(Received for publication, May 4, 1995; and in revised form, August 25, 1995)
From the
Extracellular sphingosylphosphorylcholine (SPC) and
galactosylsphingosine (psychosine) induced Ca mobilization in a dose-dependent manner in HL60 leukemia cells.
The rapid and transient increase in intracellular Ca
concentration ([Ca
]
)
elicited by SPC and psychosine at concentrations lower than 30
µM was inhibited by treatment of the cells with pertussis
toxin (PTX) and U73122, a phospholipase C inhibitor, as was the case
for UTP, a P
-purinergic agonist. The increase in
[Ca
]
induced by these
lysosphingolipids was associated with inositol phosphate production,
which was also sensitive to PTX and U73122. The inositol phosphate
response is not secondary to the increase in
[Ca
]
as evidenced by
the observation that thapsigargin and ionomycin, Ca
mobilizing agents, never induced inositol phosphate production
and, unlike lysosphingolipids, the
[Ca
]
rise by these
agents was totally insensitive to PTX and U73122. When HL60 cells were
differentiated into neutrophil-like cells by dibutyryl cyclic AMP,
inositol phosphate and Ca
responses to
AlF
were enhanced, probably reflecting an
increase in the amount of G
and G
compared
with undifferentiated cells. In the neutrophil-like cells, however, the
responses to SPC and psychosine were markedly attenuated. This may
exclude the possibility that the lysosphingolipids activate rather
directly PTX-sensitive GTP-binding proteins or the phospholipase C
itself. Other lysosphingolipids including glucosylsphingosine
(glucopsychosine) and sphingosylgalactosyl sulfate (lysosulfatides) at
30 µM or lower concentrations also showed PTX- and
U73122-sensitive Ca
mobilization and inositol
phosphate response in a way similar to SPC and psychosine. However,
platelet-activating factor and lysoglycerophospholipids such as
lysophosphatidylcholine and lysophosphatidic acid were less effective
than these lysosphingolipids in the induction of Ca
mobilization. Taken together, the results indicate that a group
of lysosphingolipids at appropriate doses induces Ca
mobilization through inositol phosphate production by
phospholipase C activation. The lysosphingolipids-induced enzyme
activation may be mediated by PTX-sensitive GTP-binding protein-coupled
receptors, which may be different from previously identified
platelet-activating factor receptor or lysophosphatidic acid receptor.
Sphingolipids have recently been shown to be important
participants in the regulation of a variety of cellular processes (1, 2, 3) . Sphingosine, one of the
metabolites of sphingolipids, was in its early studies demonstrated as
a potent endogenous inhibitor of protein kinase C (1, 4) and has been implicated to be a negative
regulator for a few signaling processes(1, 4) .
Further studies, however, revealed that the exogenous sphingosine also
induces various types of positive biological actions, e.g. activation of phospholipase D(5) , stimulation of cell
proliferation(6) , regulation of Ca mobilization from the internal pool (7, 8, 9, 10, 11) , and
inhibition of Ca
influx through the plasma
membranes(12) . These actions seem to be exerted through
phosphatidate (5) or a phosphorylated product of sphingosine,
sphingosine 1-phosphate (S1P) (
)(7, 8, 13, 14, 15) ;
many of them were suggested to be independent of protein kinase C. S1P
was reported to act directly on the internal Ca
pool
resulting in Ca
mobilization in a way similar to
inositol 1,4,5-trisphosphate(8, 15) . This
lysosphingolipid has also been proposed as a second messenger of
platelet-derived growth factor and serum on cell proliferation in
fibroblasts(16) . In the brain and other peripheral tissues of
inherited sphingolipid disorders, it has been shown that any one of
lysosphingolipids, e.g. sphingosylphosphorylcholine (SPC),
galactosylsphingosine (psychosine), or glucosylsphingosine
(glucopsychosine), is
accumulated(4, 17, 18, 19) . These
lysosphingolipids might be responsible for the respective
pathogenesis(4, 17, 18, 19) . SPC
has recently been shown, similarly to S1P, to be a potent
Ca
releaser from the internal pool and suggested to
cause the Ca
release from the
1,4,5-trisphosphate-sensitive pool in various cell
types(7, 8, 9, 10) .
