RESUMO
The Esophageal Cancer Related Gene 4 (ECRG4) is a highly conserved tumour suppressor gene encoding various peptides (augurin, CΔ16 augurin, ecilin, argilin, CΔ16 argilin) which can be processed and secreted. In the present work, we examined ECRG4 expression and location in a wide range of rat organs and reviewed the available literature. ECRG4 mRNA was identified in all examined tissues by quantitative PCR (qPCR). ECRG4 immunoreaction was mainly cytoplasmic, and was detected in heart and skeletal muscles, smooth muscle cells showing only weak reactions. In the digestive system, ECRG4 immunostaining was stronger in the esophageal epithelium, bases of gastric glands, hepatocytes and pancreatic acinar epithelium. In the lymphatic system, immunoreactive cells were detectable in the thymus cortex, lymph node medulla and splenic red pulp. In the central and peripheral nervous systems, different neuronal groups showed different reaction intensities. In the endocrine system, ECRG4 immunoreaction was detected in the hypothalamic paraventricular and supraoptic nuclei, hypophysis, thyroid and parathyroid glands, adrenal zona glomerularis and medulla and Leydig cells, as well as in follicular and luteal cells of the ovary. In the literature, ECRG4 has been reported to inhibit cell proliferation and increase apoptosis in various cell types. It is down-regulated, frequently due to hypermethylation, in esophageal, prostate, breast and colon cancers, together with glioma (oncosuppressor function), although it is up-regulated in papillary thyroid cancer (oncogenic role). ECRG4 expression is also higher in non-proliferating cells of the lymphatic system. In conclusion, our identification of ECRG4 in many structures suggests the involvement of ECRG4 in the tumorigenesis of other organs and also the need for further research. In addition, on the basis of the location of ECRG4 in neurons and endocrine cells and the fact that it can be secreted, its role as a neurotransmitter/neuromodulator and endocrine factor must be examined in depth in the future.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genética , Sequência de Aminoácidos , Animais , Glândulas Endócrinas/citologia , Glândulas Endócrinas/metabolismo , Feminino , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Neurônios/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
KISS1 and its receptor, KISS1R, have both been found to be expressed in central nervous system, but few data are present in the literature about their distribution in peripheral nervous structures. Thus, the aim of the present study was to investigate, through immunohistochemistry, the expression and distribution of KISS1 and KISS1R in the rat and human carotid bodies and superior cervical ganglia, also with particular reference to the different cellular populations. Materials consisted of carotid bodies and superior cervical ganglia were obtained at autopsy from 10 adult subjects and sampled from 10 adult Sprague-Dawley rats. Immunohistochemistry revealed diffuse expression of KISS1 and KISS1R in type I cells of both human and rat carotid bodies, whereas type II cells were negative. In both human and rat superior cervical ganglia positive anti-KISS1 and -KISS1R immunostainings were also selectively found in ganglion cells, satellite cells being negative. Endothelial cells also showed moderate immunostaining for both KISS1 and KISS1R. The expression of both kisspeptins and kisspeptin receptors in glomic type I cells and sympathetic ganglion cells supports a modulatory role of KISS1 on peripheral chemoreception and sympathetic function. Moreover, local changes in blood flow have been considered to be involved in carotid body chemoreceptor discharge and kisspeptins and kisspeptin receptors have also been found in the endothelial cells. As a consequence, a possible role of kisspeptins in the regulation of carotid body blood flow and, indirectly, in chemoreceptor discharge may also be hypothesized.
Assuntos
Corpo Carotídeo/metabolismo , Regulação da Expressão Gênica/fisiologia , Kisspeptinas/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Gânglio Cervical Superior/metabolismo , Adulto , Animais , Corpo Carotídeo/citologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Coelhos , Ratos Sprague-Dawley , Receptores de Kisspeptina-1 , Gânglio Cervical Superior/citologiaRESUMO
Both ghrelin and obestatin are derived from preproghrelin by post-translational processing. The two peptides are secreted into the blood but circulating levels of these peptides have not been assessed in women with ovarian tumours. Therefore, the purpose of this study was to evaluate peripheral blood concentrations of active and total ghrelin and obestatin in patients with benign ovarian tumours and those with ovarian cancer. The studies were conducted on 22 patients operated due to benign ovarian tumours, and 31 patients operated due to ovarian cancer. A control group consisted of 32 women, 24 to 65 years of age. Both in women with benign ovarian tumours and those with ovarian cancer blood concentrations of active ghrelin and obestatin were higher than in the control group (active ghrelin: 90 +/- 4, 84 +/- 4 and 56 +/- 9 pg/ml, respectively, obestatin: 660 +/- 36; 630 +/- 30 and 538 +/- 31 ng/ml (x +/- SE), respectively). In contrast, total ghrelin concentrations in blood were similar in the studied groups. The alterations resulted in increased values of active to total ghrelin concentration ratio in the peripheral blood of patients with benign ovarian tumours or with ovarian cancer (0.79 +/- 0.02 and 0.93 +/- 0.05, respectively vs 0.58 +/- 0.02 in the control group). Due to the absence of any convincing proof for the presence of a functional GHS-R-1a receptor for ghrelin in human ovaries it did not seem probable that the observed elevated levels of active ghrelin and obestatin were directly linked to development of ovarian tumours.
