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A W Wolkoff

Publications and source records attributed to A W Wolkoff.

At least 19 recordsLinked to original sources
PubMed1992

A role for microtubules in sorting endocytic vesicles in rat hepatocytes.

The vectorial nature of hepatocyte receptor-mediated endocytosis (RME) and its susceptibility to cytoskeletal disruptors has suggested that a polarized network of microtubules plays a vital role in directed movement during sorting. Using as markers a well-known ligand, asialoorosomucoid, and its receptor, we have isolated endocytic vesicles that bind directly to and interact with stabilized endogenous hepatocyte microtubules at specific times during a synchronous, experimentally initiated, single wave of RME. Both ligand- and receptor-containing vesicles copelleted with microtubules in the absence of ATP but did not pellet under similar conditions when microtubules were not polymerized. When 5 mM ATP was added to preparations of microtubule-bound vesicles, ligand-containing vesicles were released into the supernatant, while receptor-containing vesicles remained immobilized on the microtubules. Release of ligand-containing vesicles from microtubules was prevented by monensin treatment during the endocytic wave. Several proteins, including the microtubule motor protein cytoplasmic dynein, were present in these preparations and were released from microtubule pellets by ATP addition concomitantly with ligand. These results suggest that receptor domains within the endosome can be immobilized by attachment to microtubules so that, following monensin-sensitive dissociation of ligand from receptor, ligand-containing vesicles can be pulled along microtubules away from the receptor domains by a motor molecule, such as cytoplasmic dynein, thereby delineating sorting.

Adenosine Triphosphate↗
PubMed1991

Role of chloride and intracellular pH on the activity of the rat hepatocyte organic anion transporter.

Previous studies in cultured rat hepatocytes revealed that initial uptake of sulfobromophthalein (BSP) was markedly reduced upon removal of Cl- from the medium. In the present study, unidirectional Cl- gradients were established in short-term cultured rat hepatocytes and their effect on BSP uptake was determined. These investigations revealed that BSP uptake requires external Cl- and is not stimulated by unidirectional Cl- gradients, suggesting that BSP transport is not coupled to Cl- transport. In contrast, BSP transport is stimulated by an inside-to-outside OH- gradient, consistent with OH- exchange or H+ cotransport. As the presence of Cl- is essential for but not directly coupled to BSP transport, binding of 35S-BSP to hepatocytes was determined at 4 degrees C. This revealed an approximately 10-fold higher affinity of cells for BSP in the presence as compared to the absence of Cl- (Ka = 3.2 +/- 0.8 vs. 0.42 +/- 0.09 microM-1; P less than 0.02). Affinity of BSP for albumin was Cl(-)-independent, and was approximately 10% of its affinity for cells in the presence of Cl-. These results indicate that extracellular Cl- modulates the affinity of BSP for its hepatocyte transporter.

Animals↗
PubMed1991

Expression of the hepatocellular chloride-dependent sulfobromophthalein uptake system in Xenopus laevis oocytes.

The expression of the basolateral chloride-activated organic anion uptake system of rat hepatocytes has been studied in Xenopus laevis oocytes. Injection of oocytes with rat liver poly(A)+RNA resulted in the functional expression of chloride-dependent sulfobromophthalein (BSP) uptake within 3-5 d. This expressed chloride-dependent BSP uptake system exhibited saturation kinetics (apparent Km approximately 6.2 microM) and efficiently extracted BSP from its binding sites on BSA. Furthermore, the chloride-activated portion of BSP uptake was inhibited by bilirubin (10 microM; inhibition 53%), 4,4'-diisothiocyano-2,2-disulfonic acid stilbene (DIDS, 100 microM; 80%), taurocholate (100 microM; 80%), and cholate (200 microM; 95%). In contrast to results with total rat liver mRNA, injection of mRNA derived from the Na+/bile acid cotransporter cDNA (Hagenbuch, B., B. Stieger, M. Foguet, H. Lübbert, and P. J. Meier. 1991. Proc. Natl. Acad. Sci. USA. In press.) had no effect on BSP uptake into oocytes. Size fractionation of total rat liver mRNA revealed that a 2.0- to 3.5-kb size-class mRNA was sufficient to express the hepatic chloride-dependent BSP uptake system. These data indicate that "expression cloning" in oocytes represents a promising approach to ultimately clone the cDNA coding for the hepatocyte high affinity, chloride-dependent organic anion uptake system. Furthermore, the results confirm that the Na+/bile acid cotransport system does not mediate BSP uptake.

