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.
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, People's Republic of China.
Hong
H
Wang
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, People's Republic of China.
Zhong-Kun
ZK
Wang
Research Centre of Biological Science, Taishan Medical University, Tai'an 271000, People's Republic of China.
Peng
P
Jiao
College of Urban & Rural Development, Beijing University of Agriculture, Beijing 102206, People's Republic of China.
Xu-Ping
XP
Zhou
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, People's Republic of China.
Du-Bao
DB
Yang
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, People's Republic of China.
Zhen-Yong
ZY
Wang
College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, People's Republic of China. Electronic address: wanglin2013@sdau.edu.cn.
Lin
L
Wang
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D006706:Homeostasis; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000187:drug effects / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009942:Organometallic Compounds / Q000633:toxicity*; D061251:Primary Cell Culture; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D001896:Boron Compounds; D002614:Chelating Agents; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009942:Organometallic Compounds; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; C109986:2-aminoethoxydiphenyl borate; C008261:lead acetate; D002118:Calcium
Apoptosis; Lead; Proximal tubular cells; Redistribution of subcellular calcium