©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Ca Mobilizing Action of Sphingosine in Jurkat Human Leukemia T Cells
EVIDENCE THAT SPHINGOSINE RELEASES Ca FROM INOSITOL TRISPHOSPHATE- AND PHOSPHATIDIC ACID-SENSITIVE INTRACELLULAR STORES THROUGH A MECHANISM INDEPENDENT OF INOSITOL TRISPHOSPHATE (*)

(Received for publication, August 24, 1995; and in revised form, February 13, 1996)

Shoji Sakano Haruo Takemura (§) Keiko Yamada (2) Kenshi Imoto Masamitsu Kaneko (1) Hideyo Ohshika

From the  (1)Departments of Pharmacology andEmergency and Critical Care Medicine, School of Medicine and the (2)School of Health Sciences, Sapporo Medical University, S.1, W.17, Sapporo 060, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Effects of sphingosine on Ca mobilization in the human Jurkat T cell line were examined. Sphingosine increased the cytoplasmic Ca concentration ([Ca]) in a dose-dependent manner with an ED of around 8 µM. Sphingosine and OKT3, a CD3 monoclonal antibody, transiently increased [Ca], which declined to the resting level in the absence of extracellular Ca. Under the same conditions, pretreatment with sphingosine inhibited but did not abolish an increase in [Ca] induced by the subsequent addition of OKT3 and vice versa. However, pretreatment with sphingosine did not affect an increase in [Ca] induced by OKT3 in the presence of Ca. OKT3 increased IP(3) formation, but sphingosine did not affect the level of IP(3) by itself nor did it cause IP(3) formation induced by OKT3. In permeabilized Jurkat cells, the addition of IP(3) released Ca from nonmitochondrial intracellular stores, but the addition of sphingosine did not. Sphingosine, stearylamine, and psychosine increased [Ca] and diacylglycerol (DG) kinase activation; however, ceramide did not, whereas sphingosine 1-phosphate slightly activated DG kinase without elevation of [Ca]. Pretreatment with R59022, a DG kinase inhibitor, abolished the peak but did not affect the sustained response of [Ca] to sphingosine. Phosphatidic acid (PA) elevated [Ca], after which it declined to a resting level even in the presence of extracellular Ca. In accordance with this, PA did not stimulate Ca uptake into cells, but sphingosine and OKT3 did. Pretreatment with PA partially inhibited a rise in [Ca] induced by the subsequent addition of sphingosine and vice versa in the absence of extracellular Ca. Under similar conditions, pretreatment with PA affected an elevation of [Ca] induced by OKT3 less, after which the subsequent addition of sphingosine did not increase [Ca]. In permeabilized Jurkat cells, the addition of IP(3) did not release Ca, but PA did in the presence of heparin. Pretreatment with thapsigargin, a microsomal Ca-ATPase inhibitor, abolished the rises of [Ca] induced by the subsequent addition of sphingosine, OKT3, and PA in the absence of extracellular Ca. The present results suggest that at least two kinds of intracellular Ca stores exist in Jurkat cells, both of which are IP(3)- and PA-sensitive, and that sphingosine mobilizes Ca from both stores in an IP(3)-independent manner. Furthermore, the IP(3)- but not the PA-sensitive intracellular Ca store seems to regulate Ca entry induced by sphingosine.


INTRODUCTION

Sphingosine is a major sphingolipid involved in a variety of cell types and is thought to be a second messenger in the sphingomyelin signal transduction pathway(1) . The major action of sphingosine has been shown to be a potent and specific inhibition of protein kinase C (2, 3, 4) . Sphingosine and its derivatives also have actions other than protein kinase C inhibition such as inhibition of Na,K-ATPase(5) , activation of phospholipase C(6, 7, 8) , activation of phospholipase D(9, 10) , inhibition of calmodulin kinase(11) , activation of casein kinase II(12) , activation of tyrosine kinase(13) , activation of DG kinase (14, 15) , and others(16) . In addition, recent reports have shown that sphingosine mobilizes Ca in smooth muscle cells(17) , parotid acinar cells(18) , skeletal muscle(19) , pancreatic acinar cells(20) , neutrophils(21) , and thyroid cells(22) . Thus, sphingosine may function as an endogenous modulator of cell function and as a second messenger(1) .

