RESUMEN
OBJECTIVES: To characterize the splicing machinery (SM) of leukocytes from primary antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE) and antiphospholipid syndrome with lupus (APS + SLE) patients, and to assess its clinical involvement. METHODS: Monocytes, lymphocytes and neutrophils from 80 patients (22 APS, 35 SLE and 23 APS + SLE) and 50 HD were purified, and 45 selected SM components were evaluated by qPCR-microfluidic array. Relationship with clinical features and underlying regulatory mechanisms were assessed. RESULTS: APS, SLE and APS + SLE leukocytes displayed significant and specific alterations in SM-components (SMC), associated with clinical features [autoimmune profiles, disease activity, lupus nephritis (LN), and CV-risk markers]. A remarkable relationship among dysregulated SMC in monocytes and the presence of LN in SLE was highlighted, revealing a novel pathological mechanism, which was further explored. Immunohistology analysis of renal biopsies highlighted the pathological role of the myeloid compartment in LN. Transcriptomic analysis of monocytes from SLE-LN(+) vs SLE-LN(-) identified 271 genes differentially expressed, mainly involved in inflammation and IFN-signaling. Levels of IFN-related genes correlated with those of SMC in SLE-LN(+). These results were validated in two external SLE-LN(+) datasets of whole-blood and kidney biopsies. In vitro, SLE-LN(+)-serum promoted a concomitant dysregulation of both, the IFN signature and several SMC, further reversed by JAKinibs treatment. Interestingly, IFNs, key inflammatory cytokines in SLE pathology, also altered SMC. Lastly, the over/down-expression of selected SMC in SLE-monocytes reduced the release of inflammatory cytokines and their adhesion capacity. CONCLUSION: Overall, we have identified, for the first time, a specific alteration of SMC in leukocytes from APS, SLE and APS + SLE patients that would be responsible for the development of distinctive clinical profiles.
Asunto(s)
Síndrome Antifosfolípido , Lupus Eritematoso Sistémico , Nefritis Lúpica , Humanos , Inflamación , CitocinasRESUMEN
In addition to its essential role in the physiological control of longitudinal growth, growth-hormone (GH) is endowed with relevant metabolic functions, including anabolic actions in muscle, lipolysis in adipose-tissue and glycemic modulation. Adult obesity is known to negatively impact GH-axis, thereby promoting a vicious circle that may contribute to the exacerbation of the metabolic complications of overweight. Yet, to what extent early-overnutrition sensitizes the somatotropic-axis to the deleterious effects of obesity remains largely unexplored. Using a rat-model of sequential exposure to obesogenic insults, namely postnatal-overfeeding during lactation and high-fat diet (HFD) after weaning, we evaluated in both sexes the individual and combined impact of these nutritional challenges upon key elements of the somatotropic-axis. While feeding HFD per se had a modest impact on the adult GH-axis, early overnutrition had durable effects on key elements of the somatotropic-system, which were sexually different, with a significant inhibition of pituitary gene expression of GH-releasing hormone-receptor (GHRH-R) and somatostatin receptor-5 (SST5) in males, but an increase in pituitary GHRH-R, SST2, SST5, GH secretagogue-receptor (GHS-R) and ghrelin expression in females. Notably, early-overnutrition sensitized the GH-axis to the deleterious impact of HFD, with a significant suppression of pituitary GH expression in both sexes and lowering of circulating GH levels in females. Yet, despite their similar metabolic perturbations, males and females displayed rather distinct alterations of key somatotropic-regulators/ mediators. Our data document a synergistic effect of postnatal-overnutrition on the detrimental impact of HFD-induced obesity on key elements of the adult GH-axis, which is conducted via mechanisms that are sexually-divergent.
