Hydrogen peroxide attenuates store-operated calcium entry and enhances calcium extrusion in thyroid FRTL-5 cells

Redox modulation participates in the regulation of intracellular free calcium concentration ([Ca²⁺]_i) in several cell types. In thyroid cells, including FRTL-5 cells, changes in [Ca²⁺]_i regulate several important functions, including the production of H₂O₂ (hydrogen peroxide). As H₂O₂ is of crucial importance for the production of thyroid hormones, we investigated the effects of H₂O₂ on [Ca²⁺]_i in thyroid FRTL-5 cells. H₂O₂ itself did not modulate basal [Ca²⁺]_i. However, H₂O₂ attenuated store-operated calcium entry evoked by thapsigargin, both in a sodium-containing buffer and in a sodium-free buffer. The effect of H₂O₂ was abrogated by the reducing agent β-mercaptoethanol. H₂O₂ also attenuated the thapsigargin-evoked entry of barium and manganese. The effect of H₂O₂ was, at least in part, mediated by activation of protein kinase C (PKC), as H₂O₂ enhanced the binding of [³H]phorbol 12,13-dibutyrate. H₂O₂ also stimulated the translocation of the isoenzyme PKCε from the cytosolic fraction to the particulate fraction. Furthermore, H₂O₂ did not attenuate store-operated calcium entry in cells treated with staurosporine or calphostin C, or in cells with down-regulated PKC. H₂O₂ depolarized the membrane potential in bisoxonol-loaded cells and when patch-clamp in the whole-cell mode was used. The depolarization was attenuated in cells with down-regulated PKC. This depolarization, at least in part, explained the H₂O₂-evoked inhibition of calcium entry. In addition, H₂O₂ enhanced the extrusion of calcium from cells stimulated with thapsigargin and this effect was abolished in cells with down-regulated PKC and after treatment of the cells with the reducing agent β-mercaptoethanol. In conclusion H₂O₂ attenuates an increase in [Ca²⁺]_i. As H₂O₂ is produced in thyroid cells in a calcium-dependent manner, our results suggest that H₂O₂ may participate in the regulation of [Ca²⁺]_i in these cells via a negative-feedback mechanism involving activation of PKC.