RESUMO
The pathway of renin secretion has been defined in the mouse submandibular gland (SMG). Renin is first synthesized as a prorenin, rapidly cleaved to a one-chain renin, and then very slowly processed to a two-chain form which is stored in mature granules. In pulse-labeling experiments of minced SMG, the swift appearance in the culture medium of radiolabeled one-chain renin, before granule formation, suggested that this form was secreted by a constitutive pathway independent of the granules, possibly directly from the Golgi. Supporting this hypothesis, phenylephrine, which stimulates the secretion of granules, causes a 4-fold increase in the two-chain renin, with little or no effect on the secretion of one-chain renin. Confirming evidence for the existence of the constitutive pathway was provided by the action of monensin, an ionophore that inhibits transport through the Golgi. Monensin inhibited the appearance of radiolabeled newly synthesized renin in the granules and medium while causing accumulation of the newly synthesized one-chain renin in the microsomes. Analysis of secreted renin by Western blot showed that monensin selectively inhibited the secretion of one-chain renin while not affecting the secretion of the stored two-chain renin. Taken together, our data suggest that one-chain renin is primarily secreted soon after synthesis by a pathway that bypasses the granules, while two-chain renin is secreted predominantly from the granules.
Assuntos
Renina/metabolismo , Glândula Submandibular/enzimologia , Animais , Grânulos Citoplasmáticos/enzimologia , Feminino , Cinética , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos , Microssomos/enzimologia , Monensin/farmacologia , Valores de Referência , Renina/isolamento & purificaçãoRESUMO
Although hypoosmotic stress-induced cell swelling activates phosphatidylinositol-3-kinase, its impact on the downstream signal protein kinase B and cell growth is unknown. Activator protein-1 is in part phosphatidylinositol-3-kinase dependent, and is important in proliferation. We hypothesized that cell swelling modulates proliferation in HepG2 cells via the protein kinase B-dependent activation of activator protein-1. HepG2 cells pretreated with or without LY294002 were exposed for up to 30 minutes to hypoosmotic medium (160 mOsm/L). Tumor necrosis factor-alpha (1.4 nmol/L) or normoosmolar medium (270 mOsm/L) served as positive and negative controls, respectively. Western immunoblots measured cytoplasmic phosphorylated and total protein kinase B. Electromobility shift assays measured nuclear activator protein-1. Methylene blue assays measured cell proliferation at 24, 48, and 72 hours after stimulation. Hypoosmotic stress phosphorylated protein kinase B by 10 minutes. Subsequently, hypoosmotic exposure stimulated activator protein-1 by 30 minutes. Pulse exposure to hypoosmotic stress potentiated HepG2 proliferation by 72 hours as compared to both negative controls and LY-inhibited cells (n = 4 per group, P = 0.009 and P = 0.004, respectively; P <0.001 analysis of variance. All three activation events were abolished with LY294002 pretreatment. In HepG2 cells, hypoosmotic stress-induced swelling stimulates proliferation via protein kinase B-mediated activation of activator protein-1. These data delineate a possible mechanism linking changes in cell volume to growth in human liver cancer.
Assuntos
Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/fisiologia , Fator de Transcrição AP-1/metabolismo , Animais , Western Blotting , Divisão Celular , Cromonas/farmacologia , Meios de Cultura , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Morfolinas/farmacologia , Pressão Osmótica , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt , Ratos , Sistemas do Segundo Mensageiro , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In previous immunohistochemical studies, chronic venous insufficiency (CVI) ulcers have been shown to display positive staining for interleukin-10 (IL-10), while other wounds (including autologous donor wound tissue) show a reduced staining level. IL-10 inhibits the synthesis of many proinflammatory cytokines, while also inhibiting antigen presentation by antigen-presenting cells. It is possible that abnormally high amounts of IL-10 in chronic wounds may be related to the failure of these wounds to progress to final wound healing. The purpose of this study was to quantify the levels of IL-10 in CVI ulcers and autologous donor tissue using Western blotting. Extracts were prepared from frozen wound tissue samples and equal amounts of protein were concentrated by immune-precipitation for Western blot analysis. Densitometric analysis was performed on nonsaturated chemilumigraphs and normalized to an IL-10 standard run on each gel. The quantity of IL-10 in CVI ulcers was found to be 490% of the quantity in autologous donor tissue. This study provides confirmatory quantitative data which supports previous immunohistochemical findings showing elevated levels of IL-10 in CVI ulcers.
