Vol. 186, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
July 31, 1992
Pages 790-795
REGULATION
OF l’HOSPHOINOSITIDE BY SPHINGOSINE
HYDROLYSIS IN CULTURED AND PSYCHOSINE
ASTROCYTES
Terry Ritchie, Abraham Rosenberg, and Ernest P. Noble Alcohol Research Center, Department of Psychiatry and Biobehavioral Brain Research Institute University of California, Los Angeles, CA 90024-1759 Received
June
8,
Sciences
1992
SUMMARY: The effects of sphingosine and psychosine on phosphoinositide hydrolysis in primary cultured astrocytes were determined. Exposure to sphingosine produced a dose-dependent stimulation of’ phosphoinositide hydrolysis requiring the presence of external Ca++ for optimal activity. The addition of 10 pM norepinephrine resulted in a stimulation additional to that with sphingosine. The al-antagonist prazosin completely inhibited norepinephrine-induced phosphoinositide hydrolysis but had no effect on that produced by sphingosine. Psychosine (108 PM), when co-incubated with sphingosine, produced complete inhibition of sphingosine-induced phosphoinositide hydrolysis at all doses of sphingosine tested (33-668 FM). Likewise, psychosine totally inhibited norepinephrineinduced phosphoinositide hydrolysis. The protein kinase C inhibitor staurosporine (1 PM) had no effect on sphingosine-induced phosphoinositide hydrolysis. These findings suggest that lysosphingolipids such as sphingosine and psychosine may play an important role in the regulation of phosphoinositide turnover in astrocytes by a mechanism dependent on extracellular Caf+ and independent of the al-adrenergic receptor and protein kinase C. o 1s~ ~~~~~~~~ press, I”~.
The agonist-induced generating
breakdown
diacylglycerol
(l-3).
Inositol
of Ca++ while diacylglycerol potent inhibitors
of PKC activation
(4,5). Sphingosine
the action of activators
have been attributed
It has thus been hypothesized of PKC activity providing
(PI) second messenger system. However, related
lysosphingolipids
of intracellular
stores
Recent investigations
and phorbol may function
mechanism
esters.
Most of the cellular
as an endogenous
of PKC negative
for one branch of the phosphoinositide
the full range of physiological
has not yet been determined.
into to be
of PKC by competitively
to a direct consequence of its inhibition
that sphingosine a regulatory
and
and related lysosphingolipids
blocks the activation
such as diacylglycerol
C functions as a signal 1,4,5+isphosphate
in the mobilization
this system have shown sphingosine
responses elicited by sphingosine modulator
functions
inositol
is a key activator of protein kinase C (PKC).
regulating
(4,5).
by phospholipase
of two second messengers,
1,4,5-trisphosphate
the mechanisms inhibiting
of phosphoinositides
system via the production
activities of sphingosine
Some activities
of sphingosine
and
such as
Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; G protein, guanine nucleotide binding PBS, phosphate buffered saline; PKC, protein kinase C; PI, protein; NE, norepinephrine; phosphoinositide. 0006-291X/92
Copyright All rights
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
790
Vol.
BIOCHEMICAL
186. No. 2, 1992
inhibition
of thyrotropin
releasing
hormone
AND BlOPHYStCAL
binding
to pituitary
receptor tyrosine kinase activity (7) may occur independently
RESEARCH COMMUNlCATlONS
cells (6) and activation
of PKC. Herein, we describe the potent
stimulation
of PI hydrolysis by sphingosine
by a mechanism that occurs independently
is inhibited
by the endogenous
analog psychosine (galactosylsphingosine).
sphingosine
of the EGF
of PKC and that
METHODS Primary cultures of astrocytes were prepared from the brains of I-Z-day-old rat pups essentially as described by McCarthy and de Vellis (B), with the exception that a mechanical rather than an enzymatic dissociation of cells was used. For the experiments, astrocytes were seeded in 24well plates at a density of 1.5 X 105 cells per well and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. These cultures were used for the PI hydrolysis assays within 7-10 days. The stimulated hydrolysis of PI’s was measured by a modification of the procedure of Berridge et al. (9) as previously described (IO). Cells in 24-well plates were incubated overnight with 0.5 ml of DMEM containing 1 @Zi of [3H]myo-inositol per well to prelabel membrane PI’s. The cells were washed twice with 1 ml of Dulbecco’s phosphate buffered saline (PBS) followed by the addition of 0.5 ml of salt dissolved in water, PBS containing 4.5 g/l glucose. Sphingosine, prepared as the hydrochloride was then added to the desired concentration to specific wells and the 24-well plates were shaken on a rotary shaker for 5 min followed by incubation at 23’C for 1 hr. The cells were then washed twice with 1 ml of PBS per wash. In experiments where the effects of psychosine were evaluated, cells were treated likewise with psychosine. The cells were then preincubated for 15 min at 37oC with 0.5 ml of PBS containing 10 mM LiCl and specific substances to be tested. At the end of this period, the preincubation buffer was removed and the reaction was initiated by the addition of 0.5 ml of substances to be tested in PBS containing 4.5 g/l glucose and 10 mM LiCl. After an incubation period of 30 min at 37oC, the reaction was terminated by the addition of 1 ml of ice-cold absolute methanol to each well. The cells were then scraped and the entire contents of each well were transferred to polypropylene tubes containing 0.4 ml water and 1 ml chloroform. The samples were thoroughly vortexed and centrifuged at 500 x g for 5 min to separate the aqueous and chloroform phases. A 1.5 ml aliquot of the upper aqueous phase was applied to a mini-column containing Bio-Rad AGl-X8 resin (formate form) for each sample. Free [3H]inositol and [3H]glycerophosphoinositol were washed through the columns with 5 ml of buffer containing 5 mM sodium borate and 60 mM sodium formate. The [3H]inositol phosphates were then eluted for counting with 3 ml of buffer containing 1.0 M ammonium formate and 0.1 M formic acid. A 0.5 ml aliquot from the chloroform layer from each sample was counted for radioactivity to determine the amount of [‘Hlinositol incorporated into lipids. The radioactivity of all samples was measured by liquid scintillation counting. PI hydrolysis is based on the amount of [3H]inositol phosphates formed and is expressed as the percentage of total [3H]inositol incorporated into lipids (dpm ammonium formate fraction + dpm chloroform fraction) converted into [3H]inositol phosphates. In experiments assessing the effect of the PKC inhibitor staurosporine, cells were preincubated for 15 min at 37’C with 1 FM staurosporine prior to the addition of sphingosine. All subsequent incubations throughout the assay contained staurosporine at this concentration.
RESULTS Exposure to sphingosine stimulation
of PI hydrolysis (Fig. IA).
to psychosine, an inhibition concentration inhibition
of astrocytes prelabeled
with [3H]inositol
When cells exposed to sphingosine
of sphingosine-induced
were simultaneously
exposed
PI hydrolysis occurred that was dependent
of psychosine (Fig. 1B). Psychosine, at a concentration
of sphingosine-induced
produced a dose-dependent
PI hydrolysis at all concentrations
of 108 PM, produced
of sphingosine
on the a potent
tested (33-668 PM)
(Fig. 1A). In previous studies, we have found that PI hydrolysis in primary cultured ashocytes occurred primarily
by stimulation
of the al-adrenergic
that induced by exposure to sphingosine
receptor (10,111. As with NE-stimulated
PI hydrolysis,
requires the presence of external Ca++ for optimal 791
activity, as
Vol.
BIOCHEMICAL
186, No. 2, 1992
AND BIOPHYSICAL
60
RESEARCH COMMUNICATIONS
-
45 m 0
0 pM Psychosine 108 @A Psychosine
A
50
m 0
40
T
84 uM Sphingosine 0 uM Sphingosine
35
40
30 25
30 20 20
15 10
10 5 0
0
33
84
167
Sphingosine
250
334
Concentration
0
666
0
(PM)
22
54
Psychosine
108
162
Concentration
216
(@A)
Fie. Effects of sphingosine and psychosine on PI hydrolysis in cultured astrocytes. (A) Astrocytes prelabeled with 13H]inositol were exposed to various concentrations of sphingosine with or without the addition of 108 pM psychosine. (B) Astrocytes prelabeled with [3Hlinositol were exposed to 84 nM sphingosine with or without the addition of various concentrations of psychosine. For both (A) and (8) the accumulation of 13H1inositol phospates was subsequently determined as described in METHODS. Values are the mean + SEM for a typical experiment. Similar results were obtained in at least three other experiments for both (A) and (B).
evidenced by a reduction
in the accumulation
of [ 3H]inositol
phosphates in the presence of 1 mM EGTA
(0 mM Ca++) versus that obtained with 0.9 mM external Ca++(TabIe Fig. 2A shows the additive combination.
PI hydrolysis
When astrocytes exposed to 84 ~Msphingosine
the accumulation
of [3H1inositol
phosphates
induced by either substance alone. producing
effect of stimulated
a maximal stimulation
1).
was additive
by sphingosine
were further stimulated with
the stimulation
When cells were exposed to 334 pM sphingosine,
a concentration
of PI hydrolysis,
when
compared
and NE in
with 10 pM NE,
10 pM NE failed to produce any further stimulation
(data not shown).
Table 1. Effect of extracellular Ca++ on phosphoinositide hydrolysis in cultured astrocytes 13HlInositol Phosphates (% 13H]PI’s Converted) 1 mM ECTA (0 n-&i Ca++) + 10 pM Norepinephrine 1 mM ECTA (0 mM Ca++) + 167 PM Sphingosine
12.45 25.35 24.23
0.9 mM
18.54 k 0.21
1 mM EGTA (0 mM Ca++)
Ca++
0.9 n&l Ca++ + 10 pM Norepinephrine 0.9 mM Caf++ 167 UM Suhingosine
39.73 37.32
f 0.28 + 2.43 + 1.26
+ 0.56 + 0.62
Ashocytes prelabeled with 13HJinositol were exposed to vehicle or 167 nM sphingosine as described in METHODS. The accumulation of [3H]inositol phos hates was measured after stimulation with the indicated typical
substances
in medium
containing
0 mM Ca +r or 0.9 n-M Ca*.
