Asialoglycoprotein Research Articles

C-src Tyrosine Kinase Is Associated with the Asialoglycoprotein Receptor in Human Hepatoma Cells

The asialoglycoprotein (ASGP) receptor is expressed on hepatocytes and liver-derived cell lines and is responsible for the endocytosis of galactose-terminal glycoproteins via the coated pit pathway. Prior data showed that tyrosine kinase activity plays an important role in this endocytic process, though the critical kinase(s) responsible for this effect are unknown. We have detected a 60-kDa protein which coprecipitates with ASGP receptor in detergent-solubilized lysates of HepG2 cells. This protein autophosphorylates and binds radioactive ATP. It comigrates with authentic pp60 c-src and is recognized by a specific anti-src monoclonal antibody. The kinase associated with the ASGP receptor retains the ability to phosphorylate exogenous substrates on tyrosine. In conclusion, the tyrosine kinase c-src associates with the ASGP receptor, a protein of the coated pit pathway of endocytosis.

In vitro gene transfer with a novel galactosylated spermine bolaamphiphile.

We describe the synthesis of a alpha-galacto-omega-spermine bolaamphiphile (GalSper) and report on the gene transfer mediated with lipoplexes it forms either when used alone or in conjunction with DOPE or with DOGS (Transfectam). Lipofection with GalSper was investigated with human HepG2 or murine BNL-CL2 hepatocytes expressing the asialo-glycoprotein (ASGP) receptor, which displays a high affinity for galactosyl residues, or with A549 cells which do not express ASGP. Although lower luciferase expression levels in BNL-CL2 and in HepG2 cells were obtained with GalSper/DOPE N/P 2.5 lipoplexes as compared with control DOGS/DOPE N/P 2.5 particles or with the more positively charged N/P 5 particles (yet through a different mechanism), specific receptor-mediated endocytosis of DNA can be achieved with this targeted cationic GalSper bolaamphiphile presenting a single galactose residue. The present work suggests that GalSper-based DNA formulations appear as promising synthetic vectors for specific gene delivery to ASGP(+) cells.

Hepatocyte Asialoglycoprotein Receptor Assay Using Stable Isotopes and Neutron Activation Analysis

Since the initial studies of receptor-mediated endocytosis on LDL (1)(2) and asialoglycoprotein (ASGP) (3)(4), many internalized ligands and their receptors have been identified and characterized (5). It was soon recognized that these receptors could be exploited for pharmaceutical and medical imaging applications (6). In the case of the ASGP receptor (ASGP-R), delivery of diagnostic or therapeutic agents to hepatocytes was achieved by attachment of the agent to a variety of macromolecules possessing the terminal galactose signal necessary to bind the ASGP-R (6). These moieties include radioactive isotopes (7), drugs (8), and DNA (9). Considering the effort devoted to developing drug delivery systems utilizing the ASGP-R, surprisingly little effort has been devoted to developing simple, easy-to-use assays to measure this receptor activity in vivo. Much of the technology associated with receptor-based diagnostic agents has arisen in fields associated with magnetic resonance and single photon emission computed tomography imaging. One class of magnetic resonance compounds includes iron chelates, which appear to be absorbed by hepatocytes by an unknown biochemical mechanism (10). Saini et al. (11) and others have demonstrated the promise of ferrite particles as a magnetic resonance contrast agent for liver imaging. These commercially available micron-sized particles are taken up by the Kupffer cells of liver and do not measure ASGP-R activity. Sato et al. (12) reported on the ability of the ASGP-R to take up galactose-terminated, protein-coated ferrite particles in vivo. These particles are taken up preferentially by hepatocytes (70%) and largely avoid uptake by the reticular endothelial system. For a crystal-based material to bind the ASGP-R of hepatocytes, it must penetrate pores of the 100-nm fenestrae characteristic of endothelial cells that lie between the hepatocyte and the circulatory compartment (13). Particles smaller than 100 nm are needed to serve as hepatocyte-directed diagnostic agents. Josephson …

Reduction of plasma angiotensin II to normal levels by antisense oligodeoxynucleotides against liver angiotensinogen cannot completely attenuate vascular remodeling in spontaneously hypertensive rats.

