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First published online 1 April 2003
doi: 10.1242/jcs.00420

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Apoptotic insults impair Na⁺, K⁺-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidant stress

Xue Qing Wang, Ai Ying Xiao, Christian Sheline, Krzystztof Hyrc, Aizhen Yang, Mark P. Goldberg, Dennis W. Choi^* and Shan Ping Yu^,

Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, St Louis, MO 63110, USA
^* Merck Research Labs, West Point, PA 19486, USA
Department of Pharmaceutical Sciences, School of Pharmacy, Medical University of South Carolina, 280 Calhoun Street, Charleston, SC 29425 USA

View larger version (15K): [in a new window]   Fig. 1. Na+, K+-pump currents in cortical neurons and its ATP dependence. Identification of the membrane current associated with activities of the Na+, K+-ATPase. (A) Whole-cell recordings of the Na+, K+-pump current, Ipump, in cultured cortical neurons. At the holding potential of –60 mV, an inward current was triggered by an acute application of the Na+, K+-pump inhibitor ouabain (1 mM) or strophanthidin (500 µM), corresponding to the block of a tonic activity of the Na+, K+-pump. The effect of strophanthidin was reversible, an outward current appeared owing to reactivation of the Na+, K+-ATPase. (B) The membrane current associated with the Na+, K+-pump was highly dependent on the presence of intracellular Na+ ([Na+]i) and extracellular K+ ([K+]o). When Na+ was removed from the pipette internal solution or K+ was removed from the extracellular solution, little membrane current was observed upon application of 500 µM strophanthidin. (C) The I-V relationship of Na+, K+-pump current obtained by subtracting I-V curves constructed by membrane current responses to various voltage steps from the –100 mV holding potential in the presence and absence of 1 mM ouabain. The reversal potential of the pump current estimated from the I-V curve is about –133 mV. The relationship showed an outward rectification at depolarized potentials and a decrease of the current at very positive potentials, both are consistent with previous reports of the pump current (De Weer et al., 1988). Voltage pulses were applied every 4 seconds with increments of 20 mV starting at –100 mV. n=10. (D) The ATP-dependence of Ipump was revealed by comparison of the currents recorded with microelectrodes containing either 5 mM ATP or ATP-free internal solution. In the presence of ATP, there was no time-dependent decline of Ipump; without ATP in the internal solution, Ipump was greatly diminished 10 minutes after establishing the whole-cell configuration. n=10 and 8 for ATP and ATP-free groups, respectively. *P<0.05 compared with the current at 1-2 minutes.

View larger version (35K): [in a new window]   Fig. 2. Suppression of the Na+, K+-pump activity by apoptotic insults. Ipump was recorded at –60 mV in cortical neurons during exposure to the apoptotic insult serum deprivation or staurosporine, which induced neuronal apoptosis in 24-48 hours. (A) Ipump was gradually suppressed after 5 hours in a serum-free medium; a 9-hour serum deprivation profoundly blocked the Na+, K+-pump (n=16). Pyruvate (5 mM) and succinate (5 and 20 mM) preserved the pump activity during serum deprivation. Notably, succinate showed less effect than pyruvate at 5 mM concentration; 20 mM succinate protected the pump current similarly as 5 mM pyruvate did. n=6-21 cells. (B) Incubation with 0.1 µM staurosporine for 30 minutes did not show any effect on the current; prolonged incubation, however, resulted in progressive depression of Ipump. By 12 hours, there was little strophanthidin-sensitive current detected. Pyruvate (5 mM) or succinate (20 mM), co-applied with staurosporine, was able to retain the pump current at around control levels even after 12-hour exposure. Succinate at 5 mM also attenuated the suppression of Ipump. n=7-17. (C) C2-ceramide (25 µM) gradually suppressed Ipump; co-applied pyruvate (5 mM), however, could not prevent the Na+, K+-pump failure (n=8). Note that the effect of C2-ceramide on the pump activity was slower and milder than that of serum deprivation and staurosporine, in agreement with less cell death induced by C2-ceramide (see Fig. 7D). (D) Top: Representative inward Na+, K+-pump current induced by 500 µM strophanthidin in control cells and cells exposed to staurosporine (0.1 µM, 10 hours). Bar graph: control Na+, K+-pump current and the current in cells undergoing apoptosis (0.1 µM staurosporine, 9-10 hours); the Na+, K+-pump current in staurosporine-treated cells was significantly higher after 10-minute dialysis with an internal solution containing 10 mM ATP. n=5 for control group and the staurosporine group without ATP dialysis; n=6 for the staurosporine group with ATP dialysis. (E) Top: Representative outward current generated by activation of the Na+, K+-pump in control cells and cells exposed to 0.1 µM staurosporine (10 hours). Whole-cell recordings were established in regular external solution of 3 mM K+; cells were exposed to a K+-free extracellular solution for 10 seconds followed by 5 seconds exposure to 4 mM K+. The outward current triggered by external K+ was fully blocked by strophanthidin (data not shown), consistent with a pump current. Bar graph: The apoptotic insult staurosporine (0.1 µM, 9-10 hours) markedly suppressed the pump current (n=8). *P<0.01 compare with controls at time zero; #P<0.01 compare with apoptotic insult alone at same time point.