Values are the mean f SEM for a
experiment. Similar results were obtained in three other experiments. 792
Vol.
BIOCHEMICAL
186, No. 2, 1992 60
AND BIOPHYSICAL
r
RESEARCH COMMUNICATIONS
60
B1
m Control
50
0 [z9
50
10 pM Prazosin 108 pk.4 Psychosine
40
30
20
10
Control
10 jdA NE
84 1Jn sphingodnb
0
10 /AM NE + 84 /LM sphingosins
L
Control
10 /AM NE
334 pM sphingosine
F&& Comparison of NE- and sphingosine-stimulated PI hydrolysis in cultured astrocytes. (A) Additive effect of NE and sphingosine on PI hydrolysis. (B) Effects of prazosin and psychosine on NEand sphingosine-stimulated PI hydrolysis. Astrocytes prelabeled with 13Hlinositol were exposed to the indicated amounts of sphingosine as described in METHODS. The accumulation of [3H]inositol phosphates was measured after stimulation with the indicated substances. Values are the mean * SEM for a typical experiment. For both (A) and (B) similar results were obtained with at least one other exoeriment.
To determine receptor,
the effect
prazosin
was
accumulation sphingosine
whether
sphingosine
of the al-adrenergic
found
to completely
of ]3H]inositoI is exerting
inhibit
phosphates
its effects
inhibition
whether
induced
PI hydrolysis
inhibition
stimulated
PI hydrolysis
determined
(Table 2). Staurosporine,
than
to inhibiting
of NE-stimulated
To investigate
be inducing inhibitor
observed,
PI hydrolysis
prazosin
NE-stimulated
at a site other
effect of psychosine which, in addition complete
may receptor
PI hydrolysis,
the adrenergic
receptor.
sphingosine-induced
of PKC by sphingosine
may
the effect of the potent
on Phosphoinositide
15 min Similar
prelabeted prior results
with
13H]inositol
were incubated
of sphingosine. Values in a second experiment.
793
with vehicle are
While
no effect
on the
suggesting
This
contrasts
that
with
the
the
indirectly
the
mean
be responsible
in Cultured
(control) f SEM
for the
staurosporine
with sphingosine,
14.01 15.29 52.21 58.92
+ 1 pM Staurosporine
to the addition were obtained
2B).
PI hydrolysis, produced
PKC inhibitor
Hydrolysis
PBS ControI
Astrocytes
al-adrenergic
2B).
13HlInositol f% 13HlPl’s
1 FM Staurosporine 334 uM Sphingosine 334 uM Sphingosine
it had
to sphingosine
(Fig.
the
(Fig.
by exposure
both alone and in combination
Table 2. Effect of Staurosporine
via
was determined
was
was found to
Astrocytes
Phosphates Converted) f f +_ k
0.41 0.87 0.59 1.29
or 1 uM staurosporine for
a single
experiment.
for
Vol.
BIOCHEMICAL
186, No. 2, 1992
have no effect on PI hydrolysis, any bearing on the stimulation
indicating
AND RIOPHYSICAL RESEARCH COMMUNICATIONS
that in cultured
astrocytes the activity of PKC has little if
of PI hydrolysis by sphingosine.
DISCUSSION While
most investigations
lysosphingolipids inhibition
delving
have generally
shown
into the biological
astrocytes by sphingosine
Recent evidence indicating
the stimulation
via a mechanism
of PKC by sphingosine
hydrolysis observed.
the inability
been reported
However,
in this study would
with rat parotid
Psychosine hydrolysis.
of a major second messenger system in
that is not dependent
on the activity of PKC.
proved
to inhibit
of the PKC inhibitor
acinar cells where sphingosine
in astrocytes
staurosporine
stimulated
psychosine’s relationship
Similar
results have
the accumulation
of inositol
of PKC activity (17).
to be a potent
acted similarly
to sphingosine
PI
to produce any effect
antagonist
to sphingosine-stimulated
This contrasts with findings from a previous study where both sphingosine
as well as other lysosphingolipids,
PI hydrolysis
as a plausible mechanism for the stimulated
indicate that PKC is likely not involved.
phosphates by a mechanism also independent
and other
a result of sphingosine’s
that PKC may function in a feedback mechanism
(12-16) would suggest inhibition on PI hydrolysis
of sphingosine
these responses to be primarily
of PKC, this study has demonstrated
cultured
activities
PI
and psychosine,
in producing
a strong inhibition
PI hydrolysis
is quite different from that with PKC
regarding
of PKC (18). Thus
activity. The precise mechanism by which sphingosine unclear.
