RESUMO
BACKGROUND AND AIMS: Epicatechin (EC) intake has been suggested to be beneficial for the prevention of cardiovascular disorders, and it is well known that adipose tissue inflammation is one of the major risk factors for coronary heart diseases. The purpose of the present study was to determine the in vitro and in vivo effects of EC on adipose tissue inflammation and obesity. METHODS AND RESULTS: DNA microarray analysis was performed to evaluate the effects of EC on gene expression in adipocytes co-cultured with bacterial endotoxin-stimulated macrophages. To determine the in vivo effects of the catechin, C57BL/6 mice were fed either a high-fat diet (HFD) or HFD combined with EC, and metabolic changes were observed EC suppressed the expression of many inflammatory genes in the adipocytes co-cultured with endotoxin-stimulated macrophages. Specifically, EC markedly suppressed chemokine (CC motif) ligand 19 (CCL19) expression. The target cell of EC appeared to macrophages. The in vivo study indicated that mice fed the EC-supplemented HFD were protected from diet-induced obesity and insulin resistance. Accordingly, the expression levels of genes associated with inflammation in adipose tissue and in the liver were downregulated in this group of mice. CONCLUSIONS: EC exerts beneficial effects for the prevention of adipose tissue inflammation and insulin resistance. Since we previously reported that mice deficient in the CCL19 receptor were protected from diet-induced obesity and insulin resistance, it can be concluded that the beneficial effects of EC could be mediated, at least in part, by marked suppression of CCL19 expression.
Assuntos
Adipócitos/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Catequina/farmacologia , Quimiocina CCL19/metabolismo , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina , Obesidade/prevenção & controle , Paniculite/prevenção & controle , Células 3T3-L1 , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Quimiocina CCL19/genética , Técnicas de Cocultura , Modelos Animais de Doenças , Regulação para Baixo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/genética , Obesidade/metabolismo , Paniculite/etiologia , Paniculite/genética , Paniculite/metabolismo , Células RAW 264.7 , Fatores de TempoRESUMO
Epidemiological studies suggest that the severity of periodontitis is higher in people with diabetes than in healthy individuals. Insulin resistance might play a crucial role in the pathogenesis of multiple diabetic complications and is reportedly induced in the gingiva of rodents with type 2 diabetes; however, the molecular mechanisms underlying the pathogenesis of diabetes-related periodontitis remain unclear. Therefore, we aimed to investigate whether endothelial insulin resistance in the gingiva may contribute to the pathogenesis of periodontitis as well as elucidate its underlying molecular mechanisms. We demonstrated that insulin treatment downregulated lipopolysaccharide (LPS)-induced or tumor necrosis factor α (TNFα)-induced VCAM1 expression in endothelial cells (ECs) via the PI3K/Akt activating pathway, resulting in reduced cellular adhesion between ECs and leukocytes. Hyperglycemia-induced selective insulin resistance in ECs diminished the effect of insulin on LPS- or TNFα-stimulated VCAM1 expression. Vascular endothelial cell-specific insulin receptor knockout (VEIRKO) mice exhibited selective inhibition of the PI3K/Akt pathway in the gingiva and advanced experimental periodontitis-induced alveolar bone loss via upregulation of Vcam1, Tnfα, Mcp-1, Rankl, and neutrophil migration into the gingiva compared with that in the wild-type (WT) mice despite being free from diabetes. We also observed that insulin-mediated activation of FoxO1, a downstream target of Akt, was suppressed in the gingiva of VEIRKO and high-fat diet (HFD)-fed mice, hyperglycemia-treated ECs, and primary ECs from VEIRKO. Further analysis using ECs transfected with intact and mutated FoxO1, with mutations at 3 insulin-mediated phosphorylation sites (T24A, S256D, S316A), suggested that insulin-mediated regulation of VCAM1 expression and cellular adhesion of ECs with leukocytes was attenuated by mutated FoxO1 overexpression. These results suggest that insulin resistance in ECs may contribute to the progression of periodontitis via dysregulated VCAM1 expression and cellular adhesion with leukocytes, resulting from reduced activation of the PI3K/Akt/FoxO1 axis.
