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.
- calcium entry
↵1 These authors contributed equally to this study.
Abbreviations used: 2-APB, 2-aminoethoxydiphenylborate; [Ca2+]i, intracellular free calcium concentration; C1P, C2-ceramide 1-phosphate; C8-C1P, C8-ceramide 1-phosphate; DHS, dihydrosphingosine; DMS, dimethylsphingosine; IP3, inositol 1,4,5-trisphosphate; lysoPA, lysophosphatidic acid; PLC, phospholipase C; SOC, store-operated calcium; SPC, sphingosylphosphorylcholine; S1P, sphingosine 1-phosphate; TSH, thyrotropin.
- The Biochemical Society, London ©2003