Na+-dependent sources of intra-axonal Ca2+ release in rat optic nerve during in vitro chemical ischemia.
The contribution of intracellular stores to axonal Ca2+ overload during chemical ischemia in vitro was examined by confocal microscopy. Ca2+ accumulation was measured by fluo-4 dextran (low-affinity dye, KD approximately 4 microM) or by Oregon Green 488 BAPTA-1 dextran (highaffinity dye, KD approximately 450 nM). Axonal Na+ was measured using CoroNa Green. Ischemia in CSF containing 2 mM Ca2+ caused an approximately 3.5-fold increase in fluo-4 emission after 30 min, indicating a large axonal Ca2+ rise well into the micromolar range. Axonal Na+ accumulation was enhanced by veratridine and reduced, but not abolished, by TTX. Ischemia in Ca2+-free (plus BAPTA) perfusate resulted in a smaller but consistent Ca2+ increase monitored by Oregon Green 488 BAPTA-1, indicating release from intracellular sources. This release was eliminated in large part when Na+ influx was reduced by replacement with N-methyl-D-glucamine (NMDG+; even in depolarizing high K+ perfusate), Li+, or by the application of TTX and significantly increased by veratridine. Intracellular release also was reduced significantly by neomycin or 1-(6-[(17beta-methoxyestra-1,3,5 [10]-trien-17-yl) amino] hexyl)-1H-pyrrole-2,5-dione (U73122 [GenBank]) (phospholipase C inhibitors), heparin [inositol trisphosphate (IP3) receptor blocker], or 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157; mitochondrial Na+/Ca2+ exchange inhibitor) as well as ryanodine. Combining CGP37157 with U73122 [GenBank] or heparin decreased the response more than either agent alone and significantly improved electrophysiological recovery. Our conclusion is that intra-axonal Ca2+ release during ischemia in rat optic nerve is mainly dependent on Na+ influx. This Na+ accumulation stimulates three distinct intra-axonal sources of Ca2+: (1) the mitochondrial Na+/Ca2+ exchanger driven in the Na+ import/Ca2+ export mode, (2) positive modulation of ryanodine receptors, and (3) promotion of IP3 generation by phospholipase C.
Nikolaeva
Maria A
MA
Division of Neuroscience, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, K1Y 4K9, Canada.
Mukherjee
Ballari
B
Stys
Peter K
PK
D000779:Anesthetics, Local / Q000494:pharmacology; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004347:Drug Interactions; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D005947:Glucose / Q000172:deficiency; D066298:In Vitro Techniques; D007511:Ischemia / Q000209:etiology / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods; D009584:Nitrogen / Q000008:administration & dosage; D009900:Optic Nerve / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D020318:Rats, Long-Evans; D012964:Sodium / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology; D013997:Time Factors; D014701:Veratridine / Q000494:pharmacology
D000779:Anesthetics, Local; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C003320:sodium nitride; D013779:Tetrodotoxin; D004533:Egtazic Acid; D014701:Veratridine; D012964:Sodium; D005947:Glucose; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D009584:Nitrogen; D002118:Calcium