RESUMO
Over the last two decades, the understanding of the mechanisms involved in pituitary ontogenesis has largely increased. Since the first description of POU1F1 human mutations responsible for a well-defined phenotype without extra-pituitary malformation, several other genetic defects of transcription factors have been reported with variable degrees of phenotype-genotype correlations. However, to date, despite the identification of an increased number of genetic causes of isolated or multiple pituitary deficiencies, the etiology of most (80-90 %) congenital cases of hypopituitarism remains unsolved. Identifying new etiologies is of importance as a post-natal diagnosis to better diagnose and treat the patients (delayed pituitary deficiencies, differential diagnosis of a pituitary mass on MRI, etc.), and as a prenatal diagnosis to decrease the risk of early death (undiagnosed corticotroph deficiency for instance). The aim of this review is to summarize the main etiologies and phenotypes of combined pituitary hormone deficiencies, associated or not with extra-pituitary anomalies, and to suggest how the identification of such etiologies could be improved in the near future.
Assuntos
Hipopituitarismo/diagnóstico , Hipopituitarismo/etiologia , Animais , Previsões , Humanos , Hipopituitarismo/genética , Mutação/genética , Fenótipo , Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Hormônios Hipofisários/genética , Hormônios Hipofisários/metabolismoRESUMO
Testicular germ cell cancers are the most common solid malignancies in young men, but their pathogenesis remains undetermined although some epidemiological data have implicated both environmental and genetic factors. Glial cell-derived neurotrophic factor (GDNF) is secreted by Sertoli cells, and promotes germ stem cell proliferation by activating RET, a tyrosine kinase receptor. Over-expression of GDNF in adult transgenic mice induces the development of testicular tumours that mimic human seminoma, the most frequent testicular germ cell tumour. Activating mutations of RET were previously reported in several types of cancer, including thyroid, pituitary, adrenal and melanoma cancer. Both mouse experimental model and clinical studies suggested that mutations or selective polymorphisms of RET might be associated with human seminoma. To verify this hypothesis, we conducted this study in a French University Hospital and carried out an association study using tissue samples from 66 paraffin-embedded seminoma tumours. The most frequently mutated exons of the RET proto-oncogene were sequenced to identify mutations or selective polymorphisms. No somatic mutations were identified. The polymorphic variants frequencies did not differ from those in a control Caucasian population. Human classical seminoma that occurs in young men does not appear to be linked with mutations or relevant polymorphisms of RET.
Assuntos
Neoplasias Embrionárias de Células Germinativas/genética , Proteínas Proto-Oncogênicas c-ret/genética , Seminoma/genética , Animais , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Masculino , Camundongos , Proto-Oncogene Mas , Neoplasias Testiculares/genéticaRESUMO
CONTEXT: LHX4 is a LIM homeodomain transcription factor involved in pituitary ontogenesis. Only a few heterozygous LHX4 mutations have been reported to be responsible for congenital pituitary hormone deficiency. SUBJECTS AND METHODS: A total of 136 patients with congenital hypopituitarism associated with malformations of brain structures, pituitary stalk, or posterior pituitary gland was screened for LHX4 mutations. RESULTS: Three novel allelic variants that cause predicted changes in the protein sequence of LHX4 (2.3%) were found (p.Thr99fs, p.Thr90Met, and p.Gly370Ser). On the basis of functional studies, p.Thr99fs mutation was responsible for the patients' phenotype, whereas p.Thr90Met and p.Gly370Ser were likely polymorphisms. Patients bearing the heterozygous p.Thr99fs mutation had variable phenotypes: two brothers presented somato-lactotroph and thyrotroph deficiencies, with pituitary hypoplasia and poorly developed sella turcica; the youngest brother (propositus) also had corpus callosum hypoplasia and ectopic neurohypophysis; their father only had somatotroph deficiency and delayed puberty with pituitary hyperplasia. Functional studies showed that the mutation induced a complete loss of transcriptional activity on POU1F1 promoter and a lack of DNA binding. Cotransfection of p.Thr99fs mutant and wild-type LHX4 failed to evidence any dominant negative effect, suggesting a mechanism of haploinsufficiency. We also identified prolactin and GH promoters as potential target genes of LHX4 and found that the p.Thr99fs mutant was also unable to transactivate these promoters. CONCLUSIONS: The present report describes three new exonic LHX4 allelic variants with at least one being responsible for congenital hypopituitarism. It also extends the phenotypical heterogeneity associated with LHX4 mutations, which includes variable anterior pituitary hormone deficits, as well as pituitary and extrapituitary abnormalities.
