RESUMO
Pulsatile release of GnRH is essential for proper reproductive function, but little information is available on the molecular processes underlying this intermittent activity. Recently, GnRH gene expression (GnRH-GE) episodes and exocytotic pulses have been identified separately in individual GnRH-expressing cells, raising the exciting possibility that both activities are linked functionally and are fundamental to the pulsatile process. To explore this, we monitored GnRH-GE (using a GnRH promoter-driven luciferase reporter) and exocytosis (by FM1-43 fluorescence) in the same, living GT1-7 cells. Our results revealed a strong temporal association between exocytotic pulses and GnRH-GE episodes. To determine whether a functional link existed, we blocked one process and evaluated the other. Transcriptional inhibition with actinomycin D had only a modest influence on exocytosis, suggesting that exocytotic pulse activity was not dictated acutely by episodes of gene expression. In contrast, blockage of exocytosis with anti-SNAP-25 (which obstructs secretory granule fusion) abolished GnRH-GE pulse activity, indicating that part of the exocytotic process is responsible for triggering episodes of GnRH-GE. When taken together, our findings suggest that a careful balance is maintained between release and biosynthesis in GT1-7 cells. Such a property may be important in the hypothalamus to ensure that GnRH neurons are in a constant state of readiness to respond to changes in reproductive function.
Assuntos
Exocitose/fisiologia , Expressão Gênica/fisiologia , Hormônio Liberador de Gonadotropina/genética , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Transformada , Exocitose/efeitos dos fármacos , Corantes Fluorescentes/farmacocinética , Expressão Gênica/efeitos dos fármacos , Genes Reporter/fisiologia , Luciferases/genética , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Fluxo Pulsátil , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , Proteína 25 Associada a SinaptossomaRESUMO
Functional heterogeneity within populations of breast cancer cells contribute to the seemingly paradoxical effects of antiestrogens and the development of antiestrogen "resistance." Our objectives were to determine the degree to which T-47D cells may respond inappropriately (positively) to the antiestrogen 4-hydroxytamoxifen (HOT) alone, and whether all cells that respond to the stimulatory effects of estradiol-17beta (E2) are inhibited by the addition of HOT. Single, living T-47D cells were transfected by microinjection with an estrogen response element (ERE)-driven luciferase reporter plasmid. Transfected cells were then treated with medium alone, HOT, E2 or a combination thereof on consecutive days, exposed to the substrate luciferin and subjected to quantification of photonic emissions reflective of ERE-stimulated activity. This analysis revealed a subpopulation of cells that exhibited increased ERE-driven photonic activity in response to HOT. In companion studies, E2-stimulated ERE activity was reversed (on average) with HOT addition. However, analysis of individual cells revealed that although HOT reduced photonic activity in the majority (89.2%) of E2-responsive cells, there was a small subset (10.8% of the population) that was stimulated by E2 + HOT cotreatment. Our data support the hypothesis that these cells possess an intrinsic "resistance" to antiestrogenic agents, and that this could contribute to the remodeling of tumor cell populations toward a "resistant" phenotype.
Assuntos
Antagonistas de Estrogênios/farmacologia , Estrogênios/farmacologia , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Neoplasias da Mama , Resistência a Medicamentos , Estradiol/farmacologia , Luciferina de Vaga-Lumes/metabolismo , Genes Reporter , Humanos , Luciferases/genética , Fótons , Elementos de Resposta/genética , Transfecção , Células Tumorais CultivadasRESUMO
Periodic secretion of GnRH from the hypothalamus is the driving force for the release of gonadotropic hormones from the pituitary, but the roles of individual neurons in the context of this pulse generator are not known. In this study we used FM1-43 to monitor the membrane turnover associated with exocytosis in single GT1-7 neurons and found an intrinsic secretory pulsatility (frequency, 1.4 +/- 0.1/h; pulse duration, 17.3 +/- 0.6 min) that, during time in culture, became progressively synchronized among neighboring cells. Voltage-gated calcium channels and gap junctional communication each played a major role in synchronized pulsatility. An L-type calcium channel inhibitor, nimodipine, abolished synchronized pulsatility. In addition, functional gap junction communication among adjacent cells was detected, but only under conditions where pulsatile synchronization was also observed, and the gap junction inhibitor octanol abolished both without affecting pulse frequency or duration. Our results, therefore, provide strong evidence that the GnRH pulse generator in GT1-7 cells arises from a single cell oscillator mechanism that is synchronized through network signaling involving voltage-gated calcium channels and gap junctions.
