| Author: | To, Yu |
| Title: | Mechanisms of iron uptake by cultured brain astrocytes in rats |
| Degree: | M.Phil. |
| Year: | 1999 |
| Subject: | Rats -- Nervous system Brain -- Physiology Iron -- Metabolism Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | 193 leaves : ill. (some col.) ; 30 cm |
| Language: | English |
| Abstract: | The experiments described in this thesis were performed to investigate the effect of iron on the development of rat brain astrocytes in vitro and the mechanisms of transferrin-bound and transferrin-free iron uptake by cells. This thesis consists of 7 chapters, starting with a general introduction, followed by general methods, then 4 chapters (Chapter 3-6) describing the experimental works involved, and finally concluded with a general discussion. Chapter 1 Chapter 1 presents a review of the literature. The recent development and current knowledge in some related aspects of brain iron metabolism to this thesis are briefly described. These aspects include: (1) The history of iron in the brain; (2) Brain iron distribution and function; (3) Iron mobilization and storage in the brain; (4) Abnormal brain iron metabolism and central nervous system diseases; and (5) Astrocytes and brain iron metabolism. Chapter 2 Chapter 2 describes the materials and methods used in this study. Other methods used specially for certain experiments are described within the methods section of the relevant chapter. Apparatuses used in the investigation are described in this chapter as well. Chapter 3 Chapter 3 describes experiments for examining the effects of ferric nitrilotriacetate (Fe-NTA) at vary concentrations on brain astrocytes in culture. One of the aims of these experiments is to check whether the experimental system to be used for studying membrane transport of iron in astrocytes could induce free radical production and lipid peroxidation. In addition to morphologic studies, 3-(4,5-dimehtylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the cell number. The result demonstrated that high concentration of chelated ferric iron had an adverse effect on astrocytes. Significant reduction of astrocyte population was found at concentrations of 20uM to 5000uM, p values being smaller than 0.05, 0.01, or 0.001 respectively. The effects of Fe-NTA were both concentration and duration dependent. However, no significant difference of morphology was found between 5uM and 10uM groups and the controls. In contrast, when a low iron concentration (5uM) was added to dishes, the cultures could be kept in normal condition for three weeks. Results also showed that the cultured astrocytes were more resistant to high concentration of chelated iron exposure than the cultured cerebellar granule cells. These results demonstrated that the cultured brain astrocytes could be utilized as an experimental model for investigating the mechanisms of iron transport across the membrane of brain glial cells when the concentration used and incubation time are less than 10uM and 3 weeks respectively. Chapter 4 In this chapter, the binding of iron saturated 125I-transferrin to the cultured rat astrocytes was investigated at pH 7.4 and 4C. The results demonstrated that cortical astrocytes in suspension bound 125I-transferrin by a saturable and specific manner, indicating the presence of a receptor for transferrin on the membrane of the cells. Scatchard and Hill plot analysis showed that the dissociation constant (Kd) of the binding was about 3.45 x l0-8 M and the number of receptor was about 7.09 x 104/cell. The receptor was specific for rat and human transferrin. The binding of 125I -rat transferrin could be competitively and specifically inhibited by unlabeled iron saturated rat and human transferrin and no difference was found between the interaction of rat and human transferrin with this receptor. Whereas the interaction of duck or camel transferrin with this receptor was very weak. The Hill coefficient was 0.9877, almost no different from 1, indicating the absence of cooperativity. It suggested that 125I-transferrin bound to a single class site on the membrane of astrocytes. Chapter 5 The aim of the experiments described in this chapter was to investigate some aspects of mechanisms of transferrin-bound iron uptake by the cultured brain astrocytes in rats. After 15 days of culture, the astrocytes were exposed to 1uM of double-labeled transferrin at 37C or 4C for varying time. The cellular transferrin-bound iron and transferrin uptake was analyzed by measuring the intracellular radioactivity with 帠- counter. The results showed that transferrin iron uptake kept increasing in a linear manner up to at least 30 min. In contrast to transferrin iron uptake, the internalization of transferrin into the cells was most rapid only during the first 10 min and then slowed to a plateau level, indicating that transferrin internalization into the cells is a saturable process. Both transferrin and transferrin iron uptake were temperature-dependent. The uptake at 37C was significantly higher than that at 4C. The addition of either methylamine or ammonium chloride, blockers of transferrin iron uptake via inhibiting iron release from transferrin within endosomes, significantly decreased the cellular transferrin-bound iron uptake but had no significant effect on transferrin internalization. The pretreatment of cells with trypsin significantly inhibited the cellular transferrin-bound iron and transferrin uptake. These results suggested that transferrin-bound iron transport across the membrane of astrocytes was a transferrin receptor-mediated process and the pattern generally paralleled characteristics of transferrin-bound iron uptake in other mammalian cells outside of the brain. Chapter 6 In this chapter, the mechanism of transferrin-free iron uptake by the cultured brain astrocytes was investigated. Effects of incubation time, iron concentration, pH, temperature and some other divalent metals on the cellular transferrin-free uptake were determined. After 15 days of plating, the cells were incubated with transferrin-free iron in isotonic sucrose solution at different temperatures for certain time. The cellular transferrin-free iron uptake was analysed by measuring the cellular radioactivity with y-counter. The result showed that the cultured astrocytes had the capacity to acquire transferrin-free iron. The iron uptake by cells increased with incubation time in a linear manner at a rate 5.776 fmol/103 cell per mm within the 20 min of incubation period. The uptake was time and temperature dependent, iron concentration saturable, and inhibited by several divalent metal ions, including Co2+, Zn2+, Mn2+, Ni2+ and Ca2+. The uptake was also pH sensitive, the pH optimum being 6.5. These characteristics of transferrin-free iron uptake by the cultured astrocytes, similar to those obtained from cells outside of the brain and the cultured cerebellar granule cells, implied that a carrier-mediated iron transport system might be present on the membrane of this type of brain glial cells. Chapter 7 This chapter presents a general discussion of the methods and results of the experiments described in this thesis. Some relevant aspects for future research are suggested. |
| Rights: | All rights reserved |
| Access: | open access |
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