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1.
Reprod Fertil Dev ; 25(2): 343-50, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22950963

RESUMO

The hypoxic microenvironment that occurs in fast-growing tissue such as the corpus luteum (CL) is a major contributor to its ability to survive via the induction of an intricate vascular network. Cellular responses to hypoxia are mediated by hypoxia-inducible factor-1 (HIF-1), an oxygen-regulated transcriptional activator. HIF-1, a heterodimer consisting of a constitutively-expressed ß subunit and an oxygen-regulated α subunit, binds to the hypoxia responsive element (HRE) present in the promoter regions of responsive genes. This review summarises evidence for the involvement of hypoxia and HIF-1α in CL development and function. Special emphasis is given to hypoxia-induced, luteal cell-specific expression of multiple genes (vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF-2), prokineticin receptor 2 (PK-R2), stanniocalcin 1 (STC-1) and endothelin 2 (EDN-2) that participate in the angiogenic process during CL formation.


Assuntos
Hipóxia Celular/fisiologia , Corpo Lúteo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Fisiológica/fisiologia , Ovário/irrigação sanguínea , Ovário/embriologia , Corpo Lúteo/embriologia , Endotelina-2/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hormônios Gastrointestinais/metabolismo , Glicoproteínas/metabolismo , Humanos , Modelos Biológicos , Neovascularização Fisiológica/genética , Neuropeptídeos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Life Sci ; 91(13-14): 703-9, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22727793

RESUMO

AIMS: To examine the levels of endothelin system components in granulosa lutein cells (GLCs) of women with PCOS and compare them to normally ovulating women undergoing In Vitro Fertilization (IVF). Polycystic ovary syndrome (PCOS) is one of the most common endocrine-metabolic disorders in women of reproductive age. Endothelins are locally produced by endothelial and granulosa cells of the preovulatory follicle. Abnormal expression or production of endothelins may be a contributing factor in PCOS pathogenesis. MAIN METHODS: Follicular aspirates containing GLCs were obtained from PCOS and normally ovulating patients undergoing oocyte retrieval during the IVF cycle. RNA was extracted and endothelin system components were quantified using real-time PCR. GLCs were cultured in basal media for 7 days, and then challenged with various luteinizing agents (luteinizing hormone, hCG, or forskolin) for 24 h. KEY FINDINGS: In GLCs from women with PCOS, Endothelin-1 mRNA expression was elevated (2.2-fold) as compared with normally ovulating women, whereas endothelin-2 mRNA was reduced (1.8-fold). ET receptors and endothelin-converting enzyme showed the same expression levels in the two groups. In vitro modeling showed that although the steroidogenic response was preserved in GLC, endothelin expression levels were not exhibited in vitro in their original pattern. SIGNIFICANCE: Dysregulation of ovarian endothelin expression may induce a pathologic ovulation pattern characteristic of PCOS.


Assuntos
Endotelina-1/genética , Endotelina-2/genética , Regulação da Expressão Gênica , Células Lúteas/metabolismo , Síndrome do Ovário Policístico/fisiopatologia , Adulto , Ácido Aspártico Endopeptidases/genética , Estudos de Casos e Controles , Gonadotropina Coriônica/farmacologia , Colforsina/farmacologia , Enzimas Conversoras de Endotelina , Feminino , Fertilização in vitro , Humanos , Hormônio Luteinizante/farmacologia , Metaloendopeptidases/genética , Recuperação de Oócitos , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Receptores de Endotelina/genética , Fatores de Tempo , Adulto Jovem
3.
Biol Reprod ; 86(3): 92, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22174022