Assuntos
Proteínas de Homeodomínio/genética , Hipopituitarismo/genética , Mutação , Fatores de Transcrição/genética , Adulto , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Genótipo , Humanos , Hipopituitarismo/congênito , Íntrons , Proteínas com Homeodomínio LIM , Masculino , Pessoa de Meia-Idade , Linhagem , FenótipoRESUMO
The aim of this paper is to report an atypical presentation of MEN2A, in a patient carrying the C634R mutation of the RET-protooncogene. A 41-year-old Tunisian woman was admitted to our department with newly diagnosed hyperglycemia. She had a history of bilateral urinary stone recurrence, managed successfully on two occasions. On physical examination a thyroid node of 1cm on the left side was found. Laboratory evaluation and imaging findings confirmed the diagnosis of primary hyperparathyroidism. During cervicotomy, the parathyroid adenoma was resected and the thyroid node was suspected to be a carcinoma. Total thyroidectomy, with appropriate neck nodal resection, was performed. Histological examination confirmed the diagnosis of parathyroid adenoma and revealed a multifocal and bilateral medullary carcinoma. These findings led to the diagnosis of multiple endocrine neoplasia. DNA-analysis demonstrated a germline Cys634Arg mutation in the RET-protooncogene. During the postoperative follow-up, blood pressure as well as the level of urinary methoxylated metabolites increased progressively. Imaging findings were compatible with the diagnosis of bilateral pheochromocytoma. In conclusion, this case report of MEN 2A linked to a 634 RET mutation was peculiar by its revelation mode (1) hyperparathyroidism moreover linked to an adenoma and (2) associated with diabetes, mechanisms of which are probably multifactorial (familial type 2 diabetes, hypercalcemia, catecholamines excess).
Assuntos
Adenoma/diagnóstico , Adenoma/genética , Carcinoma Medular/diagnóstico , Carcinoma Medular/genética , Neoplasia Endócrina Múltipla Tipo 2a/diagnóstico , Neoplasia Endócrina Múltipla Tipo 2a/genética , Feocromocitoma/diagnóstico , Feocromocitoma/genética , Proteínas Proto-Oncogênicas c-ret/genética , Adenoma/cirurgia , Adulto , Carcinoma Medular/cirurgia , DNA/genética , Diabetes Mellitus/etiologia , Feminino , Humanos , Hiperglicemia/diagnóstico , Hiperglicemia/etiologia , Neoplasia Endócrina Múltipla Tipo 2a/cirurgia , Esvaziamento Cervical , Neoplasias das Paratireoides/diagnóstico , Neoplasias das Paratireoides/patologia , Feocromocitoma/cirurgia , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/patologia , TireoidectomiaRESUMO
DEFINITION: Congenital hypopituitarism is characterized by multiple pituitary hormone deficiency, including somatotroph, thyrotroph, lactotroph, corticotroph or gonadotroph deficiencies, due to mutations of pituitary transcription factors involved in pituitary ontogenesis. INCIDENCE: Congenital hypopituitarism is rare compared with the high incidence of hypopituitarism induced by pituitary adenomas, transsphenoidal surgery or radiotherapy. The incidence of congenital hypopituitarism is estimated to be between 1:3000 and 1:4000 births. CLINICAL SIGNS: Clinical presentation is variable, depending on the type and severity of deficiencies and on the age at diagnosis. If untreated, main symptoms include short stature, cognitive alterations or delayed puberty. DIAGNOSIS: A diagnosis of combined pituitary hormone deficiency (CPHD) must be suspected when evident causes of hypopituitarism (sellar tumor, postsurgical or radioinduced hypopituitarism...) have been ruled out. Clinical, biological and radiological work-up is very important to better determine which transcription factor should be screened. Confirmation is provided by direct sequencing of the transcription factor genes. AETIOLOGY: Congenital hypopituitarism is due to mutations of several genes encoding pituitary transcription factors. Phenotype varies with the factor involved: PROP1 (somatolactotroph, thyrotroph, gonadotroph and sometimes corticotroph deficiencies; pituitary hyper and hypoplasia), POU1F1 (somatolactotroph and thyrotroph deficiencies, pituitary hypoplasia), HESX1 (variable pituitary deficiencies, septo-optic dysplasia), and less frequently LHX3 (somatolactotroph, thyrotroph and gonadotroph deficiencies, limited head and neck rotation) and LHX4 (variable pituitary deficiencies, ectopic neurohypophysis, cerebral abnormalities). MANAGEMENT: An appropriate replacement of hormone deficiencies is required. Strict follow-up is necessary because patients develop new deficiencies (for example late onset corticotroph deficiency in patients with PROP1 mutations). GENETIC COUNSELLING: Type of transmission varies with the factor and the mutation involved (recessive transmission for PROP1 and LHX3, dominant for LHX4, autosomal or recessive for POU1F1 and HESX1). PROGNOSIS: It is equivalent to patients without pituitary deficiencies if treatment is started immediately when diagnosis is confirmed, and if a specialized follow-up is performed.
