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1.
Yamashita M Shinohara T Tsuji S Myrvik QN Nishiyama A Henriksen RA Shibata Y 《Journal of immunology (Baltimore, Md. : 1950)》2007,179(10):7072-7078
Over 25 years ago, it was observed that peritoneal macrophages (Mphi) isolated from mice given heat-killed Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG) i.p. did not release PGE(2). However, when peritoneal Mphi from untreated mice are treated with HK-BCG in vitro, cyclooxygenase 2 (COX-2), a rate-limiting enzyme for PGE(2) biosynthesis, is expressed and the release of PGE(2) is increased. The present study of peritoneal Mphi obtained from C57BL/6 mice and treated either in vitro or in vivo with HK-BCG was undertaken to further characterize the cellular responses that result in suppression of PGE(2) release. The results indicate that Mphi treated with HK-BCG in vivo express constitutive COX-1 and inducible COX-2 that are catalytically inactive, are localized subcellularly in the cytoplasm, and are not associated with the nuclear envelope (NE). In contrast, Mphi treated in vitro express catalytically active COX-1 and COX-2 that are localized in the NE and diffusely in the cytoplasm. Thus, for local Mphi activated in vivo by HK-BCG, the results indicate that COX-1 and COX-2 dissociated from the NE are catalytically inactive, which accounts for the lack of PGE(2) production by local Mphi activated in vivo with HK-BCG. Our studies further indicate that the formation of catalytically inactive COX-2 is associated with in vivo phagocytosis of HK-BCG, and is not dependent on extracellular mediators produced by in vivo HK-BCG treatment. This attenuation of PGE(2) production may enhance Mphi-mediated innate and Th1-acquired immune responses against intracellular infections which are suppressed by PGE(2). 相似文献
2.
Stuhr LE Reith A Lepsøe S Myklebust R Wiig H Reed RK 《American journal of physiology. Cell physiology》2003,285(5):C1101-C1108
Previous studies indicated that connective tissue cells in dermis are involved in control of interstitial fluid pressure (Pif). We wanted to develop and characterize an in vitro model representative of loose connective tissue to study dynamic changes in fluid pressure (Pf) over a time course of a few minutes. Pf was measured with micropipettes in human dermal fibroblast cell aggregates of varying size (<100- and >100-µm diameter) and age (days 1-4) kept at different temperatures (15, 25, and 35°C). Pressures were measured at different depths of micropipette penetration and after treatment with prostaglandin E1 isopropyl ester (PGE1), latanoprost (PGF2), and ouabain. Pf was positive (more than +2 mmHg) during control conditions and increased with increasing aggregate size (day 2), age (day 4 vs. day 1), temperature, and depth of micropipette penetration. Pf decreased from 2.9 to 2.0 mmHg during the first 10 min after application of 10 µl of 1 mM PGE1 (P < 0.001). Pf increased from 3.0 to 4.8 mmHg (P < 0.01) after administration of 10 µl of 1.4 µM ouabain and from 3.1 to 4.4 mmHg after addition of 5 µl of 1.42 mM PGF2 (P > 0.05). In conclusion, we have developed and validated a new in vitro method for studying fluid pressure in loose connective tissue elements with the advantage of allowing reliable and rapid screening of substances that have a potential to modify Pf and studying in more detail specific cell types involved in control of Pf. This study also provides evidence that fibroblasts in the connective tissue can actively modulate Pf. micropuncture; prostaglandin E1; prostaglandin F2; ouabain; integrins 相似文献
3.
