RESUMO
KEY POINTS: Two-pore channels (TPCs) were identified as a novel family of endolysosome-targeted calcium release channels gated by nicotinic acid adenine dinucleotide phosphate, as also as intracellular Na(+) channels able to control endolysosomal fusion, a key process in autophagic flux. Autophagy, an evolutionarily ancient response to cellular stress, has been implicated in the pathogenesis of a wide range of cardiovascular pathologies, including heart failure. We report direct evidence indicating that TPCs are involved in regulating autophagy in cardiomyocytes, and that TPC knockout mice show alterations in the cardiac lysosomal system. TPC downregulation implies a decrease in the viability of cardiomyocytes under starvation conditions. In cardiac tissues from both humans and rats, TPC transcripts and protein levels were higher in females than in males, and correlated negatively with markers of autophagy. We conclude that the endolysosomal channels TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes, and also that they are differentially expressed in male and female hearts. ABSTRACT: Autophagy participates in physiological and pathological remodelling of the heart. The endolysosomal two-pore channels (TPCs), TPC1 and TPC2, have been implicated in the regulation of autophagy. The present study aimed to investigate the role of TPC1 and TPC2 in basal and induced cardiac autophagic activity. In cultured cardiomyocytes, starvation induced a significant increase in TPC1 and TPC2 transcripts and protein levels that paralleled the increase in autophagy identified by increased LC3-II and decreased p62 levels. Small interfering RNA depletion of TPC2 alone or together with TPC1 increased both LC3II and p62 levels under basal conditions and in response to serum starvation, suggesting that, under conditions of severe energy depletion (serum plus glucose starvation), changes in the autophagic flux (as assessed by use of bafilomycin A1) occurred either when TPC1 or TPC2 were downregulated. The knockdown of TPCs diminished cardiomyocyte viability under starvation and simulated ischaemia. Electron micrographs of hearts from TPC1/2 double knockout mice showed that cardiomyocytes contained large numbers of immature lysosomes with diameters significantly smaller than those of wild-type mice. In cardiac tissues from humans and rats, TPC1 and TPC2 transcripts and protein levels were higher in females than in males. Furthermore, transcript levels of TPCs correlated negatively with p62 levels in heart tissues. TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes (i.e. there is a negative effect on cell viability under stress conditions in their absence) and they are differentially expressed in male and female human and murine hearts, where they correlate with markers of autophagy.
Assuntos
Autofagia/fisiologia , Canais de Cálcio/fisiologia , Lisossomos/fisiologia , Miócitos Cardíacos/fisiologia , Caracteres Sexuais , Idoso , Animais , Animais Recém-Nascidos , Apêndice Atrial/fisiologia , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Seipin/BSCL2 mutations can cause type 2 congenital generalised lipodystrophy (BSCL) or dominant motor neurone diseases. Type 2 BSCL is frequently associated with some degree of intellectual impairment, but not to fatal neurodegeneration. In order to unveil the aetiology and pathogenetic mechanisms of a new neurodegenerative syndrome associated with a novel BSCL2 mutation, six children, four of them showing the BSCL features, were studied. METHODS: Mutational and splicing analyses of BSCL2 were performed. The brain of two of these children was examined postmortem. Relative expression of BSCL2 transcripts was analysed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in different tissues of the index case and controls. Overexpressed mutated seipin in HeLa cells was analysed by immunofluorescence and western blotting. RESULTS: Two patients carried a novel homozygous c.985C>T mutation, which appeared in the other four patients in compound heterozygosity. Splicing analysis showed that the c.985C>T mutation causes an aberrant splicing site leading to skipping of exon 7. Expression of exon 7-skipping transcripts was very high with respect to that of the non-skipped transcripts in all the analysed tissues of the index case. Neuropathological studies showed severe neurone loss, astrogliosis and intranuclear ubiquitin(+) aggregates in neurones from multiple cortical regions and in the caudate nucleus. CONCLUSIONS: Our results suggest that exon 7 skipping in the BSCL2 gene due to the c.985C>T mutation is responsible for a novel early onset, fatal neurodegenerative syndrome involving cerebral cortex and basal ganglia.
Assuntos
Subunidades gama da Proteína de Ligação ao GTP/genética , Lipodistrofia Generalizada Congênita/genética , Mutação , Criança , Éxons/genética , Evolução Fatal , Feminino , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Genótipo , Células HeLa , Humanos , Lipodistrofia Generalizada Congênita/patologia , Lipodistrofia Generalizada Congênita/fisiopatologia , Masculino , Fenótipo , Splicing de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Animal studies suggest that the endocannabinoid system (ECS) plays a role in the regulation of myocardial contractility and in the pathogenesis of heart failure. The current study aimed to proof the existence of endocannabinoid receptors on human ventricular myocardium and to determine whether human chronic heart failure (CHF) is associated with changes in endocannabinoid receptor expression and distribution. Expression of cannabinoid receptor 1 (CB1) and cannabinoid receptor (CB2) on human heart was assessed by means of real-time PCR and immunohistochemistry. On healthy human left ventricular myocardium, mRNA transcripts of CB1 and CB2 receptors were expressed in an almost equal proportion. In patients with CHF, mRNA expression of CB1 receptors was shown to be downregulated 0.7-fold (0.7.+/-0.15, n=12, p<0.01), whereas expression of CB2 receptors was upregulated more than 11-fold (11.6+/-4.5; n=12; p<0.005). Corresponding results were obtained by immunohistochemistry. Blood levels of endocannabinoids were significantly elevated (anandamide 3.5-fold (p<0.001); 2-AG 7-fold (p=0.02)) in patients with CHF, as compared to healthy volunteers. Both CB1 and CB2 receptors are present on healthy human left ventricular myocardium in a balanced distribution. Patients suffering from CHF exhibit a shift of the CB1-CB2 receptor ratio towards expression of CB2 receptors combined with significantly elevated peripheral blood levels of endocannabinoids indicating an activation of the ECS. These results might open up new perspectives regarding the role of endocannabinoid signalling in CHF and its potential as a target for pharmacological modulation.
Assuntos
Moduladores de Receptores de Canabinoides/metabolismo , Endocanabinoides , Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Receptor CB2 de Canabinoide/sangue , Adulto , Ácidos Araquidônicos/sangue , Moduladores de Receptores de Canabinoides/sangue , Estudos de Casos e Controles , Glicerídeos/sangue , Ventrículos do Coração/patologia , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Miocárdio/patologia , Alcamidas Poli-Insaturadas/sangue , Receptor CB1 de Canabinoide/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Merkel cells are neuroendocrine cells associated to a neural sensitive ending and localized primarily in the epidermis, although they are also found in oral mucosa. Sox2 or SRY-box2 is a key transcription factor important in the maintenance of embryonic neural crest stem cell pluripotency. Sox2 has been described in Merkel cells of skin and in Merkel cell carcinomas, but not specifically in oral Merkel cells. The aims of the present study were to analyze the density of Merkel cells in human oral mucosa and to study the expression of Sox2 in these cells. For these purposes, immunohistochemical analyses for Sox2 and CK20 (the best marker for Merkel cells) were automatically performed on sections of normal human oral mucosa. Double immunofluorescence for Sox2 and CK20 was also performed. To analyze the density of Merkel cells, CK20 positive cells were counted in each sample and the length of the epithelial apical edge was measured (cells/mm). Merkel cells, demonstrated by CK20 immunoreactivity, were found in 95% of oral mucosa specimens studied (n=21). Mean density of Merkel cells in oral mucosa was 1.71±2.34 cells/mm. Sox2 immunoreactivity was found in the nuclei of scattered cells located at the basal layer. Serial sections immunostained for Sox2 and CK20 showed that Sox2-positive cells of oral mucosa coexpressed CK20, confirming that they were Merkel cells. Immunofluorescence for Sox2 and CK20 showed colocalization of both markers, demonstrating that virtually all oral Merkel cells expressed Sox2. This transcription factor could play a role in Merkel cell maturation and maintenance.