Asunto(s)
Dieta Alta en Grasa , Hormona del Crecimiento/metabolismo , Obesidad/etiología , Hipernutrición/complicaciones , Caracteres Sexuales , Animales , Peso Corporal , Femenino , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Modelos Biológicos , Obesidad/genética , Especificidad de Órganos , Hipernutrición/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Receptores de Somatotropina/genética , Receptores de Somatotropina/metabolismoRESUMEN
The anterior pituitary gland is a key organ involved in the control of multiple physiological functions including growth, reproduction, metabolism and stress. These functions are controlled by five distinct hormone-producing pituitary cell types that produce growth hormone (somatotropes), prolactin (lactotropes), adrenocorticotropin (corticotropes), thyrotropin (thyrotropes) and follicle stimulating hormone/luteinizing hormone (gonadotropes). Classically, the synthesis and release of pituitary hormones was thought to be primarily regulated by central (neuroendocrine) signals. However, it is now becoming apparent that factors produced by pituitary hormone targets (endocrine and non-endocrine organs) can feedback directly to the pituitary to adjust pituitary hormone synthesis and release. Therefore, pituitary cells serve as sensors to integrate central and peripheral signals in order to fine-tune whole-body homeostasis, although it is clear that pituitary cell regulation is species-, age- and sex-dependent. The purpose of this review is to provide a comprehensive, general overview of our current knowledge of both central and peripheral regulators of pituitary cell function and associated intracellular mechanisms, focusing on human and non-human primates.
Asunto(s)
Hipófisis/metabolismo , Primates/metabolismo , Transducción de Señal , Animales , Humanos , Modelos AnimalesRESUMEN
Somatostatin (SST) and cortistatin (CORT) are two highly related neuropeptides involved in the regulation of various endocrine secretions. In particular, SST and CORT are two primary negative regulators of GH secretion. Consequently, single SST or CORT knockout mice exhibit elevated GH levels; however, this does not lead to increased IGF-1 levels or somatic growth. This apparent lack of correspondence has been suggested to result from compensatory mechanisms between both peptides. To test this hypothesis, in this study we explored, for the first time, the consequences of simultaneously deleting endogenous SST and CORT by generating a double SST/CORT knockout mouse model and exploring its endocrine and metabolic phenotype. Our results demonstrate that simultaneous deletion of SST and CORT induced a drastic elevation of endogenous GH levels, which, surprisingly, did not lead to changes in growth rate or IGF-1 levels, suggesting the existence of additional factors/systems that, in the absence of endogenous SST and CORT, could counteract GH actions. Notably, elevation in circulating GH levels were not accompanied by changes in pituitary GH expression or by alterations in the expression of its main regulators (GHRH and ghrelin) or their receptors (GHRH receptor, GHS receptor, or SST/CORT receptors) at the hypothalamic or pituitary level. However, although double-SST/CORT knockout male mice exhibited normal glucose and insulin levels, they had improved insulin sensitivity compared with the control mice. Therefore, these results suggest the existence of an intricate interplay among the known (SST/CORT), and likely unknown, inhibitory components of the GH/IGF-1 axis to regulate somatic growth and glucose/insulin homeostasis.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Neuropéptidos/uso terapéutico , Somatostatina/uso terapéutico , Animales , Femenino , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
CONTEXT: Desmopressin is a synthetic agonist of vasopressin receptors (AVPRs). The desmopressin stimulation test is used in the diagnosis and postsurgery prognosis of Cushing disease (CD). However, the cellular and molecular mechanisms underlying the desmopressin-induced ACTH increase in patients with CD are poorly understood. OBJECTIVE: The objectives of this study were to determine, for the first time, whether desmopressin acts directly and exclusively on pituitary corticotropinoma cells to stimulate ACTH expression/release and to elucidate the cellular and molecular mechanisms involved in desmopressin-induced ACTH increase in CD. DESIGN: A total of 8 normal pituitaries (NPs), 23 corticotropinomas, 14 nonfunctioning pituitary adenomas, 17 somatotropinomas, and 3 prolactinomas were analyzed for AVPR expression by quantitative real-time RT-PCR. Primary cultures derived from corticotropinomas, nonfunctioning pituitary adenomas, somatotropinomas, prolactinomas, and NPs were treated with desmopressin, and ACTH secretion/expression, [Ca(2+)]i kinetics, and AVPR expression and/or proliferative response were evaluated. The relationship between AVPR expression and plasma adrenocorticotropin/cortisol levels obtained from desmopressin tests was assessed. RESULTS: Desmopressin affects all functional parameters evaluated in corticotropinoma cells but not in NPs or other pituitary adenomas cells. These effects might be due to the dramatic elevation of AVPR1b expression levels found in corticotropinomas. In line with this notion, the use of an AVPR1b antagonist completely blocked desmopressin stimulatory effects. Remarkably, only AVPR1b expression was positively correlated with elevated plasma adrenocorticotropin levels in corticotropinomas. CONCLUSIONS: The present results provide a cellular and molecular basis to support the desmopressin stimulation test as a reliable, specific test for the diagnosis and postsurgery prognosis of CD. Furthermore, our data indicate that AVPR1b is responsible for the direct/exclusive desmopressin stimulatory pituitary effects observed in CD, thus opening the possibility of exploring AVPR1b antagonists as potential therapeutic tools for CD treatment.
