RESUMO
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.
RESUMO
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.
Assuntos
Apoptose/fisiologia , Genes tat , HIV-1/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/virologia , Leucemia de Células T/patologia , Leucemia de Células T/virologia , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro , Inibidores Enzimáticos/farmacologia , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia de Células T/tratamento farmacológico , Potenciais da Membrana , Mitocôndrias/fisiologia , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Protoporfirinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transfecção , Células Tumorais Cultivadas/virologia , Proteína X Associada a bcl-2 , Proteína bcl-X , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
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.
Assuntos
Hormônio do Crescimento Humano/metabolismo , Adeno-Hipófise/metabolismo , Animais , Células Cultivadas , Hormônio do Crescimento Humano/administração & dosagem , Humanos , Immunoblotting/métodos , Masculino , Microscopia Eletrônica , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos Wistar , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Hormônio do Crescimento/metabolismo , Oligopeptídeos/farmacologia , Animais , Peso Corporal , Divisão Celular , Feminino , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Processamento de Imagem Assistida por Computador , Immunoblotting , Imuno-Histoquímica , Ratos , Ratos WistarRESUMO
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.
Assuntos
Adenoma/ultraestrutura , Bromocriptina/farmacologia , Hormônio do Crescimento/biossíntese , Octreotida/farmacologia , Neoplasias Hipofisárias/ultraestrutura , Hormônio Liberador de Tireotropina/farmacologia , Adenoma/metabolismo , Adulto , Feminino , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Modelos Biológicos , Neoplasias Hipofisárias/metabolismo , Células Tumorais CultivadasAssuntos
Síndrome de Cushing/diagnóstico por imagem , Fluordesoxiglucose F18 , Neoplasias da Coluna Vertebral/diagnóstico por imagem , Teratoma/diagnóstico por imagem , Síndrome de ACTH Ectópico/complicações , Síndrome de ACTH Ectópico/diagnóstico por imagem , Hormônio Adrenocorticotrópico/fisiologia , Adulto , Síndrome de Cushing/etiologia , Feminino , Humanos , Tomografia por Emissão de Pósitrons , Região Sacrococcígea/diagnóstico por imagem , Região Sacrococcígea/patologia , Neoplasias da Coluna Vertebral/complicações , Neoplasias da Coluna Vertebral/metabolismo , Teratoma/complicações , Teratoma/metabolismoRESUMO
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.
Assuntos
Hormônio Adrenocorticotrópico/sangue , Desamino Arginina Vasopressina , Hipersecreção Hipofisária de ACTH/diagnóstico , Hipersecreção Hipofisária de ACTH/metabolismo , Receptores de Vasopressinas/metabolismo , Adenoma/sangue , Adenoma/diagnóstico , Adenoma/cirurgia , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Hipersecreção Hipofisária de ACTH/cirurgia , Testes de Função Hipofisária , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Neoplasias Hipofisárias/sangue , Neoplasias Hipofisárias/diagnóstico , Neoplasias Hipofisárias/cirurgia , PrognósticoRESUMO
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.
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Hormônio do Crescimento/metabolismo , Hipófise/metabolismo , Somatostatina/metabolismo , Animais , Feminino , Expressão Gênica , Imuno-Histoquímica/métodos , Hipófise/patologia , Radioimunoensaio/métodos , Ratos , Ratos WistarRESUMO
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.
Assuntos
Separação Celular/métodos , Dopamina , Hipófise/citologia , Animais , Cromatografia de Afinidade , Feminino , Ligantes , Ratos , Ratos EndogâmicosRESUMO
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.