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Biomedical subjects

A Miyake

Publications and source records attributed to A Miyake.

At least 109 records · Page 6Linked to original sources
PubMed1995

Interleukin-6 stimulates cell proliferation of rat pituitary clonal cell lines in vitro.

We investigated the effect of recombinant human IL-6 (rhIL-6) on cell proliferation using the MtT/E rat pituitary tumor cell line, which was recently established by Inoue et al. This cell line expresses the homeodomain protein Pit-1/GHF 1 and does not produce any significant amount of pituitary hormones, but retains its tumorigenicity by back-transplantation into rats, resulting in production of prolactin. MtT/E cells were seeded into Falcon 24-well plates at a density of 2 x 10(4) cells/well in a cultured medium, containing 10% horse serum and 2.5% fetal bovine, with test drug. After four-days (12 days for the time-course study) incubations, the cells were counted using a hemocytometer. Incubation for 4 days with rhIL-6 caused concentration-dependent stimulation of MtT/E cell growth and [3H]-thymidine incorporation into MtT/E cells. Addition of 20 ng/ml rhIL-6 to the culture medium stimulated MtT/E cell growth in a time-dependent manner, withdrawal of rhIL-6 from the culture medium reduced MtT/E cell growth, and re-addition of rhIL-6 to the culture medium again stimulated MtT/E cell growth. Among the cytokines tested, granulocyte colony-stimulating factor (rh G-CSF) also showed a slight but significant mitogenic activity on the MtT/E cells. Analysis of 125I-rhIL-6 binding to the MtT/E cells indicated a dissociation constant of 0.953 x 10(-9) mmol/l and the presence of 968 binding sites per cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Simultaneous measurements of exocytosis and intracellular calcium concentration with fluorescent indicators in single pituitary gonadotropes.

Previously, we established a method for the estimation of exocytosis in single gonadotropes using an impermeable fluorescent membrane probe, TMA-DPH. In this study, we have developed a method for the simultaneous measurement of exocytosis and intracellular free Ca2+ concentration ([Ca2+]i) by double-labeling with TMA-DPH and the intracellular Ca2+ probe, Fura-2/AM, using a fluorescence microscope with a 3-wavelength excitation and 2-wavelength emission system. We, therefore, clarified the relationship between spontaneous [Ca2+]i oscillation or gonadotropin releasing hormone (GnRH)-induced intracellular Ca2+ mobilization and exocytosis in gonadotropes. Under resting conditions, some gonadotropes showed various types of spontaneous [Ca2+]i oscillations, while others did not, but all showed basal exocytosis. Each [Ca2+]i peak oscillation did not cause Ca(2+)-regulated exocytosis, and even complete blockage of the [Ca2+]i increase by the intracellular Ca2+ chelator BAPTA/AM had no effect on basal exocytosis. Both GnRH-induced intracellular Ca2+ mobilization and regulated exocytosis showed a similar pattern of peaks and plateaus. Blockage of the [Ca2+]i increase by BAPTA/AM almost completely inhibited the GnRH-stimulated exocytosis. These results show that spontaneous [Ca2+]i oscillations under resting conditions are not linked to regulated or basal exocytosis, and that intracellular Ca2+ mobilization is essential for GnRH-stimulated exocytosis.

Animals↗
PubMed1995

Transient expression of FGF receptor-4 mRNA in the rat cerebellum during postnatal development.

The fibroblast growth factor (FGF) receptor-4 mRNA in the adult rat brain is expressed preferentially in the medial habenular nucleus. In this paper, we examined the expression of FGFR-4 mRNA in the brain during postnatal development. Interestingly, in addition to the persistent expression of FGFR-4 mRNA in the medial habenular nucleus, FGFR-4 mRNA was transiently expressed in the proliferative zone of the external granule layer of the developing cerebellum. The localization and transient expression of FGFR-4 mRNA in the developing cerebellum suggest that FGFR-4 mRNA was expressed by proliferative granule cells. The present findings indicate that FGFR-4 in the brain has an important role in the postnatal development of the cerebellar cortex.

Animals↗
PubMed1995

Perimenopausal changes in serum lipids and lipoproteins: a 7-year longitudinal study.