Asunto(s)
Hormona Adrenocorticotrópica/sangre , Desamino Arginina Vasopresina , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/diagnóstico , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/metabolismo , Receptores de Vasopresinas/metabolismo , Adenoma/sangre , Adenoma/diagnóstico , Adenoma/cirugía , Anciano , Humanos , Masculino , Persona de Mediana Edad , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT)/cirugía , Pruebas de Función Hipofisaria , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Neoplasias Hipofisarias/sangre , Neoplasias Hipofisarias/diagnóstico , Neoplasias Hipofisarias/cirugía , PronósticoRESUMEN
Digital investigation methods are becoming more and more important due to the proliferation of digital crimes and crimes involving digital evidence. Network forensics is a research area that gathers evidence by collecting and analysing network traffic data logs. This analysis can be a difficult process, especially because of the high variability of these attacks and large amount of data. Therefore, software tools that can help with these digital investigations are in great demand. In this paper, a novel approach to analysing and visualising network traffic data based on growing hierarchical self-organising maps (GHSOM) is presented. The self-organising map (SOM) has been shown to be successful for the analysis of highly-dimensional input data in data mining applications as well as for data visualisation in a more intuitive and understandable manner. However, the SOM has some problems related to its static topology and its inability to represent hierarchical relationships in the input data. The GHSOM tries to overcome these limitations by generating a hierarchical architecture that is automatically determined according to the input data and reflects the inherent hierarchical relationships among them. Moreover, the proposed GHSOM has been modified to correctly treat the qualitative features that are present in the traffic data in addition to the quantitative features. Experimental results show that this approach can be very useful for a better understanding of network traffic data, making it easier to search for evidence of attacks or anomalous behaviour in a network environment.
Asunto(s)
Redes de Comunicación de Computadores/legislación & jurisprudencia , Crimen , Ciencias Forenses , Internet/legislación & jurisprudencia , Algoritmos , Análisis por Conglomerados , Redes Neurales de la Computación , Neuronas/fisiología , Dinámicas no Lineales , Programas InformáticosRESUMEN
Somatostatin receptors (sst1-5) are present in different types of tumors, where they inhibit key cellular processes such as proliferation and invasion. Although ssts are densely expressed in breast cancer, especially sst2, their role and therapeutic potential remain uncertain. Recently, we identified a new truncated sst5 variant, sst5TMD4, which is related to the abnormal response of certain pituitary tumors to treatment with somatostatin analogs. Here, we investigated the possible role of sst5TMD4 in breast cancer. This study revealed that sst5TMD4 is absent in normal mammary gland, but is abundant in a subset of poorly differentiated human breast tumors, where its expression correlated to that of sst2. Moreover, in the MCF-7 breast cancer model cell, sst5TMD4 expression increased malignancy features such as invasion and proliferation abilities (both in cell cultures and nude mice). This was likely mediated by sst5TMD4-induced increase in phosphorylated extracellular signal-regulated kinases 1 and 2 and p-Akt levels, and cyclin D3 and Arp2/3 complex expression, which also led to mesenchymal-like phenotype. Interestingly, sst5TMD4 interacts physically with sst2 and thereby alters its signaling, enabling disruption of sst2 inhibitory feedback and providing a plausible basis for our findings. These results suggest that sst5TMD4 could be involved in the pathophysiology of certain types of breast tumors.
