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Modulatory effect of protein kinase C on thapsigargin-induced calcium entry in thyroid FRTL-5 cells.

ABSTRACT: The aim of the present study was to investigate the regulation of calcium influx in thyroid FRTL-5 cells. Stimulating Fura 2-loaded cells with thapsigargin rapidly increased the cytosolic Ca2+ concentration ([Ca2+]i), which then stabilized at a new elevated plateau level. The initial increase in [Ca2+]i consisted mainly of the release of sequestered Ca2+. The plateau phase was totally dependent on extracellular Ca2+. The influx of Ca2+ was blocked by Ni2+ and was decreased in depolarized cells. The importance of protein kinase C in regulating influx of Ca2+ was then evaluated. Addition of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate prior to thapsigargin significantly decreased the influx of extracellular Ca2+. Studies with bisoxonol to measure membrane potential showed that TPA depolarized the plasma membrane in FRTL-5 cells. In cells where protein kinase C was downregulated or was inhibited by staurosporine, the thapsigargin-induced influx of Ca2+ was enhanced. The results indicate that emptying intracellular Ca2+ pools is sufficient to induce influx of Ca2+ in FRTL-5 cells, and that protein kinase C has a modulatory effect on this process.

SUBMITTER: Tornquist K

PROVIDER: S-EPMC1132293 | biostudies-other | 1993 Mar

REPOSITORIES: biostudies-other

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Project description:Redox modulation participates in the regulation of intracellular free calcium concentration ([Ca(2+)](i)) in several cell types. In thyroid cells, including FRTL-5 cells, changes in [Ca(2+)](i) regulate several important functions, including the production of H(2)O(2) (hydrogen peroxide). As H(2)O(2) is of crucial importance for the production of thyroid hormones, we investigated the effects of H(2)O(2) on [Ca(2+)](i) in thyroid FRTL-5 cells. H(2)O(2) itself did not modulate basal [Ca(2+)](i). However, H(2)O(2) attenuated store-operated calcium entry evoked by thapsigargin, both in a sodium-containing buffer and in a sodium-free buffer. The effect of H(2)O(2) was abrogated by the reducing agent beta-mercaptoethanol. H(2)O(2) also attenuated the thapsigargin-evoked entry of barium and manganese. The effect of H(2)O(2) was, at least in part, mediated by activation of protein kinase C (PKC), as H(2)O(2) enhanced the binding of [(3)H]phorbol 12,13-dibutyrate. H(2)O(2) also stimulated the translocation of the isoenzyme PKCepsilon from the cytosolic fraction to the particulate fraction. Furthermore, H(2)O(2) did not attenuate store-operated calcium entry in cells treated with staurosporine or calphostin C, or in cells with down-regulated PKC. H(2)O(2) 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 H(2)O(2)-evoked inhibition of calcium entry. In addition, H(2)O(2) 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 beta-mercaptoethanol. In conclusion H(2)O(2) attenuates an increase in [Ca(2+)](i). As H(2)O(2) is produced in thyroid cells in a calcium-dependent manner, our results suggest that H(2)O(2) may participate in the regulation of [Ca(2+)](i) in these cells via a negative-feedback mechanism involving activation of PKC.

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Modulatory effect of protein kinase C on thapsigargin-induced calcium entry in thyroid FRTL-5 cells.

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