Ca Mobilizing Action of Sphingosine in Jurkat Human Leukemia T Cells

Effects of sphingosine on Ca mobilization in the human Jurkat T cell line were examined. Sphingosine increased the cytoplasmic Ca concentration ([Ca ] ) in a dose-dependent manner with an ED of around 8 μM. Sphingosine and OKT3, a CD3 monoclonal antibody, transiently increased [Ca ] , which declined to the resting level in the absence of extracellular Ca . Under the same conditions, pretreatment with sphingosine inhibited but did not abolish an increase in [Ca ] induced by the subsequent addition of OKT3 and vice versa . However, pretreatment with sphingosine did not affect an increase in [Ca ] induced by OKT3 in the presence of Ca . OKT3 increased IP formation, but sphingosine did not affect the level of IP by itself nor did it cause IP formation induced by OKT3. In permeabilized Jurkat cells, the addition of IP released Ca from nonmitochondrial intracellular stores, but the addition of sphingosine did not. Sphingosine, stearylamine, and psychosine increased [Ca ] and diacylglycerol (DG) kinase activation; however, ceramide did not, whereas sphingosine 1-phosphate slightly activated DG kinase without elevation of [Ca ] . Pretreatment with R59022, a DG kinase inhibitor, abolished the peak but did not affect the sustained response of [Ca ] to sphingosine. Phosphatidic acid (PA) elevated [Ca ] , after which it declined to a resting level even in the presence of extracellular Ca . In accordance with this, PA did not stimulate Ca uptake into cells, but sphingosine and OKT3 did. Pretreatment with PA partially inhibited a rise in [Ca ] induced by the subsequent addition of sphingosine and vice versa in the absence of extracellular Ca . Under similar conditions, pretreatment with PA affected an elevation of [Ca ] induced by OKT3 less, after which the subsequent addition of sphingosine did not increase [Ca ] . In permeabilized Jurkat cells, the addition of IP did not release Ca , but PA did in the presence of heparin. Pretreatment with thapsigargin, a microsomal Ca -ATPase inhibitor, abolished the rises of [Ca ] induced by the subsequent addition of sphingosine, OKT3, and PA in the absence of extracellular Ca . The present results suggest that at least two kinds of intracellular Ca stores exist in Jurkat cells, both of which are IP - and PA-sensitive, and that sphingosine mobilizes Ca from both stores in an IP -independent manner. Furthermore, the IP - but not the PA-sensitive intracellular Ca store seems to regulate Ca entry induced by sphingosine.