Introduction

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Recent studies indicate that sphingolipid metabolites function as a new class of intra- and intercellular second messengers, involved in a large variety of cellular processes. Besides ceramide and sphingosine, sphingosine-1-phosphate (SPP), which results from the phosphorylation of sphingosine by sphingosine kinase, has been in the focus of recent interest. Two distinct cellular actions of SPP have been proposed, namely, as agonist ligand for plasma membrane receptors and as intracellular second messenger (Meyer zu Heringdorf et al., 1997). Specific G protein-coupled SPP receptors, first characterized by functional studies (van Koppen et al., 1996a), were recently identified as members of the Edg receptor family (Goetzl and An, 1998; Lee et al., 1998; Okamoto et al., 1998; Zondag et al., 1998; Ancellin and Hla, 1999). The evidence for an intracellular action of SPP is based on the following major findings. First, activation of various plasma membrane receptors, such as the platelet-derived growth factor receptor (Olivera and Spiegel, 1993), the FcRI and FcRI antigen receptors (Choi et al., 1996; Melendez et al., 1998a,b), and the tumor necrosis factor- receptor (Xia et al., 1998), was found to rapidly increase intracellular SPP production by sphingosine kinase. Second, inhibition of sphingosine kinase with the competitive inhibitor DL-threo-dihydrosphingosine (tDHS) strongly reduced or even prevented cellular events triggered by these tyrosine kinase-linked receptors, such as activation of mitogen-activated protein (MAP) kinases, specifically of the extracellular signal-regulated kinases (Erks), stimulation of DNA synthesis, Ca²⁺ mobilization, and vesicular trafficking (Olivera and Spiegel, 1993; Choi et al., 1996; Pyne et al., 1996; Rani et al., 1997; Melendez et al., 1998a,b; Xia et al., 1998). Finally, intracellular SPP was found to mimic some of the receptor responses, i.e., it was shown to mobilize Ca²⁺ from internal stores and induce activation of Erks and DNA synthesis (Ghosh et al., 1994; Mattie et al., 1994; Qiao et al., 1998; Xia et al., 1998; van Brocklyn et al., 1998).

Recently, we have provided evidence that intracellular SPP production is apparently involved in Ca²⁺ signaling of some G protein-coupled receptors. Specifically, activation of M₂ and M₃ muscarinic acetylcholine receptors expressed in HEK-293 cells was found to rapidly increase intracellular SPP formation. Moreover, intracellular injection of SPP specifically and rapidly mobilized Ca²⁺ in intact HEK-293 cells, and inhibition of sphingosine kinase markedly inhibited Ca²⁺ mobilization by these and other G protein-coupled receptors (Meyer zu Heringdorf et al., 1998). In a comparable manner, we have recently reported that intracellular SPP formation apparently participates in Ca²⁺ signaling and Ca²⁺-dependent enzyme release, but not superoxide production, triggered by the G_i-coupled formyl peptide receptor in human leukemia (HL-60) granulocytes (Alemany et al., 1999). Most important, the Ca²⁺-mobilizing action of intracellular SPP was independent of plasma membrane SPP receptors. In HEK-293 cells, their action was prevented by pertussis toxin (PTX) treatment (Meyer zu Heringdorf et al., 1998), whereas in HL-60 cells extracellular SPP was inactive (van Koppen et al., 1996b; Alemany et al., 1999; present work). Furthermore, inhibition of Ca²⁺ signaling by the sphingosine kinase inhibitors did not affect phospholipase C stimulation by the G protein-coupled receptors or inositol 1,4,5-trisphosphate-induced Ca²⁺ release, and was not due to inhibition of protein kinase C (Meyer zu Heringdorf et al., 1998; Alemany et al., 1999).

In this study, we examined intracellular SPP formation by the G protein-coupled P2Y₂ receptor in HL-60 cells and whether this reaction is involved in Ca²⁺ mobilization and MAP kinase activation by the purinergic receptor. The P2Y₂ receptor, formerly termed P_2U receptor, is expressed in promyelocytic and myeloid differentiated HL-60 cells and mediates its effects largely via PTX-insensitive G proteins, most likely G₁₆ (Klinker et al., 1996; Baltensberger and Porzig, 1997). We report herein that P2Y₂ receptor activation induces rapid SPP production in HL-60 cells. Moreover, evidence is provided suggesting that intracellular SPP formation apparently represents an amplification system for receptor-mediated Ca²⁺ mobilization, but is not required for MAP kinase activation in HL-60 cells.

	Experimental Procedures

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Materials. 1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)/AM, ionomycin, and A-23187 were from Calbiochem (Schwalbach, Germany). D-erythro-[³H]Sphingosine (18 Ci/mmol) was from New England Nuclear (Brussels, Belgium). Rabbit anti-phosphospecific MAP kinase antibodies and rabbit anti-Erk antibodies were purchased from New England Biolabs (Schwalbach, Germany) and Santa Cruz (Heidelberg, Germany), respectively. All other materials were from previously described sources (Meyer zu Heringdorf et al., 1998; Alemany et al., 1999). Before use, tDHS was directly diluted in Hanks' balanced salt solution (HBSS) (118 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 5 mM D-glucose, and 15 mM HEPES, pH 7.4), containing in addition 1 mg/ml fatty-acid-free BSA. The respective solvent was used as vehicle control.

Cell Culture. Human promyelocytic HL-60 cells, provided by Dr. T. Wieland (Institut für Pharmakologie, Universität Hamburg), were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 150 U/ml penicillin, and 150 µg/ml streptomycin in 5% CO₂. For differentiation into neutrophil-like cells, HL-60 cells were cultured for 48 h in the presence of 0.5 mM dibutyryl cAMP. For PTX treatment, cells were incubated for 20 h with 100 ng/ml of the toxin. HEK-293 cells stably expressing the M₃ muscarinic acetylcholine receptor were cultured as reported in Meyer zu Heringdorf et al. (1998).

Measurement of [Ca²⁺]_i. [Ca²⁺]_i was determined with the fluorescent Ca²⁺ indicator dye Fura-2 in a Hitachi spectrofluorimeter as described in Alemany et al. (1999). Briefly, cells were loaded with 1 µM Fura-2/AM for 1 h at room temperature in HBSS. Thereafter, cells were washed twice, resuspended at a density of 1 × 10⁶ cells/ml, and used for fluorescence measurements. In some experiments, [Ca²⁺]_i was determined in the absence of extracellular Ca²⁺ and/or after treatment of cells for 1 min with 10 µM tDHS.

Assay of SPP Formation. Formation of SPP in HEK-293 cells and HL-60 cells was determined as reported previously (Alemany et al., 1999; Meyer zu Heringdorf et al., 1999). In brief, HL-60 cells (1.8 × 10⁶ cells) equilibrated in HBSS/BSA for 5 min at 37°C were incubated with 0.1 µCi [³H]sphingosine (~10⁵ cpm/tube; ~30 nM final concentration) and the agents indicated for the indicated periods of time at 37°C in a total volume of 200 µl. The reactions were stopped by addition of 2 ml of ice-cold methanol, followed by 1 ml of chloroform. After pelleting particulate material, the supernatant was evaporated to dryness and redissolved in 20 µl of methanol. Then, the samples were spotted onto silica gel 60 thin layer chromatography plates, together with authentic unlabeled sphingosine and SPP. Separation of the products was achieved with 1-butanol:acetic acid:water (3:1:1) as solvent system. Sphingosine and SPP spots visualized by staining with ninhydrin spray were scraped off, and radioactivity was measured by liquid scintillation counting. Formation of [³H]SPP is expressed as counts per minute per 1.8 × 10⁶ cells and corrected for time 0 values, amounting to ~100 cpm. The assay of [³H]SPP formation in HEK-293 cells was as in HL-60 cells, except that 0.7 × 10⁶ cells were present in the assay and that lipid extraction was performed on cells filtered over glass fiber filters to stop the reaction (Meyer zu Heringdorf et al., 1999).

Assay of MAP Kinase Activation. HL-60 cells (5 × 10⁶ cells) serum-starved overnight in growth medium were incubated for 5 min at 37°C in HBSS without and with 30 µM tDHS, followed by stimulation for 1 min with the indicated agonists. The reactions were stopped by addition of 0.5 ml of lysis buffer, containing 1% SDS and 10 mM Tris-HCl, pH 7.4, and heating of the lysates for 5 min at 95°C. After five passages through a 25-gauge needle, insoluble material was pelleted and the supernatant diluted with lysis buffer. Aliquots of the diluted samples (100 µl) were mixed with 200 µl of 3-fold concentrated electrophoresis sample buffer (250 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% 2-mercaptoethanol) and boiled for another 10 min. Equal amounts of protein (~20 µg, determined by the bicinchoninic acid method) of each sample were subjected to SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels and then blotted onto nitrocellulose filters. Nitrocellulose was then blocked with 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 5% BSA (fraction V; Sigma, Deisenhofen, Germany). After washing three times for 5 min in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.2% Tween 20, phosphorylated Erk1, Erk2, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 MAP kinase were detected on the blots by incubating with rabbit anti-phosphospecific Erk1/Erk2 antibodies (1 h at room temperature), anti-phosphospecific SAPK/JKN antibodies, and anti-phosphospecific p38 MAP kinase antibodies (each overnight at 4°C), respectively (dilution of the antibodies 1:1000). After three washes for 5 min, the blots were incubated with goat peroxidase-conjugated anti-rabbit antibodies (Sigma). After 1 h, the blots were washed again and immunoreactivity was visualized by enhanced chemiluminescence (Amersham, Freiburg, Germany). Total amount of Erk was detected with a rabbit anti-ERK1 antibody (0.1 µg/ml).

Data Presentation and Analysis. Unless otherwise stated, results are presented as mean ± S.E. of at least three independent experiments, each performed in duplicate or triplicate. Curve fitting was done by using iterative nonlinear regression analysis with the Prism program (GraphPad, San Diego, CA). Statistical analysis was performed by Student's two-tailed t test for unpaired data.

	Results

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Stimulation of SPP Production by the P2Y₂ Receptor in HL-60 Cells. To study whether the P2Y₂ receptor stimulates intracellular SPP production in HL-60 cells, formation of [³H]SPP from [³H]sphingosine was determined in the presence of the P2Y₂ receptor agonists UTP and ATP. Basal conversion of [³H]sphingosine to [³H]SPP in promyelocytic HL-60 cells was rapid, and within 3 to 5 min a plateau of [³H]SPP accumulation was reached (data not shown). Activation of P2Y₂ receptors by UTP (100 µM), which was applied simultaneously with [³H]sphingosine and which did not affect its cellular uptake, induced a rapid and transient increase in [³H]SPP production (Fig. 1A). After 2 min, the increase in [³H]SPP accumulation reached a maximum of 60 to 80% above basal values and then declined, approaching basal values after 5 min. Half-maximal and maximal stimulation of [³H]SPP production was observed at 0.6 ± 0.2 µM and 10 to 100 µM UTP, respectively (Fig. 1B). P2Y₂ receptor activation with ATP induced a similar rapid and transient increase in [³H]SPP accumulation, reaching 75% above basal values after a 2-min stimulation with 100 µM ATP. Furthermore, [³H]SPP production induced by UTP and ATP was rather similar in promyelocytic and myeloid differentiated HL-60 cells (data not shown and see below). When the measurements were performed in the presence of the sphingosine kinase inhibitor tDHS (Buehrer and Bell, 1992), basal and UTP-stimulated [³H]SPP formation was strongly reduced (Fig. 2). At 10 µM tDHS, basal [³H]SPP formation was reduced by about 80%, and that stimulated by UTP was completely suppressed, indicating that sphingosine kinase is responsible for P2Y₂ receptor-mediated SPP production in HL-60 cells.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   UTP-stimulated SPP production in HL-60 cells. Formation of [3H]SPP from [3H]sphingosine was measured in promyelocytic HL-60 cells for the indicated periods of time in the absence and presence of 100 µM UTP (A) or for 2 min with UTP at the indicated concentrations (B) as described under Experimental Procedures. Values are expressed as percentage of [3H]SPP production relative to unstimulated cells, amounting to 3400 to 3900 cpm/1.8 × 106 cells at 1 to 5 min of incubation. *, significantly different from basal SPP formation (P < .05, 4 to 12 experiments).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Inhibition of basal and UTP-stimulated SPP production by tDHS. Basal and UTP (100 µM)-stimulated [3H]SPP formation was measured for 2 min in HL-60 cells in the absence and presence of tDHS at the indicated concentrations. Values are expressed as percentage of [3H]SPP production relative to unstimulated control cells, amounting to 2480 ± 194 cpm/1.8 × 106 cells (three to five experiments).

