scholarly journals Plasminogen binding to rat hepatocytes in primary culture and to thin slices of rat liver

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 729-736 ◽  
Author(s):  
SL Gonias ◽  
LL Braud ◽  
WA Geary ◽  
SR VandenBerg
Keyword(s):  
Primary Culture ◽  
Rat Liver ◽  
Anticoagulant Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rat Hepatocytes ◽  
Aminocaproic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thin Slices ◽  
The Media ◽  
Epsilon Aminocaproic Acid

Human 125I-plasminogen bound readily to rat hepatocytes in primary culture at 4 degrees C and at 37 degrees C. Binding was inhibited by lysine and reversed by lysine, epsilon-aminocaproic acid, or nonradiolabeled plasminogen. The Kd for binding of 125I-plasminogen to hepatocytes was 0.59 +/- 0.16 mumol/L, as determined from the saturation isotherm by nonlinear regression (r2 = 0.99) and the Scatchard transformation by linear regression (r2 = 0.93). The number of sites per cell was 14.1 +/- 1.1 x 10(6). Fibrinogen synthesis and secretion by hepatocytes was insufficient to account for the major fraction of plasminogen binding, as determined by enzyme-linked immunosorbent assay (ELISA). Polyacrylamide gel electrophoresis and trichloroacetic acid precipitation studies demonstrated that plasminogen is neither activated nor degraded when bound to hepatocytes at 37 degrees C. Thin slices of whole rat liver (500 microns), isolated and prepared totally at 4 degrees C, bound 125I-plasminogen. Binding was inhibited by lysine. 125I-albumin binding to liver slices was minimal and not inhibited by lysine. Activation of plasminogen by tissue plasminogen activator (t-PA) was enhanced by hepatocytes in primary culture. When lysine was included in the media, the enhanced rate of activation was no longer observed. After activation with t-PA, much of the plasmin remained associated with hepatocyte surfaces and was partially protected from inhibition by alpha 2-antiplasmin. These studies suggest that hepatocyte plasminogen binding sites may provide important surface anticoagulant activity.

scholarly journals Plasminogen binding to rat hepatocytes in primary culture and to thin slices of rat liver

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 729-736 ◽  
Author(s):  
SL Gonias ◽  
LL Braud ◽  
WA Geary ◽  
SR VandenBerg
Keyword(s):  
Primary Culture ◽  
Rat Liver ◽  
Anticoagulant Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rat Hepatocytes ◽  
Aminocaproic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thin Slices ◽  
The Media ◽  
Epsilon Aminocaproic Acid

Abstract Human 125I-plasminogen bound readily to rat hepatocytes in primary culture at 4 degrees C and at 37 degrees C. Binding was inhibited by lysine and reversed by lysine, epsilon-aminocaproic acid, or nonradiolabeled plasminogen. The Kd for binding of 125I-plasminogen to hepatocytes was 0.59 +/- 0.16 mumol/L, as determined from the saturation isotherm by nonlinear regression (r2 = 0.99) and the Scatchard transformation by linear regression (r2 = 0.93). The number of sites per cell was 14.1 +/- 1.1 x 10(6). Fibrinogen synthesis and secretion by hepatocytes was insufficient to account for the major fraction of plasminogen binding, as determined by enzyme-linked immunosorbent assay (ELISA). Polyacrylamide gel electrophoresis and trichloroacetic acid precipitation studies demonstrated that plasminogen is neither activated nor degraded when bound to hepatocytes at 37 degrees C. Thin slices of whole rat liver (500 microns), isolated and prepared totally at 4 degrees C, bound 125I-plasminogen. Binding was inhibited by lysine. 125I-albumin binding to liver slices was minimal and not inhibited by lysine. Activation of plasminogen by tissue plasminogen activator (t-PA) was enhanced by hepatocytes in primary culture. When lysine was included in the media, the enhanced rate of activation was no longer observed. After activation with t-PA, much of the plasmin remained associated with hepatocyte surfaces and was partially protected from inhibition by alpha 2-antiplasmin. These studies suggest that hepatocyte plasminogen binding sites may provide important surface anticoagulant activity.

Plasminogen Interactions with Immobilized Fibrinogen

1989 ◽  
Vol 62 (04) ◽  
pp. 1078-1082 ◽  
Author(s):  
Burt Adelman ◽  
Patricia Ouynn
Keyword(s):  
Immunosorbent Assay ◽  
Aminocaproic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Regions ◽  
Dose Dependent Fashion ◽  
Epsilon Aminocaproic Acid ◽  
Dose Dependent

SummaryThis report describes the binding of plasminogen to fibrinogen adsorbed onto polystyrene wells. Binding was determined by enzyme linked immunosorbent assay. Both glu- and lys-plasminogen bound to immobilized fibrinogen in a dose-dependent fashion. However, more lys- than glu-plasminogen bound when equal concentrations of either were added to immobilized fibrinogen. Plasminogen binding was inhibited by epsilon aminocaproic acid indicating that binding was mediated via lysine-binding regions of plasminogen. Soluble fibrinogen added in excess of immobilized fibrinogen did not compete for plasminogen binding but fibrinogen fragments produced by plasmin digestion of fibrinogen did. Treatment of immobilized fibrinogen with thrombin caused a small but significant (p <0.01) increase in plasminogen binding. These studies demonstrate that immobilized fibrinogen binds both glu- and lys-plasminogen and that binding is mediated via lysine-binding regions. These interactions may facilitate plasminogen binding to fibrinogen adsorbed on to surfaces and to cells such as platelets which bind fibrinogen.

