RESUMEN
Endocrine cells in the mammalian pituitary are arranged into three-dimensional homotypic networks that wire the gland and act to optimise hormone output by allowing the transmission of information between cell ensembles in a temporally precise manner. Despite this, the structure-function relationships that allow cells belonging to these networks to display coordinated activity remain relatively uncharacterised. This review discusses the recent technological advances that have allowed endocrine cell network structure and function to be probed and the mathematical models that can be used to analyse and present the resulting data. In particular, we focus on the mechanisms that allow endocrine cells to dynamically function as a population to drive hormone release as well as the experimental and theoretical methods that are used to track and model information flow through the network.
Asunto(s)
Modelos Teóricos , Hipófisis/fisiología , Animales , Humanos , Neuronas/fisiología , Hipófisis/citologíaRESUMEN
The hormones secreted by the anterior pituitary gland regulate major functions such as reproduction, as well as body growth and metabolism. Their efficiency of action highly depends on their temporal profile of release in the blood stream. This review summarises the recent evidence suggesting that the circadian clock genes that pace our daily rhythms may also contribute to the regulation of pituitary pulsatility, even in the non 24-h range. This inter-relation between molecular circadian oscillators and endocrine rhythmicities is discussed in light of the longstanding literature that has considered the involvement of the central circadian pacemaker located within the suprachiasmatic nuclei. Other arguments that suggest a role for circadian clock genes outside the suprachiasmatic nuclei are also presented, with a special emphasis on endocrine pituitary cells and hypothalamic neuroendocrine neurones that directly pace pituitary secretion rates.
Asunto(s)
Relojes Biológicos/fisiología , Ritmo Circadiano/fisiología , Sistema Hipotálamo-Hipofisario/fisiología , Adenohipófisis/fisiología , Animales , Relojes Biológicos/genética , Ritmo Circadiano/genética , Cricetinae , Femenino , Humanos , Masculino , Ratones , Ratas , Núcleo Supraquiasmático/fisiologíaRESUMEN
In stable transfection experiments in the GH-producing GC cell line, a construct containing the entire signal peptide and the first 22 residues of human GH linked in frame with enhanced green fluorescent protein (eGFP), produced brightly fluorescent cells with a granular distribution of eGFP. This eGFP reporter was then inserted into a 40-kb cosmid transgene containing the locus control region for the hGH gene and used to generate transgenic mice. Anterior pituitaries from these GH-eGFP transgenic mice showed numerous clusters of strongly fluorescent cells, which were also immunopositive for GH, and which could be isolated and enriched by fluorescence-activated cell sorting. Confocal scanning microscopy of pituitary GH cells from GH-eGFP transgenic mice showed a markedly granular appearance of fluorescence. Immunogold electron microscopy and RIA confirmed that the eGFP product was packaged in the dense cored secretory vesicles of somatotrophs and was secreted in parallel with GH in response to stimulation by GRF. Using eGFP fluorescence, it was possible to identify clusters of GH cells in acute pituitary slices and to observe spontaneous transient rises in their intracellular Ca2+ concentrations after loading with Ca2+ sensitive dyes. This transgenic approach opens the way to direct visualization of spontaneous and secretagogue-induced secretory mechanisms in identified GH cells.
Asunto(s)
Hormona de Crecimiento Humana/biosíntesis , Proteínas Luminiscentes/genética , Adenohipófisis/metabolismo , Animales , Calcio/análisis , Cósmidos , Gránulos Citoplasmáticos/química , Citosol/química , Citometría de Flujo , Expresión Génica , Proteínas Fluorescentes Verdes , Hormona Liberadora de Hormona del Crecimiento/farmacología , Hormona de Crecimiento Humana/genética , Humanos , Inmunohistoquímica , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Microscopía Confocal , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Adenohipófisis/ultraestructuraRESUMEN
Endocrine cells isolated from the anterior pituitary fire intracellular Ca2+ ([Ca2+]i) transients due to voltage-gated Ca2+ entry. However, the patterns of [Ca2+]i transients within the glandular parenchyma of the anterior pituitary are unknown. Here we describe, using real-time confocal laser microscopy, several spontaneous patterns of calcium signaling in acute pituitary slices prepared from male as well as cycling and lactating female rats. Forty percent of the cells demonstrated a spontaneous bursting mode, consisting of an active period of [Ca2+]i transients firing at a constant frequency, followed by a rest period during which cells were either silent or randomly active. The remaining recordings from endocrine cells either demonstrated random [Ca2+]i transients or were silent. These rhythmic bursts of [Ca2+]i transients, which required extracellular calcium, were detected in lactotrophs, somatotrophs, and corticotrophs within the acute slices. Of significance was the finding that the bursting mode could be adjusted by hypothalamic factors. In slices prepared from lactating rats, TRH recruited more bursting cells and finely adjusted the average duty cycle of [Ca2+]i bursts such that cells fired patterned bursts for approximately 70% of the recording period. Eighty-six percent of these cells were lactotrophs. Thus, the rhythmic [Ca2+]i bursts and their tuning by secretagogues may provide timing information that could encode for one or more cellular functions (e.g. exocytosis and/or gene expression) critical for the release of hormones by endocrine cells in the intact gland.
