RESUMO
The protective effects of insulin-like growth factor I on the somatostatin (SRIF) system in the temporal cortex after beta-amyloid (Abeta) injury may be mediated through its N-terminal tripeptide glycine-proline-glutamate (GPE). GPE is cleaved to cyclo[Pro-Gly] (cPG), a metabolite suggested to mediate in neuroprotective actions. We evaluated the effects of GPE and cPG in the temporal cortex of Abeta25-35-treated rats on SRIF and SRIF receptor protein and mRNA levels, adenylyl cyclase activity, cell death, Abeta25-35 accumulation, cytosolic calcium levels ([Ca(2+)](c)) and the intracellular signaling mechanisms involved. GPE and cPG did not change Abeta25-35 levels, but GPE partially restored SRIF and SRIF receptor 2 protein content and mRNA levels and protected against cell death after Abeta25-35 insult, which was coincident with Akt activation and glycogen synthase kinase 3beta inhibition. In addition, GPE displaced glutamate from NMDA receptors and blocked the glutamate induced rise in cytosolic calcium in isolated rat neurons and moderately increased Ca(2+) influx per se. Our findings suggest that GPE, but not its metabolite, mimics insulin-like growth factor I effects on the SRIF system through a mechanism independent of Abeta clearance that involves modulation of calcium and glycogen synthase kinase 3beta signaling.
Assuntos
Sinalização do Cálcio/fisiologia , Quinase 3 da Glicogênio Sintase/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Oligopeptídeos/fisiologia , Somatostatina/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Células Cultivadas , Feminino , Glicogênio Sintase Quinase 3 beta , Fator de Crescimento Insulin-Like I/química , Fragmentos de Peptídeos/toxicidade , Gravidez , Ratos , Ratos Wistar , Transdução de Sinais/fisiologiaRESUMO
"Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA) has been shown to be hepatotoxic for human users, but molecular mechanisms involved in this effect remained poorly understood. MDMA-induced cell damage is related to programmed cell death in serotonergic and dopaminergic neurons. However, until now there has been no evidence of apoptosis induced by MDMA in liver cells. Here we demonstrate that exposure to MDMA caused apoptosis of freshly isolated rat hepatocytes and of a cell line of hepatic stellate cells (HSC), as shown by chromatin condensation of the nuclei and accumulation of oligonucleosomal fragments in the cytoplasm. In both cell types, apoptosis correlated with decreased levels of bcl-x(L), release of cytochrome c from the mitochondria and activation of caspase 3. In HSC, but not in hepatocytes, MDMA induced poly(ADP-ribose)polymerase (PARP) proteolysis. These results suggest that apoptosis of liver cells could be involved in the hepatotoxicity of MDMA.
Assuntos
Apoptose , Hepatócitos/efeitos dos fármacos , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Animais , Benzimidazóis , Caspase 3 , Caspases/análise , Células Cultivadas , Cromatina/química , Grupo dos Citocromos c/análise , Citoplasma/efeitos dos fármacos , Fragmentação do DNA , Relação Dose-Resposta a Droga , Hepatócitos/química , Hepatócitos/patologia , Masculino , N-Metil-3,4-Metilenodioxianfetamina/toxicidade , Poli(ADP-Ribose) Polimerases/análise , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-bcl-2/análise , Ratos , Ratos Wistar , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
To test whether a turnlike arrangement is involved in the bioactive conformation of CCK4 analogues upon CCK1 receptor recognition, we describe the preparation of two series of CCK4 derivatives, in which the central dipeptide Met-Asp has been replaced by recognized beta-turn mimetics {(2S,5S,11bR)- and (2R,5R,11bS)-2-amino-5-carboxy-3-oxo-2,3,5,6,11,11b-hexahydro-1H-indolizino[8,7-b]indole (IBTM) and beta-turn dipeptide, 2-oxo-7-thio-1-azabicyclo[4.3.0]nonane (BTD)}. The incorporation of the indolizinoindole IBTM type II beta-turn mimetic is preferred over its type II' counterpart for efficient CCK1 receptor recognition, while BTD derivatives were completely inactive. The structure-conformation-activity relationship study in the IBTM series has shown some essential requirement of these CCK4 derivatives to favorably interact with CCK1 receptors: (a) the adoption of turnlike conformations, (b) the presence of an L-Phe residue and a C-terminal carboxamide moiety, and (c) the indole ring of the IBTM skeleton. Moreover, the existence of pi-pi interactions between the phenyl ring of d-Phe residues and the indole ring of IBTM framework is detrimental for binding affinity. A series of potent and selective CCK1 receptor antagonists, exemplified by compounds 8a and 8b, emerges among these IBTM-containing derivatives.