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1.
Am J Physiol Endocrinol Metab ; 304(6): E614-22, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23321474

RESUMO

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) direct the activation of distinct signaling pathways that determine cell fate. In this study, the pathways activated and the mechanisms by which ROS and RNS control the viability of pancreatic ß-cells were examined. Although both nitric oxide and hydrogen peroxide (H2O2) induce DNA damage, reduce cell viability, and activate AMPK, the mechanisms of AMPK activation and cell death induction differ between each reactive species. Nitric oxide activates the unfolded protein and heat shock responses and MAPK kinase signaling, whereas H2O2 stimulates p53 stabilization and poly(ADP-ribose) polymerase (PARP) activation but fails to induce the unfolded protein or heat shock responses or MAPK activation. The control of cell fate decisions is selective for the form of stress. H2O2-mediated reduction in ß-cell viability is controlled by PARP, whereas cell death in response to nitric oxide is PARP independent but associated with the nuclear localization of GAPDH. These findings show that both ROS and RNS activate AMPK, induce DNA damage, and reduce cell viability; however, the pathways controlling the responses of ß-cells are selective for the type of reactive species.


Assuntos
Resposta ao Choque Térmico , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Sistema de Sinalização das MAP Quinases , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Resposta a Proteínas não Dobradas , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular , Dano ao DNA , Secreção de Insulina , Células Secretoras de Insulina/citologia , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Transporte Proteico , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Técnicas de Cultura de Tecidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Eur J Pharmacol ; 682(1-3): 12-20, 2012 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-22381068

RESUMO

Sigma-1 receptors are associated with Alzheimer's disease, major depressive disorders, and schizophrenia. These receptors show progrowth/antiapoptotic properties via their chaperoning functions to counteract ER (endoplasmic reticulum) stress, to block neurodegeneration, and to regulate neuritogenesis. The sigma-1 receptor knock out mouse offered an opportunity to assess possible mechanisms by which the sigma-1 receptor modulates cellular oxidative stress. Nuclear magnetic resonance (NMR) metabolomic screening of the WT (wild type) and sigma-1 KO (knockout) livers was performed to investigate major changes in metabolites that are linked to oxidative stress. Significant changes in protein levels were also identified by two-dimensional (2D) gel electrophoresis and mass spectrometry. Increased levels of the antioxidant protein peroxiredoxin 6 (Prdx6), and the ER chaperone BiP (GRP78) compared to WT littermates were detected. Oxidative stress was measured in WT and sigma-1 KO mouse liver homogenates, in primary hepatocytes and in lung homogenates. Furthermore, sigma-1 receptor mediated activation of the antioxidant response element (ARE) to upregulate NAD(P)H quinone oxidoreductase 1 (NQO1) and superoxide dismutase 1 (SOD1) mRNA expression in COS cells was shown by RT PCR. These novel functions of the sigma-1 receptor were sensitive to well-known sigma ligands via their antagonist/agonist properties.


Assuntos
Antioxidantes/metabolismo , Estresse Oxidativo , Receptores sigma/metabolismo , Elementos de Resposta/genética , Animais , Células COS , Chlorocebus aethiops , Chaperona BiP do Retículo Endoplasmático , Técnicas de Inativação de Genes , Cobaias , Camundongos , Estresse Oxidativo/genética , Proteômica , Receptores sigma/deficiência , Receptores sigma/genética , Receptor Sigma-1
3.
Curr Pharm Des ; 18(7): 920-9, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22288412

RESUMO

The sigma-1 receptor is a 26 kDa endoplasmic reticulum resident membrane protein that has been shown to have chaperone activity in addition to its promiscuous binding to pharmacological agents. Ligand binding domain(s) of the sigma-1 receptor have been identified using the E. coli expressed and purified receptor protein and novel radioiodinated azido photoaffinity probes followed by proteolytic and chemical cleavage strategies. The outcome of these experiments indicates that the sigma-1 receptor ligand binding regions are formed primarily by juxtaposition of its second and third hydrophobic domains, regions where the protein shares considerable homology with the fungal enzyme, sterol isomerase that is essential for the biosynthesis of ergosterol. Data indicate that these hydrophobic steroid binding domain like (SBDL) regions on the sigma-1 receptor are likely to interact selectively with N-alkyl amines such as the endogenous sphingolipids and with synthetic N-alkylamines and N-aralkylamines derivatives. A proposed model for the sigma-1 receptor is presented.