Assuntos
Diabetes Mellitus Tipo 2 , Hiperglicemia , Resistência à Insulina , Periodontite , Animais , Camundongos , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais , Hiperglicemia/complicações , Insulina/metabolismo , Resistência à Insulina/fisiologia , Lipopolissacarídeos/farmacologia , Periodontite/complicações , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Myxobolus wulii (=Myxosoma magna) was first described from the gills of goldfish, Carassius auratus auratus, in China. Subsequently, a myxosporean infecting the hepatopancreas of allogynogenetic gibel carp, C. auratus gibelio, was designated as a different species, Myxobolus guanqiaoensis, although the morphological features were almost identical to those of M. wulii. In Japan, an unidentified Myxobolus sp. was found in the gills and hepatopancreas of goldfish. Morphological and molecular analyses in the present study identified these myxosporeans as M. wulii, which was thus shown to use different habitats in the host fish. Phylogenetic analyses of small subunit ribosomal RNA gene sequences showed that M. wulii is closely related to two gill-infecting Myxobolus species, M. ampullicapsulatus and M. longisporus. Fish infected with M. wulii in the hepatopancreas exhibit swollen abdomens and chronic mortality. Hepatopancreas tissues are virtually destroyed and replaced with plasmodia of M. wulii. A remarkable difference in susceptibility to M. wulii between two clones of allogynogenetic gibel carp was observed, suggesting that resistance to the myxosporean infection was established in a clone of fish bred by allogynogenesis.
Assuntos
Doenças dos Peixes/parasitologia , Carpa Dourada/parasitologia , Interações Hospedeiro-Parasita , Myxozoa/fisiologia , Animais , Feminino , Brânquias/parasitologia , Hepatopâncreas/parasitologia , Myxozoa/classificação , Doenças Parasitárias em Animais/parasitologia , Partenogênese , Filogenia , Especificidade da EspécieRESUMO
The present study evaluates the influence of anesthesia on the parasitic fauna of monogenea fish parasites, as its intensity and viability. Two experiments were conducted: Evaluation of an anesthetic method by sprinkling eugenol directly on gills and evaluation of monogenea motility and viability; Comparison of immersion and directly sprinkling on the gills with benzocaine and eugenol followed by evaluation on parasite intensity. The results suggest that the anesthetic sprinkling didn't interfere in the parasite motility, morphology and body surface integrity analyzed by fluorescence method. The monogenean intensity in the gills was lower in fish anesthetized by immersion method compared to the sprinkling method and the control group. This method of anesthesia can be used in parasitological studies.
Assuntos
Anestésicos/farmacologia , Benzocaína/farmacologia , Caraciformes/fisiologia , Caraciformes/parasitologia , Eugenol/farmacologia , Platelmintos/efeitos dos fármacos , Anestesia/veterinária , Animais , Brânquias/efeitos dos fármacos , Brânquias/parasitologia , Brânquias/fisiologiaAssuntos
Endoscopia Gastrointestinal/efeitos adversos , Perfuração Esofágica/etiologia , Seio Piriforme/lesões , Adulto , Endoscopia Gastrointestinal/métodos , Perfuração Esofágica/diagnóstico por imagem , Humanos , Masculino , Seio Piriforme/diagnóstico por imagem , Seio Piriforme/cirurgia , RadiografiaRESUMO
The cellular mechanisms involved in the accelerated bone loss occurring in association with estrogen deprivation as seen following the menopause have not been fully understood. Insulin-like growth factor-I (IGF-I) is the local regulator of osteoblasts and one of its binding protein, insulin-like growth factor binding protein-4 (IGFBP-4), binds to IGF-I and suppresses its biological activity. We have therefore studied the effects of 17 beta-estradiol and parathyroid hormone (PTH) on binding activity of IGFBP-4 and osteoblastic activity using SaOS-2 osteoblastic cells. DNA and collagen synthesis were enhanced by IGF-I or 17 beta-estradiol and suppressed by PTH in a concentration dependent manner. The inhibitory effect of PTH on DNA and collagen synthesis was abolished by the presence of IGF-I or 17 beta-estradiol. The binding activity of IGFBP-4 was stimulated 2-fold by 10 nM PTH, while this PTH-induced IGFBP-4 production was completely inhibited by the addition of 17 beta-estradiol. The stimulated DNA and collagen synthesis by IGF-I or 17 beta-estradiol were inhibited by IGFBP-4 in a concentration dependent manner. The simplest explanation is that 17 beta-estradiol suppressed the inhibitory effect of PTH on osteoblastic activity by inhibiting the PTH-induced increment of IGFBP-4 binding activity in SaOS-2 cells.