Assuntos
Grelina/sangue , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Hormônios Peptídicos/sangue , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Ghrelin and obestatin originate from the same peptide precursor, preproghrelin. Both peptides are secreted in the blood. We investigated serum active and total ghrelin and obestatin concentrations in women with uterine myomatosis. Serum concentrations of active ghrelin in uterine leiomyoma were significantly higher compared to women in the control group (86 +/- 3 vs 56 +/- 9 pg/ml, respectively; p < 0.02). On the other hand, serum concentrations of total ghrelin and obestatin in uterine leiomyoma did not differ from those in the control group. In the control group the ratio of active to total ghrelin concentrations amounted to 0.62, while in women with uterine myoma it was 0.95, pointing to a prevalence of the active form of ghrelin in women with uterine myoma. Also the ratio of active ghrelin concentration to obestatin concentration was higher in the latter group while the ratio of total circulating ghrelin to obestatin concentrations was similar in the two groups. The data may suggest a role of active ghrelin in the development of a myoma. Moreover, the results indicate that increased blood ratios of active to total ghrelin and to obestatin concentrations are not specific for cachexia.
Assuntos
Grelina/sangue , Leiomioma/sangue , Neoplasias Uterinas/sangue , Adulto , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Expression of the intermediate filament, nestin, was long believed to be restricted to neuroectodermal stem cells. However, nestin expression has recently been detected in several tumors. Since adrenocortical carcinoma, a tumor entity still very difficult to classify, may gain the ability to aberrantly express neuroectodermal proteins including chromogranin A and synaptophysin, we asked the question whether nestin might also be detected in adrenocortical carcinomas, and if so, whether it might serve as a tool for clinical pathology. Therefore, we studied the expression of nestin in normal adrenal glands, adrenocortical adenomas, and adrenocortical cancers using specific immunohistochemistry and semi-quantitative reverse transcriptase-polymerase chain reaction. Immunostaining was nestin-positive in 1 out of 9 normal adrenal glands (11%), 2 out of 20 adrenocortical adenomas (10%), and 13 out of 16 adrenocortical carcinomas (81%). Expression of nestin mRNA could be detected in all microdissected tissues, independently of their grade of dedifferentiation. We conclude that our findings provide further evidence that nestin, as a marker, is not restricted to neuronal stem cells and nestin expression is worth to be studied in adrenocortical tumors.
Assuntos
Neoplasias do Córtex Suprarrenal/patologia , Adenoma Adrenocortical/classificação , Carcinoma Adrenocortical/patologia , Biomarcadores Tumorais/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Glândulas Suprarrenais/metabolismo , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Feminino , Expressão Gênica , Humanos , Proteínas de Filamentos Intermediários/genética , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , NestinaRESUMO
AIM: To test if treatment with GnRH analogue, which leads to a significant reduction in myoma volume, changes expression of leptin genes and gene coding leptin receptor isoforms in uterine myomas and in the surrounding unaltered myometrium. METHODS: Using RT-PCR, expression of leptin genes and leptin receptor genes was studied in myomas and in the surrounding myometrium in women with uterine myomas, untreated or treated with GnRH analogue. In the randomly selected cases presence of leptin protein and of leptin receptor proteins was examined also by Western blotting. RESULTS: Expression of leptin genes was demonstrated both in myomas and in the surrounding myometrium, and a similar pattern of expression was found for leptin receptor isoforms. The results of RT-PCR were confirmed by Western blotting, which documented the identical distribution of leptin proteins and leptin receptor proteins in studied tissues. Treatment with GnRH analogue had no effect on the expression pattern of studied genes. CONCLUSION: The results of the present study on the administration of GnRH analogue to females with myomas suggest that no direct or immediate inter-relationship exists between expression of leptin genes in uterine myomas on one hand and estrogen, progesterone and leptin levels in the blood on the other. Expression seems to be of a more durable nature but factors that induce such expression remain unknown.
