Cytosolic Ca2+ changes during in vitro ischemia in rat hippocampal slices: major roles for glutamate and Na+-dependent Ca2+ release from mitochondria

Abstract

This work determined Ca2+ transport processes that contribute to the rise in cytosolic Ca2+ during in vitro ischemia (deprivation of oxygen and glucose) in the hippocampus. The CA1 striatum radiatum of rat hippocampal slices was monitored by confocal microscopy of calcium green-1. There was a 50-60% increase in fluorescence during 10 min of ischemia after a 3 min lag period. During the first 5 min of ischemia the major contribution was from Ca2+ entering via NMDA receptors; most of the fluorescence increase was blocked by MK-801. Approximately one-half of the sustained increase in fluorescence during 10 min of ischemia was caused by activation of Ca2+ release from mitochondria via the mitochondrial 2Na+-Ca2+ exchanger. Inhibition of Na+ influx across the plasmalemma using lidocaine, low extracellular Na+, or the AMPA/kainate receptor blocker CNQX reduced the fluorescence increase by 50%. The 2Na+-Ca2+ exchange blocker CGP37157 also blocked the increase, and this effect was not additive with the effects of blocking Na+ influx. When added together, CNQX and lidocaine inhibited the fluorescence increase more than CGP37157 did. Thus, during ischemia, Ca2+ entry via NMDA receptors accounts for the earliest rise in cytosolic Ca2+. Approximately 50% of the sustained rise is attributable to Na+ entry and subsequent Ca2+ release from the mitochondria via the 2Na+-Ca2+ exchanger. Sodium entry is also hypothesized to compromise clearance of cytosolic Ca2+ by routes other than mitochondrial uptake, probably by enhancing ATP depletion, accounting for the large inhibition of the Ca2+ increase by the combination of CNQX and lidocaine.

Figures

Fig. 1.

Ca2+ fluorescence before and during ischemia. Rat hippocampal slices (300 μm) were loaded with 10 μm calcium green-1 AM for 45 min at 33°C and washed in normal buffer for at least 30 min before the experiments. In vitro ischemia was achieved by switching the normal buffer to glucose-free buffer equilibrated with 95% N2/5% CO2. A, B, Representative images taken in the CA1 area before and at the end of 10 min of ischemia, respectively. Scale bar, 100 μm. C, One typical recording of Ca2+ fluorescence during and after ischemia (horizontal line) from s. pyramidale and s. radiatum highlighted by rectangles inA. There was no correction for photo bleaching (curve-fitting) in this experiment. Points a andb represent the time points for A andB, respectively. The increase in fluorescence intensity was larger in s. radiatum than in s. pyramidale.

Fig. 2.

Changes in [Ca2+]i levels in s. radiatum of the hippocampal CA1 area induced by 5–10 min of ischemia, NMDA, and KCl. A baseline signal was collected for 10–20 min and was used to determine the control values of fluorescence (F) during ischemia via a curve-fitting program using Microsoft Excel. Values were expressed as the percentage change of fluorescence over the control value [(ΔF/F) × 100]. All experiments were performed at 37°C. A, Average fluorescence changes during 5 min (n = 4) and 10 min (n = 6) of ischemia (Isch).B, Average fluorescence change caused by 5 min exposure to 200 μm NMDA (horizontal line) and the effect of MK-801 (10 μm) during normoxia (n = 4). C, Summary of the change in fluorescence at different time points during NMDA exposure with or without MK-801. D, Average fluorescence change induced by 5 min exposure to 50 mm KCl (horizontal line) in 1.2 mm Ca2+(n = 4) or Ca2+-free (n = 3) buffer. E, Summary of KCl effects in normal Ca2+ buffer at different time points of exposure. Note that the lag time for the rise of [Ca2+]i was much shorter (30 sec) in the presence of NMDA or KCl than during ischemia (∼3 min).

Fig. 3.

Effect of replacing Cl− with gluconate on [Ca2+]i during ischemia.A, There was normally a 2.5–3.5 min delay in the onset of fluorescence change (control curve). Replacing extracellular Cl− did not affect the basal fluorescence but resulted in a much earlier fluorescence increase. The fluorescence rose as early as 0.5 min after the onset of ischemia. B, The average lag time for both control and gluconate-treated slices is shown. The latter was significantly different from the control condition (*p < 0.001; n = 6 for each trace).

Fig. 4.

Effect of MK-801 on [Ca2+]i during 10 min of ischemia. MK-801 (10 μm) was added to the buffer 30 min before and during ischemia. A, Average traces of fluorescence for the control and MK-801 groups during 10 min of ischemia (horizontal line). B, The change in fluorescence levels after 5 and 10 min of ischemia with or without MK-801. MK-801 reduced the ΔF/F from 43.7 ± 9.7 to 12.3 ± 6.3% at 5 min of ischemia but showed no significant effect at the end of 10 min of ischemia (n = 6).

Fig. 5.

Left, Middle, Effects of nimodipine, ω-conotoxin GVIA, and benzamil on [Ca2+]i during 5 and 10 min of ischemia are shown. None of the drugs inhibited the fluorescence increase during ischemia [n = 5, 4, and 6 for nimodipine (20 μm), ω-conotoxin GVIA (2 μm), and benzamil (100 μm) experiments, respectively]. Right, Rat hippocampal slices were exposed to 50 mm KCl for 5 min under nonischemic conditions. Nimodipine significantly suppressed the elevation of cytosolic Ca2+ during KCl exposure, demonstrating the efficacy of this drug in the present system.

Fig. 6.

Effects of various drugs and low Na+ buffer on [Ca2+]i during ischemia (horizontal line). A, CGP37157 (10 μm). B, CNQX (10 μm) with and without CGP37157 (CGP) and GYKI52466 (GYKI; 50 μm). C, Lidocaine (50 μm). All drugs reduced the fluorescence increase during 10 min of ischemia. D, Combination of CNQX and lidocaine showing a stronger inhibition of the fluorescence increase during ischemia than is seen with either drug alone (n = 6). E, The additive effects of MK-801 (10 μm) and CNQX (10 μm).F, Low external Na+. NaCl in the buffer was replaced withN-methyl-d-glucamine. This substitution reduced the fluorescence increase during ischemia, and CGP37157 had no further effect (n = 6).

Fig. 7.

Intracellular Na+ content after 7.5 min of ischemia in whole hippocampal slices and in CA1 s. radiatum. The measurement of intracellular Na+ in the slice was as described elsewhere (Kass et al., 1993). After each experiment, slices were washed in ice-cold isotonic sucrose for 10 min to remove extracellular Na+ and then dried at 80°C overnight. The tissue was weighed and extracted in 0.1N nitric acid overnight, and total Na+ in the supernatant was measured with a flame photometer. A, Intracellular Na+ content in the whole slice. CGP37157 (CGP; 10 μm; n = 4), CNQX (20 μm; n = 4), and lidocaine (50 μm; n = 6) were added to the normal buffer 30 min before ischemia (*p < 0.05, compared with control ischemia). B, Effect of CNQX on intracellular Na+ content at the end of 7.5 min of ischemia in the CA1 s. radiatum. CNQX showed no significant effect on intracellular Na+ content in this region (n = 7).

Fig. 8.

[Ca2+]i during ischemia in Ca2+-free medium: effects of mitochondrial exchange blocker and Na+ entry blockers. A, The increase in fluorescence in Ca2+-free medium was comparable with that in normal Ca2+ buffer. The horizontal barrepresents the duration of ischemia (10 min). B, The fluorescence increase during ischemia in Ca2+-free medium was suppressed by the same manipulations that were effective in normal Ca2+ buffer except that MK-801 did not show any effect in Ca2+-free medium. Drug treatments were as follows (from left to right): control, CGP37157, CNQX, GYKI52466, lidocaine, CNQX + lidocaine, low Na+, low Na+ + CGP37157, and MK-801, respectively (*p < 0.05 and **p < 0.001, compared with each control value;n = 5–8).