摘要:

AbstractTransforming growth factor beta (TGF‐β), a potent modulator of cell growth, differentiation, and the expression of extracellular matrix components in a variety of cell types, exists as two distinct homodimers (TGF‐β1 and TGF‐β2), sharing 71% sequence homology. Radioreceptor and previously described radioimmunological assays using rabbit antibodies have not been able to distinguish between these two forms. We have developed antisera in turkeys against native TGF‐β1 and TGF‐β2, each of which specifically blocks both the receptor binding and biological activity of each of these peptides. With these immunological reagents we describe sensitive and specific immunological assays for TGF‐β1 and TGF‐β2 in complex biological fluids. Using these assays we show that both TGF‐β1 and TGF‐β2 are secreted by a variety of cultured cells, but that some cells secrete predominantly either TGF‐β1 or TGF‐β2 while others secrete both peptides in nearly equal amounts. Our results demonstrate that the expression of each of the two forms of

ISSN:0021-9541    

DOI:10.1002/jcp.1041380112

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

年代:1989

数据来源: WILEY

摘要:

AbstractTo examine the importance of topological constraints on DNA during erythroid development, we measured the effects of camptothecin and teniposide, two tumoricidal agents which are also specific inhibitors of type I and type II topoisomerases respectively, on the formation of hematopoietic colonies by cultured human bone marrow cells. When added to bone marrow culture, each inhibitor alone impairs the formation of early BFU‐E‐derived colonies, late CFU‐E‐derived colonies and mixed hematopoietic (CFU‐GEMM‐derived) colonies by up to 100%. Inhibition of colony formation is directly related to the time of inhibitor addition and the inhibitor concentration tested. Although either inhibitor alone reduces colony formation by 90%, when added together at a submaximal concentration, camptothecin and teniposide exert a synergistic suppressive effect. Furthermore, addition of topoisomerase inhibitors to culture impairs hemoglobinization of colony erythroblasts in a time‐dependent fashion. In contrast to the effects of topoisomerase inhibitors, the antiproliferative agent aphidicolin reduces erythroid colony number and size without altering hemoglobinization of colony erythroblasts. Since neither topoisomerase inhibitor alters the morphology of cultured cells, the capacity of cells to exclude trypan blue or the potential to form erythroid colonies through the interval required for the first progenitor cell division, it is unlikely that camptothecin or teniposide are cytotoxic to hematopoietic cells. Human mononuclear cells enriched in bone marrow lymphocytes and nucleated erythroblasts from both human and mouse sources release DNA into the detergent soluble fraction. Release requires functional topoisomerases and is altered by acute exposure to topoisomerase inhibitors. Our results suggest that topoisomerases are critical not only to proliferation but also to differentiation of human marrow erythroid progenitor cells and stem cel

ISSN:0021-9541    

DOI:10.1002/jcp.1041380113

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

年代:1989

数据来源: WILEY

摘要:

AbstractMesangial cells are smooth muscle‐like cells of the renal glomerulus which contract and produce prostaglandins in response to vasopressin and angiotensin. These responses serve to regulate the glomerular capillary filtering surface area. We have used the membrane potential‐sensitive fluorescent dye bis‐oxonol and the intracellular fluorescent calcium‐sensitive probe Indo‐1 to study the changes in membrane potential (Em) and intracellular free calcium concentration ([Ca2+]i) in cultured rat mesangial cells in response to vasoconstrictor hormones. Basal [Ca2+]iwas 227 ± 4 nM, and stimulation by maximal concentrations of either vasopressin or angiotensin resulted in a transient 4–6‐fold rise. Resting membrane potential was 45.8 ± 0.9 mV and vasoconstrictor hormones caused a depolarization of 14–18 mV. The following extracellular ion substitutions indicated that chloride efflux was the predominant ion flux responsible for depolarization: (1) depolarization persisted when sodium in the medium was substituted with N‐methylglucamine; (2) substitution of medium sodium chloride with sodium gluconate, which enhances the gradient for chloride efflux, augmented vasoconstrictor‐stimulated depolarization; (3) suspension of cells in potassium chloride medium resulted in depolarization, following which, stimulation by either vasopressin or angiotensin resulted in hyperpolarization; and (4) this hyperpolarization did not occur when potassium gluconate medium was used to depolarize the cells. The calcium ionophore ionomycin also resulted in membrane depolarization. However, prevention of the rise in [Ca2+]iby prior exposure to ionomycin in calcium‐free medium or by loading mesangial cells with the intracellular calcium buffer BAPTA did not abrogate the depolarization response to vasoconstrictor hormones. This indicates that a rise in intracellular calcium is not necessary for depolarization. In contrast, prior depolarization of the cells using varying concentrations of KCl in the external medium, which dissipated the electrochemical gradient for chloride efflux, resulted in a corresponding prolongation of the transient calcium response to vasopressin and angiotensin. These findings indicate that angiotensin and vasopressin depolarize mesangial cells by activating chloride channels and that this activation can occur by both calcium‐dependent and ‐independent mechanisms. In addition, activation of chloride channels with resulting depolarization may serve to

