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Plate was centrifuged for 1 min at 500 × g and placed in 37°C humidified incubator while cell suspension was prepared. HEKD2 cells were dissociated and collected using 0.25% trypsin-EDTA (Life Technologies, Grand Island, NY), diluted to 10 ml with growth media, centrifuged at 500 × g for 5 min, and resuspended in 10 ml growth medium. Cells were counted and diluted to 5 × 10 5 cells/ml in growth medium, and 30 µl of this suspension was added to wells of EnSpire plate to achieve a final density of 15 000 cells/well and a total volume of 50 µl/well. Cells were incubated in a humidified incubator until 95% confluent, typically 16–24 h.
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Fgf2 −/− mice similarly have deficient recovery of club cell secretory protein + bronchial epithelium in response to naphthalene. We conclude that FGF2 is not required for bleomycin-induced pulmonary fibrosis, but rather is essential for epithelial repair and maintaining epithelial integrity after bleomycin-induced lung injury in mice. These data identify that FGF2 acts as a protective growth factor after lung epithelial injury, and call into question the role of FGF2 as a profibrotic growth factor in vivo. Clinical Relevance Fibroblast growth factors (FGFs) and, in particular, FGF2, are implicated in the pathogenesis of pulmonary fibrosis and recovery from airway epithelial cell (AEC) injury; however, the in vivo requirement for FGF2 in the development of pulmonary fibrosis is not known. In this study, we have found that mice lacking FGF2 do not show altered levels of fibrosis in response to bleomycin, indicating that FGF2 is dispensable for the generation of bleomycin-induced pulmonary fibrosis. In addition, we show that FGF2 knockout mice have increased mortality and weight loss in response to bleomycin, accompanied by deficient recovery of mature alveolar epithelium and epithelial barrier function.
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FGF2 protein levels are also increased in bronchoalveolar lavage (BAL) from patients with IPF (), and immunohistochemistry for FGF2 localizes to mast cells in IPF lungs (). Other studies have shown FGF2 staining in IPF lungs localized to the endothelium and areas of angiogenesis (). In response to bleomycin, immunohistochemical staining of mouse lungs localized FGF2 to inflammatory cells, including macrophages (, ) and mast cells (, –), as well as airway epithelial cells (–).
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Try out full version of GrandVJ in DEMO mode Installers below can also run without a license in DEMO mode. The software will be fully functional with a DEMO banner displayed on the output except saving is disabled. Activating the software will leave the DEMO mode. To order ArKaos products directly, please visit the GrandVJ 2.6.2 This is the latest stable version. Version: 2.6.2 File size: 344MB Language: English Date added: 30 Nov. 2018 Version: 2.6.2 File size: 337MB Language: English Date added: 30 Nov.
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Prolonged inflammation in Fgf2 −/− mice compared with wild-type mice after bleomycin. A single dose of intranasal bleomycin (4 mg/kg) was administered to Fgf2 −/− or wild-type littermate controls, and mice were collected at 7, 14, or 21 days after treatment.
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To induce sensitization, the DRD2 ligand was added and cells were pre-incubated in a 37°C humidified incubator for 2 h. Following the 2 h incubation ( i.e. Sensitization period), forskolin (300 nM final concentration) containing 0.5 mM IBMX was added in the presence of 1 µM spiperone (to inhibit acute ligand activation of the DRD2). Cells were incubated at room temperature for 1 h and cAMP accumulation was measured as described above. 2.6 Dynamic Mass Redistribution For DMR assays, 20 µl per well of HEK-D2 growth medium was added to an EnSpire- LFC 384 fibronectin coated plate (PerkinElmer, Waltham, MA).
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Data shown represent the average and S.E.M. Of three independent experiments conducted in duplicate. 3.4 Aripiprazole’s effects on dynamic mass redistribution (DMR) Another approach used to measure aripiprazole’s effects on the DRD2 employed a labelfree holistic approach.
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3.3 Aripiprazole’s effects on heterologous sensitization of AC5 Because aripiprazole was a partial agonist for Gαi/o activation and an antagonist for Gβγ subunits, a more complex G protein-dependent signaling pathway was analyzed. Heterologous sensitization of adenylyl cyclase is a cellular adaptive response that occurs following chronic activation of Gαi/o-coupled receptors [].
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The dopamine D 2 receptor (DRD2) is a GPCR that is targeted in the therapies of Parkinson’s disease and schizophrenia. The DRD2 couples to inhibitory heterotrimeric G proteins leading to activation of Gα subunits that inhibit adenylyl cyclases, as well as promoting Gβγ signaling. Additional adaptive signaling responses of the DRD2 include β-arrestin recruitment and heterologous sensitization of adenylyl cyclase [, ]. The drugs used in the therapies for Parkinson’s disease (e.g.
