Quantification of rat hepatocyte transferrin receptors with poly- and monoclonal antibodies and protein A

Rudolph, John R.; Regoeczi, E.; Southward, Suzanne M.R.

doi:10.1007/bf00493303

Cited by 14 publications

(4 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A specific activity of 10 to 16 pCi/pg transferrin was routinely obtained. Human transferrin was used because the human protein has a higher affinity for the rat TfR than the homologous protein (19)(20)(21)(22).…”

Section: Tfr Assaymentioning

confidence: 99%

Transferrin receptor expression in rat liver: Immunohistochemical and biochemical analysis of the effect of age and iron storage

et al. 1990

View full text Add to dashboard Cite

Hepatic transferrin receptors were studied in normal male rats at 1 to 59 wk after weaning, using immunohistochemical and biochemical techniques. The number of transferrin receptors measured and the intensity of the staining in situ decreased rapidly during the first 10 wk of life and more slowly thereafter. Immunohistochemistry further demonstrated changes in the topographical and (sub)cellular localization of the transferrin receptor. In the young rat livers, staining was almost exclusively present on hepatocytes in acinar zone 2 + 3 in a honeycomb to sinusoidal pattern. With aging, a panacinar heterogeneous and mainly sinusoidal staining of hepatocytes was more frequent. Kupffer cell positivity was more obvious as compared with the young rat livers. The observed changes in transferrin receptor expression may partly be explained by age-dependent alterations in DNA synthesis and proliferative potential of the liver cells. A series of rats were iron loaded with carbonyl iron up to 39 wk and "unloaded" by administration of a normal diet during 20 wk. In these animals, serial histochemical studies showed predominantly parenchymal (7 to 14 wk), mixed parenchymal and reticuloendothelial (39 wk) and almost exclusive reticuloendothelial siderosis (59 wk). In the siderotic livers transferrin receptor numbers tended to be lower than in the controls with significant differences after 14 and 39 wk. Immunohistochemistry showed decreased parenchymal but increased reticuloendothelial transferrin receptor expression with iron load. After the period of unloading, parenchymal transferrin receptors were virtually absent despite the negligible siderosis of these cells. In contrast, siderotic reticuloendothelial cells were intensely positive. These findings support down-regulation of parenchymal transferrin receptor resulting from iron storage. However, the positivity of siderotic reticuloendothelial cells and the absence of re-emergence of parenchymal receptors in conditions of minimal parenchymal and prominent reticuloendothelial siderosis need further elucidation.

show abstract

Section: Tfr Assaymentioning

confidence: 99%

Transferrin receptor expression in rat liver: Immunohistochemical and biochemical analysis of the effect of age and iron storage

et al. 1990

View full text Add to dashboard Cite

show abstract

“…Isolated hepatocytes were prepared and incubated to allow for re-expression of cell-surface components exactly as described previously (Sharma et al, 1989). This was identical to a previous study of the binding of ' 'I-Fe: + -transferrin to isolated hepatocytes (Rudolph et al, 1988). …”

Section: Methodsmentioning

confidence: 99%

Fe³⁺₂‐transferrin and Fe³⁺₂‐asialotransferrin deliver iron to hepatocytes by an identical mechanism

Sharma

Grant

1991

European Journal of Biochemistry

View full text Add to dashboard Cite

We have been unable to demonstrate the unequivocal presence of transferrin receptors on rat hepatocytes. The binding and presumed internalisation of lZ5I-Fe2 +-transferrin by freshly isolated hepatocytes was only partially inhibited by up to a 104-fold molar excess of unlabelled ligand and was virtually insensitive to chloroquine. There would appear to be only a weak association between this ligand and some component of the hepatocyte cell surface. These results were not compatible with the commitment of Fe;+-transferrin to either a receptormediated endocytotic pathway nor to a rapid recycling pathway through sorting endosomes. Desialylation of the biantennary oligosaccharide side chains of Fe:+-transferrin engendered a low affinity (Kd 2 0.25 pM) for the asialoglycoprotein receptor. 1251-Fe: +-asialotransferrin was only superficially internalised by isolated hepatocytes but this was characteristic of ligands for the asialoglycoprotein receptor which have only biantennary (or single triantennary) side chains. This was also incompatible with the delivery of the ligand to sorting endosomes where the release of iron has been presumed to occur. Despite the different properties of the two ligands, the rates of iron uptake from 59Fe: +-transferrin and 59Fe: +-asialotransferrin were identical, suggesting a common mechanism for the translocation of iron across the plasma (or possibly endosomal) membrane such as a transmembrane oxidoreductase. Competition studies with unlabelled ligand or impermeable ferric ammonium citrate gave an IC50 of 1 -15 pg Fe3' /ml for this process. The absence of transferrin receptor from the surface of the terminally differentiated, quiescent hepatocyte would be compatible with the dual roles suggested for transferrin as an iron transport protein and as a growth factor. The release of iron at the hepatocyte cell surface would effectively uncouple the two functions and render the hepatocyte unresponsive to growth stimulation by transferrin.A healthy liver contains about 30% of total body iron which is stored in hepatocytes as a complex with ferritin. The net retention or release of iron from this store is determined by the serum iron concentration, which is set by the level of transferrin saturation. This in turn is set by the balance between the synthesis in the bone marrow, degradation by the reticuloendothelial system and absorption of iron from the gut (see Bothwell et al., 1979). The hepatic iron store is, therefore, the buffer for the maintenance of iron haemostasis and primarily responds to variations in iron requirement by the bone marrow.Despite its physiological importance, the mechanism of iron accumulation by hepatocytes remains a paradox. Since the expression of transferrin receptors is a characteristic of cell growth and proliferation (Huebers and Finch, 1987) the terminally differentiated, quiescent hepatocyte would be expected to show only a basal level of expression or even a complete absence of this receptor. At the same time, this cell continuously accumulates and releases ir...

