RESUMO
BACKGROUND AND PURPOSE: To assess the efficacy and safety of olesoxime, a molecule with neuroprotective properties, in patients with amyotrophic lateral sclerosis (ALS) treated with riluzole. METHODS: A double-blind, randomized, placebo-controlled, multicenter trial of 18 months' duration was conducted in 512 subjects, with probable or definite ALS and a slow vital capacity (SVC) ≥70%, receiving 330 mg olesoxime daily or matching placebo and 50 mg riluzole twice a day in all. The primary intention-to-treat (ITT) outcome analysis was 18 months' survival. Secondary outcomes were rates of deterioration of the revised ALS functional rating scale (ALSFRS-R), focusing on the 9-month assessment, SVC and manual muscle testing. Blood levels, safety and tolerability of olesoxime were also assessed. RESULTS: At 18 months, 154 of the 512 ITT patients had died (79 of 253 placebo, 75 of 259 olesoxime). Estimated overall survival according to Kaplan-Meier analysis was 67.5% (95% CI 61.0%-73.1%) in the placebo group and 69.4% (95% CI 63.0%-74.9%) in the olesoxime group; hence survival was not significantly different between treatment arms (P = 0.71, stratified bulbar/spinal log-rank). The other efficacy end-points evaluated were also negative, with the exception of a small difference in ALSFRS-R global score at 9 months in favor of olesoxime but not sustained after 18 months' treatment nor evident in either the stratified bulbar or spinal subpopulations. Treatment did not raise any safety concerns. CONCLUSIONS: Olesoxime, although well tolerated, did not show a significant beneficial effect in ALS patients treated with riluzole.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Colestenonas/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Adulto , Idoso , Estudos de Casos e Controles , Método Duplo-Cego , Europa (Continente) , Feminino , Humanos , Cooperação Internacional , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
We have used antibodies to identify Schwann cells and oligodendrocytes and to study the expression of myelin-specific glycolipids and proteins in these cells isolated from perinatal rats. Our findings suggest that only Schwann cells which have been induced to myelinate make detectable amounts of galactocerebroside (GC), sulfatide, myelin basic protein (BP), or the major peripheral myelin glycoprotein (P0). When rat Schwann cells were cultured, they stopped making detectable amounts of these myelin molecules, even when the cells were associated with neurites in short-term explant cultures of dorsal root ganglion. In contrast, oligodendrocytes in dissociated cell cultures of neonatal optic nerve, corpus callosum, or cerebellum continued to make GC, sulfatide and BP for many weeks, even in the absence of neurons. These findings suggest that while rat Schwann cells require a continuing signal from appropriate axons to make detectable amounts of myelin-specific glycolipids and proteins, oligodendrocytes do not. Schwann cells and oligodendrocytes also displayed very different morphologies in vitro which appeared to reflect their known differences in myelinating properties in vivo. Since these characteristic morphologies are maintained when Schwann cells and oligodendrocytes were grown together in mixed cultures and in the absence of neurons, we concluded that they are intrinsic properties of these two different myelin-forming cells.
Assuntos
Glicolipídeos/biossíntese , Proteínas da Mielina/biossíntese , Neuroglia/metabolismo , Oligodendroglia/metabolismo , Células de Schwann/metabolismo , Envelhecimento , Animais , Células Cultivadas , Galactosilceramidas/biossíntese , Gânglios Espinais/citologia , Gânglios Simpáticos/citologia , Oligodendroglia/citologia , Ratos , Células de Schwann/citologia , Nervo Isquiático/citologia , Sulfoglicoesfingolipídeos/metabolismoRESUMO
Activation of kainate receptors causes Co2+ influx into neurons, type-2 astrocytes, and O-2A progenitor cells. Agonist-activated Co2+ uptake can be performed using cultured cells or fresh tissue slices. Based on the pattern of response to kainate, glutamate, and quisqualate, three functionally different kainate-activated ion channels (K1, K2, and K3) can be discriminated. Co2+ uptake through the K1 receptor was only activated by kainate. Both kainate and glutamate activated Co2+ uptake through the K2 receptor. Co2+ uptake through the K3 receptor was activated by all three ligands: kainate, glutamate, and quisqualate. Co2+ uptake occurred through a nonselective cation entry pathway permeable to Co2+, Ca2+, and Mn2+. The agonist-dependent activation of divalent cation influx through different kainate receptors could be correlated with expression of certain kainate receptor subunit combinations. These results are indicative of kainate receptors that may contribute to excitatory amino acid-mediated neurotoxicity.
Assuntos
Cobalto/metabolismo , Canais Iônicos/fisiologia , Neurônios/fisiologia , Receptores de Neurotransmissores/fisiologia , Animais , Transporte Biológico , Cálcio/metabolismo , Células Cultivadas , Cerebelo/fisiologia , Glutamatos/farmacologia , Hipocampo/fisiologia , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Ácido Caínico/farmacologia , Manganês/metabolismo , N-Metilaspartato/farmacologia , Ácido Quisquálico/farmacologia , Ratos , Ratos Endogâmicos , Receptores de Ácido CaínicoRESUMO
BACKGROUND AND PURPOSE: Parkinson's disease (PD) is usually diagnosed clinically from classical motor symptoms, while definitive diagnosis is made postmortem, based on the presence of Lewy bodies and nigral neuron cell loss. α-Synuclein (ASYN), the main protein component of Lewy bodies, clearly plays a role in the neurodegeneration that characterizes PD. Additionally, mutation in the SNCA gene or copy number variations are associated with some forms of familial PD. Here, the objective of the study was to evaluate whether olesoxime, a promising neuroprotective drug can prevent ASYN-mediated neurotoxicity. EXPERIMENTAL APPROACH: We used here a novel, mechanistically approachable and attractive cellular model based on the inducible overexpression of human wild-type ASYN in neuronally differentiated human neuroblastoma (SHSY-5Y) cells. This model demonstrates gradual cellular degeneration, coinciding temporally with the appearance of soluble and membrane-bound ASYN oligomers and cell death combining both apoptotic and non-apoptotic pathways. KEY RESULTS: Olesoxime fully protected differentiated SHSY-5Y cells from cell death, neurite retraction and cytoplasmic shrinkage induced by moderate ASYN overexpression. This protection was associated with a reduction in cytochrome c release from mitochondria and caspase-9 activation suggesting that olesoxime prevented ASYN toxicity by preserving mitochondrial integrity and function. In addition, olesoxime displayed neurotrophic effects on neuronally differentiated SHSY-5Y cells, independent of ASYN expression, by promoting their differentiation. CONCLUSIONS AND IMPLICATIONS: Because ASYN is a common underlying factor in many cases of PD, olesoxime could be a promising therapy to slow neurodegeneration in PD.
Assuntos
Colestenonas/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , alfa-Sinucleína/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Humanos , Neurônios/citologia , Neurônios/metabolismoRESUMO
The neuropeptide galanin (GAL) is widely distributed throughout the diffuse neuroendocrine system, and is coexpressed with acetylcholine, norepinephrine, prolactin, and a variety of other messenger substances in different cell types. Bovine chromaffin cells in primary culture synthesize and store GAL along with catecholamines, chromogranin A, and enkephalin peptides, as well as other neurosecretory products, and secrete all these molecules in response to nicotinic stimulation. The regulation of GAL biosynthesis and secretion were studied by measuring changes in messenger RNA (mRNA(GAL], and peptide immunoreactivity, 24-72 h after stimulation of secretion (40 mM potassium or 10 microM veratridine), or exposure to stimulators of protein kinase C (100 nM phorbol myristate acetate) and protein kinase A (25 microM forskolin). Depolarization-induced stimulation of GAL biosynthesis, like that of enkephalin and other neuropeptides, was calcium dependent, suggesting that calcium generally mediates both exocytotic release and new peptide synthesis thus coordinating maintenance of neuropeptide levels in chromaffin cells. GAL and mRNA(GAL) were also upregulated by stimulation of protein kinase A with forskolin. Treatment with PMA increased GAL and mRNA(GAL) to an even greater extent than depolarization. Thus GAL expression can be regulated by three distinct signal transduction systems in chromaffin cells: depolarization-stimulated calcium influx, activation of protein kinase C and activation of protein kinase A, which in addition differentially up- and down-regulate the expression of several other neurosecretory proteins and peptides resulting in different patterns of GAL/neuropeptide coexistence in bovine chromaffin cells. GAL coexistence with diverse neuroendocrine substances may reflect the relative activity of these three signalling systems in other neuroendocrine cell types as well.
