Short-Term Evaluation of Left Ventricular Mass and Function in Children With Growth Hormone Deficiency After Replacement Treatment.

Background: Our study was designed to assess the effects of GHD on nutritional and metabolic parameters, brain natriuretic peptide (BNP) levels, and left ventricular mass (LVM) in prepubertal children and after short-term GH replacement therapy. Materials and Methods: This prospective study enrolled 81 children. We compared 40 GHD children (16 males and 24 females) to 41 healthy children (control group) (18 males and 23 females). All subjects were at Tanner Stage I (aged 7-11 years). At the baseline, a blood sample was drawn and echocardiographic images were obtained. These tests were repeated on the GHD subjects after 6 months of GH replacement therapy. Body surface, weight, size, blood pressure, heart rate, glucose, insulin, HOMA-IR, HOMA-ß, QUICKI, cholesterol, HDLc, LDLc, triglycerides, IGF1, and IGFBP3 were measured. Indexed LVM, diastolic and systolic diameter (dD-sD), diastolic and systolic LV function, isovolumic relaxation time, right ventricle function, and BNP levels were obtained through echocardiography. These parameters were correlated to growth factors. Data were analyzed using Student's t-test or U-Mann-Whitney-test and Pearson's correlation, considering p < 0.05 to be significant. Results: Indexed LVM was smaller in GHD patients than in controls, whereas diastolic and systolic functions, BNP, metabolic, and nutritional profiles were similar. After treatment, nutritional and metabolic profiles significantly improved, though diastolic and systolic functions did not seem to have changed. There was a significant increase in LVM. Indexed LVM was similar to that of controls. Significant correlations were obtained between LVM-IGF1 and sD-IGFBP3. Conclusions: GHD in childhood is associated with a lower indexed LVM. In the short-term, GH increases the indexed LVM, while maintaining normal systolic and diastolic functions, BNP, and an improved lipid profile.

A cellular and molecular basis for the selective desmopressin-induced ACTH release in Cushing disease patients: key role of AVPR1b receptor and potential therapeutic implications.

CONTEXT: Desmopressin is a synthetic agonist of vasopressin receptors (AVPRs). The desmopressin stimulation test is used in the diagnosis and postsurgery prognosis of Cushing disease (CD). However, the cellular and molecular mechanisms underlying the desmopressin-induced ACTH increase in patients with CD are poorly understood. OBJECTIVE: The objectives of this study were to determine, for the first time, whether desmopressin acts directly and exclusively on pituitary corticotropinoma cells to stimulate ACTH expression/release and to elucidate the cellular and molecular mechanisms involved in desmopressin-induced ACTH increase in CD. DESIGN: A total of 8 normal pituitaries (NPs), 23 corticotropinomas, 14 nonfunctioning pituitary adenomas, 17 somatotropinomas, and 3 prolactinomas were analyzed for AVPR expression by quantitative real-time RT-PCR. Primary cultures derived from corticotropinomas, nonfunctioning pituitary adenomas, somatotropinomas, prolactinomas, and NPs were treated with desmopressin, and ACTH secretion/expression, [Ca(2+)]i kinetics, and AVPR expression and/or proliferative response were evaluated. The relationship between AVPR expression and plasma adrenocorticotropin/cortisol levels obtained from desmopressin tests was assessed. RESULTS: Desmopressin affects all functional parameters evaluated in corticotropinoma cells but not in NPs or other pituitary adenomas cells. These effects might be due to the dramatic elevation of AVPR1b expression levels found in corticotropinomas. In line with this notion, the use of an AVPR1b antagonist completely blocked desmopressin stimulatory effects. Remarkably, only AVPR1b expression was positively correlated with elevated plasma adrenocorticotropin levels in corticotropinomas. CONCLUSIONS: The present results provide a cellular and molecular basis to support the desmopressin stimulation test as a reliable, specific test for the diagnosis and postsurgery prognosis of CD. Furthermore, our data indicate that AVPR1b is responsible for the direct/exclusive desmopressin stimulatory pituitary effects observed in CD, thus opening the possibility of exploring AVPR1b antagonists as potential therapeutic tools for CD treatment.

Recruiting of somatotroph cells after combined somatostatin, GHRH and growth hormone (GH) secretagogue stimulation in a study of pituitary GH reserve in prepuberal female rats.

