Iron uptake and heme synthesis by isolated rat liver mitochondria. Diferric transferrin as iron donor and the effect of pyrophosphate

Isolated rat liver mitochondria accumulate iron from fully saturated transferrin at neutral pH. With 5 microM iron as diferric transferrin, accumulation at 30 degrees C amounts to approx. 40 pmol/mg protein per h. With access to a suitable porphyrin substrate, 70-80% of the amount of iron accumulated is recovered in heme. Mobilization of iron and synthesis of heme both depend on a functioning respiratory chain. Vacant iron-binding sites on mono- and apotransferrin compete with the mitochondria for iron mobilized from transferrin. Pyrophosphate at concentrations in the range 10-50 microM enhances mobilization of iron, counterbalances the inhibitory effect of mono- and apotransferrin and enhances metallochelatase activity. The results emphasize the putative suitability of pyrophosphate as an intracellular iron-transport ligand in situ.     

Pathways in the binding and uptake of ferritin by hepatocytes

The binding and uptake of rat liver ferritin by primary cultures of rat liver hepatocytes was studied in order to assess the relative importance of saturable, high-affinity pathways and nonspecific processes in the incorporation of the protein by the cells. To minimize artifacts, ferritin not subjected to heat treatment and labeled in vivo with 59Fe was used. Binding to cell membranes was estimated from incubations performed at 4 degrees C. After 2 h, when a steady state in cell-associated ferritin had been achieved, approx. 4-10(4) binding sites per cell were observed, with an affinity constant for ferritin of 1 x 10(9) M-1. At 37 degrees C, the maximal uptake from these sites was 1.3 x 10(5) ferritin molecules/cell per h. For ferritin molecules bearing an average of 2400 iron atoms, this uptake amounts to 5 x 10(6) iron atoms/cell per min. Half-maximal uptake was achieved at a ferritin concentration, or KM1, of 3 x 10(-9) M. Although uptake rates at least a thousand times greater could be achieved by binding to the much larger number of low-affinity sites, the apparent KM2 for such 'nonspecific' uptake was 4 x 10(-7) M. At ferritin concentrations up to 2 nM, at least 90% of ferritin bound and taken up by hepatocytes involves saturable, high-affinity sites, presumably true ferritin receptors.     

Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

The effects of an antibody to the rat transferrin receptor and of rat serum albumin on the uptake of diferric transferrin by rat hepatocytes

The role of high-affinity specific transferrin receptors and low-affinity, non-saturable processes in the uptake of transferrin and iron by hepatocytes was investigated using fetal and adult rat hepatocytes in primary monolayer culture, rat transferrin, rat serum albumin and a rabbit anti-rat transferrin receptor antibody. The intracellular uptake of transferrin and iron occurred by saturable and non-saturable mechanisms. Treatment of the cells with the antibody almost completely eliminated the saturable uptake of iron but had little effect on the non-saturable process. Addition of albumin to the incubation medium reduced the endocytosis of transferrin by the cells but had no significant effect on the intracellular accumulation of iron. The maximum effect of rat serum albumin was observed at concentrations of 3 mg/ml and above. At a low incubation concentration of transferrin (0.5 microM), the presence of both rat albumin and the antibody decreased the rate of iron uptake by the cells to about 15% of the value found in their absence, but to only 40% when the diferric transferrin concentration was 5 microM. These results confirm that the uptake of transferrin-bound iron by both fetal and adult rat hepatocytes in culture occurs by a specific, receptor-mediated process and a low-affinity, non-saturable process. The low-affinity process increases in relative importance as the iron-transferrin concentration is raised.     

Selective inhibition of long-chain fatty acid uptake in short-term cultured rat hepatocytes by an antibody to the rat liver plasma membrane fatty acid-binding protein

