Biomedical subjects

R J Stockert

Publications and source records attributed to R J Stockert.

77 records · Page 5Linked to original sources

PubMedDate not supplied

Origins of biliary copper.

We tested the hypothesis that the copper present in bile--the major route of elimination of the metal from the body--is derived exclusively from hepatocytes by administering radiocopper (64Cu or 67Cu)-labeled ionic Cu, desialylated (AsCPN) or intact human ceruloplasmin (CPN), intravenously, to rats with cannulated bile ducts. The rates of appearance and the total amounts of radiolabeled isotope recovered in bile were measured. The three vehicles chosen for the delivery of radiocopper interact differently with hepatocytes: ionic Cu is taken up by a passive process (Schmitt, R. C. et al., Am. J. Physiol. 1983; 244:G183-G191); AsCPN is promptly cleared from the circulation by specific receptor-mediated endocytosis, and CPN largely remains in the circulation for the duration of the experiment. Similar amounts of radiocopper were recovered following injections of ionic Cu (3.1%) or CPN (2%), but substantially larger amounts (8.1%) were excreted after administration of AsCPN. Using an antibody to CPN which reacts also with AsCPN, we found about 70% of the bile radioactivity to be immunoprecipitable following injections of either glycoprotein, indicating that a fraction of these copper proteins had entered the bile essentially unmodified. Our observations indicate that in addition to the lysosomal compartment which catabolizes a portion of the AsCPN in hepatocytes, there appears to be a direct route for AsCPN from hepatocellular sinusoids to the bile canaliculi. Since CPN does not interact significantly with hepatocytes, its presence in bile suggests transcytosis via the biliary epithelium.

Animals↗

PubMedDate not supplied

Activity and regulation of low density lipoprotein receptors in a human hepatoblastoma cell line.

The responsiveness of low density lipoprotein (LDL) binding and uptake was measured in a human hepatoblastoma cell line, Hep G2. These cells exhibited both saturable, high-affinity and nonsaturable low-affinity components similar to that described for LDL binding in other mammalian cells. In addition, receptor-mediated uptake of [125I]LDL was dependent on the presence of Ca++ and required intact lysine residues in LDL as evidenced by abolition of binding following reductive methylation of LDL. The receptor activity was rapidly responsive to changes made in LDL concentrations in the medium, up-regulating by 200% in the absence of LDL, and down-regulating by 56% in the presence of LDL, both changes occurring within 2 hr. Cholesterol synthesis was estimated by measuring 3-hydroxymethylglutaryl coenzyme A reductase activity. The activity of this enzyme was also found to increase rapidly in the absence of LDL and decrease rapidly in the presence of LDL in the media. The human cell line Hep G2 possesses an active and responsive receptor for LDL and provides a useful model for the study of lipoprotein uptake and metabolism in intact human liver.

Carcinoma, Hepatocellular↗

PubMedDate not supplied

Reduced transport of bilirubin and asialoorosomucoid in regenerating rat liver is a microtubule-independent event.

In previous studies, we found that uptake of bilirubin and asialoorosomucoid is depressed in regenerating rat liver. To determine what role the hepatic cytoskeleton plays in this modulation of uptake, animals were treated with colchicine, an inhibitor of microtubular polymerization. Normal unoperated rats or rats following two-thirds hepatectomy or sham surgery were injected with colchicine, lumicolchicine (50 micrograms per 100 gm of body weight, i.p.) or normal saline. Lumicolchicine, an analog of colchicine, has no effect on microtubules and was used as a control. At 12 hr after surgery, sham-operated and unoperated animals received a second equal dose. Partially hepatectomized animals received one-third the initial dose. At 24 hr after surgery, livers were perfused in situ and single-pass multiple indicator dilution studies were performed. Colchicine as compared to lumicolchicine pretreatment reduced apparent influx of bilirubin and asialoorosomucoid in regenerating liver by 50% but had no effect in liver from normal or sham-operated rats. Analysis of indicator dilution curves revealed that reduced influx in colchicine-treated liver was attributable to an increased vascular volume of distribution. These results suggest that microtubules may play a role in maintenance of normal hepatic vascular architecture during regeneration. Lack of effect of colchicine on modulation of bilirubin and asialoorosomucoid uptake during regeneration suggests that other, as yet unknown, factors result in down-regulation of the specific hepatocellular transport systems for these two ligands.

Animals↗

PubMedDate not supplied

Transport and intracellular distribution of copper in a human hepatoblastoma cell line, HepG2.

The uptake of radiocopper by HepG2 cells is a saturable, temperature-dependent and cellular energy-independent process with a Vmax of 7.1 +/- 0.2 pmoles min-1 mg protein-1 and an estimated Km of 3.3 +/- 0.5 microM. The rate of copper uptake is reduced at an equimolar concentration of albumin and is unaffected by zinc at a 10-fold molar excess. Approximately 70% of the newly incorporated radiocopper binds to membranes and organelles, while 30% is recovered in the cytosol. The soluble fraction can be resolved into two copper-binding protein peaks. Incubation of HepG2 with nonisotopic copper results in displacement of radiocopper associated with the proteins contained in the lower molecular weight peak. Exposure of the cells to cycloheximide inhibits the incorporation of the isotope into this fraction.

Biological Transport↗

PubMedDate not supplied

Selective regulation of intrinsic membrane proteins in HepG2.

Expression of three hepatocellular membrane proteins--the asialoglycoprotein receptor (hepatic binding protein) the insulin receptor and organic anion binding protein--have been studied in the HepG2 cell line. HepG2 grown in minimal essential medium supplemented with 10% fetal bovine serum maximally expressed hepatic binding protein and insulin receptor only at confluence while organic anion binding protein appeared independent of the state of cellular proliferation. When cells were grown in medium supplemented with dialyzed fetal bovine serum, adult bovine serum or in chemically defined medium, expression of hepatic binding protein and insulin receptor but not organic anion binding protein was reduced by 60 to 80%. Immunoblotting techniques revealed that cells grown in dialyzed fetal bovine serum contained virtually no mature, glycosylated 45 kD hepatic binding protein, but a small amount of 36 kD protein. Metabolic labeling of cells grown in dialyzed fetal bovine serum with [35S] methionine indicated reduced synthesis of the 45 kD hepatic binding protein and the absence of the 36 kD protein. Restoration of normal expression of hepatic binding protein and insulin receptor was achieved by addition to dialyzed fetal bovine serum of: fetal bovine serum; 2,000 dalton ultrafiltrate of fetal bovine serum, or 300 to 350 dalton fraction of the ultrafiltrate. The normal concentration of organic anion binding protein demonstrable in cells grown in dialyzed fetal bovine serum indicates that the low molecular weight factor(s) is not a generalized modulator of plasma membrane biogenesis. However, its effect on the steady-state level of the hepatic binding protein and insulin receptor, characteristic of mature hepatocytes, suggests a role for this fetal serum factor in hepatocellular differentiation.

Anion Transport Proteins↗