ISSN:0021-9541    

DOI:10.1002/jcp.1041380114

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

年代:1989

数据来源: WILEY

摘要:

AbstractOVCA 433 human ovarian carcinoma cells secrete both mammalian plasminogen activators (PAs) urokinase (UK) and tissue‐type PA (tPA). Treatment of cells with 4β‐phorbol‐12‐myristate‐13‐acetate (PMA), a stimulator of protein kinase C (PKC), leads to large increases in the secretion rates of both PA types. PA stimulation by PMA is time‐ and concentration‐dependent, with maximal effects occurring between 12 and 24 h at PMA concentrations of 1–10 ng/ml. The PMA effect is mimicked by mezerein, another known PKC stimulator, but not by 4α‐phorbol or 4α‐phorbol‐12,13‐didecanoate, two phorbol compounds that do not stimulate PKC. PA activity is virtually unaffected by 1‐oleoyl‐2‐acetylglycerol (OAG), a synthetic diacylglycerol that stimulates PKC in vitro but has variable effects on whole cells. PMA stimulation of PA activity is blocked by both actinomycin D and cycloheximide, indicating requirements for new RNA and protein synthesis. When analyzed individually, the relative PMA‐induced increases in UK and tPA activities are identical. Increased UK activity is fully accounted for by increased UK antigen secretion, whereas increased tPA secretion accounts for only about one‐half of the increased tPA activity. Similarly, PMA induces large increases in steady‐state UK mRNA levels, while its effects on tPA mRNA levels are only modest. Thus, while increases in secretion rates and mRNA levels can completely account for UK stimulation, other mechanisms augmenting these processes must exist specifically for tPA. Since the relative increases in UK and tPA activities are identical despite the probable existence of multiple mechanisms contributing to tPA regulation, our data suggest the possibility of interrelationships between the two pathways such that equivalent degrees of UK and tPA acti

ISSN:0021-9541    

DOI:10.1002/jcp.1041380115

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

年代:1989

数据来源: WILEY

摘要:

AbstractThe basic fibroblast growth factor (FGF) is a potent mitogen that has vascular endothelium as one of its principle target cells. Recent work has provided both the complete amino acid sequence of basic FGF and the nucleotide sequence of the genes for both human and bovine basic FGF. Although capillary endothelial cells have been shown to produce basic FGF in vitro and to deposit basic FGF in their extracellular matrix in vitro as well, no direct evidence yet exists for the distribution of basic FGF in vivo. Antipeptide antibodies were prepared against a 15‐amino‐acid sequence from the amino terminus of basic FGF in order to avoid cross‐reactivity with acidic FGF, a protein with 55% overall homology to basic FGF. After affinity purification, these antisera were used to localize the basic fibroblast growth factor in the fetal and adult bovine retina. Immunoreactive material was found in capillaries of the inner nuclear layer, a capillary network undergoing development during the third trimester in the fetal bovine eye. Although the resolution of the technique does not permit a unique assignment of cellular localization, the presence of stain immediately adjacent to the lumen of capillaries suggests that capillary endothelial cells may produce the basic fibroblast growth factor in vivo during vascular develo