These
observations suggest that, in addition to protein kinase C inhibition,
intracellular Ca mobilization is an important action
of lysosphingolipids, which may have pathological and physiological
significance. This raises the question of whether the Ca
mobilization is caused by the activation of the phospholipase
C-Ca
signal transduction pathway. In fact, recent
studies demonstrated that extracellular S1P in Swiss 3T3 fibroblasts (15) and sphingosine in Swiss 3T3 fibroblasts (5) ,
astrocytes(20) , and foreskin fibroblasts (21) can
induce inositol phosphate production, probably reflecting activation of
phospholipase C. Although the S1P-induced
[Ca
]
increase in the
cells has been suggested to occur independently of the enzyme
activation(15) , at least a part of the sphingosine-induced
Ca
mobilization as well as phospholipase C activation
in foreskin fibroblasts was sensitive to PTX, showing some similarity
to a typical feature of PTX-sensitive G-protein-mediated activation of
the phospholipase C-Ca
pathway (21) . If this
is the case, we might be allowed to imagine the presence of a
receptor(s) for the lysosphingolipids which lead to the activation of
phospholipase C, although the previous findings have not excluded the
possibility that the lipids penetrate into the cells and act on the
pathway inside the cells.
In the present paper, our study was
focussed on the Ca mobilizing actions of SPC and
other lysosphingolipids which are accumulated in the respective
sphingolipidosis, especially on the mechanisms of their actions. We
found that, in HL60 leukemia cells, extracellularly added
lysosphingolipids at 30 µM or less induced a rapid and
transient increase in
[Ca
]
, the features of
which are indistinguishable from those of the Ca
response induced by UTP, a P
-purinergic agonist, in
the same cells. The transient
[Ca
]
rises were
associated with inositol phosphate production, and both Ca
and inositol phosphate responses were inhibited by the treatments
of cells with PTX and U73122, a potent phospholipase C inhibitor. Our
results suggest that extracellular lysosphingolipids at appropriate
doses induce a [Ca
]
rise due to the activation of the phospholipase C being
mediated by a putative receptor(s) coupled to a PTX-sensitive
G-protein(s).
Figure 2:
Effects of sphingomyelinase treatment and
TLC purification of SPC and psychosine on Ca
mobilization. In A, 3 mM SPC and psychosine were
incubated at 37 °C for 1 h with or without sphingomyelinase (SMase, 0.2 units) in 10 mM Tris-HCl buffer (pH 7.4),
containing 2 mM MgCl
, 10% MeOH, 0.8% glycerol in a
final volume of 100 µl. A part (2 µl) of the reaction mixture
was analyzed by Silica Gel 60 TLC in solvent I as described under
``Experimental Procedures.'' Lane 1, SPC (without
SMase); lane 2, SPC (with SMase); lane 3, SPC (with
SMase) plus authentic sphingosine; lane 4, authentic
sphingosine; lane 5, TLC-purified SPC; lane 6,
psychosine (without SMase); lane 7, psychosine (with SMase).
In B, unpurified psychosine (lane 1) and TLC-purified
psychosine (lane 2) were analyzed by Silica Gel 60 TLC in
solvent I as described under ``Experimental Procedures.'' In A and B, the position of origin (O), front (F), SPC, psychosine (PSY), or sphingosine was marked
as an arrow. In C and D,
[Ca
]
response to SPC
or psychosine (PSY) treated with or without SMase,
TLC-purified SPC, or TLC-purified psychosine was measured as indicated.
Concentration would be 30 µM unless degradation occurred
during enzyme treatment.
Figure 7:
Differentiation into neutrophil-like cells
attenuates phospholipase C and the subsequent Ca
mobilization in response to SPC and psychosine. In A, cell
membranes were prepared from undifferentiated cells (a) and
neutrophil-like cells differentiated by dibutyryl cyclic AMP (b). Their cholate extracts were subjected to a
SDS-polyacrylamide gel electrophoresis, transferred to an Immobilon
sheet, and then probed with G
- or
G
-specific antiserum as described under
``Experimental Procedures.'' In B and C,
representative traces of
[Ca
]
changes in
undifferentiated cells (B) and neutrophil-like cells (C) (non-treated (a and b) or treated (c) with PTX) are shown. At arrows, 10 nM formyl-Met-Leu-Phe (FMLP) or 30 µM SPC was
added to the incubation medium, as indicated. In D,
[Ca
]
changes caused
for 2 min by 30 µM SPC and psychosine, and for 10 min by
AlF
(10 mM NaF plus 10
µM AlCl
) in differentiated cells
(neutrophil-like cells) were compared with those in undifferentiated
cells. The results are expressed as percentages of those in
undifferentiated cells. In an inset, a typical trace of
[Ca
]
change by
AlF
in undifferentiated (a) and
neutrophil-like (b) cells is shown. In E,
undifferentiated cells (open column or
) and
neutrophil-like cells (closed column or
) both labeled
with [
H]inositol were incubated for 1 min without
or with formyl-Met- Leu-Phe (10 nM), SPC (30 µM),
or psychosine (30 µM). Production of IP
+
IP
was measured. Results are expressed as percentages of
the respective basal value obtained without any addition. Normalized
basal values (cpm) were 580 ± 35 and 472 ± 73 for
undifferentiated cells and the neutrophil-like cells, respectively. In
the inset, time courses of AlF
(10 mM NaF plus 10 µM
AlCl
)-induced response (IP
+
IP
) are shown. Results are expressed as percentages of the
respective initial value. Data are means ± S.E. of three
separate experiments.