Assuntos
Hormônio Liberador de Gonadotropina/farmacologia , Leiomioma/genética , Leptina/genética , Mioma/genética , Miométrio/metabolismo , Receptores de Superfície Celular/genética , Neoplasias Uterinas/genética , Adulto , Western Blotting , Estudos de Casos e Controles , Primers do DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Hormônio Liberador de Gonadotropina/análogos & derivados , Humanos , Leiomioma/metabolismo , Leptina/metabolismo , Pessoa de Meia-Idade , Mioma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores para Leptina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Uterinas/metabolismoRESUMO
AIM: Examination of the potential role of leptin in the development of uterine myomas. Expression of the leptin gene and leptin receptor gene was tested in the myometrium of healthy women, and in myomas and the surrounding myometrium of women with benign tumors. METHODS: Using RT-PCR, expression of the leptin gene and leptin receptor gene were studied in myomas and in the surrounding myometrium in 30 women with uterine myomas at various phases of the menstrual cycle, and in the myometrium of ten women in a control group. Presence of leptin gene proteins and leptin receptor gene proteins in the women was also examined by Western blotting. RESULTS: Using RT-PCR, expression of the leptin gene was demonstrated both in myomas and in the surrounding myometrium. In contrast, expression of the gene could not be detected in the myometrium of healthy women. The results were confirmed by Western blotting, which documented the identical distribution of leptin proteins and leptin receptor proteins in studied tissues. CONCLUSION: Demonstration of the expression of leptin genes and leptin proteins in uterine myomas and in the surrounding myometrium, and their absence in the myometrium of healthy women suggests the involvement of leptin in the development of uterine myomas.
Assuntos
Leiomioma/metabolismo , Leptina/metabolismo , Miométrio/metabolismo , Receptores de Superfície Celular/metabolismo , Neoplasias Uterinas/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Primers do DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Receptores para Leptina , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Orexins A and B are hypothalamic peptides that originate from the proteolytic cleavage of preproorexin and act through two subtypes of receptors, named OX1-R and OX2-R. OX1-R almost exclusively binds orexin-A, whereas OX2-R is nonselective for both orexins. We previously found that orexin-A, via the OX1-R, stimulates cortisol secretion from dispersed human adrenocortical cells. In this study, we demonstrate that six of eight cortisol-secreting adenomas expressed preproorexin mRNA, and seven of 10 adenomas contained measurable amounts of orexin-A but not orexin-B. Normal adrenal cortexes neither expressed preproorexin nor contained orexins. All adenomas expressed OX1-R and OX2-R mRNAs, and real-time PCR showed that the expression of both receptors was up-regulated in adenomas, compared with normal adrenal cortex. Orexin-A concentration-dependently raised basal cortisol secretion from freshly dispersed normal and adenomatous cells, minimal and maximal effective concentrations being 10(-10) and 10(-8) m, and the peptide efficacy (percent increase elicited by 10(-8) m orexin-A) was significantly higher in adenomas than in the normal adrenal cortex. Orexin-B was ineffective, thereby indicating that orexin secretagogue action is mediated by the OX1-R. In contrast, both orexins (10(-8) m) raised the proliferative activity of cultured normal and adenomatous cells, suggesting that this effect is mediated by OX2-R or both receptor subtypes. Collectively, our findings allow us to conclude that the orexin system is overexpressed in cortisol-secreting adenomas and suggest that orexin-A may act as an autocrine-paracrine regulator of the secretory activity and growth of some of these adrenal tumors.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Hidrocortisona/metabolismo , Receptores de Neuropeptídeos/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Primers do DNA , DNA Complementar/genética , Amplificação de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Neuropeptídeos/farmacologia , Receptores de Orexina , Orexinas , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Receptores Acoplados a Proteínas G , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Neuropeptides B and W (NPB and NPW) are regulatory peptides that act via two subtypes of G protein-coupled receptors, named GPR7 and GPR8. RT-PCR demonstrated the expression of these receptors in both zona glomerulosa and zona fasciculata-reticularis (ZF/R) cells of the human adrenal cortex. NPB and NPW did not affect aldosterone secretion from dispersed zona glomerulosa cells but enhanced cortisol production from ZF/R cells, NPB being more effective than NPW. NPB evoked sizable cAMP and inositol triphosphate responses from ZF/R cells, which were abrogated by the adenylate cyclase inhibitor SQ-22536 and the phospholipase C inhibitor U-73122, respectively. Cortisol response to NPB was lowered by either SQ-22536 and the protein kinase (PK) A inhibitor H-89 or U-73122 and the PKC inhibitor calphostin-C and abolished by the simultaneous exposure to H-89 and calphostin-C. NPW elicited only a rise in cAMP production from dispersed ZF/R cells, and its cortisol response was suppressed by both SQ-22536 and H-89. PreproNPB and preproNPW mRNAs were detected in human adrenal cortexes. We conclude that: 1) NPB and NPW exert a secretagogue action on human ZF/R cells, probably acting in an autocrine-paracrine manner; and 2) the effect of NPB is mediated by both the adenylate cyclase/PKA and the phospholipase C/PKC cascades, whereas that of NPW involves only the activation of the former signaling pathway.