SPP Production and MAP Kinase Activation. To study whether intracellular SPP formation participates in MAP kinase activation, the effect of the sphingosine kinase inhibitor tDHS on P2Y₂ receptor-mediated activation of various MAP kinases was examined. In the absence of tDHS, addition of 100 µM UTP induced a strong and rapid (maximum at 1 min) increase in phosphorylation states of Erk1 and Erk2 in promyelocytic HL-60 cells, as detected with phosphospecific antibodies against these MAP kinases (Fig. 3A). Pretreatment of the cells for 5 min with 30 µM tDHS, fully blocking UTP-induced SPP formation, did not alter basal Erk phosphorylation and had no effect on phosphorylation of Erk1 and Erk2 induced by UTP. Total amount of Erk, measured with an anti-Erk1 antibody, was not affected by either UTP or tDHS (data not shown). tDHS also did not inhibit phosphorylation of Erk1 and Erk2 by the phorbol ester phorbol-12-myristate-13-acetate (PMA, 1 µM), inducing after 1-min stimulation an increase in ERK phosphorylation equivalent to that seen with 100 µM UTP (Fig. 3A). Similar to promyelocytic cells, treatment of myeloid differentiated HL-60 cells with tDHS (30 µM, 5 min) had no effect on phosphorylation of Erk1 and Erk2 induced by UTP (100 µM), PMA (1 µM), or N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10 µM) (data not shown). P2Y₂ receptor activation by UTP (100 µM, 1 min) also induced distinct increases in phosphorylation states of SAPK/JNK (p46) and p38 MAP kinase in promyelocytic and differentiated HL-60 cells, respectively (Fig. 3, B and C). Similar to receptor-induced Erk phosphorylation, pretreatment of the cells with tDHS (30 µM, 5 min) did not affect UTP-induced phosphorylation of SAPK/JNK or p38 MAP kinase.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   tDHS does not inhibit activation of MAP kinases in HL-60 cells. Promyelocytic (A and B) and myeloid differentiated HL-60 cells (C) (5 × 106 cells each) were incubated for 5 min at 37°C in the absence (Control) and presence of 30 µM tDHS and then stimulated for 1 min with 100 µM UTP or 1 µM PMA as indicated. After SDS gel electrophoresis and transfer to nitrocellulose membranes, phosphorylation of Erk1 and Erk2 (A), JNK/SAPK (B), and p38 MAP kinase (C) was detected by immunoblotting with antiphosphospecific MAP kinase antibodies. The phosphorylated Erk1 (p44), Erk2 (p42), JNK/SAPK (p46), and p38 MAP kinase are marked by the arrows. Shown is a representative experiment repeated twice.

SPP Production and Ca²⁺ Mobilization. Previous studies with sphingosine kinase inhibitors in myeloid differentiated HL-60 cells suggested that intracellular SPP formation plays a major role in Ca²⁺ mobilization by the G_i-coupled formyl peptide receptor (Alemany et al., 1999). Therefore, P2Y₂ receptor-induced [Ca²⁺]_i increases and inhibition of this response by tDHS were studied. Half-maximal and maximal [Ca²⁺]_i increases were observed at ~0.5 µM and 10 to 100 µM UTP, respectively (data not shown), thus, at concentrations very similar to those required for stimulation of SPP formation (Fig. 1B). To study specifically the effect of tDHS on Ca²⁺ release from intracellular stores, we measured [Ca²⁺]_i transients in the absence of extracellular Ca²⁺. Figure 4A illustrates typical changes in [Ca²⁺]_i in HL-60 cells stimulated with 10 µM UTP under this condition. There was a rapid [Ca²⁺]_i increase, by about 200 nM, which declined to basal levels within 1 to 2 min. Pretreatment of HL-60 cells for 1 min with 10 µM tDHS, which by itself did not alter [Ca²⁺]_i, suppressed the UTP-induced [Ca²⁺]_i increase (Fig. 4B). Under the same condition, tDHS did not inhibit the increase in [Ca²⁺]_i induced by thapsigargin (1 µM), amounting to 149 ± 38 and 146 ± 12 nM above basal in the presence of vehicle and 10 µM tDHS, respectively. Most important, extracellularly applied SPP (10 µM) had no effect on [Ca²⁺]_i in HL-60 cells (Fig. 4C). tDHS (10-30 µM) also strongly inhibited (by 80-90%) UTP- or ATP-induced [Ca²⁺]_i increases measured in the presence of extracellular Ca²⁺ (data not shown). These data, thus, suggested that sphingosine kinase-catalyzed SPP formation plays a major role in Ca²⁺ mobilization by the P2Y₂ receptor in HL-60 cells, similarly as reported for the formyl peptide receptor (Alemany et al., 1999).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Inhibition of UTP-induced Ca2+ mobilization by tDHS and lack of effect of extracellular SPP on [Ca2+]i. Shown are typical traces of UTP (10 µM)-induced [Ca2+]i increase determined in HL-60 cells pretreated for 1 min with vehicle (A) or 10 µM tDHS (B). Ca2+ was omitted from the extracellular medium, and 50 µM EGTA was added 2 min before stimulus exposure. C, typical trace of [Ca2+]i after the extracellular application of SPP (10 µM) in the presence of 1 mM extracellular Ca2+. Addition of vehicle (BSA), tDHS, UTP, and SPP is indicated by the arrows. Shown are representative experiments repeated five times.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Involvement of intracellular Ca2+ in receptor-mediated [3H]SPP formation. Myeloid differentiated HL-60 cells (A and B) or HEK-293 cells (C) were pretreated for 30 min without (Control) and with 20 µM BAPTA/AM, followed by measurement of [3H]SPP formation in the absence (Basal) and presence of 10 µM fMLP or 100 µM ATP for 2 min (A), 10 µM fMLP for the indicated periods of time (B) or 100 µM carbachol for 30 s (C). In A, values are expressed as percentage of [3H]SPP production relative to unstimulated control cells, amounting to 1642 ± 320 cpm/1.8 × 106 cells. In C, values are mean ± S.D. from a representative experiment performed three times. *, significantly different from basal SPP formation (P < .05, three to four experiments).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Ca2+ influx is not required for UTP-induced SPP production. UTP (100 µM)-induced increases in [Ca2+]i and [3H]SPP production were measured in HL-60 cells either in the presence of extracellular Ca2+ (Control) or in cells treated for 2 min with 5 mM EGTA in the absence of extracellular Ca2+. Shown are basal and UTP-stimulated [3H]SPP production measured for 2 min (left) and maximal UTP-induced [Ca2+]i increases (right). *, significantly different from basal SPP formation (P < .05, four to five experiments).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Increase in SPP production by Ca2+ ionophores. [3H]SPP formation was measured for 2 min in myeloid differentiated HL-60 cells in the presence of 1 mM extracellular Ca2+ without (Basal) and with fMLP, ionomycin, A-23187 (10 µM each) or combinations of fMLP with ionomycin or A-23187 as indicated. *, significantly different from basal SPP formation (P < .05, three to four experiments).

