RESUMO
Whereas PNS neurons in culture are intrinsically responsive to peptide trophic factors, retinal ganglion cells (RGCs) are not unless they are depolarized, or their intracellular levels of cyclic AMP (cAMP) are elevated. We show here that depolarization increases cAMP in cultured RGCs sufficiently to enhance their responsiveness and that the trophic responsiveness of developing RGCs in intact retinas depends on physiological levels of activity and cAMP elevation. Responsiveness is lost after axotomy but is restored by cAMP elevation. The death of axotomized RGCs can be prevented if they are simultaneously stimulated by several trophic factors together with cAMP elevation. Thus, the death of RGCs after axotomy is not caused solely by the loss of retrograde trophic stimuli but also by a profound loss of trophic responsiveness.
Assuntos
Fatores de Crescimento Neural/fisiologia , Células Ganglionares da Retina/fisiologia , Inibidores de Adenilil Ciclases , Animais , Axotomia , Morte Celular , Núcleo Celular/enzimologia , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Eletrofisiologia , Imunofluorescência , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Although 3',5'-cyclic AMP (cAMP) is known to regulate oligodendrocyte development in vitro, little is known about the identity of agonists that induce cAMP synthesis in oligodendroglia. To identify such agonists, we used a novel immunohistochemical method of visualizing cAMP within single cells to screen compounds that are known to activate cAMP synthesis in other cellular systems. Calcitonin, corticotropin-releasing hormone, and vasoactive intestinal peptide elevated cAMP in oligodendroglial cells but not in other cell types present in the cultures (i.e., astrocytes and microglia). In contrast, prostaglandins and the beta-adrenergic agonist isoproterenol, which have previously been reported to induce modest increases in oligodendroglial cell cAMP from biochemical assay of cell homogenates, did not induce a detectable cAMP response in individually identified oligodendroglial cells but instead induced a robust cAMP response in nonoligodendroglial cells.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Calcitonina/farmacologia , Hormônio Liberador da Corticotropina/farmacologia , AMP Cíclico/metabolismo , Oligodendroglia/efeitos dos fármacos , Prostaglandinas/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Calcitonina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Sistema Nervoso Central/citologia , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Técnicas de Cocultura , Hormônio Liberador da Corticotropina/metabolismo , Substâncias de Crescimento/farmacologia , Imuno-Histoquímica , Isoproterenol/farmacologia , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Prostaglandinas/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
Adenosine 3':5'-cyclic monophosphate (cAMP) is a key second messenger in signaling pathways governing many cellular processes. To define the subcellular localization and relative abundance of cAMP, we developed a novel immunochemical approach based on acrolein fixation to visualize cAMP within cells. We describe here the fixation and immobilization of cAMP within cells and the production of specific, high titer polyclonal antibodies that recognize cAMP. Relative levels of cAMP immunofluorescence were quantitated in glial cells (oligodendrocytes, astrocytes, Schwann cells, and glioma cells) that were either untreated or treated with activators of endogenous adenylyl cyclase to raise cAMP levels. In treated cells, cAMP immunofluorescence is strongly localized in the perinuclear cytoplasm.
Assuntos
AMP Cíclico/análise , Acroleína , Adenilil Ciclases/metabolismo , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Astrócitos/química , AMP Cíclico/imunologia , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Técnica Direta de Fluorescência para Anticorpo , Glioma/química , Indicadores e Reagentes , Neuroglia/química , Coelhos , Células de Schwann/químicaRESUMO
The short-term survival of highly purified embryonic spinal motor neurons (SMNs) in culture can be promoted by many peptide trophic factors, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), glial-derived neurotrophic factor (GDNF), and hepatocyte growth factor (HGF). We have asked whether these peptides are sufficient to promote the long-term survival of purified E15 SMNs. Contrary to previous reports, we find that when SMNs are cultured in serum-free medium containing a single peptide trophic factor only approximately one-third of the cells survive for 3 d in culture. When multiple factors are combined, additive effects on survival are observed transiently, but by 7 d of culture the majority of SMNs has died. Surprisingly, when cAMP levels are elevated, the majority of SMNs extend processes and survive for 1 week in culture in the absence of peptide trophic factors, even in low-density cultures. A combination of five peptide trophic factors, together with cAMP elevation, promotes the long-term survival of most of the SMNs in serum-free culture for 3 weeks. These findings provide useful culture conditions for studying the properties of SMNs and have implications for the treatment of motor neuron diseases.
Assuntos
AMP Cíclico/metabolismo , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Fatores de Crescimento Neural , Medula Espinal/citologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Sobrevivência Celular/fisiologia , Fator Neurotrófico Ciliar , Colforsina/farmacologia , Estradiol/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Fator de Crescimento de Hepatócito/farmacologia , Hidrocortisona/farmacologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Neurônios Motores/efeitos dos fármacos , Degeneração Neural/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Oxigênio/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Progesterona/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tiroxina/farmacologia , Tretinoína/farmacologiaRESUMO
Canine discoid lupus erythematosus (DLE) and mucocutaneous pyoderma (MCP) have overlapping clinical and histopathological changes, often making diagnosis difficult. Histopathological features of 27 nasal planum biopsies were scored to determine whether DLE and MCP were histopathologically distinguishable. Long-term follow-up, enabling assessment of clinical diagnoses, was available on 15 cases; 11/15 cases were immunomodulatory responsive (ImR) and 4/15 were antibiotic responsive (AbR). Clinical diagnosis, determined by response to treatment for 15/27 cases, was not predictable based on scoring of histopathological features. Distinct histopathological patterns were observed: 2/11 ImR cases had a lymphocyte-rich interface dermatitis. All other cases had the same histopathological changes: a band-like diffuse superficial plasmacytic to lymphoplasmacytic dermatitis +/- focal basal cell damage, but different clinical diagnoses (4/4 AbR, 9/11 ImR). German shepherd dogs/crosses were over-represented (44.4% of the cases) and tended to have more multifocal lesions (41.7% vs. 26.7% of all other breeds). Longer duration of disease was associated with a preponderance of plasmacytic infiltrate (P = 0.026).