(Received for publication, September 5, 1996, and in revised form, December 6, 1996)
From The Biomembrane Institute, Seattle, Washington 98119 and ¶ Department of Pathobiology, University of Washington, Seattle, Washington 98195
Sphingosine 1-phosphate (Sph-1-P) has been implicated as an intracellular second messenger in many studies. We investigated the metabolism of Sph-1-P and the mechanism by which Sph-1-P induces activation in enucleated and highly differentiated platelets. Platelets lack Sph-1-P lyase activity, possess persistently active sphingosine (Sph) kinase, and abundantly store Sph-1-P. Although exogenous Sph-1-P activated platelets, intracellular Sph-1-P, formed from exogenously added Sph by cytosolic Sph kinase, failed to do so. To support the notion that exogenous Sph-1-P stimulates platelets from outside, contact of platelet surfaces with immobilized Sph-1-P covalently linked to glass particles resulted in platelet activation. Furthermore, we detected the specific binding sites for radiolabeled Sph-1-P on the platelet surface, suggesting extracellular effects of Sph-1-P on plasma membrane receptors. This specific Sph-1-P binding was inhibited not by other sphingolipids but by lysophosphatidic acid (LPA), and platelet aggregation response to LPA was specifically desensitized by prior addition of Sph-1-P. Finally, internally stored Sph-1-P is released extracellularly upon stimulation, and the release correlated well with protein kinase C activation in intact platelets. These results suggest that Sph-1-P acts not intracellularly but intercellularly, following discharge from activated platelets, and shares a platelet surface receptor with LPA.
Sphingolipid metabolites have been implicated as modulators of
membrane signal transduction systems and shown to be involved in
diverse cellular processes (1-5). The phosphorylated sphingoid base
sphingosine 1-phosphate (Sph-1-P)1 is the
initial product of catabolism of sphingosine (Sph) by Sph kinase and,
generally, is then cleaved by Sph-1-P lyase to ethanolamine phosphate
and fatty aldehyde (1, 5, 6). Sph-1-P has several important physiologic
functions in addition to its role as a metabolite of Sph. Although
originally proposed as a mitogenic messenger (7-10), Sph-1-P has been
shown to be involved in a variety of cellular functions, including
regulation of cell motility (11, 12), activation of muscarinic
K+ current in atrial myocytes (13), mediation of FcRI
antigen receptor signaling (14), and neurite retraction (15). In
nonproliferative, terminally differentiated platelets, Sph-1-P induces
shape change and aggregation reactions by itself and synergistically
elicits aggregation in combination with weak platelet agonists such as epinephrine and ADP (16).
The Sph-1-P level in cells is generally low because of its degradation by Sph-1-P lyase, and Sph kinase is considered to be the rate-limiting factor in Sph catabolism (7, 8, 14, 17). Sph kinase activity is rapidly stimulated, and Sph-1-P level is transiently increased by specific stimuli. Sph-1-P has, therefore, been proposed as an intracellular second messenger (8, 14), mobilizing Ca2+ from an internal source via an inositol trisphosphate-independent pathway (18). Many studies have supported this notion (an intracellular site of Sph-1-P action). An intracellular Ca2+-permeable channel is reportedly located in the endoplasmic reticulum, which may be gated by Sph-1-P (18-22). Furthermore, the endoplasmic reticulum contains the kinase that produces Sph-1-P (20). However, involvement of pertussis toxin-sensitive GTP-binding proteins in Sph-1-P-induced signaling has been reported recently (9, 13, 23, 24). Very recently, it was demonstrated that Sph-1-P only acts from the extracellular surface of the plasma membrane (13, 15). These phenomena suggest the existence of a cell surface receptor for Sph-1-P, and that the site of Sph-1-P action is extracellular.
We now report here investigation of Sph-1-P biology in platelets, which lack a nucleus and do not proliferate, yet play important roles in physiological or pathophysiological phenomena such as thrombosis, hemostasis, and atherosclerosis. These highly differentiated cells are quite unique in terms of Sph-1-P-related metabolism, and this phospholipid acts not intracellularly but intercellularly. Furthermore, a specific binding site for Sph-1-P exists on the platelet surface, and this is recognized also by lysophosphatidic acid (LPA), a glycerolipid that is similar to Sph-1-P in structure and capable of inducing a multiplicity of biological effects (25, 26).
