Activation of the plasma membrane Na⁺/H⁺ exchanger NHE1 contributes importantly to ischemic/anoxic cell damage, yet the mechanisms involved are unclear. In NIH3T3 cells, PCR studies confirmed the expression of NHE1 and -8, yet not NHE2, -3, and -4. Chemical anoxia (10 mM azide, 10 min) was associated with a decrease in pH_i which was exacerbated by the NHE1 inhibitor EIPA (5 µM). Reperfusion (azide washout) elicited a rapid, EIPA-sensitive alkalinization to 7.60 ± 0.057 (n=6), compared to a starting pH_i of 7.49 ± 0.032 (n=6). Cell survival was reduced by prolonged chemical anoxia (to 87% at 3 h and 41% at 24 h, MTT assay), an effect counteracted by EIPA at early ( 6 h) time points. Chemical anoxia was furthermore associated with: (i) a rapid ( 10 min) and transient phosphorylation of p38 MAPK, which was abolished by NHE1 inhibitors (EIPA, cariporide, 5 µM); (ii) increased phosphorylation of the tumor suppressor p53, which was maximal at 3 h and unaffected by EIPA and by the p38 MAPK inhibitor SB203580; (iii) a transient reduction in phosphorylated ezrin-radixin-moesin (ERM) protein, which appeared to be counteracted by EIPA. In contrast, neither chemical anoxia nor EIPA affected Akt phosphorylation.

In summary, in NIH3T3 cells, chemical anoxia was associated with decreased pH_i, and activation of NHE1, p38 MAPK, and p53, reduced levels of phosphorylated ERM proteins, and reduced survival. Suggesting an important role of NHE1 upstream of p38 MAPK in anoxic cell death, NHE1 inhibition blocked p38 MAPK activation and increased survival after chemical anoxia.

Funding: Danish Natural Sciences Research Council (grant 21-01-0507, 21-04-0535)