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Gene induction during differentiation of human pulmonary type II cells in vitro.
Wade KC, Guttentag SH, Gonzales LW, Maschhoff KL, Gonzales J, Kolla V, Singhal S, Ballard PL
(2006) Am J Respir Cell Mol Biol 34: 727-37
MeSH Terms: 8-Bromo Cyclic Adenosine Monophosphate, Cell Differentiation, Cells, Cultured, Dexamethasone, Epithelial Cells, Fatty Acid Synthases, Fetal Proteins, Fetus, Gene Expression Profiling, Gene Expression Regulation, Developmental, Glycerol Kinase, Humans, Infant, Infant, Newborn, Lipoprotein Lipase, Lung, Lysosome-Associated Membrane Glycoproteins, Phospholipids, RNA, Messenger, Subcellular Fractions, Time Factors, Transcriptional Activation, Transferases (Other Substituted Phosphate Groups)
Show Abstract · Added January 20, 2015
Mature alveolar type II cells that produce pulmonary surfactant are essential for adaptation to extrauterine life. We profiled gene expression in human fetal lung epithelial cells cultured in serum-free medium containing dexamethasone and cyclic AMP, a treatment that induces differentiation of type II cells. Microarray analysis identified 388 genes that were induced > 1.5-fold by 72 h of hormone treatment. Induced genes represented all categories of molecular function and subcellular location, with increased frequency in the categories of ionic channel, cell adhesion, surface film, lysosome, extracellular matrix, and basement membrane. In time-course experiments, self-organizing map analysis identified a cluster of 17 genes that were slowly but highly induced (5- to approximately 190-fold) and represented four functional categories: surfactant-related (SFTPC, SFTPA, PGC, SFTPB, LAMP3, LPL), regulatory (WIF2, IGF2, IL1RL1, NR4A2, HIF3A), metabolic (MAOA, ADH1B, SEPP1), and transport (SCNN1A, CLDN18, AQP4). Induction of both mRNA and protein for these genes, which included nine newly identified regulated genes, was confirmed, and cellular localization was determined in both fetal and postnatal tissue. Induction of lysosomal-associated membrane protein 3 required both hormones, and expression was localized to limiting membranes of lamellar bodies. Hormone-induced differentiation of human type II cells is associated with genome-wide increased expression of genes with diverse functions.
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23 MeSH Terms
Developmental regulation of claudin localization by fetal alveolar epithelial cells.
Daugherty BL, Mateescu M, Patel AS, Wade K, Kimura S, Gonzales LW, Guttentag S, Ballard PL, Koval M
(2004) Am J Physiol Lung Cell Mol Physiol 287: L1266-73
MeSH Terms: 1-Methyl-3-isobutylxanthine, 8-Bromo Cyclic Adenosine Monophosphate, Dexamethasone, Endocytosis, Fetus, Gene Expression Regulation, Developmental, Humans, Lung, Membrane Proteins, Sodium Chloride, Tight Junctions
Show Abstract · Added January 20, 2015
Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.
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11 MeSH Terms
Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic.
Gonzales LW, Angampalli S, Guttentag SH, Beers MF, Feinstein SI, Matlapudi A, Ballard PL
(2001) Pediatr Pathol Mol Med 20: 387-412
MeSH Terms: 1-Methyl-3-isobutylxanthine, 8-Bromo Cyclic Adenosine Monophosphate, Animals, Cell Culture Techniques, Cell Differentiation, Cells, Cultured, Collagenases, Coloring Agents, Culture Media, Serum-Free, Cyclic AMP, DNA, Complementary, Dexamethasone, Epithelial Cells, Glucocorticoids, Glycoproteins, Humans, Immunoblotting, Immunohistochemistry, Lung, Microscopy, Electron, Oxazines, Phosphatidylcholines, Phosphodiesterase Inhibitors, Phospholipids, Plastics, Precipitin Tests, Proteolipids, Pulmonary Surfactant-Associated Protein A, Pulmonary Surfactant-Associated Protein D, Pulmonary Surfactant-Associated Proteins, Pulmonary Surfactants, RNA, Messenger, Surface-Active Agents, Time Factors, Transfection, Trypsin
Show Abstract · Added January 20, 2015
We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day) 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (approximately 34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.
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36 MeSH Terms
cAMP-dependent protein kinase inhibits mGluR2 coupling to G-proteins by direct receptor phosphorylation.