Although cross-sectional studies suggest considerable influence of menopause on serum lipids and lipoproteins in women, it is not exactly clear. During our 7-year longitudinal study, serum concentrations of total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides and low-density lipoprotein (LDL) cholesterol were measured in 16 healthy perimenopausal women (aged 47-56 years at menopause) who had undergone annual examinations 4 years before and 3 years after menopause under a health examinations system in Osaka. Longitudinal design enabled us to study the natural course of serum lipids and lipoproteins. The results show that from 4 years before to 1 year after menopause, the serum concentration of total cholesterol and LDL cholesterol increased on average by 25 mg/dl (14%) and 20 mg/dl (19%), respectively. Serum concentrations of triglycerides and of HDL cholesterol remained virtually unchanged during the perimenopausal and postmenopausal periods. It was concluded that serum lipids and lipoproteins are thus significantly altered as a consequence of menopause, resulting in a more atherogenic profile in the postmenopausal period.

Arteriosclerosis↗
PubMed1995

Correlation between serum prolactin levels and hepatocellular tumorigenesis induced by 3'-methyl-4-dimethylaminoazobenzene in mice.

Ovariectomy at 1 month of age promotes development of hepatocellular adenomatous nodules in female C57BL/6 x DS-F1 mice treated neonatally with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). Implantation of oestradiol-17 beta (E2) pellets at 1 month of age suppresses nodule development. Since E2 increases serum levels of prolactin, high serum levels of prolactin in mice that have received implants of E2 pellets may play a role in the suppression of hepatocellular tumorigenesis. Therefore, to investigate the role of prolactin in hepatocellular tumorigenesis, we examined development of adenomatous nodules in female mice that had been treated neonatally with 3'-Me-DAB and had undergone ovariectomy at 1 month of age, under various serum levels of prolactin. Treatment of these mice with perphenazine (dopamine antagonist) from 6 months of age or transplantation of pituitary glands under the renal capsule at 6 months of age markedly increased serum levels of prolactin and significantly suppressed the incidence of adenomatous nodules at 12 months of age. Implantation of E2 pellets at 1 month of age increased serum levels of prolactin to a greater extent and further decreased the incidence of adenomatous nodules. Treatment of mice that had received implants of E2 pellets at 1 month of age with bromocriptine (dopamine agonist) from 6 months of age decreased serum levels of prolactin, and was accompanied by an increase in the incidence of nodules. The present results showed that an increase in serum levels of prolactin was accompanied by a decrease in incidence of liver tumours induced by 3'-Me-DAB in mice, suggesting a suppressive effect of prolactin on liver tumorigenesis in mice. Thus, it is possible that the suppressive effect of oestrogen on liver tumorigenesis in mice is mediated, at least in part, by prolactin.

Adenoma↗
PubMed1995

Mouse growth hormone-releasing factor secretion is activated by inhibin and inhibited by activin in placenta.

We investigated the effects of activin and inhibin on the regulation of mouse growth hormone-releasing factor (mGHRF) secretion by primary placental cells harvested at Day 12 of pregnancy. Activin-A, an activator of FSH secretion, inhibited mGHRF secretion. In contrast, inhibin, an inhibitor of FSH secretion, activated mGHRF secretion. The lowest concentrations of activin-A and inhibin that significantly affected mGHRF secretion were 2 nM. Follistatin, a binding protein of activin, completely eliminated the ability of activin to inhibit mGHRF secretion. The steady-state level of mGHRF mRNA, as assessed by Northern analysis, was reduced by incubation of placental cells with activin-A. All activin and inhibin subunit mRNAs were expressed in mouse placenta, and their expressions increased during gestation. These findings suggest that activin and inhibin have opposite effects on mGHRF secretion as compared with FSH secretion and that they regulate mGHRF secretion in an autocrine or paracrine manner in the mouse placenta in vivo.

Activins↗
PubMed1995

Posttranscriptional inhibition of mouse placental lactogen-II secretion by transforming growth factor beta 1: synergistic effects with epidermal growth factor and interleukin-6.

