RESUMO
Two alkalinizing mechanisms coexist in cardiac myocytes to maintain intracellular pH: sodium/bicarbonate cotransporter (electroneutral isoform NBCn1 and electrogenic isoform NBCe1) and sodium/proton exchanger (NHE1). Dysfunction of these transporters has previously been reported to be responsible for the development of cardiovascular diseases. The aim of this study was to evaluate the contribution of the downregulation of the NBCe1 to the development of cardiac hypertrophy. To specifically reduce NBCe1 expression, we cloned shRNA into a cardiotropic adeno-associated vector (AAV9-shNBCe1). After 28 days of being injected with AAV9-shNBCe1, the expression and the activity of NBCe1 in the rat heart were reduced. Strikingly, downregulation of NBCe1 causes significant hypertrophic heart growth, lengthening of the action potential in isolated myocytes, an increase in the duration of the QT interval and an increase in the frequency of Ca2+ waves without any significant changes in Ca2+ transients. An increased compensatory expression of NBCn1 and NHE1 was also observed. We conclude that reduction of NBCe1 is sufficient to induce cardiac hypertrophy and modify the electrical features of the rat heart.
Assuntos
Bicarbonatos , Simportadores de Sódio-Bicarbonato , Ratos , Animais , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Bicarbonatos/metabolismo , Cardiomegalia/genética , Cardiomegalia/metabolismo , Sódio/metabolismo , Isoformas de Proteínas/metabolismo , Concentração de Íons de HidrogênioRESUMO
BACKGROUND: Many parallels exist between growth and development of the placenta and that of cancer. One parallel is shared expression of antigens that may have functional importance and may be recognized by the immune system. Here, we characterize expression and regulation of one such antigen, Trophoblast glycoprotein (TPGB; also called 5T4), in the placenta across gestation, in placentas of preeclamptic (PE) pregnancies, and in purified microvesicles and exosomes. METHODS: Trophoblast glycoprotein expression was analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. Regulation of 5T4 in cytotrophoblast cells was examined under either differentiating conditions of epidermal growth factor or under varying oxygen conditions. Microvesicles and exosomes were purified from supernatant of cultured and perfused placentas. RESULTS: Trophoblast glycoprotein expression was prominent at the microvillus surface of syncytiotrophoblast and on the extravillous trophoblast cells, with minimal expression in undifferentiated cytotrophoblasts and normal tissues. Trophoblast glycoprotein expression was elevated in malignant tumors. In cytotrophoblasts, 5T4 was induced by in vitro differentiation, and its messenger RNA (mRNA) was increased under conditions of low oxygen. PE placentas expressed higher 5T4 mRNA than matched control placentas. Trophoblast glycoprotein was prominent within shed placental microvesicles and exosomes. CONCLUSION: Given the potential functional and known immunological importance of 5T4 in cancer, these studies reveal a class of proteins that may influence placental development and/or sensitize the maternal immune system. In extravillous trophoblasts, 5T4 may function in epithelial-to-mesenchymal transition during placentation. The role of syncytiotrophoblast 5T4 is unknown, but its abundance in shed syncytial vesicles may signify route of sensitization of the maternal immune system.