Figure 1:
Effect
of PTX on SPC and psychosine-induced increase in
[Ca]
. A,
control cells, non-treated with PTX, and B, PTX-treated cells,
show traces of time-dependent
[Ca
]
changes each
representing the changes induced by UTP (1 µM) or the
indicated doses (µM) of SPC, psychosine (PSY) or
sphingosine. C-E show dose-dependent increase in
[Ca
]
(=peak value - basal value) with SPC (C), PSY (D), and sphingosine (E) in control
cells (
) and PTX-treated cells (
). Results are means
± S.E. of five separate experiments. F shows
[Ca
]
obtained by
1 µM UTP in a control (open column) and
PTX-treated (closed column) cells. Results are means ±
S.E. of seven separate experiments.
As mentioned in the
Introduction, another well documented action of lysosphingolipids is
protein kinase C inhibition, especially in the earlier period of the
studies(4) . SPC and psychosine therefore might induce
Ca mobilization as a result of the enzyme inhibition.
To examine this possibility, we also used sphingosine which is a
similar or more potent inhibitor of protein kinase C than SPC or
psychosine(4) . Sphingosine also increased
[Ca
]
; however, the time course
was very slow and the net increase was much less than that induced by
similar doses of SPC and psychosine (Fig. 1A).
Moreover, the sphingosine-induced
[Ca
]
increase was totally
insensitive to PTX (Fig. 1, B and E). We also
examined the effect of another protein kinase C inhibitor,
1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), but this drug never
increased [Ca
]
(data not
shown). In fibroblasts(11, 21) , the
sphingosine-induced [Ca
]
rise
has been suggested to be protein kinase C-independent. These results
suggest that at least the PTX-sensitive increase in
[Ca
]
by SPC and psychosine is
independent of the protein kinase C inhibition.
TLC analysis of the
SPC sample showed the presence of a small, but detectable, amount of
unknown compound that is positive with ninhydrin at R = 0.26 (Fig. 2A, lane 1).
However, it was confirmed that SPC itself elicited the Ca
response and the contaminated unknown compound is inactive to
induce the response (Fig. 2). Sphingomyelinase from B. cereus almost completely converted SPC to sphingosine, but did
not influence the unknown compound (Fig. 2A, lanes
2-4). The enzyme-treated SPC never elicited a rapid and
transient [Ca
]
increase which
is a characteristic to the untreated SPC, instead induced a rather slow
increase probably due to sphingosine (Fig. 2C). The
enzyme was rather specific to SPC; psychosine was tolerable to the
enzyme (Fig. 2A, lanes 6 and 7) and
the lipid-induced [Ca
]
increase
was unaffected by its treatment (Fig. 2C). Furthermore,
the TLC-purified SPC sample, which is free from the unknown compound (Fig. 2A, lane 5), induced the Ca
response to an extent similar to that of the unpurified SPC (Fig. 2C).
Although psychosine obtained from the
drug company showed a single spot that is positive with ninhydrin and
anthrone/HSO
on TLC using two solvent systems,
we further purified the psychosine by TLC (Fig. 2B, lanes 1 and 2). The purified psychosine also induced
a rapid and transient [Ca
]
increase as effectively as the unpurified psychosine did (Fig. 2D). Since the active compound to induce
Ca
mobilization was demonstrated to be SPC or
psychosine itself and furthermore, there was no appreciable difference
in the ability to induce Ca
response between purified
and unpurified products, we performed the following experiments without
further purification of the lipids.
Figure 3:
Effects of extracellular
Ca and U73122 on SPC, psychosine, and UTP-induced
increase in [Ca
]
.
Representative traces of [Ca
]
change from three or four separate experiments by 30
µM SPC (A), 30 µM psychosine (B), and 1 µM UTP (C) in the absence or
presence of 2.5 mM EGTA (as shown with
``E'' in the panel) or 2.5 µM U73122
(as shown with ``U73'' in the panel) are
shown.
Figure 4:
Time-dependent effect of SPC, psychosine,
and UTP on inositol phosphate production. The cells labeled with
[H]inositol were incubated for the indicated
times without (
) or with 30 µM SPC (
), 30
µM psychosine (
), or 1 µM UTP (
)
in the cells non-treated (A, C, and E) or
treated (B, D, and F) with PTX. Results are
expressed as percentages of the respective initial value. Normalized
initial values (cpm) in control cells were 465 ± 16, 206
± 6, and 473 ± 16 for IP, IP
, and
IP
, respectively. These values were not significantly
changed by PTX treatment. All data are means ± S.E. of three
separate experiments.
Figure 5:
Dose-response curves of SPC and psychosine
on inositol phosphate production and its suppression by PTX and U73122.
The cells labeled with [H]inositol were incubated
for 1 min with the indicated doses of SPC (A) and psychosine
(PSY) (B) in the cells non-treated (
,
) and
treated (
) with PTX. In some experiments, U73122 (
) (2.5
µM) was added to the incubation medium 2 min before SPC
and PSY addition. Production of IP
plus IP
was
measured. Results are expressed as percentages of the basal values
obtained without test agents. Normalized basal values (cpm) were 554
± 16 and 552 ± 16 for the cells non-treated and treated
with PTX, respectively. Data are means ± S.E. of three separate
experiments.
Figure 6:
Thapsigargin and ionomycin induced
[Ca]
increase in a
manner independent of phospholipase C and insensitive to PTX.
[Ca
]
change in the
cells non-treated (A) and treated (B) with PTX was
monitored. At arrows, 300 nM thapsigargin as shown
with ``TG,'' 1 µM ionomycin or 2.5
µM U73122 as shown with ``U73'' were
added. The results shown are representative of three separate
experiments. In C, the cells labeled with
[
H]inositol were incubated for the indicated
times without (
), with 300 nM thapsigargin (
), 1
µM ionomycin (
), or 30 µM SPC
(
). Results (IP
+ IP
production) are
expressed as percentages of initial values. Data are means ±
S.E. of three separate experiments.
As shown in Fig. 7A, the contents of G and G
were actually increased by a dibutyryl cyclic AMP treatment of
the cells as evidenced from increases in immunodetectable
G
and G
. The dibutyryl cyclic
AMP-treated cells also showed a PTX-sensitive
formyl-Met-Leu-Phe-induced [Ca
] increase (Fig. 7C) and inositol phosphate production (Fig. 7E), currently recognized to be differentiation
markers. AlF
, a non-selective G-protein
activator, induces phospholipase C activation and the subsequent
Ca
mobilization in many types of
cells(34, 36) . These AlF
actions shown in Fig. 7, D and E, are
very slow, but significant, and are slightly stronger in the
differentiated cells than in the undifferentiated ones, probably
reflecting higher contents of G
proteins in the
neutrophil-like differentiated cells than in the undifferentiated cells (Fig. 7, D and E). Unexpectedly, however, the
SPC-induced Ca
mobilization was markedly attenuated
in the neutrophil-like cells (Fig. 7, B and C). The Ca
response to psychosine was also
decreased (Fig. 7D). In parallel with the
Ca
response, the inositol phosphate response to SPC
and psychosine was clearly attenuated by differentiation, suggesting
that the lipids signaling of the PTX-sensitive G-protein-coupled
phospholipase C-Ca
pathway is blocked before a
G-protein step in the neutrophil-like cells (Fig. 7E).
Figure 8:
Effect of glucopsychosine and
lysosulfatides on [Ca]
and inositol phosphate production. In A (control
cells non-treated with PTX) and B (PTX-treated cells),
representative traces of [Ca
]
changes by the indicated doses of glucopsychosine (GlcPSY) and lysosulfatides (LSF) are shown. Where
indicated, U73122 (2.5 µM) was added 2 min before addition
of the respective lysosphingolipid. In C (control cells) and D (PTX-treated cells), the cells labeled with
[
H]inositol were incubated for the indicated
times without (
), with 30 µM lysosulfatides (
),
or with 30 µM glucopsychosine (
). Results are
expressed as percentages of initial values. Data are means ±
S.E. of three separate experiments.
