Korean J Physiol Pharmacol.  2013 Aug;17(4):275-281. 10.4196/kjpp.2013.17.4.275.

Expression and Activity of the Na-K ATPase in Ischemic Injury of Primary Cultured Astrocytes

• Kim MJ
• Hur J
• Ham IH
• Yang HJ
• Kim Y
• Park S
• Cho YW

• KMID: 2285461
• DOI: http://doi.org/10.4196/kjpp.2013.17.4.275

Abstract

Astrocytes are reported to have critical functions in ischemic brain injury including protective effects against ischemia-induced neuronal dysfunction. Na-K ATPase maintains ionic gradients in astrocytes and is suggested as an indicator of ischemic injury in glial cells. Here, we examined the role of the Na-K ATPase in the pathologic process of ischemic injury of primary cultured astrocytes. Chemical ischemia was induced by sodium azide and glucose deprivation. Lactate dehydrogenase assays showed that the cytotoxic effect of chemical ischemia on astrocytes began to appear at 2 h of ischemia. The expression of Na-K ATPase alpha1 subunit protein was increased at 2 h of chemical ischemia and was decreased at 6 h of ischemia, whereas the expression of alpha1 subunit mRNA was not changed by chemical ischemia. Na-K ATPase activity was time-dependently decreased at 1, 3, and 6 h of chemical ischemia, whereas the enzyme activity was temporarily recovered to the control value at 2 h of chemical ischemia. Cytotoxicity at 2 h of chemical ischemia was significantly blocked by reoxygenation for 24 h following ischemia. Reoxygenation following chemical ischemia for 1 h significantly increased the activity of the Na-K ATPase, while reoxygenation following ischemia for 2 h slightly decreased the enzyme activity. These results suggest that the critical time for ischemia-induced cytotoxicity of astrocytes might be 2 h after the initiation of ischemic insult and that the increase in the expression and activity of the Na-K ATPase might play a protective role during ischemic injury of astrocytes.

Keyword

Chemical ischemia; Na-K ATPase; Primary cultured astrocytes

MeSH Terms

Adenosine Triphosphatases
Astrocytes
Brain Injuries
Glucose
Ischemia
L-Lactate Dehydrogenase
Neuroglia
Neurons
RNA, Messenger
Sodium Azide
Adenosine Triphosphatases
Glucose
L-Lactate Dehydrogenase
RNA, Messenger
Sodium Azide

Figure

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  Fig. 1 Primary cultures of astrocytes. Immunohistochemistry of primary astrocytes and Na-K ATPase. DAPI, blue (A); GFAP, red (B); Na-K ATPase, green (C); Merged image (D). Scale bar=200 µm.

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  Fig. 2 Effect of chemical ischemia on the cytotoxicity and viability of cultured astrocytes. Cytotoxicity and cell viability was measured by LDH and MTT assays, respectively. The levels of LDH release (A) and MTT reduction (B) were quantified and compared to the control at each time point. Each value indicates the mean±S.E.M. normalized to the control of each time point. Data were obtained from five experiments. *p<0.05, **p<0.01, and ***p<0.001.

• 

  Fig. 3 Effect of chemical ischemia on the expression of Na-K ATPase α1 subunit and Na-K ATPase activity in cultured astrocytes. (A) Western blot analysis shows the expression of Na-K ATPase α1 subunit protein following chemical ischemia for 1, 2, 3, and 6 h. Data were obtained from five experiments. (B) Real-time PCR analysis shows the expression of Na-K ATPase α1 subunit mRNA in cultured astrocytes following chemical ischemia for 1, 2, 3, and 6 h. Data were obtained from four experiments. (C) The activity of Na-K ATPase in the membrane fraction of cultured astrocytes after chemical ischemia for 1, 2, 3, and 6 h. Data were obtained from five experiments. For all experiments, each value indicates the mean±S.E.M. normalized to the control of each time point. *p<0.05, **p<0.01.

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  Fig. 4 Effects of reoxygenation on the changes in the cytotoxicity, viability, and activity of the Na-K ATPase. The levels of LDH release (A) and MTT reduction (B) were quantified at 24 h of reoxygenation following chemical ischemia for 1, 2, 3, and 6 h. (C) Activity of Na-K ATPase was measured at 24 h of reoxygenation following chemical ischemia for 1 and 2 h. Data show the mean± S.E.M. of the relative values obtained from five animals. *p<0.05, **p<0.01, and ***p<0.001.

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