RESUMO
BACKGROUND: Electroconvulsive therapy (ECT) is a widely used and efficient treatment modality in psychiatry, although the basis for its therapeutic effect is still unknown. Past research has shown seizure activity to be a regulator of neurogenesis in the adult brain. This study examines the effect of a single and multiple electroconvulsive seizures on neurogenesis in the rat dentate gyrus. METHODS: Rats were given either a single or a series of 10 electroconvulsive seizures. At different times after the seizures, a marker of proliferating cells, Bromodeoxyuridine (BrdU), was administered to the animals. Subsequently, newborn cells positive for BrdU were counted in the dentate gyrus. Double staining with a neuron-specific marker indicated that the newborn cells displayed a neuronal phenotype. RESULTS: A single electroconvulsive seizure significantly increased the number of new born cells in the dentate gyrus. These cells survived for at least 3 months. A series of seizures further increased neurogenesis, indicating a dose-dependent mechanism. CONCLUSIONS: We propose that generation of new neurons in the hippocampus may be an important neurobiologic element underlying the clinical effects of electroconvulsive seizures.
Assuntos
Giro Denteado/crescimento & desenvolvimento , Eletroconvulsoterapia , Neurônios/fisiologia , Animais , Antimetabólitos , Apoptose , Bromodesoxiuridina , Contagem de Células , Divisão Celular , Giro Denteado/citologia , Técnica Direta de Fluorescência para Anticorpo , Imuno-Histoquímica , Masculino , Microscopia Confocal , Fenótipo , Ratos , Ratos WistarRESUMO
The effect of two isoforms of platelet-derived growth factor (PDGF), PDGF-AA and PDGF-BB, was tested on dissociated cell cultures of ventral mesencephalon from rat and human embryos. PDGF-BB but not PDGF-AA reduced the progressive loss of tyrosine hydroxylase- (TH)-positive neurons in rat and human cell cultures. The mean number of TH-positive cells in the PDGF-BB-treated rat culture was 64% and 106% higher than in the control cultures after 7 and 10 days in vitro, respectively. Corresponding figures for human TH-positive neurons were 90% and 145%. The influence of PDGF-BB was specific for TH-positive neurons and not a general trophic effect, since no change of either total cell number or metabolic activity was found. In PDGF-BB-treated cultures of human but not rat tissue the TH-positive neurons had longer neurites than observed in control or PDGF-AA-treated cultures. These data indicate that PDGF-BB may act as a trophic factor for mesencephalic dopaminergic neurons and suggest that administration of PDGF-BB could ameliorate degeneration and possibly promote axonal sprouting of these neurons in vivo.
Assuntos
Dopamina/fisiologia , Mesencéfalo/citologia , Neurônios/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Imuno-Histoquímica , Neuritos/efeitos dos fármacos , Gravidez , Ratos , Sais de Tetrazólio , Tiazóis , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
We have examined the effects of three macrophage-derived cytokines, platelet-derived growth factor (PDGF), transforming growth factor-beta 1 (TGF-beta 1) and interleukin-1 alpha (IL-1 alpha) on the contraction of collagen type I gels populated by human foreskin fibroblasts. Contraction was quantified as loss in gel weight. Both PDGF-AA and PDGF-BB were found to induce a rapid collagen-gel contraction. TGF-beta 1 also stimulated gel contraction but with a delayed onset and at a slower rate than the PDGF-stimulated contraction. Rabbit polyclonal IgGs recognizing PDGF-AA and PDGF-BB, respectively, specifically inhibited the effects of the corresponding PDGF isoforms. However, the stimulatory effect of TGF-beta 1 was not affected by any of the anti-PDGF antibodies. The ability of PDGF to stimulate contraction became less pronounced in collagen gel cultures grown in the absence of growth factors over periods of several days. Under the same conditions, the stimulatory effect of TGF-beta 1 was not reduced. The reduced response to PDGF may be due to reduced tension on fibroblasts growing in collagen gels, since fibroblasts on free-floating gels showed a marked reduction in PDGF-BB-induced PDGF beta-receptor aggregates when compared to fibroblasts on attached collagen gels. IL-1 alpha inhibited initial collagen gel contraction, and at later stages induced a visible degradation of the collagen gels, presumably due to the generation of collagenase activity. The combination of IL-1 alpha and PDGF-BB stimulated initial collagen gel contraction, although less effectively than PDGF-BB alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Colágeno/química , Fibroblastos/fisiologia , Interleucina-1/fisiologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Bovinos , Células Cultivadas , Géis , Humanos , Imunoglobulina G/imunologia , Cinética , Fator de Crescimento Derivado de Plaquetas/imunologia , Fatores de TempoRESUMO