We studied the effect of transforming growth factor beta 1 (TGF beta 1) on mouse placental lactogen (mPL)-I and mPL-II secretion by primary cultures of placental cells from Days 7, 9, and 12 of pregnancy. We also studied the effects of co-incubation of epidermal growth factor (EGF) or interleukin-6 (IL-6) with TGF beta 1 on mPL-I and mPL-II secretion. TGF beta 1 at 10 ng/ml did not affect mPL-I secretion by cells from Days 7 or 9 of pregnancy or mPL-II secretion by cells from Day 7 of pregnancy but significantly inhibited mPL-II secretion by cells from Days 9 or 12 of pregnancy. The lowest concentration of TGF beta 1 that significantly inhibited mPL-II secretion by cells from Days 9 or 12 of pregnancy was 1 ng/ml. Immunocytochemistry for mPL-II indicated that treatment of placental cells from Day 12 of pregnancy with 10 ng/ml TGF beta 1 significantly reduced the number of mPL-II-containing cells. Inhibition of mPL-II secretion by TGF beta 1 was eliminated completely by addition of an anti-TGF beta 1 antibody. Northern analysis showed that steady state levels of mPL-II mRNA were not reduced by incubation of placental cells from Day 12 of pregnancy with 10 ng/ml TGF beta 1 for 5 days. EGF at 10 ng/ml significantly inhibited mPL-II secretion by cells from Day 7 of pregnancy, and addition of 10 ng/ml TGF beta 1, which did not itself inhibit mPL-II secretion by those cells, enhanced the inhibition by EGF of mPL-II secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Cytokines that use gp130 as a signal transducer stimulate mouse placental lactogen-I (mPL-I) but inhibit mPL-II production in vitro.

Interleukin-11 (IL-11), leukemia inhibitory factor (LIF), and oncostatin M (OM), all of which use gp130 as a signal transducer, significantly inhibited mouse placental lactogen-II (mPL-II) secretion by cultured placental cells from Days 7, 9, and 12 of pregnancy. These cytokines significantly stimulated mPL-I secretion by cells from Day 9, but not Day 7, of pregnancy. An antibody to LIF completely blocked the stimulatory and inhibitory effects of LIF on mPL-I and mPL-II secretion, respectively. LIF and OM decreased the abundance of mPL-II mRNA in placental cells. Double immunocytochemistry for mPL-I and mPL-II indicated that LIF, OM, and IL-11 significantly increased the number of giant cells containing only mPL-I or both mPL-I and mPL-II but decreased the number of giant cells containing only mPL-II. IL-6, which also uses gp130 as a signal transducer, inhibits mPL-II secretion only after midpregnancy; however, addition of soluble IL-6 receptor (sIL-6R) together with IL-6 resulted in a significant inhibition of mPL-II secretion before midpregnancy. Treatment of cells from Day 12 of pregnancy with IL-6 during the first 2 days of culture resulted in significant inhibition of mPL-II secretion by the third day of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Cyclic adenosine-3',5'-monophosphate stimulates mouse placental lactogen-I (mPL-I) secretion but inhibits mPL-II secretion at midpregnancy.

To determine whether cAMP regulates mouse placental lactogen-I (mPL-I) and mPL-II secretion at midpregnancy in vitro, mouse placental tissue from day 9 of pregnancy was dispersed with collagenase, cells were fractionated on a Percoll gradient, and the purified trophoblast cells were plated in a serum-free medium. The cells were then incubated with various agents that increased the intracellular cAMP level for 5 days. 8-Bromo-cAMP stimulated mPL-I secretion, but inhibited mPL-II secretion in a time- and dose-dependent manner without changing the amount of newly synthesized trichloroacetic acid-precipitable proteins. Cholera toxin and forskolin, which increase intracellular cAMP accumulation, also regulated mPL-I and mPL-II secretion in the same manner. 8-Bromo-cAMP increased the intracellular mPL-I concentration, decreased the intracellular mPL-II concentration, and increased the immunoprecipitable newly synthesized mPL-I concentration in both the medium and cells. 8-Bromo-cAMP increased the expression of mPL-I messenger RNA and decreased the expression of mPL-II messenger RNA. The sequential reverse hemolytic plaque assay and double immunocytochemistry indicated that 8-bromo-cAMP regulates the subpopulation of giant cells containing and releasing mPL. These findings suggest that an increase in intracellular cAMP stimulates mPL-I secretion, but inhibits mPL-II secretion by changing the subpopulation of giant cells containing and releasing mPL.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Oxytocin stimulates mitogen-activated protein kinase activity in cultured human puerperal uterine myometrial cells.

