Depletion of intracellular Ca2+ store itself may be a major factor in thapsigargin-induced ER stress and apoptosis in PC12 cells.

The mechanisms of intracellular calcium store depletion and store-related Ca(2+) dysregulation in relation to apoptotic cell death in PC12 cells were investigated at physiological temperatures with a leak-resistant fluorescent indicator dye Fura-PE3/AM by a cooled CCD imaging analysis system. Electron microscopic observations have shown thapsigargin (TG; 100 nM)-induced apoptosis in PC12 cells. Thorough starvation of stored Ca(2+) by BAPTA/AM (50 microM), or La(3+) (100 microM) enhanced while dantrolene (100 microM) attenuated the TG-induced apoptosis by preventing a calcium release from internal stores. An immunoblotting analysis revealed an enhanced expression of GRP78, the hallmark of endoplasmic reticulum (ER) stress when cells were treated by TG along with BAPTA/AM. These results indicate that the depletion of the intracellular Ca(2+) stores itself induces the ER stress and apoptosis in PC12 cells without any involvement of the capacitative calcium entry (CCE) or a sustained elevation of intracellular Ca(2+) concentrations ([Ca(2+)](i)).

Amyloid-beta-protein (A beta) (25-35)-associated free radical generation is strongly influenced by the aggregational state of the peptides.

We investigated whether or not the Amyloid-beta-protein (A beta) itself spontaneously generates free radicals using electron spin resonance (ESR) spectroscopy while also monitoring the aggregational state of A beta and A beta-induced cytotoxicity. The present results demonstrated a four-line spectrum in the presence of A beta25-35 with N-tert-butyl-alpha-phenylnitrone (PBN) but not in the presence of PBN alone in phosphate-buffered saline (PBS). The fact that the four-line spectrum obtained for the A beta25-35/PBN in PBS was completely abolished in the presence of the iron-chelating agent Desferal demonstrated the observed four-line spectrum to be iron-dependent. On the other hand, A beta25-35 with PBN in phosphate buffer (PB) did not produce any definite four-line spectrum. the present results showed the amyloid fibril formation of A beta25-35 in PBS to be much higher than that of A beta25-35 in PB. Moreover, A beta-induced cytotoxicity assays showed A beta incubated in PBS to be more cytotoxic than that incubated in PB. These results thus demonstrate that A beta(25-35)-associated free radical generation is strongly influenced by the aggregational state of the peptides.