The exact role of angiotensinogen (AGT) in vascular remodeling has yet to be determined. In the present study, we examined the effects of reducing plasma AGT by intravenous injections with antisense oligodeoxynucleotides (ODNs) against AGT targeted to the liver on vascular remodeling in spontaneously hypertensive rats (SHRs). The ODNs against rat AGT were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for regulating liver gene expression. Male SHRs (n = 18) and age-matched male Wistar- Kyoto (WKY) rats (n = 4) were used for this study. All animals were fed a standard rat diet throughout the experiment At 10 weeks of age, the SHRs were divided into three groups (n = 6); systolic blood pressure (SBP) was similar in each group. The control group received saline, the sense group was injected with the sense ODN complex and the antisense group was injected with the antisense ODN complex. WKY rats were fed for the same period of time. The ASOR-poly(L)lysine-ODN complex was injected into the tail veins twice a week. At the end of the treatment, a reduction in AGT mRNA levels in the liver and plasma AGT was observed only in the animals injected with antisense ODNs. Antisense ODNs significantly reduced the plasma angiotensin II (Ang II) concentrations to levels similar to those observed in WKY rats. Antisense ODNs significantly reduced the SBP (180.7 +/- 4.4 mmHg) and media cross-sectional areas of the aorta (1.11 +/- 0.02 mm2), which were still larger than those seen in WKY rats (140.3 +/- 2.1 mmHg, 0.84 +/- 0.02 mm2), compared with the SHRs injected with sense ODNs (225.2 +/- 4.4 mmHg, 1.24 +/- 0.02 mm2) and control SHRs (223.7 +/- 4.8 mmHg, 1.25 +/- 0.02 mm2). The aortic angiotensin-converting enzyme (ACE) activity and collagen concentrations, which were significantly higher than those seen in WKY rats, did not significantly change among the SHR groups. The aortic AGT, ACE, angiotensin II type 1 (AT1) receptor and angiotensin II type 2 (AT2) receptor mRNA also did not significantly change among the SHR groups. On the basis of these findings, plasma AGT is thus considered to play a role in the development of hypertrophy of smooth muscle in the aorta of SHRs, it is thought to have only a slight effect, however, on the remodeling of the matrix tissue when the suppression of hypertension is insufficient.

Lactoferrin binding to the rat asialoglycoprotein receptor requires the receptor’s lectin properties

Lactoferrin binds to rat hepatic lectin 1 (RHL1), the major subunit of the asialoglycoprotein (ASGP) receptor, with high affinity, by a galactose-independent mechanism. To better understand the molecular basis of this novel interaction, we compared the binding of lactoferrin and asialo-orosomucoid (ASOR) to isolated rat hepatocytes and to purified ASGP receptors as a function of pH, Ca(2+) and receptor acylation. Binding of (125)I-lactoferrin and (125)I-ASOR to isolated rat hepatocytes at 4 degrees C decreased sharply at pH<6, following similar titration curves. Binding of (125)I-lactoferrin and (125)I-ASOR to hepatocytes was Ca(2+)-dependent. Binding increased progressively at > or =300 microM CaCl(2), in the presence of 1 mM EDTA. Monensin treatment of hepatocytes, which causes hepatocytes to accumulate inactive ASGP receptors, reduced surface binding of (125)I-lactoferrin and (125)I-ASOR by 46 and 49%, respectively, with only a 16% loss of immunodetectable receptor protein from the cell surface. Finally, deacylation of purified ASGP receptors in vitro with 1 M hydroxylamine abolished receptor lectin activity as reflected by the loss of (125)I-ASOR binding as well as the complete loss of specific (125)I-lactoferrin binding. Treatment with 1 M Tris had no effect on binding of either ligand. We conclude from these data that galactose-independent lactoferrin binding to the ASGP receptor requires the receptor's carbohydrate-recognition domain to be in an active configuration. An active configuration is promoted by neutral pH and Ca(2+), and also requires the receptor subunits to be acylated.

Lactoferrin binding to the rat asialoglycoprotein receptor requires the receptor's lectin properties

Lactoferrin binds to rat hepatic lectin 1 (RHL1), the major subunit of the asialoglycoprotein (ASGP) receptor, with high affinity, by a galactose-independent mechanism. To better understand the molecular basis of this novel interaction, we compared the binding of lactoferrin and asialo-orosomucoid (ASOR) to isolated rat hepatocytes and to purified ASGP receptors as a function of pH, Ca2+ and receptor acylation. Binding of 125I-lactoferrin and 125I-ASOR to isolated rat hepatocytes at 4 °C decreased sharply at pH &amp;lt; 6, following similar titration curves. Binding of 125I-lactoferrin and 125I-ASOR to hepatocytes was Ca2+-dependent. Binding increased progressively at ≥ 300 μM CaCl2, in the presence of 1 mM EDTA. Monensin treatment of hepatocytes, which causes hepatocytes to accumulate inactive ASGP receptors, reduced surface binding of 125I-lactoferrin and 125I-ASOR by 46 and 49%, respectively, with only a 16% loss of immunodetectable receptor protein from the cell surface. Finally, deacylation of purified ASGP receptors in vitro with 1 M hydroxylamine abolished receptor lectin activity as reflected by the loss of 125I-ASOR binding as well as the complete loss of specific 125I-lactoferrin binding. Treatment with 1 M Tris had no effect on binding of either ligand. We conclude from these data that galactose-independent lactoferrin binding to the ASGP receptor requires the receptor's carbohydrate-recognition domain to be in an active configuration. An active configuration is promoted by neutral pH and Ca2+, and also requires the receptor subunits to be acylated.