Stimulation of human fibroblasts by platelet-derived growth factor (PDGF)-BB leads to a down-regulation of PDGF beta-receptors and a concomitant appearance of intracellular granular accumulations of receptors, as determined by stainings with the mAb PDGFR-B2. The granules contained both the ligand and PDGF beta-receptors, as revealed by double-immunofluorescence staining, and were formed in response to PDGF-BB but not in response to other cytokines tested. The formation of intracellular PDGF beta-receptor granules was dependent on PDGF-BB concentration and time of stimulation. The granular PDGF beta-receptor staining on cells treated with PDGF-BB for 1 h at 37 degrees C was used to investigate the effects of macrophage-derived cytokines on PDGF beta-receptor expression. The number of PDGF beta-receptor granules was found to be reduced in fibroblasts grown for 48 h in the presence of PDGF-BB, TNF-alpha, or IL-1; PDGF-AA under the same conditions had no effect. The reduction observed was paralleled by a decrease in cell surface expression of PDGF beta-receptors, measured as binding of 125I-PDGF-BB and of the PDGFR-B2 antibody. Furthermore, both TNF-alpha and IL-1 decreased the detergent-extractable pool of PDGF-beta receptors in the fibroblasts, as revealed by immunoblotting of detergent cell extracts. Finally, the decrease in PDGF beta-receptors after culturing of the cells in the presence of TNF-alpha and IL-1 was accompanied by a decreased incorporation of [3H]thymidine in response to PDGF-BB stimulation. In conclusion, our data suggest that certain macrophage-derived cytokines can modulate the expression of PDGF beta-receptors by cultured fibroblasts, which may contribute in part to their reduced responsiveness to PDGF.
Assuntos
Interleucina-1/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Receptores de Superfície Celular/análise , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , DNA/biossíntese , Regulação para Baixo , Fibroblastos/química , Humanos , Técnicas Imunoenzimáticas , Octoxinol , Polietilenoglicóis/farmacologia , Receptores do Fator de Crescimento Derivado de Plaquetas , Proteínas Recombinantes/farmacologiaRESUMO
Platelet-derived growth factor (PDGF) beta-receptor expression in normal and rheumatoid synovia was investigated by double immunofluorescence staining of frozen sections and by in situ hybridization. In the inflamed synovia, PDGF beta-receptor mRNA was present in vascular cells, as well as in discrete stromal cells. PDGF beta-receptor expressing cells in rheumatoid synovia were characterized by double immunofluorescence staining using the PDGFR-B2 monoclonal antibody at a concentration at which this antibody merely stained granular accumulations of PDGF beta-receptors. Granular accumulations of PDGF beta-receptors were articulate in blood vessel cells, but also appeared in discrete stromal cells. Thus, the overall distribution of cells having granular accumulations of PDGF beta-receptors was similar to the distribution of cells expressing PDGF beta-receptor mRNA. Double immunofluorescence stainings showed that: (a) a majority (greater than 90%) of resident macrophages did not express granular PDGF beta-receptor staining, but macrophages were often juxtaposed to PDGF beta-receptor-positive cells; (b) T lymphocytes did not express PDGF beta-receptors, but these cells were frequently found in the proximity of cells stained by PDGFR-B2; (c) in some blood vessels both HLA-DR expressing cells and PDGF beta-receptor expressing cells could be visualized, whereas in other blood vessels, cells expressing only one of these activation markers could be detected; (d) smooth muscle cells in blood vessels contained PDGF beta-receptors; and (e) capillary endothelial cells in the inflamed synovia recurrently displayed granular PDGF beta-receptor staining. The granular accumulations of PDGF beta-receptors may reflect internalization of the receptor as a result of paracrine or autocrine ligand stimulation. In support of such a possibility are the findings that elevated levels of PDGF B chain mRNA were detected by in situ hybridization in the inflamed synovia, and that cells expressing PDGF B chain mRNA were distributed similarly to cells expressing PDGF beta-receptor mRNA. Taken together, the results indicate that PDGF has a role in the inflammatory process in rheumatoid synovitis, most likely by stimulating proliferative events in the vasculature.