Assuntos
Células de Merkel/metabolismo , Mucosa Bucal/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Queratina-20/metabolismo , Masculino , Pessoa de Meia-Idade , Células-Tronco PluripotentesRESUMO
Mesenchymal stem cells (MSCs) are a promising clinical therapy for ischemic stroke. However, critical parameters, such as the most effective administration route, remain unclear. Intravenous (i.v.) and intraarterial (i.a.) delivery routes have yielded varied outcomes across studies, potentially due to the unknown MSCs distribution. We investigated whether MSCs reached the brain following i.a. or i.v. administration after transient cerebral ischemia in rats, and evaluated the therapeutic effects of both routes. MSCs were labeled with dextran-coated superparamagnetic nanoparticles for magnetic resonance imaging (MRI) cell tracking, transmission electron microscopy and immunohistological analysis. MSCs were found in the brain following i.a. but not i.v. administration. However, the i.a. route increased the risk of cerebral lesions and did not improve functional recovery. The i.v. delivery is safe but MCS do not reach the brain tissue, implying that treatment benefits observed for this route are not attributable to brain MCS engrafting after stroke.
Assuntos
Isquemia Encefálica/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Animais , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/reabilitação , Isquemia Encefálica/terapia , Rastreamento de Células , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Dextranos , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestrutura , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Ratos , Recuperação de Função FisiológicaRESUMO
Superparamagnetic iron oxide nanoparticles (MNPs) together with magnetic resonance imaging (MRI) are the preferred tools for monitoring the fate and biodistribution of administered cells in stem cell therapy studies. Commercial MNPs need transfection agents and long incubation times for sufficient cell labeling and further in vivo cell detection. In this work, we have synthesized MNPs coated with pluronic F127 and tetronic 908, and validated their applicability as contrast agents for MRI cell detection on two different cell types: rat mesenchymal stem cells (MSCs) and multipotent neural progenitor cell line from mice (C17.2). No transfection agent was needed for a complete MNP internalization, and the uptake was only dependent on MNP concentration in medium and limited on the incubation time. By combining in vivo MRI and ex vivo histology microscopy, we have demonstrated the MRI signal detected corresponded exclusively to labeled cells and not to free particles. Pluronic F127- and tetronic 908-coated MNPs represent promising contrast agents for stem cell tracking due to their ease of use in preparation, their efficiency for cell labeling, and their high sensitivity for in vivo cell detection.
Assuntos
Meios de Contraste/química , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/química , Polímeros/química , Animais , Encéfalo/diagnóstico por imagem , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Masculino , Células-Tronco Mesenquimais/citologia , Microscopia Eletrônica de Transmissão , Ratos , Ratos Sprague-Dawley , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer.
Assuntos
Adenocarcinoma/genética , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Gástricas/genética , Proliferação de Células , Feminino , Humanos , Masculino , Transdução de SinaisRESUMO
Orexin-A and -B are hypothalamic peptides derived from a precursor called prepro-orexin and related with the regulation of the energy balance and arousal. They act on G protein receptors named orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). In the present study, we used immunohistochemical techniques to detect the distribution of OXR in normal human adrenal gland and adrenal tumours (adrenocortical adenomas and pheochromocytomas). OX1R was expressed in the cortex of the normal human adrenal gland (glomerulosa, fasciculata and reticular zones) and OX2R was located in the medulla (epinephrine and norepinephrine cells). By the double immunofluorescence techniques, we demonstrated that virtually all medullar cells (epinephrine and norepinephrine cells) expressed OX2R. As was expected, according to the results obtained in normal tissues, cortical tumours (adrenocortical adenomas) were positive for OX1R but not for OX2R and conversely, medullar tumours (pheochromocytomas) expressed only OX2R.
