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1

Melanocortins induce interleukin 6 gene expression and secretion through melanocortin receptors 2 and 5 in 3T3-L1 adipocytes  

PubMed Central

Interleukin 6 (IL6) is a pleiotropic cytokine that not only affects the immune system, but also plays an active role in many physiological events in various organs. Notably, 35% of systemic IL6 originates from adipose tissues under noninflammatory conditions. Here, we describe a previously unknown function of melanocortins in regulating Il6 gene expression and production in 3T3-L1 adipocytes through membrane receptors which are called melanocortin receptors (MCRs). Of the five MCRs that have been cloned, MC2R and MC5R are expressed during adipocyte differentiation. ?-Melanocyte-stimulating hormone (?-MSH) or ACTH treatment of 3T3-L1 adipocytes induces Il6 gene expression and production in a time- and concentration-dependent manner via various signaling pathways including the protein kinase A, p38 mitogen-activated protein kinase, cJun N-terminal kinase, and I?B kinase pathways. Specific inhibition of MC2R and MC5R expression with short interfering Mc2r and Mc5r RNAs significantly attenuated the ?-MSH-induced increase of intracellular cAMP and both the level of Il6 mRNA and secretion of IL6 in 3T3-L1 adipocytes. Finally, when injected into mouse tail vein, ?-MSH dramatically increased the Il6 transcript levels in epididymal fat pads. These results suggest that ?-MSH in addition to ACTH may function as a regulator of inflammation by regulating cytokine production.

Jun, Dong-Jae; Na, Kyung-Yoon; Kim, Wanil; Kwak, Dongoh; Kwon, Eun-Jeong; Yoon, Jong Hyuk; Yea, Kyungmoo; Lee, Hyeongji; Kim, Jaeyoon; Suh, Pann-Gill; Ryu, Sung Ho; Kim, Kyong-Tai

2010-01-01

2

Melanocortins induce interleukin 6 gene expression and secretion through melanocortin receptors 2 and 5 in 3T3-L1 adipocytes.  

PubMed

Interleukin 6 (IL6) is a pleiotropic cytokine that not only affects the immune system, but also plays an active role in many physiological events in various organs. Notably, 35% of systemic IL6 originates from adipose tissues under noninflammatory conditions. Here, we describe a previously unknown function of melanocortins in regulating Il6 gene expression and production in 3T3-L1 adipocytes through membrane receptors which are called melanocortin receptors (MCRs). Of the five MCRs that have been cloned, MC2R and MC5R are expressed during adipocyte differentiation. alpha-Melanocyte-stimulating hormone (alpha-MSH) or ACTH treatment of 3T3-L1 adipocytes induces Il6 gene expression and production in a time- and concentration-dependent manner via various signaling pathways including the protein kinase A, p38 mitogen-activated protein kinase, cJun N-terminal kinase, and IkappaB kinase pathways. Specific inhibition of MC2R and MC5R expression with short interfering Mc2r and Mc5r RNAs significantly attenuated the alpha-MSH-induced increase of intracellular cAMP and both the level of Il6 mRNA and secretion of IL6 in 3T3-L1 adipocytes. Finally, when injected into mouse tail vein, alpha-MSH dramatically increased the Il6 transcript levels in epididymal fat pads. These results suggest that alpha-MSH in addition to ACTH may function as a regulator of inflammation by regulating cytokine production. PMID:20089716

Jun, Dong-Jae; Na, Kyung-Yoon; Kim, Wanil; Kwak, Dongoh; Kwon, Eun-Jeong; Yoon, Jong Hyuk; Yea, Kyungmoo; Lee, Hyeongji; Kim, Jaeyoon; Suh, Pann-Gill; Ryu, Sung Ho; Kim, Kyong-Tai

2010-04-01

3

Triiodothyronine regulates distribution of thyroid hormone receptors by activating AMP-activated protein kinase in 3T3-L1 adipocytes and induces uncoupling protein-1 expression.  

PubMed

The purposes of this study were to examine whether thermogenesis in 3T3-L1 adipocytes is related to variations in thyroid hormone receptors (TRs) that are differently regulated by triiodothyronine (T3), and the possible role of AMP-activated protein (AMPK) in thermogenesis after cell differentiation. Differentiated 3T3-L1 adipocytes were maintained under four conditions: normal control group, T3 treatment group, AMPK agonist (5-aminoimidazole-4-carboxamide-1-?-D-ribofuranoside) treatment group, and T3 and AMPK inhibitor (Compound C) treatment group. Real-time polymerase chain reaction was then performed to evaluate the changes in TR? and TR? mRNA levels in the cells, as well as marker genes for brown adipose tissue including uncoupling protein (UCP)-1 and Cidea. Western blotting was carried out for the cells to detect the expressions of TR?, TR?, and AMPK protein levels. After T3 treatment, the mRNA and protein levels of TR? decreased compared with the control group, while TR? mRNA and protein levels increased markedly at the same time. We also found elevated mRNA levels of UCP-1 and Cidea after exposure to T3. However, the distribution of TRs was reversed by Compound C. AMPK protein levels were clearly activated by T3. Our results suggest that the distribution of TRs is related to thermogenesis, and AMPK may participate in the alterations. PMID:24771016

Wang, Cheng-Zhi; Wei, Dan; Guan, Mei-Ping; Xue, Yao-Ming

2014-08-01

4

Diacylglycerol and ceramide formation induced by dopamine D2S receptors via Gbeta gamma -subunits in Balb/c-3T3 cells.  

PubMed

Diacylglycerol (DAG) and ceramide are important second messengers affecting cell growth, differentiation, and apoptosis. Balb/c-3T3 fibroblast cells expressing dopamine-D2S (short) receptors (Balb-D2S cells) provide a model of G protein-mediated cell growth and transformation. In Balb-D2S cells, apomorphine (EC(50) = 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at 1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicating G(i)/G(o) proteins. To address which G proteins are involved, Balb-D2S clones expressing individual PTX-insensitive Galpha(i) proteins were treated with PTX and tested for apomorphine-induced responses. Neither PTX-insensitive Galpha(i2) nor Galpha(i3) rescued D2S-induced DAG or ceramide formation. Both D2S-induced DAG and ceramide signals required Gbetagamma-subunits and were blocked by inhibitors of phospholipase C [1-(6-[([17beta]-3-methoxyestra-1,2,3[10]-trien- 17yl)amino]hexyl)-1H-pyrrole-2,5-dione (U-73122) and partially by D609]. The similar G protein specificity of D2S-induced calcium mobilization, DAG, and ceramide formation indicates a common Gbetagamma-dependent phospholipase C-mediated pathway. Both D2 agonists and ceramide specifically induced mitogen-activated protein kinase (ERK1/2), suggesting that ceramide mediates a novel pathway of D2S-induced ERK1/2 activation, leading to cell growth. PMID:12431910

Liu, Gele; Ghahremani, Mohammad H; Banihashemi, Behzad; Albert, Paul R

2003-03-01

5

Interleukin-2 (IL-2) dependent expression of biologically relevant IL-2 receptors: uncoupling of anti-T3 induced receptor expression with cyclosporin  

SciTech Connect

Human peripheral blood T cell expression of IL-2 receptors (IL-2R), detected by both immunocytofluorometry and /sup 125/I-IL-2 binding, was studied using lymphocytes stimulated with monoclonal anti-T3 antibodies (Leu-4, OKT3). Lymphocytes, isolated from healthy individuals, were prescreened and classified as Leu-4 responders or non-responders according to 72 h /sup 3/H-thymidine incorporation experiments. Leu-4 non-responder lymphocytes, though capable of normal IL-2R expression and IL-2 secretion when cultured with OKT3 (IgG2a), expressed little to no IL-2R nor secreted IL-2 when stimulated with Leu-4 (IgG1). In addition, the amount of IL-2 secreted by Leu-4 stimulated, Leu-4 responder cells, was one-third- to one-fifth of that detected when OKT3 was used as the stimulant. The addition of recombinant IL-2 (rIL-2) to a Leu-4 stimulated, Leu-4 non-responder lymphocyte culture, resulted in the expression of IL-2R and cellular proliferation, indicating that IL-2 upregulated its biologically relevant receptor. As expected, cyclosporin-A (CSA) inhibited the secretion of IL-2 and subsequent proliferation of Leu-4 stimulated, Leu-4 responder cells. Unexpectedly, however, the expression of IL-2R was also blocked. Exogenous rIL-2 partially reversed the effect of CSA on IL-2R expression and proliferation. The results indicate that IL-2 may provide an additional, required signal for optimal IL-2R expression.

Gitter, A.B.D.; Labus, J.M.; Butler, L.D.

1986-03-01

6

Thyroid Hormone T3 Counteracts STZ Induced Diabetes in Mouse  

PubMed Central

This study intended to demonstrate that the thyroid hormone T3 counteracts the onset of a Streptozotocin (STZ) induced diabetes in wild type mice. To test our hypothesis diabetes has been induced in Balb/c male mice by multiple low dose Streptozotocin injection; and a group of mice was contemporaneously injected with T3. After 48 h mice were tested for glucose tolerance test, insulin serum levels and then sacrified. Whole pancreata were utilized for morphological and biochemical analyses, while protein extracts and RNA were utilized for expression analyses of specific molecules. The results showed that islets from T3 treated mice were comparable to age- and sex-matched control, untreated mice in number, shape, dimension, consistency, ultrastructure, insulin and glucagon levels, Tunel positivity and caspases activation, while all the cited parameters and molecules were altered by STZ alone. The T3-induced pro survival effect was associated with a strong increase in phosphorylated Akt. Moreover, T3 administration prevented the STZ-dependent alterations in glucose blood level, both during fasting and after glucose challenge, as well as in insulin serum level. In conclusion we demonstrated that T3 could act as a protective factor against STZ induced diabetes.

Madaro, Luca; Ranieri, Danilo; Lupoi, Lorenzo; Stigliano, Antonio; Torrisi, Maria Rosaria; Bouche, Marina; Toscano, Vincenzo; Misiti, Silvia

2011-01-01

7

T3 glycoprotein is functional although structurally distinct on human T-cell receptor. gamma. T lymphocytes  

SciTech Connect

The T-cell receptor (TCR) ..gamma.. gene product occurs in association with T3 (CD3) polypeptides on the surface of human T lymphocytes. TCR ..gamma.. lymphocytes express arrays of T3 polypeptides distinct from those typically observed on TCR ..cap alpha beta.. lymphocytes. This report demonstrates that identical T3 ..gamma.., delta, and element of polypeptides are synthesized by TCR ..gamma.. lymphocytes and TCR ..cap alpha beta.. lymphocytes. However, the processing of T3 delta oligosaccharides is distinct in the two cell types. This observation may suggest distinct quaternary structures of these receptor complexes. Despite these structural differences, the T3 molecule on TCR ..gamma.. lymphocytes is functional. It is associated with and comodulates with TCR ..gamma.. and it serves as a substrate from protein kinase C-mediated phosphorylation. Anti-T3 monoclonal antibodies induce a rapid increase in cytoplasmic free calcium, indicating that the receptor complex is involved in signal transduction and triggering of TCR ..gamma.. lymphocytes.

Krangel, M.S.; Bierer, B.E.; Devlin, P.; Clabby, M.; Strominger, J.L.; McLean, J.; Brenner, M.B.

1987-06-01

8

Acylated and unacylated ghrelin protect MC3T3-E1 cells against tert-butyl hydroperoxide-induced oxidative injury: pharmacological characterization of ghrelin receptor and possible epigenetic involvement.  

PubMed

Increasing evidence suggests a role for oxidative stress in age-related decrease in osteoblast number and function leading to the development of osteoporosis. This study was undertaken to investigate whether ghrelin, previously reported to stimulate osteoblast proliferation, counteracts tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in MC3T3-E1 osteoblastic cells as well as to characterize the ghrelin receptor (GHS-R) involved in such activity. Pretreatment with ghrelin (10(-7)-10(-11 )M) significantly increased viability and reduced apoptosis of MC3T3-E1 cells cultured with t-BHP (250 ?M) for three hours at the low concentration of 10(-9 )M as shown by MTT assay and Hoechst-33258 staining. Furthermore, ghrelin prevented t-BHP-induced osteoblastic dysfunction and changes in the cytoskeleton organization evidenced by the staining of the actin fibers with Phalloidin-FITC by reducing reactive oxygen species generation. The GHS-R type 1a agonist, EP1572 (10(-7)-10(-11 )M), had no effect against t-BHP-induced cytotoxicity and pretreatment with the selective GHS-R1a antagonist, D-Lys(3)-GHRP-6 (10(-7 )M), failed to remove ghrelin (10(-9) M)-protective effects against oxidative injury, indicating that GHS-R1a is not involved in such ghrelin activity. Accordingly, unacylated ghrelin (DAG), not binding GHS-R1a, displays the same protective actions of ghrelin against t-BHP-induced cytotoxicity. Preliminary observations indicate that ghrelin increased the trimethylation of lys4 on histones H3, a known epigenetic mark activator, which may regulate the expression of some genes limiting oxidative damage. In conclusion, our data demonstrate that ghrelin and DAG promote survival of MC3T3-E1 cell exposed to t-BHP-induced oxidative damage. Such effect is independent of GHS-R1a and is likely mediated by a common ghrelin/DAG binding site. PMID:24705647

Dieci, Elisa; Casati, Lavinia; Pagani, Francesca; Celotti, Fabio; Sibilia, Valeria

2014-07-01

9

Characterization of melanocortin receptor subtype expression in murine adipose tissues and in the 3T3-L1 cell line.  

PubMed

It has been known for many years that adipocytes express high affinity ACTH and alpha-melanocyte stimulating hormone (MSH) binding sites, and that ACTH, alpha-MSH, and beta-lipotropin are potent lipolytic hormones. We show here that the adipocyte response to the melanocortin peptides results from the expression of both the MC2 (ACTH) receptor as well as the newly discovered MC5 receptor. Using RT-PCR and Northern blot hybridization, high levels of MC2 receptor messenger RNA (mRNA) were found in all adipose tissues examined in the mouse, whereas MC5 receptor mRNA was found in a subset of these. Both receptors mRNAs were also found in the 3T3-L1 cell line but only after the cells had been induced to differentiate into adipocytes. This cell line was then used to characterize the pharmacological properties of the MC2 and MC5 receptor sites in situ. The MC2 receptor exhibits properties similar to the ACTH receptor characterized in adrenocortical cells, coupling to activation of adenylyl cyclase with an EC50 of approximately 1 nM. An MSH binding site characterized in these cells is presumably the MC5 receptor, based on the observation that this is the only other melanocortin receptor mRNA detected in these cells. The MC5 receptor in the 3T3-L1 adipocyte activated adenylyl cyclase in response to alpha-MSH stimulation. Interestingly, Nle4, D-Phe7-alpha-MSH (NDP-MSH), a commonly used synthetic alpha-MSH agonist, was a potent antagonist of the MC5 receptor expressed in the 3T3-L1 cell line. Although the agouti signaling peptide is a potent antagonist of NDP-MSH binding to the MC1 and MC4 melanocortin receptors, agouti was unable to block NDP-MSH binding in the 3T3-L1 adipocyte. PMID:8612546

Boston, B A; Cone, R D

1996-05-01

10

Thyroid hormone (T3) inhibits ciprofibrate-induced transcription of genes encoding beta-oxidation enzymes: cross talk between peroxisome proliferator and T3 signaling pathways.  

PubMed Central

Peroxisome proliferators cause rapid and coordinated transcriptional activation of genes encoding peroxisomal beta-oxidation system enzymes by activating peroxisome proliferator-activated receptor (PPAR) isoform(s). Since the thyroid hormone (T3; 3,3',5-triiodothyronine) receptor (TR), another member of the nuclear hormone receptor superfamily, regulates a subset of fatty acid metabolism genes shared with PPAR, we examined the possibility of interplay between peroxisome proliferator and T3 signaling pathways. T3 inhibited ciprofibrate-induced luciferase activity as well as the endogenous peroxisomal beta-oxidation enzymes in transgenic mice carrying a 3.2-kb 5'-flanking region of the rat peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene fused to the coding region of luciferase. Transfection assays in hepatoma H4-II-E-C3 and CV-1 cells indicated that this inhibition is mediated by TR in a ligand-dependent fashion. Gel shift assays revealed that modulation of PPAR action by TR occurs through titration of limiting amounts of retinoid X receptor (RXR) required for PPAR activation. Increasing amounts of RXR partially reversed the inhibition in a reciprocal manner; PPAR also inhibited TR activation. Results with heterodimerization-deficient TR and PPAR mutants further confirmed that interaction between PPAR and TR signaling systems is indirect. These results suggest that a convergence of the peroxisome proliferator and T3 signaling pathways occurs through their common interaction with the heterodimeric partner RXR. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7

Chu, R; Madison, L D; Lin, Y; Kopp, P; Rao, M S; Jameson, J L; Reddy, J K

1995-01-01

11

Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells  

SciTech Connect

Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

2006-07-01

12

RADIATION INDUCED OVERAGING OF A 2024-T3 ALUMINUM ALLOY  

Microsoft Academic Search

Annealing experiments on both irradiated and unirradiated samples of a ; 2024-T3 aluminum alloy were followed by metallographic examination, hardness ; measurements, and an analysis of x-ray back reflection patterns. The severely ; overaged condition after neutron irradiation previously reported by Wallack is ; shown to be primarily radioinduced and not due only to a high ambient temperature. ; This

A. M. Adair; R. E. Hook; H. J. Garrett

1961-01-01

13

Mechanisms of resveratrol-induced inhibition of clonal expansion and terminal adipogenic differentiation in 3T3-L1 preadipocytes.  

PubMed

We show that resveratrol prevents clonal expansion and terminal adipogenesis in 3T3-L1 preadipocytes. An early resveratrol effect was the inhibition of AKT and mitogen-activated protein kinase signaling, accompanied by down regulation of cyclin D1 expression, abrogation of retinoblastoma protein hyperphosphorylation, and subsequent inhibition of cell cycle reentry and clonal expansion, as indicated by cyclin A2 repression. Resveratrol inhibited terminal adipogenesis at the level of peroxisome proliferator-activated receptor-?2 expression and activity. This was independent from the preceding inhibition of clonal expansion. Peroxisome proliferator-activated receptor-?2 overexpression and activation partially restored fatty acid-binding protein 4 induction in resveratrol-treated 3T3-L1. Resveratrol activated AMP-activated protein kinase (AMPK) but did not induce PPAR-? co-activator 1? (PGC1?) and mitochondrial biogenesis in 3T3-L1. Treatment with the Sirt1 inhibitor splitomicin augmented downregulation of peroxisome proliferator-activated receptor-?2 and fatty acid-binding protein 4 expressions in resveratrol-treated 3T3-L1 and did not prevent the inhibition of terminal adipogenesis. Moreover, splitomicin could not obviate resveratrol-induced cyclin D1 repression, retinoblastoma protein hypophosphorylation, and inhibition of clonal expansion. Our data suggest that resveratrol inhibits clonal expansion and terminal adipogenesis in 3T3-L1 by several mechanisms. PMID:23525482

Mitterberger, Maria C; Zwerschke, Werner

2013-11-01

14

Functional expression of 5-HT{sub 2A} receptor in osteoblastic MC3T3-E1 cells  

SciTech Connect

In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT{sub 2} receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT{sub 2A} and 5-HT{sub 2C} receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT{sub 2A} receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT{sub 2A} receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.

Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan)] [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan); Nishio, Hiroaki, E-mail: nishio@fupharm.fukuyama-u.ac.jp [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan)] [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan)

2010-05-28

15

Interaction of human beta 1 thyroid hormone receptor and its mutants with DNA and retinoid X receptor beta. T3 response element-dependent dominant negative potency.  

PubMed Central

Mutations in the human beta thyroid hormone receptor (h-TR beta) gene are associated with the syndrome of generalized resistance to thyroid hormone. We investigated the interaction of three h-TR beta 1 mutants representing different types of functional impairment (kindreds ED, OK, and PV) with different response elements for 3,3',5-triiodothyronine (T3) and with retinoid X receptor beta (RXR beta). The mutant receptors showed an increased tendency to form homodimers on a palindromic T3-response element (TREpal), a direct repeat (DR + 4), and an inverted palindrome (TRElap). On TRElap, wild type TR binding was decreased by T3, while the mutant receptors showed a variably decreased degree of dissociation from TRElap in response to T3. The extent of dissociation was proportional to their T3 binding affinities. RXR beta induced the formation of h-TR beta 1:RXR beta heterodimers equally well for mutants and the wild type h-TR beta 1 on these T3 response elements. However, the T3-dependent increase in heterodimerization with RXR beta was absent or reduced for the mutant TRs. Transient transfection studies indicated that the dominant negative potency was several-fold more pronounced on the TRElap as compared to TREpal or DR + 4. In CV-1 and HeLa cells, transfection of RXR beta could not reverse the dominant negative action. These results demonstrate that the binding of mutant h-TRs to DNA, as well as their dominant negative potency, are TRE dependent. In addition, competition for DNA binding, rather than for limiting amounts of RXR beta, is likely to mediate the dominant negative action. Images

Meier, C A; Parkison, C; Chen, A; Ashizawa, K; Meier-Heusler, S C; Muchmore, P; Cheng, S Y; Weintraub, B D

1993-01-01

16

Biological activity of bovine placental lactogen in 3T3-F442A preadipocytes is mediated through a somatogenic receptor.  

PubMed

Bovine placental lactogen (bPL) exhibited antimitogenic differentiation-promoting biological activity in 3T3-F442A preadipocytes. Competitive binding studies and affinity labelling revealed bPL activity to be mediated through a somatogenic type of receptor that recognizes human growth hormone (hGH) and bovine GH, but not ovine prolactin or human PL. The bioactivity of bPL was sixfold lower than that of hGH despite that bPL is binding to the somatogenic receptors with fivefold higher affinity. This discrepancy may result from the relatively low ability of bPL to induce post-receptoral effects such as receptor dimerization. PMID:1618336

Vashdi, D; Elberg, G; Sakal, E; Gertler, A

1992-06-29

17

An angiotensin II AT 1 receptor antagonist, telmisartan augments glucose uptake and GLUT4 protein expression in 3T3-L1 adipocytes  

Microsoft Academic Search

Evidence has accumulated that some of the angiotensin II AT1 receptor antagonists have insulin-sensitizing property. We thus examined the effect of telmisartan on insulin action using 3T3-L1 adipocytes. With standard differentiation inducers, a higher dose of telmisartan effectively facilitated differentiation of 3T3-L1 preadipocytes. Treatment of both differentiating adipocytes and fully differentiated adipocytes with telmisartan caused a dose-dependent increase in mRNA

Muneya Fujimoto; Hiroaki Masuzaki; Tomohiro Tanaka; Shintaro Yasue; Tsutomu Tomita; Kayoko Okazawa; Junji Fujikura; Hideki Chusho; Ken Ebihara; Tatsuya Hayashi; Kiminori Hosoda; Kazuwa Nakao

2004-01-01

18

Effect of microsomal enzyme inducers on the biliary excretion of triiodothyronine (T(3)) and its metabolites.  

PubMed

It has been postulated that inducers of UDP-glucuronosyltransferase (UGT) decrease circulating thyroid hormone concentrations by increasing their biliary excretion. The inducers pregnenolone-16 alpha-carbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) are each effective at reducing serum thyroxine concentrations. However, only PCN treatment produces a marked increase in serum levels of thyroid-stimulating hormone (TSH), whereas 3MC and PCB cause little to no increase in TSH. Excessive TSH elevation is considered the primary stimulus for thyroid tumor development in rats, yet the mechanism by which enzyme induction leads to TSH elevation is not fully understood. Whereas PCN, 3MC, and PCB all increase microsomal UGT activity toward T(4), only PCN causes an increase in T(3)-UGT activity in vitro. The purpose of this study was to determine whether PCN, which increases serum TSH, causes an increase in the glucuronidation and biliary excretion of T(3) in vivo. Male rats were fed control diet or diet containing PCN (1000 ppm), 3MC (250 ppm), or PCB (100 ppm) for 7 days. Animals were then given [(125)I]-T(3), i.v., and bile was collected for 2 h. Radiolabeled metabolites in bile were analyzed by reverse-phase HPLC with gamma-detection. The biliary excretion of total radioactivity was increased up to 75% by PCN, but not by 3MC or PCB. Of the T(3) excreted into bile, approximately 75% was recovered as T(3)-glucuronide, with remaining amounts represented as T(3)-sulfate, T(2)-sulfate, T(3), and T(2). Biliary excretion of T(3)-glucuronide was increased up to 66% by PCN, while neither 3MC nor PCB altered T(3)-glucuronide excretion. These findings indicate that PCN increases the glucuronidation and biliary excretion of T(3) in vivo, and suggest that enhanced elimination of T(3) may be the mechanism responsible for the increases in serum TSH caused by PCN. PMID:11812922

Vansell, Nichole R; Klaassen, Curtis D

2002-02-01

19

Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines  

Microsoft Academic Search

Curcumin, which is a widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis. We report that curcumin induces cell shrinkage, chromatin condensation, and DNA fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene?transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT?29,

Ming?Chung Jiang

1996-01-01

20

Withaferin A induces apoptosis and inhibits adipogenesis in 3T3-L1 adipocytes.  

PubMed

Withaferin A (WA), a highly oxygenated steroidal lactone that is found in the medicinal plant Withania somnifera (also called ashwagandha) has been reported to have anti-tumor, anti-angiogenesis, and pro-apoptotic activity. We investigated the effects of WA on viability, apoptosis and adipogenesis in 3T3-L1 adipocytes. Pre- and post-confluent preadipocytes and mature adipocytes were treated with WA (1-25 microM) up to 24 hrs. Viability and apoptosis were measured by CellTiter-Blue Cell Viability Assay and single strand DNA ELISA Assay, respectively. WA decreased viability and induced apoptosis in all stages of cells. Induction of apoptosis by WA in mature adipocytes was mediated by increased ERK1/2 phosphorylation and altered Bax and Bcl2 protein expression. The effect of WA on adipogenesis was examined by AdipoRed Assay after treating with WA (0.1-1 microM) during the differentiation period. WA decreased lipid accumulation in a dose-dependent manner and decreased the expression of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha and adipocyte fatty acid binding protein. The effects on apoptosis and lipid accumulation were also confirmed with Hoechst staining and Oil Red O staining, respectively. These results show that WA acts on adipocytes to reduce cell viability and adipogenesis and also induce apoptosis. PMID:19346589

Park, Hea Jin; Rayalam, Srujana; Della-Fera, Mary Anne; Ambati, Suresh; Yang, Jeong-Yeh; Baile, Clifton A

2008-01-01

21

CREB Activation Induces Adipogenesis in 3T3-L1 Cells  

PubMed Central

Obesity is the result of numerous, interacting behavioral, physiological, and biochemical factors. One increasingly important factor is the generation of additional fat cells, or adipocytes, in response to excess feeding and/or large increases in body fat composition. The generation of new adipocytes is controlled by several “adipocyte-specific” transcription factors that regulate preadipocyte proliferation and adipogenesis. Generally these adipocyte-specific factors are expressed only following the induction of adipogenesis. The transcription factor(s) that are involved in initiating adipocyte differentiation have not been identified. Here we demonstrate that the transcription factor, CREB, is constitutively expressed in preadipocytes and throughout the differentiation process and that CREB is stimulated by conventional differentiation-inducing agents such as insulin, dexamethasone, and dibutyryl cAMP. Stably transfected 3T3-L1 preadipocytes were generated in which we could induce the expression of either a constitutively active CREB (VP16-CREB) or a dominant-negative CREB (KCREB). Inducible expression of VP16-CREB alone was sufficient to initiate adipogenesis as determined by triacylglycerol storage, cell morphology, and the expression of two adipocyte marker genes, peroxisome proliferator activated receptor gamma 2, and fatty acid binding protein. Alternatively, KCREB alone blocked adipogenesis in cells treated with conventional differentiation-inducing agents. These data indicate that activation of CREB was necessary and sufficient to induce adipogenesis. Finally, CREB was shown to bind to putative CRE sequences in the promoters of several adipocyte-specific genes. These data firmly establish CREB as a primary regulator of adipogenesis and suggest that CREB may play similar roles in other cells and tissues.

Reusch, Jane E. B.; Colton, Lilliester A.; Klemm, Dwight J.

2000-01-01

22

Endothelin-1 inhibits TNF alpha-induced iNOS expression in 3T3-F442A adipocytes.  

PubMed

1. Endothelin-1 (ET-1) and tumor necrosis factor alpha (TNFalpha) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNFalpha induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNFalpha-induced iNOS expression and to determine whether or not ET-1 could influence TNFalpha-induced iNOS expression and NO production. 2. In differentiated 3T3-F442A, treatment with TNFalpha (20 ng ml(-1)) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription-polymerase chain reaction and Northern blot analysis. TNFalpha-induced iNOS expression requires nuclear factor kappaB activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38-MAP kinase pathways. 3. ET-1, but not ET-3, inhibited the TNFalpha-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA(2) 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. 4. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. 5. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNFalpha, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. 6. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNFalpha-induced iNOS expression. PMID:12839867

Mérial-Kieny, Christelle; Lonchampt, Michel; Cogé, Francis; Verwaerde, Patrick; Galizzi, Jean-Pierre; Boutin, Jean A; Lafontan, Max; Levens, Nigel; Galitzky, Jean; Félétou, Michel

2003-07-01

23

Endothelin-1 inhibits TNF?-induced iNOS expression in 3T3-F442A adipocytes  

PubMed Central

Endothelin-1 (ET-1) and tumor necrosis factor ? (TNF?) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNF? induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNF?-induced iNOS expression and to determine whether or not ET-1 could influence TNF?-induced iNOS expression and NO production. In differentiated 3T3-F442A, treatment with TNF? (20 ng ml?1) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription–polymerase chain reaction and Northern blot analysis. TNF?-induced iNOS expression requires nuclear factor ?B activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38–MAP kinase pathways. ET-1, but not ET-3, inhibited the TNF?-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA2 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNF?, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNF?-induced iNOS expression.

Merial-Kieny, Christelle; Lonchampt, Michel; Coge, Francis; Verwaerde, Patrick; Galizzi, Jean-Pierre; Boutin, Jean A; Lafontan, Max; Levens, Nigel; Galitzky, Jean; Feletou, Michel

2003-01-01

24

Folate receptor {alpha} regulates cell proliferation in mouse gonadotroph {alpha}T3-1 cells  

SciTech Connect

We have previously found that the mRNA and protein levels of the folate receptor alpha (FR{alpha}) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FR{alpha} has not fully been determined. We investigated the effect of FR{alpha} over-expression in the mouse gonadotroph {alpha}T3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FR{alpha} were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FR{alpha} promotes cell proliferation. These effects were abrogated in the same {alpha}T3-1 cells when transfected with a mutant FR{alpha} cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FR{alpha} over-expressing cells. In summary, our data suggests that FR{alpha} regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.

Yao, Congjun; Evans, Chheng-Orn [Department of Neurosurgery and Laboratory of Molecular Neurosurgery and Biotechnology, Emory University, School of Medicine, Atlanta, Georgia (United States)] [Department of Neurosurgery and Laboratory of Molecular Neurosurgery and Biotechnology, Emory University, School of Medicine, Atlanta, Georgia (United States); Stevens, Victoria L. [Epidemiology and Surveillance Research, American Cancer Society, Atlanta, Georgia (United States)] [Epidemiology and Surveillance Research, American Cancer Society, Atlanta, Georgia (United States); Owens, Timothy R. [Emory University, School of Medicine, Atlanta, Georgia (United States)] [Emory University, School of Medicine, Atlanta, Georgia (United States); Oyesiku, Nelson M., E-mail: noyesik@emory.edu [Department of Neurosurgery and Laboratory of Molecular Neurosurgery and Biotechnology, Emory University, School of Medicine, Atlanta, Georgia (United States)

2009-11-01

25

Pear pomace water extract inhibits adipogenesis and induces apoptosis in 3T3-L1 adipocytes  

PubMed Central

Obesity occurs when a person's calorie intake exceeds the amount of energy burns, which may lead to pathologic growth of adipocytes and the accumulation of fat in the tissues. In this study, the effect and mechanism of pear pomace extracts on 3T3-L1 adipocyte differentiation and apoptosis of mature adipocytes were investigated. The effects of pear pomace extract on cell viability and the anti-adipogenic and proapoptotic effects were investigated via MTT assay, Oil red O staining, western blot analysis and apoptosis assay. 3T3-L1 preadipocytes were stimulated with DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 µg/ml insulin and 1 µM dexamethasone for differentiation to adipocytes. 3T3-L1 cells were cultured with PBS or water extract of pear pomace. Water extract of pear pomace effectively inhibited lipid accumulations and expressions of PPAR-? and C/EBP? in 3T3-L1 cells. It also increased expression of p-AMPK and decreased the expression of SREBP-1c and FAS in 3T3-L1 cells. The induction of apoptosis was observed in 3T3-L1 cells treated with pear pomace. These results indicate that pear pomace water extract inhibits adipogenesis and induces apoptosis of adipocytes and thus can be used as a potential therapeutic substance as part of prevention or treatment strategy for obesity.

Rhyu, Jin; Kim, Min Sook; You, Mi-Kyoung; Bang, Mi-Ae

2014-01-01

26

Methylglyoxal induces oxidative stress and mitochondrial dysfunction in osteoblastic MC3T3-E1 cells.  

PubMed

Methylglyoxal is a reactive dicarbonyl compound produced by glycolytic processing and identified as a precursor of advanced glycation end products. The elevated methylglyoxal levels in patients with diabetes are believed to contribute to diabetic complications, including bone defects. The objective of this study was to evaluate the effect of methylglyoxal on the function of osteoblastic MC3T3-E1 cells. The data indicated that methylglyoxal decreased osteoblast differentiation and induced osteoblast cytotoxicity. Pretreatment of MC3T3-E1 cells with aminoguanidine (a carbonyl scavenger), Trolox (an antioxidant), and cyclosporin A (a blocker of the mitochondrial permeability transition pore) prevented methylglyoxal-induced cytotoxicity in MC3T3-E1 cells. However, BAPTA/AM (an intracellular Ca(2+) chelator) and dantrolene (an inhibitor of endoplasmic reticulum Ca(2+) release) did not reverse the cytotoxic effect of methylglyoxal. Methylglyoxal increased the formation of intracellular reactive oxygen species, mitochondrial superoxide, and cardiolipin peroxidation in osteoblastic MC3T3-E1 cells. Methylglyoxal also decreased the mitochondrial membrane potential and intracellular ATP and nitric oxide levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity contributes to the cytotoxicity of methylglyoxal. Furthermore, the results demonstrated that methylglyoxal induced protein adduct formation, inactivation of glyoxalase I, and activation of glyoxalase II. Aminoguanidine reversed all aforementioned effects of methylglyoxal. Taken together, these data support the notion that high methylglyoxal concentrations have detrimental effects on osteoblasts through a mechanism involving oxidative stress and mitochondrial dysfunction. PMID:24164256

Suh, K S; Choi, E M; Rhee, S Y; Kim, Y S

2014-02-01

27

Decreased anterior pituitary T3 nuclear receptors in a Walker 256 carcinoma-bearing rat model of nonthyroidal disease.  

PubMed

Rats bearing the Walker 256 carcinoma have decreased pituitary nuclear T3 but normal pituitary TSH content and response to experimental hypothyroidism. To elucidate further the role of T3 receptor occupancy and biological response in the tumor-bearing rat model of nonthyroidal disease, we measured the concentration of T3 nuclear receptors, rTSH and rGH and beta-TSH mRNA and GH mRNA in the anterior pituitary of euthyroid rats bearing the Walker 256 carcinoma. The abundance of T3 nuclear receptors was decreased in tumor-bearing rats and was associated with a decrease in mRNA content for beta-TSH and GH. alpha-tubulin mRNA was decreased to a comparable degree. The pituitary content of rTSH and rGH was, however, the same as in control animals. Since tumor rats have normal regulation of TSH secretion by thyroid hormone, the present findings suggest that TSH secretion in T rats is maintained by a lower T3 nuclear receptor occupancy than in controls. The decrease in beta-TSH mRNA may precede a decrease in TSH synthesis and changes in pituitary TSH stores. Since the decrease in GH mRNA was comparable to the decrease in alpha-tubulin mRNA, it does not appear to be specifically related to decreased T3 nuclear receptor occupancy. We conclude that, in the tumor-bearing rat model of nonthyroidal disease, decreases in beta-TSH mRNA occur despite a decreased T3 receptor occupancy. Both thyroid-dependent and thyroid-independent factors may be involved in regulating beta-TSH mRNA. PMID:2609901

Hupart, K H; DeFesi, C R; Katz, C P; Shapiro, L E; Surks, M I

1989-12-01

28

Morphological transformation induced by glass fibers in BALB/c-3T3 cells.  

PubMed

Studies were conducted to determine whether 1) glass fibers can induce morphological transformation in BALB/c-3T3 cells, 2) the transforming activity of glass fibers is related to fiber size, and 3) transformed cells induced by glass fibers possess neoplastic properties. In the transformation assay, BALB/c-3T3 cells were treated with three different types of glass fibers: Manville code 100 (JM-100, Manville Corp., Denver, CO), Owens-Corning AAA-10 (AAA-10, Owens-Corning Corp., Toledo, OH), and Owens-Corning general building insulation (ISL, Owens-Corning Corp.) fibers. The neoplastic properties were investigated using the soft agar cloning and gene transfection methods. All three different glass fibers were cytotoxic at high concentrations and induced dose-related increases in morphological transformation. The transforming activity was inversely related to fiber size, with AAA-10 showing higher activity than JM-100 and JM-100 showing higher activity than ISL fiber. Transformed cells induced by glass fibers exerted anchorage-independent growth (90%) and DNA transfection-mediated transformation (100%). These results indicate that glass fibers are capable of transforming mammalian (BALB/c-3T3) cells in vitro as a function of their physical properties and that glass fiber-induced transformed cells possess preneoplastic characteristics. PMID:8525469

Gao, H G; Whong, W Z; Jones, W G; Wallace, W E; Ong, T

1995-01-01

29

Pituitary resistance to thyroid hormone syndrome is associated with T3 receptor mutants that selectively impair beta2 isoform function.  

PubMed

Resistance to thyroid hormone (RTH) syndrome is an inherited inability to respond appropriately to T3 hormone. In generalized RTH, the T3 response of both the pituitary and periphery is disrupted. In pituitary (or central) RTH, the ability of the pituitary to sense (and down-regulate) elevated T3 is selectively impaired, whereas the periphery remains relatively T3 responsive, resulting in peripheral thyrotoxicity. Both forms of disease are linked to mutations in thyroid hormone receptor (TR)-beta. TRbeta is expressed by alternate mRNA splicing as two isoforms: TRbeta2, found primarily in the pituitary/hypothalamus, and TRbeta1, expressed broadly in many tissues. We report here that the wild-type TRbeta2 isoform displays an enhanced T3 response relative to the TRbeta1 isoform. Mutations associated with generalized RTH (P453S, G345S) impair both TRbeta2 and TRbeta1 function proportionally, whereas mutations associated with pituitary-specific RTH (R338L, R338W, R429Q) disproportionately disrupt TRbeta2 function. We propose that in the normal organism, and in generalized RTH, TRbeta2 in the pituitary can sense rising T3 levels in advance of TRbeta1 in the periphery, preventing thyrotoxicity. In contrast, the TRbeta mutations associated with pituitary RTH disproportionately disrupt the pituitary's ability to sense and suppress elevated T3 levels in advance of the periphery, producing symptoms of thyrotoxicity. PMID:15802373

Wan, Wei; Farboud, Behnom; Privalsky, Martin L

2005-06-01

30

Butyrate modulates the expression of. beta. -adrenergic receptor subtype in 3T3-L1 cells  

SciTech Connect

In mouse 3T3-L1 fibroblasts, the glucocorticoid dexamethasone (dex) affects a switch in ..beta..-adrenergic receptor (..beta..AR) subtype expression from ..beta../sub 1/AR to ..beta../sub 2/AR and increases total ..beta..AR number. They now demonstrate a similar effect by sodium butyrate (B) and find that the combined effect of these two gene-activating agents is greater than additive suggesting different mechanisms of action on the ..beta..AR. ..beta..AR are assayed in membranes prepared from 3T3-L1 cells using the radiolabeled ..beta..AR-specific antagonist (/sup 125/I)-cyanopindolol. ..beta..AR subtype is determined by competition binding of the ..beta../sub 2/AR-selective antagonist ICI 118.551 for the radioligand. B (2-10mM) causes a dose-dependent increase in total ..beta..AR number (up to 2-fold over control) and the proportion of ..beta../sub 2/AR. B (5mM) causes a time-dependent increase in total ..beta..AR number (2-fold) and the proportion of ..beta../sub 2/AR up to 24 hr. Dex maximally increases total ..beta..AR number (2-fold) when treated for 48 hr at concentrations greater than or equal to 100nM. B (2 or 5mM) together with dex (250nM) have a greater than additive effect on total ..beta..AR number at 24 hr (1.7-fold) and at 48 hr (1.4-2.4-fold, using 5 or 10mM B and dex greater than or equal to 10nM). The proportion of ..beta../sub 2/AR is also greater when both compounds are added together. In comparison with proprionate and valerate, B increases total ..beta..AR number and the proportion of ..beta../sub 2/AR to a greater extent and at lower concentrations. To determine a functional correlate to these findings, cells were pre-treated for 48 hr with B and/or dex, intracellular ATP labeled with /sup 3/H-adenine, followed by treatment with forskolin (10..mu..M) and ..beta..AR agonists. B caused a dramatic increase in /sup 3/H-cAMP produced compared to control and dex treatments and a greater than additive effect was again achieved when B and dex were added together.

Poksay, K.S.; Nakada, M.T.; Crooke, S.T.; Stadel, J.M.

1986-03-05

31

High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells  

SciTech Connect

In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10/sup 6/ receptors per cell. The cell line with the highest /sup 125/I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10/sup 6/ receptors with a K/sub d/ of 10/sup -9/ M. This level was not dependent on exposure to metals but could be increased further to 2 x 10/sup 7/ receptors per cell by addition of sodium butyrate to the culture medium. The ..cap alpha.. and ..beta.. subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the ..cap alpha.. and ..beta.. subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

1987-08-01

32

Human papillomavirus type 16 DNA-induced malignant transformation of NIH 3T3 cells.  

PubMed Central

A biological function for human papillomavirus 16 (HPV 16) DNA was demonstrated by transformation of NIH 3T3 cells. HPV 16 DNA has been found frequently in genital cancer and has been classified as a papillomavirus on the basis of DNA homology. A recombinant HPV 16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full-length HPV 16 genome, induced morphologic transformation; the transformed cells were tumorigenic in nude mice. Expression of transforming activity was unique because of the long latency period (more than 4 weeks) required for induction of morphologic transformation and because the transfected DNA existed primarily in a multimeric form with some rearrangements. Furthermore, virus-specific RNAs were expressed in the transformants. The transformation of NIH 3T3 cells provides a model for analyzing the functions of HPV 16, which is associated with cervical carcinomas. Images

Yasumoto, S; Burkhardt, A L; Doniger, J; DiPaolo, J A

1986-01-01

33

Drinking water disinfection byproduct iodoacetic acid induces tumorigenic transformation of NIH3T3 cells.  

PubMed

Iodoacetic acid (IAA) and iodoform (IF) are unregulated iodinated disinfection byproducts (DBPs) found in drinking water. Their presence in the drinking water of China has not been documented. Recently, the carcinogenic potential of IAA and IF has been a concern because of their mutagenicity in bacteria and genotoxicity in mammalian cells. Therefore, we measured their concentrations in Shanghai drinking water and assessed their cytotoxicity, genotoxicity, and ability to transform NIH3T3 cells to tumorigenic lines. The concentrations of IAA and IF in Shanghai drinking water varied between summer and winter with maximum winter levels of 2.18 ?g/L IAA and 0.86 ?g/L IF. IAA with a lethal concentration 50 (LC50) of 2.77 ?M exhibited more potent cytotoxicity in NIH3T3 cells than IF (LC50 = 83.37 ?M). IAA, but not IF, induced a concentration-dependent DNA damage measured by ?-H2AX staining and increased tail moment in single-cell gel electrophoresis. Neither IAA nor IF increased micronucleus frequency. Prolonged exposure of NIH3T3 cells to IAA increased the frequencies of transformed cells with anchorage-independent growth and agglutination with concanavalin A. IAA-transformed cells formed aggressive fibrosarcomas after inoculation into Balb/c nude mice. This study demonstrated that IAA has a biological activity that is consistent with a carcinogen and human exposure should be of concern. PMID:23641915

Wei, Xiao; Wang, Shu; Zheng, Weiwei; Wang, Xia; Liu, Xiaolin; Jiang, Songhui; Pi, Jingbo; Zheng, Yuxin; He, Gengsheng; Qu, Weidong

2013-06-01

34

Prednisolone induces the Wnt signalling pathway in 3T3-L1 adipocytes  

PubMed Central

Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids.

Fleuren, Wilco W. M.; Linssen, Margot M. L.; Toonen, Erik J. M.; van der Zon, Gerard C. M.; Guigas, Bruno; de Vlieg, Jacob; Dokter, Wim H. A.; Ouwens, D. Margriet

2013-01-01

35

R-Ras induces malignant, but not morphologic, transformation of NIH3T3 cells.  

PubMed

Although previous studies have not identified transforming properties of the Ras-related protein R-Ras, two recent observations have prompted our further evaluation of R-Ras function. First, we observed that mutant forms of the closely related R-Ras2/TC21 protein (approximately 70% identity) exhibited the same potent transforming activity as oncogenic Ras proteins. Second, R-Ras association with Bcl-2 suggested a possible role for R-Ras in apoptotic growth control. Therefore, we have performed a detailed analysis of R-Ras transforming potential in NIH3T3 cells. Whereas expression of a mutant R-Ras protein (38V; analogous to the 12V activating Ras mutation) did not induce morphologic transformation of NIH3T3 cells, R-Ras(38V)-expressing cells proliferated in low serum, formed colonies in soft agar, and formed progressive tumors in nude mice. Like Ras-transformed cells, R-Ras(38V)-transformed cells exhibited constitutively activated mitogen activated protein kinases. Furthermore, R-Ras(38V) stimulated transcriptional activation of Ras-responsive promoter elements, and this activity (and transformation) was blocked by Raf dominant negative proteins. Finally, whereas co-expression of Bcl-2 did not cause significant alteration in wild type or mutant R-Ras transforming activity, coexpression of v-Myc and R-Ras(38V) induced a striking morphologic transformation of NIH3T3 cells. Taken together, these observations suggest that aberrant R-Ras function may stimulate malignant transformation, in the absence of morphologic transformation, via up-regulation of part of the Ras signal transduction pathway. PMID:7936652

Cox, A D; Brtva, T R; Lowe, D G; Der, C J

1994-11-01

36

Localization of transferrin receptors and insulin-like growth factor II receptors in vesicles from 3T3-L1 adipocytes that contain intracellular glucose transporters  

Microsoft Academic Search

Transferrin receptors in detergent extracts of subcellular membrane fractions prepared from 3T3-L1 adipocytes were measured by a binding assay. There was a small but significant increase (1.2-fold) in the amount of receptor in a crude plasma membrane frac- tion and a 40% decrease in the number of transferrin receptors in microsomal membranes prepared from insulin-treated cells, when compared with correspond-

Laura I. Tanner; Gustav E. Lienhard

1989-01-01

37

Anandamide induced PPAR? transcriptional activation and 3T3-L1 preadipocyte differentiation  

Microsoft Academic Search

We investigated the effects of anandamide on peroxisome proliferator-activated receptor ? (PPAR?) activity. In two different transactivation systems using either full-length or only the ligand binding domain of PPAR?, we showed that anandamide, but not palmitoylethanolamide induced transcriptional activation of PPAR? in a dose dependent manner with an EC50 of 8 ?M. In addition, competition binding experiments showed that anandamide

Monsif Bouaboula; Sandrine Hilairet; Jean Marchand; Lluis Fajas; Gerard Le Fur; Pierre Casellas

2005-01-01

38

The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation.  

PubMed

While the role of p75(NTR) signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75(NTR) on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75(NTR)-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75(NTR) in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75(NTR), as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75(NTR)-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75(NTR) signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation. PMID:22906707

Mikami, Yoshikazu; Suzuki, Shinnosuke; Ishii, Yumiko; Watanabe, Nobukazu; Takahashi, Tomihisa; Isokawa, Keitaro; Honda, Masaki J

2012-12-01

39

Proliferative and morphological changes induced by vanadium compounds on Swiss 3T3 fibroblasts.  

PubMed

Vanadium compounds are shown to have a mitogenic effect on fibroblast cells. The effects of vanadate, vanadyl and pervanadate on the proliferation and morphological changes of Swiss 3T3 cells in culture are compared. Vanadium derivatives induced cell proliferation in a biphasic manner, with a toxic-like effect at doses over 50 microM, after 24 h of incubation. Vanadyl and vanadate were equally potent at 2.5-10 microM. At 50 microM vanadate inhibited cell proliferation, whereas slight inhibition was observed at 100 microM of vanadyl. At 10 microM pervanadate was as potent as vanadate and vanadyl in stimulating fibroblast proliferation, but no effect was observed at lower concentrations. A pronounced cytotoxic-like effect was induced by pervanadate at 50 microM. All of these effects were accompanied by morphological changes: transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation; and loss of lamellar processes. The magnitude of these transformations correlates with the potency of vanadium derivatives to induce a cytotoxic-like effect: pervanadate > vanadate > vanadyl. These data suggest that the oxidation state and coordination geometry of vanadium determine the degree of the cytotoxicity. PMID:9210295

Cortizo, A M; Sálice, V C; Vescina, C M; Etcheverry, S B

1997-04-01

40

Expression of progesterone receptor B is associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH3T3 cells  

SciTech Connect

Previously, we found a significant reduction of progesterone receptor B (PR-B) expression levels in the Ras-mediated NIH3T3 cell transformation, and re-expression of exogenous PR-B eliminated the tumorigenic potential. We hypothesized that this reduction is of biological significance in cell transformation. In the present study, we determined the correlation between PR-B expression and cell cycle progression. In synchronized NIH3T3 cells, we found an increase in PR-B protein and p27 CDK inhibitor levels in the G0/G1 phase and a reduction due to redistribution in the S and G2/M phases. The MEK inhibitor or cAMP stimulation arrested NIH3T3 cells in the G0/G1 phase of the cell cycle. The expression of PR-B and p27 CDK inhibitors was up-regulated by treatment with both the MEK inhibitor and cAMP. Treatment of synchronized cells with a PKA inhibitor in the presence of 1% calf serum resulted in a significant reduction in both PR-B and p27 levels. The decrease in the PR-B levels caused by anti-sense oligomers or siRNA corresponded to the reduction in p27 levels. PR-B overexpression by adenovirus infection induced p27 and suppressed cell growth. Finally, we showed that PR-B modulation involved in the regulation of NIH3T3 cell proliferation was independent of nuclear estrogen receptor (ER) activity but dependent on non-genomic ER activity.

Horiuchi, Shinji [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan) and Department of Obstetrics and Gynecology, School of Medicine, Fukuoka University, Jyounanku Fukuoka 814-0180 (Japan); Kato, Kiyoko [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan)]. E-mail: kkatoh@tsurumi.beppu.kyushu-u.ac.jp; Suga, Shin [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Takahashi, Akira [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Ueoka, Yousuke [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Arima, Takahiro [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Nishida, Jun-ichi [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan); Hachisuga, Toru; Kawarabayashi, Tatsuhiko [Department of Obstetrics and Gynecology, School of Medicine, Fukuoka University, Jyounanku Fukuoka 814-0180 (Japan); Wake, Norio [Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, Oita, 874-0838 (Japan)

2005-05-01

41

Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes  

Microsoft Academic Search

This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1

Michelle N. Yarmo; Anne Landry; André S. D. Molgat; AnneMarie Gagnon; Alexander Sorisky

2009-01-01

42

Critical role of leukotriene B4 receptor signaling in mouse 3T3-L1 preadipocyte differentiation  

PubMed Central

Background Various inflammatory mediators related to obesity might be closely related to insulin resistance. Leukotrienes (LTs) are involved in inflammatory reactions. However, there are few reports regarding the role of LTs in adipocyte differentiation. Therefore, we investigated the role of leukotriene B4 (LTB4)-leukotriene receptor (BLT) signaling in mouse 3T3-L1 fibroblastic preadipocyte differentiation to mature adipocytes. Methods Mouse 3T3-L1 preadipocytes were treated with lipoxygenase (LOX) inhibitors, BLT antagonist, and small interfering RNA (siRNA) for BLT1 and BLT2 to block the LTB4-BLT signaling pathway, then the adipocyte differentiation such as lipid accumulation and the increase in triglyceride was evaluated. Results Blockade of BLT signaling by treatment with a LOX inhibitor or a BLT antagonist suppressed preadipocyte differentiation into mature adipocytes. In addition, knockdown of BLT1 and BLT2 by siRNAs dramatically inhibited differentiation. These results indicate the LTB4-BLT signaling pathway may positively regulate preadipocyte differentiation and be a rate-limiting system to control adipocyte differentiation. Conclusions The LTB4-BLT signaling pathway provides a potent regulatory signal that accelerates the differentiation of mouse 3T3-L1 preadipocytes. Further investigations are necessary to confirm the exact role of LTB4 and BLTs signaling pathways in preadipocyte differentiation.

2013-01-01

43

Monoclonal antibodies to the insulin receptor mimic metabolic effects of insulin but do not stimulate receptor autophosphorylation in transfected NIH 3T3 fibroblasts  

SciTech Connect

The metabolic actions of insulin and anti-insulin receptor monoclonal antibodies were compared with their effects on insulin receptor phosphorylation in mouse NIH 3T3 fibroblasts transfected with human insulin receptor cDNA. In serum-starved NIH 3T3 HIR3.5 cells, uptake of 2-deoxy-({sup 3}H)glucose was stimulated up to 2-fold after 30 min with insulin, with a half-maximal effect at 0.1 nM insulin. Incorporation of ({sup 3}H)thymidine was stimulated {approx} 12-fold after a 16-hr preincubation with insulin, with a half-maximal effect at 2 nM insulin. Phosphorylation of insulin receptor {beta}-subunit in cells prelabeled with ({sup 32}P)phosphate was increased 10- to 20-fold within 5 min of adding insulin. Monoclonal antibodies reacting with four different epitopes on the insulin receptor mimicked the effect of insulin on 2-deoxyglucose uptake. These antibodies also stimulated thymidine incorporation, although the maximum stimulation was only {approx} 30% that of insulin. It is concluded that the insulin-like metabolic effects of antibodies involve a mechanism of receptor activation that is independent of autophosphorylation and hence that receptor autophosphorylation is not an essential step in triggering at least some events in the insulin signaling pathway.

Soos, M.A.; O'Brien, R.M.; Brindle, N.P.J.; Stigter, J.M.; Okamoto, A.K.; Whittaker, J.; Siddle, K. (Univ. of Cambridge (England))

1989-07-01

44

Serum-induced G0/G1 transition in chemically transformed 3T3 cells  

SciTech Connect

Quiescent, chemically transformed (benzo-a-pyrene) BALB/c 3T3 cells (BP A31) enter the cell division cycle when exposed to complete medium containing 10% fetal calf serum (FCS); the number of cells recruited is a function of the duration of serum exposure. The recruitment of cells by short (<4 h) serum pulses is not inhibited by simultaneous exposure to cycloheximide (CH), and therefore the initial commitment does not require protein synthesis. The cells enter S phase with a constant delay following the removal of CH, even if CH exposure has been continued for as long as 20 h after the end of the serum pulse. The cell recruitment by serum pulses was inhibited by 5,6-dichloro-1-..beta..-D-ribofuranosyl-benzimidazole (DRB), an inhibitor of cytoplasmic mRNA accumulation. These data suggest that serum exposure produces a stable memory that is necessary and sufficient for the eventual progression through G1 to S phase that occurs when protein synthesis is resumed after the removal of CH; this memory probably consists of mRNA species that are induced by serum and that are stable in the absence of protein synthesis. Unexpectedly, pretreatment of quiescent BP A31 cells with CH (8-24 h) dramatically increased the fraction of the total cell population that is recruited by a serum pulse of fixed duration.

Gray, H.E.; Buchou, T.; Mester, J.