Diagnostic confirmation of growth hormone (GH) deficiency in children and adults is based on stimulation tests designed to assess the pituitary reserve by measuring the amount of GH released into the bloodstream; however, the results obtained by this means cannot provide any direct indication of the amount of GH actually produced by pituitary somatotroph cells. The present paper sought to test the hypothesis that release of GH following administration of specific stimuli does not accurately reflect the somatotroph cell response, and that the amount of GH released into the bloodstream may often be greater or smaller than the amount synthesized. GH release and changes in the proportion of somatotroph cells were charted in prepuberal female Wistar rats, following administration of several different GH stimuli: GHRH (1 microg/kg), GHRP-6 (1 microg/kg), GHRELIN (1 microg/kg) and combined GHRH-based treatments, with or without SRIH pretreatment (1 microg/kg) 90 minutes earlier. Peak serum GH values were recorded 15 minutes after administration of GHRH+GHRELIN and GHRH+GHRP-6; maximum stimulation in terms of an increased proportion of somatotroph cells occurred 15 minutes after combined adminstration of GHRH + GHRELIN. SRIH pretreatment (- 90 min) inhibited GH release, with a subsequent "escape" and lack of response to stimulation which lasted at least 30 minutes except following administration of GHRH. However, combined administration of GHRH+GHRELIN maintained stimulation of the somatotroph cell population. In conclusion, the results suggest that the enhanced GH release prompted by stimulation tests used to diagnose GH deficiency in prepuberal female rats does not fully reflect somatroph cell dynamics, and that not all the GH produced and stored by somatotroph cells is released into the bloodstream.

Influence of chronic treatment with the growth hormone secretagogue Ipamorelin, in young female rats: somatotroph response in vitro.

Growth hormone (GH) is secreted in the anterior pituitary gland by the somatotroph cells. Secretion is regulated by growth hormone releasing hormone (GHRH) and somatostatin. Morever, GH secretagogues (GHS) can exert a considerable effect on GH secretion. In order to determine the effects of chronic treatment with the GHS Ipamorelin on the composition of the somatotroph cell population and on somatotroph GH content, an in vitro analysis was performed of the percentage of somatotroph cells (% of total), the ratio of different GH cell types (strongly/weakly-staining) and individual GH content, in pituitary cell cultures obtained from young female rats receiving Ipamorelin over 21 days (Ipamorelin group) and the effects were compared with those of GHRH (GHRH group) or saline (saline group). The ultrastructure of somatotroph cells did not change, but the volume density of secretion granules was increased (P<0.05) by previous in vivo Ipamorelin or GHRH treatment. In 3-day basal pituitary cell monolayer cultures, the percentage of somatotroph cells showed no modifications between groups, nor was there any change in the ratio of strongly/weakly immunostaining GH cells. In the Ipamorelin group alone, in vitro treatment with Ipamorelin (10(-8) M), or GHRP 6 (10(-8) M), or GHRH (10(-8) M) for 4 hours, increased the percentage of somatotroph cells, without modifying the ratio of strongly/weakly immunostained GH cells. Basal intracellular GH content in somatotroph cells over 4 hours was lower in the Ipamorelin group and the GHRH group than in the saline group. Only in the Ipamorelin group did Ipamorelin (10(-8) M), GHRP 6 (10(-8) M) and GHRH (10(-8) M) prompt increased intracellular GH content. These data suggest that, at least in the young female rat, the GHS Ipamorelin is able to exert a dynamic control effect on the somatotroph population and on GH hormone content.

Chronic in vivo Ipamorelin treatment stimulates body weight gain and growth hormone (GH) release in vitro in young female rats / El tratamiento crónico in vivo con ipamorelin estimula el aumento de peso corporal y la liberación de hormona de crecimiento (GH) in vitro en ratas hembras jóvenes

The pentapeptide Ipamorelin stimulates growth hormone (GH) release in pigs, sheep, dogs, rats and humans. In order to determine the effects of chronic treatment with Ipamorelin on the in vitro GH response, Ipamorelin was administered daily for 21 days to 60-day old female rats and the effects compared with those of growth hormone-releasing hormone (GHRH) (10 ?g/kg body weight). Daily monitoring revealed a higher % weight gain in the Ipamorelin-pretreated group (IPG) and the GHRH-pretreated group (GPG) than in the vehicle-pretreated group (VPG). In 3-day pituitary cell monolayer cultures, basal GH release was greater in IPG and GPG than in VPG. After 3-day culture, basal GH release from individual cells was lower in IPG than in VPG and GPG. The intracellular GH content was lower in IPG and GPG than in VPG (P<0.05). In 3-day monolayer cultures, Ipamorelin (10-8 M) and GHRH (10-8 M) increased GH release in VPG and IPG, but not in GPG. Similarly, Ipamorelin (10-8 M) and GHRH (10-8 M) stimulated individual GH release in VPG and IPG. In conclusion, these data show that chronic administration of Ipamorelin to young female rats is effective in increasing body weight gain and enhancing in vitro basal and Ipamorelin or GHRH-stimulated GH release. In the same conditions, GHRH increases body weight gain and maintains basal GH at levels similar to those produced by Ipamorelin, but without prompting any in vitro enhancement of the GH response to subsequent stimuli. These results suggest that Ipamorelin does not lead to desensitization of the GH response in young female rats, whilst GHRH produces desensitization of GH release in vitro (AU)