RESUMO

We recently compared prostaglandin F2alpha (PG)-induced global gene expression profiles in PG-refractory, bovine corpus luteum (CL) collected on Day 4 of the estrous cycle, versus PG-responsive, Day 11 CL. Transcriptome analyses led us to study the regulation of angiogenesis-related genes by PG and their functions in luteal endothelial cells (ECs). We found that PG regulated angiogenesis-modulating factors in a luteal stage-dependent way. A robust increase in FGF2 expression (mRNA and protein) occurred in the PG-refractory Day 4 CL promoting CL survival and function. Inhibitors of FGF2 action, thrombospondin 1 and 2, their receptor (CD36), and PTX3 were upregulated by PG specifically in Day 11 CL undergoing luteolysis. VEGF mRNA decreased 4 h post-PG in both Day 4 and Day 11 CL. The resulting destabilization of blood vessels in Day 11 CL is expected to weaken the gland and reduce its hormonal output. These genes were expressed in dispersed luteal ECs and steroidogenic cells; however, thrombospondin 1 and FGF2 were more abundant in luteal ECs. Expression of such genes and their ability to modulate FGF2 actions were investigated. Similar to its in vivo effect, PG, in vitro, stimulated the expression of thrombospondins and PTX3 genes in several luteal cell models. Importantly, these factors influenced the angiogenic properties of luteal ECs. FGF2 dose-dependently enhanced cell migration and proliferation, whereas thrombospondin 1 and PTX3 inhibited FGF2 actions in luteal ECs. Collectively, the data presented here suggest that, by tilting the balance between pro- and antiangiogenic factors, PG can potentially control the ability of the CL to resist or advance toward luteolysis.


Assuntos
Bovinos/fisiologia , Corpo Lúteo/fisiologia , Dinoprosta/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Luteólise/fisiologia , Neovascularização Fisiológica/fisiologia , Animais , Proteína C-Reativa/genética , Proteína C-Reativa/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica/fisiologia , Fase Luteal/fisiologia , Modelos Animais , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/fisiologia , Trombospondina 1/genética , Trombospondina 1/fisiologia , Trombospondinas/genética , Trombospondinas/fisiologia
4.
Endocrinology ; 151(4): 1914-22, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20176726

RESUMO

The pattern and regulation of endothlin-2 (EDN2) expression and its putative roles in bovine ovaries were investigated. EDN2 mRNA was determined in corpus luteum (CL) and during folliculoluteal transition induced by GnRH in vivo. EDN2 was elevated only in the early CL and was not present in older CL. In the young CL, EDN2 mRNA was identified mainly in luteal cells but not endothelial cells that expressed the EDN1 gene. Similarly, in preovulatory follicles, EDN2 was expressed in the granulosa cells (GCs) and not in the vascular theca interna. LH and hypoxia are two major stimulants of CL formation. Therefore, GCs were cultured with bovine LH, under hypoxic conditions. GCs incubated with bovine LH resulted in increased EDN2 mRNA 42 h later. CoCl2, a hypoxia-mimicking agent, elevated EDN2 in GCs in a dose-dependent manner. Incubation of the human GC line (Simian virus 40 large T antigen) under low oxygen tension (1%) augmented EDN2 6 and 24 h later. In these two cell types, along with EDN2, hypoxia augmented VEGF. EDN2 induced in GCs changes that characterize the developing CL: cell proliferation as well as up-regulation of vascular endothelial growth factor and cyclooxygenase-2 (mRNA and protein levels). Human chorionic gonadotropin also up-regulated these two genes. Small interfering RNA targeting EDN-converting enzyme-1 effectively reduced its mRNA levels. This treatment, expected to lower the mature EDN2 peptide production, inhibited VEGF mRNA levels and GC numbers. Together these data suggest that elevated EDN2 in the early bovine CL, triggered by LH surge and hypoxia, may facilitate CL formation by promoting angiogenesis, cell proliferation, and differentiation.


Assuntos
Corpo Lúteo/crescimento & desenvolvimento , Endotelina-2/metabolismo , Células da Granulosa/metabolismo , Hipóxia/metabolismo , Hormônio Luteinizante/metabolismo , Análise de Variância , Animais , Western Blotting , Bovinos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cobalto/farmacologia , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/metabolismo , Relação Dose-Resposta a Droga , Endotelina-2/genética , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Hormônio Liberador de Gonadotropina/farmacologia , Células da Granulosa/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Endocrinology ; 150(1): 413-21, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18818292