Assuntos
Comunicação Celular , Exocitose , Junções Comunicantes/fisiologia , Hormônio Liberador de Gonadotropina/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/fisiologia , Ciclo Celular , Linhagem Celular , Camundongos , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismoRESUMO
The diverse role of milk in the neonate is slowly expanding beyond nutritional and immunological borders to include possible developmental roles for the numerous hormones and growth factors found in this medium. Yet, despite the growing list of milk-borne factors, the precise impact of each of these on the neonate remains to be elucidated. The focus of this review is to summarize studies from our laboratory which demonstrate clearly that milk-borne factors play an obligatory role in the postnatal development of at least one organ, the anterior pituitary gland.
Assuntos
Crescimento , Leite/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Substâncias de Crescimento/metabolismo , Hormônios/metabolismo , Humanos , Adeno-Hipófise/citologia , Adeno-Hipófise/crescimento & desenvolvimento , Adeno-Hipófise/metabolismo , Prolactina/metabolismoRESUMO
We previously showed that primary rat mammotropes exhibited four distinct patterns of 'spontaneous' free intracellular calcium ([Ca2+]i) oscillatory behavior: a quiescent state A and three oscillatory states B,C&D, which differed in frequency/amplitude characteristics. When [Ca2+]i was monitored in 10 min windows separated by several hours, these phenotypes were frequently found to interconvert, raising the question about whether these transitions were random or ordered events. We reasoned that if such activity were random, then neither episode duration nor transitional probabilities should differ among phenotypes. We tested this logic in the current study by making long-term, continuous measurements of [Ca2+]i in mammotropes microinjected with Fura-2-dextran and identified by their ability to express a prolactin promoter-driven reporter plasmid. We found that transitions occurred in ~25% of cells (n = 36 from 9 independent experiments) once every 1-5 h and demarcated phenotype episodes of different duration (A, 1.04 +/- 0.2 h; B, 1.64 +/- 0.3 h; C, 2.45 +/- 0.62 h; D, 0.90 +/- 0.2 h, mean +/- SEM). Moreover, some transitions occurred more frequently than others and linked specific phenotypes into a common pattern: C to B to A. Our results demonstrate that the seemingly spontaneous nature of [Ca2+]i phenotype transitions are, in fact, ordered and support the view that they comprise a structured 'code' like that proposed to underlie calcium-dependent regulation of exocytosis and gene expression.
Assuntos
Sinalização do Cálcio , Adeno-Hipófise/citologia , Prolactina/metabolismo , Animais , Células Cultivadas , Dextranos , Feminino , Corantes Fluorescentes , Fura-2 , Processamento de Imagem Assistida por Computador , Medições Luminescentes , Microscopia de Fluorescência , Adeno-Hipófise/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Firefly luciferase is used widely as a reporter enzyme for studies of gene regulation and expression. The recent development of new technologies that combine luciferase reporter technology and digital imaging microscopy has enabled multiple measurements of gene expression in the same living cell. Although this approach has already provided new insights about expression dynamics, its future utility is limited by the three- to four-hour half-life of firefly luciferase in mammalian cells. Because of this, rapid increases or decreases in gene expression may not be detected, owing to the accumulation of residual luciferase. Accordingly, the goal of the present study was to develop a luciferase reporter with a reduced functional half-life. This was accomplished by adding a synthetic fragment to the firefly luciferase-coding sequence that encoded the proteolytic "PEST" signal from mouse ornithine decarboxylase. When placed under the control of estrogen response elements and expressed in human breast cancer T-47D cells, the modified luciferase protein (LUCODC-DA) displayed a functional half-life of 0.84 h compared to 3.68 h for the wild-type enzyme. As anticipated, the overall rate of photonic emissions in cells expressing the destabilized luciferase was about sevenfold lower than that of their wild-type counterparts, presumably because of the reduction of steady-state luciferase accumulation. Even so, the photonic activity derived from LUCODC-DA was still sufficient to enable real-time measurements of gene expression in single living cells.