Assuntos
Alcanos/metabolismo , Aminas/metabolismo , Sítios de Ligação , Marcadores de Fotoafinidade/metabolismo , Receptores sigma/química , Receptores sigma/metabolismo , Esfingosina/metabolismo , Alcanos/química , Aminas/química , Humanos , Marcadores de Fotoafinidade/química , Esfingosina/análogos & derivados , Esfingosina/química , Receptor Sigma-1
4.
Am J Physiol Cell Physiol ; 300(2): C328-37, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21084640

RESUMO

σ-Receptors are integral membrane proteins that have been implicated in a number of biological functions, many of which involve the modulation of ion channels. A wide range of synthetic ligands activate σ-receptors, but endogenous σ-receptor ligands have proven elusive. One endogenous ligand, dimethyltryptamine (DMT), has been shown to act as a σ-receptor agonist. Progesterone and other steroids bind σ-receptors, but the functional consequences of these interactions are unclear. Here we investigated progesterone binding to σ(1)- and σ(2)-receptors and evaluated its effect on σ-receptor-mediated modulation of voltage-gated Na(+) channels. Progesterone binds both σ-receptor subtypes in liver membranes with comparable affinities and blocks photolabeling of both subtypes in human embryonic kidney 293 cells that stably express the human cardiac Na(+) channel Na(v)1.5. Patch-clamp recording in this cell line tested Na(+) current modulation by the σ-receptor ligands ditolylguanidine, PB28, (+)SKF10047, and DMT. Progesterone inhibited the action of these ligands to varying degrees, and some of these actions were reduced by σ(1)-receptor knockdown with small interfering RNA. Progesterone inhibition of channel modulation by drugs was consistent with stronger antagonism of σ(2)-receptors. By contrast, progesterone inhibition of channel modulation by DMT was consistent with stronger antagonism of σ(1)-receptors. Progesterone binding to σ-receptors blocks σ-receptor-mediated modulation of a voltage-gated ion channel, and this novel membrane action of progesterone may be relevant to changes in brain and cardiovascular function during endocrine transitions.


Assuntos
Progesterona/metabolismo , Receptores sigma/antagonistas & inibidores , Canais de Sódio/metabolismo , Animais , Células Cultivadas , Guanidinas/farmacologia , Células HEK293 , Humanos , Fígado/efeitos dos fármacos , N,N-Dimetiltriptamina/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.5 , Fenazocina/análogos & derivados , Fenazocina/farmacologia , Piperazinas/farmacologia , Progesterona/farmacologia , RNA Interferente Pequeno/farmacologia , Ratos , Receptores sigma/metabolismo , Receptor Sigma-1
5.
Bioorg Med Chem ; 18(12): 4397-404, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20493718

RESUMO

The sigma-1 receptor is a unique non-opioid, non-PCP binding site that has been implicated in many different pathophysiological conditions including psychosis, drug addiction, retinal degeneration and cancer. Based on the structure of fenpropimorph, a high affinity (K(i)=0.005 nM)(1) sigma-1 receptor ligand and strong inhibitor of the yeast sterol isomerase (ERG2), we previously deduced a basic sigma-1 receptor pharmacophore or chemical backbone composed of a phenyl ring attached to a di-substituted nitrogen atom via an alkyl chain.(2) Here, we report the design and synthesis of various N,N-dialkyl or N-alkyl-N-aralkyl derivatives based on this pharmacophore as well as their binding affinities to the sigma-1 receptor. We introduce three high affinity sigma-1 receptor compounds, N,N-dibutyl-3-(4-fluorophenyl)propylamine (9), N,N-dibutyl-3-(4-nitrophenyl)propylamine (3), and N-propyl-N'-4-aminophenylethyl-3-(4-nitrophenyl)propylamine (20) with K(i) values of 17.7 nM, 0.36 nM, and 6 nM, respectively. In addition to sigma receptor affinity, we show through cytotoxicity assays that growth inhibition of various tumor cell lines occurs with our high affinity N,N-dialkyl or N-alkyl-N-aralkyl derivatives.


Assuntos
Compostos de Anilina/química , Antineoplásicos/síntese química , Ligantes , Morfolinas/química , Propilaminas/química , Propilaminas/síntese química , Receptores sigma/antagonistas & inibidores , Compostos de Anilina/síntese química , Compostos de Anilina/toxicidade , Antineoplásicos/química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Morfolinas/síntese química , Morfolinas/toxicidade , Propilaminas/toxicidade , Ligação Proteica , Receptores sigma/metabolismo , Receptor Sigma-1
6.
Science ; 323(5916): 934-7, 2009 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-19213917

RESUMO

The sigma-1 receptor is widely distributed in the central nervous system and periphery. Originally mischaracterized as an opioid receptor, the sigma-1 receptor binds a vast number of synthetic compounds but does not bind opioid peptides; it is currently considered an orphan receptor. The sigma-1 receptor pharmacophore includes an alkylamine core, also found in the endogenous compound N,N-dimethyltryptamine (DMT). DMT acts as a hallucinogen, but its receptor target has been unclear. DMT bound to sigma-1 receptors and inhibited voltage-gated sodium ion (Na+) channels in both native cardiac myocytes and heterologous cells that express sigma-1 receptors. DMT induced hypermobility in wild-type mice but not in sigma-1 receptor knockout mice. These biochemical, physiological, and behavioral experiments indicate that DMT is an endogenous agonist for the sigma-1 receptor.


