Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes |
| |
Authors: | Meimaridou Eirini Jacobson Jake Seddon Alan M Noronha-Dutra Alberto A Robertson William G Hothersall John S |
| |
Institution: | aCentre for Prevention and Treatment of Urinary Stones, Institute of Urology and Nephrology, University College London, London W1W 7EJ, UK bDepartment of Molecular Pathogenesis, Institute of Neurology, Queens Square, London, UK cSchool of Life Sciences, Kingston University, Kingston upon Thames, UK |
| |
Abstract: | We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O2?) 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. O2? 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 O2? in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O2? or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm2. Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O2? (+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 O2?, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψm) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca2+ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca2+ from internal stores. Thus, COM-induced mitochondrial O2? requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O2? production, which is probably regulated by Δψm. |
| |
Keywords: | Calcium oxalate Crystal Superoxide Mitochondria Chemiluminescence Kidney Free radicals |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|