Calcium dynamics and resting transcriptional activity regulates prolactin gene expression

Producción CientíficaResearch on the regulation of hormone gene expression by calcium signaling is hampered by the difficulty of monitoring both parameters within the same individual, living cells. Here we achieved concurrent, dynamic measurements of both intracellular Ca2+ concentration ([Ca2+]i) and prolactin (PRL) gene promoter activity in single, living pituitary cells. Cells were transfected with the luciferase reporter gene under control of the PRL promoter and subjected to bioluminescence and fluorescence imaging before and after presentation of TSH-releasing hormone (TRH), a prototypic regulator of PRL secretion and gene expression that induces a transient Ca2+ release, followed by sustained Ca2+ influx. We found that cells displaying specific photonic emissions (i.e. mammotropes) showed heterogeneous calcium and transcriptional responses to TRH. Transcriptionally responsive cells always exhibited a TRH-induced [Ca2+]i increase. In addition, transcriptional responses were related to the rate of Ca2+ entry but not Ca2+ release. Finally, cells lacking transcriptional responses (but showing [Ca2+]i rises) exhibited larger levels of resting PRL promoter activity than transcriptionally responsive cells. Thus, our results suggest that the sustained entry of Ca2+ induced by TRH (but not the Ca2+ release) regulates transcriptional responsiveness. Superimposed on this regulation, the previous, resting PRL promoter activity also controls transcriptional responses.National Institutes of Health (grant DK-38215)Fondo de Investigaciones Sanitarias (grant FIS 01/0769

Heavy Quarks and Heavy Quarkonia as Tests of Thermalization

We present here a brief summary of new results on heavy quarks and heavy quarkonia from the PHENIX experiment as presented at the "Quark Gluon Plasma Thermalization" Workshop in Vienna, Austria in August 2005, directly following the International Quark Matter Conference in Hungary.Comment: 8 pages, 5 figures, Quark Gluon Plasma Thermalization Workshop (Vienna August 2005) Proceeding

Vulnerable Children, Young People, and Families: Policy, Practice, and Social Justice in England and Scotland

This chapter begins by highlighting the rise of vulnerability as a term in social policy, and the three-level approach that is used to examine it. The first level is definitional, examining the possibility of defining vulnerability and vulnerabilities through a consideration of relevant literature and a number of recent policy documents. The second looks at how policy developments in Scotland and England have diverged, particularly since 2010, and how vulnerability has become more central to education policy in England. The third level focuses on practice, presenting research undertaken by the authors into a programme developed to support vulnerable children, young people, and families in Northern England as a case study exemplifying some of the factors affecting the effectiveness of programmes in which schools played an important but not central part. This practice perspective is still too often overlooked in discussions of policy and definition, and it is suggested that its inclusion will contribute to the ongoing debate about both how best to support vulnerable families and the implications for education and social justice

Dynamics of stimulus-expression coupling as revealed by monitoring of prolactin promoter-driven reporter activity in individual, living mammotropes

Producción CientíficaSingle-cell paradigms have greatly expanded our knowledge about stimulus-secretion coupling, but the understanding of stimulus-gene expression coupling has lagged behind for lack of a dynamic model sufficiently sensitive to provide single-cell resolution. In the present study, we made continuous indirect measurements within individual, living cells of expression dynamics both before and after treatment with a gene-activating secretagogue. This was accomplished by transfecting (via microinjection) individual, primary mammotropes with a PRL promoter-driven luciferase reporter plasmid, and then quantifying the rate of photonic emissions (reflective of endogenous gene activity). We found that individual cells exhibit spontaneous, random, short-term fluctuations of basal reporter activity and are extremely heterogeneous in terms of responses to a stimulatory agent (TRH). In addition, we found that responses are affected by several factors including the secretory status of the pituitary donor, the manner in which the stimulus is presented, and by the initial level of reporter activity. Moreover, the responsiveness of an individual cell can fluctuate dramatically over time. These results invite speculation that a given cell can “sense” its gene activation state and regulate its response accordingly to satisfy requirements for the corresponding secretory product.National Institutes of Health (grant DK-38215

Effects of cellular interactions on calcium dynamics in prolactin-secreting cells