An increase in the cytoplasmic free Ca concentration ([Ca]) contributes to an essential triggering signal for T cell activation in the immune system(23, 24) . This increase in [Ca] is due to the release of Ca from intracellular stores and is sustained by the influx of extracellular Ca(24, 25) . Some lymphokines such as tumor necrosis factor alpha, interleukin 1-beta, and -interferon are thought to increase the metabolism of sphingomyelin in lymphocytes(26) . These facts led us to speculate that sphingosine could have an important role in T cell function due to Ca mobilization. However, there are no reports about the effect of sphingosine alone on Ca mobilization in lymphocytes. We report here that sphingosine mobilizes Ca from PA-sensitive (^1)as well as IP(3)-sensitive intracellular stores in Jurkat cells independently of inositol phosphates.


MATERIALS AND METHODS

Reagents

[-P]ATP (1000-3000 Ci/mmol) and CaCl(2) (5-50 mCi/mgCa) were from DuPont NEN; fura-2 acetoxymethyl ester (fura-2/AM) and fura-2 pentapotassium salt were from Molecular Probes (Eugene, OR). Inositol 1,4,5-trisphosphate was from Dojindo Co. (Kumamoto, Japan), beta-octylglucoside was from Calbiochem, and sphingosine-1-phosphate was from Biomol (Plymouth Meeting, PA). OKT3 and R59022 were kind gifts from Dr. N. Satoh (Sapporo Medical University) and Dr. D. de Chaffoy de Courcelles (Janssen Company), respectively. All other reagents were obtained from Sigma.

Cell Culture

Jurkat T cells were donated by Fujisaki Cell Center (Okayama, Japan). The cells were maintained under 5% CO(2)/95% O(2) in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (50 units/ml), streptomycin (50 µg/ml), and L-glutamine (300 µg/ml) as described previously(14) .

Measurement of [Ca](i)

Measurement of [Ca](i) was performed as described previously (27) . Briefly, a Jurkat T cell suspension (around 2 times 10^6 cells/ml) was loaded with 2 µM fura-2/AM for 30 min at room temperature. Following the loading period, the T cells were washed twice with Krebs-Ringer-HEPES medium (KRH) containing 0.2% bovine serum albumin. The composition of KRH was as follows: 120 mM NaCl, 5.4 mM KCl, 1.0 mM CaCl(2), 0.8 mM MgCl(2), 11.1 mM glucose, and 20 mM HEPES (pH 7.4). Fluorescence of the fura-2-loaded cell suspension was monitored with a Jasco CAF-110 (Japan Spectroscopic Co., Tokyo, Japan) in a cuvette at 35 °C. The excitation wavelengths were 340 and 380 nm, and emission was measured at 510 nm. [Ca](i) was estimated as described by Grynkiewicz et al.(28) . For the experiments in the absence of extracellular Ca, 3 mM EGTA was added to the cell suspension 3 min before drug stimulation to chelate extracellular Ca.

Measurement of IP(3)

Jurkat cells (10^7 cells/ml) were washed twice with KRH and suspended in KRH containing 10 mM LiCl and preincubated for 10 min at 37 °C. The mixture after the addition of drugs was further incubated for 30 s. The reaction was then terminated by an equal volume of 15% trichloroacetic acid. The preparations were kept on ice for 30 min and then centrifuged. The supernatant was washed three times with 10 volumes of water-saturated diethyl ether to remove trichloroacetic acid from the solution. The resultant solutions were neutralized by titration of pH 7.5 with NaHCO(3). After centrifugation, 100 µl of supernatant was assayed using IP(3) assay system (Amersham Corp.).