Assuntos
Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Diferenciação Celular , Feminino , Humanos , Glicoproteínas de Membrana/genética , Placentação/fisiologia , Gravidez , Primeiro Trimestre da Gravidez/metabolismo , Segundo Trimestre da Gravidez/metabolismoRESUMO
INTRODUCTION: Maternal T-cells reactive towards paternally inherited fetal minor histocompatibility antigens are expanded during pregnancy. Placental trophoblast cells express at least four fetal antigens, including human minor histocompatibility antigen 1 (HA-1). We investigated oxygen as a potential regulator of HA-1 and whether HA-1 expression is altered in preeclamptic placentas. METHODS: Expression and regulation of HA-1 mRNA and protein were examined by qRT-PCR and immunohistochemistry, using first, second, and third trimester placentas, first trimester placental explant cultures, and term purified cytotrophoblast cells. Low oxygen conditions were achieved by varying ambient oxygen, and were mimicked using cobalt chloride. HA-1 mRNA and protein expression levels were evaluated in preeclamptic and control placentas. RESULTS: HA-1 protein expression was higher in the syncytiotrophoblast of first trimester as compared to second trimester and term placentas (P<0.01). HA-1 mRNA was increased in cobalt chloride-treated placental explants and purified cytotrophoblast cells (P = 0.04 and P<0.01, respectively) and in purified cytotrophoblast cells cultured under 2% as compared to 8% and 21% oxygen (P<0.01). HA-1 mRNA expression in preeclamptic vs. control placentas was increased 3.3-fold (P = 0.015). HA-1 protein expression was increased in syncytial nuclear aggregates and the syncytiotrophoblast of preeclamptic vs. control placentas (P = 0.02 and 0.03, respectively). DISCUSSION: Placental HA-1 expression is regulated by oxygen and is increased in the syncytial nuclear aggregates and syncytiotrophoblast of preeclamptic as compared to control placentas. Increased HA-1 expression, combined with increased preeclamptic syncytiotrophoblast deportation, provides a novel potential mechanism for exposure of the maternal immune system to increased fetal antigenic load during preeclampsia.
Assuntos
Antígenos de Histocompatibilidade Menor/metabolismo , Oligopeptídeos/metabolismo , Oxigênio/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Trofoblastos/metabolismo , Adulto , Cobalto/farmacologia , Feminino , Expressão Gênica , Humanos , Antígenos de Histocompatibilidade Menor/genética , Oligopeptídeos/genética , Placenta/efeitos dos fármacos , Pré-Eclâmpsia/genética , Gravidez , Trofoblastos/efeitos dos fármacosRESUMO
Involvement of the maternal and fetal immune systems in the events of pregnancy was generally overlooked by reproductive biologists until the mid-twentieth century when many landmark explorations were reported. Now, more than half a century later, it is well understood that with the initiation of pregnancy, immune cells in mammalian uteri are reprogrammed, losing their cytotoxic potential and providing an immunosuppressive environment suitable for harboring the genetically different fetus. We propose that it is the placenta that is mainly responsible for this conversion and maintenance throughout pregnancy. Studies in our laboratory indicate that trophoblast-derived soluble HLA-G has a subtle but well defined role in programming uterine placental macrophages, a potentially destructive immune cell population. Thus, placental HLA-G plays a critical role in assuring that the developing fetus emerges unscathed at parturition.
Assuntos
Desenvolvimento Fetal/imunologia , Troca Materno-Fetal/imunologia , Reprodução/imunologia , Distinções e Prêmios , Feminino , Antígenos HLA-G/fisiologia , Humanos , Tolerância Imunológica/genética , Tolerância Imunológica/imunologia , Imunidade Inata/fisiologia , GravidezRESUMO
The semiallogenic fetus is tolerated by the maternal immune system through control of innate and adaptive immune responses. Trophoblast cells secrete nanometer scale membranous particles called exosomes, which have been implicated in modulation of the local and systemic maternal immune system. Here we investigate the possibility that exosomes secreted from the first trimester and term placenta carry HLA-G and B7 family immunomodulators. Confocal microscopy of placental sections revealed intracellular co-localization of B7-H1 with CD63, suggesting that B7-H1 associates with subcellular vesicles that give rise to exosomes. First trimester and term placental explants were then cultured for 24 h. B7H-1 (CD274), B7-H3 (CD276) and HLA-G5 were abundant in pelleted supernatants of these cultures that contained microparticles and exosomes; the latter, however, was observed only in first trimester pellets and was nearly undetectable in term explant-derived pellets. Further purification of exosomes by sucrose density fractionation confirmed the association of these proteins specifically with exosomes. Finally, culture of purified trophoblast cells in the presence or absence of EGF suggested that despite the absence of HLA-G5 association with term explant-derived exosomes, it is present in exosomes secreted from mononuclear cytotrophoblast cells. Further, differentiation of cytotrophoblast cells reduced the presence of HLA-G5 in secreted exosomes. Together, the results suggest that the immunomodulatory proteins HLA-G5, B7-H1 and B7-H3, are secreted from early and term placenta, and have important implications in the mechanisms by which trophoblast immunomodulators modify the maternal immunological environment.