Assuntos
Interleucina-10/análise , Úlcera/metabolismo , Insuficiência Venosa/metabolismo , Western Blotting , Doença Crônica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Humanos , Testes de Precipitina , CicatrizaçãoRESUMO
A guinea pig model of halothane hepatitis was used to explore the humoral immune response induced by multiple halothane exposures and the potential role this response might play in contributing to liver damage. Three different strains of guinea pigs (Strain 2, Amana, and Hartley) were exposed to 1% halothane under either 21 or 80% oxygen for 4 hr at 2-week intervals. In each strain, halothane induced the appearance of an antibody cross-reactive with trifluoroacetylated guinea pig serum albumin (TFA-GSA). Three of six Strain 2 guinea pigs demonstrated an association between antibody titer and serum glutamate pyruvate transaminase levels. However, the possible cause and effect relationship between these two factors requires more investigation. Hartley guinea pigs had a 4- to 11-fold higher level of anti-TFA antibody than the other two strains because of either a "higher responder" genetic background or exposure conditions that favored oxidative metabolism of halothane. Immunization of Amana guinea pigs with TFA-GSA evoked a specific anti-TFA antibody response. However, the presence of this antibody before halothane exposure did not potentiate the transient liver damage induced by exposure. Thus, these results demonstrate that in guinea pigs multiple exposures to halothane induce the formation of an antibody that recognizes a reactive intermediate of halothane formed during the anesthetic's metabolism.
Assuntos
Formação de Anticorpos , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Halotano/imunologia , Animais , Antígenos , Feminino , Cobaias , Masculino , Albumina Sérica/imunologia , Especificidade da Espécie , Ácido Trifluoracético/imunologiaRESUMO
Multiple halothane exposures in rabbits generate modified liver proteins or antigens that appear to incorporate the metabolic intermediate of halothane, trifluoroacetyl halide (TFA), as identified by specific anti-TFA antibody. These halothane-induced antigens are most prevalent throughout the second to fourth days following a single halothane exposure and are in highest concentration after the second and third exposure. In addition, five consecutive halothane exposures at 2-week intervals caused the sustained expression of these halothane-induced antigens throughout the first 4 days following the last exposure. By the seventh day, however, antigen expression began to decline. Although there is great heterogeneity in the molecular weights of the halothane-induced antigens, the predominant proteins appear to be 85k, 58k, 53k, 37k and 24k. These liver proteins could reflect self proteins altered by trifluoroacetylation by halothane metabolites and may be potential immunogens in the initiation of a halothane-induced immune response.
Assuntos
Antígenos/análise , Halotano/imunologia , Animais , Formação de Anticorpos , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Relação Dose-Resposta Imunológica , Fluoracetatos , Fígado/imunologia , Masculino , Peso Molecular , Coelhos , Albumina Sérica/imunologia , Fatores de Tempo , Ácido Trifluoracético/imunologiaRESUMO
Multiple or single halothane exposure of rabbits or guinea pigs induces an antibody reactive with trifluoroacetylated (TFA) proteins. The antigen that initiates this immune response was investigated in halothane-exposed rabbits and guinea pigs for its anatomical location in the liver, the chronology of its expression in situ and exposure conditions which would modulate its expression. Using an immuno-staining technique, binding by an anti-TFA antibody to the antigen was detected in liver tissue from all halothane-exposed rabbits and guinea pigs. Antigen could be detected only in the centrilobular area around the central vein where staining intensity was concentrated in an area seven to nine cells deep. In halothane-exposed rabbits, the appearance of TFA antigen was most predominant on the first and second days following a single exposure. Multiple exposures induced TFA antigen in a larger area around the central vein than did a single exposure. Though maximal expression of TFA antigen occurred following two or three exposures, subsequent exposures did not potentiate antigen expression. In halothane-exposed guinea pigs, exposure to deuterated halothane, which reduces the extent and metabolites of oxidative halothane metabolism, elicited the appearance of TFA antigen around the central veins, although to a lesser extent than during halothane exposure. Halothane-induced antigen was evident in guinea pigs as early as 6 h post-exposure and was still apparent 90 h later. Thus, halothane exposure by inhalation elicits the appearance of TFA protein conjugates which may, in turn, evoke the anti-TFA immune response.
