Amiloride selectively inhibits the urokinase‐type plasminogen activator

Vassalli, Jean‐Dominique; Belin, Dominique

doi:10.1016/0014-5793(87)80039-x

Cited by 381 publications

(240 citation statements)

References 27 publications

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“…Amiloride (1 mM) was also present in solution with the plasminogen and VLK-pNA in some experiments. Amiloride is a selective inhibitor of uPA that does not inhibit tissue-type plasminogen activator (46).…”

Section: Proteins and Reagents-[glumentioning

confidence: 99%

Embryonic Fibroblasts That Are Genetically Deficient in Low Density Lipoprotein Receptor-related Protein Demonstrate Increased Activity of the Urokinase Receptor System and Accelerated Migration on Vitronectin

Weaver

Hussaini

Mazar

et al. 1997

Journal of Biological Chemistry

100

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Low density lipoprotein receptor-related protein (LRP) mediates the endocytosis of diverse ligands, including urokinase plasminogen activator (uPA) and its receptor, uPAR, which have been implicated in cellular migration. The purpose of this study was to determine whether LRP affects cellular migration. Murine embryonic fibroblasts (MEF) that are LRP-deficient due to targeted gene disruption and exotoxin selection (MEF-2), heterozygous fibroblasts (PEA-10), and wild-type fibroblasts (MEF-1) were compared. When cultures were denuded of cells in a 1-mm-wide strip, all three cell types migrated into the denuded area. The MEF-2 cells migrated nearly twice as rapidly as the MEF-1 cells or PEA-10 cells. The difference in migration velocity was duplicated in culture wells that were precoated with serum or vitronectin and partially duplicated in wells coated with fibronectin but not in wells coated with type I collagen or Matrigel. uPA was detected in MEF-2 conditioned medium (CM) at a concentration of 0.30 ؎ 0.02 nM, which was 13-fold higher than the level detected in MEF-1 CM or PEA-10 CM, suggesting one potential mechanism for the enhanced migration of MEF-2 cells. uPAR was also increased on MEF-2 cells by 4 -5-fold, as determined by PI-PLC release, and by 2.5-fold, as determined by a uPA/uPAR activity assay. Mannosamine treatment, which down-regulates cell-surface uPAR, decreased MEF-2 migration by 40% without significantly affecting MEF-1 migration. MEF-2 CM, which is uPArich, increased the rate of MEF-1 migration, and MEF-1 CM did not. These studies demonstrate alterations in cellular migration and in the activity of the uPA/uPAR system which accompany complete deficiency of LRP expression in fibroblasts. We propose that uPA and uPAR form an autocrine loop for promoting fibroblast migration and that LRP counteracts the activity of this system.

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Section: Proteins and Reagents-[glumentioning

confidence: 99%

Embryonic Fibroblasts That Are Genetically Deficient in Low Density Lipoprotein Receptor-related Protein Demonstrate Increased Activity of the Urokinase Receptor System and Accelerated Migration on Vitronectin

Weaver

Hussaini

Mazar

et al. 1997

Journal of Biological Chemistry

100

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“…The overlay was covered with a glass cover slide held on standard spacers. For the determination of in situ tPA and uPA activity, the same protocol was performed applying an overlay mixture containing 1 mm amiloride, a specific uPA inhibitor (Vassalli et al, 1987), and polyclonal goat anti-human tPA immunoglobulins G (0.2 mg ml-1) (Biopool, Ume'a, Sweden) respectively. Overlay prepared in the absence of plasminogen was used as a control for non-specific caseinolytic activity.…”

Section: Semiquantitative Histozymographymentioning

confidence: 99%

Alterations in plasminogen activation correlate with epithelial cell dysplasia grading in colorectal adenomas