Asunto(s)
Señalización del Calcio/fisiología , Adenohipófisis/fisiología , Animales , Calcio/metabolismo , Electrofisiología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Fura-2 , Técnicas In Vitro , Masculino , Microscopía Confocal , Adenohipófisis/citología , Adenohipófisis/efectos de los fármacos , Ratas , Ratas Wistar , Hormona Liberadora de Tirotropina/farmacologíaRESUMEN
In order to map the molecular determinants that dictate the subcellular localization of human protein kinase C alpha (hPKCalpha), full-length and deletion mutants of hPKCalpha were tagged with the green fluorescent protein (GFP) and transiently expressed in GH3B6 cells. We found that upon thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate stimulation, hPKCalpha-GFP was localized exclusively in regions of cell-cell contacts. Surprisingly, PKCalpha failed to translocate in single cells despite the presence of TRH receptors, as attested by the TRH-induced rise in intracellular calcium concentration in these cells. TRH-stimulated translocation of hPKCalpha-GFP from the cytoplasm to cell-cell contacts was a biphasic process: a fast (measured in seconds) and transient phase, followed by a slower (approximately 1 hour) and long lasting phase. The latter and the translocation induced by phorbol 12-myristate 13-acetate absolutely required the N-terminal V1 region. In contrast to the full-length hPKCalpha, the N-terminal regulatory domain alone or associated with the V3 hinge region was spontaneously and uniformly localized at the plasma membrane of single and apposed cells. However, treatment with the calcium chelator BAPTA/AM induced a differential cytoplasmic/nuclear redistribution of the regulatory domain, depending on its association with V3, which suggests the existence of a mechanism controlling the cytoplasmic sequestration of inactive hPKCalpha and involving the V3 region. By using other deletion mutants, we were able to map the sequence required for this sequestration to the C2+V3 regions. This work points to the existence of a complex interplay between membrane targeting and cytoplasmic sequestration in the control of the spatiotemporal localization of hPKCalpha.
Asunto(s)
Membrana Celular/enzimología , Citoplasma/enzimología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Calcio/metabolismo , Catálisis , Fraccionamiento Celular , Línea Celular , Eliminación de Gen , Humanos , Inmunohistoquímica , Microscopía Confocal , Hipófisis/citología , Hipófisis/metabolismo , Plásmidos , Pruebas de Precipitina , Proteína Quinasa C-alfa , Proteínas Recombinantes de Fusión/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Hormona Liberadora de Tirotropina/farmacología , Factores de Tiempo , TransfecciónRESUMEN
We investigated the organization of spontaneous rises in cytosolic free Ca2+ concentration ([Ca2+]i) due to electrical activity in acute pituitary slices. Real time confocal imaging revealed that 73% of the cells generated fast peaking spontaneous [Ca2+]i transients. Strikingly, groups of apposing cells enhanced their [Ca2+]i in synchrony with a speed of coactivation >1,000 microm/s. Single-cell injection of Neurobiotin or Lucifer yellow labeled clusters of cells, which corresponded to coactive cells. Halothane, a gap junction blocker, markedly reduced the spread of tracers. Coupling between excitable cells was mainly homologous in nature, with a prevalence of growth hormone-containing cells. We conclude that spontaneously active endocrine cells are either single units or arranged in synchronized gap junction-coupled assemblies scattered throughout the anterior pituitary gland. Synchrony between spontaneously excitable cells may help shape the patterns of basal secretion.