Genetic analysis and cAMP measurement: comparison between lean and obese anovulating mice / Análise genética e dosagem de AMP cíclico: comparação entre camundongos fêmeas anovuladoras magras e obesas

PURPOSE: To evaluate genes differentially expressed in ovaries from lean (wild type) and obese (ob/ob) female mice and cyclic AMP production in both groups. METHODS: The expression on messenger RNA levels of 84 genes concerning obesity was analyzed through the PCR array, and cyclic AMP was quantified by the enzyme immunoassay method. RESULTS: The most downregulated genes in the Obesity Group included adenylate cyclase-activating polypeptide type 1, somatostatin, apolipoprotein A4, pancreatic colipase, and interleukin-1 beta. The mean decrease in expression levels of these genes was around 96, 40, 9, 4.2 and 3.6-fold, respectively. On the other hand, the most upregulated genes in the Obesity Group were receptor (calcitonin) activity-modifying protein 3, peroxisome proliferator activated receptor alpha, calcitonin receptor, and corticotropin-releasing hormone receptor 1. The increase means in the expression levels of such genes were 2.3, 2.7, 4.8 and 6.3-fold, respectively. The ovarian cyclic AMP production was significantly higher in ob/ob female mice (2,229±52 fMol) compared to the Control Group (1,814±45 fMol). CONCLUSIONS: Obese and anovulatory female mice have reduced reproductive hormone levels and altered ovogenesis. Several genes have their expression levels altered when leptin is absent, especially adenylate cyclase-activating polypeptide type 1. .

OBJETIVO: Avaliar os genes diferencialmente expressos em ovários de camundongos fêmeas magras (tipo selvagem) e obesas (ob/ob) e a produção de AMP cíclico em ambos os grupos. MÉTODOS: A expressão nos níveis de RNA mensageiro de 84 genes relacionados à obesidade foi analisada por PCR Array, e o AMP cíclico foi quantificado por método imunoenzimático. RESULTADOS: Os genes que mais sofreram diminuição da expressão no Grupo Obesidade incluíram o tipo 1 de polipeptídeo ativador da adenilato ciclase, o da somatostatina, da apolipoproteína A4, da colipase pancreática e da beta interleucina 1. A média de redução na expressão desses genes foi de aproximadamente 96, 40, 9, 4,2 e 3,6 vezes, respectivamente. Por outro lado, os genes que mais tiveram aumento na expressão no Grupo Obesidade foram o gene da proteína modificadora da atividade do receptor de calcitonina 3, do proliferador de peroxissomos ativados por proteína alfa, do receptor de calcitonina e do receptor para hormônio liberador de corticotropinas 1. As médias de acréscimo nos níveis de expressão de tais genes foram de 2,3, 2,7, 4,8 e 6,3 vezes, respectivamente. A produção de AMP cíclico ovariana foi significantemente aumentada em camundongos fêmeas ob/ob (2.229±52 fMol) quando comparada ao Grupo Controle (1.814±45 fMol). CONCLUSÕES: Camundongos fêmeas obesas e anovuladoras possuem níveis de hormônio reprodutivo reduzidos e ovulogênese alterada. Vários genes mostram níveis de expressão alterados quando a leptina está ausente, principalmente o tipo 1 de polipeptídeo ativador da adenilato ciclase. .

An inhibitory compound against the interaction between Galpha(s) and the third intracellular loop region of serotonin receptor subtype 6 (5-HT(6)) disrupts the signaling pathway of 5-HT(6)

Serotonin receptor subtype 6 (5-HT(6)) is a neurotransmitter receptor, which is involved in various brain functions such as memory and mood. It mediates signaling via the interaction with a stimulatory G-protein. Especially, the third intracellular loop (iL3) of 5-HT(6) and the alpha subunit of stimulatory G protein (Galpha(s)) are responsible for the signaling process of 5-HT(6). Chemical compounds that could inhibit the interaction between the iL3 region of 5-HT(6) and Galpha(s) were screened from a chemical library consisted of 5,600 synthetic compounds. One of the identified compounds bound to Galpha(s) and effectively blocked the interaction between Galpha(s) and the iL3 region of 5-HT(6). The identified compound was further shown to reduce the serotonin-induced accumulation of cAMP in 293T cells transformed with 5-HT(6) cDNA. It also lowered the Ca2+ efflux induced by serotonin in cells expressing 5-HT(6) and chimeric Galpha(s5/q). These results indicate that the interaction between the iL3 of 5-HT(6) and Galpha(s) can be exploited for screening of regulatory compounds against the signaling pathway of 5-HT(6).

