Abstract
This project sought to establish how lanthanide metals (Ln) bind to cell membranes, whether they are transported into cells and how they are distributed in vivo. Europium (Eu), chosen as a representative of the Ln series, as the chloride salt, formed stable aggregates (microprecipitates) in solution at neutral pH, even in phosphate- and bicarbonate-free conditions. The uptake of EuCI3 by intestinal brush-border membrane vesicles (BBMV) was difficult to determine because the aggregates interfered with the BBMV uptake assay and the concentration of available Eu3+ was not known. When tested as Eu-citrate, Eu-nitrilotriacetate and other complexes (ratio of Eu:ligand, 1:2) of intermediate stability constants (log KS of about 7 - 12) Eu was available for uptake by BBMV in phosphate- and bicarbonate-free conditions. Complexes with lower log KS did not keep Eu in solution whilst those of higher stability did not donate it to BBMV. The relationship, expressed as a reverse-sigmoidal curve, between log Ks and uptake of Eu by BBMV was consistent for most complexes with the exception of Eu-catechol complex. Eu bound to BBMV at two sites, as judged by displacement with the powerful ligand diethylenetriaminepentaacetic acid (DTPA). Binding to the DTP A-sensitive (membrane exterior) site predominated at 20°C. Uptake by the DTPA-resistant site was markedly enhanced at 37°C, suggesting a strong association with the membrane. Only small amounts (< 10 %) of bound Eu were released into the vesicle interior, confirming the poor membrane penetration of Ln ions. Thus Ln binding may be responsible for cytoprotective effects and changes in BBMV shape which were observed with Ln. Eu precipitation from most complexes was promoted in solutions containing phosphate and bicarbonate. However, the Eu-DTPA complex was stable in physiological solutions. Pre-formed Eu-albumin, despite a relatively low log KS (6. 4), was more stable in physiological solutions than complexes of log KS of 7 - 12. Eu precipitation in physiological solutions could also be prevented by increasing ligand:Eu ratio. When injected intravenously into rats, EuCI3 was sequestered by the liver and spleen. Eu in the liver was present in parenchymal and non-parenchymal cells. Eu injected as the Eu-albumin and Eu-DTPA complexes was rapidly excreted in the urine; a small but significant amount of Eu was retained in the kidney. EuCl3 did not effect Ca2+ uptake by BBMV and HoCI3 increased the calcium content of isolated hepatocytes, suggesting that Ln might not inhibitors of Ca2+ transport in all biological systems. These results question the validity of ubiquitously using Ln as analogues of calcium.