RESUMO
Chromogranin A (CGA), produced by human and rat myocardium, generates several biologically active peptides processed at specific proteolytic cleavage sites. A highly conserved cleavage N-terminal site is the bond 64-65 that reproduces the native rat CGA sequence (rCGA1-64), corresponding to human N-terminal CGA-derived vasostatin-1. rCGA1-64 cardiotropic activity has been explored in rat cardiac preparations. In Langendorff perfused rat heart, rCGA1-64 (from 33 nM) induced negative inotropism and lusitropism as well as coronary dilation, counteracting isoproterenol (Iso) - and endothelin-1 (ET-1) -induced positive inotropic effects and ET-1-dependent coronary constriction. rCGA1-64 also depressed basal and Iso-induced contractility on rat papillary muscles, without affecting calcium transients on isolated ventricular cells. Structure-function analysis using three modified peptides on both rat heart and papillary muscles revealed the disulfide bridge requirement for the cardiotropic action. A decline in Iso intrinsic activity in the presence of the peptides indicates a noncompetitive antagonistic action. Experiments on rat isolated cardiomyocytes and bovine aortic endothelial cells indicate that the negative inotropism observed in rat papillary muscle is probably due to an endothelial phosphatidylinositol 3-kinase-dependent nitric oxide release, rather than to a direct action on cardiomyocytes. Taken together, our data strongly suggest that in the rat heart the homologous rCGA1-64 fragment exerts an autocrine/paracrine modulation of myocardial and coronary performance acting as stabilizer against intense excitatory stimuli.
Assuntos
Cromogranina A/metabolismo , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Músculos Papilares/metabolismo , Vasodilatação/fisiologia , Animais , Aorta/citologia , Aorta/metabolismo , Comunicação Autócrina/efeitos dos fármacos , Comunicação Autócrina/fisiologia , Cálcio/metabolismo , Cardiotônicos/farmacologia , Bovinos , Cromogranina A/farmacologia , Células Endoteliais/citologia , Endotelina-1/farmacologia , Humanos , Isoproterenol/farmacologia , Masculino , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Músculos Papilares/citologia , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Vasodilatação/efeitos dos fármacosRESUMO
Actin antibodies were purified by affinity chromatography from the serum of rabbits immunized with actin isolated from bovine skeletal myofibrils. By the indirect immunofluorescence technique the pattern of organization of actin was studied in cultured astroglial cells prepared from cerebral hemispheres of newborn rats. Labelling with monospecifc actin antibody revealed that the flat epitheloid astrocytes contained bundles of actin fibres arranged in characteristic patterns. Actin fibres disaggregated in round cells produced by trypsinization and reorganized when cells returned to their elongate or polygonal form. The retraction of cell bodies produced by treatment with cytochalasin B was associated with the disappearance of actin filament bundles. Reversion to the former flattened morphology and organization of fibres occurred after removal of the drug. In response to either dibutyryl adenosine 3'5'-monophosphate, 3-isobutyl-l-methylxanthine, rat brain extract or to lesser extent to norepinephrine, flat epitheloid cells took on a stellate appearance with extensive processes, resembling more closely mature astrocytes. The conversion of flat epitheloid cells into stellate cells was associated with the disappearance of immunofluorescent fibres and the appearance of diffuse immunofluorescence indicating the structural reorganization of actin from a highly organized fibrillar state into a loosely organized actin network. It is concluded that the respect to actin patterns, the behaviour of flat epithelioid astrocytes in culture does not differ from that of culture non-muscle cells of mesenchymal origin. The pattern of organization of actin in stellate process-bearing astrocytes is compatible with the involvement of actin in typical astroglial motility and in establishment of characteristic stellate shape.
Assuntos
Actinas/metabolismo , Astrócitos/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Encéfalo , Bucladesina/farmacologia , Células Cultivadas , Citocalasina B/farmacologia , Imunofluorescência , Norepinefrina/farmacologia , Ratos , Extratos de Tecidos/farmacologiaRESUMO
PURPOSE: The quantitative and qualitative ganglioside composition of retinal photoreceptor cells is unknown. The aim of this study was to analyze the lipid, especially ganglioside, make-up of photoreceptors compared to other retinal cells. METHODS: Retinas from adult normal rats were mechanically separated into outer (photoreceptors) and inner (other retinal neurons and glia) halves be planar vibratome sectioning. Total lipids were extracted, and each fraction (neural, phospholipids, and glycosphingolipids) was eluted sequentially by column chromatography and quantitated through high-performance thin layer chromatogram analysis. Similar analyses were performed on entire retinas from adult normal rats, adult dystrophic rats lacking photoreceptors (RCS-rdy-p+ strain), and isolated photoreceptor outer segments. RESULTS: Whereas phospholipids were distributed equally between the two halves, inner retina contained significantly more cholesterol (68% total) and gangliosides (74% total) than outer retina on a unit protein basis. The distribution on a percent molar basis of specific gangliosides also was significantly different between the two halves: Outer retina was dominated by GD3 (45% total ganglioside) and contained only trace amounts (<4%) of complex species (GT1b and GQ1b); inner retina was more typical of mature brain tissue exhibiting substantial amounts (approximately 25%) of more complex species. These data were supported by lipid compositional analyses of mutant photoreceptor-less retina. However, isolated outer segments resembled whole retina in containing higher levels of complex gangliosides. CONCLUSIONS: These data indicate that, compared to other central nervous system-derived neurons, photoreceptor cell body membranes exhibit a highly unusual simplified ganglioside composition. Such an unusual neuronal lipid composition may reflect structural adaptations to their specialized function.
Assuntos
Gangliosídeos/análise , Neurônios/química , Células Fotorreceptoras/química , Retina/química , Animais , Química Encefálica , Cromatografia em Camada Fina , Imunofluorescência , Gangliosídeos/isolamento & purificação , Lipídeos/análise , Lipídeos/isolamento & purificação , Fosfolipídeos/análise , Fosfolipídeos/isolamento & purificação , Ratos , Ratos Mutantes , Ratos Wistar , Degeneração Retiniana/etiologia , Degeneração Retiniana/genéticaRESUMO
Polypeptide growth factors and gangliosides can both be considered as trophic agents involved in almost all stages of neural cell development, differentiation, survival, and pathology. In most cases their physiological roles are still not clear due to the considerable complexity in their regulation. Several growth factors [e.g., basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF)] and one species of ganglioside (GM1) have been shown to exert interactions with each other and also to exhibit neuroprotective effects against retinal ischemia in vivo and cerebral excitotoxicity in vitro. Different experimental models are used to investigate their relevance to ischemic and excitotoxic conditions in the retina, and it is shown that (1) both bFGF and EGF show very effective neuroprotection for rat retinal neurones exposed to toxic levels of glutamate or its nonphysiological agonist kainate in vitro; (2) GM1 (10(-5M) used under the same conditions does not afford protection; (3) retinal glial cells also suffer morphological perturbations following glutamate or kainate treatment, but this effect is dependent on neuron-glial interactions, indicating the existence of intermediate neuron-derived messenger molecules; (4) these glial changes can be corrected by posttreatment with either bFGF or EGF in vitro; (5) using an in vivo animal model involving anterior chamber pressure-induced ischemia in adult rats, it is shown that either pretreatment by intraperitoneal injection of GM1, or posttreatment by intraocular injection of the same ganglioside, reduces significantly histological damage to inner nuclear regions; and (6) in cultured retinal Müller glial cells the existence of molecular and metabolic interactions between both types of trophic factors is demonstrated. Hence both these groups of trophic molecules show interesting features for retinal ischemic treatment.
Assuntos
Gangliosídeos/farmacologia , Substâncias de Crescimento/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores , Retina/fisiologia , Vasos Retinianos/fisiologia , Animais , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Gangliosídeo G(M1)/farmacologia , Isquemia/patologia , Isquemia/fisiopatologia , Isquemia/prevenção & controle , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Ratos , Retina/citologia , Retina/efeitos dos fármacos , Vasos Retinianos/citologia , Vasos Retinianos/efeitos dos fármacosRESUMO
Ganglioside profiles in spinal cord from 13-day mouse fetuses, 21-day postnatal and adult mice were compared with those harvested from organotypic cross-sections of fetal mouse spinal cord grown for 28 days in vitro in a serum-free medium. All the major species of gangliosides reported for brain were present both in the in vivo tissue and cultured spinal cord, though not necessarily at each developmental stage examined. Fresh tissues showed increases and decreases in various gangliosides as have been reported for higher brain centers at similar stages of development in mammals and birds. However, qualitative and quantitative differences exist between fresh spinal cord and cultured cord explants as well as between galactose-grown and galactose-free cultures. Spinal cord explants grown in the presence of galactose showed measurable amounts of GM2 and GM3 which were not detected in the control-defined medium-grown cultures. The differences between the two culture groups may be related to interneuronal connectivity patterns.
