Na+-dependent sources of intra-axonal Ca2+ release in rat optic nerve during in vitro chemical ischemia

Abstract

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.

Figure 1.

Confocal images of live optic nerve before and during ischemia. Axons were coloaded with red ion-insensitive Alexa Fluor 594 dextran for visualization of axonal profiles and with either low-affinity fluo-4 dextran (K_D, 4 μm; top row) to demonstrate larger increases in axonal Ca²⁺ (A, before ischemia; B, 30 min in ischemic buffer; C, D, Ca²⁺-sensitive fluorescence in pseudocolor) or high-affinity Oregon Green 488 BAPTA-1 dextran (K_D, 500 nm; middle row) to demonstrate contributions of intracellular Ca²⁺ stores in 0 Ca²⁺ (plus BAPTA) conditions (E, pre-ischemia image; F, same image in pseudocolor; G, after 7 min in 0 Ca/BAPTA ischemic buffer; H, after 15 min in Ca²⁺-replete ischemic buffer). The bottom row demonstrates an Na⁺-sensitive CoroNa Green fluorescence increase in ischemia (I, before ischemia; J, 30 min in ischemic buffer; K, L, Na⁺-dependent fluorescence in pseudocolor). The solid green structure (asterisk) is a dye-filled capillary.

Figure 2.

Time course of normalized axonal green/red fluorescence ratio showing a 3.5-fold Ca²⁺-dependent fluorescence increase over baseline after 30 min of ischemia in 2 mm Ca²⁺ with fluo-4 as the Ca²⁺ indicator (A). In 0 Ca²⁺ (plus BAPTA) perfusate, the ischemia evoked a 19% increase with Oregon Green 488 BAPTA-1 dextran as the Ca²⁺ indicator (B), indicating a release of this ion from intra-axonal compartments. The inset demonstrates a fluorescence decrease in control experiments without ischemia, indicating that perfusion with the 0 Ca²⁺/BAPTA solution gradually reduced axoplasmic [Ca²⁺].

Figure 3.

Time course of normalized green/red (Na⁺-sensitive CoroNa Green/Na⁺-insensitive) fluorescence ratio. A, Approximately linear decrease in axoplasmic fluorescence in nonischemic conditions (each point represents the mean from ∼30 axons). B, Reversal of the fluorescence decline during chemical ischemia. C, Fluorescence changes adjusted for loss of the Na⁺-sensitive dye, demonstrating the net rise of axoplasmic [Na⁺] during ischemia (traces represent the means from 60-90 axons). Activating Na⁺ channels with veratridine further exacerbated axonal Na⁺ loading during ischemia, whereas blocking these channels with TTX reduced, but did not abolish completely, axonal Na⁺ accumulation. Dashed lines indicate baseline fluorescence.

Figure 4.

Effects of Na⁺ influx on Ca²⁺-dependent fluorescence changes during ischemia in 0 Ca²⁺ (plus BAPTA) perfusate. Combining 0 Ca²⁺ with 0 Na⁺ (126 mm of NaCl replaced with the impermeant NMDG⁺ ion and 26 mm of NaHCO₃ with choline bicarbonate) in the perfusate or the application of 1 μm TTX in large part prevented any Ca²⁺ increase. Preapplication of Li⁺-substituted 0 Na⁺ perfusate (126 mm of NaCl replaced with LiCl and 26 mm of NaHCO₃ with choline bicarbonate) in 0 Ca²⁺ (plus BAPTA), which allows axonal depolarization to occur (in contrast to the impermeable NMDG ion), also prevented ischemic Ca²⁺ rise. Veratridine (which increased Na⁺ influx) (Fig. 3) markedly increased the ischemic Ca²⁺ response. Forcing depolarization with 40 mm K⁺ in the absence of Na⁺ did not promote ischemic Ca²⁺ rise, indicating that it is mainly the Na⁺ influx, and not axonal depolarization, that triggers the release of Ca²⁺ from intracellular compartments. The dashed line indicates baseline fluorescence.

Figure 5.

Bar graph summarizing the effects of ER and mitochondrial Ca²⁺ release blockers (measured at the point of maximum Ca²⁺-dependent fluorescence increase, which occurred within 5-10 min after chemical ischemia was applied). Neomycin (300-500 μm),  U73122 (20 μm), heparin (85 mg/ml), ryanodine (60 μm), and CGP37157 (20 μm) all significantly suppressed ischemic Ca²⁺ increases (all in 0 Ca²⁺ plus BAPTA perfusate; ^*p < 0.01 compared with drug-free control). Combining  U73122 or heparin with CGP37157 reduced the Ca²⁺ rise even more. White numbers within the bars represent the number of individual axons analyzed for each treatment. Error bars indicate SD.