Assuntos
Estradiol/farmacologia , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Osteoblastos/metabolismo , Hormônio Paratireóideo/farmacologia , Células Cultivadas , Replicação do DNA/efeitos dos fármacos , Feminino , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Osteoblastos/patologia , Osteoporose Pós-Menopausa/metabolismo , Osteoporose Pós-Menopausa/patologiaRESUMO
A radioactive photoaffinity label for the GnRH receptor was prepared by derivatization of radiodinated [D-Lys6] des-Gly10-GnRH N-ethylamide with the heterobifunctional photolabile reagent N-hydroxysuccinimidyl-4-azido-benzoate. This high affinity photoreactive analog was employed for radiolabeling and characterization of pituitary GnRH receptors in rat, rabbit, mouse, sheep, and cow adenohypophyses and gonadal GnRH receptors in the rat ovary and testis. In rat, rabbit, and mouse pituitary glands, analysis of the GnRH receptor-ligand complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed two labeled components, both of which were displaced by unlabeled GnRH agonist and antagonist analogs. The larger receptor component was a relatively broad band, with mol wt in rat, rabbit, and mouse of 59,000 +/- 1,900, 62,000 +/- 700, and 60,000 +/- 800, respectively. In the rat pituitary gland, the larger component was composed of 63,000 and 52,000 mol wt components, of which the latter was more heavily labeled and was predominant in purified pituitary gonadotrophs. The mol wts of the smaller components were 40,000 +/- 1,600, 43,000 +/- 1,200, and 41,000 +/- 1,000, respectively. In bovine and ovine pituitary glands, the photolabeled GnRH receptor was a single band with mol wt of 42,000 +/- 1,200 and 39,000 +/- 500, respectively. In the rat ovary and testis, photolabeled GnRH receptors were similar to those in the rat pituitary gland, with two distinct components of 53,000 +/- 1,000 and 42,000 +/- 1,000 mol wt. These findings demonstrate that the pituitary receptors that mediate similar physiological actions of GnRH in different species possess broadly similar structural properties, with minor variations between species. It is also evident that the divergent actions of GnRH in different tissues of the same species, as in the rat pituitary and gonads, are expressed through receptors of similar structure.
Assuntos
Marcadores de Afinidade/metabolismo , Azidas/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Células Intersticiais do Testículo/metabolismo , Adeno-Hipófise/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Dietilestilbestrol/farmacologia , Feminino , Hipofisectomia , Cinética , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Peso Molecular , Coelhos , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/isolamento & purificação , Receptores LHRHRESUMO
The effects of insulin-like growth factor binding proteins (IGFBPs) on human CG (hCG)-induced oocyte maturation, ovulation, steroidogenesis, and intrafollicular plasminogen activator (PA) activity were investigated in rabbit ovaries perfused in vitro. The addition of IGFBP-3, but not IGFBP-1, to the perfusate dose dependently inhibited hCG-induced ovulation, whereas ovulation failed to occur in any ovaries perfused with medium or IGFBP-3 alone. IGFBP-3 (100 ng/ml) significantly inhibited the resumption of meiosis in ovulated ova and follicular oocytes in hCG-treated ovaries, as well as the hCG-stimulated production of estradiol (E2), but not progesterone, by the perfused ovaries. Intrafollicular PA activity increased significantly within 1 h after exposure to hCG, reaching a maximum at 4 h; IGFBP-3 significantly inhibited hCG-stimulated intrafollicular PA activity. The blockade of hCG-induced ovulation by IGFBP-3 correlated with the reduction in intrafollicular PA activity. Treatment with hCG induced a 2.5-fold increase in intrafollicular IGF-I messenger RNA levels at 4 h. Although ovulation failed to occur in ovaries treated with IGF-I (100 ng/ml) in the absence of gonadotropin, IGF-I significantly increased the mean diameter of preovulatory follicles and stimulated the resumption of meiosis in follicular oocytes. These effects of IGF-I on follicular growth and oocyte maturation were significantly inhibited by IGFBP-3 (100 ng/ml). Furthermore, IGFBP-3 significantly inhibited the IGF-I-stimulated production of E2. In conclusion, IGFBP-3, but not IGFBP-1, blocked the stimulatory effects of hCG in the ovulatory process. These findings suggest that IGFBP-3 may contribute to the regulation of intrafollicular PA activity during follicular development and ovulation evoked by gonadotropin exposure, at least in part, via neutralizing endogenously produced IGF-I.
Assuntos
Gonadotropina Coriônica/farmacologia , Estradiol/biossíntese , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Oócitos/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovulação , Animais , Senescência Celular , Feminino , Humanos , Fator de Crescimento Insulin-Like I/genética , Oócitos/fisiologia , Ovário/metabolismo , Ovário/fisiologia , RNA Mensageiro/metabolismo , CoelhosRESUMO
The present study was undertaken to investigate the effects of GH on follicular growth, oocyte maturation, ovulation, and production of insulin-like growth factor-I (IGF-I) in the in vitro perfused rabbit ovaries. Ovulation did not occur in any ovaries perfused with GH at a concentration of 1, 10, 100, or 200 ng/ml, but the addition of GH to the perfusate increased the follicle diameter in a dose-dependent manner. The production of IGF-I by ovaries perfused with medium alone was very low throughout the perfusion period. The addition of 100 ng/ml GH to the perfusate significantly increased ovarian production of IGF-I at 4, 6, 8, and 12 h compared with the contralateral control ovaries. Changes in the tissue concentrations of IGF-I in ovaries perfused with 100 ng/ml GH paralleled those triggered by exposure to 50 IU human CG (hCG). When the effect of GH on the tissue concentration of IGF-I was determined at 4 h, GH stimulated the tissue concentration of IGF-I in perfused rabbit ovaries in a dose-dependent manner. The percent increase in follicle diameter in ovaries treated with GH was significantly correlated with the intraovarian IGF-I content. The mean number of ovulations per ovary and the ovulatory efficiency were significantly reduced in ovaries perfused with 5 IU hCG, compared with those in ovaries perfused with 50 IU hCG. The addition of 100 ng/ml GH to the perfusate significantly increased the ovulatory efficiency and follicle diameter in the 5 IU hCG-treated ovaries. Exposure to GH significantly stimulated the resumption of meiosis in the follicular oocytes compared with that in ovaries perfused with medium alone. Furthermore, GH significantly stimulated the resumption of meiosis in ovulated ova and follicular oocytes in ovaries treated with 5 IU hCG. Thus, exposure to GH-stimulated follicular growth, oocyte maturation, and production of IGF-I in the in vitro perfused rabbit ovaries, which indicates that the ovary is in fact a site of GH reception and action. Additionally, GH enhanced the effects of gonadotropins, acting synergistically to promote the ovulatory process. These observations suggest that GH may amplify gonadotropin actions in the process of follicular development and ovulation, at least in part, by stimulating ovarian IGF-I production.