Assuntos
Hipopituitarismo/genética , Hormônios Hipofisários/deficiência , Diagnóstico Diferencial , Proteínas de Homeodomínio/genética , Terapia de Reposição Hormonal , Humanos , Hipopituitarismo/congênito , Hipopituitarismo/diagnóstico , Hipopituitarismo/terapia , Hormônios Hipofisários/uso terapêutico , Fator de Transcrição Pit-1/genéticaRESUMO
In pituitary cells, activation of the cAMP pathway by specific G protein-coupled receptors controls differentiative functions and proliferation. Constitutively active forms of the alpha subunit of the heterotrimeric G(s) protein resulting from mutations at codon 201 or 227 (gsp oncogene) were first identified in 30-40% of human GH-secreting pituitary adenomas. This rate of occurrence suggests that the gsp oncogene is not responsible for initiating the majority of these tumors. Moreover, there is a large overlap between the clinical phenotypes observed in patients with tumors bearing the gsp oncogene and those devoid of this oncogene. To explore the role of G(s)alpha in GH-secreting adenomas, we obtained somatolactotroph GH4C1 cell lines by performing doxycycline-dependent conditional overexpression of the wild-type G(s)alpha protein and expression of the gsp oncogene. Although the resulting adenylyl cyclase and cAMP levels were 10-fold lower in the wild-type G(s)alpha-overexpressing cell line, a sustained MAPK ERK1/2 activation was observed in both cell lines. Overexpression of the wild-type G(s)alpha protein as the gsp oncogene initiated chronic activation of endogenous prolactin synthesis and release, as well as chronic activation of ERK1/2-sensitive human prolactin and GH promoters.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Regulação Enzimológica da Expressão Gênica , Hormônio do Crescimento/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Prolactina/metabolismo , Somatotrofos/metabolismo , Adenilil Ciclases/metabolismo , Animais , Linhagem Celular , Cromograninas , AMP Cíclico/metabolismo , Doxiciclina/farmacologia , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/genética , Proteínas Oncogênicas/genética , Prolactina/genética , Ratos , Somatotrofos/enzimologia , Fatores de Tempo , Transfecção , Transgenes/efeitos dos fármacosRESUMO
Approximately 40% of growth hormone-secreting pituitary adenomas have somatic mutations in the GNAS1 gene (the so-called gsp oncogene). These mutations at codon 201 or codon 227 constitutively activate the alpha subunit of the adenylate cyclase-stimulating G protein G(s). GNAS1 is subject to a complex pattern of genomic imprinting, its various promoters directing the production of maternally, paternally, and biallelically derived gene products. Transcripts encoding G(s)alpha are biallelically derived in most human tissues. Despite this, we show here that in 21 out of 22 gsp-positive somatotroph adenomas, the mutation had occurred on the maternal allele. To investigate the reason for this allelic bias, we also analyzed GNAS1 imprinting in the normal adult pituitary and found that G(s)alpha is monoallelically expressed from the maternal allele in this tissue. We further show that this monoallelic expression of G(s)alpha is frequently relaxed in somatotroph tumors, both in those that have gsp mutations and in those that do not. These findings imply a possible role for loss of G(s)alpha imprinting during pituitary somatotroph tumorigenesis and also suggest that G(s)alpha imprinting is regulated separately from that of the other GNAS1 products, NESP55 and XLalphas, imprinting of which is retained in these tumors.
Assuntos
Acromegalia/etiologia , Acromegalia/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Impressão Genômica , Adenoma/complicações , Adenoma/genética , Adulto , Alelos , Sequência de Bases , Primers do DNA/genética , Feminino , Humanos , Masculino , Hipófise/metabolismo , Neoplasias Hipofisárias/complicações , Neoplasias Hipofisárias/genética , Polimorfismo GenéticoRESUMO
Despite the existence of interspecies phenotypic variability, animal models have yielded valuable insights into human pituitary diseases. Studies on Snell and Jackson mice known to have growth hormone, prolactin and thyroid-stimulating hormone deficiencies involving the hypoplastic pituitary gland have led to identifying alterations of the pituitary specific POU homeodomain Pit-1 transcription factor gene. The human phenotype associated with rare mutations in this gene was found to be similar to that of these mice mutants. Terminal differentiation of lactotroph cells and direct regulation of the prolactin gene both require interactions between Pit-1 and cell type specific partners, including panpituitary transcriptional regulators such as Pitx1 and Pitx2. Synergistic activation of the prolactin promoter by Pitx factors and Pit-1 is involved not only in basal condition, but also in responsiveness to forskolin, thyrotrophin-releasing-hormone and epidermal growth factor. In corticotroph cells, Pitx1 interacts with Tpit. Tpit mutations have turned out to be the main molecular cause of neonatal isolated adrenocorticotrophin deficiency. This finding supports the idea that Tpit plays an essential role in the differentiation of the pro-opiomelanocortin pituitary lineage. The effects of Pit-1 are not restricted to hormone gene regulation because this factor also contributes to cell division and protects the cell from programmed cell death. Lentiviral vectors expressing a Pit-1 dominant negative mutant induced time- and dose-dependent cell death in somatotroph and lactotroph adenomas in vitro. Gene transfer by lentiviral vectors should provide a promising step towards developing an efficient specific therapeutic approach by which a gene therapy programme for treating human pituitary adenomas could be based.
