Stimulation of sphingosine-1-phosphate formation by the P2Y(2) receptor inHL-60 cells: Ca2+ requirement and implication in receptor-mediated Ca2+ mobilization, but not MAP kinase activation

Sphingosine-1-phosphate (SPP), produced by sphingosine kinase, has recently been reported to act as an intracellular second messenger for Ca2+ and mit ogenic responses triggered by membrane receptors and as an extracellular li gand for specific SPP receptors. Here, we investigated the signaling pathwa y leading to SPP production by the G protein-coupled P2Y(2) receptor and it s functional implication in human leukemia (HL-60) cells, which do not resp ond to extracellular SPP. P2Y(2) receptor activation by UTP or ATP resulted in rapid and transient production of SPP, which was insensitive to pertuss is toxin and blocked by the sphingosine kinase inhibitor, DL-threo-dihydros phingosine. Treatment of HL-60 cells with this inhibitor did not affect act ivation of mitogen-activated protein kinases, but suppressed Ca2+ mobilizat ion by the P2Y(2) receptor. However, receptor-induced SPP production appare ntly required an increase in intracellular Ca2+ concentration, but not Ca2 influx, and was mimicked by exposure of cells to Ca2+ ionophores. Taken to gether, activation of the P2Y(2) receptor stimulates SPP production in HL-6 0 cells, a process apparently not required for mitogen-activated protein ki nase activation, but most likely representing an amplification system for r eceptor-mediated Ca2+ signaling.