Ca Release from Permeabilized Cells

Permeabilized Jurkat T cells were prepared and the Ca release was determined as described previously(27) . Briefly, Jurkat T cells (around 10^7 cells/ml) were incubated in a cuvette at 35 °C in medium of the following composition: 20 mM NaCl, 100 mM KCl, 5 mM MgSO(4), 20 mM HEPES (pH 7.2) supplemented with 10 µM antimycin A and 10 µg/ml oligomycin. Fura-2 free acid (1 µM), 3 mM ATP, 10 mM phosphocreatine, 10 units/ml creatine phosphokinase, and 30 µg/ml saponin were added. When fluorescence declined to a stable state, drugs were added.

The Assay for DG Kinase

DG kinase was assayed as described previously(15) . Briefly, Jurkat cells were disrupted by brief sonication in 10 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 50 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol. After centrifugation at 200 times g for 10 min, the cytosolic and membrane fractions were obtained by a further centrifugation at 100,000 times g for 30 min. The DG kinase activity of the supernatant was measured by the octylglucoside mixed micellar assay(29) . The reaction mixture (50 µl) contained 50 mM MOPS (pH 7.2), 50 mM octylglucoside, 1 mM dithiothreitol, 20 mM NaF, 2 mM (8 mol%) DG, 10 mM (40 mol%) sphingosine or sphingosine analogues, 10 mM MgCl(2), 500 µM [-P]ATP, and enzyme. After the incubation for 2 min at 30 °C, the lipids were extracted and analyzed by thin-layer chromatography.

Ca Uptake

Drugs and Ca (1 µCi) were added to 0.2-ml aliquots of cell suspension (2 times 10^6 cells/ml). After 5 min, Ca uptake was determined by the addition of the aliquot to 1 ml of iced incubation solution containing 2 mM LaCl(3). The cells were separated by centrifugation and then washed twice with another 1 ml of the iced incubation solution containing 1 mM EGTA by alternate centrifugation and resuspension. The cells were then resuspended in distilled water and Ca in the cells was measured using standard liquid scintillation counting techniques.


RESULTS AND DISCUSSION

Ca Mobilization of Sphingosine in Intact Jurkat Cells

The addition of sphingosine (20 µM) gradually increased [Ca](i) with a latency of about 30 s (Fig. 1A). This lag period for Ca mobilization caused by sphingosine is consistent with those in DDT(1)MF-2 smooth muscle cells(17) , 3T3 fibroblasts (30) , and human fibroblasts(8) . [Ca](i) elevated by sphingosine slightly decreased and then reached a sustained, higher level of [Ca](i). Fig. 1B shows the dose-response relation for the peak response of [Ca](i) to sphingosine. [Ca](i) increased at 2 µM sphingosine and reached the maximum response at 12 µM sphingosine. The EC value for sphingosine was approximately 8 µM. Sphingosine stimulates Ca mobilization in smooth muscle cell line DTT(1)MF-2(17) , rat parotid acinar cells(18) , skinned rabbit skeletal muscle fiber(19) , pancreatic acinar cells(20) , human neutrophils(21) , and thyroid FRTL-5 cells(31) . However, the concentration of sphingosine needed to mobilize Ca in these cells is rather high. The sphingosine concentration in cardiac and skeletal muscle cytosol is around 1 µM(32) . Thus, the concentration of sphingosine we used for Ca mobilization would be more physiological. Tumor necrosis factor alpha activates sphingomyelin turnover in Jurkat T cells(33) . Furthermore, sphingomyelin turnover may be an important signaling mechanism transducing the actions of tumor necrosis factor alpha and -interferon with a specific function in differentiation of HL-60 cells(34) . In addition, immune cell activation failure is suggested to be explained by the inadequacy of Ca signaling in mononuclear cells (35) and T cells(36) . It is tempting to speculate that sphingosine may potentiate immune function via an enhancement of Ca signaling.


Figure 1: Effect of 20 µM sphingosine on [Ca] (A) and the dose-response curve (B) of [Ca] for sphingosine in Jurkat T cells. Jurkat cells were loaded with 2 µM fura-2/AM for 30 min at room temperature, washed, and then suspended with fresh Krebs-Ringer-HEPES medium in a cuvette at 35 °C. [Ca] was estimated as explained under ``Materials and Methods.'' The increase in [Ca] is expressed as a net change in the peak of [Ca] from the resting level of [Ca]. Each point in B is the mean ± S.E. of four determinations.



