Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes

• Published in: Free radical biology & medicine 2005-06, Vol.38 (12), p.1553-1564
• Main Authors: Meimaridou, Eirini, Jacobson, Jake, Seddon, Alan M., Noronha-Dutra, Alberto A., Robertson, William G., Hothersall, John S.
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Calcium oxalate; Calcium Oxalate - pharmacology; Calcium Phosphates - pharmacology; Cell Line; Chemiluminescence; Crystal; Crystallization; Dicyclohexylcarbodiimide - pharmacology; Dogs; Durapatite - pharmacology; Free radicals; Intracellular Membranes - drug effects; Ion Channels - drug effects; Kidney; Kidney - metabolism; Membrane Potentials - drug effects; Microscopy, Electron, Scanning; Mitochondria; Mitochondria - drug effects; Mitochondria - ultrastructure; Nigericin - pharmacology; Potassium-Hydrogen Antiporters - antagonists & inhibitors; Superoxide; Superoxides - metabolism
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2005.02.020

We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O 2 − ) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O 2 − was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [ 14C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O 2 − in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O 2 − or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm 2. Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O 2 − (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O 2 − , implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca 2+ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca 2+ from internal stores. Thus, COM-induced mitochondrial O 2 − requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O 2 − production, which is probably regulated by Δψ m.