Ceramide 1-phosphate increases intracellular free calcium concentrations in thyroid FRTL-5 cells:: evidence for an effect mediated by inositol 1,4,5-trisphosphate and intracellular sphingosine 1-phosphate

Sphingolipid (SP) derivatives have diverse effects on the regulation of intracellular free calcium concentrations ([Ca2+](i)) in a multitude of non-excitable cells. In the present investigation, the effect of C-2-ceramide I-phosphate (C1P) on [Ca2+](i) was investigated in thyroid FRTL-5 cells. C1P evoked a concentration-dependent increase in [Ca2+](i) both in a calcium-containing and a calcium-free buffer. A substantial part of the C I P-evoked increase in [Ca2+](i) was due to calcium entry. The effect of C1P was attenuated by overnight pretreatment of the cells with pertussis toxin. Similar results were obtained with C-8-ceramide I-phosphate, although the magnitude of the responses was smaller than with C1P. The phospholipase C inhibitor U73122 attenuated the effect of C1P. C1P invoked a small, but significant, increase in inositol 1,4,5-trisphosphate (IP2). How ever, the effect of C1P on [Ca2+](i) was inhibited by neither Xestospongin C, 2-aminoethoxydiphenylborate nor neomycin. C1P mobilized calcium from an IP3-sensitive calcium store, as C1P did not increase [Ca2+](i) in cells pretreated with thapsigargin. The effect of C1P on [Ca2+](i)was potently attenuated by dihydrosphingosine and dimethylsphingosine, two inhibitors of sphingosine kinase, but not by the inactive SP-derivative N-acetyl sphingosine. Stimulating the cells with C I P evoked an increase in the production of intracellular sphingosine I-phosphate. C I P did not modulate DNA synthesis or the forskolin-evoked production of cAMP. The results indicate that C I P may be an important SP participating in cellular signalling.