RESUMO

Follicular development, follicular rupture, and corpus luteum (CL) formation are accompanied by extensive tissue remodeling. We examined whether heparanase (HPSE), which cleaves heparan sulfate glycosaminoglycans, is induced during these processes. Prostaglandin F2alpha injection, which initiated luteolysis and the development of a preovulatory follicle, moderately increased HPSE mRNA in bovine granulosa cells (GCs). GnRH, used to induce gonadotropin surge, markedly augmented HPSE mRNA levels 12 h after its injection. The temporal pattern of HPSE gene expression in follicular-luteal transition was further examined in follicles collected before, and 4, 10, 20, 25, and 60 h after GnRH injection. HPSE mRNA increased transiently 10-20 h after GnRH injection to levels 10-fold higher than in untreated heifers. HPSE protein levels were similarly elevated 20 h after GnRH injection in GCs, but not in the theca layer. Cyclooxygenase-2 (PTGS2) mRNA peaked before ovulation when HPSE levels returned to baseline levels. HPSE mRNA abundance also remained low in the CLs. The antiprogesterone, RU-486, elevated HPSE levels in GC culture, suggesting that progesterone secreted by CLs may inhibit HPSE. HPSE immunostaining was more abundant in GCs than thecae. In cultured GCs, LH induced a transient increase in HPSE mRNA 3-6 h after its addition, but not at 24 h. However, PTGS2 mRNA was clearly induced at this time. These findings suggest that: 1) HPSE may play a role in ovulation but much less so during CL development, and 2) GC-derived HSPE may be a novel member of the LH-induced extracellular matrix-degrading enzyme family and may contribute to follicular rupture.


Assuntos
Glucuronidase/genética , Células da Granulosa/enzimologia , Hormônio Luteinizante/farmacologia , Animais , Aromatase/genética , Bovinos , Feminino , Regulação da Expressão Gênica , Glucuronidase/biossíntese , Hormônio Liberador de Gonadotropina/fisiologia , Células da Granulosa/efeitos dos fármacos , Lactação/fisiologia , Hormônio Luteinizante/fisiologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Ovulação , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética
6.
Cancer Res ; 68(22): 9265-73, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19010899

RESUMO

Endothelin-1 (ET-1) has been implicated in the progression of various cancers, including ovarian carcinoma. We found that the ovarian carcinoma cell lines ES2 and OVCAR3 and tumors from different anatomic sites expressed ET-1 system members [ET receptor A and ET-converting enzyme-1 (ECE-1)]. However, only ECE-1 was significantly higher in the solid tumors compared with effusions. We therefore investigated the effect of RNA interference-induced knockdown of ECE-1, the key enzyme in ET-1 production, on these two ovarian carcinoma cell lines. Small interfering RNA (siRNA) targeting of ECE-1 markedly reduced ECE-1 mRNA and protein levels, which subsequently led to 80% to 90% inhibition of ET-1 peptide secretion by the cells. ECE-1 silencing also profoundly affected the behavior of tumor cells compared with cells treated with scrambled siRNA. Silenced cells exhibited (a) reduced ET-1-dependent p44/42 mitogen-activated protein kinase phosphorylation; (b) decreased invasiveness and matrix metalloproteinase-2 activity; (c) improved adhesion to basal lamina proteins, laminin-1, and collagen IV; and (d) increased E-cadherin, an epithelial adhesion molecule, and reduced N-cadherin expression, a mesenchymal marker. Altered cell adherence is one of the hallmarks of the transformed phenotype, often characterized by the loss of the epithelial features and the gain of a mesenchymal phenotype. ECE-1 ablation did not, however, alter viable ovarian carcinoma cell numbers. Addition of exogenous ET-1 reversed the effects cited above. Taken together, these data indicate that siRNA is an effective tool for manipulating ECE-1 expression, ET-1 biosynthesis, and invasiveness of ovarian carcinoma. ECE-1 silencing may therefore develop into a promising novel anticancer therapy.


Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Endotelina-1/antagonistas & inibidores , Metaloendopeptidases/antagonistas & inibidores , Neoplasias Ovarianas/terapia , RNA Interferente Pequeno/farmacologia , Ácido Aspártico Endopeptidases/genética , Linhagem Celular Tumoral , Endotelina-1/biossíntese , Enzimas Conversoras de Endotelina , Feminino , Humanos , Metaloendopeptidases/genética , Invasividade Neoplásica , Neoplasias Ovarianas/patologia , RNA Mensageiro/análise , Receptor de Endotelina A/genética
7.
Biol Reprod ; 76(5): 749-58, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17229935

RESUMO

Prokineticin 1 (PROK1), also termed endocrine gland-derived vascular endothelial growth factor (endocrine gland-derived VEGF), is a newly identified protein assigned with diverse biologic functions. It binds two homologous G protein-coupled receptors, PROKR1 and PROKR2. To better understand the roles of PROK1 and its receptors in ovarian function, their expression was determined in follicles and corpora lutea (CLs) at different developmental stages. PROK1 mRNA levels were low at early luteal stage and midluteal stage, but increased sharply during natural or induced luteolysis. High PROK1 mRNA levels also were found in atretic follicles. This profile of PROK1 expression was opposite to that of the well-established angiogenic factor VEGF. Of the two receptor-type expressions, PROKR1 but not PROKR2 was correlated positively with its ligand. Immunohistochemical staining revealed that PROK1 was located mainly within the muscular layer of arterioles, and during regression it also was localized to macrophages and steroidogenic cells. The expression pattern of ITGB2 mRNA, a leukocyte cell marker, overlapped that of PROK1, thus suggesting that leukocyte infiltration may explain the elevated expression of PROK1 in atretic follicles and regressing CL. Indeed, flow cytometry analyses showed that nearly all beta-2 integrin chain (ITGB2)-positive cells also were stained with anti-PROK1 and that significantly more ITGB2/PROK1 double-stained cells were present in degenerating follicles and CL. Furthermore, when challenged in vitro with PROK1, adherent, mononuclear cell numbers and TNF levels were elevated, indicating that PROK1 triggers monocyte activation. Together, these data suggest that PROK1, acting via PROKR1, may be involved in the recruitment of monocytes to regressing CL and atretic follicles and their consequent activation therein.


Assuntos
Corpo Lúteo/crescimento & desenvolvimento , Ciclo Estral/metabolismo , Atresia Folicular/fisiologia , Ovário/metabolismo , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/biossíntese , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/genética , Animais , Bovinos , Contagem de Células , Ceramidas/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/biossíntese , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Dinoprosta/biossíntese , Dinoprosta/genética , Escherichia coli/metabolismo , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese
8.
Cell Physiol Biochem ; 18(6): 315-26, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17170518

RESUMO

Prokineticins (PKs), multifunctional secreted proteins, activate two endogenous G protein-coupled receptors (R) termed PK-R1 and PK-R2. It was suggested that PK1 acts selectively on the endothelium of endocrine glands, yet PK-Rs were also found in endothelial cells (EC) derived from other tissues. Therefore we examined here the characteristics of PK - system in EC derived from different vascular beds. Corpus luteum (CL)-derived EC (LEC) expressed both PK-R1 and PK-R2. In contrast, EC from the aorta (BAEC) only expressed PK-R1. Interestingly, also EC from brain capillaries (BCEC) expressed only PK-R1. The distinct pattern of PK-R expression may define EC phenotypic heterogeneity. Regulation of receptor expression also differed in BAEC and LEC: TNFalpha markedly reduced PK-R1 only in BAEC, but serum removal decreased PK-R1 in both cell types. Therefore, if cells were initially serum-starved, the anti-apoptotic effect of PKs was retained only in LEC. Yet, addition of PKs concomitant with serum removal enhanced the proliferation and survival of both BAEC and LEC. Immunohistochemical staining showed that in CL and aorta PK1 was expressed in smooth muscle cells in vessel walls, suggesting a paracrine mode of action. PK1 enhanced the net paracellular transport (measured by electrical resistance and Mannitol transport) in LEC but not in BAEC or BCEC. Collectively, these findings indicate that PKs serve as mitogens and survival factors for microvascular (LEC) and macrovascular (BAEC) EC. However, the distinct expression and function of PK receptors suggest different physiological roles for these receptors in various EC types.


Assuntos
Endotélio Vascular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismo , Animais , Aorta/química , Aorta/citologia , Aorta/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Encéfalo/metabolismo , Capilares/citologia , Capilares/metabolismo , Bovinos , Células Cultivadas , Corpo Lúteo/irrigação sanguínea , Corpo Lúteo/citologia , Corpo Lúteo/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/química , Endotélio Vascular/citologia , Feminino , Imuno-Histoquímica , Permeabilidade , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética
9.
Endocrinology ; 147(11): 5228-35, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16887911

RESUMO

Endothelin-1 (ET-1) and nitric oxide (NO) play pivotal roles in corpus luteum (CL) function. The present study examined the interplay between NO and ET-1 synthesis in the bovine CL. We found similar inducible and endothelial NO synthase (iNOS and eNOS, respectively) activities in the young CL (d 1-5) expressing the highest levels of both eNOS and iNOS mRNA. These values later declined at mid-cycle (d 8-15) and remained low at later stages (d 16-18). Luteolysis, initiated by prostaglandin F2alpha analog administration, further reduced NOS mRNA and by 24 h, NOS values dropped to approximately 15% of those at mid-cycle. eNOS protein levels followed a similar pattern to its mRNA. Because endothelial cells (ECs) are the main site for ET-1 and NO production in the CL, we examined the direct effects of the NO donor, NONOate on luteal ECs (LECs). Elevated NO levels markedly decreased ET-1 mRNA, and peptide concentrations in cultured and freshly isolated LECs in a dose-dependent manner. In agreement, NOS inhibitor, NG-nitro-l-arginine methyl ester, stimulated ET-1 mRNA expression in these cells. Interestingly, NO also up-regulated prostaglandin F2alpha receptors in LECs. These data show that there is an inverse relationship between NOS and ET-1 throughout the CL life span, and imply that this pattern may be the result of their interaction within the resident LECs. NOS are expressed in a physiologically relevant manner: elevated NO at an early luteal stage is likely to play an important role in angiogenesis, whereas reduced levels of NO during luteal regression may facilitate the sustained up-regulation of ET-1 levels during luteolysis.


Assuntos
Corpo Lúteo/metabolismo , Células Endoteliais/metabolismo , Endotelina-1/biossíntese , Óxido Nítrico Sintase Tipo III/fisiologia , Óxido Nítrico Sintase Tipo II/fisiologia , Animais , Bovinos , Dinoprosta/farmacologia , Feminino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/fisiologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III/genética , RNA Mensageiro/análise
10.
Exp Biol Med (Maywood) ; 231(6): 723-8, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16740988

RESUMO

Endothelin-converting enzyme (ECE)-1 cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (ECE-1a, 1b, 1c, and 1d) have been identified to date, they differ only in their amino terminus and share the catalytic domain located in the C-terminal end. In addition to full-length ECE-1 forms, we identified novel, alternatively spliced messenger RNAs (mRNAs) of ECE-1b, 1c, and 1d. These splice variants (SVs) lack exon 3', which codes for the transmembrane (TM) region and is present in full-length forms. SV mRNAs were highly expressed in endothelial cells (EC) derived from macrovascular and microvascular beds. Analyses of ECE-1d and its SV forms in stably transfected human embryonic kidney (HEK)-293 cells revealed that both proteins were recognized by antibodies to C-terminal ECE-1, but an antibody to the N-terminal only bound ECE-1d. The novel protein, designated ECE-1sv, has an apparent molecular weight of 75 kDa. ECE-1sv lacks the TM sequence (or signal peptide) and, therefore, is expected to remain cytosolic. Presence of ECE-1sv in different cellular compartments than the full-length forms of the ECE-1 may suggest a distinct physiologic role for these proteins.