The failure of the al-adrenergic
PI hydrolysis
and psychosine affect PI hydrolysis
receptor antagonist prazosin to inhibit
suggests that sphingosine’s
site of action is different
liver have found that a major portion of the free sphingosine (19) where it could interact with various membrane alter the physical properties the observation combination
with
NE-stimulated
from that of NE. Studies with rat
is associated with the plasma membrane
components.
While sphingolipids
did not enhance NE-stimulated
NE suggests
PI hydrolysis
otherwise.
The potent
that omission
It is possible
and
for extracellular
in light of evidence
can attenuate and even in some cases
with appropriate
of sphingosine-stimulated
G proteins.
activity
and psychosine
was competitive
binding
of PI hydrolysis
exert their actions on PI hydrolysis whether
in nature, analog
this enzyme may account 794
nucleotide
stimulation
It is also possible that sphingosine
by such a close structural with
of specific guanine
C. While it was not determined
PI hydrolysis
interaction
the involvement
receptor activation.
mechanism for receptor-mediated
that sphingosine
interact directly with phospholipase sphingosine-induced
by the requirement
This is not surprising
medium
at a site distal to membrane
Recent evidence has demonstrated
agonist-antagonist
of both sphingosine-
inositol lipid turnover in the brain (20-22). Thus, it seems likely that sphingosine
proteins (G proteins) in the transduction interaction
PI hydrolysis.
of Ca ++ from the incubation
is acting to stimulate PI hydrolysis
(23-26).
of receptor activity,
by psychosine indicates that activity induced by both substances occurs
Ca++ by both NE- and sphingosine-stimulated abolish stimulated
may serve to
activity but instead was additive in
inhibition
through a common mechanism of action. This is further supported showing
sphingosine-induced
of regions of membrane and thus serve as modulators
that sphingosine
is at present
and psychosine
via may
psychosine antagonism
the strong inhibition
suggests the possibility for the observed
results.
of the that an Such a
Vol.
186,
possibility with
No.
BIOCHEMICAL
2, 1992
is strengthened
the failure
by the inhibition
of the al-adrenergic
AND
BIOPHYSICAL
of NE-stimulated receptor
antagonist
RESEARCH
PI hydrolysis prazosin
COMMUNICATIONS
by psychosine
coupled
to affect stimulation
of PI
hydrolysis by sphingosine. The finding affect PI hydrolysis activities,
in particular
physiologically. higher
that endogenous
baseline levels of inositol modulating
showing
suggests
phosphate
the
that
that
turnover.
and diacylglycerol its stimulation
function
by tonically
may function regulating
the
second messengers as well as maintaining
by diacylglycerol.
They may subsequently
activity of these systems in response to stimulation
The full physiological
has other cellular
these substances
substances and could very well act as second messengers themselves sphingolipid
and psychosine severely
sphingosine
and psychosine used in this study are likely much
levels, these substances still may
resting activity of PKC by regulating in
such as sphingosine
that of PKC inhibition,
While the amounts of sphingosine
than physiological
important
lysosphingolipids
coupled with previous investigations
significance
psychosine should become much clearer when their regulation
as a result of alterations
of the activities
be
by other bioactive of sphingosine
in and
at the cellular level is better known.
ACKNOWLEDGMENTS We wish to thank Ruth Cole for the technical assistance in the preparation of the astrocyte cultures used in this investigation. This work was supported in part by the Pike Professorship for Alcohol Studies (EPN) and USPHS grant AA 07653.
REFERENCES 1. Berridge, M.J., and Irvine, R.E (1984) Nature 312,315321. 2. Majerus, P W., Connolly, T.M., Deckmyn, H., Ross, T.S., Boss, T.E., Ishii, H., Bansal, VS., and Wilson, O.B. (1986) Science 234, 1519-1526. 3. Nishizuka, LJ. (1984) Science 225, 1365-1370. 4. Hannum, Y.A., and Bell, R.M. (1989) Science 243,500-507. 5. Merrill, A.H. (1991) J. Bioenerg. Biomembr. 23,83-104. 6. Winicov, I., and Gershengom, M. (1988) J. Biol. Chem. 263,12179-12182. 7. Davis, R., Girones, N., and Faucher, M. (1988) J. Biol. Chem. 263,5373-5379. 8. McCarthy, K.D., and de Vellis, J. (1980) J. Cell. Biol. 85, 890-902. 9. Berridge, M.J., Downes, C.P., and Hanley, M.R. (1982) Biochem. J. 206,587-595. 10. Ritchie, T., Kim, H.-S., Cole, R., de Vellis, J., and Noble, E.P. (1988) Alcohol 5, 183-187. 11. Ritchie, T., Cole, R., Kim, H.-S., de Vellis, J., and Noble, E.P. (1987) Life Sci. 41, 31-39. 12. Orellana, S.A., Solski, EA., and Brown, J.H. (1985) J. Biol. Chem. 260,5236-5239. 13. Orellana, S., Solski, P.A., and Brown, J.H. (1987) J. Biol. Chem. 262,1638-1643. 14. Pearce, B., Morrow, C., and Murphy, S. (1988) J. Neurochem. 50,936-944. 15. Ryu, S.H., Kim, U.-H., Wahl, M.I., Brown, A.B., Carpenter, G., Huang, K.-P., and Rhee, S.G. (1990) J. Biol. Chem. 265,17941-17945. 16. Vincentini, L.M., Di Virgilio, F., Ambrosini, A., Pozzan, T, and Meldolesi, J. (1985) Biochem. Biophys. Res. Con-m. 127,310-317. 17. Sugiya, H., and Furuyama, S. (1990) Cell Calcium 11,469475. 18. Hannum, Y.A., and Bell, R.M. (1987) Science 235, 670-674. 19. Slife, C.W., Wang, E., Hunter, R., Wang, S., Burgess, C., Liotta, D.C., and Merrill, A.H. (1989) J. Biol. Chem. 264, 10371-10377. 20. Pearce, B., Cambray-Deakin, M., Morrow, C., Grimble, J., and Murphy, S., (1985) J. Neurochem. 45, 15X-1540. 21. Griffin, H.D., Hawthorne, J.N., and Sykes, M. (1979) Biochem. Pharmacol. 28, 1143-1147. 22. Gonzales, R.A., and Crews, ET. (1984) J. Neurosci. 4,3120-3127. 23. Litosch, I., Wallis, C., and Fain, J.N. (1985) J. Biol. Chem. 260,5464-5471. 24. Uhing, R.J., Prpic, V., Jiang, H., and Exton, J.H. (1986) J. I)iol. Chem. 261, 2140-2146. 25. Straub, R.E., and Gershengorn, M.C. (1986) J. Biol. Chem. 261,2712-2717. 26. Hepler, J.R., and Harden, T.K. (1986) Biochem. J. 239,141-146. 795
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
July 31, 1992
Pages 790-795
REGULATION
OF l’HOSPHOINOSITIDE BY SPHINGOSINE
HYDROLYSIS IN CULTURED AND PSYCHOSINE
ASTROCYTES
Terry Ritchie, Abraham Rosenberg, and Ernest P. Noble Alcohol Research Center, Department of Psychiatry and Biobehavioral Brain Research Institute University of California, Los Angeles, CA 90024-1759 Received
June
8,
Sciences
1992
SUMMARY: The effects of sphingosine and psychosine on phosphoinositide hydrolysis in primary cultured astrocytes were determined. Exposure to sphingosine produced a dose-dependent stimulation of’ phosphoinositide hydrolysis requiring the presence of external Ca++ for optimal activity. The addition of 10 pM norepinephrine resulted in a stimulation additional to that with sphingosine. The al-antagonist prazosin completely inhibited norepinephrine-induced phosphoinositide hydrolysis but had no effect on that produced by sphingosine. Psychosine (108 PM), when co-incubated with sphingosine, produced complete inhibition of sphingosine-induced phosphoinositide hydrolysis at all doses of sphingosine tested (33-668 FM). Likewise, psychosine totally inhibited norepinephrineinduced phosphoinositide hydrolysis. The protein kinase C inhibitor staurosporine (1 PM) had no effect on sphingosine-induced phosphoinositide hydrolysis. These findings suggest that lysosphingolipids such as sphingosine and psychosine may play an important role in the regulation of phosphoinositide turnover in astrocytes by a mechanism dependent on extracellular Caf+ and independent of the al-adrenergic receptor and protein kinase C. o 1s~ ~~~~~~~~ press, I”~.
The agonist-induced generating
breakdown
diacylglycerol
(l-3).
Inositol
of Ca++ while diacylglycerol potent inhibitors
of PKC activation
(4,5). Sphingosine
the action of activators
have been attributed
It has thus been hypothesized of PKC activity providing
(PI) second messenger system. However, related
lysosphingolipids
of intracellular
stores
Recent investigations
and phorbol may function
mechanism
esters.
Most of the cellular
as an endogenous
of PKC negative
for one branch of the phosphoinositide
the full range of physiological
has not yet been determined.
into to be
of PKC by competitively
to a direct consequence of its inhibition
that sphingosine a regulatory
and
and related lysosphingolipids
blocks the activation
such as diacylglycerol
C functions as a signal 1,4,5+isphosphate
in the mobilization
this system have shown sphingosine
responses elicited by sphingosine modulator
functions
inositol
is a key activator of protein kinase C (PKC).
regulating
(4,5).
by phospholipase
of two second messengers,
1,4,5-trisphosphate
the mechanisms inhibiting
of phosphoinositides
system via the production
activities of sphingosine
Some activities
of sphingosine
and
such as
Abbreviations used: DMEM, Dulbecco’s modified Eagle’s medium; G protein, guanine nucleotide binding PBS, phosphate buffered saline; PKC, protein kinase C; PI, protein; NE, norepinephrine; phosphoinositide. 0006-291X/92
Copyright All rights
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
790
Vol.