1987-03-01

45

Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells  

NASA Technical Reports Server (NTRS)

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Butters, R. R. Jr; Sugimoto, T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

1998-01-01

46

Esculetin Induces Apoptosis and Inhibits Adipogenesis in 3T3-L1 Cells  

Microsoft Academic Search

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3-L1 adipocyte apoptosis and adipogenesis.Research Methods and Procedures: 3T3-L1 pre-confluent preadipocytes and lipid-filled adipocytes were incubated with esculetin (0 to 800 ?M) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis

Jeong-Yeh Yang; Mary Anne Della-Fera; Diane L. Hartzell; Cass Nelson-Dooley; Dorothy B. Hausman; Clifton A. Baile; A. Baile

2006-01-01

47

Complete Activation of Thyroid Hormone Receptor ? by T3 is Essential for Normal Cochlear Function and Morphology in Mice  

PubMed Central

Background/Aims: Thyroid hormones (THs) regulate many developmental processes, including the developmental onset of cochlear differentiation and function. TH action is mediated mostly by triiodothyronine (T3) bound to thyroid hormone nuclear receptors (TRs). At positive regulated genes and in the absence of THs, nuclear co-repressors are bound to TRs and decrease basal transcription rate. Ligand (T3) binding results in the dissociation of co-repressors and the recruitment of co-activators to the complex, which results in full transcriptional activation. Methods: We measured cochlear function in two knock-in mouse models: TR? E457A/E457A, with the TR? co-activator binding surface (AF-2) disrupted to prevent co-activator binding; and TR? ?337T/?337T, which is unable to bind T3. Cochlear morphology and function were analyzed in 10-week-old normal and mutated mice. Cochlear function was determined by measuring auditory brainstem responses, cochlear tuning and compound action potential (CAP) thresholds. Results: All TR? ?337T/?337T and 85% of the TR?E457A/E457A mice presented elevated CAP thresholds (P < 0.05 or less). Five percent of the TR?E457A/E457A mice presented normal CAP thresholds with broadened cochlear tuning. TR?E457A/E457A and TR? ?337T/?337T presented developmental defects that led to a decreased width (P < 0.01) and an increased thickness (P<0.01) of the tectorial membrane. In addition, TR? ?337T/?337T animals showed an increased tectorial membrane area (P<0.01). Conclusion: Both mutations were deleterious to tectorial membrane development and led to important alterations in cochlear morphology and loss of cochlear function.

Richter, Claus-Peter; Munscher, Adrian; Santana Machado, Danielle; Wondisford, Fredric E.; Ortiga-Carvalho, Tania M.

2011-01-01

48

Paraquat-induced oxidative stress represses phosphatidylinositol 3-kinase activities leading to impaired glucose uptake in 3T3-L1 adipocytes.  

PubMed

Accumulated evidence indicates that oxidative stress causes and/or promotes insulin resistance; however, the mechanism by which this occurs is not fully understood. This study was undertaken to elucidate the molecular mechanism by which oxidative stress induced by paraquat impairs insulin-dependent glucose uptake in 3T3-L1 adipocytes. We confirmed that paraquat-induced oxidative stress decreased glucose transporter 4 (GLUT4) translocation to the cell surface, resulting in repression of insulin-dependent 2-deoxyglucose uptake. Under these conditions, oxidative stress did not affect insulin-dependent tyrosine phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and -2, or binding of the phosphatidylinositol 3'-OH kinase (PI 3-kinase) p85 regulatory subunit or p110alpha catalytic subunit to each IRS. In contrast, we found that oxidative stress induced by paraquat inhibited activities of PI 3-kinase bound to IRSs and also inhibited phosphorylation of Akt, the downstream serine/threonine kinase that has been shown to play an essential role in insulin-dependent translocation of GLUT4 to the plasma membrane. Overexpression of active form Akt (myr-Akt) restored inhibition of insulin-dependent glucose uptake by paraquat, indicating that paraquat-induced oxidative stress inhibits insulin signals upstream of Akt. Paraquat treatment with and without insulin treatment decreased the activity of class Ia PI 3-kinases p110alpha and p110beta that are mainly expressed in 3T3-L1 adipocytes. However, paraquat treatment did not repress the activity of the PI 3-kinase p110alpha mutated at Cys(90) in the p85 binding region. These results indicate that the PI 3-kinase p110 is a possible primary target of paraquat-induced oxidative stress to reduce the PI 3-kinase activity and impaired glucose uptake in 3T3-L1 adipocytes. PMID:20430890

Shibata, Michihiro; Hakuno, Fumihiko; Yamanaka, Daisuke; Okajima, Hiroshi; Fukushima, Toshiaki; Hasegawa, Takashi; Ogata, Tomomi; Toyoshima, Yuka; Chida, Kazuhiro; Kimura, Kumi; Sakoda, Hideyuki; Takenaka, Asako; Asano, Tomoichiro; Takahashi, Shin-Ichiro

2010-07-01

49

Characterization of a platelet-derived growth factor receptor on Swiss 3T3 cells by affinity crosslinking.  

PubMed

Crosslinking experiments with various bifunctional reagents were used to investigate the nature and fate of the platelet growth factor (PDGF) receptor on Swiss mouse 3T3 cells. With ethylene glycol bis succinimidyl succinate (EGS) two bands with Mr 205,000 and Mr 190,000 were labeled at equal intensity, while with disuccinimidyl suberate (DSS) and the photoactivatable p-azidophenylglyoxal (pAPG) almost exclusively the latter band was labeled, when analyzed by SDS polyacrylamide gel electrophoresis under reducing conditions. Evidence is presented that the Mr 190,000 band represents a Mr 175,000 receptor protein crosslinked to a single chain of the PDGF-dimer and the Mr 205,000 species the same Mr 175,000 protein crosslinked to both chains of PDGF. Pretreatment of cells with tunicamycin generated a third labeled band with Mr 150,000, while pretreatment with neuraminidase resulted in a shift of the Mr 205,000 and 190,000 bands by 5,000. This shows that the PDGF receptor is a sialoglycoprotein, consisting of a Mr approximately 135,000 proteinaceous core and a Mr approximately 40,000 carbohydrate moiety containing sialic acid. The virtually unchanged labeling intensity seen with tunicamycin and neuraminidase pretreated cells further suggests that the carbohydrate portion of the receptor is not required for PDGF binding. Finally, the crosslinking technique was used to show that at 37 degrees C performed 125I-PDGF receptor complexes disappear from the cell surface with a t1/2 approximately 8 min. PMID:2838625

Hosang, M

1988-01-01

50

Inhibitory effect on protein kinase Ctheta by Crocetin attenuates palmitate-induced insulin insensitivity in 3T3-L1 adipocytes.  

PubMed

Epidemiologic and experimental studies have pointed to an etiologic role of elevated plasma free fatty acids in insulin resistance, which is frequently associated with a state of low-grade inflammation. In this study, we investigated the effects of Crocetin, a unique carotenoid, on insulin resistance induced by palmitate in 3T3-L1 adipocytes. Exposure of palmitate led to an increase in insulin receptor substrate-1 (IRS-1) serine(307) phosphorylation as well as activation of c-Jun NH(2)-terminal kinase (JNK) and inhibitor kappaB kinase beta (IKKbeta), concomitantly with reductions of IRS-1 function and glucose metabolism. Interestingly, pretreatment with Crocetin almost reversed all of these abnormalities in a dose-dependent manner. IRS-1 serine(307) phosphorylation was significantly reduced by JNK or IKKbeta inhibitor, especially by combination of these two inhibitors. Moreover, palmitate treatment induced activation of protein kinase Ctheta (PKCtheta) while blocking PKCtheta significantly inhibited JNK and IKKbeta activation induced by palmitate or phorbol 12-myristate 13-acetate (PKC activator, PMA), and attenuated the palmitate-induced defects in insulin action. Crocetin demonstrated an impressive suppression in the activation of PKCtheta induced not only by palmitate but also by PMA in a dose-dependent manner. Taken together, Crocetin inhibited JNK and IKKbeta activation via suppression of PKCtheta phosphorylation, attenuating insulin insensitivity induced by palmitate in 3T3-L1 adipocytes. PMID:20541543

Yang, Lina; Qian, Zhiyu; Ji, Hui; Yang, Ruhui; Wang, Yuhuan; Xi, Liang; Sheng, Liang; Zhao, Bohua; Zhang, Xiaoming

2010-09-10

51

Characterization of PHA and anti-T3 induced transduction mechanisms in a human T-cell leukaemia line.  

PubMed

Stimulation of the T-cell line JURKAT with PHA or anti-T3 antibody leads to a rapid and sustained rise of cytosolic free Ca2+, as determined by quin2 fluorescence measurements. Pertussis toxin and N-ethylmaleimide, substances known to inactivate a regulatory N-protein, caused partial to complete inhibition of the cytosolic free Ca2+ response induced both by anti-T3 or PHA. The high cytosolic free Ca2+ level induced by anti-T3 or PHA declined more rapidly after addition of phorbol ester, phorbol myristate acetate (PMA). PMA did not affect cytosolic free Ca2+ changes induced by ionomycin indicating that the effect of PMA is due to a direct inhibitory effect on a transduction mechanism and not to activation of Ca2+ extrusion. Our data suggest that a regulatory N-protein is involved in the transduction of the PHA and anti-T3 response into a rapid and sustained elevation of cytosolic free Ca2+. Activation of protein kinase C by PMA modulates the calcium response in JURKAT cells, suggesting that protein kinase C may be involved in feedback regulation of the transduction mechanism. PMID:3495499

Ng, J; Fredholm, B; Jondal, M; Andersson, T

1987-01-01

52

Protective effect of tetrahydroxystilbene glucoside against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells.  

PubMed

Oxidative stress can induce apoptosis and decrease activities of osteoblasts. 2,3,5,4'-tetrahydroxystilbene-2-O-?-D-glucoside (TSG), is a potent antioxidant derived from a Chinese herb Polygonum multiflorum Thunb. To evaluate the protective effect provided by TSG to osteoblastic MC3T3-E1 cells, the cells were pretreated with TSG for 24h before being treated with 0.3mM hydrogen peroxide (H(2)O(2)) for 24 h, then some markers of osteoblast function and oxidative damage of the cells were examined. Our data demonstrated that TSG significantly (P< 0.05) increased cell survival, alkaline phosphatase (ALP) activity, calcium deposition, and the mRNA expression of ALP, collagen I (COL-I) and osteocalcin (OCN) in the presence of H(2)O(2). In addition, TSG decreased the production of receptor activator of nuclear factor-?B ligand (RANKL), interleukin-6 (IL-6), intracellular reactive oxygen species and malondialdehyde (MDA) of osteoblastic MC3T3-E1 cells induced by H(2)O(2). Taken together, these results demonstrated that the protective effect provided by TSG to osteoblastic MC3T3-E1 cells was mediated, at least in part, via inhibition of the release of bone-resorbing mediators and oxidative damage of the cells. Our results indicated that TSG may be effective in providing protection against osteoporosis associated with oxidative stress. PMID:22683865

Zhang, Jin-Kang; Yang, Liu; Meng, Guo-Lin; Fan, Jing; Chen, Jian-Zong; He, Qi-Zhen; Chen, Shi; Fan, Jin-Zhu; Luo, Zhuo-Jing; Liu, Jian

2012-08-15

53

Expression and function of insulin/insulin-like growth factor I hybrid receptors during differentiation of 3T3-L1 preadipocytes.  

PubMed Central

During the assembly of cell surface receptors, insulin proreceptors are sometimes joined to insulin-like growth factor (IGF) receptor precursors to form covalently linked hybrid receptors. To address the biological consequences of hybrid receptor formation, we studied 3T3-L1 cells known to undergo a 50-70-fold increase in insulin binding while maintaining nearly constant levels of IGF-I binding during differentiation from preadipocytes into adipocytes. The presence of insulin/IGF receptor hybrids in 3T3-L1 adipocytes was demonstrated by the immunoprecipitation of phosphorylated receptors and a novel enzyme-linked immunoassay. Hybrid receptor levels were very low in the early stages of differentiation and increased rapidly between days 4 and 6, reaching a level about 100-fold higher in the mature adipocyte. Coincident with the hybrid assembly, the formation of archetypal (alpha2,beta2) IGF receptors decreased. In fully differentiated adipocytes, virtually all of the IGF receptors were in hybrid form. Stimulation by IGF-I of receptors isolated from mature adipocytes caused autophosphorylation of IGF receptor beta subunits in hybrid complexes, whereas autophosphorylated IGF holoreceptors were not demonstrable. Insulin and IGF-I were equipotent in stimulating glucose uptake in the differentiated adipocytes, leading to the conclusion that hybrid insulin/IGF receptors can transduce a transmembrane signal when activated by IGF-I. We conclude that hybrid formation constitutes a novel post-translational mechanism whereby increased synthesis of insulin receptors limits the cell surface expression of the homologous IGF receptor. Furthermore, biological actions in 3T3-L1 adipocytes, previously attributed to archetypal IGF receptors, are in fact mediated through hybrid receptors.

Modan-Moses, D; Janicot, M; McLenithan, J C; Lane, M D; Casella, S J

1998-01-01

54

Pleiotrophin Transforms NIH 3T3 Cells and Induces Tumors in Nude Mice  

NASA Astrophysics Data System (ADS)

The pleiotrophin (PTN) gene (Ptn) encodes an 18-kDa protein that is highly conserved among mammalian species and that functions as a weak mitogen and promotes neurite-outgrowth activity in vitro. To further investigate the role PTN plays in regulating cell growth, we overexpressed the bovine PTN cDNA and now show that PTN phenotypically transforms NIH 3T3 cells, as evidence by increased cell number at confluence, focus formation, anchorage-independent growth, and tumor formation in the nude muse. The results demonstrate that the Ptn gene has the potential to regulate NIH 3T3 cell growth and suggest that PTN may influence abnormal cell growth in vivo.

Chauhan, Anil K.; Li, Yue-Sheng; Deuel, Thomas F.

1993-01-01

55

Conjugated linoleic acid suppresses triglyceride accumulation and induces apoptosis in 3T3-L1 preadipocytes  

Microsoft Academic Search

Four sets of experiments were conducted to examine the influence of conjugated linoleic acid (CLA) isomers during proliferation\\u000a and differentiation of cultures of 3T3-L1 preadipocytes using physiological culturing conditions. Cultures treated with either\\u000a albumin [bovine serum albumin (BSA) vehicle] or linoleic acid (LA) served as controls. For the proliferation study (Expt.\\u000a 1), cells were cultured in media containing a crude

M. Evans; C. Geigerman; J. Cook; L. Curtis; B. Kuebler; M. McIntosh

2000-01-01

56

Microconstituent-Induced Pitting Corrosion in Aluminum Alloy 2024-T3  

Microsoft Academic Search

Free corrosion immersion experiments were conducted on a commercial airframe material, Al 2024-T3 (UNS A92024), in 0.5 M sodium chloride (NaCl) solution to investigate the role of microconstituents in pitting corrosion. The alloy was found to contain numerous constituent particles (> 300,000 per cm [> 2 million per in.]), and pitting corrosion essentially was attributable to these particles. Two types

G. S. Chen; M. S. Gao; R. P. Wei

1996-01-01

57

Expression of glucagon-like peptide-1 receptor and glucose?dependent insulinotropic polypeptide receptor is regulated by the glucose concentration in mouse osteoblastic MC3T3-E1 cells.  

PubMed

Glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon-like peptide-1 receptor (GLP?1R) are incretin receptors that play important roles in regulating insulin secretion from pancreatic ? cells. Incretin receptors are also thought to play a potential role in bone metabolism. Osteoblasts in animals and humans express GIPR; however, the presence of GLP-1R in these cells has not been reported to date. Thus, the aim of this study was to determine whether GLP-1R and GIPR are expressed in osteoblastic cells, and whether their expression levels are regulated by the extracellular glucose concentration. Mouse osteoblastic MC3T3-E1 cells were cultured in medium containing normal (5.6 mM) or high (10, 20 or 30 mM) glucose concentrations, with or without bone morphogenetic protein-2 (BMP-2). RT-PCR, western blot analysis and immunofluorescence were carried out to determine GIPR and GLP-1R mRNA and protein expression levels. Cell proliferation was also assessed. The GLP-1R and GIPR mRNA expression levels were higher in the MC3T3-E1 cells cultured in medium containing high glucose concentrations with BMP-2 compared with the cells cultured in medium containing normal glucose concentrations with or without BMP-2. GLP-1R protein expression increased following culture in high-glucose medium with BMP-2 compared with culture under normal glucose conditions. However, the cellular localization of GLP-1R was not affected by either glucose or BMP-2. In conclusion, our data demonstrate that the expression of GLP-1R and GIPR is regulated by glucose concentrations in MC3T3-E1 cells undergoing differentiation induced by BMP-2. Our results reveal the potential role of incretins in bone metabolism. PMID:24866833

Aoyama, Emina; Watari, Ippei; Podyma-Inoue, Katarzyna Anna; Yanagishita, Masaki; Ono, Takashi

2014-08-01

58

Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity.  

PubMed

Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. PMID:22180388

Choi, Eun Mi

2013-06-01

59

Effect of 635 nm irradiation on high glucose-boosted inflammatory responses in LPS-induced MC3T3-E1 cells.  

PubMed

Hyperglycemia occurs in patients with poorly controlled diabetes mellitus and contributes to bone resorption and increased susceptibility to bacterial infections. Hyperglycemia can incite low-grade inflammation that can contribute to the resorption of bone, especially the periodontal bone. The increased susceptibility to periodontal infections can contribute to bone resorption through the activation of osteoclasts. In this study, the osteoblastic, clonal cell line, MC3T3-E1, was used in an in vitro model of hyperglycemia and lipopolysaccharide-induced reactive oxygen species generation to determine the potential anti-inflammatory effect of 635 nm light-emitting diode (LED) irradiation or whether 635 nm LED irradiation can be a potential anti-inflammatory treatment. LED irradiation of MC3T3-E1 cells stimulated with lipopolysaccharide in a high glucose-containing medium decreased the level of cyclooxygenase gene and protein expression and reduced the level of prostaglandin E2 expression by decreasing the amount of reactive oxygen species generation. LED irradiation also inhibited the osteoclastogenesis in MC3T3-E1 cells by regulating the receptor activator of nuclear factor kappa-B ligand and osteoprotegerin. These findings reveal the mechanisms which are important in the pathogenesis of diabetic periodontitis and highlight the beneficial effects of 635 nm LED irradiation in reducing the adverse effects of diabetic periodontitis. PMID:22699799

Kwon, HyukIl; Lim, WonBong; Kim, JiSun; Jeon, SangMi; Kim, SangWoo; Karna, Sandeep; Cha, HyunRok; Kim, OkJoon; Choi, HongRan

2013-05-01

60

Paprika Pigments Attenuate Obesity-Induced Inflammation in 3T3-L1 Adipocytes  

PubMed Central

Obesity is related to various diseases, such as diabetes, hyperlipidemia, and hypertension. Adipocytokine, which is released from adipocyte cells, affects insulin resistance and blood lipid level disorders. Further, adipocytokine is related to chronic inflammation in obesity condition adipocyte cells. Paprika pigments (PPs) contain large amounts of capsanthin and capsorubin. These carotenoids affect the liver and improve lipid disorders of the blood. However, how these carotenoids affect adipocyte cells remains unknown. Present study examined the effects of PP on adipocytokine secretion, which is related to improvement of metabolic syndrome. In addition, suppressive effects of PP on chronic inflammation in adipocyte cells were analyzed using 3T3-L1 adipocyte cells and macrophage cell coculture experiments. PP promoted 3T3-L1 adipocyte cells differentiation upregulated adiponectin mRNA expression and secretion. Further, coculture of adipocyte and macrophage cells treated with PP showed suppressed interleukin-6 (IL-6), tumor necrosis factor-? (TNF-?), monocyte chemotactic protein-1 (MCP-1), and resistin mRNA expression, similarly to treatment with troglitazone, which is a PPAR? ligand medicine. Conclusion. These results suggest that PP ameliorates chronic inflammation in adipocytes caused by obesity. PP adjusts adipocytokine secretion and might, therefore, affect antimetabolic syndrome diseases.

Maeda, Hayato; Saito, Shuuichi; Nakamura, Nozomi; Maoka, Takashi

2013-01-01

61

Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors  

PubMed Central

The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38? cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 ± 5.2 and 50.5 ± 8.0 pmol/mg protein). P2Y receptor stimulation of CD38? cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave.

Bruzzone, Santina; Kunerth, Svenja; Zocchi, Elena; De Flora, Antonio; Guse, Andreas H.

2003-01-01

62

Six new chalcones from Angelica keiskei inducing adiponectin production in 3T3-L1 adipocytes.  

PubMed

Angelica keiskei (Ashitaba in Japanese), a traditional herb in Japan, contains abundant prenylated chalcones. It has been reported that the chalcones from A. keiskei showed such bioactivities as anti-bacterial, anti-cancer and anti-diabetic effects. Xanthoangelol, 4-hydroxyderricin and six new chalcones were isolated in this study from an ethanol extract of A. keiskei by octadecyl silyl (ODS) and silica gel chromatography, and identified by 1D- and 2D-nuclear magnetic resonance (NMR) and high-resolution mass spectrometric analyses. The chalcones from A. keiskei markedly increased the expression of the adiponectin gene and the production of adiponectin in 3T3-L1 adipocytes. These results suggest that the chalcones from A. keiskei might be useful for preventing the metabolic syndrome. PMID:22738967

Ohnogi, Hiromu; Kudo, Yoko; Tahara, Kenichi; Sugiyama, Katsumi; Enoki, Tatsuji; Hayami, Shoko; Sagawa, Hiroaki; Tanimura, Yuko; Aoi, Wataru; Naito, Yuji; Kato, Ikunoshin; Yoshikawa, Toshikazu

2012-01-01

63

Tyrosine phosphorylation and morphological transformation induced by four vanadium compounds on MC3T3E1 cells.  

PubMed

The present study was performed to determine the phosphotyrosine-protein levels induced by insulin and by four vanadium derivatives in MC3T3E1 osteoblast-like cells. We have also attempted to associate these patterns with the vanadium-induced growth and morphological changes of such cells. Vanadate (Vi), vanadyl (VO), bis(maltolato)oxovanadium (IV) (BMOV) and bis(maltolato)dioxovanadium (V) (BMV) stimulate cell growth in a narrow range of concentration, but are also inhibitors for the cells at high concentrations. Vanadium-treated cells displayed clear changes in their morphology after overnight incubation. However, BMV was the least cytotoxic and the weakest inducer of morphological changes. All the compounds promote the phosphorylation of tyrosine residues in several proteins. This effect was more pronounced at low than at high doses. At low doses (10 microM), BMV showed a phosphorylation pattern similar to that of insulin, while Vi, VO and BMOV induced strong phosphorylation of cell proteins. The present findings suggest that the vanadium-induced growth regulation and morphological changes in MC3T3E1 osteoblast-like cells are associated with the ability of these agents to increase the phosphotyrosine protein levels and to inhibit phosphotyrosine phosphatases. These properties are dependent on the oxidation state as well as on the organic ligand which coordinates the vanadium atom. PMID:10497886

Sálice, V C; Cortizo, A M; Gómez Dumm, C L; Etcheverry, S B

1999-08-01

64

AICAR inhibits adipocyte differentiation in 3T3L1 and restores metabolic alterations in diet-induced obesity mice model  

PubMed Central

Background Obesity is one of the principal causative factors involved in the development of metabolic syndrome. AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. The role of AMP-activated protein kinase in adipocyte differentiation is not completely understood, therefore, we examined the effect of 5-aminoimidazole-4-carboxamide-1-?-D-ribofuranoside (AICAR), a pharmacological activator of AMP-activated protein kinase (AMPK) on adipocyte differentiation in 3T3L1 cells and in a mouse Diet induced obesity (DIO) model. Methods To examine the effect of AICAR on adipocyte differentiation in 3T3L1 cells and in a mouse Diet induced obesity (DIO) model, 3T3L1 cells were differentiatied in the presence or absence of different concentration of AICAR and neutral lipid content and expression of various adipocyte-specific transcription factors were examined. In vivo study, treated and untreated mice with AICAR (0.1–0.5 mg/g body weight) were fed high-fat diet (60% kcal% fat) to induce DIO and several parameters were studied. Results AICAR blocked adipogenic conversion in 3T3L1 cells along with significant decrease in the neutral lipid content by downregulating several adipocyte-specific transcription factors including peroxisome proliferators-activated receptor ? (PPAR?), C/EBP? and ADD1/SREBP1, which are critical for adipogenesis in vitro. Moreover, intraperitoneal administration of AICAR (0.5 mg g/body weight) to mice fed with high-fat diet (60% kcal% fat) to induce DIO, significantly blocked the body weight gain and total content of epididymal fat in these mice over a period of 6 weeks. AICAR treatment also restored normal adipokine levels and resulted in significant improvement in glucose tolerance and insulin sensitivity. The reduction in adipose tissue content in AICAR treated DIO mice was due to reduction in lipid accumulation in the pre-existing adipocytes. However, no change was observed in the expression of PPAR?, C/EBP? and ADD1/SREBP1 transcription factors in vivo though PGC1? expression was significantly induced. Conclusion This study suggests that AICAR inhibits adipocyte differentiation via downregulation of expression of adipogenic factors in vitro and reduces adipose tissue content in DIO mice by activating expression of PGC1? without inhibiting adipocyte-specific transcription factors in DIO mice.

Giri, Shailendra; Rattan, Ramandeep; Haq, Ehtishamul; Khan, Mushfiquddin; Yasmin, Rifat; Won, Je-song; Key, Lyndon; Singh, Avtar K; Singh, Inderjit

2006-01-01

65

Functional proteomic analysis of long-term growth factor stimulation and receptor tyrosine kinase coactivation in Swiss 3T3 fibroblasts.  

PubMed

In Swiss 3T3 fibroblasts, long-term stimulation with PDGF, but not insulin-like growth factor 1 (IGF-1) or EGF, results in the establishment of an elongated migratory phenotype, characterized by the formation of retractile dendritic protrusions and absence of actin stress fibers and focal adhesion complexes. To identify receptor tyrosine kinase-specific reorganization of the Swiss 3T3 proteome during phenotypic differentiation, we compared changes in the pattern of protein synthesis and phosphorylation during long-term exposure to PDGF, IGF-1, EGF, and their combinations using 2DE-based proteomics after (35)S- and (33)P-metabolic labeling. One hundred and five differentially regulated proteins were identified by mass spectrometry and some of these extensively validated. PDGF stimulation produced the highest overall rate of protein synthesis at any given time and induced the most sustained phospho-signaling. Simultaneous activation with two or three of the growth factors revealed both synergistic and antagonistic effects on protein synthesis and expression levels with PDGF showing dominance over both IGF-1 and EGF in generating distinct proteome compositions. Using signaling pathway inhibitors, PI3K was identified as an early site for signal diversification, with sustained activity of the PI3K/AKT pathway critical for regulating late protein synthesis and phosphorylation of target proteins and required for maintaining the PDGF-dependent motile phenotype. Several proteins were identified with novel PI3K/Akt-dependent synthesis and phosphorylations including eEF2, PRS7, RACK-1, acidic calponin, NAP1L1, Hsp73, and fascin. The data also reveal induction/suppression of key F-actin and actomyosin regulators and chaperonins that enable PDGFR to direct the assembly of a motile cytoskeleton, despite simultaneous antagonistic signaling activities. Together, the study demonstrates that long-term exposure to different growth factors results in receptor tyrosine kinase-specific regulation of relatively small subproteomes, and implies that the strength and longevity of receptor tyrosine kinase-specific signals are critical in defining the composition and functional activity of the resulting proteome. PMID:22956732

Nagano, Kohji; Akpan, Akunna; Warnasuriya, Gayathri; Corless, Steven; Totty, Nick; Yang, Alice; Stein, Robert; Zvelebil, Marketa; Stensballe, Allan; Burlingame, Al; Waterfield, Michael; Cramer, Rainer; Timms, John F; Naaby-Hansen, Søren

2012-12-01

66

Functional Proteomic Analysis of Long-term Growth Factor Stimulation and Receptor Tyrosine Kinase Coactivation in Swiss 3T3 Fibroblasts*  

PubMed Central

In Swiss 3T3 fibroblasts, long-term stimulation with PDGF, but not insulin-like growth factor 1 (IGF-1) or EGF, results in the establishment of an elongated migratory phenotype, characterized by the formation of retractile dendritic protrusions and absence of actin stress fibers and focal adhesion complexes. To identify receptor tyrosine kinase-specific reorganization of the Swiss 3T3 proteome during phenotypic differentiation, we compared changes in the pattern of protein synthesis and phosphorylation during long-term exposure to PDGF, IGF-1, EGF, and their combinations using 2DE-based proteomics after 35S- and 33P-metabolic labeling. One hundred and five differentially regulated proteins were identified by mass spectrometry and some of these extensively validated. PDGF stimulation produced the highest overall rate of protein synthesis at any given time and induced the most sustained phospho-signaling. Simultaneous activation with two or three of the growth factors revealed both synergistic and antagonistic effects on protein synthesis and expression levels with PDGF showing dominance over both IGF-1 and EGF in generating distinct proteome compositions. Using signaling pathway inhibitors, PI3K was identified as an early site for signal diversification, with sustained activity of the PI3K/AKT pathway critical for regulating late protein synthesis and phosphorylation of target proteins and required for maintaining the PDGF-dependent motile phenotype. Several proteins were identified with novel PI3K/Akt-dependent synthesis and phosphorylations including eEF2, PRS7, RACK-1, acidic calponin, NAP1L1, Hsp73, and fascin. The data also reveal induction/suppression of key F-actin and actomyosin regulators and chaperonins that enable PDGFR to direct the assembly of a motile cytoskeleton, despite simultaneous antagonistic signaling activities. Together, the study demonstrates that long-term exposure to different growth factors results in receptor tyrosine kinase-specific regulation of relatively small subproteomes, and implies that the strength and longevity of receptor tyrosine kinase-specific signals are critical in defining the composition and functional activity of the resulting proteome.

Nagano, Kohji; Akpan, Akunna; Warnasuriya, Gayathri; Corless, Steven; Totty, Nick; Yang, Alice; Stein, Robert; Zvelebil, Marketa; Stensballe, Allan; Burlingame, Al; Waterfield, Michael; Cramer, Rainer; Timms, John F.; Naaby-Hansen, S?ren

2012-01-01

67

Epidermal Growth Factor Receptor (EGFR) Test Utilization in the United States: A Case Study of T3 Translational Research  

PubMed Central

Purpose We examined hospital use of the epidermal growth factor receptor (EGFR) assay for lung cancer patients. Our goal was to inform the development of a model to predict T3 translation of guideline-directed, molecular diagnostic tests. Methods This was a retrospective observational study. Using logistic regression, we analyzed the association between likelihood to order the EGFR assay and hospital’s institutional and regional characteristics. Results Significant institutional predictors included: Affiliation with an academic medical center (odds ratio [OR], 1.48; 95% CI, 1.20–1.83), Participation in an NCI clinical research cooperative group (OR, 2.06, 1.66–2.55), PET scan (OR, 1.44, 1.07–1.94) and cardio thoracic surgery (OR, 1.90, 1.52–2.37) services. Significant regional predictors included: Metropolitan county (OR, 2.08, 1.48 to 2.91), Above average education (OR, 1.46, 1.09 to 1.96), Above average income (OR, 1.46, 1.04–2.05). Distance from an NCI cancer center was a negative predictor (OR, 0.996, 0.995–0.998), a 34% decrease in likelihood for every 100 miles. Conclusion In 2010, 12% of US acute care hospitals ordered the EGFR assay, suggesting most lung cancer patients did not have access to this test. This case study illustrated the need for: 1) Increased dissemination and implementation research. 2) Interventions to improve adoption of guideline-directed, molecular diagnostic tests by community hospitals.

Lynch, Julie A.; Khoury, Muin J.; Borzecki, Ann; Cromwell, Jerry; Hayman, Laura L.; Ponte, Pat Reid; Miller, Glenn A.; Lathan, Christopher S.

2013-01-01

68

?-Mangostin induces apoptosis and suppresses differentiation of 3T3-L1 cells via inhibiting fatty acid synthase.  

PubMed

?-Mangostin, isolated from the hulls of Garcinia mangostana L., was found to have in vitro cytotoxicity against 3T3-L1 cells as well as inhibiting fatty acid synthase (FAS, EC 2.3.1.85). Our studies showed that the cytotoxicity of ?-mangostin with IC(50) value of 20 µM was incomplicated in apoptotic events including increase of cell membrane permeability, nuclear chromatin condensation and mitochondrial membrane potential (??m) loss. This cytotoxicity was accompanied by the reduction of FAS activity in cells and could be rescued by 50 µM or 100 µM exogenous palmitic acids, which suggested that the apoptosis of 3T3-L1 preadipocytes induced by ?-mangostin was via inhibition of FAS. Futhermore, ?-mangostin could suppress intracellular lipid accumulation in the differentiating adipocytes and stimulated lipolysis in mature adipocytes, which was also related to its inhibition of FAS. In addition, 3T3-L1 preadipocytes were more susceptible to the cytotoxic effect of ?-mangostin than mature adipocytes. Further studies showed that ?-mangostin inhibited FAS probably by stronger action on the ketoacyl synthase domain and weaker action on the acetyl/malonyl transferase domain. These findings suggested that ?-mangostin might be useful for preventing or treating obesity. PMID:22428036

Quan, Xiaofang; Wang, Yi; Ma, Xiaofeng; Liang, Yan; Tian, Weixi; Ma, Qingyun; Jiang, Hezhong; Zhao, Youxing

2012-01-01

69

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.  

PubMed Central

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells. Images

Pietrzkowski, Z; Sell, C; Lammers, R; Ullrich, A; Baserga, R

1992-01-01

70

Autophagy may protect MC3T3-E1 cells from fluoride-induced apoptosis.  

PubMed

Fluoride is an essential trace element for all mammalian species; however, excess fluoride intake is known to be toxic to cells in animals and humans. The toxicity of fluoride is mainly exerted via induction of apoptosis. Autophagy is induced by numerous cytotoxic stimuli; however, it is often unclear whether, under specific conditions, autophagy has a pro?survival or a pro?apoptotic role. To answer this critical question, the present study assessed autophagy and apoptosis simultaneously in single cells. It was demonstrated that fluoride was able to inhibit cell proliferation and induce apoptosis and autophagy, whereas autophagy appeared to be protective. Further analysis revealed that MAPK/JNK?dependent autophagy may be protective in fluoride?induced apoptosis. It is anticipated that the presented single?cell approach may be a powerful tool for gaining a quantitative understanding of the complex regulation of autophagy, its effect on cell fate and its association with other cellular pathways. PMID:24682525

Wei, Min; Duan, Dongmei; Liu, Yujie; Wang, Zhigang; Li, Zhongli

2014-06-01

71

GH induced lipolysis stimulation in 3T3-L1 adipocytes stably expressing hGHR: analysis on signaling pathway and activity of 20K hGH  

Microsoft Academic Search

We have constructed a cell line of 3T3-L1 which can efficiently express human GHR (3T3-L1-hGHR) after differentiation to adipocytes. The expressed hGHR was detected as two bands with approximate molecular sizes of 120K by Western analysis using hGHR specific monoclonal antibody. Maximum lipolytic activity induced by hGH in the 3T3-L1-hGHR was enhanced 10-fold as compared to that in 3T3-L1, suggesting

N Asada; Y Takahashi; M Wada; N Naito; H Uchida; M Ikeda; M Honjo

2000-01-01

72

RELAXIN enhances differentiation and matrix mineralization through Relaxin/insulin-like family peptide receptor 2 (Rxfp2) in MC3T3-E1 cells in vitro.  

PubMed

RELAXIN (RLN) is a polypeptide hormone of the insulin-like hormone family; it facilitates birth by softening and widening the pubic symphysis and cervix in many mammals, including humans. The role of RLN in bone metabolism was recently suggested by its ability to induce osteoclastogenesis and activate osteoclast function. RLN binds to RELAXIN/INSULIN-LIKE FAMILY PEPTIDE 1 (RXFP1) and 2 (RXFP2), with varying species-specific affinities. Young men with mutated RXFP2 are at high risk for osteoporosis, as RXFP2 influences osteoblast metabolism by binding to INSULIN-LIKE PEPTIDE 3 (INSL3). However, there have been no reports on RLN function in osteoblast differentiation and mineralization or on the functionally dominant receptors for RLN in osteoblasts. We previously described Rxfp1 and 2 expression patterns in developing mouse oral components, including the maxillary and mandibular bones, Meckel's cartilage, tongue, and tooth primordia. We hypothesized that Rln/Rxfp signaling is a key mediator of skeletal development and metabolism. Here, we present the gene expression patterns of Rxfp1 and 2 in developing mouse calvarial frontal bones as determined by in situ hybridization. In addition, RLN enhanced osteoblastic differentiation and caused abnormal mineralization and extracellular matrix metabolism through Rxfp2, which was predominant over Rxfp1 in MC3T3-E1 mouse calvarial osteoblasts. Our data suggest a novel role for Rln in craniofacial skeletal development and metabolism through Rxfp2. PMID:24857857

Duarte, Carolina; Kobayashi, Yukiho; Kawamoto, Tatsuo; Moriyama, Keiji

2014-08-01

73

?-Tocotrienol induced cell cycle arrest and apoptosis via activating the Bax-mediated mitochondrial and AMPK signaling pathways in 3T3-L1 adipocytes.  

PubMed

This study aimed to examine the anti-proliferative effects of ?-, ?- and ?-tocotrienols (?T3, ?T3 and ?T3), and ?-tocopherol on 3T3-L1 adipocytes. Results showed that compared with other vitamin E analogues, ?T3 demonstrated the most potent anti-proliferative effect on 3T3-L1 cells. It significantly caused a reduction in mitochondrial membrane potential (??m) and an increase in ROS formation, as well as inducing cell apoptosis and cell cycle arrest at S phase. Further studies showed that it down-regulated Bcl-2 and PPAR-? expression, suppressed Akt and ERK activation and phosphorylation, and caused cytochrome c release from mitochondria to cytosol, whereas it up-regulated CD95 (APO-1/CD95) and Bax expression, and caused caspase-3 and JNK activation, PARP cleavage and AMPK phosphorylation. Pretreatments with caspase-3 (z-DEVD-fmk) and AMPK (CC) inhibitors significantly suppressed the ?T3-induced ROS production and cell death. Caspase-3 inhibitor also efficiently blocked CD95 (APO-1/CD95) and Bax expression, caspase-3 activation and PARP cleavage, whereas antioxidant N-acetyl-l-cysteine, AMPK inhibitor and AMPK siRNA effectively blocked the AMPK phosphorylation. Taken together, these results conclude that the potent anti-proliferative and anti-adipogenic effects of ?T3 on 3T3-L1 adipocytes could be through the Bax-mediated mitochondrial and AMPK signaling pathways. PMID:23816832

Wu, Shu-Jing; Huang, Guang-Yu; Ng, Lean-Teik

2013-09-01

74

Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells  

PubMed Central

Background Insulin receptor substrate (IRS)-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS) are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels. Methods and results In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO) provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3), aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress-induced autophagy and cell death. Conclusion Our results suggest that overexpression of IRS-1 promotes cells growth, inhibits basal autophagy, reduces oxidative stress-induced autophagy, and diminishes oxidative stress-mediated autophagy-dependent cell death. ROS-mediated autophagy may occur via inhibition of IRS-1/phosphatidylinositol 3-kinase/mTOR signaling. Our data afford a plausible explanation for IRS-1 involvement in tumor initiation and progression.

2012-01-01

75

The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts  

PubMed Central

Wnt proteins play important roles in regulating cell differentiation, proliferation and polarity. Wnts have been proposed to play roles in tissue repair and fibrosis, yet the gene expression profile of fibroblasts exposed to Wnts has not been examined. We use Affymetrix genome-wide expression profiling to show that a 6-h treatment of fibroblasts of Wnt3a results in the induction of mRNAs encoding known Wnt targets such as the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2). Wnt3a also induces mRNAs encoding potent pro-fibrotic proteins such as TGF? and endothelin-1 (ET-1). Moreover, Wnt3a promotes genes associated with cell adhesion and migration, vasculature development, cell proliferation and Wnt signaling. Conversely, Wnt3a suppresses gene associated with skeletal development, matrix degradation and cell death. Results were confirmed using real-time polymerase chain reaction of cells exposed to Wnt3a and Wnt10b. These results suggest that Wnts induce genes promoting fibroblast differentiation towards angiogenesis and matrix remodeling, at the expense of skeletal development.

Chen, Shaoqiong; McLean, Sarah; Carter, David E.

2008-01-01

76

Tissue inhibitor of metalloproteinase 1 expression and secretion are induced by beta-adrenergic stimulation in 3T3-L1 adipocytes.  

PubMed

Tissue inhibitor of metalloproteinase (TIMP)-1 is an adipocytokine upregulated in obesity which might promote adipose tissue development. In the current study, the impact of the beta-adrenergic agonist isoproterenol on TIMP-1 gene expression and secretion was determined in 3T3-L1 adipocytes. Interestingly, isoproterenol increased TIMP-1 secretion 2.7-fold. Furthermore, isoproterenol induced TIMP-1 mRNA in a time- and dose-dependent fashion with significant effects observed as early as 1 h after effector addition and at concentrations as low as 1 microM isoproterenol. Significant isoproterenol-induced upregulation of TIMP-1 mRNA could also be found in immortalized brown adipocytes. Inhibitor experiments confirmed that the positive effect of isoproterenol on TIMP-1 is mediated via beta-adrenergic receptors and protein kinase A. Moreover, increasing cAMP levels with forskolin or dibutyryl-cAMP was sufficient to stimulate TIMP-1 synthesis. Insulin induced basal TIMP-1 mRNA, but did not significantly influence forskolin-induced TIMP-1 expression. Taken together, we demonstrate that TIMP-1 expression and secretion are selectively upregulated in adipocytes by beta-adrenergic agonists via a classic Gs-protein-coupled pathway. PMID:16731796

Kralisch, S; Lossner, U; Bluher, M; Paschke, R; Stumvoll, M; Fasshauer, M

2006-06-01

77

Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes  

SciTech Connect

This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1 adipogenesis, was reduced in the presence of MacCM (89%; n = 3; p < 0.001), and BrdU incorporation was impaired by 55% (n = 3; p < 0.01). Activation of ERK1/2 was not affected by MacCM, and neither was the expression of p27{sup kip1}, a cyclin-dependent kinase inhibitor. However, phosphorylation of the retinoblastoma protein (Rb), required for cell cycle progression, was impaired by MacCM (94% inhibition; n = 3; p < 0.01). Differentiation-dependent expression, nuclear localization, and DNA binding ability of C/EBP{beta} were not inhibited by MacCM. Alterations in cell cycle-associated proteins may be important with respect to the anti-adipogenic action of MacCM.

Yarmo, Michelle N.; Landry, Anne; Molgat, Andre S.D.; Gagnon, AnneMarie [Department of Medicine, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Sorisky, Alexander [Department of Medicine, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada)], E-mail: asorisky@ohri.ca

2009-02-01

78

Regulation of Apelin and Its Receptor Expression in Adipose Tissues of Obesity Rats with Hypertension and Cultured 3T3-L1 Adipocytes.  

PubMed

The apelin/APJ system has been implicated in obesity-related hypertension. We investigated the mechanism responsible for the pathogenesis of obesity-related hypertension with a special focus on the crosstalk between AngII/its type 1 receptor (AT1R) signaling and apelin/APJ expression. Sprague-Dawley rats fed a high-fat (obesity-related hypertension, OH) or normal-fat diet (NF) for 15 weeks were randomly assigned to one of two groups and administered vehicle or perindopril for 4 weeks. Compared to the NF rats, the OH rats showed lower levels of plasma apelin and apelin/APJ mRNAs of perirenal adipose tissues, and these changes were restored by perindopril. Administration of the AT1R antagonist olmesartan resulted in the restoration of the reduction of apelin and APJ expressions induced by AngII for 48 h in 3T3-L1 adipocytes. Among several inhibitors for extracellular signal-regulated kinases 1/2 (ERK1/2) PD98059, p38 mitogen-activated protein kinase (p38MAPK) SB203580 and phosphatidylinositol 3-kinase (PI3K) LY294002, the latter showed an additive effect on AngII-mediated inhibitory effects. In addition, the levels of p-Akt, p-ERK and p38MAPK proteins were decreased by long-term treatment with AngII (120 min), and these changes were restored by Olmesartan. Apelin/APJ appears to be impaired in obesity-related hypertension. The AngII inhibition-mediated beneficial effects are likely attributable, at least in part, to restoration of p38/ERK-dependent apelin/APJ expression in diet-induced obesity-related hypertension. PMID:24770651

Wu, Hongxian; Cheng, Xian Wu; Hao, Changning; Zhang, Zhi; Yao, Huali; Murohara, Toyoaki; Dai, Qiuyan

2014-01-01

79

Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions  

NASA Technical Reports Server (NTRS)

Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

Pavalko, F. M.; Chen, N. X.; Turner, C. H.; Burr, D. B.; Atkinson, S.; Hsieh, Y. F.; Qiu, J.; Duncan, R. L.

1998-01-01

80

PPAR? agonist fenofibrate attenuates TNF-?-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway.  

PubMed

The ligand-activated transcription factor peroxisome proliferator-activated receptor-? (PPAR?) participates in the regulation of cellular inflammation. More recent studies indicated that sirtuin1 (SIRT1), a NAD(+)-dependent deacetylase, regulates the inflammatory response in adipocytes. However, whether the role of PPAR? in inflammation is mediated by SIRT1 remains unclear. In this study, we aimed to determine the effect of PPAR? agonist fenofibrate on the expressions of SIRT1 and pro-inflammatory cytokine CD40 and underlying mechanisms in 3T3-L1 adipocytes. We found that fenofibrate inhibited CD40 expression and up-regulated SIRT1 expression in tumor necrosis factor-? (TNF-?)-stimulated adipocytes, and these effects of fenofibrate were reversed by PPAR? antagonist GW6471. Moreover, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or knockdown of SIRT1 could attenuate the effect of fenofibrate on TNF-?-induced CD40 expression in adipocytes. Importantly, NF-?B inhibitor pyrrolidine dithiocarbamate (PDTC) augmented the effect of fenofibrate on CD40 expression in adipocytes. Further study found that fenofibrate decreased the expression of acetylated-NF-?B p65 (Ac-NF-?B p65) in TNF-?-stimulated adipocytes, and the effect of fenofibrate was abolished by SIRT1 inhibition. In addition, fenofibrate up-regulated SIRT1 expression through AMPK in TNF-?-stimulated adipocytes. Taken together, these findings indicate that PPAR? agonist fenofibrate inhibits TNF-?-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway. PMID:23603572

Wang, Weirong; Lin, Qinqin; Lin, Rong; Zhang, Jiye; Ren, Feng; Zhang, Jianfeng; Ji, Meixi; Li, Yanxiang

2013-06-10

81

Gene expression changes in BALB/3T3 transformants induced by poly(L-lactic acid) or polyurethane films.  

PubMed

We performed DNA microarray analysis on two BALB/3T3 transformants (A5 and A6) induced by polyurethane (PU) film, two (L11 and L21) induced by biodegradable poly(L-lactic acid) (PLLA) film, and the parental cells. The transforming ability of the cells was in the order A5 < A6 < L21 < L11. In all, 1176 cancer-related genes were up- or down-regulated in at least one transformant. Those that were markedly up-regulated were c-fos protooncogene, FBJ osteosarcoma oncogene B, and Jun oncogene; those markedly down-regulated were pleiotrophin, histidine triad nucleotide-binding protein, protein kinase C iota, and large multifunctional protease 7. A common function of proteins encoded by genes that underwent marked expression changes was bone formation. The genes were c-fos, FBJ osteosarcoma, Jun, pleiotrophin, a disintegrin-like and metalloprotease with TS-1 motif protein 1. This finding was consistent with the tumor formation in the 2-year PLLA or PU subcutaneous implantation into rats. The number of genes that underwent marked expression change in each transformant was consistent with its malignancy. PLLA induced more malignant transformants than PU, especially in relation to osteosarcoma-like gene expression. PMID:14704980

Matsuoka, Atsuko; Tsuchiya, Toshie

2004-02-01

82

Identification of a receptor for peptides of the bombesin family in Swiss 3T3 cells by affinity cross-linking.  

PubMed

The cross-linking agent ethylene glycol-bis(succinimidyl succinate) was used to covalently link 125I-labeled gastrin releasing peptide (125I-GRP) to an Mr 75,000-85,000 surface protein in Swiss 3T3 cells that displays many characteristics of a specific receptor for peptides of the bombesin family. This protein was not present in other cell lines which do not exhibit receptors for bombesin-like peptides. Unlabeled GRP competed for affinity labeling of the Mr 75,000-85,000 protein in a concentration-dependent manner, and other bombesin-related peptides also inhibited the cross-linking of 125I-GRP to this component. In contrast, high concentrations of a variety of other peptide hormones and mitogens had no effect. Affinity labeling of the Mr 75,000-85,000 protein was dependent on the concentration of 125I-GRP and exhibited saturability. 125I-GRP affinity labeling of this protein was also demonstrated by two-dimensional gel electrophoresis. These studies suggest that an Mr 75,000-85,000 surface protein with an isoelectric point of 6.0 to 6.5 is a major component of the receptor for peptides of the bombesin family in Swiss 3T3 cells. PMID:3031056

Zachary, I; Rozengurt, E

1987-03-25

83

Effect of Turmeric and its Active Principle Curcumin on T3-Induced Oxidative Stress and Hyperplasia in Rat Kidney: A Comparison  

PubMed Central

The present study was designed to compare the potential of turmeric and its active principle curcumin on T3-induced oxidative stress and hyperplasia. Adult male Wistar strain rats were rendered hyperthyroid by T3 treatment (10 ?g · 100 g?1 · day?1 intraperitoneal for 15 days in 0.1 mM NaOH) to induce renal hyperplasia. Another two groups were treated similarly with T3 along with either turmeric or curcumin (30 mg kg?1 body weight day?1 orally for 15 days). The results indicate that T3 induces both hypertrophy and hyperplasia in rat kidney as evidenced by increase in cell number per unit area, increased protein content, tubular dilation and interstitial edema. These changes were accompanied by increased mitochondrial lipid peroxidation and superoxide dismutase activity without any change in catalase activity and glutathione content suggesting an oxidative predominance. Both turmeric and curcumin were able to restore the level of mitochondrial lipid peroxidation and superoxide dismutase activity in the present dose schedule. T3-induced histo-pathological changes were restored with turmeric treatment whereas curcumin administration caused hypoplasia. This may be due to lower concentration of curcumin in the whole turmeric. Thus it is hypothesized that regulation of cell cycle in rat kidney by T3 is via reactive oxygen species and curcumin reveres the changes by scavenging them. Although the response trends are comparable for both turmeric and curcumin, the magnitude of alteration is more in the later. Turmeric in the current dose schedule is a safer bet than curcumin in normalizing the T3-induced hyperplasia may be due to the lower concentration of the active principle in the whole spice.

Panigrahi, Jogamaya; Bhanja, Shravani; Chainy, Gagan B. N.

2010-01-01

84

The Effects of Propionate and Valerate on Insulin Responsiveness for Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myotubes via G Protein-Coupled Receptor 41  

PubMed Central

Since insulin resistance can lead to hyperglycemia, improving glucose uptake into target tissues is critical for regulating blood glucose levels. Among the free fatty acid receptor (FFAR) family of G protein-coupled receptors, GPR41 is known to be the G?i/o-coupled receptor for short-chain fatty acids (SCFAs) such as propionic acid (C3) and valeric acid (C5). This study aimed to investigate the role of GPR41 in modulating basal and insulin-stimulated glucose uptake in insulin-sensitive cells including adipocytes and skeletal muscle cells. Expression of GPR41 mRNA and protein was increased with maximal expression at differentiation day 8 for 3T3-L1 adipocytes and day 6 for C2C12 myotubes. GPR41 protein was also expressed in adipose tissues and skeletal muscle. After analyzing dose-response relationship, 300 µM propionic acid or 500 µM valeric acid for 30 min incubation was used for the measurement of glucose uptake. Both propionic acid and valeric acid increased insulin-stimulated glucose uptake in 3T3-L1 adipocyte, which did not occur in cells transfected with siRNA for GPR41 (siGPR41). In C2C12 myotubes, these SCFAs increased basal glucose uptake, but did not potentiate insulin-stimulated glucose uptake, and siGPR41 treatment reduced valerate-stimulated basal glucose uptake. Therefore, these findings indicate that GPR41 plays a role in insulin responsiveness enhanced by both propionic and valeric acids on glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes, and in valerate-induced increase in basal glucose uptake in C2C12 myotubes.

Han, Joo-Hui; Kim, In-Su; Jung, Sang-Hyuk; Lee, Sang-Gil; Son, Hwa-Young; Myung, Chang-Seon

2014-01-01

85

Low molecular weight molecules of oyster nacre induce mineralization of the MC3T3-E1 cells.  

PubMed

The nacre layer from the pearl oyster shell is considered as a promising osteoinductive biomaterial. Nacre contains one or more signal molecules capable of stimulating bone formation. The identity and the mode of action of these molecules on the osteoblast differentiation were analyzed. Water-soluble molecules from nacre were fractionated according to dialysis, solvent extraction, and reversed-phase HPLC. The activity of a fraction composed of low molecular weight molecules in the mineralization of the MC3T3-E1 extracellular matrix was investigated. Mineralization of the preosteoblast cells was monitored according to alizarin red staining, Raman spectroscopy, scanning electron microscopy, and quantitative RT-PCR. Molecules isolated from nacre, ranging from 50 to 235 Da, induced a red alizarin staining of the preosteoblasts extracellular matrix after 16 days of culture. Raman spectroscopy demonstrated the presence of hydroxyapatite (HA) in samples treated with these molecules. Scanning electron microscopy pictures showed at the surface of the treated cells the occurrence of clusters of spherical particles resembling to HA. The treatment of cells with nacre molecules accelerated expression of collagen I and increased the mRNA expression of Runx2 and osteopontin. This study indicated that the nacre molecules efficient in bone cell differentiation are certainly different from proteins, and could be useful for in vivo bone repair. PMID:17729263

Rousseau, Marthe; Boulzaguet, Hélène; Biagianti, Julie; Duplat, Denis; Milet, Christian; Lopez, Evelyne; Bédouet, Laurent

2008-05-01

86

Chlamydia Induces Anchorage Independence in 3T3 Cells and Detrimental Cytological Defects in an Infection Model  

PubMed Central

Chlamydia are Gram negative, obligate intracellular bacterial organisms with different species causing a multitude of infections in both humans and animals. Chlamydia trachomatis is the causative agent of the sexually transmitted infection (STI) Chlamydia, the most commonly acquired bacterial STI in the United States. Chlamydial infections have also been epidemiologically linked to cervical cancer in women co-infected with the human papillomavirus (HPV). We have previously shown chlamydial infection results in centrosome amplification and multipolar spindle formation leading to chromosomal instability. Many studies indicate that centrosome abnormalities, spindle defects, and chromosome segregation errors can lead to cell transformation. We hypothesize that the presence of these defects within infected dividing cells identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation. Here we demonstrate that infection with Chlamydia trachomatis is able to transform 3T3 cells in soft agar resulting in anchorage independence and increased colony formation. Additionally, we show for the first time Chlamydia infects actively replicating cells in vivo. Infection of mice with Chlamydia results in significantly increased cell proliferation within the cervix, and in evidence of cervical dysplasia. Confocal examination of these infected tissues also revealed elements of chlamydial induced chromosome instability. These results contribute to a growing body of data implicating a role for Chlamydia in cervical cancer development and suggest a possible molecular mechanism for this effect.

Knowlton, Andrea E.; Fowler, Larry J.; Patel, Rahul K.; Wallet, Shannon M.; Grieshaber, Scott S.

2013-01-01

87

cis9, trans11-Conjugated Linoleic Acid Differentiates Mouse 3T3-L1 Preadipocytes into Mature Small Adipocytes through Induction of Peroxisome Proliferator-activated Receptor ?  