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Dipeptídeos/química , Indóis/química , Tetragastrina/análogos & derivados , Tetragastrina/síntese química , Amilases/metabolismo , Animais , Córtex Cerebral/metabolismo , Técnicas In Vitro , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mimetismo Molecular , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Estrutura Secundária de Proteína , Ensaio Radioligante , Ratos , Receptor de Colecistocinina A/antagonistas & inibidores , Relação Estrutura-Atividade , Tetragastrina/química , Tetragastrina/farmacologiaRESUMO
With the aim of reversing selectivity or antagonist/agonist functionality in the 5-(tryptophylamino)-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-derived potent and highly selective CCK(1) antagonists, a series of 4-benzyl and 4-methyl derivatives have been synthesized. Whereas the introduction of the benzyl group led, in all cases, to complete loss of the binding affinity, the incorporation of the methyl group gave a different result depending on the stereochemistry of the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine scaffold. Thus, the introduction of the methyl group into the (4aS,5R)-diastereoisomers, giving a (4S)-configuration, produced a 3-fold increase in the CCK(1) binding potency and selectivity. However, the same structural manipulation in the opposite (4aR,5S)-stereochemistry, leading to a (4R,4aR,5S)-configuration, produced reversal of the selectivity for CCK(1) to the CCK(2) receptors. The replacement of the Boc group at the tryptophan moiety by a 2-adamantyloxycarbonyl group also contributed to that reversal. The resulting compounds displayed moderate CCK(2) antagonist activity in rat and human receptors, and a very small partial agonist effect on the production of inositol phosphate in COS-7 cells transfected with the wild-type human CCK(2) receptor.
Assuntos
Piridinas/síntese química , Pirimidinas/síntese química , Receptor de Colecistocinina B/antagonistas & inibidores , Receptores de Quimiocinas/antagonistas & inibidores , Amilases/metabolismo , Animais , Ligação Competitiva , Células COS , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Humanos , Técnicas In Vitro , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Piridinas/química , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Ensaio Radioligante , Ratos , Receptores CCR1 , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
"Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA), is a derivative of amphetamine with hepatotoxic effects that has been shown to induce apoptosis of cultured liver cells. In the present work, we studied the role played by oxidative stress in the apoptotic response caused by MDMA on a cell line of hepatic stellate cells (HSC). MDMA-treatment provoked oxidative stress determined as reactive oxygen species (ROS) accumulation and decrease of intracellular reduced glutathione levels. Pre-treatment with the antioxidant pyrrolidine dithiocarbamate blocked ROS production but did not prevent MDMA-induced apoptosis of HSC. The pro-oxidant menadione induced in HSC ROS production and apoptosis that were prevented by pyrrolidine dithiocarbamate, showing HSC to be susceptible to oxidative stress-induced apoptosis. Addition of exogenous GSH or its precursor NAC potentiated the apoptotic action of MDMA but blocked apoptosis induced by menadione. Pre-treatment of HSC with the cytochrome P450 inhibitor quinine diminished the extent of apoptosis caused by MDMA, suggesting the involvement of a metabolic derivative of MDMA on its apoptotic effect. Nuclear factor NF-kappaB was activated by MDMA in a oxidative stress independent fashion and played a protective role in the apoptotic response, since inhibition of NF-kappaB by treatment with parthenolide or by viral infection with a dominant-negative form of NIK (Ad5dnNIK) resulted in an increase of MDMA-induced cell death. In summary, MDMA-induced apoptosis of HSC is accompanied, but not caused by oxidative stress; a metabolic derivative of the drug is responsible for the apoptotic effect of MDMA, which is partially blocked by NF-kappaB activation.