The Effect of alpha MSH Analogues on Rat Bones

Melanocortin is the downstream mediator of leptin signaling and absence of leptin signaling in ob/ob and db/db mice revealed the enhancement of bone formation through the central regulation. While alpha-melanocyte-stimulating hormone (alpha MSH) inhibits the secretion of interleukin-1 alpha and tumor necrosis factor-alpha from the inflammatory cells, alpha MSH can also enhance clonal expansion of pro B cells linked to stimulation of osteoclastogenesis. Therefore, we tested the effect of melanocortin on bones. alpha MSH analogues [6His] alpha MSH-ND and [6Asn] alpha MSH-ND were synthesized and the radio-ligand receptor binding- and cyclic AMP generating activity were analyzed in China Hamster Ovary cell line over- expressing melanocortin receptors. The EC50 of [6His] alpha MSH-ND measured from melanocortin-1, 3, 4 and 5 receptors were 0.008 0.0045, 1.523 0.707, 0.780 0.405, and 250.320 42.234 nM, respectively, and the EC50 of [6Asn] alpha MSH-ND were 16.8 6.94, 271.8 21.95, 8.0 1.21, and 1132.5 635.46 nM, respectively. Four weeks after the subcutaneous injection of the analogues, the body weights in the [6His] alpha MSH-ND and the [6Asn] alpha MSH-ND treated groups (346.0 20.63 g vs. 350.0 13.57 g) were lower than that of the vehicle treated group (375.8 17.31 g, p 0.05). There was no difference in the total femoral BMD measured by dual x-ray absorptiometry among the three groups. Among the three groups, there were no differences in the total numbers of crystal violet positive- or alkaline phosphatase positive colonies, in the expression of Receptor Activator of Nuclear Factor Kappa-B ligand on the tibia and the total number of multinucleated osteoclast-like cells differentiated from primary cultured bone marrow cells. From the above results, no evidence of bone gain or loss was found after treatment of the alpha MSH analogues peripherally.

Altered Renal Expression of Aquaporin-2 Water Channels in Rats with Experimental Two-Kidney, One Clip Hypertension

The present study was aimed at examining the regulation of aquaporin (AQP)-2 water channels in the kidney in two-kidney, one clip (2K1C) hypertension. Rats were made 2K1C hypertensive for 6 weeks, and their expression of AQP2 channel proteins was determined in the clipped and contralateral kidneys. To examine the upstream affecting AQP2 channels, adenylyl cyclase activity was also determined. Along with the hypertension, in the clipped kidney, the abundance of AQP2 proteins was significantly decreased in the cortex, outer and inner medulla, while their trafficking remained unaltered. Concomitantly with the reversal of the blood pressure at 24 hours following removal of the clip, the AQP2 abundance also returned to the control level. The arginine vasopressin-evoked generation of cAMP was decreased in the clipped kidney, which again was reversed to the control level following removal of the clip. In contrast, the expression of AQP2 channels as well as the activity of adenylyl cyclase remained unaltered in the contralateral kidney. These results indicate an altered regulation of AQP2 water channels in the clipped kidney in 2K1C hypertension.

Correlación entra la proliferación celular y la expresión de receptores beta adrenérgicos en linfocitos T activados / Relation between cell proliferation and beta adrenergic receptors expression in activated T lymphocytes

Se caracterizó la presencia de receptores (R)ß adrenérgicos em linfocitos murinos normales estimulados com Concanavalina A (Con A) y en una línea celular hiperproliferativa (BW5147) mediante la unión específica del radioligando 125Iodo-cianopindolol (125I/CYP)) a las células. La funcionalidad de los R se midió a través de cambios en la produccón de AMPc en presencia del agonista ß adrenérgico isoproterenol (ISO). Ambos tipos celulares mostraron un número disminuido de R ß adrenérgicos por unión específica al 125I-CYP. Se midieron los niveles intracelulares de AMPc en presencia de un agonista ß adrenérgico, las células BW5147 no mostraron un incremento significativo en los niveles de AMPc mientras que las células con Con A mostraron un incremento aunque menor al que presentan linfocitos normales, sin embargo, ambos tipos celulares respondieron a la prostaglandina E1(PGE1) produciendo un incremento en la produción de AMPc. Podemos concluir que la línea celular hiperproliferativa BW5147 no posee R funcionales, en este trabajo se analiza la posible implicancia de un camino intracelular alternativo y su control neuroendocrino

Antibodies against beta adrenoceptors in mothers of children with congenital heart block

En este trabajo se empleó un modelo experimental "in vitro" en el cual se midió la frecuencia de las contracciones, la producción de AMPc y la la unión de ligandos específicos al receptor ß adrenérgicos en las aurículas de ratas neonatales y adultas; a fin de, determinar si anticuerpos con actividad ß adrenérgica interactuaban con receptores ß adrenérgicos cardíacos y alteraban su compartimiento fisiológico. Los sueros y la fracción IgG de madres de infantes con bloqueo cardíaco neonatal congénito incrementaron la frecuencia de las contracciones y la producciín de AMPc en el miocardio auricular neonato. Este efecto fue inhibido por propranolol, puntalizando una participación ß adrenérgica. La IgG también compitió con el radiologando específico por los receptores ß adrenérgicos (3H-CGP) en las membranas purificadas de miocardio neonatal. Ni los efectos biológicos ni los ensayos de unión específica fueron modificados cuando se usó miocardio de rata adulta. La reactividad contra adrenoreceptores cardíacos por parte de anticuerpos provenientes de madres de niños con bloqueo neonatal congénito, podría ser otro factor sérico que debería considerarse en la patofisiología del desarrollo del bloqueo neonatal congénito

