Richard J. Stockert

Hepatocellular copper toxicity and its attenuation by zinc.

We studied the mechanisms by which excess copper exerts, and zinc mitigates, toxic effects on HepG2 cells. Survival and cell growth were reduced in media containing greater than 500 microM copper chloride for 48 h; LD50 was 750 microM. At 1,000 microM copper for 1 h, there was a general reduction of protein synthesis, and no recognizable changes in cellular ultrastructure. Incubation of cells with 200 microM zinc acetate before exposure to copper, raised the LD50 for confluent cells to 1,250 microM copper chloride, improved protein synthesis, and increased synthesis of a 10-kD protein, apparently metallothionein. The mitigation, by zinc, of coppers toxicity may in part be mediated through induction of this protein in the hepatocyte.

Regulation of early endocytic vesicle motility and fission in a reconstituted system.

We previously established conditions to reconstitute kinesin-dependent early endocytic vesicle motility and fission on microtubules in vitro. The present study examined the question whether motility and fission are regulated in this system. Screening for proteins by immunofluorescence microscopy revealed that the small G protein, Rab4, was associated with 80% of hepatocyte-derived early endocytic vesicles that contain the ligand asialoorosomucoid (ASOR). By contrast, other markers for early endocytic vesicles including clathrin, Rab5 and EEA1 were present in the preparation but did not colocalize with the ASOR vesicles. Guanine nucleotides exchanged into the Rab4 present on the vesicles as shown by solubilization of Rab4 by Rab-GDI; solubilization was inhibited by incubation with GTP-γ-S and promoted by GDP. Pre-incubation of vesicles with GDP increased the number of vesicles moving on microtubules and markedly increased vesicle fission. This increase in motility from GDP was shown to be towards the minus end of microtubules, possibly through activation of the minus-end-directed kinesin, KIFC2. Pre-incubation of vesicles with GTP-γ-S, by contrast, repressed motility. Addition of exogenous GST-Rab4- GTP-γ-S led to a further repression of motility and fission. Repression was not seen with addition of GST-Rab4-GDP. Treatment of vesicles with Rab4 antibody also repressed motility, and repression was not seen when vesicles were pre-incubated with GDP. Based on these results we hypothesize that endogenous Rab4-GTP suppresses motility of ASOR-containing vesicles in hepatocytes and that conversion of Rab4-GTP to Rab4-GDP serves as a molecular switch that activates minus-end kinesin-based motility, facilitating early endosome fission and consequent receptor-ligand segregation.

Effect of nafenopin on the uptake of bilirubin and sulfobromophthalein by isolated perfused rat liver.

Hepatic uptake of bilirubin and sulfobromophthalein has kinetic characteristics suggesting facilitated diffusion. Because these compounds demonstrate mutual competition for uptake, a shared uptake mechanism has been presumed. Previous studies in isolated perfused regenerating liver revealed depressed uptake of bilirubin, sulfobromophthalein, and asialoorosomucoid, a desialylated glycoprotein which enters hepatocytes by receptor-mediated endocytosis. This study was designed to determine whether or not depressed transport seen in liver regeneration occurs in other states of hepatocellular proliferation. Rats were pretreated with nafenopin (200 mg/kg . day x 2), a drug that causes rapid hepatocellular proliferation similar to that seen in regeneration. Twenty-four hours after nafenopin treatment, liver weight increased by 40%. Influx, efflux, and sequestration rate constants in isolated perfused liver were quantitated by a computer fit to the model of Goresky. Results 1 day after nafenopin treatment revealed no change in transport parameters for bilirubin and asialoorosomucoid, but 55% and 49% reductions in influx of sulfobromophthalein and conjugated bilirubin, respectively. These studies suggest that hepatocellular proliferation alone is not responsible for the transport alterations seen during liver regeneration. Nafenopin effectively unmasks differences in uptake of bilirubin and other more water soluble organic anions such as sulfobromophthalein and conjugated bilirubin, suggesting that their uptake mechanisms are partially independent.

The rat hepatocyte plasma membrane organic anion binding protein is immunologically related to the mitochondrial F1 adenosine triphosphatase beta-subunit.

A 55-kD organic anion binding protein (OABP) was identified previously in liver cell plasma membrane sinusoidal subfractions. Although this protein was localized to the surface of hepatocytes by immunofluorescence, immunoblot analysis revealed reactivity toward both plasma membrane and mitochondrial fractions. To clarify these findings, an immunoreactive clone from a rat liver cDNA expression library was isolated, the 1,500-base pair cDNA insert was sequenced, and the corresponding beta-galactosidase fusion protein was expressed and purified. The resulting sequence corresponded to that of the rat mitochondrial F1-adenosine triphosphatase (F1-ATPase) beta-subunit. This protein and OABP are of similar size and are mutually immunologically cross-reactive. That the antigen was present on the cell surface as well as in mitochondria was suggested from studies of immunoprecipitation after cell-surface iodination, and light- and electron-microscopic immunocytochemistry. Photoaffinity labeling of bovine F1-ATPase with high-specific-activity [35S]sulfobromophthalein revealed binding only to the beta-subunit. Hepatocyte uptake of bilirubin and sulfobromophthalein requires cellular ATP and mitochondria also transport these organic anions, which at high doses inhibit respiration. The presence of an organic anion binding site on the F1-ATPase beta-subunit suggests that it may play a role in these processes.

Unique antigen of cultured Hodgkin's cells. A putative sialyltransferase

Hodgkins disease-derived giant cell lines (HD-cells) express high levels of ectosialyltransferase activity presumed to be a galactose-specific lectin recognizing the desialylated 3-fucosyl-N-acetyllactosamine structure (X-hapten). Both the anti-X-hapten monoclonal antibody VIM-D5 and a polyclonal antiserum to another galactose-lectin, the hepatic asialoglycoprotein receptor (HBP), recognize a 55,000-mol wt HD-cell protein (Paietta, E., R. J. Stockert, A. G. Morell, V. Diehl, and P. H. Weirnik. 1986. Proc. Natl. Acad. Sci. USA. 83:3451-3455.) That the expression of the 55,000-mol wt protein is restricted to HD-cells among X-hapten positive cells lines is confirmed in this study. The 55,000-mol wt protein is shown to be present on the cell surface and intracellularly, where an additional immunocrossreactive 150,000-mol wt protein is recognized. Extraction of the 55,000 mol wt protein from HD-cell lysates by affinity chromatography results in the loss of sialyltransferase activity. While evidence for a single protein possessing both the antigenic and the enzymatic activity is not direct, these results suggest that the ectosialyltransferase unique to HD-cells is a 55,000-mol wt membrane glycoprotein possessing the X-hapten oligosaccharide.

Cellular and molecular biology of the liver.

Recently, several lines of investigation focused on basic mechanisms governing cellular and molecular aspects of liver biology have intersected at the study of the hepatic stem cell. Despite years of study, the very question of the existence of the hepatic stem cell has yet to be unequivocally established. A second field of investigation into the cellular and molecular aspects of liver biology is aimed at liver-directed gene therapy in which several new vehicles have been devised to mediate gene transfer. Gene therapy is no longer thought of in the limited framework of a means to correct inherited disorders; it is now expanding into new therapeutic applications. A third major area of investigation includes studies of mechanisms that regulate membrane protein traffic necessary to maintain the integrity of differentiated liver cell function. In this review, some of the most recent advances and applications in these three areas are highlighted, and, where appropriate, points of interaction and potential therapeutic importance are emphasized.