Assuntos
Envelhecimento/metabolismo , Gangliosídeos/metabolismo , Medula Espinal/metabolismo , Animais , Galactose/farmacologia , Técnicas In Vitro , Camundongos , Microscopia Eletrônica de Varredura , Medula Espinal/embriologia , Medula Espinal/crescimento & desenvolvimentoRESUMO
Ganglioside (GG) and neurotrophic growth factor (GF) interactions in retinal neuronal and glial cells have been very little studied. Rat retinas were mechanically separated into outer (photoreceptor or PR) and inner (other neurons, IR) halves by planar vibratome sectioning and retinal Müller glial (RMG) cells were isolated and cultured according to previously published methods. The distribution on a percent molar basis of individual GG was different between the two halves: PR were dominated by GD3 (48% total GG) and contained only trace amounts (< 4%) of complex species (GT1b, GQ); IR was more typical of mature brain tissue, exhibiting substantial amounts (approximately 25%) of more complex GG. The GG profile of RMG cells was also simple, dominated by GM3 (60%) and GD1a (20%). A single addition to the medium of 500 pM bFGF or EGF for 48 hr to cultured RMG cells led to significant increases in total GG levels of 30-40%. Such treatments by both growth factors induced increases in GM3, whereas longer exposure (96 hr) of confluent RMG to these factors additionally stimulated synthesis of more complex GG. Incubations of RMG with [3H]-glucosamine showed that GG synthesis was 2-fold stimulated by growth factors. We also tested the effect of GM3 on one of the bFGF receptor transduction pathways, namely PI-3 kinase activation. To our knowledge these data constitute the first demonstration of neurotrophic factor stimulation of GG levels in cells of CNS in vitro. Such complex interactions may have particularly important consequences for neural physiopathology.
Assuntos
Gangliosídeos/metabolismo , Fatores de Crescimento Neural/farmacologia , Retina/efeitos dos fármacos , Animais , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Metabolismo dos Lipídeos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Retina/citologia , Retina/metabolismoAssuntos
Actinina/metabolismo , Grânulos Cromafim/enzimologia , Sistema Cromafim/enzimologia , Dopamina beta-Hidroxilase/metabolismo , Proteínas Musculares/metabolismo , Medula Suprarrenal/enzimologia , Animais , Bovinos , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Miosinas/metabolismoRESUMO
Neurotrophic factors such as basic fibroblast and epidermal factor (bFGF and EGF respectively) are known to influence many differentiative processes, but their effects on an important group of glycosylated signalling molecules involved in neural differentiation, the gangliosides, are unknown. To study this possibility, we analyzed the effects of exogenously added bFGF and EGF upon the amount and type of endogenous gangliosides extracted from purified cultures of retinal Müller glial cells. A single addition of 500 pM bFGF or EGF for 48 h to such cultures led to significant increases in total ganglioside levels of 30-40%. Analysis of the distribution of specific ganglioside species within control and growth factor treated cells revealed that the precursor form GM3 formed 50-60% of the total ganglioside pool in all cases, the remainder being composed principally of GD1a (20%) with no detectable tri-sialogangliosides. Growth factor treatment for 48 h led to increases mainly in GM3, whereas longer exposure (96 h) of confluent glial cultures to growth factors additionally stimulated synthesis of GT1b. Furthermore, growth factor-induced ganglioside increases were dose-dependent, reaching maximal stimulation at 500 pM for bFGF. Incorporation of radiolabelled [3H]-glucosamine into glial cultures showed that ganglioside synthesis was stimulated 2-fold by the growth factors. To our knowledge these data constitute the first demonstration of neurotrophic factor stimulation of ganglioside levels in cells of central nervous system origin. Such complex interactions between peptide growth factors and gangliosides, if occurring in vivo, could have important consequences for retinal cell behaviour.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Gangliosídeos/metabolismo , Neuroglia/metabolismo , Retina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Gangliosídeo G(M3)/metabolismo , Metabolismo dos Lipídeos , Neuroglia/efeitos dos fármacos , Neuroglia/ultraestrutura , Ratos , Ratos Wistar , Retina/citologia , Retina/efeitos dos fármacos , Estimulação QuímicaRESUMO
Currently available techniques concerning extraction and characterization of the different lipids from biological specimens are designed for particular families and do not address consecutive isolation of lipid constituents in their globality. We describe here a simple, nondestructive chromatographic procedure that allows efficient elution and further analysis of the major lipid classes (neutral lipids, phospholipids, nonsialylated sphingolipids, and gangliosides) in their natural states from the same starting material. The procedure describes the use of solvent mixtures adapted to silicic acid column chromatography and permits 90-97% recovery of each of the above lipid groups. We have particularly concentrated on optimizing the efficient recovery of the diverse minor forms of gangliosides, free of other contaminants, from relatively small amounts of neural tissue. As model systems we have used in vivo and in vitro preparations of mammalian retina for which only fragmentary data are available on lipid composition. We show that relative to brain, retina contains, for example, twofold more sphingomyelin and sixfold more GD3 ganglioside. In turn, cultured retinal glial cells contain twofold higher levels of globoside and eightfold higher amounts of GM3 ganglioside with respect to intact retina. Compared to previously published techniques, we obtain improved total ganglioside recovery, with enrichment of poly-sialogangliosides. The technique presented here should be widely applicable to analyze global lipid composition of diverse biological samples.
Assuntos
Química Encefálica/fisiologia , Gangliosídeos/isolamento & purificação , Lipídeos/isolamento & purificação , Neuroglia/química , Retina/química , Animais , Células Cultivadas , Fracionamento Químico , Cromatografia em Camada Fina , Fosfolipídeos/isolamento & purificação , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Esfingolipídeos/isolamento & purificação , SuínosRESUMO
Previous work from this and other laboratories has shown that the neuritogenic effect due to exogenous gangliosides on primary neurons in culture is accompanied by several morphological and biochemical modifications. The present results indicate that the treatment of these neurons with gangliosides, under the experimental conditions which are known to produce a sprouting effect, inhibited the influx of 45Ca2+ and increased the release of 45Ca2+ from the cells. No significant differences were noted using concentrations of gangliosides (10(-8)-10(-5) M) either below or above the critical micellar concentrations. No apparent specificity was observed among various species of individual sialocompounds (GM1, GD1a). Moreover the presence or absence of fetal calf serum in the culture medium influenced the levels of 45Ca2+ fluxes. This study confirms the hypothesis that gangliosides may be considered as Ca2+ flux modulators in neuronal cells.
Assuntos
Cálcio/metabolismo , Gangliosídeos/farmacologia , Neurônios/metabolismo , Animais , Bovinos/sangue , Bovinos/embriologia , Células Cultivadas , Meios de Cultura , Sangue Fetal , Fatores de TempoRESUMO
The relative effects of medium chain (MCT) and long chain triglycerides (LCT) on intestinal morphology and functions were compared. Adult rats received intragastrically for 10 days an isoenergetic mixture containing either 50% MCT/50% LCT or 100% LCT. The other constituents of the diets were identical, and animals fed a standard diet orally were used as a reference group. Animals who were given the MCT/LCT diet showed a higher mucosal mass and protein content and increased villus length and crypt depth in the proximal part of the small intestine compared with the LCT and control diet groups. Administration of [3H] thymidine 12 hours before death resulted in a significant increase in the incorporation of the precursor into cellular DNA in the jejunum of rats given MCT. In rats given LCT as the only fat, the free fatty acid content of the microvillus membrane showed a 20 fold increase and at the same time there was a significant drop in the cholesterol content and in the cholesterol/protein ratio. Differences in the lipid composition of enterol diet or in the microvillus membrane did not effect adversely membrane bound hydrolase activities. These findings suggest that MCT in the diet confers advantages in addition to the provision of rapidly available energy.