Biomedical subjects

L S Frawley

Publications and source records attributed to L S Frawley.

At least 37 records · Page 2Linked to original sources

PubMed1998

Phytoestrogens have agonistic and combinatorial effects on estrogen-responsive gene expression in MCF-7 human breast cancer cells.

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.

Breast Neoplasms↗

PubMed1997

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 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.

Animals↗

PubMed1997

Real-time monitoring of estrogen-regulated gene expression in single, living breast cancer cells: a new paradigm for the study of molecular dynamics.

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.

Breast Neoplasms↗

PubMed1997

Octylphenol (OP), an environmental estrogen, stimulates prolactin (PRL) gene expression.

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.

Animals↗

PubMed1997

Phenotypic characterization and functional correlation of alpha-MSH binding to pituitary cells.

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.

Animals↗

PubMed1997

Discordance of prolactin gene transcription, mRNA storage, and hormone release in individual mammotropes.

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.

Animals↗

PubMed1997

Extracellular ATP as an autocrine/paracrine regulator of prolactin release.

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.

Adenosine Triphosphate↗

PubMed1996

Dual expression of p80 type I and p68 type II interleukin-I receptors on anterior pituitary cells synthesizing growth hormone.

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.

Animals↗

PubMed1996

Intercellular communication: relative importance of cellular adhesion and paracrine signaling to hormonal gene expression.

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.

Animals↗

PubMed1996

Dynamic monitoring and quantification of gene expression in single, living cells: a molecular basis for secretory cell heterogeneity.

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.

Animals↗

PubMed1996

Role of guanine nucleotide-binding proteins, Gi alpha 3 and Gs alpha, in dopamine and thyrotropin-releasing hormone signal transduction: evidence for competition and commonality.

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.

Analysis of Variance↗

PubMed1995

Individual lactotropes release prolactin in a temporally divergent and sexually dimorphic pattern.

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.

Animals↗

PubMed1994

Stimulation of lactotrope differentiation in vitro by fibroblast growth factor.

We have reported previously that differentiation of PRL-secreting cells in rats is regulated by a maternal peptide transferred to the neonatal circulation after ingestion of mothers' milk. Inasmuch as milk contains numerous hormones and biologically active peptides, the present study was designed to test the capacity of various growth factors and hypothalamic peptides at inducing the differentiation of PRL cells in vitro. Anterior pituitary cells from 1-day-old rat pups were cultured in a serum-free system for 6 days with a wide concentration range of each test peptide. After this culture period, lactotrope differentiation was assessed by subjecting the anterior pituitary cells to reverse hemolytic plaque assays for PRL. Our efforts were focused on those growth factors and hypophysiotropic peptides found in milk and/or known to regulate pituitary function. Included among these were TRH, GH-releasing factor, somatostatin, vasoactive intestinal peptide, angiotensin-II, insulin-like growth factor-I and -II, LH-releasing hormone, arginine vasopressin, and acidic and basic fibroblast growth factors (aFGF and bFGF, respectively). Of these peptides, only aFGF and bFGF were capable of stimulating lactotrope differentiation. Specifically, we found that maximally effective concentrations of aFGF and bFGF increased the percentage of PRL-releasing cells by almost 8-fold, from about 0.5% to over 4% of all pituitary cells. In addition, bFGF was found to be about 10-fold more potent than aFGF at inducing the differentiation of PRL secretors, with minimum effective doses approaching 10(-11) and 10(-10) M for bFGF and aFGF, respectively. These results suggest that bFGF is a strong candidate to subserve a role in regulating the differentiation of lactotropes in vivo.

Animals↗

PubMed1994

Evidence that stimulatory dopamine receptors may be involved in the regulation of prolactin secretion.

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.

Animals↗

PubMed1994

Real time measurement of gene expression in living endocrine cells.

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.

Animals↗

PubMed1994

Paradoxical effects of dopamine (DA): Gi alpha 3 mediates DA inhibition of PRL release while masking its PRL-releasing activity.

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.

Animals↗

PubMed1994

Dynamic fluctuations in the secretory activity of individual lactotropes as demonstrated by a modified sequential plaque assay.

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.

Animals↗

PubMed1994

Secretory characteristics and phenotypic plasticity of growth hormone- and prolactin-producing cell lines.

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)

Animals↗