Assuntos
Medula Suprarrenal/fisiologia , Cálcio/fisiologia , Regulação da Expressão Gênica , Neuropeptídeos/genética , Peptídeos/genética , Proteínas Quinases/metabolismo , Transdução de Sinais , Animais , Bovinos , Células Cultivadas , Encefalinas/análise , Imunofluorescência , Galanina , Peptídeos/análise , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Sistemas do Segundo MensageiroRESUMO
We showed that insulin appears in the chick embryo before beta-cells are recognizable, as well as in the egg constituents even before fertilization. Acid-ethanol extracts of 2- to 8-day-old embryos were gel-filtered on Sephadex G-50. The peak of immunoreactive insulin chromatographed in a position corresponding to that of authentic insulin. The immunoreactive insulin extracted from embryos was approximately 2 ng/g wet wt during early embryogenesis (days 2, 3, and 4), with a 2- to 3- fold increase by days 5 and 6, in conjunction with pancreatic development. The heads of the embryos contributed 22-23% of the total insulin on days 3 and 4, but only 5% by day 5. Based on its reactivity in a pork insulin RIA, chicken insulin RIA, and rat adipocyte bioassay, we concluded that the material is very similar to avian (chicken or turkey) rather than mammalian type insulin. Similar immunological and biological insulin-like activity [but at much lower concentrations (0.2-0.8 ng/ml)] were recovered from the gel-filtered acid-ethanol extracts of yolk and white of unfertilized and fertilized eggs. This study, which shows that insulin is present at a very early stage in ontogeny, extends observations that insulin is native to organisms that lack pancreatic islets, including flies, worms, and microbes.
Assuntos
Embrião de Galinha/análise , Insulina/análise , Óvulo/análise , Tecido Adiposo/efeitos dos fármacos , Animais , Bioensaio , Cromatografia em Gel , Clara de Ovo/análise , Gema de Ovo/análise , Feminino , Insulina/farmacologia , Radioimunoensaio , Ratos , Ratos EndogâmicosRESUMO
Two neuropeptides, enkephalin and vasoactive intestinal polypeptide (VIP), are simultaneously increased in cultures of bovine chromaffin cells after diverse treatments including elevation of cAMP, application of nicotine, or chronic depolarization. We now show that phorbol esters can specifically elevate VIP in cultured chromaffin cells without changing the amount of enkephalin. Peptide histidine isoleucine, a VIP-related peptide, is also expressed concomitantly with VIP after treatment with phorbol ester. Immunocytochemical examination of drug-treated cells defines a subpopulation of chromaffin cells which are responsive to phorbol ester stimulation. The unique ability of phorbol esters to selectively regulate VIP expression indicates the presence of independent mechanisms for controlling the expression of individual neuropeptides in chromaffin cells.
Assuntos
Grânulos Cromafim/metabolismo , Sistema Cromafim/metabolismo , Forbóis/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Peptídeo Intestinal Vasoativo/biossíntese , Animais , Bovinos , Células Cultivadas , Grânulos Cromafim/efeitos dos fármacos , Colforsina , Diterpenos/farmacologia , Relação Dose-Resposta a Droga , Encefalina Metionina/biossíntese , Encefalinas/genética , Biossíntese Peptídica , Peptídeo PHI , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
We have isolated clones from a human brain cDNA library coding for two splice variants of mGluR1. The combined sequences account for 6864 base pairs (bp) of the approximately 7 kilobase (kb) transcript seen on northern blots of human brain mRNA. The distribution of mGluR1 mRNA in human brain is similar to that found in rat brain and the gene for mGluR1 is localized on human chromosome 6. The mRNA for mGluR1 alpha contains an open reading frame that codes for a 1194 amino acid protein, which is slightly smaller than rat mGluR1 alpha. The major structural difference noted between the human and rat mGluR1 is a deletion of 21 nucleotides which would result in the loss of seven amino acids in the middle of a proline- and glutamine-rich region of the C-terminal tail of mGluR1 alpha. The 85 bp exon that results in the HmGluR1 beta splice variant was found to code for a protein with two amino acid differences compared to the rat receptor. Functional and pharmacological characterization of heterologously expressed human mGluR1 was performed using electrophysiological recordings from Xenopus oocytes and calcium imaging in HEK-293 cells. No major differences were found in the response of human mGluR1 to typical agonists and antagonists compared to the rat, or in the behavior of the two splice variants.