In the present paper we have shown that lysosphingolipids
(SPC, psychosine, glucopsychosine, and lysosulfatides) at doses lower
than 30 µM induce phospholipase C activation and the
subsequent Ca mobilization in a manner sensitive to
PTX and U73122, a phospholipase C inhibitor. This is, to our knowledge,
the first indication that these lysosphingolipids activate the
phospholipase C-Ca
system possibly through receptors
coupling to a PTX-sensitive G-protein(s). The putative receptors may be
different from the previously identified platelet-activating factor
receptor (37) and lysophosphatidic acid receptor(38) .
As far as extracellular SPC-induced intracellular Ca mobilization is concerned, a few studies on fibroblasts (39) and FRTL-5 thyroid cells (40) have been reported.
However, no significant production of inositol phosphate was observed
in these experiments(39, 40) , despite the fact that
Ca
mobilizing receptor agonists, such as bradykinin,
induced not only Ca
mobilization to an extent similar
to that with SPC but also phospholipase C activation under the same
conditions(39) . In addition, in other studies, SPC mobilized
Ca
from permeabilized
cells(7, 8, 9, 10) and purified
endoplasmic reticulum membrane vesicles(8) . On the basis of
these previous results, the SPC actions have been currently considered
to occur inside the cells by the incorporated SPC molecules, without
activating phospholipase C. The present results, however, suggest that
at least the early phase of the Ca
mobilization
induced by lower than 30 µM SPC or other lysosphingolipids
in intact HL60 cells is mediated by the activation of the enzyme. This
suggestion is based on the following findings. First, SPC and other
lysosphingolipids at doses lower than 30 µM induced
immediate activation of phospholipase C. Second, U73122, a potent
phospholipase C inhibitor, suppressed at least the early phase of the
lysosphingolipids-induced increase in
[Ca
]
. Third, treatment of the
cells with either PTX or dibutyryl cyclic AMP attenuated both the
lysosphingolipid-induced phospholipase C activation and Ca
mobilization. Finally, phospholipase C activation is not a
secondary response to the increase in
[Ca
]
; agents such as
thapsigargin and ionomycin, which primarily increase
[Ca
]
, never activated the
enzyme in HL60 cells under the present conditions (Fig. 6).
Several findings in the present study suggest that the
lysosphingolipids signaling is performed through G-protein-coupled
receptors. The pattern and kinetics of
[Ca]
increase and inositol
phosphate production by the lysosphingolipids were very similar to
those of the responses to a G-protein-coupled receptor agonist, UTP (a
P
-purinergic agonist) (Fig. 1, Fig. 3, and Fig. 4). Furthermore, as stronger evidence for the involvement
of G-protein coupled receptors, the lysosphingolipid actions are
suppressed by prior treatment of the cells with PTX which, as is well
known, ADP-ribosylates G
-proteins and thereby blocks
communication between receptors and effector enzymes. Similar PTX
sensitivity has already been shown in the phospholipase C activation
induced by several receptor agonists such as formyl-Met-Leu-Phe and UTP
in leukocytes such as HL60 cells and neutrophils. This finding has been
concluded to reflect the fact that receptors coupling to PTX-sensitive
G-proteins mediate the phospholipase C
activation(24, 25, 26, 32) . In this
analogy, it is reasonable to assume that the lysosphingolipid actions
are mediated via G
-protein-coupled receptors. It is still
possible, however, that amphipathic lysosphingolipids penetrate into
the cells and then directly activate G
-proteins. If this
was the case, PTX would block the lipid-induced actions. This
possibility is excluded from the experiments shown in Fig. 7.
Dibutyryl cyclic AMP-induced differentiation into neutrophil-like cells
enhanced AlF
(a nonspecific G-protein
activator)-induced phospholipase C activation, probably reflecting the
increase in the amount of G
-proteins. This suggests that in
the differentiated cells, the downstream region of the
G-protein-mediated signaling cascade leading to phospholipase C
activation and Ca
mobilization is rather fortified by
the increase in PTX-sensitive G-proteins. On the contrary, the SPC and
psychosine-induced enzyme activation was seriously suppressed by
differentiation of the cells. This suggests that differentiation
impairs the process between the action sites of lipids (or receptors)
and G-proteins and hence may rule out the possibility that these
lysosphingolipids directly activate G-proteins. Thus, the present
pharmacological study suggests the existence of G-protein-coupled
receptors for lysosphingolipids, although conclusive evidence for the
existence of the receptors will have to await their molecular cloning.