Asunto(s)
Neoplasias de la Mama/metabolismo , Variación Genética , Receptores de Somatostatina/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Humanos , MAP Quinasa Quinasa 1/metabolismo , Ratones , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias , Proteína Oncogénica v-akt/metabolismo , Fosforilación , Pronóstico , Somatostatina/fisiologíaRESUMEN
Somatostatin and cortistatin have been shown to act directly on pituitary somatotrophs to inhibit growth hormone (GH) release. However, previous results from nonprimate species indicate that these peptides can also directly stimulate GH secretion, at low concentrations. The relevance of this phenomenon in a nonhuman primate model was investigated in the present study by testing the impact of somatostatin/cortistatin on GH release in primary pituitary cell cultures from baboons. High doses (> 10(-10) m) of somatostatin/cortistatin did not alter basal GH secretion but blocked GH-releasing hormone (GHRH)- and ghrelin-induced GH release. However, at low concentrations (10(-17)-10(-13) m), somatostatin/cortistatin dramatically stimulated GH release to levels comparable to those evoked by GHRH or ghrelin. Use of somatostatin receptor (sst) specific agonists/antagonists, and signal transduction blockers indicated that sst2 and sst1 activation via intact adenylate cylcase and mitogen-activated protein kinase systems mediated the inhibitory actions of high-concentration somatostatin. By contrast, the stimulatory actions of low-dose somatostatin on GH release were mediated by sst5 signalling through adenylate cylcase/cAMP/protein kinase A and intracellular Ca(2+) pathways, and were additive with ghrelin (not GHRH). Notably, low-concentrations of somatostatin, similar to sst5-agonists, inhibited prolactin release. These results clearly demonstrate that the ultimate impact of somatostatin/cortistatin on hormone release is dose-dependent, cell type-selective and receptor-specific, where the stimulatory effects of low-concentration somatostatin/cortistatin on GH release extend to primates, thereby supporting the notion that this action is relevant in regulating GH secretion in humans.
Asunto(s)
AMP Cíclico/fisiología , Hormona del Crecimiento/metabolismo , Hipófisis/efectos de los fármacos , Receptores de Somatostatina/fisiología , Somatostatina/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Papio , Hipófisis/citología , Hipófisis/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
CONTEXT: There is concern that pegvisomant could be associated with a higher risk of tumor growth. The rate and possible determinants of this tumor growth are unknown. OBJECTIVE: The objective of the study was to investigate the clinical, immunohistological, and molecular factors conditioning tumor growth in patients taking pegvisomant. DESIGN AND SETTING: This was a cross-sectional study performed from 2004 to 2010 in four university hospitals in Spain. PATIENTS: Seventy-five acromegalic patients with active disease resistant to somatostatin analogs treated with pegvisomant were followed up for a mean of 29 ± 20 months. MAIN OUTCOME MEASURES: Magnetic resonance images before initiation of pegvisomant, at 6 months, and then yearly were examined in all patients. Immunohistological and molecular studies were performed in tumors that grew. RESULTS: A significant increase in tumor size was observed in five patients (6.7%). Absence of previous irradiation (P = 0.014) and shorter duration of prepegvisomant somatostatin analog therapy (P < 0.001) were associated with an increased risk of tumor growth. A stepwise multivariate linear regression analysis (R(2) = 0.334, P < 0.001) identified the duration of somatostatin analog therapy prior to pegvisomant (beta = -4.509, P = 0.014) as the only significant predictor of tumor growth. In those tumors that grew, GH expression and insulin receptor expression were higher (P = 0.033 in both cases) than in the control group. CONCLUSIONS: No previous radiotherapy, shorter duration of prepegvisomant somatostatin analog therapy, and higher tumor expression of GH and insulin receptor could be risk factors for tumor growth during pegvisomant therapy.
Asunto(s)
Adenoma/tratamiento farmacológico , Adenoma Hipofisario Secretor de Hormona del Crecimiento/tratamiento farmacológico , Hormona de Crecimiento Humana/análogos & derivados , Receptores de Somatotropina/antagonistas & inhibidores , Acromegalia/diagnóstico por imagen , Acromegalia/tratamiento farmacológico , Acromegalia/etiología , Adenoma/genética , Adenoma/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Terapia Combinada , Estudios Transversales , Progresión de la Enfermedad , Femenino , Adenoma Hipofisario Secretor de Hormona del Crecimiento/genética , Adenoma Hipofisario Secretor de Hormona del Crecimiento/patología , Hormona de Crecimiento Humana/metabolismo , Hormona de Crecimiento Humana/uso terapéutico , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Adhesión en Parafina , Hipófisis/patología , Hipófisis/cirugía , Radiografía , Análisis de Regresión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Adulto JovenRESUMEN
It is well established that reproductive function is gated by the state of energy reserves of the organism; conditions of metabolic stress and energy insufficiency being frequently coupled to disturbed reproductive maturation and/or infertility. In addition, obesity is also commonly linked to altered puberty onset and reproductive impairment. Such an impact of energy status on the reproductive axis is conveyed through a number of neuropeptide hormones and metabolic cues, whose nature and mechanisms of action have begun to be deciphered only in recent years. In this context, the emergence of kisspeptins, encoded by the KiSS-1 gene, and their receptor, GPR54, as indispensable signals for normal pubertal maturation and gonadal function, has raised the possibility that the KiSS-1/GRP54 system might also participate in coupling body energy status and reproduction. We revise herein the experimental evidence, gathered in rodent models, supporting the contention that the hypothalamic KiSS-1 system operates as a central conduit for conveying metabolic information onto the centers governing reproductive function, through a putative leptin-kisspeptin-GnRH pathway. Admittedly, key aspects of this 'metabolic' network involving the KiSS-1 system, such as its different peripheral regulators and central effectors, have not been fully elucidated. Nonetheless, the proposed hypothalamic circuitry, responsible for transmitting metabolic information onto the reproductive axis through KiSS-1 neurons, might explain, at least in part, the mechanisms for the well-known alterations of fertility linked to conditions of disturbed energy balance in humans, from anorexia nervosa to morbid obesity.
