SPHINGOSINE 1-PHOSPHATE MOBILIZES SEQUESTERED CALCIUM, ACTIVATES CALCIUM-ENTRY, AND STIMULATES DEOXYRIBONUCLEIC-ACID SYNTHESIS IN THYROID FRTL-5 CELLS

Sphingosine 1-phosphate (SPP) potently mobilizes sequestered calcium a nd is a mitogen in several cell types. In the present investigation, w e have evaluated the effect of SPP on intracellular free calcium conce ntration ([Ca2+](i)) and synthesis of DNA in thyroid FRTL-5 cells. SPP rapidly and transiently mobilized sequestered calcium and stimulated entry of extracellular calcium. The entry of calcium, but not the mobi lization, was in part inhibited by pretreatment with pertussis toxin ( Ptx), and by activation of protein kinase C. SPP did not stimulate the production of inositol 1,4,5-trisphosphate. SPP stimulated the incorp oration of H-3-thymidine in a time- and dose-dependent manner. The eff ect was not inhibited by Ptx. Furthermore, SPP stimulated the activati on of the proto-oncogene c-fos. SPP rapidly tyrosine-phosphorylated an approximately 66 kDa protein. This phosphorylation persisted for at l east 1 h. Pretreatment of the cells with genistein abolished the SPP-e voked tyrosine phosphorylation, and attenuated the SPP-evoked increase in [Ca2+](i). Furthermore, the SPP-evoked activation of Na+-H+ exchan ge was inhibited by genistein. The phosphorylation was not attenuated by pretreatment of the cells with Ptx. SPP per se did not affect cellu lar cAMP levels but attenuated the TSH-evoked increase in cAMP. As the effect of SPP might be due to activation of phospholipase D, we teste d whether phosphatidic acid (PA) mobilized calcium or stimulated the i ncorporation of H-3-thymidine. PA mobilized sequestered calcium but di d not stimulate calcium entry. PA very modestly enhanced the incorpora tion of H-3-thymidine. Our results suggest, that SPP stimulates DNA sy nthesis and activates entry of calcium in FRTL-5 cells. The effect on calcium entry appears to be dependent, at least in part, on one or sev eral tyrosine kinases.