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Biomedical subjects

E H Morgan

Publications and source records attributed to E H Morgan.

At least 73 records · Page 4Linked to original sources
PubMed1990

Effect of cellular iron concentration on iron uptake by hepatocytes.

The effect of intracellular iron content on transferrin and iron uptake by cultured hepatocytes isolated from fetal rat liver was examined with ferric ammonium citrate and the iron chelator desferrioxamine (DFO). Incubation of the cells with ferric ammonium citrate for 24 h significantly increased the cellular nonheme iron level, whereas the number of transferrin binding sites and the uptake of transferrin and iron were reduced. In contrast, when iron-treated cells were incubated with DFO for 24 h, the cellular nonheme iron level was not altered, but the number of transferrin binding sites was increased. Treatment of the cells with exogenous iron and/or DFO did not affect the uptake of transferrin and iron by the nonsaturable processes. These results indicated that, in cultured hepatocytes, transferrin receptor expression and the subsequent uptake of transferrin and iron are regulated by the size of an intracellular, chelatable iron pool, whereas the uptake of iron by the nonsaturable processes is dependent on the extracellular transferrin concentration.

Animals↗
PubMed1989

Calcium chelators induce association with the detergent-insoluble cytoskeleton and functional inactivation of the transferrin receptor in reticulocytes.

Incubation of reticulocytes with EDTA, EGTA (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), but not with desferrioxamine B, at temperatures above 20 degrees C resulted in the loss of their ability to take up iron in a temperature-, time- and concentration-dependent manner. No inhibition of transferrin or iron uptake occurred if the incubations were performed at 20 degrees C or below. At higher temperatures, the inhibition was attributable to loss of functional transferrin receptors, not to altered affinity or endocytosis of the remaining receptors. The changes could not be reversed by washing the cells and reincubation in the presence of Ca2+, Mg2+ or Zn2+. However, they could be completely prevented by performing the initial incubation with chelators in the presence of diferric transferrin and partly prevented by the use of apotransferrin. Incubation with the chelators resulted in much less reduction in the ability of the cells to bind anti-transferrin receptor immunoglobulin than transferrin. The fate of the receptor was studied by polyacrylamide gel electrophoresis of reticulocyte membrane proteins before and after extraction with Triton X-100, and by immunological staining of Western blots for the transferrin receptor. Treatment of the cells with EDTA led to a loss of the ability of Triton X-100 to solubilize the receptor and its retention in the Triton-insoluble cytoskeletal matrix of the cells. It is concluded that incubation of reticulocytes with the chelators at temperatures above 20 degrees C causes an altered interaction of the transferrin receptor with the cytoskeleton. This change, which is probably due to chelation of Ca2+ in the cell membrane, is accompanied by an irreversible loss of the receptor's ability to bind transferrin.

Animals↗
PubMed1989

Transformation-induced changes in transferrin and iron metabolism in myogenic cells.

The uptake of transferrin and iron by cultured myogenic cells transformed with a temperature-sensitive strain of the Rous sarcoma virus (tsLA24) was compared with that of normal developing myogenic cells which were proliferating at the same rate as the transformed cells. The mechanism of transferrin and iron uptake was the same in the transformed cells as in normal myogenic cells and involved receptor-mediated endocytosis of transferrin. However, there were differences in transferrin receptor numbers and receptor function. The number of receptors in transformed cells was more than twice as great as in the normal cells largely due to increased surface receptor numbers. Despite this, the rate of iron uptake increased by only 20% in the transformed cells due to less efficient cycling of the transferrin receptors and less efficient release of iron from transferrin to intracellular sites. Some internalized iron was released from the transformed cells still bound to transferrin. A fast and a slow rate of transferrin exocytosis were identified in transformed cells, as in normal cells, indicating that there were at least two intracellular pathways for transferrin. The fast pathway predominated in the transformed cells, compared with an equal importance of the two pathways in the normal cells.

Animals↗
PubMed1989

A method for infection of cultured myogenic cells with Rous sarcoma virus using polybrene.