Regulation of PGE(2) and PGI(2) release from human umbilical vein endothelial cells by actin cytoskeleton 总被引:2,自引:0,他引:2
Sawyer SJ Norvell SM Ponik SM Pavalko FM 《American journal of physiology. Cell physiology》2001,281(3):C1038-C1045
Disruption of microfilaments in human umbilical vein endothelialcells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA)resulted in a 3.3- to 5.7-fold increase in total synthesis ofprostaglandin E2 (PGE2) and a 3.4- to 6.5-foldincrease in prostacyclin (PGI2) compared with controlcells. Disruption of the microtubule network with nocodazole orcolchicine increased synthesis of PGE2 1.7- to 1.9-fold andPGI2 1.9- to 2.0-fold compared with control cells.Interestingly, however, increased release of PGE2 andPGI2 from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold morePGE2 and 3.8-fold more PGI2 released from HUVECcompared with control cells; latA treatment resulted in 17.7-fold more PGE2 and 11.2-fold more PGI2 released comparedwith control cells. Both increased synthesis and release ofprostaglandins in response to all drug treatments were completelyinhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2).Disruption of either microfilaments using cytD or latA or ofmicrotubules using nocodazole or colchicine resulted in a significantincrease in COX-2 protein levels, suggesting that the increasedsynthesis of prostaglandins in response to drug treatments may resultfrom increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest thatthe cytoskeleton plays an important role in maintenance of endothelialbarrier function by regulating prostaglandin synthesis and release from HUVEC. 相似文献
4.
COX-2 expression and cell cycle progression in human fibroblasts 总被引:4,自引:0,他引:4
Cyclooxygenase-2 (COX-2) is continuously expressed in mostcancerous cells where it appears to modulate cellular proliferation andapoptosis. However, little is known about the contribution oftransient COX-2 induction to cell cycle progression or programmed celldeath in primary cells. In this study we determined whether COX-2regulates proliferation or apoptosis in human fibroblasts. COX-2 mRNA, protein, and prostaglandin E2(PGE2) were not detected in quiescent cells but wereexpressed during the G0/G1 phase of the cellcycle induced by serum. Inhibition of COX-2 did not alter G0/G1 to S phase transition or induceapoptosis at concentrations that diminished PGE2.Addition of interleukin-1 to serum enhanced COX-2 expression andPGE2 synthesis over that by serum alone but had no effecton the progression of these cells into S phase. Furthermore,platelet-derived growth factor drove the G0 fibroblasts into the cell cycle without inducing detectable levels of COX-2 orPGE2. Collectively, these data show that transient COX-2expression in primary human fibroblasts does not influence cell cycle progression. 相似文献
5.
Influence of increased mechanical loading by hypergravity on the microtubule cytoskeleton and prostaglandin E2 release in primary osteoblasts 总被引:1,自引:0,他引:1
Searby ND Steele CR Globus RK 《American journal of physiology. Cell physiology》2005,289(1):C148-C158
Cells respond to a wide range of mechanical stimuli such as fluid shear and strain, although the contribution of gravity to cell structure and function is not understood. We hypothesized that bone-forming osteoblasts are sensitive to increased mechanical loading by hypergravity. A centrifuge suitable for cell culture was developed and validated, and then primary cultures of fetal rat calvarial osteoblasts at various stages of differentiation were mechanically loaded using hypergravity. We measured microtubule network morphology as well as release of the paracrine factor prostaglandin E2 (PGE2). In immature osteoblasts, a stimulus of 10x gravity (10 g) for 3 h increased PGE2 2.5-fold and decreased microtubule network height 1.12-fold without affecting cell viability. Hypergravity (3 h) caused dose-dependent (550 g) increases in PGE2 (5.3-fold at 50 g) and decreases (1.26-fold at 50 g) in microtubule network height. PGE2 release depended on duration but not orientation of the hypergravity load. As osteoblasts differentiated, sensitivity to hypergravity declined. We conclude that primary osteoblasts demonstrate dose- and duration-dependent sensitivity to gravitational loading, which appears to be blunted in mature osteoblasts. mechanotransduction; differentiation; bone 相似文献
6.