Functional evaluation of poly-(N-ρ-vinylbenzyl-O-β-Dgalactopyranosyl-[1-4]-D-gluconamide)(PVLA) as a liver specific carrier

Hepatocytes express the specific C-type lectin, asialoglycoprotein (ASGP) receptor, on the surface to remove the ligand-bearing proteins from circulation. The specific expression and ligand specificity are thought to be the ideal characters for the target of drug or gene delivery. Various galactose-bearing molecules were synthesized for this purpose. However, the biological or functional interaction of these molecules with the ASGP receptor still remains to be elucidated. In this study, we evaluated the functional ability of synthetic galactose polymer ligand, poly-(N-ρ-vinylbenzyl-O-β-Dgalactopyranosyl-[1-4]-D-gluconamide) (PVLA), to interact with recombinant ASGP receptors using mouse ASGP receptor (mouse hepatic lectin; MHL) gene-transfected CHO cells. PVLA-coated beads bound to and were endocytosed by the whole (MHL-1/-2) ASGP receptor-expressing CHO cells like hepatocytes while PVMA (poly-(N-ρ-vinylbenzyl-O-β-D-glucopyranosyl-[1-4]-D-gluconamide) did not. Interestingly, PVLA-coated beads were also endocytosed by either MHL-1 or MHL-2 alone expressing cells, which are known to be incapable of endocytosing natural ligands. In addition, the endocytosis of PVLA-coated beads by MHL-expressing CHO cells or primary hepatocytes was inhibited only by soluble PVLA but not by the same galactose molecular concentration of soluble asialofetuin. Furthermore, PVLA-coated beads were endocytosed by primary hepatocyte to a significantly higher degree than asialofetuin-coated beads in vitro. These results suggest that PVLA has higher affinity to the ASGP receptor than the natural ligands in blood. Consistently, it was demonstrated that intravenously injected FITC-labeled PVLA but not PVMA drastically accumulated in parenchymal cells of the liver in vivo. Taken together, PVLA exhibiting higher affinity with hepatocytes than natural ligands is thought to be an attractive and practical carrier-ligand for liver targeting.

Chronic antisense therapy for angiotensinogen on cardiac hypertrophy in spontaneously hypertensive rats.

We examined the effect of the suppression of plasma angiotensinogen (AGT) by the intravenous injection of antisense oligodeoxynucleotides (ODNs) against AGT targeted to the liver on cardiac remodeling in spontaneously hypertensive rats (SHR). The ODNs against rat AGT were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for regulating liver gene expression. Male SHR (n = 18), and age-matched male Wistar-Kyoto rats (WKY, n = 6) were used for this study. At 10 weeks of age, the SHR were divided into three groups (each group n = 6), and the systolic blood pressure (SBP) did not significantly change among them. The control SHR and WKY groups received saline, the sense SHR group was injected with the sense ODNs complex and the antisense SHR group was injected with the antisense ODNs complex, from 10 to 20 weeks of age. ASOR-poly(L)lysine-ODNs complex was injected via the tail veins twice a week. At the end of the treatment, a reduction of hepatic AGT mRNA, cardiac angiotensin II type 1 receptor mRNA and the plasma AGT concentration was only observed in the antisense-injected SHR but not in the other groups of SHR and WKY. This antisense therapy did not significantly change the mRNA expression for angiotensin converting enzyme, angiotensin II type 2 receptor and AGT in the left ventricle (LV) among all three groups. Although the plasma angiotensin II (Ang II) concentration significantly decreased to the level of WKY after the antisense therapy, the SBP, LV to body weight ratio and % collagen volume fraction also showed a reduction, however, these findings were still larger than in the WKY than in either the sense-injected SHR or control SHR. The plasma AGT is considered to play a role in the development of cardiac hypertrophy in SHR, but it has not a complete effects on cardiac remodeling even if the plasma Ang II levels are inhibited because of an insufficient suppression of hypertension.

Multicomponent DNA carrier with a vesicular stomatitis virus G-peptide greatly enhances liver-targeted gene expression in mice.

