epidermal-growth-factor and dibutyryl-cyclic-3--5--cytidine-monophosphate

The lacrimal gland is primarily responsible for the aqueous portion of the tear film. Simultaneous addition of cholinergic agonists or growth factors with cAMP-dependent agonists potentiates secretion. Recent investigations revealed that cAMP decreases p44/p42 mitogen-activated protein kinase (MAPK) activity stimulated by cholinergic agonists and growth factors that could account for this potentiation. In this study the authors identify the signal transduction pathway used by cAMP to inhibit MAPK activity.. Rat lacrimal gland acini were incubated with H89, an inhibitor of protein kinase A, before the addition of dibutyryl cAMP (dbcAMP, 10(-3) M) for 30 minutes. Basal MAPK and CREB activity and MAPK activity after stimulation with the cholinergic agonist carbachol (Cch) or epidermal growth factor (EGF) for 5 minutes was determined. The effect of dbcAMP on EGF receptor activity and basal and stimulated Ras, Raf-1, mitogen-activated protein kinase kinase (MEK), and MAPK activity was determined. The effect of a Rap-1 inhibitor, GGTI-298, on MAPK activity after the addition of dbcAMP was also determined.. H89 relieved the inhibition of cAMP on MAPK activity and inhibited CREB activity. Incubation with dbcAMP did not have any effect either on the EGF receptor or on Ras but significantly inhibited both basal and Raf-1 and MEK activity stimulated with Cch or EGF. GGTI-298 did not have any effect on cAMP-dependent decrease in MAPK activity.. The authors conclude that cAMP mediates the inhibition of MAPK by PKA in a Raf-1-dependent manner.

Topics: Animals; Blotting, Western; Carbachol; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cyclic CMP; Epidermal Growth Factor; ErbB Receptors; Isoquinolines; Lacrimal Apparatus; Male; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-raf; Rats; Rats, Sprague-Dawley; Sulfonamides

Testicular Leydig cells express estrogen receptors and are the predominant source of the male sex steroid hormone testosterone (T). Previous studies demonstrated that genistein acts through estrogen receptors in Leydig cells. In the present study, pre-treatment of Leydig cells isolated from 35 day-old male Long Evans rats with the epidermal growth factor receptor (EGFR) kinase inhibitor AG 1478 abrogated genistein inhibition of T biosynthesis. Also, incubation of Leydig cells in culture medium containing epidermal growth factor (EGF) decreased T secretion (control: 255+/-16; EGF: 190+/-17ng/10(6) cells, 24h) (P<0.05). However, T secretion by genistein-treated Leydig cells (0.1nM, 10muM; 24h) was rescued by post-treatment incubation with forskolin (control: 275+/-28 versus 325+/-35; 780+/-85; ng/10(6) cells, 3h) and dibutyryl cyclic adenosine 3'-5'-monophosphate (dbcAMP) (control: 370+/-65 versus 580+/-75; 2500+/-200; ng/10(6) cells, 3h) (P>0.05). Furthermore, post-treatment incubation with cholera toxin, an activator of G proteins, caused genistein-treated Leydig cells to produce similar T amounts as untreated control (control: 55+/-5 versus 52+/-2 and 47+/-4; ng/10(6) cells, 3h) (P>0.05). These observations imply that genistein action interferes with coupling of transmembrane luteinizing hormone receptors (LHR) with G proteins. Uncoupling of LHR from G proteins adversely affects adenylate cyclase function and impacts LH-dependent stimulation of Leydig cells. These findings have implications for testicular steroidogenesis in individuals exposed to genistein and soy-based products.