Assuntos
Glândulas Suprarrenais/metabolismo , Adenoma Adrenocortical/metabolismo , Feocromocitoma/metabolismo , Receptores de Neuropeptídeos/metabolismo , Adolescente , Córtex Suprarrenal/metabolismo , Medula Suprarrenal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Receptores de Orexina , Receptores Acoplados a Proteínas G , Receptores de Neuropeptídeos/biossíntese , Distribuição Tecidual/fisiologiaRESUMO
BACKGROUND: Adiponectin is an anti-inflammatory adipocytokine believed to be involved in the pathogenesis of chronic heart failure (CHF). We aimed to characterize the expression of adiponectin and its receptors in CHF and to assess the impact of microRNAs on the cardiac adiponectin system. METHODS: Expression of adiponectin and adiponectin receptors (ADIPOR1 and ADIPOR2) was studied by qPCR and immunohistochemistry in myocardial tissues of patients with end-stage CHF and control subjects. MicroRNA binding was evaluated by cloning of an ADIPOR2 3´-untranslated-region reporter construct and subsequent transfection experiments. Effects of miRNA transfection were analyzed in cardiomyocyte cell cultures by qPCR and Western blotting. Gene silencing of ADIPOR2 was performed by siRNA transfection, and the effects of hypoxia/serum starvation were analyzed by flow cytometry. RESULTS: Although CHF patients displayed elevated plasma adiponectin levels, myocardial adiponectin expression generally was very low. In CHF, cardiac ADIPOR1 expression increased by >4-fold, whereas the increase in ADIPOR2 was less than 2-fold. Reporter gene assays on constructs containing the ADIPOR2-3'-untranslated region suggest that microRNA-150 specifically repressed ADIPOR2 expression. Transfection of cardiomyocytes with premiR-150 precursor molecules resulted in 60% down-regulation of ADIPOR2 mRNA and a significant reduction of ADIPOR2 protein expression. MicroRNA-150 was substantially expressed in both normal and CHF myocardium, with a 1.7-fold higher expression in CHF. Finally, knock-down experiments elucidated a stress-protective role of ADIPOR2 in cardiomyocytes. CONCLUSIONS: MicroRNA-150 counteracts ADIPOR2 up-regulation in CHF and thus may contribute to adiponectin resistance. Targeting microRNA-150 may be a future strategy to restore cardioprotective adiponectin effects.
Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , MicroRNAs/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Receptores de Adiponectina/metabolismo , Adiponectina/metabolismo , Adulto , Células Cultivadas , Doença Crônica , Inativação Gênica/efeitos dos fármacos , Insuficiência Cardíaca/patologia , Humanos , Pessoa de Meia-Idade , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/patologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores de Adiponectina/efeitos dos fármacos , Receptores de Adiponectina/genética , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Galectins are a family of soluble lectins expressed in a variety of tissues, which play many important regulatory roles in inflammation, immunity, and cancer. The up-regulation of galectin-3 in hypertrophied hearts and the development of fibrosis have been shown in experimental studies. Increased galectin-3 levels are associated with poor long-term survival in end-stage heart failure (HF). We examined the relationship between plasma galectin-3 levels and the myocardial tissue expression of galectin-3 in patients with end-stage HF. MATERIAL AND METHODS: Expression of galectin-3 was assessed by real-time PCR and immunohistochemistry in left ventricle and atrial myocardium of patients (n=12) with end-stage HF undergoing heart transplantation. All patients gave informed consent. Serum expression of galectin-3 was assessed by ELISA in serum from 20 patients with end-stage HF and in 20 healthy volunteers who served as controls. RESULTS: Expression of galectin-3 was similar in the myocardium of patients in comparison to the control group, independently of the anatomical area (HF vs. healthy ventricle: 1.73E-02 vs. 1.50 E-02; HF vs. healthy atrium: 1.32E-02 vs. 1.16E-02). However, serum expression of galectin-3 was significantly higher in the end-stage HF patients compared to the healthy controls (13.02±10.6 vs. 3.7±1.3 ng/ml; p<0.05). CONCLUSIONS: Plasma galectin-3 levels correlate with the ejection fraction and are elevated in patients with HF. However, the myocardial expression of galectin-3 does not correlate with the ventricular ejection fraction. Our data support the use of galectin-3 as a marker of heart insufficiency.
