sq-23377 and sphingosine-phosphorylcholine

1. The increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) following repetitive stimulation with ATP or sphingosylphosphorylcholine (SPC) in single porcine aortic smooth muscle cells was investigated using the Ca(2+) indicator, fura-2. 2. The ATP-induced [Ca(2+)](i) increase resulted from both Ca(2+) release and Ca(2+) influx. The former was stimulated by phospholipase C activation, while the latter occurred predominantly via the receptor-operated Ca(2+) channels (ROC), rather than the store-operated Ca(2+) channels (SOC) or the voltage-operated Ca(2+) channel (VOC). Furthermore, the P2X(5) receptor was shown to be responsible for the ATP-induced Ca(2+) influx. 3. A reproducible [Ca(2+)](i) increase was induced by repetitive ATP stimulation, but was abolished by removal of extracellular Ca(2+) or inhibition of intracellular Ca(2+) release using U-73122 or thapsigargin, and was restored by Ca(2+) readdition in the former case. 4. SPC only caused Ca(2+) release, and the amplitude of the repetitive SPC-induced [Ca(2+)](i) increases declined gradually. However, a reproducible [Ca(2+)](i) increase was seen in cells in which protein kinase C being inhibited, which increased the SPC-induced Ca(2+) influx, rather than IP(3) generation. 5. In conclusion, although the amplitude of the ATP-induced Ca(2+) release, measured when Ca(2+) influx was blocked, or of the Ca(2+) influx when Ca(2+) release was blocked, progressively decreased following repetitive stimulation, the overall [Ca(2+)](i) increase for each stimulation under physiological conditions remained the same, suggesting that the Ca(2+) stores were replenished by an influx of Ca(2+) during stimulation. The SPC-induced [Ca(2+)](i) increase resulted solely from Ca(2+) release and decreased gradually following repetitive stimulation, but the decrease could be prevented by stimulating Ca(2+) influx, further supporting involvement of the intracellular Ca(2+) stores in Ca(2+) signalling.

Topics: Adenosine; Adenosine Triphosphate; Animals; Aorta; Calcium; Calcium Channel Blockers; Calcium Signaling; Cells, Cultured; Cyclic AMP; Egtazic Acid; Estrenes; Imidazoles; Ionomycin; Manganese; Muscle, Smooth, Vascular; Phosphorylcholine; Pyrrolidinones; Sphingosine; Staurosporine; Swine; Thapsigargin; Thionucleosides; Thionucleotides; Virulence Factors, Bordetella

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.

Topics: Biological Transport; Calcium; Cell Compartmentation; Cell Death; Dose-Response Relationship, Drug; Edetic Acid; Fluorescent Dyes; Fura-2; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Ionomycin; Manganese; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pertussis Toxin; Phosphorylcholine; Saponins; Sphingosine; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella