(Received for publication, August 24, 1995; and in revised form, February 13, 1996)
From the
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
formation, but sphingosine did not affect the level of IP
by itself nor did it cause IP
formation induced by
OKT3. In permeabilized Jurkat cells, the addition of IP
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
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
- and PA-sensitive, and
that sphingosine mobilizes Ca
from both stores in an
IP
-independent manner. Furthermore, the IP
- but
not the PA-sensitive intracellular Ca
store seems to
regulate Ca
entry induced by sphingosine.
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
, interleukin 1-
, 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 (
)as well
as IP
-sensitive intracellular stores in Jurkat cells
independently of inositol phosphates.
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
]
, which returned to the
resting level, and the sustained increase in
[Ca
]
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
]
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
]
. The present results
clearly showed that sphingosine mobilized intracellular Ca
in Jurkat T cells. We examined whether sphingosine releases
Ca
from IP
-sensitive intracellular stores
by using the anti-CD3 antibody OKT3, an IP
-generating drug.
In the absence of extracellular Ca
, pretreatment with
sphingosine inhibited but did not abolish
[Ca
]
induced by the subsequent
addition of OKT3; likewise the rise in
[Ca
]
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
-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.
Figure 3:
Effects of 20 µM sphingosine (SPH) and 10 µg/ml OKT3 on IP formation.
Jurkat cells (10
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
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
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
, 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
(data not
shown).
Figure 4:
Ca release from the
nonmitochondrial intracellular Ca
store in
permeabilized Jurkat cells. Jurkat cells (around 10
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
and sphingosine (SPH) were added as indicated by arrows. Each trace
is a representative one from at least three
experiments.
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 g for 10 min, the cytosolic and membrane fractions were obtained by
further centrifugation at 100,000
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
, 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]
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
]
to sphingosine but did not
affect the sustained increase in [Ca
]
induced by sphingosine. These results suggested that sphingosine
might increase [Ca
]
via
formation of PA. This was confirmed by the observation that the
addition of PA (10 µM) elevated
[Ca
]
by itself (Fig. 7C). However, the increase in
[Ca
]
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
-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
-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.
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
- 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
, 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
- 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.
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
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
-insensitive intracellular store. Both
stores have Ca
-ATPase, which is blocked by
thapsigargin. Sphingosine releases Ca
from the
IP
-sensitive intracellular store via an unknown
messenger(s) and activates DG kinase. Depletion of Ca
in the IP
-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
-sensitive intracellular store
activates Ca
entry, which causes a sustained increase
in [Ca
]
. Sphingomyelin turnover
may be an important signaling mechanism transducing the actions of
tumor necrosis factor
and
-interferon with a specific
function in cell differentiation(34) , and Ca
entry causing a sustained increase in
[Ca
]
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
-sensitive Ca
intracellular store
followed by the sustained activation of Ca
entry. On
the other hand, an increase in [Ca
]
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.