Assuntos
Regulação da Expressão Gênica/fisiologia , Terapia Genética , Doenças da Hipófise/genética , Adeno-Hipófise/metabolismo , Hormônios Hipofisários/metabolismo , Fator de Transcrição Pit-1/metabolismo , Animais , Técnicas de Transferência de Genes , Hormônio do Crescimento/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Mutantes Neurológicos , Mutação/genética , Doenças da Hipófise/fisiopatologia , Doenças da Hipófise/terapia , Adeno-Hipófise/citologia , Adeno-Hipófise/fisiopatologia , Hormônios Hipofisários/deficiência , Hormônios Hipofisários/genética , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/fisiopatologia , Neoplasias Hipofisárias/terapia , Prolactina/metabolismo , Proteínas com Domínio T , Tireotropina/metabolismo , Fator de Transcrição Pit-1/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Homeobox PITX2RESUMO
OBJECTIVE: This study compared the potency of a somatostatin receptor (sstr)2-sstr5 analog, BIM-23244, of an sstr2-dopamine D2 receptor (sstr2-DAD2) molecule, BIM-23A387 and of new somatostatin-dopamine chimeric molecules with differing, enhanced affinities for sstr2, sstr5 and DAD2, BIM-23A758, BIM-23A760 and BIM-23A761, to suppress GH and prolactin (PRL) from 18 human GH adenomas that are partially responsive to octreotide or lanreotide. MATERIALS AND METHODS: The sstr2, sstr5 and DAD2 mRNA levels were determined by RT-PCR. The effect of drugs was tested in cell cultures at various concentrations. RESULTS: In all tumors, the sstr2, sstr5 and DAD2 mRNA levels were coexpressed (mean levels+/-s.e.m. 0.4+/-0.1, 5.3+/-1.9 and 2.0+/-0.4 copy/copy beta-glucuronidase). In 13 tumors, the maximal suppression of GH secretion produced by BIM-23A387 (30+/-3%) and BIM-23244 (28+/-3%) was greater than that produced by octreotide (23+/-3%). In six out of 13 tumors, BIM-23A758, BIM-23A760 and BIM- 23A761 produced greater maximal suppression of GH secretion than octreotide (33+/-5, 38+/-2 and 41+/-2 vs 24+/-2%). Their EC(50) values were 10, 2 and 4 pmol/l. BIM-23A761 was more effective than BIM-23A387 in GH suppression (41+/-2 vs 32+/-4%). The new chimeric molecules produced maximal PRL suppression greater than octreotide (62+/-8 to 74+/-5 vs 46+/-11%). CONCLUSIONS: Novel dopamine-somatostatin chimeric molecules with differing, enhanced activity at sstr2, sstr5 and DAD2, consistently produced significatly greater suppression of GH and PRL than either octreotide or single-receptor-interacting ligands in tumors from patients classified as only partially responsive to octreotide therapy. The higher efficacy of the chimeric compounds was, at least partially, linked to their high affinity for sstr2 (IC50 1-10 pmol/l). The other mechanisms by which such molecules produce an enhanced inhibition of GH remain to be elucidated.