Genes can be targeted to hepatocytes in vitro and in vivo by the use of asialoorosomucoid-polylysine conjugates. After systemic application, this nonviral vector is recognized by highly selective asialoglycoprotein (AsGP) receptors on the sinusoidal liver cell membrane and is taken up via receptor-mediated endocytosis. As most of the DNA is rapidly transferred to lysosomes where it is degraded, transfection efficiency is low and gene expression transient. To address this problem, we incorporated a pH-dependent synthetic hemolytic peptide derived of the G-protein of Vesicular Stomatitis Virus (VSV) into the gene transfer system, to increase endosomal escape of internalized DNA. The multicomponent carrier binds DNA in a nondamaging way, is still recognized by the AsGP receptor, and is targeted to the liver in vivo. Injection of DNA complexes containing a luciferase marker gene resulted in luciferase expression of 29 000 pg/g liver which corresponded to an increase of a factor of 10(3) overexpression after injection of DNA complexes without endosomolytic peptide. Furthermore, the amount of intact transgene within isolated liver cell nuclei was increased by a factor of 10(1)-10(2) by the use of the multicomponent carriers. These results demonstrate that incorporation of a hemolytic peptide into a nonviral vector can greatly increase gene expression while retaining cell type targetability in vivo.

Secretion of preβ HDL increases with the suppression of cholesteryl ester transfer protein in Hep G2 cells

Preβ HDL are small, protein rich lipoproteins that are predominantly composed of apo A-I, without apo A-II. Preβ HDL are secreted from the liver as nascent HDL and/or are produced in the incubated plasma by cholesteryl ester transfer protein (CETP). However, the role of CETP in the secretion of HDL from the liver has yet to be determined. In the present study, we examined the effect of the suppression of hepatic CETP by antisense oligodeoxynucleotides (ODNs) against CETP targeted to the liver on the secretion of apo A-I using a Hep G2 cell culture. The ODNs against CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for the regulation of liver gene expression. Hep G2 cells were cultured in DMEM supplemented with 10% FBS. After 2 days, the medium was changed to DMEM with EGF and the cells were divided into three groups. The control group received saline, while the sense group was mixed with the sense ODNs complex and the antisense group was mixed with the antisense ODNs complex, respectively, for 2 days. Both the hepatic CETP mRNA and the CETP mass in the medium in the antisense group decreased significantly more than in the sense and the control groups (CETP mass: 1.697±0.410 ng/mg cell protein vs. 2.367±0.22 and 2.360±0.139, n=3 in each determination). In contrast, both the hepatic apo A-I mRNA and the apo A-I mass in the medium in the antisense group were significantly higher than those in the sense and the control groups (apo A-I mass; 1.877±0.215 μg/mg cell protein vs. 1.213±0.282 and 1.097±0.144, n=3 in each determination). The increase in apo A-I was mainly due to the increase in preβ apo A-I. These findings may partly explain why HDL and apo A-I increase in patients with CETP deficiency, while also indicating the possibility that the original level of preβ HDL is sufficient in such patients.

Enhanced inhibition of hepatitis B virus production by asialoglycoprotein receptor-directed interferon.

Most chronic carriers of hepatitis B virus (HBV) do not respond to interferon (IFN) treatment. This limitation of IFN therapy may be due in part to scant expression of IFN receptor in the liver. Because the asialoglycoprotein (ASGP) receptor is specifically expressed in the liver at high density, the ASGP receptor-binding domain was generated within an N-glycosylated human IFN-beta molecule by the removal of sialic acid to direct this cytokine to the liver. This modified IFN (asialo-IFN-beta) demonstrated greater inhibition of HBV production in ASGP receptor-positive human liver cells transfected with a replication-competent HBV construct than did conventional IFN-alpha or IFN-beta. Furthermore, the enhanced antiviral effect of asialo-IFN-beta was supported by induction of the 2'-5' oligoadenylate synthetase, an indicator of IFN activity, at a level significantly higher than that produced by conventional IFN-beta. Moreover, mouse asialo-IFN-beta profoundly reduced viremia in vivo in HBV-transfected athymic nude mice, in contrast to conventional IFN-beta, which had no substantial effect. These experiments demonstrate that directing IFN to ASGP receptor facilitates its signaling in the liver and augments its antiviral effect, and is therefore useful in overcoming the limited antiviral effect of conventional IFNs.

Expression of asialoglycoprotein receptor in human fetal liver.

