Uptake, Internalization and Degradation of the Novel Plasminogen Activator Reteplase (BM 06.022) in the Rat

1995 ◽  
Vol 74 (06) ◽  
pp. 1501-1510 ◽  
Author(s):  
J Kuiper ◽  
H van de Bilt ◽  
U Martin ◽  
Th J C van Berkel
Keyword(s):  
Plasminogen Activator ◽  
Liver Cells ◽  
The Novel ◽  
Uptake Mechanism ◽  
Liver Uptake ◽  
Lysosomal Compartment ◽  
Β Phase ◽  
Α Phase ◽  
Parenchymal Liver

SummaryThe catabolism of the novel plasminogen activator reteplase (BM 06.022) was described. For this purpose BM 06.022 was radiolabelled with l25I or with the accumulating label l25I-tyramine cellobiose (l25I-TC).BM 06.022 was injected at a pharmacological dose of 380 μg/kg b.w. and it was cleared from the plasma in a biphasic manner with a half-life of about 1 min in the α-phase and t1/2of 20-28 min in the β-phase. 28% and 72% of the injected dose was cleared in the α-phase and β-phase, respectively. Initially liver, kidneys, skin, bones, lungs, spleen, and muscles contributed mainly to the plasma clearance. Only liver and the kidneys, however, were responsible for the uptake and subsequent degradation of BM 06.022 and contributed for 75% to the catabolism of BM 06.022. BM 06.022 was degraded in the lysosomal compartment of both organs. Parenchymal liver cells were responsible for 70% of the liver uptake of BM 06.022. BM 06.022 associated rapidly to isolated rat parenchymal liver cells and was subsequently degraded in the lysosomal compartment of these cells. BM 06.022 bound with low-affinity to the parenchymal liver cells (550 nM) and the binding of BM 06.022 could be displaced by t-PA (IC50 5.6 nM), indicating that the low-density lipoprotein receptor-related protein (LRP) could be involved in the binding of BM 06.022. GST-RAP, which is an inhibitor of LRP, could in vivo significantly inhibit the uptake of BM 06.022 in the liver.It is concluded that BM 06.022 is metabolized primarily in the liver and the kidneys. These organs take up and degrade BM 06.022 in the lysosomes. The uptake mechanism of BM 06.022 in the kidneys is unknown, while LRP is responsible for a low-affinity binding and uptake of BM 06.022 in parenchymal liver cells.

scholarly journals Interaction of mutants of tissue-type plasminogen activator with liver cells: effect of domain deletions

1996 ◽  
Vol 313 (3) ◽  
pp. 775-780 ◽  
Author(s):  
Johan KUIPER ◽  
Anita VAN'T HOF ◽  
Marlies OTTER ◽  
Erik A. L. BIESSEN ◽  
Dingeman C. RIJKEN ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Plasma Clearance ◽  
Specific Binding ◽  
Liver Cells ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Parenchymal Liver

The fibrin-specific thrombolyticum tissue-type plasminogen activator (t-PA) has proven to be a potent drug in several clinical trials, but its clinical application is complicated by the rapid clearance of t-PA from the circulation. The rapid plasma clearance of t-PA results from the uptake of t-PA in the liver. t-PA consists of several domains which may be involved in the interaction with the liver. Three domain-deletion mutants, which were produced by the use of a cassette gene system, were studied in vivo and in vitro for their capacity to bind to the various types of rat liver cells. The three mutants lacked, in comparison to control t-PA, the epidermal growth factor (G) domain, the finger (F) domain or the G domain plus the first kringle (K1). The plasma clearance of the three mutants was slower than that of control t-PA. The slower plasma clearance resulted from a decreased liver uptake: 50 and 80% for t-PA mutants and control t-PA respectively. It was found that the K1 domain was of major importance for the uptake of t-PA by liver endothelial cells in vivo and in vitro. The high-affinity binding of t-PA (and t-PA mutants) to parenchymal liver cells depended largely on the presence of the G domain. Other domain(s), like the F, K2 or protease domain, may be responsible for low-affinity, t-PA-specific binding to rat parenchymal liver cells.