Assuntos
Artrite Reumatoide/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Membrana Sinovial/irrigação sanguínea , Artrite Reumatoide/patologia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Humanos , Fator de Crescimento Derivado de Plaquetas/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas , Valores de Referência , Membrana Sinovial/metabolismoRESUMO
C-CAM (Cell-CAM 105) is a cell surface glycoprotein that is involved in cell-cell adhesion of rat hepatocytes in vitro. To elucidate the adhesion mechanism the binding properties of purified C-CAM were investigated. Using proteins immobilized on nitrocellulose it was found that radiolabeled C-CAM bound to C-CAM but not to a variety of other proteins. Partitioning in Triton X-114 showed that C-CAM has hydrophobic properties. In accordance with this, C-CAM was effectively incorporated into phosphatidylcholine liposomes by dialysis from octylglucoside-containing solutions. The C-CAM-containing liposomes bound specifically to isolated hepatocytes. This binding was blocked by Fab fragments of anti-C-CAM antibodies. Furthermore, preincubation of hepatocytes with anti-C-CAM antibodies followed by washing of the cells blocked binding of C-CAM-containing liposomes. At increasing C-CAM contents in the reconstituted liposomes a marked self-aggregation of the liposomes occurred. This aggregation was blocked by Fab fragments of anti-C-CAM antibodies and by alkaline pH. After neutralization a rapid reaggregation occurred. Neither C-CAM binding to C-CAM immobilized on nitrocellulose nor C-CAM-liposome aggregation required calcium ions. Liposomes reconstituted with C-CAM-depleted membrane glycoproteins did not self-aggregate or bind to hepatocytes. Thus, it is concluded that C-CAM can bind specifically to C-CAM in a homophilic binding reaction that does not require calcium. Accordingly, C-CAM has the potential of directly mediating cell-cell adhesion via C-CAM-C-CAM binding between adjacent cells.
Assuntos
Adenosina Trifosfatases , Moléculas de Adesão Celular/metabolismo , Animais , Antígenos CD , Colódio , Técnicas In Vitro , Lipossomos , Fígado/citologia , Fígado/metabolismo , Ligação Proteica , Ratos , Ratos EndogâmicosRESUMO
The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent beta 1 integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-beta 1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin beta 1-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-beta 1 integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the beta 1 integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.
Assuntos
Colágeno/metabolismo , Fibroblastos/fisiologia , Integrinas/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Actinas/análise , Sequência de Aminoácidos , Animais , Sangue , Linhagem Celular , Fibroblastos/análise , Fibronectinas/imunologia , Fibronectinas/farmacologia , Géis , Humanos , Imunoglobulina G/farmacologia , Integrinas/análise , Integrinas/imunologia , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Ratos , Proteínas Recombinantes , Fatores de Crescimento Transformadores/farmacologiaAssuntos
Coração/embriologia , Estimulação Física , Animais , Células Cultivadas , Colágeno , Membranas Artificiais , Contração Miocárdica , RatosRESUMO
The cellular location of cellCAM 105 was studied by indirect immunofluorescence microscopy of primary rat hepatocytes grown in monolayer culture. Staining corresponding to cellCAM 105 was seen both in cell-cell contact areas and on the upper surfaces of the cells. In the cell-cell contact areas the antigen was not accessible to the antibodies unless the cells were either permeabilized with detergent or incubated in a calcium-free medium. Removal of calcium from the medium caused the cells to separate from each other. Within a few minutes wide intercellular clefts were formed, and upon further incubation the cells became stellate-shaped and finally remained in contact with each other only via thin cellular processes. These processes were cellCAM 105-positive and at sites where they attached to the bodies of the contracted cells a granular fluorescence pattern appeared. After 24-48 h of culture, intercellular channels resembling bile canaliculi were sometimes formed in the hepatocyte monolayers. The membranes of these intercellular channels were stained for cellCAM 105. After culture for several days the hepatocytes lost their polygonal shape and gradually acquired a more fibroblast-like morphology. This morphological change was accompanied by a decrease in cellCAM 105-specific fluorescence, both in the cell-cell contact areas and on the free cell surfaces.