In the absence of extracellular Ca, sphingosine caused a transient increase in [Ca](i), which returned to the resting level, and the sustained increase in [Ca](i) induced by sphingosine was abolished (Fig. 2A), suggesting that sphingosine mobilized Ca from intracellular Ca stores as well as from extracellular medium. In Jurkat cells, Breittmayer et al.(37) have reported that sphingosine does not modify Ca release from intracellular stores as judged from indirect observations but inhibits the Ca influx induced by OKT3, thapsigargin, and ionomycin. We checked whether sphingosine inhibits [Ca](i) evoked by OKT3 in the presence of extracellular Ca. As shown in Fig. 2(C and D), sphingosine did not affect Ca mobilization induced by OKT3 and vice versa. The reason for the difference between their results and ours is unknown. However, they did not examine the effects of sphingosine alone on [Ca](i). The present results clearly showed that sphingosine mobilized intracellular Ca in Jurkat T cells. We examined whether sphingosine releases Ca from IP(3)-sensitive intracellular stores by using the anti-CD3 antibody OKT3, an IP(3)-generating drug. In the absence of extracellular Ca, pretreatment with sphingosine inhibited but did not abolish [Ca](i) induced by the subsequent addition of OKT3; likewise the rise in [Ca](i) induced by sphingosine was decreased but not abolished after the pretreatment with OKT3 (Fig. 2, A and B). This suggested that sphingosine partially mobilized Ca from IP(3)-sensitive intracellular stores.


Figure 2: Effects of sphingosine (SPH) and OKT3 on [Ca] in the absence (A and B) and presence (C and D) of extracellular Ca. Jurkat cells were treated with fura-2/AM as described in Fig. 1. [Ca] was estimated as explained under ``Materials and Methods.'' In A and B, EGTA (3 mM) was added 3 min before the addition of the first drug. Each trace is a representative one from at least three experiments.



Independent Action of Ca Mobilization of Sphingosine on IP(3)

Sphingosine could release Ca from IP(3)-sensitive intracellular stores via inositol phosphate formation because sphingosine stimulates hydrolysis of phosphatidylinositol in rat parotid cells(38) , rat astrocytes(39) , and human fibroblasts(8) , although sphingosine and sphingomyelin inhibit phospholipase C to catalyze hydrolysis of phosphatidylinositol in liposomes(7) . As shown in Fig. 3, OKT3 increased IP(3) in Jurkat cells in the presence of 10 mM LiCl. However, the addition of sphingosine did not cause IP(3) formation by itself nor did it affect the level of IP(3) induced by OKT3. The results suggested that inositol phosphate formation might not be involved in Ca mobilization caused by sphingosine and that the reduction of Ca release induced by OKT3 in the presence of sphingosine (Fig. 2A) is not caused by inhibition of IP(3) formed by OKT3. In accordance with this hypothesis, sphingosine releases Ca in an IP(3)-independent manner in rat pancreatic acinar cells (20) and human neutrophils(21) . Chao et al.(8) suggested that sphingosine enhances phosphatidylinositol turnover by stimulating phospholipase C activity resulting from modulation by G-protein interaction. However, stimulation of the T cell receptor through tyrosine kinase, but not through G-protein, activates phospholipase C-1, which yields IP(3) and DG from hydrolysis of phosphatidylinositol bisphosphate(40) . Sphingosine inhibits tyrosine kinase of the insulin receptor(13) . Thus, sphingosine may not activate tyrosine kinase to activate phospholipase C-1 in Jurkat cells.


Figure 3: Effects of 20 µM sphingosine (SPH) and 10 µg/ml OKT3 on IP(3) formation. Jurkat cells (10^7 cells/ml) were washed twice with KRH and suspended in KRH containing 10 mM LiCl and preincubated for 10 min at 37 °C. The mixture after the addition of drugs was further incubated for 30 s, and then the reaction was then terminated by 15% trichloroacetic acid. The supernatant was washed three times with water-saturated diethyl ether to remove trichloroacetic acid from the solution. The resultant solutions were neutralized, and then after centrifugation, supernatant was assayed using IP(3) assay system. Each bar is the mean ± S.E. of five determinations.