Assuntos
Processamento Alternativo , Encéfalo/metabolismo , Cromossomos Humanos Par 6 , Receptores de AMPA/biossíntese , Receptores de AMPA/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Cicloleucina/análogos & derivados , Cicloleucina/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Variação Genética , Ácido Glutâmico/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Oócitos/fisiologia , Fases de Leitura Aberta , RNA Mensageiro/metabolismo , Ratos , Receptores de AMPA/fisiologia , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transfecção , XenopusRESUMO
Anti-IFA, a panspecific monoclonal antibody which was raised against human glial fibrillary acidic protein (GFAP), recognizes a determinant common to GFAP and all other intermediate filament proteins. This antibody can be used to identify intermediate filament proteins from both vertebrate and invertebrate tissues. Its utility in immunoblot studies of intermediate filament proteins is enhanced by using cytoskeleton extracts and protease treatment to facilitate the transfer of high molecular weight (greater than 70 000) proteins from gels to nitrocellulose membranes.
Assuntos
Proteínas de Filamentos Intermediários/análise , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Química Encefálica , Colódio , Citoesqueleto/análise , Eletroforese em Gel de Poliacrilamida , Imunoquímica , Proteínas de Filamentos Intermediários/imunologia , Pronase , Ratos , Medula Espinal/análiseRESUMO
I have prepared a number of monoclonal antibodies to chromaffin cell membranes. One of these antibodies recognizes a number of antigenically related proteins that are present in all tissues examined. In the adrenal, these proteins are completely excluded from chromaffin granules but are present in other subcellular membrane fractions. This non-granule membrane-specific antibody has been designated NG3. A second antibody, CG7, binds to a single protein which segregates specifically into chromaffin granules. The protein recognized by CG7 is cytochrome b561, or chromomembrin B, one of the major protein components of chromaffin granule membranes. CG7 also labels a protein (the identical cytochrome b561) in bovine posterior pituitary neurosecretory vesicle membranes indicating that it functions in both peptidergic and catecholaminergic secretory granules. These two monoclonal antibodies provide useful probes of both granule and extra-granule membrane proteins for studies of membrane trafficking in chromaffin cells.
Assuntos
Anticorpos Monoclonais/imunologia , Grânulos Cromafim/imunologia , Sistema Cromafim/imunologia , Proteínas de Membrana/imunologia , Medula Suprarrenal/inervação , Animais , Especificidade de Anticorpos , Bovinos , Grupo dos Citocromos b/imunologia , Células Enterocromafins/imunologia , Epitopos/imunologiaRESUMO
CG7 is a monoclonal antibody that recognizes cytochrome b561, a major protein component of adrenal medullary chromaffin granules. Immunocytochemical studies using this antibody have demonstrated that cytochrome b561 is present in many neural and endocrine tissues and that its distribution is correlated with the presence of either catecholamines or amidated peptides in the tissue. These tissues include neuronal cell bodies and/or fibers in the gut, blood vessels, retina, and posterior pituitary, endocrine cells of the gut, anterior and intermediate lobes of the pituitary, heart muscle, and all adrenal medullary chromaffin cells. The discovery of cytochrome b561 in many neuropeptide-containing tissues regardless of the presence of catecholamines is consistent with a general role for cytochrome b561 as a secretory granule membrane electron carrier. Its expression may be linked to an ascorbic acid requirement by both catecholamine and neuropeptide biosynthetic enzymes.
Assuntos
Medula Suprarrenal/inervação , Anticorpos Monoclonais , Grupo dos Citocromos b/metabolismo , Sistema Nervoso/anatomia & histologia , Animais , Sistema Cardiovascular/inervação , Bovinos , Grânulos Cromafim/ultraestrutura , Imunofluorescência , Intestinos/inervação , Hipófise/anatomia & histologia , Retina/anatomia & histologiaRESUMO
We show that cultured bovine adrenal chromaffin cells can be stimulated to produce atrial natriuretic peptide-like immunoreactivity (ANP). ANP levels increased 5-fold in response to either forskolin or phorbol ester treatment, and 17-fold after depolarization by 40 mM potassium. These agents appear to act through distinct second messenger mediated pathways to cause increased accumulation of ANP. When forskolin and phorbol ester were added simultaneously, their effects were synergistic: ANP levels increased 30-fold, more than the product of the increases achieved by treatment with either drug alone. The ANP present in bovine chromaffin cell extracts appeared immunologically identical to human ANP(1-28). By gel filtration, the immunoreactive ANP extracted from chromaffin cells was in a high molecular weight form, although HPLC fractionation revealed that a portion of the total immunoreactivity could be eluted with the same retention time as synthetic ANP standards.