PGE2 reduces arachidonic acid release in murine podocytes: evidence for an autocrine feedback loop 总被引:5,自引:0,他引:5
Lemieux LI Rahal SS Kennedy CR 《American journal of physiology. Cell physiology》2003,284(2):C302-C309
Increased glomerularprostaglandin E2 (PGE2) production isassociated with the progression of diseases such as membranous nephropathy, nephrotic syndrome, and anti-Thy1 nephritis. Weinvestigated the signaling pathways that regulate the synthesis andactions of PGE2 in glomerular podocytes. To study itsactions, we assessed the ability of PGE2 to regulate theproduction of its own precursor, arachidonic acid (AA), in a mousepodocyte cell line. PGE2 dose-dependently reduced phorbolester (PMA)-mediated AA release. Inhibition of PMA-stimulated AArelease by PGE2 was found to be cAMP/PKA-dependent, becausePGE2 significantly increased levels of this secondmessenger, whereas the inhibitory actions of PGE2 werereversed by PKA inhibition and reproduced by the cAMP-elevating agentsforskolin and IBMX. PGE2 synthesis in this podocyte cellline increased fourfold at 60 min in response to PMA, coinciding withupregulation of cyclooxygenase (COX)-2 but not COX-1 levels. However,PGE2 synthesis was significantly reduced by COX-1-selectiveinhibition, yet to a lesser extent by COX-2-selective inhibition. Ourfindings suggest that PMA-stimulated PGE2 synthesis inmouse podocytes requires both basal COX-1 activity and induced COX-2expression, and that PGE2 reduces PMA-stimulated AA releasein a cAMP/PKA-dependent manner. Such an autocrine regulatory loop mighthave important consequences for podocyte and glomerular function in thecontext of renal diseases involving PGE2 synthesis. 相似文献
7.
S. Nakatsugi N. Sugimoto M. Furukawa 《Prostaglandins, leukotrienes, and essential fatty acids》1996,55(6):451-457
Lipopolysaccharide (LPS) stimulated prostaglandin E2 (PGE2) formation and induction of cyclooxygenase-2 (COX-2) expression without changing the levels of COX-1 protein in rat peritoneal macrophages. Non-steroidal anti-inflammatory drugs (NSAIDs) (nimesulide, indomethacin and ibuprofen) strongly inhibited LPS-stimulated PGE2 production without any effect on COX-2 protein expression, suggesting that NSAIDs are active in inhibiting the ability of COX-2 to convert arachidonic acid (AA) endogenously released in response to LPS stimulation. Exogenous AA can be converted to PGE2 by both COX isoforms even in LPS-stimulated macrophages. NSAIDs inhibited PGE2 production from exogenous AA mediated by both COX-1 and COX-2. However, the two isoforms interacted differentially with different NSAIDs. Furthermore, NSAIDs were distinctly more active in inhibiting PGE2 production from endogenous AA than that from exogenous AA. These data suggest that PGE2 production through COX-2 from exogenous AA may not be subject to the same regulatory processes as that from endogenous AA and the two metabolic processes may be differentially sensitive to different NSAIDs. 相似文献
8.