Assuntos
Galectina 3/metabolismo , Insuficiência Cardíaca/metabolismo , Transplante de Coração , Miocárdio/metabolismo , Adulto , Biomarcadores/sangue , Biomarcadores/metabolismo , Feminino , Galectina 3/sangue , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Volume SistólicoRESUMO
BACKGROUND: Autosomal recessive congenital ichthyosis (ARCI) is a rare, nonsyndromic, heterogeneous disorder of cornification. It is divided into three clinical subtypes: lamellar ichthyosis (LI); congenital ichthyosiform erythroderma; and harlequin ichthyosis. In the majority of patients, LI is caused by transglutaminase-1 (TGase1) deficiency resulting from mutations in both copies of the transglutaminase 1 (TGM1) gene in chromosome 14. CASE REPORT: We report a patient with a severe LI phenotype who has a homozygous putative splicing mutation in the TGM1 gene. Our aim is to assess the pathologic effect of the TGM1 c.984+1G>A by splicing assays and bioinformatic tools. RESULTS: c.984+1G>A mutation created two alternative TGM1 mRNA splice variants that included 30 or 32 nucleotides of the 5' of intron 6. At the protein level, the partial in-frame aberrant transcript retaining 30 bp of intron 6 led to the insertion of 10 amino acids (p.Met329_Val330ins10) at the catalytic core domain of TGM1 protein (codons 247-572), whereas the transcript with the insertion of 32 nucleotides is predicted to encode a truncated protein (p.Val330MetfsX12). CONCLUSION: Our splicing assay, together with bioinformatic prediction tools, supports the pathological effect of the recently identified c.984+1G>A mutation in the TGM1 gene and unravels the molecular mechanism by which c.984+1G>A acts.
Assuntos
Ictiose Lamelar/genética , Mutação , Transglutaminases/genética , Portador Sadio , Criança , Análise Mutacional de DNA , Feminino , Humanos , Transglutaminases/deficiênciaRESUMO
Ghrelin is a peptide hormone mainly produced by the stomach, which strongly stimulates the release of growth hormone (GH) via the GH secretagogue receptor 1a (GHSR-1a) located in the hypothalamus. It has been reported to exert performance-enhancing effects on myocardial function, and as both ghrelin and GHSR-1a are expressed in myocardial tissues, the ghrelin system may have a direct GH-independent impact on cardiac function. We intended to investigate the expression of ghrelin and its receptor GHSR-1a in different myocardial areas of patients with chronic heart failure (CHF) as compared to heart-healthy subjects to better define the role of the ghrelin signaling system in the networks regulating cardiac function and its potential as a target for diagnosis and/or treatment of CHF. Myocardium biopsies of 12 patients undergoing heart transplantation and suffering from CHF were obtained. Expression of both ghrelin and GHSR-1a was assessed by means of immunohistochemistry and real-time PCR. Expression of ghrelin was significantly decreased in CHF hearts both in atrium and ventricles in comparison to the control hearts (p<0.05). The expression of the GHS-1a receptor was significantly increased in the CHF biopsies as compared to controls (p<0.05). No significant differences were found between the anatomical areas studied. Expression of myocardial ghrelin and GHSR-1a is directly associated with myocardial function: CHF hearts exhibit an impaired ghrelin production which might reflect maladaptive processes and an - probably compensatory - increase in GHSR-1a expression. These findings may open up new perspectives regarding the potential of ghrelin signaling as a target for pharmacological modulation.
Assuntos
Grelina/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Receptores de Grelina/metabolismo , Adulto , Grelina/genética , Insuficiência Cardíaca/genética , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Pessoa de Meia-Idade , Receptores de Grelina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ghrelin, the endogenous ligand of the growth hormone secretagogue receptor (GHS-R), is a 28 amino acid peptide originally isolated from rat stomach that has been primarily involved in the central neuroregulation of GH secretion and food intake. Previous studies demonstrated that ghrelin has a widespread expression in different normal cells and tissues, as well as in gastric, thyroid, testicular, breast and lung neoplasias. In the current study, we use molecular biology to detect ghrelin transcripts expression in rats, and immunohistochemical techniques to investigate the cellular distribution of this peptide in rat and human thyroid and parathyroid glands and tumours. Ghrelin was localized in thyroid C cells and in parathyroid cells. Thyroid carcinomas (medullar, follicular and papillary) and parathyroid adenomas also showed intense and diffuse immunostaining for ghrelin. These data provide direct morphological evidence that ghrelin may well be acting in a paracrine fashion in the regulation of thyroid follicular cell function. The diffuse ghrelin immunostaining found in the parathyroid gland opens up the possibility of its secretion to the bloodstream or its involvement in the regulation of the parathyroid function. Overall, expression of ghrelin in human and rat thyroid and parathyroid glands is highly suggestive of a conserved role of this molecule in the regulation of thyroid and parathyroid cell function.
Assuntos
Glândulas Paratireoides/metabolismo , Neoplasias das Paratireoides/metabolismo , Hormônios Peptídicos/metabolismo , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , Feminino , Expressão Gênica , Grelina , Humanos , Imuno-Histoquímica , Masculino , Neoplasias das Paratireoides/genética , Hormônios Peptídicos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Glândula Tireoide/genéticaRESUMO
Orexins (OXA and OXB) are peptides derived from a common precursor called prepro-orexin. They act through G-protein receptors named orexin 1 receptor (OX(1)R) and orexin 2 receptor (OX(2)R). Orexins were first demonstrated in neurons of the lateral hypothalamus and found to be related to the control of food intake. However, it has been shown that they are widely distributed in both the nervous system and peripheral tissues, including endocrine organs such as the pituitary and adrenal glands. Merkel cells are neuroendocrine cells situated in the epidermis, tactile hairs and oral mucosa, and act as mechanoreceptors. Up to the present, various neuropeptides have been detected in these cells. The aim of the present study was to detect the presence of prepro-orexin and orexin receptors (OX(1)R and OX(2)R) in porcine Merkel cells using immunohistochemistry. Prepro-orexin was expressed in the cytoplasm of Merkel cells in the skin of the pig snout. Immunoreactivity for prepro-orexin was more intense in the mature side of the cell, where the dense-cored granules are accumulated. Epidermal nerve terminals associated with Merkel cells and dermal nerve fibres showed no immunostaining. Both orexin receptors (OX(1)R and OX(2)R) were also demonstrated in the cytoplasm of Merkel cells of pig snout skin. The finding of orexins and their receptors in Merkel cells suggests that they have an autocrine function. Further studies are needed to ascertain the significance of this function.
Assuntos
Células de Merkel/química , Neuropeptídeos/análise , Precursores de Proteínas/análise , Receptores de Neuropeptídeos/análise , Animais , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intracelular , Receptores de Orexina , Orexinas , Receptores Acoplados a Proteínas G , SuínosRESUMO
Orexins A and B are hypothalamic peptides derived from a precursor called prepro-orexin and are associated with the stimulation of food intake and arousal. There is evidence that orexins act on some pituitary functions. Since no studies have been done concerning the presence of orexins in human pituitary, it is unclear whether the local effect of these peptides is due to orexins synthesized in the pituitary or to circulating-derived orexins. To define a possible paracrine regulatory role of orexins on pituitary cell function, we have sought to characterize the expression of orexins in the human adenohypophysis as well as to identify the cell types that express these proteins. In the present study, we used immunohistochemistry and double labeling to detect the presence of orexin A and orexin B in human pituitary. Orexin A was localized in 33% of pituitary cells. With double immunofluorescence techniques we demonstrated that orexin A is present in PRL (82 +/- 5.3%), TSH (18 +/- 2.3%), GH (10 +/- 2.3%), FSH (8 +/- 2.6%), and LH (7 +/- 3.2%) cells, but not in corticotroph cells. Orexin B was found in virtually all corticotrophs cells of the anterior pituitary. These results demonstrate that lactotroph cells are the main source of orexin A and corticotroph cells of orexin B. In summary the present findings provide the first evidence that orexins A and B are expressed in specific human pituitary cell types. Our data provide the cellular basis for a paracrine role of orexins in human pituitary cell function and further our understanding regarding the mechanisms by which orexins influence neuroendocrine function.