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 C₂-ceramide 1-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 C1P-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 C₈-ceramide 1-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 (IP₃). However, 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 C1P evoked an increase in the production of intracellular sphingosine 1-phosphate. C1P did not modulate DNA synthesis or the forskolin-evoked production of cAMP. The results indicate that C1P may be an important SP participating in cellular signalling.