there is evidence that increasing cAMP levels through PDEIV inhibition improves the symptoms of these disorders. In the present study, the selective PDEIV inhibitors rolipram and Ro 20-1724, labeled with C-11, were evaluated in vivo in rats, as potential radioligands for imaging PDEIV enzymes with positron emission tomography (PET). Biodistribution experiments revealed a greater than threefold increased regional brain retention of [11C]rolipram as compared to [11C]Ro 20-1724. [nC]Rolipram uptake was higher in rat brain areas (e.g., cortical regions and olfactory system) showing higher expression of PDEIV enzymes, as determined previously using [3H]rolipram autoradiography or molecular genetic techniques. Binding of [n1C]rolipram and [11C]Ro 20-1724 was specific, since coadministration of high doses of unlabeled rolipram (10 mg/Kg, i.v.) or Ro 20-1724 (30 mg/Kg with [11C]rolipram and 10 mg/Kg with [11C]Ro 20-1724, i.v.) reduced radioactivity uptake in brain regions. Pretreatment with high doses of the PDEI selective inhibitor vinpocetine (10 mg/Kg, i.p., 15 min prior), or the noradrenaline reuptake inhibitor desipramine (10 mg/Kg, i.p., 30 min prior), or coinjection with the adenylyl cyclase activator forskolin (6.5 or 15 mg/Kg, i.v.), did not inhibit [11C]rolipram uptake in brain areas, suggesting binding selectivity for PDEIV enzymes. We conclude that [11C]rolipram, but not [11C]Ro 20-1724, is a promising radioligand for imaging the PDEIV enzymes with PET.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , 4-(3-Butoxi-4-metoxibenzil)-2-imidazolidinona/metabolismo , Inibidores de Fosfodiesterase/metabolismo , Pirrolidinonas/metabolismo , Tomografia Computadorizada de Emissão , Animais , Radioisótopos de Carbono , Masculino , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Rolipram , Especificidade por Substrato , Distribuição TecidualRESUMO
BACKGROUND: Previous studies have found altered receptor/G protein-modulated adenylyl cyclase (AC) activity in subjects with mood disorders. METHODS: To investigate whether these effects are associated with altered levels of specific isoforms of AC, we measured AC isoform I, IV and V/VI immunoreactivities in postmortem temporal cortex from nine depressed suicide victims, nine subjects with bipolar disorder (BD) and 18 age-matched non-psychiatric controls. Basal, GTPgammaS- and forskolin-stimulated AC activities were measured in the temporal cortex from the nine depressed suicide victims and their controls. RESULTS: Western blotting revealed significant reductions in immunolabeling in AC type IV (-49%; p < 0.05) in depressed suicide subjects compared to age-matched controls, but no differences were found in AC type I or type V/VI. There were no statistically significant differences in AC type I, IV or V/VI immunoreactivities between BD and matched control subjects. Functionally, there was a significant reduction in forskolin-stimulated AC activity in depressed suicide subjects compared to controls, which may be, in part, related to higher basal AC activity in the former group. LIMITATIONS: Our sample size was small with diverse subject characteristics. CONCLUSIONS: These preliminary findings suggest altered levels and/or function in AC type IV may contribute to disturbances in the postreceptor cAMP signaling cascade in depression.
Assuntos
Adenilil Ciclases/análise , AMP Cíclico/metabolismo , Transtorno Depressivo/fisiopatologia , Suicídio , Lobo Temporal/enzimologia , Adenilil Ciclases/imunologia , Adenilil Ciclases/metabolismo , Adolescente , Adulto , Idoso , Autopsia , Western Blotting , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas , Transdução de SinaisRESUMO
Elevated G protein abundance and/or function has been implicated in the pathophysiology and pharmacotherapy of bipolar affective disorder. To test the interactions between chronic lithium and carbamazepine on behavioral changes induced by cholera toxin (CTX), which catalyzes ADP-ribosylation and constitutively activates G alphas/olf, rats were given chronic dietary lithium, carbamazepine (CBZ), or regular food (REG) and injected bilaterally in the nucleus accumbens (nACC) with CTX (400 ng/ml/side) or vehicle. Locomotor activity was tested daily for 2 weeks after the injection. CTX increased locomotor activity, but a significant interaction between drug treatment and CTX reflected a two- to threefold increase of CTX-induced hyperactivity in the lithium-treated group. In contrast, on day 1, the CBZ-CTX group was significantly more active than the the LI-CTX and REG-CTX groups, both of which had suppressed locomotor activity. There was a significant reduction in CTX-catalyzed ADP ribosylation of G alphas (52 kDa and 45 kDa) in the nucleus accumbens in all three CTX-treated groups. The potentiation of the behavioral effect of CTX by lithium supports the hypothesis that lithium interacts with G proteins; however, the mechanism of interaction appears to be more complex than direct attenuation of G alphas function, as previously suggested.