Asunto(s)
Reproducción/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Metabolismo Energético , Fertilidad/fisiología , Gónadas/fisiología , Humanos , Sistema Hipotálamo-Hipofisario/fisiología , Leptina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
The presence of ghrelin and its receptor, growth hormone (GH) secretagogue receptor, in the hypothalamus and pituitary, and its ability to stimulate GH release in vivo and in vitro, strongly support a significant role for this peptide in the control of somatotroph function. We previously demonstrated that ghrelin elicits GH secretion directly in somatotrophs by activating two major signalling cascades, which involve inositol phosphate and cAMP. In as much as nitric oxide (NO) and its mediator cGMP have been recently shown to contribute substantially to the response of somatotrophs to key regulatory hormones, including GH-releasing hormone, somatostatin and leptin, we investigated the possible role of this signalling pathway in ghrelin-induced GH release in vitro. Accordingly, cultures of pituitary cells from prepuberal female pigs were challenged with ghrelin (10(-8) m, 30 min) in the absence or presence of activators or blockers of key steps of the NO synthase (NOS)/NO/guanylate cyclase (GC)/cGMP route and GH secretion was measured. Two distinct activators of the NO route, S-nitroso-N-acetylpenicillamine (SNAP) (5 x 10(-4) m) and L-arginine methyl ester hydrochloride (L-AME) (10(-3) m), comparably stimulated GH secretion when applied alone. The presence of L-AME enhanced ghrelin-stimulated GH secretion, whereas SNAP did not alter its effect. Conversely, two different NOS/NO pathway inhibitors, N(w)-nitro-L-arginine methyl ester hydrochloride (10(-5) m) or haemoglobin (20 microg/ml), similarly blocked ghrelin-induced (but not basal) GH release, thus indicating that NO contributes critically to ghrelin action in somatotrophs. Moreover, incubation with a permeable cGMP analogue, 8-Br-cGMP (10(-8) m) stimulated GH secretion, but did not modify the stimulatory action of ghrelin, suggesting that cGMP could mediate the action of NO. Indeed, inhibition of GC by 10 microm LY-53,583 did not alter basal GH secretion but abolished the GH-releasing action of ghrelin. Taken together, our results provide novel evidence indicating that ghrelin requires activation of the NOS/NO route, and its subsequent GC/cGMP signal transduction pathway, as necessary steps to induce GH secretion from somatotrophs.
