Search

Research 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 Ca(2+) concentration ([Ca(2+)](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 Ca(2+) release, followed by sustained Ca(2+) 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 [Ca(2+)](i) increase. In addition, transcriptional responses were related to the rate of Ca(2+) entry but not Ca(2+) release. Finally, cells lacking transcriptional responses (but showing [Ca(2+)](i) rises) exhibited larger levels of resting PRL promoter activity than transcriptionally responsive cells. Thus, our results suggest that the sustained entry of Ca(2+) induced by TRH (but not the Ca(2+) release) regulates transcriptional responsiveness. Superimposed on this regulation, the previous, resting PRL promoter activity also controls transcriptional responses.

PRL gene expression in the anterior pituitary has been the focus of intensive investigation for many years, but very little information is available on the actual dynamics by which this process occurs in individual mammotrope cells. Here, we used single cell bioluminescent imaging microscopy and a recently refined reporter gene strategy to measure PRL promoter-driven gene expression (PRL-GE) in individual living primary mammotropes. Using this approach we report a new phenomenon involving repetitive on/off gene expression bursts that occurred in a distinctly noncircadian oscillatory pattern. Furthermore, we demonstrate a functional basis for these gene expression oscillations, inasmuch as PRL-GE pulses were sensitive to calcium-dependent modulation, which we show arose exclusively as changes in the shape of individual pulse episodes. Our results provide the first clear evidence that PRL-GE, in its homologous cell environment, displays oscillatory bursts of activity. Moreover, they strongly support the idea that these discrete on/off bursts of activity serve as an important determinant of the timing and level of PRL-GE under both basal and stimulated conditions.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 Ca2+ ([Ca2+]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 [Ca2+]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 [Ca2+]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.

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.

Recent evidence demonstrates that ATP, costored with a number of hormones and neurotransmitters in secretory granules, is coreleased during exocytosis of these agents. Here, we explored the possibility that extracellular ATP subserves an autocrine and/or paracrine role in the regulation of prolactin (PRL) release by subjecting rat pituitary cells to various experimental manipulations aimed at evaluating putative interactions between ATP and mammotropes. Our results strongly support the view that ATP functions as a local regulator of PRL secretion. To be more specific, we observed that ATP is released in a predictable manner by physiologically relevant secretagogues that are reasonably targeted to mammotropes. Moreover, we found that ATP can act directly on pituitary cells to stimulate the release of PRL from most (if not all) mammotropes. Finally, we determined that antagonism or removal of ATP leads to a diminution of PRL export from pituitary cells cultured under basal or thyrotropin-releasing hormone-stimulated conditions. On the basis of these results, we propose that ATP acts locally to amplify and prolong the PRL secretory response elicited by a more traditional hypophysiotropic signal.

The mammotrope has traditionally been a favored model for studies of hormonal gene expression, biosynthesis, and release. However, the primary site(s) at which these processes are coordinated and integrated remains to be established. Because there is considerable indirect evidence to suggest that the rate of prolactin (PRL) secretion is dictated, in large part, at the level of transcription, the relative contribution of other putative regulatory foci has received less attention. The purpose of the present study was to test the primacy of transcriptional regulation at the single-cell level. To this end, we quantified within individual mammotropes the relationship between PRL gene transcription, mRNA storage, and hormone release. This was accomplished by the combined application of "real-time" measurement of gene expression, in situ hybridization cytochemistry, and reverse hemolytic plaque assay, respectively. Our results demonstrate a quantitative dissociation among these variables, suggesting that control mechanisms besides transcription play a primary role in integrating and coordinating flow through the PRL secretory pathway.