The Role of the Low-Density Lipoprotein Receptor-Related Protein (LRP) in the Plasma Clearance and Liver Uptake of Recombinant Single-chain Urokinase-type Plasminogen Activator in Rats

1997 ◽  
Vol 77 (04) ◽  
pp. 710-717 ◽  
Author(s):  
Marieke E van der Kaaden ◽  
Dingeman C Rijken ◽  
J Kar Kruijt ◽  
Theo J C van Berkel ◽  
Johan Kuiper
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Single Chain ◽  
Liver Uptake ◽  
Type Plasminogen Activator ◽  
Parenchymal Liver ◽  
Urokinase Type Plasminogen Activator ◽  
Density Lipoprotein Receptor

SummaryUrokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91 % and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= deltal25- rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-deltal25-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

scholarly journals Carrier-Mediated Delivery of 9-(2-Phosphonylmethoxyethyl)Adenine to Parenchymal Liver Cells: a Novel Therapeutic Approach for Hepatitis B

2000 ◽  
Vol 44 (3) ◽  
pp. 477-483 ◽  
Author(s):  
Remco L. A. de Vrueh ◽  
Erik T. Rump ◽  
Erika van de Bilt ◽  
Richard van Veghel ◽  
Jan Balzarini ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Lithocholic Acid ◽  
Hepatic Uptake ◽  
Liver Cells ◽  
Asialoglycoprotein Receptor ◽  
Parenchymal Liver ◽  
Lipophilic Prodrug ◽  
Acid Labile

ABSTRACT Our aim is to selectively deliver 9-(2-phosphonylmethoxyethyl)adenine (PMEA) to parenchymal liver cells, the primary site of hepatitis B virus (HBV) infection. Selective delivery is necessary because PMEA, which is effective against HBV in vitro, is hardly taken up by the liver in vivo. Lactosylated reconstituted high-density lipoprotein (LacNeoHDL), a lipid particle that is specifically internalized by parenchymal liver cells via the asialoglycoprotein receptor, was used as the carrier. PMEA could be incorporated into the lipid moiety of LacNeoHDL by attaching, via an acid-labile bond, lithocholic acid-3α-oleate to the drug. The uptake of the lipophilic prodrug (PMEA-LO) by the liver was substantially increased after incorporation into LacNeoHDL. Thirty minutes after injection of [3H]PMEA-LO-loaded LacNeoHDL into rats, the liver contained 68.9% ± 7.7% of the dose (free [3H]PMEA, <5%). Concomitantly, the uptake by the kidney was reduced to <2% of the dose (free [3H]PMEA, >45%). The hepatic uptake of PMEA-LO-loaded LacNeoHDL occurred mainly by parenchymal cells (88.5% ± 8.2% of the hepatic uptake). Moreover, asialofetuin inhibited the liver association by >75%, indicating uptake via the asialoglycoprotein receptor. The acid-labile linkage in PMEA-LO, designed to release PMEA during lysosomal processing of the prodrug-loaded carrier, was stable at physiological pH but was hydrolyzed at lysosomal pH (half-life, 60 to 70 min). Finally, subcellular fractionation indicates that the released PMEA is translocated to the cytosol, where it is converted into its active diphosphorylated metabolite. In conclusion, lipophilic modification and incorporation of PMEA into LacNeoHDL improves the biological fate of the drug and may lead to an enhanced therapeutic efficacy against chronic hepatitis B.

Characterization of the Interaction of a Complex of Tissue-type Plasminogen Activator and Plasminogen Activator Inhibitor Type 1 with Rat Liver Cells

1995 ◽  
Vol 74 (05) ◽  
pp. 1298-1304 ◽  
Author(s):  
J Kuiper ◽  
M Otter ◽  
A H Voorschuur ◽  
A J van Zonneveld ◽  
D C Rijken ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Liver Cells ◽  
Tissue Type ◽  
Type Plasminogen Activator ◽  
Parenchymal Liver ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Pai 1

SummaryThe present study was undertaken in order to determine the recognition site for tissue-type plasminogen activator-plasminogen activator inhibitor type 1 [t-PA-PAI-1] complexes in rat liver in vivo and in vitro. After intravenous injection into rats t-PA-PAI-1 complexes were rapidly removed from the plasma and the liver took up 80% of the injected dose. Within the liver parenchymal and endothelial liver cells contributed mainly to the uptake of t-PA-PAI-1, and were responsible for 62% and 24% of the liver uptake, respectively. The interaction of t-PA- PAI-1 with isolated rat parenchymal liver cells was of high affinity (Kd 17 nM). A well-known antagonist of the α2-macroglobulin receptor (α2MR/low-density lipoprotein receptor-related protein (LRP), GST-39kDa protein (GST-39kDaP) efficiently inhibited the binding (IC50 0.7 nM) of t-PA-PAI-1 to rat parenchymal liver cells. The interaction of t-PA-PAI-1 with LRP on rat parenchymal liver cells was not Ca2+-dependent and is most probably mediated by a specific determinant on PAI-1, since an anti-PAI-1 monoclonal antibody inhibited the binding of t-PA-PAI-1, where as free t-PA did not. The binding of t-PA-PAI-1 to rat hepatocytes could not be inhibited by a complex of plasmin and α2-antiplasmin nor by various other ligands of LRP like β-VLDL and lactoferrin. Binding of t-PA-PAI-1 to rat parenchymal liver cells was followed by internalization and subsequent degradation in the lysosomal compartment.It is concluded that parenchymal and endothelial liver cells mediate the removal of t-PA-PAI-1 complexes from the circulation. LRP on rat parenchymal liver cells is responsible for the uptake and degradation of t-PA-PAI-1 and may therefore be important for the regulation of the t-PA levels in the circulation.

scholarly journals Uptake of mannose-terminated glycoproteins in isolated rat liver cells. Evidence for receptor-mediated endocytosis in hepatocytes

1984 ◽  
Vol 223 (1) ◽  
pp. 151-160 ◽  
Author(s):  
H Tolleshaug ◽  
T Berg ◽  
R Blomhoff
Keyword(s):  
Binding Capacity ◽  
Liver Cells ◽  
Receptor Mediated Endocytosis ◽  
Parenchymal Liver ◽  
Yeast Invertase ◽  
Rat Liver Cells ◽  
Specific Uptake ◽  
Parenchymal Cells ◽  
Ribonuclease B

Even though most of the hepatic binding capacity for mannose-terminated glycoproteins has previously been shown to reside in the hepatocytes (not in the non-parenchymal cells), detailed evidence for the specific uptake of mannose-terminated ligands has been lacking. In the present studies, yeast invertase, a large glycoprotein (Mr 270 000) containing about 50% mannose, was shown to be taken up into hepatocytes by receptor-mediated endocytosis. The uptake was saturable and could be specifically inhibited by mannosides or by a Ca2+ chelator. The asialo-glycoprotein receptor was not involved. The low-Mr (13 000) ligand ribonuclease B, which contains a single high-mannose glycan, was not taken up by hepatocytes; however, it was taken up as fast as invertase by non-parenchymal liver cells. After injection of 131I-invertase into a rat in vivo, about one-half of the labelled protein was recovered in the hepatocytes. On a per-cell basis, each endothelial cell contained 3-4 times as much radioactivity as did the hepatocytes. On fractionation of hepatocytes in sucrose gradients, invertase showed a different intracellular distribution from that of asialo-fetuin, in that invertase moved much faster into that region of the gradient where the lysosomes were recovered. This indicates that invertase and asialo-fetuin are not transported intracellularly by identical mechanisms.

Presence of HIV-1 in human parenchymal and non-parenchymal liver cells in vivo

1993 ◽  
Vol 19 (2) ◽  
pp. 252-258 ◽  
Author(s):  
Chantal Housset ◽  
Eugénia Lamas ◽  
Valérie Courgnaud ◽  
Olivier Boucher ◽  
Pierre-Marie Girard ◽  
...  
Keyword(s):  
Liver Cells ◽  
Parenchymal Liver ◽  
Hiv 1

scholarly journals Recognition of lactoferrin and aminopeptidase M-modified lactoferrin by the liver: involvement of proteoglycans and the remnant receptor

1996 ◽  
Vol 313 (1) ◽  
pp. 289-295 ◽  
Author(s):  
Gijsbertus J. ZIERE ◽  
J. Kar KRUIJT ◽  
Martin K. BIJSTERBOSCH ◽  
Theo J. C. van BERKEL
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Liver Cells ◽  
Recognition Site ◽  
Low Density ◽  
Liver Uptake ◽  
Aminopeptidase M ◽  
Parenchymal Liver ◽  
Parenchymal Cells ◽  
Initial Binding