The regulation of mitogen-activated protein (MAP) kinase by oxytocin in cultured human uterine myometrial cells was investigated. Oxytocin caused the rapid stimulation of MAP kinase activity detected in 32P incorporation of MAP-2. Oxytocin also stimulated the phosphorylation of MAP kinase detected in incorporation of [32P]orthophosphate into MAP kinase. Furthermore, oxytocin induced the tyrosine phosphorylation of MAP kinase. The oxytocin-dependent increase in the tyrosine phosphorylation of MAP kinase displayed a transient time course and was dependent on the concentration of oxytocin applied to the cells. Furthermore, we examined the mechanism by which oxytocin induced MAP kinase phosphorylation. Islet-activating protein (100 ng/ml), which inactivates Gi/Go proteins, blocked the oxytocin-induced phosphorylation of MAP kinase. Moreover, 1 microM ritodrine, which is known to relax uterine muscle contraction, attenuated oxytocin-induced MAP kinase activity and phosphorylation. These results provide evidence that oxytocin acutely activates MAP kinase through an islet-activating protein-sensitive G-protein in human uterine myometrial cells, suggesting that this new pathway may play an important role in the biological action of oxytocin on these cells.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

Cyclic adenosine 3',5'-monophosphate stimulation of placental proliferin and proliferin-related protein secretion.

To identify factors that regulate proliferin (PLF) and PLF-related protein (PRP) secretion by the mouse placenta, placental cells from day 9 of pregnancy were cultured for up to 5 days, and PLF and PRP release into the medium was assessed by RIA. Transforming growth factor-alpha, interleukin-1 alpha, and interleukin-6 did not regulate either PLF or PRP secretion. However, treatment of primary placental cell cultures with 8-bromo-cAMP, cholera toxin, or forskolin resulted in 2- to 3-fold increases in the percentages of PLF- and PRP-producing cells in the population and corresponding increases in both PLF and PRP messenger RNA and secreted protein. The increase in the number of PLF-producing cells was accompanied by an increase in the number of cells expressing both PLF and mouse placental lactogen-I. These data suggest that cAMP levels can regulate trophoblast giant cell differentiation and, consequently, the amount of PLF and PRP secretion.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Expression of messenger ribonucleic acid for epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and EGF receptor in human amnion cells: possible role of TGF alpha in prostaglandin E2 synthesis and cell proliferation.

The amnion plays important structural and functional roles in the maintenance of pregnancy and the initiation of parturition. Recently, we reported that epidermal growth factor (EGF) activates prostaglandin (PG) production and cell growth in cultured amnion cells. In this study, we showed the expression of EGF, transforming growth factor-alpha (TGF alpha), and EGF receptor protein and messenger ribonucleic acid in amnion cells, using an immunofluorescence technique and the reverse transcription-polymerase chain reaction. Next, we studied the effect of TGF alpha on intracellular Ca2+ mobilization and PGE2 production in amnion cells. TGF alpha induced an increase in the intracellular Ca2+ concentration in amnion cells, and this increase was significantly reduced when the cells were incubated with cobalt chloride (a Ca2+ channel blocker; 2.5 mmol/L) or EGTA (a Ca2+ chelator; 5 mmol/L). TGF alpha enhanced PGE2 production, and this increase was significantly inhibited when the cells were incubated with indomethacin (a cyclooxygenase inhibitor; 10 mumol/L), cobalt chloride (2.5 mmol/L), or EGTA (5 mmol/L). We also investigated the effect of TGF alpha on the growth of cultured human amnion cells by using flow cytometric analysis of the DNA content. TGF alpha induced DNA synthesis by human amnion cells, and indomethacin inhibited the TGF alpha-induced DNA synthesis. These results suggest that 1) EGF/TGF alpha are expressed and produced in amnion cells; 2) these endogenous factors may regulate the proliferation of amnion cells in an autocrine or paracrine manner; and 3) these growth factors may exert their effects via intracellular Ca2+ mobilization and PGE2 production.

Amnion↗
PubMed1995

Synthetic studies on condensed-azole derivatives. I. Synthesis and anti-asthmatic activities of omega-substituted alkylthioimidazo[1,2-b]pyridazines.

A series of novel omega-substituted alkylthioimidazo[1,2-b]pyridazines was designed and synthesized in an effort to find a novel anti-asthmatic agent. The anti-asthmatic activity of these compounds was evaluated on the basis of their ability to inhibit thromboxane A2 synthetase and platelet activating factor (PAF)-induced bronchoconstriction in guinea pigs. None of these compounds significantly inhibited thromboxane A2 synthetase, though, sulfonamide derivatives potently inhibited PAF-induced bronchoconstriction. Among them, 3-(imidazo[1,2-b]pyridazin-6-yl)thiopropanesulfonamide (5) showed the most potent inhibitory effect. The anti-asthmatic effects of compound 5 in experimental models were superior to those of theophylline.