PubMed Central

Dietary conjugated linoleic acid (CLA) has been reported to exhibit a number of therapeutic effects in animal models and patients, such as anti-hypertensive, anti-hyperlipidemic, anti-arteriosclerotic, anti-carcinogenic, and anti-diabetic effects. However, the underlying mechanism is not well-characterized. In the present study, the effects of cis(c)9, trans(t)11-CLA on the differentiation of mouse 3T3-L1 preadipocytes into mature adipocytes were examined. Treatment with c9, t11-CLA in the presence of insulin, dexamethasone, and 3-isobutyl-1-methyl-xanthine (differentiation cocktail) significantly stimulated the accumulation of triacylglycerol. The microscopic observation of cells stained by Oil Red O demonstrated that c9, t11-CLA increases the amount and proportion of small mature adipocytes secreting adiponectin, a benign adipocytokine, when compared to the differentiation cocktail alone. Furthermore, c9, t11-CLA increased bioactive peroxisome proliferator-activated receptor ? (PPAR?) levels in a nuclear extract of 3T3-L1 cells, suggesting the enhancing effect of this fatty acid on the nuclear transmission of PPAR?, a master regulator of adipocyte differentiation, in 3T3-L1 cells. These results suggest that the therapeutic effects of c9, t11-CLA on lifestyle-related diseases are partially due to the enhanced formation of small adipocytes from preadipocytes via PPAR? stimulation.

Sakuma, Satoru; Nishioka, Yuki; Imanishi, Ryohta; Nishikawa, Kenji; Sakamoto, Hirotada; Fujisawa, Junji; Wada, Koichiro; Kamisaki, Yoshinori; Fujimoto, Yohko

2010-01-01

88

Ox-LDL Induces ER Stress and Promotes the adipokines Secretion in 3T3-L1 Adipocytes  

PubMed Central

Adipocytes behave as a rich source of adipokines, which may be the link between obesity and its complications. Endoplasmic reticulum (ER) stress in adipocytes can modulate adipokines secretion. The aim of this study is to evaluate the effect of oxidized low density lipoprotein?ox-LDL?treatment on ER stress and adipokines secretion in differentiated adipocytes. 3T3-L1 pre-adipocytes were cultured and differentiated into mature adipocytes in vitro. Differentiated adipocytes were incubated with various concentrations of ox-LDL (0-100 µg/ml) for 48 hours; 50µg/ml ox-LDL for various times (0-48 hours) with or without tauroursodeoxycholic acid (TUDCA) (0-400µM) pre-treatment. The protein expressions of ER stress markers, glucose regulated protein 78(GRP78) and CCAAT/enhancer binding protein [C/EBP] homologous protein (CHOP) in adipocytes were detected by Western blot. The mRNA expressions of visfatin and resistin were measured by real-time PCR and the protein release of visfatin and resistin in supernatant were determined by ELISA. Treatment with ox-LDL could increase the cholesterol concentration in adipocytes. Ox-LDL induced the expressions of GRP78 and CHOP protein in adipocytes and promoted visfatin and resistin secretion in culture medium in dose and time-dependent manner. TUDCA could attenuate the effect of ox-LDL on GRP78 and CHOP expressions and reduce visfatin and resistin at mRNA and protein level in dose-dependent manner. In conclusion, ox-LDL promoted the expression and secretion of visfatin and resistin through its activation of ER stress, which may be related to the increase of cholesterol load in adipocytes.

Chen, Yaqin; Chen, Mingjie; Wu, Zhihong; Zhao, Shuiping

2013-01-01

89

Ox-LDL induces ER stress and promotes the adipokines secretion in 3T3-L1 adipocytes.  

PubMed

Adipocytes behave as a rich source of adipokines, which may be the link between obesity and its complications. Endoplasmic reticulum (ER) stress in adipocytes can modulate adipokines secretion. The aim of this study is to evaluate the effect of oxidized low density lipoprotein (ox-LDL) treatment on ER stress and adipokines secretion in differentiated adipocytes. 3T3-L1 pre-adipocytes were cultured and differentiated into mature adipocytes in vitro. Differentiated adipocytes were incubated with various concentrations of ox-LDL (0-100 µg/ml) for 48 hours; 50 µg/ml ox-LDL for various times (0-48 hours) with or without tauroursodeoxycholic acid (TUDCA) (0-400 µM) pre-treatment. The protein expressions of ER stress markers, glucose regulated protein 78(GRP78) and CCAAT/enhancer binding protein [C/EBP] homologous protein (CHOP) in adipocytes were detected by Western blot. The mRNA expressions of visfatin and resistin were measured by real-time PCR and the protein release of visfatin and resistin in supernatant were determined by ELISA. Treatment with ox-LDL could increase the cholesterol concentration in adipocytes. Ox-LDL induced the expressions of GRP78 and CHOP protein in adipocytes and promoted visfatin and resistin secretion in culture medium in dose and time-dependent manner. TUDCA could attenuate the effect of ox-LDL on GRP78 and CHOP expressions and reduce visfatin and resistin at mRNA and protein level in dose-dependent manner. In conclusion, ox-LDL promoted the expression and secretion of visfatin and resistin through its activation of ER stress, which may be related to the increase of cholesterol load in adipocytes. PMID:24278099

Chen, Yaqin; Chen, Mingjie; Wu, Zhihong; Zhao, Shuiping

2013-01-01

90

Differential expression of mutant and normal beta T3 receptor alleles in kindreds with generalized resistance to thyroid hormone.  

PubMed Central

Thyroid hormone resistance (THR) is primarily an autosomal dominant inherited disease characterized by resistance of pituitary and peripheral tissues to the action of thyroid hormone. We investigated whether the heterogeneous phenotypic features that occur not only among kindreds but also within the same kindred might be due to the expression of differing ratios of mutant and normal receptors in tissues. Using an allele-specific primer extension method, we determined the relative expression of normal and mutant mRNAs from the fibroblasts of affected and unaffected members of two kindreds with TRH: A-H and N-N. While two affected members of A-H, as expected, had nearly equal amounts of normal and mutant hTR beta mRNA, two other members had mutant mRNA levels that accounted for at least 70% of the hTR beta mRNA. Phenotypic variability within and between kindreds with generalized resistance to thyroid hormone GRTH may be due to this differential expression of the mutant and wild type mRNA. Furthermore, when several clinical parameters of THR were compared in several affected members from two kindreds with GRTH, we found that two cases in one kindred exhibited a high mutant-to-normal hTR beta ratio and had considerably more bone resistance during their development. In certain kindreds with THR, differing ratios of normal and mutant hTR receptors may be age and growth related and may account for the reported attenuation of phenotypic symptoms with age. Images

Mixson, A J; Hauser, P; Tennyson, G; Renault, J C; Bodenner, D L; Weintraub, B D

1993-01-01

91

Regulation of the beta-adrenergic receptor-adenylate cyclase complex of 3T3-L1 fibroblasts by sodium butyrate  

SciTech Connect

Mouse 3T3-L1 fibroblasts contain beta-adrenergic receptors (BAR), predominantly of the B/sub 1/ subtype. Incubation of these cells with 2-10 mM sodium butyrate (SB) for 24-48 hr results in a switch in the BAR subtype from B/sub 1/ to B/sub 2/ and promotes a 1.5 to 2.5 fold increase in total BAR number. Other short chain acids were not as effective as SB in promoting changes in BAR. BAR were assayed in membranes prepared from the 3T3-L1 cells using the radiolabeled antagonist (/sup 125/I)-cyanopindolol and the B/sub 2/ selective antagonist ICI 118.551. BAR subtype switch was confirmed functionally by measuring cellular cAMP accumulation in response to agonists. The structure and amount of the alpha subunits of the guanine nucleotide regulatory proteins N/sub s/ and N/sub i/ were determined by ADP-ribosylation using /sup 32/P-NAD and either cholera toxin or pertussis toxin for labeling of the respective subunits. Preincubation of cells with 5 mM SB for 48 hr resulted in a 2-3 fold increase in the labeling of the alpha subunits of both N/sub s/ and N/sub i/. A protein of M/sub r/ = 44,000 showed enhanced labeling by cholera toxin following SB treatment of the cells. These data indicate SB concomitantly regulates expression of BAR subtype and components of the adenylate cyclase in 3T3-L1 cells.

Stadel, J.M.; Poksay, K.S.; Nakada, M.T.; Crooke, S.T.

1986-05-01

92

Triiodothyronine (T3) induces proinsulin gene expression by activating PI3K: possible roles for GSK-3? and the transcriptional factor PDX-1.  

PubMed

Thyroid hormone (TH) activates PI3K and Akt, leading to glucose uptake in rat skeletal muscle cells and proliferation of insulinoma cells, respectively. However, TH actions on pancreatic beta cells have been little explored, which lead us to evaluate the TH eff ects on proinsulin gene expression, and the involvement of PI3K/Akt/GSK-3? signaling pathway, and a transcriptional factor for insulin (PDX-1). INS-1E cells were sorted into 3 groups: control and TH-depleted treated or not with T3 for 30 min. Cells were also previously treated with actinomycin D (ActD), cycloheximide (CHX), wortmannin or Akt inhibitor. Proinsulin mRNA expression was evaluated by real time PCR, and pGSK-3? and PDX-1 protein content was analyzed by Western blotting. TH depletion decreased proinsulin mRNA content, which was restored after acute T3 treatment. ActD, CHX and wortmannin, but not Akt inhibitor, prevented the rapid stimulatory eff ect of T3 on proinsulin mRNA expression. TH depletion did not affect the phosphorylated GSK-3? and PDX-1 protein content; but T3 treatment led to an increase in the content of these proteins. These data indicate that T3 acutely increases proinsulin mRNA expression, by mechanisms which depends on the activation of PI3K, but not of Akt, and may involve the inactivation of GSK-3? by phosphorylation. Since GSK-3? enhances PDX-1 degradation rate, the GSK-3? inactivation could explain the increase of PDX-1 content in T3-treated cells. Considering that PDX-1 is one of the most important transcriptional factors for proinsulin gene expression, its enhancement may underlie the increased proinsulin mRNA content acutely induced by T3. PMID:23147208

Goulart-Silva, F; Serrano-Nascimento, C; Texeira, S S; Nunes, M T

2013-01-01

93

ER stress-inducible ATF3 suppresses BMP2-induced ALP expression and activation in MC3T3-E1 cells.  

PubMed

Endoplasmic reticulum (ER) stress suppresses osteoblast differentiation. Activating transcription factor (ATF) 3, a member of the ATF/cAMP response element-binding protein family of transcription factors, is induced by various stimuli including cytokines, hormones, DNA damage, and ER stress. However, the role of ATF3 in osteoblast differentiation has not been elucidated. Treatment with tunicamycin (TM), an ER stress inducer, increased ATF3 expression in the preosteoblast cell line, MC3T3-E1. Overexpression of ATF3 inhibited bone morphogenetic protein 2-stimulated expression and activation of alkaline phosphatase (ALP), an osteogenic marker. In addition, suppression of ALP expression by TM treatment was rescued by silencing of ATF3 using shRNA. Taken together, these data indicate that ATF3 is a novel negative regulator of osteoblast differentiation by specifically suppressing ALP gene expression in preosteoblasts. PMID:24315873

Park, Jae-kyung; Jang, Hoon; Hwang, SeongSoo; Kim, Eun-Jung; Kim, Dong-Ern; Oh, Keon-Bong; Kwon, Dae-Jin; Koh, Jeong-Tae; Kimura, Kumi; Inoue, Hiroshi; Jang, Won-Gu; Lee, Jeong-Woong

2014-01-01

94

T3RU test  

MedlinePLUS

Resin T3 uptake; T3 resin uptake; Thyroid hormone-binding ratio ... This test is done to check your thyroid function. Thyroid function is complex and depends on the action of many different hormones, including thyroid-stimulating hormone (TSH), T3, and T4. This ...

95

Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin  

SciTech Connect

Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.

Murayama, T.; Ui, M.

1985-06-25

96

Cyanidin 3-glucoside protects 3T3-L1 adipocytes against H2O2- or TNF-alpha-induced insulin resistance by inhibiting c-Jun NH2-terminal kinase activation.  

PubMed

Anthocyanins are naturally occurring plant pigments and exhibit an array of pharmacological properties. Our previous study showed that black rice pigment extract rich in anthocyanin prevents and ameliorates high-fructose-induced insulin resistance in rats. In present study, cyanidin 3-glucoside (Cy-3-G), a typical anthocyanin most abundant in black rice was used to examine its protective effect on insulin sensitivity in 3T3-L1 adipocytes exposed to H(2)O(2) (generated by adding glucose oxidase to the medium) or tumor necrosis factor alpha (TNF-alpha). Twelve-hour exposure of 3T3-L1 adipocytes to H(2)O(2) or TNF-alpha resulted in the increase of c-Jun NH(2)-terminal kinase (JNK) activation and insulin receptor substrate 1 (IRS1) serine 307 phosphorylation, concomitantly with the decrease in insulin-stimulated IRS1 tyrosine phosphorylation and cellular glucose uptake. Blocking JNK expression using RNA interference efficiently prevented the H(2)O(2)- or TNF-alpha-induced defects in insulin action. Pretreatment of cells with Cy-3-G reduced the intracellular production of reactive oxygen species, the activation of JNK, and attenuated H(2)O(2)- or TNF-alpha-induced insulin resistance in a dose-dependent manner. In parallel, N-acetyl-cysteine, an antioxidant compound, did not exhibit an attenuation of TNF-alpha-induced insulin resistance. Taken together, these results indicated that Cy-3-G exerts a protective role against H(2)O(2)- or TNF-alpha-induced insulin resistance in 3T3-L1 adipocytes by inhibiting the JNK signal pathway. PMID:18179781

Guo, Honghui; Ling, Wenhua; Wang, Qing; Liu, Chi; Hu, Yan; Xia, Min

2008-03-15

97

Ameliorating Effects of Fermented Rice Bran Extract on Oxidative Stress Induced by High Glucose and Hydrogen Peroxide in 3T3-L1 Adipocytes  

Microsoft Academic Search

In this study, we investigated whether fermented rice bran (FRB) can ameliorate the oxidative stress induced by high glucose\\u000a and hydrogen peroxide (H2O2) in 3T3-L1 adipocytes by analyzing reactive oxygen species (ROS), oil red O staining, as well as the expression of mRNAs\\u000a related to glucose homeostasis and adipogenesis. It was first confirmed that rice bran fermented by Issatchenkia orientalis

Dongyeop Kim; Gi Dong Han

98

Nano-hydroxyapatite particles induce apoptosis on MC3T3-E1 cells and tissue cells in SD rats  

NASA Astrophysics Data System (ADS)

While the advantages of nanomaterials are being increasingly recognized, their potential toxicity is drawing more and more attention and concern. In this study, we explore the toxicity mechanism of 20-30 nm rod-shaped hydroxyapatite (HA) nanoparticles in vitro and in vivo. The nanoparticles were prepared by precipitation and characterized by IR, XRD and TEM. Concentrations of 0 ?g mL-1, 10 ?g mL-1, 100 ?g mL-1, 1 mg mL-1, and 10 mg mL-1 were applied to the MC3T3-E1 cells for viability (MTT-test). Based on the characteristic differences of the two methods of cell death, the morphological features of the MC3T3-E1 cell line co-cultured with nano-hydroxyapatite (n-HA) (10 mg mL-1) for 24 h were also observed by TEM. Furthermore, important serum biochemical markers and histopathological examinations were used to evaluate the potential toxicological effect of n-HA on the major organs of SD rats injected intraperitoneally with n-HA (33.3 mg kg-1 body weight). In the results, we found cell growth inhibition and apoptosis in MC3T3-E1 cells co-cultured with n-HA. Moreover, apoptosis but not necrosis was illustrated in liver and renal tissue by using histopathology slices and serum biochemical markers. It suggests that apoptosis may be the possible mechanism of n-HA toxicity and provides a better understanding of the biocompatibility of nanomaterials applied in human bone repair.

Wang, Liting; Zhou, Gang; Liu, Haifeng; Niu, Xufeng; Han, Jingyun; Zheng, Lisha; Fan, Yubo

2012-04-01

99

Myricetin, a naturally occurring flavonoid, prevents 2-deoxy-D-ribose induced dysfunction and oxidative damage in osteoblastic MC3T3-E1 cells.  

PubMed

Myricetin, a naturally occurring flavonoid, was investigated to determine whether it could protect osteoblasts from 2-deoxy-d-ribose induced dysfunction and oxidative damage in the MC3T3-E1 cells. MC3T3-E1 cells were incubated with 2-deoxy-d-ribose and/or myricetin, and markers of osteoblast function and oxidative damage were examined. Compared with control incubation, 2-deoxy-d-ribose significantly (P<0.05) inhibited alkaline phosphatase (ALP) activity, collagen content, and calcium deposition at the concentration of 20 mM. Cellular malondialdehyde (MDA), protein carbonyl, and advanced oxidation protein products contents were significantly (P<0.05) increased in the presence of 2-deoxy-d-ribose (20 mM). Myricetin significantly (P<0.05) increased cell survival, ALP activity, collagen, osteocalcin, osteoprotegerin, and calcium deposition and decreased MDA, protein carbonyl, and advanced oxidation protein products contents of osteoblastic MC3T3-E1 cells in the presence of 20 mM 2-deoxy-d-ribose. These results demonstrate that myricetin attenuates 2-deoxy-d-ribose induced damage, suggesting that myricetin may be a useful dietary supplement for minimizing oxidative injury in diabetes related bone diseases. PMID:18599037

Lee, Kyung-Hee; Choi, Eun-Mi

2008-09-01

100

A possible role of oxidative stress in the vanadium-induced cytotoxicity in the MC3T3E1 osteoblast and UMR106 osteosarcoma cell lines.  

PubMed

The cytotoxicity and free radical production induced by vanadium compounds were investigated in an osteoblast (MC3T3E1) and an osteosarcoma (UMR106) cell lines in culture. Vanadate induced cell toxicity, reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) increased in a concentration-dependent manner (0.1-10 mM) after 4 h. The concentration-response curve of vanadate-induced cytotoxicity and oxidative stress in MC3T3E1 cells was shifted to the left of the UMR106 curve, suggesting a greater sensitivity of the non-transformed cells in comparison to the osteosarcoma UMR106 cells. Supplementing with vitamin E acetate (80 microM) significantly inhibited ROS and TBARS formation but did not improve the vanadate-dependent decrease in cell number. Other vanadium compounds (vanadyl, pervanadate, and VO/Aspi, a complex of vanadyl(IV) with aspirin) showed different degrees of cell toxicity and induced oxidative stress. Altogether these results suggest that oxidative stress is involved in vanadium induced osteoblastic cytotoxicity, although the mechanism is unknown. PMID:10874156

Cortizo, A M; Bruzzone, L; Molinuevo, S; Etcheverry, S B

2000-06-01

101

Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells  

SciTech Connect

Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with (35S)methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of (32P)orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.

Nakai, A.; Hirayama, C.; Ohtsuka, K.; Hirayoshi, K.; Nagata, K. (Kyoto Univ. (Japan))

1990-06-01

102

The ?-SiC Nanowires (~100 nm) Induce Apoptosis via Oxidative Stress in Mouse Osteoblastic Cell Line MC3T3-E1  

PubMed Central

Silicon carbide (SiC), a compound of silicon and carbon, with chemical formula SiC, the beta modification (?-SiC), with a zinc blende crystal structure (similar to diamond), is formed at temperature below 1700°C. ?-SiC will be the most suitable ceramic material for the future hard tissue replacement, such as bone and tooth. The in vitro cytotoxicity of ?-SiC nanowires was investigated for the first time. Our results indicated that 100?nm long SiC nanowires could significantly induce the apoptosis in MC3T3-E1 cells, compared with 100??m long SiC nanowires. And 100?nm long SiC nanowires increased oxidative stress in MC3T3-E1 cells, as determined by the concentrations of MDA (as a marker of lipid peroxidation) and 8-OHdG (indicator of oxidative DNA damage). Moreover, transmission electron microscopy (TEM) was performed to evaluate the morphological changes of MC3T3-E1 cells. After treatment with 100?nm long SiC nanowires, the mitochondria were swelled and disintegrated, and the production of ATP and the total oxygen uptake were also decreased significantly. Therefore, ?-SiC nanowires may have limitations as medical material.

Xie, Weili; Xie, Qi; Jin, Meishan; Huang, Xiaoxiao; Zhang, Xiaodong; Shao, Zhengkai; Wen, Guangwu

2014-01-01

103

The ? -SiC Nanowires (~100 nm) Induce Apoptosis via Oxidative Stress in Mouse Osteoblastic Cell Line MC3T3-E1.  

PubMed

Silicon carbide (SiC), a compound of silicon and carbon, with chemical formula SiC, the beta modification ( ? -SiC), with a zinc blende crystal structure (similar to diamond), is formed at temperature below 1700°C. ? -SiC will be the most suitable ceramic material for the future hard tissue replacement, such as bone and tooth. The in vitro cytotoxicity of ? -SiC nanowires was investigated for the first time. Our results indicated that 100?nm long SiC nanowires could significantly induce the apoptosis in MC3T3-E1 cells, compared with 100? ? m long SiC nanowires. And 100?nm long SiC nanowires increased oxidative stress in MC3T3-E1 cells, as determined by the concentrations of MDA (as a marker of lipid peroxidation) and 8-OHdG (indicator of oxidative DNA damage). Moreover, transmission electron microscopy (TEM) was performed to evaluate the morphological changes of MC3T3-E1 cells. After treatment with 100?nm long SiC nanowires, the mitochondria were swelled and disintegrated, and the production of ATP and the total oxygen uptake were also decreased significantly. Therefore, ? -SiC nanowires may have limitations as medical material. PMID:24967352

Xie, Weili; Xie, Qi; Jin, Meishan; Huang, Xiaoxiao; Zhang, Xiaodong; Shao, Zhengkai; Wen, Guangwu

2014-01-01

104

The signaling potential of the receptors for insulin and insulin-like growth factor I (IGF-I) in 3T3-L1 adipocytes: comparison of glucose transport activity, induction of oncogene c-fos, glucose transporter mRNA, and DNA-synthesis.  

PubMed

The receptors for insulin and insulin-like growth factor I (IGF-I) have in common a high sequence homology and diverse overlapping functions, (e.g., the stimulation of acute metabolic events and the induction of cell growth.). In the present study, we have compared the potential of insulin and IGF-I receptors in stimulating glucose transport activity, glucose transporter gene expression, DNA-synthesis, and expression of proto-oncogene c-fos in 3T3-L1 adipocytes which express high levels of both receptors. Binding of both hormones to their own receptors was highly specific as compared with binding to the respective other receptor (insulin receptor: KD = 3.6 nM, KI of IGF-I greater than 500 nM; IGF-I receptor, KD = 1.1 nM, KI of insulin = 191 nM). Induction of proto-oncogene c-fos mRNA by insulin and IGF-I paralleled their respective receptor occupancy and was thus induced by both hormones via their own receptor (EC50 of insulin, 3.7; IGF-I, 3.9 nM). Similarly, both insulin and IGF-I increased DNA synthesis (EC50 of insulin, 5.8 nM; IGF-I, 4.0 nM), glucose transport activity (EC50 of insulin, 1.7 nM; IGF-I, 1.4 nM), and glucose transporter (GLUT4) mRNA levels in concentrations corresponding with their respective receptor occupancy. These data indicate that in 3T3-L1 cells the alpha-subunits of insulin and IGF-I receptors have an equal potential to stimulate a metabolic and a mitogenic response. PMID:1660482

Weiland, M; Bahr, F; Höhne, M; Schürmann, A; Ziehm, D; Joost, H G

1991-12-01

105

A commercial formulation of glyphosate inhibits proliferation and differentiation to adipocytes and induces apoptosis in 3T3-L1 fibroblasts.  

PubMed

Glyphosate-based herbicides are extensively used for weed control all over the world. Therefore, it is important to investigate the putative toxic effects of these formulations which include not only glyphosate itself but also surfactants that may also be toxic. 3T3-L1 fibroblasts are a useful tool to study adipocyte differentiation, this cell line can be induced to differentiate by addition of a differentiation mixture containing insulin, dexamethasone and 3-isobutyl-1-methylxanthine. We used this cell line to investigate the effect of a commercial formulation of glyphosate (GF) on proliferation, survival and differentiation. It was found that treatment of exponentially growing cells with GF for 48h inhibited proliferation in a dose-dependent manner. In addition, treatment with GF dilution 1:2000 during 24 or 48h inhibited proliferation and increased cell death, as evaluated by trypan blue-exclusion, in a time-dependent manner. We showed that treatment of 3T3-L1 fibroblasts with GF increased caspase-3 like activity and annexin-V positive cells as evaluated by flow cytometric analysis, which are both indicative of induction of apoptosis. It was also found that after the removal of GF, remaining cells were able to restore proliferation. On the other hand, GF treatment severely inhibited the differentiation of 3T3-L1 fibroblasts to adipocytes. According to our results, a glyphosate-based herbicide inhibits proliferation and differentiation in this mammalian cell line and induces apoptosis suggesting GF-mediated cellular damage. Thus, GF is a potential risk factor for human health and the environment. PMID:22546541

Martini, Claudia N; Gabrielli, Matías; Vila, María del C

2012-09-01

106

Biochanin A stimulates osteoblastic differentiation and inhibits hydrogen peroxide-induced production of inflammatory mediators in MC3T3-E1 cells.  

PubMed

Phytoestrogens are plant chemicals that are structurally analogous to estrogen and are known to affect estrogenic activity. Biochanin A, a naturally occurring isoflavone, has been identified and detected in various diets and plant species. We examined the effects of biochanin A on the differentiation of osteoblastic MC3T3-E1 cells and the production of local factors in osteoblasts. Biochanin A (1-50 microM) caused a significant elevation of cell growth, alkaline phosphatase (ALP) activity, collagen content, and osteocalcin secretion in osteoblastic MC3T3-E1 cells (p<0.05). The effect of biochanin A (10 microM) in increasing ALP activity and collagen content was completely prevented by the presence of 10(-6) M cycloheximide and 10(-6) M tamoxifen, suggesting that biochanin A's effect results from a newly synthesized protein component and might be partly involved in estrogen action. We then examined the effect of biochanin A on the H2O2-induced production of inflammatory mediators in osteoblasts. Biochanin A (1-10 microM) decreased the 0.2 mM H2O2-induced production of TNF-alpha, IL-6 and NO in osteoblasts. These results suggest that biochanin A may be useful as potential phytoestrogens, which play important physiological roles in the prevention of postmenopausal osteoporosis. PMID:16204952

Lee, Kyung-Hee; Choi, Eun-Mi

2005-10-01

107

Melatonin rescues 3T3-L1 adipocytes from FFA-induced insulin resistance by inhibiting phosphorylation of IRS-1 on Ser307.  

PubMed

Melatonin is biosynthesized in the pineal gland and secreted into the bloodstream. Evidences indicate a role of melatonin in the regulation of glucose metabolism. The objective of this study was to investigate the effect of melatonin on insulin sensitivity in insulin resistant adipocytes. Following a preincubation with melatonin or vehicle for 30 min, insulin resistant cells of 3T3-L1 adipocytes were induced by palmitic acids (300 ?M, 6 h). Our results showed that palmitic acids inhibited both the basal and insulin-stimulated uptake of [(3)H]-2-Deoxyglucose, down-regulated the levels of IRS-1 and GLUT-4. However, compared to the vehicle group, melatonin pre-treatment increased significantly the uptake of [(3)H]-2-Deoxyglucose as well as the level of GLUT-4, and decreased phosphorylated IRS-1 (Ser307) although total IRS-1 did not change significantly. These data suggest that palmitic acids impair insulin signal via down-regulating the expressions of IRS-1 and GLUT-4; whereas melatonin can ameliorate insulin sensitivity by inhibiting Ser307 phosphorylation in IRS-1 and increasing GLUT-4 expressions in insulin resistant 3T3-L1 adipocytes. We conclude that melatonin regulates the insulin sensitivity and glucose homeostasis via inhibiting Ser-phosphorylation and improving function of IRS-1. PMID:24846082

She, Meihua; Hou, Hongjie; Wang, Zongbao; Zhang, Chi; Laudon, Moshe; Yin, Weidong

2014-08-01

108

Down-regulation of cyclic-nucleotide phosphodiesterase 3B in 3T3-L1 adipocytes induced by tumour necrosis factor alpha and cAMP.  

PubMed Central

We have used murine 3T3-L1 cells, which differentiate in culture and acquire morphological and biochemical features of mature adipocytes, as a model for studying the expression of cyclic-nucleotide phosphodiesterase (PDE) 3B activity, protein and mRNA during differentiation and during long-term treatment of the cells with tumour necrosis factor alpha (TNF-alpha), a cytokine associated with insulin resistance, and a cAMP analogue, N(6),2'-O-dibutyryl cAMP (dbcAMP). PDE3B activity, protein and mRNA could be detected 4 days after the initiation of differentiation of 3T3-L1 preadipocytes. Treatment of 3T3-L1 adipocytes with 10 ng/ml TNF-alpha for 24 h produced a maximal (50%) decrease in PDE3B activity, protein and mRNA, which was well correlated with both activation of protein kinase A (PKA) and stimulation of lipolysis, presumably reflecting an increase in intracellular cAMP concentration. To investigate the effect of cAMP on PDE3B we treated 3T3-L1 adipocytes with dbcAMP. After 4 h with 0.5 mM dbcAMP, PDE3B activity was decreased by 80%, which was also correlated with a decrease in PDE3B protein and mRNA. This effect was abolished in the presence of N-[2-(bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide] (H-89), a specific PKA inhibitor. We conclude that the lipolytic effect of TNF-alpha involves the down-regulation of PDE3B, which is associated with increased activation of PKA, presumably owing to increased levels of cAMP. In addition, the PKA activation induced by dbcAMP resulted in the down-regulation of PDE3B. These results, which suggest that PDE3B is a novel target for long-term regulation by TNF-alpha and cAMP, could contribute to the understanding of the mechanisms of insulin resistance.

Rahn Landstrom, T; Mei, J; Karlsson, M; Manganiello, V; Degerman, E

2000-01-01

109

Induction of AP1 activity associated with c-Jun and JunB is required for mitogenesis induced by insulin and vanadate in SV40-transformed 3T3T cells  

Microsoft Academic Search

Insulin and vanadate function as complete mitogens for SV40-transformed murine 3T3T (CSV3-1) cells but not for nontransformed 3T3T cells. Mitogenesis induced by insulin and vanadate in CSV3-1 cells is associated with the induction of the expression of protooncogenes c-Jun and junB, two major AP-1 transcription factor components. We now report that both insulin and vanadate induce a significant increase in

Hanlin Wang; Zirong Xie; Robert E. Scott

1997-01-01

110

Effects of paeoniflorin on tumor necrosis factor-?-induced insulin resistance and changes of adipokines in 3T3-L1 adipocytes.  

PubMed

TNF? plays an important role in the adipocyte dysfunction, including lipolysis acceleration, insulin resistance and changes of adipokines. Recently, we showed that paeoniflorin attenuates adipocyte lipolysis and inhibits the phosphorylation of ERK, JNK, IKK stimulated by TNF?. However, the effects of paeoniflorin on adipocytes insulin resistance and changes of adipokines remain unknown. The aim of the current study was to investigate the role of paeoniflorin in preventing insulin resistance or inflammation in 3T3-L1 adipocytes treated with TNF?. Our results showed that paeoniflorin restored insulin-stimulated [(3)H]2-DOG uptake, which was reduced by TNF?, with concomitant restoration in serine phosphorylation of IRS-1 and insulin-stimulated phosphorylation of AKT in adipocytes. Paeoniflorin attenuated TNF?-mediated suppression of the expressions of PPAR? and PPAR? target genes, and the improvement of paeoniflorin on TNF?-induced insulin resistance was attenuated by GW9662, an antagonist of PPAR? activity. Moreover, paeoniflorin could inhibit the expressions and secretions of IL-6 and MCP-1 from adipocytes induced by TNF?. These results, together with our previous data, indicate that paeoniflorin exerts a beneficial effect on adipocytes to prevent TNF?-induced insulin resistance and inflammatory adipokine release. Our studies provide important evidence for an ability of paeoniflorin in amelioration of TNF?-induced adipocyte dysfunction, which would be helpful to clarify its potential role in the treatment of obesity. PMID:23978582

Kong, Poren; Chi, Rongxiang; Zhang, Linlin; Wang, Ningjian; Lu, Yingli

2013-12-01

111

The fungal chimerolectin MOA inhibits protein and DNA synthesis in NIH/3T3 cells and may induce BAX-mediated apoptosis.  

PubMed

The Marasmius oreades mushroom agglutinin (MOA) is a blood group B-specific lectin carrying an active proteolytic domain. Its enzymatic activity has recently been shown to be critical for toxicity of MOA toward the fungivorous soil nematode Caenorhabditis elegans. Here we present evidence that MOA also induces cytotoxicity in a cellular model system (murine NIH/3T3 cells), by inhibiting protein synthesis, and that cytotoxicity correlates, at least in part, with proteolytic activity. A peptide-array screen identified the apoptosis mediator BAX as a potential proteolytic substrate and further suggests a variety of bacterial and fungal peptides as potential substrates. These findings are in line with the suggestion that MOA and related proteases may play a role for host defense. PMID:24747075

Cordara, Gabriele; Winter, Harry C; Goldstein, Irwin J; Krengel, Ute; Sandvig, Kirsten

2014-05-16

112

Quantification of cell adhesion force with AFM: distribution of vitronectin receptors on a living MC3T3-E1 cell  

Microsoft Academic Search

Distribution of vitronectin (VN) receptors on a living murine osteoblastic cell was successfully measured by atomic force microscopy (AFM). First, the distribution of the integrin ?5 subunit which constitutes a part of the VN receptor on the cell was confirmed by conventional immunohistochemistry after fixing the cell. To visualize the distribution of the receptor on a living cell by an

H. Kim; Hideo Arakawa; Toshiya Osada; Atsushi Ikai

2003-01-01

113

SPARC is over-expressed in adipose tissues of diet-induced obese rats and causes insulin resistance in 3T3-L1 adipocytes.  

PubMed

Secreted protein acidic and rich in cysteine (SPARC) is a secretory multifunctional matricellular glycoprotein. High circulating levels of SPARC have been reported to be associated with obesity and insulin resistance. The aim of the present study was to investigate whether SPARC induces insulin resistance and mitochondrial dysfunction in adipocytes. Our results showed that feeding high fat diet to rats for 12 weeks significantly increased SPARC expression in adipose tissues at both mRNA and protein levels. Moreover, SPARC overexpression in stably transfected 3T3-L1 cells induced insulin resistance and mitochondrial dysfunction, as evidenced by inhibition of insulin-stimulated glucose transport, lower ATP synthesis and mitochondrial membrane potential, reduced expression of glucose transporter 4 (GLUT4), and increased levels of reactive oxygen species (ROS) in mature adipocytes. Finally, overexpression of SPARC also modulated the expression levels of several inflammatory cytokines, which play important roles in insulin resistance, glucose and lipid metabolism during adipogenesis. In conclusion, our data suggest that SPARC is involved in obesity-induced adipose insulin resistance and may serve as a potential target in the treatment of obesity and obesity-related insulin resistance. PMID:23910024

Shen, Yang; Zhao, Yuyan; Yuan, Lizhi; Yi, Wei; Zhao, Rui; Yi, Qianru; Yong, Tongwu

2014-01-01

114

Effects of streptozocin-induced diabetes and food restriction on quantities and source of T4 and T3 in rat tissues.  

PubMed

Diabetes mellitus and fasting are both associated with low plasma thyroid hormone concentrations and loss of body weight. To discriminate between the separate effects of energy shortage and insulin, we studied control rats, diabetic rats (DM), DM rats treated with insulin (DMI), and rats after modified fasting (MF1 and MF2; 70 and 30% of normal daily food intake, respectively). In double-isotopic equilibrium experiments, we determined the tissue thyroxine (T4) and triiodothyronine (T3) concentrations and the contribution of local T4-to-T3 conversion to total T3 in rat tissues; thyroidal T4 and T3 secretion and extrathyroidal T3 production were calculated. In DM and DMI rats, plasma T4 and T3 decreased; in MF1 and MF2 rats, only plasma T4 decreased. Thyroidal T4 secretion decreased, whereas that of T3 remained normal. The decrease in tissue T4 in MF and DM rats paralleled the decrease in plasma T4. Although plasma T3 did not differ in DM and DMI rats, total T3 concentrations in all tissues were not the same due to changed uptake of T3 from plasma and local T4-to-T3 conversion; these changes were not found in several tissues of MF1 and MF2 rats. Our results suggest that the decrease in tissue T4 during diabetes mellitus is due to the decrease in plasma T4 caused by the decreased thyroidal secretion, possibly due to intracellular energy shortage. The changes in tissue T3 during diabetes mellitus are only partly attributable to the same phenomenon; in several tissues, the decrease in T3 seems more related to the lack of insulin. PMID:1733802

Schröder-van der Elst, J P; van der Heide, D

1992-02-01

115

Luteolin protects osteoblastic MC3T3-E1 cells from antimycin A-induced cytotoxicity through the improved mitochondrial function and activation of PI3K\\/Akt\\/CREB  

Microsoft Academic Search

Luteolin is a flavonoid found in many herbal extracts including celery, green pepper, parsley, perilla leaf and seeds, and chamomile. Antimycin A (AMA) is an inhibitor of the mitochondrial electron transport chain. In the present study, the protective effect of luteolin on AMA-induced cell damage was investigated in osteoblastic MC3T3-E1 cells. Luteolin significantly increased the viability of MC3T3-E1 cells in

Eun Mi Choi

116

Rosiglitazone Induces Mitochondrial Biogenesis in Differentiated Murine 3T3-L1 and C3H/10T1/2 Adipocytes  

PubMed Central

Growing evidence indicates that PPAR? agonists, including rosiglitazone (RSG), induce adipose mitochondrial biogenesis. By systematically analyzing mitochondrial gene expression in two common murine adipocyte models, the current study aimed to further establish the direct role of RSG and capture temporal changes in gene transcription. Microarray profiling revealed that in fully differentiated 3T3-L1 and C3H/10T1/2 adipocytes treated with RSG or DMSO vehicle for 1, 2, 4, 7, 24, and 48?hrs, RSG overwhelmingly increased mitochondrial gene transcripts time dependently. The timing of the increases was consistent with the cascade of organelle biogenesis, that is, initiated by induction of transcription factor(s), followed by increases in the biosynthesis machinery, and then by increases in functional components. The transcriptional increases were further validated by increased mitochondrial staining, citrate synthase activity, and O2 consumption, and were found to be associated with increased adiponectin secretion. The work provided further insight on the mechanism of PPAR?-induced mitochondrial biogenesis in differentiated adipocytes.

Rong, James X.; Klein, Jean-Louis D.; Qiu, Yang; Xie, Mi; Johnson, Jennifer H.; Waters, K. Michelle; Zhang, Vivian; Kashatus, Jennifer A.; Remlinger, Katja S.; Bing, Nan; Crosby, Renae M.; Jackson, Tymissha K.; Witherspoon, Sam M.; Moore, John T.; Ryan, Terence E.; Neill, Sue D.; Strum, Jay C.

2011-01-01

117

Focal adhesion kinase (p125FAK) and paxillin are substrates for sphingomyelinase-induced tyrosine phosphorylation in Swiss 3T3 fibroblasts.  

PubMed Central

We examined the effect of sphingomyelinase on tyrosine phosphorylation of intracellular proteins in mouse Swiss 3T3 fibroblasts. Incubation of the cells with bacterial sphingomyelinase resulted in the elevation of tyrosine phosphorylation of multiple cellular proteins of 190, 130, 120, 97 and 70 kDa within minutes. The 120 and 70 kDa tyrosine-phosphorylated peptides were identified as p125 focal adhesion kinase (p125FAK) and paxillin respectively by the use of specific antibodies against the proteins. Tyrosine kinase activity associated with anti-p125FAK immunoprecipitate was stimulated by incubation of cells with sphingomyelinase. Cytochalasin D, which selectively disrupts the network of actin filaments, inhibited sphingomyelinase-induced tyrosine phosphorylation of p125FAK and elevation of tyrosine kinase activity in the anti-p125FAK immunoprecipitates. Sphingomyelinase-induced phosphorylation of p125FAK was not inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. This was in sharp contrast with a wortmannin-sensitive phosphorylation of p125FAK observed in platelet-derived growth factor (PGDF)-stimulated cells. Thus hydrolysis of sphingomyelin is considered to regulate the tyrosine kinase cascade including p125FAK and paxillin by a mechanism distinct from PDGF.

Sasaki, T; Hazeki, K; Hazeki, O; Ui, M; Katada, T

1996-01-01

118

mDia2 Induces the Actin Scaffold for the Contractile Ring and Stabilizes Its Position during Cytokinesis in NIH 3T3 Cells  

PubMed Central

mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.

Watanabe, Sadanori; Ando, Yoshikazu; Yasuda, Shingo; Hosoya, Hiroshi; Watanabe, Naoki; Ishizaki, Toshimasa

2008-01-01

119

A short pulse of mechanical force induces gene expression and growth in MC3T3-E1 osteoblasts via an ERK 1/2 pathway  

NASA Technical Reports Server (NTRS)

Physiological mechanical loading is crucial for maintenance of bone integrity and architecture. We have calculated the strain caused by gravity stress on osteoblasts and found that 4-30g corresponds to physiological levels of 40-300 microstrain. Short-term gravity loading (15 minutes) induced a 15-fold increase in expression of growth-related immediate early gene c-fos, a 5-fold increase in egr-1, and a 3-fold increase in autocrine bFGF. The non-growth-related genes EP-1, TGF-beta, and 18s were unaffected by gravity loading. Short-term physiological loading induced extracellular signal-regulated kinase (ERK 1/2) phosphorylation in a dose-dependent manner with maximum phosphorylation saturating at mechanical loading levels of 12g (p < 0.001) with no effect on total ERK. The phosphorylation of focal adhesion kinase (FAK) was unaffected by mechanical force. g-Loading did not activate P38 MAPK or c-jun N-terminal kinase (JNK). Additionally, a gravity pulse resulted in the localization of phosphorylated ERK 1/2 to the nucleus; this did not occur in unloaded cells. The induction of c-fos was inhibited 74% by the MEK1/2 inhibitor U0126 (p < 0.001) but was not affected by MEK1 or p38 MAPK-specific inhibitors. The long-term consequence of a single 15-minute gravity pulse was a 64% increase in cell growth (p < 0.001). U0126 significantly inhibited gravity-induced growth by 50% (p < 0.001). These studies suggest that short periods of physiological mechanical stress induce immediate early gene expression and growth in MC3T3-E1 osteoblasts primarily through an ERK 1/2-mediated pathway.

Hatton, Jason P.; Pooran, Milad; Li, Chai-Fei; Luzzio, Chris; Hughes-Fulford, Millie

2003-01-01

120

Inhibitory effects of a tryptamine derivative on ultraviolet radiation-induced apoptosis in MC3T3-E1 mouse osteoblasts.  

PubMed

MS-IPA1 is a new synthetic compound that is synthesized from tryptamine. Recently, our group demonstrated that SST-VED-I-1, which has a similar chemical structure to MS-IPA1, inhibits starvation-induced apoptosis in osteoblasts. However, the effects of MS-IPA1 on apoptosis in osteoblasts have not yet been examined. Therefore, this study examined the effects of this compound on apoptosis in osteoblasts. In this study, MC3T3-E1 mouse osteoblasts were used and apoptosis was induced by ultraviolet radiation (UV). We investigated the effect of MS-IPA1 on apoptosis by analyzing caspase3/7 activity, translocation of phosphatidylserine (PS), and mRNA expression levels of Bcl-2 and Bax. In addition, it was investigated whether MS-IPA1 affects cell proliferation and cell cycle progression. We found that MS-IPA1 had no effect on cell proliferation or cell cycle progression. However, MS-IPA1 suppressed UV-induced cell death in a dose-dependent manner, which was accompanied with the inhibition of caspase activation and translocation of PS. Furthermore, after UV exposure, Bcl-2 expression was increased in the MS-IPA1-treated cells as compared to that in the vehicle-treated cells. In contrast, Bax expression was decreased in the MS-IPA1-treated cell as compared to that in the vehicle-treated cells. These results suggest that MS-IPA1 has an inhibitory effect on apoptosis in osteoblasts through a Bcl-2 family-dependent signaling pathway. PMID:21282935

Mikami, Yoshikazu; Senoo, Motoki; Lee, Mio; Yamada, Kiyoshi; Ochiai, Kuniyasu; Honda, Masaki J; Watanabe, Eri; Watanabe, Nobukazu; Somei, Masanori; Takagi, Minoru

2011-01-01

121

Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for G protein regulation of phospholipase A/sub 2/  

SciTech Connect

In Swiss 3T3 fibroblasts bradykinin stimulated inositol phosphate (InsP) formation and prostaglandin E/sub 2/ (PGE/sub 2/) synthesis. The EC/sub 50/ values for stimulation of PGE/sub 2/ synthesis and InsP formation by bradykinin were similar, 200 pM and 275 pM, respectively. Guanosine-5'-(..gamma..-thio)triphosphate stimulated PGE/sub 2/ synthesis and InsP formation, and guanosine-5'-(..beta..-thio)diphosphate inhibited both PGE/sub 2/ synthesis and InsP formation stimulated by bradykinin. Neither bradykinin-stimulated PGE/sub 2/ synthesis nor InsP formation was sensitive to pertussis toxin. Phorbol ester, dexamethasone, and cycloheximide distinguished between bradykinin-stimulated PGE/sub 2/ synthesis and InsP formation. Phorbol 12-myristate 13-acetate enhanced bradykinin-stimulated PGE/sub 2/ synthesis but inhibited bradykinin-stimulated InsP formation. Pretreatment of cells with dexamethasone for 24 hr inhibited bradykinin-stimulated PGE/sub 2/ synthesis but was without effect on bradykinin-stimulated InsP formation. Cycloheximide inhibited on bradykinin-stimulated InsP formation. When bradykinin was added to cells prelabeled with (/sup 3/H) choline, the phospholipase A/sub 2/ products lysophosphatidylcholine and glycerophosphocholine were generated. The data suggest that bradykinin receptors are coupled by GTP-binding proteins to both phospholipase C and phospholipase A/sub 2/ and that phospholipase A/sub 2/ is the enzyme that catalyzes release of arachidonate for prostaglandin synthesis.

Burch, R.M.; Axelrod, J.

1987-09-01

122

I Lost Weight, but I Became Weak and Cannot Walk-A Case of Nutraceutical (T3)-Induced Thyrotoxic Periodic Paralysis.  

PubMed

Thyrotoxic periodic paralysis (TPP) is a rare reversible cause of paralysis and cramping. TPP is usually precipitated by common causes of thyrotoxicosis such as Grave disease or multinodular goiter. TPP precipitated by exogenous triiodothyronine (T3) intake is an extremely rare occurrence with only 3 cases reported to date. We now report a 24-year-old healthy manual laborer who developed quadriparesis during a period of rest after heavy exertion and carbohydrate intake. He had severe hypokalemia (potassium level 1.9 mmole/L). Correction of his hypokalemia reversed the paralysis without rebound hyperkalemia. After a detailed history review, he reported that he had been consuming nutraceuticals containing T3 for 1 month to lose weight, and laboratory studies confirmed factitious T3 toxicosis. There was no evidence of renal or gastrointestinal potassium wasting. This episode of TPP was the first manifestation of thyrotoxicosis in this patient, and avoidance of T3 intake prevented more episodes. PMID:23567793

Panikkath, Ragesh; Nugent, Kenneth

2013-04-01

123

The thyroid hormone receptor ? induces DNA damage and premature senescence  

PubMed Central

There is increasing evidence that the thyroid hormone (TH) receptors (THRs) can play a role in aging, cancer and degenerative diseases. In this paper, we demonstrate that binding of TH T3 (triiodothyronine) to THRB induces senescence and deoxyribonucleic acid (DNA) damage in cultured cells and in tissues of young hyperthyroid mice. T3 induces a rapid activation of ATM (ataxia telangiectasia mutated)/PRKAA (adenosine monophosphate–activated protein kinase) signal transduction and recruitment of the NRF1 (nuclear respiratory factor 1) and THRB to the promoters of genes with a key role on mitochondrial respiration. Increased respiration leads to production of mitochondrial reactive oxygen species, which in turn causes oxidative stress and DNA double-strand breaks and triggers a DNA damage response that ultimately leads to premature senescence of susceptible cells. Our findings provide a mechanism for integrating metabolic effects of THs with the tumor suppressor activity of THRB, the effect of thyroidal status on longevity, and the occurrence of tissue damage in hyperthyroidism.

Zambrano, Alberto; Garcia-Carpizo, Veronica; Gallardo, Maria Esther; Villamuera, Raquel; Gomez-Ferreria, Maria Ana; Pascual, Angel; Buisine, Nicolas; Sachs, Laurent M.; Garesse, Rafael

2014-01-01

124

Selection and characterization of T-cell variants lacking molecules involved in T-cell activation (T3 T-cell receptor, T44, and T11): analysis of the functional relationship among different pathways of activation  

SciTech Connect

A clone of the interleukin 2-producing Jurkat leukemia cell line termed JA3 (surface phenotype, T3/sup +/, Ti/sup +/, T44/sup +/, T11/sup +/, T40/sup +/) has been used to induce and select cell variants lacking surface molecules involved in T-cell activation. Following 200 rad of ..gamma..-radiation (1 rad = 0.01 Gy), cells were treated with monoclonal antibodies (mAbs) directed to T3, Ti, T44, or T11 antigen and complement. After growth of the residual cells in culture, negative cells were cloned under limiting conditions. Depending on the specificity of the mAb used for the immunoselection, three groups of variants were obtained. (i) The use of mAbs directed to T3 or Ti resulted in cell variants that expressed the T3/sup -/ Ti/sup -/T44/sup +/ Leu1/sup +/ T11/sup +/ T40/sup +/ 4F2/sup +/ HLA class I/sup +/ surface phenotype. (ii) Immunoselection with anti-T44 mAb resulted in 2 variants that shared the T3/sup -/ Ti/sup -/ T44/sup -/ Leu1/sup -/ T11/sup -/ T40/sup -/ 4F2/sup -/ HLA class I/sup +/ phenotype. (iii) Cell treatment with anti-T11 mAb resulted in 15 variants characterized by the lack of T11 antigen expression and of all the other T-cell-specific surface antigens. Therefore, it appears that the different sets of JA3 cell variants, like T cells at discrete stages of intrathymic differentiation, may follow a coordinated expression of surface differentiation antigens. Analysis of the functional responsiveness of the three distinct groups of JA3 cell variants to different stimuli showed that all produced interleukin 2 in response to A23187 calcium ionophore plus phorbol 12-myristate 13-acetate.

Moretta, A.; Poggi, A.; Olive, D.; Bottino, C.; Fortis, C.; Pantaleo, G.; Moretta, L.

1987-03-01

125

Extracellular calcium-sensing-receptor (CaR)-mediated opening of an outward K(+) channel in murine MC3T3-E1 osteoblastic cells: evidence for expression of a functional CaR  

NASA Technical Reports Server (NTRS)

The existence in osteoblasts of the G-protein-coupled extracellular calcium (Ca(o)(2+))-sensing receptor (CaR) that was originally cloned from parathyroid and kidney remains controversial. In our recent studies, we utilized multiple detection methods to demonstrate the expression of CaR transcripts and protein in several osteoblastic cell lines, including murine MC3T3-E1 cells. Although we and others have shown that high Ca(o)(2+) and other polycationic CaR agonists modulate the function of MC3T3-E1 cells, none of these actions has been unequivocally shown to be mediated by the CaR. Previous investigations using neurons and lens epithelial cells have shown that activation of the CaR stimulates Ca(2+)-activated K(+) channels. Because osteoblastic cells express a similar type of channel, we have examined the effects of specific "calcimimetic" CaR activators on the activity of a Ca(2+)-activated K(+) channel in MC3T3-E1 cells as a way of showing that the CaR is not only expressed in those cells but is functionally active. Patch-clamp analysis in the cell-attached mode showed that raising Ca(o)(2+) from 0.75 to 2.75 mmol/L elicited about a fourfold increase in the open state probability (P(o)) of an outward K(+) channel with a conductance of approximately 92 pS. The selective calcimimetic CaR activator, NPS R-467 (0.5 micromol/L), evoked a similar activation of the channel, while its less active stereoisomer, NPSS-467 (0.5 micromol/L), did not. Thus, the CaR is not only expressed in MC3T3-E1 cells, but is also functionally coupled to the activity of a Ca(2+)-activated K(+) channel. This receptor, therefore, could transduce local or systemic changes in Ca(o)(2+) into changes in the activity of this ion channel and related physiological processes in these and perhaps other osteoblastic cells.

Ye, C. P.; Yamaguchi, T.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

2000-01-01

126

Synergy between the T3/Ti complex and Tp44 in human T cell activation  

SciTech Connect

Ligands, including antigen, which interact with the T3/T cell antigen receptor (Ti) induce an increase in cytoplasmic free calcium ((Ca/sup + +/)/sub i/). However, such an increase in (Ca/sup + +/)/sub i/ is not sufficient to result in T cell activation. Activation can occur if such ligands, which increase (Ca/sup + +/)/sub i/, are added in the presence of phorbol myristate acetate (PMA). This suggests that interactions involving other T cell surface receptors might function in a manner analogous to PMA and play an important role in T cell activation. Using the human leukemic T cell line Jurkat, they demonstrated that 9.3, a monoclonal antibody specific for a 90 kD homodimer expressed on human T cells (Tp44), synergized with some ligands interacting with T3/Ti on Jurkat in the induction of IL-2 production from Jurkat. Thus, 9.3 synergized with PHA, ConA, or sepharose beads coupled with anti-T3 or anti-Ti but not with calcium ionophores or soluble anti-T3 or anti-Ti. Furthermore, at high concentrations only, 9.3 could also synergize with PMA in the activation of Jurkat and T3/Ti negative mutants of Jurkat. No detectable activation of protein kinase C was induced by 9.3 alone. These studies suggest that T cell activation may result from the simultaneous triggering of several distinct T cell surface molecules.

Weiss, A.; Manger, B.; Shields, R.; Imboden, J.

1986-03-05

127

Role of c-myc in Simian Virus 40 Large Tumor Antigen-Induced DNA Synthesis in Quiescent 3T3-L1 Mouse Fibroblasts  

Microsoft Academic Search

Stably transfected NIH 3T3-L1 mouse fibroblasts (L1 cells) expressing the simian virus 40 large tumor antigen (LTAg) maintain c-myc expression and proliferation in low serum, whereas cells expressing the mutant form LTAg-K1, defective in binding of the retinoblastoma suppressor gene product pRb, showed reduced levels of c-myc RNA and only background levels of DNA synthesis in low serum. The role

Heiko Hermeking; Dieter A. Wolf; Franz Kohlhuber; Achim Dickmanns; Marc Billaud; Ellen Fanning; Dirk Eick

1994-01-01

128

Cytotoxic Necrotizing Factor 1 from Escherichia coli and Dermonecrotic Toxin from Bordetella bronchiseptica Induce p21 rho -dependent Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin in Swiss 3T3 Cells  

Microsoft Academic Search

Treatment of Swiss 3T3 cells with cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli and dermonecrotic toxin (DNT) from Bordetella bronchiseptica, which directly target and activate p21(rho), stimulated tyrosine phosphorylation of focal adhesion kinase (p125(fak)) and paxillin. Tyrosine phosphorylation induced by CNF1 and DNT occurred after a pronounced lag period (2 h), and was blocked by either lysosomotrophic agents or

Hadriano M. Lacerda; Gill D. Pullinger; Alistair J. Laxi; Enrique Rozengurt

1997-01-01

129

Functional Redundancy of Type II BMP Receptor and Type IIB Activin Receptor in BMP2-Induced Osteoblast Differentiation  

PubMed Central

Signaling pathways for bone morphogenetic proteins (BMPs) are important in osteoblast differentiation. Although the precise function of type I BMP receptors in mediating BMP signaling for osteoblast differentiation and bone formation has been characterized previously, the role of type II BMP receptors in osteoblasts is to be well clarified. In this study, we investigated the role of type II BMP receptor (BMPR-II) and type IIB activin receptor (ActR-IIB) in BMP2-induced osteoblast differentiation. While osteoblastic 2T3 cells expressed BMPR-II and ActR-IIB, loss-of-function studies, using dominant negative receptors and siRNAs, showed that BMPR-II and ActR-IIB compensated each other functionally in mediating BMP2 signaling and BMP2-induced osteoblast differentiation. This was evidenced by two findings. First, unless there was loss of function of both type II receptors, isolated disruption of either BMPR-II or ActR-IIB did not remove BMP2 activity. Second, in cells with loss of function of both receptors, restoration of function of either BMPR-II or ActR-IIB by transfection of the wild-type forms, restored BMP2 activity. These findings suggest a functional redundancy between BMPR-II and ActR-IIB in osteoblast differentiation. Results from experiments to test the effects of transforming growth factor b (TGF-?), activin, and fibroblast growth factor (FGF) on osteoblast proliferation and differentiation suggest that inhibition of receptor signaling by double-blockage of BMPR-II and ActR-IIB is BMP-signaling specific. The observed functional redundancy of type II BMP receptors in osteoblasts is novel information about the BMP signaling pathway essential for initiating osteoblast differentiation.