Assuntos
Estabilidade Enzimática , Expressão Gênica , Luciferases/genética , Luciferases/metabolismo , Animais , Neoplasias da Mama , Clonagem Molecular , Besouros , Meia-Vida , Humanos , Camundongos , Microinjeções , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Transfecção , Células Tumorais CultivadasRESUMO
It 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 ofintracellular 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, 100 microM) 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.
Assuntos
Cálcio/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/fisiologia , Animais , Linhagem Celular , Embrião de Mamíferos , Exocitose/efeitos dos fármacos , Hipotálamo , Camundongos , Microscopia de Fluorescência , N-Metilaspartato/farmacologia , Periodicidade , Potássio/farmacologiaRESUMO
Dynamic intracellular processes in endocrine cells are usually controlled by the coordinated modulation of two or more functionally related genes. Attempts to gain a more complete understanding of these processes would be facilitated greatly by a method enabling activity measurements of two genes at the same time. Here we describe how we developed such a system and used it to determine indirectly whether individual, living pituitary cells could concurrently express both the growth hormone (GH) and prolactin (PRL) genes. Our results demonstrate that coexpression of these genes is indeed possible. Moreover, our findings provide a general paradigm for future "real-time" analysis of other interrelated genes involved in the regulation of endocrine processes.
Assuntos
Hormônio do Crescimento/genética , Luciferases/genética , Biologia Molecular/métodos , Hipófise/citologia , Prolactina/genética , Animais , Células Cultivadas , Clonagem Molecular , Cnidários , Besouros , Feminino , Expressão Gênica/fisiologia , Genes Reporter , Lactação , Hipófise/fisiologia , Regiões Promotoras Genéticas/fisiologia , RatosRESUMO
Single-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.
Assuntos
Hipófise/citologia , Prolactina/genética , Hormônio Liberador de Tireotropina/farmacologia , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Lactação , Luciferases/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Masculino , Mamíferos , Fótons , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Prolactina/efeitos dos fármacos , Prolactina/metabolismo , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Fatores Sexuais , Hormônio Liberador de Tireotropina/metabolismo , Fatores de Tempo , Ativação Transcricional , TransfecçãoRESUMO
Somatotropes comprise two morphologically and functionally distinct subpopulations of low (LD) and high (HD) density cells. We recently reported that GRF induces different patterns of increase in the cytosolic free Ca2+ concentration in single porcine LD and HD somatotropes, which for LD cells required not only Ca2+ influx but also intracellular Ca2+ mobilization. This suggested that GRF may activate multiple signaling pathways in pig LD and HD somatotropes to stimulate GH secretion. To address this question, we first assessed the direct GRF effect on second messenger activation in cultures of LD and HD cells by measuring cAMP levels and [3H]myo-inositol incorporation. Secondly, to determine the relative importance of cAMP- and inositol phosphate (IP)-dependent pathways, and of intra- and extracellular Ca2+, GRF-induced GH release from cultured LD and HD somatotropes was measured in the presence of specific blockers. GRF increased cAMP levels in both subpopulations, whereas it only augmented IP turnover in LD cells. Accordingly, adenylate cyclase inhibition by MDL-12,330A abolished GRF-stimulated GH release in both subpopulations, whereas phospholipase C inhibition by U-73122 only reduced this effect partially in LD cells. Likewise, blockade of Ca2+ influx with Cl2Co reduced GRF-stimulated GH secretion in both LD and HD somatotropes, whereas depletion of thapsigargin-sensitive intracellular Ca2+ stores only decreased the secretory response to GRF in LD cells. These results demonstrate that GRF specifically and differentially activates multiple signaling pathways in two somatotrope subpopulations to stimulate GH release. Thus, although the prevailing signaling cascade employed by GRF in both subpopulations is adenylate cyclase/cAMP/extracellular Ca2+, the peptide also requires activation of the phospholipase C/IP/intracellular Ca2+ pathway to exert its full effect in porcine LD somatotropes.