Assuntos
Processamento Alternativo , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Endotélio Vascular/citologia , Metaloendopeptidases/química , Metaloendopeptidases/genética , Animais , Bovinos , Linhagem Celular , Células Cultivadas , Corpo Lúteo/irrigação sanguínea , Enzimas Conversoras de Endotelina , Feminino , Humanos , Isoenzimas/química , Isoenzimas/genética , Peso Molecular , Estrutura Terciária de Proteína , RNA Mensageiro/análise , Veias Umbilicais/citologia
11.
J Biol Chem ; 280(49): 40867-74, 2005 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-16186113

RESUMO

Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (a-d) have been identified to date; they differ only in their NH(2) terminus but share the catalytic domain located in the COOH-terminal end. Using quantitative PCR, we found ECE-1d to be the most abundant type in several endothelial cells (EC) types. In addition to full-length ECE-1 forms we have identified novel, alternatively spliced mRNAs of ECE-1 b-d. These splice variants (SVs) lack exon 3', which codes for the transmembrane region and is present in full-length forms. SVs mRNA were highly expressed in EC derived from macro and microvascular beds but much less so in other, non-endothelial cells expressing ECE-1, which suggests that the splicing mechanism is cell-specific. Analyses of ECE-1d and its SV form in stably transfected HEK-293 cells revealed that both proteins were recognized by anti COOH-terminal ECE-1 antibodies, but anti NH(2)-terminal antibodies only bound ECE-1d. The novel protein, designated ECE-1 sv, has an apparent molecular mass of 75 kDa; by using site-directed mutagenesis its start site was identified in a region common to all ECE-1 forms suggesting that ECE-1 b-d SV mRNAs are translated into the same protein. In agreement with the findings demonstrating common COOH terminus for ECE-1sv and ECE-1d, both exhibited a similar catalytic activity. However, immunofluorescence staining and differential centrifugation revealed a distinct intracellular localization for these two proteins. The presence of ECE-1sv in different cellular compartments than full-length forms of the enzyme may suggest a distinct physiological role for these proteins.


Assuntos
Processamento Alternativo , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Metaloendopeptidases/química , Metaloendopeptidases/genética , Isoformas de Proteínas/análise , Animais , Aorta , Ácido Aspártico Endopeptidases/fisiologia , Sequência de Bases , Sítios de Ligação , Bovinos , Fracionamento Celular , Linhagem Celular , Membrana Celular/enzimologia , Células Cultivadas , Cricetinae , Cricetulus , Células Endoteliais/enzimologia , Enzimas Conversoras de Endotelina , Imunofluorescência , Expressão Gênica , Humanos , Metaloendopeptidases/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
12.
Domest Anim Endocrinol ; 29(2): 318-28, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15927442

RESUMO

A dense network of capillaries irrigates the corpus luteum (CL) allowing an intricate cross talk between luteal steroiodgenic and endothelial cell (EC) types. Indeed, luteal endothelial cells (LEC) play pivotal roles throughout the entire CL life-span. Microvascular endothelial cells are locally specialized to accommodate the needs of individual tissues, therefore unraveling the characteristics of LEC is imperative in CL physiology. Numerous studies demonstrated that endothelium-derived endothelin-1 (ET-1) is upregulated by the luteolytic hormone-prostaglandin F2alpha (PGF2alpha) and functions as an important element of the luteolytic cascade. To have a better insight on its synthesis and action, members of ET system (ET-1, ET converting enzyme -ECE-1 and ET(A) and ET(B) receptors) were quantified in LEC. The characteristic phenotype of these cells, identified by high ET-1 receptor expression (both ET(A), ET(B)) and low ET-1 and ECE-1 levels, was gradually lost during culture suggesting that luteal microenvironment sustains the selective phenotype of its resident endothelial cells. Proper vascularization and endothelial cell activity per se are essential for normal CL function. Therefore, factors affecting vascular growth are expected to play major role in the regulation of luteal function. Concomitantly with the angiogenic process, luteal PGF2alpha and its receptors (PGFR) are induced and maintained during most of the CL life-span, suggesting a possible role of PGF2alpha in LEC proliferation and function. Dispersed LEC expressed PGFR and incubation with the prostaglandin stimulated mitogen-activated protein kinase (MAPK) signaling cascade. PGF2alpha activated p42/44 MAPK phosphorylation also in long-term cultured LEC. In this cell type, PGF2alpha increased cell number, 3H-Thymidine incorporation and cell survival. Additionally, PGF2alpha rapidly and transiently stimulated the expression of immediate-early response genes, i.e. c-fos and c-jun mRNA, further suggesting a mitogenic effect for this prostaglandin in LEC. These data imply that PGF2alpha may assume different and perhaps opposing roles depending on luteal microenvironment.