Sph-1-P was prepared from sphingosylphosphocholine with bacterial phospholipase D, as described previously (27). C2-ceramide (Cer) (28) and N,N-dimethylsphingosine (29) were synthesized as described previously. [3-3H]Sph-1-P was prepared by ATP-dependent phosphorylation of [3-3H]Sph catalyzed by Sph kinase obtained from Balb/c 3T3 fibroblasts (30).
CMK cells were kindly provided by Dr. T. Sato (Chiba University, Chiba, Japan). K562 cells were from the American Type Culture Collection (Rockville, MD). Bovine and rat livers were obtained from Pelfreez (Rogers, AR).
The following materials were obtained from the indicated suppliers: [3-3H]Sph (22.0 Ci/mmol) and [3H]acetic anhydride (50 mCi/mmol) (DuPont NEN); Sph, Cer (type III), 12-O-tetradecanoylphorbol 13-acetate (TPA), 1-oleoyl-2-acetyl-glycerol, and bovine serum albumin (essentially fatty acid free) (Sigma); thrombin, collagen, and epinephrine (Chrono-Log, Havertown, PA); staurosporine and prostaglandin E1 (Biomol, Plymouth Meeting, PA); and Fura-2/AM (Molecular Probes, Eugene, OR).
Preparation of PlateletsCitrated venous blood was obtained from healthy adult volunteers. Washed platelets were prepared and handled as described previously (16), unless stated otherwise. Bovine serum albumin (fatty acid-free) (1%) was added when indicated. For Sph-1-P lyase activity assay, outdated platelet concentrates obtained from the Oregon Red Cross (Portland, OR) were used.
Sph-1-P Lyase Activity AssaySph-1-P lyase activity was measured as described previously (6) except that [3-3H]Sph-1-P instead of [4,5-3H]dihydrosphingosine-1-phosphate was used as a substrate. The reaction products were applied to silica gel 60 HPTLC plates (Merck, Darmstadt, Germany), and the plates were developed in chloroform:methanol:acetic acid (50:50:1). In this polar solvent, all radioactive lyase metabolites (palmitaldehyde, palmitic acid, and palmitol) ran closely together near the front, and the whole region was scraped off and counted by liquid scintillation counting.
Metabolism of [3H]Sph in PlateletsPlatelet suspensions (0.5 ml) were incubated with 1 µM [3H]Sph (0.2 µCi). At the indicated time points, the reaction was terminated by the addition of 1.875 ml of ice-cold chloroform:methanol:concentrated HCl (100:200:1), and lipids were extracted from the cell suspensions and analyzed for [3H]Sph metabolism as described previously (16). Portions of lipids obtained from the lower chloroform phase were applied to silica gel 60 HPTLC plates, and the plates were developed in butanol:acetic acid:water (3:1:1), followed by autoradiography. Silica gel areas containing radiolabeled sphingolipids were scraped off and counted by liquid scintillation counting.
Quantitative Measurement of Sph and Sph-1-PSph (31) and Sph-1-P (32) were quantitatively measured by N-acylation with [3H]acetic anhydride into [3H]C2-Cer (N-[3H]acetylated Sph) and [3H]C2-Cer-1-P (N-[3H]acetylated Sph-1-P), respectively, as described previously.
Platelet Shape ChangePlatelet shape change and aggregation were determined turbidometrically (33), as described (16). Calibration was performed with zero light transmission defined for platelet suspension and 100% transmission for the buffer.
Measurement of Intracellular Ca2+ ConcentrationIntracellular Ca2+ concentration was measured using the Ca2+-sensitive fluorophore Fura-2 as described (16). Values of peak increases after addition of an agent were quantified.
Preparation and Application of Sph-1-P or Sph Immobilized on a Solid SupportSph or Sph-1-P was immobilized on the surface of
controlled-pore glass particles with a long chain alkylamine (CPG,
Inc., Fairfield, NJ). Sph-1-P or Sph derivative possessing -carboxyl group was synthesized and linked to a long chain alkylamine of the
glass particles through an amide linkage, as described previously (34).
The long chain alkylamine acts as a spacer arm and avoids low steric
availability of Sph-1-P or Sph immobilized on glass, enabling them to
mimic parent (free) Sph-1-P or Sph. For control glass particles, the
amino group was blocked by N-acetylation. Sph-1-P and Sph
contents of the conjugates were 22 and 39 µmol/g of dry glass
particles, respectively. Extensive washing of the Sph-1-P- and
Sph-attached glass particles with chloroform:methanol (1:2) did not
result in dissolution of free lipids, which was confirmed by thin layer
chromatography. The glass particles were added into platelet
suspensions under constant stirring at 1000 rpm, with the Sph-1-P or
Sph concentration at 40 µM, followed by scanning electron
microscopy (16) and measurement of intracellular Ca2+
concentration.
