Distribution of iron in the brain varies with region, cell type, and age. Furthermore, some neurological diseases are accompanied by an abnormal accumulation of iron in specific areas of the CNS. These findings implicate a mobile intracerebral iron pool; however, transport of iron across the blood‐brain barrier and its regulation are largely unknown. In an extensive series of experiments in primary cultures of porcine blood‐brain barrier endothelial cells, we separately quantified surface‐bound and total cellular transferrin receptor pools. Although 90% of all transferrin receptors were located inside the cell, only 10% of these intracellular receptors actively took part in the endocytic cycle. This large “inactive” intracellular transferrin receptor pool could either function as a storage site for spare receptors or be activated by the cell to increase its capacity for iron transport. Data were corrected for nonspecific binding by a separate biochemical assessment using a 100‐fold excess of unlabeled ligand. Data were also analyzed in a nonlinear curve‐fit program. This resulted in a less elaborate and less biased estimate of nonspecific binding.
Iron is essential in the cellular metabolism of all mammalian tissues, including the brain. Intracerebral iron concentrations vary with age and in several (neurological) diseases. Although it is evident that endothelial cells lining the capillaries in the brain are of importance, factors governing the regulation of intracerebral iron concentration are unknown. To investigate the role of blood‐brain barrier endothelial cells in cerebral iron regulation, primary cultures of porcine blood‐brain barrier endothelial cells were grown in either iron‐enriched or iron‐depleted medium. Iron‐enriched cells showed a reduction in surface‐bound and total transferrin receptor numbers compared with iron‐depleted cells. Transferrin receptor kinetics showed that the transferrin receptor internalization rate in iron‐enriched cultures was higher, whereas the transferrin receptor externalization rate in iron‐enriched cultures was lower than the rate in iron‐depleted cultures. Moreover, blood‐brain barrier endothelial cells cultured in iron‐enriched medium were able to accumulate more iron intracellularly, which underlines our kinetic data on transferrin receptors. Our results agree with histopathological studies on brain tissue of patients with hemochromatosis, suggesting that at high peripheral iron concentrations, the rate of iron transport across the blood‐brain barrier endothelial cells is to some extent proportional to the peripheral iron concentration.
Electron spectroscopic imaging (ESI) with the energy-filtering transmission electron microscope enables the investigation of chemical elements in ultrathin biological sections. An analysis technique has been developed to calculate elemental maps and quantitative distributions from ESI sequences. Extensive experience has been obtained with a practical implementation of this technique. A procedure for more robust element detection has been investigated and optimized. With the use of Fe-loaded Chelex beads, the measurement system has been evaluated with respect to the linearity of the element concentration scale, the reproducibility of the measurements and the visual usage of image results. In liver specimens of a patient with an iron storage disease the detectability of iron was tested and we tried to characterize iron-containing components. The concentration measurement scale is approximately linear up to a relative section thickness of approximately equal to 0.5. Monitoring of this parameter is therefore considered to be important. The reproducibility was measured in an experiment with Fe-Chelex. The iron concentration differed by 6.4% between two serial measurements. Element distributions are in many applications interpreted visually. For this purpose the frequently used net-intensity distributions are regarded as unsuitable. For the quantification and visual interpretation of concentration differences mass thickness correction has to be performed. By contrast, for the detection of elements the signal-to-noise ratio is the appropriate criterion. Application of ESI analysis demonstrated the quantitative chemical capabilities of this technique in the investigation of iron storage diseases. Based on an assumed ferritin iron loading in vivo, different iron components can be discerned in liver parenchymal cells of an iron-overloaded patient.
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