Assuntos
Dopamina/análogos & derivados , Neoplasias Hipofisárias/tratamento farmacológico , Prolactinoma/tratamento farmacológico , Receptores de Dopamina D2/administração & dosagem , Receptores de Somatostatina/administração & dosagem , Somatostatina/análogos & derivados , Acromegalia/sangue , Acromegalia/tratamento farmacológico , Adulto , Antineoplásicos Hormonais/administração & dosagem , Dopamina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Feminino , Hormônio do Crescimento Humano/sangue , Hormônio do Crescimento Humano/metabolismo , Humanos , Masculino , Octreotida/administração & dosagem , Neoplasias Hipofisárias/sangue , Neoplasias Hipofisárias/genética , Prolactina/sangue , Prolactina/metabolismo , Prolactinoma/sangue , Prolactinoma/genética , RNA Mensageiro/análise , Receptores de Dopamina D2/genética , Receptores de Somatostatina/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Somatostatina/administração & dosagem , Células Tumorais CultivadasRESUMO
In order to study the activation mechanism of heterotrimeric G-proteins by agonist-liganded receptors, GTP gamma S binding to membranes was measured in rat adenohypophyseal cells after addition of dopamine (DA) or vasoactive intestinal peptide (VIP), which, respectively, inhibit and activate pituitary adenylyl cyclase. G-protein subunit present in anterior pituitary cells was characterized by either ADP-ribosylation catalysed by Bordetella pertussis and cholera toxins or by immunoblot using specific antisera. Binding of GTP gamma S was found to depend upon GTP gamma S and Mg2+ concentrations; it was sensitive to pretreatment of the cells with cholera and Bordetella pertussis toxins (IAP). DA increased binding of the nucleotide. Paradoxically, VIP decreased the rate of GTP gamma S binding; the effect was suppressed by prior treatment of the cells with either cholera toxin or IAP. VIP also increased [33P]ADPribose incorporation in Gi/Go-proteins catalysed by IAP. Forskolin was also able to decrease GTP gamma S binding, thus suggesting that the binding of forskolin with the adenylyl cyclase catalytic unit might activate Gs proteins through an increased interaction between Gs and adenylyl cyclase. Taken together, these results suggest that VIP, as well as forskolin, may both accelerate the activation of Gs and suppress the inhibitory effect of activated Gi/Go-proteins. Interactions between Gs and Gi/Go subunits mediated by beta gamma and/or adenylyl cyclase might thus result in a kinetic coupling of transduction pathways involving distinct G-proteins.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Adeno-Hipófise/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Toxina Adenilato Ciclase , Animais , Sítios de Ligação/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas/efeitos dos fármacos , Toxina da Cólera/farmacologia , Colforsina/farmacologia , Dopamina/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Proteínas de Ligação ao GTP/química , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Trifosfato/análogos & derivados , Magnésio/farmacologia , Modelos Biológicos , Toxina Pertussis , Ratos , Ratos Sprague-Dawley , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The pituitary-specific transcription factor Pit-1 (pituitary-specific factor 1) is known to play a key role in the differentiation of PRL-, GH-, and TSH-secreting cells, and in the regulation of expression of the corresponding genes. In recent years, 12 distinct mutations of the Pit-1 gene have been shown to be responsible for a phenotype of multiple congenital pituitary hormone deficiency involving PRL, GH, and TSH. We had previously identified, in four siblings with GH, PRL, and TSH deficiencies, a mutation (F135C) resulting in a single amino acid change within the POU-specific binding domain of the Pit-1 molecule. In the present report, we have explored the functional effect of the F135C mutation. In vitro activity tests performed by transfection in human HeLa cells showed decreased transactivation capacity on the PRL, GH, and Pit-1 genes. The DNA binding experiments performed by gel shift showed that the F135C mutation generated a protein capable of binding to DNA response elements. To analyze how the F135C mutation might affect functionality of the transcription factor despite a normal DNA binding, we used a structure modelization approach and also analyzed two other Pit-1 mutant proteins (F135A and F135Y). The loss of functionality in these two mutants was similar to that of F135C. This finding was in keeping with our molecular modeling studies. According to structural data derived from the crystallographic analysis of the DNA/Pit-1 POU domain complex, the conformation of the first helix of the F135C-mutated POU-specific domain could be perturbed to such an extent that any interaction with other transcription cofactors might be definitively prevented.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Hormônios Hipofisários/deficiência , Fatores de Transcrição/química , Fatores de Transcrição/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hormônio do Crescimento/genética , Hormônio do Crescimento/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fenilalanina , Hormônios Hipofisários/metabolismo , Prolactina/genética , Prolactina/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Fator de Transcrição Pit-1 , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
The non-invasive electromagnetic blood flowmeter described in this paper allows us to measure pulsatile flow through a limb. The limb is placed in a magnetic field and the blood flow rate induces electromagnetic forces which are detected at the skin surface with ECG electrodes (Faraday's law). A special computer technique is necessary to isolate the signal from artefacts (local ECG, BCG, EMG). In vitro calibration is performed using a circulatory model and in vivo using mongrel dogs. Its validity is assessed by comparing the results with the responses obtained from the invasive electromagnetic flowmeter. Sources of error in the measurement such as blood composition (Na+, K+), haematocrit (45% to 29%), and venous flow are reported here. The results indicate that the method is reliable, easy to utilise and offers a unique non-invasive way of measuring true pulsatile blood flow rate in humans. Various clinical applications are discussed for possible use of the device.