BIOCHEMICAL
186. No. 2, 1992
inhibition
of thyrotropin
releasing
hormone
AND BlOPHYStCAL
binding
to pituitary
receptor tyrosine kinase activity (7) may occur independently
RESEARCH COMMUNlCATlONS
cells (6) and activation
of PKC. Herein, we describe the potent
stimulation
of PI hydrolysis by sphingosine
by a mechanism that occurs independently
is inhibited
by the endogenous
analog psychosine (galactosylsphingosine).
sphingosine
of the EGF
of PKC and that
METHODS Primary cultures of astrocytes were prepared from the brains of I-Z-day-old rat pups essentially as described by McCarthy and de Vellis (B), with the exception that a mechanical rather than an enzymatic dissociation of cells was used. For the experiments, astrocytes were seeded in 24well plates at a density of 1.5 X 105 cells per well and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. These cultures were used for the PI hydrolysis assays within 7-10 days. The stimulated hydrolysis of PI’s was measured by a modification of the procedure of Berridge et al. (9) as previously described (IO). Cells in 24-well plates were incubated overnight with 0.5 ml of DMEM containing 1 @Zi of [3H]myo-inositol per well to prelabel membrane PI’s. The cells were washed twice with 1 ml of Dulbecco’s phosphate buffered saline (PBS) followed by the addition of 0.5 ml of salt dissolved in water, PBS containing 4.5 g/l glucose. Sphingosine, prepared as the hydrochloride was then added to the desired concentration to specific wells and the 24-well plates were shaken on a rotary shaker for 5 min followed by incubation at 23’C for 1 hr. The cells were then washed twice with 1 ml of PBS per wash. In experiments where the effects of psychosine were evaluated, cells were treated likewise with psychosine. The cells were then preincubated for 15 min at 37oC with 0.5 ml of PBS containing 10 mM LiCl and specific substances to be tested. At the end of this period, the preincubation buffer was removed and the reaction was initiated by the addition of 0.5 ml of substances to be tested in PBS containing 4.5 g/l glucose and 10 mM LiCl. After an incubation period of 30 min at 37oC, the reaction was terminated by the addition of 1 ml of ice-cold absolute methanol to each well. The cells were then scraped and the entire contents of each well were transferred to polypropylene tubes containing 0.4 ml water and 1 ml chloroform. The samples were thoroughly vortexed and centrifuged at 500 x g for 5 min to separate the aqueous and chloroform phases. A 1.5 ml aliquot of the upper aqueous phase was applied to a mini-column containing Bio-Rad AGl-X8 resin (formate form) for each sample. Free [3H]inositol and [3H]glycerophosphoinositol were washed through the columns with 5 ml of buffer containing 5 mM sodium borate and 60 mM sodium formate. The [3H]inositol phosphates were then eluted for counting with 3 ml of buffer containing 1.0 M ammonium formate and 0.1 M formic acid. A 0.5 ml aliquot from the chloroform layer from each sample was counted for radioactivity to determine the amount of [‘Hlinositol incorporated into lipids. The radioactivity of all samples was measured by liquid scintillation counting. PI hydrolysis is based on the amount of [3H]inositol phosphates formed and is expressed as the percentage of total [3H]inositol incorporated into lipids (dpm ammonium formate fraction + dpm chloroform fraction) converted into [3H]inositol phosphates. In experiments assessing the effect of the PKC inhibitor staurosporine, cells were preincubated for 15 min at 37’C with 1 FM staurosporine prior to the addition of sphingosine. All subsequent incubations throughout the assay contained staurosporine at this concentration.
RESULTS Exposure to sphingosine stimulation
of PI hydrolysis (Fig. IA).
to psychosine, an inhibition concentration inhibition
of astrocytes prelabeled
with [3H]inositol
When cells exposed to sphingosine
of sphingosine-induced
were simultaneously
exposed
PI hydrolysis occurred that was dependent
of psychosine (Fig. 1B). Psychosine, at a concentration
of sphingosine-induced
produced a dose-dependent
PI hydrolysis at all concentrations
of 108 PM, produced
of sphingosine
on the a potent
tested (33-668 PM)
(Fig. 1A). In previous studies, we have found that PI hydrolysis in primary cultured ashocytes occurred primarily
by stimulation
of the al-adrenergic
that induced by exposure to sphingosine
receptor (10,111. As with NE-stimulated
PI hydrolysis,
requires the presence of external Ca++ for optimal 791
activity, as
Vol.
BIOCHEMICAL
186, No. 2, 1992
AND BIOPHYSICAL
60
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-
45 m 0
0 pM Psychosine 108 @A Psychosine
A
50
m 0
40
T
84 uM Sphingosine 0 uM Sphingosine
35
40
30 25
30 20 20
15 10
10 5 0
0
33
84
167
Sphingosine
250
334
Concentration
0
666
0
(PM)
22
54
Psychosine
108
162
Concentration
216
(@A)
Fie. Effects of sphingosine and psychosine on PI hydrolysis in cultured astrocytes. (A) Astrocytes prelabeled with 13H]inositol were exposed to various concentrations of sphingosine with or without the addition of 108 pM psychosine. (B) Astrocytes prelabeled with [3Hlinositol were exposed to 84 nM sphingosine with or without the addition of various concentrations of psychosine. For both (A) and (8) the accumulation of 13H1inositol phospates was subsequently determined as described in METHODS. Values are the mean + SEM for a typical experiment. Similar results were obtained in at least three other experiments for both (A) and (B).
evidenced by a reduction
in the accumulation
of [ 3H]inositol
phosphates in the presence of 1 mM EGTA
(0 mM Ca++) versus that obtained with 0.9 mM external Ca++(TabIe Fig. 2A shows the additive combination.