Protiva

Sordat²,

Chaubert³

et al. 1998

Br J Cancer

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Summary Proteases are important for neoplastic invasion but a specific role for the plasminogen activator system in the progression of colorectal epithelial dysplasia to adenomatous lesions remains unclear. Consecutive tissue cryosections of 51 adenomas, 49 distant mucosa samples and five mucosa samples from control subjects were histopathologically analysed for dysplasia grade and tissue type, urokinase plasminogen activator levels and plasminogen activator inhibitor type 1 (PAI-1) using immunosorbent methods. Plasminogen activation and urokinase-mediated proteolytic activity levels were assessed using in situ zymography. Plasminogen activation and tissue-type activator levels were lower in adenomas than in mucosae (P < 0.001). PAI-1 concentration and urokinase levels were higher in adenomas than in mucosae (P < 0.001 and P < 0.001 respectively). In adenomas, urokinase concentration increased in parallel with PAI-1, but only the urokinase levels correlated with the dysplasia grade (P < 0.01). Thus, the alterations in plasminogen activation correlated with epithelial cell dysplasia grading. In the mucosa to adenoma transition, a marked decrease in tissue-type plasminogen activator occurred. In adenomas, this decrease was accompanied by a concomitant increase in urokinase and PAI-1. The urokinase level only continued to rise in parallel with the dysplasia grade. Resulting protease-antiprotease imbalance in high-grade dysplasia may represent the phenotypic change associated with malignant transformation and invasive behaviour.Keywords: colorectal adenoma; epithelial cell dysplasia; plasminogen activator inhibitor type 1; tissue-type plasminogen activator; urokinase-type plasminogen activator A series of phenotypic changes are associated with the malignant conversion of the colorectal epithelium. The grade of epithelial cell dysplasia is a hallmark of malignant potential in the process known as the adenoma-carcinoma sequence (Muto et al, 1975;O'Brien et al, 1990;Hamilton, 1992). While the morphological changes taking place during the development and progression of colorectal dysplasia can readily be detected by histopathology, the proteolytic alterations underlying these multiple events remain unclear.It has been shown that components of the plasminogen activation system are key participants in the regulation of extracellular matrix turnover during cell migration, tissue modelling and malignant invasion (Konishi et al, 1982;Vassalli et al, 1991;Blasi, 1993). Previous studies have shown that tissue extracts from colorectal carcinomas have increased levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors (PAI) and decreased levels of tissue-type plasminogen activator (tPA) compared with morphologically normal adjacent mucosa (Gelister et al, 1986;Sier et al, 1991a). In adenomas, the levels of activators and inhibitors appear to be between those found in normal mucosa and those found in carcinoma (Gelister et al, 1986;Bruin et al, 1987;Sim et al, 1988;Suzumiya et al, 1988;Sier et al, ...

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“…After a heating at 50Â°C,80 or 130 /j.1of this mixture were applied over prewarmed cryostat tissue sections and evenly spread under 22-x 22-or 24-x 32-mm glass coverslips. The slides were incubated in a humid chamber at 37Â°Cfor 3-6 h. The same procedure was performed applying a mixture without plasminogen, containing 1 mM amiloride (20), a uPA inhibitor, or 0.2 mg/ml of anti-human iPA goat immunoglobulins (American Diagnostica. Inc., Greenwich, CT).…”

Section: Immunohistochemistrymentioning

confidence: 99%

Plasminogen activation in melanocytic neoplasia

et al. 1993

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A large body of experimental evidence suggests that plasminogen acti vators provide tumoral cells with efficient means to degrade extracellular matrix constituents and thereby facilitate their dissemination to distant sites. Melanocytic neoplasia encompass a spectrum of lesions exhibiting diverse clinical behavior that remain difficult to predict with current histopathological evaluations. Little information concerning the contribu tion of plasminogen activation in diagnostic specimens of human melanocytic tumors is presently available. We thus analyzed biopsy specimens of pigmented skin lesions by histolÃ³gica! techniques that identify the cellular sites of synthesis of plasminogen activators and of their inhibitors and that localize the sites of plasminogen activators-catalyzed enzymatic activities. We found that urokinase-type plasminogen activators (uPA) and plasmin ogen activator inhibitor type 1 mRNAs accumulate in atypical nevocytes and in melanoma cells, but not in benign nevocytes. However, uPAcatalyzed proteolytic activity was detected exclusively in melanomas.These observations suggest that up-regulation of the uPA gene is an early feature of melanocyte transformation and that unbalanced enzyme/ inhibitor activity is associated with the malignant phenotype. By support ing a role for uPA in melanoma invasiveness, they provide a novel tool for the evaluation of atypia in nevi.

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Amiloride selectively inhibits the urokinase‐type plasminogen activator

Cited by 381 publications

References 27 publications

Embryonic Fibroblasts That Are Genetically Deficient in Low Density Lipoprotein Receptor-related Protein Demonstrate Increased Activity of the Urokinase Receptor System and Accelerated Migration on Vitronectin

Embryonic Fibroblasts That Are Genetically Deficient in Low Density Lipoprotein Receptor-related Protein Demonstrate Increased Activity of the Urokinase Receptor System and Accelerated Migration on Vitronectin

Alterations in plasminogen activation correlate with epithelial cell dysplasia grading in colorectal adenomas

Plasminogen activation in melanocytic neoplasia

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