Uptake of long-chain fatty acids by short-term cultured hepatocytes was studied. Rat hepatocytes, which were cultured for 16 h on plastic dishes (3.6 X 10(6) cells/dish), were incubated with [3H]oleate in the presence of various concentrations of bovine serum albumin as a function of the concentration of unbound [3H]oleate in the medium. At 37 degrees C initial uptake velocity (V0) was saturable (Km = 9 X 10(-8) M; Vmax = 835 pmol/min per mg protein). V0 was temperature dependent with an optimum at 37 degrees C and markedly reduced at 4 degrees C and 70 degrees C. To evaluate the biologic significance of a previously isolated rat liver plasma membrane fatty acid-binding protein as putative carrier protein in the hepatocellular uptake of fatty acids, cultured hepatocytes were treated with a monospecific rabbit antibody (IgG-fraction) to this membrane protein or the IgG-fraction of the pre-immune serum as controls. Uptake kinetics of [3H]oleate in antibody pretreated short-term cultured hepatocytes revealed a depression of Vmax by 70%, while Km was only reduced by 16% compared to controls, indicating a predominant non-competitive type of inhibition. V0 of a variety of long-chain fatty acids (oleic acid, arachidonic acid, palmitic acid, stearic acid) was reduced by 56-69%, while V0 of [35S]sulfobromophthalein, [3H]cholic acid and [14C]taurocholic acid remained unaltered. These data support the concept that in the system of cultured hepatocytes, uptake of long-chain fatty acids is mediated by the rat liver plasma membrane fatty acid-binding protein.     

Endothelial binding of transferrin in fractionated liver cell suspensions

Several studies using crude liver cell suspensions incubated with labeled transferrin have led to a conclusion that hepatocytes have transferrin receptors. When a visual probe, which permits evaluation of transferrin binding to individual cells, was used, the binding was unexpectedly found to be limited to endothelial cells in liver cell suspensions. Neither hepatocytes nor Kupffer cells contained transferrin receptors. In the present study, we fractionated liver cell suspensions using metrizamide gradients and centrifugal elutriation to obtain hepatocytes, Kupffer cell and endothelial cell fractions of high purity. Incubation of these fractions with 125I- or 59Fe-labeled transferrin led to exclusive binding to endothelial cells but not hepatocytes nor Kupffer cells. Kinetic analysis demonstrated Kd of 1.9 X 10(-7) M, Bmax of 3.1 pmol/10(6) cells per min, corresponding to 2.1 X 10(5) molecules/cell per min. At 4 degrees C, the binding reached a steady-state plateau within 5 min. Comparison of our data with those of previous investigators demonstrates a consistency if we consider that crude liver cell suspensions are contaminated with 2-3% endothelial cells. Thus, the previously reported findings may be entirely due to the contamination of crude liver cell suspensions with a small number of endothelial cells.     

Transferrin endocytosis and the mechanism of iron uptake by reticulocytes in the toad (Bufo marinus)

Reticulocytosis was induced in toads (Bufo marinus) by treatment with phenylhydrazine. Iron and transferrin uptake and transferrin endocytosis and exocytosis by these cells were measured. The mean number of transferrin receptors per cell was found to be 4.5 X 10(5) and the affinity constant of transferrin to receptors was 0.2 X 10(7) M-1. Iron and transferrin uptake were temp.-dependent processes. An inflection point occurred at 15-16 degrees C in the Arrhenius plots of endocytosis and iron uptake. The activation energies of these two processes above and below the inflection temperature were 31 and 71 kJ/mol. It is concluded that iron uptake by immature toad erythroid cells occurs by receptor-mediated endocytosis which still functions at temps as low as 5 degrees C.     

Hepatocellular transport of cyclosomatostatins: evidence for a carrier system related to the multispecific bile acid transporter.

The uptake of the cyclopeptide c(Phe-Thr-Lys-Trp-Phe-D-Pro) (008), an analog of somatostatin with retro sequence, was studied in isolated hepatocytes. 008 is taken up by hepatocytes in a concentration-, time-, energy- and temperature- dependent manner. Since 008 is hydrophobic, it binds rapidly to liver cells. This is evident by the positive intercept at the gamma-axis in the uptake curves. At higher concentrations, a minor part of the transport occurs by diffusion at a rate of 8.307.10(-6) cm/s. This part of diffusion is measured at 4 degrees C and can be subtracted from the uptake at 37 degrees C resulting in the carrier mediated part of uptake which is saturable. Kinetic parameters for the saturable part of uptake are Km 1.5 microM and Vmax 40.0 pmol/mg per min. The transport is decreased in the absence of oxygen and in the presence of metabolic inhibitors. Uptake is accelerated at temperatures above 20 degrees C. The activation energy was determined to be 30.77 kJ/mol. The membrane potential and not a sodium gradient is the main driving force for 008 transport. Cholate (a typical substrate of the multispecific bile acid transporter) and taurocholate are mutual competitive inhibitors of 008 uptake. Phalloidin, antamanide and iodipamide, typical foreign substrates of the transporter, interfere with the uptake of 008. AS 30D ascites hepatoma cells, known to be unable to transport bile acids, phalloidin and iodipamide, are also unfit to transport 008. Interestingly, sulfobromophthalein (BSP) but not rifampicin, both foreign substrates of the bilirubin carrier, inhibits the transport of 008 in a competitive manner.     