Assuntos
Hormônio do Crescimento/farmacologia , Fator de Crescimento Insulin-Like I/biossíntese , Folículo Ovariano/fisiologia , Ovário/metabolismo , Animais , Gonadotropina Coriônica/farmacologia , Meios de Cultura , Feminino , Meiose/efeitos dos fármacos , Oócitos/citologia , Concentração Osmolar , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovulação , Perfusão , Coelhos , Fatores de TempoRESUMO
The properties of GnRH receptor sites in the human placenta were analyzed by binding studies performed in particulate and solubilized receptor preparations. The binding affinities (Ka) of the membrane receptor of term placenta for the GnRH superagonists [D-Lys6]- and [D-Ala6]des-Gly10- GnRH-N-ethylamide were 1.6 X 10(6) and 5.4 X 10(5) M-1, respectively. The binding affinities of native GnRH and a potent GnRH antagonist were similar to those of the superagonists (1.1 and 2.0 X 10(6) M-1, respectively). The placental sites could be solubilized by extraction with the detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane sulfonate, with retention of 40-45% of the specific binding activity, though with a significant decrease in binding affinity. The sites were also solubilized with sodium dodecyl sulfate after covalent labeling with a photoreactive 125I-labeled [D-Lys6] GnRH agonist derivative. Analysis of the solubilized hormone-receptor complex on polyacrylamide gradient gels showed a single band of 53,700 mol wt, similar to the mol wt of the pituitary GnRH receptor in other species. It is clear that the placental receptor differs markedly from the GnRH receptor of the pituitary gland in its low binding affinity and lack of selectivity for GnRH analogs. However, it is possible that the human placental receptor for GnRH could serve as a low affinity regulatory site for locally formed GnRH or related low affinity regulatory site for locally formed GnRH or related peptides within the placenta, and that the placental GnRH system has a significant role in the maintenance of pregnancy.
Assuntos
Córion/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Microvilosidades/metabolismo , Placenta/metabolismo , Receptores de Superfície Celular/metabolismo , Ligação Competitiva , Membrana Celular/metabolismo , Feminino , Humanos , Cinética , Peso Molecular , Gravidez , Receptores de Superfície Celular/isolamento & purificação , Receptores LHRH , Especificidade por SubstratoRESUMO
Interleukin-6 (IL-6) may play an important role in human CG (hCG) production by activating the IL-6-receptor (-R) system on human trophoblasts. Trophoblasts produced hCG in response to rIL-6 as well as to 8-bromo cAMP (8-Br-cAMP), 12-O-tetradecanoyl phorbol-13-acetate (TPA), and calcium ionophore A23187. To determine whether the signal transduction pathway activated by the IL-6-R system depends on protein kinases such as protein kinase A, protein kinase C, and Ca2+/calmodulin-dependent kinase, trophoblasts were stimulated with recombinant (r-) IL-6 in the presence or absence of protein kinase inhibitors such as N(2-methyl-aminoethyl)-5-isoquinoline sulfonamide dihydrochloride (H8), and 1-(5-isoquinolinesulfomyl)-2-methylpiperazine (H7) and a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1- napthalenesulfonamide (W7), H8, H7, and W7 failed to suppress rIL-6-induced hCG production but completely inhibited hCG production induced by 8-Br-cAMP, TPA, and the GnRH agonist (GnRHa), respectively. In contrast, genistein, a tyrosine kinase inhibitor, completely suppressed rIL-6-induced hCG production but failed to inhibit hCG production induced by 8-Br-cAMP, TPA, and A23187. Genistein also did not suppress GnRH-induced hCG production. The addition of genistein to rIL-1- and rTNF-alpha-stimulated trophoblasts inhibited rIL-1-induced and rTNF-alpha induced hCG production but maintained rIL-1- and rTNF-alpha-induced IL-6 production. These results show that the IL-6/IL-6-R system-induced signal transduction pathway in the placenta probably stimulates hCG production by activating a tyrosine kinase pathway. The experiment with genistein shows that the GnRH/GnRH-R system activates a signal transduction pathway distinct from that activated by the IL-6/IL-6-R system.