Assuntos
Carbamazepina/farmacologia , Toxina da Cólera/farmacologia , Hipercinese/induzido quimicamente , Lítio/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Psicotrópicos/farmacologia , Adenosina Difosfato Ribose/metabolismo , Análise de Variância , Animais , Peso Corporal/efeitos dos fármacos , Catálise , Sinergismo Farmacológico , Proteínas de Ligação ao GTP/metabolismo , Hipercinese/metabolismo , Masculino , Atividade Motora/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Higher basal concentrations of intracellular calcium Ca2+ in platelets and lymphocytes from subjects with bipolar affective disorder than in unipolar depressed and healthy subjects implicate abnormal intracellular Ca2+ signaling in bipolar disorder. The purpose of this study was to clarify whether these intracellular Ca2+ abnormalities are trait related. METHOD: Basal Ca2+ concentration was measured by using ratiometric fluorescence assay with fura-2 for T lymphocytes and Epstein-Barr-virus-immortalized B lymphoblasts from physically healthy subjects with DSM-IV diagnoses of bipolar mood disorder (bipolar I, N = 28; bipolar II, N = 11) or major depressive disorder (N = 14), mixed psychiatric patients with non-mood disorders (N = 14), and health subjects (N = 20). Phytohemagglutinin-stimulated (10 micrograms/ml) intracellular Ca2+ levels were also determined in T lymphocytes. RESULTS: The basal Ca2+ concentration was significantly higher in the B lymphoblasts from patients with bipolar I disorder, but not bipolar II disorder or major depression, than in healthy subjects or psychiatric patients with nonmood disorders. There was a significant interaction between gender and diagnosis in the effect on basal Ca2+ levels in T lymphocytes. Contrasts of diagnoses within gender revealed significantly higher basal Ca2+ concentrations in T lymphocytes in male bipolar I patients, but not mean with bipolar II disorder or major depression, than in healthy male comparison subjects. Phytohemagglutinin-stimulated Ca2+ concentrations did not differ among groups, but the percent differences from basal Ca2+ levels were lower in bipolar I and depressed patients than in healthy subjects. CONCLUSIONS: These findings support the occurrence of abnormal calcium homeostasis in bipolar disorder and suggest that trait-dependent factors account, at least partly, for the higher basal lymphocyte Ca2+ concentration in bipolar I subjects.
Assuntos
Transtorno Bipolar/sangue , Cálcio/análise , Linfócitos/química , Adulto , Transtorno Bipolar/metabolismo , Transtorno Bipolar/fisiopatologia , Plaquetas/química , Cálcio/fisiologia , Linhagem Celular Transformada , Transformação Celular Viral , Transtorno Depressivo/sangue , Transtorno Depressivo/metabolismo , Transtorno Depressivo/fisiopatologia , Feminino , Humanos , Ativação Linfocitária , Masculino , Linfócitos T/químicaRESUMO
BACKGROUND: Magnetic resonance spectroscopy studies have suggested above-normal turnover of membrane phospholipids in brains of patients with schizophrenia. One possible explanation for these findings is increased activity of the phospholipid-catabolizing enzyme phospholipase A2 (PLA2). However, attempts to demonstrate higher PLA2 activity in the serum of subjects with schizophrenia have led to conflicting results. We hypothesized that this was due to serum PLA2 activity consisting of a family of different enzymes, with each group of investigators measuring activity of different PLA2 forms. DESIGN: Activity of PLA2 in serum samples obtained from 24 individuals with schizophrenia was compared with serum obtained from 33 age- and sex-matched control subjects, using both fluorometric and radiometric assays with different substrates. Each method had previously yielded conflicting results concerning the status of the enzyme in schizophrenia. RESULTS: With the fluorometric assay, serum PLA2 activity in individuals with schizophrenia was markedly increased by 49% compared with control subjects (P < .001). In contrast, radiometric assay of the same serum samples resulted in PLA2 activity not significantly different between patients and control subjects. Further investigations demonstrated that, whereas the radiometric assay measured activity of a calcium-dependent enzyme, the fluorometric assay detected a calcium-insensitive enzyme possessing an acid-neutral pH optimum. CONCLUSIONS: Increased calcium-independent PLA2 activity was seen in the serum of patients with schizophrenia. This change, if present also in the brain, may well explain the increased levels of phosphodiesters observed using magnetic resonance spectroscopy and therefore may contribute to the pathophysiological features of the disorder.
