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1.
Phytother Res ; 20(9): 725-31, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16775808

RESUMO

CKBM is an herbal formula composed of five Chinese medicinal herbs (Panax ginseng, Schisandra chinensis, Fructus crataegi, Ziziphus jujuba and Glycine max) supplemented with processed Saccharomyces cerevisiae. It has been demonstrated that CKBM is capable of triggering the release of IL-6 and TNFalpha from human peripheral blood mononuclear cells. In this report, T-lymphocytic Sup-T1 cells and B-lymphocytic Ramos cells were utilized as cellular models to investigate how CKBM regulates intracellular signaling as well as the production of cytokines. CKBM stimulated the three major subgroups of mitogen-activated protein kinase (i.e. ERK, JNK and p38) in Sup-T1 cells, but only triggered the activation of ERK and p38 in Ramos cells. The induction of mitogen-activated protein kinases (MAPK) activations varied with the duration of treatment, as well as with the dosage of CKBM. In terms of cytokine production, treatment of CKBM alone did not trigger the release of IL-1beta and IFNgamma, but it suppressed the LPS-induced IFNgamma production from both Sup-T1 cells and Ramos cells. In view of the therapeutic effects of traditional Chinese medicines in inflammatory and autoimmune disorders, the results suggest that CKBM may exhibit its immuno-modulatory effects by regulating intracellular signaling as well as cytokine production in different lymphocytic cell types.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Interferon gama/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Lipopolissacarídeos , Fatores de Tempo
3.
J Pineal Res ; 32(2): 97-105, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12071474

RESUMO

Using 2[125I]iodomelatonin as the radioligand, we characterized 2[125I]iodomelatonin binding sites in guinea pig platelet membrane preparations. Saturation radioreceptor studies indicated that these 2[125I]iodomelatonin binding sites were of picomolar affinity and femtomolar density. The dissociation constant (Kd) and maximum number of receptor sites (Bmax) were 42.5 +/- 1.79 pM and 11.8 +/- 0.8 fmol/mg protein (n = 6), respectively. 2[125I]Iodomelatonin competition studies with indoles or drugs indicate the following rank order of potency: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin > 5-methoxytryptophol, whereas serotonin and its analogs had less than 20% inhibition at 0.1 mM. Guanosine 5'-O-(3-thiotriphosphate) significantly increased the Kd by twofold suggesting that these binding sites are coupled to the guanine nucleotide binding proteins. Immunoblotting studies using anti-MT(1) IgG demonstrated one peptide blockable band with an apparent molecular mass of 37 kDa. Melatonin had no effect on prostacyclin or forskolin-stimulated intracellular 3',5'-cyclic adenosine monophosphate accumulation. A diurnal variation in binding density, which was abolished after the animals were adapted to constant light conditions, was observed. Age related studies demonstrated that Bmax increased as the animal matured. Physiological melatonin concentrations potentiated whereas those at pharmacological levels inhibited adenosine diphosphate- or arachidonic acid-stimulated platelet aggregation. Our study demonstrated G-protein coupled, saturable, reversible and highly specific picomolar affinity 2[125I]iodomelatonin binding sites in guinea pig platelets. Pharmocological and physiological data indicate that they may be different from the nanomolar [3H]melatonin binding sites in human platelets previously reported.


Assuntos
Plaquetas/metabolismo , Melatonina/análogos & derivados , Melatonina/metabolismo , Difosfato de Adenosina/farmacologia , Fatores Etários , Animais , Sítios de Ligação , Ligação Competitiva , Plaquetas/efeitos dos fármacos , AMP Cíclico/metabolismo , Feminino , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cobaias , Radioisótopos do Iodo , Luz , Masculino , Melatonina/farmacologia , Agregação Plaquetária , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Melatonina , Serotonina/metabolismo , Fatores Sexuais
4.
J Pineal Res ; 32(4): 243-52, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11982794

RESUMO

2[125I]Iodomelatonin ([125I]Mel) binding sites were characterized on membrane preparations of young chick hearts. [125I]Mel binding was rapid, saturable, stable, reversible, specific and of picomolar affinity and femtomolar density. Guanosine 5'-O-(3-thiotriphosphate) significantly lowered the binding affinity by one- to twofold, supporting G-protein linkage of melatonin receptors. Binding was detected as early as embryonic day-9 (E9), and increased steadily peaking at E13 before it slowly declined to about 15% of the peak level a week posthatch. Specific [125I]Mel binding was significantly increased by in ovo administration of inotropic agents dopamine and isoproterenol. Melatonin or 2-iodo-N-butanoyl-tryptamine inhibited isoproterenol-stimulated cAMP accumulation in primary heart cell cultures and the effect was attenuated after pretreatment with pertussis toxin (PTX). Localization of melatonin receptors using autoradiography showed intense labeling in the coronary arteries in all age groups whereas those in the myoblasts decreased as the heart matured. While the myoblasts and undifferentiated developing coronary arteries expressed melatonin MT1 receptor subtype in E11 hearts as detected by immunostaining with anti-MT1 receptor serum, immunoreactivities were observed mostly on the endothelium/subendothelium and smooth muscle cells of the well developed coronary vessels in posthatch hearts. Collectively, our data suggest the presence of PTX-sensitive, G protein-coupled melatonin receptors, whose expression is up-regulated by dopamine and isoproterenol, in the chick heart. Activation of these receptors, which include MT1 subtype, may modulate beta-adrenergic receptor-mediated cAMP signaling in the control of chick heart and coronary artery physiology.


