Endocrine control of DNA synthesis in renal, adrenal and vascular tissues of the rat in vivo

The aims of this study were firstly to investigate the influence of the renin-angiotensin system on growth of various target tissues (kidney, adrenal gland and blood vessels). Secondly, to determine whether changes in growth were a direct consequence of trophic actions of angiotensin II (AII) or secondary to changes in blood pressure. Thirdly to investigate the interactions of the renin-angiotensin system with other factors (nitric oxide synthesis, glucocorticoid hormone) in the control of growth of target tissues. In each group of rats bromodeoxyuridine (BrdUrd), a thymidine analogue, was given by subcutaneous infusion continuously for two weeks during various treatments. The animals were killed and DNA synthesis was assessed histologically by calculating a BrdUrd index for each cell type. Kidney sections were immunocytochemically stained for renin content. Various morphometric measurements were made in adrenal and vascular tissues. The renin-angiotensin system was manipulated in three ways: i) by infusing AII subcutaneously, ii) by feeding a low or supplemented sodium diet, iii) by administering captopril in the drinking water. The role of nitric oxide in the regulation of DNA synthesis was investigated by treating rats for up to four weeks with an inhibitor of arginine synthase, L-NAME. The effect of glucocorticoid hormone was tested by implanting a dexamethasone pellet subcutaneously. Blood Vessels: In mesenteric blood vessels, AII infusion increased blood pressure and stimulated DNA synthesis in the endothelium, media and adventitia of arteries. Compared with rats fed a sodium supplemented diet, rats given low sodium food had higher BrdUrd indices in all parts of the blood vessels. Captopril lowered the BrdUrd index in the media and adventitia but had no effect on the endothelium. Neither L-NAME nor dexamethasone significantly affected DNA synthesis in vascular smooth muscle cells. DNA synthesis in mesenteric veins was not affected by any treatment. Medial area in transverse sections of arteries was greater after AII treatment. None of the other treatments caused vascular hypertrophy. Adrenal Gland: The BrdUrd indices of epithelial and non-epithelial cells were assessed separately in six areas of the adrenal gland: the zona glomerulosa, the zona intermedia, the outer and inner fasciculata, the zona reticularis and the adrenal medulla. AII and low dietary sodium increased the BrdUrd index significantly in the zona glomerulosa and in the zona reticularis but had no effect on the zona fasciculata. Treatment with captopril caused zona glomerulosa atrophy but had no effect on the BrdUrd index. Captopril reduced DNA synthesis in the reticularis. There was hypertrophy of the zona glomerulosa after treatment with either AII or low sodium whereas, high sodium, L-NAME, dexamethasone and captopril caused atrophy. Changes in the BrdUrd index in the medulla appeared to be controlled in a compensatory manner by blood pressure. Treatments which tend to increase blood pressure (AII, dexamethasone, L-NAME) reduced the BrdUrd index whereas captopril caused an increase. Kidney: The number of renin secreting cells in the kidney was increased by low sodium and also by captopril. Despite the increase in the number of cells, there was no evidence of DNA synthesis in any renin-secreting cell. BrdUrd indices were calculated for glomerular, tubular and interstitial cells. AII increased the BrdUrd index in glomerular cells, captopril caused an decrease but low and high dietary sodium, L-NAME and dexamethasone had no effect. Dexamethasone increased the BrdUrd index in tubule cells and L-NAME increased DNA synthesis in interstitial cells. AII, captopril or dietary sodium manipulation had no effect on DNA synthesis in tubules or interstitial cells. Conclusions: The control of growth of the cardiovascular system is complex, involving direct actions of the renin-angiotensin system. Angiotensin II increases DNA synthesis of many cell types in vivo. Angiotensin II directly regulates DNA synthesis in the adrenal cortex and in blood vessels. Some of the effects of AII are compensatory. Many of the effects of AII on growth are influenced by blood pressure and by interactions with other factors such as nitric oxide and glucocorticoid hormones

Duplicación, organización genómica y regulación transcripcional de los receptores de la hormona del crecimiento de peces. Aspectos básicos y aplicados en dorada Sparus aurata.