Assuntos
Corpo Lúteo/irrigação sanguínea , Células Endoteliais/fisiologia , Sequência de Aminoácidos , Animais , Dinoprosta/fisiologia , Endotelina-1/química , Endotelina-1/fisiologia , Feminino , Homeostase , Humanos , Dados de Sequência Molecular , Receptores de Endotelina/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia
13.
Reproduction ; 128(4): 463-73, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15454641

RESUMO

Endothelium-derived endothelin-1 (ET-1) and nitric oxide (NO) are pivotal regulators of corpus luteum (CL) function. To have a better insight into their synthesis and action, members of the ET system (ET-1, ET converting enzyme (ECE-1) isoforms a-d, ETA and ETB receptors) along with NO synthase (NOS) isoforms--endothelial (e)NOS and inducible (i)NOS--were quantified in CL-derived endothelial cells (CLEC). The expression of these genes in microvascular CLEC, obtained by lectin-coated magnetic beads, was compared with cells removed from the luteal microenvironment and maintained in culture for different durations, and with endothelial cells (EC) derived from a large blood vessel (i.e. bovine aortic endothelial cells, BAEC). The profile of gene expression in the different EC types was determined by quantitative real-time PCR. Freshly isolated EC from mid-cycle CL exhibited high ET-1 receptor expression (both ETA and ETB), low ET-1 synthesizing ability (both prepro (pp) ET-1 and ECE-1), but elevated iNOS - the high throughput NOS isoform. The distinct phenotype of CLEC was lost soon after an overnight culture. ETA and ETB receptor levels declined, ppET-1 levels increased while iNOS was reduced. These changes were extenuated during long-term culture of CLEC. The general pattern of gene expression in BAEC and long-term cultured CLEC was similar yet some differences, reminiscent of freshly isolated CLEC, remained: ECE-1c, ETB receptor and NOS isoforms were expressed differently in BAEC as compared with lines of CLEC. This study suggests that the luteal microenvironment is necessary to sustain the selective phenotype of its resident endothelial cells. The inverse relationship between ppET-1 and iNOS observed in freshly isolated CLEC and in cultured cells is physiologically significant and suggests that ET-1 and NO may modulate the production of each other.


Assuntos
Corpo Lúteo/metabolismo , Células Endoteliais/química , Endotelina-1/análise , Endotélio Vascular/metabolismo , Óxido Nítrico/análise , Animais , Aorta , Ácido Aspártico Endopeptidases/genética , Bovinos , Separação Celular/métodos , Células Cultivadas , Corpo Lúteo/citologia , Endotelina-1/genética , Enzimas Conversoras de Endotelina , Endotélio Vascular/citologia , Fator VIII/análise , Feminino , Metaloendopeptidases , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Isoformas de Proteínas/genética , RNA Mensageiro/análise , Receptor TIE-2/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
14.
Vaccine ; 22(3-4): 493-502, 2004 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-14670332

RESUMO

Oral antigens administered to newly hatched chicks induce oral tolerance. Some of the antigens encountered via the gut during this period are pathogen-derived, and should not be tolerogenic. As chicks are protected in early life by maternal antibodies, we assumed that the same antibodies also served to prevent tolerance by blocking the relevant antigen. We used bovine serum albumin (BSA) as a model antigen, and initially showed that tolerance was invariably generated in chicks younger than 3 days of age. We then showed that tolerance and its prevention were due to circulatory BSA: intravenous BSA induced tolerance, BSA was present in serum of previously fed chicks, and tolerance was completely blocked in chicks containing high levels of maternal anti-BSA. These findings indicate that tolerance in the young chick is probably generated in central lymphoid organs, and that maternal antibodies block access of antigen to these organs, thereby preserving immune competence.


Assuntos
Anticorpos/farmacologia , Galinhas/imunologia , Tolerância Imunológica/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Anticorpos/administração & dosagem , Embrião de Galinha , Feminino , Imunização , Imunoglobulina G/biossíntese , Intestinos/imunologia , Soroalbumina Bovina/imunologia
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