Ghosh et al.(17) have reported that sphingosine directly releases Ca from permeabilized smooth muscle cells. We examined whether sphingosine directly released Ca from intracellular Ca stores (Fig. 4). The addition of 10 µM IP(3) released Ca from the nonmitochondrial intracellular Ca store; however, 20 µM sphingosine did not release Ca by itself nor did it affect the Ca release induced by IP(3), suggesting that the Ca-mobilizing action of sphingosine from intracellular stores was mediated by a messenger or metabolite. In addition, the longer pretreatment with sphingosine did not affect the release of Ca induced by IP(3) (data not shown).


Figure 4: Ca release from the nonmitochondrial intracellular Ca store in permeabilized Jurkat cells. Jurkat cells (around 10^7 cells/ml) were incubated in a cuvette at 35 °C in the intracellular medium as explained under ``Materials and Methods.'' Fura-2 free acid (1 µM), an ATP regenerating system, and 30 µg/ml saponin were added. When fluorescence declined to a stable state, IP(3) and sphingosine (SPH) were added as indicated by arrows. Each trace is a representative one from at least three experiments.



Effects of Sphingosine and Its Derivatives on [Ca](i) and DG Kinase Activation

SPP has been recently reported to be a putative second messenger that mobilizes Ca in 3T3 fibroblasts (30, 41) and DDT(1)MF-2 smooth muscle cells (42) rather than sphingosine. We examined the effects of sphingosine and its analogues, SPP, psychosine, stearylamine, and ceramide (N-acyl-D-erythro-sphingosine) on [Ca](i) (Fig. 5). However, SPP (20 µM) did not elevate [Ca](i) in Jurkat cells, although a lower concentration of SPP (5 µM) increased it in NIH/3T3 fibroblasts (data not shown). Psychosine (20 µM) and stearylamine (20 µM) also increased [Ca](i) almost like sphingosine. On the other hand, 10 µg/ml ceramide did not increase [Ca](i). We used a long chain ceramide, which might be impermeable to Jurkat cells. We further examined whether the short chain cell-permeant C(2)-ceramide (N-acetyl-D-erythro-sphingosine) mobilizes Ca; however, 20 µM of this compound did not elevate [Ca](i) (data not shown). Furthermore, SPP might not enter Jurkat cells, although it is lipophilic. However, this is unlikely because SPP had only a weak stimulation of DG kinase, as assayed in cytosolic and membrane fractions (Fig. 6). Furthermore, stearylamine and psychosine, which are not precursors of SPP, elevated [Ca](i) (Fig. 5) and released Ca from both IP(3)- and PA-sensitive intracellular stores like sphingosine in the absence of extracellular Ca (data not shown).


Figure 5: Effects of sphingosine (SPH), SPP, stearylamine (STL), psychosine (PSC), and ceramide (CRM) on [Ca] in Jurkat cells. Jurkat cells were treated with fura-2/AM as described in Fig. 1. [Ca] was estimated as explained under ``Materials and Methods.'' Each trace is a representative one from at least three experiments.




Figure 6: Effects of sphingosine (SPH), SPP, stearylamine (STL), psychosine (PSC), and ceramide (CRM) on DG kinase in Jurkat cells. Jurkat cells were disrupted by brief sonication in 10 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 50 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol. After centrifugation at 200 times g for 10 min, the cytosolic and membrane fractions were obtained by further centrifugation at 100,000 times g for 30 min. The DG kinase activity of the supernatant was measured by the octylglucoside mixed micellar assay. The reaction mixture (50 µl) contained 50 mM MOPS (pH 7.2), 50 mM octylglucoside, 1 mM dithiothreitol, 20 mM NaF, 2 mM (8 mol%) DG, 10 mM (40 mol%) sphingosine or sphingosine analogues, 10 mM MgCl(2), 500 µM [-P]ATP, and enzyme. The incubation was done for 2 min at 30 °C, and the lipids were extracted and analyzed by thin-layer chromatography. The ordinate expresses fold above control. Each bar is an average of representative assays in duplicate from at least three experiments.