1. Lactoferrin and aminopeptidase M-modified lactoferrin (APM-lactoferrin; which lacks its 14 N-terminal amino acids) inhibit the liver uptake of lipoprotein remnants. In the present study, the role of proteoglycans in the initial interaction of β-migrating very-low-density lipoprotein (β-VLDL), native and APM-lactoferrin with isolated rat parenchymal liver cells was investigated. Treatment of the cells with chondroitinase lowered the Kd of lactoferrin binding (from 10 to 2.4 μM), and the number of sites/cell (from 20×106 to 7×106), while heparinase treatment did not affect the binding. The binding characteristics of APM-lactoferrin and β-VLDL were not altered by treatment of the cells with chondroitinase or heparinase. It is concluded that proteoglycans are not involved in the initial binding of APM-lactoferrin and β-VLDL to parenchymal cells, while chondroitin sulphate proteoglycans are mainly responsible for the massive, low-affinity binding of native lactoferrin. 2. The binding of lactoferrin, APM-lactoferrin and β-VLDL to parenchymal liver cells was not influenced by the glutathione S-transferase-receptor-associated protein (GST-RAP) (97.2±4.0%, 95.5±3.7% and 98.5% of the control binding), while the binding of α2-macroglobulin was fully blocked at 10 μg/ml GST-RAP (1.8±0.5% of the control binding). Since GST-RAP blocks the binding of all the known ligands to the low-density lipoprotein (LDL)-receptor-related protein (LRP), it is concluded that LRP is not the initial primary recognition site for lactoferrin, APM-lactoferrin and β-VLDL on parenchymal liver cells. 3. We showed earlier that APM-lactoferrin, as compared with lactoferrin, is a more effective inhibitor of the liver uptake of lipoprotein remnants (49.4±4.0% versus 80.8±4.8% of the control at 500 μg/ml respectively). We found in the present study that β-VLDL is able to inhibit the binding of APM-lactoferrin to parenchymal liver cells significantly (74.9±3.3% of the control; P < 0.002), while the lactoferrin binding was unaffected. It is concluded that a still unidentified specific recognition site (the putative remnant receptor) is responsible for the initial binding of remnants to parenchymal cells and it is suggested that the partial cross-competition between APM-lactoferrin and β-VLDL may be of further help in the elucidation of the molecular nature of this recognition site.

Abstract 30: Apolipoprotein CIII Sialylation is a Critical Determinant of Liver Triglyceride Metabolism

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Hussein N Yassine ◽  
Ambika Ramrakhiani ◽  
Aarushi Parekh ◽  
Ryan Walker ◽  
Michael Goran ◽  
...  
Keyword(s):  
Plasma Concentrations ◽  
Liver Cells ◽  
Liver Fat ◽  
Lipid Uptake ◽  
Liver Uptake ◽  
Critical Determinant ◽  
Triglyceride Metabolism ◽  
Apo E

Apolipoprotein (apo) CIII inhibits lipoprotein lipase (LPL) activity and remnant particle uptake in the liver by the lipolysis stimulated receptor (LSR); and therefore modulates liver and systemic lipid metabolism. Although post-translational modifications of apo CIII lead to asialylated, mono- or di-sialylated variants, the biological relevance of these sialylations is not known. Our objectives were to determine the association of apo C-III sialylation with plasma triglycerides and liver fat content in vivo, and to evaluate the effect of apo CIII sialylation on lipid uptake in liver cells or LPL activity in vitro. In 209 obese non-diabetic adolescent Hispanic participants, apo CIII variants in plasma were measured using mass spectrometric immunoassay. Increased plasma apo C-III sialylation (di- to mono-sialylated apo CIII ratio) was associated with lower triglyceride levels (r=-0.43, p<0.001) and liver fat (by MRI, r=-0.27, p<0.001) independent of total apo CIII concentrations. Higher plasma concentrations of the mono-, but not the di-sialylated variant, were associated with higher triglycerides concentrations (r=0.53, p<0.001). In HepG2 liver cells, immunoprecipitated apo CIII from VLDL of participants in the upper quartile of plasma apo CIII sialylation less effectively inhibited LSR-mediated VLDL uptake compared to apo CIII from those in the lower quartile of apo CIII sialylation (28% difference, p=0.02). This difference in inhibition was abolished by removal of sialic acid with neuraminidase. Apo CIII isolated from VLDL of participants with different level of apo CIII sialylation showed similar capacity to inhibit fatty acid release by LPL of control VLDL. VLDL particles with higher apo CIII sialylation also demonstrated a two-fold increase in ApoE. We conclude that a greater apo CIII sialylation together with particle enrichment in apo E allow for more efficient VLDL liver uptake and less liver fat accumulation. The measurement of plasma apo CIII sialylation may be a useful index of triglyceride metabolism and risk of fatty liver.

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