It is clear that alpha-melanocyte-stimulating hormone (alpha-MSH), released by the hypophysial neurointermediate lobe, is a mediator of suckling-induced prolactin release, but several questions surrounding its role remain unresolved. Accordingly, the objectives of the present study were 1) to establish whether alpha-MSH could bind in a reversible manner to a specific secretory type cell within the adenohypophysis (AP), 2) to resolve the issue of whether the peptide could compete with dopamine for the same receptor binding site, and 3) to seek a functional signaling correlate for alpha-MSH binding. In pursuit of these objectives, we subjected pituitary cells from lactating rats to alpha-MSH receptor autoradiography, AP hormone immunocytochemistry, or digital imaging fluorescence microscopy with fura 2 as a calcium-sensitive probe. Our results show that alpha-MSH binding is restricted to mammotropes and that a specific subpopulation of these express functional alpha-MSH receptors that are coupled to a Ca2+ signaling pathway. Moreover, alpha-MSH does not compete with dopamine antagonists/agonists for the same binding site.

A growing concern about "endocrine disrupters" and their impact on estrogen-dependent-phenomena led us to investigate the effects of OP, an environmental estrogen, on PRL gene expression. To this end, we found that OP stimulates the PRL gene to a similar degree as estradiol (E2), although E2 is a 1000-fold more potent in this regard. We then sought to confirm that OP exerts its effects through E2-receptors. Accordingly, we treated cells with tamoxifen (TAM), an anti-estrogen, and found that it blunted the response induced by OP, suggesting that E2-receptors mediated this response. Finally, using "real time" measurements of gene expression in living cells, we found that a large fraction of mammotropes were OP-responsive. These results clearly demonstrate that OP affects PRL gene expression in a manner identical to that of E2. Inasmuch as PRL subserves an obligatory role in the regulation of lactation as well as in neonatal development, we believe that environmental estrogens such as OP have the potential to adversely affect the maternal-infant unit by altering PRL gene expression.

Interleukin-1 alpha (IL-1 alpha) and IL-1 beta bind to either the p80 type I IL-1 receptor (IL-1RI) or the p68 type II IL-1R (IL-1RII) on both T and B lymphocytes. We and others have previously shown that the anterior pituitary gland also has specific high affinity binding sites for IL-1 alpha (Kd = approximately 1 nM) and expresses transcripts for both isoforms of the IL-1R. However, the identity of cells in the anterior pituitary gland that express the IL-1R and whether different populations of adenohypophyseal cells express different isoforms of the IL-1R remain unknown. Here we have used a combination of immunohistochemistry and histochemistry to localize IL-1RI and IL-1RII to specific target cells in the mouse anterior pituitary gland. Perfusion-fixed, paraffin-embedded sections of anterior pituitary gland were immunolabeled with well characterized monoclonal antibodies to either IL-1RI or IL-1RII and counterstained using a modified Gomori's method to document acidophils and basophils. Immunolabeling demonstrated that both IL-1RI and IL-1RII were abundantly expressed on a single population of anterior pituitary gland cells and that these cells could be classified on the basis of histochemical staining as a subpopulation of acidophils. The distribution, morphology, and proportion of cells immunolabeled for IL-1RI and IL-1RII were consistent with GH-synthesizing cells. To confirm this hypothesis, a modified indirect avidin-biotin complex, sequential peroxidase/alkaline phosphatase technique was used to label anterior pituitary gland cells with antibodies to IL-1RI followed by antibodies to IL-1RII, GH, PRL, or ACTH. The IL-1RI-positive cells predominately coexpressed IL-1RII and GH, but very little, if any, PRL or ACTH. These data establish that the predominant cell population in the murine anterior pituitary gland that constitutively expresses IL-1R stain as acidophils histochemically, is round to oval with dense granular cytoplasm and eccentric nuclei, synthesizes GH, and simultaneously expresses IL-1RI and IL-1RII isoforms.

Although there is a consensus that a cell's microenvironment can have a dramatic influence on its ability to express a particular gene, the relative contribution of physical interaction (cell to cell adhesion) and paracrine signaling to this phenomenon has been difficult to discern. Here, we addressed this problem in mammotropes by making "real-time" measurements of prolactin (PRL) gene expression followed by immunocytochemistry (for post facto identification of a neighbor's phenotype). Our results show that it is the nature (phenotype) rather than the physical presence of a neighboring cell that dictates the degree to which the PRL gene is expressed.