We previously reported that sphingosine increases PA accumulation via DG kinase activation in Jurkat cells(14, 15) . PA formation results from the activation of phospholipase D, inhibition of phosphatidate phosphohydrolase, or activation of DG kinase. Sphingosine or SPP activates phospholipase D in fibroblasts (9, 43) and arterial endothelial cells(10) . On the other hand, sphingosine inhibits phosphatidate phosphohydrolase in NG108-15 cells (44) and human neutrophils(45) . We examined the effects of sphingosine and its analogues on DG kinase activation (Fig. 6). Sphingosine, stearylamine, and psychosine activated DG kinase, consistent with an increase in [Ca](i) induced by these drugs (Fig. 5). SPP (20 µM) slightly increased DG kinase activation, but ceramide did not activate DG kinase. We previously reported that sphingosine activates DG kinase rather than phospholipase D in Jurkat cells(14) . Again, the present results showed that sphingosine activated DG kinase (Fig. 6). Furthermore, 100 µM propranolol did not affect Ca mobilization (data not shown), showing that PA formation induced by sphingosine does not involve the inhibition of phosphatidate phosphohydrolase. Thus, sphingosine seems to activate DG kinase to result in PA formation in Jurkat cells. DG kinase was activated by sphingosine, stearylamine, and psychosine but not by ceramide. SPP had a weak stimulatory effect on DG kinase. However, this weak activation of DG seemed not to cause Ca mobilization. Why sphingosine, stearylamine, and psychosine activate DG kinase is unclear. We have recently demonstrated that 80-kDa DG kinase is an EF hand-type Ca-binding protein (46) and that this isozyme can be activated by Ca-dependent interaction with phospholipids(47) . This suggests the possibility that sphingosine mobilizes Ca first and then activates DG kinase. However, this is unlikely because SPP slightly activated DG kinase without affecting Ca mobilization ( Fig. 5and Fig. 6), and DG kinase activation by sphingosine is independent of Ca(14) .

To further examine the contribution of DG kinase to the sphingosine effect on Ca mobilization, we used R59022, a DG kinase inhibitor. We reported that R59022 inhibited DG kinase activated by sphingosine in a dose-dependent manner(48) , and we used 25 µM R59022, which showed about 75% inhibition of DG kinase activated by sphingosine, in the present experiment. As shown in Fig. 7(A and B), pretreatment with this inhibitor abolished the peak response of [Ca](i) to sphingosine but did not affect the sustained increase in [Ca](i) induced by sphingosine. These results suggested that sphingosine might increase [Ca](i) via formation of PA. This was confirmed by the observation that the addition of PA (10 µM) elevated [Ca](i) by itself (Fig. 7C). However, the increase in [Ca](i) induced by PA declined to the resting level even in the presence of extracellular Ca in accordance with a previous report(49) . Thus, PA formed by sphingosine would be responsible for the IP(3)-insensitive release of Ca. This is supported by the observation that the incubation with R59022 would completely inhibit Ca release induced by sphingosine after the release of Ca from the IP(3)-sensitive store induced by OKT3 (Fig. 7D).


Figure 7: Effects of 20 µM sphingosine (SPH), 25 µM R59022, 10 µM PA, and 10 µg/ml OKT3 on [Ca] in the presence (A, B, and C) and the absence (D) of extracellular Ca. Jurkat cells were treated with fura-2/AM as described in the legend to Fig. 1. [Ca] was estimated as explained under ``Materials and Methods.'' In D, EGTA (3 mM) was added 3 min before the addition of the first drug. Each trace is a representative one from at least three experiments.