Assuntos
Antígenos B7/metabolismo , Antígeno B7-H1/metabolismo , Exossomos/metabolismo , Antígenos HLA-G/metabolismo , Fatores Imunológicos/metabolismo , Placenta/metabolismo , Placentação , Biomarcadores/metabolismo , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Células Cultivadas , Exossomos/ultraestrutura , Feminino , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Placenta/citologia , Placenta/imunologia , Gravidez , Primeiro Trimestre da Gravidez , Terceiro Trimestre da Gravidez , Isoformas de Proteínas/metabolismo , Tetraspanina 30/metabolismo , Técnicas de Cultura de Tecidos , Trofoblastos/citologia , Trofoblastos/imunologia , Trofoblastos/metabolismoRESUMO
Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2011 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology: 1) immunology; 2) epigenetics; 3) comparative placentation; 4) trophoblast differentiation; 5) stem cells.
Assuntos
Nível de Saúde , Placenta/fisiologia , Animais , Pesquisa Biomédica/tendências , Diferenciação Celular , Epigênese Genética , Feminino , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imunomodulação , Masculino , MicroRNAs/fisiologia , Fisiologia Comparada/tendências , Placenta/citologia , Placenta/imunologia , Placentação , Gravidez , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Transplante de Células-Tronco/tendências , Células-Tronco/citologia , Células-Tronco/imunologia , Trofoblastos/citologia , Trofoblastos/imunologiaRESUMO
Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 there were twelve themed workshops, six of which are summarized in this report. 1. The immunology workshop focused on normal and pathological functions of the maternal immune system in pregnancy. 2. The transport workshop dealt with regulation of ion and water transport across the syncytiotrophoblast of human placenta. 3. The epigenetics workshop covered DNA methylation and its potential role in regulating gene expression in placental development and disease. 4. The vascular reactivity workshop concentrated on methodological approaches used to study placental vascular function. 5. The workshop on epitheliochorial placentation covered current advances from in vivo and in vitro studies of different domestic species. 6. The proteomics workshop focused on a variety of techniques and procedures necessary for proteomic analysis and how they may be implemented for placental research.
Assuntos
Feto/fisiologia , Placenta/fisiologia , Trofoblastos/fisiologia , Animais , Educação , Epigênese Genética/fisiologia , Feminino , Feto/irrigação sanguínea , Feto/citologia , Feto/imunologia , Humanos , Transporte de Íons/fisiologia , Troca Materno-Fetal/fisiologia , Placenta/irrigação sanguínea , Placenta/citologia , Placenta/imunologia , Placentação/fisiologia , Gravidez , Proteômica/métodos , Trofoblastos/citologia , Trofoblastos/imunologiaRESUMO
Pregnancy induces priming of the maternal cellular and humoral immune systems. The paternally-inherited fetal antigens that influence maternal T and B cells include both major and minor histocompatibility antigens - the same antigens that are problematic in allotransplantation. Animal models have facilitated our understanding of the lymphocyte responses to fetal antigens, and our appreciation of the parallel response in pregnant women is increasing. The physiologic properties of the placenta as well as trafficking of cells between mother and fetus allow ample opportunity for sampling of fetal proteins by the maternal immune system. Here, the current state of knowledge of fetal antigen-specific lymphocyte responses in pregnancy is reviewed.