Schaffhauser H, Cai Z, Hubalek F, Macek TA, Pohl J, Murphy TJ, Conn PJ
(2000) J Neurosci 20: 5663-70
MeSH Terms: 8-Bromo Cyclic Adenosine Monophosphate, Adenine, Amino Acid Sequence, Animals, Anticonvulsants, CHO Cells, Cricetinae, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases, Cyclopropanes, Dentate Gyrus, Enzyme Inhibitors, Excitatory Postsynaptic Potentials, GTP-Binding Proteins, Glutamic Acid, Glycine, Guanosine 5'-O-(3-Thiotriphosphate), Isoquinolines, Molecular Sequence Data, Mutagenesis, Neurons, Perforant Pathway, Phosphorylation, Protein Binding, Rats, Receptors, Metabotropic Glutamate, Serine, Sulfonamides, Transfection
Show Abstract · Added February 19, 2015
One of the primary physiological roles of group II and group III metabotropic glutamate receptors (mGluRs) is to presynaptically reduce synaptic transmission at glutamatergic synapses. Interestingly, previous studies suggest that presynaptic mGluRs are tightly regulated by protein kinases. cAMP analogs and the adenylyl cyclase activator forskolin inhibit the function of presynaptic group II mGluRs in area CA3 of the hippocampus. We now report that forskolin has a similar inhibitory effect on putative mGluR2-mediated responses at the medial perforant path synapse and that this effect of forskolin is blocked by a selective inhibitor of cAMP-dependent protein kinase (PKA). A series of biochemical and molecular studies was used to determine the precise mechanism by which PKA inhibits mGluR2 function. Our studies reveal that PKA directly phosphorylates mGluR2 at a single serine residue (Ser(843)) on the C-terminal tail region of the receptor. Site-directed mutagenesis combined with biochemical measures of mGluR2 function reveal that phosphorylation of this site inhibits coupling of mGluR2 from GTP-binding proteins
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29 MeSH Terms
Activation of mitogen-activated protein kinase pathways by cyclic GMP and cyclic GMP-dependent protein kinase in contractile vascular smooth muscle cells.
Komalavilas P, Shah PK, Jo H, Lincoln TM
(1999) J Biol Chem 274: 34301-9
MeSH Terms: 8-Bromo Cyclic Adenosine Monophosphate, Alkaloids, Animals, Aorta, Carbazoles, Cells, Cultured, Cyclic GMP, Cyclic GMP-Dependent Protein Kinases, Enzyme Activation, Enzyme Inhibitors, Gene Expression, Indoles, JNK Mitogen-Activated Protein Kinases, MAP Kinase Kinase 4, MAP Kinase Kinase Kinases, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinase Kinases, Mitogen-Activated Protein Kinases, Muscle Contraction, Muscle, Smooth, Vascular, Platelet-Derived Growth Factor, Protein-Serine-Threonine Kinases, Pyrroles, Rats, Rats, Sprague-Dawley, Recombinant Fusion Proteins, Thionucleotides, Time Factors, Transfection, Tumor Necrosis Factor-alpha, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added March 11, 2015
Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.
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32 MeSH Terms
Antibodies to a segment of tyrosine hydroxylase phosphorylated at serine 40.
Goldstein M, Lee KY, Lew JY, Harada K, Wu J, Haycock JW, Hokfelt T, Deutch AY
(1995) J Neurochem 64: 2281-7
MeSH Terms: 8-Bromo Cyclic Adenosine Monophosphate, Animals, Antibodies, Antibody Specificity, Colforsin, Corpus Striatum, Cyclic AMP-Dependent Protein Kinases, Enzyme-Linked Immunosorbent Assay, Immunoblotting, Immunohistochemistry, Immunosorbent Techniques, Male, PC12 Cells, Phosphorylation, Phosphoserine, Rats, Rats, Sprague-Dawley, Recombinant Proteins, Synaptosomes, Tyrosine 3-Monooxygenase
Show Abstract · Added May 27, 2014
A synthetic peptide corresponding to residues 32-47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser40 and used to generate antibodies in rabbits. Reactivity of the anti-pTH32-47 antibodies with phospho- and dephospho-Ser40 forms of TH protein and peptide TH32-47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody-capture ELISAs, anti-pTH32-47 was more reactive with the phospho-TH than with the dephospho-TH forms. Conversely, antibodies against the nonphosphorylated peptide reacted preferentially with the dephospho-TH forms. In western blots, labeling of the approximately 60-kDa TH band by anti-pTH32-47 was readily detectable in lanes containing protein kinase A-phosphorylated native TH at 10-100 ng/lane. In blots of supernatants prepared from striatal synaptosomes, addition of a phosphatase inhibitor was necessary to discern labeling of the TH band with anti-pTH32-47. Similarly, anti-pTH32-47 failed to immunoprecipitate TH activity from supernatants prepared from untreated tissues, whereas prior treatment with either 8-bromoadenosine 3',5'-cyclic monophosphate or forskolin enabled removal of TH activity by anti-pTH32-47. Lastly, in immunohistochemical studies, anti-pTH32-47 selectively labeled catecholaminergic cells in tissue sections from perfusion-fixed rat brain.
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20 MeSH Terms
Induction of synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin by follicle-stimulating hormone, 8-bromo-cyclic AMP, and low density lipoprotein in cultured bovine granulosa cells.
Funkenstein B, Waterman MR, Simpson ER
(1984) J Biol Chem 259: 8572-7
MeSH Terms: 8-Bromo Cyclic Adenosine Monophosphate, Adrenodoxin, Animals, Cattle, Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P-450 Enzyme System, Enzyme Induction, Female, Follicle Stimulating Hormone, Granulosa Cells, Kinetics, Lipoproteins, LDL, Oxidoreductases, Progesterone
Show Abstract · Added February 12, 2015
The actions of follicle-stimulating hormone (FSH), 8-bromo-cyclic AMP (8-Br-cAMP), and low density lipoprotein (LDL) to stimulate the production of progesterone and the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450ssc) and adrenodoxin were investigated in bovine granulosa cells maintained in primary monolayer culture. Treatment of granulosa cells in culture with FSH resulted in an increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc in a concentration-dependent fashion with a maximal effect being obtained at an FSH concentration of 500 ng/ml. Treatment of granulosa cells with FSH also resulted in the induction of synthesis of adrenodoxin. The cyclic AMP analog, 8-Br-cAMP, induced the synthesis of both cytochrome P-450scc and adrenodoxin to a greater extent than did FSH. LDL also stimulated the synthesis of both cytochrome P-450scc and adrenodoxin, when added to cells maintained in the presence of lipoprotein-poor serum. The presence of FSH or 8-Br-cAMP together with LDL resulted in a higher rate of enzyme synthesis than that observed with each effector alone. FSH, 8-Br-cAMP, and LDL also stimulated progesterone production by cultured granulosa cells. The results of this study offer a possible mechanism whereby granulosa cells undergo cytodifferentiation in vivo into luteal cells. The concentration of LDL in follicular fluid is very low. Following ovulation, vascularization of the follicle occurs and thus the granulosa cells are exposed to high levels of LDL, allowing for provision of substrate cholesterol, as well as stimulation of the synthesis of the enzymes involved in cholesterol side chain cleavage.
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14 MeSH Terms
Induction of synthesis of mitochondrial steroidogenic enzymes of bovine adrenocortical cells by analogs of cyclic AMP.
Kramer RE, Rainey WE, Funkenstein B, Dee A, Simpson ER, Waterman MR
(1984) J Biol Chem 259: 707-13
MeSH Terms: 8-Bromo Cyclic Adenosine Monophosphate, Adrenal Cortex, Adrenocorticotropic Hormone, Adrenodoxin, Animals, Bucladesine, Butyrates, Butyric Acid, Cattle, Cyclic AMP, Cytochrome P-450 Enzyme System, Enzyme Induction, Mitochondria, Pregnenolone
Show Abstract · Added February 12, 2015
The long term action of cyclic AMP analogs to stimulate the synthesis of cytochromes P-450scc, P-45011 beta, and adrenodoxin has been studied utilizing confluent monolayers of adult bovine adrenocortical cells maintained for periods of time up to 72 h in the absence or presence of dibutyryl cyclic AMP (1 mM), 8-bromo cyclic AMP (1 mM), or ACTH (adrenocorticotropin) (10(-6) M). The synthesis of these proteins was examined by radiolabeling cellular proteins with [35S]methionine or else by translating RNA extracted from such cells in a cell-free system in the presence of [35S]methionine. In each case, the protein under study was immunoprecipitated utilizing specific antisera, or IgG fractions prepared from such antisera. ACTH and both analogs of cyclic AMP caused an increase in the synthesis of cytochrome P-450scc which reached a maximum 36-48 h after addition, and then declined. On the other hand, butyric acid (1 mM) had no effect on the synthesis of cytochrome P-450scc. Cytochrome P-450scc activity measured as pregnenolone production by both intact cells or isolated mitochondria from such cells was increased following incubation of cells with either dibutyryl cyclic AMP or ACTH. The binding of rabbit anti-cytochrome P-450scc IgG was also increased in cells incubated with dibutyryl cyclic AMP or ACTH as estimated by immunofluorescence microscopy using fluorescein-tagged anti-rabbit IgG. Furthermore, dibutyryl cyclic AMP and ACTH both increased the synthesis of adrenodoxin and of cytochrome P-45011 beta, as well as the activity of 11 beta-hydroxylase. In addition, ACTH stimulated the secretion of cyclic AMP in a time- and concentration-dependent fashion. Thus, it is concluded that analogs of cyclic AMP can mimic the long term actions of ACTH to induce the synthesis of steroidogenic enzymes, and that this action of ACTH is likely mediated by cyclic AMP.
