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

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 (IP3R) 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 IP3 levels with up-regulated IP3R-1 and IP3R-2 (mRNA and protein) levels were revealed in Pb-exposed rPT cells. In summary, IP3R-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.