Assuntos
Feto/imunologia , Tolerância Imunológica/imunologia , Gravidez/imunologia , Animais , Feminino , Humanos , Isoantígenos/imunologia , Masculino , Placenta/imunologiaRESUMO
Trophoblast expression of immunomodulatory proteins in the human placenta is among the mechanisms that are critical for ensuring lymphocyte tolerance to the semi-allogeneic fetus. High levels of B7-H1 on trophoblast cells together with the known role of this protein in establishment of peripheral tolerance suggest that B7-H1 mediates immunological protection of the placenta during gestation. In this study, we investigated the molecular mechanisms of regulation of B7-H1 in trophoblast cells by epidermal growth factor (EGF), a key regulator of trophoblast cell differentiation. EGF increased B7-H1 protein levels within 24 h and mRNA levels within 4h of the initiation of treatment; by 24 h B7-H1 mRNA levels were similar between control and EGF-treated cells. Analysis of two different potential promoter regions revealed strong promoter activity in response to IFN-gamma. In contrast, no promoter activity could be induced by EGF, suggesting that this cytokine regulates B7-H1 expression post-transcriptionally in trophoblast cells. EGF-induced B7-H1 protein expression was completely blocked in the presence of inhibitors of the PI3Kinase/Akt/mTOR pathway, a pathway known to regulate gene expression at the translational level. Finally, analysis of monosomal and polysomal mRNA fractions of untreated and EGF-treated term trophoblast cells revealed that EGF induces a shift towards the translatable fractions and away from the untranslated fractions. These results highlight a novel mechanism for regulation of B7 family proteins in the placenta.
Assuntos
Antígenos CD/metabolismo , Diferenciação Celular/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Trofoblastos/citologia , Adulto , Antígenos CD/genética , Antígenos CD/imunologia , Antígeno B7-H1 , Fusão Celular , Separação Celular , Células Cultivadas , Vilosidades Coriônicas , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Feminino , Expressão Gênica , Humanos , Interferon gama/farmacologia , Gravidez , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/metabolismo , Trofoblastos/metabolismo , Adulto JovemRESUMO
The placenta utilizes both active and passive mechanisms to evade rejection by the maternal immune system. Recently, the mRNA for two newly cloned members of the B7 family of immunomodulatory cell-associated proteins have been identified in the human term placenta. In this article, we review the current knowledge of the B7 family member B7-H1, and discuss how it may participate in modulation of the maternal immune system at the maternal-fetal interface. B7-H1 has been found to possess immunostimulatory or immunoinhibitory properties, and immunohistological examination of first trimester and term placenta has revealed that this protein is abundant in the placenta. B7-H1 is highly expressed by both the syncytiotrophoblast and extravillous cytotrophoblast, both of which lie in direct contact with maternal blood and tissue. Further, treatment of the choriocarcinoma cell line, JEG-3, with recombinant human interferon (IFN)-gamma resulted in a dose-dependent increase in the abundance of the message for B7-H1, suggesting that IFN-gamma could regulate expression of B7-H1 by the trophoblast. These studies document that the positioning of B7-H1 at the maternal-fetal interface is such that it could participate in suppression of activated maternal leukocytes.
Assuntos
Adjuvantes Imunológicos/fisiologia , Antígeno B7-1/imunologia , Proteínas Sanguíneas , Troca Materno-Fetal/fisiologia , Peptídeos , Adulto , Antígenos CD , Antígeno B7-1/genética , Antígeno B7-H1 , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Idade Gestacional , Humanos , Interferon gama/farmacologia , Glicoproteínas de Membrana , Gravidez , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Elementos de Resposta/efeitos dos fármacos , Elementos de Resposta/genética , Trofoblastos/imunologia , Células Tumorais CultivadasRESUMO
In the cat ventricle angiotensin II exerts a positive inotropic effect produced by an increase in intracellular calcium associated with a prolongation of relaxation. The signaling cascades involved in these effects as well as the subcellular mechanisms of the negative lusitropic effect are still not clearly defined. The present study was directed to investigate these issues in cat papillary muscles and isolated myocytes. The functional suppression of the sarcoplasmic reticulum (SR) with either 0.5 microm ryanodine or 0.5 microm ryanodine plus 1 microm thapsigargin or the preincubation of the myocytes with the specific inhibitor of the inositol 1,4,5-triphosphate (IP3) receptors [diphenylborinic acid, ethanolamine ester (2-APB), 5-50 microm] did not prevent the positive inotropic effect and the increment in Ca2+ transient produced by 1 microm angiotensin II. In contrast, protein kinase C (PKC) inhibitors, chelerythrine (20 microm) and calphostin C (1 microm) completely inhibited both, the angiotensin II-induced increase in L-type calcium current and positive inotropic effect. The prolongation of half relaxation time produced by 0.5 microm angiotensin II [207+/-15.4 msec (control) to 235+/-19.98 msec (angiotensin II), P<0.05] was completely blunted by PKC inhibition. This antirelaxant effect, which was independent of intracellular pH changes, was associated with a prolongation of the action potential duration and was preserved after either the inhibition of the SR and the SR Ca2+ ATPase (ryanodine plus thapsigargin) or of the reverse mode of the Na+/Ca2+ exchanger (KB-R7943, 5 microm). We conclude that in feline myocardium the positive inotropic and negative lusitropic effects of angiotensin II are both entirely mediated by PKC without any significant participation of the IP3 limb of the phosphatidylinositol/phospholipase C cascade. The results suggest that the antirelaxant effect of angiotensin II might be determined by the decrease in Ca2+ efflux through the Na+/Ca2+ exchanger produced by the angiotensin II-induced prolongation of the action potential duration.