Distinct K+ conductive pathways are required for Cl- and K+ secretion across distal colonic epithelium 总被引:1,自引:0,他引:1
Secretion of Cl and K+ in the colonic epithelium operates through a cellular mechanism requiring K+ channels in the basolateral and apical membranes. Transepithelial current [short-circuit current (Isc)] and conductance (Gt) were measured for isolated distal colonic mucosa during secretory activation by epinephrine (Epi) or PGE2 and synergistically by PGE2 and carbachol (PGE2 + CCh). TRAM-34 at 0.5 µM, an inhibitor of KCa3.1 (IK, Kcnn4) K+ channels (H. Wulff, M. J. Miller, W. Hänsel, S. Grissmer, M. D. Cahalan, and K. G. Chandy. Proc Natl Acad Sci USA 97: 81518156, 2000), did not alter secretory Isc or Gt in guinea pig or rat colon. The presence of KCa3.1 in the mucosa was confirmed by immunoblot and immunofluorescence detection. At 100 µM, TRAM-34 inhibited Isc and Gt activated by Epi (4%), PGE2 (30%) and PGE2 + CCh (60%). The IC50 of 4.0 µM implicated involvement of K+ channels other than KCa3.1. The secretory responses augmented by the K+ channel opener 1-EBIO were inhibited only at a high concentration of TRAM-34, suggesting further that KCa3.1 was not involved. Sensitivity of the synergistic response (PGE2 + CCh) to a high concentration TRAM-34 supported a requirement for multiple K+ conductive pathways in secretion. Clofilium (100 µM), a quaternary ammonium, inhibited Cl secretory Isc and Gt activated by PGE2 (20%) but not K+ secretion activated by Epi. Thus Cl secretion activated by physiological secretagogues occurred without apparent activity of KCa3.1 channels but was dependent on other types of K+ channels sensitive to high concentrations of TRAM-34 and/or clofilium. epinephrine; prostaglandin E2; cholinergic; Kcnn4; TRAM-34; clofilium 相似文献
9.
10.
Prostaglandin E2 activates outwardly rectifying Cl(-) channels via a cAMP-dependent pathway and reduces cell motility in rat osteoclasts 总被引:1,自引:0,他引:1
Okamoto F Kajiya H Fukushima H Jimi E Okabe K 《American journal of physiology. Cell physiology》2004,287(1):C114-C124
We examined changes in electrical and morphological properties of rat osteoclasts in response to prostaglandin (PG)E2. PGE2 (>10 nM) stimulated an outwardly rectifying Cl current in a concentration-dependent manner and caused a long-lasting depolarization of cell membrane. This PGE2-induced Cl current was reversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and tamoxifen. The anion permeability sequence of this current was I > Br Cl > gluconate. When outwardly rectifying Cl current was induced by hyposmotic extracellular solution, no further stimulatory effect of PGE2 was seen. Forskolin and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) mimicked the effect of PGE2. The PGE2-induced Cl current was inhibited by pretreatment with guanosine 5'-O-2-(thiodiphosphate) (GDPS), Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS), N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide dihydrochloride (H-89), and protein kinase A inhibitors. Even in the absence of nonosteoclastic cells, PGE2 (1 µM) reduced cell surface area and suppressed motility of osteoclasts, and these effects were abolished by Rp-cAMPS or H-89. PGE2 is known to exert its effects through four subtypes of PGE receptors (EP1EP4). EP2 and EP4 agonists (ONO-AE1-259 and ONO-AE1-329, respectively), but not EP1 and EP3 agonists (ONO-DI-004 and ONO-AE-248, respectively), mimicked the electrical and morphological actions of PGE2 on osteoclasts. Our results show that PGE2 stimulates rat osteoclast Cl current by activation of a cAMP-dependent pathway through EP2 and, to a lesser degree, EP4 receptors and reduces osteoclast motility. This effect is likely to reduce bone resorption. prostanoid receptor agonists; electrophysiology; motile activity; bone resorption 相似文献
11.
Age-associated increase in PGE2 synthesis and COX activity in murine macrophages is reversed by vitamin E 总被引:8,自引:0,他引:8
Wu Dayong; Mura Casilda; Beharka Alison A.; Han Sung Nim; Paulson K. Eric; Hwang Daniel; Meydani Simin Nikbin 《American journal of physiology. Cell physiology》1998,275(3):C661
We previouslyshowed that increased macrophage andPGE2 production with age is due toenhanced cyclooxygenase (COX) activity and COX-2 expression. This studydetermined the effect of vitamin E supplementation on macrophagePGE2 synthesis in young and old mice and its underlying mechanism. Mice were fed 30 or 500 parts permillion vitamin E for 30 days. Lipopolysaccharide (LPS)-stimulated macrophages from old mice produced significantly morePGE2 than those from young mice.Vitamin E supplementation reversed the increasedPGE2 production in old mice buthad no effect on macrophage PGE2production in young mice. In both LPS-stimulated and unstimulated macrophages, COX activity was significantly higher in old than in youngmice at all intervals. Vitamin E supplementation completely reversedthe increased COX activity in old mice to levels comparable to those ofyoung mice but had no effect on macrophage COX activity of young miceor on COX-1 and COX-2 protein or COX-2 mRNA expression in young or oldmice. Thus vitamin E reverses the age-associated increase in macrophagePGE2 production and COX activity.Vitamin E exerts its effect posttranslationally, by inhibiting COXactivity. 相似文献
12.
Yan J. Jiang Tian-Rui Xu Biao Lu David Mymin Edwin A. Kroeger Tom Dembinski Xi Yang Grant M. Hatch Patrick C. Choy 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2003,1633(1):51-60
Cyclooxygenase (COX) is the rate-limiting enzyme for the biosynthesis of prostaglandins in monocytes/macrophages. The COX-1 is constitutively expressed in most tissues and may be involved in cellular homeostasis, whereas the COX-2 is an inducible enzyme that may play an important role in inflammation and mitogenesis. When U937 monocytic cells were incubated with retinoic acid (RA) for 48 h, cell differentiation took place with concomitant increases in prostaglandin E2 (PGE2) production and COX activity. In this study, the mechanism of RA (all-trans- or 9-cis-RA)-induced enhancement of PGE2 biosynthesis in U937 cells was examined. Treatment of cells with all-trans- or 9-cis-RA up to 48 h caused an increase in PGE2 production in a time- and dose-dependent manner. Both RA isomers caused the enhancement of PGE2 production and the up-regulation of COX-1 expression at the protein and mRNA levels. The increase in COX-1 mRNA was found to precede the increase in COX-1 protein expression. Interestingly, the COX-2 protein and COX-2 mRNA were not detected in U937 cells, and their levels remained undetectable during the entire course of RA treatment. We conclude that treatment of U937 cells by RA for 48 h caused the initiation of cell differentiation, which was found to be concomitant with a significant increase in PGE2 production mediated via the up-regulation of COX-1 mRNA and protein expression. 相似文献
13.
5-lipoxygenase and cyclooxygenase regulate wound closure in NIH/3T3 fibroblast monolayers 总被引:1,自引:0,他引:1
Green JA Stockton RA Johnson C Jacobson BS 《American journal of physiology. Cell physiology》2004,287(2):C373-C383
Wound healing involves multiple cell signaling pathways, including those regulating cell-extracellular matrix adhesion. Previous work demonstrated that arachidonate oxidation to leukotriene B4 (LTB4) by 5-lipoxygenase (5-LOX) signals fibroblast spreading on fibronectin, whereas cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) formation facilitates subsequent cell migration. We investigated arachidonate metabolite signaling in wound closure of perturbed NIH/3T3 fibroblast monolayers. We found that during initial stages of wound closure (0120 min), all wound margin cells spread into the wound gap perpendicularly to the wound long axis. At regular intervals, between 120 and 300 min, some cells elongated to project across the wound and meet cells from the opposite margin, forming distinct cell bridges spanning the wound that act as foci for later wound-directed cell migration and resulting closure. 5-LOX inhibition by AA861 demonstrated a required LTB4 signal for initial marginal cell spreading and bridge formation, both of which must precede wound-directed cell migration. 5-LOX inhibition effects were reversible by exogenous LTB4. Conversely, COX inhibition by indomethacin reduced directed migration into the wound but enhanced early cell spreading and bridge formation. Exogenous PGE2 reversed this effect and increased cell migration into the wound. The differential effects of arachidonic acid metabolites produced by LOX and COX were further confirmed with NIH/3T3 fibroblast cell lines constitutively over- and underexpressing the 5-LOX and COX-2 enzymes. These data suggest that two competing oxidative enzymes in arachidonate metabolism, LOX and COX, differentially regulate sequential aspects of fibroblast wound closure in vitro. leukotriene B4; prostaglandin E2; spreading; migration; bridges 相似文献
14.