PI hydrolysis
When astrocytes exposed to 84 ~Msphingosine
the accumulation
of [3H1inositol
phosphates
induced by either substance alone. producing
effect of stimulated
a maximal stimulation
1).
was additive
by sphingosine
were further stimulated with
the stimulation
When cells were exposed to 334 pM sphingosine,
a concentration
of PI hydrolysis,
when
compared
and NE in
with 10 pM NE,
10 pM NE failed to produce any further stimulation
(data not shown).
Table 1. Effect of extracellular Ca++ on phosphoinositide hydrolysis in cultured astrocytes 13HlInositol Phosphates (% 13H]PI’s Converted) 1 mM ECTA (0 n-&i Ca++) + 10 pM Norepinephrine 1 mM ECTA (0 mM Ca++) + 167 PM Sphingosine
12.45 25.35 24.23
0.9 mM
18.54 k 0.21
1 mM EGTA (0 mM Ca++)
Ca++
0.9 n&l Ca++ + 10 pM Norepinephrine 0.9 mM Caf++ 167 UM Suhingosine
39.73 37.32
f 0.28 + 2.43 + 1.26
+ 0.56 + 0.62
Ashocytes prelabeled with 13HJinositol were exposed to vehicle or 167 nM sphingosine as described in METHODS. The accumulation of [3H]inositol phos hates was measured after stimulation with the indicated typical
substances
in medium
containing
0 mM Ca +r or 0.9 n-M Ca*.
Values are the mean f SEM for a
experiment. Similar results were obtained in three other experiments. 792
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186, No. 2, 1992 60
AND BIOPHYSICAL
r
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60
B1
m Control
50
0 [z9
50
10 pM Prazosin 108 pk.4 Psychosine
40
30
20
10
Control
10 jdA NE
84 1Jn sphingodnb
0
10 /AM NE + 84 /LM sphingosins
L
Control
10 /AM NE
334 pM sphingosine
F&& Comparison of NE- and sphingosine-stimulated PI hydrolysis in cultured astrocytes. (A) Additive effect of NE and sphingosine on PI hydrolysis. (B) Effects of prazosin and psychosine on NEand sphingosine-stimulated PI hydrolysis. Astrocytes prelabeled with 13Hlinositol were exposed to the indicated amounts of sphingosine as described in METHODS. The accumulation of [3H]inositol phosphates was measured after stimulation with the indicated substances. Values are the mean * SEM for a typical experiment. For both (A) and (B) similar results were obtained with at least one other exoeriment.
To determine receptor,
the effect
prazosin
was
accumulation sphingosine
whether
sphingosine
of the al-adrenergic
found
to completely
of ]3H]inositoI is exerting
inhibit
phosphates
its effects
inhibition
whether
induced
PI hydrolysis
inhibition
stimulated
PI hydrolysis
determined
(Table 2). Staurosporine,
than
to inhibiting
of NE-stimulated
To investigate
be inducing inhibitor
observed,
PI hydrolysis
prazosin
NE-stimulated
at a site other
effect of psychosine which, in addition complete
may receptor
PI hydrolysis,
the adrenergic
receptor.
sphingosine-induced
of PKC by sphingosine
may
the effect of the potent
on Phosphoinositide
15 min Similar
prelabeted prior results
with
13H]inositol
were incubated
of sphingosine. Values in a second experiment.
793
with vehicle are
While
no effect
on the
suggesting
This
contrasts
that
with
the
the
indirectly
the
mean
be responsible
in Cultured
(control) f SEM
for the
staurosporine
with sphingosine,
14.01 15.29 52.21 58.92
+ 1 pM Staurosporine
to the addition were obtained
2B).
PI hydrolysis, produced
PKC inhibitor
Hydrolysis
PBS ControI
Astrocytes
al-adrenergic
2B).
13HlInositol f% 13HlPl’s
1 FM Staurosporine 334 uM Sphingosine 334 uM Sphingosine
it had
to sphingosine
(Fig.
the
(Fig.
by exposure
both alone and in combination
Table 2. Effect of Staurosporine
via
was determined
was
was found to
Astrocytes
Phosphates Converted) f f +_ k
0.41 0.87 0.59 1.29
or 1 uM staurosporine for
a single
experiment.
for
Vol.