Assuntos
Gonadotropina Coriônica/biossíntese , Hormônio Liberador de Gonadotropina/farmacologia , Interleucina-6/fisiologia , Proteínas Tirosina Quinases/metabolismo , Receptores Imunológicos/fisiologia , Transdução de Sinais/fisiologia , Trofoblastos/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Calcimicina/farmacologia , Calmodulina/antagonistas & inibidores , Ativação Enzimática , Feminino , Genisteína , Humanos , Interleucina-1/farmacologia , Interleucina-6/biossíntese , Interleucina-6/farmacologia , Isoflavonas/farmacologia , Inibidores de Proteínas Quinases , Receptores de Interleucina-6 , Proteínas Recombinantes/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Trofoblastos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Plasma CRH levels are considerably higher in women during the third trimester of pregnancy than in non-pregnant women. Most of plasma CRH in pregnant women is bound to CRH-binding protein (CRH-BP). To gain further insight into CRH physiology during pregnancy, we measured the responses of plasma ACTH and cortisol and the changes in bound and free forms of CRH in plasma after human CRH administration (2 micrograms/kg) in five pregnant (39-40 weeks of pregnancy) and five nonpregnant women. The mean basal plasma ACTH and cortisol levels in the pregnant women were higher than those in the nonpregnant women. However, the maximum increments in plasma ACTH and cortisol levels and the integrated ACTH and cortisol responses, after subtraction of the basal levels after CRH administration, were similar in the two groups. The plasma CRH half-time in the pregnant group was similar to that in the nonpregnant group. The mean basal plasma CRH level in the nonpregnant women was 1.5 +/- 0.2 (+/- SE) pmol/L, and that in the pregnant women was 360 +/- 35 pmol/L. On gel filtration chromatography, almost all of the CRH in the plasma was protein bound (320 +/- 30 pmol/L) in the pregnant women; no CRH peaks were detected in nonpregnant women because of the low plasma CRH levels. After CRH administration, the level of the bound form of plasma CRH was highest at 5 min, and then declined to a plateau at 15 min and 30 min in the pregnant women. In the nonpregnant women, protein-bound CRH also was highest at 5 min, but it progressively declined thereafter. The disappearance rate of the bound CRH in plasma from the nonpregnant women was similar to that of the second compartment of the plasma decay curves of the free CRH from both groups. We conclude that the plasma ACTH and cortisol responses to exogenous CRH are similar in pregnant and nonpregnant women, the effect of CRH-BP on the disappearance of plasma CRH is minimal, and plasma CRH-BP in pregnant women has the capacity to bind additional CRH.
Assuntos
Hormônio Adrenocorticotrópico/sangue , Hormônio Liberador da Corticotropina/farmacocinética , Hidrocortisona/sangue , Gravidez/sangue , Adulto , Sítios de Ligação , Cromatografia em Gel , Hormônio Liberador da Corticotropina/sangue , Feminino , Humanos , RadioimunoensaioRESUMO
A human plasma CRH-binding protein (CRH-BP) was identified and characterized by chemical cross-linking of 125I-Tyr-hCRH to human plasma using disuccinimidyl suberate. The apparent mol wt of the cross-linked complex determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography was approximately 43,000. The mol wt was slightly lower in the nonreduced state, suggesting the presence of intramolecular disulfide bonds. Subtracting the mol wt of 125I-Tyr-CRH, the BP appeared to have a mol wt of approximately 38,000. Binding was specific since the appearance of the 43,000 dalton band was not affected by unlabeled ACTH, vasopressin, serum albumin, or gamma-globulin, but was inhibited by unlabeled hCRH dose dependently. Pretreatment of plasma with 0.1 mol/L HCl, 0.01 mol/L NaOH, 10 mmol/L dithiothreitol, or trypsin before cross-linking abolished its ability to bind 125I-Tyr-hCRH. Rat, rabbit, or goat plasma or human cerebrospinal fluid did not bind 125I-Tyr-CRH. It is unlikely that CRH-BP is a CRH receptor, because the estimated mol wt of the CRH-BP is smaller than the reported size of CRH receptors, and the CRH-BP did not bind to ovine CRH. The binding of 125I-Tyr-CRH to CRH-BP decreased in the third trimester of pregnancy, when plasma CRH levels were markedly elevated. However, after dissociating endogenous CRH from the CRH-BP, the binding was almost the same as in nonpregnant subjects. In addition, CRH-BP inhibited CRH-induced ACTH secretion from cultured rat anterior pituitary cells. We conclude that most of the increased plasma CRH found in pregnant women is bound to CRH-BP, and so is inactive, therefore plasma ACTH levels do not increase to above the normal range.