Assuntos
Moléculas de Adesão Celular/análise , Fígado/citologia , Animais , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/fisiologia , Comunicação Celular , Membrana Celular/análise , Membrana Celular/metabolismo , Células Cultivadas , Imunofluorescência , Fígado/análise , Fígado/metabolismo , Microscopia de Fluorescência , Ratos , Ratos EndogâmicosRESUMO
The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.
Assuntos
Fibroblastos/metabolismo , Músculo Liso/metabolismo , Receptores de Superfície Celular/biossíntese , Animais , Anticorpos Monoclonais , Células Cultivadas/metabolismo , Fibroblastos/citologia , Humanos , Immunoblotting , Imuno-Histoquímica , Músculo Liso/citologia , Hibridização de Ácido Nucleico , Fosforilação , Receptores do Fator de Crescimento Derivado de Plaquetas , SuínosRESUMO
Expression of B-type receptors for platelet-derived growth factor (PDGF) in frozen sections of blood vessels from tissues affected by abnormal vascular cell proliferation was investigated by immunohistochemical techniques and compared with expression of these receptors in blood vessels of normal tissues. Receptors were not expressed, or expressed at low levels, in vessels of normal tissues. In contrast, a pronounced expression of PDGF B-type receptors was seen on vascular smooth muscle cells in atherosclerotic plaques, rejected kidneys, and chronic synovitis. These observations suggest induction of PDGF B-type receptors on vascular smooth muscle cells in inflamed tissues, which would render such cells responsive to growth stimulation by PDGF released from captured platelets, or produced locally (eg, by inflammatory cells or smooth muscle cells). Autocrine or paracrine stimulation of cell growth caused by the effect of PDGF on cells with induced receptors may be important in the formation of the proliferative lesions found in atherosclerosis and in certain forms of chronic inflammation.
Assuntos
Arteriosclerose/metabolismo , Músculo Liso Vascular/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Superfície Celular/metabolismo , Anticorpos Monoclonais , Arteriosclerose/patologia , Divisão Celular , Rejeição de Enxerto , Humanos , Imuno-Histoquímica , Transplante de Rim , Músculo Liso Vascular/patologia , Receptores do Fator de Crescimento Derivado de Plaquetas , Sinovite/metabolismoRESUMO
Cell-CAM 105, a glycoprotein that is involved in recognition and adhesion between isolated rat hepatocytes in vitro, was purified to homogeneity by a combination of immunoaffinity chromatography, gel-exclusion chromatography and ion-exchange chromatography. Electrophoretic, compositional and enzymic analyses were performed and the glycoprotein was shown to consist of two peptide chains, of apparent Mr 110,000 and 105,000 respectively, that are glycosylated to similar extents. Carbohydrate analyses demonstrated the presence of sialic acid, galactose, mannose, fucose and glucosamine, but no galactosamine, indicating that only N-linked oligosaccharides occurred. The total content of carbohydrate amounted to 33%. Peptide mapping indicated that the two peptide chains were structurally very similar. After incubation of cultured hepatocytes with [32P]Pi, phosphorylated cell-CAM 105 could be isolated. Both peptide chains were labelled and phospho-amino-acid analysis demonstrated that serine residues had become phosphorylated. A significant feature of cell-CAM 105 was a susceptibility to autolytic degradation that was difficult to inhibit. The major degradation products had apparent Mr 90,000 and 70,000, respectively. The effect of purified cell-CAM 105 on cell-cell adhesion of re-aggregating hepatocytes was studied. A significant inhibition was observed, indicating that the protein is directly involved in intercellular adhesion of these cells.