Assuntos
Alucinógenos/metabolismo , N,N-Dimetiltriptamina/metabolismo , Receptores sigma/metabolismo , Animais , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Cobaias , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Ratos , Receptores sigma/agonistas , Receptores sigma/antagonistas & inibidores , Triptaminas/metabolismo , Receptor Sigma-1
7.
Biochemistry ; 47(27): 7205-17, 2008 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-18547058

RESUMO

Radioiodinated photoactivatable photoprobes can provide valuable insights regarding protein structure. Previous work in our laboratory showed that the cocaine derivative and photoprobe 3-[ (125)I]iodo-4-azidococaine ([ (125)I]IACoc) binds to the sigma-1 receptor with 2-3 orders of magnitude higher affinity than cocaine [Kahoun, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. Using this photoprobe, we demonstrated the insertion site for [ (125)I]IACoc to be Asp188 [Chen, Y. (2007) Biochemistry 46, 3532-3542], which resides in the proposed steroid binding domain-like II (SBDLII) region (amino acids 176-194) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. An additional photoprobe based on the sigma-1 receptor ligand fenpropimorph, 1- N-(2-3-[ (125)I]iodophenyl)propane ([ (125)I]IAF), was found to label a peptide in both the SBDLII and steroid binding domain-like I (SBDLI) (amino acids 91-109) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. In this report, we describe two novel strategically positioned carrier-free, radioiodinated photoaffinity labels specifically designed to probe the putative "nitrogen interacting region" of sigma-1 receptor ligands. These two novel photoprobes are (-)-methyl 3-(benzoyloxy)-8-2-(4-azido-3-[ (125)I]iodobenzene)-1-ethyl-8-azabicyclo[3.2.1]octane-2-carboxylate ([ (125)I]-N-IACoc) and N-propyl- N-(4-azido-3-iodophenylethyl)-3-(4-fluorophenyl)propylamine ([ (125)I]IAC44). In addition to reporting their binding affinities to the sigma-1 and sigma-2 receptors, we show that both photoaffinity labels specifically and covalently derivatized the pure guinea pig sigma-1 receptor (26.1 kDa) [Ramachandran, S. (2007) Protein Expression Purif. 51, 283-292]. Cleavage of the photolabeled sigma-1 receptor using Endo Lys C and cyanogen bromide (CNBr) revealed that the [ (125)I]-N-IACoc label was located primarily in the N-terminus and SBDLI-containing peptides of the sigma-1 receptor, while [ (125)I]IAC44 was found in peptide fragments consistent with labeling of both SBDLI and SBDLII.


Assuntos
Marcadores de Fotoafinidade/metabolismo , Receptores sigma/química , Animais , Autorradiografia , Sítios de Ligação , Cocaína/análogos & derivados , Cocaína/síntese química , Cocaína/química , Brometo de Cianogênio/metabolismo , Cobaias , Metaloendopeptidases/metabolismo , Peso Molecular , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Ratos , Receptores sigma/metabolismo , Receptor Sigma-1
8.
Mol Pharmacol ; 72(4): 921-33, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17622576

RESUMO

sigma Receptors, once considered a class of opioid receptors, are now regarded as a unique class of receptors that contain binding sites for a wide range of ligands, including the drug 1-N(2',6'-dimethylmorpholino)3-(4-t-butylpropylamine) (fenpropimorph), a yeast sterol isomerase inhibitor. Because fenpropimorph has high-binding affinity to the sigma-1 receptor, we have synthesized a series of fenpropimorph-like derivatives with varying phenyl ring substituents and have characterized their binding affinities to the sigma-1 receptor. In addition, we have synthesized a carrier-free, radioiodinated fenpropimorph-like photoaffinity label, 1-N-(2',6'-dimethyl-morpholino)-3-(4-azido-3-[(125)I]iodo-phenyl)propane ([(125)I]IAF), which covalently derivatized the sigma-1 receptor (25.3 kDa) in both the rat liver and guinea pig liver membranes and the sigma-2 receptor (18 kDa) in rat liver membranes with high specificity. Furthermore, after cleaving the specific [(125)I]IAF-photolabeled sigma-1 receptor in guinea pig and rat liver membranes and the pure guinea pig sigma-1 receptor with EndoLys-C and cyanogen bromide, the [(125)I]IAF label was identified both in a peptide containing steroid binding domain-like I (SBDLI) (amino acids 91-109) and in a peptide containing steroid binding domain-like II (SBDLII) (amino acids 176-194). Because a single population of binding sites (R(2) = 0.992) for [(125)I]IAF interaction with the sigma-1 receptor was identified by (+)-[(3)H]pentazocine competitive binding with nonradioactive [(127)I]IAF, it was concluded that SBDLI (amino acids 91-109) and SBDLII (amino acids 176-194) comprises, at least in part, regions of the sigma-1 receptor ligand binding site(s).


Assuntos
Sondas Moleculares , Marcadores de Fotoafinidade , Receptores sigma/metabolismo , Animais , Sítios de Ligação , Cobaias , Ligantes , Microssomos Hepáticos/metabolismo , Morfolinas/metabolismo , Morfolinas/farmacologia , Ressonância Magnética Nuclear Biomolecular , Ratos , Receptor Sigma-1
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