Alzheimer's amyloid-beta peptide inhibits sodium/calcium exchange measured in rat and human brain plasma membrane vesicles
Section snippets
Preparation of rat and human plasma membrane vesicles
Frozen tissue sections from both normal and Alzheimer's disease brain frontal cortex were obtained from the National Neurological Research Bank (VAMA Wadsworth, Los Angeles, CA). Plasma membrane vesicles from both rat and human brain were prepared as described previously.[55]
Preparation of Aβ peptides
Three amyloid peptides were used in this study. Purified Aβ1–40 (H2N-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV-OH) was obtained from Quality Controlled Biochemicals (Hopkinton, MA), while Aβ25–35 (H2N-GSNKGAIIGLM-OH) and Aβ
Aggregation of synthetic Aβ25–35 in 10 mM HCl solution
Aβ25–35 was used to study aggregation kinetics, while non-aggregating peptides Aβscrambled and substance P were used as controls. The peptides were dissolved in 10 mM HCl at a concentration around 3–10 mg/ml, then incubated at 37°C. The reason for using 10 mM HCl was to slow aggregation of Aβ25–35 such that the effects of unaggregated peptide on Na+/Ca2+ exchange could be studied. Fig. 1 shows the influence of incubation time upon aggregation as measured by thioflavine T fluorescence (ThT)
Do aggregated Aβ peptides inhibit Ca2+ transport by the Na+/Ca2+ exchanger?
Inhibition of vesicular Ca2+ accumulation during Na+-dependent Ca2+ uptake and Ca2+/Ca2+ exchange, as shown in this study, would be expected if aggregated Aβ peptides inhibited Ca2+ transport by the exchanger. Perhaps the most attractive alternative explanation for the mechanism of aggregated Aβ peptide effects on vesicular Ca2+ content is an increase in membrane ion permeability. Two findings from the present study argue against increases in Ca2+ permeability as the underlying mechanism of the
Conclusions
The exact mechanisms of Aβ-induced neurotoxicity in cell culture has yet to be solved. Accumulation of free radicals play a necessary role, but disruptions of Ca2+ homeostasis are also important. In the present study it was shown that exposure to aggregated Aβ25–35 and Aβ1–40 produced a partial reduction in Na+-dependent Ca2+ accumulation by plasma membrane vesicles. These data are consistent with a proposed mechanism where aggregated Aβ peptides directly interact with hydrophobic surfaces of
Unlinked References
4, 5, 9, 24, 31, 43
Acknowledgements
This work was supported by a grant from the National Institute of Neurological Disorders and Stroke, No. NS300384 and the Alzheimer's Association.
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