Assuntos
Adenilil Ciclases/metabolismo , Córtex Suprarrenal/fisiologia , Hidrocortisona/metabolismo , Neuropeptídeos/farmacologia , Receptores de Neuropeptídeos/genética , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/metabolismo , Aldosterona/metabolismo , AMP Cíclico/metabolismo , Primers do DNA , Humanos , RNA Mensageiro/genética , Receptores Acoplados a Proteínas G , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroide 11-beta-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/genética , Zona Fasciculada/fisiologia , Zona Glomerulosa/fisiologia , Zona Reticular/fisiologiaRESUMO
Pneumadin (PNM) is a decapeptide originally isolated from mammalian lungs, and exerts a potent antidiuretic action by stimulating arginine-vasopressin release. We have recently developed a sensitive and specific radioimmunoassay (RIA) for rat PNM and detected high concentrations of PNM--not only in the rat lungs, but also in the prostate. Hence, we investigated whether prostate PNM content is regulated by sex hormones. Male adult rats were orchidectomized or sham-operated and given a subcutaneous injection of testosterone or estradiol (40 and 5 mg/kg), respectively. The animals were decapitated one week after surgery, and their ventral prostates were promptly removed and weighed. PNM concentration and localization in the prostate were investigated by RIA and immunocytochemistry (ICC). Orchidectomy resulted in significant decreases in the prostate weight and PNM concentration, and testosterone administration prevented these effects. Estradiol administration to sham-operated rats caused prostate atrophy without changing PNM concentration. ICC localized PNM immunoreactivity (IR) exclusively in the epithelial cells of the ventral prostate. Orchidectomy markedly reduced PNM-IR concentration, while testosterone abolished this effect. Estradiol did not modify PNM-IR concentration in the atrophic prostate of sham-operated rats. We conclude that PNM content of rat prostate is dependent on the presence of adequate levels of circulating testosterone. The possibility that PNM plays a key role in the maintenance of the prostate growth is unlikely since estradiol-induced gland atrophy is not associated with any decrease in PNM concentration. The localization of PNM in the epithelial cells could suggest that this peptide may be involved in the regulation of some testosterone-dependent secretory functions of the rat prostate.
Assuntos
Hormônios Esteroides Gonadais/fisiologia , Oligopeptídeos/metabolismo , Próstata/metabolismo , Animais , Estradiol/administração & dosagem , Estradiol/farmacologia , Imuno-Histoquímica , Injeções Subcutâneas , Masculino , Orquiectomia , Concentração Osmolar , Radioimunoensaio , Ratos , Ratos Wistar , Testosterona/administração & dosagem , Testosterona/farmacologia , Distribuição TecidualRESUMO
Cholecystokinin (CCK) IS a regulatory peptide that acts via two receptor subtypes, CCK1-R and CCK2-R. RT-PCR demonstrated the expression of both CCK1-R and CCK2-R in the zona glomerulosa (ZG), but not zona fasciculata-reticularis cells of the human adrenal cortex. CCK and the CCK2-R agonist pentagastrin enhanced basal aldosterone secretion from ZG cells without affecting cortisol production from zona fasciculata-reticularis cells. The aldosterone response to CCK and pentagastrin was suppressed by a CCK2-R antagonist, but not by a CCK1-R antagonist. Pentagastrin evoked a sizeable cAMP, but not inositol triphosphate, response from ZG cells, whereas CCK plus CCK2-R antagonist was ineffective. The cAMP response to pentagastrin was abrogated by CCK2-R antagonist or the adenylate cyclase inhibitor SQ-22536, and the aldosterone response was abolished by both SQ-22536 and the protein kinase A inhibitor H-89. Both CCK and pentagastrin increased steroidogenic acute regulatory protein mRNA expression in ZG cells; the effect was abrogated by CCK2-R antagonist. We conclude that CCK exerts secretagogue action on human ZG cells, acting through CCK2-Rs coupled to the adenylate cyclase/protein kinase A signaling cascade, which, in turn, stimulates the expression of steroidogenic acute regulatory protein, the rate-limiting step of steroidogenesis.