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14 MeSH Terms
Evidence for the presence of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in fresh granulosa cells. Effects of follicle-stimulating hormone and cyclic AMP on cholesterol side chain cleavage cytochrome P-450 synthesis and activity.
Funkenstein B, Waterman MR, Masters BS, Simpson ER
(1983) J Biol Chem 258: 10187-91
MeSH Terms: 8-Bromo Cyclic Adenosine Monophosphate, Adrenodoxin, Animals, Bucladesine, Cattle, Cyclic AMP, Cytochrome P-450 Enzyme System, Female, Follicle Stimulating Hormone, Granulosa Cells, Isoenzymes, NADPH-Ferrihemoprotein Reductase
Show Abstract · Added February 12, 2015
The synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and adrenodoxin was studied both in freshly harvested bovine granulosa cells and in granulosa cells maintained in primary monolayer culture. In addition, the action of follicle-stimulating hormone (FSH) and cyclic AMP analogs to stimulate the synthesis of cytochrome P-450scc was investigated in cultured cells. Precursor forms of cytochrome P-450scc and adrenodoxin were immunoisolated from a cell-free translation system directed by RNA prepared from freshly obtained granulosa cells that were not luteinized. Furthermore, the presence of cytochrome P-450scc in lysates of granulosa cells freshly obtained from very small follicles (containing less than 0.1 ml of follicular fluid) and in mitochondria of freshly obtained granulosa cells was demonstrated by using an immunoblotting technique. Continuous treatment of cultured granulosa cells with FSH or with cyclic AMP analogs (dibutyryl cyclic AMP or 8-bromo cyclic AMP) for 72 h increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc. Moreover, FSH, dibutyryl cyclic AMP, and 8-bromo cyclic AMP stimulated pregnenolone production by cultured granulosa cells (2.3-, 4.0-, and 7.5-fold increase over control, respectively), indicative of an increase in cholesterol side chain cleavage activity. The results of this study demonstrate for the first time the presence of two components of the cholesterol side chain cleavage system in freshly obtained granulosa cells, and provide direct evidence for the trophic effect of FSH and its presumed mediator, cyclic AMP, on the synthesis of cytochrome P-450scc in granulosa cells.
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12 MeSH Terms
The Bulla ocular circadian pacemaker. I. Pacemaker neuron membrane potential controls phase through a calcium-dependent mechanism.
McMahon DG, Block GD
(1987) J Comp Physiol A 161: 335-46
MeSH Terms: 8-Bromo Cyclic Adenosine Monophosphate, Animals, Calcium, Circadian Rhythm, Light, Membrane Potentials, Mollusca, Ocular Physiological Phenomena, Retina, Serotonin
Show Abstract · Added March 18, 2020
In an effort to understand the cellular basis of entrainment of circadian oscillators we have studied the role of membrane potential changes in the neurons which comprise the ocular circadian pacemaker of Bulla gouldiana in mediating phase shifts of the ocular circadian rhythm. We report that: 1. Intracellular recording was used to measure directly the effects of the phase shifting agents light, serotonin, and 8-bromo-cAMP on the membrane potential of the basal retinal neurons. We found that light pulses evoke a transient depolarization followed by a smaller sustained depolarization. Application of serotonin produced a biphasic response; a transient depolarization followed by a sustained hyperpolarization. Application of a membrane permeable analog of the intracellular second messenger cAMP, 8-bromo-cAMP, elicited sustained hyperpolarization, and occasionally a weak phasic depolarization. 2. Changing the membrane potential of the basal retinal neurons directly and selectively with intracellularly injected current phase shifts the ocular circadian rhythm. Both depolarizing and hyperpolarizing current can shift the phase of the circadian oscillator. Depolarizing current mimics the phase shifting action of light, while hyperpolarizing current produces phase shifts which are transposed approximately 180 degrees in circadian time to depolarization. 3. Altering BRN membrane potential with ionic treatments, depolarizing with elevated K+ seawater or hyperpolarizing with lowered Na+ seawater, produces phase shifts similar to current injection. 4. The light-induced depolarization of the basal retinal neurons is necessary for phase shifts by light. Suppressing the light-induced depolarization with injected current inhibits light-induced phase shifts. 5. The ability of membrane potential changes to shift oscillator phase is dependent on extracellular calcium. Reducing extracellular free Ca++ from 10 mM to 1.3 X 10(-7) M inhibits light-induced phase shifts without blocking the photic response of the BRNs. The results indicate that changes in the membrane potential of the pacemaker neurons play a critical role in phase shifting the circadian rhythm, and imply that a voltage-dependent and calcium-dependent process, possibly Ca++ influx, shifts oscillator phase in response to light.
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MeSH Terms