Regulation of rat Na+/Pi cotransporter-1 gene expression: the roles of glucose and insulin

1996 ◽  
Vol 271 (6) ◽  
pp. E1021-E1028 ◽  
Author(s):  
H. Li ◽  
P. Ren ◽  
M. Onwochei ◽  
R. J. Ruch ◽  
Z. Xie
Keyword(s):  
Gene Expression ◽  
Glucose Metabolism ◽  
Primary Culture ◽  
Rat Liver ◽  
Inorganic Phosphate ◽  
Rat Hepatocytes ◽  
Cdna Clones ◽  
Homeostatic Regulation ◽  
Liver And Kidney ◽  
Streptozotocin Induced Diabetes

Cytosolic inorganic phosphate (P(i)) is important for glucose metabolism. It plays a role in homeostatic regulation of glucose by insulin and glucagon. Recently, we isolated two cDNA clones for rat Na+/P(i) cotransporter-1 (rNaPi-1) and demonstrated that they are expressed primarily in the rat liver and kidney. We now report that the expression of rNaPi-1 in these tissues is regulated by fasting and streptozotocin-induced diabetes. Using rat hepatocytes in primary culture, we also demonstrate that glucose and insulin upregulate rNaPi-1 expression, whereas glucagon and elevated intracellular adenosine 3',5'-cyclic monophosphate levels downregulate its expression. Because 2-deoxyglucose exhibits no effect on rNaPi-1 gene expression, we suggest that some metabolite accumulated during glucose metabolism may be responsible for the effects of glucose and insulin on rNaPi-1 gene expression. Our data also reveal that other known Na+/P(i) cotransporter genes, NaPi-2 and Ram-1 (a receptor for amphotropic murine retrovirus), are not regulated by insulin and glucose. It is therefore proposed that various subtypes of Na+/P(i) cotransporters are differentially regulated and that each subtype may be involved in a specific cellular function, rNaPi-1 may be responsible for Pi uptake by liver and kidney for glucose metabolism, whereas NaPi-2 may play a key role in P(i) reabsorption in the kidney.

scholarly journals Rat liver pyruvate carboxylase. Purification, detection and quantification of apo and holo forms by immuno-blotting and by an enzyme-linked immunosorbent assay

1986 ◽  
Vol 236 (2) ◽  
pp. 527-533 ◽  
Author(s):  
F Ahmad ◽  
P M Ahmad ◽  
A Mendez
Keyword(s):  
Rat Liver ◽  
Immunosorbent Assay ◽  
Pyruvate Carboxylase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rat Liver Mitochondria ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enzyme Preparations ◽  
Detection And Quantification

A simple scheme for the purification of pyruvate carboxylase from rat liver mitochondria is described. It is rapid and provides high-purity pyruvate carboxylase with excellent yield and reproducibility. The final enzyme preparations appear to be homogeneous by the following criteria: elution behaviour on molecular-sizing matrix, SDS/polyacrylamide-gel electrophoresis, Ouchterlony double-diffusion analysis and Western blotting. Detection and quantification of nanogram amounts of pyruvate carboxylase (apo and holo forms) in total tissue homogenates by immuno-blotting and by enzyme-linked immunosorbent assay are described. The data provided suggest that under normal physiological conditions (both in vivo and in vitro) essentially all the pyruvate carboxylase molecules are biotinylated.

scholarly journals A role of asialoglycoproteins for plasma-membrane-induced inhibition of the switching from α1 to β subtypes in adrenergic response during primary culture of rat hepatocytes

1996 ◽  
Vol 316 (3) ◽  
pp. 743-749 ◽  
Author(s):  
Yasuo KAJIYAMA ◽  
Yutaka SANAI ◽  
Michio UI
Keyword(s):  
Primary Culture ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Prior Treatment ◽  
Adult Rats ◽  
Adult Rat ◽  
Liver Membranes ◽  
Young Rat

