RESUMO
In the mouse medulla oblongata, we characterized binding properties and functional responses of two recognition sites for imidazoline compounds: I(1)-imidazoline and alpha(2)-adrenergic receptors. The mouse medulla expresses a higher density of I(1)-receptors than in the rat, whereas alpha(2)-receptor densities were similar between the two species. In anesthetized, ventilated and paralyzed mice, we tested the hypotensive actions of the I(1)/alpha(2) agonist moxonidine, determined its central site of its actions, and the relative roles of I(1) and alpha(2)-receptors. Experiments were performed in C(57)Bl(6) wild type and alpha(2A)-adrenergic receptor deficient mice. In both types of mice, neuronal activation within the rostral ventrolateral medulla (RVLM) region by glutamate microinjection elicited increases in arterial pressure. Moxonidine (0.5 nmol/site/10 nl) microinjected bilaterally into this vasopressor region decreased arterial pressure by 30% and heart rate by 11% in wild type mice. Efaroxan, the I(1)/alpha(2) antagonist (0.4 nmol) when microinjected into the RVLM elevated blood pressure itself and abolished the action of moxonidine, whereas alpha(2)-blockade with SK&F 86466 had no significant effect on blood pressure and did not attenuate moxonidine's effect. To more definitively test the role of alpha(2)-adrenergic receptors in the action of moxonidine, moxonidine was microinjected into the RVLM of alpha(2A)-adrenergic deficient mice. The decreases in arterial pressure were nearly identical to those of wild type mice, whereas bradycardia was attenuated. Thus, in the mouse moxonidine acts within the RVLM region to lower arterial pressure mainly through the I(1)-imidazoline receptor independent of alpha(2)-adrenergic receptors.
Assuntos
Anti-Hipertensivos/farmacologia , Imidazóis/farmacologia , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos alfa 2/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 2 , Antagonistas Adrenérgicos alfa/farmacologia , Tonsila do Cerebelo/química , Tonsila do Cerebelo/metabolismo , Animais , Benzofuranos/farmacologia , Ligação Competitiva , Pressão Sanguínea/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Receptores de Imidazolinas , Injeções Intravenosas , Bulbo/química , Bulbo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microinjeções , Ponte/química , Ponte/metabolismo , Receptores de Droga/análise , Receptores de Droga/antagonistas & inibidores , Receptores de Droga/metabolismoRESUMO
En este trabajo se desarrolló la metodología para establecer las condiciones óptimas para el estudio de la unión del complejo receptor-cortisol. Se utilizaron en est estudio 200 hipófisis provenientes de cadáveres. Las condiciones estudiadas fueron: la concentración de proteínas, el pH de los amortiguadores, la temperatura, el tiempo de incubación y la saturación. Los resultados mostraron que la máxima unión específica se logró a una concentración de proteínas de entre 8.5 y 10 mg/ml citosol. La temperatura que se logró la mejor unión fue de 0C. Las incubaciones realizadas mostraron como el tiempo óptimo par la unión, el de 18 hs. El pH óptimo del amortiguador fue entre 7.3 y 7.6, la saturación se logró a una concentración de 3nM de 3H-cortisol, con un exceso de 200 veces ((200x) de cortisol "frío". El método de separación entre el esteroide libre y el unido, utilizando carbón y dextrán, se obtuvo con una eficiencia de 98.7%. La estandarización de estas condiciones es necesaria para el estudio eficiente de las constantes fisico-químicas derivadas de la unión receptor-hormona. Se considera que los resultados del presente trabajo además de contribuir al conocimiento del receptor de cortisol ofrecen un marco de referencia de las condiciones óptimas de tiempo de incubación, temperatura, concentración de proteínas etc., requeridas para su estudio. Esta información facilita el estudio de las constantes de disociación (Kd), asociación (Ka) y número de sitios de unión (n) de una hipófisis aislada en donde se cuenta con poca cantidad de tejido, (1 gramo) lo que no permite establecer todas las condiciones de estudio en cada caso
Assuntos
Hipófise/metabolismo , Hidrocortisona/análise , Técnicas In Vitro , Receptores de Droga/análise , Técnicas HistológicasRESUMO
In this work we continue the studies on the presence of an endogenous digitalis-like factor in mammals. A water extract from guinea pig heart was partially purified by liquid chromatography. The extract displaced (3H)ouabain specific binding to guinea pig heart Na,K-ATPase yielding a displacement curve parallel to that observed when digoxin was used as displacing agent. The data suggest that the activity of the extract was due to the presence of a factor that recognizes the digitalis binding site but not to ions nor lipids. Furthermore, the extract cross-reacted with antidigoxin antibodies in a manner similar to the specific antigens. These results support the hypothesis of the existence of an endogenous ligand of the digitalis receptor in mammals that could be a physiological regulator of the sodium pump.
Assuntos
Glicosídeos Digitálicos/análise , Miocárdio/análise , Receptores de Droga/análise , ATPase Trocadora de Sódio-Potássio , Animais , Cromatografia Líquida , Cobaias , Miocárdio/metabolismo , Ouabaína/metabolismo , Radioimunoensaio , Receptores de Droga/metabolismoRESUMO
Labeling of lectin receptors during the cell cycle. (Localizabión de receptores para lectinas durante el ciclo celular). Arch. Biol. Med. Exper. 10: 100-104, 1976. The topographic distribution of specific cell surface receptors for concanavalin A and wheat germ agglutinin was studied by ultrastructural labeling in the course of the cell cycle. C12TSV5 cells were synchronized by double thymidine block or mechanical selection (shakeoff). They were labeled by means of lectin-peroxidase techniques while in G1 S, G2 and M phases of the cycle. The results obtained were similar for both lectins employed. Interphase cells (G1 S, G2) present a stlihtly discontinous labeling pattern that is similar to the one observed on unsynchronized cells of the same line. Cells in mitosis, on the contrary, present a highly discontinous distribution of reaction product. This pattern disappears after the cells enters G1 and is not present on mitotic cells fixed in aldehyde prior to labeling.