ISSN:0021-9541    

DOI:10.1002/jcp.1041380116

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

年代:1989

数据来源: WILEY

摘要:

AbstractRat pheochromocytoma PC12 cells respond to the binding of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) by extending neurites in a manner resembling sympathetic neurons. This response requires cell attachment to an appropriate substratum (Fujii et al., J. Neurosci., 2:1157, 1982); attachment factors which function in this capacity include the adhesive proteins fibronectin and laminin. Incubating PC12 cells with a ployclonal antiserum directed against a putative 140‐kDa fibroblast cell surface fibronectin receptor (anti‐gp140) perturbed spreading but not attachment of the cells to fibronectin and laminin substrates. However, in the presence of anti‐gp 140 or its Fab fragments, NGF‐stimulated neurite outgrowth was dramatically reduced. The antibody also caused a retraction of previously extended neurites. SDS‐PAGE analysis of immunoprecipitates of PC12 cells surface labeled with125I identified a prominent 120–140‐kDa band, suggesting that the site of anti‐gp 140 action in PC12 cells is also through a fibro

ISSN:0021-9541    

DOI:10.1002/jcp.1041380117

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

年代:1989

数据来源: WILEY

摘要:

AbstractCyclic AMP arrests T lymphocytes in the G1 phase of the cell cycle, and prolonged exposure results in cytolysis. Both of these effects require cyclic AMP‐dependent protein kinase. We recently observed that some S49 mouse T lymphoma cell lines selected for hydroxyurea resistance were not arrested in G1 by cyclic AMP. Further analysis revealed that these cell lines were cyclic AMP‐dependent protein kinase deficient, and conversely, other cyclic AMP‐dependent protein kinase deficient cell lines not selected for hydroxyurea resistance were two‐ to threefold more hydroxyurea resistant. However, hydroxyurea is a specific inhibitor of ribonucleotide reductase and does not inhibit this kinase. We subsequently showed that cyclic AMP‐dependent protein kinase will phosphorylate the M2 but not the M1 subunit of ribonucleotide reductase in vitro, and this phosphorylation will diminish CDP reductase activity. In vivo phosphorylation of M2 occurred under conditions similar to those that generate cell cycle arrest. We conclude that the M2 subunit of ribonucleotide reductase can be a target of cyclic AMP‐dependent protein kinase. The phosphorylated enzyme has diminished activity, and this may play a role in cyclic AMP‐induced lymphocyte cell

ISSN:0021-9541    

DOI:10.1002/jcp.1041380118

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

年代:1989

数据来源: WILEY

摘要:

AbstractSmooth muscle cell proliferation is regulated through the coordinated action of growth inhibitors and growth factors/mitogens; a specific heparin‐epidermal growth factor (EGF) complementation has been proposed (Reilly et al., 1987, J. Cell. Physiol.,131:149–157). In culture, vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) proliferate more rapidly than VSMC from control Wistar Kyoto rats (WKY). We observed that, compared with WKY‐derived VSMC, cells from SHR were markedly less susceptible to growth inhibition both by heparin and its homopolysaccharide analogue pentosan polysulphate (PPS). SHR‐derived VSMC exhibited a reduced capacity for binding of [3H]heparin to specific extracellular surface receptors, whereas affinities for heparin were comparable between both VSMC isolates. The early (0–2 hr at 37°C) kinetics of internalization did not differ between SHR‐ and WKY‐derived VSMC, but both internalized equivalent proportions (∼ 10%) of initially surface‐bound heparin. VSMC from SHR exhibited a greater capacity, without a changed affinity, for [I125]EGF binding than VSMC from WKY. Pre‐exposure of VSMC to heparin or PPS decreased, in a time‐dependent manner, the EGF binding capacity for both SHR and WKY (by 40–50% after 72 hr). However, in absolute terms, the EGF‐binding capacity of VSMC from SHR exposed to heparinoids was similar to that o