Progress in understanding the dynamics of gene expression has been hampered by lack of a strategy for continuously monitoring this process within normal, living cells. Here, we employed a modification of conventional luciferase technology to make single and repeated real-time measurements of PRL gene expression from individual, living lactotropes from nursing rats. Cells were individually transfected by microinjection with a PRL promoter/luciferase reporter construct. Levels of PRL gene transcription were quantified by photonic imaging in the same cells before and after 24 h of culture in the presence or absence of the dopamine agonist bromocryptine or epidermal growth factor, two well known regulators of PRL gene transcription. We found these cells to be remarkably heterogeneous with respect to basal PRL gene expression and that the degree of activity within a single cell could fluctuate greatly over time under basal culture conditions. Treatment with bromocryptine or epidermal growth factor induced predictable and reversible changes in the average responses observed, yet individual cells displayed marked differences in response to these agents. These findings demonstrate the utility of this paradigm for monitoring dynamics of gene expression within normal, living cells of any type. Moreover, they provide a molecular basis for the secretory heterogeneity and plasticity that have come to be known as hallmarks of lactotrope cell function.

It is clear that dopamine (DA) at high concentrations (> 100 nmol/l) inhibits the release of prolactin (PRL). Paradoxically, this monoamine at low concentrations (< 10 nmol/l) has also been shown to augment PRL secretion. One possible explanation for these divergent effects is that DA binds receptors capable of interacting with multiple G protein subtypes that recruit opposing intracellular signaling pathways within lactotropes. To identify G proteins which couple DA receptor activation to PRL secretion, we have selectively immunoneutralized the activity of Gi alpha 3 and Gs alpha in primary cultures of rat pituitaries and subsequently tested the ability of these cultures to respond to high and low dose DA. Specifically, permeabilized pituitary cell cultures from random-cycling female rats were treated with control immunoglobulins (IgGs; 50 micrograms/ml) purified from preimmune serum (PII) or IgGs directed against the C-terminal portion of Gi alpha 3 or Gs alpha. After immunoneutralization of these G proteins, cells were challenged with 10 or 1000 nmol DA/l and the relative amount of PRL released was assessed by reverse hemolytic plaque assay. Results were expressed as % of basal values and compared. Under control conditions (PII), 1000 nmol DA/l inhibited (61.4 +/- 7.6% of basal values; mean +/- S.E.M.) while 10 nmol DA/l augmented (120.0 +/- 7.0%) PRL release in five separate experiments. Treatment of cells with anti-Gi alpha 3 attenuated the inhibitory effect of high dose DA (87.3 +/- 14.5%). However, elimination of Gi alpha 3 activity did not significantly alter the PRL stimulatory effect of 10 nmol DA/l (121.0 +/- 5.2%). Interestingly, immunoneutralization of Gs alpha resulted in a reciprocal shift in the activity of the lower dose of DA from stimulatory to inhibitory (69.7 +/- 7.3%) while combined treatment of anti-Gi alpha 3 and anti-Gs alpha abrogated the responsiveness of pituitary cell cultures to either DA treatment (1000 nmol/l, 70.7 +/- 12.5% and 10 nmol/l, 87.5 +/- 21.4%). These data reveal that ligand-activated DA receptors can interact with both Gi alpha 3 and Gs alpha. Elimination of the stimulatory component (Gs alpha) favors the DA receptor activation of the inhibitory pathway (Gi alpha 3) suggesting a competition between negative and positive intracellular signaling mechanisms in normal lactotropes. In addition to DA treatment, we also challenged permeabilized pituitary cells with 100 nmol thyrotropin-releasing hormone (TRH)/l as a positive control for secretory integrity. As anticipated, TRH stimulated PRL release to 188.0 +/- 31.0% of basal values under control conditions. Unexpectedly, immunoneutralization of Gs alpha completely blocked the ability of TRH to induce PRL release (101.8 +/- 12.0%). This neutralizing effect was specific to Gs alpha in that blockade of Gi alpha 3 activity had no significant effect on TRH-stimulated PRL release (166.2 +/- 13.1%). These data are the first to support a direct role of Gs alpha in TRH signal transduction within PRL-secreting cells.

Recent studies reveal that individual lactotropes vary in the amount of prolactin (PRL) released from day to day and that the magnitude of these variations is gender specific. In an attempt to further elucidate the dynamics of hormone secretion by single cells, we have investigated short-term variations in PRL release from male and lactating female rat lactotropes. To this end, hormone release from individual lactotropes was monitored at 12-min intervals for up to 2 h, using a modified version of the reverse hemolytic plaque assay. We found that pituitary cells from both genders could release PRL in either an intermittent or continuous mode, but that the former pattern of secretion predominated for male lactotropes. Interestingly, the mode of hormone secretion was tightly coupled (P < 0.001) to the amount of hormone released (continuous >> intermittent), regardless of the gender of the pituitary donor. The existence of gender-specific differences in secretory modalities indicates that this fluctuating pattern of hormone secretion by individual cells is not random but regulated, perhaps through entrainment in vivo by gonadal steroids.

The existence of differences in the amount of hormone released by individual lactotropes from the same pituitary gland has been unequivocally established. Such secretory heterogeneity could reflect the existence of lactotrope subtypes that differ quantitatively in their capacity to release constant amounts of hormone over time. Alternatively, the same pattern could arise from the intermittent secretion of hormone by individual cells. In an attempt to distinguish between these possibilities, we used a novel version of a sequential plaque assay to monitor daily the amount of PRL secreted by the same individual cells over a period of 4 days. When more than 1200 cells from male and lactating female rats were analyzed in this manner, we found that the average amount of hormone released over time was relatively constant within gender and that (as anticipated) cells from females released 2.6-fold more PRL than did those from males. However, assessment of secretory variability by individual lactotropes revealed that male cells were considerably more irregular in their pattern of PRL release than were their female counterparts. Indeed, only 11.8 +/- 2.5% (mean +/- SEM) of male cells secreted relatively constant amounts of hormone over the 4-day period, whereas 43.4 +/- 8.4% of the female cells exhibited a similar profile (assessed by comparing coefficients of variation for individual cells). Interestingly, this variability in secretion seemed to be inversely related to the amount of hormone released; constant secretors had a much higher average plaque size (2.5-fold greater; P < 0.01) than irregular secretors. Taken together, our results demonstrate that individual lactotropes from the same animal can release hormone in either a constant or episodic manner, and that the gender of the pituitary donor can have a strong influence on the relative abundance of each phenotype. Moreover, our data suggest the former phenotype makes a greater contribution to overall PRL secretion than the latter.

In an attempt to elucidate the mechanism(s) underlying the paradoxical (inhibitory vs. stimulatory) effects of DA on PRL release, we treated permeabilized pituitary cells with antibodies directed against the carboxyl-terminus of the alpha-subunit of the guanine nucleotide binding protein, Gi3. Immunoneutralization of Gi alpha 3 completely blocked the inhibitory effect of 1000 nM DA on PRL release, as assessed by reverse hemolytic plaque assays. In contrast, DA at a 100-fold lower concentration (10 nM) had no effect on PRL release under control conditions, but elicited a stimulatory response in the presence of anti-Gi alpha 3. Examination of the frequency distribution of plaque sizes indicated that suppression and augmentation of PRL secretion by DA was not attributable to distinct subpopulations of lactotropes. Taken together, these data suggest that all pituitary lactotropes have the potential to respond to the inhibitory and stimulatory activities of DA. However, the stimulatory action of this monoamine is normally masked by tonic activation of Gi alpha 3 which couples DA to its inhibitory pathway.

The mechanisms underlying hormonal gene expression are complex, and key regulatory events are often temporally separated. Our understanding of these dynamic processes has been hindered in part by the lack of a method suitable for making dynamic measurements of gene expression. In an attempt to develop such a system, we modified conventional luciferase reporter technology to enable real time measurement of gene expression in living endocrine cells.