Topics: Adenylyl Cyclases; Animals; Cells, Cultured; Cholera Toxin; Colforsin; Cyclic CMP; Dose-Response Relationship, Drug; Down-Regulation; Endocrine Disruptors; Enzyme Activators; Epidermal Growth Factor; ErbB Receptors; Genistein; Leydig Cells; Luteinizing Hormone; Male; Protein Kinase Inhibitors; Quinazolines; Rats; Rats, Long-Evans; Receptors, LH; Signal Transduction; Testosterone; Tyrphostins

We previously found that addition of cAMP and a Ca(2+)/PKC-dependent agonist causes synergism or potentiation of protein secretion from rat lacrimal gland acini. In the present study we determined whether cAMP decreases p44/p42 mitogen-activated protein kinase (MAPK) activity in the lacrimal gland. Since we know that activation of MAPK attenuates protein secretion stimulated by Ca(2+)- and PKC-dependent agonists, we also determined whether this activation causes potentiation of secretion. Freshly prepared rat lacrimal gland acinar cells were incubated with dibutyryl cAMP (DBcAMP), carbachol (a cholinergic agonist), phenylephrine (an alpha(1)-adrenergic agonist), or epidermal growth factor (EGF). The latter three agonists are known to activate p44/p42 MAPK. p44/p42 MAPK activity and protein secretion were measured. As measured by Western blot analysis, DBcAMP inhibited both basal and agonist-stimulated p44/p42 MAPK activity. Cellular cAMP levels were increased by 1) using two different cell-permeant cAMP analogs, 2) activating adenylyl cyclase (L-858051), or 3) activation of G(s)-coupled receptors (VIP). The cell-permeant cAMP analogs, L-858051, and VIP inhibited basal p44/p42 MAPK activity by 50, 40, and 40%, respectively. DBcAMP and VIP inhibited carbachol- and EGF-stimulated MAPK activity. cAMP, but not VIP, inhibited phenylephrine-stimulated MAPK activity. Potentiation of secretion was detected when carbachol, phenylephrine, or EGF was simultaneously added with DBcAMP. We conclude that increasing cellular cAMP levels inhibits p44/p42 MAPK activity and that this could account for potentiation of secretion obtained when cAMP was elevated and Ca(2+) and PKC were increased by agonists.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Adrenergic alpha-Agonists; Animals; Blotting, Western; Calcium; Carbachol; Cholinergic Agonists; Colforsin; Cyclic AMP; Cyclic CMP; Diterpenes; Dose-Response Relationship, Drug; Enzyme Activators; Epidermal Growth Factor; In Vitro Techniques; Lacrimal Apparatus; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Peroxidases; Phenylephrine; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Phosphorylation; Protein Kinase C; Rats; Rats, Sprague-Dawley; Signal Transduction; Time Factors; Vasoactive Intestinal Peptide

Follicular fluid meiosis-activating sterol (FF-MAS) is regarded as an important compound relevant to meiotic resumption in mammalian oocytes. The objective of this study was to investigate the influence of FF-MAS on germinal vesicle breakdown (GVBD) and first polar body (PBI) extrusion with regard to culture conditions, state of the oocyte and mouse strain. Denuded oocytes (DO) and cumulus-enclosed oocytes (CEO) were retrieved from PMSG-primed Quackenbush or C57BL/6J x DBA/2 (C57) mice and cultured for 20 h in alpha-MEM medium under the following conditions: (i) 250 micromol/l dibutyryl cAMP (dbcAMP) +/- EGF, 1 ng/ml or FF-MAS, 20 micromol/l; (ii) 4 mmol/l hypoxanthine (HX) +/- EGF or FF-MAS; (iii) HX + EGF + FF-MAS; and (iv) HX + FF-MAS 5 h priming and subsequent culture with HX + EGF. Oocyte GVBD and PBI emission were recorded and stained with Hoechst 33342. Very limited meiotic inhibition was observed in Quackenbush mice in comparison with C57 mice. FF-MAS promoted maturation in C57 DO and CEO and Quackenbush DO. In Quackenbush DO and CEO and C57 DO a significant increase in atypical PBI extrusion occurred, but not in C57 CEO as well as in EGF-treated Quackenbush CEO primed or co-cultured with FF-MAS. These results support a meiosis resumption function for FF-MAS and suggest that in its presence, the quality of the MII oocytes retrieved appears to be influenced by the strain of the mice, the state of the oocyte and the presence or absence of growth factors in the culture medium.