Asunto(s)
GMP Cíclico/fisiología , Ghrelina/farmacología , Hormona del Crecimiento/metabolismo , Óxido Nítrico/fisiología , Animales , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Femenino , Guanilato Ciclasa/fisiología , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/fisiología , S-Nitroso-N-Acetilpenicilamina/farmacología , Transducción de Señal/fisiología , Somatotrofos/metabolismo , PorcinosRESUMEN
Cortistatin is a recently discovered neuropeptide that is structurally related to somatostatin, the classic inhibitor of growth hormone (GH) release. Cortistatin binds with high affinity to all five somatostatin receptors (sst1-5), and, like somatostatin, cortistatin inhibits in vivo GH release in man and rats. In this report, we compared the in vitro actions of cortistatin and somatostatin using primary pig pituitary cell cultures. In this species, we have previously reported that somatostatin not only inhibits GH-releasing hormone (GHRH)-stimulated GH release at high doses, but also stimulates basal GH release at low (pM) doses, a dual response that is markedly dependent on the subpopulation of pituitary somatotropes examined. Results reported herein demonstrate that cortistatin closely mimics the dose-dependent inhibitory and stimulatory effects of somatostatin on GH secretion. As cortistatin, unlike somatostatin, binds to the human receptor for ghrelin/GH secretagogs (GHS-R), we also investigated whether cortistatin stimulates GH release through this receptor by using a synthetic, short form of cortistatin, cortistatin-8 (CST8), which lacks the sst-binding capacity of full-length cortistatin but retains its GHS-R-binding capacity. Interestingly, CST8 stimulated GH release only at low doses (10(-15) M), and did not reduce GH secretion stimulated by GHRH, ghrelin, or low-dose, full-length cortistatin, yet it counteracted that induced by a nonpeptidyl GHS, L-163 255. Taken together, our results indicate that the dual, inhibitory and stimulatory effects of cortistatin on GH release closely parallel those of somatostatin and are probably mediated by the same receptor(s) and signaling pathway(s) for both peptides. Furthermore, they suggest that the pathway(s) activated by cortistatin (and somatostatin) to stimulate GH release are not initiated by GHS-R activation.
Asunto(s)
Hormona del Crecimiento/metabolismo , Neuropéptidos/metabolismo , Neuropéptidos/farmacología , Somatostatina/metabolismo , Somatotrofos/efectos de los fármacos , Somatotrofos/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Hormona Liberadora de Hormona del Crecimiento/genética , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Humanos , Neuropéptidos/genética , Péptidos/genética , Péptidos/metabolismo , Péptidos/farmacología , Piperidinas/metabolismo , Piperidinas/farmacología , Hipófisis/citología , Ratas , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Ghrelina , Transducción de Señal/fisiología , Somatostatina/genética , Somatotrofos/citología , Compuestos de Espiro/metabolismo , Compuestos de Espiro/farmacología , PorcinosRESUMEN
Once thought to act only as a somatotropin release-inhibiting factor (SRIF), SRIF is currently viewed as a pleiotropic neuroendocrine factor controlling secretion, gene expression, apoptosis and signalling in many different targets. Actually, despite the numerous studies that have characterized SRIF action on somatotropes, new facets are continuously being discovered which help enlightening the biology of this cell type. As an example, ten years ago we demonstrated that SRIF exerts a dual, inhibitory/stimulatory effect on GH release from cultured pig somatotropes, which depends on the concentration of the peptide and on a divergent responsiveness of the two main cell subsets comprising the somatotrope population. Specifically, very low, picomolar doses of SRIF were found to stimulate GH release in vitro from intact cultures of dispersed pig pituitary cells and from purified somatotrope subpopulations. Conversely, higher (10(-7)M) SRIF concentrations inhibited, as expected, GHRH-induced GH release from intact pituitary cells and from one of the somatotrope subtypes; yet, at this same dose, it stimulated GH release from the other somatotrope subset. Analysis of second messenger pathways revealed that cAMP is the main signal conveying the stimulatory effects of low-dose SRIF. This peptide also exerts a distinct, dose-dependent regulation of the expression of three of its receptor subtypes (sst1, sst2 and sst5) at the pituitary. Indeed, acute in vitro treatment with a high SRIF dose increased mRNA levels of all three subtypes, whereas a low SRIF concentration only increased that of sst5. Interestingly, short term treatment with GHRH or ghrelin reduced the expression of sst5, and not that of sst1 and sst2. Hopefully, ongoing studies on cloning and individual characterization of porcine sst will help to unravel the complex and exciting response of somatotropes to SRIF.