LIU, HONGBIN; ZHANG, RONGRONG; CHEN, DI; OYAJOBI, BABATUNDE O.; ZHAO, MING

2013-01-01

130

Accepting the T3D  

SciTech Connect

In April, a 128 PE Cray T3D was installed at Los Alamos National Laboratory`s Advanced Computing Laboratory as part of the DOE`s High-Performance Parallel Processor Program (H4P). In conjunction with CRI, the authors implemented a 30 day acceptance test. The test was constructed in part to help them understand the strengths and weaknesses of the T3D. In this paper, they briefly describe the H4P and its goals. They discuss the design and implementation of the T3D acceptance test and detail issues that arose during the test. They conclude with a set of system requirements that must be addressed as the T3D system evolves.

Rich, D.O.; Pope, S.C.; DeLapp, J.G.

1994-10-01

131

Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin ? gene promoter  

SciTech Connect

Highlights: •MED1 is a bona fide T3-dependent coactivator on TSHB promoter. •Mice with LxxLL-mutant MED1 have attenuated TSH? mRNA and thyroid hormone levels. •MED1 activates TSHB promoter T3-dependently in cultured cells. •T3-dependent MED1 action is enhanced when SRC1/SRC2 or HDAC2 is downregulated. •MED1 is also a T3-independent GATA2/Pit1 coactivator on TSHB promoter. -- Abstract: The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor ? (TR?) on the TSH? gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSH? gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSH? gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSH? gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSH? gene promoter.

Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan)] [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States)] [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan) [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan)

2013-10-11

132

Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential.  

PubMed

In this work we investigated the toxicological effects of nude and chemically functionalised (-NH(2), -OH and -COOH groups) multiwall carbon nanotubes (mwCNTs) using immortalised mouse fibroblasts cell line (Balb/3T3) as in vitro model, alternative to the use of animals, to assess basal cytotoxicity, carcinogenic potential, genotoxicity and cell interaction of nanomaterials (NM). Combining in vitro tests such as cell transformation assay and micronucleus with physicochemical and topological analysis, we obtained results showing no cytotoxicity and genotoxicity. Carcinogenic potential and mwCNTs interaction with cells were instead evident. We stressed the importance that different toxicological end points have to be considered when studying NM, therefore, assays able to detect long-term effects, such as carcinogenicity, must be taken into account together with a panel of tests able to detect more immediate effects like basal cytotoxicity or genotoxicity. PMID:22279961

Ponti, Jessica; Broggi, Francesca; Mariani, Valentina; De Marzi, Laura; Colognato, Renato; Marmorato, Patrick; Gioria, Sabrina; Gilliland, Douglas; Pascual Garcìa, César; Meschini, Stefania; Stringaro, Annarita; Molinari, Agnese; Rauscher, Hubert; Rossi, François

2013-03-01

133

Trans-cinnamic acid increases adiponectin and the phosphorylation of AMP-activated protein kinase through G-protein-coupled receptor signaling in 3T3-L1 adipocytes.  

PubMed

Adiponectin and intracellular 5'adenosine monophosphate-activated protein kinase (AMPK) are important modulators of glucose and fat metabolism. Cinnamon exerts beneficial effects by improving insulin sensitivity and blood lipids, e.g., through increasing adiponectin concentrations and AMPK activation. The underlying mechanism is unknown. The Gi/Go-protein-coupled receptor (GPR) 109A stimulates adiponectin secretion after binding its ligand niacin. Trans-cinnamic acid (tCA), a compound of cinnamon is another ligand. We hypothesize whether AMPK activation and adiponectin secretion by tCA is transmitted by GPR signaling. Differentiated 3T3-L1 cells were incubated with pertussis toxin (PTX), an inhibitor of G(i)/G(o)-protein-coupling, and treated with different tCA concentrations. Treatment with tCA increased adiponectin and the pAMPK/AMPK ratio (p ? 0.001). PTX incubation abolished the increased pAMPK/AMPK ratio and adiponectin secretion. The latter remained increased compared to controls (p ? 0.002). tCA treatment stimulated adiponectin secretion and AMPK activation; the inhibitory effect of PTX suggests GPR is involved in tCA stimulated signaling. PMID:24557583

Kopp, Christina; Singh, Shiva P; Regenhard, Petra; Müller, Ute; Sauerwein, Helga; Mielenz, Manfred

2014-01-01

134

5-Hydroxytryptamine type 7 receptor neuroprotection against NMDA-induced excitotoxicity is PDGF? receptor dependent.  

PubMed

The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the hippocampus. Long-term (2-24 h) activation of 5-HT7 receptors regulates growth factor receptor expression, including the expression of platelet-derived growth factor (PDGF) ? receptors. Direct activation of PDGF? receptors in primary hippocampal and cortical neurons inhibits NMDA receptor activity and attenuates NMDA receptor-induced neurotoxicity. Our objective was to investigate whether the 5-HT7 receptor-induced increase in PDGF? receptor expression would be similarly neuroprotective. We demonstrate that 5-HT7 receptor agonist treatment in primary hippocampal neurons also increases the expression of phospholipase C (PLC) ?, a downstream effector of PDGF? receptors associated with the inhibition of NMDA receptor activity. To determine if the up-regulation of PDGF? receptors is neuroprotective, primary hippocampal neurons were incubated with the 5-HT7 receptor agonist, LP 12, for 24 h. Indeed, LP 12 treatment prevented NMDA-induced neurotoxicity and this effect was dependent on PDGF? receptor kinase activity. Treatment of primary neurons with LP 12 also differentially altered NMDA receptor subunit expression, reducing the expression of NR1 and NR2B, but not NR2A. These findings demonstrate the potential for providing growth factor receptor-dependent neuroprotective effects using small-molecule ligands of G protein-coupled receptors. PMID:23336565

Vasefi, Maryam S; Kruk, Jeff S; Heikkila, John J; Beazely, Michael A

2013-04-01

135

Molecular mechanism of 9-cis-retinoic acid inhibition of adipogenesis in 3T3-L1 cells  

SciTech Connect

Highlights: ? We examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1. ? 9-cis-RA inhibited lipid accumulation in adipogenetically-induced 3T3-L1 cells. ? A RXR pan-antagonist suppressed the inhibitory effects of 9-cis-RA on adipogenesis. ? This antagonist had no effects on RXR? and PPAR? levels in 9-cis-RA-treated cells. ? 9-cis-RA-induced decrease in both RXR? and PPAR? was independent of RXR activation. -- Abstract: Retinoic acid (RA) signaling is mediated by specific nuclear hormone receptors. Here we examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1 cells. 9-cis-RA inhibits the lipid accumulation of adipogenetically induced 3T3-L1 cells. The complex of retinoid X receptor ? (RXR?) with peroxisome proliferator-activated receptor ? (PPAR?) is a major transcription factor in the process of adipogenesis, and the levels of these molecules were decreased by 9-cis-RA treatment. A RXR pan-antagonist suppressed 9-cis-RA’s inhibitory effects on adipogenesis, but not on the intracellular levels of both RXR? and PPAR?. These results suggest that 9-cis-RA could inhibit adipogenesis by activating RXR, and decrease both RXR and PPAR?s levels in a RXR activation-independent manner.

Sagara, Chiaki; Takahashi, Katsuhiko [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan)] [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan); Kagechika, Hiroyuki [School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda, Tokyo 101-0062 (Japan)] [School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda, Tokyo 101-0062 (Japan); Takahashi, Noriko, E-mail: t-noriko@hoshi.ac.jp [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan)] [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan)

2013-03-29

136

Endoplasmic reticulum stress suppresses lipin-1 expression in 3T3-L1 adipocytes  

SciTech Connect

Highlights: ? Lipin-1 involves lipid metabolism, adipocyte differentiation, and inflammation. ? Adipose lipin-1 expression is reduced in obesity. ? ER stress suppresses lipin-1 expression in 3T3-L1 adipocytes. ? Activation of PPAR-? recovers ER stress-induced lipin-1 reduction. -- Abstract: Lipin-1 plays crucial roles in the regulation of lipid metabolism and cell differentiation in adipocytes. In obesity, adipose lipin-1 mRNA expression is decreased and positively correlated with systemic insulin sensitivity. Amelioration of the lipin-1 depletion might be improved dysmetabolism. Although some cytokines such as TNF-? and interleukin-1? reduces adipose lipin-1 expression, the mechanism of decreased adipose lipin-1 expression in obesity remains unclear. Recently, endoplasmic reticulum (ER) stress is implicated in the pathogenesis of obesity. Here we investigated the role of ER stress on the lipin-1 expression in 3T3-L1 adipocytes. We demonstrated that lipin-1 expression was suppressed by the treatment with ER stress inducers (tunicamycin and thapsigargin) at transcriptional level. We also showed that constitutive lipin-1 expression could be maintained by peroxisome proliferator-activated receptor-? in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-? recovered the ER stress-induced lipin-1 suppression. These results suggested that ER stress might be involved in the pathogenesis of obesity through lipin-1 depletion.

Takahashi, Nobuhiko, E-mail: ntkhs@hoku-iryo-u.ac.jp [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan) [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Yoshizaki, Takayuki [Innovation Center, Kagoshima University, 1-21-40, Korimoto, Kagoshima 890-0065 (Japan)] [Innovation Center, Kagoshima University, 1-21-40, Korimoto, Kagoshima 890-0065 (Japan); Hiranaka, Natsumi; Suzuki, Takeshi [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yui, Tomoo; Akanuma, Masayoshi [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Kanazawa, Kaoru [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yoshida, Mika; Naito, Sumiyoshi [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Fujiya, Mikihiro; Kohgo, Yutaka [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan)] [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Ieko, Masahiro [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)

2013-02-01

137

Promoter Polymorphisms of the Interferon-? Receptor Gene and Development of Interferon-Induced Depressive Symptoms in Patients with Chronic Hepatitis C: Preliminary Findings  

Microsoft Academic Search

Background: Interferon (IFN)-? treatment frequently induces depression, which can impair quality of life and reduce treatment adherence. Defining relevant risk factors for IFN-?-induced depression is essential for designing preventative treatment strategies. Objective: The purpose of the present study was to determine whether promoter polymorphisms of –408C\\/T, –3C\\/T and GT repeat dinucleotide microsatellite in the IFN-?\\/? receptor 1 (IFNAR1) gene are

Keizo Yoshida; Oyetunde Alagbe; Xiaohong Wang; Bobbi Woolwine; Marie Thornbury; Charles L. Raison; Andrew H. Miller

2005-01-01

138

Antiobesity Effects of an Edible Halophyte Nitraria retusa Forssk in 3T3-L1 Preadipocyte Differentiation and in C57B6J/L Mice Fed a High Fat Diet-Induced Obesity  

PubMed Central

Nitraria retusa is an edible halophyte, used in Tunisia for several traditional medicine purposes. The present study investigated the antiobesity effects of Nitraria retusa ethanol extract (NRE) in 3T3-L1 cells using different doses and in high-fat diet-induced obesity in mice. Male C57B6J/L mice were separately fed a normal diet (ND) or a high-fat diet (HFD) and daily administrated with NRE (50, 100?mg/kg) or one for 2 days with Naringenin (10?mg/kg). NRE administration significantly decreased body weight gain, fat pad weight, serum glucose, and lipid levels in HFD-induced obese mice. To elucidate the mechanism of action of NRE, the expression of genes involved in lipid and carbohydrate metabolism were measured in liver. Results showed that mice treated with NRE demonstrated a significant decrease in cumulative body weight and fat pad weight, a significant lowering in glucose and triglycerides serum levels, and an increase in the HDL-cholesterol serum level. Moreover mRNA expression results showed an enhancement of the expression of genes related to liver metabolism. Our findings suggest that NRE treatment had a protective or controlling effect against a high fat diet-induced obesity in C57B6J/L mice through the regulation of expression of genes involved in lipolysis and lipogenesis and thus the enhancement of the lipid metabolism in liver.

Zar Kalai, Feten; Han, Junkyu; Ksouri, Riadh; El Omri, Abdelfatteh; Abdelly, Chedly; Isoda, Hiroko

2013-01-01

139

Anti-obesity effect of Blumea balsamifera extract in 3T3-L1 preadipocytes and adipocytes.  

PubMed

Obesity, the leading metabolic disease in the world, is a serious health problem in industrialized countries. We investigated the anti-obesity effect of Blumea balsamifera extract on adipocyte differentiation of 3T3-L1 preadipocytes and anti-obesity effect of 3T3-L1 adipocytes. We found that treatment with an extract of Blumea balsamifera suppressed lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity without affecting cell viability in 3T3-L1 preadipocytes and adipocytes. Furthermore, Blumea balsamifera extract brought significant attenuation of expressions of key adipogenic transcription factors, including peroxisome proliferator-activated receptor (PPAR)gamma, CCAAT element binding protein (C/EBPs) and leptin, however, induced up-regulation of adiponectin at the protein level in 3T3-L1 preadipocytes and adipocytes. These results suggest that Blumea balsamifera extract may block adipogenesis, at least in part, by decreasing key adipogenic transcription factors in 3T3-L1 preadipocytes and may have antiatherogenic, anti-inflammatory, and antidiabetic effects through up-regulation of adiponectin in 3T3-L1 adipocytes. PMID:19885945

Kubota, Hiroaki; Kojima-Yuasa, Akiko; Morii, Risako; Huang, Xuedan; Norikura, Toshio; Rho, Sook-Nyung; Matsui-Yuasa, Isao

2009-01-01

140

Chronic exposure to stress hormones promotes transformation and tumorigenicity of 3T3 mouse fibroblasts  

PubMed Central

Epinephrine and norepinephrine are produced during psychological stress and can directly bind to cells to induce DNA damage. These effects may have more long-lasting consequences such as DNA mutations resulting in an increased potential for cellular transformation and/or tumor progression. This study examined the molecular effects of a chronic (24 h) in vitro exposure to these stress hormones on murine 3T3 cells. Long exposures (24 h) in dose–response experiments with norepinephrine or epinephrine induced significant increases in DNA damage in treated cells compared to that of untreated controls as measured by the alkaline comet assay. Pre-treatment with a blocking agent (the ?-adrenergic receptor antagonist propranolol) eliminated this increase in damage. In addition, both norepinephrine and epinephrine increased cellular transformation, as assessed by growth in soft agar, and 3T3 cells pre-treated with either norepinephrine or epinephrine induced a more rapid onset of tumors and more aggressive tumor growth in nude mice. In summary, incubation of 3T3 cells with catecholamines results in long-term DNA damage as measured by increased transformed phenotypes and tumor progression, indicating that they are important mediators of stress effects on genomic instability and vulnerability to tumor formation.

FLINT, MELANIE S.; BAUM, ANDREW; EPISCOPO, BRITTENY; KNICKELBEIN, KELLY Z.; LIEGEY DOUGALL, ANGELA J.; CHAMBERS, WILLIAM H.; JENKINS, FRANK J.

2013-01-01

141

The thyroid hormone receptor (TR) beta-selective agonist GC-1 inhibits proliferation but induces differentiation and TR beta mRNA expression in mouse and rat osteoblast-like cells.  

PubMed

Previous studies showed anabolic effects of GC-1, a triiodothyronine (T3) analogue that is selective for both binding and activation functions of thyroid hormone receptor (TR) beta1 over TRalpha1, on bone tissue in vivo. The aim of this study was to investigate the responsiveness of rat (ROS17/2.8) and mouse (MC3T3-E1) osteoblast-like cells to GC-1. As expected, T3 inhibited cellular proliferation and stimulated mRNA expression of osteocalcin or alkaline phosphatase in both cell lineages. Whereas equimolar doses of T3 and GC-1 equally affected these parameters in ROS17/2.8 cells, the effects of GC-1 were more modest compared to those of T3 in MC3T3-E1 cells. Interestingly, we showed that there is higher expression of TRalpha1 than TRbeta1 mRNA in rat (approximately 20-90%) and mouse (approximately 90-98%) cell lineages and that this difference is even higher in mouse cells, which highlights the importance of TRalpha1 to bone physiology and may partially explain the modest effects of GC-1 in comparison with T3 in MC3T3-E1 cells. Nevertheless, we showed that TRbeta1 mRNA expression increases (approximately 2.8- to 4.3-fold) as osteoblastic cells undergo maturation, suggesting a key role of TRbeta1 in mediating T3 effects in the bone forming cells, especially in mature osteoblasts. It is noteworthy that T3 and GC-1 induced TRbeta1 mRNA expression to a similar extent in both cell lineages (approximately 2- to 4-fold), indicating that both ligands may modulate the responsiveness of osteoblasts to T3. Taken together, these data show that TRbeta selective T3 analogues have the potential to directly induce the differentiation and activity of osteoblasts. PMID:19280098

Beber, Eduardo H; Capelo, Luciane P; Fonseca, Tatiana L; Costa, Cristiane C; Lotfi, Claudimara F; Scanlan, Thomas S; Gouveia, Cecilia H A

2009-04-01

142

Suppressive effects of chlorophyllin on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) and tumor promoter-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells.  

PubMed

The potentially protective role of chlorophyllin, the sodium and copper salt of chlorophyll a against the initiation and promotion stages in carcinogenesis was studied by in vitro short-term assays. Chlorophyllin showed a dose-dependent suppressive effect on 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1)-induced umu C gene expression of Salmonella typhimurium (TA 1535/pSK 1002) in the presence of metabolizing enzyme mixture. The similar inhibitory effect of chlorophyllin was detected in mitomycin C (MMC)-dependent umu C gene expression in the absence of metabolizing enzyme mixture. Furthermore chlorophyllin also exhibited a dose-dependent inhibition on 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity of 3T3 fibroblast cells at the same concentrations. However, when chlorophyll a isolated from Japanese tea leaves was applied on the same assay systems as a comparative experiment, chlorophyll a showed much weaker activity compared with that of chlorophyllin. The significance of this finding is discussed from the viewpoint of the protective role of chlorophyllin against carcinogenesis. PMID:8830802

Okai, Y; Higashi-Okai, K; Yano, Y; Otani, S

1996-08-01

143

Modulation of Cell-Substrate Adhesion by Arachidonic Acid: Lipoxygenase Regulates Cell Spreading and ERK1/2-inducible Cyclooxygenase Regulates Cell Migration in NIH-3T3 Fibroblasts  

PubMed Central

Adhesion of cells to an extracellular matrix is characterized by several discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading, migration, and immobilization. We find that although arachidonic acid release is rate-limiting in the overall process of adhesion, its oxidation by lipoxygenase and cyclooxygenases regulates, respectively, the cell spreading and cell migration stages. During the adhesion of NIH-3T3 cells to fibronectin, two functionally and kinetically distinct phases of arachidonic acid release take place. An initial transient arachidonate release occurs during cell attachment to fibronectin, and is sufficient to signal the cell spreading stage after its oxidation by 5-lipoxygenase to leukotrienes. A later sustained arachidonate release occurs during and after spreading, and signals the subsequent migration stage through its oxidation to prostaglandins by newly synthesized cyclooxygenase-2. In signaling migration, constitutively expressed cyclooxygenase-1 appears to contribute ?25% of prostaglandins synthesized compared with the inducible cyclooxygenase-2. Both the second sustained arachidonate release, and cyclooxygenase-2 protein induction and synthesis, appear to be regulated by the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2. The initial cell attachment-induced transient arachidonic acid release that signals spreading through lipoxygenase oxidation is not sensitive to ERK1/2 inhibition by PD98059, whereas PD98059 produces both a reduction in the larger second arachidonate release and a blockade of induced cyclooxygenase-2 protein expression with concomitant reduction of prostaglandin synthesis. The second arachidonate release, and cyclooxygenase-2 expression and activity, both appear to be required for cell migration but not for the preceding stages of attachment and spreading. These data suggest a bifurcation in the arachidonic acid adhesion-signaling pathway, wherein lipoxygenase oxidation generates leukotriene metabolites regulating the spreading stage of cell adhesion, whereas ERK 1/2-induced cyclooxygenase synthesis results in oxidation of a later release, generating prostaglandin metabolites regulating the later migration stage.

Stockton, Rebecca A.; Jacobson, Bruce S.

2001-01-01

144

Illudins C2 and C3 Stimulate Lipolysis in 3T3-L1 Adipocytes and Suppress Adipogenesis in 3T3-L1 Preadipocytes.  

PubMed

The secondary metabolites illudins C2 (1) and C3 (2), obtained from the culture broth of Coprinus atramentarius, have been shown to possess antimicrobial activity. In the present study, we discovered novel biological activities of 1 and 2 in lipolysis of differentiated 3T3-L1 adipocytes and adipogenesis of 3T3-L1 preadipocytes. Compounds 1 and 2 exhibit a dose-dependent increase in glycerol release and thereby reduce intracellular lipid accumulation. The stimulatory effects of 1 and 2 on lipolysis are prevented by cAMP-dependent protein kinase (PKA) and extracellular signal-regulated kinase (ERK) inhibitors. Compounds 1 and 2 down-regulated perilipin and also affected the mRNA and protein levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). However, 1 and 2 treatment leads to a significant increase in PKA-mediated phosphorylation of HSL at S563 and S660. In addition, 1 and 2 treatment in 3T3-L1 preadipocytes induces down-regulation of the critical transcription factors, CCAAT/enhancer binding protein ? and ? (C/EBP? and C/EBP?), and peroxisome proliferator activated receptor ? (PPAR?), which are required for adipogenesis, and accordingly inhibits adipogenesis. These results suggest that 1 and 2 might be useful for treating obesity due to their modulatory effects on fat by affecting adipocyte differentiation and fat mobilization. PMID:24597820

Kim, Sun-Ok; Sakchaisri, Krisada; Asami, Yukihiro; Ryoo, In-Ja; Choo, Soo-Jin; Yoo, Ick-Dong; Soung, Nak-Kyun; Kim, Young Sang; Jang, Jae-Hyuk; Kim, Bo Yeon; Ahn, Jong Seog

2014-04-25

145

Binding of amyloid ? peptide to ?2 adrenergic receptor induces PKA-dependent AMPA receptor hyperactivity  

PubMed Central

Progressive decrease in neuronal function is an established feature of Alzheimer’s disease (AD). Previous studies have shown that amyloid ? (A?) peptide induces acute increase in spontaneous synaptic activity accompanied by neurotoxicity, and A? induces excitotoxic neuronal death by increasing calcium influx mediated by hyperactive ?-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors. An in vivo study has revealed subpopulations of hyperactive neurons near A? plaques in mutant amyloid precursor protein (APP)-transgenic animal model of Alzheimer’s disease (AD) that can be normalized by an AMPA receptor antagonist. In the present study, we aim to determine whether soluble A? acutely induces hyperactivity of AMPA receptors by a mechanism involving ?2 adrenergic receptor (?2AR). We found that the soluble A? binds to ?2AR, and the extracellular N terminus of ?2AR is critical for the binding. The binding is required to induce G-protein/cAMP/protein kinase A (PKA) signaling, which controls PKA-dependent phosphorylation of GluR1 and ?2AR, and AMPA receptor-mediated excitatory postsynaptic currents (EPSCs). ?2AR and GluR1 also form a complex comprising postsynaptic density protein 95 (PSD95), PKA and its anchor AKAP150, and protein phosphotase 2A (PP2A). Both the third intracellular (i3) loop and C terminus of ?2AR are required for the ?2AR/AMPA receptor complex. A? acutely induces PKA phosphorylation of GluR1 in the complex without affecting the association between two receptors. The present study reveals that non-neurotransmitter A? has a binding capacity to ?2AR and induces PKA-dependent hyperactivity in AMPA receptors.—Wang, D., Govindaiah, G., Liu, R., De Arcangelis, V., Cox, C. L., Xiang, Y. K. Binding of amyloid ? peptide to ?2 adrenergic receptor induces PKA-dependent AMPA receptor hyperactivity.

Wang, Dayong; Govindaiah, G.; Liu, Ruijie; De Arcangelis, Vania; Cox, Charles L.; Xiang, Yang K.

2010-01-01

146

Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes  

SciTech Connect

Highlights: •Nebivolol may act as a partial agonist of ?3-adrenergic receptor (AR). •Nebivolol stimulates mitochondrial DNA replication and protein expression. •Nebivolol promotes mitochondrial synthesis via activation of eNOS by ?3-AR. -- Abstract: Nebivolol is a third-generation ?-adrenergic receptor (?-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24 h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-? coactivator-1? (PGC-1?), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-L-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and ?3-AR blocker SR59230A markedly attenuated PGC-1?, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of ?3-AR receptors.

Huang, Chenglin; Chen, Dongrui; Xie, Qihai [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)] [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Yang, Ying, E-mail: yangying_sh@yahoo.com [Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)] [Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Shen, Weili, E-mail: weili_shen@hotmail.com [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)] [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)

2013-08-16

147

Extract of Chaga mushroom (Inonotus obliquus) stimulates 3T3-L1 adipocyte differentiation.  

PubMed

Chaga mushroom (Inonotus obliquus) has long been used as a folk medicine due to its numerous biological functions such as antibacterial, antiallergic, antiinflammatory and antioxidative activities. In the present study, it was found that the I. obliquus hot water extract (IOWE) activated adipogenesis of 3T3-L1 preadipocytes. Even in the absence of adipogenic stimuli by insulin, the IOWE strongly induced adipogenesis of 3T3-L1 preadipocytes. The major constituent of IOWE was glucose-rich polysaccharides with a molecular mass of 149? kDa. IOWE enhanced the differentiation of 3T3-L1 preadipocytes, increasing TG (triacylglycerol) accumulation that is critical for acquisition of the adipocyte phenotype, in a dose-dependent manner. IOWE stimulated gene expression of C/EBP? (CCAAT/enhancer-binding protein ?) and PPAR? (peroxisome proliferator-activated receptors ?) during adipocyte differentiation, and induced the expression of PPAR? target genes such as aP2 (adipocyte protein 2), LPL (lipoprotein lipase) and CD36 (fatty acid translocase). Immunoblot analysis revealed that IOWE increased the expression of adipogenic makers such as PPAR? and GLUT4 (glucose transporter 4). The luciferase reporter assay demonstrated that IOWE did not exhibit PPAR? ligand activity. Although these results require further investigation, the ability of natural mushroom product to increase PPAR? transcriptional activities may be expected to be therapeutic targets for dyslipidemia and type 2 diabetes. PMID:21031614

Joo, Jeong In; Kim, Dong Hyun; Yun, Jong Won

2010-11-01

148

Exendin-4, a GLP-1 receptor agonist, directly induces adiponectin expression through protein kinase A pathway and prevents inflammatory adipokine expression  

Microsoft Academic Search

Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist that has been used as a drug injected subcutaneously for treatment of type 2 diabetes. Many studies have revealed molecular targets of Ex-4, but its influence on adipokines has not been determined. Our study showed that Ex-4 induced secretion of adiponectin into the culture medium of 3T3-L1 adipocytes. This effect of

Le Thi Kim Chung; Toshio Hosaka; Masaki Yoshida; Nagakatsu Harada; Hiroshi Sakaue; Tohru Sakai; Yutaka Nakaya

2009-01-01

149

Thyroid Hormones, T3 and T4, in the Brain  

PubMed Central

Thyroid hormones (THs) are essential for fetal and post-natal nervous system development and also play an important role in the maintenance of adult brain function. Of the two major THs, T4 (3,5,3?,5?-tetraiodo-l-thyronine) is classically viewed as an pro-hormone that must be converted to T3 (3,5,3?-tri-iodo-l-thyronine) via tissue-level deiodinases for biological activity. THs primarily mediate their effects by binding to thyroid hormone receptor (TR) isoforms, predominantly TR?1 and TR?1, which are expressed in different tissues and exhibit distinctive roles in endocrinology. Notably, the ability to respond to T4 and to T3 differs for the two TR isoforms, with TR?1 generally more responsive to T4 than TR?1. TR?1 is also the most abundantly expressed TR isoform in the brain, encompassing 70–80% of all TR expression in this tissue. Conversion of T4 into T3 via deiodinase 2 in astrocytes has been classically viewed as critical for generating local T3 for neurons. However, deiodinase-deficient mice do not exhibit obvious defectives in brain development or function. Considering that TR?1 is well-established as the predominant isoform in brain, and that TR?1 responds to both T3 and T4, we suggest T4 may play a more active role in brain physiology than has been previously accepted.

Schroeder, Amy C.; Privalsky, Martin L.

2014-01-01

150

Non-NMDA receptor antagonist-induced drinking in rat  

NASA Technical Reports Server (NTRS)

Glutamate has been implicated in the central control of mechanisms that maintain body fluid homeostasis. The present studies demonstrate that intracerebroventricular (i.c.v.) injections of the non-N-methyl-d-aspartate (NMDA) receptor antagonists 6, 7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX) induce drinking in rats. The dipsogenic effect of i.c.v. DNQX was antagonized by the non-NMDA receptor agonist alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). The water intake induced by DNQX was also blocked by pretreatment with a NMDA receptor antagonist, MK-801, but not by angiotensin type 1 (AT1) or acetylcholine muscarinic receptor antagonists (losartan and atropine). The results indicate that non-NMDA receptors may exert a tonic inhibitory effect within brain circuits that control dipsogenic activity and that functional integrity of NMDA receptors may be required for the non-NMDA receptor antagonists to induce water intake. Copyright 1998 Published by Elsevier Science B.V.

Xu, Z.; Johnson, A. K.

1998-01-01

151

G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors*  

PubMed Central

We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB2 receptors, ?-arrestin 2, and ERK1/2 signaling because treatment with CB2 shRNA lentiviral particles, ?-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB2 receptors and increased interaction between ?-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB2 receptors, which up-regulates 5-HT2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB2 receptors; and the ?-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.

Franklin, Jade M.; Carrasco, Gonzalo A.

2013-01-01

152

Human mesangial cells express inducible macrophage scavenger receptor  

Microsoft Academic Search

Human mesangial cells express inducible macrophage scavenger receptor.BackgroundType A scavenger receptors (Scr) mediate the uptake of modified low-density lipoproteins by macrophages. The accumulation of lipids via this process is thought to lead to foam cell formation in atherosclerotic plaques. Human mesangial cells (HMCs) have not been previously shown to express Scr in normal culture. We therefore investigated whether there is

Xiong Z. Ruan; Zac Varghese; Stephen H. Powis; John F. Moorhead

1999-01-01

153

T3/rT3-ratio is associated with insulin resistance independent of TSH.  

PubMed

Thyroid dysfunction has been shown to be associated with insulin resistance (IR). This may involve peripheral thyroid hormone metabolism, which is assumed to be reflected by the ratio triiodothyronine/reverse triiodothyronine (T3/rT3-ratio). To explore a potential association between the T3/rT3-ratio and IR we investigated pairs which differed in IR, but were matched by sex, age, body mass index (BMI), and thyroid stimulating hormone (TSH). For this purpose, matched pair analyses were embedded into a cross sectional study group. 22 pairs were matched from either the first or the third tertile of HOMA%S of a cohort of 353 euthyroid subjects with normal glucose metabolism who did not take any medication. The T3/rT3-ratio was compared in the matched pairs. The T3/rT3-ratio was significantly increased in the insulin resistant subjects compared to their insulin sensitive partners (8.78 ± 0.47 vs. 7.33 ± 0.33, p=0.019). Furthermore the T3/rT3-ratio was lower in men compared to women (p for the within-subject effect=0.046) both in the insulin sensitive and the insulin resistant subjects. Here we show that the T3/rT3-ratio, which is supposed to reflect the tissue thyroid hormone metabolism, is significantly increased in insulin resistant subjects. This further supports a link between thyroid function and IR. PMID:21104580

Ruhla, S; Arafat, A M; Weickert, M O; Osterhoff, M; Isken, F; Spranger, J; Schöfl, C; Pfeiffer, A F H; Möhlig, M

2011-02-01

154

Salvia miltiorrhiza induces VEGF expression and regulates expression of VEGF receptors in osteoblastic cells.  

PubMed

This study investigated in vitro whether Salvia Miltiorrhiza Bunge (SM) induces vascular endothelial growth factor (VEGF) expression and regulates expression of VEGF receptors 1 (VEGFR-1) and 2 (VEGFR-2) on osteoblasts. MC3T3-E1 cells were cultured with SM and VEGF at points 24, 48 and 72 h. A blank control was included. The mRNA expression of VEGF, VEGFR-1 and VEGFR-2 was examined using real-time polymerase chain reaction. VEGF protein expression was examined using enzyme linked immunosorbent assay. SM increased VEGF mRNA expression by 21% at 24 h (p?receptors in MC3T3-E1 cells. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23873436

Wenden, Alex; Yang, Yanqi; Chai, Lei; Wong, Ricky W K

2014-05-01

155

A 350-kb Cosmid Contig in 3p14.2 That Crosses the t(3;8) Hereditary Renal Cell Carcinoma Translocation Breakpoint and 17 Aphidicolin-Induced FRA3B Breakpoints  

Microsoft Academic Search

The constitutive fragile site at human chromosomal band 3p14.2, FRA3B, has been described as the most active common fragile site in the human genome. FRA3B is cytologically indistinguishable from the chromosome 3 breakpoint observed in the hereditary renal cell carcinoma (hRCC) translocation t(3;8) (p14.2;q24.13). Previous work demonstrated that a 1330-kb YAC clone, YC850A6, spans both the t(3;8) translocation and FRA3B

William Paradee; Charles M. Wilke; Liang Wang; Ravi Shridhar; Chadwick M. Mullins; Ann Hoge; Thomas W. Glover; David I. Smith

1996-01-01

156

Potentiation of morphine-induced mechanical antinociception by ?? receptor inhibition: role of peripheral ?? receptors.  

PubMed

We studied the modulation of morphine-induced mechanical antinociception and side effects by ?? receptor inhibition. Both wild-type (WT) and ?? receptor knockout (??-KO) mice showed similar responses to paw pressure (100-600 g). The systemic (subcutaneous) or local (intraplantar) administration of ?? antagonists (BD-1063, BD-1047, NE-100 and S1RA) was devoid of antinociceptive effects in WT mice. However, ??-KO mice exhibited an enhanced mechanical antinociception in response to systemic morphine (1-16 mg/kg). Similarly, systemic treatment of WT mice with ?? antagonists markedly potentiated morphine-induced antinociception, and its effects were reversed by the selective ?? agonist PRE-084. Although the local administration of morphine (50-200 ?g) was devoid of antinociceptive effects in WT mice, it induced dose-dependent antinociception in ??-KO mice. This effect was limited to the injected paw. Enhancement of peripheral morphine antinociception was replicated in WT mice locally co-administered with ?? antagonists and the opioid. None of the ?? antagonists tested enhanced morphine-antinociception in ??-KO mice, confirming a ??-mediated action. Morphine-induced side-effects (hyperlocomotion and inhibition of gastrointestinal transit) were unaltered in ??-KO mice. These results cannot be explained by a direct interaction of ?? ligands with ?-opioid receptors or adaptive changes of ?-receptors in ??-KO mice, given that [(3)H]DAMGO binding in forebrain, spinal cord, and hind-paw skin membranes was unaltered in mutant mice, and none of the ?? drugs tested bound to ?-opioid receptors. These results show that ?? receptor inhibition potentiates morphine-induced mechanical analgesia but not its acute side effects, and that this enhanced analgesia can be induced at peripheral level. PMID:23524304

Sánchez-Fernández, Cristina; Nieto, Francisco Rafael; González-Cano, Rafael; Artacho-Cordón, Antonia; Romero, Lucía; Montilla-García, Ángeles; Zamanillo, Daniel; Baeyens, José Manuel; Entrena, José Manuel; Cobos, Enrique José

2013-07-01

157

Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts  

SciTech Connect

When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of (3H)thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of (3H)thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.

Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y. (Osaka Univ. Medical School (Japan))

1991-03-01

158

Deletion of the thyroid hormone receptor 1 prevents the structural alterations of the cerebellum induced by hypothyroidism  

Microsoft Academic Search

Thyroid hormone (T3) controls critical aspects of cerebellar development, such as migration of postmitotic granule cells and terminal differentiation of Purkinje cells. T3 acts through nuclear receptors (TR) of two types, TR1 and TR, that either repress or activate gene expression. We have analyzed the cerebellar structure of developing mice lacking the TR1 isoform, which normally accounts for about 80%

Beatriz Morte; Jimena Manzano; Thomas Scanlan; Björn Vennström; Juan Bernal

2002-01-01

159

Forced swim-induced musculoskeletal hyperalgesia is mediated by CRF2 receptors but not by TRPV1 receptors.  

PubMed

The exacerbation of musculoskeletal pain by stress in humans is modeled by the musculoskeletal hyperalgesia in rodents following a forced swim. We hypothesized that stress-sensitive corticotropin releasing factor (CRF) receptors and transient receptor vanilloid 1 (TRPV1) receptors are responsible for the swim stress-induced musculoskeletal hyperalgesia. We confirmed that a cold swim (26 °C) caused a transient, morphine-sensitive decrease in grip force responses reflecting musculoskeletal hyperalgesia in mice. Pretreatment with the CRF2 receptor antagonist astressin 2B, but not the CRF1 receptor antagonist NBI-35965, attenuated this hyperalgesia. Desensitizing the TRPV1 receptor centrally or peripherally using desensitizing doses of resiniferatoxin (RTX) failed to prevent the musculoskeletal hyperalgesia produced by cold swim. SB-366791, a TRPV1 antagonist, also failed to influence swim-induced hyperalgesia. Together these data indicate that swim stress-induced musculoskeletal hyperalgesia is mediated, in part, by CRF2 receptors but is independent of the TRPV1 receptor. PMID:23624287

Abdelhamid, Ramy E; Kovacs, Katalin J; Pasley, Jeffrey D; Nunez, Myra G; Larson, Alice A

2013-09-01

160

Spurious t3 thyrotoxicosis unmasking multiple myeloma.  

PubMed

Objective. To document a case of spurious T3 thyrotoxicosis in a 54-year-old woman. Methods. We present the diagnostic approach of a patient with euthyroid hypertri-iodothyronemia. Results. A 54-year-old, clinically euthyroid woman without personal or family history of thyroid disease referred to endocrinology for possible T3 thyrotoxicosis, after thyroid function tests revealed total T3 > 800?ng/dL (reference range 60-181), normal TSH, and T4. The laboratory data were not compatible with the clinical picture, so thyroid binding globulin abnormalities were suspected. Additional laboratory studies confirmed the diagnosis of multiple myeloma. Conclusion. Monoclonal gammopathy is characterized by the presence of a monoclonal immunoglobulin in the serum or urine, occurring in multiple myeloma, and can cause assay interference and spurious results. We identify a newly recognized cause of euthyroid hypertri-iodothyronemia, due to binding of T3 to monoclonal immunoglobulins in the setting of multiple myeloma. Our case is the only one to date suggesting that monoclonal immunoglobulins from multiple myeloma may exhibit binding to T3 only. PMID:23984117

Antonopoulou, Marianna; Silverberg, Arnold

2013-01-01

161

TRPA1 receptors mediate environmental irritant-induced meningeal vasodilatation.  

PubMed

The TRPA1 receptor is a member of the transient receptor potential (TRP) family of ion channels expressed in nociceptive neurons. TRPA1 receptors are targeted by pungent compounds from mustard and garlic and environmental irritants such as formaldehyde and acrolein. Ingestion or inhalation of these chemical agents causes irritation and burning in the nasal and oral mucosa and respiratory lining. Headaches have been widely reported to be induced by inhalation of environmental irritants, but it is unclear how these agents produce headache. Stimulation of trigeminal neurons releases CGRP and substance P and induces neurogenic inflammation associated with the pain of migraine. Here we test the hypothesis that activation of TRPA1 receptors is the mechanistic link between environmental irritants and peptide-mediated neurogenic inflammation. Known TRPA1 agonists and environmental irritants stimulate CGRP release from dissociated rat trigeminal ganglia neurons and this release is blocked by a selective TRPA1 antagonist, HC-030031. Further, TRPA1 agonists and environmental irritants increase meningeal blood flow following intranasal administration. Prior dural application of the CGRP antagonist, CGRP(8-37), or intranasal or dural administration of HC-030031, blocks the increases in blood flow elicited by environmental irritants. Together these results demonstrate that TRPA1 receptor activation by environmental irritants stimulates CGRP release and increases cerebral blood flow. We suggest that these events contribute to headache associated with environmental irritants. PMID:21075522

Kunkler, Phillip Edward; Ballard, Carrie Jo; Oxford, Gerry Stephen; Hurley, Joyce Harts

2011-01-01

162

[alpha ]-Lipoic acid prevents the development of glucose-induced insulin resistance in 3T3-L1 adipocytes and accelerates the decline in immunoreactive insulin during cell incubation  

Microsoft Academic Search

Oxidative stress has been implicated in glucose toxicity. We tested the hypothesis that certain antioxidants may prevent insulin-resistant glucose transport that develops in adipocytes after sustained exposure to high glucose, provided insulin is present. The antioxidant [alpha ]-lipoic acid has been proposed as an insulin sensitizer. 3T3-L1 adipocytes were preincubated 18 hours in media containing insulin (0.6 nmol\\/L) with low

Eddie L. Greene; Bryce A. Nelson; Katherine A. Robinson; Maria G. Buse

2001-01-01

163

The metabolism of T4 and T3 in cultured chick-embryo heart cells.  

PubMed

Thyroxine (T4) and tri-iodothyronine (T3) were metabolized in cultured chick-embryo heart cells via inner-ring de-iodination and O-sulfation. The products of T3 metabolism were 3,3'T2, 3'T1, and sulfated esters of T3 and 3,3'T2. The major product of T4 degradation was 3,3',5'-T3 (rT3). ONo T3 was produced from T4. Propylthioracil inhibited the metabolism of T3. Pretreatment of cultures with T3 or T4 enhanced the metabolism of both hormones; actinomycin D and cycloheximide inhibited the stimulatory effect of T3. The stimulation by T3 was linearly related to the log of the concentration of T3 in cells grown in normal chick serum or in cells grown in dehormonized serum. These results suggest that thyroid hormones induced an increased synthesis of the enzymes involved in their metabolism and therefore may regulate their own disposal rate. PMID:7439522

Dickstein, Y; Schwartz, H; Gross, J; Gordon, A

1980-10-01

164

Artepillin C, as a PPAR? ligand, enhances adipocyte differentiation and glucose uptake in 3T3-L1 cells.  

PubMed

The nuclear receptor peroxisome proliferator-activated receptor (PPAR) ? plays an important role in adipocyte differentiation. Its ligands, including thiazolidinediones, improve insulin sensitivity in type 2 diabetes. We investigated the effects of artepillin C, an ingredient of Baccharis dracunculifolia, on adipogenesis and glucose uptake using 3T3-L1 cells. In PPAR? ligand-binding assays, artepillin C exhibited binding affinity toward PPAR?. Artepillin C dose-dependently enhanced adipocyte differentiation of 3T3-L1 cells. As a result of the artepillin C-induced adipocyte differentiation, the gene expression of PPAR? and its target genes, such as aP2, adiponectin and glucose transporter (GLUT) 4, was increased. These increases were abolished by cotreatment with GW9662, a PPAR? antagonist. In mature 3T3-L1 adipocytes, artepillin C significantly enhanced the basal and insulin-stimulated glucose uptake. These effects were decreased by cotreatment with a PI3K inhibitor. Although artepillin C had no effects on the insulin signaling cascade, artepillin C enhanced the expression and plasma membrane translocation of GLUT1 and GLUT4 in mature adipocytes. In conclusion, these findings suggest that artepillin C promotes adipocyte differentiation and glucose uptake in part by direct binding to PPAR?, which could be the basis of the pharmacological benefits of green propolis intake in reducing the risk of type 2 diabetes. PMID:21219874

Choi, Sun-Sil; Cha, Byung-Yoon; Iida, Kagami; Lee, Young-Sil; Yonezawa, Takayuki; Teruya, Toshiaki; Nagai, Kazuo; Woo, Je-Tae

2011-04-01

165

TRPV1 receptor inhibition decreases CCL2-induced hyperalgesia.  

PubMed

Modulation of nociceptive synaptic transmission in the spinal cord is implicated in the development and maintenance of several pathological pain states. The chemokine CCL2 (C-C motif ligand 2) was shown to be an important factor in the development of neuropathic pain after peripheral nerve injury. In our experiments we have studied the effect of CCL2 application and TRPV1 (transient receptor potential vanilloid 1) receptor activation on nociceptive signaling and the modulation of synaptic transmission. Intrathecal drug application in behavioral experiments and patch-clamp recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs, eEPSCs) from superficial dorsal horn neurons in acute rat spinal cord slices were used. The intrathecal application of CCL2 induced thermal hyperalgesia and mechanical allodynia, while pretreatment with the TRPV1 receptor antagonist SB366791 diminished the thermal but not the mechanical hypersensitivity. Patch-clamp experiments showed an increase of sEPSC and mEPSC (124.5 ± 12.8% and 161.2 ± 17.3%, respectively) frequency in dorsal horn neurons after acute CCL2 application. This CCL2-induced increase was prevented by SB366791 pretreatment (89.4 ± 6.0%, 107.5 ± 14.2%). CCL2 application increased the amplitude of eEPSCs (188.1 ± 32.1%); this increase was significantly lower in experiments with SB366791 pretreatment (120.8 ± 17.2%). Our results demonstrate that the activation of spinal TRPV1 receptors plays an important role in the modulation of nociceptive signaling induced by CCL2 application. The mechanisms of cooperation between the CCL2 activated receptors and TRPV1 receptors on the central branches of primary afferent fibers may be especially important during different pathological pain states and need to be further investigated. PMID:24495396

Spicarova, Diana; Adamek, Pavel; Kalynovska, Nataliia; Mrozkova, Petra; Palecek, Jiri

2014-06-01

166

Insulin-induced loss of the insulin receptor in IM-9 lymphocytes. A biological process mediated through the insulin receptor.  

PubMed

Exposure of cultured lymphocytes of the IM-9 line to insulin results in a rapid, time-dependent reduction in the number of insulin receptors to a new steady state concentration. Both the rate of loss and the net loss of receptors were directly related to the ambient insulin concentration. The insulin-induced loss of receptors was mediated by binding of insulin to the receptor itself; insulins, which varied 200-fold in biopotency, produced receptor loss in direct proportion to the ability of each insulin to occupy the receptor. The residual insulin receptors were normal following insulin-mediated receptor loss by a variety of sensitive binding criteria. While insulin binding to its receptors was a necessary condition to induce receptor loss, it was not sufficient. Thus, reduction in the temperature of the preincubation from 37 degrees C to 20 degrees C (which enhanced the total amount of insulin bound to the receptor) abolished the loss of insulin receptors. Likewise, cycloheximide prevented the insulin-induced loss of receptors. Furthermore, turkey erythrocytes, which lack active macromolecular synthesis, had no change in the concentration of insulin receptors when exposed to insulin for similar periods. Interestingly, the turkey erythrocytes, when exposed to insulin or to proinsulin, showed a time- and concentration-dependent increase in the affinity of the insulin receptor over a restricted part of the insulin-binding isotherm, which was reversed over a period of several hours following removal of hormone. The insulin-mediated decrease in receptor number on IM-9 lymphocytes was reversible. Following removal of insulin from the growth medium, about one-half of the receptors were restored within 10 h and the full complement of insulin receptors was restored within 24 h. Cycloheximide prevented restoration of the insulin receptor. PMID:7000764

Kosmakos, F C; Roth, J

1980-10-25

167

Genistein inhibits the proliferation and differentiation of MCF-7 and 3T3-L1 cells via the regulation of ER? expression and induction of apoptosis  

PubMed Central

The present study investigated the effect of the phytochemical genistein on the proliferation and differentiation of MCF-7 and 3T3-L1 cells via the regulation of estrogen receptor-? (ER?) expression and the induction of apoptosis. When MCF-7 human breast cancer cells were treated with 50, 100, 150 and 200 ?M genistein for 24, 48 or 72 h, cell growth was significantly decreased in a concentration-dependent manner. Notably, the patterns of ER? expression and proliferation in MCF-7 cells treated with genistein were similar. Furthermore, ER? expression in differentiating 3T3-L1 cells was significantly inhibited by 48 h treatment with 50 ?M genistein, which was selected based on the results of cytotoxicity assays on 3T3-L1 preadipocytes [lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays]. Under the same conditions, genistein-induced apoptotic features were observed in MCF-7 and differentiating 3T3-L1 cells. This observation is supported by the finding that B-cell lymphoma 2 (Bcl-2) expression was reduced while that of Bcl-2-associated X protein (Bax) was induced by genistein. The results of the present study suggest that an ER?-related pathway and the induction of apoptosis are involved in the proliferation of MCF-7 cells and the differentiation of 3T3-L1 cells.

CHOI, EUN JEONG; JUNG, JAE YEON; KIM, GUN-HEE

2014-01-01

168

Inhibitory effect of andrographolide in 3T3-L1 adipocytes differentiation through the PPAR? pathway.  

PubMed

Andrographolide (AG), an active compound found in Andrographis paniculate Nees, has been shown to exert anti-inflammatory, anticancer and anti-hyperglycemic effects. However, its biological activities against obesity have not been reported. The purpose of this study was to investigate the effect of AG on the differentiation of 3T3-L1 preadipocytes. We found AG significantly inhibited not only on adipocyte differentiation induced by standard adipogenic agents and MDI, but also on the adipogenesis-related transcription factor, peroxisome proliferator-activated receptor ? (PPAR?), as well as the expressions of the PPAR? targeted genes, such as CD36, LPL, FAS and other adiocyte markers. Taken together, our data showed AG inhibited the early stage of adipogenic differentiation, in part via the inhibition of PPAR?-dependent mechanisms. PMID:22449851

Jin, Lina; Fang, Wenqiang; Li, Bo; Shi, Guojun; Li, Xiaoying; Yang, Ying; Yang, Jian; Zhang, Zhiguo; Ning, Guang

2012-07-01

169

A primer on cytokines: sources, receptors, effects, and inducers.  

PubMed Central

Protection against pathogens is a prerequisite for survival of most organisms. To cope with this continuous challenge, complex defense mechanisms have evolved. The construction, adaptation, and maintenance of these mechanisms are under control of an extensive network of regulatory proteins called cytokines. A great number of cytokines have been described over the last 2 decades. This review consists of an overview of cytokines that are involved in immune responses and describes some historical and general aspects as well as prospective clinical applications. Major biological effects together with information on cytokine receptors, producers, inducers, and biochemical and molecular characteristics are listed in tables. In addition, some basic information is given on cytokine receptor signal transduction. Finally, the recent discoveries of cytokine receptors functioning as coreceptors in the pathogenesis of human immunodeficiency virus are summarized.

Curfs, J H; Meis, J F; Hoogkamp-Korstanje, J A

1997-01-01

170

Crosslinking-Induced Endocytosis of Acetylcholine Receptors by Quantum Dots  

PubMed Central

In a majority of patients with myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies target postsynaptic AChR clusters and thus compromise the membrane integrity of neuromuscular junctions (NMJs) and lead to muscle weakness. Antibody-induced endocytosis of AChRs in the postsynaptic membrane represents the initial step in the pathogenesis of MG; however, the molecular mechanisms underlying AChR endocytosis remain largely unknown. Here, we developed an approach to mimic the pathogenic antibodies for inducing the crosslinking and internalization of AChRs from the postsynaptic membrane. Using biotin-?-bungarotoxin and quantum dot (QD)-streptavidin, cell-surface and internalized AChRs could be readily distinguished by comparing the size, fluorescence intensity, trajectory, and subcellular localization of the QD signals. QD-induced AChR endocytosis was mediated by clathrin-dependent and caveolin-independent mechanisms, and the trafficking of internalized AChRs in the early endosomes required the integrity of microtubule structures. Furthermore, activation of the agrin/MuSK (muscle-specific kinase) signaling pathway strongly suppressed QD-induced internalization of AChRs. Lastly, QD-induced AChR crosslinking potentiated the dispersal of aneural AChR clusters upon synaptic induction. Taken together, our results identify a novel approach to study the mechanisms of AChR trafficking upon receptor crosslinking and endocytosis, and demonstrate that agrin-MuSK signaling pathways protect against crosslinking-induced endocytosis of AChRs.

Geng, Lin; Peng, H. Benjamin

2014-01-01

171

Vasopressin-induced sensitization: involvement of neurohypophyseal peptide receptors.  

PubMed

Rats pretreated with an intracerebroventricular (i.c.v.) injection of 10 pmol of vasopressin or vasopressin analogs, including deamino-D-vasopressin, [pGlu4,Cyt6]vasopressin, [pGlu-Asn-Cys(Cys)]Pro-Leu-Gly-NH2, des-Gly-NH9(2)-vasopressin, Pro-Leu-Gly-NH2, Pro-Arg-Gly-NH2, became markedly hyper-responsive to the motor effects, 24 h later, to a subsequent challenge dose of vasopressin, but not vasopressin-related peptides. A vasopressin V1 receptor antagonist, [d(CH2)1(5),Tyr(Me)2]vasopressin, but not the vasopressin V2 receptor antagonist, [d(CH2)1(5),Tyr(Et)2,Val4]vasopressin, or a more selective vasopressin V2 receptor antagonist, [d(CH2)1(5),D-Ile2,Ile4]vasopressin, or the oxytocin receptor antagonist, [d(CH2)1(5),Tyr(Me)2,Thr4,Orn8,Tyr-NH9(2)]vasotocin ([d(CH2)1(5),Tyr(Me)2,Thr4,Tyr-NH9(2)]OVT), blocked vasopressin and vasopressin analog-induced sensitization. Furthermore, both vasopressin V2 receptor antagonists were found to sensitize the brain to a subsequent vasopressin injection. This vasopressin V2 receptor antagonist-induced sensitization was also blocked by the vasopressin V1 receptor antagonist. Next, we wanted to determine if this sensitization process could involve the release of endogenous vasopressin in the brain as reflected in an amplification of vasopressin mRNA expression. However pretreatment of rats with an i.c.v. vasopressin injection was not associated with an increase in vasopressin mRNA expression in the bed nucleus of the stria terminalis, medial amygdala or the paraventricular nucleus of the hypothalamus when measured 0, 1, 3, 7, 12, or 24 h after the first vasopressin injection. As many vasopressin analogs can induce sensitization, we suggest that a novel type of receptor may be involved in the sensitization process. PMID:8788413

Poulin, P; Szot, P; Dorsa, D M; Pittman, Q J

1995-12-27

172

Impact of stress hormone on adipogenesis in the 3T3-L1 adipocytes.  

PubMed

Stress hormone is known to play a vital role in lipolysis and adipogenesis in fat cells. The present study was carried out to evaluate the effect of epinephrine on adipogenesis in the 3T3-L1 cells. The investigation on adipogenesis was done in both mono and co-cultured 3T3-L1 cells. 3T3-L1 preadipocytes and C2C12 cells were grown independently on transwell plates and transferred to differentiation medium. Following differentiation, C2C12 cells transferred to 3T3-L1 plate and treated with medium containing 10 ?g/ml of epinephrine. Adipogenic markers such as fatty acid binding protein 4, peroxisome proliferator activating receptor, CCAAT/enhancer-binding protein, adiponectin, lipoprotein lipase and fatty acid synthase mRNA expressions were evaluated in the 3T3-L1 cells. Epinephrine treatment reduced adipogenesis, evidenced by reducing adipogenic marker mRNA expression in the 3T3-L1 cells. In addition, glycerol accumulation and oil red-O staining supported the reduced rate of adipogenesis. Taking all together, it is concluded that the stress hormone, epinephrine reduces the rate of adipogenesis in the mono and co-cultured 3T3-L1 cells. In addition, the rate of adipogenesis is much reduced in the co-cultured 3T3-L1 cells compared monocultured 3T3-L1 cells. PMID:23943061

Pandurangan, Muthuraman; Ravikumar, Sambandam

2014-08-01

173

Dark chocolate receptors: epicatechin-induced cardiac protection is dependent on delta-opioid receptor stimulation.  

PubMed

Epicatechin, a flavonoid, is a well-known antioxidant linked to a variety of protective effects in both humans and animals. In particular, its role in protection against cardiovascular disease has been demonstrated by epidemiologic studies. Low-dose epicatechin, which does not have significant antioxidant activity, is also protective; however, the mechanism by which low-dose epicatechin induces this effect is unknown. Our laboratory tested the hypothesis that low-dose epicatechin mediates cardiac protection via opioid receptor activation. C57BL/6 mice were randomly assigned to 1 of 10 groups: control, epicatechin, naloxone (nonselective opioid receptor antagonist), epicatechin + naloxone, naltrindole (?-specific opioid receptor antagonist), epicatechin + naltrindole, norbinaltorphimine (nor-BNI, ?-specific opioid receptor antagonist), epicatechin + nor-BNI, 5-hydroxydecanoic acid [5-HD, ATP-sensitive potassium channel antagonist], and epicatechin + 5-HD. Epicatechin (1 mg/kg) or other inhibitors (5 mg/kg) were administered by oral gavage or intraperitoneal injection, respectively, daily for 10 days. Mice were subjected to 30 min coronary artery occlusion followed by 2 h of reperfusion, and infarct size was determined via planimetry. Whole heart homogenates were assayed for downstream opioid receptor signaling targets. Infarct size was significantly reduced in epicatechin- and epicatechin + nor-BNI-treated mice compared with control mice. This protection was blocked by naloxone, naltrindole, and 5-HD. Epicatechin and epicatechin + nor-BNI increased the phosphorylation of Src, Akt, and I?B?, while simultaneously decreasing the expression of c-Jun NH(2)-terminal kinase and caspase-activated DNase. All signaling effects are consistent with opioid receptor stimulation and subsequent cardiac protection. Naloxone, naltrindole, and 5-HD attenuated these effects. In conclusion, epicatechin acts via opioid receptors and more specifically through the ?-opioid receptor to produce cardiac protection from ischemia-reperfusion injury. PMID:20833967

Panneerselvam, Mathivadhani; Tsutsumi, Yasuo M; Bonds, Jacqueline A; Horikawa, Yousuke T; Saldana, Michelle; Dalton, Nancy D; Head, Brian P; Patel, Piyush M; Roth, David M; Patel, Hemal H

2010-11-01

174

Agonist-induced endocytosis of rat somatostatin receptor 1.  

PubMed

Somatostatin-receptor 1 (sst1) is an autoreceptor in the central nervous system that regulates the release of somatostatin. Sst1 is present intracellularly and at the cell surface. To investigate sst1 trafficking, rat sst1 tagged with epitope was expressed in rat insulinoma cells 1046-38 (RIN-1046-38) and tracked by antibody labeling. Confocal microscopic analysis revealed colocalization of intracellularly localized rat sst1-human simplex virus (HSV) with Rab5a-green fluorescent protein and Rab11a-green fluorescent protein, indicating the distribution of the receptor in endocytotic and recycling organelles. Somatostatin-14 induced internalization of cell surface receptors and reduction of binding sites on the cell surface. It also stimulated recruitment of intracellular sst1-HSV to the plasma membrane. Confocal analysis of sst1-HSV revealed that the receptor was initially transported within superficial vesicles. Prolonged stimulation of the cells with the peptide agonist induced intracellular accumulation of somatostatin-14. Because the number of cell surface binding sites did not change during prolonged stimulation, somatostatin-14 was internalized through a dynamic process of continuous endocytosis, recycling, and recruitment of intracellularly present sst1-HSV. Accumulated somatostatin-14 bypassed degradation via the endosomal-lysosomal route and was instead rapidly released as intact and biologically active somatostatin-14. Our results show for the first time that sst1 mediates a dynamic process of endocytosis, recycling, and re-endocytosis of its cognate ligand. PMID:17170097

Roosterman, Dirk; Kreuzer, Oliver J; Brune, Nicole; Cottrell, Graeme S; Bunnett, Nigel W; Meyerhof, Wolfgang; Steinhoff, Martin

2007-03-01

175

Light-inducible receptor tyrosine kinases that regulate neurotrophin signalling.  

PubMed

Receptor tyrosine kinases (RTKs) are a family of cell-surface receptors that have a key role in regulating critical cellular processes. Here, to understand and precisely control RTK signalling, we report the development of a genetically encoded, photoactivatable Trk (tropomyosin-related kinase) family of RTKs using a light-responsive module based on Arabidopsis thaliana cryptochrome 2. Blue-light stimulation (488?nm) of mammalian cells harbouring these receptors robustly upregulates canonical Trk signalling. A single light stimulus triggers transient signalling activation, which is reversibly tuned by repetitive delivery of blue-light pulses. In addition, the light-provoked process is induced in a spatially restricted and cell-specific manner. A prolonged patterned illumination causes sustained activation of extracellular signal-regulated kinase and promotes neurite outgrowth in a neuronal cell line, and induces filopodia formation in rat hippocampal neurons. These light-controllable receptors are expected to create experimental opportunities to spatiotemporally manipulate many biological processes both in vitro and in vivo. PMID:24894073

Chang, Ki-Young; Woo, Doyeon; Jung, Hyunjin; Lee, Sangkyu; Kim, Sungsoo; Won, Joungha; Kyung, Taeyoon; Park, Hyerim; Kim, Nury; Yang, Hee Won; Park, Jae-Yong; Hwang, Eun Mi; Kim, Daesoo; Heo, Won Do

2014-01-01

176

Vasoactive Intestinal Polypeptide and Muscarinic Receptors: Supersensitivity Induced by Long-Term Atropine Treatment  

NASA Astrophysics Data System (ADS)

Long-term treatment of rats with atropine induced large increases in the numbers of muscarinic receptors and receptors for vasoactive intestinal polypeptide in the salivary glands. Since receptors for vasoactive intestinal polypeptide coexist with muscarinic receptors on the same neurons in this preparation, the results suggest that a drug that alters the sensitivity of one receptor may also affect the sensitivity of the receptor for a costored transmitter and in this way contribute to the therapeutic or side effects of the drug.

Hedlund, Britta; Abens, Janis; Bartfai, Tamas

1983-04-01

177

Heterologous desensitization of bombesin-induced mitogenesis by prolonged exposure to vasopressin: a post-receptor signal transduction block.  

PubMed Central

Prolonged exposure of quiescent Swiss 3T3 cells to vasopressin prevents mitogenic stimulation on subsequent addition of bombesin. This heterologous desensitization is selective and can be mimicked by vasopressin agonists, including [Lys8]vasopressin and oxytocin but not by the V1-type-specific vasopressin receptor antagonist [Pmp1,O-Me-Tyr2,Arg8]vasopressin [where Pmp is 1-(beta-mercapto-beta,beta-cyclopenthamethylene propionic acid)]. Furthermore, vasopressin-induced loss of responsiveness to bombesin can be blocked by addition of this antagonist, indicating that heterologous desensitization is mediated through the vasopressin receptor. Desensitization requires prolonged incubation (half-maximal desensitization occurring after approximately 20 hr of pretreatment) and continuous protein synthesis. Bombesin responsiveness is restored by incubation in the absence of vasopressin. Pretreatment does not alter the number, affinity, or internalization capacity of the bombesin receptors. However, the induction of the protooncogene c-fos by bombesin is profoundly inhibited after vasopressin pretreatment. We suggest that the coupling of the activated bombesin receptor to the generation of its early signals is impaired in desensitized cells. Images

Millar, J B; Rozengurt, E

1989-01-01

178

[Oxidized glutathione induces activation of the epidermal growth factor receptor and MAP kinases ERK 1,2].  

PubMed

Ligand-independent activation ("transactivation") of the epidermal growth factor receptor (EGFR) was demonstrated upon cell stimulation with cytokines, activators of G-protein-coupled receptors and various stressors. Recently, we showed transactivation of EGFR and activation of transcription factor STAT3, rather than STAT1, induced by glutathione disulfide (GSSG) and glutoxim in epidermoid carcinoma A431 cells (Burova et al., Dokl. Akad. Nauk., 2005, 404: 1-3). Glutoxim (PHARMA-VAM, Moscow) is a pharmacological synthetic analogue of GSSG, whose therapeutic use as an immunomodulator has been permitted. In this study, we investigated dynamics of EGFR activation upon A431 cell stimulation with GSSG and glutoxim. The time course of activation has a sinuous pattern. It has been shown that the intrinsic EGFR tyrosine kinase is responsible for the receptor phosphorylation induced by GSSG and glutoxim. Here, we also demonstrated the activation of ERK 1,2 upon treatment of A431 cells and HER14 cells (HIN 3T3 fibroblasts transfected with full-length EGFR) with these drugs. ERK 1,2 activation was abolished by AG1478, a pharmacological inhibitor of EGFR tyrosine kinase, implicating intrinsic EGFR tyrosine kinase in this process. PMID:16893056

Vasilenko, K P; Burova, E B; Antonov, V G; Nikol'ski?, N N

2006-01-01

179

Zinc-chelated Vitamin C Stimulates Adipogenesis of 3T3-L1 Cells  

PubMed Central

Adipose tissue development and function play a critical role in the regulation of energy balance, lipid metabolism, and the pathophysiology of metabolic syndromes. Although the effect of zinc ascorbate supplementation in diabetes or glycemic control is known in humans, the underlying mechanism is not well described. Here, we investigated the effect of a zinc-chelated vitamin C (ZnC) compound on the adipogenic differentiation of 3T3-L1 preadipocytes. Treatment with ZnC for 8 d significantly promoted adipogenesis, which was characterized by increased glycerol-3-phosphate dehydrogenase activity and intracellular lipid accumulation in 3T3-L1 cells. Meanwhile, ZnC induced a pronounced up-regulation of the expression of glucose transporter type 4 (GLUT4) and the adipocyte-specific gene adipocyte protein 2 (aP2). Analysis of mRNA and protein levels further showed that ZnC increased the sequential expression of peroxisome proliferator-activated receptor gamma (PPAR?) and CCAAT/enhancer-binding protein alpha (C/EBP?), the key transcription factors of adipogenesis. These results indicate that ZnC could promote adipogenesis through PPAR? and C/EBP?, which act synergistically for the expression of aP2 and GLUT4, leading to the generation of insulin-responsive adipocytes and can thereby be useful as a novel therapeutic agent for the management of diabetes and related metabolic disorders.

Ghosh, Chiranjit; Yang, Seung Hak; Kim, Jong Geun; Jeon, Tae-Il; Yoon, Byung Hyun; Lee, Jai Young; Lee, Eun Young; Choi, Seok Geun; Hwang, Seong Gu

2013-01-01

180

Morphine induces ? opioid receptor endocytosis in guinea pig enteric neurons following prolonged receptor activation  

PubMed Central

Background & Aims The ? opioid receptor (?OR) undergoes rapid endocytosis following acute stimulation with opioids and most opiates, but not with morphine. We investigated whether prolonged activation of ?OR affects morphine’s ability to induce receptor endocytosis in enteric neurons. Methods We compared the effects of morphine, a poor ?OR-internalizing opiate, and [D-Ala2, MePhe4,Gly-ol5] enkephalin (DAMGO), a potent ?OR-internalizing agonist, on ?OR trafficking in enteric neurons and on the expression of dynamin and ?-arrestin immunoreactivity in the ileum of guinea pigs rendered tolerant by chronic administration of morphine. Results Morphine (100 µM) strongly induced endocytosis of ?OR in tolerant but not naïve neurons (55.7%±9.3% vs. 24.2%±7.3%, P<0.001) whereas DAMGO (10 µM) strongly induced internalization of ?OR in neurons from tolerant and naïve animals (63.6%±8.4% and 66.5%±3.6%). Morphine- or DAMGO-induced ?OR endocytosis resulted from direct interactions between the ligand and the ?OR, because endocytosis was not affected by tetrodotoxin, a blocker of endogenous neurotransmitter release. Ligand-induced ?OR internalization was inhibited by pretreatment with the dynamin inhibitor, dynasore. Chronic morphine administration resulted in a significant increase in dynamin and translocation of dynamin immunoreactivity from the intracellular pool to the plasma membrane, but did not affect ? arrestin immunoreactivity. Conclusion Chronic activation of ?ORs increases the ability of morphine to induce ?OR endocytosis in enteric neurons, which depends on the level and cellular localization of dynamin, a regulatory protein that has an important role in receptor-mediated signal transduction in cells.

Patierno, Simona; Anselmi, Laura; Jaramillo, Ingrid; Scott, David; Garcia, Rachel; Sternini, Catia

2010-01-01

181

GPER mediates the inhibitory actions of estrogen on adipogenesis in 3T3-L1 cells through perturbation of mitotic clonal expansion.  

PubMed

G-protein-coupled estrogen receptor 1 (GPER) mediates non-genomic signaling of estrogenic events. Here we showed for the first time that Gper/GPER is expressed in Swiss 3T3 mouse embryo preadipocytes 3T3-L1, and that Gper/GPER is up-regulated during differentiation of the cells induced by monocyte differentiation-inducing (MDI) cocktail. Activation of GPER by the natural ligand 17?-estradiol (E2), and the specific agonist G1, was shown to inhibit lipid accumulation in 3T3-L1 cells, while such inhibition was reversed upon knockdown of GPER using specific siRNA. GPER was also found to mediate perturbation of mitotic clonal expansion (MCE) in these cells by inhibiting cell cycle arrest during MDI cocktail-induced differentiation. Persistent activation of cell cycle regulating factors cyclin-dependant kinase (CDK) 4, CDK6 and cyclin D1, and phosphorylation of retinoblastoma (Rb) protein at serine 795 was observed in the G1-treated cells. Taken together, our results indicate that E2-GPER signaling leads to an inhibition of adipogenesis in 3T3-L1 cells via perturbation of MCE. PMID:23871778

Zhu, Pei; Yuen, Jacky M L; Sham, Kathy W Y; Cheng, Christopher H K

2013-11-01

182

Feedback-Inducible Nuclear-Receptor-Driven Reporter Gene Expression in Transgenic Mice  

Microsoft Academic Search

Understanding nuclear receptor signaling in vivo would be facilitated by an efficient methodology to determine where a nuclear receptor is active. Herein, we present a feedback-inducible expression system in transgenic mice to detect activated nuclear receptor effector proteins by using an inducible reporter gene. With this approach, reporter gene induction is not limited to a particular tissue, and, thus, this

Alexander Mata de Urquiza; Ludmila Solomin; Thomas Perlmann

1999-01-01

183

Advanced glycation end-products (AGEs) induce concerted changes in the osteoblastic expression of their receptor RAGE and in the activation of extracellular signal-regulated kinases (ERK).  

PubMed

An increase in the interaction between advanced glycation end-products (AGEs) and their receptor RAGE is believed to contribute to the pathogenesis of chronic complications of Diabetes mellitus, which can include bone alterations such as osteopenia. We have recently found that extracellular AGEs can directly regulate the growth and development of rat osteosarcoma UMR106 cells, and of mouse calvaria-derived MC3T3E1 osteoblasts throughout their successive developmental stages (proliferation, differentiation and mineralisation), possibly by the recognition of AGEs moieties by specific osteoblastic receptors which are present in both cell lines. In the present study we examined the possible expression of RAGE by UMR106 and MC3T3E1 osteoblastic cells, by immunoblot analysis. We also investigated whether short-, medium- or long-term exposure of osteoblasts to extracellular AGEs, could modify their affinity constant and maximal binding for AGEs (by 125I-AGE-BSA binding experiments), their expression of RAGE (by immunoblot analysis) and the activation status of the osteoblastic ERK 1/2 signal transduction mechanism (by immunoblot analysis for ERK and P-ERK). Our results show that both osteoblastic cell lines express readily detectable levels of RAGE. Short-term exposure of phenotypically mature osteoblastic UMR106 cells to AGEs decrease the cellular density of AGE-binding sites while increasing the affinity of these sites for AGEs. This culture condition also dose-dependently increased the expression of RAGE and the activation of ERK. In proliferating MC3T3E1 pre-osteoblasts, 24-72 h exposure to AGEs did not modify expression of RAGE, ERK activation or the cellular density of AGE-binding sites. However, it did change the affinity of these binding sites forAGEs, with both higher- and lower-affinity sites now being apparent. Medium-term ( 1 week) incubation of differentiated MC3T3E1 osteoblasts with AGEs, induced a simultaneous increase in RAGE expression and in the relative amount of P-ERK. Mineralising MC3T3E1 cultures grown for 3 weeks in the presence of extracellular AGEs showed a decrease both in RAGE and P-ERK expression. These results indicate that, in phenotypically mature osteoblastic cells, changes in ERK activation closely follow the AGEs-induced regulation of RAGE expression. Thus, the AGEs-induced biological effects that we have observed previously in osteoblasts, could be mediated by RAGE in the later stages of development, and mediated by other AGE receptors in the earlier pre-osteoblastic stage. PMID:12962137

Cortizo, Ana M; Lettieri, María G; Barrio, Daniel A; Mercer, Natalia; Etcheverry, Susana B; McCarthy, Antonio D

2003-08-01

184

RIC-3 differentially modulates ?4?2 and ?7 nicotinic receptor assembly, expression, and nicotine-induced receptor upregulation  

PubMed Central

Background Recent work has shown that the chaperone resistant to inhibitors of acetylcholinesterase (RIC-3) is critical for the folding, maturation and functional expression of a variety of neuronal nicotinic acetylcholine receptors. ?7 nicotinic receptors can only assemble and functionally express in select lines of cells, provided that RIC-3 is present. In contrast, ?4?2 nicotinic receptors can functionally express in many cell lines even without the presence of RIC-3. Depending on the cell line, RIC-3 has differential effects on ?4?2 receptor function – enhancement in mammalian cells but inhibition in Xenopus oocytes. Other differences between the two receptor types include nicotine-induced upregulation. When expressed in cell lines, ?4?2 receptors readily and robustly upregulate with chronic nicotine exposure. However, ?7 nicotinic receptors appear more resistant and require higher concentrations of nicotine to induce upregulation. Could the coexpression of RIC-3 modulate the extent of nicotine-induced upregulation not only for ?7 receptors but also ?4?2 receptors? We compared and contrasted the effects of RIC-3 on assembly, trafficking, protein expression and nicotine-induced upregulation on both ?7 and ?4?2 receptors using fluorescent protein tagged nicotinic receptors and Förster resonance energy transfer (FRET) microscopy imaging. Results RIC-3 increases assembly and cell surface trafficking of ?7 receptors but does not alter ?7 protein expression in transfected HEK293T cells. In contrast, RIC-3 does not affect assembly of ?4?2 receptors but increases ?4 and ?2 subunit protein expression. Acute nicotine (30 min exposure) was sufficient to upregulate FRET between ?4 and ?2 subunits. Surprisingly, when RIC-3 was coexpressed with ?4?2 receptors nicotine-induced upregulation was prevented. ?7 receptors did not upregulate with acute nicotine in the presence or absence of RIC-3. Conclusions These results provide interesting novel data that RIC-3 differentially regulates assembly and expression of different nicotinic receptor subunits. These results also show that nicotine-mediated upregulation of ?4?2 receptors can be dynamically regulated by the presence of the chaperone, RIC-3. This could explain a novel mechanism why high affinity ?4?2 receptors are upregulated in specific neuronal subtypes in the brain and not others.

2013-01-01

185

Homotypic and Heterotypic Adhesion Induced by Odorant Receptors and the ?2-Adrenergic Receptor  

PubMed Central

In the mouse olfactory system regulated expression of a large family of G Protein-Coupled Receptors (GPCRs), the Odorant Receptors (ORs), provides each sensory neuron with a single OR identity. In the wiring of the olfactory sensory neuron projections, a complex axon sorting process ensures the segregation of >1,000 subpopulations of axons of the same OR identity into homogeneously innervated glomeruli. ORs are critical determinants in axon sorting, and their presence on olfactory axons raises the intriguing possibility that they may participate in axonal wiring through direct or indirect trans-interactions mediating adhesion or repulsion between axons. In the present work, we used a biophysical assay to test the capacity of ORs to induce adhesion of cell doublets overexpressing these receptors. We also tested the ?2 Adrenergic Receptor, a non-OR GPCR known to recapitulate the functions of ORs in olfactory axon sorting. We report here the first evidence for homo- and heterotypic adhesion between cells overexpressing the ORs MOR256-17 or M71, supporting the hypothesis that ORs may contribute to olfactory axon sorting by mediating differential adhesion between axons.

Fouquet, Coralie; Dubacq, Caroline; Boggetto, Nicole; Pincet, Frederic; Gourier, Christine; Trembleau, Alain

2013-01-01

186

AMPA receptor potentiation can prevent ethanol-induced intoxication.  

PubMed

We present a substantial series of behavioral and imaging experiments, which demonstrate, for the first time, that increasing AMPA receptor-mediated neurotransmission via administration of potent and selective biarylsulfonamide AMPA potentiators LY404187 and LY451395 reverses the central effects of an acutely intoxicating dose of ethanol in the rat. Using pharmacological magnetic resonance imaging (phMRI), we observed that LY404187 attenuated ethanol-induced reductions in blood oxygenation level dependent (BOLD) in the anesthetized rat brain. A similar attenuation was apparent when measuring local cerebral glucose utilization (LCGU) via C14-2-deoxyglucose autoradiography in freely moving conscious rats. Both LY404187 and LY451395 significantly and dose-dependently reversed ethanol-induced deficits in both motor coordination and disruptions in an operant task where animals were trained to press a lever for food reward. Both prophylactic and acute intervention treatment with LY404187 reversed ethanol-induced deficits in motor coordination. Given that LY451395 and related AMPA receptor potentiators/ampakines are tolerated in both healthy volunteers and elderly patients, these data suggest that such compounds may form a potential management strategy for acute alcohol intoxication. PMID:17851540

Jones, Nicholas; Messenger, Marcus J; O'Neill, Michael J; Oldershaw, Anna; Gilmour, Gary; Simmons, Rosa M A; Iyengar, Smriti; Libri, Vincenzo; Tricklebank, Mark; Williams, Steve C R

2008-06-01

187

Endothelin-1 regulates adiponectin gene expression and secretion in 3T3-L1 adipocytes via distinct signaling pathways.  

PubMed

Adiponectin, which is specifically and highly expressed in adipose tissue, has pleiotropic insulin-sensitizing effects. Endothelin-1 (ET-1) is a potent vasoconstrictive peptide mainly produced by endothelial cells. We previously showed that ET-1 can induce insulin resistance in vitro and in vivo and proposed that it might regulate adiponectin expression and secretion, thus affecting the homeostasis of whole-body energy metabolism. In the present study, we explored the regulatory effects of ET-1 on adiponectin expression and secretion and the underlying mechanisms in 3T3-L1 adipocytes using Northern blotting and ELISA. ET-1 was found to cause a significant time- and dose-dependent decrease in adiponectin expression, and this effect was inhibited by the ET type A receptor (ETAR) antagonist BQ-610 but not by the ETBR antagonist BQ-788. To explore the underlying mechanism, we examined the involvement of the cAMP-dependent protein kinase A-, phospholipase A2-, protein kinase C-, and MAPK-mediated pathways using inhibitors and found that only PD98059 and U0126, inhibitors that blocked MAPK/ERK kinase's ability to activate the ERKs, prevented ET-1-induced down-regulation of adiponectin. Furthermore, acute ET-1 treatment significantly stimulated adiponectin secretion by 3T3-L1 adipocytes, and this effect was inhibited by the ETAR antagonist BQ-610, the inositol-1,4,5-triphosphate receptor blocker 2-APB, and phospholipase C inhibitor U73122, showing that the release of adiponectin stimulated by ET-1 was mediated through the ETAR and the inositol-1,4,5-triphosphate pathway. In conclusion, ET-1 regulates adiponectin expression and secretion by two different signaling pathways in 3T3-L1 adipocytes. These findings suggested that the cardiovascular system affects adipocyte physiology by regulating the expression of adipocytokines and, consequently, energy homeostasis via vasoactive factors, such as ET-1. PMID:17194742

Juan, Chi-Chang; Chuang, Tung-Yueh; Chang, Chih-Ling; Huang, Seng-Wong; Ho, Low-Tone

2007-04-01

188

General anesthetic-induced channel gating enhancement of 5-hydroxytryptamine type 3 receptors depends on receptor subunit composition.  

PubMed

5-Hydroxytryptamine (serotonin) (5-HT) type 3 (5-HT(3)) receptors are members of an anesthetic-sensitive superfamily of Cys-loop ligand-gated ion channels that can be formed as homomeric 5-HT(3A) or heteromeric 5-HT(3AB) receptors. When the efficacious agonist 5-HT is used, the inhaled anesthetics halothane and chloroform (at clinically relevant concentrations) significantly reduce the agonist EC(50) for 5-HT(3A) receptors but not for 5-HT(3AB) receptors. In the present study, we used dopamine (DA), a highly inefficacious agonist for 5-HT(3) receptors, to determine whether the difference in sensitivity between 5-HT(3A) and 5-HT(3AB) receptors to the potentiating effects of halothane and chloroform is due to differential modulation of agonist affinity, channel gating, or both. Using the two-electrode voltage-clamp technique with 5-HT(3A) and 5-HT(3AB) receptors expressed in Xenopus oocytes, we found that chloroform and halothane enhanced currents evoked by receptor-saturating concentrations of DA for both receptor subtypes in a concentration-dependent manner but that the magnitude of enhancement was substantially greater for 5-HT(3A) receptors than for 5-HT(3AB) receptors. Isoflurane induced only a small enhancement of currents evoked by receptor-saturating concentrations of DA for 5-HT(3A) receptors and no enhancement for 5-HT(3AB) receptors. For both receptor subtypes, none of the three test anesthetics significantly altered the agonist EC(50) for DA, implying that these anesthetics do not affect agonist binding affinity. Our results show that chloroform, halothane, and (to a much lesser degree) isoflurane enhance channel gating for 5-HT(3A) receptors and that the incorporation of 5-HT(3B) subunits to produce heteromeric 5-HT(3AB) receptors markedly attenuates the ability of these anesthetics to enhance channel gating. PMID:16081679

Solt, Ken; Stevens, Renna J; Davies, Paul A; Raines, Douglas E

2005-11-01

189

Nuclear Receptors: Mediators And Modifiers Of Inflammation-Induced Cholestasis  

PubMed Central

Inflammation-induced cholestasis (IIC) is a frequently occurring phenomenon. A central role in its pathogenesis is played by nuclear receptors (NRs). These ligand-activated transcription factors not only regulate basal expression of hepatobiliary transport systems, but also mediate adaptive responses and possess anti-inflammatory characteristics. The latter two functions may be exploited in the search for new treatments for IIC and likely for cholestasis in general as well. Current knowledge of the pathogenesis of IIC and the dual role NRs in this process are reviewed. Special interest is given to the use of NRs as potential targets for intervention.

Mulder, Jaap; Karpen, Saul J.; Tietge, Uwe J.F.; Kuipers, Folkert

2014-01-01

190

ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)  

EPA Science Inventory

ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845) Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

191

Isoproterenol stimulates monocyte chemoattractant protein-1 expression and secretion in 3T3-L1 adipocytes.  

PubMed

Recently, monocyte chemoattractant protein (MCP)-1 has been characterized as a novel adipocytokine upregulated in obesity and insulin resistance which impairs insulin signaling in muscle and fat in vitro. Growing evidence, on the other hand, suggests that increased activity of the sympathetic nervous system is an integral part in the development of insulin resistance. In the current study, the impact of the beta-adrenergic agonist isoproterenol on MCP-1 mRNA synthesis and secretion was determined in 3T3-L1 adipocytes. Interestingly, isoproterenol increased MCP-1 secretion 3-fold. Furthermore, 10 microM isoproterenol acutely induced MCP-1 mRNA by up to 5.3-fold in a time-dependent fashion with significant stimulation seen at concentrations as low as 0.3 microM effector. Studies using pharmacological inhibitors suggested that basal and isoproterenol-induced MCP-1 expressions are mediated via beta-adrenergic receptors and protein kinase A. Moreover, acute activation of adenylyl cyclase by forskolin was sufficient to mimic the effects of isoproterenol. Taken together, our results demonstrate that isoproterenol induces MCP-1 expression and secretion via a classical GS-protein-coupled pathway and support the notion that MCP-1 might be an interesting novel candidate linking obesity and insulin resistance. PMID:16644035

Kralisch, Susan; Klein, Johannes; Lossner, Ulrike; Bluher, Matthias; Paschke, Ralf; Stumvoll, Michael; Fasshauer, Mathias

2006-07-15

192

Echinacea purpurea root extract enhances the adipocyte differentiation of 3T3-L1 cells.  

PubMed

Echinacea purpurea has been shown to have anti-diabetic activities; for example, it activates peroxisome proliferator-activated receptor ? (PPAR?) and increases insulin-stimulated glucose uptake. Adipogenesis has been used to study the insulin signaling pathway and to screen anti-diabetic compounds. The present study was conducted to investigate the effects of an ethanol extract of E. purpurea (EEEP) and its constituents on the insulin-induced adipocyte differentiation of 3T3-L1 preadipocytes. When adipocyte differentiation was induced with insulin plus 3-isobutyl-1-methylxanthine and dexamethasone, the accumulation of lipid droplets and the cellular triglyceride content were significantly increased by EEEP. The expressions of PPAR? and C/EBP? in adipocytes treated with EEEP were gradually increased as compared with control cells. Fat accumulation and triglyceride content of adipocytes treated with dodeca-2(E),4(E)-dienoic acid isobutylamide were significantly increased as compared with control cells. The expressions of PPAR? and C/EBP? in adipocytes treated with dodeca-2(E),4(E)-dienoic acid isobutylamide were significantly higher than in control cells. These results suggest EEEP promotes the adipogenesis that is partially induced by insulin and that dodeca-2(E),4(E)-dienoic acid isobutylamide appears to be responsible for EEEP-enhanced adipocyte differentiation. PMID:24085629

Shin, Dong-Mi; Choi, Kyeong-Mi; Lee, Youn-Sun; Kim, Wonkyun; Shin, Kyong-Oh; Oh, Seikwan; Jung, Jae-Chul; Lee, Mi Kyeong; Lee, Yong-Moon; Hong, Jin Tae; Yun, Yeo-Pyo; Yoo, Hwan-Soo

2014-06-01

193

Protein Kinase C? Mediates ?-Opioid Receptor-induced Cross-desensitization of Chemokine Receptor CCR5*  

PubMed Central

We have previously shown that the ?-opioid receptor (MOR) is capable of mediating cross-desensitization of several chemokine receptors including CCR5, but the biochemical mechanism of this process has not been fully elucidated. We have carried out a series of functional and biochemical studies and found that the mechanism of MOR-induced cross-desensitization of CCR5 involves the activation of PKC?. Inhibition of PKC? by its pseudosubstrate inhibitor, or its siRNA, or dominant negative mutants suppresses the cross-desensitization of CCR5. Our results further indicate that the activation of PKC? is mediated through a pathway involving phosphoinositol-dependent kinase-1 (PDK1). In addition, activation of MOR elevates the phosphorylation level and kinase activity of PKC?. The phosphorylation of PKC? can be suppressed by a dominant negative mutant of PDK1. We observed that following MOR activation, the interaction between PKC? and PDK1 is immediately increased based on the analysis of fluorescent resonance energy transfer in cells with the expression of PKC?-YFP and PDK1-CFP. In addition, cells expressing PKC? kinase motif mutants (Lys-281, Thr-410, Thr-560) fail to exhibit full MOR-induced desensitization of CCR5 activity. Taken together, we propose that upon DAMGO treatment, MOR activates PKC? through a PDK1-dependent signaling pathway to induce CCR5 phosphorylation and desensitization. Because CCR5 is a highly proinflammatory receptor, and a critical coreceptor for HIV-1, these results may provide a novel approach for the development of specific therapeutic agents to treat patients with certain inflammatory diseases or AIDS.

Song, Changcheng; Rahim, Rahil T.; Davey, Penelope C.; Bednar, Filip; Bardi, Giuseppe; Zhang, Lily; Zhang, Ning; Oppenheim, Joost J.; Rogers, Thomas J.

2011-01-01

194

Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPAR{gamma} activation  

SciTech Connect

Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPAR{gamma} by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPAR{gamma} target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPAR{gamma} and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-il; Uemura, Taku; Hirai, Shizuka [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)] [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)] [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)

2009-12-25

195

Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization.  

PubMed

We have developed a quantitative method to evaluate the interaction between cell surface receptors and the endocytic apparatus. This method exploits occupancy-dependent changes in internalization rates that occur in cells expressing high numbers of receptors. We found that constitutive internalization of the transferrin receptor behaves as a simple, first order process that is unaltered by ligand. Internalization of the epidermal growth factor (EGF) receptor, however, behaves as a saturable, second order process that is induced by receptor occupancy. Internalization of EGF receptors occurs through at least two distinct pathways: a low capacity pathway that has a relatively high affinity for occupied receptors, and a low affinity pathway that has a much higher capacity. The high affinity pathway was observed in all cells having receptors with intrinsic tyrosine kinase activity. Mutant EGF receptors lacking kinase activity could not utilize the high affinity pathway and were internalized only through the low affinity one. Mutated receptors with decreased affinity for kinase substrates were also internalized at decreased rates through the high affinity, inducible pathway. In the case of vitellogenin receptors in Xenopus oocytes, occupied receptors competed more efficiently for internalization than empty ones. Insulin increased the endocytic capacity of oocytes for vitellogenin receptors. Similarly, serum increased the capacity of the inducible pathway for EGF receptors in mammalian cells. These data are consistent with a model of internalization in which occupied receptors bind to specific cellular components that mediate rapid internalization. Ligand-induced internalization results from an increase in the affinity of occupied receptors for the endocytic apparatus. Hormones can also indirectly regulate endocytosis by increasing the number of coated pits or their rate of internalization. The ability to dissect receptor-specific effects from cell-specific ones should be very useful in investigating the molecular mechanisms of receptor mediated endocytosis. PMID:1975591

Lund, K A; Opresko, L K; Starbuck, C; Walsh, B J; Wiley, H S

1990-09-15

196

An extract of Lagerstroemia speciosa L. has insulin-like glucose uptake-stimulatory and adipocyte differentiation-inhibitory activities in 3T3-L1 cells.  

PubMed

The effects of extracts isolated from Lagerstroemia speciosa L. (banaba) on glucose transport and adipocyte differentiation in 3T3-L1 cells were studied. Glucose uptake-inducing activity of banaba extract (BE) was investigated in differentiated adipocytes using a radioactive assay, and the ability of BE to induce differentiation in preadipocytes was examined by Northern and Western blot analyses. The hot water BE and the banaba methanol eluent (BME) stimulated glucose uptake in 3T3-L1 adipocytes with an induction time and a dose-dependent response similar to those of insulin. Furthermore, there were no additive or synergistic effects found between BE and insulin on glucose uptake, and the glucose uptake activity of insulin could be reduced to basal levels by adding increasing amounts of BE. Unlike insulin, BE did not induce adipocyte differentiation in the presence of 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone (DEX). BE inhibited the adipocyte differentiation induced by insulin plus IBMX and DEX (IS-IBMX-DEX) of 3T3-L1 preadipocytes in a dose-dependent manner. The differences in the glucose uptake and differentiation inhibitory activities between untreated cells and those treated with BE were significant (P < 0.01). The inhibitory activity was further demonstrated by drastic reductions of peroxisome proliferator-activated receptor gamma2 (PPARgamma2) mRNA and glucose transporter-4 (GLUT4) protein in cells induced from preadipocytes with IS-IBMX-DEX in the presence of BE. The unique combination of a glucose uptake stimulatory activity, the absence of adipocyte differentiation activity and effective inhibition of adipocyte differentiation induced by IS-IBMX-DEX in 3T3-L1 cells suggest that BE may be useful for prevention and treatment of hyperglycemia and obesity in type II diabetics. PMID:11533261

Liu, F; Kim, J; Li, Y; Liu, X; Li, J; Chen, X

2001-09-01

197

Icariin induces osteoblast proliferation, differentiation and mineralization through estrogen receptor-mediated ERK and JNK signal activation.  

PubMed

Icariin, the main active flavonoid glucoside isolated from Herba epimedii (HEF), is an anabolic agent in bone that has been reported to prevent bone loss in ovariectomized rats and postmenopausal women. However, the molecular mechanism for this anabolic action of Icariin remain largely unknown. Here, we found that Icariin could promote MC3T3-E1 osteoblastic cell proliferation and reduce cell apoptosis, associated with increased mRNA levels of positive regulators of cell cycle gene Cyclin E and proliferating cell nuclear antigen (PCNA), decreaed mRNA level of negative regulator gene, Cyclin-dependent kinase 4 inhibitor B (Cdkn2B), and reduced caspase-3 activity. Icariin also enhanced MC3T3-E1 cell differentiation and mineralization demonstrated by increased the expression of differentiation markers, alkaline phosphatase (ALP) and collagen type I (Col I), and bone nodule formation via Alizarin red S staining. To characterize the underlying mechanisms, we examined the effect of Icariin on mitogen-activated protein kinase (MAPK) signaling. Icariin treatment rapidly induced extracellular signal-regulated kinase (ERK) and c-Jun N terminal kinase (JNK) activation but showed no effect on activation of p38 kinase. Furthermore, Icariin-mediated effects on osteoblasts were dramatically attenuated by treatment with specific inhibitors of MAPKs, U0126 (ERK inhibitor) and SP600125 (JNK inhibitor). Interestingly, treatment of osteoblasts with estrogen receptor antagonist ICI182780 attenuated Icariin-mediated effect of proliferation and mineralization, associated with suppression of ERK and JNK phosphorylation. These observations provide a potential mechanism of anabolic actions of Icariin involving ERK and JNK pathway by estrogen receptor. PMID:23764463

Song, Lige; Zhao, Jiashen; Zhang, Xiuzhen; Li, Hong; Zhou, Yun

2013-08-15

198

Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury.  

PubMed

Cannabinoid receptor type 1 (CB1)-induced suppression of transient receptor potential vanilloid type 1 (TRPV1) activation provides a therapeutic option to reduce inflammation and pain in different animal disease models through mechanisms involving dampening of TRPV1 activation and signaling events. As we found in both mouse corneal epithelium and human corneal epithelial cells (HCEC) that there is CB1 and TRPV1 expression colocalization based on overlap of coimmunostaining, we determined in mouse corneal wound healing models and in human corneal epithelial cells (HCEC) if they interact with one another to reduce TRPV1-induced inflammatory and scarring responses. Corneal epithelial debridement elicited in vivo a more rapid wound healing response in wildtype (WT) than in CB1(-/-) mice suggesting functional interaction between CB1 and TRPV1. CB1 activation by injury is tenable based on the identification in mouse corneas of 2-arachidonylglycerol (2-AG) with tandem LC-MS/MS, a selective endocannabinoid CB1 ligand. Suppression of corneal TRPV1 activation by CB1 is indicated since following alkali burning, CB1 activation with WIN55,212-2 (WIN) reduced immune cell stromal infiltration and scarring. Western blot analysis of coimmunoprecipitates identified protein-protein interaction between CB1 and TRPV1. Other immunocomplexes were also identified containing transforming growth factor kinase 1 (TAK1), TRPV1 and CB1. CB1 siRNA gene silencing prevented suppression by WIN of TRPV1-induced TAK1-JNK1 signaling. WIN reduced TRPV1-induced Ca(2+) transients in fura2-loaded HCEC whereas pertussis toxin (PTX) preincubation obviated suppression by WIN of such rises caused by capsaicin (CAP). Whole cell patch clamp analysis of HCEC showed that WIN blocked subsequent CAP-induced increases in nonselective outward currents. Taken together, CB1 activation by injury-induced release of endocannabinoids such as 2-AG downregulates TRPV1 mediated inflammation and corneal opacification. Such suppression occurs through protein-protein interaction between TRPV1 and CB1 leading to declines in TRPV1 phosphorylation status. CB1 activation of the GTP binding protein, G(i/o) contributes to CB1 mediated TRPV1 dephosphorylation leading to TRPV1 desensitization, declines in TRPV1-induced increases in currents and pro-inflammatory signaling events. PMID:23142606

Yang, Y; Yang, H; Wang, Z; Varadaraj, K; Kumari, S S; Mergler, S; Okada, Y; Saika, S; Kingsley, P J; Marnett, L J; Reinach, P S

2013-02-01

199

Dopamine and noradrenaline receptor stimulation: Reversal of reserpine-induced suppression of motor activity  

Microsoft Academic Search

The motor activity of reserpine treated mice was recorded after drug treatments causing stimulation of dopamine or noradrenaline receptors or both. The dopamine receptor stimulating agent apomorphine elicited an activation with stereotypies whereas the noradrenaline receptor stimulating agent clonidine was inefficient. Combined treatment with apomorphine and clonidine induced marked stimulation with jumping. Biochemically, clonidine did not significantly interfere with the

Nils-Erik Andén; Ulf Strömbom; Torgny H. Svensson

1973-01-01

200

IL-1-induced murine osteoblast IL-6 production is mediated by the type 1 IL-1 receptor and is increased by 1,25 dihydroxyvitamin D3.  

PubMed Central

IL-1-induced osteoblast IL-6 production represents one possible mechanism by which IL-1 augments bone resorption. In this report, we show that the murine osteoblastic cell line (MC3T3-E1) expresses type 1 IL-1 receptors based on 125I-HrIL1 alpha binding, blocked by type 1 IL-1R antibodies (35F5), and analysis of MC3T3 RNA by reverse transcription (RT)-DNA amplification and Northern analysis. MC3T3 cells do not express detectable type 2 IL-1R mRNA by RT-DNA amplification. IL-1 induces (IL-1 ED50, 0.1 pM) IL-6 production through the type 1 IL-1R as 35F5 antibodies block IL-1-stimulated IL-6 production. Vitamin D3 increases IL-1R expression dose- and metabolite-dependently, with 1,25-(OH)2D3 having the greatest potency, and also enhances IL-1's capacity to stimulate IL-6 production at low IL-1 levels. Both IL-1 and 1,25-(OH)2D3 induce type 1 IL-1R and not type 2 IL-1R upregulation based on ligand binding and RT-DNA amplification. Increased IL-1R expression requires a 5-7-h treatment and is protein/RNA synthesis dependent. These observations imply that IL-1-induced IL-6 production in osteoblasts is mediated by type 1 IL-1Rs and that increased IL-1R expression could play a role in mediating IL-1-induced skeletal responses. Images

Lacey, D L; Grosso, L E; Moser, S A; Erdmann, J; Tan, H L; Pacifici, R; Villareal, D T

1993-01-01

201

Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes  

SciTech Connect

Highlights: ? Notch4IC modulates the ERK pathway and cell cycle to promote 3T3-L1 proliferation. ? Notch4IC facilitates 3T3-L1 differentiation by up-regulating proadipogenic genes. ? Notch4IC promotes proliferation during the early stage of 3T3-L1 adipogenesis. ? Notch4IC enhances differentiation during subsequent stages of 3T3-L1 adipogenesis. -- Abstract: Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBP? and PPAR? was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBP?, PPAR?, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.

Lai, Peng-Yeh [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China)] [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China); Tsai, Chong-Bin [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China) [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China); Department of Ophthalmology, Chiayi Christian Hospital, Chiayi 600, Taiwan, ROC (China); Tseng, Min-Jen, E-mail: biomjt@ccu.edu.tw [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China)] [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China)

2013-01-18

202

?-Adrenergic Receptor Mediation of Stress-Induced Reinstatement of Extinguished Cocaine-Induced Conditioned Place Preference in Mice: Roles for ?1 and ?2 Adrenergic Receptors  

PubMed Central

Stress can trigger the relapse of drug use in recovering cocaine addicts and reinstatement in rodent models through mechanisms that may involve norepinephrine release and ?-adrenergic receptor activation. The present study examined the role of ?-adrenergic receptor subtypes in the stressor-induced reinstatement of extinguished cocaine-induced (15 mg/kg i.p.) conditioned place preference in mice. Forced swim (6 min at 22°C) stress or activation of central noradrenergic neurotransmission by administration of the selective ?2 adrenergic receptor antagonist 2-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole (BRL-44,408) (10 mg/kg i.p.) induced reinstatement in wild-type, but not ?- adrenergic receptor-deficient Adrb1/Adrb2 double-knockout, mice. In contrast, cocaine administration (15 mg/kg i.p.) resulted in reinstatement in both wild-type and ?-adrenergic receptor knockout mice. Stress-induced reinstatement probably involved ?2 adrenergic receptors. The ?2 adrenergic receptor antagonist -(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI-118,551) (1 or 2 mg/kg i.p.) blocked reinstatement by forced swim or BRL-44,408, whereas administration of the nonselective ?-adrenergic receptor agonist isoproterenol (2 or 4 mg/kg i.p.) or the ?2 adrenergic receptor-selective agonist clenbuterol (2 or 4 mg/kg i.p.) induced reinstatement. Forced swim-induced, but not BRL-44,408-induced, reinstatement was also blocked by a high (20 mg/kg) but not low (10 mg/kg) dose of the ?1 adrenergic receptor antagonist betaxolol, and isoproterenol-induced reinstatement was blocked by pretreatment with either ICI-118,551 or betaxolol, suggesting a potential cooperative role for ?1 and ?2 adrenergic receptors in stress-induced reinstatement. Overall, these findings suggest that targeting ?-adrenergic receptors may represent a promising pharmacotherapeutic strategy for preventing drug relapse, particularly in cocaine addicts whose drug use is stress related.

Vranjkovic, Oliver; Hang, Shona; Baker, David A.

2012-01-01

203

Loss of Differentiation Control in Transformed 3T3 T Proadipocytes1  

Microsoft Academic Search

Nontransformed 3T3 T mesenchymal\\/proadipocyte stem cells can be readily induced to differentiate, yet previous work has shown that 3T3 T cells that are spontaneously or virally transformed not only lose their normal growth control mechanisms but also lose the ability to differenti ate. Loss of growth control can be due to autocrine mechanisms in some transformed cells, but the mechanisms

Rodney L. Sparks; Blake J. Allen; Andrea I. Zygmunt; Ethan E. Strauss

204

Overexpression of Banna mini-pig inbred line fatty acid binding protein 3 promotes adipogenesis in 3T3-L1 preadipocytes.  

PubMed

Fatty acid binding protein 3 (H-FABP, FABP3) has been significantly associated with intramuscular fat (IMF) content in pigs, which is positively correlated with palatability of pork. However, its underlying function is not fully elucidated. We have investigated the effects of overexpression of the FABP3 gene on differentiation and adipogenesis of 3T3-L1 preadipocytes in the fat Banna mini-pig inbred line (fBMIL). Eukaryotic vectors that expressed the FABP3 protein were constructed, and stably established in the 3T3-L1 preadipocytes cell line. Cells were induced in a standard differentiation cocktail. Morphological changes and the degree of adipogenesis were measured by Oil Red O staining assay and triacylglycerol content measurement, respectively. mRNA expression levels of triacylglycerol metabolism-related genes were measured by qPCR. FABP3 significantly promoted differentiation of 3T3-L1 cells and enhanced triacylglycerol levels (P?receptor ? (PPAR?), adipocyte fatty acid binding protein (422/aP2) and glycerol-3-phosphate dehydrogenase (GPDH) gene increased markedly (P?T3-L1 preadipocytes primarily by upregulating lipogenic PPAR?, 422/aP2 and GPDH genes. PMID:24737696

Yi, Bao; Wang, Jigui; Wang, Shuang; Yuan, Daoli; Sun, Jiazeng; Li, Zhili; Mao, Yaping; Hou, Qiang; Liu, Weiquan

2014-08-01

205

Low magnitude and high frequency mechanical loading prevents decreased bone formation responses of 2T3 preosteoblasts.  

PubMed

Bone loss due to osteoporosis or disuse such as in paraplegia or microgravity is a significant health problem. As a treatment for osteoporosis, brief exposure of intact animals or humans to low magnitude and high frequency (LMHF) mechanical loading has been shown to normalize and prevent bone loss. However, the underlying molecular changes and the target cells by which LMHF mechanical loading alleviate bone loss are not known. Here, we hypothesized that direct application of LMHF mechanical loading to osteoblasts alters their cell responses, preventing decreased bone formation induced by disuse or microgravity conditions. To test our hypothesis, preosteoblast 2T3 cells were exposed to a disuse condition using the random positioning machine (RPM) and intervened with an LMHF mechanical load (0.1-0.4 g at 30 Hz for 10-60 min/day). Exposure of 2T3 cells to the RPM decreased bone formation responses as determined by alkaline phosphatase (ALP) activity and mineralization even in the presence of a submaximal dose of BMP4 (20 ng/ml). However, LMHF mechanical loading prevented the RPM-induced decrease in ALP activity and mineralization. Mineralization induced by LMHF mechanical loading was enhanced by treatment with bone morphogenic protein 4 (BMP4) and blocked by the BMP antagonist noggin, suggesting a role for BMPs in this response. In addition, LMHF mechanical loading rescued the RPM-induced decrease in gene expression of ALP, runx2, osteomodulin, parathyroid hormone receptor 1, and osteoglycin. These findings suggest that preosteoblasts may directly respond to LMHF mechanical loading to induce differentiation responses. The mechanosensitive genes identified here provide potential targets for pharmaceutical treatments that may be used in combination with low level mechanical loading to better treat osteoporosis or disuse-induced bone loss. PMID:19125415

Patel, Mamta J; Chang, Kyungh Hwa; Sykes, Michelle C; Talish, Roger; Rubin, Clinton; Jo, Hanjoong

2009-02-01

206

Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes  

SciTech Connect

Highlights: •Long-term hypoxia decreased the size of LDs and lipid storage in 3T3-L1 adipocytes. •Long-term hypoxia increased basal lipolysis in 3T3-L1 adipocytes. •Hypoxia decreased lipid-associated proteins in 3T3-L1 adipocytes. •Hypoxia decreased basal glucose uptake and lipogenic proteins in 3T3-L1 adipocytes. •Hypoxia-mediated lipogenesis may be an attractive therapeutic target against obesity. -- Abstract: Background: A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function. Methods: Differentiated 3T3-L1 adipocytes were incubated at 5% O{sub 2} for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC). Results: HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p < 0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p < 0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p < 0.01). Conclusion: We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via decreased glucose uptake and lipogenic protein expression and increased basal lipolysis. Such an hypoxia-induced decrease in lipogenesis may be an attractive therapeutic target against lipid-associated metabolic diseases.

Hashimoto, Takeshi, E-mail: thashimo@fc.ritsumei.ac.jp [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)] [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Yokokawa, Takumi; Endo, Yuriko [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)] [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Iwanaka, Nobumasa [Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)] [Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Higashida, Kazuhiko [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan) [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Faculty of Sport Science, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359-1192 (Japan); Taguchi, Sadayoshi [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)] [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)

2013-10-11

207

Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts.  

PubMed Central

Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires phosphatidylinositol 3-kinase (PI3K) activity since it is abrogated both by the specific inhibitor wortmannin and by overexpression of the dominant negative P13K p85 subunit. Consistently, Gas6 activates the P13K downstream targets S6K and Akt, whose activation is abrogated by addition of wortmannin. Moreover, rapamycin treatment blocks Gas6-induced entry into the S phase of serum-starved NIH 3T3 cells. We also demonstrate the requirement of Src tyrosine kinase for Gas6 signalling since stable or transient expression of a catalytically inactive form of Src significantly inhibited Gas6-stimulated entry into the S phase. Accordingly, Gas6 addition to serum-starved NIH 3T3 cells causes activation of the intrinsic Src kinase activity. When specifically analyzed in a survival assay, these elements were found to be required for the survival effect of Gas6. Taken together, the evidence presented here identifies elements involved in the Gas6 transduction pathway that are responsible for its antiapoptotic effect and suggests that Src is involved in the events regulating cell survival.

Goruppi, S; Ruaro, E; Varnum, B; Schneider, C

1997-01-01

208

Sciadopitysin alleviates methylglyoxal-mediated glycation in osteoblastic MC3T3-E1 cells by enhancing glyoxalase system and mitochondrial biogenesis.  

PubMed

Methylglyoxal (MG) is a precursor of advanced glycation end products, which contribute to diabetic complications, including bone defects. In the present study, the effect of sciadopitysin on MG-induced cytotoxicity was investigated using osteoblastic MC3T3-E1 cells. Pretreatment of MC3T3-E1 cells with sciadopitysin prevented the MG-induced cell death and protein adducts formation. Sciadopitysin restored the MG-induced change in glyoxalase activity almost to the control level and increased glutathione levels. In addition, sciadopitysin decreased MG-induced formation of intracellular reactive oxygen species (ROS), mitochondrial superoxide, and cardiolipin peroxidation. These findings suggest that sciadopitysin provides a protective action against MG-induced glycation by increasing MG detoxification system and by reducing oxidative stress. Pretreatment with sciadopitysin prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential (MMP) dissipation and adenosine triphosphate (ATP) loss. The nitric oxide (NO) level was decreased by MG treatment, but it was significantly increased by sciadopitysin, suggesting that sciadopitysin may induce NO-dependent mitochondrial biogenesis. Furthermore, sciadopitysin treatment increased the levels of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1?), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (TFAM). These findings indicate that sciadopitysin might exert its therapeutic effects via upregulation of mitochondrial biogenesis. Therefore, sciadopitysin may prevent the development of diabetic osteopathy. PMID:24628445

Choi, E M; Suh, K S; Rhee, S Y; Kim, Y S

2014-07-01

209

Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors.  

PubMed

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors. PMID:11152677

Chaturvedi, K; Bandari, P; Chinen, N; Howells, R D

2001-04-13

210

Central serotonergic mechanisms on head twitch response induced by benzodiazepine receptor agonists.  

PubMed

Intraperitoneal injection of benzodiazepine receptor agonists (estazolam, zopiclone, triazolam: 0.03-0.24 mmol/kg) induces the head twitch response (HTR). The present study was undertaken to examine the possible participation of the serotonergic system in the mechanism of head twitches induced by benzodiazepine receptor agonists (BZ-RAs). The HTR induced by BZ-RAs was suppressed by pretreatment with ketanserine (1 mg/kg, i.p.), a selective 5-HT(2) receptor antagonist. Pretreatment with fluoxetine (10 mg/kg, i.p.), a 5-HT reuptake inhibitor, and 8-hydroxy-2-(di-n-propylamino)tetralin, a 5-HT(1A) receptor agonist, also suppressed the HTR induced by BZ-RAs. These results suggest that the HTR induced by BZ-RAs may be the result of an activation of postsynaptic 5-HT(2) receptors, probably due to direct action. PMID:11287817

Tadano, T; Hozumi, M; Satoh, N; Oka, R; Hishinuma, T; Mizugaki, M; Arai, Y; Yasuhara, H; Kinemuchi, H; Niijima, F; Nakagawasai, O; Tan-no, K; Kisara, K

2001-01-01

211

Shikonin induces programmed necrosis-like cell death through the formation of receptor interacting protein 1 and 3 complex.  

PubMed

An alternative cell demise programmed necrosis has also been proposed when apoptotic machinery is impaired or blocked during tumor necrosis factor alpha (TNF?) stimulation. Shikonin (SKN), an herbal extract from the Chinese plant, has been reported to induce either apoptosis or necrosis depending on cell types or its concentrations. In this presentation, SKN caused cell death of NIH3T3 in a dose-dependent manner. Intriguingly, SKN-mediated cell death was in part protected by necrostatin-1 (Nec-1), a specific inhibitor of programmed necrosis, but not zVAD a pan-caspase inhibitor. SKN directly mediated cell death via receptor interacting protein1 and 3 (RIP1-RIP3) complex formation, which is required for TNF?-mediated programmed necrosis. Additionally, SKN-caused cell death was reversed by a reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) whereas TNF?-mediated necrosis was successfully protected by butylated hydroxyanisole (BHA), implying that ROS may be differentially derived from death inducing agents. Concurrently with the protective effect of the ROS scavenger or Nec-1 on TNF? or SKN, the RIP1-RIP3 complex was significantly affected in the presence of those agents. Here, it is highlighted that SKN as well as TNF? can directly mediate cell death via a pronecrotic complex, but ROS were generated via different routes depending on cell death-inducing agents. PMID:23261677

Park, Seungyeon; Shin, Heesuk; Cho, Youngsik

2013-05-01

212

Epac, not PKA catalytic subunit, is required for 3T3-L1 preadipocyte differentiation  

PubMed Central

Cyclic AMP plays a critical role in adipocyte differentiation and maturation. However, it is not clear which of the two intracellular cAMP receptors, exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor or protein kinase A/cAMP-dependent protein kinase, is essential for cAMP-mediated adipocyte differentiation. In this study, we utilized a well-defined adipose differentiation model system, the murine preadipocyte line 3T3-L1, to address this issue. We showed that knocking down Epac expression in 3T3-L1 cells using lentiviral based small hairpin RNAs down-regulated peroxisome proliferator-activated receptor gamma expression and dramatically inhibited adipogenic conversion of 3T3-L1 cells while inhibiting PKA catalytic subunit activity by two mechanistically distinct inhibitors, heat stable protein kinase inhibitor and H89, had no effect on 3T3-L1 adipocyte differentiation. Moreover, cAMP analog selectively activating Epac was not able to stimulate adipogenic conversion. Our study demonstrated that while PKA catalytic activity is dispensable, activation of Epac is necessary but not sufficient for adipogenic conversion of 3T3-L1 cells.

Ji, Zhenyu; Mei, Fang C.; Cheng, Xiaodong

2009-01-01

213

Epac, not PKA catalytic subunit, is required for 3T3-L1 preadipocyte differentiation.  

PubMed

Cyclic AMP plays a critical role in adipocyte differentiation and maturation. However, it is not clear which of the two intracellular cAMP receptors, exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor or protein kinase A/cAMP-dependent protein kinase, is essential for cAMP-mediated adipocyte differentiation. In this study, we utilized a well-defined adipose differentiation model system, the murine preadipocyte line 3T3-L1, to address this issue. We showed that knocking down Epac expression in 3T3-L1 cells using lentiviral based small hairpin RNAs down-regulated peroxisome proliferator-activated receptor gamma expression and dramatically inhibited adipogenic conversion of 3T3-L1 cells while inhibiting PKA catalytic subunit activity by two mechanistically distinct inhibitors, heat stable protein kinase inhibitor and H89, had no effect on 3T3-L1 adipocyte differentiation. Moreover, cAMP analog selectively activating Epac was not able to stimulate adipogenic conversion. Our study demonstrated that while PKA catalytic activity is dispensable, activation of Epac is necessary but not sufficient for adipogenic conversion of 3T3-L1 cells. PMID:20036887

Ji, Zhenyu; Mei, Fang C; Cheng, Xiaodong

2010-01-01

214

Proteinase-activated receptor 2 sensitizes transient receptor potential vanilloid 1, transient receptor potential vanilloid 4, and transient receptor potential ankyrin 1 in paclitaxel-induced neuropathic pain.  

PubMed

Paclitaxel chemotherapy is limited by a long-lasting painful neuropathy that lacks an effective therapy. In this study, we tested the hypothesis that paclitaxel may release mast cell tryptase, which activates protease-activated receptor 2 (PAR2) and, subsequently, protein kinases A and C, resulting in mechanical and thermal (both heat and cold) hypersensitivity. Correlating with the development of neuropathy after repeated administration of paclitaxel, mast cell tryptase activity was found to be increased in the spinal cord, dorsal root ganglia, and peripheral tissues in mice. FSLLRY-amide, a selective PAR2 antagonist, blocked paclitaxel-induced neuropathic pain behaviors in a dose- and time-dependent manner. In addition, blocking downstream signaling pathways of PAR2, including phospholipase C (PLC), protein kinase A (PKA), and protein kinase C? (PKC), effectively attenuated paclitaxel-induced mechanical, heat, or cold hypersensitivity. Furthermore, sensitized pain response was selectively inhibited by antagonists of transient receptor potential (TRP) V1, TRPV4, or TRPA1. These results revealed specific cellular signaling pathways leading to paclitaxel-induced neuropathy, including the activation of PAR2 and downstream enzymes PLC, PKC?, and PKA and resultant sensitization of TRPV1, TRPV4, and TRPA1. Targeting one or more of these signaling molecules may present new opportunities for the treatment of paclitaxel-induced neuropathy. PMID:21763756

Chen, Y; Yang, C; Wang, Z J

2011-10-13

215

Alpha-2 adrenergic and imidazoline receptor agonists prevent cue-induced cocaine-seeking  

PubMed Central

Background Drug-associated cues can elicit stress-like responses in addicted individuals, indicating thatcue- and stress-induceddrug relapse may share some neural mechanisms.It is unknown whether?2 adrenergic receptor agonists, which are known toattenuate stress-induced reinstatement of drug-seeking in rats,also reduce cue-induced reinstatement. Methods Rats were tested for reinstatement of drug-seekingfollowing cocaine self-administration and extinction. We first evaluated the effects ofclonidine, an agonist at ?2 and imidazoline-1 (I1) receptors, on relapse to cocaine-seeking. To explore possible mechanisms of clonidine’s effects, we then tested more specific ?2 or I1 agonists, post-synaptic adrenergic receptor (?1 and ?) antagonists, andcorticotropin-releasing factor receptor-1 (CRF R1) antagonists. Results We found that clonidine,andthe more selective ?2 agonists UK-14,304 and guanfacine, decreased cue-induced reinstatement of cocaine-seeking.The specific I1 receptor agonist moxonidine reduced cue-induced as well as cocaine-induced reinstatement.Clonidine or moxonidine effects on cue-induced reinstatementwere reversed by the selective ?2 receptor antagonist RS-79948, indicatinga role for ?2 receptors.Prazosin and propranolol, antagonists at the ?1and ? receptor, respectively, reducedcue-induced reinstatement only when administered in combination. Finally, the CRF R1 antagonist CP-154,526reduced cue-induced reinstatement, as previouslyobservedfor stress-induced reinstatement, indicating possible overlap between stress and cue mechanisms. Conclusions These results indicate that ?2 and I1 receptor agonists are novel therapeutic options for prevention of cue-induced cocaine relapse. Given that ?2 receptor stimulation is associated with sedation in humans, the I1agonist moxonidineseems to have substantial potential for treating addictive disorders.

Smith, Rachel J.; Aston-Jones, Gary

2011-01-01

216

Agarwood induced laxative effects via acetylcholine receptors on loperamide-induced constipation in mice.  

PubMed

Agarwood (Aquilaria sinensis, Aquilaria crasna) is well known as an incense in the oriental region such as Thailand, Taiwan, and Cambodia, and is used as a digestive in traditional medicine. We investigated the laxative effects and mechanism of agarwood leaves extracted with ethanol (EEA-1, Aquilaria sinensis; EEA-2, Aquilaria crasna). EEA-1, EEA-2, the main constituents of EEAs (mangiferin, and genkwanin-5-O-primeveroside), and senna increased the frequency and weight of stools in loperamide-induced constipation model mice. EEA-1 and EEA-2 did not induce diarrhea as a side effect, but senna induced severe diarrhea. EEA-1 and senna increased gastro-intestinal (GI) transit in the model mice. EEA-1, but not senna, also increased the intestinal tension of isolated jejunum and ileum in guinea pigs, and the tension increase was blocked by atropine, a muscarinic receptor antagonist, but not by other inhibitors (granicetron, pyrilamine, or bradykinin-antagonist peptide). Furthermore, the increase in frequency and weight of stools induced by EEA-1 were blocked by pre-administration of atropine in the model mice. These findings indicate that EEAs exerted a laxative effect via acetylcholine receptors in the mouse constipation model. PMID:20699592

Kakino, Mamoru; Izuta, Hiroshi; Ito, Tetsuro; Tsuruma, Kazuhiro; Araki, Yoko; Shimazawa, Masamitsu; Oyama, Masayoshi; Iinuma, Munekazu; Hara, Hideaki

2010-01-01

217

NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs) during inflammation induced visceral hypersensitivity  

PubMed Central

Background Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs), co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG) neurons expressing the transient receptor potential vanilloid-1 (TRPV1) receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX) prior to inflammation and behavioural testing. Results CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs. Conclusion Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned animals. Therefore, these data suggest that CANs contribute to visceral hypersensitivity during inflammation.

Suckow, Shelby K; Caudle, Robert M

2009-01-01

218

Interleukin 8 (IL-8) induces the expression of kinin B1 receptor in human lung fibroblasts.  

PubMed

Kinin B1 receptors are induced by various inflammatory mediators. The aim of the present study was to investigate the effect of the CXC chemokine IL-8 on kinin B1 receptor expression in IMR-90 cells, by performing binding studies and Northern blot analysis of B1 receptor mRNA levels. We demonstrated here that the density of the kinin B1 receptors could be increased by the chemokine IL-8 in a concentration- and time-dependent manner. IL-8 also increased the kinin B1 receptor mRNA level in IMR-90 cells. IL-8-induced B1 receptor expression could be totally abolished by pretreatment with the metabolic inhibitors. Furthermore, expression was markedly reduced by antibodies to human IL-1alpha. In conclusion, IL-8 increased the expression of kinin B1 receptors in IMR-90 cells and this effect is likely to be secondary to the production of IL-1beta. PMID:9918799

Bastian, S; Paquet, J L; Robert, C; Cremers, B; Loillier, B; Larrivée, J F; Bachvarov, D R; Marceau, F; Pruneau, D

1998-12-30

219

Central antinociception induced by ketamine is mediated by endogenous opioids and ?- and ?-opioid receptors.  

PubMed

It is generally believed that NMDA receptor antagonism accounts for most of the anesthetic and analgesic effects of ketamine, however, it interacts at multiple sites in the central nervous system, including NMDA and non-NMDA glutamate receptors, nicotinic and muscarinic cholinergic receptors, and adrenergic and opioid receptors. Interestingly, it was shown that at supraspinal sites, ketamine interacts with the ?-opioid system and causes supraspinal antinociception. In this study, we investigated the involvement of endogenous opioids in ketamine-induced central antinociception. The nociceptive threshold for thermal stimulation was measured in Swiss mice using the tail-flick test. The drugs were administered via the intracerebroventricular route. Our results demonstrated that the opioid receptor antagonist naloxone, the ?-opioid receptor antagonist clocinnamox and the ?-opioid receptor antagonist naltrindole, but not the ?-opioid receptor antagonist nor-binaltorphimine, antagonized ketamine-induced central antinociception in a dose-dependent manner. Additionally, the administration of the aminopeptidase inhibitor bestatin significantly enhanced low-dose ketamine-induced central antinociception. These data provide evidence for the involvement of endogenous opioids and ?- and ?-opioid receptors in ketamine-induced central antinociception. In contrast, ?-opioid receptors not appear to be involved in this effect. PMID:24675031

Pacheco, Daniela da Fonseca; Romero, Thiago Roberto Lima; Duarte, Igor Dimitri Gama

2014-05-01

220

Ursolic Acid Inhibits Adipogenesis in 3T3-L1 Adipocytes through LKB1/AMPK Pathway  

PubMed Central

Background Ursolic acid (UA) is a triterpenoid compound with multiple biological functions. This compound has recently been reported to possess an anti-obesity effect; however, the mechanisms are less understood. Objective As adipogenesis plays a critical role in obesity, the present study was conducted to investigate the effect of UA on adipogenesis and mechanisms of action in 3T3-L1 preadipocytes. Methods and Results The 3T3-L1 preadipocytes were induced to differentiate in the presence or absence of UA for 6 days. The cells were determined for proliferation, differentiation, fat accumulation as well as the protein expressions of molecular targets that regulate or are involved in fatty acid synthesis and oxidation. The results demonstrated that ursolic acid at concentrations ranging from 2.5 µM to 10 µM dose-dependently attenuated adipogenesis, accompanied by reduced protein expression of CCAAT element binding protein ? (C/EBP?), peroxisome proliferator-activated receptor ? (PPAR?), CCAAT element binding protein ? (C/EBP?) and sterol regulatory element binding protein 1c (SREBP-1c), respectively. Ursolic acid increased the phosphorylation of acetyl-CoA carboxylase (ACC) and protein expression of carnitine palmitoyltransferase 1 (CPT1), but decreased protein expression of fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Ursolic acid increased the phosphorylation of AMP-activated protein kinase (AMPK) and protein expression of (silent mating type information regulation 2, homolog) 1 (Sirt1). Further studies demonstrated that the anti-adipogenic effect of UA was reversed by the AMPK siRNA, but not by the Sirt1 inhibitor nicotinamide. Liver kinase B1 (LKB1), the upstream kinase of AMPK, was upregulated by UA. When LKB1 was silenced with siRNA or the inhibitor radicicol, the effect of UA on AMPK activation was diminished. Conclusions Ursolic acid inhibited 3T3-L1 preadipocyte differentiation and adipogenesis through the LKB1/AMPK pathway. There is potential to develop UA into a therapeutic agent for the prevention or treatment of obesity.

He, Yonghan; Li, Ying; Zhao, Tiantian; Wang, Yanwen; Sun, Changhao

2013-01-01

221

Adenosine A1 receptors mediate chronic ethanol-induced increases in receptor-stimulated cyclic AMP in cultured hepatocytes.  

PubMed Central

Cellular responses to adenosine depend on the distribution of the two adenosine receptor subclasses. In primary cultures of rat hepatocytes, adenosine receptors were coupled to adenylate cyclase via A1 and A2 receptors which inhibit and stimulate cyclic AMP production respectively. R-(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA), the adenosine A1 receptor-selective agonist, inhibited glucagon-stimulated cyclic AMP production with an IC50 of 19 nM. This inhibition was blocked by the A1-specific antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPDX). 5'-N- Ethylcarboxamidoadenosine (NECA), an agonist which stimulates A2 receptors, increased cyclic AMP production with an EC50 of 0.6 microM. Treatment of primary cultures of rat hepatocytes with 100 mM ethanol for 48 h decreases the quantity and function of the inhibitory guanine-nucleotide regulatory protein (G(i)), resulting in a sensitization of receptor-stimulated cyclic AMP production [Nagy and deSilva (1992) Biochem. J. 286, 681-686]. When cells were cultured with 2 units/ml adenosine deaminase, to degrade extracellular adenosine, ethanol-induced increases in cyclic AMP production were completely prevented. Moreover, the specific A1-receptor antagonist, CPDX, also blocked the chronic effects of ethanol on receptor-stimulated cyclic AMP production. Treatment with adenosine deaminase or CPDX also prevented the decrease in quantity of the alpha subunit protein of G(i) observed in hepatocytes after chronic treatment with ethanol. Taken together, these results suggest that activation of adenosine A1 receptors on primary cultures of hepatocytes is involved in the development of chronic ethanol-induced sensitization of receptor-stimulated cyclic AMP production. Images Figure 3

Nagy, L E; DeSilva, S E

1994-01-01

222

Regulated renin release from 3T3-L1 adipocytes.  

PubMed

Whereas adipose tissue possesses a local renin-angiotensin system, the synthesis and regulated release of renin has not been addressed. To that end, we utilized differentiating 3T3-L1 cells and analyzed renin expression and secretion. Renin mRNA expression and protein enzymatic activity were not detectable in preadipocytes. However, upon differentiation, renin mRNA and both intracellular and extracellular renin activity were upregulated. In differentiated adipocytes, forskolin treatment resulted in a 28-fold increase in renin mRNA, whereas TNFalpha treatment decreased renin mRNA fourfold. IL-6, insulin, and angiotensin (Ang) II were without effect. In contrast, forskolin and TNFalpha each increased renin protein secretion 12- and sevenfold, respectively. Although both forskolin and TNFalpha induce lipolysis in adipocytes, fatty acids, prostaglandin E(2), and lipopolysaccharide had no effect on renin mRNA or secretion. To evaluate the mechanism(s) by which forskolin and/or TNFalpha are able to regulate renin secretion, a general lipase inhibitor (E600) and PKA inhibitor (H89) were used. Both inhibitors attenuated forskolin-induced renin release, whereas they had no effect on TNFalpha-regulated secretion. In contrast, E600 potentiated forskolin-stimulated renin mRNA levels, whereas H89 had no effect. Neither inhibitor had any influence on TNFalpha regulation of renin mRNA. Relative to lean controls, renin expression was reduced 78% in the epididymal adipose tissue of obese male C57Bl/6J mice, consistent with TNFalpha-mediated downregulation of renin mRNA in the culture system. In conclusion, the expression and secretion of renin are regulated under a complex series of hormonal and metabolic determinants in mature 3T3-L1 adipocytes. PMID:19293336

Fowler, Jason D; Johnson, Nathan D; Haroldson, Thomas A; Brintnall, Joy A; Herrera, Julio E; Katz, Stephen A; Bernlohr, David A

2009-06-01

223

Receptor-mediated methylprednisolone pharmacodynamics in rats: Steroid-induced receptor down-regulation  

Microsoft Academic Search

Several approaches to receptor down-regulation were examined to extend previous receptor\\/ genemediated pharmacokinetic\\/dynamic models of corticosteroids. Down-regulation of the glucocorticoid receptor was considered as an instantaneous event or as a gradual steroid-receptor-mediated process. Concentrations of plasma methylprednisolone, free hepatic cytosolic receptors, and the activity of hepatic tyrosine aminotransferase (TAT) enzyme were measured for 16 hr following administration of 0, 10,

David B. Haughey; William J. Jusko

1992-01-01

224

Genistein induces oestrogen receptor-? gene expression in osteoblasts through the activation of mitogen-activated protein kinases/NF-?B/activator protein-1 and promotes cell mineralisation.  

PubMed

Oestrogen and oestrogen receptors (ER) play critical roles in the maintenance of bone remodelling. Genistein, structurally similar to 17?-oestradiol, is a phyto-oestrogen that may be beneficial for treating osteoporosis. In the present study, we evaluated the effects of genistein on the regulation of ER? gene expression and osteoblast mineralisation using MC3T3-E1 cells and primary rat calvarial osteoblasts as our experimental models. Exposure of MC3T3-E1 cells and primary rat osteoblasts to genistein at ? 10 ?m for 24 h did not affect the cell morphology or viability. However, treatment of MC3T3-E1 cells with 10 ?m-genistein enhanced the phosphorylation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase 1/2 in a time-dependent manner. Sequentially, genistein increased the translocation of NF-?B and c-Jun from the cytoplasm to the nucleus. Consequently, exposure of MC3T3-E1 cells to genistein induced ER? mRNA expression in concentration- and time-dependent manners. In parallel, the amounts of cytosolic and nuclear ER? in MC3T3-E1 cells were increased following genistein administration. Additionally, genistein also increased the levels of ER? mRNA and nuclear ER? protein in rat calvarial osteoblasts. A bioinformatic search revealed that there are several ER?-specific DNA-binding elements in the 5'-promoter regions of the bone morphogenetic protein-6, collagen type I and osteocalcin genes. As a result, genistein could induce the expressions of these osteoblast differentiation-related genes in primary rat osteoblasts. Co-treatment with genistein and traditional differentiation reagents synergistically increased osteoblast mineralisation. Therefore, the present study showed that genistein can induce ER? gene expression via the activation of MAPK/NF-?B/activator protein-1 and accordingly stimulates differentiation-related gene expressions and osteoblast mineralisation. PMID:23829885

Liao, Mei-Hsiu; Tai, Yu-Ting; Cherng, Yih-Giun; Liu, Shing-Hwa; Chang, Ya-An; Lin, Pei-I; Chen, Ruei-Ming

2014-01-14

225

PACAP-induced ERK activation in HEK cells expressing PAC1 receptors involves both receptor internalization and PKC signaling.  

PubMed

The pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor (Adcyap1r1) is a G protein-coupled receptor (GPCR) that activates adenylyl cyclase and PLC. Similar to many other GPCRs, our previous studies showed that the PAC1 receptor is internalized after ligand binding to form signaling endosomes, which recruit additional second messenger pathways. Using a human embryonic kidney (HEK 293) PAC1Hop1-EGFP receptor cell line, we have examined how different PAC1 receptor signaling mechanisms contribute to MEK/ERK activation. Unlike PAC1 receptor-stimulated adenylyl cyclase/cAMP production in the plasma membrane, PACAP-mediated ERK phosphorylation was partly dependent on receptor internalization, as determined by treatment with pharmacological inhibitors of endocytosis or temperature reduction, which also suppressed receptor internalization. Stimulation of cAMP generation by forskolin or exposure to the cell-permeable cAMP analogs 8-bromo-cAMP and dibutyryl cAMP had minimal effects on ERK phosphorylation in this system. The ability of reduced temperature (24°C) to consistently suppress ERK activation to a greater extent than the endocytosis inhibitors Pitstop 2 and dynasore indicated that other mechanisms, in addition to PAC1 internalization/endosome activation, were involved. Inhibition of PAC1 receptor-stimulated PLC/diacylglycerol/PKC signaling by bisindoylmaleimide I also attenuated ERK phosphorylation, and direct PKC activation with phorbol ester increased ERK phosphorylation in a temperature-dependent manner. Inhibition of PAC1 receptor endocytosis and PKC activation completely blocked PACAP-stimulated ERK activation. PACAP augmented phosphorylated ERK staining uniformly over the cytoplasm and nucleus, and PKC signaling facilitated nuclear phosphorylated ERK translocation. In sum, our results show that PACAP/PAC1 receptor endocytosis and PLC/diacylglycerol/PKC activation represent two complementary mechanisms contributing to PACAP-induced ERK activation. PMID:24696141

May, Victor; Buttolph, Thomas R; Girard, Beatrice M; Clason, Todd A; Parsons, Rodney L

2014-06-01

226

Renal D3 dopamine receptor stimulation induces natriuresis by endothelin B receptor interactions  

PubMed Central

Dopaminergic and endothelin systems participate in the control blood pressure by regulating sodium transport in the renal proximal tubule. Disruption of either the endothelin B receptor (ETB) or D3 dopamine receptor gene in mice produces hypertension. To examine whether these two receptors interact we studied the Wistar–Kyoto (WKY) and spontaneously hypertensive (SHR) rats by selectively infusing reagents into the right kidney of anesthetized rats. The D3 receptor agonist (PD128907) caused natriuresis in WKY rats which was partially blocked by the ETB receptor antagonist. In contrast, PD128907 blunted sodium excretion in the SHRs. We found using laser confocal microscopy that the ETB receptor was mainly located in the cell membrane in control WKY cells. Treatment with the D3 receptor antagonist caused its internalization into intracellular compartments that contained the D3 receptors. Combined use of D3 and ETB antagonists failed to internalize ETB receptors in cells from WKY rats. In contrast in SHR cells, ETB receptors were found mainly in internal compartments under basal condition and thus were likely prevented from interacting with the agonist-stimulated, membrane-bound D3 receptors. Our studies suggest that D3 receptors physically interact with proximal tubule ETB receptors and that the blunted natriuretic effect of dopamine in SHRs may be explained, in part, by abnormal D3/ETB receptor interactions.

Zeng, Chunyu; Asico, Laureano D.; Yu, Changqing; Villar, Van Anthony M.; Shi, Weibin; Luo, Yingjin; Wang, Zheng; He, Duofen; Liu, Yan; Huang, Lan; Yang, Chengming; Wang, Xukai; Hopfer, Ulrich; Eisner, Gilbert M.; Jose, Pedro A.

2014-01-01

227

St. John's Wort Promotes Adipocyte Differentiation and Modulates NF-?B Activation in 3T3-L1 Cells.  

PubMed

St. John's wort (SJW), or Hypericum perforatum, is a perennial herb that has been used in the treatment of depression in several countries. Though its therapeutic effect on depression has been extensively studied, its influence on metabolic syndrome is yet to be fully characterized. Therefore, we investigated the effect of SJW extract on adipocyte differentiation and its anti-inflammatory effects by using 3T3-L1 preadipocytes. Oil Red O staining indicated that SJW promotes adipocyte differentiation, while immunoblots indicated that SJW increases the expression of peroxisome proliferator activated receptor ? (PPAR?), a nuclear receptor regulating adipocyte differentiation, and adiponectin, an anti-inflammatory adipokine. Furthermore, the anti-inflammatory activity of SJW was demonstrated by its inhibition of the activation of nuclear factor-?B (NF-?B), an inflammatory transcription factor. Stimulation of mature 3T3-L1 adipocytes by tumor necrosis factor-? (TNF-?) decreased the expression of the NF-?B inhibitor I?B?, and increased its phosphorylation. Treatment with SJW further decreased the TNF-?-induced perturbation in I?B? expression and phosphorylation, which indicated that SJW mediated the inhibition of NF-?B activation. In addition, SJW decreased the TNF-?-induced increase in the mRNA levels of pro-inflammatory adipokines, interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Collectively, our results indicate that SJW treatment could promote adipocyte differentiation probably through its anti-inflammatory activity, which in turn suggests that SJW has the potential to minimize the risk factors of metabolic syndrome. PMID:24989005

Hatano, Tomoko; Sameshima, Yuka; Kawabata, Mami; Yamada, Shizuo; Shinozuka, Kazumasa; Nakabayashi, Toshikatsu; Mizuno, Hideya

2014-01-01

228

Functional selectivity induced by mGlu? receptor positive allosteric modulation and concomitant activation of Gq coupled receptors.  

PubMed

Metabotropic glutamate receptors (mGlus) are a group of Family C Seven Transmembrane Spanning Receptors (7TMRs) that play important roles in modulating signaling transduction, particularly within the central nervous system. mGlu(4) belongs to a subfamily of mGlus that is predominantly coupled to G(i/o) G proteins. We now report that the ubiquitous autacoid and neuromodulator, histamine, induces substantial glutamate-activated calcium mobilization in mGlu(4)-expressing cells, an effect which is observed in the absence of co-expressed chimeric G proteins. This strong induction of calcium signaling downstream of glutamate activation of mGlu(4) depends upon the presence of H(1) histamine receptors. Interestingly, the potentiating effect of histamine activation does not extend to other mGlu(4)-mediated signaling events downstream of G(i/o) G proteins, such as cAMP inhibition, suggesting that the presence of G(q) coupled receptors such as H(1) may bias normal mGlu(4)-mediated G(i/o) signaling events. When the activity induced by small molecule positive allosteric modulators of mGlu(4) is assessed, the potentiated signaling of mGlu(4) is further biased by histamine toward calcium-dependent pathways. These results suggest that G(i/o)-coupled mGlus may induce substantial, and potentially unexpected, calcium-mediated signaling events if stimulation occurs concomitantly with activation of G(q) receptors. Additionally, our results suggest that signaling induced by small molecule positive allosteric modulators may be substantially biased when G(q) receptors are co-activated. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. PMID:22426233

Yin, Shen; Zamorano, Rocio; Conn, P Jeffrey; Niswender, Colleen M

2013-03-01

229

Inflammation and peripheral 5-HT7 receptors: the role of 5-HT7 receptors in carrageenan induced inflammation in rats.  

PubMed

The aim of this study was: (1) to investigate possible role for 5-HT7 receptors in carrageenan induced inflammatory paw oedema in rats; (2) to determine the presence of 5-HT7 receptors in rat paw tissue; (3) to observe the effects of 5-HT7 receptor agonist and antagonist administration on inflammation; and (4) to determine a unique mechanism for inflammatory processes via 5-HT7 receptors. Effects of 5-HT7 receptor agonist, antagonist and indomethacin were investigated in carrageenan induced paw oedema in rats. Blood and tissue samples were collected and evaluated biochemically for serum cytokine levels, tissue oxidant-antioxidant balance and histopathologically for inflammatory cell accumulation. We performed Real Time PCR analyses for tissue 5-HT7 receptor and COX mRNA expressions. The 5-HT7 receptor agonist AS-19 exerted significant anti-inflammatory effect both alone and in combination with indomethacin. Antagonist, SB269970, did not affect inflammation alone but decreased the effects of agonist when co-administered. 5-HT7 mRNA levels were higher in the carrageenan group than healthy control. Carrageenan+indometacin group decreased the mRNA expression of 5-HT7 when compared to carrageenan group. While agonist administration decreased 5-HT7 mRNA expression when compared to carrageenan group. Agonist decreased paw COX expression. Agonist also decreased serum cytokine levels and tissue oxidative stress. In conclusion, this study demonstrated for the first time that 5-HT7 receptors are expressed in rat paw tissue and that this expression responds to inflammatory stimuli. The 5-HT7 receptor may be a promising new therapeutic target for prevention of inflammation and inflammatory disorders and may also provide a new glimpse into inflammation pathophysiology. PMID:23742863

Albayrak, Abdulmecit; Halici, Zekai; Cadirci, Elif; Polat, Beyzagul; Karakus, Emre; Bayir, Yasin; Unal, Deniz; Atasoy, Mustafa; Dogrul, Ahmet

2013-09-01

230

Pituitary adenylate cyclase polypeptide (PACAP) stimulates cyclic AMP formation in pituitary fibroblasts and 3T3 tumor fibroblasts: lack of enhancement by protein kinase C activation.  

PubMed

A number of neuropeptides were shown to produce potent mitogenic effects on Swiss 3T3 fibroblasts by activating the phospholipase C pathway. Here we provide evidence for the activation by PACAP of the adenylate cyclase pathway in 3T3, as well as in non-tumoral pituitary fibroblasts, similarly to what was seen in pituitary endocrine cells. In these cells, PACAP triggered elevation of both intracellular and extracellular contents of cAMP and the effect was time- and dose-dependent, with half-maximal stimulations being induced with about 0.1 nM. Following activation of protein kinase C (PKC) by the phorbol ester phorbol 12-myristate 13-acetate (PMA), PACAP-induced cAMP production was amplified in pituitary endocrine cells, but was either unchanged or dampened in 3T3 and pituitary fibroblasts, respectively. Pretreatment of cells with pertussis toxin (PT) failed to change the effect of PMA on PACAP-stimulated adenylate cyclase activity, irrespective of the cell type being used. However, PT dramatically reduced the potentiation by PMA of cAMP production enhanced by forskolin in 3T3 cells. These results provide new evidence pointing to the presence in fibroblasts of receptors for PACAP, coupled to cAMP production, which may play a role in the modulation of the mitogenic signal. They also indicate that, compared with pituitary endocrine cells, PKC activation in fibroblasts differentially affected PACAP-induced cAMP formation and that these effects were unaltered upon inhibition by PT of Gi-like proteins. PMID:1280235

Koch, B; Lutz-Bucher, B

1992-09-01

231

14,15-Epoxyeicosatrienoic acid induces vasorelaxation through the prostaglandin EP 2 receptors in rat mesenteric artery  

Microsoft Academic Search

Epoxyeicosatrienoic acids (EETs) induce vasorelaxation, probably through G protein-coupled receptors. The identity of these receptors is unclear, but it has been reported that EETs may bind to peroxisome proliferator activated receptors (PPARs) and E-prostanoid (EP) receptors. Therefore, we studied whether PPARs or EP receptors were involved in 14,15-EET-induced vasorelaxation. Isometric tensions of rat mesenteric arteries were measured. The vasorelaxant effect

Cui Yang; Yiu-Wa Kwan; Alice Lai-Shan Au; Christina Chui-Wa Poon; Qian Zhang; Shun-Wan Chan; Simon Ming-Yuen Lee; George Pak-Heng Leung

2010-01-01

232

Effect of Cannabinoid Receptor Agonists on Streptozotocin-Induced Hyperalgesia in Diabetic Neuropathy  

Microsoft Academic Search

The effect of CB-1 and CB-2 receptor agonists, as well as an influence of a non-selective inhibitor of nitric oxide synthase (NOS), L-NOArg, and an inhibitor acting preferentially on cyclooxygenase-1 (COX-1), indomethacin, on the action of cannabinoid receptor agonists in a streptozotocin (STZ)-induced neuropathic model was investigated. When administered alone, a non-selective cannabinoid receptor agonist, WIN 55,212-2, a potentially selective

Magdalena Bujalska

2008-01-01

233

Peripheral benzodiazepine receptor ligand Ro5-4864 inhibits isoprenaline-induced cardiac hypertrophy in rats  

Microsoft Academic Search

Oxidative stress plays a significant role in the pathogenesis of cardiac hypertrophy. Peripheral benzodiazepine receptors are ubiquitously expressed in various tissues, including the heart. Peripheral benzodiazepine receptors have been reported to be involved in the protection of cells against oxygen radical damage. The present study was designed to determine whether Ro5-4864 (a peripheral benzodiazepine receptor ligand) can inhibit isoprenaline-induced cardiac

Amardeep Jaiswal; Santosh Kumar; Rajesh Enjamoori; Sandeep Seth; Amit Kumar Dinda; Subir Kumar Maulik

2010-01-01

234

Distinct Mechanisms of Agonist-induced Endocytosis for Human Chemokine Receptors CCR5 and CXCR4  

Microsoft Academic Search

Desensitization of the chemokine receptors, a large class of G protein-coupled receptors, is mediated in part by agonist-driven receptor endocytosis. However, the exact pathways have not been fully defined. Here we demonstrate that the rate of ligand-induced endocytosis of CCR5 in leukocytes and expression systems is significantly slower than that of CXCR4 and requires prolonged agonist treatment, suggesting that these

Sundararajan Venkatesan; Jeremy J. Rose; Robert Lodge; Philip M. Murphy; John F. Foley; Cell Biology

2003-01-01

235

The role of hypothalamic H1 receptor antagonism in antipsychotic-induced weight gain.  

PubMed

Treatment with second generation antipsychotics (SGAs), notably olanzapine and clozapine, causes severe obesity side effects. Antagonism of histamine H1 receptors has been identified as a main cause of SGA-induced obesity, but the molecular mechanisms associated with this antagonism in different stages of SGA-induced weight gain remain unclear. This review aims to explore the potential role of hypothalamic histamine H1 receptors in different stages of SGA-induced weight gain/obesity and the molecular pathways related to SGA-induced antagonism of these receptors. Initial data have demonstrated the importance of hypothalamic H1 receptors in both short- and long-term SGA-induced obesity. Blocking hypothalamic H1 receptors by SGAs activates AMP-activated protein kinase (AMPK), a well-known feeding regulator. During short-term treatment, hypothalamic H1 receptor antagonism by SGAs may activate the AMPK-carnitine palmitoyltransferase 1 signaling to rapidly increase caloric intake and result in weight gain. During long-term SGA treatment, hypothalamic H1 receptor antagonism can reduce thermogenesis, possibly by inhibiting the sympathetic outflows to the brainstem rostral raphe pallidus and rostral ventrolateral medulla, therefore decreasing brown adipose tissue thermogenesis. Additionally, blocking of hypothalamic H1 receptors by SGAs may also contribute to fat accumulation by decreasing lipolysis but increasing lipogenesis in white adipose tissue. In summary, antagonism of hypothalamic H1 receptors by SGAs may time-dependently affect the hypothalamus-brainstem circuits to cause weight gain by stimulating appetite and fat accumulation but reducing energy expenditure. The H1 receptor and its downstream signaling molecules could be valuable targets for the design of new compounds for treating SGA-induced weight gain/obesity. PMID:23640535

He, Meng; Deng, Chao; Huang, Xu-Feng

2013-06-01

236

Serotonin 5-HT2 Receptors Induce a Long-Lasting Facilitation of Spinal Reflexes Independent of Ionotropic Receptor Activity  

PubMed Central

Dorsal root-evoked stimulation of sensory afferents in the hemisected in vitro rat spinal cord produces reflex output, recorded on the ventral roots. Transient spinal 5-HT2C receptor activation induces a long-lasting facilitation of these reflexes (LLFR) by largely unknown mechanisms. Two Sprague-Dawley substrains were used to characterize network properties involved in this serotonin (5-HT) receptor-mediated reflex plasticity. Serotonin more easily produced LLFR in one substrain and a long-lasting depression of reflexes (LLDR) in the other. Interestingly, LLFR and LLDR were bidirectionally interconvertible using 5-HT2A/2C and 5-HT1A receptor agonists, respectively, regardless of substrain. LLFR was predominantly A? afferent fiber mediated, consistent with prominent 5-HT2C receptor expression in the A? fiber projection territories (deeper spinal laminae). Reflex facilitation involved an unmasking of polysynaptic pathways and an increased receptive field size. LLFR emerged even when reflexes were evoked three to five times/h, indicating an activity independent induction. Both the NMDA and AMPA/kainate receptor-mediated components of the reflex could be facilitated, and facilitation was dependent on 5-HT receptor activation alone, not on coincident reflex activation in the presence of 5-HT. Selective blockade of GABAA and/or glycine receptors also did not prevent reflex amplification and so are not required for LLFR. Indeed, a more robust response was seen after blockade of spinal inhibition, indicating that inhibitory processes serve to limit reflex amplification. Overall we demonstrate that the serotonergic system has the capacity to induce long-lasting bidirectional changes in reflex strength in a manner that is nonassociative and independent of evoked activity or activation of ionotropic excitatory and inhibitory receptors.

Shay, Barbara L.; Sawchuk, Michael; Machacek, David W.; Hochman, Shawn

2009-01-01

237

Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes.  

PubMed

Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBP? and PPAR? was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBP?, PPAR?, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis. PMID:23237809

Lai, Peng-Yeh; Tsai, Chong-Bin; Tseng, Min-Jen

2013-01-18

238

TRPM7 channels regulate proliferation and adipogenesis in 3T3-L1 preadipocytes.  

PubMed

Transient receptor potential melastatin-7 (TRPM7) channels are involved in many cellular physiological and pathological processes. The present study was designed to investigate the expression of TRPM7 channels and the potential role in regulating cell proliferation and adipogenesis in 3T3-L1 preadipocytes with approaches of whole-cell patch voltage-clamp, molecular biology, cell proliferation, adipogenesis, etc. We found that a TRPM7-like current was recorded with Mg(2+) -free pipette solution in 3T3-L1 preadipocytes, and the current was inhibited by intercellular free Mg(2+) . The TRPM7-like current was potentiated by acidic pH and inhibited by 2-aminoethoxydiphenyl borate (2-APB). RT-PCR, Western blot and immunocytochemistry revealed that gene and protein of TRPM7 channels were abundant in 3T3-L1 preadipocytes. Blockade of TRPM7 channels with 2-APB inhibited cell proliferation in 3T3-L1 cells. In addition, knockdown of TRPM7 with specific siRNA inhibited both proliferation and adipogenesis. The present study demonstrates for the first time that TRPM7 channels regulate cell cycle and adipogenesis of 3T3-L1 preadipocytes. PMID:23765921

Chen, Kui-Hao; Xu, Xiao-Hui; Liu, Yi; Hu, Yan; Jin, Man-Wen; Li, Gui-Rong

2014-01-01

239

Counterregulation of nuclear 3,5,3'-triiodo-L-thyronine (T3) binding by oxidized and reduced-nicotinamide adenine dinucleotide phosphates in the presence of cytosolic T3-binding protein in vitro  

SciTech Connect

The role of cytosolic T3-binding protein (CTBP) in the regulation of nuclear T3 binding was studied in vitro. Nuclear (125I)T3 binding was observed in the presence of 1.0 mM dithiothreitol (DTT). When the nuclei prepared from rat kidney were incubated with inactive form of CTBP which was also prepared from rat kidney, (125I)T3 binding to nuclei was not affected. When the nuclei were incubated with inactive form of CTBP in the presence of NADP, (125I)T3 binding to nuclei was increased, whereas binding was diminished when nuclei were incubated with CTBP in the presence of NADPH. The inactive form of CTBP was activated by NADPH. NADP also activated CTBP in the presence of DTT. Both active forms of CTBP were again inactivated by extraction with charcoal, and these inactive forms were reactivated by NADPH or by NADP and DTT, but not by NADP alone. Although the nuclei treated with 0.3 M NaCl lost the binding activity for (125I)T3 in the absence of NADP, the nuclei retained the binding activity for (125I)T3 in the presence of NADP and the inactive form of CTBP. Treatment of the nuclei with 0.5 M NaCl lost the binding activity for (125I)T3 not only in the absence but also in the presence of NADP and CTBP. These results suggested that NADP and NADPH play roles as counterregulatory factors for nuclear T3 binding in the presence of CTBP. Further, it was speculated that binding sites for the T3-CTBP complex, which is generated in the presence of NADP and DTT, are present in nuclei, and that binding sites for the complex are different from nuclear T3 receptors.

Hashizume, K.; Miyamoto, T.; Yamauchi, K.; Ichikawa, K.; Kobayashi, M.; Ohtsuka, H.; Sakurai, A.; Suzuki, S.; Yamada, T.

1989-04-01

240

Mechanistic distinctions between agrin and laminin-1 induced aggregation of acetylcholine receptors  

PubMed Central

Background One of the earliest steps in synaptogenesis at the neuromuscular junction is the aggregation of nicotinic acetylcholine receptors at the postsynaptic membrane. This study presents quantitative analyses of receptor and ?-Dystroglycan aggregation in response to agrin and laminin-1, alone or in combination. Results Both laminin and agrin increased overall expression of receptors on the plasma membrane. Following a 24 hour exposure, agrin increased the number of receptor aggregates but did not affect the number of ?-Dystroglycan aggregates, while the reverse was true of laminin-1. Laminin also increased receptor concentration within aggregates, while agrin had no such effect. Finally, the spatial distribution of aggregates was indistinguishable from random in the case of laminin, while agrin induced aggregates were closer together than predicted by a random model. Conclusions Agrin and laminin-1 both increase acetylcholine receptor aggregate size after 24 hours, but several lines of evidence indicate that this is achieved via different mechanisms. Agrin and laminin had different effects on the number and density of receptor and ?-Dystroglycan aggregates. Moreover the random distribution of laminin induced (as opposed to agrin induced) receptor aggregates suggests that the former may influence aggregate size by simple mass action effects due to increased receptor expression.

Lee, Lara K; Kunkel, Dennis D; Stollberg, Jes

2002-01-01

241

ETHANOL INDUCES HIGHER BEC IN CB1 CANNABINOID RECEPTOR KNOCKOUT MICE WHILE DECREASING ETHANOL PREFERENCE  

Microsoft Academic Search

Abstract — Aims: Previous studies have shown that CB1 cannabinoid receptors are involved in the behavioural effects induced by chronic ethanol administration in Wistar rats by using SR 141716, a CB1 cannabinoid receptor antagonist. These studies have now been extended to investigate the effect of acute and chronic alcoholization on blood ethanol concentration (BEC) and ethanol preference in CB1 knockout

F. LALLEMAND; P. De Witte

2004-01-01

242

Theophylline and Dexamethasone Induce Peroxisome Proliferator-Activated Receptor-? Expression in Human Eosinophils  

Microsoft Academic Search

Eosinophils are major effector cells in allergic diseases including asthma. Peroxisome proliferator-activated receptor-? (PPAR?) is a nuclear receptor that regulates immune reaction. We have previously demonstrated that human eosinophils express PPAR? and that stimulation with a synthetic agonist for PPAR? attenuated the factor-induced eosinophil survival and chemotaxis. However, the modulator of the eosinophil PPAR? expression has not yet been studied.

Atsuko Usami; Shigeharu Ueki; Wataru Ito; Yoshiki Kobayashi; Takahito Chiba; Gulixian Mahemuti; Hajime Oyamada; Yumiko Kamada; Miyoshi Fujita; Hikari Kato; Norihiro Saito; Hiroyuki Kayaba; Junichi Chihara

2006-01-01

243

Can We Predict Real T3 Stage Prostate Cancer in Patients with Clinical T3 (cT3) Disease before Radical Prostatectomy?  

PubMed Central

Purpose Down-staging of clinical T3 (cT3) prostate cancer after radical prostatectomy (RP) is not uncommon due to the inaccuracy of the currently available staging modalities, although selected down-staged cT3 patients can be a candidate for definitive RP. We identified the significant predictors for down-staging of cT3 after RP. Materials and Methods We included 67 patients with cT3 stage prostate cancer treated with radical perineal prostatectomy (RPP) between 1998 and 2006 and reviewed their medical records retrospectively. The clinical stage was obtained according to the DRE, the prostate biopsy findings, and the prostate MRI. Results Fifty three (79%) patients with cT3 prostate cancer were down-staged to pT2 after RP. The percent of positive cores had the strongest association with down-staging of cT3 [p = 0.01, odds ratio (OR) = 6.3], followed by baseline prostate specific antigen (PSA) (p = 0.03, OR = 5.0), the biopsy Gleason sum (GS) (p = 0.03, OR = 4.7), and the maximum tumor volume of the positive cores (p = 0.05, OR = 4.0). When the cut-off points of significant parameters which were a PSA < 10 ng/mL, a percent of positive cores ? 30%, a maximum tumor volume of the positive cores ? 75% and GS ? 7 were combined, the sensitivity, specificity, and positive predictive value were 0.25%, 1.00%, and 100%, respectively. Conclusion The percent of positive cores ? 30%, serum PSA < 10 ng/mL, the biopsy GS ? 7, and the maximum tumor volume of the positive cores ? 75% were the significant predictors of down-staging cT3 disease after RP.

Lee, Hye Won; Jeon, Seong Soo; Lee, Hyun Moo; Choi, Han Yong

2010-01-01

244

Toward a Consensus on the Operation of Receptor-Induced Calcium Entry Signals  

NSDL National Science Digital Library

Receptor-induced Ca2+ signals involve both Ca2+ release from intracellular stores and extracellular Ca2+ entry across the plasma membrane. The channels mediating Ca2+ entry and the mechanisms controlling their function remain largely a mystery. Here we critically assess current views on the Ca2+ entry process and consider certain modifications to the widely held hypothesis that Ca2+ store emptying is the fundamental trigger for receptor-induced Ca2+ entry channels. Under physiological conditions, receptor-induced store depletion may be quite limited. A number of distinct channel activities appear to mediate receptor-induced Ca2+ entry, and their activation is observed to occur through quite diverse coupling processes.

Donald L. Gill (University of Maryland School of Medicine;Department of Biochemistry and Molecular Biology REV); Randen L. Patterson (Pennsylvania State University;Mueller Laboratory REV)

2004-07-27

245

Endothelin subtype A receptor antagonist induces osteopenia in growing rats  

Microsoft Academic Search

Previous studies suggested that endothelin (ET) peptides are involved in bone metabolism. We examined the effects of long-term blockade of the ETA receptor, a receptor subtype primarily involved in the anabolic actions of ET, on bone mineral status in growing rats. Eight-week-old rats injected intraperitoneally with FR139317 50 mg\\/kg body weight, a specific ETA receptor antagonist, for 2 or 4

Hirokazu Tsukahara; Chikahide Hori; Masahiro Hiraoka; Kazutaka Yamamoto; Yasushi Ishii; Mitsufumi Mayumi

1998-01-01

246

Receptor potential and light-induced mitochondrial activation in blowfly photoreceptor mutants  

Microsoft Academic Search

1.Simultaneous measurements of the receptor potential and the light-induced mitochondrial activation were performed in white-eyed blowflies Calliphora vicina, mutant chalky, and Lucilia cuprina, mutants wFand w'nss. The intensity dependence and the temporal dynamics were investigated.2.The characteristic curve of the light-induced mitochondrial activation vs. log intensity has an S-like shape, which is much steeper than the characteristic curve of the receptor

M. H. Mojet; J. Tinbergen; D. G. Stavenga

1991-01-01

247

?-opioid receptor stimulation inhibits cardiac hypertrophy induced by ? 1-adrenoceptor stimulation in the rat  

Microsoft Academic Search

To test the hypothesis that ?-opioid receptor stimulation inhibits cardiac hypertrophy induced by ?1-adrenoceptor stimulation, we determined the effects of trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective ?-opioid receptor agonist, on cardiac hypertrophy induced by isoprenaline, a selective ?-adrenoceptor agonist, in neonatal ventricular myocytes upon blockade of ?2-adrenoceptor. Hypertrophy of cardiomyocytes was determined by increases in (i) total protein content; (ii)

Dan Shan; Hongxin Wang; Yuhong Su; Yang Jing; Tak Ming Wong

2007-01-01

248

Renal hemodynamics in radiocontrast medium-induced renal dysfunction: A role for dopamine-1 receptors  

Microsoft Academic Search

Renal hemodynamics in radiocontrast medium-induced renal dysfunction: A role for dopamine-1 receptors.BackgroundRadiocontrast medium (RCM) administration induces a transient increase in renal blood flow (RBF), followed by a prolonged vasoconstriction. This vasoconstrictor phase in RBF is accompanied by a decrement in glomerular filtration rate (GFR). Nonselective dopamine (DA) receptor stimulation is known to increase RBF and GFR. Clinical studies, however, fail

George L. Bakris; Nancy A. Lass; Dana Glock

1999-01-01

249

Angiotensin II type 2 receptor antagonist reduces bleomycin-induced pulmonary fibrosis in mice  

Microsoft Academic Search

BACKGROUND: The role of angiotensin II type 2 receptor (AT2) in pulmonary fibrosis is unknown. To evaluate the influence of angiotensin II type 1 receptor (AT1) and AT2 antagonists in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis. METHODS: We examined effects of the AT1 antagonist (AT1A) olmesartan medoxomil (olmesartan) and the AT2 antagonist (AT2A) PD-123319 on BLM-induced pulmonary fibrosis,

Yuko Waseda; Masahide Yasui; Yoriko Nishizawa; Kanako Inuzuka; Hazuki Takato; Yukari Ichikawa; Atsuro Tagami; Masaki Fujimura; Shinji Nakao

2008-01-01

250

A water-soluble extract of Petalonia binghamiae inhibits the expression of adipogenic regulators in 3T3-L1 preadipocytes and reduces adiposity and weight gain in rats fed a high-fat diet.  

PubMed

We previously showed that an ethanolic extract of the edible brown algae Petalonia binghamiae promotes the differentiation of 3T3-L1 preadipocytes and decreases hyperglycemia in streptozotocin-induced diabetic mice. Here, we report that a water-soluble extract of P. binghamiae thalli, prepared by enzymatic digestion, inhibits preadipocyte differentiation and adipogenesis in a dose-dependent manner. In differentiating 3T3-L1 preadipocytes, the extract (designated PBEE) decreased the expression of peroxisome proliferator-activated receptor ?, CCAAT/enhancer-binding proteins ? and ?, and fatty acid-binding protein aP2. It also inhibited the mitotic clonal expansion process of adipocyte differentiation, and it inhibited insulin-stimulated uptake of glucose into mature 3T3-L1 adipocytes by reducing phosphorylation of insulin receptor substrate-1. In rats with high-fat diet (HFD)-induced obesity, PBEE exhibited potent anti-obesity effects. In this animal model, increases in body weight and fat storage were suppressed by the addition of PBEE to the drinking water at 500 mg/L for 30 days. PBEE supplementation reduced serum levels of glutamic pyruvic and glutamic oxaloacetic transaminases and increased the serum level of high-density lipoprotein cholesterol. Moreover, it significantly decreased the accumulation of lipid droplets in liver tissue, suggesting a protective effect against HFD-induced hepatic steatosis. Taken together, these data demonstrate that PBEE inhibits preadipocyte differentiation and adipogenesis in cultured cells and in rodent models of obesity. PMID:20332066

Kang, Seong-Il; Kim, Moo-Han; Shin, Hye-Sun; Kim, Hyo-Min; Hong, Youn-Suk; Park, Ji-Gweon; Ko, Hee-Chul; Lee, Nam-Ho; Chung, Wan-Seok; Kim, Se-Jae

2010-12-01

251

Contribution of PGE2 EP1 receptor in hemin-induced neurotoxicity  

PubMed Central

Although hemin-mediated neurotoxicity has been linked to the production of free radicals and glutamate excitotoxicity, the role of the prostaglandin E2 (PGE2)-EP1 receptor remains unclear. Activation of the EP1 receptor in neurons results in increased intracellular calcium levels; therefore, we hypothesize that the blockade of the EP1 receptor reduces hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wild-type (WT) and EP1?/? mice, we investigated the EP1 receptor role in hemin neurotoxicity measured by lactate dehydrogenase (LDH) cell survival assay. Hemin (75 ?M) induced greater release of LDH in WT (34.7 ± 4.5%) than in EP1?/? (27.6 ± 3.3%) neurons. In the presence of the EP1 receptor antagonist SC-51089, the hemin-induced release of LDH decreased. To further investigate potential mechanisms of action, we measured changes in the intracellular calcium level [Ca2+]i following treatment with 17-phenyl trinor PGE2 (17-pt-PGE2) a selective EP1 agonist. In the WT neurons, 17-pt-PGE2 dose-dependently increased [Ca2+]i. However, in EP1?/? neurons, [Ca2+]i was significantly attenuated. We also revealed that hemin dose-dependently increased [Ca2+]i in WT neurons, with a significant decrease in EP1?/? neurons. Both 17-pt-PGE2 and hemin-induced [Ca2+]i were abolished by N-methyl-D-aspartic (NMDA) acid receptor and ryanodine receptor blockers. These results suggest that blockade of the EP1 receptor may be protective against hemin neurotoxicity in vitro. We speculate that the mechanism of hemin neuronal death involves [Ca2+]i mediated by NMDA acid receptor-mediated extracellular Ca2+ influx and EP1 receptor-mediated intracellular release from ryanodine receptor-operated Ca2+ stores. Therefore, blockade of the EP1 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin.

Mohan, Shekher; Glushakov, Alexander V.; deCurnou, Alexander; Narumiya, Shuh; Dore, Sylvain

2013-01-01

252

Roles of ?-opioid receptors and nociceptin/orphanin FQ peptide receptors in buprenorphine-induced physiological responses in primates.  

PubMed

Buprenorphine is known as a ?-opioid peptide (MOP) receptor agonist, but its antinociception is compromised by the activation of nociceptin/orphanin FQ peptide (NOP) receptors in rodents. The aim of this study was to investigate the roles of MOP and NOP receptors in regulating buprenorphine-induced physiological responses in primates (rhesus monkeys). The effects of MOP antagonist (naltrexone), NOP antagonist [(±)-1-[(3R*,4R*)-1-(cyclooctylmethyl)-3-(hydroxymethyl)-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397)], and NOP agonists [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5] decan-4-one (Ro 64-6198) and 3-endo-8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510)] on buprenorphine were studied in three functional assays for measuring analgesia, respiratory depression, and itch in primates. Over the dose range of 0.01 to 0.1 mg/kg, buprenorphine dose-dependently produced antinociception, respiratory depression, and itch/scratching responses, and there was a ceiling effect at higher doses (0.1-1 mg/kg). Naltrexone (0.03 mg/kg) produced similar degrees of rightward shifts of buprenorphine's dose-response curves for all three endpoints. Mean pK(B) values of naltrexone (8.1-8.3) confirmed that MOP receptors mediated mainly buprenorphine-induced antinociception, respiratory depression, and itch/scratching. In contrast, J-113397 (0.1 mg/kg) did not change buprenorphine-induced physiological responses, indicating that there were no functional NOP receptors in buprenorphine-induced effects. More importantly, both NOP agonists, Ro 64-6198 and SCH 221510, enhanced buprenorphine-induced antinociception without respiratory depression and itch/ scratching. The dose-addition analysis revealed that buprenorphine in combination with the NOP agonist synergistically produced antinociceptive effects. These findings provided functional evidence that the activation of NOP receptors did not attenuate buprenorphine-induced antinociception in primates; instead, the coactivation of MOP and NOP receptors produced synergistic antinociception without other side effects. This study strongly supports the therapeutic potential of mixed MOP/NOP agonists as innovative analgesics. PMID:22743574

Cremeans, Colette M; Gruley, Erin; Kyle, Donald J; Ko, Mei-Chuan

2012-10-01

253

Roles of ?-Opioid Receptors and Nociceptin/Orphanin FQ Peptide Receptors in Buprenorphine-Induced Physiological Responses in Primates  

PubMed Central

Buprenorphine is known as a ?-opioid peptide (MOP) receptor agonist, but its antinociception is compromised by the activation of nociceptin/orphanin FQ peptide (NOP) receptors in rodents. The aim of this study was to investigate the roles of MOP and NOP receptors in regulating buprenorphine-induced physiological responses in primates (rhesus monkeys). The effects of MOP antagonist (naltrexone), NOP antagonist [(±)-1-[(3R*,4R*)-1-(cyclooctylmethyl)-3-(hydroxymethyl)-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397)], and NOP agonists [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5] decan-4-one (Ro 64-6198) and 3-endo-8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510)] on buprenorphine were studied in three functional assays for measuring analgesia, respiratory depression, and itch in primates. Over the dose range of 0.01 to 0.1 mg/kg, buprenorphine dose-dependently produced antinociception, respiratory depression, and itch/scratching responses, and there was a ceiling effect at higher doses (0.1–1 mg/kg). Naltrexone (0.03 mg/kg) produced similar degrees of rightward shifts of buprenorphine's dose-response curves for all three endpoints. Mean pKB values of naltrexone (8.1–8.3) confirmed that MOP receptors mediated mainly buprenorphine-induced antinociception, respiratory depression, and itch/scratching. In contrast, J-113397 (0.1 mg/kg) did not change buprenorphine-induced physiological responses, indicating that there were no functional NOP receptors in buprenorphine-induced effects. More importantly, both NOP agonists, Ro 64-6198 and SCH 221510, enhanced buprenorphine-induced antinociception without respiratory depression and itch/ scratching. The dose-addition analysis revealed that buprenorphine in combination with the NOP agonist synergistically produced antinociceptive effects. These findings provided functional evidence that the activation of NOP receptors did not attenuate buprenorphine-induced antinociception in primates; instead, the coactivation of MOP and NOP receptors produced synergistic antinociception without other side effects. This study strongly supports the therapeutic potential of mixed MOP/NOP agonists as innovative analgesics.

Cremeans, Colette M.; Gruley, Erin; Kyle, Donald J.

2012-01-01

254

Receptor-induced death in human natural killer cells: involvement of CD16  

PubMed Central

Propriocidal regulation of T cells refers to apoptosis induced by interleukin 2 (IL-2) activation with subsequent antigen receptor stimulation. We examined whether natural killer (NK) cells exhibited cytokine- and ligand-induced death similar to activated T cells. Peripheral NK cells were examined for ligand-induced death using antibodies to surface moieties (CD2, CD3, CD8, CD16, CD56), with and without prior activation of IL-2. Only those NK cells stimulated first with IL-2 and then with CD16 exhibited ligand-induced death; none of the other antibody stimuli induced this phenomenon. Next we examined various cytokines (IL-2, IL-4, IL-6, IL-7, IL-12, IL-13, interferon alpha and gamma) that can activate NK cells and determined if CD16- induced killing occurred. Only IL-2 and IL-12 induced NK cell death after occupancy of this receptor by aggregated immunoglobulin or by cross-linking with antireceptor antibody. The CD16-induced death was inhibited by herbimycin A, indicating that cell death was dependent upon protein tyrosine kinases. Identical to T cells, the form of cell death for NK cells was demonstrated to be receptor-induced apoptosis. Overall these data indicate that highly activated NK cells mediate ligand-induced apoptosis via signaling molecules like CD16. Whereas the propriocidal regulation of T cells is antigen specific, this is not the case for NK cells due to the nature of the receptor. The clinical implications of this finding are considered.

1995-01-01

255

Differential role of protein kinase C in desensitization of muscarinic receptor induced by phorbol esters and receptor agonists  

SciTech Connect

PKC, a phorbol ester receptor, copurified with specific binding sites of ({sup 3}H)phorbol-12,13,-dibutyrate (({sup 3}H)PDBu). The specific binding of ({sup 3}H)PDBu to intact cells was saturable to a single class of binding sites. The PKC and phorbol ester receptors in N1E-115 cells can be down regulated by prolonged phorbol ester incubation. Phorbol 12-myristate 13-acetate (PMA) suppressed muscarinic receptor-mediated cyclic GMP response in a time-dependent and a concentration-dependent fashion and the suppressive effect of PMA could be attenuated by a protein kinase inhibitor, H-7, as well as by down-regulation of the PKC through long-term incubation with PDBu. Exposure of the cells to the muscarinic agonist carbamylcholine also desensitized subsequent CBC-mediated cyclic GMP response. However, pretreatment with carbamylcholine did not desensitize histamine-induced cyclic GMP formation while treatment with PMA suppressed this histamine-mediated response. Preincubation of the cells with CBC, but not with phorbol ester, resulted in down-regulation of muscarinic receptors. The loss of muscarinic receptors induced by agonist even occurred when the phosphoinositide hydrolysis response was suppressed.

Lai, Wi Sheung.

1989-01-01

256

Brain CB1 receptor expression following lipopolysaccharide-induced inflammation  

PubMed Central

Cannabinoid 1 receptors (CB1) are highly expressed on presynaptic terminals in the brain where they are importantly involved in the control of neurotransmitter release. Alteration of CB1 expression is associated with a variety of neurological and psychiatric disorders. There is now compelling evidence that peripheral inflammatory disorders are associated with depression and cognitive impairments. These can be modeled in rodents with peripheral administration of lipopolysaccharide (LPS), but central effects of this treatment remain to be fully elucidated. As a reduction in endocannabinoid tone is thought to contribute to depression, we asked whether the expression of CB1 in the central nervous system (CNS) is altered following LPS treatment. CD1 mice received LPS (0.1–1 mg/kg, ip) and 6 hours later activated microglial cells were observed only in circumventricular organs and only at the higher dose. At 24 hours, activated microglial cells were identified in other brain regions, including the hippocampus, a structure implicated in some mood disorders. Immunohistochemistry and real-time PCR were utilized to evaluate the change of CB1 expression 24 hours after inflammation. LPS induced an increase of CB1 mRNA in hippocampus and brainstem. Subsequent immunohistochemical analysis revealed reduced CB1 in hippocampus, especially in CA3 pyramidal layer. Analysis of co-localization with markers of excitatory and inhibitory terminals indicated that the decrease in CB1 expression was restricted to glutamatergic terminals. Despite widespread microglial activation, these results suggest that peripheral LPS treatment leads to limited changes in CB1 expression in the brain.

Hu, Huangming; Ho, Winnie; Mackie, Ken; Pittman, Quentin J.; Sharkey, Keith A.

2012-01-01

257

Protective effect of ?-lipoic acid on islet cells co-cultured with 3T3L1 adipocytes  

PubMed Central

Obesity and ?-cell dysfunction due to oxidative stress impact the pathogenesis of type 2 diabetes mellitus. We co-cultured 3T3L1 adipocytes and islet cells in the presence or absence of the antioxidant ?-lipoic acid (LA) and assayed the effects of the adipocytes and LA on the secretion of insulin by the islet cells and on the activities of factors involved in secretion and oxidative stress. At low glucose concentrations (2.8 mmol/l), the presence of adipocytes (co-culture) increased insulin secretion compared with islet cells cultured alone (control) and this increase was diminished by LA (co-culture plus LA). At high glucose concentrations (22 mmol/l), insulin secretion levels were similar for all islet groups, resulting in a restoration of the stimulation index in the presence of LA. The mRNA levels of the glucose-stimulated insulin secretion (GSIS) genes glucokinase, glucose transporter 2 and Kir6.2 were downregulated under co-culture and co-culture plus LA conditions. Protein and tyrosine phosphorylation levels of insulin receptor-? and insulin receptor substrate-1 were decreased under co-culture conditions and were restored by LA treatment. Cellular malondialdehyde levels increased in the co-cultured islets and this increase was blocked by LA. The mRNA levels of superoxide dismutase and catalase were reduced under co-culture conditions and these reductions were eliminated by the addition of LA. In conclusion, 3T3L1 adipocytes disturb insulin secretion and induce islet dysfunction. The effects may be mediated by multiple pathways, which include downregulation of GSIS gene expression, suppression of islet cell insulin signaling and the induction of oxidative stress. LA may protect islet cells via activation of islet cell insulin signaling and the mRNA expression of antioxidant enzymes.

WANG, YUFAN; DONG, WEIPING; DING, XIAOYING; WANG, FENG; WANG, YUFEI; CHEN, XINYA; YU, LONG; LI, XIAOHUA; ZHANG, AIFANG; PENG, YONGDE

2012-01-01

258

Enhancement of D2 receptor agonist-induced inhibition by D1 receptor agonist in the ventral tegmental area.  

PubMed Central

1. A microiontophoretic study was performed on chloral hydrate-anaesthetized rats to examine the role of D1 receptors in the ventral tegmental area (VTA) neurones, which are inhibited by autoreceptor and D2 receptor agonists. 2. Inhibition by microiontophoretic application of quinpirole (a D2 agonist) of antidromic spikes elicited by stimulation of the nucleus accumbens in dopaminergic neurones of the VTA, was significantly enhanced by simultaneous application of SKF 38393 (D1 agonist), although SKF 38393 alone had little effect on the neurones. 3. In addition, quinpirole-induced inhibition was antagonized by iontophoretic application of domperidone (D2 antagonist), but was not affected by SCH 23390 (D1 antagonist). 4. Furthermore, SKF 38393-induced enhancement of inhibition by quinpirole was antagonized by simultaneous application of SCH 23390. 5. These results suggest that activation of D1 receptors located on the VTA dopaminergic neurones or on non-dopaminergic nerve terminals is not essential for inducing inhibition of the dopaminergic neurones, but enhances D2 receptor-mediated inhibition directly or indirectly via inhibitory neurones. Images Figure 1

Momiyama, T.; Sasa, M.; Takaori, S.

1993-01-01

259

Role and Efforts of T3C in Corrosion Economics.  

National Technical Information Service (NTIS)

The basic purpose of T3C activity is to show how to acquire specific corrosion cost information so that overall costs for doing business can be reduced. The scope of T3C is to accumulate data, appraise methods, develop recommended practices, promote knowl...

L. D. Perrigo B. R. Appleman R. I. Pamer J. L. Thompson

1979-01-01

260

The kinin B1 receptor: an inducible G protein coupled receptor.  

PubMed

The B1 receptor, selectively stimulated by des-Arg9 fragments of native kinins, has a place in the vast family of G protein coupled receptors. We discuss a series of six criteria useful for comparing the B1 receptor with the more prominent and studied bradykinin B2 receptor. The B1 receptor has attracted interest because it is rapidly upregulated in biological systems following some types of tissue injury, notably the injection of bacterial materials to rabbits, rats, or pigs. A fast and specific genetic program recruits the expression of what we know now to be a G protein coupled receptor in smooth muscle cells, endothelial cells, fibroblasts, and a few other cell types. The cytokine network has been linked to B1 receptor expression in functional experiments, and this may be related to the recent finding of potential cytokine response elements in the proposed gene promoter of the human B1 receptor gene. The experimental approach of B1 receptor mRNA transcriptional regulation, protein synthesis, and maturation is illustrated, based on the biochemical (Northern blot) and functional analysis of isolated organs from rabbits injected with bacterial lipopolysaccharide or incubated in vitro with or without interleukin-1. PMID:9276155

Marceau, F; Larrivée, J F; Saint-Jacques, E; Bachvarov, D R

1997-06-01

261

Aldosterone deficiency and mineralocorticoid receptor antagonism prevent angiotensin II-induced cardiac, renal, and vascular injury  

PubMed Central

Angiotensin II causes cardiovascular injury in part by aldosterone-induced mineralocorticoid receptor activation, and it can also activate the mineralocorticoid receptor in the absence of aldosterone in vitro. Here we tested whether endogenous aldosterone contributes to angiotensin II/salt-induced cardiac, vascular, and renal injury by the mineralocorticoid receptor. Aldosterone synthase knockout mice and wild type littermates were treated with angiotensin II or vehicle plus the mineralocorticoid receptor antagonist spironolactone or regular diet while drinking 0.9- saline. Angiotensin II/salt caused hypertension in both the knockout and wild type mice; an effect significantly blunted in the knockout mice. Either genetic aldosterone deficiency or mineralocorticoid receptor antagonism reduced cardiac hypertrophy, aortic remodeling, and albuminuria, as well as cardiac, aortic, and renal plasminogen activator inhibitor-1 mRNA expression during angiotensin II treatment. Mineralocorticoid receptor antagonism reduced angiotensin II/salt-induced glomerular hypertrophy, but aldosterone deficiency did not. Combined mineralocorticoid receptor antagonism and aldosterone deficiency reduced blood urea nitrogen and restored nephrin immunoreactivity. Angiotensin II/salt also promoted glomerular injury through the mineralocorticoid receptor in the absence of aldosterone. Thus, mineralocorticoid antagonism may have protective effects in the kidney beyond aldosterone synthase inhibition.

Luther, James M.; Luo, Pengcheng; Wang, Zuofei; Cohen, Samuel E.; Kim, Hyung-Suk; Fogo, Agnes B.; Brown, Nancy J.

2012-01-01

262

Desensitization of GABAergic receptors as a mechanism of zolpidem-induced somnambulism.  

PubMed

Sleepwalking is a frequently reported side effect of zolpidem which is a short-acting hypnotic drug potentiating activity of GABA(A) receptors. Paradoxically, the most commonly used medications for somnambulism are benzodiazepines, especially clonazepam, which also potentiate activity of GABA(A) receptors. It is proposed that zolpidem-induced sleepwalking can be explained by the desensitization of GABAergic receptors located on serotonergic neurons. According to the proposed model, the delay between desensitization of GABA receptors and a compensatory decrease in serotonin release constitutes the time window for parasomnias. The occurrence of sleepwalking depends on individual differences in receptor desensitization, autoregulation of serotonin release and drug pharmacokinetics. The proposed mechanism of interaction between GABAergic and serotonergic systems can be also relevant for zolpidem abuse and zolpidem-induced hallucinations. It is therefore suggested that special care should be taken when zolpidem is used in patients taking at the same time selective serotonin reuptake inhibitors. PMID:21565448

Juszczak, Grzegorz R

2011-08-01

263

GRK2 Protein-mediated Transphosphorylation Contributes to Loss of Function of ?-Opioid Receptors Induced by Neuropeptide FF (NPFF2) Receptors*  

PubMed Central

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of ?-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF2 receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF2 receptor by [d-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of Gi/o proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-d-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF2 receptor activation was sufficient to recruit ?-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.

Mouledous, Lionel; Froment, Carine; Dauvillier, Stephanie; Burlet-Schiltz, Odile; Zajac, Jean-Marie; Mollereau, Catherine

2012-01-01

264

GRK2 protein-mediated transphosphorylation contributes to loss of function of ?-opioid receptors induced by neuropeptide FF (NPFF2) receptors.  

PubMed

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of ?-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit ?-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex. PMID:22375000

Moulédous, Lionel; Froment, Carine; Dauvillier, Stéphanie; Burlet-Schiltz, Odile; Zajac, Jean-Marie; Mollereau, Catherine

2012-04-13

265

ADP-ribosyl cyclase and ryanodine receptors mediate endothelin ETA and ETB receptor-induced renal vasoconstriction in vivo  

PubMed Central

ADP-ribosyl cyclase (ADPR cyclase) and ryanodine receptors (RyR) participate in calcium transduction in isolated afferent arterioles. We hypothesized that this signaling pathway is activated by ETA and ETB receptors in the renal vasculature to mediate vasoconstriction in vivo. To test this, we measured acute renal blood flow (RBF) responses to ET-1 in anesthetized rats and mice in the presence and absence of functional ADPR cyclase and/or RyR. Inhibitors of ADPR cyclase (nicotinamide) or RyR (ruthenium red) reduced RBF responses to ET-1 by 44% (P < 0.04 for both) in Sprague-Dawley rats. Mice lacking the predominant form of ADPR cyclase (CD38?/?) had RBF responses to ET-1 that were 47% weaker than those seen in wild-type mice (P = 0.01). Selective ETA receptor stimulation (ET-1+BQ788) produced decreases in RBF that were attenuated by 43 and 56% by nicotinamide or ruthenium red, respectively (P < 0.02 for both). ADPR cyclase or RyR inhibition also reduced vasoconstrictor effects of the ETB receptor agonist sarafotoxin 6c (S6c; 77 and 54%, respectively, P < 0.02 for both). ETB receptor stimulation by ET-1 + the ETA receptor antagonist BQ123 elicited responses that were attenuated by 59 and 60% by nicotinamide and ruthenium red, respectively (P < 0.01 for both). Nicotinamide attenuated RBF responses to S6c by 54% during inhibition of nitric oxide synthesis (P = 0.001). We conclude that in the renal microcirculation in vivo 1) ET-1-induced vasoconstriction is mediated by ADPR cyclase and RyR; 2) both ETA and ETB receptors activate this pathway; and 3) ADPR cyclase participates in ETB receptor signaling independently of NO.

Thai, Tiffany L.; Arendshorst, William J.

2008-01-01

266

Prolonged inhibition of 5-HT3 receptors by palonosetron results from surface receptor inhibition rather than inducing receptor internalization  

PubMed Central

Background and Purpose The 5-HT3 receptor antagonist palonosetron is an important treatment for emesis and nausea during cancer therapy. Its clinical efficacy may result from its unique binding and clearance characteristics and receptor down-regulation mechanisms. We investigated the mechanisms by which palonosetron exerts its long-term inhibition of 5-HT3 receptors for a better understanding of its clinical efficacy. Experimental Approach Cell surface receptors (recombinantly expressed 5HT3A or 5HT3AB in COS-7 cells) were monitored using [3H]granisetron binding and ELISA after exposure to palonosetron. Receptor endocytosis was investigated using immunofluorescence microscopy. Key Results Chronic exposure to palonosetron reduced the number of available cell surface [3H]granisetron binding sites. This down-regulation was not sensitive to either low temperature or pharmacological inhibitors of endocytosis (dynasore or nystatin) suggesting that internalization did not play a role. This was corroborated by our observation that there was no change in cell surface 5-HT3 receptor levels or increase in endocytic rate. Palonosetron exhibited slow dissociation from the receptor over many hours, with a significant proportion of binding sites being occupied for at least 4 days. Furthermore, our observations suggest that chronic receptor down-regulation involved interactions with an allosteric binding site. Conclusions and Implications Palonosetron acts as a pseudo-irreversible antagonist causing prolonged inhibition of 5-HT3 receptors due to its very slow dissociation. In addition, an irreversible binding mode persists for at least 4 days. Allosteric receptor interactions appear to play a role in this phenomenon.

Hothersall, J Daniel; Moffat, Christopher; Connolly, Christopher N

2013-01-01

267

Monocyte chemotactic peptide receptor. Functional characteristics and ligand-induced regulation.  

PubMed Central

Monocytes, macrophages, and neutrophils will demonstrate several important cellular functions in response to synthetic formylated oligopeptides. N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine (fNLPNTL) was a potent chemoattractant for human blood monocytes; a 1.0-nM concentration induced a maximal chemotactic response. Binding of 125I-labeled fNLPNTL to the monocyte formyl peptide receptor was rapid, specific, and saturable at 4, 24, or 37 degrees C. At 4 degrees C, monocytes from several different donors demonstrated between 10,000 and 18,000 receptors/cell with a dissociation constant (Kd) of 1.7-2.7 nM. The association of the 125I peptide with the cells was irreversible at the elevated temperatures and exceeded the amount of surface receptor by approximately four-fold, suggesting receptor-mediated peptide endocytosis. Processing of rhodamine-labeled fNLPNTL by monocytes was observed directly by video intensification microscopy. At 37 degrees C, diffuse membrane fluorescence was seen initially, followed by rapid aggregation and internalization of the peptide. Monocytes incubated with fNLPNTL displayed a temperature dependent loss of surface binding capacity (receptor down-regulation). This decrease was due to a decrease in surface receptor number rather than to a decrease in receptor affinity. A dose-response curve for peptide-induced receptor down-regulation correlated with a dose-response curve for 125I-labeled fNLPNTL uptake, suggesting that each uptake event led to the loss of one surface receptor. Surface receptor replenishment following down-regulation was rapid and not dependent on new protein synthesis, but was inversely related to both the time and peptide concentration used to induce down-regulation. An exact correlation between receptor down-regulation and functional deactivation of the chemotactic response could not be demonstrated. Images

Weinberg, J B; Muscato, J J; Niedel, J E

1981-01-01

268

Monocyte chemotactic peptide receptor. Functional characteristics and ligand-induced regulation.  

PubMed

Monocytes, macrophages, and neutrophils will demonstrate several important cellular functions in response to synthetic formylated oligopeptides. N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine (fNLPNTL) was a potent chemoattractant for human blood monocytes; a 1.0-nM concentration induced a maximal chemotactic response. Binding of 125I-labeled fNLPNTL to the monocyte formyl peptide receptor was rapid, specific, and saturable at 4, 24, or 37 degrees C. At 4 degrees C, monocytes from several different donors demonstrated between 10,000 and 18,000 receptors/cell with a dissociation constant (Kd) of 1.7-2.7 nM. The association of the 125I peptide with the cells was irreversible at the elevated temperatures and exceeded the amount of surface receptor by approximately four-fold, suggesting receptor-mediated peptide endocytosis. Processing of rhodamine-labeled fNLPNTL by monocytes was observed directly by video intensification microscopy. At 37 degrees C, diffuse membrane fluorescence was seen initially, followed by rapid aggregation and internalization of the peptide. Monocytes incubated with fNLPNTL displayed a temperature dependent loss of surface binding capacity (receptor down-regulation). This decrease was due to a decrease in surface receptor number rather than to a decrease in receptor affinity. A dose-response curve for peptide-induced receptor down-regulation correlated with a dose-response curve for 125I-labeled fNLPNTL uptake, suggesting that each uptake event led to the loss of one surface receptor. Surface receptor replenishment following down-regulation was rapid and not dependent on new protein synthesis, but was inversely related to both the time and peptide concentration used to induce down-regulation. An exact correlation between receptor down-regulation and functional deactivation of the chemotactic response could not be demonstrated. PMID:6268661

Weinberg, J B; Muscato, J J; Niedel, J E

1981-09-01

269

Protective effect of liquiritigenin against methylglyoxal cytotoxicity in osteoblastic MC3T3-E1 cells.  

PubMed

Methylglyoxal (MG), a reactive dicarbonyl compound, is a metabolic byproduct of glycolysis and elevated MG levels contribute to diabetic complications. Glycation reactions of MG with amino acids can induce oxidative stress, leading to subsequent cytotoxicity. In the present study, the effect of liquiritigenin on MG-induced cytotoxicity was investigated using osteoblastic MC3T3-E1 cells. Pretreatment of MC3T3-E1 cells with liquiritigenin prevented the MG-induced cell death and production of protein adduct, intracellular reactive oxygen species, mitochondrial superoxide, cardiolipin peroxidation, and TNF-? in osteoblastic MC3T3-E1 cells. In addition, liquiritigenin increased the activity of glyoxalase I inhibited by MG. These findings suggest that liquiritigenin provides a protective action against MG-induced cell damage by reducing oxidative stress and by increasing MG detoxification. Pretreatment with liquiritigenin prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss. Additionally, the nitric oxide and PGC-1? levels were significantly increased by liquiritigenin, suggesting that liquiritigenin may induce mitochondrial biogenesis. Our findings indicate that liquiritigenin might exert its therapeutic effects via enhancement of glyoxalase I activity and mitochondrial function, and anti-oxidant and anti-inflammatory activities. Taken together, liquiritigenin has potential as a preventive agent against the development of diabetic osteopathy related to MG-induced oxidative stress in diabetes. PMID:24789098

Suh, Kwang Sik; Rhee, Sang Youl; Kim, Young Seol; Choi, Eun Mi

2014-06-25

270

Percutaneous bipolar radiofrequency T3 sympathicotomy in Raynaud's disease -A case report-  

PubMed Central

A 54-year-old female was suffering from cold-induced Raynaud's attacks in her both hands with symptoms most severe in her left hand. As the patient did not respond to previous medical treatments and endoscopic thoracic sympathectomy, we performed percutaneous bipolar radiofrequency thoracic sympathicotomy at the left T3 vertebral level. After the procedure, the patient obtained a long duration of symptom relief over 3 years. Percutaneous bipolar radiofrequency T3 sympathicotomy is minimally invasive and effective technique by creating large continuous strip lesion.

Kang, Sang-Soo; Park, Jung-Chan; Hong, Sung-Jun; Yoon, Young-Jun

2012-01-01

271

Allopregnanolone prevents dieldrin-induced NMDA receptor internalization and neurotoxicity by preserving GABA(A) receptor function.  

PubMed

Dieldrin is an endocrine disruptor that accumulates in mammalian adipose tissue and brain. It induces convulsions due to its antagonism of the ?-aminobutyric acid A receptor (GABA(A)R). We have previously reported that long-term exposure to dieldrin causes the internalization of the N-methyl-D-aspartate receptor (NMDAR) as a result of persistent GABA(A)R inhibition. Because the neurosteroids 17?-estradiol (E2) and allopregnanolone are known to modulate the function and trafficking of GABA(A)R and NMDAR, we examined the effects of E2 and allopregnanolone on dieldrin-induced GABA(A)R inhibition, NMDAR internalization, and neuronal death in cortical neurons. We found that 1 nM E2 increased the membrane expression of NR1/NR2B receptors and postsynaptic density 95 but did not induce their physical association. In contrast, 10 nM E2 had no effect on these proteins but reduced NR2A membrane expression. We also found that exposure to 60 nM dieldrin for 6 d in vitro caused the internalization of NR1 and NR2B but not NR2A. Treatment with either 1 nM E2 or 10 ?M allopregnanolone prevented the dieldrin-induced reduction in membrane levels of the NR1/NR2B receptors. Furthermore, prolonged exposure to 200 nM dieldrin down-regulated the expression of NR2A; this was inhibited only by allopregnanolone. Although both hormones restored NMDAR function, as measured by the NMDA-induced rise in intracellular calcium, allopregnanolone (but not E2) reversed the inhibition of GABA(A)R and neuronal death caused by prolonged exposure to dieldrin. Our results indicate that allopregnanolone protects cortical neurons against the neurotoxicity caused by long-term exposure to dieldrin by maintaining GABA(A)R and NMDAR functionality. PMID:22166974

Briz, Víctor; Parkash, Jyoti; Sánchez-Redondo, Sara; Prevot, Vincent; Suñol, Cristina

2012-02-01

272

Purine receptor P2Y6 mediates cellular response to ?-ray-induced DNA damage.  

PubMed

We previously showed that nucleotide P2 receptor agonists such as ATP and UTP amplify ?-ray-induced focus formation of phosphorylated histone H2A variant H2AX (?H2AX), which is considered to be an indicator of DNA damage so far, by activating purine P2Y6 and P2Y12 receptors. Therefore, we hypothesized that these P2 receptors play a role in inducing the repair response to ?-ray-induced DNA damage. In the present study, we tested this idea by using human lung cancer A549 cells. First, reverse-transcription polymerase chain reaction (RT-PCR) showed that P2Y6 receptor is highly expressed in A549 cells, but P2Y12 receptor is only weakly expressed. Next, colony formation assay revealed that P2Y6 receptor antagonist MRS2578 markedly reduced the survival rate of ?-ray-exposed A549 cells. The survival rate was also significantly reduced in P2Y6-knock-down cells, compared with scramble siRNA-transfected cells. Since it has reported that phosphorylation of ERK1/2 after activation of EGFR via P2Y6 and P2Y12 receptors is involved in the repair response to ?-ray-induced DNA damage, we next examined whether ?-ray-induced phosphorylation of ERK1/2 was also inhibited by MRS2578 in A549 cells. We found that it was. Taken together, these findings indicate that purinergic signaling through P2Y6 receptor, followed by ERK1/2 activation, promotes the cellular repair response to ?-ray-induced DNA damage. PMID:24418705

Ide, Shunta; Nishimaki, Naoko; Tsukimoto, Mitsutoshi; Kojima, Shuji

2014-01-01

273

Coactivation of NMDA receptors by glutamate and -serine induces dilation of isolated middle cerebral arteries  

PubMed Central

N-methyl--aspartate (NMDA) receptors are glutamate-gated cation channels that mediate excitatory neurotransmission in the central nervous system. In addition to glutamate, NMDA receptors are also activated by coagonist binding of the gliotransmitter, -serine. Neuronal NMDA receptors mediate activity-dependent blood flow regulation in the brain. Our objective was to determine whether NMDA receptors expressed by brain endothelial cells can induce vasodilation of isolated brain arteries. Adult mouse middle cerebral arteries (MCAs) were isolated, pressurized, and preconstricted with norepinephrine. N-methyl--aspartate receptor agonists, glutamate and NMDA, significantly dilated MCAs in a concentration-dependent manner in the presence of -serine but not alone. Dilation was significantly inhibited by NMDA receptor antagonists, -2-amino-5-phosphonopentanoate and 5,7-dichlorokynurenic acid, indicating a response dependent on NMDA receptor glutamate and -serine binding sites, respectively. Vasodilation was inhibited by denuding the endothelium and by selective inhibition or genetic knockout of endothelial nitric oxide synthase (eNOS). We also found evidence for expression of the pan-NMDA receptor subunit, NR1, in mouse primary brain endothelial cells, and for the NMDA receptor subunit NR2C in cortical arteries in situ. Overall, we conclude that NMDA receptor coactivation by glutamate and -serine increases lumen diameter in pressurized MCA in an endothelial and eNOS-dependent mechanism.

LeMaistre, Jillian L; Sanders, Samuel A; Stobart, Michael J; Lu, Lingling; Knox, J David; Anderson, Hope D; Anderson, Christopher M

2012-01-01

274

Melanocortin 3 receptors control crystal-induced inflammation  

Microsoft Academic Search

In this study we have characterized the anti-inflammatory profile of a selective melanocortin type 3 receptor (MC3-R) ligand (D-Trp8)--MSH, vali- dating in vitro results with analyses in mice deficient for this receptor subtype. In wild-type (WT) macrophages, (D-Trp8)--MSH activated MC3-R (as tested by accumu- lation of cyclic AMP) and inhibited (50%) the release of interleukin (IL)-1 and the chemokine KC

Connie W. Lam; Airu S. Chen; Paolo Grieco; Mauro Perretti

2006-01-01

275

Lentiviral vectors efficiently transduce quiescent mature 3T3-L1 adipocytes.  

PubMed

Obesity is associated with many serious afflictions such as cardiovascular disease, cancer, and diabetes. One of the main cellular systems used to study the underlying physiological and biological processes is the 3T3-L1 preadipocyte differentiation model. However, studies on 3T3-L1 adipocytes are hampered by the fact that genetic modification of mature adipocytes is notoriously difficult. In this report, we evaluated the use of lentivirus-mediated gene transfer into 3T3-L1 mature adipocytes. We demonstrate that quiescent, fully differentiated 3T3-L1 adipocytes as well as 3T3-L1 preadipocytes can be efficiently transduced with HIV-1-derived lentiviral vectors. Upon transduction using LV-PGK-GFP lentiviral vector at 100 ng p24 per 10(5) cells, more than 95% of the 3T3-L1 adipocytes in the culture expressed the GFP reporter gene. There were no overt signs of toxicity or cytopathogenicity in the cultures. Furthermore, modification of undifferentiated preadipocytes did not affect their capacity to differentiate. In addition, insulin-induced glucose uptake was not affected by the procedure. In contrast, adenoviral-mediated gene transfer into 3T3-L1 adipocytes is associated with marked cytopathogenicity. From these data, we conclude that lentiviral vectors are the gene-transfer system of choice for genetic modification of mature adipocytes. The availability of an efficient vector system may stimulate the use of adipose tissue as a target for gene therapy in obesity and other disorders. PMID:14759805

Carlotti, Françoise; Bazuine, Merlijn; Kekarainen, Tuija; Seppen, Jurgen; Pognonec, Philippe; Maassen, J Antonie; Hoeben, Rob C

2004-02-01

276

Localization of type I interferon receptor limits interferon-induced TLR-3 in epithelial cells  

EPA Science Inventory

This study aimed to expand on the role of type I IFNs in the influenza-induced upregulation of TLR3 and determine whether and how the localization of the IFN-alpha/beta receptor (IFNAR) in respiratory epithelial cells could modify IFN-induced responses. Using differentiated prima...

277

Inhibition of Methylcholanthrene-induced Carcinogenesis by an InterferonReceptor-dependent Foreign Body Reaction  

Microsoft Academic Search

The foreign body reaction is one of the oldest host defense mechanisms against tissue damage which involves inflammation, scarring, and encapsulation. The chemical carcinogen methyl- cholanthrene (MCA) induces fibrosarcoma and tissue damage in parallel at the injection site. Tumor development induced by MCA but not due to p53-deficiency is increased in inter- feron- ? receptor (IFN- ? R)-deficient mice. In

Zhihai Qin; Hye-Jung Kim; Jens Hemme; Thomas Blankenstein

278

Growth changes of 3T3 cells in the presence of mineral fibers  

SciTech Connect

The relationship between exposure to asbestos fibers and the development of mesothelioma or bronchial carcinoma prompted many countries to ban its use from commercial products. The biological mechanism by which asbestos induces or promotes mesothelioma or carcinoma is still unknown. In order to study the influence of fibers on the cell surface, 3T3 fibroblasts were cultured in the presence of various mineral fibers. The acute cytotoxicity produced by mineral fibers was evaluated by the trypan blue dye exclusion method; growth of 3T3 cells was measured as well as the maximum cell density at saturation. It was found that growth of 3T3 cells was slower in the presence of chrysotile while light microscopy revealed an increased cellular chromogenicity and a modification of the cell-cell arrangement in the presence of this fiber. An assay is described in which chrysotile causes an increase in the maximum cell density at saturation.

Dumas, L.; Page, M.

1986-01-01

279

Cell cycle dependency of murine cytomegalovirus replication in synchronized 3T3 cells.  

PubMed Central

Synchronized murine 3T3 cells have been used to investigate the possible dependency of murine cytomegalovirus replication upon the cell cycle. The normal latent period of 12 h characteristic of asynchronous 3T3 cells was protracted to more than 24 h after an early G1 infection in synchronous cells. In this case viral progeny were not detected until after the initiation of the host S-phase. Cells maintained in the G1 phase did not replicate virus. This failure could not be explained by a decrease in virus penetration but was apparently due to a requirement for an event associated with the host S-phase. Thymidine-induced inhibition of cell cycle traverse also blocked virus replication. Viral DNA synthesis did not initiate until after the initiation of host DNA. In contrast, herpes simplex virus type 1 replicated in 3T3 cells independently of the cell cycle.

Muller, M T; Hudson, J B

1977-01-01

280

Gamma-hydroxybutyrate (GHB) induces cognitive deficits and affects GABAB receptors and IGF-1 receptors in male rats.  

PubMed

In recent years, the abuse of the club drug gamma-hydroxybutyrate (GHB) has become increasingly popular among adolescents. The drug induces euphoria but can also result in sedation, anaesthesia as well as short-term amnesia. In addition, the abuse of GHB causes cognitive impairments and the mechanism by which GHB induces these impairments is not clarified. The present study investigates the impact of GHB treatment on spatial learning and memory using a water maze (WM) test in rats. Furthermore, the behavioural data is combined with an autoradiographic analysis of the GABAB and the IGF-1 receptor systems. The results demonstrate that the animals administered with GHB display an impaired performance in the WM test as compared to controls. In addition, significant alterations in GABAB and IGF-1 receptor density as well as GABAB receptor functionality, were observed in several brain regions associated with cognitive functions e.g. hippocampus. To conclude, our findings suggest that GHB treatment can affect spatial learning and memory, and that this outcome at least to some extent is likely to involve both GABAB and IGF-1 receptors. PMID:24786330

Johansson, Jenny; Grönbladh, Alfhild; Hallberg, Mathias

2014-08-01

281

Assembly and structure of the T3SS.  

PubMed

The Type III Secretion System (T3SS) is a multi-mega Dalton apparatus assembled from more than twenty components and is found in many species of animal and plant bacterial pathogens. The T3SS creates a contiguous channel through the bacterial and host membranes, allowing injection of specialized bacterial effector proteins directly to the host cell. In this review, we discuss our current understanding of T3SS assembly and structure, as well as highlight structurally characterized Salmonella effectors. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. PMID:24512838

Burkinshaw, Brianne J; Strynadka, Natalie C J

2014-08-01

282

Neuropeptide S attenuates neuropathological, neurochemical and behavioral changes induced by the NMDA receptor antagonist MK-801.  

PubMed

Neuropeptide S (NPS) and its cognate receptor were reported to mediate anxiolytic-like and arousal effects. NPS receptors are predominantly expressed in the brain, especially in limbic structures, including amygdala, olfactory nucleus, subiculum and retrosplenial cortex. In contrast, the NPS precursor is expressed in only a few brainstem nuclei where it is co-expressed with various excitatory transmitters, including glutamate. The current study investigates interactions of the NPS system with glutamatergic neurotransmission. It has been suggested that dysfunctions in glutamatergic neurotransmission via N-methyl-D-aspartate (NMDA) receptors might be involved in the pathophysiology of schizophrenia since NMDA receptor antagonists, such as MK-801, have been shown to induce psychotic-like behavior in humans and animal models. Also, MK-801 is known to produce histological changes such as cytoplasmic vacuoles in retrosplenial cortex neurons where NPS receptors are highly expressed. In this study we show that NPS is able to alleviate neuropathological, neurochemical and behavioral changes produced by NMDA receptor antagonists. NPS treatment attenuated MK-801-induced vacuolization in the rat retrosplenial cortex in a dose-dependent manner that can be blocked by an NPS receptor-selective antagonist. NPS also suppressed MK-801-induced increases of extracellular acetylcholine levels in the retrosplenial cortex. In the prepulse inhibition (PPI) assay, animals pretreated with NPS recovered significantly from MK-801-induced disruption of PPI. Our study suggests that NPS may have protective effects against the neurotoxic and behavioral changes produced by NMDA receptor antagonists and that NPS receptor agonists may elicit antipsychotic effects. PMID:19576911

Okamura, Naoe; Reinscheid, Rainer K; Ohgake, Shintaro; Iyo, Masaomi; Hashimoto, Kenji

2010-01-01

283

Functional Relevance of the Switch of VEGF Receptors/Co-Receptors during Peritoneal Dialysis-Induced Mesothelial to Mesenchymal Transition  

PubMed Central

Vascular endothelial growth factor (VEGF) is up-regulated during mesothelial to mesenchymal transition (MMT) and has been associated with peritoneal membrane dysfunction in peritoneal dialysis (PD) patients. It has been shown that normal and malignant mesothelial cells (MCs) express VEGF receptors (VEGFRs) and co-receptors and that VEGF is an autocrine growth factor for mesothelioma. Hence, we evaluated the expression patterns and the functional relevance of the VEGF/VEGFRs/co-receptors axis during the mesenchymal conversion of MCs induced by peritoneal dialysis. Omentum-derived MCs treated with TGF-?1 plus IL-1? (in vitro MMT) and PD effluent-derived MCs with non-epithelioid phenotype (ex vivo MMT) showed down-regulated expression of the two main receptors Flt-1/VEGFR-1 and KDR/VEGFR-2, whereas the co-receptor neuropilin-1 (Nrp-1) was up-regulated. The expression of the Nrp-1 ligand semaphorin-3A (Sema-3A), a functional VEGF competitor, was repressed throughout the MMT process. These expression pattern changes were accompanied by a reduction of the proliferation capacity and by a parallel induction of the invasive capacity of MCs that had undergone an in vitro or ex vivo MMT. Treatment with neutralizing anti-VEGF or anti-Nrp-1 antibodies showed that these molecules played a relevant role in cellular proliferation only in naïve omentum-derived MCs. Conversely, treatment with these blocking antibodies, as well as with recombinant Sema-3A, indicated that the switched VEGF/VEGFRs/co-receptors axis drove the enhanced invasion capacity of MCs undergoing MMT. In conclusion, the expression patterns of VEGFRs and co-receptors change in MCs during MMT, which in turn would determine their behaviour in terms of proliferation and invasion in response to VEGF.

Perez-Lozano, Maria Luisa; Sandoval, Pilar; Rynne-Vidal, Angela; Aguilera, Abelardo; Jimenez-Heffernan, Jose Antonio; Albar-Vizcaino, Patricia; Majano, Pedro L.; Sanchez-Tomero, Jose Antonio; Selgas, Rafael; Lopez-Cabrera, Manuel

2013-01-01

284

Exendin-4, a GLP-1 receptor agonist, directly induces adiponectin expression through protein kinase A pathway and prevents inflammatory adipokine expression.  

PubMed

Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist that has been used as a drug injected subcutaneously for treatment of type 2 diabetes. Many studies have revealed molecular targets of Ex-4, but its influence on adipokines has not been determined. Our study showed that Ex-4 induced secretion of adiponectin into the culture medium of 3T3-L1 adipocytes. This effect of Ex-4 is due to increased adiponectin mRNA level through the GLP-1R. Both forskolin and 3-isobutyl-1-methylxanthine (IBMX), which may finally elevate cyclic adenosine monophosphate (cAMP) concentration, prevented the induction of adiponectin expression by Ex-4. Moreover, H89, a protein kinase A inhibitor, blocked the effect of Ex-4 on adiponectin. On the other hand, Ex-4 decreased the mRNA levels of inflammatory adipokines. The results indicate that Ex-4 directly promotes adiponectin secretion via the protein kinase A pathway in 3T3-L1 adipocytes and may ameliorate insulin resistance. PMID:19850014

Kim Chung, Le Thi; Hosaka, Toshio; Yoshida, Masaki; Harada, Nagakatsu; Sakaue, Hiroshi; Sakai, Tohru; Nakaya, Yutaka

2009-12-18

285

Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors  

PubMed Central

Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP3 prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP3 receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP3 receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP3 receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP3 prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP3 receptor as a target to induce laxative effects.

Tunaru, Sorin; Althoff, Till F.; Nusing, Rolf M.; Diener, Martin; Offermanns, Stefan

2012-01-01

286

Angiotensin II type 2 receptor mediated angiotensin II and high glucose induced decrease in renal prorenin\\/renin receptor expression  

Microsoft Academic Search

Activation of prorenin\\/renin receptor [(P)RR] mediates non-enzymatic pathway for the physiological function of prorenin\\/renin. It also plays a role in the pathogenesis of diabetic nephropathy. However, the mechanisms of regulating (P)RR expression are only partially understood. In the present study, we examine the change of (P)RR under hyperglycemic conditions in the kidneys of streptozotocin (STZ)-induced diabetic (DM) rats and cultured

Ming He; Lin Zhang; Ying Shao; Hong Xue; Li Zhou; Xiao-Fang Wang; Chen Yu; Tai Yao; Li-Min Lu

2010-01-01

287

Pharmacodynamic effects of serotonin (5HT) receptor ligands in pigs: stimulation of 5HT 2 receptors induces malignant hyperthermia  

Microsoft Academic Search

In pigs, the serotonin-2 (5-HT2) receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), 0.8 mg\\/kg, induced “psychotic” behaviour (e. g., grimacing, backward locomotion, blank stare) and a muscular syndrome, which is known as malignant hyperthermia (MH) in pigs and humans. This syndrome is characterized by generalized skeletal muscle rigidity, leading to an increase in body temperature, marked acidosis, hyperkaliaemia, cyanosis and elevation of lactate,

Wolfgang Liischer; Ulrike Witte; Gabriele Fredow; Martin Ganter; Klaus Bickhardt

1990-01-01

288

Oxytocin receptor ligands induce changes in cytoskeleton in neuroblastoma cells.  

PubMed

Aim of the present study was to evaluate effects of ligands of oxytocin receptors on gene expression of neurofilament proteins (nestin and microtubule-associated protein 2 (MAP2)) associated with neuronal differentiation and growth factors (brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)) related to neuronal growth. Fluorescent staining of F-actin was used to observe morphology of cells. Co-treatment with oxytocin and oxytocin receptor antagonist--atosiban--resulted in significant increase of MAP2 gene expression in SK-N-SH cells. There was no effect of oxytocin on gene expression of growth factors BDNF and NGF. Surprisingly, oxytocin with atosiban significantly increased mRNA levels for both BDNF and NGF. Gene expression of vasopressin receptor (V1aR) significantly decreased in response to vasopressin. Atosiban decreased mRNA levels for oxytocin receptor (OXTR) and V1aR. Oxytocin significantly decreased OXTR and nestin mRNA levels and increased mRNA levels for BDNF and NGF in U-87 MG cells. The densest recruitment of F-actin filaments was observed in apical parts of filopodia in SK-N-SH cells incubated in oxytocin presence. Present data demonstrate complex role of ligands of oxytocin receptors in regulation of gene expression of intermediate filaments and thus, oxytocin might be considered as a growth factor in neuronal type of cells. PMID:23335033

Bakos, Jan; Strbak, Vladimir; Paulikova, Helena; Krajnakova, Lucia; Lestanova, Zuzana; Bacova, Zuzana

2013-07-01

289

Processing and ligand-induced modifications of the V2 vasopressin receptor.  

PubMed

Synthesis, processing and agonist-induced modifications of the V2 vasopressin receptor were examined in stably or transiently transfected HEK293 cells. Metabolic labeling with S methionine for 30 min revealed a predominant precursor protein which subsequently gave rise to the mature receptor on the cell surface. Maturation of the receptor was unrelated to glycosylation suggesting that it was the consequence of protein refolding. In addition to monomeric forms of V2 receptor protein, oligomers of the precursor protein were also detected in SDS-PAGE. These oligomers seemed to be dimers and tetrameres, and were more apparent in transiently transfected cells that produced higher quantities of protein then stably transfected cells. No oligomers of the mature receptor were detected, and co-transfection of the wild type with a mutant V2 receptor lacking G-protein coupling activity did not alter the function of the wild type receptor. These results indicated that the formation of oligomeric was most likely a consequence of overproduction of the protein and not a required step for receptor function. Addition of vasopressin promoted phosphorylation and sequestration of the wild type receptor, and of the R137H mutant receptor which lacks coupling to G proteins. Activation of protein kinases A or C did not result in phosphorylation of un-occupied receptor. Phosphate incorporated into the protein was stable in the continuous presence of the ligand despite sequestration of the receptor protein. Deletion of the last 14 amino acids abolished receptor phosphorylation but not sequestration and desensitization, indicating that these two processes are not dependent on protein phosphorylation. Additionally, phosphorylation and sequestration of the R137H mutant receptor revealed that phosphorylation and sequestration does not require coupling to Gs. The wild type V2 vasopressin receptor was found to be palmitoylated at two cysteines at the carboxyl terminus. Either cysteine could be palmitoylated independently of each other and the presence of at least one was required to obtain receptor expression similar to the wild type. The turnover of the palmitic acid incorporated into the receptor was not altered by the addition of vasopressin demonstrating that this post-translational modification of the receptor was not altered by the ligand-promoted phosphorylation of the protein. PMID:10026823

Sadeghi, H M; Innamorati, G; Esqueda, E; Birnbaumer, M

1998-01-01

290

Garcinol Potentiates TRAIL-Induced Apoptosis through Modulation of Death Receptors and Antiapoptotic Proteins  

PubMed Central

Whether garcinol, the active component from Garcinia indica, can modulate the sensitivity of cancer cells to TRAIL, a cytokine currently in phase II clinical trial, was investigated. We found that garcinol potentiated TRAIL-induced apoptosis of cancer cells as indicated by intracellular esterase activity, DNA strand breaks, accumulation of the membrane phospholipid phosphatidylserine, mitochondrial activity, and activation of caspase-8, -9, and -3. We found that garcinol, independent of the cell type, induced both of the TRAIL receptors, death receptors (DR)-4 and DR5. Garcinol neither induced the receptors on normal cells, nor sensitized them to TRAIL. Deletion of DR5 or DR4 by small interfering RNA significantly reduced the apoptosis induced by TRAIL and garcinol. In addition, garcinol downregulated various cell survival proteins including survivin, bcl-2, XIAP and cFLIP; and induced bid cleavage, bax and cytochrome c release. Induction of DRs by garcinol was found to be independent of modulation of CHOP, p53, bax, ERK or JNK. The effect of garcinol was mediated through the generation of reactive oxygen species, in as much as both induction of DRs, modulation of antiapoptotic and proapoptotic proteins and potentiation of TRAIL-induced apoptosis were abolished by N-acetyl cysteine and glutathione. Interestingly, garcinol also converted TRAIL-resistant cells to TRAIL-sensitive. Overall, our results indicate that garcinol can potentiate TRAIL-induced apoptosis through upregulation of death receptors and downregulation of antiapoptotic proteins.

Prasad, Sahdeo; Ravindran, Jayaraj; Sung, Bokyung; Pandey, Manoj K; Aggarwal, Bharat B.

2010-01-01

291

Glucose-induced phosphorylation of the insulin receptor. Functional effects and characterization of phosphorylation sites.  

PubMed Central

Elevated glucose concentrations have been reported to inhibit insulin receptor kinase activity. We studied the effects of high glucose on insulin action in Rat1 fibroblasts transfected with wild-type human insulin receptor (HIRcB) and a truncated receptor lacking the COOH-terminal 43 amino acids (delta CT). In both cell lines, 25 mM glucose impaired receptor and insulin receptor substrate-1 phosphorylation by 34%, but IGF-1 receptor phosphorylation was unaffected. Phosphatidylinositol 3-kinase activity and bromodeoxyuridine uptake were decreased by 85 and 35%, respectively. This was reversed by coincubation with a protein kinase C (PKC) inhibitor or microinjection of a PKC inhibitor peptide. Phosphopeptide mapping revealed that high glucose or PMA led to serine/threonine phosphorylation of similar peptides. Inhibition of the microtubule-associated protein (MAP) kinase cascade by the MAP kinase kinase inhibitor PD98059 did not reverse the impaired phosphorylation. We conclude that high glucose inhibits insulin action by inducing serine phosphorylation through a PKC-mediated mechanism at the level of the receptor at sites proximal to the COOH-terminal 43 amino acids. This effect is independent of activation of the MAP kinase cascade. Proportionately, the impairment of insulin receptor substrate-1 tyrosine phosphorylation is greater than that of the insulin receptor resulting in attenuated phosphatidylinositol 3-kinase activation and mitogenic signaling.

Pillay, T S; Xiao, S; Olefsky, J M

1996-01-01

292

Enhancement of leptin receptor signaling by SOCS3 deficiency induces development of gastric tumors in mice.  

PubMed

Leptin acts on its receptor (ObR) in the hypothalamus to inhibit food intake and energy expenditure. Leptin and ObR are also expressed in the gastrointestinal tract; however, the physiological significance of leptin signaling in the gut remains uncertain. Suppressor of cytokine signaling 3 (SOCS3) is a key negative feedback regulator of ObR-mediated signaling in the hypothalamus. We now show that gastrointestinal epithelial cell-specific SOCS3 conditional knockout (T3b-SOCS3 cKO) mice developed gastric tumors by enhancing leptin production and the ObRb/signal transducer and activator of transcription 3 (STAT3) signaling pathway. All T3b-SOCS3 cKO mice developed tumors in the stomach but not in the bowels by 2 months of age, even though the SOCS3 deletion occurred in both the epithelium of stomach and bowels. The tumors developed in the absence of the inflammatory response and all cKO mice died within 6 months. These tumors displayed pathology and molecular alterations, such as an increase in MUC2 (Mucin 2, oligomeric mucus/gel-forming) and TFF3 (trefoil factor 3), resembling human intestinal-type gastric tumors. Administration of antileptin antibody to T3b-SOCS3 cKO mice reduced hyperplasia of gastric mucosa, which is the step of the initiation of gastric tumor. These data suggest that SOCS3 is an antigastric tumor gene that suppresses leptin overexpression and ObRb/STAT3 hyperactivation, supporting the hypothesis that the leptin/ObRb/STAT3 axis accelerates tumorigenesis and that it may represent a new therapeutic target for the treatment of gastric cancer. PMID:23178499

Inagaki-Ohara, K; Mayuzumi, H; Kato, S; Minokoshi, Y; Otsubo, T; Kawamura, Y I; Dohi, T; Matsuzaki, G; Yoshimura, A

2014-01-01

293

3',4'-Dimethoxythioflavone induces endothelium-dependent vasorelaxation through activation of epidermal growth factor receptor.  

PubMed

It is of interest to investigate whether synthetic thioflavonoids have vasorelaxant actions as natural flavonoids. We tested the hypothesis that 3',4'-dimethoxythioflavone induces endothelium-dependent vasorelaxation through activation of epidermal growth factor (EGF) receptor. Rat aortic rings were mounted in organ baths and subjected to relaxation upon contraction. 3',4'-Dimethoxythioflavone induced endothelium-dependent vasorelaxation, which was attenuated by pretreatment with either L-N (?)-nitroarginine methyl ester, an inhibitor of nitric oxide synthase, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. 3',4'-Dimethoxythioflavone-induced vasorelaxation was not affected by pretreatment with a general estrogen receptor antagonist ICI 182,780, a selective estrogen receptor-? antagonist methyl-piperidino-pyrazole dihydrochloride, or a G protein-coupled receptor 30 antagonist G15. However, pretreatment with EGF receptor blockers AG1478 or DAPH, but not with a phosphatidylinositol-3 kinase inhibitor LY294002 or an Akt1/2 kinase inhibitor Akt inhibitor VIII, attenuated 3',4'-dimethoxythioflavone-induced vasorelaxation. In addition, pretreatment with a Src inhibitor PP2 or an ERK inhibitor U0126 also attenuated vascular relaxation induced by the cumulative addition of 3',4'-dimethoxythioflavone. However, neither a mitochondrial electron transport inhibitor rotenone, an NADPH oxidase inhibitor apocynin, nor a superoxide dismutase mimetic MnTMPyP affected the vascular relaxation induced by the cumulative addition of 3',4'-dimethoxythioflavone. In conclusion, 3',4'-dimethoxythioflavone induces endothelium-dependent vasorelaxation through activation of EGF receptor and Src/ERK pathway in rat aorta. PMID:23232926

Jang, Eun Jin; Seok, Young Mi; Lee, Jae In; Cho, Hyun Min; Sohn, Uy Dong; Kim, In Kyeom

2013-04-01

294

Tranexamic acid induces kaolin intake stimulating a pathway involving tachykinin neurokinin 1 receptors in rats.  

PubMed

Tranexamic acid suppresses post-partum haemorrhage and idiopathic menorrhagia through its anti-fibrinolytic action. Although it is clinically useful, it is associated with high risks of side effects such as emesis. Understanding the mechanisms underlying tranexamic acid-induced emesis is very important to explore appropriate anti-emetic drugs for the prevention and/or suppression of emesis. In this study, we examined the receptors involved in tranexamic acid-induced kaolin intake in rats, which reflects the drug's clinical emetogenic potential in humans. Further, we examined the brain regions activated by administration of tranexamic acid and elucidated pivotal pathways of tranexamic acid-induced kaolin intake. We examined the effects of ondansetron, a 5-hydroxytryptamine 3 receptor antagonist, domperidone, a dopamine 2 receptor antagonist, and aprepitant, a tachykinin neurokinin 1 (NK1) receptor antagonist, on tranexamic acid-induced kaolin intake in rats. Then, we determined the brain regions that showed increased numbers of c-Fos immunoreactive cells. Finally, we examined the effects of an antagonist(s) that reduced tranexamic acid-induced kaolin intake on the increase in c-Fos immunoreactive cells. Aprepitant significantly decreased tranexamic acid-induced kaolin intake. However, neither ondansetron nor domperidone decreased kaolin intake. Tranexamic acid significantly increased c-Fos immunoreactive cells by approximately 5.5-fold and 22-fold in the area postrema and nucleus of solitary tract, respectively. Aprepitant decreased the number of c-Fos immunoreactive cells in both areas. Tranexamic acid induced kaolin intake possibly via stimulation of tachykinin NK1 receptors in rats. The tachykinin NK1 receptor could be targeted to prevent and/or suppress emesis in patients receiving tranexamic acid. PMID:24333477

Kakiuchi, Hitoshi; Kawarai-Shimamura, Asako; Kuwagata, Makiko; Orito, Kensuke

2014-01-15

295

Injury-induced expression of glial androgen receptor in the zebra finch brain.  

PubMed

Astrogliosis occurs following injury to the zebra finch brain. To date, only estrogen synthase (aromatase) has been identified in injury-induced astrocytes. The expression of other steroidogenic enzymes or their receptors remains unknown in the avian brain. However, in mammals, an upregulation of androgen receptors has been identified in glial cells. The aim of this study was to determine if the androgen receptor is upregulated following injury in adult zebra finches. Finches were given a single penetrating injury and brain tissue was collected 24 or 72?h later. Expression of androgen receptor was examined using immunohistochemistry and quantified using quantitative polymerase chain reaction (qPCR) analysis. Androgen receptors were localized to astrocytes versus neurons, further solidifying the role for astrocytes in neural recovery. PMID:23819447

Duncan, Kelli A; Moon, John; Vartosis, Dante; Zee, Isabelle

2013-11-15

296

Curcumin prevents corticosterone-induced neurotoxicity and abnormalities of neuroplasticity via 5-HT receptor pathway.  