Assuntos
Anticorpos , Antígenos/análise , Halotano/imunologia , Fígado/imunologia , Animais , Anticorpos/imunologia , Reações Antígeno-Anticorpo , Deutério , Esquema de Medicação , Fluoracetatos , Cobaias , Halotano/administração & dosagem , Halotano/metabolismo , Masculino , Coelhos , Albumina Sérica/imunologia , Ácido Trifluoracético/imunologiaRESUMO
In the male CD-1 mouse submandibular gland (SMG) renin activity increases markedly with puberty. We have reported that this is, in part, due to an androgen-mediated increase in renin gene transcription. In this study, we examined whether posttranslational processing is also influenced by androgen. We studied secretion and processing of active renin in the CD-1 male mouse SMG which secretes primarily the Ren-2 renin isozyme before and after puberty and also compared findings with the adult female CD-1 mouse. Maturation increases renin level and secretion rate in the male SMG but much less in the female. In addition, Western blot analysis of molecular forms of renin in SMG tissue and media shows a predominance of 1-chain intermediate form of renin before puberty but of the 2-chain mature form thereafter. Adult female SMG contains and secretes much less active renin than the male organ. Administration of testosterone to the female CD-1 mouse induces an adult male level and pattern of secretion, with high concentrations of active 2-chain renin being secreted. These data suggest that SMG renin is androgen regulated, in part by an androgen-responsive enzyme that processes 1-chain renin to the 2-chain form.
Assuntos
Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Renina/metabolismo , Glândula Submandibular/enzimologia , Testosterona/farmacologia , Envelhecimento/metabolismo , Animais , Western Blotting , Feminino , Masculino , Camundongos , Renina/análiseRESUMO
Four hapten-carrier conjugates were synthesized to evaluate any potential antigenic similarities between these synthetic compounds and the immunogens induced in vivo by the anesthetic, halothane and, thus, be used eventually as a more sensitive probe to detect the presence of these halothane-induced antibodies in halothane-exposed individuals. In this study, antibodies from five halothane hepatitis patients were used to evaluate these antigenic alterations since the specificity of these antibodies would most accurately reflect the antigenic structure of halothane-induced immunogens. Quantitation of antibody binding to these synthetic proteins was determined in an enzyme linked immunosorbent assay and immunoblot techniques. Trifluoroacetylated rabbit serum albumin was 5 times more reactive with these antibodies and thus more antigenic than the homologous acetylated moiety confirming the importance of the trifluoromethyl moiety as an epitope in the immunogen in vivo. Insertion of a spacer arm, aminocaproic acid, between the hapten and carrier moieties and an epitope density of 40% acetylation also increased antigenicity. Through these structural alterations produced in vitro, antigenic compounds have been produced which may resemble more closely the immunogen elicited in vivo and which may ultimately serve as more sensitive probes for halothane-induced antibodies from exposed individuals.
Assuntos
Aminocaproatos/síntese química , Fluoracetatos , Halotano/imunologia , Albumina Sérica/síntese química , Vacinas Sintéticas , Acetatos/síntese química , Acetatos/imunologia , Acetilação , Aminocaproatos/química , Aminocaproatos/imunologia , Animais , Anticorpos/imunologia , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Epitopos/imunologia , Feminino , Flúor/imunologia , Halotano/efeitos adversos , Halotano/metabolismo , Haptenos/imunologia , Humanos , Immunoblotting , Coelhos , Albumina Sérica/química , Albumina Sérica/imunologia , Ácido Trifluoracético/síntese química , Ácido Trifluoracético/química , Ácido Trifluoracético/imunologia , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: Following hypoosmotic stress-induced cell volume change, the actin cytoskeleton reorganizes itself. The role of this reorganization in the activation of the phosphatidylinositol 3-OH-kinase/protein kinase B/activator protein 1 (PI-3-K/PKB/AP-1) proliferative signaling cascade is unknown. Focal adhesion kinase (FAK) participates in the cytoskeleton-based activation of PI-3-K. We hypothesized that hypoosmotic stress-induced activation of PKB and AP-1 in HepG2 cells is dependent on an intact actin cytoskeleton and subsequent FAK phosphorylation. METHODS: HepG2 cells were incubated for 1 h with or without 20 microM cytochalasin D, an actin disrupter, and were then exposed for up to 30 min to hypoosmotic medium (200 mOsm/L) to induce swelling. Tumor necrosis factor alpha (1.4 nM) and medium alone served as positive and negative controls, respectively. Western blots measured cytoplasmic phosphorylated or total FAK and PKB. EMSAs measured nuclear AP-1. All experiments were performed in triplicate. RESULTS: Exposure to hypoosmotic stress resulted in activation of the following signaling messengers in a sequential fashion: (1) phosphorylation of FAK occurred by 2 min, (2) phosphorylation of PKB occurred by 10 min, (3) nuclear translocation of AP-1 occurred by 30 min. All three signaling events were abolished when these cells were pretreated with cytochalasin D. CONCLUSION: Actin reorganization following hypoosmotic stress is essential for the FAK-mediated activation of the PI-3-K/PKB/AP-1 proliferative cascade. These data delineate a possible mechanism by which the cell swelling-induced cytoskeletal changes can initiate proliferative signal transduction in human liver cancer.