Specifically, we measured Gα signaling, Gβγ signaling, heterologous sensitization of adenylyl cyclase, and dynamic mass redistribution (DMR). Aripiprazole displayed a unique profile for activation of G protein signaling, behaving as a robust antagonist of Gβγ subunits, while partially activating Gα. Additionally, aripiprazole was a weak partial agonist/antagonist for heterologous sensitization of adenylyl cyclase and in cell-based DMR experiments. The unique antagonist profile of aripiprazole may provide additional opportunities for developing targeted antipsychotic agents. 2.2 Cell culture Human embryonic kidney (HEK) cells stably expressing the long isoform of the DRD2 (HEK-D2) and adenylyl cyclase 2 (HEK-AC2/D2) or adenylyl cyclase 5 (HEK-AC5/D2) were cultured in Dubelcco’s Modified Eagle Medium (Life Technologies, Grand Island, NY) supplemented with 5% bovine calf serum (Hyclone, Logan, UT), 5% fetal clone I (Hyclone, Logan, UT), and Antibiotic-Antimycotic (Life Technologies, Grand Island, NY), and puromycin (Sigma-Aldrich, St. Louis, MO) (HEK-D2), or zeocin (Life Technologies, Grand Island, NY) and G418 (Invivogen, San Diego, CA) (HEK-AC2/D2), or puromycin and G418 (HEK-AC5/ D2). Cells were grown to confluency in 15 cm disses, harvested with Cell Dissociation Buffer (Life Technologies, Grand Island, NY), and resuspended in 5 ml of fetal bovine serum (Hyclone, Logan, UT) containing 10% dimethyl sulfoxide (Sigma-Aldrich, St.
Recent studies describe decreased levels of Fgf2 mRNA in lungs from a large cohort of patients with IPF (), but the temporal and spatial pattern of Fgf2 expression and signaling remains unclear in early versus late stages of disease. In primary human lung fibroblasts, administration of TGF-β1 induces FGFR expression () and FGF2 expression and secretion into culture media (, ).
Delayed recovery of E-cadherin and surfactant protein (SP) C in Fgf2 −/− mice after bleomycin. A single dose of intranasal bleomycin (4 mg/kg) was administered to Fgf2 −/− or wild-type littermate controls. Immunohistochemistry for E-cadherin was performed on wild-type ( A) or Fgf2 −/− ( B) lungs 21 days after bleomycin. Immunohistochemistry for pro-SPC was performed on wild-type ( C) or Fgf2 −/− ( D) lungs 21 days after bleomycin. Scale bars, 600 μM ( A and B), 400 μM ( C and D).
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This ectodomain inhibits multiple FGFs known to bind to the IIIc splice variant of FGFR2, including FGFs 1, 2, 4, 6, 8, 9, 17, and 18 (). Despite this, the cell-specific role of FGF signaling in the development of pulmonary fibrosis remains uncertain. FGF2 is a potent alveolar type II cell mitogen (), as well as a potent mediator of pulmonary fibroblast proliferation in vitro ().
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Western blotting for phosphorylated extracellular signal–regulated kinase (ERK) 1/2, total ERK1/2, and β-tubulin is shown. ( B) Densitometric analysis of phosphorylated ERK1/2, normalized to both total ERK1/2 and β-tubulin. Data are presented as mean ± SEM of fold increase from PBS-treated mice. FGF2 Is Not Required for Pulmonary Fibrosis in Mice Given the importance of FGF2 for fibroblast proliferation in response to profibrotic stimuli in vitro, we hypothesized that FGF2 would be required for fibrosis in vivo. Fgf2 −/− mice have not been tested in models of acute lung injury or pulmonary fibrosis, although Fgf1 −/−, Fgf2 −/− mice have decreased carbon tetrachloride–induced liver fibrosis ().
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Mortality is expressed as a Kaplan-Meier survival plot. Fgf2 −/− and wild-type mice have similar fibrosis after bleomycin.
The first generation agents, and to some extent the second generation antipsychotics, also antagonize other GPCRs in the central nervous system (e.g. Muscarinic and histamine as well as other dopamine and serotonin receptors) resulting in a number of side effects [].
The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Pramipexole, ropinirole) are agonists of the Gα response of the DRD2, whereas those used for schizophrenia are either antagonists or partial agonists (e.g. Haloperidol, aripiprazole, respectively) [].
Blog Article: MediaMaster Pro 4.2.5 This is the latest stable version. Version: 4.2.5 File size: 393MB Language: English Date added: 28 Oct. 2016 Version: 4.2.5 File size: 338MB Language: English Date added: 28 Oct. 2016 Dongle driver for OS X Yosemite [Also included in the installer] User Guide [PDF] [Also included in the installer] Release Notes [PDF] [Also included in the installer] This software uses the NDI™ library.
In this phenomenon, prolonged Gαi/o agonist exposure results in a marked enhancement of subsequent drug-stimulated cAMP accumulation. Mechanistically, it has been shown that both Gα and Gβγ subunits of G proteins are involved in this cellular response []. Heterologous sensitization of AC5 was measured in HEK cells stably expressing AC5 and the DRD2 by pre-incubating the cells with the DRD2 ligands for 2 h before stimulating cAMP accumulation with forskolin. Pretreatment with dopamine or quinpirole resulted in dose-dependent enhancements in the forskolin-stimulated cAMP accumulation with EC50 values of approximately 8 nM (). Pramipexole, ropinirole, and rotigotine also led to heterologous sensitization of AC5 and had maximal responses greater than 100% (). In contrast, aripiprazole displayed a weak partial agonist response in this assay, resulting in only 18% of dopamine’s response ().