Asialoglycoprotein (ASGP) receptor, which exists in hepatic parenchymal cells, plays important roles in the clearance of desialylated glycoproteins from the circulation. To study the expression of ASGP receptor in human fetal liver, specimens were obtained after therapeutic/spontaneous abortion or at autopsy with informed consent. Reverse transcription polymerase chain reaction and immunohistochemical staining showed that ASGP receptor was present as early as 15 weeks of gestation in human fetal liver. In liver at 15 weeks-gestation it was stained uniformly in cytoplasm as seen in HepG2 cells. As gestation proceed the ASGP receptor was frequently observed on the cell surface and intracellular staining showed a spotted distribution. We conclude that the expression of ASGP receptor is an early event in the developmental process of fetal liver. Furthermore, the distribution of ASGP receptor changes as the liver develops.

A DNA delivery system containing listeriolysin O results in enhanced hepatocyte-directed gene expression.

AIM:To determine whether incorporation of the pH dependent bacterial toxin listeriolysin O (LLO) into the DNA carrier system could increase the endosomal escape of internalized DNA and result gene expression.METHODS:A multi component delivery system was prepared consisting of asialoglycoprotein (ASG), poly L-lysine(PL), and LLO.Two marker genes, luciferase and beta galactosidase in plasmids were complexed and administered in vitro to Huh7(ASG receptor (+) and SK Hep1(ASG receptor (-) cells. Purity, hemolytic activity, gene expression, specificity, and toxicity were evaluated.RESULTS:An LLO containing conjugate retained cell target-ing specificity and membranolytic activity. In ASG receptor (+) cells,luciferase gene expression was enhanced by more than 7 fold over that of conjugates without the incorporation of listeriolysin O. No significant expression occurred in ASG receptor (-) cells. Enhancement of betagalactosidase gene expression was less, but still significantly increased over controls. There was no detectable toxicity at concentrations shown to be effective in transfection studies.CONCLUSIONS:ASOR-PL can be coupled to LLO using disulfide bonds, and successfully target and increase the gene expression of foreign DNA.

Iron loading of isolated rat hepatocytes inhibits asialoglycoprotein receptor dynamics and induces formation of rat hepatic lectin-1 [correction of leptin-1] (RHL-1) oligomers.

The major subunit [rat hepatic lectin-1 (RHL-1)] of the asialoglycoprotein (ASGP) receptor mediates endocytosis of the iron-binding protein lactoferrin (Lf) by isolated rat hepatocytes, yet iron loading of cultured adult rat hepatocytes increases the binding and endocytosis of Lf while greatly inhibiting the uptake of desialylated ligand. In the present study, we determined whether the iron-induced Lf-binding site is RHL-1 and examined the nature of the iron-induced block in ASGP receptor endocytic function. Isolated rat hepatocytes increased their non-haem iron content from 70 to 470 p.p. b. following incubation with ferric ammonium citrate (<=100 microgram/ml). These conditions blocked internalization of 125I-asialo-orosomucoid (ASOR) by approximately 90% but increased 125I-Lf endocytosis by 40%. ASOR and anti-RHL-1 sera blocked the binding and endocytosis of 125I-Lf on control cells but not on iron-loaded cells, indicating that the iron-induced Lf-binding site on hepatocytes is not RHL-1. Iron-loading of hepatocytes in the presence or absence of excess ASOR did not significantly alter the number of active ASGP receptors on the cell surface. In contrast, iron-loading decreased the number of active intracellular receptors by 40% and blocked the uptake of 125I-ASOR prebound to the cells by approximately 80%. Under these conditions, we found an iron-dependent evolution of 88 and 140 kDa RHL-1-containing, beta-mercaptoethanol-sensitive multimers that constituted up to 34 and 23%, respectively, of total immunodetectable RHL-1. We propose that iron-induced formation of cystinyl-linked RHL-1-containing multimers inhibits ASGP receptor movement between cell surface and interior and disrupts acylation of intracellular receptors.

Effect of Antisense Oligonucleotides against Cholesteryl Ester Transfer Protein on the Development of Atherosclerosis in Cholesterol-fed Rabbits