Assuntos
Proteínas de Transporte/sangue , Hormônio Liberador da Corticotropina/sangue , Gravidez/sangue , Hormônio Adrenocorticotrópico/metabolismo , Adulto , Animais , Ligação Competitiva , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/fisiologia , Células Cultivadas , Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Adeno-Hipófise/metabolismo , Ligação Proteica , Radioimunoensaio , RatosRESUMO
The Rho family of small GTPases occupies a key position in the control of cell motility and morphology in response to extracellular stimuli. Rho proteins trigger the formation of contractile stress fibers, resulting in regulation of cell motility. We explored the expression and function of RhoA in human endometrium and decidua. RhoA immunoreactivity had a predominantly glandular epithelial distribution in the proliferative phase and midsecretory phase. In decidua, the expression of RhoA was more pronounced in the stromal cells as well as in the glandular epithelium. RhoA protein levels in proliferative phase and midsecretory phase endometrium as well as decidua were evaluated by immunoblotting; a single band of RhoA protein with a molecular mass of 21 kDa was detected in all cell lysates. Cultured human decidual cells were found to have few actin stress fibers. Decidual cells lost their actin stress fibers by the treatment with C3, an exoenzyme produced by Clostridium botulinum, whereas new actin stress fibers appeared in human decidual cells stimulated with lysophosphatidic acid (LPA). Mouse blastocysts became attached to cultured human decidual cells after embryos hatched from the zona pellucida. The majority of hatched blastocysts attached to human decidual cells within 24 h. Blastocysts attached to decidual cells exhibited extensive outgrowth after 48 h in culture. Treatment of decidual cells with C3 exoenzyme or LPA did not affect the rates of hatching and attachment of blastocysts, but outgrowth of embryos on decidual cells was inhibited by C3 exoenzyme treatment in a dose-dependent manner. Contrariwise, addition of LPA to decidual cells dose dependently increased the outgrowth of embryos on decidual cells. These findings suggest that RhoA in decidual cells is important for embryonic development and differentiation after attachment.
Assuntos
Toxinas Botulínicas , Implantação do Embrião/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , ADP Ribose Transferases/metabolismo , Actinas/metabolismo , Adulto , Animais , Blastocisto/metabolismo , Western Blotting , Adesão Celular , Células Cultivadas , Decídua/metabolismo , Endométrio/citologia , Endométrio/efeitos dos fármacos , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Indicadores e Reagentes , Lisofosfolipídeos/farmacologia , Camundongos , GravidezRESUMO
Specific receptors for gonadotrophin-releasing hormone (GnRH) were extracted from the rat pituitary gland with several detergents and characterized by binding studies with the potent GnRH antagonist [Ac-D-pCl-Phe1.2, D-Trp3, D-Lys6, D-Ala10]-GnRH (GnRHant). The particulate GnRH receptors were most effectively solubilized with 5 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS), which extracted 63% of the original membrane binding activity when assayed with 125I-labelled GnRHant. The binding affinities of particulate and CHAPS-solubilized receptors analysed with 125I-labelled GnRHant were 1.5 +/- 0.4 x 10(9) M-1 and 1.2 +/- 0.2 x 10(9) M-1 respectively. Gel filtration of the CHAPS-solubilized receptor revealed a major peak of specific binding activity with Mr of about 700,000. A hormone-receptor complex of similar Mr was observed when CHAPS-solubilized receptors were labelled with photoreactive radioiodinated [D-Lys6]-des-Gly10-GnRH-N-ethyl-amide and then analysed by gel chromatography. However, when pituitary particles were photolabelled and solubilized in 2% Triton X-100 before analysis on Sephacryl S-300, the Mr of the receptor was approximately 250,000, similar to the value obtained by sucrose density gradient centrifugation of the CHAPS-solubilized receptor. After solubilization in sodium dodecyl sulphate (SDS) the photolabelled receptor was eluted from Sephacryl S-300 as a 60 kDa peak which on SDS-gel electrophoresis contained a 52 kDa component, corresponding to the major binding subunit extracted directly from photolabelled pituitary membranes. The difference in higher molecular weight forms observed under non-denaturing and denaturing conditions could reflect the need for additional membrane components to maintain the active conformation of the GnRH receptor site. Whereas the minimum Mr of the solubilized receptor is about 250,000 under non-denaturing conditions, analysis of the photolabelled GnRH-receptor complex by SDS chromatography and electrophoresis indicates that a binding subunit with Mr of 50,000-60,000 is present in the GnRH holoreceptor.
Assuntos
Hipófise/metabolismo , Receptores LHRH/isolamento & purificação , Marcadores de Afinidade , Animais , Azidas , Ligação Competitiva , Fenômenos Químicos , Físico-Química , Ácidos Cólicos , Detergentes , Masculino , Peso Molecular , Ensaio Radioligante , Ratos , Ratos Endogâmicos , Receptores LHRH/metabolismoRESUMO
The cellular mechanisms involved in the accelerated bone loss occurring in association with estrogen deprivation as seen following the menopause are not fully understood. Insulin-like growth factor-I (IGF-I) is the local regulator of osteoblasts and one of its binding proteins, insulin-like growth factor-binding protein-4 (IGFBP-4), binds to IGF-I and suppresses biological activity. Previous studies have shown that the binding activity of IGFBP-4 in the conditioned medium of parathyroid hormone (PTH)-treated SaOS-2 osteoblastic-like cells is enhanced twofold and that this PTH-enhanced IGFBP-4 binding activity is abolished by 17 beta-estradiol. Levels of IGFBP-4 in the conditioned medium have been reported to be regulated not only at the level of production but also at the level of degradation which is catalyzed by a protease that specifically cleaves IGFBP-4. We have, therefore, studied the effects of 17 beta-estradiol and PTH on IGFBP-4 protease activity using SaOS-2 cells. SaOS-2 cells produce a protease that specifically cleaves IGFBP-4 into two fragments of approximately 18 and 14 kilodaltons. IGFBP-4 protease activity in the conditioned medium from PTH-treated cells was suppressed, while this PTH-induced suppression of protease activity was reversed by the addition of 17 beta-estradiol to the cultures. IGFBP-4 proteolytic activity was stimulated by IGF-I or IGF-II added exogenously and was inhibited by EDTA or protease inhibitors. IGFBP-4 proteolyzed in the conditioned medium from cells treated with PTH and 17 beta-estradiol was less effective at inhibiting IGF-I-stimulated [3H]thymidine incorporation into DNA compared with that proteolyzed in the conditioned medium from PTH-treated cells. The simplest explanation is that 17 beta-estradiol suppressed the inhibitory effect of PTH on osteoblastic activity by inhibiting the PTH-induced suppression of IGFBP-4 protease activity.
Assuntos
Estradiol/farmacologia , Metaloendopeptidases/metabolismo , Osteoblastos/enzimologia , Osteoporose Pós-Menopausa/etiologia , Hormônio Paratireóideo/farmacologia , Linhagem Celular , DNA/biossíntese , Depressão Química , Feminino , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/farmacologia , Pessoa de Meia-Idade , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoporose Pós-Menopausa/metabolismo , Proteína Plasmática A Associada à Gravidez , Timidina/metabolismoRESUMO
We have examined the biological effects of insulin-like growth factor-binding proteins (IGFBPs) on insulin-like growth factor (IGF)-activated glucose consumption in a BALB/c 3T3 subline. The method employed was a colorimetric measurement of glucose consumption, allowing the detection of changes from the initial glucose concentration in conditioned medium, following the addition of IGFs and IGFBPs. Human IGFBP-1, purified from amniotic fluid, inhibited IGF-activated glucose consumption, although it had no effect on insulin-activated glucose consumption. The median effective dose (ED50) of IGFBP-1 to cause inhibitory effects on IGF-activated glucose consumption was 100-200 micrograms/l and was similar for both IGF-I and IGF-II at a concentration of 1.0 microgram IGF/l. Therefore, at IGF concentrations of comparable activity, the inhibitory effects of IGFBP-1 were greater for IGF-I than for IGF-II, because of the higher activity of IGF-I in this assay. Recombinant human IGFBP-3 also inhibited IGF-activated glucose consumption, without affecting insulin-stimulated glucose consumption. The inhibitory effects of IGFBP-3 were greater for IGF-II than for IGF-I when IGFBP-3 was coincubated with either of the IGFs, at both IGF concentrations of comparable activity and equivalent molar concentrations. Thus, it became clear that the inhibitory effects of these IGFBPs on IGF biological action depended primarily upon their affinity for the specific IGF ligand and molar ratio of IGFBP/IGF peptide. Interestingly, when cells were pretreated with IGFBP-3, prior to the simultaneous addition of IGFs and IGFBP-3, the inhibitory effect was higher for IGF-I than for IGF-II. Either no effect or a minor inhibitory effect on IGF-activated glucose consumption was detected with IGFBP pretreatment alone. When the ED50 for inhibition of IGF action by IGFBPs in this in-vitro assay was compared with the physiological concentrations of IGFs and IGFBPs in normal human serum and in amniotic fluid, it was estimated that the IGFBP-1 concentration present in serum was not sufficient to modulate IGF action effectively while the concentration in amniotic fluid was enough for effective suppression. IGFBP-3 exhibited an ED50 low enough to suppress IGF-II and possibly IGF-I action when cells were pretreated with IGFBP-3. Thus, our data suggested that IGFBP-1 in amniotic fluid and IGFBP-3 in serum could be a potent inhibitor for IGF action. IGFBP-1 in serum, however, may not be able to function as a direct inhibitor under physiological conditions but, rather, may modulate IGF action together with other IGFBPs.