Adrenergic responses of rat hepatocytes were studied by measuring Ins(1,4,5)P3 (for the response via α1-subtype receptors) and cAMP (for β-subtype response) generation during brief incubation of cells with respective agonists. Hepatocytes from young rats with an age of 1 week displayed a very high β response without a significant α1 response. The β response decreased and the α1 response increased progressively as the age increased; the response was almost exclusively via α1 receptors in hepatocytes of adult rats 9 weeks or more old. The β response developed, again at the expense of the α1 response, in hepatocytes from adult rats during the primary culture at low cell densities [(1–2.5)×104 cells/cm2]. Such ‘α1 to β subtype switching’ of adrenergic responses in vitro was totally inhibited by adding plasma membranes prepared from adult rat liver into the low-cell-density culture, but not inhibited at all by membranes from young rat liver. The inhibitory effect of adult rat liver membranes was lost when the membranes had been exposed to endoglycosidase F or β-galactosidase but was not affected by prior treatment with sialidase. On the contrary, young rat liver membranes became inhibitory to ‘α1 to β subtype switching’ after prior treatment with sialidase. Thus glycoproteins with unsialylated galactosyl termini on the surface of adult rat hepatocytes are likely to function as a determinant of the relative development of α1/β subtypes of adrenergic responses; the β response is predominant in hepatocytes in the juvenile, presumably as a result of sialylation of the galactosyl termini of the functional glycoproteins.

A Comparison of Plasminogen Activators Derived from Rat Plasma, Primary Rat Hepatocytes and Isolated Perfused Rat Liver

1982 ◽  
Vol 47 (02) ◽  
pp. 166-172 ◽  
Author(s):  
Yoav Sharoni ◽  
Maria C Topal ◽  
Patricia R Tuttle ◽  
Henry Berger
Keyword(s):  
Rat Liver ◽  
Isoelectric Point ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Plasminogen Activators ◽  
Rat Plasma ◽  
Isolated Perfused Rat Liver ◽  
Perfused Rat Liver ◽  
Molecular Weights ◽  
Physiological Relevance

SummaryOf the two cell types it was possible to culture from the dissociated rat liver, hepatocytes and Kupffer cells, only the former were fibrinolytically active. Rat hepatocytes during the first 24 hr in culture secreted two plasminogen activators with molecular weights identical to those found in rat plasma, an 80,000-dalton form (PA-80) and a 45,000-dalton form (PA-45). Partially purified preparations of plasminogen activators from both sources were subjected to isoelectric focusing (IEF) to compare characteristics further. There were three distinct peaks of PA-45 in each preparation with isoelectric points of 7.1, 7.2 and 7.4; all electrophoretic forms had the same low affinity to fibrin. PA-80 from both sources displayed similar IEF profiles with forms ranging from pH values of 7 to 8, all with the same high affinity to fibrin. The major form of PA-80 in the plasma preparation had an isoelectric point of 7.9 whereas that in the hepatocyte preparation had an isoelectric point of 7.6. The isolated perfused rat liver was also shown to produce both PA-80 and PA-45 emphasizing the physiological relevance of the findings with hepatocytes. It is concluded that in the rat hepatocytes contribute to the plasma profile with regard to the plasminogen activator content.

Vanadate induction of L-type pyruvate kinase mRNA in adult rat hepatocytes in primary culture

Diabetes ◽  
1991 ◽  
Vol 40 (4) ◽  
pp. 462-464 ◽  
Author(s):  
M. Miralpeix ◽  
J. F. Decaux ◽  
A. Kahn ◽  
R. Bartrons
Keyword(s):  
Primary Culture ◽  
Pyruvate Kinase ◽  
Rat Hepatocytes ◽  
Adult Rat ◽  
Adult Rat Hepatocytes

Determination of Epsilon Aminocaproic Acid Based on Charge Transfer Complexation with p-Nitrophenlol by Spectrophotometry

2020 ◽  
Vol 16 ◽  
Author(s):  
Sheng-Yun Li ◽  
Fang Tian
Keyword(s):  
Charge Transfer ◽  
Aminocaproic Acid ◽  
Concentration Limit ◽  
Official Method ◽  
Good Precision ◽  
Pharmaceutical Dosage Forms ◽  
Relative Standard ◽  
A Charge ◽  
Epsilon Aminocaproic Acid

: A spectrophotometry was investigated for the determination of epsilon aminocaproic acid (EACA) with p-nitrophenol (PNP). The method was based on a charge transfer (CT) complexation of this drug as n-electron donor with π-acceptor PNP. Experiment indicated that the CT complexation was carried out at room temperature for 10 minutes in dimethyl sulfoxide solvent. The spectrum obtained for EACA/PNP system showed the maximum absorption band at wavelength of 425 nm. The stoichiometry of the CT complex was found to be 1:1 ratio by Job’s method between the donor and the acceptor. Different variables affecting the complexation were carefully studied and optimized. At the optimum reaction conditions, Beer’s law was obeyed in a concentration limit of 1～6 µg mL-1. The relative standard deviation was less than 2.9%. The apparent molar absoptivity was determined to be 1.86×104 L mol-1cm-1 at 425 nm. The CT complexation was also confirmed by both FTIR and 1H NMR measurements. The thermodynamic properties and reaction mechanism of the CT complexation have been discussed. The developed method could be applied successfully for the determination of the studied compound in its pharmaceutical dosage forms with a good precision and accuracy compared to official method as revealed by t- and F-tests.

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