摘要:

AbstractWe investigated the effect of interleukin 6 (IL‐6) on the migration of rabbit corneal epithelial in vitro and on the attachment of dissociated corneal epithelial cells to a fibronectin matrix. When corneal blocks were cultured with IL‐6 for 24 hours, the length of the path of epithelial migration over exposed corneal stroma increased significantly (p<0.005 at the concentration of 10 ng/ml) in proportion to the concentrations of IL‐6 (0.1–10.0 ng/ml). The addition of antiserum against fibronectin or of GRGDSP abolished the stimulatory effect of IL‐6 on epithelial migration. When corneal epithelial cells were cultured with various concentrations of IL‐6, suspended, and plated on wells coated with fibronectin (10 μg/ml), the number of cells attached to the wells increased in a dose‐dependent manner. The presence of antibody against fibronectin or of GRGDSP during the attachment assay decreased the number of cells attached to the fibronectin matrix, regardless of the fact that the cells had been cultured with IL‐6 or not. IL‐6 stimulated the attachment of corneal epithelial cells to collagen type IV and to laminin matrices. However, the presence of GRGDSP did not affect the cell attachment to collagen type IV and to laminin. These findings strongly indicate that IL‐6 stimulates epithelial migration in the cornea by a fibronectin‐dependent mechanism, presumably the increased expression of fibronectin receptors.

ISSN:0021-9541    

DOI:10.1002/jcp.1041530102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractAmylin is a 37 amino acid peptide produced mainly by β‐cells of the endocrine pancreas. Human amylin has 43% homology with human calcitonin gene‐related peptide (CGRP) and 13% homology with human calcitonin (CT). Amylin and CGRP have been reported to have CT‐like hypocalcemic activity in vivo. To investigate the role of amylin in bone, we examined the mechanisms of action of human amylin, CGRP, and CT in osteoclasts and osteoblasts. Both human amylin and CGRP inhibited 1α, 25‐dihydroxyvitamin D3[1α, 25(OH)2D3]‐induced bone resorption in an organ culture system, and the potencies of the two peptides were similarly ∼60‐fold lower than that of human CT. Using a recently developed procedure for preparing large numbers of osteoclast‐like multinucleated cells (MNCs) formed in co‐cultures of mouse osteoblasts and bone marrow cells in the presence of 1α, 25(OH)2D3, we found that both human amylin and CGRP stimulated cAMP production in osteoclast‐like MNCs, but only at 60‐fold higher concentrations than human CT. Specific binding of [125I]‐human CT to osteoclast‐like MNCs was detected (dissociation constant, 3 × 10−8M; binding sites, 3 × 107per cell). To displace the bound [125I]‐human CT from osteoclast‐like MNCs, about 170‐fold higher concentrations of human amylin and CGRP were required. No specific bindings of [125I]‐CGRP to osteoclast‐like MNCs could be detected. Human CGRP stimulated cAMP production both in established mouse osteoblast‐like cells (KS‐4) and in mouse primary osteoblast‐like cells. Amylin was a weak agonist for cAMP production in KS‐4 cells. The increment in cAMP production induced by CGRP and amylin was abolished by the addition of human CGRP(8–37), a selective antagonist for CGRP receptors. CT did not stimulate cAMP production in KS‐4 cells. Amylin, but not CT, displaced the bound [125I]‐human CGRP from rat brain membranes. These results indicate that amylin binds not only to CT receptors in osteoclast‐like MNCs but also to CGRP receptors in osteoblasts. The relative potencies of these compounds to induce cAMP production was CT>amylin ≒ CGRP in osteoclast‐like MNCs and CG

ISSN:0021-9541    

DOI:10.1002/jcp.1041530103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractOur results show that an insulin‐like growth factor binding protein, IGFBP‐3, purified from rat serum, is an inhibitor of chick embryo fibroblast (CEF) growth. It abolished DNA synthesis in CEF stimulated by IGF‐l as well as by human serum. Rat IGFBP‐3 and IDF45 (an inhibitory diffusible factor secreted by mouse cells) had the same activities, confirming that they have an intrinsic capacity to inhibit serum stimulation and may be considered as growth inhibitors. Our data show that inhibition by IGFBP‐3 of serum stimulation was not simply the result of its inhibition of IGF present in the serum: 1) While anti‐I lgG was able to completely inhibit stimulation induced by added IGF‐I, it did not decrease stimulation induced by 1% human serum. Anti‐IGF‐II IgG inhibited the stimulation induced by added IGF‐II, but only 25% decreased the stimulation induced by 0.7% serum. The percent inhibition was not significantly increased when the concentration of serum was decreased to 0.2%, which induced 140% stimulation of DNA synthesis; 2) stimulation by 0.2% serum was much more inhibited by IGFBP‐3 than by IgG anti IGF‐II; 3) after separation of IGF‐I and IGF‐II from serum by chromatography of acidified serum proteins on BioGel P150, the remaining serum proteins (with a molecular mass greater than 45 kDa) which were depleted in IGF‐I and ‐II (verified by RIA determination) still stimulated DNA synthesis, and this stimulation was 80% inhibited by

ISSN:0021-9541    

DOI:10.1002/jcp.1041530104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWe have shown previously that OK cells recover from an acid load in a medium nominally CO2‐free by extruding H via a Na/H exchanger and a passive H‐conductive pathway. In this work, the regulation of cell pH (pHi) was studied after additon or withdrawal of CO2/HCO3(5%CO2, 95 mM HCO3, pH = 8) using the fluoroprobe BCECF. In the presence of Na and amiloride to inhibit Na/H exchange, the recovery of pHiafter CO2entry and CO2exit were found to depend in part on HCO3entry and exit, respectively. Efflux of H per se also contributed to restoring pHiafter CO2addition, whereas H influx may have played a smaller role to normalize pHiafter CO2removal. DIDS, 0.5mM, significantly inhibited both recovery phases of pHi. Removal of Na failed to inhibit the recovery of pHiafter CO2addition and removal. Cl removal also failed to inhibit pHirecovery after CO2removal. Cell depolarization in the presence of Na moderately stimulated the pHirecovery rate after CO2addition whereas it markedly inhibited the normalization of pHiafter CO2removal. Cell depolarization in the absence of sodium had only a slight effect to increase pHirecovery after CO2addition but markedly prevented the pHirecovery after CO2removal. These results indicate that OK cells lack Na or Cl‐dependent HCO3transport systems. The OK cell possesses a novel stilbene‐sensitive electrogenic HCO3transport system that is involved in the regulation of cell pH. © 1992 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041530105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractTransforming growth factor‐beta 1 (TGF‐β1) has been implicated in a variety of responses associated with wound healing and inflammation. Thus, TGF‐β1 enhances production of several extracellular matrix proteins both in vitro and in vivo, is chemotactic for monocytes, and alters the functioning of lymphocytes. We have examined the ability of TGF‐β1 to affect the behavior of human THP‐1 promonocytic leukemia cells, a cell line with the capacity to differentiate into macrophage‐like cells. TGF‐β1 reduces the growth rate of these cells, induces morphologic changes, and promotes adherence to culture surfaces. In addition, the adherent cell population expresses high levels of esterase activity, acquires the ability to ingest latex beads, and releases elevated levels of interleukin 1. TGF‐β1‐treated cells also express elevated levels of the β2family of integrins. Taken together, these results suggest that TGF‐β1 is capable of promoting the maturation of promonocytic cells into macrophages. This outcome has implications at wound sites where TGF‐β1 and a myriad of other factors interact with many cell types to facilitate he

ISSN:0021-9541    

DOI:10.1002/jcp.1041530106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe cellular constituents of the placenta are important participants in the recruitment and trafficking of inflammatory cells within the placenta. In infectioninduced labor, gestational tissues synthesize and release a variety of inflammatory cytokines whose effects include increased prostaglandin biosynthesis and the initiation of uterine contractions. Interleukin‐8 (IL‐8), a potent neutrophil chemoattractant, has been recently described as being elevated in the amniotic fluid of mothers with chorioamnionitis. We investigated the biosynthesis of IL‐8 by human amnion cells and its regulation by other inflammatory cytokines. Cultured amnion cells obtained from normal term placentae were found to produce IL‐8 in response to pathophysiologic concentrations of interleukin 1β (IL‐1β) and tumor necrosis factor alpha (TNF‐α). Treatment of amnion cells stimulated by IL‐1β with cycloheximide resulted in increased IL‐8 production, while incubation of IL‐1β treated amnion cells with actinomycin D resulted in a concentration‐dependent decrease in detectable amounts of IL‐8. Northern blot analysis of cultured amnion cells stimulated with IL‐1β demonstrated a rapid increase in IL‐8 mRNA which peaked at 2–4 hr. These in vitro result suggest inflammation of gestational tissues in vivo may result in locally produced IL‐8 and, in association with other inflammatory mediators, may be important in the pathophysiology of infection‐

ISSN:0021-9541    

DOI:10.1002/jcp.1041530107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe oxygen consumption rate, proliferative activity, and morphology of EMT6/Ro mouse mammary sarcoma cells in monolayer and multicellular spheroid culture have been investigated in a comparative study. During the transition of monolayer cells from the exponential into the plateau growth phase, there is a distinct decrease in the cellular volume that is associated with a corresponding decrease in the proliferative and respiratory activity of the cells. The decline in cell volume is mainly due to a decrease in the content of cytoplasm, whereas the size of the nucleus is only slightly reduced. A concomitant decrease in the number of mitochondria per cell obviously accounts for the reduction in cellular oxygen uptake. Despite a continuous decrease of cell proliferation from the surface to interior regions of EMT6 spheroids reflected by a gradient in tritiated thymidine labeling, volume‐related oxygen consumption is rather uniform in viable regions of these aggregates. The finding can be explained by the results of the morphometric evaluation showing a uniform volume density of mitochondria, i.e., of oxygenconsuming sites within these spheroids. © 1992 Wiley‐Liss,

ISSN:0021-9541    

DOI:10.1002/jcp.1041530108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractIschemia‐reperfusion is observed in various diseases such as myocardium infarct. Different theories have been proposed to explain the reperfusion injury, among them that the free radical generation plays a crucial role. To study the mechanisms of the reperfusion injury, a hypoxia (H)‐reoxygenation (R) model upon human umbilical vein endothelial cells in culture was developed in order to mimic the in vivo situation. Different parameters were quantified and compared under H or H/R, and we found that oxygen readmission led to damage amplification after a short hypoxia period. To estimate the importance of various causes of toxicity, the effects of various protective molecules were compared. Different antioxidant molecules, iron‐chelating agent, xanthine oxidase inhibitors, and energy‐supplying molecules were very efficient protectors. Synergy could also be observed between the antioxidants and the energy‐supplying molecules or the xanthine oxidase inhibitors. The toxic effect of O2·(−) could be lowered by the presence of SOD or glutathione peroxidase in the culture medium, whereas glutathione peroxidase was the most efficient enzyme when injected into the cells. The production of O2·(−) and of H2O2by endothelial cells was directly estimated to be, respectively, of 0.17 and 0.035 μmol/min/mg prot during the R period. O2·(−) production was completely inhibited when allopurinol was added during H and R. In addition, a xanthine oxidase activity of 21.5 10−6U/mg prot could be observed by a direct assay in cells after H but not in control cells, thus confirming the previous conclusions of xanthine oxidase as a potent source of free radicals in these conditions. Thanks to the use of cultured human endothelial cells, a clear picture was obtained of the overall process leading to cell degenerescence during the reoxygenation process. We particularly could stress the importance of the low energetic state of these cells, which is a critical factor acting synergistically with the oxidant molecules to injure the cells. These results also open new possibilities for the development of new therapeutics for ischemia.