Assuntos
Adenilil Ciclases/metabolismo , Córtex Suprarrenal/enzimologia , Aldosterona/metabolismo , Colecistocinina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor de Colecistocinina B/metabolismo , Córtex Suprarrenal/citologia , Córtex Suprarrenal/efeitos dos fármacos , Adulto , AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Pessoa de Meia-Idade , Pentagastrina/farmacologia , RNA Mensageiro/análise , Receptor de Colecistocinina A/genética , Receptor de Colecistocinina A/metabolismo , Receptor de Colecistocinina B/genética , Transdução de Sinais/fisiologiaRESUMO
VIP and pituitary adenylate cyclase-activating polypeptide (PACAP) are two regulatory peptides that possess remarkable amino acid sequence homology and act through common receptors, named PAC(1), VPAC(1), and VPAC(2). PAC(1) receptor is selective for PACAP, whereas VPAC(1) and VPAC(2) receptors bind both VIP and PACAP. We have investigated the expression and function of VIP, PACAP, and their receptors in the zona glomerulosa (ZG), zonae fasciculata and reticularis, and adrenal medulla (AM) of the human adrenal cortex. RT-PCR and RIA detected VIP and PACAP expression exclusively in AM cells. RT-PCR demonstrated the presence of PAC(1) mRNA only in AM and of VPAC(1) and VPAC(2) mRNAs in both ZG and AM cells. VIP and PACAP concentration-dependently increased aldosterone and catecholamine secretion from cultured ZG and AM cells. The catecholamine response to both peptides was higher than the aldosterone response, and the secretagogue action of PACAP was more intense than that of VIP. The aldosterone response of cultured ZG cells to VIP or PACAP was unaffected by the PAC(1) receptor antagonist PACAP-(6-38) (PAC(1)-A), but was significantly decreased by the VPAC(1) receptor antagonist [Ac-His(1),D-Phe(2),Lys(15),Arg(16)]VIP-(3-7),GH-releasing factor-(8-27)-NH(2) (VPAC(1)-A). The catecholamine response of cultured AM cells to VIP was lowered by VPAC(1)-A and unaffected by PAC(1)-A; conversely, the catecholamine response to PACAP was reduced by both PAC(1)-A and VPAC(1)-A. Simultaneous exposure to both antagonists did not abolish the catecholamine response to PACAP. Collectively, our findings allow us to conclude that in human adrenals 1) VIP and PACAP biosynthesis exclusively occurs in AM cells; 2) ZG cells are provided with functional VPAC(1) and VPAC(2) receptors, whose activation by VIP or PACAP elicits a moderate aldosterone response; 3) AM cells possess PAC(1), VPAC(1), and VPAC(2) receptors, whose activation evokes a marked catecholamine response; and 4) the catecholamine response to PACAP is more intense than that to VIP, because it is mediated by all subtypes of VIP/PACAP receptors.
Assuntos
Glândulas Suprarrenais/fisiologia , Neuropeptídeos/fisiologia , Receptores do Hormônio Hipofisário/fisiologia , Receptores de Peptídeo Intestinal Vasoativo/fisiologia , Peptídeo Intestinal Vasoativo/metabolismo , Córtex Suprarrenal/metabolismo , Medula Suprarrenal/metabolismo , Adulto , Aldosterona/metabolismo , Catecolaminas/metabolismo , Humanos , Pessoa de Meia-Idade , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Distribuição Tecidual , Zona Glomerulosa/metabolismoRESUMO
Adrenomedullin (ADM) is a hypotensive peptide, that derives from the proteolytic cleavage of pro(p)ADM and acts through two subtypes of receptors, called L1-receptor (L1-R) and calcitonin receptor-like receptor (CRLR). CRLR may function as a calcitonin gene-related peptide or a selective ADM receptor depending on the expression of the subtype 1 or the subtypes 2 and 3 of a family of proteins, named receptor-activity modifying proteins (RAMPs). Reverse transcription (RT)-polymerase chain reaction (PCR) allowed the detection of pADM mRNA in dispersed cells of eight Conn's adenomas (aldosteronomas). These cells also expressed peptidyl-glycine alpha-amidating monooxigenase, the enzyme converting immature ADM to the mature form, and contained sizeable amounts of ADM-immunoreactivity as measured by radioimmunoassay. RT-PCR also demonstrated the presence in aldosteronoma cells of the specific mRNAs of L1-R, CRLR and RAMPs 1-3. ADM (10(-8) M) inhibited angiotensin-II (10(-9) M)-simulated aldosterone secretion from cultured aldosteronoma cells, without affecting basal production. ADM (10(-8) M) also enhanced basal proliferation rate of cultured cells, as estimated by the 5-bromo-2'-deoxyuridine immunocytochemical technique. Both effects of ADM were annulled by the ADM-receptor selective antagonist ADM22-52 (10(-7) M). In conclusion, our study provides evidence that aldosteronoma cells express both ADM and ADM22-52-sensitive receptors. These findings, coupled with the demonstration that ADM exerts an aldosterone antisecretagogue action and a proliferogenic effect on cultured aldosteronoma cells, make it likely that endogenous ADM system plays a potentially important role in the paracrine or autocrine functional control of Conn's adenomas.