A method for efficiently infecting primary myogenic cultures with a temperature-sensitive variant of the Prague strain of Rous sarcoma virus (RSV, tsLA24) to obtain a high yield of transformed myogenic cells is reported. It incorporates the use of an amorphous polymer of polycations, Polybrene, to enhance the absorption of the virus by the muscle cells. In addition, other steps which were shown to be important were a) to allow cell attachment before infection, b) to infect at 35 degrees C in low protein medium, c) to use a density of 1 to 1.5 X 10(6) cells/60-mm dish, d) gentle agitation during infection, and e) to minimize the number of passages after infection. The use of the temperature-sensitive virus provided a means of confirming the presence of myogenic cells in transformed cultures. When infected cells were maintained at 35 degrees C (the permissive temperature for virus activity) they exhibited the characteristics of transformed cells. These characteristics included altered cell morphology, the absence of contact-inhibited growth, growth in semisolid medium, and expression of the src oncogene. In contrast, when infected cells were maintained at 41 degrees C (the nonpermissive temperature for virus activity) they do not express src and showed normal myogenic development and ultimately formed myotubes.

Animals↗
PubMed1989

Uptake and distribution of transferrin and iron in perfused, iron-deficient rat liver.

Uptake of transferrin and iron by the rat liver was investigated by perfusion in vitro with 125I-59Fe-labeled rat transferrin and subcellular fractionation on sucrose density gradients. Most of the 125I-transferrin was located in a low-density vesicle fraction. The 59Fe was in three peaks, of lower, the same, and higher densities than the transferrin peak. Iron deficiency resulted in a large increase in transferrin and iron uptake into all subcellular fractions. When livers were perfused with increasing concentrations of transferrin the uptake into the different peaks of transferrin and iron increased in a curvilinear fashion, which indicated that uptake occurred by saturable and nonsaturable processes, both of which increased in iron deficiency. In contrast, the uptake of 131I-labeled rat serum albumin increased linearly with concentration, and there was no difference between control and iron-deficient livers. It is concluded that iron deficiency leads to an increase in the number of high-affinity transferrin receptors and receptor-mediated endocytosis of transferrin. It also increases a nonsaturable transferrin uptake process that is probably due to adsorptive, but selective, endocytosis of transferrin.

Animals↗
PubMed1988

Differences in transferrin receptor function between normal developing and transformed myogenic cells as revealed by differential effects of phorbol ester on receptor distribution and rates of iron uptake.

The effects of the tumor promotor, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), on the intra- and extracellular distribution of transferrin receptors and rates of iron uptake were studied in normal developing myogenic cells and myogenic cells transformed with a temperature-sensitive strain of the Rous sarcoma virus. In normal developing cells PMA was found to increase the rate of iron uptake by 15-30%. There was, however, no effect on transferrin receptor distribution, suggesting that the increase in iron uptake was due to stimulation of the rate of receptor cycling. In contrast, in transformed myogenic cells, PMA had no effect even at concentrations 10 times those effective in normal myogenic cells. The specificity of PMA was demonstrated by comparison with 4 alpha-phorbol which had no effect compared with the control cells which were incubated with dimethyl sulfoxide, the solvent used to dissolve the phorbols. These results indicate a functional difference in the transferrin receptor between normal and transformed myogenic cells. The data for normal myogenic cells are similar to those previously reported for normal erythroid cells, but differ from those for some transformed cell lines in which phorbol esters were shown to cause internalization of transferrin receptors.

Animals↗
PubMed1988

Membrane transport of non-transferrin-bound iron by reticulocytes.

The transport of non-transferrin-bound iron into rabbit reticulocytes was investigated by incubating the cells in 0.27 M sucrose with iron labelled with 59Fe. In most experiments the iron was maintained in the reduced state, Fe(II), with mercaptoethanol. The iron was taken up by cytosolic, haem and stromal fractions of the cells in greater amounts than transferrin-iron. The uptake was saturable, with a Km value of approx. 0.2 microM and was competitively inhibited by Co2+, Mn2+, Ni2+ and Zn2+. It ceased when the reticulocytes matured into erythrocytes. The uptake was pH and temperature sensitive, the pH optimum being 6.5 and the activation energy for iron transport into the cytosol being approx. 80 kJ/mol. Ferric iron and Fe(II) prepared in the absence of reducing agents could also be transported into the cytosol. Sodium chloride inhibited Fe(II) uptake in a non-competitive manner. Similar degrees of inhibition was found with other salts, suggesting that this effect was due to the ionic strength of the solution. Iron chelators inhibited Fe(II) uptake by the reticulocytes, but varied in their ability to release 59Fe from the cells after it had been taken up. Several lines of evidence showed that the uptake of Fe(II) was not being mediated by transferrin. It is concluded that the reticulocyte can transport non-transferrin-bound iron into the cytosol by a carrier-mediated process and the question is raised whether the same carrier is utilized by transferrin-iron after its release from the protein.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

Effect of osmolar and ionic composition of the extracellular fluid on transferrin endocytosis and exocytosis and iron uptake by reticulocytes.

The effects of osmolar and ionic factors on endocytosis and exocytosis were investigated using rabbit reticulocytes and 125I-59Fe labelled transferrin. Endocytosis and exocytosis of transferrin and the uptake of iron were inhibited by increasing the osmolality or decreasing the ionic strength or pH of the cell incubation medium. However, elevation of the pH above 8.0 inhibited endocytosis but not exocytosis. Replacement of the NaCl in the incubation medium by Nal, NaF, NaSCN, NaClO4, Na2SO4, Na phosphate, or Na Hepes inhibited endocytosis and iron uptake but only Nal, NaF, and NaSCN inhibited exocytosis. Transferrin exocytosis was insensitive to inhibitors of anion or cation transport, but endocytosis and iron uptake were inhibited by several anion transport inhibitors. Overall, transferrin endocytosis was more sensitive than exocytosis to most of the factors which were investigated, and the effects on the rates of endocytosis and iron uptake were quantitatively very similar. The results provide strong support for the concept that transferrin endocytosis is a necessary step in iron uptake by reticulocytes. They do not support the chemiosmotic models of exocytosis in their present formulations, but do not rule out the possible role of an osmotic event in exocytosis.

Ammonium Chloride↗
PubMed1988

Transferrin endocytosis and iron uptake in developing myogenic cells in culture: effects of microtubular and metabolic inhibitors, sulphydryl reagents and lysosomotrophic agents.

The experiments described in this study were designed to investigate receptor-mediated endocytosis of transferrin and its role in iron uptake by cultured chick presumptive myoblasts (dividing and non-dividing) and myotubes. The effects of a variety of inhibitors on the internalization of transferrin and iron were investigated and three main effects were found: (i) sulphydryl reagents and microtubular inhibitors reduced the rate of transferrin and iron internalization to similar degrees, (ii) metabolic inhibitors reduced the rate of iron uptake more than that of transferrin endocytosis, and (iii) lysosomotrophic agents almost completely abolished iron accumulation by the cells without any effect on the rate of transferrin internalization. The results suggest that metabolic energy is required not only for the endocytosis of transferrin but also for subsequent steps in the iron uptake process, and that iron release from transferrin occurs in acidified endosomes. Overall, these experiments show that all or virtually all of the iron taken up by developing muscle cells from transferrin occurs as a consequence of receptor-mediated endocytosis of the protein.

2,4-Dinitrophenol↗
PubMed1988

Species specificity of transferrin binding, endocytosis and iron internalization by cultured chick myogenic cells.

The ability of unlabelled heterologous transferrin to interact with transferrin receptors on developing chick myogenic cells was investigated by measuring their capacity to inhibit the surface-binding and internalization of 125I- and 59Fe-labelled ovotransferrin. Transferrins from rat, rabbit, human, and a species of kangaroo (Macropus fuliginosus) were unable to inhibit either surface-binding or internalization of labelled ovotransferrin even at concentrations ten times the molar concentration of the ovotransferrin. Transferrins isolated from the serum of a toad (Bufo marinus) and a lizard (Teliqua rugosa), when added at high concentrations, were found to reduce surface-binding of 125I-Tf by 20-25% but did not inhibit internalization of either 125I-Tf or 59Fe. This suggests that the effects of toad and lizard transferrins are due to non-specific binding to the myogenic cells. In contrast, inhibition of both surface-binding and internalization of labelled ovotransferrin was found when myogenic cells were incubated in the presence of the homologous transferrin (ovotransferrin). The species-specificity of transferrin binding, endocytosis and iron internalization did not vary with the state of proliferation or differentiation of the myogenic cells. However, the intracellular iron utilization was found to differ between differentiating presumptive and terminally differentiated myotubes. Internalized 59Fe was fractioned by gel filtration. In dividing and non-dividing presumptive myoblasts 59Fe was found to elute in three peaks, two with elution volumes corresponding to ferritin and transferrin and one at greater elution volume than that of myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Iron-binding properties and amino acid composition of marsupial transferrins: comparison with eutherian mammals and other vertebrates.

1. Some physicochemical properties of transferrin from three marsupials, viz a possum (Trachosurus vulpecula), a kangaroo (Macropus fuliginosus) and the quokka (Setonix brachyurus) were studied and compared with those of transferrins from mammalian and non-mammalian vertebrate species. 2. The molecular weight of the marsupial transferrins fell within the range of 76,000-79,000 daltons. 3. The marsupial transferrins were similar to the transferrins of eutherian mammals with respect to optical spectral properties, iron binding capacity and the pH-dependence of iron binding, and iron release mediated by 2,3-DPG. 4. The amino acid compositions of the marsupial transferrins were compared with each other and with the transferrins from the other vertebrate species. The compositions of the marsupial transferrin were closely related to each other, and also showed similarities with transferrins from eutherian mammals and chicken ovotransferrin.

Amino Acids↗
PubMed1987

Transferrin and ferritin endocytosis and recycling in guinea-pig reticulocytes.

Transferrin and ferritin endocytosis and exocytosis by guinea-pig reticulocytes were studied using incubation with pronase at 4 degrees C to distinguish internalized and membrane-bound protein. Internalization of both transferrin and ferritin occurred in a time- and temperature-dependent fashion. Transferrin endocytosis was more rapid than that of ferritin. Transferrin binding to receptors was not altered, but transferrin endocytosis was decreased in the presence of ferritin. Iron accumulation from transferrin was inhibited by ferritin to a greater extent than could be accounted for by the decreased rate of endocytosis. In pulse-chase experiments, almost all of the transferrin was released intact from reticulocytes, but only about 50% of the total internalized ferritin was released, of which 85% was intact. The endocytosis of transferrin by rabbit reticulocytes was 2- to 2.5-times faster than guinea-pig reticulocytes. These data suggest that ferritin and transferrin are internalized by receptor-mediated endocytosis, possibly involving the same coated pits and vesicles, but that the proteins are recycled only partly in common.

Animals↗
PubMed1987

Transferrin receptor numbers and transferrin and iron uptake in cultured chick muscle cells at different stages of development.

The mechanism of iron uptake and the changes which occur during cellular development of muscle cells were investigated using primary cultures of chick embryo breast muscle. Replicating presumptive myoblasts were examined in exponential growth and after growth had plateaued. These were compared to the terminally differentiated cell type, the myotube. All cells, regardless of the state of growth or differentiation, had specific receptors for transferrin. Presumptive myoblasts in exponential growth had more transferrin receptors (3.78 +/- 0.24 X 10(10) receptors/micrograms DNA) than when division had ceased (1.70 +/- 0.14 X 10(10) receptors/micrograms DNA), while myotubes had 3.80 +/- 0.26 X 10(10) receptors/micrograms DNA. Iron uptake occurred by receptor-mediated endocytosis of transferrin. While iron was accumulated by the cells, apotransferrin was released in an undegraded form. There was a close correlation between the molar rates of endocytosis of transferrin and iron. Maximum rates of iron uptake were significantly higher in myotubes than in presumptive myoblasts in either exponential growth or after growth had plateaued. There were two rates of exocytosis of transferrin, implying the existence of two intracellular pathways for transferrin. These experiments demonstrate that iron uptake by muscle cells in culture occurs by receptor-mediated endocytosis of transferrin and that transferrin receptor numbers and the kinetics of transferrin and iron uptake vary with development of the cells.

Animals↗
PubMed1987

Transferrin-receptor interaction and iron uptake by reticulocytes of vertebrate animals--a comparative study.

Transferrin-receptor interactions and iron uptake were studied in eleven different species of vertebrate animals (3 eutherian mammals, 3 marsupials, 2 reptiles and 1 bird, amphibian and bony fish). In the initial experiments it was shown that the uptake of transferrin-bound iron by immature erythroid cells from marsupial and reptilian species occurs by receptor-mediated endocytosis as in other vertebrate animals. Reticulocytes were incubated with 125I-59Fe-labelled transferrins from heterologous species and the results for iron and transferrin uptake compared with those obtained with the homologous protein. Cells from eutherian mammals were able to take up transferrin and iron from other eutherians and from the bob-tailed lizard but not from marsupials and other submammalian species. With marsupials and reptiles a similar specificity was observed, and the marsupial cells could also utilize chicken transferrin but not vice versa. The results were extended by performing competition experiments in which the cells were incubated with radiolabelled homologous transferrin in the presence of increasing concentrations of non-radioactive heterologous transferrins. From the ability of the heterologous proteins to inhibit uptake of the homologous protein relative association constants (Ka1) for the transferrin-receptor interactions could be calculated. These Ka1 values reflected the patterns observed in the first series of experiments. These studies demonstrate that, although specificity exists in transferrin-receptor interactions throughout the range of vertebrate animals, in several instances reactivity between widely divergent species is also observed. Hence, structural similarities have been maintained throughout evolution. Nevertheless, no evidence of interaction between transferrin and its receptor from the two divisions of the Mammalia, the eutherians and the marsupials, was observed.

Animals↗