CA2-CELLS - EVIDENCE THAT SPHINGOSINE RELEASES CA2+ FROM INOSITOL TRISPHOSPHATE-SENSITIVE AND PHOSPHATIDIC-ACID-SENSITIVE INTRACELLULAR STORES THROUGH A MECHANISM INDEPENDENT OF INOSITOL TRISPHOSPHATE( MOBILIZING ACTION OF SPHINGOSINE IN JURKAT HUMAN LEUKEMIA T)

Effects of sphingosine on Ca2+ mobilization in the human Jurkat T cell line mere examined. Sphingosine increased the cytoplasmic Ca2+ concen tration ([Ca2+](i)) in a dose-dependent manner with an ED(50) of aroun d 8 mu M. Sphingosine and OKT3, a CD3 monoclonal antibody, transiently increased [Ca2+](i), which declined to the resting level in the absen ce of extracellular Ca2+, Under the same conditions, pretreatment with sphingosine inhibited but did not abolish an increase in [Ca2+](i) in duced by the subsequent addition of OKT3 and vice versa, However, pret reatment with sphingosine did not affect an increase in [Ca2+](i) indu ced by OKT3 in the presence of Ca2+. OKT3 increased IP3 formation, but sphingosine did not affect the level of IP3 by itself nor did it caus e IP3 formation induced by OKT3, In permeabilized Jurkat cells, the ad dition of IP3 released Ca2+ from nonmitochondrial intracellular stores , but the addition of sphingosine did not. Sphingosine, stearylamine, and psychosine increased [Ca2+](i) and diacylglycerol (DG) kinase acti vation; however, ceramide did not, whereas sphingosine 1-phosphate sli ghtly activated DG kinase without elevation of [Ca2+](i). Pretreatment with R59022, a DG kinase inhibitor, abolished the peak but did not af fect the sustained response of [Ca2+](i) to sphingosine. Phosphatidic acid (PA) elevated [Ca2+](i), after which it declined to a resting lev el even in the presence of extracellular Ca2+. In accordance with this , PA did not stimulate Ca-45(2+) uptake into cells, but sphingosine an d OKT3 did, Pretreatment with PA partially inhibited a rise in [Ca2+]( i) induced by the subsequent addition of sphingosine and vice versa in the absence of extracellular Ca2+. Under similar conditions, pretreat ment with PA affected an elevation of [Ca2+](i) induced by OKT3 less, after which the subsequent addition of sphingosine did not increase [C a2+](i). In permeabilized Jurkat cells, the addition of IP3 did not re lease Ca2+, but PA did in the presence of heparin. Pretreatment with t hapsigargin, a microsomal Ca2+-ATPase inhibitor, abolished the rises o f [Ca2+](i) induced by the subsequent addition of sphingosine, OKT3, a nd PA in the absence of extracellular Ca2+. The present results sugges t that at least two binds of intracellular Ca2+ stores exist in Jurkat cells, both of which are IP3- and PA-sensitive, and that sphingosine mobilizes Ca2+ from both stores in an IP3-independent manner, Furtherm ore, the IP3- but not the PA-sensitive intracellular Ca2+ store seems to regulate Ca2+ entry induced by sphingosine.