RESUMO
Enkephalins, vasoactive intestinal polypeptide, and chromogranin A are all contained in the secretory vesicles of chromaffin cells in culture, and are all released from this compartment by secretagogues in a calcium-dependent way. The biosynthesis of each of these peptides, however, is under quite independent regulation. The synthesis and secretion of enkephalin is tightly coupled to acetylcholine and elevated potassium stimulation by calcium influx. Once calcium enters the cell, calcium acts at pharmacologically distinct sites to elicit secretion and enhanced biosynthesis of Metenkephalin. This is demonstrated by the calcium-independent stimulation of enkephalin secretion by 1 mM barium, in contrast to the dependence on extracellular calcium of barium-stimulated biosynthesis of this peptide. The synthesis and secretion of VIP is also coupled to acetylcholine and elevated potassium stimulation by calcium influx. Treatment with barium demonstrates that calcium acts at distinct sites to stimulate secretion and biosynthesis of this peptide; however induction of VIP by barium and veratridine shows greater sensitivity to the calcium channel blocker methoxyverapamil (D600) than does the induction of Met-enkephalin by these agents. These differences in D600 sensitivity may be due to differences in calcium metabolism or voltage-dependent calcium channels in enkephalin-producing and VIP-inducible subpopulations of chromaffin cells. Chromogranin A levels are essentially unaffected by any of the agents which increase enkephalin and VIP levels, although it is secreted in parallel with enkephalins and catecholamines from chromaffin cells in response to secretagogues. We suggest that peptide hormones such as VIP and enkephalins are regulated by calcium-dependent stimulus-secretion-synthesis coupling in the chromaffin cell. Cyclic AMP is a positive regulator of enkephalin and VIP biosynthesis, but does not affect acute release of these peptides. The cAMP/protein kinase A system may be a distal mediator of peptide biosynthesis stimulated by secretagogues. Alternatively, cAMP may be involved in early developmental establishment of phenotype or long-term regulation of peptide biosynthesis by other hormones or neurotransmitters. Chromogranin A may represent a class of intravesicular, soluble proteins that are expressed constitutively by the chromaffin cell in the presence or absence of positive regulators of other systems. The biosynthesis of chromogranin A may be coupled to the production or assembly of the secretory vesicle itself.
Assuntos
Sistema Cromafim/metabolismo , Cromograninas/biossíntese , Encefalina Metionina/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Peptídeo Intestinal Vasoativo/biossíntese , Animais , Cálcio/fisiologia , Bovinos , Sistema Cromafim/efeitos dos fármacos , Cromogranina A , AMP Cíclico/fisiologiaRESUMO
An extensive system of neuropeptide Y (NPY) containing neurons has recently been identified in the central and peripheral nervous system. In addition, NPY and a structurally related peptide, peptide YY (PYY), containing endocrine cells have been identified in the periphery. The NPY system is of particular interest as the peptide coexists with catecholamines in the central and sympathetic nervous system and adrenal medulla. Evidence has been presented which indicates that NPY may play important roles in regulating autonomic function.
Assuntos
Glândulas Endócrinas/fisiologia , Hormônios Gastrointestinais/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Peptídeos/fisiologia , Animais , Encéfalo/fisiologia , Encéfalo/ultraestrutura , Proteínas do Tecido Nervoso/análise , Neuropeptídeo Y , Peptídeo YY , Peptídeos/análise , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/fisiologia , Distribuição TecidualRESUMO
We have found that Neuropeptide Y is colocalized with enkephalin in bovine adrenal chromaffin cells. The two peptides can be found in the same granules in those cells where they coexist. These cells correspond to the adrenergic subpopulation of chromaffin cells since they contain the epinephrine synthetic enzyme, phenylethanolamine N-methyltransferase. Despite their coexistence, production of the two peptides is independently regulated. Enkephalin levels are doubled after nicotinic depolarization (which increases enkephalin synthesis) or after treatment with reserpine (which increases enkephalin precursor processing). Neither of these treatments, acting by different mechanisms, has any effect on the levels of Neuropeptide Y.
Assuntos
Sistema Cromafim/fisiologia , Encefalinas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Glândulas Suprarrenais/citologia , Animais , Bovinos , Células Cultivadas , Grânulos Cromafim/metabolismo , Sistema Cromafim/citologia , Histocitoquímica , Imunoquímica , Microscopia Eletrônica , Microscopia de Fluorescência , Neuropeptídeo YRESUMO
Cell populations containing vasoactive intestinal polypeptide (VIP), enkephalins, and catecholamines were identified in bovine adrenal medullary cultures by immunofluorescence and radioimmunoassay. Addition of forskolin to the culture medium increased the cellular levels of both VIP and the enkephalins. These changes resulted from an increase in the number of VIP-positive cells and an increase in cellular enkephalin content.
Assuntos
Medula Suprarrenal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Colforsina , Diterpenos/farmacologia , Encefalina Leucina/metabolismo , Encefalina Metionina/metabolismo , Imunofluorescência , Técnicas In Vitro , Feniletanolamina N-Metiltransferase/metabolismo , Radioimunoensaio , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
Typical insulin receptors are present on neuroblastoma cell lines. High affinity binding for insulin was present in membrane preparations from NG108 (a hybrid mouse neuroblastoma-rat glioma) as well as in membranes from SK-N-MC and SK-N-SH, two human neuroblastoma cell lines. Specific [125I]insulin binding was 24.4% for NG108, 16.9% for SK-N-MC and 5.2% for SK-N-SH at membrane protein concentrations of 0.4 mg/ml. IC50 for [125I]insulin binding was 3.4 nM in NG108 membrane preparations and 0.9 nM for SK-N-SH and 1.8 nM in SK-N-MC membranes. Apparent mol. wt. for the alpha subunits (identified by specific immunoprecipitation using the anti-insulin receptor antiserum B10) on SDS PAGE was 134 kDa for NG108; 124 kDa for SK-N-MC and 120 kDa for SK-N-SH. Neuraminidase digestion increased the mobility of the alpha subunit from both NG108 and SK-N-MC receptors to 120 kDa, whereas that from SK-N-SH were unaffected. Endoglycosidase H and endoglycosidase F digestions increased the mobility of the alpha subunits of all 3 cell lines to varying degrees, suggesting the presence of N-linked glycosylation. Insulin induced autophosphorylation of the insulin receptor beta subunit in WGA-purified membranes from all 3 cell lines. In addition, phosphorylation of a protein with an apparent mol. wt. 105 kDa was stimulated by insulin in WGA purified membranes from NG108. Tyrosine-specific kinase activity was present in the membranes from each cell line and was stimulated by insulin in a dose-dependent manner from 10(-9) to 10(-6) M. Proinsulin was about 100 times less potent in stimulating phosphorylation of the artificial substrate poly (Glu, Tyr)4:1 when compared to insulin in accordance with its lower binding affinity to the insulin receptor. Hexose transport was stimulated by insulin in all 3 cell lines. These results indicate that neuroblastoma cells contain specific insulin receptors and that they may be useful as models for studying the role of insulin in nervous tissue.