Assuntos
Actinas/metabolismo , Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas Serina-Treonina Quinases , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/metabolismo , Núcleo Celular/metabolismo , Citocalasina D/farmacologia , Citoesqueleto/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Pressão Osmótica , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
An animal model of halothane-induced liver injury has been developed in the rabbit to study the production of humoral immunity towards a biotransformation intermediate of halothane. Rabbits exposed many times to halothane in a 75% O2/25% N2 atmosphere produce an antibody that cross-reacts with the trifluoroacetyl moiety of trifluoroacetylated rabbit serum albumin (TFA-RSA). The generation of this halothane-induced immunogen is dependent upon high oxygen tension as shown by the minimal anti-TFA antibody response seen in rabbits exposed to halothane in a 14% O2/86% N2 atmosphere. In addition, halothane exposure of rabbits specifically immunized with the metabolite-carrier complex, TFA-RSA, induces a secondary antibody response toward the immunogen. In rabbits, either immunized with TFA-RSA or not, multiple halothane exposures induce populations of antibodies with varying specificities. Evidence suggests that predominance of the metabolic intermediate, the ensuing immunogen, and the subsequent antibody response depends upon the oxygen tension during successive exposures to halothane. These successive exposures could potentially generate many different immunogens resulting in varied antibody specificities.
Assuntos
Formação de Anticorpos/efeitos dos fármacos , Halotano/imunologia , Animais , Especificidade de Anticorpos , Ligação Competitiva , Fluoracetatos , Esquemas de Imunização , Memória Imunológica , Masculino , Oxigênio/farmacologia , Coelhos , Albumina Sérica/imunologia , Ácido Trifluoracético/imunologiaRESUMO
How local renin expression is regulated in many tissues has yet to be defined. In the present studies the ontogeny of renin in submandibular gland (SMG) and kidney of CD-1 mice was examined in order to determine whether renal and extrarenal renin are similarly expressed. In males, submandibular gland (SMG) renin and renin secretory rate increase at puberty as androgen rises. The ratio of secreted forms (1-chain renin cf. 2-chain) seen on Western blots shows predominance of 1-chain prior to puberty and 2-chain thereafter. This androgen influence on renin processing and secretion was supported by reversion to prepubertal patterns with castration of adult males and by conversion to male pattern in androgen treated females which otherwise have low renin levels. In contrast, renal renin remains unchanged throughout development. The influence of ontogeny on renal and SMG renin mRNA was examined by Northern analysis using renin cDNA: SMG renin mRNA increases from near zero to high levels at puberty while renal renin mRNA level is high throughout. Taken together, these data suggest that SMG renin is influenced by androgens, whereas renal renin is not. Apparent differences in tissue renin regulation may have important implications for local function of this enzyme.