PubMed

Curcumin, a major active component of Curcuma longa, possesses antioxidant and neuroprotective activities. The present study explores the mechanisms underlying the neuroprotective effect of curcumin against corticosterone and its relation to 5-hydroxy tryptamine (5-HT) receptors. Exposure of cortical neurons to corticosterone results in decreased mRNA levels for three 5-HT receptor subtypes, 5-HT(1A), 5-HT(2A) and 5-HT(4), but 5-HT(1B,) 5-HT(2B), 5-HT(2C), 5-HT(6) and 5-HT(7) receptors remain unchanged. Pre-treatment with curcumin reversed this effect on mRNA for the 5-HT(1A) and 5-HT(4) receptors, but not for the 5-HT(2A) receptor. Moreover, curcumin exerted a neuroprotective effect against corticosterone-induced neuronal death. This observed effect of curcumin was partially blocked by either 5-HT(1A) receptor antagonist p-MPPI or 5-HT(4) receptor antagonist RS 39604 alone; whereas, the simultaneous application of both antagonists completely reversed the effect. Curcumin was also found to regulate corticosterone-induced morphological changes such as increases in soma size, dendritic branching and dendritic spine density, as well as elevate synaptophysin expression in cortical neurons. p-MPPI and RS 39604 reversed the effect of curcumin-induced change in neuronal morphology and synaptophysin expression of corticosterone-treated neurons. In addition, an increase in cyclic adenosine monophosphate (cAMP) level was observed after curcumin treatment, which was further prevented by RS 39604, but not by p-MPPI. However, curcumin-induced elevation in protein kinase A activity and phosphorylation of cAMP response element-binding protein levels were inhibited by both p-MPPI and RS 39604. These findings suggest that the neuroprotection and modulation of neuroplasticity exhibited by curcumin might be mediated, at least in part, via the 5-HT receptor-cAMP-PKA-CREB signal pathway. PMID:21689105

Xu, Ying; Li, Shan; Vernon, Matthew M; Pan, Jianchun; Chen, Ling; Barish, Philip A; Zhang, Yuan; Acharya, Abhinav P; Yu, Jie; Govindarajan, Subramaniam S; Boykin, Erin; Pan, Xiaoyu; O'Donnell, James M; Ogle, William O

2011-09-01

297

Role of periaqueductal grey prostaglandin receptors in formalin-induced hyperalgesia  

Microsoft Academic Search

In this study we have investigated the role of periaqueductal grey prostaglandin receptors in formalin-induced hyperalgesia in mice. Glutamate and GABA release changes have been monitored by in vivo microdialysis. Intra-periaqueductal grey microinjections of misoprostol, a non-selective prostaglandin receptor agonist, increased nociceptive responses in the formalin test only during the late phase. Prostanoid EP1 (L-335677), EP2 (AH 6809), EP3 (L-826266)

Patrizia Oliva; Liberato Berrino; Vito de Novellis; Enza Palazzo; Ida Marabese; Dario Siniscalco; Mariantonietta Scafuro; Loredana Mariani; Francesco Rossi; Sabatino Maione

2006-01-01

298

Tumor Necrosis Factor Receptor Family Member RANK Mediates Osteoclast Differentiation and Activation Induced by Osteoprotegerin Ligand  

Microsoft Academic Search

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically

Hailing Hsu; David L. Lacey; Colin R. Dunstan; Irina Solovyev; Anne Colombero; Emma Timms; Hong-Lin Tan; Gary Elliott; Michael J. Kelley; Ildiko Sarosi; Ling Wang; Xing-Zhong Xia; Robin Elliott; Laura Chiu; Tabitha Black; Sheila Scully; Casey Capparelli; Sean Morony; Grant Shimamoto; Michael B. Bass; William J. Boyle

1999-01-01

299

Stimulation of 131 Integrins on Fibroblasts Induces PDGF Independent Tyrosine Phosphorylation of PDGF (3Receptors  

Microsoft Academic Search

We report that integrin-mediated signaling induces a rapid and transient tyrosine phosphorylation of platelet-derived growth factor (PDGF) 13-receptors in human diploid foreskin AG 1518 fibroblasts. A tran- sient tyrosine phosphorylation of PDGF i3-receptors was evident one and two hours after cells had been plated on collagen type I and fibronectin, as well as on immobilized anti-integrin subunit IgG, but not

Christian Sundberg; Kristofer Rubin

1996-01-01

300

Endothelial P2Y 2 receptor regulates LPS-induced neutrophil transendothelial migration in vitro  

Microsoft Academic Search

Previous studies showed that P2 receptors are involved in neutrophil migration via stimulation of chemokine release and by facilitating chemoattractant gradient sensing. Here, we have investigated whether these receptors are involved in LPS-induced neutrophil transendothelial migration (TEM) using a Boyden chamber where neutrophils migrated through a layer of lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs). In line with a

Filip Kukulski; Fethia Ben Yebdri; Fariborz Bahrami; Michel Fausther; Alain Tremblay; Jean Sévigny

2010-01-01

301

Drug-induced mild therapeutic hypothermia obtained by administration of a transient receptor potential vanilloid type 1 agonist  

PubMed Central

Background The use of mechanical/physical devices for applying mild therapeutic hypothermia is the only proven neuroprotective treatment for survivors of out of hospital cardiac arrest. However, this type of therapy is cumbersome and associated with several side-effects. We investigated the feasibility of using a transient receptor potential vanilloid type 1 (TRPV1) agonist for obtaining drug-induced sustainable mild hypothermia. Methods First, we screened a heterogeneous group of TRPV1 agonists and secondly we tested the hypothermic properties of a selected candidate by dose-response studies. Finally we tested the hypothermic properties in a large animal. The screening was in conscious rats, the dose-response experiments in conscious rats and in cynomologus monkeys, and the finally we tested the hypothermic properties in conscious young cattle (calves with a body weight as an adult human). The investigated TRPV1 agonists were administered by continuous intravenous infusion. Results Screening: Dihydrocapsaicin (DHC), a component of chili pepper, displayed a desirable hypothermic profile with regards to the duration, depth and control in conscious rats. Dose-response experiments: In both rats and cynomologus monkeys DHC caused a dose-dependent and immediate decrease in body temperature. Thus in rats, infusion of DHC at doses of 0.125, 0.25, 0.50, and 0.75 mg/kg/h caused a maximal ?T (°C) as compared to vehicle control of -0.9, -1.5, -2.0, and -4.2 within approximately 1 hour until the 6 hour infusion was stopped. Finally, in calves the intravenous infusion of DHC was able to maintain mild hypothermia with ?T > -3°C for more than 12 hours. Conclusions Our data support the hypothesis that infusion of dihydrocapsaicin is a candidate for testing as a primary or adjunct method of inducing and maintaining therapeutic hypothermia.

2010-01-01

302

Therapeutics Based On The Induced Internalization Of Surface Receptors  

Cancer.gov

The National Cancer Institute, Laboratory of Cellular Oncology is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize therapeutics for diseases or conditions associated with target receptors, such as cancer, angiogenesis, or HIV infections.

303

Paradoxical neostigmine-induced TOFfade: on the role of presynaptic cholinergic and adenosine receptors.  

PubMed

Neuromuscular transmission is clinically monitored using the train-of-four ratio (TOFratio), which is the quotient between twitch tension produced by the fourth (T4) and by the first (T1) stimulus within a train-of-four stimulation at 2Hz. Neostigmine causes a paradoxical depression of the TOFratio (TOFfade) that is amplified by intra-arterial atropine in cats. This led us to question the usefulness of the TOFratio as a sole testing element to control neostigmine-induced reversal of neuromuscular transmission block caused by non-depolarizing agents. We hypothesized that the inhibition of cholinesterase activity might increase acetylcholine bioavailability and consequently cholinoceptor activation, leading to concomitant adenosine release from nerve endings and skeletal muscle fibers. The involvement of presynaptic muscarinic and adenosine receptors in neostigmine-induced TOFfade in the rat phrenic nerve diaphragm was investigated. Blockade of adenosine A2A receptors with ZM241385 and of muscarinic M2 receptors with methoctramine or atropine amplified neostigmine-induced TOFfade. Notwithstanding TOFfade amplification, the blockade of M2 or A2A receptors increased both T1 and T4 twitch tensions above control during the first 3min of neostigmine application. Beyond that period, the T1 twitch tension returned to baseline, whereas T4 decreased further until the control value with neostigmine alone. Blockade of M1 receptors by pirenzepine did not change neostigmine-induced TOFfade, unless A2A receptors were concurrently blocked with ZM241385. Data indicate that the paradoxical neostigmine-induced fade involves presynaptic mechanisms that regulate transmitter release and synaptic adenosine accumulation, including the activation of adenosine A2A and muscarinic M2 receptors. PMID:24247035

de Paula Ramos, Edivan; Antônio, Marilia Bordignon; Ambiel, Celia Regina; Correia-de-Sá, Paulo; Alves-Do-Prado, Wilson

2014-01-15

304

CB1 receptors mediate rimonabant-induced pruritic responses in mice: investigation of locus of action  

PubMed Central

Rationale Cannabinoids have recently been identified as potential neuronal modulators of pruritic response, representing a potential target in the treatment of itch associated with a variety of pathophysiologic conditions. While the selective CB1 receptor antagonist rimonabant is an established pruritic agent in both animal and clinical testing, its receptor mechanism of action and anatomical loci remain unclear. Objective The purpose of this study was to determine whether CB1 receptor blockade is critical to rimonabant-induced scratching and to identify differences in scratching response based on different routes of administration. Furthermore, experiments were designed to elucidate any evidence as to whether rimonabant elicits scratching behavior through common immunologic hypersensitivity mechanisms. Results Rimonabant was equally effective at producing scratching via intraperitoneal and local subcutaneous injection. This compound also produced an intense scratching response when administered intrathecally, but had no effects after intracerebroventricular administration. Repeated administration of rimonabant led to a decreased magnitude of scratching. While rimonabant-induced scratching was not attenuated either by pretreatment with the H1 receptor antagonist loratadine or in mast cell-deficient mice, it lacked efficacy in CB1 (?/?) mice. Conclusions Rimonabant is a potent and fully effective pruritogen when administered spinally or systemically and requires CB1 receptors to induce scratching, suggesting an important spinal CB1 receptor component of action. The lack of responsiveness to H1 antagonism or mast cell deficiency supports previous findings that cannabinoids modulate itch through neuronal mechanisms, and not by traditional hypersensitivity activation.

Schlosburg, Joel E.; O'Neal, Scott T.; Conrad, Daniel H.

2012-01-01

305

Bacterial Chaperone Protein, Skp, Induces Leukocyte Chemotaxis via C5a Receptor  

PubMed Central

C5a receptor has been identified as a leukocyte chemotactic receptor to two intrinsic chemical mediators, C5a and the S19 ribosomal protein dimer, so far. We found an Escherichia coli protein that also induced the chemotactic responses of monocytes and polymorphonuclear leukocytes via the C5a receptor. We identified the E. coli-derived chemoattractant to be Skp by the molecular size and the N-terminal amino acid sequence. Skp is a periplasmic chaperone protein widely present in gram-negative bacterial species. Immunoabsorption experiments indicated that Skp was the major leukocyte chemotactic factor in the E. coli extract. Receptor-antagonizing experiments with analogue peptides of S19 ribosomal protein and of C5a suggested that Skp induced the receptor activation by the two-step binding mechanism as in the cases of the intrinsic mediators, sharing the ligand-binding site of the receptor among them at each binding step. The C5a receptor would play a role in the host defense directly recognizing the bacteria-derived protein, besides identifying the signals of the intrinsic chemical mediators.

Shrestha, Arjun; Shi, Lei; Tanase, Sumio; Tsukamoto, Makiko; Nishino, Norikazu; Tokita, Kazutaka; Yamamoto, Tetsuro

2004-01-01

306

Gs-Coupled Adenosine Receptors Differentially Limit Antigen-Induced Mast Cell Activation  

PubMed Central

Mast cell activation results in the immediate release of proinflammatory mediators prestored in cytoplasmic granules, as well as initiation of lipid mediator production and cytokine synthesis by these resident tissue leukocytes. Allergen-induced mast cell activation is central to the pathogenesis of asthma and other allergic diseases. Presently, most pharmacological agents for the treatment of allergic disease target receptors for inflammatory mediators. Many of these mediators, such as histamine, are released by mast cells. Targeting pathways that limit antigen-induced mast cell activation may have greater therapeutic efficacy by inhibiting the synthesis and release of many proinflammatory mediators produced in the mast cell. In vitro studies using cultured human and mouse mast cells, and studies of mice lacking A2B receptors, suggest that adenosine receptors, specifically the Gs-coupled A2A and A2B receptors, might provide such a target. Here, using a panel of mice lacking various combinations of adenosine receptors, and mast cells derived from these animals, we show that adenosine receptor agonists provide an effective means of inhibition of mast cell degranulation and induction of cytokine production both in vitro and in vivo. We identify A2B as the primary receptor limiting mast cell degranulation, whereas the combined activity of A2A and A2B is required for the inhibition of cytokine synthesis.

Hua, Xiaoyang; Chason, Kelly D.; Jania, Corey; Acosta, Tatiana; Ledent, Catherine

2013-01-01

307

The Role of Purinergic Receptors in Cancer-Induced Bone Pain  

PubMed Central

Cancer-induced bone pain severely compromises the quality of life of many patients suffering from bone metastasis, as current therapies leave some patients with inadequate pain relief. The recent development of specific animal models has increased the understanding of the molecular and cellular mechanisms underlying cancer-induced bone pain including the involvement of ATP and the purinergic receptors in the progression of the pain state. In nociception, ATP acts as an extracellular messenger to transmit sensory information both at the peripheral site of tissue damage and in the spinal cord. Several of the purinergic receptors have been shown to be important for the development and maintenance of neuropathic and inflammatory pain, and studies have demonstrated the importance of both peripheral and central mechanisms. We here provide an overview of the current literature on the role of purinergic receptors in cancer-induced bone pain with emphasis on some of the difficulties related to studying this complex pain state.

Falk, Sarah; Uldall, Maria; Heegaard, Anne-Marie

2012-01-01

308

Modifying Ligand-Induced and Constitutive Signaling of the Human 5-HT4 Receptor  

PubMed Central

G protein–coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their in vivo functions have been hampered by the molecular and functional diversity of GPCRs and the paucity of ligands with specific signaling effects. To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT4b receptor, a GPCR with high constitutive Gs signaling and strong ligand-induced G-protein activation of the Gs and Gs/q pathways. The first receptor in this series, 5-HT4-D100A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced Gs signaling, but only a few (e.g., zacopride) also induced signaling via the Gq pathway. Zacopride-induced Gq signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT2C receptor. Additional point mutations (D66A and D66N) blocked constitutive Gs signaling and lowered ligand-induced Gq signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT1A conferred ligand-mediated Gi signaling. This Gi-coupled RASSL, Rs1.3, exhibited no measurable signaling to the Gs or Gq pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection.

Chang, Wei Chun; Ng, Jennifer K.; Nguyen, Trieu; Pellissier, Lucie; Claeysen, Sylvie; Hsiao, Edward C.; Conklin, Bruce R.

2007-01-01

309

Involvement of glucocorticoid receptor activation on anti-inflammatory effect induced by peroxisome proliferator-activated receptor ? agonist in mice.  

PubMed

Glucocorticoids are effective anti-inflammatory agents widely used for the treatment of acute and chronic inflammatory diseases. Recent in vitro studies have proposed that glucocorticoid receptor (GR) activation is involved in peroxisome proliferator-activated receptor ? (PPAR?) agonist-induced effects. In this study, to examine the involvement of the GR in PPAR? agonist- and retinoid X receptor (RXR) agonist-mediated anti-inflammatory effects in vivo, we tested the anti-inflammatory effects of dexamethasone (a GR agonist) with pioglitazone (a PPAR? agonist) or 6-[N-ethyl-N-(3-isopropoxy-4-isopropylphenyl)-amino] nicotinic acid (NEt-3IP; an RXR agonist) by using an experimental model of carrageenan-induced inflammation. We also evaluated the effects of a GR antagonist on PPAR? agonist- or RXR agonist-induced anti-inflammatory effects. Results showed that the GR antagonist RU486 reduced the anti-inflammatory effects of GR or PPAR? agonists but not those of the RXR agonist. In addition, combinations of GR and PPAR? agonists or GR and RXR agonists had no effect on carrageenan-induced paw edema. Moreover, the PPAR? antagonist GW9662 and RXR antagonist 6-[N-4-(trifluoromethyl)-benzenesulfonyl-N-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)-amino] nicotinic acid (NS-4TF) had no effect on the anti-inflammatory effect of the GR agonist dexamethasone. Therefore, it is suggested that GR activation in vivo does not play a direct role in PPAR?/RXR heterodimer signaling. In contrast, pioglitazone showed a partial anti-inflammatory effect via GR activation. These data provide evidence for the pro-inflammatory activity of pioglitazone. PMID:24975659

Yamamoto, Atsuki; Kakuta, Hiroki; Sugimoto, Yukio

2014-09-01

310

Sympathetically induced changes in the responses of guard hair and type II receptors in the cat.  

PubMed Central

1. Sympathetic effects on the activity of single guard hair receptors and slowly adapting, type II mechanoreceptors were studied in anaesthetized cats. 2. Spontaneous activity in type II receptors and mechanically evoked activity in both type II and guard hair receptors was recorded both without and with electrical stimulation of the lumbar sympathetic trunk. 3. Almost all guard hair receptors were desensitized by sympathetic stimulation, the mean effect being an increase in threshold to 2.2 times the unconditioned value. 4. One third of the more slowly adapting guard hair units showed sympathetically induced spontaneous activity. 5. These sympathetic effects on guard hair receptors appear not to be related to pilomotor responses or to changes in central arterial pressure. 6. Individual type II receptors were found to be excited by skin stretch along one of two axes: either parallel or perpendicular to the long axis of the leg. Intermediate orientations were not observed. 7. 'Perpendicular' units showed marked increases in resting discharge but no change in mechanical sensitivity during sympathetic stimulation. 'Parallel' units were unaffected. 8. The sympathetic effect appeared not to be related to changes in blood pressure or skin tension. 9. It is suggested that the location and orientation of stretch sensitivity of these receptors make them well suited for detection of limb movements; however, no satisfactory functional explanation was found for the pronounced sympathetic facilitation of only one subset of the receptors. Images Plate 1

Pierce, J P; Roberts, W J

1981-01-01

311

The epidermal growth factor-like domain of recombinant human thrombomodulin exhibits mitogenic activity for Swiss 3T3 cells.  

PubMed

Thrombomodulin (TM) is an anticoagulant endothelial cell surface glycoprotein containing six tandem epidermal growth factor (EGF)-like structures. We prepared a recombinant TM peptide (rTME1-6, from R214GHWA to DSGK466 of native TM) composed of these six EGF-like structures and investigated the effect of rTME1-6 peptide on the growth of the Swiss 3T3 fibroblast cell line. It was found that rTME1-6 induced proliferation of Swiss 3T3 cells and accelerated [3H]thymidine uptake into their DNA. [3H]Thymidine uptake increased in a dose-dependent manner, plateauing at 50 ng/mL rTME1-6, which was 1.8 times the control level. rTME1-6 peptide (50 ng/mL) also accelerated the DNA synthesis of human dermal fibroblasts (HDFs), A549 (a human lung cancer cell line), HepG2 (a human hepatocarcinoma cell line), and U937 cells (a human monocytic cell line) to 1.5, 1.6, 1.4, and 1.2 times the control level, respectively. The magnitude of the acceleration of DNA synthesis in Swiss 3T3 induced by rTME1-6 was approximately 20% of that of EGF on a molar basis. The uptake of [3H]thymidine was accelerated synergistically by coculture of the cells with rTME1-6 and insulin, similar to the coculture with EGF and insulin. The effects of rTME1-6 were abolished by addition of polyclonal antihuman TM IgG, whereas the actions of insulin and EGF were not influenced. Glucose uptake in Swiss 3T3 cells also increased 1.6 times over control levels by culture with 50 ng/mL rTME1-6 (1.25 nmol/L), compared with 2.7 times by 10 ng/mL EGF (1.66 nmol/L). Binding of [125I]EGF (0.5 ng/mL, 0.083 nmol/L) by the cells was inhibited by about 60% by addition of an eight-fold molar excess of nonlabeled EGF (0.664 nmol/L), whereas no inhibition of [125I]EGF binding was observed, even in the presence of a 1,000-fold molar excess (83 nmol/L) of rTME1-6. Specific binding of [125I]rTME1-6 on the cells showed a saturation curve, and the apparent concentration of rTME1-6 required for half maximum binding of the peptide on the cells was calculated to be 31.5 ng/mL. Thus, the overall results indicated that the rTME1-6 peptide had mitogenic activity for Swiss 3T3 cells, accelerated DNA synthesis and glucose uptake, and that the mitogenic activity might be mediated by binding of the peptide to a specific site different from the EGF receptor. PMID:7795228

Hamada, H; Ishii, H; Sakyo, K; Horie, S; Nishiki, K; Kazama, M

1995-07-01

312

Tazarotene-Induced Gene 1 (TIG1), a Novel Retinoic Acid Receptor-Responsive Gene in Skin  

Microsoft Academic Search

Retinoids exert their effect through ligand-dependent transcription factors, retinoic acid receptors (RAR&?, ?, and ?) and retinoid X receptor (RXR?, ?, and ?), which belong to the superfamily of steroid\\/ thyroid\\/vitamin D3 nuclear receptors. Using a subtraction hybridization approach, we have identified a cDNA sequence, Tazarotene Induced Gene 1 (TIG1), which is highly upregulated in skin raft cultures by an

Sunil Nagpal; Sheetal Patel; Arisa T. Asano; Alan T. Johnson; Madeleine Duvic; Roshantha A. S. Chandraratna

1996-01-01

313

Fluoroquinolones reduce carrageenan-induced edema in rats and the involvement of the glucocorticoid receptor system.  

PubMed

We studied the effect of fluoroquinolones (FQs) on carrageenan-induced edema in the rat footpad. Ciprofloxacin, gatifloxacin, sparfloxacin, norfloxacin, and enoxacin (s.c., 100 mg/kg), which have piperazinyl and/or cyclopropyl groups, inhibited carrageenan-induced edema, whereas levofloxacin, tosufloxacin, and pazufloxacin did not. The reduction of edema by ciprofloxacin, sparfloxacin, and enoxacin was abolished by pretreatment with mifepristone, an antagonist of the glucocorticoid receptor. These results suggest that FQs with piperazinyl and/or cyclopropyl groups can modify biological responses through enhancing the glucocorticoid-glucocorticoid receptor system. PMID:19396522

Ogino, Hiromi; Yamada, Kaori; Yuhara, Mizuki; Tsuchida, Saori; Maezawa, Kayoko; Kizu, Junko; Hori, Seiji

2009-04-01

314

Expression of human epidermal growth factor pressures cDNA in transfected mouse NIH 3T3 cells  

SciTech Connect

Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 ..mu..M ZnCl/sub 2/. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approx. = 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with /sup 125/I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.

Mroczkowski, B.; Reich, M.; Whittaker, J.; Bell, G.I.; Cohen, S.

1988-01-01

315

1?-Hydroxy-2-oxopomolic acid isolated from Agrimonia pilosa extract inhibits adipogenesis in 3T3-L1 cells.  

PubMed

In order to determine anti-adipogenic effect, this study investigated 1?-hydroxy-2-oxopomolic acid (HOA) isolated from Agrimonia pilosa inhibits adipocyte differentiation and expression of adipogenic marker genes, such as peroxisome proliferator activated receptor ? (PPAR?), CCAAT-enhancer-binding protein ? (C/EBP?), glucose transporter 4 (GLUT4), adiponectin, adipocyte fatty acid-binding protein 2 (aP2), adipocyte determination and differentiation factor 1/sterol regulatory element binding protein 1c (ADD1/SREBP1c), resistin, and fatty acid synthase (Fas) in 3T3-L1 preadipocyte. We demonstrated that HOA induced a significant decrease in lipid accumulation and expression of adipogenic marker genes in a dose-dependent manner. In addition, HOA reduced the transcripitional activity of PPAR? induced by troglitazone, a potent diabetes agent; it also suppressed expression of PPAR? and C/EBP? protein levels. Our data suggest that HOA isolated from Agrimonia pilosa inhibits adipocyte differentiation through downregulation of various adipocytokines by blocking PPAR? and C/EBP? expression. PMID:22687396

Ahn, Eun-Kyung; Lee, Jung A; Seo, Dong-Wan; Hong, Seong Su; Oh, Joa Sub

2012-01-01

316

Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor.  

PubMed

We have isolated a c-erbA cDNA clone from a GH3 cell library. The clone, denoted erb62, is 4.5 kilobases long and encodes a 461-amino acid beta-type c-erbA protein. This c-erbA protein binds 3,5,3'-triiodothyronine (T3) and T3 analogs with affinities similar to those of the authentic T3 receptor. By RNA gel blot analysis, erb62 hybridizes to a 6-kilobase RNA found in organs that express T3 receptors--e.g., heart, kidney, and brain. A COS-cell transient cotransfection system was used to show that erb62 encodes a biologically active T3 receptor. An oligonucleotide, corresponding to a portion of the rat growth hormone gene 5'-flanking region that contains a T3 response element, was inserted on the 5' side of the herpes simplex virus thymidine kinase promoter in a chloramphenicol acetyltransferase-expressing plasmid. Reporter gene expression directed by this hybrid promoter was T3 inducible only if this plasmid was cotransfected with an erb62-expressing plasmid. PMID:2899322

Koenig, R J; Warne, R L; Brent, G A; Harney, J W; Larsen, P R; Moore, D D

1988-07-01

317

Drug-induced graves disease from CTLA-4 receptor suppression.  

PubMed

Monoclonal antibody, ipilimumab, useful for treatment of metastatic melanoma, blocks CTLA-4 mediated T-cell suppression and can also cause a Graves ophthalmopathy like syndrome. Epidemiologic study has linked variant polymorphisms of CTLA-4 receptor gene to the presence of thyroid eye disease. The combination of these observations suggests CTLA-4 mediated T-cell functions are important to the pathogenesis of thyroid-associated eye disease. PMID:21242854

Borodic, Gary; Hinkle, David M; Cia, Yihong

2011-01-01

318

Estrogen related receptor ?-induced adipogenesis is PGC1?-dependent  

Microsoft Academic Search

Previous report showed that Estrogen related receptor ? (ERR?) knockout mice had a significant reduction in adipose tissue deposition. Although it was reported that ERR? could promote\\u000a adipogenesis in several immortalized preadipocytes cell lines, the mechanism behind which is still unclear to date. Besides,\\u000a the expression pattern of ERR? in white adipose tissue is rarely examined. Here, we show that

Dapeng JuJingjing; Jingjing He; Lili Zhao; Xueli Zheng; Gongshe Yang

319

Oxysterol 22(R)-Hydroxycholesterol Induces the Expression of the Bile Salt Export Pump through Nuclear Receptor Farsenoid X Receptor but Not Liver X Receptor  

PubMed Central

Oxysterols are intermediates in the synthesis of bile acids and steroid hormones from cholesterol and function as ligands for liver X receptor (LXR). Bile salt export pump (BSEP) is responsible for canalicular secretion of bile acids and is tightly regulated by its substrates bile acids through nuclear receptor farnesoid X receptor (FXR). In a microarray study using human hepatocytes, BSEP was markedly induced not only by chenodeoxycholic acid (CDCA) but also by oxysterol 22(R)-hydroxycholesterol [22(R)-OHC]. We hypothesized that the expression of BSEP was induced by oxysterols through activation of LXR. To test the hypothesis, human primary hepatocytes or hepatoma cells were treated with 22(R)-OHC, and expression of BSEP was determined. The level of BSEP mRNA was increased as much as 5-fold upon oxysterol induction. In contrast to our hypothesis, the oxysterol-induced up-regulation of BSEP is mediated through FXR but not LXR. BSEP promoter activity was markedly induced by 22(R)-OHC in the presence of FXR but not LXRs. Mutation of the FXR element IR1 in the BSEP promoter significantly reduced its ability to respond to oxysterol induction. To determine whether 22(R)-OHC and CDCA bind to similar structural features of FXR, site-directed mutagenesis was performed in the FXR ligand binding domain. Mutation of residues R331 and I352 abolished activation mediated by CDCA and 22(R)-OHC. In contrast, substitution of residues L340 and R351 differentiated CDCA- and 22(R)-OHC-mediated activation. In conclusion, oxysterol 22(R)-OHC functions as an FXR ligand to induce BSEP expression and differs in the binding with FXR from CDCA.

Deng, Ruitang; Yang, Dongfang; Yang, Jian; Yan, Bingfang

2014-01-01

320

Peripheral Glutamate Receptors Are Required for Hyperalgesia Induced by Capsaicin  

PubMed Central

Transient receptor potential vanilloid1 (TRPV1) and glutamate receptors (GluRs) are located in small diameter primary afferent neurons (nociceptors), and it was speculated that glutamate released in the peripheral tissue in response to activation of TRPV1 might activate nociceptors retrogradely. But, it was not clear which types of GluRs are functioning in the nociceptive sensory transmission. In the present study, we examined the c-Fos expression in spinal cord dorsal horn following injection of drugs associated with glutamate receptors with/without capsaicin into the hindpaw. The subcutaneous injection of capsaicin or glutamate remarkably evoked c-Fos expression in ipsilateral sides of spinal cord dorsal horn. This capsaicin evoked increase of c-Fos expression was significantly prevented by concomitant administration of MK801, CNQX, and CPCCOEt. On the other hand, there were not any significant changes in coinjection of capsaicin and MCCG or MSOP. These results reveal that the activation of iGluRs and group I mGluR in peripheral afferent nerves play an important role in mechanisms whereby capsaicin evokes/maintains nociceptive responses.

Jin, You-Hong; Takemura, Motohide; Furuyama, Akira; Yonehara, Norifumi

2012-01-01

321

Peripheral glutamate receptors are required for hyperalgesia induced by capsaicin.  

PubMed

Transient receptor potential vanilloid1 (TRPV1) and glutamate receptors (GluRs) are located in small diameter primary afferent neurons (nociceptors), and it was speculated that glutamate released in the peripheral tissue in response to activation of TRPV1 might activate nociceptors retrogradely. But, it was not clear which types of GluRs are functioning in the nociceptive sensory transmission. In the present study, we examined the c-Fos expression in spinal cord dorsal horn following injection of drugs associated with glutamate receptors with/without capsaicin into the hindpaw. The subcutaneous injection of capsaicin or glutamate remarkably evoked c-Fos expression in ipsilateral sides of spinal cord dorsal horn. This capsaicin evoked increase of c-Fos expression was significantly prevented by concomitant administration of MK801, CNQX, and CPCCOEt. On the other hand, there were not any significant changes in coinjection of capsaicin and MCCG or MSOP. These results reveal that the activation of iGluRs and group I mGluR in peripheral afferent nerves play an important role in mechanisms whereby capsaicin evokes/maintains nociceptive responses. PMID:22110945

Jin, You-Hong; Takemura, Motohide; Furuyama, Akira; Yonehara, Norifumi

2012-01-01

322

Angiotensin II Type 1 Receptor-Induced Extracellular Signal-Regulated Protein Kinase Activation Is Mediated by Ca21\\/Calmodulin-Dependent Transactivation of Epidermal Growth Factor Receptor  

Microsoft Academic Search

The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor stimulation, and recent evidence suggests that G protein- coupled receptors transactivate growth factor receptors to transmit mitogenic effects. In the present study, we report the involvement of epidermal growth factor receptor (EGF-R) in Ang II-induced extracellular signal-regulated kinase (ERK) activation, c-fos gene expression, and DNA

Satoshi Murasawa; Yasukiyo Mori; Yoshihisa Nozawa; Noriko Gotoh; Masabumi Shibuya; Hiroya Masaki; Katsuya Maruyama; Yoshiaki Tsutsumi; Yasutaka Moriguchi; Yasunobu Shibazaki; Yohko Tanaka; Toshiji Iwasaka; Mitsuo Inad; Hiroaki Matsubara

323

Angiotensin II AT1 receptor blocker candesartan prevents the fast up-regulation of cerebrocortical benzodiazepine-1 receptors induced by acute inflammatory and restraint stress  

PubMed Central

Centrally acting Angiotensin II AT1 receptor blockers (ARBs) protect from stress-induced disorders and decrease anxiety in a model of inflammatory stress, the systemic injection of bacterial endotoxin lipopolysaccharide (LPS). In order to better understand the anxiolytic effect of ARBs, we treated rats with LPS (50 µg/kg) with or without three days of pretreatment with the ARB candesartan (1 mg/kg/day), and studied cortical benzodiazepine (BZ) and corticotrophin-releasing factor (CRF) receptors. We compared the cortical BZ and CRF receptors expression pattern induced by LPS with that produced in restraint stress. Inflammation stress produced a generalized increase in cortical BZ1 receptors and reduced mRNA expression of the GABAA receptor ?2 subunit in cingulate cortex; changes were prevented by candesartan pretreatment. Moreover, restraint stress produced similar increases in cortical BZ1 receptor binding, and candesartan prevented these changes. Treatment with candesartan alone increased cortical BZ1 binding, and decreased ?2 subunit mRNA expression in the cingulate cortex. Conversely, we did not find changes in CRF1 receptor expression in any of the cortical areas studied, either after inflammation or restraint stress. Cortical CRF2 receptor binding was undetectable, but CRF2 mRNA expression was decreased by inflammation stress, a change prevented by candesartan. We conclude that stress promotes rapid and widespread changes in cortical BZ1 receptor expression; and that the stress-induced BZ1 receptor expression is under the control of AT1 receptor activity. The results suggest that the anti-anxiety effect of ARBs may be associated with their capacity to regulate stress-induced alterations in cortical BZ1 receptors.

Sanchez-Lemus, Enrique; Honda, Masaru; Saavedra, Juan M.

2012-01-01

324

5HT 7 receptor deletion enhances REM sleep suppression induced by selective serotonin reuptake inhibitors, but not by direct stimulation of 5HT 1A receptor  

Microsoft Academic Search

5-HT7 receptors are involved in REM sleep and possibly in mood disorders. REM sleep suppression and antidepressant-like behavior is observed in 5-HT7?\\/? mice and in rats treated with 5-HT7 receptor antagonists. We recently demonstrated that pharmacological blockade of 5-HT7 receptors enhances REM sleep suppression and antidepressant-like behavior induced by citalopram in rodents. It has been hypothesized that the effect of

Jonathan Shelton; Pascal Bonaventure; Xiaorong Li; Sujin Yun; Timothy Lovenberg; Christine Dugovic

2009-01-01

325

Sasa quelpaertensis Nakai extract and its constituent p-coumaric acid inhibit adipogenesis in 3T3-L1 cells through activation of the AMPK pathway.  

PubMed

In this study, we investigated the effects of Sasa quelpaertensis Nakai extract (SQE) and its main constituent, p-coumaric acid, on adipogenesis in 3T3-L1 cells. SQE markedly inhibited adipogenesis by downregulating the expression of CCAAT/enhancer-binding protein ? (C/EBP?), peroxisome proliferator-activated receptor ? (PPAR?), sterol regulatory element-binding protein-1c (SREBP-1c), and aP2. It also decreased the expression of fatty acid synthase (FAS) and adiponectin mRNAs in differentiating adipocytes. SQE increased AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation during the early phase of MDI-induced differentiation, suggesting that SQE exerted its anti-adipogenic effect via AMPK activation at an early stage of the differentiation process. p-Coumaric acid suppressed adipogenesis by attenuating the expression of C/EBP?, PPAR?, and SREBP-1c during the late phase of MDI-induced differentiation. In addition, p-coumaric acid increased the phosphorylation of AMPK and ACC, and the expression of carnitine palmitoyl transferase-1 (CPT-1) mRNA, in fully differentiated adipocytes, indicating that it promotes fatty acid ?-oxidation via AMPK signaling. Taken together, our data suggest that SQE and p-coumaric acid might have the anti-obesitic effects via AMPK pathway in 3T3-L1 cells. PMID:23810795

Kang, Seung-Woo; Kang, Seong-Il; Shin, Hye-Sun; Yoon, Seon-A; Kim, Jeong-Hwan; Ko, Hee-Chul; Kim, Se-Jae

2013-09-01

326

Methamphetamine Induces Striatal Neurokinin-1 Receptor Endocytosis Primarily in Somatostatin/NPY/NOS Interneurons and the Role of Dopamine Receptors in Mice  

PubMed Central

Methamphetamine (METH) is a psychostimulant that induces long-term deficits of dopamine terminal markers and apoptotic cell death in the striatum. Our laboratory demonstrated that pharmacological blockade of the neurokinin-1 receptor attenuated the METH-induced damage to the striatal dopamine terminals and the apoptotic cell death of some striatal neurons. Here we employed histological methods to assess the effect of METH on neurokinin-1 receptor trafficking in the striatum as an indirect index of signaling by the neuropeptide substance P (natural ligand for this receptor). Male mice received a single injection of METH (30 mg/kg, i.p.) and were sacrificed 30 minutes later. Immunohistofluorescence confocal microscopy confirmed that the neurokinin-1 receptor is located on cholinergic and somatostatin interneurons of the striatum. METH induced the trafficking of the neurokinin-1 receptor from the membrane into cytoplasmic endosomes primarily in the somatostatin/NPY/NOS interneurons and this phenomenon was attenuated by antagonists of the dopamine D1 (SCH-23390), D2 (raclopride) or neurokinin-1 (WIN-51,708) receptors. These data demonstrate that METH induces the trafficking of the striatal neurokinin-1 receptors principally in the somatostatin/NPY/NOS interneurons and that this phenomenon is dependent on the activity of dopamine D1 and D2 receptors.

Wang, Jing; Angulo, Jesus A.

2010-01-01

327

Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells  

SciTech Connect

Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of (/sup 32/P)-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions.

Knabbe, C.; Lippman, M.E.; Greene, G.L.; Dickson, R.B.

1986-05-01

328

5-HT7 receptor activation attenuates thermal hyperalgesia in streptozocin-induced diabetic mice.  

PubMed

The role of 5-HT7 receptors in the nociceptive processing received most attention during the last few years. The involvement of 5-HT? receptors in nerve injury-induced neuropathic pain states have been reported only recently; however, there are no reports on its contribution in diabetic neuropathic pain. We therefore planned to investigate the effect of 5-HT? receptor activation on the changes of nociceptive threshold in diabetic mice. Diabetes was induced by a single intraperitoneal injection of streptozocin (150 mg/kg, i.p.). The nociceptive responses in normal and diabetic animals were tested in the hot-plate and tail-flick assays. Both hot-plate and tail-flick latencies significantly shortened at 1-3/4 weeks (thermal hyperalgesia) and prolonged at 6-7 weeks (thermal hypoalgesia) after streptozocin administration. At the dose of 10 mg/kg, systemic injections of AS-19, a selective 5-HT? receptor agonist, reduced thermal hyperalgesia at early stage of diabetes, but did not influence thermal hypoalgesia at late stage. Co-administration of SB-258719, a selective 5-HT? receptor antagonist, at a dose that had no effect on its own (10 mg/kg), reversed the anti-hyperalgesic effect of AS-19. Our results indicate that systemic administration of 5-HT? receptor agonists may have clinical utility in treating diabetic neuropathic pain. PMID:22609798

Ulugol, Ahmet; Oltulu, Cagatay; Gunduz, Ozgur; Citak, Cihad; Carrara, Roberto; Shaqaqi, Mohammad Reza; Sanchez, Alicia Mansilla; Dogrul, Ahmet

2012-08-01

329

Tryptophol induces death receptor (DR) 5-mediated apoptosis in U937 cells.  

PubMed

Tryptophol is a natural component isolated from vinegar produced from the boiled extract of black soybean. We have reported that tryptophol induces apoptosis in U937 cells via activation of caspase-8 followed by caspase-3. Tryptophol, however, did not affect human peripheral blood lymphocytes (PBL). In this study, we found that tryptophol enhances formation of a death-inducing signaling complex including death receptor (DR) 5. Cell viability and induction of apoptosis by tryptophol was reduced by transfection with decoy receptor (DcR) 1. These results indicate that tryptophol induces apoptosis through DR5 and that the resistance of PBL to tryptophol-induced apoptosis might be due to competition from DcR1. PMID:17690453

Inagaki, Shyuichiro; Morimura, Shigeru; Tang, Yueqin; Akutagawa, Hiroshi; Kida, Kenji

2007-08-01

330

ErbB Receptor Activation, Cell Morphology Changes, and Apoptosis Induced by Anti-Her2 Monoclonal Antibodies  

Microsoft Academic Search

A panel of mAbs were generated against the purified soluble form of erbB2\\/Her2 receptor, corresponding to the extracellular region of the receptor, and examined for their ability to mimic the receptor ligand. Some of the mAbs strongly induced tyrosine phosphorylation of 180-185 kDa proteins, including not only Her2 but also Her3 and Her4 receptors, when they were expressed on the

Yoshiko Kita; Julia Tseng; Thomas Horan; Jie Wen; John Philo; David Chang; Barry Ratzkin; Robert Pacifici; David Brankow; Sylvia Hu; Yi Luo; Duanzhi Wen; Tsutomu Arakawa; Margery Nicolson

1996-01-01

331

Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells  

SciTech Connect

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor {gamma}2, CCAAT/enhancer-binding protein alpha (C/EBP{alpha}), sterol regulatory element binding protein-1c, and Krueppel-like factor 15, but not those of C/EBP{beta} or C/EBP{delta}, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.

Jeong, Hyun Jeong [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of)] [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of); Park, Sahng Wook [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)] [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Kim, Hojeong [Department of Anatomy, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of)] [Department of Anatomy, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of); Park, Sang-Kyu, E-mail: 49park@kd.ac.kr [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of)] [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of); Yoon, Dojun, E-mail: mozart@kd.ac.kr [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of)] [Department of Biochemistry, Kwandong University College of Medicine, Gangneung, Gangwondo 210-701 (Korea, Republic of)

2010-02-19

332

An active extract of Lindera obtusiloba inhibits adipogenesis via sustained Wnt signaling and exerts anti-inflammatory effects in the 3T3-L1 preadipocytes.  

PubMed

Obesity, the related metabolic syndrome and associated liver diseases represent an epidemic problem and demand for effective therapeutic strategies. In this regard, natural compounds derived from Oriental medicine such as green tea polyphenols influencing adipogenesis attract growing attention. In Korea, an aqueous extract from the Japanese spice bush Lindera obtusiloba is traditionally used for treatment of inflammation and prevention of liver damage. We here investigated effects of the L. obtusiloba extract on cell growth, apoptosis, Wnt signaling and differentiation of (im)mature adipocytes using 3T3-L1, an established cell line for studying adipogenesis. L. obtusiloba extract reduced the de novo DNA synthesis of 3T3-L1 preadipocytes in a concentration dependent manner with an IC(50) of ?135 ?g/ml paralleled by induction of caspase 3/7 mediated apoptosis. Hormone-induced 3T3 L1 differentiation in the presence of L. obtusiloba extract resulted in a reduced accumulation of intracellular lipid droplets by 70%, in down-regulated expression of the adipogenesis-associated proteins glucose transporter-4 and vascular endothelial growth factor, in reduced secretion of the proadipogenic matrix metalloproteinase-2, and in dampened phosphorylation of the Wnt pathway effector protein ?-catenin with subsequent diminished expression of the peroxisome proliferator-activated receptor-?. Treatment of mature adipocytes with L. obtusiloba extract also significantly reduced intracellular lipid droplets. In addition to this strong interference of L. obtusiloba extract with adipogenesis, L. obtusiloba extract exerted anti-inflammatory effects. L. obtusiloba extract significantly attenuated lipopolysaccharide- and tumor necrosis factor ?-induced secretion of IL-6 by preadipocytes, thus influencing insulin resistance and inflammatory state characterizing obesity. In conclusion, extracts of L. obtusiloba should be evaluated as a potential complementary treatment option for obesity associated with the metabolic syndrome. PMID:20092995

Freise, Christian; Erben, Ulrike; Neuman, Ulf; Kim, Kiyoung; Zeitz, Martin; Somasundaram, Rajan; Ruehl, Martin

2010-12-01

333

Ligand-induced EGF Receptor Oligomerization Is Kinase-dependent and Enhances Internalization*  

PubMed Central

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ?40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.

Hofman, Erik G.; Bader, Arjen N.; Voortman, Jarno; van den Heuvel, Dave J.; Sigismund, Sara; Verkleij, Arie J.; Gerritsen, Hans C.; van Bergen en Henegouwen, Paul M. P.

2010-01-01

334

Nanoconjugation modulates the trafficking and mechanism of antibody induced receptor endocytosis.  

PubMed

Treatment with monoclonal antibody (mAbs) is a viable therapeutic option in cancer. Recently, these mAbs such as cetuximab, herceptin, etc., have been used as targeting agents to selectively deliver chemotherapeutics to cancerous cells. However, mechanisms of nanoparticles-mAbs interactions with the target cells and its effect on intracellular trafficking and mechanism are currently unknown. In this paper, we demonstrate that the distinct patterning and dynamics of anti-EGFR (epidermal growth factor receptor) antibody cetuximab (C225)- induced EGFR internalization in pancreatic cancer cells with variable receptor expression is altered upon nanoconjugation. Nanoconjugation uniformly enhanced C225-induced EGFR endocytosis in PANC-1, AsPC-1, and MiaPaca-2 cells, influenced its compartmentalization and regulated the involvement of dynamin-2 in the endocytic processes. Receptor endocytosis and its intracellular trafficking were monitored by confocal microscopy and transmission electron microscopy. The role of dynamin-2 in EGFR endocytosis was determined after overexpressing either wild-type dynamin-2 or mutant dynamin-2 in pancreatic cancer cells followed by tracking the receptor-antibody complex internalization by confocal microscopy. Significantly, these findings demonstrate that the nanoconjugation cannot be construed as an innocuous reaction involved in attaching the targeting agent to the nanoparticle, instead it may distinctly alter the cellular processes at the molecular level, at least antibody induced receptor endocytosis. This information is critical for successful design of a nanoparticle-based targeted drug delivery system for future clinical translation. PMID:20679244

Bhattacharyya, Sanjib; Bhattacharya, Resham; Curley, Steven; McNiven, Mark A; Mukherjee, Priyabrata

2010-08-17

335

Potentiation of adenosine A1 receptor agonist CPA-induced antinociception by paeoniflorin in mice.  

PubMed

The effect of paeoniflorin (PF), a major constituent isolated from Paeony radix, on N6-Cyclopentyladenosine (CPA), a selective adenosine A1 receptor (A1 receptor) agonist, induced antinociception was examined in mice. In the tail-pressure test, CPA (0.05, 0.1, 0.2 mg/kg, s.c.) could induce antinociception in a dose-dependent manner. PF (5, 10, 20 mg/kg, s.c.) alone failed to exhibit any antinociceptive effect in mice; however, pretreatment of PF (20 mg/kg, s.c.) could significantly enhance CPA-induced antinociception. Additionally, pretreatment of 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.25 mg/kg, s.c.), a selective A1 receptor antagonist, could antagonize the antinociceptive effect of combining CPA with PF. Furthermore, in the competitive binding experiments, PF did not displace the binding of [3H]-8-Cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) but displaced that of [3H]-2-Chloro-N6-cyclopentyladenosine ([3H]-CCPA, a selective A1 receptor agonist) to the membrane preparation of rat cerebral cortex. These results suggested that PF might selectively increase the binding and antinociceptive effect of CPA by binding with A1 receptor. PMID:16880617

Liu, Da-Zhi; Zhao, Fei-Li; Liu, Jing; Ji, Xin-Quan; Ye, Yang; Zhu, Xing-Zu

2006-08-01

336

Effects of SEPYLRFamide on acetylcholine-induced currents of Helix aspersa neurones: role of ryanodine receptors.  

PubMed

The possible participation of ryanodine receptors in the modulatory effects of the endogenous Helix heptapeptide, SEPYLRFamide, on the acetylcholine-induced currents (ACh-currents) of Helix aspersa neurones was studied using the two-electrode voltage clamp technique. SEPYLRFamide (bath application) caused a reduction of the ACh-currents of D1, D2, F1, F2, F76 and F77 neurones. Ryanodine (10 microM; bath application), which modifies ryanodine-controlled Ca(2+) channels, potentiated the inhibitory effect of SEPYLRFamide on the ACh-current. An antagonist of cyclic adenosine diphosphate ribose (cADPR) and ryanodine receptors, ruthenium red (1 mM; intracellular injection), reduced the inhibitory effects of SEPYLRFamide on the ACh-current. Ryanodine (10 microM) did not change the inhibitory effect of SEPYLRFamide on the ACh-current after intracellular injection of ruthenium red. An agonist of ryanodine receptors, caffeine (5 mM; bath application), reduced the ACh-current. Ryanodine (10 microM) did not change the reduction of ACh-currents induced by the first application of caffeine but decreased the reduction of ACh-currents induced by subsequent applications of caffeine. It is proposed that ryanodine receptors are involved in the inhibitory modulatory effects of SEPYLRFamide on somatic cholinergic receptors of Helix aspersa neurones. PMID:12491070

Pivovarov, A S; Walker, R J

337

Opiate-induced constipation related to activation of small intestine opioid ?2-receptors  

PubMed Central

AIM: To investigate the role of opioid ?-receptor subtype in opiate-induced constipation (OIC). METHODS: The effect of loperamide on intestinal transit was investigated in mice. Ileum strips were isolated from 12-wk-old male BALB/c mice for identification of isometric tension. The ileum strips were precontracted with 1 ?mol/L acetylcholine (ACh). Then, decrease in muscle tone (relaxation) was characterized after cumulative administration of 0.1-10 ?mol/L loperamide into the organ bath, for a concentration-dependent study. Specific blockers or antagonists were used for pretreatment to compare the changes in loperamide-induced relaxation. RESULTS: In addition to the delay in intestinal transit, loperamide produced a marked relaxation in isolated ileum precontracted with ACh, in a dose-dependent manner. This relaxation was abolished by cyprodime, a selective opioid ?-receptor antagonist, but not modified by naloxonazine at a dose sufficient to block opioid ?-1 receptors. Also, treatment with opioid ?-1 receptor agonist failed to modify the muscle tone. Moreover, the relaxation by loperamide was attenuated by glibenclamide at a dose sufficient to block ATP-sensitive K+ (KATP) channels, and by protein kinase A (PKA) inhibitor, but was enhanced by an inhibitor of phosphodiesterase for cyclic adenosine monophosphate (cAMP). CONCLUSION: Loperamide induces intestinal relaxation by activation of opioid ?-2 receptors via the cAMP-PKA pathway to open KATP channels, relates to OIC.

Chen, Wency; Chung, Hsien-Hui; Cheng, Juei-Tang

2012-01-01

338

Streptokinase-induced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1.  

PubMed

Streptokinase activates platelets, limiting its effectiveness as a thrombolytic agent. The role of antistreptokinase antibodies and proteases in streptokinase-induced platelet activation was investigated. Streptokinase induced localization of human IgG to the platelet surface, platelet aggregation, and thromboxane A(2) production. These effects were inhibited by a monoclonal antibody to the platelet Fc receptor, IV.3. The platelet response to streptokinase was also blocked by an antibody directed against the cleavage site of the platelet thrombin receptor, protease-activated receptor-1 (PAR-1), but not by hirudin or an active site thrombin inhibitor, Ro46-6240. In plasma depleted of plasminogen, exogenous wild-type plasminogen, but not an inactive mutant protein, S(741)A plasminogen, supported platelet aggregation, suggesting that the protease cleaving PAR-1 was streptokinase-plasminogen. Streptokinase-plasminogen cleaved a synthetic peptide corresponding to PAR-1, resulting in generation of PAR-1 tethered ligand sequence and selectively reduced binding of a cleavage-sensitive PAR-1 antibody in intact cells. A combination of streptokinase, plasminogen, and antistreptokinase antibodies activated human erythroleukemic cells and was inhibited by pretreatment with IV.3 or pretreating the cells with the PAR-1 agonist SFLLRN, suggesting Fc receptor and PAR-1 interactions are necessary for cell activation in this system also. Streptokinase-induced platelet activation is dependent on both antistreptokinase-Fc receptor interactions and cleavage of PAR-1. (Blood. 2000;95:1301-1308) PMID:10666203

McRedmond, J P; Harriott, P; Walker, B; Fitzgerald, D J

2000-02-15

339

Pharmacological evaluation of SN79, a sigma (?) receptor ligand, against methamphetamine-induced neurotoxicity in vivo.  

PubMed

Methamphetamine is a highly addictive psychostimulant drug of abuse, causing hyperthermia and neurotoxicity at high doses. Currently, there is no clinically proven pharmacotherapy to treat these effects of methamphetamine, necessitating identification of potential novel therapeutic targets. Earlier studies showed that methamphetamine binds to sigma (?) receptors in the brain at physiologically relevant concentrations, where it "acts in part as an agonist." SN79 (6-acetyl-3-(4-(4-(4-florophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one) was synthesized as a putative ? receptor antagonist with nanomolar affinity and selectivity for ? receptors over 57 other binding sites. SN79 pretreatment afforded protection against methamphetamine-induced hyperthermia and striatal dopaminergic and serotonergic neurotoxicity in male, Swiss Webster mice (measured as depletions in striatal dopamine and serotonin levels, and reductions in striatal dopamine and serotonin transporter expression levels). In contrast, di-o-tolylguanidine (DTG), a well established ? receptor agonist, increased the lethal effects of methamphetamine, although it did not further exacerbate methamphetamine-induced hyperthermia. Together, the data implicate ? receptors in the direct modulation of some effects of methamphetamine such as lethality, while having a modulatory role which can mitigate other methamphetamine-induced effects such as hyperthermia and neurotoxicity. PMID:22921523

Kaushal, Nidhi; Seminerio, Michael J; Robson, Matthew J; McCurdy, Christopher R; Matsumoto, Rae R

2013-08-01

340

NOP receptor mediates anti-analgesia induced by agonist-antagonist opioids.  

PubMed

Clinical studies have shown that agonist-antagonist opioid analgesics that produce their analgesic effect via action on the kappa-opioid receptor, produce a delayed-onset anti-analgesia in men but not women, an effect blocked by co-administration of a low dose of naloxone. We now report the same time-dependent anti-analgesia and its underlying mechanism in an animal model. Using the Randall-Selitto paw-withdrawal assay in male rats, we found that nalbuphine, pentazocine, and butorphanol each produced analgesia during the first hour followed by anti-analgesia starting at ?90min after administration in males but not females, closely mimicking its clinical effects. As observed in humans, co-administration of nalbuphine with naloxone in a dose ratio of 12.5:1 blocked anti-analgesia but not analgesia. Administration of the highly selective kappa-opioid receptor agonist U69593 produced analgesia without subsequent anti-analgesia, and confirmed by the failure of the selective kappa antagonist nor-binaltorphimine to block nalbuphine-induced anti-analgesia, indicating that anti-analgesia is not mediated by kappa-opioid receptors. We therefore tested the role of other receptors in nalbuphine anti-analgesia. Nociceptin/orphanin FQ (NOP) and sigma-1 and sigma-2 receptors were chosen on the basis of their known anti-analgesic effects and receptor binding studies. The selective NOP receptor antagonists, JTC801, and J-113397, but not the sigma receptor antagonist, BD 1047, antagonized nalbuphine anti-analgesia. Furthermore, the NOP receptor agonist NNC 63-0532 produced anti-analgesia with the same delay in onset observed with the three agonist-antagonists, but without producing preceding analgesia and this anti-analgesia was also blocked by naloxone. These results strongly support the suggestion that clinically used agonist-antagonists act at the NOP receptor to produce anti-analgesia. PMID:24188792

Gear, R W; Bogen, O; Ferrari, L F; Green, P G; Levine, J D

2014-01-17

341

Protein kinase Czeta mediates micro-opioid receptor-induced cross-desensitization of chemokine receptor CCR5.  

PubMed

We have previously shown that the ?-opioid receptor (MOR) is capable of mediating cross-desensitization of several chemokine receptors including CCR5, but the biochemical mechanism of this process has not been fully elucidated. We have carried out a series of functional and biochemical studies and found that the mechanism of MOR-induced cross-desensitization of CCR5 involves the activation of PKC?. Inhibition of PKC? by its pseudosubstrate inhibitor, or its siRNA, or dominant negative mutants suppresses the cross-desensitization of CCR5. Our results further indicate that the activation of PKC? is mediated through a pathway involving phosphoinositol-dependent kinase-1 (PDK1). In addition, activation of MOR elevates the phosphorylation level and kinase activity of PKC?. The phosphorylation of PKC? can be suppressed by a dominant negative mutant of PDK1. We observed that following MOR activation, the interaction between PKC? and PDK1 is immediately increased based on the analysis of fluorescent resonance energy transfer in cells with the expression of PKC?-YFP and PDK1-CFP. In addition, cells expressing PKC? kinase motif mutants (Lys-281, Thr-410, Thr-560) fail to exhibit full MOR-induced desensitization of CCR5 activity. Taken together, we propose that upon DAMGO treatment, MOR activates PKC? through a PDK1-dependent signaling pathway to induce CCR5 phosphorylation and desensitization. Because CCR5 is a highly proinflammatory receptor, and a critical coreceptor for HIV-1, these results may provide a novel approach for the development of specific therapeutic agents to treat patients with certain inflammatory diseases or AIDS. PMID:21454526

Song, Changcheng; Rahim, Rahil T; Davey, Penelope C; Bednar, Filip; Bardi, Giuseppe; Zhang, Lily; Zhang, Ning; Oppenheim, Joost J; Rogers, Thomas J

2011-06-10

342