Sphingosine-1-Phosphate–Induced Intracellular Ca²⁺ Mobilization in Human Endothelial Cells

Abstract

The authors have studied the effect of sphingosine-1-phosphate (S1P) on Ca²⁺ release from intracellular stores in cultured human umbilical vein endothelial cells (HUVECs). In the presence of extracellular Ca²⁺, S1P increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) and this increase was partially inhibited by La³⁺ (1 μM), indicating that S1P induces Ca²⁺ influx from extracellular pool and Ca²⁺ release from intracellular stores. S1P increased [Ca²⁺]_i concentration dependently in Ca²⁺-free extracellular solution. The Hill coefficient (1.7) and EC₅₀ (420 nM) was obtained from the concentration-response relationship. When caffeine depleted Ca²⁺ store in the presence of ryanodine, S1P did not induce intracellular Ca²⁺ release. Furthermore, the Ca²⁺-induced Ca²⁺ release inhibitors ruthenium red or dantrolene completely inhibited S1P-induced intracellular Ca²⁺ release. S1P-induced intracellular Ca²⁺ release was inhibited by the phospholipase C (PLC) inhibitors neomycin and U73312, or the inositol 1,4,5-triphosphate (IP₃)-gated Ca²⁺ channel blocker aminoethoxybiphenyl borane (2-APB). In contrast, S1P-induced intracellular Ca²⁺ release was not inhibited by the mitochondrial Ca²⁺ uptake inhibitor CCCP or the mitochondrial Ca²⁺ release inhibitor cyclosporin A. These results show that S1P mobilizes Ca²⁺ from intracellular stores primarily via Ca²⁺-induced and IP₃-induced Ca²⁺ release and this Ca²⁺ mobilization is independent of mitochondrial Ca²⁺ stores.

Keywords:

• Ca²⁺-Induced Ca²⁺ Release
• Human Umbilical Vein Endothelial Cells
• IP₃-Induced Ca²⁺ Release
• Sphingosine-1-Phosphate