Assuntos
AMP Cíclico/farmacologia , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Hormônio do Crescimento/metabolismo , Fosfatos de Inositol/farmacologia , Hipófise/metabolismo , Inibidores de Adenilil Ciclases , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Contagem de Células , Células Cultivadas , AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Inositol/metabolismo , Hipófise/efeitos dos fármacos , Sistemas do Segundo Mensageiro , Suínos , Trítio , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
The rate of prolactin (PRL) secretion is influenced by the gender and physiological state of an animal, but little is known about the mechanisms involved. In the present study, we assessed possible contributions of Ca2+ dynamics and PRL gene expression to these differences. This was accomplished by monitoring spontaneous [Ca2+]i changes and PRL promotor-driven reporter activity in pituitary cultures derived from rats comprising a broad spectrum of PRL secretory capacities: male, cycling female, and lactating rats. We found that Ca2+ oscillatory activity exhibited a rank order of lactating > cycling females > males, consistent with the reported secretory capacities of mammotropes from these sources. Interestingly, we observed that the basal level of PRL promotor-driven reporter activity was the same for all three models, but that mammotropes from males were the most responsive to stimulation of PRL gene expression by elevation of [Ca2+]i. Collectively, our findings reveal gender- and state-specific differences in Ca2+ dynamics and induction of PRL gene expression. These likely contribute to reported differences in secretory capacity.
Assuntos
Cálcio/metabolismo , Expressão Gênica/fisiologia , Hipófise/metabolismo , Prolactina/metabolismo , Caracteres Sexuais , Animais , Células Cultivadas , Feminino , Expressão Gênica/efeitos dos fármacos , Lactação/fisiologia , Masculino , Prolactina/genética , Ratos , Transcrição GênicaRESUMO
Transforming growth factor-alpha (TGF-alpha) contributes to the progression of mammary carcinogenesis in part through synergistic augmentation of estradiol (E2) action. To investigate this further, we sought to determine (1) whether the duration of TGF-alpha treatment might influence the nature of the TGF-alpha/E2 interaction, and (2) whether TGF-alpha would behave in a similar manner when combined with phytoestrogens. To this end, we transfected T47-D breast cancer cells with an estrogen-responsive reporter and then treated the cells (for 4-48 h) with varying concentrations of TGF-alpha, E2, the antiestrogen 4-hydroxy-tamoxifen (HOT), and/or one of three phytoestrogens. Our findings revealed that TGF-alpha has short-term synergistic and long-term inhibitory effects on E2- and phytoestrogen-regulated gene expression. Furthermore, this secondary inhibition of E2 action by TGF-alpha was similar in magnitude to that imposed by HOT. These findings demonstrate a novel role for TGF-alpha and invite reevaluation of current models regarding TGF-alphas interactions with E2 in breast cancer cells. Our results also raise the possibility that phytoestrogens, which interact with TGF-alpha in a manner conceptually identical to that of E2, may subserve a regulatory function in breast cancer cells.
Assuntos
Adenocarcinoma/genética , Neoplasias da Mama/genética , Estrogênios não Esteroides/farmacologia , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isoflavonas , Fator de Crescimento Transformador alfa/farmacologia , Sinergismo Farmacológico , Antagonistas de Estrogênios/farmacologia , Humanos , Luciferases/metabolismo , Fitoestrógenos , Preparações de Plantas , Receptores de Estrogênio/agonistas , Receptores de Estrogênio/efeitos dos fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Transfecção/genética , Células Tumorais CultivadasRESUMO
We have shown previously that rat mammotropes produce an activity that suppresses PRL gene expression by neighboring mammotropes. Here, we tested the hypothesis that this mammotrope-derived inhibitor is transforming growth factor-beta1 (TGFbeta1). To this end, we pursued a two-pronged strategy wherein we added exogenous TGFbeta1 to primary cultures of anterior pituitary cells transfected with a rat PRL-luc construct. Measurement of luciferase activity by luminometry of extracts revealed that administration of TGFbeta1, over a range of doses shown by others to be secreted by cultures of pituitary cells, caused a significant (P < 0.05) suppression of PRL gene expression. In contrast, immunoremoval of secreted TGFbeta1 led to an elevation of PRL promoter-driven reporter activity in these cultures. In a subsequent study, we repeated these experiments with a single cell model in an attempt to determine the demographics of the cellular responses. Accordingly, we transfected (via microinjection) individual mammotropes with the rat PRL-luc construct; exposed them to TGFbeta1, its neutralizing antibody, or respective controls; and then assessed PRL gene expression in "real-time" by quantification of photons emitted by the living cells after exposure to the substrate luciferin. Our results revealed that 1) TGFbeta1 inhibited PRL gene expression in all mammotrope studied; 2) only a subgroup of mammotropes (approximately 23%) was relieved of TGFbeta1 inhibition by antibody treatment; and 3) the growth factor exerted its inhibitory effect via a paracrine, as opposed to an autocrine, mechanism. These findings identify TGFbeta1 as the paracrine agent that exerts a tonic inhibitory influence over PRL gene expression in mammotropes.