Assuntos
Adenoma/genética , Neoplasias do Córtex Suprarrenal/genética , Hiperaldosteronismo/genética , Peptídeos/genética , Receptores de Peptídeos/genética , Adenoma/patologia , Neoplasias do Córtex Suprarrenal/patologia , Adrenomedulina , Aldosterona/metabolismo , Angiotensina II/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Hiperaldosteronismo/patologia , Peptídeos/farmacologia , Peptídeos/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Radioimunoensaio , Receptores de Adrenomedulina , Receptores de Peptídeos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Orexins A and B are hypothalamic peptides, that act through two receptor subtypes, called OX1-R and OX2-R. OX1-R selectively binds orexin A, whereas OX2-R is nonselective for both orexins. High levels of OX1-R mRNA and low levels of OX2-R mRNA have been previously detected in the human adrenal cortex and medulla. Here we demonstrated by RT-PCR the expression of the OX2-R, but not the OX1-R, gene in 10 benign secreting pheochromocytomas. Both orexins A and B stimulated catecholamine secretion from pheochromocytoma slices; the maximal effective concentration was 10(-8) mol/liter. Orexins A and B (10(-8) mol/liter) increased IP3, but not cAMP production, by tumor slices, and the effect was blocked by the PLC inhibitor U-73122. The catecholamine response to 10(-8) mol/liter orexins A and B was abolished by either U-73122 or the PKC antagonist calphostin C and was unaffected by the adenylate cyclase inhibitor SQ-22536 and the PKA inhibitor H-89. Collectively, these findings suggest that orexins stimulate catecholamine secretion from human pheochromocytomas, acting through OX2-R coupled to the PLC-PKC signaling pathway.
Assuntos
Neoplasias das Glândulas Suprarrenais/metabolismo , Proteínas de Transporte/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Neuropeptídeos/farmacologia , Feocromocitoma/metabolismo , Receptores de Neuropeptídeos/genética , Relação Dose-Resposta a Droga , Estrenos/farmacologia , Humanos , Receptores de Orexina , Orexinas , Proteína Quinase C/fisiologia , Pirrolidinonas/farmacologia , RNA Mensageiro/análise , Receptores Acoplados a Proteínas G , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fosfolipases Tipo C/fisiologiaRESUMO
Adrenomedullin (AM) (10(-8) M) partially suppressed aldosterone response of dispersed rat zona glomerulosa (ZG) cells to 10 mM K+, and the nitric oxide (NO) synthase inhibitors L-NAME (10(-3) M) and 1400W (10(-4) M) effectively counteracted this effect of AM. The NO donor L-Arginine (L-Arg) (10(-5) M) decreased both basal and K+ -stimulated aldosterone secretion. The guanylate-cyclase inhibitor Ly-83583, at a concentration (10(-4) M) abolishing either the guanylate-cyclase activator guanylin- or L-Arg-induced cGMP release from dispersed ZG cells, did not affect the aldosterone antisecretagogue action of AM and L-Arg. AM (10(-8) M) evoked a moderate increase in cGMP release by dispersed ZG cells, and the effect was blocked by both 10(-4) M Ly-83583 and 10(-3) M L-NAME. Collectively, these findings allow us (1) to confirm that NO inhibits aldosterone secretion through a cGMP-independent mechanism; and (2) to suggest that stimulation of endogenous NO synthesis plays a role in the mechanisms underlying the inhibitory effect of AM on K+ -stimulated aldosterone secretion from rat ZG cells.