Assuntos
Proteínas de Transporte/farmacologia , Fibroblastos/metabolismo , Glucose/metabolismo , Somatomedinas/metabolismo , Animais , Células Cultivadas , Colorimetria , Depressão Química , Fibroblastos/efeitos dos fármacos , Insulina/farmacologia , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The effect of activation of calcium- and phospholipid-dependent protein kinase (protein kinase C) on human chorionic gonadotropin (hCG) release by cultured trophoblast cells was studied and a role of protein kinase C in the GnRH-mediated hCG release was also evaluated. Both GnRH and 1-oleoyl-2-acetylglycerol (OAG), a protein kinase C activator, stimulated hCG release after 3 h incubation in a dose-dependent manner with ED50 of 55 nmol/l and 4.0 nmol/l, respectively. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also stimulated hCG release while two non-tumor-promoting compounds, phorbol and 4 alpha-phorbol, failed to stimulate hCG release. hCG release by maximal effective dose of GnRH (10 mumol/l) or OAG (1 mumol/l) was further stimulated when cells were incubated with same concentrations of GnRH and OAG. OAG-stimulated hCG release was completely inhibited by a protein kinase C inhibitor, H-7, with ID50 of 23 nmol/l while H-7 did not affect GnRH-mediated hCG release. These results indicate that GnRH-stimulated hCG release is not mediated by protein kinase C pathway, however, the secretion of hCG is also regulated by the mechanism that involves protein kinase C activation.
Assuntos
Gonadotropina Coriônica/metabolismo , Diglicerídeos/farmacologia , Hormônio Liberador de Gonadotropina/farmacologia , Trofoblastos/efeitos dos fármacos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Isoquinolinas/farmacologia , Piperazinas/farmacologia , Gravidez , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Trofoblastos/metabolismoRESUMO
The effect of gonadal steroids on basal and GnRH-stimulated hCG release was studied using collagenase-dispersed trophoblast cells from early pregnancy. Both GnRH and a GnRH superagonist, Buserelin, stimulated hCG release with a similar dose dependency. Progesterone (0.1 to 10 micrograms/ml) inhibited GnRH-stimulated hCG release in a dose dependent manner as well as basal hCG release. Relatively high concentrations of estradiol (10 micrograms/ml) stimulated both basal and GnRH-mediated hCG release and antagonized the inhibitory effect of progesterone on hCG release at 1 micrograms/ml as well as RU486 (1 microgram/ml). These results indicate that progesterone has an important role in both basal and GnRH-mediated hCG regulatory system in the placenta.
Assuntos
Gonadotropina Coriônica/metabolismo , Hormônios Esteroides Gonadais/fisiologia , Hormônios Liberadores de Hormônios Hipofisários/fisiologia , Trofoblastos/metabolismo , Busserrelina/farmacologia , Células Cultivadas , Estradiol/fisiologia , Estrenos/farmacologia , Feminino , Humanos , Mifepristona , Hormônios Liberadores de Hormônios Hipofisários/antagonistas & inibidores , Gravidez , Progesterona/antagonistas & inibidores , Progesterona/fisiologiaRESUMO
The effect of insulin-like growth factor-I (IGF-I) and 2-methyl-3-all-trans-tetraphenyl-1,4-naphtoquinone (vitamin K2) on the synthesis of osteocalcin containing gamma-carboxyglutamic acid (Gla) residues which is the physiologically relevant form in bone metabolism was studied in cultured human osteoblast-like (MG-63) cells. Both IGF-I and vitamin K2 stimulated 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced osteocalcin containing Gla secretion in a concentration-dependent manner. This stimulatory effect of IGF-I and vitamin K2 was additive. Vitamin K2-enhanced osteocalcin containing Gla secretion was selectively suppressed by 3-(alpha-acetonyl-benzyl)-4-hydroxy-coumarin (warfarin). The stimulatory effect of IGF-I was completely abolished by the presence of cycloheximide; in contrast the effect of vitamin K2 was still observed in the presence of cycloheximide. Treatment of MG-63 cells with IGF-I caused an approximately 2.2-fold increase in osteocalcin mRNA levels (determined by reverse transcription-polymerase chain reaction). Vitamin K2 had no effect on either the stimulation of mRNA level by IGF-I or the basal level. IGF-I-stimulated osteocalcin containing Gla secretion was inhibited by one of its binding proteins (insulin-like growth factor binding protein-4) in a concentration-dependent manner. These findings suggest that the modes of action of IGF-I and vitamin K2 on 1.25(OH)2D3-induced osteocalcin containing Gla secretion in MG-63 cells are different.