Assuntos
Renina/metabolismo , Glândulas Suprarrenais/metabolismo , Fatores Etários , Animais , Feminino , Rim/metabolismo , Masculino , Camundongos , RNA Mensageiro/metabolismo , Maturidade Sexual , Glândula Submandibular/metabolismo , Testículo/metabolismoRESUMO
Free fat transplantation for soft tissue augmentation yields variable results, which may be related to the technique of fat harvest. To compare the viability of adipocytes harvested by liposuction (sal) or by excision (exc), fat harvested by both techniques from seven lipectomy patients was analyzed by glycerol-3-phosphate dehydrogenase (G3PDH) enzyme assay. Leakage of this lipogenic enzyme through the plasma membrane is a potential indicator of fat cell damage. Preliminary experiments showed this assay to be sensitive and specific for adipocyte G3PDH activity. Treatment of fat tissue with collagenase H resulted in complete release of the component fat cells for analysis with less loss of G3PDH activity, compared to other collagenase preparations. Each sample was digested and separated into three compartments: mature adipocytes-floating layer (F), acellular supernatant (S), and stromal pellet (P). Samples from each compartment were assayed for G3PDH activity, normalized to DNA content, and represented as a percentage of the whole (F + S + P). Within the subgroups, the fat cell fraction of the liposuction samples (Fsal) showed statistically more activity than the excised samples (Fexc) by paired Student's t test (P = 0.004). The supernatant (representing leaked G3PDH) and pellet fractions of excised samples revealed more G3PDH activity than the same fractions from liposuctioned tissue; the former (Sexc) to a significant degree (P = 0.036). Using this assay, the results indicate that liposuction fat harvest does not result in increased fat cell damage compared to fat harvested by excision.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo/transplante , Sobrevivência Celular , Células 3T3 , Adipócitos/enzimologia , Tecido Adiposo/cirurgia , Sequência de Aminoácidos , Animais , Membrana Celular/enzimologia , Colagenases/química , Colagenases/metabolismo , Ácido Edético , Feminino , Glicerolfosfato Desidrogenase/metabolismo , Humanos , Lipectomia , CamundongosRESUMO
Although the clinical experience with calcium alginate has been generally good, well-controlled studies examining the effect of such dressings on the processes of wound healing have not been conducted. The healing of cutaneous ulcers requires the development of a vascularized granular tissue bed, filling of large tissue defects by dermal regeneration, and the restoration of a continuous epidermal keratinocyte layer. These processes were modeled in vitro in the present study, utilizing human dermal fibroblast, microvascular endothelial cell (HMEC), and keratinocyte cultures to examine the effect of calcium alginate on the proliferation and motility of these cultures, and the formation of capillarylike structures by HMEC. This study demonstrates that the calcium alginate tested increased the proliferation of fibroblasts but decreased the proliferation of HMEC and keratinocytes. In contrast, the calcium alginate decreased fibroblast motility but had no effect on keratinocyte motility. There was no significant effect of calcium alginate on the formation of capillarylike structures by HMEC. The effects of calcium alginate on cell proliferation and migration may have been mediated by released calcium ions. These results suggest that the calcium alginate tested may improve some cellular aspects of normal wound healing, but not others.
Assuntos
Alginatos/uso terapêutico , Úlcera Cutânea/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Ácido Glucurônico , Ácidos Hexurônicos , Humanos , Queratinócitos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacosRESUMO
BACKGROUND: White cell trapping and activation occurs in the legs of patients having chronic venous insufficiency (CVI), and it is thought that this process may be important in the development of CVI ulcers. This study has compared the tissue distribution of proinflammatory (GM-CSF) and anti-inflammatory cytokines (IL-10) and inflammatory cell markers (CD68, HLA-DR) between CVI ulcers, other chronic and acute wounds, and autologous nonwounded skin to determine whether cell-mediated immunity (CMI) is impaired in CVI ulcers. METHODS: Wound and donor site tissue was obtained from 10 patients with CVI ulcers and 10 patients with other chronic and acute wounds. Serial Formalin-fixed sections were processed by standard hematoxylin and eosin staining or by indirect immunoperoxidase histochemical staining. RESULTS: HLA-DR-positive antigen-presenting cells (APC), including CD68-positive macrophages and dermal dendritic cells, were found with greater frequency in CVI ulcers than in other chronic or acute wounds (P = 0.0015) or in the autologous CVI donor site tissue (P = 0.006). CVI ulcers also demonstrated increased IL-10 staining of the entire epidermis compared to non-CVI wounds (P = 0.0019) or autologous donor site tissue (P = 0.004), whereas there was no significant change in the presence of the counteracting cytokine, GM-CSF. CONCLUSIONS: These findings suggest that although the cellular components of CMI are present in CVI ulcers, their function may be impaired by the increased level of IL-10. Future studies will examine whether IL-10-mediated suppression of CMI and/or inhibition of GM-CSF-stimulated keratinocyte proliferation may contribute to the chronic nature of CVI ulcers.