Assuntos
Aldosterona/metabolismo , Óxido Nítrico/metabolismo , Peptídeos/farmacologia , Adrenomedulina , Aldosterona/farmacologia , Amidinas/farmacologia , Aminoquinolinas/farmacologia , Animais , Arginina/farmacologia , Benzilaminas/farmacologia , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Potássio/farmacologia , Ratos , Ratos Wistar , Zona Glomerulosa/metabolismoRESUMO
Endothelin-1 (ET-1) is a 21-amino acid residue (ET-1[1-21]) hypertensive peptide, which together with its receptor subtypes A and B (ETA and ETB) is expressed in the rat adrenal cortex, where it stimulates steroid-hormone (aldosterone and corticosterone) secretion through the ETB receptor and the growth (proliferative activity) of the zona glomerulosa (ZG) through the ETA receptor. ET-1[1-21] is generated from bigET-1 by the endothelin-converting enzyme (ECE-1). However, recent evidence indicates the existence of an alternative chymase-mediated biosynthetic pathway leading to the production of an ET-1[1-31] peptide, which was found to reproduce the ETA receptor-mediated vascular effects of ET-1[1-21]. We found that ET-1[1-21], but not ET-1[1-31], concentration-dependently raised steroid secretion from dispersed rat adrenocortical cells, its effect being blocked by the ETB-receptor selective antagonist BQ-788. Both ET-1s concentration-dependently increased the number of "S-phase" cells (as detected by the 5-bromo-2'-deoxyuridine immunocytochemical method) in capsule-ZG strips within a 240 min incubation. The ZG proliferogenic action of both ET-1s was blocked by the ETA-receptor antagonist BQ-123, and ET-1[1-31] was found to be significantly more potent than ET-1[1-21]. Autoradiography showed that in the rat adrenal ET-1[1-21] displaced the binding of selective ligands to both ETA ([125I]PD-151242) and ETB receptors ([125I]BQ-3020), while ET-1[1-31] eliminates only the binding to ETA receptors. Collectively, our findings provide strong evidence that ET-1[1-31] acts in the rat adrenal glands as a selective ETA-receptor agonist, mainly involved in the stimulation of ZG proliferative activity.
Assuntos
Corticosteroides/metabolismo , Córtex Suprarrenal/metabolismo , Glândulas Suprarrenais/anatomia & histologia , Antagonistas dos Receptores de Endotelina , Endotelina-1/farmacologia , Zona Glomerulosa/efeitos dos fármacos , Córtex Suprarrenal/citologia , Córtex Suprarrenal/fisiologia , Animais , Autorradiografia , Bromodesoxiuridina/metabolismo , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores de Endotelina/fisiologia , Fase S/efeitos dos fármacos , Zona Glomerulosa/citologia , Zona Glomerulosa/fisiologiaRESUMO
BACKGROUND: Adrenal zona glomerulosa cells are provided with adrenomedullin receptors. Adrenomedullin has recently been found to enhance proliferation of cultured rat vascular smooth muscle cells and zona glomerulosa cells. OBJECTIVE: To investigate whether adrenomedullin affects rat zona glomerulosa proliferative activity through the tyrosine kinase and extracellular signal regulated kinases (ERKs) pathways. METHODS: Dispersed rat zona glomerulosa cells were cultured in vitro for 24 h and then exposed to adrenomedullin (10(-7) mol/l), alone or in the presence of tyrphostin-23 (10(-5) mol/l) or PD-98059 (10(-4) mol/l), for 24 or 48 h. To assess the rate of DNA synthesis, 5-bromo-2'-deoxyuridine (BrdU, 20 mg/ml) was also added to the medium and BrdU-positive cells were detected by immunocytochemistry. The expression of ERKs and the effect of adrenomedullin on ERKs phosphorylation and activity were assayed in dispersed zona glomerulosa cells. RESULTS: Adrenomedullin significantly increased the percentage of BrdU-positive (phase-S) zona glomerulosa cells; this effect was blocked by either the tyrosine kinase inhibitor, tyrphostin-23, or the mitogen-activated protein kinase kinase (MEK-1) inhibitor, PD-98059. Both zona glomerulosa and zona fasciculata/reticularis express ERK-1 (44 kDa) and ERK-2 (42 kDa) isoforms. However, adrenomedullin phosphorylated ERK-1 and ERK-2 only in the zona glomerulosa; this effect was blunted by the MEK-1 inhibitor, PD98059, and by the calcitonin gene-related peptide type 1 (CGRP-1) receptor antagonist, CGRP8-37, but not by the adrenomedullin C-terminal fragment, ADM22-52. CONCLUSION: Adrenomedullin stimulates the growth of rat zona glomerulosa cells through activation of CGRP-1 receptor, linked to the tyrosine kinase-MEK-1-ERKs signalling pathway. These results confirm the complex role played by this peptide in the regulation of zona glomerulosa cell physiology.
