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 calci um concentration ([Ca2+](i)) in several cell types. In thyroid cells, inclu ding FRTL-5 cells, changes in [Ca2+](i) regulate several important function s, including the production of H2O2 (hydrogen peroxide). As H2O2 is of cruc ial 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 m odulate basal [Ca2+](i). However, H2O2 attenuated store-operated calcium en try evoked by thapsigargin, both in a sodium-containing buffer and in a sod ium-free buffer. The effect of H2O2 was abrogated by the reducing agent P-m ercaptoethanol. H2O2 also attenuated the thapsigargin-evoked entry of bariu m and manganese. The effect of H2O2 was, at least in part, mediated by acti vation of protein kinase C (PKC), as H2O2 enhanced the binding of [H-3]phor bol 12,13-dibutyrate. H2O2 also stimulated the translocation of the isoenzy me PKC epsilon from the cytosolic fraction to the particulate fraction. Fur thermore, H2O2 did not attenuate store-operated calcium entry in cells trea ted with staurosporine or calphostin C, or in cells with down-regulated PKC . H2O2 depolarized the membrane potential in bisoxonol-loaded cells;and whe n patch-clamp in the whole-cell mode was used. The depolarization was atten uated in cells with downregulated PKC. This depolarization, at least in par t, 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 downregulated PKC and after tre atment of the cells with the reducing agent beta-mercaptoethanol. In conclu sion H2O2 attenuates an increase in [Ca2+](i). As H2O2 is produced in thyro id cells in a calcium-dependent manner, our results suggest that H2O2 may p articipate in the regulation of [Ca2+](i) in these cells via a negative-fee dback mechanism involving activation of PKC.