Assuntos
Vasos Coronários/fisiologia , Coração/fisiologia , Melatonina/análogos & derivados , Melatonina/metabolismo , Animais , Autorradiografia , Embrião de Galinha , Vasos Coronários/metabolismo , Feminino , Imuno-Histoquímica , Radioisótopos do Iodo , Masculino , Miocárdio/metabolismo , Ensaio Radioligante
5.
J Neurochem ; 80(5): 736-45, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11948236

RESUMO

The pineal neurohormone melatonin modulates a variety of physiological processes through different receptors. It has recently been reported that the cloned melatonin receptors (MT1, MT2 and Mel1c) exhibit differential abilities to stimulate phospholipase C (PLC) via G(16). Here we examined the molecular basis of such differences in melatonin receptor signaling. Coexpression of MT1 or MT2 with the alpha subunit of G(16) (Galpha(16) ) allowed COS-7 cells to accumulate inositol phosphates in response to 2-iodomelatonin. In contrast, Mel1c did not activate Galpha(16) even though its expression was demonstrated by radioligand binding and agonist-induced inhibition of adenylyl cyclase. As Mel1c possesses an exceptionally large C-terminal tail, we further asked if this structural feature prevented productive coupling to Galpha(16). Eleven chimeric melatonin or mutant receptors were constructed by swapping all or part of the C-terminal tail between MT1, MT2 and Mel1c. All chimeras were fully capable of binding 2-[(125) I]iodomelatonin and inhibiting adenylyl cyclase. Chimeras containing the full-length Mel1c tail were incapable of activating Galpha(16), while those that contained the complete C-terminal region of either MT1 or MT2 stimulated PLC. Incorporation of the extra portion of the C-terminal tail of Mel1c to either MT1 or MT2 completely abolished the chimeras' ability to stimulate PLC via Galpha(16). In contrast, truncation of the C-terminal tail of Mel1c allowed interaction with Galpha(16). Our results suggest that Galpha(16) can discern structural differences amid the three melatonin receptors and provide evidence for functional distinction of Mel1c from MT1 and MT2 receptors.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Melatonina/análogos & derivados , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Xenopus , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Animais , Ligação Competitiva/fisiologia , Células COS , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Radioisótopos do Iodo , Melatonina/metabolismo , Mutagênese Sítio-Dirigida , Oligopeptídeos , Peptídeos , Ligação Proteica/fisiologia , Receptores de Superfície Celular/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Melatonina , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Deleção de Sequência , Relação Estrutura-Atividade , Transfecção , Fosfolipases Tipo C/metabolismo
6.
Mol Reprod Dev ; 61(2): 192-9, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11803554

RESUMO

It has been shown that melatonin regulates uterine function. Our previous studies have demonstrated the presence of melatonin receptors in the rat uterine endometrium, indicating that melatonin may act directly on the uterus. In the present study, the histological localization of the rat uterine melatonin binding was revealed by autoradiography and the molecular subtyping was studied by in situ hybridization in the stromal cells. The signal transduction process and effects of melatonin on stromal cell proliferation was also investigated. Our autoradiograms showed that 2[(125)I]iodomelatonin binding sites were localized in the antimesometrial endometrial stroma. In situ hybridization with specific mt(1) receptor cDNA probe in the primary culture of antimesometrial stromal cells demonstrated the expression of mt(1) receptor mRNAs. Melatonin dose-dependently inhibited forskolin-stimulated cAMP accumulation, which was reversed by pertussis toxin. This indicates that the rat uterine melatonin receptors are negatively coupled to adenylate cyclase via pertussis toxin sensitive G(i) protein. Melatonin also inhibited the incorporation of [(3)H]thymidine in the rat uterine antimesometrial stromal cells, showing that melatonin has an anti-proliferative effect on the uterus. Our results suggest that melatonin may act directly on the mt(1) melatonin receptors in the rat uterine antimesometrial stromal cells to inhibit their proliferation. Its action may be mediated through a pertussis toxin-sensitive adenylate cyclase coupled G(i)-protein.


Assuntos
Endométrio/metabolismo , Melatonina/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Células Estromais/metabolismo , Toxina Adenilato Ciclase , Animais , Autorradiografia , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Endométrio/citologia , Feminino , Hibridização In Situ , Toxina Pertussis , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Melatonina , Células Estromais/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologia
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