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1.
J Pharmacol Exp Ther ; 336(2): 468-78, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21036913

RESUMO

Nitric oxide (NO) derived from neuronal nitric-oxide synthase (nNOS) and inducible nitric-oxide synthase (iNOS) plays a key role in various pain and inflammatory states. KLYP961 (4-((2-cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-difluoroquinolin-2(1H)-one) inhibits the dimerization, and hence the enzymatic activity of human, primate, and murine iNOS and nNOS (IC(50) values 50-400 nM), with marked selectivity against endothelial nitric-oxide synthase (IC(50) >15,000 nM). It has ideal drug like-properties, including excellent rodent and primate pharmacokinetics coupled with a minimal off-target activity profile. In mice, KLYP961 attenuated endotoxin-evoked increases in plasma nitrates, a surrogate marker of iNOS activity in vivo, in a sustained manner (ED(50) 1 mg/kg p.o.). KLYP961 attenuated pain behaviors in a mouse formalin model (ED(50) 13 mg/kg p.o.), cold allodynia in the chronic constriction injury model (ED(50) 25 mg/kg p.o.), or tactile allodynia in the spinal nerve ligation model (ED(50) 30 mg/kg p.o.) with similar efficacy, but superior potency relative to gabapentin, pregabalin, or duloxetine. Unlike morphine, the antiallodynic activity of KLYP961 did not diminish upon repeated dosing. KLYP961 also attenuated carrageenin-induced edema and inflammatory hyperalgesia and writhing response elicited by phenylbenzoquinone with efficacy and potency similar to those of celecoxib. In contrast to gabapentin, KLYP961 did not impair motor coordination at doses as high as 1000 mg/kg p.o. KLYP961 also attenuated capsaicin-induced thermal allodynia in rhesus primates in a dose-related manner with a minimal effective dose (≤ 10 mg/kg p.o.) and a greater potency than gabapentin. In summary, KLYP961 represents an ideal tool with which to probe the physiological role of NO derived from iNOS and nNOS in human pain and inflammatory states.


Assuntos
Anti-Inflamatórios/farmacologia , Inibidores Enzimáticos/farmacologia , Fluoroquinolonas/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Pirazinas/farmacologia , Analgésicos/farmacologia , Animais , Células Cultivadas , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/toxicidade , Fluoroquinolonas/farmacocinética , Fluoroquinolonas/toxicidade , Trânsito Gastrointestinal/efeitos dos fármacos , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Multimerização Proteica , Pirazinas/farmacocinética , Pirazinas/toxicidade
4.
BMC Cell Biol ; 5: 34, 2004 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15369603

RESUMO

BACKGROUND: Research on olfactory G-protein coupled receptors (GPCRs) has been severely impeded by poor functional expression in heterologous systems. Previously, we demonstrated that inefficient olfactory receptor (OR) expression at the plasma membrane is attributable, in part, to degradation of endoplasmic reticulum (ER)-retained ORs by the ubiquitin-proteasome system and sequestration of ORs in ER aggregates that are degraded by autophagy. Thus, experiments were performed to test the hypothesis that attenuation of ER degradation improves OR functional expression in heterologous cells. RESULTS: To develop means to increase the functional expression of ORs, we devised an approach to measure activation of the mOREG OR (Unigene # Mm.196680; Olfr73) through coupling to an olfactory cyclic nucleotide-gated cation channel (CNG). This system, which utilizes signal transduction machinery coupled to OR activation in native olfactory sensory neurons, was used to demonstrate that degradation, both by the ubiquitin-proteasome system and autophagy, limits mOREG functional expression. The stimulatory effects of proteasome and autophagy inhibitors on mOREG function required export from the ER and trafficking through the biosynthetic pathway. CONCLUSIONS: These findings demonstrate that poor functional expression of mOREG in heterologous cells is improved by blocking proteolysis. Inhibition of ER degradation may improve the function of other ORs and assist future efforts to elucidate the molecular basis of odor discrimination.


