(Received for publication, September 12, 1995; and in revised form, November 20, 1995)
From the
Sphingosine-1-phosphate (SPP) has attracted much attention as a
possible second messenger controlling cell proliferation and motility
and as an intracellular Ca-releasing agent. Here, we
present evidence that SPP activates a G protein-coupled receptor in the
plasma membrane of various cells, leading to increase in cytoplasmic
Ca
concentration
([Ca
]
), inhibition of
adenylyl cyclase, and opening of G protein-regulated potassium
channels. In human embryonic kidney (HEK) cells, SPP potently
(EC
, 2 nM) and rapidly increased
[Ca
]
in a pertussis
toxin-sensitive manner. Pertussis toxin-sensitive increase in
[Ca
]
was also observed
with sphingosylphosphorylcholine (EC
, 460 nM),
whereas other sphingolipids, including ceramide-1-phosphate, N-palmitoyl-sphingosine, psychosine, and D-erythro-sphingosine at micromolar concentrations
did not or only marginally increased
[Ca
]
. Furthermore, SPP
inhibited forskolin-stimulated cAMP accumulation in HEK cells and
increased binding of guanosine 5`-3-O-(thio)triphosphate to
HEK cell membranes. Rapid [Ca
]
responses were also observed in human transitional bladder
carcinoma (J82) cells, monkey COS-1 cells, mouse NIH 3T3 cells, Chinese
hamster ovary (CHO-K1) cells, and rat C6 glioma cells, whereas human
HL-60 leukemia cells and human erythroleukemia cells failed to respond
to SPP. In guinea pig atrial myocytes, SPP activated G
protein-regulated inwardly rectifying potassium channels.
Activation of these channels occurred strictly when SPP was applied at
the extracellular face of atrial myocyte plasma membrane as measured in
cell-attached and inside-out patch clamp current recordings. We
conclude that SPP, in addition to its proposed direct action on
intracellular Ca
stores, interacts with a high
affinity G
protein-coupled receptor in the plasma membrane
of apparently many different cell types.
Over the past years, sphingolipids have emerged as important
second messengers of cellular
signaling(1, 2, 3) . Following stimulation of
cells with nerve growth factor, tumor necrosis factor-, and
interleukin-1
, sphingomyelinases are activated leading to the
generation of ceramide, which can be further metabolized to sphingosine
and sphingosine-1-phosphate (SPP) (
)by the action of
ceramidase and sphingosine kinase, respectively. Although ceramide and
sphingosine have been the subject of extensive studies, recently,
attention has also been focused on SPP. This sphingolipid has been
demonstrated to be involved in a multitude of processes. Activation of
sphingosine kinase and enhanced formation of SPP was shown to be
induced by platelet-derived growth factor, with SPP being implicated as
an important second messenger for the promotion of DNA synthesis in
Swiss 3T3 fibroblasts(4) . DNA synthesis and cell division of
3T3 cells could also be noted when SPP was added exogenously to intact
3T3 cells(5) . At low concentrations, SPP is also able to
inhibit efficiently motility and invasiveness of various tumor cells
that cannot be mimicked by sphingosine or N-methylated
sphingosines(6) . Another important action of SPP has emerged
from the observation in various cellular systems that SPP can cause
release of Ca
from internal stores by a non-inositol
1,4,5-trisphosphate receptor-mediated
mechanism(7, 8, 9) . On the other hand, a
rapid increase in cytoplasmic Ca
concentration
([Ca
]
) could also be
obtained by direct application of SPP to intact Swiss 3T3
cells(7) .
Thus, SPP appears to be an important component in
the signaling system that is involved in Ca release
and in the regulation of cell growth and motility. However, thus far,
the molecular targets of SPP have been rather elusive. One possible
molecular target may be an intracellular Ca
-permeable
channel located in the endoplasmic reticulum, which is gated by
SPP(7, 8, 9) . This channel may be reached by
exogenously applied SPP. A unique feature of SPP response on intact
cells, however, is the immediate and transitory rise in
[Ca
]
(7) . We
therefore studied whether SPP may also activate a plasma membrane
receptor rather than surpassing the plasma membrane to activate a
putative intracellular Ca
-permeable channel in the
endoplasmic reticulum. In the present report, we provide evidence for
this hypothesis and demonstrate that SPP at nanomolar concentrations
activates pertussis toxin (PTX)-sensitive guanine nucleotide-binding
proteins (G proteins) via a plasma membrane receptor apparently present
in various cell types.
Figure 1:
Increase in
[Ca]
by SPP in HEK 293
cells and influence of EGTA and PTX pretreatment. Influence of SPP (1
µM) on [Ca
]
was determined in HEK 293 cells pretreated without and with
EGTA (5 mM, 30 s) (upper panel) or without and with
PTX (100 ng/ml, 16 h) (lower panel) by the Fura-2 method as
described under ``Experimental Procedures.'' Addition of SPP
is indicated by the arrows.
To determine the potency and specificity of SPP's
action, we performed concentration response experiments with SPP and a
variety of related sphingolipids. SPP increased
[Ca]
in HEK 293 cells with an
EC
value of 2.04 ± 0.87 nM (mean ±
S.E.), and maximal increases in [Ca
]
(300-400 nM) were observed at 1-10
µM (Fig. 2). The slope of the concentration
response curve was rather shallow, with a calculated Hill coefficient
of 0.44 ± 0.05. Out of the other sphingolipids studied,
ceramide-1-phosphate, psychosine, and N-palmitoyl-sphingosine
at 10 µM did not alter
[Ca
]
(data not shown), and D-erythro-sphingosine at 10 µM only
marginally elevated [Ca
]
. In
contrast, sphingosylphosphorylcholine increased
[Ca
]
in HEK 293 cells to the
same maximal level as SPP. Responses to sphingosylphosphorylcholine
were as fast as those to SPP but were found at much higher
concentrations (EC
, 457 ± 31 nM). Similar
to the SPP response, [Ca
]
increases induced by sphingosylphosphorylcholine were largely
blunted by PTX pretreatment (data not shown). LPA shares some
structural similarity to SPP, i.e. a long hydrocarbon chain
with a terminal phosphate group, and is known to exert many SPP-like
effects(17) . Therefore, we tested whether SPP acts by
activating LPA receptors. For this, we investigated whether
[Ca
]
transients induced by LPA
were affected by a prior challenge with SPP. As shown in Fig. 3,
this was not the case. Yet, the [Ca
]
response to SPP (1 µM) was abolished following a
first SPP challenge. In the reverse order of addition, the SPP response
was not affected following a challenge with LPA (1 µM),
while prior stimulation with LPA abolished the effect of a second LPA
challenge. From these results, it can be concluded that SPP does not
increase [Ca
]
by activating the
LPA receptor.
Figure 2:
[Ca]
responses to SPP, sphingosylphosphorylcholine (SPPC), and D-erythro-sphingosine (SPH) in HEK 293 cells.
[Ca
]
responses to SPP
(
), sphingosylphosphorylcholine (
), and D-erythro-sphingosine (
) at the indicated
concentrations were determined in HEK 293 cells. Data are presented as
percentage of the maximal [Ca
]
change in response to 10 µM SPP. There was no
further increase in [Ca
]
in response to higher concentrations of either SPP or SPPC.
Basal and maximal [Ca
]
in response to 10 µM SPP were 59 ± 18
and 386 ± 63 nM (mean ± S.D.). The Hill slopes
of the concentration response curves of SPP and SPPC were 0.44 ±
0.05 and 1.42 ± 0.13, respectively. Data are mean values
± S.E. of 3 (sphingosylphosphorylcholine, D-erythro-sphingosine) or 5 sets (SPP) of
experiments, each done in duplicate.
Figure 3:
Lack of cross-desensitization of SPP- and
LPA-induced [Ca]
increases in HEK 293 cells.
[Ca
]
increases in HEK
293 cells were determined upon addition of either 1 µM SPP
or 1 µM LPA as indicated. Addition of SPP or LPA is
indicated by the arrows.
Since the
[Ca]
-elevating action of SPP
was largely reduced by PTX treatment, we studied whether SPP by
activating PTX-sensitive G proteins inhibits adenylyl cyclase. As shown
in Fig. 4, SPP (10 µM) decreased
forskolin-stimulated cAMP accumulation in HEK 293 cells by
30-40%. This inhibitory response was completely blocked by
pretreating cells with PTX (100 ng/ml, 16 h).