Asunto(s)
Hormona del Crecimiento/metabolismo , Sistemas Neurosecretores/metabolismo , Somatostatina/metabolismo , Animales , HumanosRESUMEN
There is increasing evidence that nitric oxide (NO) produced by NO synthase (NOS), and their signalling partners, guanylyl cyclase and cGMP, play a relevant role in growth hormone (GH) secretion from somatotrophs. We previously demonstrated that both GH-releasing hormone (GHRH; 10(-8) M) and low concentrations of somatostatin (10(-15) M) stimulate pig GH release in vitro, whereas a high somatostatin concentration (10(-7) M) inhibits GHRH-induced GH secretion. To ascertain the possible contribution of the NOS-NO and guanylyl cyclase-cGMP routes to these responses, cultures of pituitary cells from prepubertal female pigs were treated (30 min) with GHRH (10(-8) M) or somatostatin (10(-7) or 10(-15) M) in the absence or presence of activators or blockers of key steps of these signalling cascades, and GH release was measured. Two distinct activators of NO route, SNAP (5x10(-4) M) or L-AME (10(-3) M), similarly stimulated GH release when applied alone (with this effect being blocked by 10(-7) M somatostatin), but did not alter the stimulatory effect of GHRH or 10(-15) M somatostatin. Conversely, two NO pathway inhibitors, NAME (10(-5) M) or haemoglobin (20 microg/ml) similarly blocked GHRH- or 10(-15) M somatostatin-stimulated GH release. 8-Br-cGMP (10(-8) to 10(-4) M) strongly stimulated GH release, suggesting that cGMP may function as a subsequent step in the NO pathway in this system. Interestingly, 10(-7) M somatostatin did not inhibit the stimulatory effect of 8-Br-cGMP. Moreover, although 8-Br-cGMP did not modify the effect of GHRH, it enhanced GH release stimulated by 10(-15) M somatostatin. Accordingly, a specific guanylyl cyclase inhibitor, LY-83, 583 (10(-5) M) did not alter 10(-15) M somatostatin-induced GH release, whereas it blocked GHRH-induced GH secretion. These results demonstrate for the first time that the NOS/NO signalling pathway contributes critically to the stimulatory effects of both GHRH and low-concentration somatostatin on GH release, and that, conversely, the subsequent guanylyl cyclase/cGMP step only mediates GHRH- and not low-concentration somatostatin-induced GH secretion from somatotrophs.
Asunto(s)
GMP Cíclico/fisiología , Hormona Liberadora de Hormona del Crecimiento/farmacología , Antagonistas de Hormonas/farmacología , Óxido Nítrico/fisiología , Hipófisis/metabolismo , Somatostatina/farmacología , Animales , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Guanilato Ciclasa/antagonistas & inhibidores , Indicadores y Reactivos , Hipófisis/citología , Hipófisis/efectos de los fármacos , PorcinosRESUMEN
Ghrelin, a recently discovered 28-aa peptide, stimulates GH release through a mechanism involving PLC- and cAMP-related signaling pathways. Recently, nitric oxide (NO) and its mediator, cGMP, have been shown to be required for the response of somatotropes to various regulators (GHRH, somatostatin, leptin). Here, we explore the possible role of the NO synthase (NOS)/NO/guanylate cyclase (GC)/cGMP signaling pathway in ghrelin-induced GH release from cultured pig somatotropes using blockers or activators of this route.
Asunto(s)
GMP Cíclico/fisiología , Hormona del Crecimiento/metabolismo , Óxido Nítrico/fisiología , Hormonas Peptídicas/fisiología , Transducción de Señal/fisiología , Animales , Células Cultivadas , Ghrelina , Ratas , PorcinosRESUMEN
Somatostatin (SRIF) is commonly regarded as an inhibitor of GH release in rodents and humans. However, in pigs, SRIF can stimulate the release of GH at low (picomolar) doses, while inhibiting GHRH-stimulated GH release at high (nanomolar) doses in primary pituitary cell cultures. One possible mechanism by which pig cells respond differently to the actions of SRIF is by differential expression and regulation of SRIF receptor subtypes. As no information is available on the homologous regulation of SRIF receptors in pigs, we examined the acute (4 h) in vitro effects of SRIF on mRNA levels of SRIF receptors sst1, sst2 and sst5 by multiplex RT-PCR. These particular sst subtypes were selected because all three have been implicated in the inhibitory effects of SRIF on GH release in both rodents and humans. At a high dose (10(-7) M), SRIF stimulated the expression of sst1, sst2 and sst5 in pig pituitary cell cultures. At a low dose (10(-13) M), SRIF markedly increased sst1, without affecting sst2 or sst5. Given that our laboratory has shown SRIF at high and low doses stimulates cAMP production in a subpopulation of pig somatotropes, we sought to determine if this signaling pathway may be responsible for the stimulatory effect of SRIF on its own receptor expression. The receptor-independent cAMP activator forskolin elevated sst1 and sst2 mRNA levels but did not affect sst5 expression, suggesting the stimulatory actions of high- and low-dose SRIF on sst1 and high-dose SRIF on sst2 mRNA levels can be mediated by activation of cAMP, whereas the stimulatory effect of high-dose SRIF on sst5 mRNA is elicited by a cAMP-independent pathway. Interestingly, both GHRH (10(-8) M) and ghrelin (10(-6) M), which release GH in pig pituitary cell cultures via cAMP-dependent mechanisms, decreased sst5 without altering sst1 or sst2 mRNA levels. Since the actions of GHRH and ghrelin on sst expression markedly contrasted with that observed for SRIF and forskolin these results clearly indicate GHRH and ghrelin are regulating sst5 mRNA levels by a cAMP-independent signaling pathway. Taken together, our results demonstrate that expression of pig SRIF receptors is under a complex, receptor subtype-selective regulation, wherein the concerted actions of key regulators of somatotrope function would play divergent and dose-dependent effects.