Oligosaccharide profiles of HIV-2 external envelope glycoprotein: dependence on host cells and virus isolates 总被引:2,自引:1,他引:1
Liedtke Steffen; Adamski Michalina; Geyer Rudolf; Pfutzner Andreas; Rubsamen-Waigmann Helga; Geyer Hildegard 《Glycobiology》1994,4(4):477-484
The glycosylation pattern of the external envelope glycoproteinof human immunodeficiency virus type 2 (HIV-2) was studied independence on host cells and virus isolates. Strains HIV-2ALT,HIV-2ROD and HIV-2D194, differing in their biological propertiesand in the amino acid sequences of their env genes, were propagatedin MOLT4, HUT78 and U937 cells, in human peripheral blood lymphocytesand monocytes/macrophages in the presence of [6-3] glucosamine.Radiolabelled viral glycoproteins were isolated from the cell-freesupernatants and digested with trypsin. Glycans were sequentiallyliberated by endo-ß-N-acetylglucosaminidase H andpeptide-N4-(N-acetyl-ß-glucosaminyl) asparagine amidaseF, and fractionated according to charge and size. Comparisonof the oligosaccharide profiles revealed that the envelope glycoproteinsof different virus isolates, propagated in the same host cells,yielded very similar glycan patterns, whereas cultivation ofan isolate in different host cells resulted in markedly divergentoligosaccharide maps. Variations concerned the proportion ofhigh-mannose-, hybrid- and complex-type substituents, as wellas the state of charge and structural parameters of the complex-typespecies. As a characteristic feature, complex-type glycans ofmacrophage-derived viral glycoprotein were almost exclusivelysubstituted by lactosamine repeats. Hence, glycosylation ofthe HIV-2 external envelope glycoprotein seems to be primarilygoverned by host cell-specific factors rather than by the aminoacid sequence of the corresponding polypeptide backbone. envelope glycoprotein glycosylation human immunodeficiency virus type 2 相似文献
15.
K+ channel KVLQT1 located in the basolateral membrane of distal colonic epithelium is not essential for activating Cl- secretion 总被引:1,自引:0,他引:1
Liao T Wang L Halm ST Lu L Fyffe RE Halm DR 《American journal of physiology. Cell physiology》2005,289(3):C564-C575
The cellular mechanism for Cl and K+ secretion in the colonic epithelium requires K+ channels in the basolateral and apical membranes. Colonic mucosa from guinea pig and rat were fixed, sectioned, and then probed with antibodies to the K+ channel proteins KVLQT1 (Kcnq1) and minK-related peptide 2 (MiRP2, Kcne3). Immunofluorescence labeling for Kcnq1 was most prominent in the lateral membrane of crypt cells in rat colon. The guinea pig distal colon had distinct lateral membrane immunoreactivity for Kcnq1 in crypt and surface cells. In addition, Kcne3, an auxiliary subunit for Kcnq1, was detected in the lateral membrane of crypt and surface cells in guinea pig distal colon. Transepithelial short-circuit current (Isc) and transepithelial conductance (Gt) were measured for colonic mucosa during secretory activation by epinephrine (EPI), prostaglandin E2 (PGE2), and carbachol (CCh). HMR1556 (10 µM), an inhibitor of Kcnq1 channels (Gerlach U, Brendel J, Lang HJ, Paulus EF, Weidmann K, Brüggemann A, Busch A, Suessbrich H, Bleich M, and Greger R. J Med Chem 44: 38313837, 2001), partially (50%) inhibited Cl secretory Isc and Gt activated by PGE2 and CCh in rat colon with an IC50 of 55 nM, but in guinea pig distal colon Cl secretory Isc and Gt were unaltered. EPI-activated K+-secretory Isc and Gt also were essentially unaltered by HMR1556 in both rat and guinea pig colon. Although immunofluorescence labeling with a Kcnq1 antibody supported the basolateral membrane presence in colonic epithelium of the guinea pig as well as the rat, the Kcnq1 K+ channel is not an essential component for producing Cl secretion. Other K+ channels present in the basolateral membrane presumably must also contribute directly to the K+ conductance necessary for K+ exit during activation of Cl secretion in the colonic mucosa. HMR1556; K+ secretion; epinephrine; prostaglandin E2; cholinergic 相似文献
16.
Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts 总被引:13,自引:0,他引:13
Chen NX Ryder KD Pavalko FM Turner CH Burr DB Qiu J Duncan RL 《American journal of physiology. Cell physiology》2000,278(5):C989-C997
Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca2+ concentration([Ca2+]i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm2). Before application of shear, cells were treatedwith two Ca2+ channel inhibitors or various blockers ofintracellular Ca2+ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe [Ca2+]i response, neithergadolinium nor nifedipine, an L-type channel Ca2+ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca2+ chelator, or thapsigargin,which empties intracellular Ca2+ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP3)-induced intracellular Ca2+ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP3-mediated intracellular Ca2+release is required for modulating flow-induced responses in MC3T3-E1 cells. 相似文献
17.
Liu HJ Wei Y Ferreri NR Nasjletti A Wang WH Fererri NR 《American journal of physiology. Cell physiology》2000,278(5):C905-C913
Vasopressin and prostaglandinE2 (PGE2) are involved in regulating NaClreabsorption in the thick ascending limb (TAL) of the rat kidney. Inthe present study, we used the patch-clamp technique to study theeffects of vasopressin and PGE2 on the apical 70 pSK+ channel in the rat TAL. Addition of vasopressinincreased the channel activity, defined asNPo, from 1.11 to 1.52 (200 pM) and 1.80 (500 pM),respectively. The effect of vasopressin can be mimicked by eitherforskolin (1-5 µM) or 8-bromo-cAMP/dibutyryl-cAMP (8-Br-cAMP/DBcAMP) (200-500 µM). Moreover, the effects of cAMP and vasopressin were not additive and application of 10 µM H-89 abolished the effect of vasopressin. This suggests that the effect ofvasopressin is mediated by a cAMP-dependent pathway. Applying 10 nMPGE2 alone had no significant effect on the channelactivity. However, PGE2 (10 nM) abolished thestimulatory effect of vasopressin. The PGE2-inducedinhibition of the vasopressin effect was the result of decreasing cAMPproduction because addition of 200 µM 8-Br-cAMP/DBcAMPreversed the PGE2-induced inhibition. In addition toantagonizing the vasopressin effect, high concentrations of PGE2 reduced channel activity in the absence of vasopressinby 33% (500 nM) and 51% (1 µM), respectively. The inhibitory effect of high concentrations of PGE2 was not the result ofdecreasing cAMP production because adding the membrane-permeant cAMPanalog failed to restore the channel activity. In contrast, inhibiting protein kinase C (PKC) with calphostin C (100 nM) abolished the effectof 1 µM PGE2. We conclude that PGE2 inhibitsapical K+ channels by two mechanisms: 1) lowconcentrations of PGE2 attenuate the vasopressin-inducedstimulation mainly by reducing cAMP generation, and 2) highconcentrations of PGE2 inhibit the channel activity by aPKC-dependent pathway. 相似文献
18.