Producción CientíficaSignals derived from other pituitary cells can have a dramatic effect on PRL gene expression and secretion by mammotropes. However, the intracellular mechanisms by which these effects are manifested on the target cell remain unexplored. Inasmuch as calcium is a key modulator of both gene expression and hormone export in mammotropes, we evaluated the effects of cell to cell contact vs. specific cellular interactions on calcium dynamics within these cells. This was accomplished by digital-imaging fluorescence microscopy of fura-2 in pituitary cells that were isolated in culture (singles) or adjoining one other cell (doublets). After calcium imaging, we then subjected cells to immunocytochemistry for PRL. Doublets were further categorized into mammotropes attached to another mammotrope (M-M) or to a nonmammotrope (M-nonM). We then calculated and compared Mean[ Ca2+]i values as well as Oscillation Indices (which reflect the oscillatory behavior of cells) in singles and doublets and found that they were not different (P> 0.05). However, the phenotype of the adjoining cell had a profound influence on both of these calcium parameters, such that the presence of one mammotrope could consistently decrease (P < 0.05) the Mean [Ca2+]i value (39.17 ± 3.83 vs. 56.24 ± 5.56 in M-nonM) and Oscillation Index (10.19 ± 1.76 vs. 21.21 ± 3.73 in M-nonM) of its neighboring counterpart. A more detailed analysis of oscillatory patterns in these cells revealed that nonoscillators were more abundant in M-M (23%) than in M-nonM (12%) doublets. Taken together, our results indicate that PRL-secreting cells convey a signal that dampens the oscillatory behavior of neighboring mammotropes. Thus, it appears that it is the phenotype rather than the physical presence of a neighbor that controls intercellular regulation of calcium dynamics among mammotropes.National Institutes of Health (grant DK-38215)USDA-Competitive Research (grant 95-37206-2438

The relationship between pulsatile secretion and calcium dynamics in single, living gonadotropin-releasing hormone neurons

Producción CientíficaIt is well established that pulsatile release of GnRH regulates the reproductive axis, but little is known about the mechanisms underlying this pulsatility. Recent findings that GT1 cells, a line derived from the mouse embryonic hypothalamus, release GnRH in a pulsatile manner indicates that this rhythmic activity is an intrinsic property of GnRH neurons. In several attempts to uncover the intracellular basis for this pulsatile phenomenon, it was revealed that intracellular calcium concentrations change in a rhythmic fashion in GnRH neurons and that cellular depolarization, which triggers a secretory event, is associated with profound calcium changes in the cells. These findings raised the intriguing possibility that periodic alterations in intracellular calcium concentrations may underlie the phenomenon of pulsatile secretion in GnRH neurons. To address this, we first adapted the use of FM1–43 fluorescence to monitor changes of secretion in individual GT1–7 cells and then combined this approach with simultaneous measurement of intracellular free calcium ([Ca2+]i, fura 2 method). In initial validation experiments, we found that stimulation of exocytosis with K+ (75 mM) or N-methyl-D-aspartate (NMDA, 100μ M) predictably evoked dynamic increases of both FM1–43 and fura 2 fluorescence. Later measurement of calcium dynamics and exocytotic activity in unstimulated cells revealed that[ Ca2+]i underwent transitions from quiescence to high oscillatory behavior, and that these shifts were frequently associated with exocytotic events. Moreover, these calcium oscillatory transitions and associated changes in secretory activity occurred synchronously among most adjacent cells and at a frequency similar to that reported for pulsatile release of GnRH by entire cultures of GnRH neurons. Taken together, these results indicate that the intrinsic secretory pulsatility of GnRH neurons appears to be a consequence of coordinated, periodic changes in the pattern of calcium oscillations within individual cells.National Institutes of Health (grants DK-38215 and HD-37657

Multi-responsiveness of single anterior pituitary cells to hypothalamic-releasing hormones: A cellular basis for paradoxical secretion

The classic view for hypothalamic regulation of anterior pituitary (AP) hormone secretion holds that release of each AP hormone is controlled specifically by a corresponding hypothalamic-releasing hormone (HRH). In this scenario, binding of a given HRH (thyrotropin-, growth hormone-, corticotropin-, and luteinizing hormone-releasing hormones) to specific receptors in its target cell increases the concentration of cytosolic Ca(2+) ([Ca(2+)](i)), thereby selectively stimulating the release of the appropriate hormone. However, “paradoxical” responses of AP cells to the four well-established HRHs have been observed repeatedly with both in vivo and in vitro systems, raising the possibility of functional overlap between the different AP cell types. To explore this possibility, we evaluated the effects of HRHs on [Ca(2+)](i) in single AP cells identified immunocytochemically by the hormone they stored. We found that each of the five major AP cell types contained discrete subpopulations that were able to respond to several HRHs. The relative abundance of these multi-responsive cells was 59% for lactotropes, 33% for thyrotropes, and in the range of 47–55% for gonadotropes, corticotropes, and somatotropes. Analysis of prolactin release from single living cells revealed that each of the four HRHs tested were able to induce hormone release from a discrete lactotrope subpopulation, the size of which corresponded closely to that in which [Ca(2+)](i) changes were induced by the same secretagogues. When viewed as a whole, our diverse functional measurements of multi-responsiveness suggest that hypothalamic control of pituitary function is more complicated than previously envisioned. Moreover, they provide a cellular basis for the so-called “paradoxical” behavior of pituitary cells to hypothalamic hypophysiotropic agents