Evaluation of β-amyloid peptide 25–35 on calcium homeostasis in cultured rat dorsal root ganglion neurons

Abstract

Accumulation of β-amyloid (Aβ) protein in brain is an important characteristic for the etiology of Alzheimer’s disease. Of all the possible processes generating the neurotoxic effects by Aβ, disruption of intracellular Ca 2+ homeostasis is the primary event. In this process, various intracellular Ca 2+ regulatory mechanisms are reported to be involved. Using patch-clamp techniques, both low and high voltage activated Ca 2+ channel currents were recorded in the cultured dorsal root ganglion (DRG) neurons. Application of Aβ protein fragment, Aβ 25–35 (2 μM), for 30 s increased the amplitude in both currents. The Aβ-triggered facilitation effect of Ca 2+ channel was found in all the depolarized potentials tested, as shown in the current–voltage relationship. Furthermore, after applying single cell Ca 2+ microfluorometric method, it was found that Aβ 25–35 alone could trigger elevations of intracellular Ca 2+ concentration ([Ca 2+] i) level in 90% of the cells tested. The elevation diminished completely by cumulatively adding CdCl 2, NiCl 2, thapsigargin (TG), FCCP and Zn 2+ in the normal bath solution. Combining pharmacological approaches, we found that voltage-dependent Ca 2+ channels, Ca 2+ stores and a putative Zn 2+-sensitive extracellular Ca 2+ entry, respectively, makes 61.0, 25.1, and 13.9% contribution to the [Ca 2+] i increase caused by Aβ. When tested in a Ca 2+-free buffer, mitochondria was found to contribute 41.3% of Aβ produced [Ca 2+] i elevation and the remaining 58.7% was attributed to endoplasmic reticulum (ER) release.