Redistribution of subcellular calcium and its effect on apoptosis in primary cultures of rat proximal tubular cells exposed to lead.

Abstract Previous studies have shown that cytosolic Ca 2+ ([Ca 2+ ] c ) overload was involved in Pb-induced apoptosis in primary cultures of rat proximal tubular (rPT) cells, but the source of elevated Ca 2+ and the effect of potential subcellular Ca 2+ redistribution on apoptosis are still unknown. In this study, variations of [Ca 2+ ] c in two culture media (Ca 2+ -containing and Ca 2+ - free) were analyzed, indicating that Pb-induced elevation of [Ca 2+ ] c was primarily generated intracellularly. Fluo-4-AM, dihydro-Rhod-2-AM and Mag-Fluo-4-AM was loaded to Pb-exposed rPT cells to monitor the imaging of Ca 2+ concentrations in the cytoplasm ([Ca 2+ ] c ), mitochondria ([Ca 2+ ] mit ) and endoplasmic reticulum (ER) ([Ca 2+ ] ER ), respectively, under the confocal microscope. Data indicate that elevations of [Ca 2+ ] c and [Ca 2+ ] mit with depletion of [Ca 2+ ] ER were revealed in Pb-treated rPT cells, but this subcellular Ca 2+ redistribution could be significantly suppressed by 2-APB, a specific inhibitor of inositol 1,4,5-trisphosphate receptor (IP 3 R) that functions to release Ca 2+ from ER stores. Simultaneously, Pb-mediated mitochondrial Ca 2+ overload can be partially suppressed by the cytosolic Ca 2+ chelator BAPTA-AM, suggesting that Ca 2+ uptake into mitochondria occurs via diverse pathways and ER Ca 2+ storage was the chief source. Furthermore, Pb-induced apoptosis was markedly inhibited by 2-APB and BAPTA-AM, respectively. Additionally, elevated IP 3 levels with up-regulated IP 3 R-1 and IP 3 R-2 (mRNA and protein) levels were revealed in Pb-exposed rPT cells. In summary, IP 3 R-mediated ER Ca 2+ release promoted the elevations of [Ca 2+ ] c and [Ca 2+ ] mit in Pb-exposed rPT cells, which played a chief role in apoptosis induced by impaired calcium homeostasis.