RESUMO
The significance of multiple growth factors acting on individual neurons in the central nervous system is presently unclear. Cultured hippocampal neurons were used in the present study to compare the neurotrophic actions of fibroblast growth factor-2 (FGF-2) with the better characterized growth factors, insulin-like growth factor (IGF)-1 and brain-derived neurotrophic factor (BDNF). Additionally, cultures were utilized to identify possible interactions between FGF-2 and the other growth factors. Activation of the ERK and Akt pro-survival pathways, as well as neuronal survival itself, were studied. The maximal magnitude of Akt activation stimulated by FGF-2 was found to be similar to that stimulated by IGF-1 and BDNF. In contrast, IGF-1 was less effective at inducing ERK activation than were BDNF and FGF-2. All three agents were found to promote survival of neurons cultured under serum-free, low-insulin conditions, with FGF-2 surprisingly being significantly more effective than the other two peptides. Co-treatment with maximal concentrations of either IGF-1 or BDNF enhanced FGF-2-stimulated Akt and ERK activation. However, no enhancement of survival beyond that stimulated by FGF-2 was observed with co-treatment. These findings suggest that FGF-2 may play an important role in promoting the survival of hippocampal neurons. Additionally, an interesting dissociation was identified between the positive interaction of FGF-2 with both IGF-1 and BDNF in activating Akt and ERK, and the lack of enhancement of FGF-2-induced neuroprotection.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipocampo/citologia , Fatores de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Ativação Enzimática/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
1: Since all 5-HT(1) receptors couple to G(i)-type G proteins and inhibit adenylyl cyclase, the functional significance of five distinct subtypes of 5-HT(1) receptors has been unclear. 2: In previous studies we have used transfected cells to demonstrate that 5-HT(1B) receptors can couple more efficiently than 5-HT(1A) receptors to activation of extracellular signal-regulated kinase (ERK) and to inhibition of adenylyl cyclase. These findings suggested the possibility that individual 5-HT(1) receptors differentially couple to isoforms of G(ialpha). 3: In the present study we utilized a model system in which pertussis toxin resistant forms of human G(ialpha1), G(ialpha2), and G(ialpha3) were used to directly compare the coupling of human 5-HT(1A), 5-HT(1B), and 5-HT(1D) receptors to each G(ialpha) in transfected human HeLa cells. 4: 5-HT(1A) receptors displayed a preference for G(ialpha1) and G(ialpha2), relative to G(ialpha3). Pertussis toxin resistant forms of G(ialpha1), G(ialpha2), and G(ialpha3) rescued 73%, 76%, and 44%, respectively, of the ERK activation stimulated by 5-HT in the absence of pertussis toxin. 5: In contrast, pertussis toxin resistant forms of G(ialpha1), G(ialpha2), and G(ialpha3) rescued 32%, 118%, and 35% of 5-HT(1B) receptor-stimulated activity, respectively, indicating that 5-HT(1B) receptors coupled primarily through G(ialpha2). A similar preference for G(ialpha2) was found in studies of the 5-HT(1D) receptor, where toxin resistant G(ialpha1), G(ialpha2), and G(ialpha3) rescued 30%, 70%, and 40% of activity, respectively. 6: In conclusion, the observed differential coupling of 5-HT(1) receptors to isoforms of G(ialpha), provides additional evidence for our previous findings that the subtypes of 5-HT(1) receptors exhibit similar, but distinct, functions.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores de Serotonina/metabolismo , Adenilil Ciclases/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Immunoblotting , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Toxina Pertussis/farmacologia , Receptor 5-HT1B de Serotonina , Receptor 5-HT1D de Serotonina , Receptores de Serotonina/genética , Receptores 5-HT1 de Serotonina , TransfecçãoRESUMO
Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) seem to play key roles in mediating neuronal plasticity in the hippocampus. In the current studies, we have used cultured hippocampal neurons to study possible interactions between the two growth factors in modulating neuronal signaling pathways. BDNF and IGF-1 were found to each effectively activate the neuroprotective Akt pathway, with the magnitude of activation being at least additive when cultures were simultaneously treated with supramaximal concentrations of peptides. Likewise, a cumulative inhibitory Akt-dependent phosphorylation of proapoptotic glycogen synthase kinase-3 was observed. Immunofluorescent studies demonstrated that a single population of neurons responded to BDNF and IGF-1. In contrast, the magnitude of BDNF-stimulated extracellular signal-regulated kinase (ERK) activation was found to be much greater than that of IGF-1-stimulated ERK, such that the difference in magnitude stimulated by BDNF in the presence and absence of IGF-1 did not reach statistical significance. Consistent with the observed agonist-stimulated activation of Akt, BDNF and IGF-1 were both found to act as neurotrophins, enhancing neuronal survival under low-insulin culture conditions. Maximal survival was achieved when both growth factors were present. These findings provide insight into the significance of multiple growth factors stimulating activation of ERK and Akt in the central nervous system. In some cases, the magnitude of activation required to elicit biological responses may be achieved only with a combination of compounds.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Hipocampo/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipocampo/metabolismo , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
5-HT(1A) receptors have been hypothesized to mediate some of the neuronal plasticity and behavioral responses stimulated by serotonin selective reuptake inhibitors. Although the cellular signaling pathways required for inducing these actions have not yet been determined, roles for the neuroprotective extracellular-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and Akt pathways have been suggested. In the current studies we have utilized primary cultures to directly determine whether hippocampal 5-HT(1A) receptors couple to activation of Akt and ERK. We found that E18 hippocampal neurons exhibit a twofold activation of Akt when exposed to nanomolar concentrations of 5-HT. The 5-HT(1/7) receptor-selective agonist 5-carboxamidotryptamine maleate (5-CT) and the 5-HT(1A/7) receptor-selective agonist 8-hydroxy-N,N-dipropyl-aminotetralin (8-OH-DPAT) maleate were found to activate Akt with equal efficacy, and similar potency, to 5-HT. p-MPPI and WAY-100635, antagonists selective for 5-HT(1A) receptors, completely inhibited 5-CT- stimulated Akt activation. Activation of Akt was also inhibited by pretreatment with pertussis toxin as well as the phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002. In contrast, the 5-HT selective antagonist, SB269970, caused no inhibition. Although the density of 5-HT(1A) receptors expressed by cultured neurons was sufficient to activate Akt, no activation of ERK was observed. These findings suggest that Akt, and not ERK, may be relevant to previous reports of hippocampal 5-HT(1A) receptors mediating neurotrophic responses.
Assuntos
Hipocampo/citologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Serotonina/análogos & derivados , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Análise de Variância , Animais , Western Blotting/métodos , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imunofluorescência/métodos , Neurônios/efeitos dos fármacos , Toxina Pertussis/farmacologia , Proteínas Proto-Oncogênicas c-akt , Ratos , Serotonina/farmacologia , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Fatores de Tempo , Transfecção/métodosRESUMO
The most commonly prescribed antidepressants, the serotonin (5-HT) selective reuptake inhibitors, increase 5-HT without targeting specific receptors. Yet, little is known about the interaction of multiple receptor subtypes expressed by individual neurons. Specifically, the effect of increases in cAMP induced by Gs-coupled 5-HT receptor subtypes on the signaling pathways modulated by other receptor subtypes has not been studied. We have, therefore, examined the activation of the extracellular-regulated kinase (ERK) and Akt pathways by Gs-coupled 5-HT7A receptors and Gq-coupled 5-HT2A receptors, which are co-expressed in discrete brain regions. Agonists for both receptors were found to activate ERK and Akt in transfected PC12 cells. 5-HT2A receptor-mediated activation of the two pathways was found to be Ca2+-dependent. In contrast, 5-HT7A receptor-mediated activation of Akt required increases in both [cAMP] and intracellular [Ca2+], while activation of ERK was inhibited by Ca2+. The activation of ERK and Akt stimulated by simultaneous treatment of cells with 5-HT2A and 5-HT7A receptor agonists was found to be at least additive. Cell-permeable cAMP analogs mimicked 5-HT7A receptor agonists in enhancing 5-HT2A receptor-mediated activation of ERK and Akt. A role was identified for the cAMP-guanine exchange factor, Epac, in this augmentation of ERK, but not Akt, activation. Our finding of enhanced activation of neuroprotective Akt and ERK pathways by simultaneous occupancy of 5-HT2A and 5-HT7A receptors may also be relevant to the interaction of other neuronally expressed Gq- and Gs-coupled receptors.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Receptores de Serotonina/metabolismo , Anfetaminas/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células PC12 , Proteínas Proto-Oncogênicas c-akt , Ratos , Receptor 5-HT2A de Serotonina/fisiologia , Receptores de Serotonina/fisiologia , Serotonina/farmacologia , Agonistas do Receptor 5-HT2 de SerotoninaRESUMO
The roles of 3',5'-cyclic adenosine monophosphate (cAMP) and protein kinase A in 5-hydroxytryptamine (5-HT)7 receptor-mediated activation of extracellular-regulated kinase (ERK) were studied in cultured hippocampal neurons and transfected PC12 cells. Activation of ERK by neuronal Gs-coupled receptors has been thought to proceed through a protein kinase A-dependent pathway. In fact we identified coupling of 5-HT7 receptors to activation of adenylyl cyclase and protein kinase A. However, no inhibition of agonist-stimulated ERK activation was found when cells were treated with H-89 and KT5720 at concentrations sufficient to completely inhibit activation of protein kinase A. However, activation of ERK was found to be sensitive to the adenylyl cyclase inhibitor 9-(tetrahydrofuryl)-adenine, suggesting a possible role for a cAMP-guanine nucleotide exchange factor (cAMP-GEF). Co-treatment of cells with 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate, a direct activator of the cAMP-GEFs Epac1 and 2, reversed the inhibition of agonist-stimulated ERK activation induced by adenylyl cyclase inhibition. Additionally, over-expression of Epac1 enhanced 5-HT7 receptor-mediated activation of ERK. These results demonstrate that the activation of ERK mediated by neuronal Gs-coupled receptors can proceed through cAMP-dependent pathways that utilize cAMP-GEFs rather than protein kinase A.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/metabolismo , Receptores de Serotonina/metabolismo , Adenilil Ciclases/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Ativadores de Enzimas/farmacologia , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Hemaglutininas/biossíntese , Hemaglutininas/genética , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Células PC12 , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
5-Hydroxytryptamine (5-HT)(2A) receptors have been implicated to play a role in both the treatment and pathophysiology of a number of psychiatric disorders. Therefore, the coupling of this receptor to signals, such as extracellular signal-regulated kinase (ERK), that elicit long-term neuronal changes may be relevant. In the present study we examined the coupling of the G(q)-coupled receptor to ERK in PC12 cells, a cell line commonly used as a neuronal model system. Activation of ERK occurred through a pathway different than the protein kinase C-dependent pathways described previously in studies of non-neuronal cells. Activation of ERK, in PC12 cells, was inhibited by both chelation of extracellular Ca(2+) and by depletion of intracellular Ca(2+) stores. Surprisingly, activation was not inhibited, but actually potentiated, by a variety of protein kinase C inhibitors covering all known protein kinase C isoforms. In contrast, the coupling of receptor to activation of ERK was found to be sensitive to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7) and N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (W13), inhibitors of calmodulin, but not to 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN62) and 2-[N-(2-hydroxyethyl)]-N-4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN93), inhibitors of calmodulin-dependent protein kinase. Additionally, the general tyrosine kinase inhibitor genistein, as well as the Src inhibitor PP1 and the epidermal growth factor receptor kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG 1478), inhibited receptor-mediated activation of ERK, suggesting a role for tyrosine kinases. In fact, 5-HT was found to stimulate tyrosine phosphorylation of a number of proteins, and this phosphorylation was inhibited by W7. 5-HT(2A) receptor-activation of ERK through a protein kinase C-independent pathway requiring Ca(2+)/calmodulin/tyrosine kinases represents a pathway distinct from those described in studies of non-neuronal cells.