ISSN:0021-9541    

DOI:10.1002/jcp.1041380119

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

年代:1989

数据来源: WILEY

摘要:

AbstractAlthough it is well known that endothelial cells transport serotonin (5‐HT) from extracellular to intracellular locations, it has been generally assumed that smooth muscle cells do not accumulate 5‐HT but, rather, respond to 5‐HT through a receptor activity unrelated to uptake of this amine or via stimulation of endothelial‐derived relaxing factor. In the present study smooth muscle cells (PASMC), isolated and cultured from bovine pulmonary artery, were evaluated for 5‐HT uptake under a variety of conditions. 5‐HT uptake was linear up to 15 min and the rate was seven‐ to eightfold higher than that by bovine pulmonary artery endothelial cells. There was intracellular metabolism of 5‐HT to 5‐hydroxy‐indoleacetic acid (5‐HIAA). The uptake was inhibited by exposure to 4°C, absence of Na+from the medium, and agents such as imipramine, verapamil, ketanserin, and methiothepin. Like that of endothelial cells, 5‐HT uptake by PASMC was stimulated by exposure of cells to anoxia for 24 hr. Unlike endothelial cells that showed no morphological changes, PASMC at early passage showed dendritic formation after 30–60 min exposure to 5‐HT at a concentration as low as 10−8M. Although this configurational change in response to 5‐HT was lost with passage of cells, transport of 5‐HT by these cells was retained. The configurational change was blocked by agents that inhibited 5‐HT uptake, such as imipramine, verapamil, ketanserin, and methiothepin; it was unaffected by inhibitors of protein kinase C, phospholipase C, and calmodulin or absence of Ca2+from the medium. We conclude that PASMC, as well as endothelial cells, accumulate 5‐HT; there appears to be a close relationship between 5‐HT uptake and configurational change of early passaged PASMC in culture. The factor(s) required for the configurational change are absent in endothelia

ISSN:0021-9541    

DOI:10.1002/jcp.1041380120

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

年代:1989

数据来源: WILEY

摘要:

AbstractSuspensions of freshly isolated rat granulosa cells were used to study endocytosis and processing of radioiodinated ovine follicle‐stimulating hormone (l‐oFSH) and to analyze the dynamics of its receptor. Ovine FSH was iodinated to a specific activity of 26 μCi/μg as determined by radioreceptor self‐displacement assays with maximum specific binding to excess membrane receptors of 46%. Radiolabeled oFSH was judged biologically equivalent to the unlabeled hormone since l‐oFSH shows saturation‐binding kinetics and stimulates steroidogenesis in a similar dose‐related manner to unlabeled oFSH. Experiments designed to study the extent and time course of degradation involved continuous exposure of isolated granulosa cells to l‐oFSH. Saturation of membrane receptors was achieved within 1.5 h of incubation, and internalization of FSH occurred in a linear manner for up to 6 h. The rate of internalization was equivalent to 2,780 FSH molecules/cell/h. Degradation of FSH became apparent after 6 h of incubation and increased to 86% of total cellular‐associated radioactivity at 22 h. FSH degradation was inhibited by 100 μM chloroquine or 0.45 mM leupeptin. The measurement of cell surface l‐oFSH binding in the combined presence of 100 μM chloroquine and 0.5 mM cycloheximide was unchanged for up to 22 h of incubation. This and other receptor binding data suggest that there is no reutilization of FSH receptors. Scatchard analyses of 4°C binding assays on intact cells indicated that a two‐site model best fit the data with association constants of K11= 1.44 (±.42) × 1010and K12= 4.35 (±.91) × 108. Receptor binding and activation studies for progesterone production yielded ED50S of 270 pM and 7.7 pM, respectively, and also indicated that 20% receptor occupancy is sufficient to stimulate maximal progesterone production. We conclude that after the initial binding event, FSH is endocytosed very slowly and is subsequently shuttled to the lysosomal compartment for degradation. The retarded rate of endocytosis may relate to novel pa

ISSN:0021-9541    

DOI:10.1002/jcp.1041380121

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

年代:1989

数据来源: WILEY