Binding assays were performed by incubating intact platelets (1 × 108 cells), suspended in phosphate-buffered saline, with 2.1 nM [3H]Sph-1-P. Reactions were initiated by the addition of the ligand and were incubated at 4 °C for 1 h unless stated otherwise. The platelets were then washed with 1 mg/ml of bovine serum albumin, 150 mM NaCl, and 50 mM Tris-HCl, pH 7.5, three times. Radioactivity of the platelets was counted by liquid scintillation counting. Under those conditions, [3H]Sph-1-P added remained intact, which was confirmed by separation with thin layer chromatography.
To examine the effect of a protease on [3H]Sph-1-P binding, platelets were treated with 0.02% of the protease (type XXV) (Sigma) at room temperature for 30 min, and assays were carried out as described above.
Total binding was defined as the amount of radioactivity bound to platelets in the absence of competing ligand. Nonspecific binding was defined as the amount of [3H]Sph-1-P binding that occurred in the presence of 50 µM Sph-1-P. Specific binding was defined as the difference between total and nonspecific binding.
Sph-1-P Release from Platelets Labeled with [3H]Sph[3H]Sph-labeled platelet suspensions, to which 1% bovine serum albumin had been added, were centrifuged for 15 s at 12,000 × g. Lipids were then extracted from the resultant medium supernatant and cell pellet and analyzed as described above. Albumin was included in the medium to prevent released Sph-1-P, a lipophilic molecule, from being nonspecifically attached to the plasma membrane surface and consequently underestimated. The percentage of Sph-1-P release into medium was calculated as 100 × ([3H]Sph-1-P in medium)/(total [3H]Sph-1-P in medium plus cell pellet).
Protein Kinase C Activation in Intact PlateletsProtein kinase C activation in intact platelets was evaluated by pleckstrin (47-kDa protein) (35) phosphorylation as described (36).
When [3H]Sph was added into platelet homogenates under established assay conditions for Sph-1-P lyase (6), no lyase products (palmitaldehyde, palmitic acid, and palmitol) were formed. Under the same conditions, high lyase activity was detected in bovine (specific activity, 11.2 ± 0.2 pmol/min·mg of protein, mean ± S.D., n = 3) and rat livers, as reported previously (6). Low but significant Sph-1-P lyase activity was detected in CMK and K562 cells (specific activities, 0.25 ± 0.04 and 0.54 ± 0.03 pmol/min·mg of protein, mean ± S.D., n = 3, respectively). CMK is a human megakaryoblastic cell line (37); K562 is a human erythroleukemia cell line capable of megakaryocytic differentiation (38). Our results confirm the absence of Sph-1-P lyase in platelets, as suggested by earlier studies (39, 40). It is likely that blood stem cells lose Sph-1-P lyase activity during late-stage differentiation into platelets.
Independence of Sph Kinase Activity from Cell Activation in PlateletsWhen 1 µM [3H]Sph was added
exogenously to platelet suspensions, the label was efficiently removed
from the medium; uptake of [3H]Sph was 96% at 5 min
after the label addition. [3H]Sph incorporated into
platelets was rapidly converted to [3H]Sph-1-P (Fig.
1A). Conversion was observed as early as at
10 s. Within 5 min, the level of Sph-1-P approached a plateau,
with 45% conversion of [3H]Sph to
[3H]Sph-1-P. At 3 h (the latest time point
observed), 42% of the [3H]Sph originally added remained
as [3H]Sph-1-P (Fig. 1A), indicating the
stability of Sph-1-P in platelets. Sph at concentrations below 10 µM did not activate platelets at all (Fig.
2B). Sph kinase, which converts
[3H]Sph into [3H]Sph-1-P, is therefore
considered to be present in platelets as an active enzyme under resting
conditions. [3H]Sph was also converted by
N-acylation into Cer at later time points (Fig.
1A); 3, 7, and 10% of added [3H]Sph was
converted into Cer at 1, 2, and 3 h, respectively. The ratio of
[3H]Sph-1-P to [3H]Sph increased rapidly to
approximately 5:1 at 20 min and stayed relatively constant at this
value until 3 h. This suggests that the cellular content of
Sph-1-P is much higher than that of Sph in resting platelets.