Assuntos
Retículo Endoplasmático/metabolismo , Receptores Odorantes/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Eugenol/metabolismo , Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Ativação do Canal Iônico , Camundongos , Odorantes , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Transporte Proteico , Proteínas Recombinantes de Fusão/biossíntese , Rodopsina , Ubiquitina/metabolismo
5.
J Biol Chem ; 283(18): 11981-94, 2008 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-18326490

RESUMO

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of alpha, beta, and gamma subunits, belongs to the ENaC/degenerin family of ion channels and forms the principal route for apical Na(+) entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the beta subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of alphabetaG37Sgamma hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na(+) flux across epithelial membranes is clinically desirable.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Indóis/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Amilorida/farmacologia , Animais , Linhagem Celular , Canais Epiteliais de Sódio/química , Espaço Extracelular , Feminino , Furina/metabolismo , Humanos , Indóis/química , Camundongos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Pseudo-Hipoaldosteronismo/metabolismo , Bibliotecas de Moléculas Pequenas/química , Xenopus
6.
Proc Natl Acad Sci U S A ; 99(7): 4692-6, 2002 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-11917125

RESUMO

The three members of the T1R class of taste-specific G protein-coupled receptors have been hypothesized to function in combination as heterodimeric sweet taste receptors. Here we show that human T1R2/T1R3 recognizes diverse natural and synthetic sweeteners. In contrast, human T1R1/T1R3 responds to the umami taste stimulus l-glutamate, and this response is enhanced by 5'-ribonucleotides, a hallmark of umami taste. The ligand specificities of rat T1R2/T1R3 and T1R1/T1R3 correspond to those of their human counterparts. These findings implicate the T1Rs in umami taste and suggest that sweet and umami taste receptors share a common subunit.


Assuntos
Ácido Glutâmico/farmacologia , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G , Paladar/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao GTP/fisiologia , Guanosina Monofosfato/farmacologia , Humanos , Inosina Monofosfato/farmacologia , Dados de Sequência Molecular , Ratos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Transfecção
7.
Proc Natl Acad Sci U S A ; 101(39): 14258-63, 2004 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-15353592

RESUMO

The T1R receptors, a family of taste-specific class C G protein-coupled receptors, mediate mammalian sweet and umami tastes. The structure-function relationships of T1R receptors remain largely unknown. In this study, we demonstrate the different functional roles of T1R extracellular and transmembrane domains in ligand recognition and G protein coupling. Similar to other family C G protein-coupled receptors, the N-terminal Venus flytrap domain of T1R2 is required for recognizing sweeteners, such as aspartame and neotame. The G protein coupling requires the transmembrane domain of T1R2. Surprisingly, the C-terminal transmembrane domain of T1R3 is required for recognizing sweetener cyclamate and sweet taste inhibitor lactisole. Because T1R3 is the common subunit in the sweet taste receptor and the umami taste receptor, we tested the interaction of lactisole and cyclamate with the umami taste receptor. Lactisole inhibits the activity of the human T1R1/T1R3 receptor, and, as predicted, blocked the umami taste of l-glutamate in human taste tests. Cyclamate does not activate the T1R1/T1R3 receptor by itself, but potentiates the receptor's response to l-glutamate. Taken together, these findings demonstrate the different functional roles of T1R3 and T1R2 and the presence of multiple ligand binding sites on the sweet taste receptor.


Assuntos
Subunidades Proteicas/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Papilas Gustativas/fisiologia , Sequência de Aminoácidos , Animais , Aspartame/farmacologia , Derivados de Benzeno/farmacologia , Sítios de Ligação , Linhagem Celular , Ciclamatos/farmacologia , Relação Dose-Resposta a Droga , Aromatizantes/farmacologia , Glutamatos/farmacologia , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Humanos , Dados de Sequência Molecular , Mutação Puntual , Estrutura Terciária de Proteína , Ratos , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Glutamato de Sódio/farmacologia , Relação Estrutura-Atividade , Sacarose/farmacologia , Paladar/efeitos dos fármacos , Paladar/fisiologia , Transfecção
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