	Discussion

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Exposure of HL-60 cells to UTP and ATP caused a rapid and transient production of SPP from sphingosine, with a time course and magnitude similar as reported for various membrane receptors in different cell types (Olivera and Spiegel, 1993; Melendez et al., 1998b; Meyer zu Heringdorf et al., 1998; Alemany et al., 1999). The potency and specificity of the nucleotides as well as the finding that UTP and ATP increased SPP production in both promyelocytic and myeloid differentiated HL-60 cells, which was blocked by the sphingosine kinase inhibitor tDHS, strongly suggest that SPP production by sphingosine kinase is stimulated by P2Y₂ receptors endogenously expressed in HL-60 cells (Klinker et al., 1996). Similar to other P2Y₂ receptor responses in HL-60 cells, UTP-induced SPP accumulation was not affected by PTX, in contrast to the formyl peptide receptor response, which was fully PTX sensitive (Alemany et al., 1999). As direct G protein activation by AlF₄ can stimulate SPP production in HL-60 cells (Alemany et al., 1999), it is thus feasible to assume that PTX-insensitive G proteins mediate this P2Y₂ receptor action.

Stimulation of SPP formation by G protein-coupled receptors in HL-60 and HEK-293 cells is apparently a Ca²⁺-dependent process. First, chelation of intracellular Ca²⁺ with BAPTA/AM eliminated SPP formation by P2Y₂ and formyl peptide receptors in HL-60 cells as well by the M₃ muscarinic receptor in HEK-293 cells. Second, depletion of intracellular Ca²⁺ stores by pretreatment of HL-60 cells with thapsigargin also prevented UTP-induced SPP formation. In contrast, removal of extracellular Ca²⁺ did not affect the P2Y₂ and formyl peptide receptor-mediated SPP accumulation. Thus, an increase in [Ca²⁺]_i caused by mobilization of intracellular Ca²⁺, but not Ca²⁺ influx, is apparently required for stimulation of SPP formation by G protein-coupled receptors in HL-60 cells. Finally, SPP formation also was increased by exposure of HL-60 cells and HEK-293 cells (our unpublished observations) to the Ca²⁺ ionophores, ionomycin, and A-23187. While this manuscript was in preparation, a study performed in TRMP canine kidney epithelial cells transfected with the platelet-derived growth factor receptor similarly concluded that sphingosine kinase activation by this tyrosine kinase receptor is a Ca²⁺-dependent event (Olivera et al., 1999). The mechanism of the Ca²⁺-dependent SPP formation is presently not clear. The sequence of the recently cloned murine sphingosine kinase enzymes contains potential phosphorylation and Ca²⁺/calmodulin-binding sites (Kohama et al., 1998). It is thus possible that enzyme activity is regulated by Ca²⁺/calmodulin and/or Ca²⁺-dependent phosphorylation, which however was not reported with the expressed enzyme.