The rapid conversion of [3H]Sph into [3H]Sph-1-P was not affected by the potent platelet activator thrombin (41) (Fig. 1B), which, under similar conditions, strongly induced platelet activation, including intracellular Ca2+ mobilization and protein kinase C activation (data not shown). Other platelet activators (collagen, TPA, and epinephrine) and the platelet activation inhibitor prostaglandin E1 (41) also had no effect on the [3H]Sph conversion (data not shown). N,N-Dimethylsphingosine, which strongly inhibits Sph kinase, suppressed conversion of [3H]Sph to [3H]Sph-1-P (Fig. 1B), as we reported previously (42).
In summary, Sph kinase is highly active, even in resting platelets, and its activity is independent of intracellular events involved in platelet activation.
Determination of Sph and Sph-1-P Levels in PlateletsWe determined the mass levels of Sph-1-P and Sph extracted from platelets. The content of Sph-1-P and Sph was calculated as 1.41 ± 0.04 nmol and 374 ± 61 pmol (mean ± S.D., n = 3 and 5), respectively, per 109 platelets. The finding that the cellular content of Sph-1-P is much higher than that of Sph is consistent with the high Sph-1-P:Sph ratio obtained in [3H]Sph labeling studies (Fig. 1A).
We measured the time course of mass changes of Sph-1-P and Sph in platelets incubated with nonradioactive Sph, the substrate of Sph kinase. Exogenous Sph was rapidly converted into Sph-1-P (Fig. 2A), as was also seen in the [3H]Sph labeling studies (Fig. 1A). Consistent with our previous finding (16), exogenous Sph-1-P induced platelet shape change and intracellular Ca2+ mobilization (Fig. 2B). These positive reactions induced by Sph-1-P were not mimicked by Sph (Fig. 2B), although Sph was rapidly converted to Sph-1-P (Figs. 1A and 2A). Sph-1-P (5 µM), but not 10 µM Sph, activated platelets (Fig. 2B), despite the fact that over 5 µM Sph-1-P was rapidly formed in platelets incubated with 10 µM Sph (Fig. 2A) by cytoplasmic Sph kinase (20, 43).
Platelet Activation Induced by Sph-1-P Immobilized on a Solid SupportThe finding that only exogenous Sph-1-P activates
platelets raises the possibility that Sph-1-P acts on these highly
differentiated cells from outside. To test this possibility, we
synthesized Sph-1-P immobilized on a solid support; a Sph-1-P
derivative possessing an -carboxyl group was covalently linked to
alkylamine-glass particles through amide linkage. Upon the addition of
the immobilized Sph-1-P, platelets underwent shape change and aggregate
formation, as determined by scanning electron microscopy (Fig.
3, C and D), and intracellular
Ca2+ mobilization (Fig. 4). Sph-bound glass
particles (Fig. 3, A and B, and Fig. 4) or
control glass particles (data not shown) had no effect on platelets.
The fact that immobilized Sph-1-P mimics free Sph-1-P in terms of
activating platelets strongly suggests that the site of Sph-1-P action
resides not inside platelets but on the surface. This hypothesis would
explain why exogenous addition of Sph-1-P but not Sph results in
platelet activation in spite of rapid Sph conversion into Sph-1-P
(which occurs intracellularly). The hypothesis is consistent with the
observation that extracellular, but not intracellular (by
microinjection), addition of Sph-1-P elicits intracellular
Ca2+ mobilization in Xenopus laevis oocytes
(44).
[3H]Sph-1-P Binding Studies
The above finding
that Sph-1-P only acts from the extracellular face of platelet surface
membranes can be best explained by the hypothesis that exogenous
Sph-1-P acts on platelets via interaction with a plasma membrane
receptor. Consequently, we performed [3H]Sph-1-P binding
studies using intact platelets. Fig. 5A shows the time course of specific binding of [3H]Sph-1-P to
platelets. The specific binding was time dependent, reached equilibrium
by 1 h, and remained constant for at least 1 h. Saturation
binding experiments were performed with [3H]Sph-1-P (Fig.
5B). The specific binding was saturated around 2 nM of the ligand when platelets were incubated at 4 °C
for 1 h with or without cold 50 µM Sph-1-P. The data
were transformed by Scatchard analysis (Fig. 5C). Platelets
were found to possess two binding sites for Sph-1-P; the
Kd values were estimated to be 110 nM
and 2.6 µM, and the numbers of the binding sites approximately 200/cell and 2400/cell, respectively. We examined the
effect of a protease on the specific binding of
[3H]Sph-1-P. Treatment of platelets with a protease (type
XXV) disrupted the specific binding of [3H]Sph-1-P almost
completely (data not shown), indicating that the binding sites are
proteins that are located on the surface of platelets. The specific
[3H]Sph-1-P binding was not detected on K562 cells, which
are capable of megakaryocytic differentiation but do not respond to
Sph-1-P in terms of Ca2+ mobilization (data not shown).
These results indicate the presence of a cell-surface receptor for
Sph-1-P on platelets.
Competition binding experiments were performed using various
sphingolipids and LPA (Fig. 6). Sph and
C2-Cer were unable to compete for [3H]Sph-1-P
binding sites on platelets. Sphingosylphosphocholine only slightly
inhibited the binding of [3H]Sph-1-P. In contrast, LPA,
which is a glycerophospholipid with a very similar structure to Sph-1-P
(26), reduced the binding of [3H]Sph-1-P to platelets as
much as unlabeled Sph-1-P did.
Desensitization of Platelet Aggregation Response to LPA by Sph-1-P
LPA is a well established platelet agonist capable of
inducing strong and irreversible aggregation response (45). Sph-1-P is
also a platelet-aggregating agent, although its effect is weaker (16).
Because the possibility that Sph-1-P and LPA may share a surface
receptor on platelets was raised, desensitization studies in platelet
aggregation were performed (Fig. 7). LPA (5 µM) induced marked platelet aggregation, which was
comparable with that induced by 5 µg/ml of collagen (41). Prior
addition of subthreshold concentrations of Sph-1-P inhibited the
platelet aggregation response to LPA. In contrast, Sph-1-P did not
affect the response to collagen. The specific desensitization of
LPA-induced platelet aggregation by Sph-1-P might be related to the
fact that Sph-1-P and LPA share a platelet surface receptor, although
we cannot completely rule out the desensitization mechanism resulting
from the modification of intracellular signaling pathways involved.
Sph-1-P Release from Activated Platelets
In view of the fact
that Sph-1-P activates platelets from outside but is abundantly stored
inside, we examined the possibility that Sph-1-P is released from
platelets and acts intercellularly as a local mediator. When platelets
were stimulated with 1 µM TPA, which can act as a
substitute for diacylglycerol and directly activates protein kinase C
(41, 46), 8, 35, 53, 57, and 60% of stored Sph-1-P was released
extracellularly 2, 5, 20, 60, and 120 min after challenge,
respectively. This protein kinase C activator was also found to release
Sph-1-P in a dose-dependent manner (Fig. 8A). The release correlated well with its
effect on protein kinase C activation in intact platelets (Fig.
8B). Under the same conditions, thrombin, which produces
diacylglycerol and activates protein kinase C as a result of
phosphatidylinositol-4,5-bisphosphate hydrolysis (41, 46), and
1-oleoyl-2-acetyl-glycerol, a membrane-permeable diacylglycerol (46),
also caused marked Sph-1-P release (Fig. 9). The Sph-1-P
release induced by these protein kinase C activators was inhibited by
staurosporine (Fig. 9), an inhibitor of protein kinases, including
protein kinase C (47). Furthermore, epinephrine, a weak platelet
stimulator incapable of activating protein kinase C by itself (41), did
not induce Sph-1-P release (data not shown). These findings indicate
that Sph-1-P can be released from activated platelets and suggest that
protein kinase C activation may be the mechanism involved.
