摘要:

AbstractOf nine biological factors (ATP, bradykinin, vasopressin, substance P, angiotensin II, norepinephrine, epinephrine, 12‐tetradecanoylphorbol 13‐acetate (TPA), and A23187 calcium ionophore) examined, bradykinin, as well as ATP, TPA, and A23187, significantly increased the phosphorylation of epidermal growth factor (EGF) receptors and reduced the binding of EGF to their high‐affinity site. The reduction in EGF binding by bradykinin, ATP, and TPA was similarly reversed by concomitant incubation with staurosporine, a protein kinase C inhibitor, implying that the phosphorylation of EGF receptors was catalyzed probably by a protein kinase C of the same or similar type in each case. This possibility was confirmed by the fact that the major phosphorylation site of EGF receptors by the stimulation with either bradykinin, ATP or TPA was the same (Thi‐654). Different from the stimulations with ATP and TPA, the effect of bradykinin of decreasing the high‐affinity EGF binding was transient (a minimum binding at 2.5 min); the reduced EGF binding was, however, sustained for up to 30 min in the presence of calyculin A, a phosphoprotein phosphatase inhibitor. Moreover, the homogenate prepared from bradykinin‐stimulated A‐431 cells had stronger dephosphorylation activity for phosphorylated EGF receptors than that from control cells. These results suggest that bradykinin stimulates both the protein kinase C system and a phosphoprotein phosphatase(s) activity in A‐431 cells. Such biphasic effects of bradykinin to phosphorylate and dephosphorylate EGF receptors via protein kinase C and a phosphoprotein phosphatase, respectively, imply a homeostatic control of receptor function in regulating phosphorylation level by the same bioactive factor. © 1993

ISSN:0021-9541    

DOI:10.1002/jcp.1041570102

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

年代:1993

数据来源: WILEY

摘要:

AbstractBacterial lipopolysaccharide (LPS) influences pulmonary vascular endothelial barrier function in vitro. We studied whether LPS regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed toEscherichia coli0111:B4 LPS 10 ng/ml or media for up to 6 h and evaluated for: (1) transendothelial14C‐albumin flux, (2) F‐actin organization with fluorescence microscopy, (3) F‐actin quantitation by spectrofluorometry, and (4) monomeric G‐actin levels by the DNAse 1 inhibition assay. LPS induced increments in14C‐albumin flux (P<0.001) and intercellular gap formation at ≥ 2–6 h. During this same time period the endothelial F‐actin pool was not significantly changed compared to simultaneous media controls. Mean (±SE) G‐actin (μg/mg total protein) was significantly (P<0.002) increased compared to simultaneous media controls at 2, 4, and 6 h but not at 0.5 or 1 h. Prior F‐actin stabilization with phallicidin protected against the LPS‐induced increments in G‐actin (P= 0.040) as well as changes in barrier function (P<0.0001). Prior protein synthesis inhibition unmasked an LPS‐induced decrement in F‐actin (P= 0.0044), blunted the G‐actin increment (P= 0.010), and increased LPS‐induced changes in endothelial barrier function (P<0.0001). Therefore, LPS induces pulmonary vascular endothelial F‐actin depolymerization, intercellular gap formation, and barrier dysfunction. Over the same time period, LPS increased total actin (P<0.0001) and new actin synthesis (P= 0.0063) which may be a compensatory endothelial cell response to LPS‐induced F‐actin d

ISSN:0021-9541    

DOI:10.1002/jcp.1041570103

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

年代:1993

数据来源: WILEY

摘要:

AbstractA precipitating factor in the development of atherosclerotic lesions is the inappropriate migration and proliferation of vascular smooth muscle cells (SMC) within the intima of the vessel wall. Focusing on the role of extracellular matrix proteins in SMC migration, we have demonstrated that thrombospondin (TSP) itself is a potent modulator of SMC motility and acts to potentiate platelet‐derived growth factor (PDGF)‐mediated SMC migration as well. Migration of SMC to TSP was dose dependent. Interestingly, maximal SMC migration to TSP exceeded that to either PDGF or basic fibroblast growth factor (bFGF). The distal COOH terminus of TSP was shown to mediate SMC migration as demonstrated by complete inhibition of the response by monoclonal antibody (mAb) C6.7. Nevertheless, proteolytic fragments of TSP were not as potent as intact TSP in mediating SMC migration. Only by combining the heparin‐binding domain (HBD) with the 140 kD COOH terminal fragment was SMC migration restored to levels seen with intact TSP. Based on antibody inhibition studies, an αv‐containing integrin receptor, but not αvβ1or αvβ3, appeared to be involved in SMC migration to TSP. The coincidental expression of PDGF and TSP at sites of vascular injury and inflammation led us to evaluate the effect of suboptimal levels of TSP on SMC responsiveness to PDGF. SMC migration in response to PDGF was enhanced nearly 60% in the presence of suboptimal concentrations of TSP. This effect was specific for PDGF and dependent on the concentration of TSP with maximal potentiation obtained between 50–100 nM TSP, concentrations tenfold lower than those necessary for SMC migration to TSP itself. mAb C6.7 completely inhibited enhancement but, as with SMC migration to TSP alone, TSP proteolytic fragments did not possess the effectiveness of the intact molecule. Additional experiments assessing SMC migration to PDGF demonstrated that PDGF stimulated SMC motility indirectly by inducing TSP synthesis. These studies suggested that TSP functions as an autocrine motility factor to modulate SMC migration, which in conjunction with PDGF could serve to aggravate and accelerate development of atherosclerotic lesions at sites of vascular injury or inflammation. © 1993

ISSN:0021-9541    

DOI:10.1002/jcp.1041570104

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

年代:1993

数据来源: WILEY

摘要:

AbstractFormer studies have linked hepatocyte growth with liver fatty acid binding protein (L‐FABP) of rat liver cytosol. In search for the roles of L‐FABP in hepatocytes, we previously stably transfected rat L‐FABP sense and antisense cDNAs into rat hepatoma HTC cells that do not contain L‐FABP RNA or protein, thereby providing a zero‐background, homologous cell model of L‐FABP‐expression suitable for controlled studies of its intracellular functions in hepatocyte‐derived cells. The present study demonstrates the abilities of L‐FABP to promote DNA synthesis and cell growth, preserve cell morphology, extend survival, and act cooperatively with unsaturated fatty acids in the transfected hepatoma cells in the absence of serum. Following removal of serum, the three control L‐FABP‐nonexpressing cell lines increased in cell lines increased in cell number for 24 hr and thereafter declined, whereas the three L‐FABP‐expressing cell lines exhibited a 39% higher rate of DNA synthesis per cell at 24 hr and grew in cell number for 48 hr. As a result, at 72 hr there were 2.5‐fold (avg.) as many L‐FABP‐expressing cells than L‐FABP‐nonexpressing cells. In addition, the L‐FABP‐expressing cells retained their original polygonal morphology at 48 hr, when in contrast most of the control nonexpressing cells were spherical in shape with membrane blebs. In an effort to identify the agonists that collaborate with L‐FABP in the growth promotion and preservation of cell morphology, various free fatty acids were examined at 48 hr for their ability to elminate the differences in behavior of the two cell types in the serum‐free medium. The unsaturated fatty acids, oleic acid (18:1 ω9), linoleic acid (18:2ω6), α‐linolenic acid (18: 3ω3), and arachidonic acid (20:4ω6), at 1 μM markedly elevated the level of DNA synthesis in the more depressed control L‐FABP‐nonexpressing cells and moderately raised it in the less depressed L‐FABP‐expressing cells. In accord, the control L‐FABP‐nonexpressing cells needed 10−6–10−5M linoleic acid to achieve the extent of DNA synthesis attained by the expressing cells in the absence of added fatty acid. At 10 μM linoleic acid, their levels of DNA synthesis were equal. In contrast, five saturated fatty acids had no detectable effect on DNA synthesis. In addition, linoleic acid at 1 μM, but not the saturated fatty acid palmitic acid (16:0), prevented the above morphological alterations in the control L‐FABP‐nonexpressing cells observed in the absence of serum, thereby retaining their original polygonal morphology and that of the expressing cells. The findings are consistent with the concept that L‐FABP improves the efficacy of the utilization of unsaturated fatty acid ligands of L‐FABP in the formation, integrity, and fluidity of cell membranes tha

ISSN:0021-9541    

DOI:10.1002/jcp.1041570105

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

年代:1993

数据来源: WILEY

摘要:

AbstractMarkers of endothelium have been studied in a new endothelial cell line derived from human umbilical cord vein cells by microinjection of a recombinant gene that includes a deletion mutant of the human vimentin gene regulatory region controlling the large T and small t antigen coding region of the SV40 virus. In culture, this immortalized venous endothelial cell line (IVEC) demonstrated morphological characteristics of endothelium; uptake of acetylated low density lipoprotein and presence of the Factor VIII‐related antigen. Treatment of IVEC cells with Interleukin‐1β (IL‐1 β) at 10 U.ml−1activates the expression of cell adhesion molecules such as endothelial leucocyte adhesion molecule (ELAM‐1), intercellular adhesion molecule‐1 (ICAM‐1), and vascular cell adhesion molecule‐1 (VCAM‐1), as observed in primary culture. Prostacyclin secretion was induced in the IVEC cells by 100 nM PMA treatment and thrombin at 0.5 U/ml. Angiotensin converting enzyme (ACE) activity detected in IVEC cells was present but lower than ACE activity in primary endothelial cells and was completely blocked by enalaprilat (1 μM), a specific ACE inhibitor. The presence of ACE mRNA was also demonstrated in IVEC cells by RT‐PCR amplification. Our data demonstrate that endothelial cells immortalized by use of this recombinant gene retain the morphological organization and numerous differentiated properties of endothelium.

ISSN:0021-9541    

DOI:10.1002/jcp.1041570106

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

年代:1993

数据来源: WILEY

摘要:

AbstractVascular smooth muscle cells (SMC) synthesize insulin‐like growth factor‐I (IGF‐I), which is a mitogen for this cell type in vitro. Since IGF binding proteins (IGFBP) modulate IGF bioactivity, we determined which IGFBPs were secreted by porcine SMC. Porcine SMC secreted 34,000 and 24,000 Mrforms of IGFBPs which were identified as IGFBP‐2 and IGFBP‐4, respectively, by immunoblotting. Northern blot analysis showed single transcripts of 1.6 kb and 2.4 kb for IGFBP‐2 and IGFBP‐4, respectively. Secretion of IGFBP‐2 was not regulated to a significant degree, with insulin, IGF‐II, IGF‐I, forskolin, and dibutyryl cyclic adenosine monophosphate (cAMP) inducing minimal changes in IGFBP‐2 secretion of less than 30% by radioimmunoassay (RIA). Insulin increased (2.8 ± 0.1‐fold) the abundance of IGFBP‐4 protein in conditioned media (CM) and increased IGFBP‐4 mRNA levels. Growth factors for SMC such as platelet‐derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor beta‐1 (TGFβ‐1) were without effect on either IGFBP‐2 or −4. IGF‐I treatment decreased the amount of IGFBP‐4 present in CM, but a corresponding decrease in IGFBP‐4 mRNA levels was not observed. In order to determine if IGFBP‐4 could modulate IGF‐I bioactivity, IGFBP‐4 was added to pSMCs with and without IGF‐I. IGF‐I alone (20 ng/ml) induced a 1.6 to threefold increase in3H‐thymidine incorporation. Addition of IGFBP‐4 (between 50 and 250 ng/ml) to cultures containing IGF‐I (20 ng/ml) had no effect on DNA synthesis compared to that observed with IGF‐I alone, while 500 ng/ml consistently caused a small decrease (15 ± 5%; mean ± SE). Immunoblotting of the CM obtained at the end of the3H‐thymidine assay showed a loss of intact IGFBP‐4 in the cultures containing IGF‐I. This corresponded with an increase in the abundance of a 16,000 Mrimmunoreactive fragment that did not bind IGF‐I. Coincubation with insulin had no effect on the amount of IGFBP‐4 that was converted to fragment, suggesting that the reaction was dependent upon IGF‐I binding to IGFBP‐4. In contrast, addition of IGFBP‐4 (500 ng/ml) to human fibroblast cultures with IGF‐I (20 ng/ml) almost completely inhibited the stimulatory effect of IGF‐I on DNA synthesis and no increase in fragment was detected in the CM. In summary, SMC secrete IGFBP‐2 and IGFBP‐4, both of which have been shown to regulate IGF‐mediated DNA synthesis. The factors that regulate the abundance of the two forms of IGFBPs differ suggesting that differential regulation of these substa

ISSN:0021-9541    

DOI:10.1002/jcp.1041570107

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe Reh cell system is suitable for evaluating events important for control of proliferation independently of mechanisms involved in differentiation, as Reh cells are unable to differentiate. In the human pre‐B cell line Reh, activation of adenylate cyclase by forskolin induces a five to tenfold rapid, transient downregulation of steady‐state c‐mycRNA within 4 hours. Concurrently, the cells are strongly growth arrested in the G1 phase of the cell cycle. To clarify if the observed growth arrest could be relieved by constitutive expression of c‐myc, an exogenous c‐mycgene under constitutive promoter control was introduced into Reh cells by electroporation. The c‐myc‐expressing construct pDMmycHyg contained human c‐mycexons 2 and 3 driven by the Mo‐MLV LTR and conferred hygromycin resistance. Exogenous c‐mycRNA transcripts and protein were constitutively expressed in the transfected clones at levels roughly twice as high as the level in nontransfected cells. Total c‐mycprotein levels were unchanged upon treatment of transfected clones with forskolin. Yet, the transfected cells were not released from growth arrest. Furthermore, the transfected Reh cells did not differentiate upon forskolin treatment. Constitutive overexpression of c‐mycis therefore not sufficient for relieving forskolin‐mediated effects on growth arrest in Reh cell

ISSN:0021-9541    

DOI:10.1002/jcp.1041570108

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

年代:1993

数据来源: WILEY

摘要:

AbstractHepatic responsiveness to β2‐adrenergic stimulation is dynamically regulated during early development as well as following hepatic injury and disease. In the present study, the molecular mechanisms that underlie the decline in the steady‐state levels of hepatic β2‐adrenergic receptor mRNA that occurs during development in the male rat were investigated. As determined by nuclear run‐on assays, an age‐associated reduction in β2‐adrenergic receptor gene transcription was observed. The transcription rate of the β2‐adrenergic receptor gene in postnatal day 18 liver was approximately 50% lower than that of fetal liver. Stability of β2‐adrenergic receptor gene transcripts was highest (t1/2≈ 6 h) in hepatocytes isolated from fetal rats and was lowest (t1/2≈ 1 h) in hepatocytes isolated from postnatal day 14 rats. In fetal hepatocytes, but not postnatal day 2 hepatocytes, cycloheximide appeared to stabilize β2‐adrenergic receptor gene transcripts in the presence of actinomycin D. These findings establish the molecular basis of reduced steady‐state levels of β2‐adrenergic receptor mRNA in liver during early postnatal development and suggest multilevel regulatory control of hepatic β2‐adrenergic receptor gene

ISSN:0021-9541    

DOI:10.1002/jcp.1041570109

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

年代:1993

数据来源: WILEY

摘要:

AbstractExogenous adenosine triphosphate (ATP) added to brush‐border membrane vesicles was rapidly degraded mainly to inosine according to the high ecto‐nucleotidase activities in these vesicles. In the absence of phosphate, inosine was slowly transformed into hypoxanthine, and xanthine oxidase and dehydrogenase activities were not detected. The presence of ecto‐adenosine deaminase and ecto‐adenosine monophosphate (AMP) nucleotidase was shown. The ecto‐adenosine deaminase was inhibited by deoxycoformycin and was also detected in rat renal brush‐border membrane vesicles. Using orthovanadate, levamisole, and α, β‐methylene adenosine diphosphate as possible inhibitors, alkaline phosphatase was shown to be the main agent responsible for ecto‐AMP nucleotidase activity. In pig renal basolateral membrane vesicles and in whole cell extracts from pig renal cortex, ecto‐AMP nucleotidase was the limiting factor in ATP degradation. Comparing the ATP catabolism in the whole cell cortical extract with the catabolism in the same sample precleared of membranes, it was shown that ectonucleotidase activity is mainly bound to the membranous components. It is also shown that the whole cell extract of pig renal cortex has hypoxanthine phosphoribosyl transferase activity, and it seems probable that the rapid and specific formation of luminal inosine and its transport into the cell in competition with adenosine may start the purine salvage pathway through the synthesis of IMP from hypoxanthine

ISSN:0021-9541    

DOI:10.1002/jcp.1041570110

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

年代:1993

数据来源: WILEY

摘要:

AbstractA deactivating factor (MDF) is released from granuloma‐like lesions of mice (giant and epithelioid macrophages) to the surrounding medium. Test cells incubated in the presence of MDF display dramatic inhibition of superoxide anion (O2−) release when stimulated. This failure to manifest O2release is observed whether PMA, all‐transretinal, or fMet‐Leu‐Phe is the stimulating agent. MDF acts on different cell types from different species; mouse macrophages as well as guinea pig, human, and mouse neutrophils. Such results suggest that it is a universal regulatory cytokine with high affinity for phagocytic lineages. The factor was subjected to various purification methods: ultrafiltration, gel chromatography, and reversed phase HPLC. A crude preparation that resulted from conditioning of medium by old macrophages (MCM) shows two peaks of activity when subjected to gel filtration. These correspond to molecular weights for the active principle of 3 and 11 kD. When the factor was obtained by extraction of the same cells after washing and sonication. Only the former peak was seen. Fractions corresponding to a MW of 3 kD from several preparations were combined and subjected to HPLC. MDF activity then appeared in a single fraction. MDF is thus putatively a modulator of the cidal activity of phagocytic cells that utilize release of reactive oxygen species for cytocidal activity. © 1993 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041570111

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

年代:1993

数据来源: WILEY