Differential Effects of Methionine and Cysteine Oxidation on [Ca2+]i in Cultured Hippocampal Neurons

Abstract

Methionine and cysteine residues in proteins are the major targets of reactive oxygen species (ROS). The present work was designed to characterize the impact of methionine and cysteine oxidation upon [Ca(2+)](i) in hippocampal neurons. We investigated the effects of H(2)O(2) and chloramine T(Ch-T) agents known to oxidize both cysteine and methionine residues, and 5, 5'-dithio-bis (2-nitrobenzoic acid) (DTNB)--a cysteine-specific oxidant, on the intracellular calcium in hippocampal neurons. The results showed that these three oxidants, 1 mM H(2)O(2), 1 mM Ch-T, and 500 microM DTNB, induced an sustained elevation of [Ca(2+)](i) by 76.1 +/- 3.9%, 86.5 +/- 5.0%, and 24.4 +/- 3.2% over the basal level, respectively. The elevation induced by H(2)O(2) and Ch-T was significantly higher than DTNB. Pretreatment with reductant DTT at 1 mM for 10 min completely prevented the action of DTNB on [Ca(2+)](i), but only partially reduced the effects of H(2)O(2) and Ch-T on [Ca(2+)](i), the reductions were 44.6 +/- 4.2% and 29.6 +/- 6.1% over baseline, respectively. The elevation of [Ca(2+)](i) induced by H(2)O(2) and Ch-T after pretreatment with DTT were statistically higher than that induced by single administration of DTNB. Further investigation showed that the elevation of [Ca(2+)](i) mainly resulted from internal calcium stores. From our data, we propose that methionine oxidation plays an important role in the regulation of intracellular calcium and this regulation may mainly be due to internal calcium stores.