Assuntos
Circulação Sanguínea , Fenômenos Eletromagnéticos , Animais , Arteriosclerose/fisiopatologia , Cães , Fenômenos Eletromagnéticos/instrumentação , Feminino , Membro Posterior/irrigação sanguínea , Humanos , Perna (Membro)/irrigação sanguínea , Masculino , Fluxo Sanguíneo Regional , ReologiaRESUMO
In lactating rats that were separated from their pups for 8 hours, suckling induced a rapid rise in plasma prolactin levels. Correlatively, it induced a depletion in dopamine (DA) and serotonin (5-HT) Concentrations within the hypothalamus as early as 5 min after the onset of suckling. This depletion lasted as long as the stimulus was maintained. A parallel increase in the level of the metabolite of 5-HT, 5-hydroxyindolacetic acid (5-HIAA), was observed. Under these conditons, the ratio of 5-HIAA/5-HT is augmented. This ratio is a good index of turnover of the transmitter. Suckling affected neither the content of noradrenaline (NA) in the hypothalamus nor the levels of any amine tested in the cerebral cortex. When pups were separated from their mother for 24 hours, suckling no longer resulted in changes in either plasma prolactin levels or hypothalamic DA, 5-HT, or 5-HIAA concentrations. We have concluded that the activation of a discrete system of 5-HT containing neurons is associated with the triggering effect of suckling on prolactin release.
Assuntos
Dopamina/metabolismo , Hipotálamo/metabolismo , Lactação , Prolactina/sangue , Serotonina/metabolismo , Animais , Castração , Feminino , Norepinefrina/metabolismo , Gravidez , Ratos , Fatores de TempoRESUMO
The role of protein kinase C (PKC) on dopamine inhibition of PRL messenger RNA (mRNA) levels was studied in anterior pituitary cells kept in primary culture. PKC was desensitized by long-term exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA). Effectiveness of PKC desensitization was confirmed by the fact that after TPA pretreatment, short-term (1-h) exposure to TPA was no longer able to trigger PRL release. In contrast, the capacity of nonreceptor-mediated secretagogues as depolarization with 48 mM K+ to release the hormone was preserved. Pretreatment with TPA did not affect basal PRL mRNA levels. In contrast, it significantly reduced the dose-dependent inhibition of PRL mRNA induced by 1 nM bromocriptine after a 4-day incubation period. Since dopamine inhibition of PRL release is mediated by several second messager pathways, including cAMP, inositol phosphates, and Ca2+, we investigated whether PKC depletion was able to interact with direct stimulation of these pathways. Pretreatment with PKC suppressed stimulation of PRL mRNA levels induced by either Forskolin (FK) or 8Br-cAMP. In parallel, it reduced basal as well as FK stimulated intracellular cAMP levels. In addition, chronic exposure to TPA completely suppressed PRL mRNA inhibition induced by nifedipine, a dihydropyridine antagonist which blocks voltage-dependent Ca2+ channels. TPA desensitization also affected the action of bromocriptine, FK or nifedipine on PRL release measured under the same conditions. The data indicate that endogenous PKC can interfere with the regulation of PRL gene expression induced by both cAMP and Ca2+ pathways, two second messengers associated with the action of dopamine in lactotroph cells.
Assuntos
Bromocriptina/farmacologia , AMP Cíclico/metabolismo , Dopamina/fisiologia , Regulação da Expressão Gênica , Adeno-Hipófise/metabolismo , Prolactina/genética , Proteína Quinase C/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Células Cultivadas , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Cinética , Nifedipino/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Potássio/farmacologia , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
Specific [125I]-Iodo-NTyr somatostatin binding sites are present in adenohypophyseal and cerebral cortical membranes. Guanine nucleotides reduce the maximal binding capacity of adenohypophyseal binding sites without significantly affecting their apparent affinity. In pituitary as well as in cortex, GTP is the most potent nucleotide followed by GDP and guanylyl imidodiphosphate (GMP-PNP). The effect appears specific of guanine nucleotides since ATP, ADP and AMP are inactive on [125I]-Iodo-NTyr somatostatin binding. These results, showing the nucleotide sensitivity of [125I]-Iodo-NTyr somatostatin binding in pituitary and cerebral cortex, are compatible with a coupling of somatostatin receptors with adenylate cyclase.