Assuntos
Angiotensina II/farmacologia , Cardiotônicos/farmacologia , Angiotensina II/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Gatos , Colagenases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Indóis/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Microscopia de Fluorescência , Miocárdio/citologia , Naftalenos/farmacologia , Músculos Papilares/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Proteína Quinase C/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Rianodina/farmacologia , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Tapsigargina/farmacologia , Fatores de TempoRESUMO
Stretching of cardiac muscle modulates contraction through the enhancement of the Ca2+ transient, but how this occurs is still not known. We found that stretching of myocytes modulates the elementary Ca2+ release process from ryanodine-receptor Ca2+-release channels (RyRCs), Ca2+ sparks and the electrically stimulated Ca2+ transient. Stretching induces PtdIns-3-OH kinase (PI(3)K)-dependent phosphorylation of both Akt and the endothelial isoform of nitric oxide synthase (NOS), nitric oxide (NO) production, and a proportionate increase in Ca2+-spark frequency that is abolished by inhibiting NOS and PI(3)K. Exogenously generated NO reversibly increases Ca2+-spark frequency without cell stretching. We propose that myocyte NO produced by activation of the PI(3)K-Akt-endothelial NOS axis acts as a second messenger of stretch by enhancing RyRC activity, contributing to myocardial contractile activation.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Óxido Nítrico/metabolismo , Proteínas Serina-Treonina Quinases , Animais , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/metabolismo , Masculino , Contração Miocárdica/efeitos dos fármacos , Miocárdio/citologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Sprague-Dawley , S-Nitroso-N-Acetilpenicilamina/farmacologia , Espectrometria de FluorescênciaRESUMO
The gut hormone, glucagon-like peptide-1 (GLP-1), which is secreted in nanomolar amounts in response to nutrients in the intestinal lumen, exerts cAMP/protein kinase A-mediated insulinotropic actions in target endocrine tissues, but its actions in heart cells are unknown. GLP-1 (10 nmol/L) increased intracellular cAMP (from 5.7+/-0.5 to 13.1+/-0.12 pmol/mg protein) in rat cardiac myocytes. The effects of cAMP-doubling concentrations of both GLP-1 and isoproterenol (ISO, 10 nmol/L) on contraction amplitude, intracellular Ca(2+) transient (CaT), and pH(i) in indo-1 and seminaphthorhodafluor (SNARF)-1 loaded myocytes were compared. Whereas ISO caused a characteristic increase (above baseline) in contraction amplitude (160+/-34%) and CaT (70+/-5%), GLP-1 induced a significant decrease in contraction amplitude (-27+/-5%) with no change in the CaT after 20 minutes. Neither pertussis toxin treatment nor exposure to the cGMP-stimulated phosphodiesterase (PDE2) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine or the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine nor the phosphatase inhibitors okadaic acid or calyculin A unmasked an ISO-mimicking response of GLP-1. In SNARF-1-loaded myocytes, however, both ISO and GLP-1 caused an intracellular acidosis (DeltapH(i) -0.09+/-0.02 and -0.08+/-0.03, respectively). The specific GLP-1 antagonist exendin 9-39 and the cAMP inhibitory analog Rp-8CPT-cAMPS inhibited both the GLP-1-induced intracellular acidosis and the negative contractile effect. We conclude that in contrast to beta-adrenergic signaling, GLP-1 increases cAMP but fails to augment contraction, suggesting the existence of functionally distinct adenylyl cyclase/cAMP/protein kinase A compartments, possibly determined by unique receptor signaling microdomains that are not controlled by pertussis toxin-sensitive G proteins or by enhanced local PDE or phosphatase activation. Furthermore, GLP-1 elicits a cAMP-dependent modest negative inotropic effect produced by a decrease in myofilament-Ca(2+) responsiveness probably resulting from intracellular acidification.