Nuevas perspectivas en el metabolismo del calcio / New perspectives in calcium metabolism

El calcio, en cuantidades pequeñas es fundamental para la actividad celular normal, pero en exceso, daña las celulas. Paradójicamente, una ingesta alta en calcio es beneficiosa en enfermedades dispares como la hipertensión, nefrolitiasis y en la prevención del cáncer de colon. Como la hormona paratiroidea (PTH) estimula el pasaje de calcio a las células y la disminución de su secreción, una dieta rica en calcio resultaría en un flujo menor de calcio a través de la membrana celular en tejidos genéticamente predispuestos. Así la ablación de las paratiroides disminuye el calcio muscular y causa mejoría histológica en la distrofia muscular, una enfermedad con calcio muscular elevado. Observaciones antropológicas sugieren que la ingesta de calcio debería ser - 1600 mg/d, mucho más alta que nuestra dieta actual. Así, el aumento de la ingesta de calcio, al disminuir la secreción de PTH, sería beneficiosa en la prevención y el tratamiento de enfermedades diversas

interrelationships between dieldrin, intestinal active transport and mucosal cyclic AMP concentrations

The effect of dieldrin on the intestinal transport of actively transported non-electrolytes and intestinal mucosal cyclic AMP concentrations was evaluated in the isolated rat jejunum and ileum. Certain dose and concentration levels of dieldrin were found to be capable for enhancing the intestinal active transport of D-glucose and L-leucine. This enhancement in the active transport of non-electrolytes by dieldrin was paralled by an increase in the mucosal cyclic AMP concentrations in both preparations. There was a positive correlation between mucosal cyclic AMP concentrations and the active transport of D-glucose and L-leucine in the isolated jejunum and ileum of rat as indication by investigating the effect of dieldrin in combiution with either forskolin or 3-isobutyl-l-methy-Lxanthine [IBMX]. on these parameters. It is suggested that cyclic AMP may act as a mediator for the enhancement effect of dieldrin on the intestinal active transport of non-electrolytes

Ultraestructura de las células adenohipofisiarias de la rata blanca sometida al efecto de una benzodiazepina en forma crónica / Ultrastructure of adenohypophyseal cells of white rats subjected to the effect of a benzodiazepine in chronic form

Se estudia la estructura de las células adenohipofisarias de la rata blanca (Rattus norvegicus) sometida al efecto de una benzodiazepina, aplicada crónicamente. Se observa que en relación con los controles, las células manotropas, somatotropas, tirotropas, gonadotropas, corticotropas, no muestran alteraciones ultra-estructurales. Se concluye que en la rata, el tratamiento crónico con diazepam, no produce alteraciones ultraestructurales visibles en las células secretoras de las hormonas adenohipofisiarias, debido probablemente al efecto de corta duración que tienen los metabolitos del diazepam en esta especie

Funçäo tireoidian e produçäo de AMP cíclico em nódulos autônomos da tiróide / Thyroid function and cyclic AMP production in autonomous thyroid nodules

Foram estudadas cinco pacientes eutiroidianas portadores de nódulos autônomos à cintilografia tireiodiana, com diàmetro superio a 3cm e näo supressíveis pela administraçäo de T, (100µg/dia durante 10 dias). Quatro das pacientes apresentaram exames laboratoriais (T4 livre, T3 TSH basal e captaçäo de 131I em 24h) normais. Fatias de tecido tireoidiano adenomatoso e paranodular obtidas pela tiroidectomia foram incubadas com concentraçöes crescentes de TSH e na presença ou näo de iodeto de potássio (KI)ou triiodotironina (T3). O incremento da produçäo de AMP cíclico (cAMP) provocado pelo TSH no tecido adenomatoso foi igual ao encontrado do tecido adjacente paranodular. No tecido paranodular a adiçäo de T3 ou KI inibiu significativamente a produçäo de cAMP, enquanto que, no tecido adenomatoso, a açäo inibitória ocorreu apenas com KI. Estes resultados sugerem: 1) a inibiçäo da secreçäo pode ocorrer na presença de níveis hormonais tiroidianos circulantes normais; 2) nâo há evidência de sensibilidade aumentada ao TSH no tecido autônomo quando avaliada pela produçäo de cAMP; 3) a ausência inibitória do T, na resposta do TSH no tecido nodular sugere alteraçäo celular ao nível de outros reguladores da geraçäo de cAMP.