Assuntos
Membrana Celular/metabolismo , Córtex Cerebral/metabolismo , Nucleotídeos de Guanina/farmacologia , Adeno-Hipófise/metabolismo , Receptores de Superfície Celular/metabolismo , Somatostatina/análogos & derivados , Animais , Guanosina Difosfato/farmacologia , Guanosina Trifosfato/farmacologia , Guanilil Imidodifosfato/farmacologia , Cinética , Masculino , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Somatostatina , Somatostatina/metabolismoRESUMO
When applied to rat anterior pituitary cells, angiotensin-II (AII) exerted two opposite effects on adenylate cyclase (AC) activity: a pertussis toxin (PTX)-sensitive inhibition of the enzyme with a maximal effect of -42 +/- 2% in crude cell membrane preparations, and, in contrast, a non-PTX-sensitive stimulation of cAMP production (maximal effect = 38 +/- 3%) in intact cells. The apparent affinity of both effects was equal to 1.8 nM. The stimulation of cAMP formation parallels the stimulation of PRL release. Under the same conditions, dopamine (DA) inhibited both membrane AC activity and cAMP formation in intact cells by a PTX-sensitive mechanism. After separation of pituitary cell types by sedimentation at unit gravity, the effects of AII and DA on intracellular cAMP and membrane AC activity coincided in the same fractions (those enriched in PRL cells). The stimulatory effect of AII on cAMP formation was about 5 times weaker than that of peptides positively coupled to AC as vasoactive intestinal peptide in total as well as in PRL-enriched cells. Since the AII receptor is also coupled to phospholipase-C (PLC) in a non-PTX-sensitive manner, we investigated whether protein kinase-C (PKC) could indirectly account for the positive effect of AII on cAMP formation. 12-O-Tetradecanolylphorbol 13-acetate (TPA), a stimulator of PKC was indeed able to increase intracellular cAMP; this effect was not additive with that of AII. conversely, application of the PKC inhibitors H7 [1-(5-isoquinolylsulfonyl)2-methyl-piperazine] and staurosporine or desensitization of PKC by long exposure of the cells to TPA abolished the cAMP response to TPA as well as that to AII. In addition, thyreoliberin, another activator of the PLC pathway, was able to stimulate cAMP formation in a PKC-dependent manner. DA inhibition of intracellular cAMP was not affected by any PKC inhibition. We conclude that in lactotroph cells, 1) the AII inhibitory coupling to AC observed in membrane preparations does not exist in intact cells, at least under basal conditions; and 2) the AII intracellular cAMP stimulation observed is not accounted for by a direct coupling with AC; it is due to a cross-talk of the PLC pathway mediated by PKC, an effect that might be shared by other PLC-stimulating mediators and may participate in the regulation of PRL release.
Assuntos
Angiotensina II/farmacologia , AMP Cíclico/metabolismo , Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Proteína Quinase C/farmacologia , Adenilil Ciclases/metabolismo , Animais , Células Cultivadas , Dopamina/farmacologia , Fosfatos de Inositol/metabolismo , Membranas Intracelulares/metabolismo , Adeno-Hipófise/citologia , Ratos , Hormônio Liberador de Tireotropina/farmacologia , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
In primary culture of anterior pituitary cells, BAY-K-8644, a calcium channel agonist, stimulated PRL secretion by 83% with EC50 of 18 nM. This effect was blocked by nifedipine, a calcium channel antagonist. The stimulations of PRL secretion induced by potassium (50 mM) and BAY-K-8644 were additive. Dopamine inhibited basal as well as BAY-K-8644-stimulated PRL secretion by 64% and 75%, respectively, and with respective EC50 values of 4.5 and 0.6 nM. In the presence of 50 mM K+, dopamine only partially blocks the dose-dependent stimulation of PRL secretion induced by the calcium channel agonist. The inhibitory dopamine effect was blocked by (+)butaclamol, a specific dopamine receptor antagonist. The dopamine response was also blocked by 1-sulpiride, a specific dopamine D2 receptor antagonist, and mimicked by RU 24926, a specific dopamine D2 receptor agonist, suggesting that the dopamine effect on BAY-K-8644-stimulated PRL secretion was mediated through a D2 dopamine receptor. Although unknown, the mechanism by which dopamine inhibited the BAY-K-8644-stimulated PRL secretion involves a GTP binding protein sensitive to Bordetella pertussis toxin. In fact, the dopamine inhibition of PRL secretion induced by the calcium channel agonist was blocked by the pretreatment of cells with the toxin. These results suggest that dopamine D2 receptors in lactotroph cells modulate calcium influx through a GTP binding protein.
Assuntos
Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Dopamina/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Toxina Pertussis , Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Fatores de Virulência de Bordetella/farmacologia , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Feminino , Cinética , Nifedipino/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Potássio/farmacologia , Prolactina/antagonistas & inibidores , Ratos , Ratos EndogâmicosRESUMO
The mechanisms by which plasma T3 and TSH decrease after a 3-day starvation period are not completely understood. In this study we tested the hypothesis of a possible modification in the sensitivity of thyrotroph cell to T3 and/or TRH. For that purpose, TRH tests were performed before and after a 3-day starvation in euthyroid, thyroidectomized, and T3-treated (75 or 175 ng/100 g BW) thyroidectomized male Wistar rats. TRH (10 to 500 ng/100 g BW) was injected iv through a chronically-implanted catheter. In another set of experiments, hypophyseal TSH content was also determined. Our results showed that after a 3-day-starvation plasma TSH decreased in all except hypothyroid rats; TSH responsiveness to TRH was unchanged in euthyroid rats but was increased in hypothyroid rats; and the T3-dependent increase in TSH responsiveness to TRH was significantly amplified. Moreover, there was a significant positive correlation between TSH responsiveness to TRH and hypophyseal TSH content. These results suggest that starvation induces an increased sensitivity of thyrotroph cell to T3.