Animals↗
PubMed1991

Organic anion transport in HepG2 cells: absence of the high-affinity, chloride-dependent transporter.

In previous studies, we identified a 55 kD organic anion-binding protein in liver cell sinusoidal plasma membrane subfractions. Other investigators identified another 55 kD bromosulfophthalein/bilirubin binding protein on the surface of rat hepatocytes and HepG2 cells and suggested that this protein served as a transporter for these ligands. In this study, transport of 35S-sulfobromophthalein by the human hepatoma cell line, HepG2, was quantified in the presence and absence of bovine serum albumin to further clarify the possible function of these plasma membrane binding proteins. In contrast to results in normal rat hepatocytes, virtually no uptake of 35S-sulfobromophthalein by HepG2 cells in the presence of bovine serum albumin was found. In the absence of albumin, HepG2 cells expressed temperature-dependent uptake of 35S-sulfobromophthalein. However, the high-affinity Cl(-)-dependent sulfobromophthalein transport that characterizes normal rat hepatocytes was absent, as indicated by an approximately 95-fold lower affinity and 170-fold higher capacity of HepG2 cells for sulfobromophthalein compared with previous results with rat hepatocytes. These results suggest that 55 kD sulfobromophthalein/bilirubin-binding protein on the liver cell surface differs from organic anion-binding protein and is not responsible for sulfobromophthalein extraction in the presence of albumin, although it may play some role in lower affinity transport by cells. Immunoblot analysis and metabolic labeling of HepG2 cells demonstrated synthesis of organic anion-binding protein. However, light microscopic immunocytochemistry and immunoprecipitation of surface iodinated rat hepatocytes and HepG2 cells with antibody to a recombinant organic anion-binding protein fusion protein indicated absence of organic anion-binding protein on the surface of HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Transport of chenodeoxycholic acid and its 3-alpha- and 7-alpha-sulfates by isolated perfused rat liver.

In patients with cholestasis, levels of sulfated bile acids rise. Sulfate esters of chenodeoxycholic acid are the most abundant of these bile acid sulfates. These compounds are taken up by the liver and excreted into bile, although their plasma clearance and biliary excretion are reduced compared with that of unsulfated bile acids. It is not clear whether this is due to differences in intrinsic hepatic uptake or biliary excretion. In the present study, single-pass transport kinetics of chenodeoxycholic acid 3-alpha-sulfate, chenodeoxycholic acid 7-alpha-sulfate and unsulfated chenodeoxycholic acid were quantified in isolated perfused rat liver. Influx of the 7-alpha- and 3-alpha-sulfated derivatives was 57% and 20% that of chenodeoxycholic acid, respectively. These three compounds bound to albumin equally well, indicating that this was not a factor in their differential uptake. Although single-pass extraction of material taken up by the liver was identical. There was no difference in bile flow or biliary excretion of material taken up by the liver was identical. There was no difference in bile flow or biliary excretion rate, regardless of which bile acid sulfate was tested. These results indicate that the low plasma elimination of sulfated bile acids previously observed by others can be explained by low hepatic influx. The diminished transport into liver resulting from sulfation could lead to enhanced elimination of bile acids by the kidney.

Animals↗
PubMed1990

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.

Affinity Labels↗
PubMed1979

Role of ligandin in transfer of bilirubin from plasma into liver.