Transferrin receptors,iron transport and ferritin metabolism in friend erythroleukemia cells

Four aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of erythroid differentiation by dimethyl sulfoxide. (1) The binding of ¹²⁵I-labeled transferrin was determined over a range of transferrin concentrations from 0.5 to 15 μM. Scatchard analysis of the binding curves demonstrated equivalent numbers of transferrin binding sites per cell: 7.78 ± 2.41 · 10⁵ in non-induced cells and 9.28 ± 1.57 · 10⁵ after 4 days of exposure to dimethyl sulfoxide. (2) The rate of iron transport was determined by measuring iron uptake from ⁵⁹Fe-labeled transferrin. Iron uptake in non-induced cells was approx. 17 000 molecules of iron/cell per min; 24 h after addition of dimethyl sulfoxide it increased to 38 000, and it rose to maximal levels of approx. 130 000 at 72 h. (3) Heme synthesis, assayed qualitatively by benzidine staining and measured quantitatively by incorporation of ⁵⁹Fe or [2-¹⁴C]glycine into cyclohexanone-extracted or crystallized heme, was not detected until 3 days after addition of dimethyl sulfoxide, when 12% of the cells were stained by benzidine and 6 pmol ⁵⁹Fe and 32 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. After 4 days, 60% of the cells were benzidine positive and 34 pmol ⁵⁹Fe and 90 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. (4) The rate of incorporation of ⁵⁹Fe into ferritin, measured by immunoprecipitation of ferritin by specific antimouse ferritin immunoglobulin G, rose from 4.4 ± 0.6 cells to 18.4 ± 1.3 pmol ⁵⁹Fe/h per 10⁸ cells 3 days after addition of dimethyl sulfoxide, and then fell to 11.6 ± 3.1 pmol 4 days after dimethyl sulfoxide when heme synthesis was maximal. These studies indicate that one or more steps in cellular iron transport distal to transferrin binding is induced early by dimethyl sulfoxide and that ferritin may play an active role in iron delivery for heme synthesis.     

A novel iron uptake mechanism mediated by GPI-anchored human p97.

The established process for iron uptake into mammalian cells involves transferrin and its receptor. Here, the role of the glycosyl-phosphatidylinositol (GPI)-linked transferrin homologue, melanotransferrin or p97, was studied using CHO cell lines defective in the transferrin receptor (TR) and transfected with human TR and/or human p97. The presence of p97 doubled the iron uptake, which could be explained by the binding of one atom of iron to one molecule of p97. The internalization of iron was shown to be temperature sensitive and saturated at a media iron concentration of 2.5 micrograms/ml with a Vmax of 0.1 pmol Fe/10(6) cell/min and a Km of 2.58 microM for p97. Treatment of the cells with either phosphatidylinositol-phospholipase C or monoclonal antibodies against p97 resulted in over a 50% reduction and a 47% increase in the iron uptake respectively. These data identify p97 as a unique cell surface GPI-anchored, iron binding protein involved in the transferrin-independent uptake of iron in mammals.     

A membrane transporter mediates access of uridine 5'-diphosphoglucuronic acid from the cytosol into the endoplasmic reticulum of rat hepatocytes: implications for glucuronidation reactions

