Sphingosine 1-phosphate, a diffusible calcium influx factor mediating store-operated calcium entry - PubMed (original) (raw)

Sphingosine 1-phosphate, a diffusible calcium influx factor mediating store-operated calcium entry

Kiyoshi Itagaki et al. J Biol Chem. 2003.

Abstract

Store-operated calcium entry (SOCE) is a fundamental mechanism of calcium signaling. The mechanisms linking store depletion to SOCE remain controversial, hypothetically involving both diffusible messengers and conformational coupling of stores to channels. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that can signal via cell surface G-protein-coupled receptors, but S1P can also act as a second messenger, mobilizing calcium directly via unknown mechanisms. We show here that S1P opens calcium entry channels in human neutrophils (PMNs) and HL60 cells without prior store depletion, independent of G-proteins and of phospholipase C. S1P-mediated entry has the typical divalent cation permeability profile and inhibitor profile of SOCE in PMNs, is fully inhibited by 1 microm Gd3+, and is independent of [Ca2+]i. Depletion of PMN calcium stores by thapsigargin induces S1P synthesis. Inhibition of S1P synthesis by dimethylsphingosine blocks thapsigargin-, ionomycin-, and platelet-activating factor-mediated SOCE despite normal store depletion. We propose that S1P is a "calcium influx factor," linking calcium store depletion to downstream SOCE.

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Figures

Fig. 1. Sphingosine 1-phosphate induces Ca2+ influx in human PMN

PMN were Fura-loaded in HEPES buffer with 1 m

Ca2+ and 0.1% BSA. Immediately prior to study, PMNs were spun for 5 s at 4500 rpm, washed once with BSA-free/Ca2+ free buffer, spun again, and resuspended in BSA-free, nominally Ca2+-free (0.3 m

EGTA) buffer for study. S1P was added at t = 30 s in the concentrations shown. For the zero S1P control, 3 _μ_l of vehicle (methanol) was added. 1 m

Ca2+ was added to the medium at t = 50 s. Dose-dependent calcium entry is immediately noted. The morphology of calcium entry is typical of SOCE in the PMN. Traces represent the mean ± S.E. of 3–4 experiments at each S1P dose.

Fig. 2. S1P does not induce emptying of PMN calcium stores

PMN were studied in BSA-free, nominally calcium-free conditions. Cells were treated with 10 μ

S1P or methanol at 30 s. The cells were then stimulated with 10 n

fMLP (EC90) at 60 s. 1 m

Ca2+ was added to the medium at t = 300 s. The store release transients seen on fMLP treatment were indistinguishable. After S1P treatment, however, SOCE was magnified severalfold. The traces shown are the mean [Ca2+]i ± S.E. from four separate experiments.

Fig. 3. Calcium mobilization by S1P is pertussis toxin (PTX)-insensitive

PMN were incubated with PTX (1 _μ_g/ml) or vehicle for 3 h at 37 °C. In the upper panel, PMN were suspended in nominal Ca2+, BSA-free buffer. At 30 s cells were treated with 5 μ

S1P. 1 m

Ca2+ was added to the medium at t = 50 s. Calcium entry in response to S1P was indistinguishable in the presence and absence of PTX. Note, again, that S1P elicits no measurable store emptying in this time frame (Fig. 2). In the lower panel, cells incubated identically with PTX were exposed to 10 n

fMLP at 30 s. PTX completely blocked the Ca2+ store release response to fMLP. The subsequent calcium entry response was limited to the level of the “leak.” (i.e. that Ca2+ entry always seen on re-calcification of the medium irrespective of prior stimulation or store depletion. PMN [Ca2+]i changes due to leak can vary from 20 to 40 n

in PMN using our methods.) PMN incubated 3 h without PTX responded normally to fMLP (lower panel, upper trace).