Los primeros receptores de hormona de crecimiento (GHR) descritos en peces se agruparon en dos grupos, las secuencias descritas en peces salmónidos y las descritas en peces no salmónidos. Las de peces no salmónidos presentaron una gran conservación estructural con las secuencias de mamíferos, pero las secuencias de peces salmónidos muestraron un bajo nivel de identidad aminoacídica con ausencia de un par de cisteínas extracelulares y un diferente patrón de tirosinas intracelulares. Llegados a este punto, se planteó la hipótesis de la existencia de dos formas alternativas para el gen GHR en el genoma de los peces: la forma de los peces no salmónidos (GHR-I) y la de los peces salmónidos (GHR-II). A la luz de esta hipótesis se plantearon los siguientes objetivos: 1) Abordar la caracterización molecular y organización genómica de los receptores de la hormona del crecimiento en peces teleósteos, 2) analizar la divergencia evolutiva de los receptores de la hormona del crecimiento en los peces teleósteos, 3) analizar en dorada el patrón de regulación transcripcional de los receptores de la hormona del crecimiento y 4) definir en dorada el uso de los receptores de la hormona del crecimiento como biomarcadores de crecimiento y estrés por confinamiento y exposición a patógeno. Para analizar la divergencia evolutiva, se diseñaron cebadores degenerados en base a las secuencias de los GHR descritos previamente en los peces salmónidos. Utilizando estos cebadores en una estrategia de RT-PCR, se determinó la existencia del GHR-II en dorada, trucha y lubina. Las nuevas secuencias de GHR-II presentaron las mismas características estructurales que las secuencias de salmónidos y se agruparon con ellas al realizar un análisis filogenético. De esta manera se definió lo que sería en grupo de secuencias de GHR-II de peces mientras que el resto de secuencias de peces corresponderían al GHR-I. La caracterización molecular de ambos GHR en dorada mostró la misma organización genómica que el GHR de mamíferos, con la salvedad de la falta de un exón homólogo al 3 de mamíferos y la presencia de un intrón adicional en el exón 10 del GHR-I. Además se determinó que ambos receptores presentaron un único sitio de inicio de la transcripción en contraposición a la situación de mamíferos: un único receptor con diferentes inicios de la transcripción. Esta diferente organización sugirió que el gen del GHR se ajusta al modelo de evolución por subfuncionalización. Para analizar su regulación transcripcional, se midió la expresión de ambos GHR junto con la de IGF-I e IGF-II bajo diferentes modelos experimentales en dorada. Los modelos estudiados fueron ayuno, estación, edad, infección por patógeno (Enteromyxum leei) y estrés por confinamiento. De estas experiencias se dedujo que la mayor de expresión de ambos GHR se dio en hígado y que los cambios en la concentración de IGF-I circulante reflejaron su expresión a nivel hepático. La expresión del GHR-I estuvo altamente correlacionada con la expresión de IGF-I e IGF-II en las experiencias de estación, edad e infección por patógeno tanto a nivel hepático como extrahepático. Sin embargo, en el modelo de estrés por confinamiento, la disminución de la expresión hepática de la IGF-I e IGF-II reflejó los cambios en la expresión del GHR-II. Esto estuvo en consonancia con los sitios de unión de factores de transcripción presentes en su región flanqueante. El modelo propuesto mostró una regulación diferencial de los GHR de dorada, aunque no se pudo excluir un cierto solapamiento funcional con independencia de que fueran promiscuos o específicos de la GH.The first fish growth hormone receptor (GHR) sequences were grouped in two major groups: salmonid fish and non-salmonid fish. Sequences of non-salmonid fish showed a high structural conservation with those of mammals. On the other hand, sequences of salmonid fish showed a low level of aminoacid identity with the lack of two extracellular cysteines and a different intracellular tyrosine pattern. At that moment, the existence of two alternative copies of the GHR gene in fish genomes, GHR-I (non-salmonid form) and GHR-II (salmonid form), was hypothesised. Following that hypothesis, we formulated the following objectives: 1) Address the molecular characterization and genomic organization of GHRs in teleost fish, 2) analyse their evolutional divergence, 3) analyse their transcriptional regulation in gilthead sea bream and 4) define their use in gilthead sea bream as biomarkers of growth and stress. The presence of GHR-II was demonstrated for the first time in gilthead sea bream, European sea bass and rainbow trout. In addition, the genomic organization of both GHR was elucidated in gilthead sea bream. Fish GHR showed the same genomic organization than human GHR except for the lack of the exon 3 and the presence of a short intron within GHR-I exon 10. Moreover, fish GHRs only contained a transcription start site whereas tetrapods’ GHR has several sites. The transcriptional regulation of gilthead sea bream GHR-I, GHR-II, IGF-I and IGF-II was assessed in basis to fasting, season, age, stress and parasite challenge (Enteromixum leei). In basis to our results, the expression of GHR-I was higher in hepatic tissue. Moreover, changes in circulating IGF-I levels reflected the IGF-I hepatic expression. GHR-I expression was highly correlated with IGF expression in season, age and parasite challenge at hepatic and extrahepatic level. However, changes in IGF expression under stress were correlated to hepatic GHR-II expression. This fact agreed with the transcription factor binding sites found in the 5’flanquing region of GHR-II. In summary, coexpression analyses suggest a key role of GHR-I in the tissue-specific regulation of IGFs in gilthead sea bream. Some functional redundancy of GHR-I and GHR-II cannot be excluded, emerging GHR-II as a stress and redox-sensitive gene