Release of Cyt c from mitochondria in astrocytes following in vitro ischemia
Normoxic control astrocytes had a punctate perinuclear pattern of Cyt c expression (). Cyt c signals were colocalized with immunostaining of mitochondrial protein Mn-superoxide dismutase (MnSOD) (thin arrow, ). At 60 min REOX following 2 h OGD, mitochondria were swollen (*, ) with punctate Cyt c distribution. By 160 min REOX, in some astrocytes, Cyt c immunostaining was absent and only MnSOD immunoreactive signals were detected in mitochondria (arrowhead, ). Moreover, Cyt c immunoreactivity was enhanced in other astrocytes in which MnSOD immunostaining signals were clearly not colocalized with Cyt c (, thick arrow). In contrast, in astrocytes treated with 10 μM bumetanide, a potent inhibitor of NKCC1, the Cyt c release at 160 min REOX was absent (, thin arrow) and accompanied with less mitochondrial swelling. Similar to the bumetanide-treated astrocytes, NKCC1−/− astrocytes exhibited more resistance to the OGD/REOX-mediated mitochondrial damage. No detectable Cyt c release at 160 min REOX was observed in NKCC1−/− astrocytes (, thin arrow). In a positive control study for Cyt c release, 10 μM FCCP exposure (4 min) triggered mitochondrial swelling and non-colocalization of Cyt c and MnSOD (, arrowhead, thick arrow), which is similar to the ischemic astrocytes.
Release of Cyt c from mitochondria in astrocytes following OGD/REOX
Quantitative analysis of Cyt c release in astrocytes
As shown in , the Rp was 0.65 ± 0.02 and the M2 was 0.72 ± 0.02 in the normoxic control astrocytes, reflecting the relative co-localization of Cyt c and MnSOD under this condition. At 60 min REOX when there was moderate Cyt c release, Rp was reduced to 0.53 ± 0.07 and M2 dropped to 0.49 ± 0.03 (p < 0.05). Moreover, when Cyt c and MnSOD became non-colocalized at 160 min REOX, Rp decreased further to 0.25 ± 0.04 (p < 0.05), accompanied with a deep drop in the M2 value (0.20 ± 0.05, p < 0.05). Interestingly, cells treated with 10 μM bumetanide or cells from NKCC1−/− mice exhibited significantly less reduction in Rp and M2 values at 160 min REOX (p < 0.05). On the other hand, in cells where Cyt c release was triggered with 10 μM FCCP, Rp and M2 values were significantly reduced (p < 0.05), similar to those at 160 min REOX.
To further investigate the role of ionic dysregulation in OGD/REOX-induced Cyt c release from mitochondria, we used SEA0400, a selective inhibitor of NCXrev activity. In astrocytes treated with the NCXrev inhibitor SEA0400 (1 μM) during REOX, the Cyt c release at 160 min REOX was absent (data not shown). The summary data in illustrate that cells treated with SEA0400 did not exhibit significant reduction in Rp (0.59 ± 0.02) and in M2 values (0.65 ± 0.01) at 160 min REOX. This was in contrast to the low Rp (0.25 ± 0.04) and M2 (0.20 ± 0.05) values in the 160 min REOX groups. These data further demonstrates a role for NCXrev in mitochondrial damage in ischemic astrocytes.
Colocalization of Cyt c
and MnSOD in astrocytes was further analyzed by plotting the distribution of Rp and M2 values. The majority of normoxic control astrocytes exhibited high Rp or M2 values with a normal distribution (Kolmogorov-Smirnov normality test, p
> 0.2, Suppl. Figure 1 A, C
). In contrast, following 2 h OGD/160 min REOX, the number of astrocytes with low Rp or M2 values were increased and the cell distribution drastically shifted to the left part of the x-axis (Suppl. Figure 1 A, C
). Moreover, the OGD/REOX-treated cells were not normally distributed (Kolmogorov-Smirnov normality test, p
< 0.05). On the other hand, either pharmacological inhibition of NKCC1 with bumetanide or genetic ablation prevented the Cyt c
release. Cells maintained a normal distribution pattern with high values of Rp or M2 (Suppl. Figure 1 B, D
). Taken together, these analyses clearly demonstrate a release of Cyt c
from mitochondria in astrocytes following OGD/REOX, and the protective effects of NKCC1 inhibition with bumetanide or by genetic ablation.
Delayed elevation in [Ca2+]cyt following OGD/REOX
We investigated the time course of changes in [Ca2+]cyt following OGD/REOX. As shown in , there were no changes in [Ca2+]cyt after 2 h OGD. Interestingly, [Ca2+]cyt started to rise at ~ 120 min REOX at a rate of ~ 8 nM/min and reached a plateau (358 ± 25 nM) between 160–180 min REOX. Moreover, the timing of the Ca2+cyt increase was close to the release of significant Cyt c at 160 min REOX. In contrast, inhibition of NKCC1 activity with bumetanide abolished the delayed rise in [Ca2+]cyt (). To test the role of NCXrev in delayed Ca2+ dysregulation, we examined whether SEA0400 (1μM), a potent inhibitor of NCXrev, can block Ca2+ rise during OGD/REOX. Inhibition of NCXrev prevented the delayed rise in [Ca2+]cyt (), which is similar to the effects of NKCC1 inhibition.