Cytosolic phospholipases A2 (cPLA2) and cyclooxygenases-1 and -2 (COX-1 and -2) play a pivotal role in the metabolism of arachidonic acid (AA) and in eicosanoid production. The coordinate regulation and expression of these enzymes is not well defined. In this study, the effect of phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor (TNF), lipopolysaccharide (LPS) and macrophage-colony stimulating factor (M-CSF) on AA release and prostaglandin E2 (PGE2) production and the expression of cPLA2 and COX-1 and -2 were investigated in U937 human pre-monocytic cells and fully differentiated macrophages. Treatment of U937 cells with PMA or macrophages with LPS increased AA release and PGE2 production. Incubation of U937 cells or macrophages for 8 h with all stimuli elevated cPLA2 expression. In contrast, cPLA2 expression was reduced upon further incubation of U937 cells or macrophages for 24 h with all stimuli indicating a bi-phasic expression pattern of this enzyme. PMA induced COX-1 expression in U937 cells whereas LPS induced COX-2 expression in macrophages. Although TNF and M-CSF induced a significant amount of AA release in both cell models, they failed to induce a comparable production of PGE2 since they were unable to induce the coordinate expression of the downstream key enzymes, COX-1 or COX-2. The results suggest that the enhancement of AA release in both U937 cells and macrophages may be caused by both increased cPLA2 activity and elevated cPLA2 protein expression. In addition, PMA stimulates PGE2 production via up-regulation of COX-1, and likely COX-2, expression in U937 cells whereas LPS stimulates PGE2 production via induction of COX-2 expression in macrophages. 相似文献
19.
《Bioorganic & medicinal chemistry letters》2020,30(4):126884
In this article, a series of 22 triarylpyrazole derivatives were evaluated for in vitro antiinflammatory activity as inhibitors of nitric oxide (NO) and prostaglandin E2 (PGE2) release induced by lipopolysaccharide (LPS) in murine RAW 264.7 macrophages. The synthesized compounds 1a-h, 2a-f and 3a-h were first examined for their cytotoxicity for determination of the non-toxic concentration for antiinflammatory screening, so that the inhibitory effects against NO and PGE2 production were not caused by non-specific cytotoxicity. Compounds 1h and 2f were the most active PGE2 inhibitors with IC50 values of 2.94 μM and 4.21 μM, respectively. Western blotting and cell-free COX-2 screening revealed that their effects were due to inhibition of COX-2 protein expression. Moreover, compound 1h exerted strong inhibitory effect on the expression of COX-2 mRNA in LPS-induced murine RAW 264.7 macrophages. 相似文献
20.
The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3-E1 osteoblasts 总被引:1,自引:0,他引:1
Malone AM Batra NN Shivaram G Kwon RY You L Kim CH Rodriguez J Jair K Jacobs CR 《American journal of physiology. Cell physiology》2007,292(5):C1830-C1836
Fluid flow due to loading in bone is a potent mechanical signal that may play an important role in bone adaptation to its mechanical environment. Previous in vitro studies of osteoblastic cells revealed that the upregulation of cyclooxygenase-2 (COX-2) and c-fos induced by steady fluid flow depends on a change in actin polymerization dynamics and the formation of actin stress fibers. Exposing cells to dynamic oscillatory fluid flow, the temporal flow pattern that results from normal physical activity, is also known to result in increased COX-2 expression and PGE2 release. The purpose of this study was to determine whether dynamic fluid flow results in changes in actin dynamics similar to steady flow and to determine whether alterations in actin dynamics are required for PGE2 release. We found that exposure to oscillatory fluid flow did not result in the development of F-actin stress fibers in MC3T3-E1 osteoblastic cells and that inhibition of actin polymerization with cytochalasin D did not inhibit intracellular calcium mobilization or PGE2 release. In fact, PGE2 release was increased threefold in the polymerization inhibited cells and this PGE2 release was dependent on calcium release from the endoplasmic reticulum. This was in contrast to the PGE2 release that occurs in normal cells, which is independent of calcium flux from endoplasmic reticulum stores. We suggest that this increased PGE2 release involves a different molecular mechanism perhaps involving increased deformation due to the compromised cytoskeleton. mechanotransduction; cell mechanics 相似文献