In addition to lysosphingolipids, platelet-activating factor and
some lysoglycerophospholipids, such as lysophosphatidylcholine and
lysophosphatidic acid, also induced Ca mobilization
in HL60 cells, but they were not as effective as SPC and other
lysosphingolipids (Table 1). Furthermore, in contrast to SPC and
psychosine effects which were attenuated in dibutyryl cAMP-induced
differentiated cells (Fig. 7), platelet-activating
factor-induced response was conversely enhanced by the induction of
differentiation, suggesting that the putative receptors for
lysosphingolipids are different from platelet-activating factor
receptor. Among lysoglycerophospholipids examined,
lysophosphatidylcholine was the most effective in the induction of
Ca
mobilization (Table 1). Similarly to the
actions of lysosphingolipids, the lysophosphatidylcholine effect was
PTX-sensitive, whereas the lysophosphatidic acid-induced response was
not (Table 1). Thus, the receptor for lysophosphatidic acid (38) appears to be different from putative receptors for
lysosphingolipids. On the other hand, it remains unclear whether
lysoglycerophospholipids (including lysophosphatidylcholine,
lysophosphatidylethanolamine, and lysophosphatidylinositol) other than
lysophosphatidic acid share with lysosphingolipids the same receptor
and signaling pathways.
Among lysosphingolipids, sphingosine and S1P
have been previously shown to induce phospholipase C activation and the
Ca mobilization in a few types of
cells(5, 15, 20, 21) . In HL60
cells, sphingosine induced the Ca
mobilization;
however, this action was PTX-insensitive (Fig. 1). Furthermore,
the [Ca
]
increase due to the
lipid was so slow that it took 1-3 min to reach a peak value (Fig. 1). Thus, the sphingosine signaling pathway seems to be
different from that of SPC and other lysosphingolipids. This also
suggests that the PTX-sensitive Ca
mobilization by
lysosphingolipids cannot be explained by the inhibition of protein
kinase C, because sphingosine is a protein kinase C inhibitor similar
to or more potent than the lysosphingolipids examined in the present
study(4) . We also preliminarily examined S1P actions on
phospholipase C and the Ca
mobilization in HL60
cells. This lipid also activated the enzyme and increased
[Ca
]
in the cells. In this
case, we could not detect any difference between S1P and SPC actions in
their sensitivity to PTX and U73122. Thus, S1P seems to share a
signaling pathway similar to that of SPC in HL60 cells. In Xenopus oocytes, however, S1P activated a Cl
channel
probably through phospholipase C activation, but SPC could not mimic
the S1P action(41) . The receptor cloning again would make it
clear whether all the lysosphingolipids and some
lysoglycerophospholipids share the same receptor or each lipid
interacts with its own receptor.
At the present stage of
investigation, the physiological roles of the lysosphingolipid-induced
activation of the phospholipase C-Ca pathway in
leukocytes have not been clarified yet. This type of lysosphingolipid
signaling was attenuated by dibutyryl cyclic AMP-induced
differentiation of HL60 cells into neutrophil-like cells (Fig. 7). In the preliminary experiments, we found that other
differentiation inducers such as dimethyl sulfoxide, retinoic acid, and
vitamin D
also diminished such lysosphingolipid signaling.
This may suggest that only under undifferentiated conditions
lysosphingolipids act as physiological and extracellular signals which
are oriented to the phospholipase C-Ca
pathway. In
the previous study in differentiated cells such as fibroblasts (39, 42) and thyroid cells (40) , SPC has been
shown to be a potent mitogen. The Ca
mobilizing
action of SPC may be involved in the cell
proliferation(39, 40) . A preliminary finding in the
undifferentiated HL60 cells, however, showed that SPC rather attenuated
the cell growth and instead facilitated cell attachment to culture
dishes. This phenomenon might reflect a physiological role of SPC as an
inducer of cell differentiation. Further study is now in progress to
clarify this point.
The possible existence of cell surface receptors for lysosphingolipids may allow consideration of a novel autocrine or paracrine regulatory mechanism operated by the lysosphingolipids in a way similar to other lipids mediators such as prostaglandins and leukotriens. At present, there are no data on the extracellular occurrence of lysosphingolipids in vivo. To establish the autocrine or paracrine role of the lipids, further studies on the problems are needed, which include characterization of intracellular and extracellular metabolic pathways and physiological functions of the lysosphingolipids as well as identification of their putative receptors.