Assuntos
Adeno-Hipófise/metabolismo , Inanição/fisiopatologia , Hormônio Liberador de Tireotropina/fisiologia , Tireotropina/metabolismo , Tri-Iodotironina/fisiologia , Animais , Privação de Alimentos , Hipotireoidismo/fisiopatologia , Masculino , Ratos , Ratos Endogâmicos , Glândula Tireoide/fisiopatologia , Hormônio Liberador de Tireotropina/sangue , Tri-Iodotironina/sangueRESUMO
17 beta-Estradiol (E2) alters different functions of pituitary cells, including cell sensitivity to several neurohormones such as LHRH, TRH, somatostatin, or dopamine, presumably by affecting receptor coupling mechanisms. Attempting to pinpoint the membrane processes underlying this modulation, we studied the effect of E2 on pituitary kinase-C (PKC) activity, a major signal transduction enzyme. The distribution of calcium- and phospholipid-dependent partially purified PKC (chromatography on DEAE-52 cellulose columns) was evaluated in membrane and cytosol fractions from anterior pituitaries of ovariectomized (OVX) or OVX plus E2-treated rats. E2 administration by implants to OVX animals increased significantly both soluble and particulate enzyme activity. The effect increased progressively from 24 h to 5 days after E2 treatment. Administration of 17 alpha-estradiol, an inactive stereoisomer of E2, was ineffective, pointing to stereospecific interaction. Total destruction of neural connections to the pituitary (complete hypothalamic lesions) did not modify the enzyme response to E2 administration, indicating a direct effect of the steroid on pituitary PKC activity. A direct E2 (10(-9) M) effect was confirmed in primary mixed cultures of pituitary cells; it was time dependent (15-96 h) and specific, and reflects a genomic E2 action. E2 treatment for shorter times had no effect on the enzyme levels or the membrane redistribution of PKC activity. In contrast, under the same experimental conditions phorbol esters (12-O-tertadecanoyl-phorbol-13-acetate (TPA] induced a rapid and sustained translocation of the enzyme. PKC activity was found in all pituitary cell types, with maximal activity in fractions of gonadotropes and thyrotropes, as evaluated in cultures enriched in certain types of pituitary cells separated by means of unit gravity gradient sedimentation. E2 treatment (10(-9) M; 72 h) significantly increased both soluble and particulate enzyme levels in all cell types. In addition, administration of E2 (10(-9) M; 72 h) to cell cultures strongly increased the TPA-evoked LH and PRL release. These results indicate that E2-induced changes in pituitary function include selective effects of the steroid on PKC activity involved at different levels in the coupling mechanisms.
Assuntos
Estradiol/farmacologia , Hipófise/enzimologia , Proteína Quinase C/metabolismo , Animais , Células Cultivadas , Feminino , Hormônio do Crescimento/metabolismo , Hormônio Luteinizante/metabolismo , Ovariectomia , Hipófise/citologia , Prolactina/metabolismo , Ratos , Ratos Endogâmicos , Acetato de Tetradecanoilforbol/farmacologia , Distribuição TecidualRESUMO
Dopamine (DA) is known to inhibit basal and hormone TRH- or angiotensin II (AngII)-stimulated PRL secretion and inositol phosphate accumulation in rat pituitary cells in primary culture. This inhibition persists when cells are incubated in a calcium-free medium (a condition in which DA could not inhibit PLC activities by blocking calcium influx) and is abolished by a Pertussis toxin treatment. These data suggest that DA receptor could be negatively coupled to PLC by a direct mechanism involving a Pertussis toxin-sensitive G protein. To demonstrate this hypothesis, we measured PLC activities on crude plasma membranes obtained from rat pituitary cells in primary culture grown in the presence of tritiated myo-inositol. We showed that 1) DA and quinpirole or RU24926 (specific D2 agonists) inhibited both basal and TRH- or AngII-stimulated membrane PLC activities. 2) Such inhibitions were completely prevented by sulpiride (specific D2 antagonist). 3) Heterotrimeric Gi1/2 proteins coupled the DA receptors to PLC because DA inhibitions were completely reversed by preincubation either with Pertussis toxin or with a specific G(alpha)i1/(alpha)i2 antibody. Such data are in favor of the existence of a direct negative coupling between DA-D2 receptor and PLC on a native physiological plasma membrane model.