show abstract

“…Parenchymal cells were isolated from the livers of Sprague-Dawley rats (200-300 g) by collagenase perfusion as outlined elsewhere (Rudolph et al, 1988). The cells combed out from the perfused organ were allowed to recover in the stir solution for 45 min a t 37°C.…”

Section: Isolation Of Hepatocytesmentioning

confidence: 99%

Interaction of rat asialotransferrin with adult rat hepatocytes: Its relevance for iron uptake and protein degradation

Rudolph

Regoeczi

1988

Journal Cellular Physiology

View full text Add to dashboard Cite

Comparative studies with rat transferrin (rTf) and asialotransferrin (asialo-rTf) were performed on suspended adult rat hepatocytes with the aim of elucidating the mechanism of enhanced hepatic Fe acquisition from asialo-rTf observed previously in vivo. At low ligand concentrations (0.05-20 micrograms/ml), the cells bound more asialo-rTf than rTf. However, the excess binding was abolished by incubation either in the presence of 1.55 mg/ml of diferric rTf or of 1 mg/ml of asialomucin. Following either treatment, asialo-rTf and rTf were bound to comparable extents. These findings indicate that both transferrin receptors and the hepatic galactose recognition system (lectin) are essential for preferential binding of asialo-rTf by hepatocytes. The possibility is considered that the lectin facilitates capture of asialo-rTf by the same binding sites that are normally available for rTf rather than that it functions as an alternative pathway. In agreement with this view, asialo-rTf could not be channeled into the lectin-mediated degradative pathway by blocking Tf receptors with human Tf. Enhanced Fe uptake from asialo-rTf was fully prevented by asialomucin and partially prevented by human Tf. The incomplete efficacy of human Tf in this regard supports reports in the literature about Fe uptake by the liver in a manner that is independent of Tf receptors. Rhesus asialo-Tf was deployed to show that no recognition mechanism exists for heterologous asialo-Tf in rat hepatocytes. The importance of using undenatured labeled proteins for studies with cells is demonstrated.

show abstract

Quantification of rat hepatocyte transferrin receptors with poly- and monoclonal antibodies and protein A

Cited by 14 publications

References 44 publications

Transferrin receptor expression in rat liver: Immunohistochemical and biochemical analysis of the effect of age and iron storage

Transferrin receptor expression in rat liver: Immunohistochemical and biochemical analysis of the effect of age and iron storage

Fe³⁺₂‐transferrin and Fe³⁺₂‐asialotransferrin deliver iron to hepatocytes by an identical mechanism

Interaction of rat asialotransferrin with adult rat hepatocytes: Its relevance for iron uptake and protein degradation

Contact Info

Product

Resources

About

Quantification of rat hepatocyte transferrin receptors with poly- and monoclonal antibodies and protein A

Cited by 14 publications

References 44 publications

Transferrin receptor expression in rat liver: Immunohistochemical and biochemical analysis of the effect of age and iron storage

Transferrin receptor expression in rat liver: Immunohistochemical and biochemical analysis of the effect of age and iron storage

Fe3+2‐transferrin and Fe3+2‐asialotransferrin deliver iron to hepatocytes by an identical mechanism

Interaction of rat asialotransferrin with adult rat hepatocytes: Its relevance for iron uptake and protein degradation

Contact Info

Product

Resources

About

Fe³⁺₂‐transferrin and Fe³⁺₂‐asialotransferrin deliver iron to hepatocytes by an identical mechanism