View larger version (21K): [in a new window]   Fig. 3. ATP synthesis and ADP/ATP ratio during apoptotic process. ATP production and ADP/ATP ratio were measured in sham control and treated cortical cultures after 9–10-hour exposures. (A) Cellular ATP level was markedly reduced by serum deprivation; pyruvate (5 mM) and succinate (20 mM) each enhanced the ATP production. n=6 measurements. (B) ADP/ATP ratio was decreased during serum deprivation, and was improved by co-applied pyruvate or succinate (n=6). (C) ATP level was reduced by 0.1 µM staurosporine; in contrast to the effect in A, pyruvate (5 mM) did not increase ATP levels: the ATP level was even lower in the presence of pyruvate. Nevertheless, succinate (20 mM) markedly enhanced the ATP level. n=8. (D) Staurosporine decreased the ADP/ATP ratio, which was improved by both pyruvate and succinate. n=8. *P<0.05 compared with controls; #P<0.05 compared with serum deprivation or staurosporine alone.

View larger version (23K): [in a new window]   Fig. 4. Phosphorylation state of the Na+, K+-pump during apoptotic process. The protein phosphorylation level of the Na+, K+-pump was assessed using the anti-phosphorylated protein antibody. (A) In the left column, cell lysates were directly subjected to SDS-PAGE; anti-Na+, K+-ATPase 3 subunit antibody (anti-3) was used as primary antibody. It demonstrated the specific binding of the anti-3 antibody to the protein. In the last two columns, an anti-3 or anti-phosphorylated protein antibody (anti-pan) was used as antibody in immunoprecipitation. Western blotting showed a clear 3 subunit protein band. (B) Cortical neurons were treated for 9 hours in control medium, serum-free medium (SD) or in 0.1 µM staurosporine (Staur). Anti-pan antibody was used to precipitate phosphorylated 3 subunit. Antibodies were linked to saturated amount of protein A-sepharose beads. Using anti-3 as primary antibody, western-blotting showed reduced phosphorylation level of the 3 subunit after serum deprivation or exposure to staurosporine. (C) Protein phosphorylation levels of the 3 subunit was measured by band relative gray intensity and corrected by corresponding protein A band intensity. Both serum deprivation (9 hours) and staurosporine exposure (9 hours) reduced the phosphorylation. As a control, more matured neuronal cultures of more than 15 days in vitro were subjected to serum deprivation (9 hours) and showed no decreased phosphorylation state (data not shown), consistent with diminished apoptosis in these cells. n=8 independent experiments for serum deprivation and n=3 for staurosporine. *P<0.05 compared with controls.