Sphingosine Releases Ca from Both IP(3)- and PA-sensitive Intracellular Stores

We examined whether sphingosine mobilizes Ca from PA-sensitive intracellular stores (Fig. 8). In the absence of extracellular Ca, the pretreatment with PA partially inhibited but did not abolish the elevation of [Ca](i) induced by the subsequent addition of sphingosine. Likewise, the pretreatment with sphingosine partially suppressed the elevation of [Ca](i) induced by the subsequent addition of PA. This suggested that sphingosine partially released Ca from PA-sensitive intracellular stores. Because it appeared that sphingosine also partially released Ca from IP(3)-sensitive intracellular stores (Fig. 2), sphingosine might release Ca from both IP(3)- and PA-sensitive stores if both stores are separated. This would be supported by the observation that the pretreatment with OKT3 did not affect the elevation of [Ca](i) induced by the second addition of PA, but then the subsequent addition of sphingosine did not increase [Ca](i) in the absence of extracellular Ca (Fig. 8C). We further examined that there would exist at least two kinds of intracellular Ca stores in Jurkat cells (Fig. 9). In permeabilized Jurkat cells, PA released Ca, which was superimposed on IP(3)-evoked Ca release and vice versa. In the presence of heparin, an antagonist of IP(3) receptor, IP(3) failed to release Ca, but PA released Ca comparable with that released by PA alone. Next, we examined whether both stores have Ca-ATPase by using thapsigargin, a microsomal Ca-ATPase inhibitor. After the pretreatment with thapsigargin in the absence of extracellular Ca, neither OKT3 nor PA nor sphingosine elevated [Ca](i) (Fig. 8D).


Figure 8: Effects of 20 µM sphingosine (SPH), 20 µM PA, 10 µg/ml OKT3, and 100 nM thapsigargin (TG) on [Ca] in the absence of extracellular Ca. Jurkat cells were treated with fura-2/AM as described in the legend to Fig. 1. [Ca] was estimated as explained under ``Materials and Methods.'' EGTA (3 mM) was added 3 min before the addition of the first drug. Each trace is a representative one from at least three experiments.




Figure 9: Ca release from the IP(3)- and PA-sensitive intracellular Ca stores in permeabilized Jurkat cells. Jurkat cells were incubated as described in the legend to Fig. 4. Fura-2 free acid (1 µM), an ATP regenerating system, and 30 µg/ml saponin were added. When fluorescence declined to a stable state, 10 µM IP(3), 10 µM PA, and 10 µg/ml heparin (HP) were added as indicated by the arrows. Each trace is a representative one from at least three experiments.



The present results suggested that there exist at least two kinds of intracellular Ca stores in Jurkat cells, both of which are IP(3)- and PA-sensitive. Breittmayer et al.(49) have reported that PA releases Ca from the intracellular store in Jurkat cells. We also reported that a cyclic AMP-sensitive intracellular store that has a role in Ca mobilization exists in Jurkat cells(27) . Guse et al.(50) suggested that at least four kinds of intracellular Ca stores exist in Jurkat cells. Thus, a diversity of intracellular Ca stores may have an important role on Ca mobilization in the immune response in T cells.

IP(3)- but Not PA-sensitive Intracellular Ca Store Couples to Ca Entry

The present results showed that sphingosine elevated [Ca](i) due to Ca entry from extracellular medium as well as Ca release from intracellular stores in Jurkat cells and that the elevation of [Ca](i) induced by PA declined to the resting level of [Ca](i) even in the presence of extracellular Ca. The mechanism of Ca entry is unknown. Putney (51) originally proposed capacitative Ca entry by which depletion of Ca in the intracellular store activates Ca entry. Using thapsigargin, we demonstrated that the intracellular Ca store regulates Ca entry at the plasma membrane(52) . This capacitative Ca entry exists in T cells(53, 54, 55) . In addition, Randriamampita and Tsien (56) recently reported that emptying of intracellular Ca stores releases a Ca influx factor to activate Ca entry at the plasma membrane in Jurkat cells. It was thus of interest to investigate whether the IP(3)- but not the PA-sensitive intracellular Ca store regulated Ca entry. To examine this possibility, we measured Ca uptake (Table 1) in the presence of sphingosine, OKT3, or PA. Sphingosine and OKT3 increased Ca uptake into the cells with the same potency, whereas Ca uptake in the presence of PA was similar to that of control. Breittmayer et al.(49) also reported that PA mobilizes Ca without any subsequent Ca influx across the plasma membrane. Thus, it is suggested that depletion of Ca in the IP(3)- but not the PA-sensitive intracellular store activates Ca entry and that Ca entry caused by sphingosine is due to capacitative Ca entry.



Concluding Remarks

The results described here support the model presented in Fig. 10whereby sphingosine interacts with two different sites to cause Ca release from intracellular stores. That is, sphingosine releases Ca from the IP(3)-sensitive intracellular store as well as the PA-sensitive intracellular store via DG kinase activation. The question of how sphingosine releases Ca from the IP(3)-sensitive intracellular store could be explained by the following three possibilities. First, the present results indicate that sphingosine did not directly release Ca from IP(3)-sensitive intracellular stores, suggesting that another metabolite or messenger rather than SPP is involved in Ca release from IP(3)-sensitive intracellular stores. Sphingosine degradation is thought to involve lytic cleavage to trans-2-hexadecanal and ethanolamine phosphate(57) . Likewise, stearylamine and psychosine have the same long chain to yield trans-2-hexadecanal. Chow and Jondal (58) reported that polyunsaturated free fatty acids increase [Ca](i) by mobilizing the IP(3)-sensitive intracellular store in Jurkat cells in a manner independent of IP(3). Second, sphingosine may modulate G-protein(8) . Some G-protein could cause Ca release from the intracellular store in Xenopus oocytes(59) . It is possible that sphingosine, stearylamine, and psychosine may release Ca from IP(3)-sensitive intracellular stores in Jurkat cells via modulation of some G-protein that leaks out from permeabilized Jurkat cells. Finally, Kim et al.(60) recently reported a sphingolipid-gated Ca-permeable channel, which seems to represent a new member of the intracellular ligand-gated Ca channel family, on intracellular stores in human endothelial cells. Thus, further study is necessary to clarify whether such a sphingolipid-gated Ca-permeable channel exists in Jurkat cells.


Figure 10: A schematic model of the mechanism of the action of sphingosine on [Ca] in Jurkat T cells. Stimulation of the T cell receptor (TCR) through tyrosine kinase activates phospholipase C- (PLC). IP(3) activates a receptor-regulated channel on the surface of the intracellular Ca store. DG is metabolized to PA by DG kinase. PA releases Ca from the IP(3)-insensitive intracellular store. Both stores have Ca-ATPase, which is blocked by thapsigargin. Sphingosine releases Ca from the IP(3)-sensitive intracellular store via an unknown messenger(s) and activates DG kinase. Depletion of Ca in the IP(3)-sensitive intracellular store activates Ca entry, possibly via a Ca influx factor (CIF).



With regard to the physiological function of the Ca signaling pathway for sphingosine, depletion of Ca in IP(3)-sensitive intracellular store activates Ca entry, which causes a sustained increase in [Ca](i). Sphingomyelin turnover may be an important signaling mechanism transducing the actions of tumor necrosis factor alpha and -interferon with a specific function in cell differentiation(34) , and Ca entry causing a sustained increase in [Ca](i) has an important role in cell differentiation(61, 62) . Thus, the function of sphingosine in lymphocytes could be via the action of sphingosine on the IP(3)-sensitive Ca intracellular store followed by the sustained activation of Ca entry. On the other hand, an increase in [Ca](i) contributes to an essential triggering signal for T cell activation(23, 24) . Thus, Ca release from the PA-sensitive intracellular store caused by sphingosine may be important for triggering signals for T cell activation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmacology, School of Medicine, Sapporo Medical University, S.1, W.17, Sapporo 060, Japan. Tel.: 81-11-611-2111; Fax: 81-11-612-5861.

(^1)
The abbreviations used are: PA, phosphatidic acid; IP(3), inositol 1,4,5-trisphosphate; DG, diacylglycerol; SPP, sphingosine-1-phosphate; MOPS, 3-[N-morpholino]propanesulfonic acid; fura-2/AM, fura-2 acetoxymethyl ester.


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