Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes.
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 microM) chemiluminescence. [(14)C]-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 microg/cm(2). Free oxalate (750 microM), 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 (deltapsi(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 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 deltapsi(m).
Meimaridou
Eirini
E
Centre for Prevention and Treatment of Urinary Stones, Institute of Urology and Nephrology, University College London, London W1W 7EJ, UK.
Jacobson
Jake
J
Seddon
Alan M
AM
Noronha-Dutra
Alberto A
AA
Robertson
William G
WG
Hothersall
John S
JS
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002129:Calcium Oxalate / Q000494:pharmacology*; D002130:Calcium Phosphates / Q000494:pharmacology; D002460:Cell Line; D003460:Crystallization; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004285:Dogs; D017886:Durapatite / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects*; D007473:Ion Channels / Q000187:drug effects; D007668:Kidney / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects*; D008855:Microscopy, Electron, Scanning; D008928:Mitochondria / Q000187:drug effects / Q000648:ultrastructure; D009550:Nigericin / Q000494:pharmacology; D027961:Potassium-Hydrogen Antiporters / Q000037:antagonists & inhibitors; D013481:Superoxides / Q000378:metabolism*
D002130:Calcium Phosphates; D007473:Ion Channels; D027961:Potassium-Hydrogen Antiporters; D013481:Superoxides; D002129:Calcium Oxalate; D004024:Dicyclohexylcarbodiimide; D017886:Durapatite; C494366:calcium phosphate, dibasic, dihydrate; D009550:Nigericin; D002118:Calcium