Multiple-indicator dilution studies of labeled bilirubin uptake were carried out on isolated perfused rat livers with variable ligandin concentrations (from normal and thyroidectomized animals with and without phenobarbital pretreatment). Ligandin concentrations, measured immunologically, increased 25% after thyroidectomy and approximately doubled after phenobarbital pretreatment but decreased to normal during perfusion in the thyroidectomized nonpretreated group. A distributed two-compartment model was fitted to the dilution data and estimates of influx, efflux, and sequestration coefficients were obtained. Influx and sequestration coefficients did not vary significantly between the groups. Efflux coefficients were significantly smaller (P less than 0.001), and hepatic ligandin concentrations were significantly larger (p less than 0.001) in phenobarbital-treated rats than in other groups. The efflux coefficient varied inversely with ligandin concentration and the volume of distribution in tissue, as perceived from the plasma space, increased in proportion to the concentration of ligandin. The increased net uptake of tracer bilirubin by the liver of phenobarbital-pretreated animals is due to decreased tracer efflux secondary to the increase in intracellular binding of bilirubin by ligandin.

Animals↗
PubMed1978

Hepatic accumulation and intracellular binding of conjugated bilirubin.

After the intravenous injection of unconjugated [(3)H]bilirubin into normal Sprague-Dawley and Wistar R rats, radiolabeled bile pigments rapidly accumulated in the liver. By 1.5 min after injection, an average of 36% of the injected isotope was present in liver homogenates. Between 3 and 15 min, 37-64% of the total intrahepatic radiolabeled bilirubin was conjugated, as demonstrated by extraction of label into the polar phase of a solvent partition system. This indicates both rapid conjugation, and accumulation of conjugated bilirubin within the liver cell. Fluorometric determination of the dissociation constants of purified bilirubin and its mono- and diglucuronides for homogeneous preparations of two human and four rat glutathione S-transferases, including ligandin, revealed avid binding of all three bile pigments to this class of proteins. Hence, the observation that the intrahepatic bile pigment pool contains substantial amounts of conjugated bilirubin can be attributed to the high binding affinities observed. Thin-layer chromatographic analysis of the (3)H-pigments produced by p-iodoaniline diazotization of homogenates and cytosol demonstrated that the intrahepatic pool of conjugated bilirubin was almost exclusively monoglucuronide. Examination of radiolabeled bilirubin conjugates excreted in bile during the first 20 min after injection of [(3)H]bilirubin showed no preferential excretion of diglucuronide. These studies indicate that (a) both bilirubin and its monoglucuronide accumulate within the liver cell as ligands with the glutathione S-transferase; and (b) bilirubin diglucuronide does not significantly accumulate within the general intrahepatocellular pool of protein-bound bile pigments. The latter observation is compatible with the formation and excretion of bilirubin diglucuronide directly from the canalicular pool of the liver cell.

Animals↗
PubMed1978

Studies of the kinetics of purified conjugated bilirubin-3H in the rat.

Radio-labeled conjugated bilirubin was purified from rat bile by affinity chromatography. Following the intravenous injection of tracer doses of this material, the initial fractional plasma disappearance rate of conjugated bilirubin-3H in the normal Sprague-Dawley rat was found to be approximately 50% faster than that for unconjugated bilirubin-3H (0.51 +/- 0.94 [SE] vs. 0.35 +/- 0.02 min.-1; P less than .001). Hepatic recovery studies in the rat indicated that, over the first four minutes after injection, disappearance of cholephilic anions from plasma is accounted for almost entirely by hepatic uptake. Hence, these studies demonstrate that hepatic uptake of conjugated bilirubin is substantially faster than that of unconjugated bilirubin. Net hepatic clearance of conjugated bilirubin in normal rats was three-fold greater than that of unconjugated bilirubin (0.94 +/- 0.16 vs. 0.30 +/- 0.03 ml./min./100 gm. body weight; P less than .001), presumably reflecting both the more rapid hepatic uptake and the ability of the former pigment to by-pass the conjugation step. Hepatic uptake of conjugated bilirubin was shown to be inhibited by unconjugated bilirubin, sulfobromophthalein and indocyanine green but not by glycocholate. These observations cannot be explained by simple diffusion and suggest that the hepatic uptake mechanism for conjugated bilirubin is the same as that for the unconjugated pigment.