Animals↗
PubMed1995

Synthetic studies on condensed-azole derivatives. III. Synthesis and anti-asthmatic activities of C-substituted alkyl side chain derivatives of omega-sulfamoylalkylthioimidazo[1,2-b]pyridazines and related compounds.

A series of novel alkylthioimidazo[1,2-b]pyridazines was synthesized and evaluated for ability to inhibit platelet activating factor (PAF)-induced bronchoconstriction in guinea pigs. Among them, 3-(imidazo[1,2-b]pyridazin-6-yl)thio-2,2-dimethylpropanesulfona mide (15) showed the most potent inhibitory effect. The structure-activity relationships in this series of compounds, in particular, the effects of conversion of the imidazopyridazine ring into other heterocyclic rings, introduction of a substituent group at the 2 or 3 position of the imidazopyridazine ring and introduction of a substituent group into the alkyl side chain, are also discussed.

Animals↗
PubMed1995

Increase in human placental glucose transporter-1 during pregnancy.

Glucose transporter-1 (GLUT1) has been found in high abundance in human placentas. The purpose of this study was to analyze the changes in the level of GLUT1 during pregnancy. We have analyzed the gestational changes in human placental GLUT1 by [3H]cytochalasin B binding assay, immunoblot analysis and Northern blot analysis. Levels of [3H]cytochalasin B binding to placental membrane in pregnancy at 7-10, 18-20 and 38-40 weeks were 4.2 +/- 0.47, 5.2 +/- 0.46 and 7.2 +/- 0.40 (mean +/- SEM, N = 4) mumol/kg protein, respectively. Amounts of 49-kD and 60-kD proteins detected in immunoblot analysis increased during pregnancy in parallel with [3H]cytochalasin B binding to placental membrane. The GLUT1 mRNA levels also increased during pregnancy. Immunohistochemical examination showed that GLUT1 was localized in the plasma membranes of trophoblast cells. These findings showed that the GLUT1 level increased during pregnancy in human placentas and suggest that GLUT1 may play an important role in fetal development.

Blotting, Northern↗
PubMed1995

Regulation of rat placental lactogen (rPL)-II secretion: cAMP inhibits rPL-II secretion in vitro.

We examined whether epidermal growth factor (EGF), interleukin (IL)-1, IL-6 and cAMP analogs, which regulate mouse placental lactogen II secretion, affect rat placental lactogen (rPL)-II secretion using a rat choriocarcinoma cell line, Rcho-1. EGF, IL-1 and IL-6 did not affect rPL-II secretion, but 8-bromo-cAMP and forskolin inhibited rPL-II secretion by the 8th day of culture. The effects were dose dependent and the lowest concentrations of 8-bromo-cAMP and forskolin that significantly inhibited rPL-II secretion were 125 and 5 mumol/l, respectively. Cholera toxin also inhibited rPL-II secretion. The reverse hemolytic plaque assay for rPL-II indicated that the cells releasing rPL-II in the culture were giant cells and that 8-bromo-cAMP decreased the number of rPL-II-releasing cells. Western blot analysis of rPL-II yielded a single band at approximately 24.5 K, and 8-bromo-cAMP treatment significantly reduced the band intensity. Northern blot analysis of rPL-II indicated that 8-bromo-cAMP also reduced rPL-II gene expression. These findings suggest that the increase of intracellular cAMP accumulation results in inhibition of rPL-II secretion by decreasing rPL-II gene expression and inhibiting giant cell differentiation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Scanning electron microscope assessment of exocytotic changes in purified gonadotropes.

These studies were undertaken to characterize the exocytotic changes in purified gonadotropes by three-dimensional imaging using scanning electron microscopy. Rat gonadotropes were purified using a fluorescence-activated cell sorter and an argon laser treatment system. The purified gonadotropes were stimulated with GnRH under various conditions and fixed for scanning electron microscopy. After the GnRH stimulation, many 'hole' structures (diameter 0.1-0.5 micron) were observed on the cell surface, and notably the population of cells with 10 or more holes was clearly increased. The pattern of the time-course of the changes in this population was perfectly consistent with the LH secretory profile of pituitary cells, and their formation of the cells with 10 or more holes was completely inhibited by pretreatment with a GnRH antagonist. Our data suggest that the hole structure represents an exocytotic opening site and that regulated exocytosis in purified gonadotropes can be evaluated by scanning electron microscopy. This method may be widely applicable to other endocrine cells.

Animals↗