Cholesteryl ester transfer protein (CETP) is the enzyme that facilitates the transfer of cholesteryl ester from high density lipoprotein (HDL) to apolipoprotein B (apoB)-containing lipoproteins. However, the exact role of CETP in the development of atherosclerosis has not been determined. In the present study, we examined the effect of the suppression of increased plasma CETP by intravenous injection with antisense oligodeoxynucleotides (ODNs) against CETP targeted to the liver on the development of atherosclerosis in rabbits fed a cholesterol diet. The ODNs against rabbit CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method to regulate liver gene expression. Twenty-two male Japanese White rabbits were used in the experiment. Eighteen animals were fed a standard rabbit chow supplemented with 0.3% cholesterol throughout the experiment for 16 weeks. At 8 weeks, they were divided into three groups (six animals in each group), among which the plasma total and HDL cholesterol concentrations did not significantly change. The control group received nothing, the sense group were injected with the sense ODNs complex, and the antisense group were injected with the antisense ODNs complex, respectively, for subsequent 8 weeks. ASOR. poly(L-lysine) ODNs complex were injected via the ear veins twice a week. Four animals were fed a standard rabbit diet for 16 weeks. The total cholesterol concentrations and the CETP mass in the animals injected with antisense ODNs were all significantly decreased in 12 and 16 weeks compared with those injected with sense ODNs and the control animals. The HDL cholesterol concentrations measured by the precipitation assay did not significantly change among the groups fed a cholesterol diet, and triglyceride concentrations did not significantly change in the four groups. However, at the end of the study, when the HDL cholesterol concentrations were measured after the isolation by ultracentrifugation and a column chromotography, they were significantly higher in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. A reduction of CETP mRNA and an increase of LDL receptor mRNA in the liver were observed in the animals injected with antisense ODNs compared with those injected with sense ODNs and the control animals. Aortic cholesterol contents and the aortic percentage lesion to total surface area were significantly lower in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. These findings showed for the first time that suppression of increased plasma CETP by the injection with antisense ODNs against CETP coupled to ASOR carrier molecules targeted to the liver could thus inhibit the atherosclerosis possibly by decreasing the plasma LDL + very low density lipoprotein (VLDL) cholesterol in cholesterol-fed rabbits.

Covalent protein-oligonucleotide conjugates for efficient delivery of antisense molecules.

Antisense oligonucleotides have been covalently attached to asialoglycoprotein (ASGP) via disulfide bond conjugation chemistry. These conjugates were characterized extensively by an array of chemical, chromatographic, and spectroscopic means. Multiple (approximately six) oligonucleotides can be conjugated to each ASGP molecule. The molecular conjugates were used to deliver antisense oligonucleotides complementary to the mRNA of the interleukin 6 signal transduction protein (gp130) to modulate the acute phase response of hepatoma (HepG2) cells in vitro. These conjugates were biologically active, as measured by inhibition of the cytokine-stimulated up-regulation of the acute phase protein haptoglobin. The level of inhibition was comparable to that found with previous technology featuring noncovalent complexes of ASGP-poly(L-lysine) and oligonucleotide. Because of the ability to control the stoichiometry of the conjugate and its unimolecular nature (as opposed to bimolecular for the noncovalent conjugates), this methodology holds great promise for further development and application.

Asialoglycoprotein/asialoglycoprotein receptor (AGP-AGPr) interaction is an important mechanism for the uptake of FK506 by hepatocytes.

Hepatic tissue concentrations of FK506 have been correlated with early acute rejection following liver transplantation. Asialoglycoproteins (AGP) reputedly bind FK506 in blood. AGP are removed from the circulation by the liver via the AGP receptor (AGPr), which resides on hepatocytes. This study was undertaken to determine if the AGP-AGPr mechanism enhances the delivery of FK506 to hepatocytes. Human orosomucoid (OM) was used as a representative AGP. asialoOM (aOM) was prepared by desialation of OM. Fresh rat hepatocytes were isolated by collagenase digestion. Tritium labeled FK506 (FK) was used to identify and quantitate FK506. Quantitation of FK in serum and culture media was by direct counting. FK in animal tissues used a method developed in our laboratory for the purpose. AGPr on resting hepatocytes was demonstrated by flow cytometry using FITC-orosomucoid and FITC-BSA controls. AGPr were enhanced by 2 g glucose/L. Two serum FK-binding fractions, 44 kD and 15 kD, were identified by gel filtration. Exogenous OM avidly bound FK and displaced FK activity from the 15 kD fraction. Serum (1%) and the 44 kD fraction enhanced the uptake of FK by hepatocytes, while serum depleted of OM-aOM by affinity chromatography was only 72.5% as effective as control serum; aOM enhanced the uptake of FK by hepatocytes to a degree similar to that of control serum but OM did not significantly affect the uptake of FK. Cold FK506 blocked the uptake and was dose dependent; cold CsA had no effect. Affinity extraction of OM from serum to which FK had previously been added removed 28.4% of FK activity. Following i.v. infusion, the kidney had the highest and liver the lowest tissue concentration of FK at 1 hr and 3 hr. In contrast, after oral administration the liver had the highest concentrations of the other tissues tested. The AGP-AGPr mechanism plays a significant role in the delivery of FK506 to hepatocytes and is likely responsible for the differences in bioavailability observed after oral and i.v. administration. Factors governing the AGP-AGPr mechanism are germane to understanding both the efficacy and toxicity of FK506 and the development optimal therapeutic strategies.