Hepatic glucuronidation of a wide variety of substrates is catalyzed by the membrane-bound UDP-glucuronosyltransferases. Uridine 5'-diphosphoglucuronic acid (UDP-GlcUA) is the essential cosubstrate for all UDP-glucuronosyltransferase-mediated reactions. The mechanism by which this bulky, hydrophilic nucleotide-sugar is transported from the cytosol (where it is synthesized) to its binding site(s) on the enzyme is unknown. To determine whether a membrane carrier mediates the access of UDP-GlcUA into the endoplasmic reticulum, the transport of uridine 5'-diphospho-D-[U-14C]glucuronic acid into vesicles of rough and smooth endoplasmic reticulum isolated from rat liver was investigated at 38 degrees C using a rapid filtration technique. Uptake of UDP-GlcUA by both rough and smooth vesicles was extremely rapid (linear for only 10-20 s) and temperature-dependent (negligible at 4 degrees C). UDP-GlcUA uptake was saturable, and similar kinetic parameters were obtained for rough and smooth vesicles (Km 1.9 microM, Vmax 443 pmol/mg protein per min, and Km 1.3 microM, Vmax 503 pmol/mg protein per min, respectively). The uptake of UDP-GlcUA also exhibited a high degree of specificity, since many related compounds, including UMP, UDP and UDP-Glc, did not influence uptake. In addition, the non-penetrating inhibitors of anion transport, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and probenecid, markedly inhibited UDP-GlcUA uptake. Finally, osmotic modulation of the intravesicular volume did not affect total uptake of UDP-GlcUA by membrane vesicles at equilibrium, indicating that this nucleotide-sugar is transported into the membrane rather than the intravesicular space. Collectively, these data provide direct evidence for a specific, carrier-mediated uptake process, which transports UDP-GlcUA from the cytosol into the endoplasmic reticulum of hepatocytes. This UDP-GlcUA transporter may be involved in the regulation of hepatic glucuronidation reactions.     

Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine

Rat liver ferritin is an effective donor of iron to rat hepatocytes. Uptake of iron from ferritin by the cells is partially inhibited by including apotransferrin in the culture medium, but not by inclusion of diferric transferrin. This inhibition is dependent on the concentration of apotransferrin, with a 30% depression in iron incorporation in the cells detected at apotransferrin concentrations above 40 micrograms/ml. However, apotransferrin does not interfere with uptake of 125I-labeled ferritin, suggesting that apotransferrin decreases retention of iron taken up from ferritin by hepatocytes by sequestering a portion of released iron before it has entered the metabolic pathway of the cells. The iron chelators desferrioxamine (100 microM), citrate (10 mM) and diethylenetriaminepentaacetate (100 microM) reduce iron uptake by the cells by 35, 25 and 8%, respectively. In contrast, 1 mM ascorbate increases iron accumulation by 20%. At a subtoxic concentration of 100 microM, chloroquine depresses ferritin and iron uptake by hepatocytes by more than 50% after 3 h incubation. Chloroquine presumably acts by retarding lysosomal degradation of ferritin and recycling of ferritin receptors.     

Effect of polyethylene glycol on lipid peroxidation in cold-stored rat hepatocytes

A mechanism suggested to cause injury to preserved organs is the generation of oxygen free radicals either during the cold-storage period or after transplantation (reperfusion). Oxygen free radicals can cause peroxidation of lipids and alter the structural and functional properties of the cell membranes. Methods to suppress generation of oxygen free radicals of suppression of lipid peroxidation may lead to improved methods of organ preservation. In this study we determined how cold storage of rat hepatocytes affected lipid peroxidation by measuring thiobarbituric acid reactive products (malondialdehyde, MDA). Hepatocytes were stored in the UW solution +/- glutathione (GSH) or +/- polyethylene glycol (PEG) for up to 96 h and rewarmed (resuspended in a physiologically balanced saline solution and incubated at 37 degrees C under an atmosphere of oxygen) after each day of storage. Hepatocytes rewarmed after storage in the UW solution not containing PEG or GSH showed a nearly linear increase in MDA production with time of storage and contained 1.618 +/- 0.731 nmol MDA/mg protein after 96 h. When the storage solution contained PEG and GSH there was no significant increase in MDA production after up to 72 h of storage and at 96 h MDA was 0.827 +/- 0.564 nmol/mg protein. When freshly isolated hepatocytes were incubated (37 degrees C) in the presence of iron (160 microM) MDA formation was maximally stimulated (3.314 +/- 0.941 nmol/mg protein). When hepatocytes were stored in the presence of PEG there was a decrease in the capability of iron to maximally stimulate lipid peroxidation. The decrease in iron-stimulated MDA production was dependent upon the time of storage in PEG (1.773 nmol/mg protein at 24 h and 0.752 nmol/mg protein at 48 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of ochratoxin A by primary cultures of rat hepatocytes.

Association of ochratoxin A with cultured rat hepatocytes occurs at 4 degrees C, and the saturation level in the medium is 0.3 mM ochratoxin A, with maximal binding after 60 min. At 37 degrees C the level of cell-associated ochratoxin A increased up to 6 h and remained at 2 nmol of toxin per mg of cell protein for 30 h. With increasing concentrations of ochratoxin A, increasing amounts of the toxin accumulated in the cells; saturation occurred at a concentration of 0.3 mM. Ochratoxin A was metabolized by hepatocytes at 37 degrees. (4R)-4-Hydroxyochratoxin A appeared in the medium at a maximal level (about 30 nmol/mg of cell protein) at an ochratoxin A concentration of 0.25 mM after 48 h of incubation. Small amounts of (4S)-4-hydroxyochratoxin A were detected only after incubation for 22 h or longer.     

Hepatocytes and reticulocytes have different mechanisms for the uptake of iron from transferrin

The uptake of iron from transferrin by isolated rat hepatocytes and rat reticulocytes has been compared. The results show the following. 1) Reticulocytes and hepatocytes express plasma membrane NADH:ferricyanide oxidoreductase activity. The activity, expressed per 10(6) cells, is approximately 60-fold higher in the hepatocyte than in the reticulocyte. 2) Hepatocyte plasma membrane NADH:ferricyanide oxidoreductase activity and uptake of iron from transferrin are stimulated by low oxygen concentration and inhibited by iodoacetate. In reticulocytes, similar changes are seen in NADH:ferricyanide oxidoreductase activity, but not on iron uptake. 3) Ferricyanide inhibits the uptake of iron from transferrin by hepatocytes, but has no effect on iron uptake by reticulocytes. 4) Perturbants of endocytosis and endosomal acidification have no inhibitory effect on hepatocyte iron uptake, but inhibit reticulocyte iron uptake. 5) Hydrophilic iron chelators effectively inhibit hepatocyte iron uptake, but have no effect on reticulocyte iron uptake. Hydrophobic iron chelators generally inhibit both hepatocyte and reticulocyte iron uptake. 6) Divalent metal cations with ionic radii similar to or less than the ferrous iron ion are effective inhibitors of hepatocyte iron uptake with no effect on reticulocyte iron uptake. The results are compatible with hepatocyte uptake of iron from transferrin by a reductive process at the cell surface and reticulocyte iron uptake by receptor-mediated endocytosis.     

Transferrin iron interactions with cultured hepatocellular carcinoma cells (PLC/PRF/5)

Hepatocellular carcinoma cells of the PLC/PRF/5 cell line had 1.9 x 10(5) transferrin receptors per tumor cell with a Kd of 1.5 x 10(-8) M. At high concentrations of transferrin the binding was not saturable. Transferrin internalization by hepatoma cells was shown by time and temperature-dependent binding studies and by pronase experiments. Transferrin recycling was confirmed by the demonstration of a progressive increase in the cellular molar ratios of iron to transferrin and by chase experiments. Ammonium chloride interfered with iron unloading. The vinca alkaloid vincristine inhibited iron and transferrin uptake. The hepatocarcinoma cells appeared to lack asialoglycoprotein receptors and therefore internalized partially desialated transferrin by the regular route. Iron uptake from transferrin was markedly inhibited by the hydrophobic ferrous chelator 2,2' bipyridine but was relatively unaffected by the hydrophilic ferric chelator desferroxamine. The implication that ferrous iron was involved in postendocytic transvesicular membrane iron transport was supported by a study in which hepatoma cells were shown to take up large amounts of ferrous iron suspended in 270 mM sucrose at pH 5.5. The interaction at this pH between surface labeled hepatoma cell extracts and ferrous iron on a Sephacryl S-300 column suggested that the postendocytic transvesicular transport of iron through the membrane was in part protein mediated. The endocytosed iron in hepatoma cells was found in association with ferritin (33%), transferrin (31%) and a low molecular weight fraction (21%).     

Inward fluxes of adenosine in erythrocytes and cultured cells measured by a quenched-flow method.

Dilazep, a vasodilator previously recognized as an inhibitor of adenosine permeation, very rapidly blocked the uptake of adenosine by cultured L5178Y cells, and accordingly was used as a quencher in a simple quenched-flow system for measuring cellular uptake of nucleosides during very short intervals. Time courses of cellular uptake of adenosine, assayed during intervals between 0.05 and 0.5s with the quenched-flow system, were linear and defined initial rates of adenosine uptake. The latter are rates of inward transport of adenosine. Kinetic constants for that process in cultured S49 cells determined with the quenched-flow procedure were similar to those determined with an assay dependent on manual timing. In studies of adenosine uptake kinetics in human erythrocytes at 22 degrees C and 37 degrees C in which the quenched-flow procedure was used, time courses of adenosine uptake were linear at both temperatures and defined initial uptake rates; kinetic constants (means +/- S.E.M.) at 22 degrees C (n = 8) were Km 25 +/- 14 microM and Vmax. 15 +/- 5 pmol/s per microliter of cell water and at 37 degrees C (n = 3) were Km 98 +/- 17 microM and Vmax. 80 +/- 9 pmol/s per microliter of cell water.     

Bile acid synthesis and secretion by rabbit hepatocytes in primary monolayer culture: comparison with rat hepatocytes

Rabbit hepatocytes isolated after liver perfusion with collagenase were maintained in primary monolayer culture for periods up to 96 h. Bile acid synthesis and secretion was measured by capillary gas-liquid chromatography and by a rapid enzymatic-bioluminescence assay. As expected from the bile acid profile of rabbit gallbladder bile, cholic acid was the only bile acid synthesized in detectable amounts and was produced at a linear rate of 170 pmol/h per mg cell protein from 24 to 96 h in culture. Ketoconazole (20 microM) inhibited cholic acid synthesis and secretion by 78%, whereas the bile acids chenodeoxycholic acid (100 microM), deoxycholic acid (100 microM) or lithocholic acid (2 microM) had no effect. When rat hepatocytes were cultured under identical conditions, the rate of bile acid synthesis was found to be only 12 pmol/h per mg cell protein, a value in agreement with previous work. The large difference in rates of bile acid synthesis between rabbit and rat hepatocytes may be due to rapid loss of cytochrome P-450 from rat hepatocytes when placed in monolayer culture. Although reportedly active in cholesterol 7 alpha-hydroxylation, form 4 cytochrome P-450 levels in rabbit hepatocytes did not correlate with rates of bile acid synthesis.     

Transferrin receptors and transferrin iron uptake by cultured human blood monocytes

Transferrin receptors have been previously found on human macrophages and it has also been shown that transferrin iron is taken up by these cells. It has therefore been inferred that the uptake is receptor mediated and involves an endocytic pathway. The subject was addressed directly in the present study in which the transferrin-iron-receptor interaction was characterized in cultured human blood monocytes. Specific, saturable diferric transferrin binding was demonstrated, with a kDa of 3.6 X 10(-8) M and a calculated receptor density of 1.25-2.5 X 10(5) receptors per cell. Incubation at 4 degrees C markedly reduced transferrin binding and completely inhibited iron uptake. Chase experiments confirmed progressive cellular loading of iron, with concomitant loss of transferrin. Inhibitors of endocytic vesicle acidification (ammonium chloride and 2,4-dinitrophenol) inhibited iron unloading from endocytosed diferric transferrin, while microtubular inhibitors (colchicine and vindesine) and a microfilament inhibitor (cytochalasin B) reduced diferric transferrin uptake but had little effect on the iron unloading pathway. A similar effect was noted with a calcium ion antagonist (verapamil) and with 2 calmodulin antagonists (chlorpromazine and imipramine). These latter findings suggest the importance of cytoskeleton-membrane interactions via a calcium, calmodulin and protein kinase C mediated system. Endocytosed iron accumulated progressively as ferritin within the cultured monocytes.     

Uptake of free hemoglobin by rat liver parenchymal cells

Parenchymal cells isolated from rat liver are capable of taking up free hemoglobin. Uptake was saturable with a concentration for half-maximal velocity of 1.35 mg/ml (1.99 X 10(-5) M) hemoglobin. At a concentration of 0.088 mg/ml, the endocytic index for hemoglobin uptake was 4.5 microliters/h per mg of cell protein. This may be compared with the rate of fluid pinocytosis by these cells of 0.025 microliter/h per mg of cell protein (determined with yeast invertase as the marker). Free beta globin chains were also taken up with an endocytic index of 26.7 microliters/h per mg of cell protein at a beta chain concentration of 0.075 mg/ml. Hemoglobin inhibited the uptake of labeled beta globin. Hemoglobin-haptoglobin complex at a concentration of 0.12 mg/ml (as hemoglobin) was cleared at a rate of 0.89 microliter/h per mg cell protein and its uptake was also inhibited by free hemoglobin. We conclude that haptoglobin serves to conserve the iron of hemoglobin by preventing its renal clearance and not by promoting its hepatic uptake.