Assuntos
Fosfolipases A/sangue , Fosfolipídeos/metabolismo , Esquizofrenia Paranoide/enzimologia , Adulto , Idoso , Encéfalo/fisiopatologia , Cloreto de Cálcio/análise , Feminino , Fluorometria , Humanos , Concentração de Íons de Hidrogênio , Masculino , Pessoa de Meia-Idade , Fosfolipases A/análise , Fosfolipases A/classificação , Fosfolipases A2 , Fosfolipídeos/fisiologia , Escalas de Graduação Psiquiátrica/estatística & dados numéricos , Radiometria , Esquizofrenia Paranoide/metabolismo , Esquizofrenia Paranoide/fisiopatologia , Psicologia do EsquizofrênicoRESUMO
Several lines of evidence implicate altered phospholipid-dependent signal transduction (PDST) in the pathophysiology of schizophrenia. Niacin induces vasodilation through mechanisms requiring intact PDST. Thus, an altered response to a challenge dose of niacin may reflect disturbances in these signalling processes in this disorder. In the present study, niacin-induced vasodilation was estimated quantitatively in schizophrenic and comparison bipolar affective disorder and healthy subjects using thermocouple sensors to measure the change in skin temperature relative to core body and ambient room temperature. Twelve (42.9%) of 28 schizophrenic subjects did not vasodilate in response to a 200-mg niacin challenge dose, whereas only 1 of 18 (6%) bipolar disorder subjects and none of 28 controls showed impaired response (Fisher's Exact Test, p < .0001). These findings support the notion that the schizophrenic syndromes are biochemically heterogeneous and suggest the existence of a subgroup of schizophrenic subjects in whom phospholipid-dependent signalling responses may be impaired.
Assuntos
Niacina , Esquizofrenia/fisiopatologia , Transdução de Sinais/fisiologia , Membranas Sinápticas/fisiologia , Adolescente , Adulto , Idoso , Transtorno Bipolar/classificação , Transtorno Bipolar/diagnóstico , Transtorno Bipolar/fisiopatologia , Feminino , Humanos , Masculino , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Fosfolipídeos/fisiologia , Escalas de Graduação Psiquiátrica , Esquizofrenia/classificação , Esquizofrenia/diagnóstico , Temperatura Cutânea/fisiologiaRESUMO
As disturbances in guanine nucleotide binding (G) protein-coupled phosphoinositide second messenger systems have been implicated in bipolar disorder, we examined whether the abundance of G alpha q/11 and phospholipase C (PLC)-beta 1 two key transducing proteins in this signaling pathway, are altered in this disorder. Compared with the controls, immunoreactive levels of G alpha q/11 were significantly elevated by 62% (p = .047) in occipital cortex of bipolar subjects. A similar increase (52%) in the PLC-beta 1 immunolabeling was also found in the occipital cortex of the bipolar subjects, but only reached marginal statistical significance (p = .07). In contrast, frontal and temporal cortex G alpha q/11 or PLC-beta 1 immunolabeling did not differ between bipolar and control subjects. Cerebral cortical immunoreactive levels of G beta 1 or G beta 2, included as a negative control, were not different between comparison groups. These findings support and extend earlier observations suggesting that disturbances in G protein-coupled second messenger signaling pathways may play an important role in the pathophysiology of bipolar affective disorder.