Assuntos
Expressão Gênica/efeitos dos fármacos , Comunicação Parácrina , Prolactina/antagonistas & inibidores , Prolactina/genética , Fator de Crescimento Transformador beta/farmacologia , Animais , Anticorpos/farmacologia , Células Cultivadas , Feminino , Comunicação Parácrina/fisiologia , Adeno-Hipófise/citologia , Adeno-Hipófise/fisiologia , Ratos , Ratos Endogâmicos , Fator de Crescimento Transformador beta/imunologiaRESUMO
Phytoestrogens can exhibit agonistic actions on estrogen-dependent gene expression in breast cancer cells. Since several different phytoestrogens may be found within a single dietary plant source, we sought to investigate whether estrogen-dependent gene expression may be further influenced by the collective treatment of breast cancer cells with multiple phytoestrogens. Accordingly, we transfected MCF-7 breast cancer cells with estrogen-responsive reporters followed by treatment with one of four phytoestrogens (genistein, daidzein, formononetin, and equol) or a combination of these in the absence of estradiol. Our results demonstrated clear-cut agonistic effects of phytoestrogens on estrogen-dependent gene expression. Moreover, combinatorial treatment consistently stimulated reporter activity above that observed for individual phytoestrogens. Inasmuch as the phytoestrogens tested are frequently found together in food sources, these combinatorial responses may more accurately reflect the consequences of in vivo exposure.
Assuntos
Neoplasias da Mama/metabolismo , Estrogênios não Esteroides/farmacologia , Estrogênios/farmacologia , Expressão Gênica/efeitos dos fármacos , Cromanos/farmacologia , Interações Medicamentosas , Equol , Genisteína/farmacologia , Humanos , Isoflavonas/farmacologia , Luciferases/genética , Fitoestrógenos , Preparações de Plantas , Transfecção , Células Tumorais CultivadasRESUMO
The existence of an intrinsic oscillator for pulsatile gonadotropin-releasing hormone (GnRH) secretion in normal and transformed GnRH neurons raises the question of whether the corresponding gene also is expressed in an episodic manner. To resolve this question, we used a modification of conventional luciferase technology, which enabled continuous monitoring of GnRH gene activity in single, living neurons. With this method, the relative rate of endogenous gene expression is estimated by quantification of photons emitted by individual neurons microinjected with a GnRH promoter-driven luciferase reporter construct. Immortalized GT1-1 neurons, which secrete the decapeptide GnRH in a pulsatile manner conceptually identical to that of their nontransformed counterparts in vivo, were chosen as the model for these studies. First, we injected individual cells with purified luciferase protein and established that the reporter half-life was sufficiently short (50 min) to enable detection of transient changes in gene expression. Next, we subjected transfected GT1-1 cells to continuous monitoring of reporter activity for 16 h and found that the majority of them exhibited spontaneous fluctuations of photonic activity over time. Finally, we established that photonic activity accurately reflected endogenous GnRH gene expression by treating transfected GT1-1 cells with phorbol 12-myristate 13 acetate (a consensus inhibitor of GnRH gene expression) and observing a dramatic suppression of photonic emissions from continuously monitored cells. Taken together, these results demonstrate the validity of our "real-time" strategy for dynamically monitoring GnRH gene activity in living neurons. Moreover, our findings indicate that GnRH gene expression as well as neuropeptide release can occur in an intermittent manner.
Assuntos
Regulação da Expressão Gênica , Genes Reporter , Hormônio Liberador de Gonadotropina/genética , Luciferases/genética , Neurônios/metabolismo , Células Cultivadas , FótonsRESUMO
Signals 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.