In most cells, Sph-1-P is degraded to ethanolamine phosphate and
fatty aldehyde by Sph-1-P lyase (1, 5, 6). Platelets lack this lyase
activity. In contrast, these enucleated cells possess a highly active
Sph kinase. Sph kinase is present in platelets as an active enzyme
under resting conditions, and its activity is not related to cell
activation by physiological agonists. It is not surprising that
platelets, which possess high Sph kinase activity and lack Sph-1-P
lyase activity, accumulate Sph-1-P abundantly. These findings on
Sph-1-P-related metabolism in enucleated platelets contrast with
previous findings on nucleated cells (7, 8, 14, 17), in which
researchers observed fast and extensive metabolism of Sph-1-P with a
relative scarcity of cellular Sph-1-P and hypothesized that Sph kinase
is the rate-limiting factor in Sph catabolism. The role of Sph-1-P as a
mitogenic signaling molecule has been extensively studied in Swiss 3T3
fibroblasts. In these cells, Sph-1-P has been shown to possess
properties that qualify it as an intracellular second messenger (7, 8,
48). Endogenous Sph-1-P is maintained at a low level, possibly because
of degradation by a lyase. Sph kinase is relatively inactive in the
resting state (only a small fraction of exogenous Sph is converted to
Sph-1-P intracellularly); Sph kinase activity is stimulated and Sph-1-P level is transiently increased by specific growth factors
(platelet-derived growth factor and serum). Recently, similar findings
were reported for FcRI-mediated signal in the rat mast cell line
(14). It seems obvious that Sph kinase regulation and hence the
functional role of Sph-1-P in nonproliferative, terminally
differentiated cells such as platelets differ from those in nucleated
cells. The possibility of Sph-1-P playing a pivotal messenger role
intracellularly is remote in platelets.
Sph-1-P is a platelet activator (16). The findings that extracellular but not intracellular Sph-1-P is capable of activating platelets, and that immobilized Sph-1-P mimics free Sph-1-P in terms of activating platelets, indicate that the site of Sph-1-P action resides not inside platelets but on the surface. The more convincing evidence for the site of Sph-1-P action being extracellular is our identification here of specific binding sites for [3H]Sph-1-P on platelets, the first demonstration of a specific [3H]Sph-1-P binding site being expressed on the surface of plasma membrane. It has been shown recently that signaling pathways of Sph-1-P are regulated by heterotrimeric GTP-binding proteins (9, 13, 23, 24), whose activation is receptor-dependent (49). Furthermore, during the course of our present study, it was reported that only exogenously (not intracellularly) added Sph-1-P induces biological responses in guinea pig atrial myocytes (13) and N1E-115 neuronal cells (15). In addition to intracellular actions after passing the plasma membrane, activation of plasma membrane receptor(s) may be a critical mechanism by which Sph-1-P exerts biological responses in various cells.
Another important finding of ours is that the putative Sph-1-P receptor may be shared by LPA, a lysoglycerophospholipid that is similar to Sph-1-P in structure (26), capable of inducing platelet aggregation (45), and released from activated platelets (50). In support of this hypothesis, we found that platelet aggregation response to LPA was specifically desensitized by Sph-1-P. These findings are not consistent with several recent studies reporting lack of cross-desensitization between Sph-1-P and LPA in various nucleated cells (13, 15, 51). In this content, it is noted that platelets are shown to possess two different levels of binding sites, a high affinity site (Kd, 110 nM) and a low affinity site (Kd, 2.6 µM), and one can assume that the low affinity site might be functional for platelets, judging from the facts that µM order of Sph-1-P concentrations are needed to induce platelet shape change and aggregation. Although the molecular mechanism of our observation in platelets remains to be solved, one possibility may be that platelets possess a unique receptor for lysophospholipids, including Sph-1-P and LPA, leading to platelet aggregation. It is already established that platelets store a variety of biologically active molecules that are secreted upon stimulation (52). The secreted molecules interact with other platelets, plasma proteins, and the vessel wall. Sph-1-P was released from platelets, as expected given that Sph-1-P activates platelets from outside, but is abundantly stored inside. The Sph-1-P release may be mediated by protein kinase C, which is also highly expressed in platelets (53). We propose that Sph-1-P be added to the list of bioactive molecules stored in platelets and released from them upon stimulation, although, at present, we do not have the direct evidence for platelet activation caused by released Sph-1-P; the fact that not only Sph-1-P but also more potent lipid mediators such as thromboxane A2 are released from activated platelets makes our trial difficult. Previous reports have suggested that sphingolipid metabolites, including Sph-1-P, constitute a new class of intracellular second messengers in cell growth regulation and signal transduction (1, 3-5). However, as shown here, a sphingolipid can act intercellularly as a local mediator through its discharge from cells to regulate cellular functions in an autocrine or paracrine fashion.
We thank Prof. S. Hakomori of University of Washington for his encouragement throughout this study. We also thank L. E. Caldwell (Fred Hutchinson Cancer Research Center) for scanning electron microscopy and Drs. S. Anderson and E. A. Sweeney for scientific editing of the manuscript.