RESUMO
Some rat melanotrophs express in vivo tyrosine hydroxylase mRNA. In this report we show, by Western-blotting, that cultures of adult rat melanotrophs, but not adenohypophyseal cells, express tyrosine hydroxylase. Immunocytochemical analyses confirmed the existence of a subpopulation of melanotrophs expressing tyrosine hydroxylase. Bromocryptine (2.5 x 10(-7) M), a D2 dopamine agonist, down-regulated melanotroph tyrosine hydroxylase expression in a time-dependent manner; initial effect was detected at 15 h and maximum at 3 days treatment (reduction to about 40% of control values). Down-regulation at 3 days was dose-dependent (ED(50) around 2 x 10(-9) M). This decrease was reversed by sulpiride, a D2 dopamine antagonist. The cell number was slightly increased by bromocryptine treatment. These data suggest tyrosine hydroxylase expression in melanotrophs being under tonic inhibitory control by dopamine innervation in vivo.
Assuntos
Dopamina/metabolismo , Hormônios Estimuladores de Melanócitos/efeitos dos fármacos , Hormônios Estimuladores de Melanócitos/metabolismo , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Bromocriptina/farmacologia , Técnicas de Cultura de Células , Agonistas de Dopamina/farmacologia , Imuno-Histoquímica , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de TempoRESUMO
External clues for neuron development include extracellular matrix (ECM) molecules. To explore ECM influence on the early development of peptide phenotype in the CNS, we have compared pro-TRH levels in primary cultures of rat hypothalamic cells plated either on poly-lysine (PL) (control) or on PL plus one of various ECM molecules at 10 microgram/ml. Fetal day 17 cells plated at a density of 1250/mm(2) were grown in a serum free medium made of Neurobasal medium supplemented with B27 (GIBCO). Cultures, consisting mainly of neurons, were analyzed at DIV 2. ECM proteins induced morphological effects in agreement with previously published studies. The amount of pro-TRH per dish, quantified by Western blotting, was increased to 275% for laminin, 191% for fibronectin and 173% for tenascin-C (control=100%); there was no effect of vitronectin. Laminin or fibronectin did not change pro-TRH mRNA or TRH levels but enhanced levels of the pro-protein convertase PC1 suggesting that the ECM molecules did regulate the translational status of pro-TRH. In conclusion, we have shown that some ECM proteins increased pro-TRH level in vitro; this may contribute to the enhancement of pro-TRH levels observed early in vivo in the hypothalamus.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Hipotálamo/citologia , Neurônios/enzimologia , Precursores de Proteínas/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Feto/citologia , Fibronectinas/análise , Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/embriologia , Técnicas In Vitro , Laminina/análise , Neurônios/química , Neurônios/citologia , Pró-Proteína Convertases , Biossíntese de Proteínas/fisiologia , Precursores de Proteínas/genética , Ácido Pirrolidonocarboxílico/análogos & derivados , RNA Mensageiro/análise , Radioimunoensaio , Ratos , Ratos Wistar , Tenascina/análise , Hormônio Liberador de Tireotropina/genética , Vitronectina/análiseRESUMO
Coculture of adult pituitary intermediate lobe (IL) cells, a target for hypothalamic dopaminergic neurons, with fetal rat hypothalamic cells accelerate differentiation of dopaminergic neurons. This involves long range diffusible as well as additional factors which may be membrane-bound. To determine whether IL membrane-bound factors contribute to the differentiating effect of IL cells, IL membranes were added to dispersed fetal hypothalamic neurons. This stimulated the outgrowth of dopaminergic neurites and elevated TH levels. Limited trypsin proteolysis of IL cell surface abolished the effect on TH levels. Addition of adenohypophyseal membranes was ineffective. Joint treatment with IL membranes, and medium conditioned (CM) over IL cells, produced the same effect on TH levels as did coculture with the same number of IL cells. The results demonstrate that IL cells express on their surface a membrane-bound factor promoting differentiation of fetal dopaminergic neurons in vitro; this factor acts in addition to diffusible activities.