Figure 4: SPP-induced inhibition of forskolin-stimulated cAMP accumulation in HEK 293 cells. cAMP levels were determined in HEK 293 cells pretreated without and with PTX (100 ng/ml, 16 h) in the presence of 50 µM forskolin (Fors) or forskolin plus 10 µM SPP as described under ``Experimental Procedures.'' Data are from one set of experiments (each done in triplicate), representative of two independent experiments that yielded similar results.
Finally, we determined
whether SPP activates G proteins, by measuring binding of the labeled
stable GTP analog GTPS to membranes of HEK 293 cells (Fig. 5). SPP (1 µM) increased binding of
[
S]GTP
S to membranes of HEK 293 cells by
about 50%. An increase of similar magnitude (about 90%) was observed in
response to carbachol (1 mM), which activates the m3
muscarinic acetylcholine receptor stably expressed in the same HEK 293
cells as described before(10) . This HEK 293 cell clone showed
identical [Ca
]
responses to SPP
as untransfected HEK 293 cells.
Figure 5:
SPP-induced GTPS binding to membranes
of HEK 293 cells. Specific binding of
[
S]GTP
S was determined in membranes of HEK
293 cells, stably expressing the human m3 muscarinic acetylcholine
receptor subtype, in the absence (control) and presence of 1 µM SPP or 1 mM carbachol as described under
``Experimental Procedures.'' Values are means ± S.E.
of five experiments, each done in
triplicate.
To study whether the PTX sensitivity of the
[Ca]
response was cell
type-dependent, we chose to test in NIH 3T3 fibroblasts whether
PTX-sensitive G proteins are involved in SPP-induced
[Ca
]
increases as well. While
in untreated NIH 3T3 cells SPP (10 µM) elevated
[Ca
]
by 130 nM, in
PTX-pretreated NIH 3T3 cells SPP-induced
[Ca
]
increase was completely
abolished (data not shown).
Figure 6:
Lack of an effect of SPP on I channel activity in atrial myocyte-attached membrane patch.
Channel current was measured using an approximately symmetrical
K
distribution across the patch, whereas the bath
solution contained 20 mM K
to
``clamp'' the resting potential (E
) at around -50 mV. Membrane
potential across the patch was E
-60 mV, resulting in a unitary inward current through open
K
channels of approximately 4 pA. This experimental
condition was used because of the strongly inward-rectifying properties
of I
channels, which pass hardly any current in the
outward direction.
Further evidence for
a receptor-mediated action of SPP was obtained from single channel
recordings in the inside-outside mode. When the pipette solution,
facing the original outer face of the plasma membrane, was supplemented
with 1 µM SPP, exposure of the inside of the membrane to
GTP (50 µM) resulted in a dramatic increase in channel
activity (Fig. 7, trace A). In the absence of SPP,
exposure to GTP alone caused a slight increase in channel activity (trace B), which is due to the fact that also basal activity
of this channel is G protein-regulated(22) . Notably, the same
low degree of channel activity was obtained when GTP (50
µM) and SPP (1 µM) were applied
simultaneously to the intracellular face of the isolated patch (trace C). Peak values (n = 4) for n*P upon exposure of the patches to GTP
were 4.6 ± 3.5 (A), 0.31 ± 0.15 (B),
and 0.26 ± 0.19 (C). In summary, the patch clamp data
clearly demonstrate that SPP acts only from the extracellular face of
the plasma membrane and requires GTP for activation of I
channels.
Figure 7:
Effects of SPP on GTP-dependent
I channel activity in inside-out patches of atrial
myocytes. Symmetrical K
distribution (150
mM/150 mM) across the membrane and patch potential of
-60 mV result in inward currents through open I
channels. SPP (1 µM) was either not added (B) or present in the pipette (A) and bath (C) solution, respectively, facing the external and internal
face of the membrane. GTP (50 µM) was present in the bath
solution for the indicated periods of time.
In the present study, we tested the hypothesis that exogenous
SPP acts on intact cells via activation of a plasma membrane receptor.