In agreement with our previous findings on formyl peptide receptor action in myeloid differentiated HL-60 cells (Alemany et al., 1999), we report herein that short-term pretreatment of the cells with the sphingosine kinase inhibitor tDHS suppresses the P2Y₂ receptor-mediated Ca²⁺ mobilization, thus arguing for a major role of SPP production in Ca²⁺ signaling by the P2Y₂ receptor. tDHS by itself did not increase [Ca²⁺]_i and did not deplete internal Ca²⁺ stores. Moreover, we reported before that suppression of Ca²⁺ signaling in HL-60 cells and HEK-293 cells by tDHS is not caused by inhibition of protein kinase C or perturbation of receptor-mediated phospholipase C stimulation (Meyer zu Heringdorf et al., 1998; Alemany et al., 1999). Because extracellularly applied SPP did not increase [Ca²⁺]_i in HL-60 cells, it is highly unlikely that intracellularly formed SPP induces Ca²⁺ mobilization by activating cell surface sphingolipids receptors after being released from the cells. Thus, SPP production appears to be essential for receptor-mediated Ca²⁺ mobilization, but an increase in [Ca²⁺]_i is apparently required for stimulation of sphingosine kinase (see above). We therefore propose that the activated P2Y₂ and formyl peptide receptors induce via increased formation of inositol 1,4,5-trisphosphate by phospholipase C a local discrete [Ca²⁺]_i increase, not detectable by the methodology used, that then activates SPP production by sphingosine kinase, ultimately leading to full Ca²⁺ mobilization. The difference between peak [³H]SPP formation and [Ca²⁺]_i elevation most likely results from the time span required for extracellularly applied [³H]sphingosine to cross the plasma membrane and reach intracellular sphingosine kinase to be converted to [³H]SPP. Thus, as also suggested for Ca²⁺ signaling by the platelet-derived growth factor receptor in TRMP cells (Olivera et al., 1999), stimulation of intracellular SPP formation by G protein-coupled receptors in HL-60 cells may represent an amplification system for Ca²⁺ signaling by these receptors that is primarily initiated by phospholipase C stimulation. In line with this hypothesis, Li et al. (2000) recently reported that peritoneal neutrophils from mice lacking both phospholipase C-2 and C-3 do not respond to fMLP with [Ca²⁺]_i increases.

Based on inhibitory effects observed with sphingosine kinase inhibitors and/or addition of extracellular SPP, SPP has been postulated to act as a second messenger of receptor-mediated mitogenic responses, including activation of MAP kinases (Su et al., 1994; Wu et al., 1995; Cuvillier et al., 1996; Pyne et al., 1996; Blakesley et al., 1997; Kozawa et al., 1997; Rani et al., 1997; van Brocklyn et al., 1998). However, treatment of HL-60 cells with the sphingosine kinase inhibitor tDHS, causing full inhibition of receptor-mediated SPP production, had no effect on P2Y₂ or formyl peptide receptor-induced activation of MAP kinases, including Erk1, Erk2, SAPK/JNK, and p38 MAP kinase. These data, thus, strongly argue against an essential role of sphingosine kinase stimulation and intracellular SPP in activation of MAP kinases by G protein-coupled receptors in HL-60 cells. In agreement with our results, several recent studies strongly suggest that some of the cellular effects formerly attributed to intracellular SPP, including Erk activation, are probably caused by an action of SPP at cell surface receptors (Sato et al., 1999; Tolan et al., 1999).

In conclusion, this study demonstrates that purinergic P2Y₂ receptors endogenously expressed in HL-60 cells stimulate SPP production by sphingosine kinase, a process apparently not required for activation of MAP kinases. However, the Ca²⁺ requirement of receptor-mediated SPP production and the inhibition of Ca²⁺ mobilization by blockade of this response suggest that intracellular SPP formation may represent an amplification system for receptor-mediated Ca²⁺ signaling and Ca²⁺-regulated cellular processes in HL-60 granulocytes.