El pentapéptido ipamorelin estimula la liberación de hormona del crecimiento (GH) en cerdos, ovejas, perros, ratas y humanos. Con el fin de determinar los efectos del tratamiento crónico con ipamorelin sobre la respuesta de GH in vitro, administramos ipamorelin diariamente, durante 21 días, a ratas hembras de 60 días y los efectos se compararon con los producidos por la administración de growth hormone-releasing hormone (GHRH) (10 µg/kg peso corporal). Hubo mayor ganancia de peso (en por ciento) en el grupo pretratado con ipamorelin (IPG) y en el grupo pretratado con GHRH (GPG), que en el grupo pretratado con salino (VPG). En los cultivos monocapa de células hipofisarias de 3 días, la liberación basal de GH fue mayor en IPG y GPG que en VPG. Tras 3 días de cultivo, la liberación basal de GH de las células hipofisarias individualmente consideradas fue menor en IPG que en VPG y GPG. El contenido intracelular de GH fue menor en IPG y GPG que en VPG (P<0.05). En los cultivos monocapa de 3 días, ipamorelin (10-8 M) y GHRH (10-8 M) incrementaron la liberación de GH en VPG e IPG, pero no en GPG. Igualmente, ipamorelin (10-8 M) estimuló la liberación individual de GH en VPG e IPG. En conclusión, estos datos muestran que la administración crónica de ipamorelin a ratas hembras jóvenes es eficaz para incrementar la ganancia de peso corporal y aumentar, in vitro, la liberación de GH, basal o estimulada por ipamorelin o por GHRH. En las mismas condiciones, GHRH incrementa la ganancia de peso corporal y mantiene la liberación basal de GH en los mismos niveles que los producidos por ipamorelin, pero sin inducir in vitro ningún incremento de la respuesta de GH a estímulos posteriores. Estos resultados sugieren que ipamorelin no produce desensibilización de la respuesta de GH en ratas hembras jóvenes, mientras que GHRH determina desensibilización de la liberación de GH in vitro (AU)

Morphological changes to somatotroph cells and in vitro individual GH release, in male rats treated with recombinant human GH.

The effect of in vivo chronic administration of recombinant human growth hormone (rhGH) on morphology and individual GH release in somatotroph cells was evaluated in young male Wistar rats. Over an 18-day period, 30-day-old male rats were injected daily with 1.5 1U rhGH/kg (GPG group) or saline (VPG group) by subcutaneous injection. Electron-immunocytochemical, ultrastructural and morphometric studies of somatotroph cells were carried out. Additionally, rat pituitary cells were dispersed and overall and individual GH release was studied by radioimmunoassay and cell immunoblot assay (quantified by image analysis), respectively. The ultrastructure and size of somatotroph cells did not change, but volume density of secretion granules was reduced (p<0.01) by previous in vivo GH treatment. At four days, basal GH release of rat pituitary cell monolayer cultures was lower in the GPG group than in the VPG group (p<0.05); after 12 hours of culture, GHRH stimulation of GH release was lower in the GPG group than in the VPG group (p<0.05), and GHRH+SRIH inhibited GH release in the GPG group (p<0.05), but not in the VPG group. The percentage of somatotroph cells was not modified, but the ratio of strongly/weakly GH-immunostained cells had changed; weakly GH-immunostained cells increased from 34% to 55%. Moreover, in vitro treatment with GHRH, SRIH, and both, easily changed the strongly/weakly GH-immunostained cell ratio. Individual GH release, however, was not changed by previous in vivo GH treatment, although GHRH preferably stimulated a subpopulation of GH cells and SRIH did not inhibit individual GH release. These data suggest that exogenous chronic rhGH treatment down-regulates somatotroph function by modifying the proportion of GH cell subpopulation.

Susceptibility of HIV-1-TAT transfected cells to undergo apoptosis. Biochemical mechanisms.

The effects of HIV-1 Tat protein on mitochondria membrane permeability and apoptosis were analysed in lymphoid cells. In this report we show that stable-transfected HIV-Tat cells are primed to undergo apoptosis upon serum withdrawal. This effect was observed in both the Jhan T cell line and the K562 cells, the latter expressing the bcr-abl chimeric gene, which confers resistance to apoptosis induced by different stimuli. Using a cytofluorimetric approach we have determined that serum withdrawal induces a disruption of the transmembrane mitochondrial potential (Deltapsim) followed by an increase of reactive oxygen species (ROS) and the subsequent DNA nuclear loss in K562-Tat cells but not in the K562-pcDNA cell line. These pre-apoptotic events were associated with the cleavage of the caspase-3, while the expression of Bcl-2, Bcl-XL and Bax proteins was not affected by the presence of Tat. Regardless of the steady state of the Bax protein, we found that in both K562 and K562-Tat cells, this protein is located in the nucleus, but after serum withdrawal its localization was mainly in the cytoplasm. The activity of caspase-3 detected in K562-Tat cells after serum withdrawal paralleled with the mitochondria permeability transition. Nevertheless, in Jhan-Tat cells the inhibition of this caspase with the specific inhibitor, z-DEVD-cmk, did not affect the disruption of the mitochondria potential induced by serum withdrawal. Interestingly, we found that HIV-Tat protein accumulates at the mitochondria in the K562-Tat cells cultured under low serum conditions, and this mitochondrial localization correlated with the Deltapsim disruption detected in these cells. In addition, HIV-1 Tat protein synergies with protoporphyrin IX (PPIX), a ligand of the mitochondrial benzodiazepine receptor, in the induction of apoptosis in both Jhan and K562 cells. Thus, HIV-1 Tat protein may induce apoptosis by a mechanism that involves mitochondrial PT and may contribute to the lymphocyte depletion seen in AIDS patients.

In vitro effects of ketoconazole on corticotrope cell morphology and ACTH secretion of two pituitary adenomas removed from patients with Nelson's syndrome.

The direct effects of ketoconazole on the secretion of ACTH by human pituitary adenoma cells from 2 patient with Nelson's syndrome were studied in vitro. Stereologically quantified, intracellular changes affect the surface density of the endoplasmic reticulum (it decreased by 73%), the volume density of the secretion granules (it decreased by 49%), and the volume density of lysosomes (it decreased by 67%). The hormone released in the culture medium decreased depending on the doses of ketoconazole used; 10 mumol/l decreased ACTH levels by 31%. These data show that ketoconazole induce marked changes on corticotrope morphology and ACTH secretion in pituitary cells obtained from patients with Nelson's syndrome.

In vitro short-term effects of SMS 201-995, bromocriptine and TRH on growth hormone cell morphology from human pituitary adenomas.

This study reports, by immunocytochemistry, ultrastructure and morphometry, the in vitro effects of SMS 201-995 (10 nM), bromocriptine (1 microM) and TRH (10 microM) on the morphology of cells from two acromegalic patient adenomas containing immunoreactive growth hormone (GH). By electron microscopy, one tumor presented numerous large secretory granules (densely granulated growth hormone cell adenoma) while they were scarce and small in the other (sparsely granulated growth hormone cell adenoma); fibrous bodies could be seen in the specimen and in vitro. In the sparsely granulated growth hormone cell adenoma, TRH produced an increase in endoplasmic reticulum surface density compared to the other cultures. Bromocriptine increased the number and decreased the secretory granule diameters, while SMS 201-995 produced no significant changes in the same time. In the densely granulated growth hormone cell adenoma, the three substances increased the number of granules. TRH increased the mitochondrial volume density and endoplasmic reticulum surface density (with respect to the other cultures). SMS 201-995 decreased the mitochondrial and lysosome volume densities and endoplasmic reticulum surface density. We conclude that 1) TRH produces in cultured cells of both adenoma types an increase in cellular activity. 2) In cultured sparsely granulated growth hormone adenoma cells, bromocriptine has a stronger inhibitory effect than SMS 201-995. In cultured densely granulated growth hormone cells adenoma, bromocriptine has smaller inhibitory effect than SMS 201-995.

[Evaluation of a technic for the isolation of mammotropic cells using dopamine as a ligand]. / Valoración de una técnica de aislamiento de células mamotropas utilizando dopamina como ligando.

Pituitary cell suspension from female Wistar rats were allowed to enter the 6MB sepharose gel with added dopamine, which acts ligand attaching the mammotrophs to the gel grains. After eluting the column with 3 different dopamine buffer concentration, 3 different cellular fractions were obtained. Fraction F1 contains 5% of mammotroph cells, fraction F2 2-4% and fraction F3 90%, as determined by immunocytochemistry. The absolute amount of mammotroph cells contained in F3 fraction increases by gradually warming up the column from room temperature to 37 degrees C. The eluted F3 fraction mammotroph cells from female lactating rats is observed to be twice the amount eluted in experiments performed with virgin rats. No ultrastructural differences between fraction F1 and fraction F3 mammotroph cells have been found.