Asunto(s)
Hipófisis/fisiología , Receptores de Somatostatina/genética , Animales , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Regulación de la Expresión Génica , Ghrelina , Hormona del Crecimiento/metabolismo , Hormona Liberadora de Hormona del Crecimiento/farmacología , Hormonas Peptídicas/farmacología , Hipófisis/citología , Hipófisis/efectos de los fármacos , Isoformas de Proteínas , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores de Somatostatina/efectos de los fármacos , Receptores de Somatostatina/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Somatostatina/metabolismo , Somatostatina/farmacología , PorcinosRESUMEN
A review is presented on progress in the research of stimulatory inputs that regulate growth hormone secretion, including recent results on the action of the hypothalamic peptides growth-hormone releasing factor (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP), as well as that of both peptidic (growth hormone-releasing hexapeptide; GHRP-6) and non-peptidyl (L-163,255) synthetic GHSs on somatotrope cell function.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/metabolismo , Hormona del Crecimiento/metabolismo , Neuropéptidos/metabolismo , Oligopéptidos/metabolismo , Piperidinas/metabolismo , Compuestos de Espiro/metabolismo , Animales , Modelos Biológicos , Péptidos/química , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Transducción de Señal , PorcinosRESUMEN
Pituitary somatotropes and melanotropes have enabled us to investigate the molecular basis and functional dynamics underlying secretory plasticity, an ability of endocrine cells to adapt their activity to the changing physiologic requirements, which generates discrete cell subpopulations within each cell hormonal type. Porcine somatotropes comprise two morphologically distinct subpopulations of low- (LD) and high-density (HD) cells, separable by Percoll gradient, that respond differently to hypothalamic regulators. In LD somatotropes, somatostatin (SRIF) inhibits growth hormone (GH)-releasing hormone (GHRH)-induced GH secretion. Conversely, SRIF alone stimulates GH release from HD somatotropes. These disparate SRIF actions entail a molecular signaling heterogeneity, in that SRIF increases cAMP levels in HD but not in LD cells as a requisite to stimulate GH release. GHRH-stimulated GH release also involves differential signaling in LD and HD cells: although it acts primarily through the cAMP/extracellular Ca2+ route in both somatotrope subsets, full response of LD somatotropes also requires the inositol phosphate/intracellular Ca2+ pathway. Amphibian melanotropes, which regulate skin adaptation to background color by secreting POMC-derived alpha-melanocyte-stimulating hormone (alphaMSH), also comprise two subpopulations with divergent secretory phenotypes. LD melanotropes show high biosynthetic and secretory activities and high responsiveness to multiple hypothalamic factors. Conversely, HD melanotropes constitute a hormone-storage subset poorly responsive to regulatory inputs. Interestingly, in black-adapted animals most melanotropes acquire the highly-secretory LD phenotype, whereas white-background adaptation, which requires less alphaMSH, converts melanotropes to the storage HD phenotype. These same interconversions can be reproduced in vitro using appropriate hypothalamic factors, thus revealing the pivotal role of the hypothalamus in regulating the functional dynamics of the secretory plasticity. Furthermore, this regulation likely involves a precise control of the secretory pathway, as suggested by the differential distribution in LD and HD melanotropes of key components of the intracellular transport, processing, and storage of secretory proteins. Hence, molecular signaling heterogeneity and unique secretory pathway components seem to relevantly contribute to the control of secretory plasticity, thereby enabling endocrine cells to finely adjust their dynamic response to the specific hormonal requirements.