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 > or = 10 microM, the rise in [Ca2+]i was biphasic; an initial phase increasing basal [Ca2+]i by 100% was succeeded by a second phase which raised [Ca2+]i to several microM. The enhanced signal was sustained and slowly approached the Fmax 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 microM, 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.
Wong
K
K
Department of Pharmacology and Therapeutics, University of Calgary, Alberta, Canada.
Kwan-Yeung
L
L
D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D007424:Intracellular Fluid / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D037342:Pertussis Toxin; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D012503:Saponins / Q000494:pharmacology; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D005456:Fluorescent Dyes; D012503:Saponins; D010566:Virulence Factors, Bordetella; C005356:sphingosine phosphorylcholine; C049925:fura-2-am; D010767:Phosphorylcholine; D008345:Manganese; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D004492:Edetic Acid; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2