Assuntos
AMP Cíclico/metabolismo , Glucagon/farmacologia , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Fragmentos de Peptídeos/farmacologia , Precursores de Proteínas/farmacologia , Animais , Cálcio/metabolismo , Cardiotônicos/farmacologia , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Peptídeo 1 Semelhante ao Glucagon , Coração/fisiologia , Concentração de Íons de Hidrogênio , Isoproterenol/farmacologia , Masculino , Contração Miocárdica/efeitos dos fármacos , Miocárdio/citologia , Ratos , Ratos Sprague-Dawley , Tionucleotídeos/farmacologia , Fatores de TempoRESUMO
Tumour necrosis factor alpha (TNF-alpha) and gamma-interferon (IFN-gamma) are cytotoxic to bovine luteal cells in vitro and may contribute to cell death during luteolysis in vivo. In this study, the mechanism by which luteal cells are killed by TNF-alpha and IFN-gamma was investigated. Luteal cells were cultured for 7 days in the presence or absence of TNF-alpha and IFN-gamma. Inhibitors of arachidonate metabolism or scavengers of free radicals were included in the culture media. In addition, the effect of IFN-beta on the viability of cytokine-treated luteal cells was tested. Lastly, untreated and cytokine-treated cells were subjected to single cell gel electrophoresis for quantification of DNA fragmentation. Neither indomethacin nor nordihydroguaiaretic acid, which are inhibitors of cyclooxygenase and lipoxygenase, respectively, were able to prevent cytokine-induced cell death. Similarly, both the phospholipase A(2) inhibitor arachidonyltrifluoromethyl ketone and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine, were largely without effect. In contrast, while vitamin C did not significantly affect viability, superoxide dismutase plus catalase increased viability of cytokine-treated cells (P < 0.05), and IFN-beta prevented cell death (P < 0.05). Finally, while control cells remained free of DNA damage, TNF-alpha plus IFN-gamma induced significant amounts of DNA damage by 48 h after initiation of treatment (P < 0.05). In conclusion, reactive oxygen species, but not arachidonate metabolism or nitric oxide, contribute to cytokine-induced luteal cell death in vitro, and the process of cell death may be via apoptosis. Furthermore, IFN-beta may confer protective effects against cytokine-induced cell death in bovine luteal cells.
Assuntos
Morte Celular , Interferon gama/farmacologia , Células Lúteas/citologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antioxidantes/farmacologia , Ácido Araquidônico/antagonistas & inibidores , Ácido Araquidônico/metabolismo , Catalase/farmacologia , Bovinos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Inibidores de Ciclo-Oxigenase/farmacologia , Dano ao DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Feminino , Sequestradores de Radicais Livres/administração & dosagem , Humanos , Indometacina/farmacologia , Interferon-alfa/farmacologia , Inibidores de Lipoxigenase/farmacologia , Células Lúteas/efeitos dos fármacos , Masoprocol/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/farmacologiaRESUMO
Angiotensin II (AngII) is a circulating peptide that produces a positive inotropic effect in the heart in several species, including humans. The subcellular mechanisms involved in producing this effect have been the focus of numerous studies; however, the results of these studies have generated considerable controversy. Although part of the controversy might arise from species and developmental differences, conflicting results have also been reported in the same species. To further complicate the understanding of the cardiac actions of AngII, the binding of the peptide to its transmembrane G-protein-coupled receptors has been shown to activate signalling cascades that involve numerous second messengers. Among these, inositol 1,4,5-triphosphate (IP3) and protein kinase C (PKC) have been shown to have the potential to modulate either one or both of the two basic mechanisms known to increase contractility: (i) an increase in the intracellular Ca2+ concentration ([Ca2+]i); or (ii) an increase in myofilament responsiveness to Ca2+. The aim of this review is to examine the effect of AngII on the fundamental components of cardiac excitation-contraction coupling: calcium currents, Na+/Ca2+ exchange, sarcoplasmic reticulum (SR)-CaZ+ release, calcium transients and contractile proteins. An answer to the following question is sought: Is the positive inotropic effect of AngII due to an increase in [Ca2+]i, to an increase in myofilament responsiveness to Ca2+, or to both?