View larger version (22K): [in a new window]   Fig. 5. Production and regulation of ROS during apoptosis. Superoxide (O-2) production was measured by the fluorescent dye DHE. The association of increased fluorescence intensity to ROS production was confirmed with the positive control xanthine/xanthine oxidase (see D). (A) Large increases in fluorescence intensity were detected after 9–10-hour treatment in the serum-free medium. This chronic increase in ROS was effectively prevented in the presence of 5 mM pyruvate or 20 mM succinate. Bar, 20 µm. (B) Superoxide production was increased after 9–10-hour serum deprivation. Pyruvate (5 mM) prevented the O2- production; succinate (20 mM) showed a less but significant inhibitory effect on superoxide stress. (C) Staurosporine (0.1 µM, 9-10 hours) induced a robust increase in O2- production; the increase was prevented or attenuated by co-applied 5 mM pyruvate and 20 mM succinate, respectively. (D) Measured in cell-free culture solution, the antioxidant scavenger property of pyruvate was confirmed by a marked reduction in DHE fluorescence intensity induced by xanthine/xanthine oxidase. Succinate was not effective. n=211-330 cells for serum deprivation experiments; n=92-148 cells for staurosporine experiments; n=128 wells in D. *P<0.05 compared with controls; #P<0.05 compared with serum deprivation or staurosporine alone.

View larger version (29K): [in a new window]   Fig. 6. Inhibitory effects of ROS on Ipump. Ipump was recorded in cortical neurons at –60 mV before and after an oxidant insult. (A) Hydrogen peroxide (0.25 mM) showed a marked inhibitory effect on Ipump after a few minutes application. This suppression of the Na+, K+-pump activity was markedly prevented in the presence of catalase (250 U/ml) or pyruvate (5 mM) added 5 minutes before and during the exposure. Succinate, on the other hand, showed no such effect even at high concentration of 20 mM (n=5-6 for each test). (B) Acute application (10 minutes) of 20 µM menadione, a stimulator of endogenous production of O2-, suppressed Ipump; this inhibition was prevented by 25 U/ml SOD or 5 mM pyruvate but not by 20 mM succinate (n6 in each test). (C) Chronic exposure to 8 µM menadione for 9-10 hours also diminished Ipump; both pyruvate (5 mM) and succinate (20 mM) protected the Na+, K+-pump activity from damage by the endogenous ROS (n=5-7). (D) Acute (20 minutes) and chronic (15-20 hours) applications of DMNQ (5-20 µM) inhibited Ipump, pyruvate (5 mM) or succinate (20 mM) antagonized the inhibition in both conditions (n=5-8).*P<0.05 compared with controls; #P<0.05 compared with hydrogen peroxide, menadione, or DMNQ alone.

View larger version (40K): [in a new window]   Fig. 7. Neuroprotection of pyruvate and succinate against apoptosis. Serum withdrawal from the culture medium resulted in 50-60% cell death in 30 hours, measured by LDH release and Trypan Blue extrusion. (A) Bright phase photos of Trypan-Blue-treated pure-neuronal cultures before and after serum deprivation. Normal cells do not show positive staining with Trypan Blue (dark color); after a 30-hour incubation in the serum-free medium many Trypan-Blue-positive cells represent the injured or dead cells lack of ability to extrude the dye from the intracellular space. Addition of 5 mM pyruvate in the serum-free medium attenuated Trypan Blue staining and cell death. Bar, 50 µm. (B) Pyruvate attenuated serum deprivation-induced cell death in a concentration-dependent manner; the neuroprotection was mostly reversed by co-applied 4-CIN (2 mM). Neuroprotective effects were achieved even when pyruvate was given up to 4 hours after the onset of serum deprivation. (C) Succinate at 5 mM showed little neuroprotective effect, increasing its concentration to 20 mM reduced serum deprivation-induced apoptosis, consistent with its effect on Ipump and ROS production at this concentration. The succinate downstream metabolite oxaloacetate (5 mM) also showed comparable neuroprotection against serum deprivation-induced apoptosis. (D) Co-applied pyruvate (5 mM) showed no attenuating effect on the neuronal death induced by C2-ceramide (25 µM, 48-hour exposure). In fact, pyruvate appeared to increase the C2-ceramide toxicity, which is consistent with a lower pump current in the presence of pyruvate and C2-ceramide (see Fig. 2C). n=8-31 cultures. *P<0.05 compared with serum deprivation alone; #P<0.05 compared with serum deprivation plus pyruvate.

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