SMase inhibited the Tg-evoked calcium entry in a concentration-dependent manner. Furthermore, an inhibition of calcium entry was obtained after preincubation of the cells with the membrane-permeable C 2 -ceramide and C 6 -ceramide analogues. The inactive ceramides dihydro-C 2 and dihydro-C 6 showed only marginal effects. Neither SMase, C 2 -ceramide, nor C 6 -ceramide affected the release of sequestered calcium. C 2 -and C 6 -ceramide also decreased the ATP-evoked calcium entry, without affecting the release of sequestered calcium. The effect of TNF-␣ and SMase was inhibited by the kinase inhibitor staurosporin and by the protein kinase C (PKC) inhibitor calphostin C but not by down-regulation of PKC. However, we were unable to measure a significant activation of PKC using TNF-␣ or C 6 -ceramide. The effect of TNF-␣ was not mediated via activation of either c-Jun N-terminal kinase or p38 kinase. We were unable to detect an increase in the ceramide (or sphingosine) content of the cells after stimulation with TNF-␣ for up to 30 min. Thus, one mechanism of action of TNF-␣, SMase, and ceramide on thyroid FRTL-5 cells is to inhibit calcium entry.

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