RESUMO
1. Cat ventricular myocytes loaded with [Ca2+]i- and pHi-sensitive probes were used to examine the subcellular mechanism(s) of the Ang II-induced positive inotropic effect. Ang II (1 microM) produced parallel increases in contraction and Ca2+ transient amplitudes and a slowly developing intracellular alkalisation. Maximal increases in contraction amplitude and Ca2+ transient amplitude were 163 +/- 22 and 43 +/- 8 %, respectively, and occurred between 5 and 7 min after Ang II administration, whereas pHi increase (0.06 +/- 0.03 pH units) became significant only 15 min after the addition of Ang II. Furthermore, the inotropic effect of Ang II was preserved in the presence of Na+-H+ exchanger blockade. These results indicate that the positive inotropic effect of Ang II is independent of changes in pHi. 2. Similar increases in contractility produced by either elevating extracellular [Ca2+] or by Ang II application produced similar increases in peak systolic Ca2+ indicating that an increase in myofilament responsiveness to Ca2+ does not participate in the Ang II-induced positive inotropic effect. 3. Ang II significantly increased the L-type Ca2+ current, as assessed by using the perforated patch-clamp technique (peak current recorded at 0 mV: -1.88 +/- 0.16 pA pF-1 in control vs. -3.03 +/- 0.20 pA pF-1 after 6-8 min of administration of Ang II to the bath solution). 4. The positive inotropic effect of Ang II was not modified in the presence of either KB-R7943, a specific blocker of the Na+-Ca2+ exchanger, or ryanodine plus thapsigargin, used to block the sarcoplasmic reticulum function. 5. The above results allow us to conclude that in the cat ventricle the Ang II-induced positive inotropic effect is due to an increase in the intracellular Ca2+ transient, an enhancement of the L-type Ca2+ current being the dominant mechanism underlying this increase.
Assuntos
Angiotensina II/farmacologia , Cardiotônicos/farmacologia , Coração/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Gatos , Eletrofisiologia , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Indóis , Potenciais da Membrana/fisiologia , Miocárdio/ultraestrutura , Técnicas de Patch-Clamp , Retículo Sarcoplasmático/fisiologia , Trocador de Sódio e Cálcio/metabolismoRESUMO
In species with hemochorial placentation, which includes humans, mice and rats, antigen-specific T and B lymphocytes which are responsible for acquired immunity are virtually absent from the maternal-fetal interface. In contrast, non-antigen specific natural killer cells and macrophages which provide innate immunity are abundant and highly specialized. Autocrine/paracrine factors such as steroid and polypeptide hormones, prostaglandins and anti-inflammatory cytokines that are present in the uterine environment during pregnancy re-program their secretory profiles. Recent studies using transgenic mice and other approaches indicate that these environmentally modified leukocytes have major pregnancy-associated functions that include facilitation of implantation, modulation of the maternal uterine vasculature, supply of growth factors to the placenta, promotion of trophoblast differentiation and facilitation of parturition.