ISSN:0021-9541    

DOI:10.1002/jcp.1041530109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractCytoskeletal protein (CSP) interactions are critical to the contractile response in muscle and non‐muscle cells. Current concepts suggest that activation of the contractile apparatus occurs through selective phosphorylation by specific cellular kinase systems. Because the Ca2+‐phospholipid‐dependent protein kinase C (PKC) is involved in the regulation of a number of key endothelial cell responses, the hypothesis that PKC modulates endothelial cell contraction and monolayer permeability was tested. Phorbol myristate acetate (PMA), a direct PKC activator, and α‐thrombin, a receptor‐mediated agonist known to increase endothelial cell permeability, both induced rapid, dose‐dependent activation and translocation of PKC in bovine pulmonary artery endothelial cells (BPAEC), as assessed by γ‐[32P]ATP phosphorylation of H1 histone in cellular fractions. This activation was temporally associated with evidence of agonist‐mediated endothelial cell contraction as demonstrated by characteristic changes in cellular morphology. Agonist‐induced activation of the contractile apparatus was associated with increases in BPAEC monolayer permeability to albumin (∼200% increase with 10−6M PMA, ∼400% increase with 10−8M α‐thrombin). To more closely examine the role of PKC in activation of the contractile apparatus, PKC‐mediated phosphorylation of two specific CSPs, the actin‐ and calmodulin‐binding protein, caldesmon77, and the intermediate filament protein, vimentin, was assessed. In vitro phosphorylation of both caldesmon and vimentin was demonstrated by addition of exogenous, purified BPAEC PKC to unstimulated BPAEC homogenates, to purified bovine platelet caldesmon77, or to purified smooth muscle caldesmon150. Caldesmon77and vimentin phosphorylation were observed in intact [32P]‐labeled BPAEC monolayers stimulated with either PMA or α‐thrombin, as detected by immunoprecipitation. In addition, BPAEC pretreatment with the PKC inhibitor, staurosporine, prevented α‐thrombin‐ and PMA‐induced phosphorylation of both cytoskeletal proteins, attenuated morphologic evidence of contraction, and abolished agonist‐induced barrier dysfunction. These results demonstrate that agonist‐stimulated PKC activity results in cytoskeletal protein phosphorylation in BPAEC monolayers, an event which occurs in concert with agonist‐mediated endothelial cell contraction and

ISSN:0021-9541    

DOI:10.1002/jcp.1041530110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractSupplemental fatty acids can modify the oxidant susceptibility of pulmonary artery endothelial cells (PAEC) in monolayer culture. In addition, in vivo dietary modifications have altered tissue and animal susceptibility to a variety of forms of oxidant stress. These modifications of oxidant injury have been attributed to changes in the numbers of fatty acid double bonds in cell lipids. We tested this hypothesis by incubating porcine PAEC in culture medium supplemented with either 0.1 mM oieic acid (18: 1ω9) or with an equivalent volume of ethanol vehicle alone (ETOH‐0.1%) for 3 h. After supplementation, PAEC were exposed to either oxidant stress, 100 μM hydrogen peroxide (H2O2) in Hanks' balanced salt solution (HBSS), or to control condition, HBSS alone, for 30 min. Supplemented PAEC were exposed to HBSS or H2O2either immediately or 24, 48, or 72 h after supplementation. Supplementation with 18:1 protected PAEC from H2O2‐induced injury at all time points. The fatty acid composition of PAEC phospholipid (PL), triglyceride (TG), and free fatty acid (FFA) subclasses was determined using thin layer and gas chromatography. The PL fraction contained the majority of PAEC fatty acids, and H2O2reduced the polyunsaturates in this fraction regardless of supplementation. Supplementation with 18:1 increased the 18:1 content of PAEC PL, TG, and FFA at all time points, modified other fatty acids to a lesser extent, but failed to alter the overall number of fatty acid double bonds at all time points. These results indicate that modification of double bond number does not fully explain the mechanisms by which changes in lipid composition can modulate oxidant injury. © 1992 Wiley‐Liss, Inc © 1992 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041530111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY