SPHINGOSINE MOBILIZES INTRACELLULAR CALCIUM IN HUMAN NEUTROPHILS

The effect of sphingosine on the cytosolic free Ca2+ concentrations, [Ca2+]i, of human neutrophils was re-examined using Fura-2 loaded cells. We found that sphingosine induced a dose-dependent elevation of [Ca2+]i. At sphingosine concentrations greater-than-or-equal-to 10 muM, the rise in [Ca2+], was biphasic; an initial phase increasing basal [Ca2+]i by 100% was succeeded by a second phase which raised [Ca2+]i to several muM. The enhanced signal was sustained and slowly approached the F(max) of Fura-2 over 10 min. Although cytotoxicity assays indicate that Fura-2 leakage contributed to the rise in fluorescence, EGTA, surprisingly, had no effect on the time course of this response. The explanation was that EGTA blocked Fura-2 leakage from and trypan blue uptake by neutrophils. Thus, in the presence of EGTA, biphasic increases in the fluorescent signal can be attributed mainly to release of intracellular Ca2+. Mn2+ quenching studies confirmed that sphingosine mobilized Ca2+ in two distinct phases and promoted the influx of Mn2+. Mn2+ entry, however, was not matched by substantial Ca2+ influx. Sphingosine elevation of [Ca2+]i was insensitive to pertussis toxin treatment of neutrophils and was not correlated with (1,4,5)IP3 formation. Studies with semi-permeabilized cells show that sphingosine, up to 80 muM, neither mobilized Ca2+ significantly nor inhibited active Ca2+ sequestration. Sphingosylphosphorylcholine induced a small but dose dependent release of Ca2+. We hypothesize that a metabolite of sphingosine may release Ca2+ directly in intact neutrophils.