Use of an asialoglycoprotein receptor-targeted magnetic resonance contrast agent to study changes in receptor biology during liver regeneration and endotoxemia in rats.

The hepatic asialoglycoprotein receptor is responsible for rapid clearance of desiaylated glycoproteins from the circulation by receptor-mediated endocytosis. Previous in vitro studies in hepatocyte preparations from rats subjected to partial hepatectomy (PH) of 70% to stimulate hepatic regeneration showed decreased asialoglycoprotein (ASGP) receptor binding. We used an ASGP receptor-targeted hepatic magnetic resonance imaging (MRI) agent, BMS 180550, to demonstrate similar changes in receptor biology in vivo. BMS 180550 is an arabinogalactan-coated ultrasmall superparamagnetic iron oxide preparation. Arabinogalactan is a ligand for the ASGP receptor and, thereby, targets the contrast agent exclusively to hepatocytes. Hepatic uptake of BMS 180550 was assessed by sequential precontrast and post-contrast MRI in rats subjected to PH of 70%. In regenerating liver 1 and 3 days after PH, the maximum decrease in hepatic signal intensity (62.2% +/- 6.1 and 59.4% +/- 3.8, respectively) was significantly less than the maximum decrease seen in sham-operated animals at 1 and 3 days postsurgery (39.5% +/- 2.5 and 44% +/- 1.0, respectively). The time necessary to reach 80% of the maximum decrease in hepatic signal intensity, (t80), was less than 2 minutes in control rats and increased to 7.5 +/- 1.3 min and 11.0 +/- 2.3 minutes at 1 and 3 days post-PH, respectively. By 7 days post-PH, contrast-enhanced MRI no longer detected a difference in regenerating liver. Because BMS 180550 is taken up exclusively by the liver, clearance of the agent from the blood was used as a measure of hepatic clearance. Concentration-time curves constructed by measuring changes in blood T2 after an intravenous dose of BMS 180550 were used to determine clearance of the agent. Blood clearance of BMS 180550 in normal liver (15.4 +/- 1.08 mL/min) obeyed first-order kinetics and did not vary over the dose range tested (10 to 100 micromol/L/kg iron). One, 3, 7, and 14 days post-PH, clearance varied with dose, suggesting ASGP receptor saturation. As regeneration proceeded, the dose of contrast agent needed to cause a deviation from first-order kinetics increased, suggesting gradual recovery of ASGP receptor function. Hepatic relaxation was determined by in vitro spectroscopy 60 minutes after administration of graded doses of BMS 180550 and showed dose-dependent increases in relaxation. Kinetic analysis at 1 day post-PH demonstrated a decrease in the apparent k(m) and the maximum response, R(max), suggesting a decrease in the number of functional asialoglycoprotein receptors with concomitant increase in receptor affinity. Systemic endotoxemia, which normally accompanies hepatic regeneration induced by PH, also decreased clearance of BMS 180550 and slowed hepatic uptake of the contrast agent. Altered BMS 180550 pharmacokinetics in endotoxin-treated rats was enhanced by prior administration of small doses of competing ligand. Our studies document the value of BMS 180550 in following changes in ASGP function that accompany PH or systemic endotoxemia. This agent may be useful in assessing the degree of hepatic regeneration in patients with clinical liver disease.

Use of an asialoglycoprotein receptor-targeted magnetic resonance contrast agent to study changes in receptor biology during liver regeneration and endotoxemia in rats

