Is calcineurin a peroxide-specific sensor in T-lymphocytes?

Abstract.

Calcineurin activity in T-lymphocytes has been shown to be sensitive to H₂O₂. In this report, we investigate the effects of H₂O₂ and other physiological oxidants on calcineurin activity both in vivo and in vitro. Intracellular calcineurin activity, as determined by NF-AT phosphorylation state and activity, is inhibited by H₂O₂ but is insensitive to superoxide and NaOCl. Similarly, treatment of T-lymphocytes with NaOCl and paraquat does not drastically alter the activity of calcineurin in crude cell lysate, while H₂O₂ causes significant inhibition. Sensitivity to H₂O₂ and NaOCl in vivo correlates with the half-life of each species in cell medium. The intracellular redox potential is unaffected by H₂O₂ (100 µM) or NaOCl (600 µM), indicating that H₂O₂ inhibits calcineurin via a direct mechanism that does not involve a change in the cytosolic redox potential. In contrast, calcineurin activity in cell lysate is inhibited by all three oxidants. H₂O₂ inactivation of calcineurin is rapid, with inactivation occurring in ≤10 s. H₂O₂ inactivation of calcineurin is reversible in T-lymphocyte lysate using dithiothreitol (DTT) alone, while in rat brain lysate, reversibility requires both DTT and Fe(II), implicating that the enzyme may exist in different metal/redox-sensitive states in different tissues. Electronic supplementary material to this paper, namely Fig. S1 showing the effect of paraquat and NaOCl on NF-AT phosphorylation, can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-002-0367-x.