Assuntos
Leucócitos/fisiologia , Útero/citologia , Animais , Citocinas/genética , Citocinas/fisiologia , Feminino , Humanos , Troca Materno-Fetal/genética , Troca Materno-Fetal/fisiologia , Camundongos , Camundongos Transgênicos , Placenta/citologia , Placenta/fisiologia , Gravidez , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/fisiologia , Útero/irrigação sanguíneaRESUMO
Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine produced by T cells and macrophages. A number of tissues also produce MIF during states of active differentiation and/or proliferation. The purpose of this study was to determine whether MIF is present in the corpus luteum (CL). The steady-state mRNA for MIF was examined in CL by Northern analysis on Day 5, Days 9-12, and Day 18 of the estrous cycle and at 0.5, 1, 4, 12, 24, and 36 h after a luteolytic injection of prostaglandin F(2alpha) (PGF(2alpha)) (n = 4 CL per time point). The greatest amount of MIF mRNA was observed in Day 5 CL compared with midcycle and Day 18 CL. Messenger RNA for MIF in CL collected 0.5 h post-PGF(2alpha) was greater than in midcycle and all other regressing CL. Immunohistochemical analysis (n = 4) revealed that MIF was present in the bovine CL throughout the estrous cycle and appeared to be localized to large luteal cells. It was concluded that MIF is produced within the bovine CL, mRNA expression is maximal in the early CL, and the protein is primarily localized to large luteal cells. The functional significance of MIF remains to be determined.
Assuntos
Corpo Lúteo/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , RNA Mensageiro/biossíntese , Animais , Northern Blotting , Western Blotting , Bovinos , Corpo Lúteo/citologia , Corpo Lúteo/crescimento & desenvolvimento , Dinoprosta/farmacologia , Estro/fisiologia , Feminino , Imuno-Histoquímica , Luteolíticos/farmacologia , Fatores Inibidores da Migração de Macrófagos/biossíntese , Fatores Inibidores da Migração de Macrófagos/genética , Progesterona/sangueRESUMO
For more than a decade, investigators have known that membrane-bound and soluble isoforms of the HLA class Ib molecule, HLA-G, are present at the maternal-fetal interface. Although it is clear that extravillous cytotrophoblast cells are major producers, other cells may also contribute. Recent studies in our laboratory raised the question of whether soluble isoforms might reach the maternal and/or fetal blood circulation. A capture enzyme-linked immunoabsorbent assay (ELISA) identified soluble HLA-G (sHLA-G) in maternal blood throughout pregnancy but failed to detect sHLA-G in cord sera. Further studies suggested that the circulating proteins may be either free heavy chain (sHLA-G1 and/or sHLA-G2) or exclusively sHLA-G2. To study the potential function(s) of the soluble isoforms to modulate local or systemic immunity in mothers, we generated recombinant sHLA-G1 and -G2 in both prokaryotic and eukaryotic systems. Preliminary experiments conducted using DNA microarray analysis suggest that sHLA-G is capable of modulating gene expression in blood mononuclear leukocytes. Potential local targets were also identified; decidual and placental macrophages but not trophoblast cells contained mRNA encoding two of the known receptors for HLA-G, ILT2 and ILT4. Collectively, the studies are consistent with the hypothesis that sHLA-G produced at the maternal-fetal interface targets to the cells of the monocyte/macrophage lineage and modulates their functions for the benefit of pregnancy.
Assuntos
Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Troca Materno-Fetal , Placenta/imunologia , Feminino , Sangue Fetal/imunologia , Antígenos HLA-G , Humanos , Leucócitos/imunologia , Macrófagos/imunologia , Gravidez , Isoformas de Proteínas/imunologiaRESUMO
Nitric oxide (NO) donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels. We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation or whether some contribution occurs via cGMP/PKG-independent mechanisms, in indo 1-loaded adult cardiac myocytes. Whereas a high concentration of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 micromol/L) significantly attenuated contraction amplitude by 24.4+/-4.5% (without changing the Ca2+ transient or total cAMP), a low concentration of SNAP (1 micromol/L) significantly increased contraction amplitude (38+/-10%), Ca2+ transient (26+/-10%), and cAMP levels (from 6.2 to 8.5 pmol/mg of protein). The negative contractile response of 100 micromol/L SNAP was completely abolished in the presence of the specific blocker of PKG KT 5823 (1 micromol/L); the positive contractile response of 1 micromol/L SNAP persisted, despite the presence of the selective inhibitor of GC 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 micromol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 micromol/L), as well as by the NO scavenger oxyhemoglobin. Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1 to 1 micromol/L) of SNAP (AC, 18% to 20% above basal activity). We conclude that NO can regulate both AC and GC in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca2+. Low levels of NO increase cAMP, at least in part, by a novel cGMP-independent activation of AC and induce a positive contractile response.