Assuntos
DNA/biossíntese , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peptídeos/farmacologia , Zona Glomerulosa/metabolismo , Adrenomedulina , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/fisiologia , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Zona Fasciculada/citologia , Zona Fasciculada/metabolismo , Zona Glomerulosa/citologiaRESUMO
BACKGROUND: 11beta-hydroxysteroid dehydrogenase Type-2 (11beta-HSD2) is an unidirectional enzyme that catalyzes the conversion of glucocorticoid hormones cortisol and corticosterone (B) into their corresponding inactive forms, cortisone, and 11-dehydrocorticosterone (DH-B). We have provided evidence that 11beta-HSD2 is expressed as messenger RNA (mRNA) and protein in human adrenocortical cells, where its activity is inhibited in vitro by the main glucocorticoid agonists, adrenocorticotropic hormone (ACTH) and angiotensin-II. It seemed worthwhile, therefore, to study the gene expression and activity of 11beta-HSD2 in cortisol-secreting adrenocortical adenomas. METHODS: Three adrenal adenomas that produced Cushing syndrome were recruited. Three normal adrenal glands were obtained from patients who underwent unilateral nephrectomy with ipsilateral adrenalectomy for renal cancer. 11beta-HSD2 gene expression was studied by reverse transcriptionpolymerase chain reaction (RT-PCR) in adenoma and normal adrenocortical tissue. Cortisol, B, cortisone, and DH-B production by adenoma and adrenal slices in vitro was assayed by quantitative high-performance liquid chromatography (HPLC), and the activity of 11beta-HSD2 was evaluated by measuring the conversion of [3H]-cortisol to [3H]-cortisone. RESULTS: RT-PCR allowed the detection of the 11beta-HSD2 mRNA in the three adrenal adenomas and normal adrenal cortices examined. Under basal conditions, adenoma slices secreted higher amounts of cortisol and B, but markedly lower amounts of cortisone and DH-B than adrenal slices. ACTH raised cortisol and B production from both specimens, and it lowered cortisone and DH-B yield. The level basal conversion of [3H]-cortisol to [3H]-cortisone was notably less in adenomas than in adrenals, and ACTH decreased it in both tissues. CONCLUSIONS: Collectively, our findings indicate that cortisol-secreting adrenal adenomas express the 11beta-HSD2 gene, but the activity of the enzyme is suppressed in adenomas when compared with the normal adrenal cortex. We advance the hypothesis that the elevated local concentration of steroid hormones that occur in adenomas down-regulates 11beta-HSD2 activity, thereby contributing to their abnormal steroidogenic function.
Assuntos
Adenoma/enzimologia , Neoplasias das Glândulas Suprarrenais/enzimologia , Hidrocortisona/metabolismo , Hidroxiesteroide Desidrogenases/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2 , Adenoma/metabolismo , Neoplasias das Glândulas Suprarrenais/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Adulto , Angiotensina II/farmacologia , Regulação Enzimológica da Expressão Gênica , Humanos , Hidroxiesteroide Desidrogenases/metabolismoRESUMO
Glucagon-receptor mRNA was detected by reverse transcription-polymerase chain reaction in three human pheochromocytomas, but not in four normal adrenal medullas. Quantitative autoradiography demonstrated the presence of abundant [(125)I-Thyr(10)]glucagon binding sites in pheochromocytomas, which were displaced by both cold glucagon and the glucagon receptor antagonist Des-His(1)[Glu(9)]glucagon amide (GR-A). Adrenal medulla was weakly labeled, and the binding was not displaced by GR-A. Glucagon enhanced epinephrine and norepinephrine release by pheochromocytoma slices, minimal and maximal effective concentrations being 10(-8) M and 10(-6) M. Adrenomedullary slices evidenced a weak catecholamine response only to 10(-5) M glucagon. GR-A abolished the secretory response to glucagon of pheochromocytomas, but not of adrenal medullas. Collectively, these findings indicate that human pheochromocytomas, but not adrenal medulla, express glucagon receptors and possess a marked secretory response to glucagon, thereby providing the rationale to explain the specificity of the glucagon provocative test in the diagnosis of pheochromocytoma.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Medula Suprarrenal/metabolismo , Glucagon/farmacologia , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Sequência de Bases , Primers do DNA , Humanos , Feocromocitoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The possible involvement of endogenous vasoactive intestinal peptide (VIP), cholecystokinin (CCK) and neurotensin (NT) in the control of thymocyte proliferation has been investigated in vivo in the immature rat. For this task, we have studied the effects of the administration of selective antagonists of the receptors of the three neuropeptides on the mitotic index (% of metaphase-arrested cells after vincristin injection) of thymocytes. Both CCK- and TN-receptor antagonists were ineffective. In contrast, two VIP receptor antagonists (VIP-As) enhanced the mitotic index of thymocytes. VIP reversed the effect of VIP-As, but when administered alone it did not alter the mitotic activity of thymocytes. In light of these findings, we conclude that endogenous VIP exerts a maximal tonic inhibitory influence on the basal proliferative activity of rat thymocytes, while endogenous CCK and NT do not play a relevant modulatory role in this process.