BIOCHEMICAL
186, No. 2, 1992
have no effect on PI hydrolysis, any bearing on the stimulation
indicating
AND RIOPHYSICAL RESEARCH COMMUNICATIONS
that in cultured
astrocytes the activity of PKC has little if
of PI hydrolysis by sphingosine.
DISCUSSION While
most investigations
lysosphingolipids inhibition
delving
have generally
shown
into the biological
astrocytes by sphingosine
Recent evidence indicating
the stimulation
via a mechanism
of PKC by sphingosine
hydrolysis observed.
the inability
been reported
However,
in this study would
with rat parotid
Psychosine hydrolysis.
of a major second messenger system in
that is not dependent
on the activity of PKC.
proved
to inhibit
of the PKC inhibitor
acinar cells where sphingosine
in astrocytes
staurosporine
stimulated
psychosine’s relationship
Similar
results have
the accumulation
of inositol
of PKC activity (17).
to be a potent
acted similarly
to sphingosine
PI
to produce any effect
antagonist
to sphingosine-stimulated
This contrasts with findings from a previous study where both sphingosine
as well as other lysosphingolipids,
PI hydrolysis
as a plausible mechanism for the stimulated
indicate that PKC is likely not involved.
phosphates by a mechanism also independent
and other
a result of sphingosine’s
that PKC may function in a feedback mechanism
(12-16) would suggest inhibition on PI hydrolysis
of sphingosine
these responses to be primarily
of PKC, this study has demonstrated
cultured
activities
PI
and psychosine,
in producing
a strong inhibition
PI hydrolysis
is quite different from that with PKC
regarding
of PKC (18). Thus
activity. The precise mechanism by which sphingosine unclear.
The failure of the al-adrenergic
PI hydrolysis
and psychosine affect PI hydrolysis
receptor antagonist prazosin to inhibit
suggests that sphingosine’s
site of action is different
liver have found that a major portion of the free sphingosine (19) where it could interact with various membrane alter the physical properties the observation combination
with
NE-stimulated
from that of NE. Studies with rat
is associated with the plasma membrane
components.
While sphingolipids
did not enhance NE-stimulated
NE suggests
PI hydrolysis
otherwise.
The potent
that omission
It is possible
and
for extracellular
in light of evidence
can attenuate and even in some cases
with appropriate
of sphingosine-stimulated
G proteins.
activity
and psychosine
was competitive
binding
of PI hydrolysis
exert their actions on PI hydrolysis whether
in nature, analog
this enzyme may account 794
nucleotide
stimulation
It is also possible that sphingosine
by such a close structural with
of specific guanine
C. While it was not determined
PI hydrolysis
interaction
the involvement
receptor activation.
mechanism for receptor-mediated
that sphingosine
interact directly with phospholipase sphingosine-induced
by the requirement
This is not surprising
medium
at a site distal to membrane
Recent evidence has demonstrated
agonist-antagonist
of both sphingosine-
inositol lipid turnover in the brain (20-22). Thus, it seems likely that sphingosine
proteins (G proteins) in the transduction interaction
PI hydrolysis.
of Ca ++ from the incubation
is acting to stimulate PI hydrolysis
(23-26).
of receptor activity,
by psychosine indicates that activity induced by both substances occurs
Ca++ by both NE- and sphingosine-stimulated abolish stimulated
may serve to
activity but instead was additive in
inhibition
through a common mechanism of action. This is further supported showing
sphingosine-induced
of regions of membrane and thus serve as modulators
that sphingosine
is at present
and psychosine
via may
psychosine antagonism
the strong inhibition
suggests the possibility for the observed
results.
of the that an Such a
Vol.
186,
possibility with
No.
BIOCHEMICAL
2, 1992
is strengthened
the failure
by the inhibition
of the al-adrenergic
AND
BIOPHYSICAL
of NE-stimulated receptor
antagonist
RESEARCH
PI hydrolysis prazosin
COMMUNICATIONS
by psychosine
coupled
to affect stimulation
of PI
hydrolysis by sphingosine. The finding affect PI hydrolysis activities,
in particular
physiologically. higher
that endogenous
baseline levels of inositol modulating
showing
suggests
phosphate
the
that
that
turnover.
and diacylglycerol its stimulation
function
by tonically
may function regulating
the
second messengers as well as maintaining
by diacylglycerol.
They may subsequently
activity of these systems in response to stimulation
The full physiological
has other cellular
these substances
substances and could very well act as second messengers themselves sphingolipid
and psychosine severely
sphingosine
and psychosine used in this study are likely much
levels, these substances still may
resting activity of PKC by regulating in
such as sphingosine
that of PKC inhibition,
While the amounts of sphingosine
than physiological
important
lysosphingolipids
coupled with previous investigations
significance
psychosine should become much clearer when their regulation
as a result of alterations
of the activities
be
by other bioactive of sphingosine
in and
at the cellular level is better known.
ACKNOWLEDGMENTS We wish to thank Ruth Cole for the technical assistance in the preparation of the astrocyte cultures used in this investigation. This work was supported in part by the Pike Professorship for Alcohol Studies (EPN) and USPHS grant AA 07653.
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