Assuntos
Apoptose/fisiologia , Glutationa/fisiologia , Hepatócitos/efeitos dos fármacos , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , NF-kappa B/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Butionina Sulfoximina/farmacologia , Células Cultivadas , Interações Medicamentosas , NF-kappa B/fisiologia , Quinina/farmacologia , Ratos , Gânglio Estrelado/citologia , Vitamina K 3/farmacologiaRESUMO
A suitable solid-phase approach, based on Fmoc/(t)Bu methodology and on the use of 2-chlorotrityl resin, allowed a rapid and efficient preparation of new GPE analogues. Most of the synthesized tripeptides displayed glutamate receptor binding affinity comparable to that of GPE, but only a few derivatives showed significant neuroprotective activity.
Assuntos
Peptídeos/síntese química , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Hipocampo/efeitos dos fármacos , Estrutura Molecular , N-Metilaspartato/toxicidade , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Peptídeos/química , Peptídeos/metabolismo , Ratos , Receptores de Glutamato/metabolismo , Relação Estrutura-Atividade , Compostos de Tritil/químicaRESUMO
The influence of several modifications on the GPE tripeptide structure upon the binding to GluRs and on their neuroprotective effects has been studied. The results indicated that the prevention of neuronal death showed by GPE and some analogues is not directly related to their affinity at glutamate receptors.
Assuntos
Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Oligopeptídeos/farmacologia , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Animais , Ligação Competitiva , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/toxicidade , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Estrutura Molecular , Neurônios/metabolismo , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Oligopeptídeos/síntese química , Oligopeptídeos/química , Ligação Proteica , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A series of GPE analogues, including modifications at the Pro and/or Glu residues, was prepared and evaluated for their NMDA binding and neuroprotective effects. Main results suggest that the pyrrolidine ring puckering of the Pro residue plays a key role in the biological responses, while the preference for cis or trans rotamers around the Gly-Pro peptide bond is not important.
Assuntos
Fármacos Neuroprotetores/síntese química , Oligopeptídeos/síntese química , Animais , Ácido Glutâmico/metabolismo , Indicadores e Reagentes , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ratos , Relação Estrutura-Atividade , Membranas Sinápticas/efeitos dos fármacos , Membranas Sinápticas/metabolismoRESUMO
Replacement of the 2-Adoc-D-alphaMeTrp residue in the non-selective CCK(1) receptor agonist PD 170292 by the Z-(2R,5R,11bS)-IBTM skeleton, able to fix a type II beta-turn-like conformation, led to a conformationally restricted dipeptoid analogue, namely 3a, which exhibited a notable increase in the CCK(1) selectivity and antagonist properties.
Assuntos
Adamantano/análogos & derivados , Adamantano/farmacologia , Citrulina/análogos & derivados , Citrulina/farmacologia , Mimetismo Molecular , Receptores da Colecistocinina/agonistas , Adamantano/metabolismo , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Citrulina/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , RatosRESUMO
2-Oxopiperazine derivatives 1 have been designed as mimetics of gamma-turn conformationally constrained tripeptides. The synthetic pathway devised for the preparation of both epimers of 1 at C(5) involves a reductive amination of cyanomethyleneamino pseudopeptides with amino acid derivatives, followed by regiospecific lactamization of the resulting C-backbone branched pseudopeptides. The versatility of this methodology is illustrated in the synthesis of analogues of the tetrapeptides Boc-[Nle(31)]-CCK-4 and Boc-[Lys(o-tolylaminocarbonyl)(31)]-CCK-4. The introduction of the new conformational restriction into these Boc-CCK-4 analogues led to a loss of 2 or 3 orders of magnitude in the affinity at CCK receptors. These results suggest the absence of a gamma-turn in the bioactive conformation of the C-terminal tripeptide of CCK-4.