Assuntos
Cálcio/metabolismo , Comunicação Celular/fisiologia , Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Animais , Células Cultivadas , Feminino , Corantes Fluorescentes , Fura-2 , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Adeno-Hipófise/citologia , RatosRESUMO
It is well known that the suckling stimulus renders mammotropes considerably more responsive to prolactin (PRL)-releasing stimuli, and the neurointermediate lobe peptide alpha-melanocyte-stimulating hormone (alpha-MSH) has been proposed to play a pivotal role in this priming. The objectives of the present study were to determine whether alpha-MSH could act directly on pituitary cells to potentiate PRL release in response to two physiologically relevant PRL secretagogues, thyrotropin-releasing hormone (TRH) and ATP, and, if so, to identify the mechanism by which this priming phenomenon is manifested. To this end, we cultured anterior pituitary cells from lactating rats overnight and then subjected them to a reverse hemolytic plaque assay for PRL to evaluate their responses to various test agents. We found that alpha-MSH, which had no effect on PRL export when tested alone, augmented by more than threefold the secretory responses to TRH and ATP. Next, we utilized digital-imaging fluorescence microscopy of fura 2 to evaluate the role of intracellular Ca2+ in this process. We found that PRL export induced by pharmacological activation of L-type voltage-operated calcium channels was also potentiated by alpha-MSH, as was Ca2+ entry induced by TRH. Our results indicate that alpha-MSH acts as a mammotrope-priming agent on a subset of mammotropes by increasing Ca2+ entry induced by PRL secretagogues.
Assuntos
Cálcio/metabolismo , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Prolactina/metabolismo , alfa-MSH/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Sinergismo Farmacológico , Feminino , Cinética , Microscopia de Fluorescência , Ratos , Hormônio Liberador de Tireotropina/farmacologiaRESUMO
Cytosolic calcium plays a critical role in the control of a number of genes, including that of the pituitary hormone PRL. Cells that secrete this hormone, termed mammotropes, display spontaneous oscillations of intracellular free calcium ([Ca2+]i) that are positively correlated to PRL release. However, the precise contribution of calcium signaling to the expression of any gene including PRL has remained obscure owing to the requirement for and lack of a strategy for monitoring both of these dynamic variables (gene expression and [Ca2+]i oscillations) in the same living cell. In the present study, we overcame this technical limitation by making real-time measurements of PRL gene expression in transfected, primary rat mammotropes previously subjected to [Ca2+]i determinations by digital imaging fluorescence microscopy of fura-2. Our results showed that the majority of mammotropes (75%) exhibited distinct oscillatory behaviors that could be subgrouped on the basis of frequency/amplitude of [Ca2+]i changes, whereas the remainder (25%) were quiescent (nonoscillatory). Interestingly, most mammotropes displayed spontaneous transitions between oscillatory and quiescent states over the course of several hours. As a consequence of this oscillatory plasticity, there was not a positive correlation between [Ca2+]i dynamics and gene expression at any point in time, as would be predicted by studies with entire populations of cells. Instead, the relationship was distinctly inverse, suggesting that dynamic changes in PRL gene expression may be regulated by temporally dissociated transitions between quiescent and oscillatory states.
Assuntos
Cálcio/metabolismo , Regulação da Expressão Gênica , Líquido Intracelular/metabolismo , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Prolactina/biossíntese , Prolactina/genética , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Microinjeções , Ratos , Ratos Sprague-DawleyRESUMO
Elucidation of the molecular mechanisms underlying the development and progression of breast cancer has been hindered by two considerations. First, mammary tumor cells exhibit a considerable degree of morphological and functional heterogeneity. Thus, the conventional strategy of measuring a population response may not provide an accurate reflection of the behavior of the functional unit: an individual cell. Second, important regulatory events are generally separated in time, yet the strategies we use to monitor them are usually static as opposed to dynamic. With these considerations in mind, it would appear that a system for studying this problem should ideally combine the resolving power of single-cell analysis with a dynamic paradigm for making multiple measurements of gene expression from the same, living cell. This report summarizes our efforts at developing, validating, and optimizing such a system for monitoring hormone-driven gene expression in T47-D human breast cancer cells.