Assuntos
Oligossacarídeos/isolamento & purificação , Receptor de Insulina/metabolismo , Ligação Competitiva , Linhagem Celular , Membrana Celular/metabolismo , Desoxiglucose/metabolismo , Humanos , Cinética , Substâncias Macromoleculares , Neuroblastoma , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/isolamento & purificaçãoRESUMO
We have used 4 cell-type-specific markers to identify individual glial and neuronal cells in dissociated cell cultures of neonatal rat sciatic nerve, dorsal root ganglia (DRG), optic nerve, cerebellum, corpus callosum, cerebral cortex and leptomeninges. Schwann cells were identified with antibodies against rat neural antigen-1 (Ran-1), neurons with tetanus toxin, astrocytes with antibody against the glial fibrillary acidic protein (GFAP) and oligodendrocytes with antibody against galactocerebroside. All of these ligands react with cell surface molecules except for anti-GFAP antibody which binds to intracellular glial filaments. Using two-fluorochrome immunofluorescence we have studied the distribution of various glycoproteins and glycolipids on these 4 major neural cell types in short-term cultures. We have found that (1) although Ran-1 is expressed on glial and neuronal tumours, it was not found on normal astrocytes, oligodendrocytes or neurons; (2) Thy-1 was present on fibroblasts and some neurons but not on the majority of leptomeningeal cells or on oligodendrocytes or astrocytes in short-term cultures (however, it was expressed on some astrocytes in longer term cultures); (3) the 'large external transformation sensitive' (LETS) protein could be detected on fibroblasts and leptomeningeal cells but not on neurons or glial cells; (4) GM1 was present on all neurons, most oligodendrocytes and approx. 50% of other cell types; sulfatide and GM3 were only detectable on oligodendrocytes, while globoside was only found on some neurons. In addition, we were able to identify putative microglial cells by the presence of cell surface receptors for IgG and by their phagocytic activity; they did not express and of the cell-type-specific defining markers.
Assuntos
Imunofluorescência , Neuralgia/imunologia , Neurônios/imunologia , Animais , Anticorpos/análise , Cerebelo/imunologia , Córtex Cerebral/imunologia , Corpo Caloso/imunologia , Técnicas de Cultura , Feminino , Gânglios Espinais/imunologia , Glicolipídeos/imunologia , Glicoproteínas/imunologia , Soros Imunes , Masculino , Meninges/imunologia , Camundongos , Proteínas do Tecido Nervoso/imunologia , Nervo Óptico/imunologia , Nervo Isquiático/imunologia , Toxina Tetânica/imunologiaRESUMO
We have identified two growth factors for cultured rat astrocytes: fibroblast growth factor, a peptide derived from either whole bovine brain, or pituitaries, and a growth factor in extracts of bovine pituitary which was previously identified as a Schwann cell mitogen. Oligodendrocytes in primary cultures derived from neonatal rat central nervous system divide only rarely if at all. These growth factors did not stimulate primary oligodendrocytes to divide. Occasionally cells found in suspension in long-term cultures of the central nervous system were enriched for cells which were identified as oligodendrocytes by the presence of galactocerebroside on their surface and myelin basic protein in their cytoplasm. When provided with a monolayer of irradiated 3T3 cells, these oligodendrocytes were able to spread out and extend elaborate branched processes typical of oligodendrocytes in the primary cultures. Unlike their counterparts in the primary cultures, these suspension-derived oligodendrocytes are capable of cell division as demonstrated by the uptake of [3H]thymidine and autoradiography.
Assuntos
Astrócitos/efeitos dos fármacos , Mitógenos/farmacologia , Mitose/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Células de Schwann/efeitos dos fármacos , Animais , Células Cultivadas , Cerebelo/citologia , Corpo Caloso/citologia , Fatores de Crescimento de Fibroblastos , Fatores de Crescimento Neural/farmacologia , Peptídeos/farmacologia , Hipófise/análise , Ratos , Nervo Isquiático/citologia , Extratos de Tecidos/farmacologiaRESUMO
We have immunized mice with enriched populations of cultured rat astrocytes and fused their spleen cells with NS-1 myeloma cells to generate antibody-secreting hybridomas. We have isolated two stable hybridoma clones which secrete monoclonal IgG2 antibodies that react with the surface of the great majority of rat astrocytes in culture. We have studied one of these antibodies in indirect immunofluorescence assays and show that it binds to the surface of rat ependymal cells, retinal Müller cells and leptomeningeal cells as well as to astrocytes, but not to cultured neurones, oligodendrocytes, Schwann cells, microglia or various non-neural cells. The antigen defined by this monoclonal antibody is protease-sensitive and rat-specific and we have called it rat neural antigen-2 (Ran-2). We also show that isolated rat ependymal cells and cultured rat Müller cells do not express other neural cell-type-specific markers, such as tetanus toxin receptors, rat neural antigen-1 (Ran-1), galactocerebroside or glial fibrillary acidic protein (GFAP). Nor do these cells express cell surface Fc receptors for IgG, phagocytose latex beads or make detectable amounts of the Thy-1 or fibronectin glycoproteins.