Delayed elevation in [Ca2+]cyt following OGD/REOX
We speculated that the delayed elevation of [Ca2+
may result from IP3
release from ER. As shown in , inhibition of IP3
R with its specific inhibitor xestospongin C (20 μM) largely blocked the delayed rise in [Ca2+
, suggesting ER Ca2+
stores as the source for the post-OGD Ca2+
rise in the cytosol. Moreover, we observed a sudden loss of fura-2 fluorescence signal in some astrocytes during 120–180 min REOX ( D-left panel, arrow). These dying cells with damaged membrane integrity failed to retain the dye. As shown in the lower panel
of D-right panel, normoxic astrocytes exhibited a basal level of cell death (6% ± 2 %). In contrast, 24 ± 3 % astrocytes died during 180 min REOX following 2 h OGD. Most importantly, a time-dependent cell death analysis revealed that the majority of cell death occurred during 125–180 min REOX, when the cytosolic Ca2+
dysregulation was triggered (Suppl. Figure 2
). Inhibition of NKCC1 or NCXrev
activity with Bu or SEA0400 prevented the OGD/REOX-induced astrocyte cell death ().
Biphasic changes in [Ca2+]ER and Ca2+m following OGD/REOX
The delayed rise in [Ca2+]cyt may result from the release of Ca2+ER. To test this hypothesis, changes of [Ca2+]ER were determined. shows that [Ca2+]ER was increased from a resting level of 27 ± 3 μM to 65 ± 3 μM at 2 h OGD (p < 0.05). During REOX, [Ca2+]ER was further augmented and reached a peak value of 130 ± 2 μM by 90 min REOX. However, after ~ 90 min REOX, Ca2+ER was abruptly released, occurring at the time when [Ca2+]cyt was increasing. Ca2+ER appeared to start refilling at 3 h REOX. These findings demonstrate that OGD/REOX causes Ca2+ER biphasic dysregulation in astrocytes. The release of Ca2+ER may in part contribute to the detrimental delayed increase in Ca2+cyt.
Biphasic changes in [Ca2+]ER and Ca2+m following OGD/REOX
Consistent with its effect on [Ca2+]cyt, inhibition of NKCC1 activity with bumetanide during REOX not only significantly attenuated REOX-mediated augmentation of Ca2+ER loading, but also blocked REOX-triggered Ca2+ER release (). To investigate whether IP3R plays a role in Ca2+ER release, inhibition of IP3R with a non-selective inhibitor 2-APB (100 μM) was tested. As shown in , 2-APB did not significantly affect accumulation of Ca2+ER during 0–90 min REOX, but it reduced the release of Ca2+ER during 2–3 h REOX. To further confirm the role of the IP3R in the OGD/REOX-mediated release of Ca2+ER, we also used the specific IP3R blocker xestospongin C (20 μM). The presence of xestospongin C during OGD/REOX did not affect Ca2+ER accumulation at 90 min REOX, but it did prevent depletion of Ca2+ER stores at 160 min REOX (). Taken together, these data suggest that REOX-triggered Ca2+ER release is in part due to IP3R activation.
To further investigate whether REOX-triggered Ca2+ER release would affect mitochondrial Ca2+ homeostasis and mitochondrial function, we next monitored the relative change in the rhod-2 florescence in mitochondria following 2 h OGD. Three hours of normoxic perfusion did not result in significant changes in Ca2+m (). However, within 15 min of REOX following 2 h OGD, there was a significant increase in Ca2+m that reached a plateau by 30 min REOX (~ 3-fold, ). Interestingly, there was a second increase in Ca2+m between 90–120 min of REOX, when Ca2+ER release and increases in [Ca2+]cyt occurred. Inhibition of NKCC1 either with bumetanide or by genetic ablation did not prevent the initial post-OGD loading of Ca2+m, but completely blocked the secondary rise in Ca2+m (). Interestingly, inhibition of IP3R with xestospongin C also abolished the second phase increase in Ca2+m, implying that the source for this second Ca2+m rise is ER (). Taken together, the data suggest that the OGD/REOX-induced released of Ca2+ER is partially buffered by mitochondria and this may facilitate mitochondrial Cyt c release.
Translocation of Cyt c from mitochondria to ER at 160 min REOX
Translocation of Cyt c into ER may play a role in IP3R activation and Ca2+ER release. We measured relative changes of Cyt c content in different subcellular fractionations (mitochondria and heavy endoplasmic reticulum; cytosol; and endoplasmic reticulum) following OGD/REOX. shows that the cytosol fraction lacked mitochondrial MnSOD and ER marker protein calnexin (CNX) expression. No MnSOD was detected in ER fraction (P3). Thus, relatively pure cytosol and ER fractions were obtained in this study. However, the P2 fraction (mitochondria and heavy endoplasmic reticulum) contained both MnSOD and CNX. The impurity of mitochondrial fraction is less relevant to this study, because our focus is on Cyt c translocation to the ER fraction (P3).
Translocation of Cyt c to ER following OGD/REOX
Under normoxic control conditions, Cyt c was largely located in mitochondria and only a trace level of Cyt c was detected in ER fraction (). At 120 min REOX after 2 h OGD, the Cyt c level in ER was elevated and further increased by 160 min REOX. The Cyt c/CNX ratio was increased by ~ 2-fold at 160 min REOX (). Moreover, inhibition of NKCC1 with 10 μM bumetanide significantly reduced the amount of Cyt c translocated to ER fraction at 160 min REOX (p < 0.05).
Changes of ER stress protein following OGD/REOX
dysregulation can lead to the unfolded protein response and ER stress development (Groenendyk and Michalak 2005
). We examined whether expression of ER stress proteins such as the phosphorylated form of eIF2α (p-eIF2α) was altered. As shown in , expression of p-eIF2α protein was increased at 2 h OGD and reached a peak level (~3.5-fold) at 120 min REOX (p < 0.05), when ER Ca2+
depletion occurred. p-eIF2α remained elevated at 3 h REOX. Inhibition of NKCC1 activity with bumetanide during 0–3 h REOX attenuated the up-regulation of p-eIF2α protein (). These results imply that Ca2+ER
dysregulation was accompanied by transient ER unfolded protein responses during 2 h OGD and 0–3 h REOX.