The hepatic asialoglycoprotein receptor is responsible for rapid clearance of desiaylated glycoproteins from the circulation by receptor- mediated endocytosis. Previous in vitro studies in hepatocyte preparations from rats subjected to partial hepatectomy (PH) of 70% to stimulate hepatic regeneration showed decreased asialoglycoprotein (ASGP) receptor binding. We used an ASGP receptor-targeted hepatic magnetic resonance imaging (MRI) agent, BMS 180550, to demonstrate similar changes in receptor biology in vivo. BMS 180550 is an arabinogalactan-coated ultrasmall superparamagnetic iron oxide preparation. Arabinogalactan is a ligand for the ASGP receptor and, thereby, targets the contrast agent exclusively to hepatocytes. Hepatic uptake of BMS 180550 was assessed by sequential precontrast and post- contrast MRI in rats subjected to PH of 70%. In regenerating liver 1 and 3 days after PH, the maximum decrease in hepatic signal intensity (62.2% +/- 6.1 and 59.4% +/- 3.8, respectively) was significantly less than the maximum decrease seen in sham-operated animals at 1 and 3 days postsurgery (39.5% +/- 2.5 and 44% +/- 1.0, respectively). The time necessary to reach 80% of the maximum decrease in hepatic signal intensity, (t80), was less than 2 minutes in control rats and increased to 7.5 +/- 1.3 min and 11.0 +/- 2.3 minutes at 1 and 3 days post-PH, respectively. By 7 days post-PH, contrast-enhanced MRI no longer detected a difference in regenerating liver. Because BMS 180550 is taken up exclusively by the liver, clearance of the agent from the blood was used as a measure of hepatic clearance. Concentration-time curves constructed by measuring changes in blood T2 after an intravenous dose of BMS 180550 were used to determine clearance of the agent. Blood clearance of BMS 180550 in normal liver (15.4 +/- 1.08 mL/min) obeyed first-order kinetics and did not vary over the dose range tested (10 to 100 micromol/L/kg iron). One, 3, 7, and 14 days post-PH, clearance varied with dose, suggesting ASGP receptor saturation. As regeneration proceeded, the dose of contrast agent needed to cause a deviation from first-order kinetics increased, suggesting gradual recovery of ASGP receptor function. Hepatic relaxation was determined by in vitro spectroscopy 60 minutes after administration of graded doses of BMS 180550 and showed dose-dependent increases in relaxation. Kinetic analysis at 1 day post-PH demonstrated a decrease in the apparent k(m) and the maximum response, R(max), suggesting a decrease in the number of functional asialoglycoprotein receptors with concomitant increase in receptor affinity. Systemic endotoxemia, which normally accompanies hepatic regeneration induced by PH, also decreased clearance of BMS 180550 and slowed hepatic uptake of the contrast agent. Altered BMS 180550 pharmacokinetics in endotoxin- treated rats was enhanced by prior administration of small doses of competing ligand. Our studies document the value of BMS 180550 in following changes in ASGP function that accompany PH or systemic endotoxemia. This agent may be useful in assessing the degree of hepatic regeneration in patients with clinical liver disease. (Hepatology 1996 Jun;23(6):1631-41)

Inhibition of asialoglycoprotein endocytosis and degradation in rat hepatocytes by protein phosphatase inhibitors.

In isolated rat hepatocytes, a radiolabelled tyramine-cellobiose conjugate of asialo-orosomucoid, 125I-TC-AOM, was rapidly taken up by receptor-mediated endocytosis and proteolytically degraded in the lysosomes, where radioactive degradation products accumulated. Okadaic acid and other protein phosphatase inhibitors (microcystin-LR, calyculin A) strongly reduced the fraction of asialoglycoprotein (ASGP) receptors localized to the cell surface, and correspondingly inhibited the uptake of 125I-TC-AOM. In addition, the inhibitors suppressed 125I-TC-AOM degradation strongly (90% at 150 nM) and potently (half-maximal effect at 20 nM okadaic acid), indicating an involvement of protein phosphorylation, and of a protein phosphatase of type 2A, in the regulation of intracellular endocytic flux. The effects of okadaic acid on 125I-TC-AOM accumulation, as well as on degradation, could be eliminated by the protein kinase inhibitor genistein. Okadaic acid prevented the transfer of 125I-TC-AOM to a non-recycling endocytic compartment, causing its retention in a recycling compartment from which about one-third of the endocytosed 125I-TC-AOM could be returned to the cell surface and detached from its receptor in the presence of EGTA. ASGP receptors recycled extensively both in the presence and absence of okadaic acid, as indicated by a sustained uptake of 125I-TC-AOM. Sucrose density gradient analysis and sedimentation studies indicated that okadaic acid caused accumulation of 125I-TC-AOM in light endosomes (1.11 g/ml), preventing its transfer to dense endosomes (1.14 g/ml) and lysosomes (1.18 g/ml). The lysosomes could be identified in density gradients by their contents of lysosomal marker enzymes and acid-soluble radioactivity, and by their sensitivity towards the lysosome-disrupting agent glycyl-L-phenylalanine-2-naphthylamide. By using endocytosed AOM-gold particles as an ultrastructural endocytic marker, it could be shown that the light endosomes accumulating ASGP in the presence of okadaic acid had the morphological appearance of small endocytic vesicles/tubules and multivesicular endosomes. Whereas in control cells 4% of the AOM-gold was in small vesicles/tubules, 55% in multivesicular endosomes and 41% in lysosomes, the corresponding figures for okadaic acid-treated cells were 17%, 73% and 11%. Our results thus indicate that protein phosphatase inhibitors have two effects on ASGP endocytosis: (1) an early inhibition of ligand uptake, due to a reduction in the fraction of ASGP receptors at the cell surface, and (2) an inhibition of ASGP transfer from a recycling compartment consisting of light, small endocytic vesicles and multivesicular endosomes, to a non-recycling compartment consisting of dense multivesicular endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)