It is well established that dopamine (DA) effectively inhibits PRL secretion from anterior pituitary mammotropes via D2-DA receptors. Paradoxically, it is reported that the monoamine can actually increase PRL release under appropriate experimental conditions. Although the mechanism underlying this stimulatory effect remains undefined, the ability of D1- and D5-DA receptors to activate adenylyl cyclase raises the possibility that a similar receptor subtype is present in the anterior pituitary and mediates the stimulatory effects of DA on PRL release. The purpose of the present study was to explore this possibility. First, we tested whether D1 and D5 receptors could couple to and stimulate PRL secretion. Subclones of GH4C1 cells (which secrete PRL, but do not express DA receptors) stably expressing human D1 or D5 receptors were treated with DA (10(-16)-10(-6) M), and the medium PRL content was measured by RIA. Subclones transfected with short or long forms of the human D2 receptor were also tested. As expected, DA (10(-6) M) inhibited PRL release from cells expressing either short or long D2 receptors by 41% and 39%, respectively (P < 0.01; n = 4 separate experiments). In contrast, comparable concentrations of DA (10(-(8) and 10(-6) M) increased PRL release from cells expressing D1 or D5 receptors by 76% and 122%, respectively (P < 0.01; n = 4). Thus, both D1 and D5 receptors were fully capable of stimulating PRL release from transfected GH4C1 cells. We next sought to determine whether the gene for at least one of these structurally similar receptors was expressed in rat anterior pituitary tissue. First strand cDNA was synthesized, using a rat D5-specific oligonucleotide primer and reverse transcriptase, from total RNA extracted from the anterior pituitary glands of five lactating female rats. The specifically primed cDNA then served as a template for 35 cycles of polymerase chain reaction amplification in which nested primers specific for the rat D5 receptor were used. Electrophoresis of the DNA resolved a 696-basepair band corresponding to a fragment of the D5 receptor in each of five anterior pituitary samples (verified by digestion with three different restriction endonucleases). Taken together, these results demonstrate that both D1 and D5 receptors are capable of mediating the stimulatory effects of DA on PRL release and that the mRNA for DA D5 receptors is present in rat anterior pituitary glands. Our findings support the view that PRL release in vivo may be modulated via one or more stimulatory DA receptors.

Considerable information regarding the regulation of GH and prolactin (PRL) release has been generated using pituitary cell lines as model systems. Inasmuch as these cultures have been derived from single cells by clonal selection it has frequently been assumed that they are composed of homogeneous populations of hormone secretors. However, experience with GH3 cells clearly demonstrates that such is not the case, since this GH- and PRL-producing line is comprised of a mixture of cells that are bihormonal, secrete only GH, or secrete neither hormone. Interestingly, the relative amount of these phenotypic subpopulations is not fixed, but can be altered by treatment with established regulators of GH and PRL secretion. This potential for secretory heterogeneity and phenotypic plasticity prompted us to examine the cellular composition of other commonly used GH- and/or PRL-secreting cell lines under control and treatment conditions. First, GH4C1, GH1, GC, MMQ and P0 cells were maintained according to established media and culture protocols and the relative abundance of GH and PRL secretors was assessed by reverse haemolytic plaque assays. As shown previously for GH3 cells, two cell lines were found to be functionally heterogeneous. Specifically, GH4C1 and GH1 were comprised of mixed populations of GH (25.9 +/- 1.1% and 51.3 +/- 6.5% (S.E.M.) respectively) and PRL (44.8 +/- 3.7 and 66.1 +/- 4.1% respectively) secretors. However, MMQ and GC cell cultures were relatively homogeneous with respect to hormone secretion in that the MMQ cells released PRL (72.2 +/- 4.9%) but not GH, while GC cells released GH (93.6 +/- 1.4%) but not PRL.(ABSTRACT TRUNCATED AT 250 WORDS)