Animals↗
PubMed1976

Rotor's syndrome. A distinct inheritable pathophysiologic entity.

Urinary total, isomer I and isomer III coproporphyrin excretion was determined in 11 patients with Rotor's syndrome, 23 phenotypically normal family members, 16 patients with the Dubin-Johnson syndrome and 20 normal control subjects. Control subjects excreted 24.8 +/- 1.3 per cent (mean SEM) of urinary coproporphyrin as isomer I. Patients with the Dubin-Johnson syndrome excreted 88.9 +/- 1.3 per cent as urinary coproporphyrin I, and patients with Rotor's syndrome excreted 64.8 +/- 2.5 per cent as urinary coproporphyrin I, significantly different from the control subjects and the patients with the Dubin-Johnson syndrome (p less than 0.001). Eight phenotypically normal parents and children of patients with Rotor's syndrome excreted 42.9 +/- 5.4 per cent as urinary coproporphyrin I, intermediate between results in patients with Rotor's syndrome and control subjects (p less than 0.001). Total urinary coproporphyrin excretion was markedly increased in patients with Rotor's syndrome (332 +/- 86 mug/g creatinine) as compared to that in control subjects (p less than 0.001) or obligate heterozygotes (p less than 0.025). With respect to urinary coproporphyrin excretion, Rotor's syndrome and Dubin-Johnson syndrome are both inherited as autosomal recessive traits and are separate pathophysiologic entities. Study of rare but distinct inheritable disorders, such as these, provide insight into the functional dissociation of hepatic transport mechanisms.

Animals↗
PubMed1976

Abnormal hepatic transport of indocyanine green in Gilbert's syndrome.

The plasma fractional disappearance rate of indocyanine green (kICG, min-1) and the absolute hepatic ICG removal rate (VICG, mg per kg per min) were determined in 26 patients with Gilbert's syndrome (GS) and 19 normal control volunteers following intravenous administration of doses of 0.5, 2.0, 3.5, and 5.0 mg per kg of dye. The diagnosis of GS was based on studies of radiobilirubin kinetics in all cases and liver biopsy in 22 cases. The patients were further classified into 3 subgroups, based on patterns of sulfobromophthalein (BSP) kinetics, as follows: GS I (15 patients) had normal BSP metabolism; GS II (5 patients) had a defect in BSP metabolism beyond the stage of initial hepatic uptake; and GS III (6 patients) had a defect in the initial hepatic uptake of BSP (Gastroenterology 63:472-481, 1972). Both kICG and VICG were significantly reduced, compared to normal controls, in the GS III group with defective BSP uptake, but did not differ significantly from normal in the GS I and GS II groups. Michaelis-Menten analysis of the data indicated that VMAX for ICG uptake in the GS III group (1.2 +/- 0.6 mg per min per kg) was significantly reduced compared to the previously established normal value of 3.6 +/- 0.6 mg per min per kg; (P less than 0.01). For the total population of 26 patients with Gilbets' syndrome, there was a highly significant correlation (r= 0.77, P less than 0.01) between kICG and lambda21BSP, the fractional hepatic uptake rate for BSP. These studies confirm previous work indicating that patients with Gilbert's syndrome constitute a heterogeneous population with regard to defects in hepatic organic anion transport, some of the defects not being attributable to glucuronyl transferese deficiency. Future studies of Gilbert's syndrome must take into account the existence of these subgroups, since they may have different underlying pathogenetic mechanisms.

Adolescent↗