Fig. 4. Calcium influx caused by S1P is insensitive to PLC inhibition

PMN were treated with 20 μ

U73122 (upper trace) for 15 min in the cuvette in the presence of 1 m

calcium and 0.1% fatty-acid free BSA. Recordings were begun at t = 0. At 30 s 10 n

fMLP was applied and no [Ca2+]i flux was detected. At 100 s 20 μ

S1P was applied. S1P caused calcium influx. In the lower trace (displaced for clarity) PMN were exposed to S1P at the time marked without prior exposure to U73122. [Ca2+]i flux is indistinguishable in the presence and absence of U73122. Representative traces are shown, three to four experiments were done per condition. No S1P-mediated [Ca2+]i flux is seen in the absence of external Ca2+. S1P is bound strongly by BSA, and this slow pattern of calcium influx is typical of PMN responses to S1P in BSA-containing medium. The BSA was required to prevent cell clumping after adding the U73122.

Fig. 5. Responses of HL-60 cells to S1P

Undifferentiated HL60 or differentiated HL60-PMN cells (see “Experimental Procedures”) were exposed to 10 n

fMLP (A) or to S1P (B). Undifferentiated cells showed no response to fMLP and a typical small Ca2+ “leak” upon re-addition of external Ca2+. HL60-PMNs show a morphologically normal Ca2+ store release transient after fMLP stimulation, followed by typical SOCE. Similar induction of Ca2+ store release was seen in response to multiple GPCR agonists (see text). In B, neither HL60 nor HL60-PMN showed any Ca2+ store release response to S1P, but brisk Ca2+ entry was seen upon re-addition of external Ca2+. Only the Ca2+ “leak” was seen in the absence of S1P, which was very similar (note the change in scale) in HL60 (A) and HL60-PMN (B). These findings demonstrate that S1P-mediated Ca2+ entry is independent of the overall degree of GPCR expression in PMN-like cells.

Fig. 6. S1P elicits both Sr2+ and Ca2+ entry in PMN

After Fura-2AM loading, PMN were spun and washed in buffer without BSA or Ca2+. Cells were spun and resuspended in cuvettes in BSA/Ca2+-free media. S1P was applied at 30 s. Sr2+ and Ca2+ were added at 300 and 450 s, respectively. S1P causes dose-dependent entry of each cation in relative proportions similar to those seen after TG, ionomycin, and PAF stimulation in our prior studies (24). Representative traces are shown, n = 3–4 per condition.

Fig. 7. Inhibition of S1P-induced calcium entry by inorganic channel blockers

PMN were stimulated with S1P in the presence of the heavy metal and lanthanide channel-blockers La3+, Ni2+, and Zn2+ (all 1 m

), and the media were re-calcified at the times shown. La3+ abolishes Ca2+ entry. Experiments performed in the presence of Zn2+ show traces overlapping those done in the presence of La3+ (data not shown). In the presence of Ni2+, [Ca2+]i traces were indistinguishable from those seen in the absence of any blockers (data not shown). Representative traces are shown, n = 3–4 per condition.

Fig. 8. Gadolinium (Gd3+) inhibits S1P-induced calcium entry

PMN were treated with the concentrations of Gd3+ shown for 1 min in the presence of calcium prior to study. 10 μ

S1P was added at t = 30 s. Gd3+ caused dose-dependent suppression of S1P-induced Ca2+ entry. Complete blockade of Ca2+ entry was seen at ∼1 μ

. Representative traces are shown, n = 3 for each concentration.

Fig. 9. Direct store depletion causes PMN S1P synthesis

PMN (2 × 106) were incubated with DMS or vehicle as indicated for 10 min. Reactions were started (t = 0) by adding [3H]sphingosine with or without TG (see text). The reactions were stopped at 0, 2, 5, and 10 min. Total [3H]S1P was collected and assayed by TLC. The mean ± S.E. of three observations at each time point is shown. TG initiated rapid synthesis of S1P. No significant S1P synthesis was seen in the absence of TG. DMS abolished S1P production in TG-treated cells to the level of unstimulated cells.

Fig. 10. DMS inhibits PMN SOCE initiated by direct store depletion

PMN were incubated for 1 min with increasing doses of DMS in calcium-free media. In A, 500 n

TG was added at 30 s. 1 m

Ca2+ was added after the completion of store release. In B, 100 n

ionomycin (Iono) was added at 30 s. Again, 1 m

calcium was added after the completion of store release. In each case, store release was unaffected by DMS, but SOCE was suppressed in a dose-dependent manner. In the presence of 15 μ

DMS, calcium entry was suppressed to the level of the “leak” current. Representative traces are shown, n = 3–4 for each concentration of DMS.

Fig. 11. DMS inhibits HL60 SOCE initiated by direct store depletion

HL60 (upperpanel) and HL60-PMN (lowerpanel) cells were studied in a like manner to the PMN studied in Fig. 10. Quantitative Ca2+ store release was unaffected by the differentiation of the cells (note the difference in scale) or by the presence of DMS. Again though, 10 μ

DMS was found to inhibit TG-initiated SOCE to the level of “leak.” SOCE response to TG (which is GPCR-independent) was markedly increased by differentiation of the cells.

Fig. 12. DMS inhibits SOCE in a calcium-independent manner

Fura-loaded PMN were incubated in 20 μ

BAPTA for 9 min at room temperature. A, cells were exposed to vehicle only (Me2SO) at 30 s. Samples were then observed in the cuvette for 10 min. At that time, a mixture of 500 μ

Mn2+ and 1.5 m

Ca2+ was added where indicated to quench Fura. In B, the cells were exposed to TG (500 n

) at 30 s. Again, 500 μ

Mn2+ and 1.5 m

Ca2+ were added at the time indicated. In C, cells were pretreated for 1 min with 10 μ

DMS and then exposed to TG (500 n

) at 30 s. Again, after allowing 10 min for store depletion, Mn2+ and Ca2+ were added. Fluorescent intensity (FI) was monitored at both 340/380 nm (not shown) and at 360 nm, as shown (FI360). No 340/380 nm [Ca2+]i release transient was seen at any time (data not shown). On Mn2+ addition, a small initial drop in total fluorescence at 360 nm is noted due to quenching of Fura in the media, but no Mn2+ entry into the cells was detected in the absence of prior store depletion (A). In contrast, prior TG treatment (B) caused rapid Mn2+ entry. TG-dependent Mn2+ entry was blocked by prior treatment with DMS (C). Linear curve-fitting (SigmaPlot) was then performed on the FI360 after Mn2+ addition (n = 3–4 independent experiments per condition). This revealed a mean slope of −2.3 ± 0.6 units/s after treatment with vehicle only. The FI360 slope decreased to −14.9 ± 0.9 units/s in TG-treated cells but was returned to unstimulated levels (−3.1 ± 0.4 units/s) by DMS pretreatment (p < 0.01, analysis of variance/Tukey's test). The data show that DMS inhibits the calcium entry response to TG store depletion in a [Ca2+]i -independent fashion.

Fig. 13. DMS inhibits G-protein-coupled receptor-initiated SOCE

PMN were exposed to 100 n

PAF at t = 30 s in calcium-free medium. In A (control) no DMS was used. The DMS vehicle (Me2SO) was applied at 150 s. In B, DMS was applied 100 s prior to PAF. In C, DMS was added at 150 s, allowing >1 min for full DMS effect. All media were re-calcified at t = 220 s to measure SOCE. The traces represent the mean [Ca2+]i ± S.E. of three experiments. Each experimental trace is shown compared with an identical “blank” control trace where only PAF vehicle (3 _μ_l of ethanol) was used at t = 30. This “blank” trace demonstrates the nonspecific calcium “leak.” DMS given prior to store depletion (B) completely eliminated PAF-initiated SOCE. DMS given after store depletion (C) caused only minimal inhibition.

Fig. 14. DMS does not block calcium influx

In this experiment PMN were incubated with DMS at the concentrations shown or with vehicle (3 _μ_l of Me2SO) for 1 min prior to recordings in Ca2+-free, BSA-free media. At t = 30 s, 10 μ

S1P was applied. Sr2+ (1 m

) was added at 300 s, and 1 m

Ca2+ was added at 450 s. DMS caused dose-dependent enhancement of the entry of each cation. Control experiments using DMS alone demonstrated no calcium entry above the normal “leak” seen on cation re-addition (data not shown). The data show that DMS does not inhibit Ca2+ entry into PMN. Rather, it suggests that a previously undescribed synergism may exist between the cellular effects of exogenous S1P and DMS. These effects remain to be studied. Representative traces are shown, n = 3 independent experiments.

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Sphingosine 1-phosphate, a diffusible calcium influx factor mediating store-operated calcium entry - PubMed (original) (raw)