EFFECTS OF NICOTINE AND COTININE ON THE SYNTHESIS AND RELEASE OF PLASMINOGEN ACTIVATOR FROM BOVINE AORTIC ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
Be-Sheng Kuo ◽  
Maciej Dryjski ◽  
Thorir D Bjornsson
Keyword(s):  
Endothelial Cells ◽  
Cigarette Smoking ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Dependent Manner ◽  
Individual Molecular Species ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Stimulation Of ◽  
Aortic Endothelial

While cigarette smoking has beenimplicated in the development of cardiovascular diseases, it has been reported to increase fibrinolytic activity in vivo. However, no data is available regarding the underlying mechanism of action. The present investigation was carried out to evaluate the effects of nicotine and its major metabolite, cotinine, on the seretion of plasminogen activator (PA) and PA inhibitor (PAI) by cultured bovine aortic endothelial cells. PA activity was determined by the fibrin plate method, and individual molecular species with PA and PAI activities were separated and visualized using SDS-PAGE with zymography and reverse fibrin autography. Both nicotine and cotinine increased PA secretion in a time- and dose-dependent manner. A maximum stimulation in PA secretion after 24-hour incubation was observed for nicotine at 10-8 M and for cotinine at 10-7> M, which corresponded to 2.5- and 2.7-foldincreases over control, respectively. These concentrations are in the range observed after cigarette smoking. The pharmacological stimulation of PA activity required both RNA and protein synthesis, as evidencedby its inhibition by cycloheximide and actinomycin D. Both control cells and cells treated with nicotine and cotinine produced multiple forms of PA and a single form of PAI. The PAI was mainly of the latent form as no quenching effect was observed on standard tissue plasminogen activator (tPA) and urokinase (UK) after they were mixed with the conditioned culture medium. The PAs werefound to consist of both tPA and UK,and the corresponding complexes with PAI, however, the UK bands were wider than the tPA bands. Both species were enhanced by nicotine and cotinine. Although activities of all species of PA were enhanced by nicotine and cotinine, these compounds had no apparent quantitative or qualitative effects on the release ofPAI. These results thus suggest that the mechanism underlying the enhanced fibrinolytic activity after cigarette smoking may be due to nicotine- and cotinine-induced stimulation of PA synthesis and subsequent release.

Influence of Nicotine and Cotinine on the Expression of Plasminogen Activator Activity in Bovine Aortic Endothelial Cells

1989 ◽  
Vol 61 (01) ◽  
pp. 070-076 ◽  
Author(s):  
Be-Sheng Kuo ◽  
Maciej Dryjski ◽  
Thorir D Bjornsson
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Dependent Manner ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Fibrin Plate ◽  
Sds Page ◽  
Aortic Endothelial

SummaryThe effects of nicotine and its major metabolite, cotinine, were evaluated on the secretion of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) in cultured bovine aortic endothelial cells. Both compounds increased PA secretion, determined by 125I-fibrin plate assay, in a time- and dose-dependent manner. Maximum effects after 24 hr incubation were observed for nicotine at 10-8 M and for cotinine at 10-7 M, which corresponded to about 2.6-fold increases over control for both compounds. The pharmacological PA stimulation required both RNA and protein syntheses, as evidenced by inhibition by acfinomycin D and cycloheximide. Both control and treated cells produced multiple forms of PA, as evaluated by SDS-PAGE zymography, and a single form of PAI, as evidenced by reverse fibrin autography. Although activities of all species of PA were enhanced by nicotine and cotinine, these compounds had no significant effects on the release of PAI. These results thus suggest that nicotine and cotinine may have fibrinolytic activity in vivo.

ROLE OF POLYAMINES IN THE REGULATION OF SYNTHESIS AND SECRETION OF PLASMINOGEN ACTIVATOR FROM BOVINE AORTIC ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
Be-Sheng Kuo ◽  
Gil Korner ◽  
Thorir D Bjornsson
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Polyamine Metabolism ◽  
Individual Species ◽  
Dependent Manner ◽  
Plate Method ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Fibrin Plate ◽  
Aortic Endothelial