Topics: Animals; Cells, Cultured; Cholestenes; Coculture Techniques; Cyclic CMP; Epidermal Growth Factor; Female; Hypoxanthine; Meiosis; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Oocytes; Species Specificity

We established selective primary cultures of neurons, astrocytes, and microglial cells from cryopreserved fetal cerebral cortex of cynomolgus monkeys (Macaca fascicularis). At 14 days in serum-containing medium, the cell cultures of the fetal cerebral cortex consisted primarily of neurons, astrocytes, and floating microglial cells. At 21 days, we observed a small number of myelin basic protein (MBP)-positive oligodendrocytes. The addition of cytosine arabinoside (a selective DNA synthesis inhibitor) at 2 days in culture eliminated proliferative glial cells, allowing adequate numbers of neurons to survive selectively. A chemically defined serum-free medium successfully supported neuronal survival at a level equivalent to that supported by the serum-containing medium. Brain-derived neurotrophic factor (BDNF) significantly affected the survival of primate neurons. Glutamate induced a significant degree of neuronal cell death against primate neurons and MK-801, a selective N-methyl-D-aspartate receptor (NMDAR) antagonist, blocked cell death, which suggests that primate cortical neurons have NMDAR and the glutamate-induced cell toxicity is mediated by NMDAR. In the serum-free medium, type-1 astrocytes responded to dibutyryl cyclic AMP and showed a process-bearing morphology. The growth of type-1 astrocytes in the serum-free medium was stimulated by epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and hydrocortisone, which are known growth factors in rat type-1 astrocytes. Cultured microglial cells expressed CD68, a monocyte marker. Macrophage-colony stimulating factor (M-CSF) stimulated microglial cell growth in the serum-free medium. These selective primary culture systems of primate cerebral cortical cells will be useful in issues involving species specificity in neuroscience.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Astrocytes; Cell Culture Techniques; Cell Death; Cell Division; Cells, Cultured; Cerebral Cortex; Culture Media, Serum-Free; Cyclic CMP; Cytarabine; Dizocilpine Maleate; Dose-Response Relationship, Drug; Epidermal Growth Factor; Excitatory Amino Acid Antagonists; Fetus; Fibroblast Growth Factors; Glial Fibrillary Acidic Protein; Glutamic Acid; Immunohistochemistry; Immunosuppressive Agents; Macaca fascicularis; Microglia; Microtubule-Associated Proteins; Myelin Basic Protein; Neurons

Excitatory amino acid transporter 1 (EAAT1) is one of the two glial glutamate transporters that clear the extracellular glutamate generated during neuronal signal transmission. Here, we cloned and characterized a 2.1-kb promoter region of human EAAT1 and investigated its function in the transcriptional regulation of the EAAT1 gene in human primary astrocytes. The full-length promoter region lacked TATA and CCAAT boxes and an initiator element, it contained several potential transcription factor-binding sites and it exhibited promoter activity in primary astrocytes and in several types of transformed cells. Consecutive 5'-deletion analysis of the EAAT1 promoter indicated the presence of negative and positive regulatory regions and a putative core promoter between -57 bp and +20 bp relative to the transcription start site (TSS). The core promoter contained a single GC-box in position -52/-39 and one E-box near the TSS and the GC-box site that was responsible for 90% of the basal promoter activity as determined by mutational analysis. Electrophoretic mobility shift, supershift and competition assays demonstrated binding of stimulating proteins (Sp) 1 and 3 to the GC-box and upstream stimulating factor (USF) 1 to the E-box. Treatment of primary human astrocytes with cellular modulators 8-bromo cyclic AMP and epidermal growth factor increased EAAT1 promoter activity in transient transfection assays and increased cellular EAAT1 mRNA expression and glutamate uptake by astrocytes. Conversely, tumor necrosis factor-alpha reduced both EAAT promoter activity and cellular EAAT1 mRNA expression. These results enable studies of transcriptional regulation of EAAT1 gene at the promoter level.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Astrocytes; Base Sequence; Blotting, Northern; Cells, Cultured; Cloning, Molecular; Cyclic CMP; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Epidermal Growth Factor; Excitatory Amino Acid Transporter 1; Fetus; Gene Expression Regulation; Glutamic Acid; Humans; Luciferases; Mice; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Rats; Regulatory Sequences, Nucleic Acid; RNA; Tetradecanoylphorbol Acetate; Time Factors; Transcription Factors; Transcription Initiation Site; Transcription, Genetic; Transcriptional Activation; Transfection; Transforming Growth Factor alpha; Upstream Stimulatory Factors