We demonstrate that SPP rapidly increases
[Ca]
in various cells, inhibits
forskolin-stimulated adenylyl cyclase in HEK 293 cells, and activates
I
channels in atrial myocytes. These responses were
either to a large extent or completely PTX-sensitive, indicating
activation of G
-type G proteins. Since SPP is a lipid that
may be readily taken up by cells, it was important to determine whether
SPP activates G proteins directly or requires a plasma membrane
receptor to activate G proteins. We could demonstrate in atrial
myocytes that SPP only acts from the extracellular face of the plasma
membrane and is not able to activate G
-regulated
I
channels when applied to the intracellular face of
the plasma membrane. We therefore conclude that SPP acts by binding to
a plasma membrane receptor that couples predominantly to PTX-sensitive
G proteins. Consistent with this conclusion is the observation that the
calcium responses to SPP varied strongly from one cell type to another,
and in some cell types no distinct calcium response to SPP was noted at
all.
During the course of this study, the groups of Spiegel and
Sturgill (23, 24) reported that exogenous SPP
decreases cellular cAMP levels, stimulates inositol phosphate
formation, increases [Ca]
,
activates mitogen-activated protein kinase, and stimulates DNA
synthesis in Swiss 3T3 fibroblasts and that these cellular actions of
SPP are largely or completely abolished by PTX pretreatment. These
observations led the authors to conclude that SPP may selectively
activate PTX-sensitive G proteins in a receptor-independent fashion or
alternatively activate a specific cell surface receptor that is coupled
to these G proteins. Although the data reported in Swiss 3T3 cells are,
at least partially, in agreement with those reported herein, there are
also important differences. First, the effects of SPP in Swiss 3T3
cells were observed at rather high concentrations (EC
values of about 2 µM) compared to about 3 orders of
magnitude lower concentrations of SPP (EC
values of about
2 nM) required to increase
[Ca
]
in HEK 293 cells and to
activate I
channels in atrial myocytes(21) .
Second and most important, these reports left open the crucial question
about the site of action of exogenous SPP.
Several observations
suggest that SPP binds to a specific SPP receptor in the plasma
membrane. SPP was effective in the nanomolar range (EC of
2 nM), whereas various other sphingolipids, including
ceramide-1-phosphate, D-erythro-sphingosine,
psychosine, and N-palmitoyl-sphingosine, did not or only
marginally increase [Ca
]
in HEK
293 cells, even at micromolar concentrations. Likewise, the
structurally related phospholipid LPA, which effectively elevated
[Ca
]
at nanomolar
concentrations, did not desensitize the SPP-induced
[Ca
]
response in HEK 293 cells
at micromolar concentrations. This lack of cross-desensitization for
induced [Ca
]
transients in HEK
293 cells is in agreement with a similar study in A431 cells described
recently(25) . On the other hand, sphingosylphosphorylcholine
fully mimicked the SPP response, although about 200-fold higher
concentrations of sphingosylphosphorylcholine than of SPP were required
for increasing [Ca
]
in HEK 293
cells. This sphingolipid also mimics SPP's actions on
Ca
release from endoplasmic reticulum, however
apparently at similar concentrations as of SPP(8, 9) .
The shallow concentration response curve of SPP compared to the rather
steep one of sphingosylphosphorylcholine for increasing
[Ca
]
in HEK 293 cells may even
suggest that distinct types of receptors are involved in SPP's
actions.
The synthesis of SPP from sphingosine is catalyzed by the enzyme sphingosine kinase. However, present information about this enzyme is rather scarce. Sphingosine kinase appears to be a cytosolic enzyme in platelets while being membrane-associated in rat brain and other tissues(26) . Since our study indicates that exogenous SPP acts at a plasma membrane-located receptor, it will be of interest to study how SPP synthesis is regulated by hormones and growth factors, how SPP is released from cells, and what physiological functions SPP plays in different tissues.
In conclusion, this study demonstrates
that SPP can regulate intracellular second messengers and membrane
channels through activation of specific receptors apparently present in
many different cell types and coupled to PTX-sensitive
G-type G proteins. SPP has also been implicated as an
intracellular second messenger releasing Ca
from
internal stores. Thus, the present study suggests that SPP has at least
two molecular targets of action, i.e. the proposed
sphingolipid-gated Ca
-permeable channel in the
endoplasmic reticulum as well as a high affinity G
protein-coupled receptor in the plasma membrane.