The effects of three polyamines, putrescine (PUT), spermidine (SPD) and spermine (SPM), were investigated on the synthesis and secretion of plasminogen activator (PA) and antiactivator (PAI) activities in confluent bovine aortic endothelial cells. PA activity was determined bythe fibrin plate method, and individual species with PA and PAI activities were separated and visualized using SDS-PAGE with zymography and reverse fibrin autography. Both control cells and cells treated with polyamines secreted PA activity in a time-dependent fashion. After 24-hour incubation, the three polyamines enhanced PA secretion in a dose-dependent manner (10-6 to 2.5 × 10-3 M), with a potency order of SPM > SPD> PUT, as estimated by the fibrin plate method. The maximum PA releases after PUT (0.5 mM), SPD (2.5 mM) and SPM (0.5 mM) were 1.7, 4.5 and 5.4 times control levels, respectively. Concentrations lower than 1 μM had essentially no effects. The enhancement of PA activity by polyamines was blocked by actino-mycin D and cycloheximide, while it was not affected by inhibitors of polyamine biosynthesis except that the enhancement by PUT (0.5 mM) was reduced by methylglyoxal bis(guanylhydrazone). These data suggest that polyamines directly stimulate PA synthesis and secretion through promotion of gene transcription and translation, and that this effect appears to be related to their position in the biosynthetic pathway of polyamines. The kinetic patterns of activities of ornithine decarboxylase and S-adenosy-methionine decarboxylase in confluent endothelial cells stimulated withfresh culture medium suggest that there is rapid turnover of intracellular polyamines. Multiple forms of secreted PA were observed and both tissue- and urokinase-type PA were enhanced by polyamines, while the PAI activity, as evaluted by reverse fibrin autograpy, was apparently reduced. These experimental results suggest that polyamines may play an important role in the regulation of the synthesis and secretion of plasminogen activators, and that this biological function could be modified by disease states and by agents that are associated with altered polyamine metabolism.

The Dexamethasone-Induced Inhibitor of Plasminogen Activator in Hepatoma Cells Is Antigenically-Related to an Inhibitor Produced by Bovine Aortic Endothelial Cells

1986 ◽  
Vol 55 (01) ◽  
pp. 008-011 ◽  
Author(s):  
David J Loskutoff ◽  
Karen Roegner ◽  
Larry A Erickson ◽  
Raymond R Schleef ◽  
Anna Huttenlocher ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Specific Inhibitor ◽  
Hepatoma Cells ◽  
Biochemical Properties ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Immunological Relationship ◽  
Relationship Of ◽  
Aortic Endothelial

SummaryGlucocorticoids decrease plasminogen activator (PA) activity in HTC rat hepatoma cells by inducing a specific inhibitor of PA activity (PAI). This inhibitor is similar in several biochemical properties to the PAI purified from bovine aortic endothelial cells (BAEs). We have used reverse fibrin autography and antiserum against BAE PAI to establish more fully the biochemical and immunological relationship of these inhibitors. Both inhibitors migrated with an apparent Mr of approximately 50,000, and the activity of both PAIs was stimulated by treatment with SDS suggesting that each of these molecules exists in both an active and a latent form. Antiserum to the BAE PAI immunoprecipi-tated all of the HTC PAI demonstrable by reverse fibrin autography. Finally, using this antiserum in a functional immunoassay, we have demonstrated that dexamethasone increases both active and latent PAI made by HTC cells. These results, indicate that HTC PAI and BAE PAI are antigenically as well as biochemically related molecules.

scholarly journals Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS

1999 ◽  
Vol 276 (4) ◽  
pp. C838-C847 ◽  
Author(s):  
Li-Hong Yeh ◽  
Young J. Park ◽  
Riple J. Hansalia ◽  
Imraan S. Ahmed ◽  
Shailesh S. Deshpande ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Detection ◽  
Pyrrolidine Dithiocarbamate ◽  
Dependent Manner ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Aortic Endothelial

The shear-induced intracellular signal transduction pathway in vascular endothelial cells involves tyrosine phosphorylation and activation of mitogen-activated protein (MAP) kinase, which may be responsible for the sustained release of nitric oxide. MAP kinase is known to be activated by reactive oxygen species (ROS), such as H2O2, in several cell types. ROS production in ligand-stimulated nonphagocytic cells appears to require the participation of a Ras-related small GTP-binding protein, Rac1. We hypothesized that Rac1 might serve as a mediator for the effect of shear stress on MAP kinase activation. Exposure of bovine aortic endothelial cells to laminar shear stress of 20 dyn/cm2for 5–30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited in a dose-dependent manner the shear-stimulated increase in total cytosolic and, specifically, MAP kinase tyrosine phosphorylation. Hence, the onset of shear stress caused an enhanced generation of intracellular ROS, as evidenced by an oxidized protein detection kit, which were required for the shear-induced total cellular and MAP kinase tyrosine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressing a dominant negative Rac1 gene product (N17rac1). We concluded that the GTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAP kinase via regulation of the flow-dependent redox changes in endothelial cells in physiological and pathological circumstances.

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