Vol. 168, No. 2, 1990 April 30, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 512-519
BIOCHEMICAL
COHPARATIVRSTIJDIESOF PEOSPEOINOSITIDl3 RYDROLYSISINDDCRDBY RNDOTRRLIN-RRLATRD PEPTIDBSIN CULTDRRD CRRRRELLAR ASTROCYTES, C6-GLIOl4AAND CRRRRBLLAR GRANDLR CELLS Wan-Wan Lin”‘,
Chen Yuan Lee’
and De-Maw
Chuangr*
‘Biological Psychiatry Branch, National Institute of Mental Health Bldg. 10, Room 3N212 Bethesda, MD 20892 2Department of Pharmacology, National Taiwan University, Received
February
27,
College of Medicine, Taipei, Taiwan, R.O.C.
1990
Summary : Effects of endothelin (ET) homologues (ET-1,2,3 and sarafotoxin S ) and its precursor (big ET-l) on phosphoinositide (PI)6turnover were compared in neurally-related cell cultures. All ET-related peptides induced a robust increase of PI turnover in cerebellar astrocytes, C6-glioma and cerebellar granule cells. The rank order of potency in stimulating PI turnover was ET-&ET-2),S >ET-3>big ET-l for granule cell neurons, while it was ET-1#-2aS >big ET-l>ET-3 for astrocytes and Cd-glioma cells. Short-te% pretreatment with phorbol dibutyrate (PDBu) attenuated the ET-l-induced PI response in all three types of cultures. However, long-term pretreatment with PDBu attenuated the response in granule cells and Cc-gliomas, but enhanced responses to ET and ATP in astrocytes. Long-term exposure of cells to pertussis toxin (PTX) attenuated the PI response to ET in astrocytes and C -gliomas, but not in granule cells. Thus, phospholipase C-couplid ET receptors are expressed in both neurons and glial cells, but they differ considerably in their pharmacological selectivity and signal transduction mechanisms in stimulating PI hydrolysis. Q 1990 Academic Press, Inc.
Endothelin-1 peptides the
(ET-l) (1).
of
three
ET-l,
2
and
peptide the
one
of
studies
the
distinct 3.
venom
smooth
members
In addition, ,
Atractaspis
engadilnsis of ET are
correspondence
this
peptide
should
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
potent
512
of
vasoactive
DNA have
the ET
family
a structurally
S
that the actions muscle cells since
*To whom all
of
most
of a human genomic
of ET, named sarafotoxin
indicated
Copyright All rights
Cloning
existence
termed
0006-291X/90
is
has been (3). not
limited
displays
be addressed.
shown (2),
related
identified
from
Recent
studies
to
vascular
several
other
Vol.
BIOCHEMICAL
168, No. 2, 1990 biological
activities
believed its
in
to exert
specific
of
second
is
the
its
messenger
to generate
the
term
system
primary
coupled this
receptors
are
have
compared
Seb)
and its
astrocytes, receptor in these
also
three
refer
cerebellar
of ET homologues
types
ET-l)
on
and cerebellar
that
of cultured
express
ET
(ET-1,2,3
PLC-coupled
ET
glial
We
in cerebellar
cells.
PLC-catalyzed examined
for
cells.
and sarafotoxin
PI turnover granule
stimulate also
and shown
(10).
demonstrated in neurally-related
were
to
4-phosphate, cells
(big
By
collectively
granule
we
C
and diacylglycerol.
of rat
which
systems,
ET action
phospholipase
previously
precursor
agonists,
the
by
is
One type
We have
present
effects
cells.
mediating (PI)
we
to PI hydrolysis
ET with
4,5-bisphosphate.
study,
C6-glioma
various
in
target
phosphatidylinositol cultures
In
of
phosphates
phosphoinoside,
phosphatidylinositol,
receptors
involved
(4-9).
by interacting
phosphoinositide
inositol
phosphatidylinositol that
systems
activities
on the surface
of
(PLC)
non-vascular
biological
receptors
hydrolysis
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A variety breakdown
their
effects
of of PI in
cells.
MATERIALSANJI METHODS Materials ET-l, 2, 3, big ET-1 and sarafotoxin S were purchased from Peptide Institute Inc. (Osaka, Japan). A23187 was obtained from Calbiochem (San Diego, CA). All other chemicals were products of Sigma Chemical Co. (St. Louis, MO).
Cell culture Cerebellar granule cells and astrocytes were prepared from 8-day old rats as previously described (11,12). For the preparation of granule cells, dissociated cells were plated at a density of 3x lo6 cells per 35-mm dish precoated with poly-L-lysine and cultured in Basal Eagle’s Medium containing 10% fetal calf serum, 2 mM glutamine, 50 ug/ml gentamicin and 25 mM KCl. After 18-24h in culture, 10 UN cytosine arabinoside was added to prevent the replication and growth of glial cells. Granule cells were used after 8 days in culture. The purity of granule cells has been shown to be greater than 90% (11). Cultures of astrocytes were prepared as described for granule cells except that KC1 was omitted in culture medium and added to the culture that cytosine arabinoside was not (depolarization by KC1 is required for the survival of granule cell neurons). Culture media were changed twice a week and cells in culture. At this were used for experiments after lo-12 days time, they had reached confluency and comprised mainly (about 90%) flat polygonal-shaped glial cells (12). Rat C6-glioma cells (American Type Culture Collection, Rockville, MD) were grown in Dulbecco’s Modified Eagle’s medium containing 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 ug/ml). Cells with passage numbers between 513
BIOCHEMICAL
Vol. 168, No. 2, 1990
AND BIOPHYSICAL
13-35 were subcultured into 35-mm dishes measured when cells reached confluency.
RESEARCH COMMUNICATIONS
and
PI turnover
was
Heasurement of PI turnover PI hydrolysis was measured as the accumulation of 3H-inositol monophosphates (IP) in the presence of 20 mM LiCl in cells prelabeled overnight with 3H-myo-inositol as described previously (10). The reaction mixtures in physiological saline solution were incubated at 37’C for 45 min after stimulation of cells with receptor agonists. The reaction was terminated by addition of ice-cold methanol and the accumulation of 3H-IP was determined by chromatography on a AG column. 1x8
RESULTS ET-l
S6,)
and its
induced
homologues
a dose-dependent granule
cerebellar (Fig.1).
In
exhibited
similar
distinct
potencies,
increase
In an
5-
hydrolysis were
observed
some
receptor
activator
5-
order
shows
the
in
these
three all
cell
three
no
of types.
but not
the
effects
of
NH,
were
observed
in
Table were
2 show
that
elicited
nonadditive,
on PI
order
responses
Carbachol
effect
in
it
elicited
Cs-glioma
activation
of
cells.
astrocytes
glutamate,
astrocytes,
but
PI responses
to
ET-induced
of
a robust effect
Similarly,
II
in the in
in astrocytes differences For example,
and neurotensin
not gliomas. effect
to ET
vasoactive intestinal more pronounced
Some notable
ATP or angiotensin 514
little
and gliomas,
ET-1
PI
effects
was seen
angiotensin
on
was
cells.
by ATP
in granule
by NE, carbachol,
rank
differential
whereas
while
increase)
noted.
cells,
between
peptides
Although
cells.
detected
These
agonists
granule also
to la-fold)
of ET-1(1.7nM)aET-
cultures,
were
Marked
and its
various
of histamine, 5-HT, glutamate, (VIP) and arg-vasopressin were gliomas
14-
a
effects peptide and
order
ET-1(37nM)>ET-3(56nM). effects
agonists
were
but
rank
ET-l
to 8-fold
with
cells
1
and
Their
(about
a rank
(about
C6 -glioma
in granule
astrocytes
peptides
increase),
cultures,
greater
with
effects
in
cells
five
to 6-fold
astrocyte
ET-1(3.3nM)JET-2(5.1nM)>SGb(6.3nM)>big Table
in
C6-glioma
ET-1(26nM)>ET-3(46nM).
marked in
accumulation
these
PI breakdown.
even
of IP accumulation
turnover
and
cells,
and sarafotoxin
ET-1(1.3nH)=ET-2(1.4nM)~S~~(l.8nM)>ET-3
elicited
induced
astrocytes (about
(66nM).
2(2.6nM)aSbb(3.9nM)>big also
ET-l
of 3H-IP
in inducing
were
3, big
increase
granule
efficacies
ET-l
homologues
cells,
cultured
and ECsO values (BnN)>big
(ET-2,
The results
and Ssb
in
in astrocytes additive to that
was II.
In C6-gliomas,
Vol.
BIOCHEMICAL
168, No. 2, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2000 b MOO t ~1600-
p 73 t s0o
,g
g 1400 -
U
%4002a 300B T 3 200-
f
1200-
!
1000 800
-
a 2 ?
600-
-i ,I
aoo200 100 1
100 -
I 9 8 Concentrcltiin (-kg
-
700-
: v 'b
600 -
1
7
6
M)
%b
I:a.
100
Fig. 1. Dose-responce curves for PI turnover induced by ET-related peptides in cerebellar granule cells (A), cerebellar astrocytes (B) and %gliomas (C). The data were expressed as mean i S.E.M. from at least three independent experiments; each performed in triplicate.
the
responses
responses
to ET-l
Table a
phorbol
ester,
other
hr)
exposure no
the
the
effects In
responses
on
contrast,
treatment
granule
with
responses same
PDBu attenuated cells
515
(13).
with
the PI response
C6-gliomas.
of ET and ATP the
shown).
to
In
to ET
Long-term
(24
in astrocytes angiotensin II,
treatment
to ET and ATP in C6-gliomas.
treatments
cerebellar
effects
not
(10 min)
decreased and
however,
nonadditive; (data
short-term
in astrocytes
enhanced
also
additive
PDBu, markedly
and NE.
and long-term in
that
significant
decreased
Ssb were
and ATP were
agonists
neurotensin
ET
and
3 illustrates
and with
to ET-l
the another
markedly Both
short-
PI response experiment,
to
Vol.
168,
No.
BIOCHEMICAL
2, 1990
TABLE various
1.
Stimulation agonists
AND
BIOPHYSICAL
RESEARCH
of ‘B-IP accumulation in cerebellar granule cells, astrocytes and Cs-gliomas IP-accumulation
Agonist
Concentration
Granule
elicited cerebellar
(4: of
cells
COMMUNICATIONS
control)
Astrocytes
Cc-gliomas --904+145(8)
ET
10 nM 30 nM
836*78(X) ---
2183+190(13) ---
Carbachol
100uM 1 ml4
2848*272(17) ---
--215*23(3)
--91+1
lOOut 1 mM
560+62(18)
--709*79(7)
--117*14(3)
1OOuM 1 mM
374+34(15)
500uM
306+33(16)
NE
Histamine
5-HT
---
---
1 mW
---
Glutamate
1OOuM
ATP
100*13(3)
---
---
151*5(3)
103+7
910+118(14)
181*14(4)
101*11(3)
1OOuM
143*10(3)
470+48(7)
1 uM
164*4(3)
1553+168(g)
Neurotensin
1 un
157*8(3)
435+59(6)
VIP
1 UM
205*11(3)
113*8(3)
Arg-vasopressin
1 UM
192*5(3)
124&15(3)
Angiotensin
II
(3)
---
122*3(3)
---
by
(4)
1263+122(4)
10226(3) 104*1(3)
98+14(3) 104*1(3)
Measurements of PI were described in the Rethods. The concentrations of receptor agonists are as indicated. The 100% values (basal activity) were 820*55 dpm/dish (n=25) in granule 1195+105 dpm/dish (n=18) in astrocytes and 1510+150 cells, dpm/dish (n=lO) in C -gliomas. Data in parentheses indicate numbers of independent gxperiments; each performed in triplicate.
TABLE
2. Additivity other receptor
by ‘H-IP agonists
accumulation in cerebellar IP
Agonist
Concentration
None ifib Carbachol ATP Angiotensin
accumulation Control 100
II
10 1 1 100 1
nM mid ml4 UM un
1482+34
635521 181*16 24Ot25
1918+43
induced astrocytes (% of
by ET
and
control)
+ET
(10
nt4)
1677+55 1690+64 2608266
1974+102 1974+15 3373*767
Measurements of PI turnover were performed as described in the Methods in the absence or presence of indicated receptor agonists. The 100% basal values were 1185*50 dpm/dish. Results are mean + S.E.M. from a triplicated experiment which was performed three times with similar results.
516
Vol.
168,
No.
BIOCHEMICAL
2, 1990
TABLE
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
3. Effects of PTX and PDBu on agonist-induced in cerebellar astrocytes and C6-gliomas 4: of response
to agonist
Astrocyte PTX (24hr)
Agonist
PI turnover
alone Cs-gliomas
PDBu (10min)
PDBu (‘24hr)
PTX (24hr)
ET (10 nM) 63*6(3) 64*5(3) 169*17(5) 75+12(4)26+5(3) 180+23(5) ATP (100 ut4) Angiotensin II 86+20(3)50_+13(3)117+24(3) (1 uM) Neurotensin 122&E(3) 47+19(3)122+12(3) (1 uM) NE (1 mM) 44*8(3) 18*6(3) 118+26(3)
PDBu (10min)
PDBu (24hr)
24&6(3) 25*3(3) 92*20(3) 7*1(3)
33+4(3) 2&9(3)
Cells pretreated with vehicle, PTX or PDBu (10 min or 24 hr as indicated) were exposed to various agonists for 45 min. The agonist-induced PI turnover in the PTX or PDBu treatment was expressed as the percentage of the agonist response measured in the absence of PDBu or PTX. The concentrations of PDBu and PTX used were 500 nM and 500 rig/ml, respectively. Basal PI turnover was 66+5(5), 92*4(3), 84&5(6) ( % of control) in astrocytes and 101+3(3), ?9+2(3) in C -gliomas after treatment with 115+9(3), PTX (24hr), PDBu (10min) and PDBb (24hr), respectively. Data in parentheses indicate numbers of independent experiments.
pretreatment to
ET,
of cells ATP
gliomas
with
and NE
(Table
neurotensin
3),
PTX for
whereas
the
in astrocytes
not
significantly
PI
hydrolysis
24
in astrocytes
responses
and
changed.
by ET
PI response response
to angiotensin
the effect
Long-term
induced
hr decreased
and ET-induced
in
II
and
of ATP in gliomas
PTX treatment
and carbachol
did
were
not
alter
in granule
cells
(13,14). DISCUSSION Although of
ET-l
PI
turnover
astrocytes
and
effects
were
differed three gliomas with kinase while gliomas Exposure response
and its in
C6 -gliomas noted.
types
in
with
of
the
to
3) cells ET
being
a robust cells,
major
differences
five
rank
(Fig.1).
same treatment
(Table
the
induced granule
peptides
order a
increase cerebellar in
examined,
of potencies
much weaker
Long-term
their they
in
these
stimulator
treatment
of
in cells
presumably
C, enhanced the
some
their
ET-3
and astrocytes PDBu, which
,
Among
considerably cell
homologues
cerebellar
resulted in depletion of protein to ET and ATP in astrocytes, responses reduced
the responses
and the
ET effect
to
for
PTX
in astrocytes
24
hr markedly
and gliomas 517
to ET and ATP in
in granule (Table
cells
(13).
inhibited 3)
the with
no
BIOCHEMICAL
Vol. 168, No. 2, 1990 effect
in granule
agonists
cells
of
suggest
that
that
cerebellar
granule in
different
coupling PTX
3),
but
classes
or
It
is
3).
This
PLC-coupled
it
may be
distinct astrocytes to that
shown
cells suggests
pools,
cells
in
these
stimulation
with
these
neurons
glial
cells,
Ross,
C.A.
related
are
to ET-induced
are
heterologous cell
of ET-l (data
(10)
subtypes.
shown)
is
2) and C6-gliomas for
(data these
cerebellar
granule
the
the synthesis personal
to determine
if
release
of
and
primary
shown)
agonists
to separate
In C6-glioma
The
not
cultures. triggers
that
in granule
subpopulations
and in
similar
receptors.
PI turnover
linked
in
and suggests of
and receptors
with
and sarafotoxin
not
cells
classes
different
Snyder,S.H.,
required
of others
with
ET promotes and
some
associated ET-1
(15)
protein.
Alternatively,
in different
but
G
cyclic
(ai)
of different
and agonist-evoked
on the same cells or
Gi
to
PLC-coupled
the effects
same population
(Table
in
PTX decreased
copresence
granule the
suggest
involved
the
the same astrocyte.
expressed
ET receptors
either
lipid
studies
ET-
astrocytes that
present
are
the
in cultures
at
(13),
of intracellular of
altering
the
in astrocytes
of ET response of
a rise
2) and C6-gliomas
between (13),
G proteins
of the effects
act
of
astrocytes
cells
astrocytes,
in
in cerebellar
peptides
in granule
without
being
(Table
additivity
in
astrocytes
The nonadditivity
both
of
may reflect
G proteins
ET the
cultures
effects
inactivation
G proteins
that
out
primary
ADP-ribosylation
responses
of
for
and rule
to contaminating
of putative
that,
agonist-induced
results
mechanisms
to PLC. The sensitivity
persistent
of interest
(Table
not
the consequence
following
three
these
types in
receptor
these
preparation.
ET receptors
protein,
cell
is due
may be due to direct
AMP
transduction
by PTX of ET-induced
(Table
of in
together,
of ET shown
cells
inhibition
gliomas
that
effect
the neuronal
The
Taken
in different
the
a variety
PI hydrolysis
1). signal
involved
possibility present
(Table
distinct
are
Moreover,
stimulated
cultures
receptors
and
(13).
differentially
types
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
are
inositol of cells,
glutamate
from
cultures
of
of DNA (Maccumber,N.W., communications). these
events
Further are
causally
PI breakdown. RJWKRENCES
1.
Yanagisawa, Kobayashi, Nature 332,
M., Kurihara, H. M., Mitsui, Y., ,Goto, 411-415. 518
Kimura, S., Tomobe, Y., K. and Masaki, T. (1988)
Vol.
168, No. 2, 1990
2.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Inoue, A., Yanagisawa, H., Kimura, S., Kasuya, Y., Miyauchi, T Goto, K. and Masaki, T. (1989) Proc. Natl. Acad. Sci. Usi
3.
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86,
2863-2867.
Takasaki, C., Tamiya, N., Bdolah, A., Wollberg, Z. and Kochva, E. (1988) Toxicon 26, 543-548. Uchida, Y., Ninomiya, H., Saotome, M., Ohtsuka, H., Yanagisawa, M., Goto, K., Hasaki, T. and Hasegawa, S. (1988) Eu. J. Pharmacol. 154, 227-228. Ishikawa, T., Yanagisawa, M., Kimura, S., Goto, K. and Masaki, T. (1988) Pflugers Arch 413, 108-110. Kozuka, M., Ito, T., Hirose, S., Takahashi, K. and Hagiwara, H. (1989) Biochem. Biophys. Res. Commun.159, 317-323. Korbmacher, C., Helbig, H., Haller, H., Erickson-Lamg, K.A. and Wiederholt, M. (1989) Biochem. Biophys. Res. Commun. 164, 1031-1039. Maggi, C.A., Giuliani, S., Patacchini, R., Santicioli, P., Turini, D., Barbanti, G. and Meli, A. (1989) Br. J. Pharmacol. 96, 755-757. Lin, W.-W. and Lee, C.Y. (1990) Eu. J. Pharmacol. in press. tin, W.-W., Lee, C.Y. and Chuang, D.-M. (1989) Eu. J. Pharmacol. 166,581-582. Gallo, V., Ciotti, M.T., Coletti, A., Aloisi, F. and Levi, G. (1982) Proc. Natl. Acad. Sci. USA 79, 7919-7923. Holopainen, I. and Kontro, P. (1984) Acta Physiol. Stand 122, 381-386. Lin, W.-W., Lee, C.Y. and Chuang, D.-M. (1989) Neuroscience Abst. 15, P658. xu, J. and Chuang, D.-M. (1987) J. Pharmacol. Exp. Ther. 242, 238-244. Lin, W.-W., Lee, C.Y. and Chuang, D.-M. (1990) FASEBJ. in press.
519
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 512-519
BIOCHEMICAL
COHPARATIVRSTIJDIESOF PEOSPEOINOSITIDl3 RYDROLYSISINDDCRDBY RNDOTRRLIN-RRLATRD PEPTIDBSIN CULTDRRD CRRRRELLAR ASTROCYTES, C6-GLIOl4AAND CRRRRBLLAR GRANDLR CELLS Wan-Wan Lin”‘,
Chen Yuan Lee’
and De-Maw
Chuangr*
‘Biological Psychiatry Branch, National Institute of Mental Health Bldg. 10, Room 3N212 Bethesda, MD 20892 2Department of Pharmacology, National Taiwan University, Received
February
27,
College of Medicine, Taipei, Taiwan, R.O.C.
1990
Summary : Effects of endothelin (ET) homologues (ET-1,2,3 and sarafotoxin S ) and its precursor (big ET-l) on phosphoinositide (PI)6turnover were compared in neurally-related cell cultures. All ET-related peptides induced a robust increase of PI turnover in cerebellar astrocytes, C6-glioma and cerebellar granule cells. The rank order of potency in stimulating PI turnover was ET-&ET-2),S >ET-3>big ET-l for granule cell neurons, while it was ET-1#-2aS >big ET-l>ET-3 for astrocytes and Cd-glioma cells. Short-te% pretreatment with phorbol dibutyrate (PDBu) attenuated the ET-l-induced PI response in all three types of cultures. However, long-term pretreatment with PDBu attenuated the response in granule cells and Cc-gliomas, but enhanced responses to ET and ATP in astrocytes. Long-term exposure of cells to pertussis toxin (PTX) attenuated the PI response to ET in astrocytes and C -gliomas, but not in granule cells. Thus, phospholipase C-couplid ET receptors are expressed in both neurons and glial cells, but they differ considerably in their pharmacological selectivity and signal transduction mechanisms in stimulating PI hydrolysis. Q 1990 Academic Press, Inc.
Endothelin-1 peptides the
(ET-l) (1).
of
three
ET-l,
2
and
peptide the
one
of
studies
the
distinct 3.
venom
smooth
members
In addition, ,
Atractaspis
engadilnsis of ET are
correspondence
this
peptide
should
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
potent
512
of
vasoactive
DNA have
the ET
family
a structurally
S
that the actions muscle cells since
*To whom all
of
most
of a human genomic
of ET, named sarafotoxin
indicated
Copyright All rights
Cloning
existence
termed
0006-291X/90
is
has been (3). not
limited
displays
be addressed.
shown (2),
related
identified
from
Recent
studies
to
vascular
several
other
Vol.
BIOCHEMICAL
168, No. 2, 1990 biological
activities
believed its
in
to exert
specific
of
second
is
the
its
messenger
to generate
the
term
system
primary
coupled this
receptors
are
have
compared
Seb)
and its
astrocytes, receptor in these
also
three
refer
cerebellar
of ET homologues
types
ET-l)
on
and cerebellar
that
of cultured
express
ET
(ET-1,2,3
PLC-coupled
ET
glial
We
in cerebellar
cells.
PLC-catalyzed examined
for
cells.
and sarafotoxin
PI turnover granule
stimulate also
and shown
(10).
demonstrated in neurally-related
were
to
4-phosphate, cells
(big
By
collectively
granule
we
C
and diacylglycerol.
of rat
which
systems,
ET action
phospholipase
previously
precursor
agonists,
the
by
is
One type
We have
present
effects
cells.
mediating (PI)
we
to PI hydrolysis
ET with
4,5-bisphosphate.
study,
C6-glioma
various
in
target
phosphatidylinositol cultures
In
of
phosphates
phosphoinoside,
phosphatidylinositol,
receptors
involved
(4-9).
by interacting
phosphoinositide
inositol
phosphatidylinositol that
systems
activities
on the surface
of
(PLC)
non-vascular
biological
receptors
hydrolysis
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A variety breakdown
their
effects
of of PI in
cells.
MATERIALSANJI METHODS Materials ET-l, 2, 3, big ET-1 and sarafotoxin S were purchased from Peptide Institute Inc. (Osaka, Japan). A23187 was obtained from Calbiochem (San Diego, CA). All other chemicals were products of Sigma Chemical Co. (St. Louis, MO).
Cell culture Cerebellar granule cells and astrocytes were prepared from 8-day old rats as previously described (11,12). For the preparation of granule cells, dissociated cells were plated at a density of 3x lo6 cells per 35-mm dish precoated with poly-L-lysine and cultured in Basal Eagle’s Medium containing 10% fetal calf serum, 2 mM glutamine, 50 ug/ml gentamicin and 25 mM KCl. After 18-24h in culture, 10 UN cytosine arabinoside was added to prevent the replication and growth of glial cells. Granule cells were used after 8 days in culture. The purity of granule cells has been shown to be greater than 90% (11). Cultures of astrocytes were prepared as described for granule cells except that KC1 was omitted in culture medium and added to the culture that cytosine arabinoside was not (depolarization by KC1 is required for the survival of granule cell neurons). Culture media were changed twice a week and cells in culture. At this were used for experiments after lo-12 days time, they had reached confluency and comprised mainly (about 90%) flat polygonal-shaped glial cells (12). Rat C6-glioma cells (American Type Culture Collection, Rockville, MD) were grown in Dulbecco’s Modified Eagle’s medium containing 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 ug/ml). Cells with passage numbers between 513
BIOCHEMICAL
Vol. 168, No. 2, 1990
AND BIOPHYSICAL
13-35 were subcultured into 35-mm dishes measured when cells reached confluency.
RESEARCH COMMUNICATIONS
and
PI turnover
was
Heasurement of PI turnover PI hydrolysis was measured as the accumulation of 3H-inositol monophosphates (IP) in the presence of 20 mM LiCl in cells prelabeled overnight with 3H-myo-inositol as described previously (10). The reaction mixtures in physiological saline solution were incubated at 37’C for 45 min after stimulation of cells with receptor agonists. The reaction was terminated by addition of ice-cold methanol and the accumulation of 3H-IP was determined by chromatography on a AG column. 1x8
RESULTS ET-l
S6,)
and its
induced
homologues
a dose-dependent granule
cerebellar (Fig.1).
In
exhibited
similar
distinct
potencies,
increase
In an
5-
hydrolysis were
observed
some
receptor
activator
5-
order
shows
the
in
these
three all
cell
three
no
of types.
but not
the
effects
of
NH,
were
observed
in
Table were
2 show
that
elicited
nonadditive,
on PI
order
responses
Carbachol
effect
in
it
elicited
Cs-glioma
activation
of
cells.
astrocytes
glutamate,
astrocytes,
but
PI responses
to
ET-induced
of
a robust effect
Similarly,
II
in the in
in astrocytes differences For example,
and neurotensin
not gliomas. effect
to ET
vasoactive intestinal more pronounced
Some notable
ATP or angiotensin 514
little
and gliomas,
ET-1
PI
effects
was seen
angiotensin
on
was
cells.
by ATP
in granule
by NE, carbachol,
rank
differential
whereas
while
increase)
noted.
cells,
between
peptides
Although
cells.
detected
These
agonists
granule also
to la-fold)
of ET-1(1.7nM)aET-
cultures,
were
Marked
and its
various
of histamine, 5-HT, glutamate, (VIP) and arg-vasopressin were gliomas
14-
a
effects peptide and
order
ET-1(37nM)>ET-3(56nM). effects
agonists
were
but
rank
ET-l
to 8-fold
with
cells
1
and
Their
(about
a rank
(about
C6 -glioma
in granule
astrocytes
peptides
increase),
cultures,
greater
with
effects
in
cells
five
to 6-fold
astrocyte
ET-1(3.3nM)JET-2(5.1nM)>SGb(6.3nM)>big Table
in
C6-glioma
ET-1(26nM)>ET-3(46nM).
marked in
accumulation
these
PI breakdown.
even
of IP accumulation
turnover
and
cells,
and sarafotoxin
ET-1(1.3nH)=ET-2(1.4nM)~S~~(l.8nM)>ET-3
elicited
induced
astrocytes (about
(66nM).
2(2.6nM)aSbb(3.9nM)>big also
ET-l
of 3H-IP
in inducing
were
3, big
increase
granule
efficacies
ET-l
homologues
cells,
cultured
and ECsO values (BnN)>big
(ET-2,
The results
and Ssb
in
in astrocytes additive to that
was II.
In C6-gliomas,
Vol.
BIOCHEMICAL
168, No. 2, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2000 b MOO t ~1600-
p 73 t s0o
,g
g 1400 -
U
%4002a 300B T 3 200-
f
1200-
!
1000 800
-
a 2 ?
600-
-i ,I
aoo200 100 1
100 -
I 9 8 Concentrcltiin (-kg
-
700-
: v 'b
600 -
1
7
6
M)
%b
I:a.
100
Fig. 1. Dose-responce curves for PI turnover induced by ET-related peptides in cerebellar granule cells (A), cerebellar astrocytes (B) and %gliomas (C). The data were expressed as mean i S.E.M. from at least three independent experiments; each performed in triplicate.
the
responses
responses
to ET-l
Table a
phorbol
ester,
other
hr)
exposure no
the
the
effects In
responses
on
contrast,
treatment
granule
with
responses same
PDBu attenuated cells
515
(13).
with
the PI response
C6-gliomas.
of ET and ATP the
shown).
to
In
to ET
Long-term
(24
in astrocytes angiotensin II,
treatment
to ET and ATP in C6-gliomas.
treatments
cerebellar
effects
not
(10 min)
decreased and
however,
nonadditive; (data
short-term
in astrocytes
enhanced
also
additive
PDBu, markedly
and NE.
and long-term in
that
significant
decreased
Ssb were
and ATP were
agonists
neurotensin
ET
and
3 illustrates
and with
to ET-l
the another
markedly Both
short-
PI response experiment,
to
Vol.
168,
No.
BIOCHEMICAL
2, 1990
TABLE various
1.
Stimulation agonists
AND
BIOPHYSICAL
RESEARCH
of ‘B-IP accumulation in cerebellar granule cells, astrocytes and Cs-gliomas IP-accumulation
Agonist
Concentration
Granule
elicited cerebellar
(4: of
cells
COMMUNICATIONS
control)
Astrocytes
Cc-gliomas --904+145(8)
ET
10 nM 30 nM
836*78(X) ---
2183+190(13) ---
Carbachol
100uM 1 ml4
2848*272(17) ---
--215*23(3)
--91+1
lOOut 1 mM
560+62(18)
--709*79(7)
--117*14(3)
1OOuM 1 mM
374+34(15)
500uM
306+33(16)
NE
Histamine
5-HT
---
---
1 mW
---
Glutamate
1OOuM
ATP
100*13(3)
---
---
151*5(3)
103+7
910+118(14)
181*14(4)
101*11(3)
1OOuM
143*10(3)
470+48(7)
1 uM
164*4(3)
1553+168(g)
Neurotensin
1 un
157*8(3)
435+59(6)
VIP
1 UM
205*11(3)
113*8(3)
Arg-vasopressin
1 UM
192*5(3)
124&15(3)
Angiotensin
II
(3)
---
122*3(3)
---
by
(4)
1263+122(4)
10226(3) 104*1(3)
98+14(3) 104*1(3)
Measurements of PI were described in the Rethods. The concentrations of receptor agonists are as indicated. The 100% values (basal activity) were 820*55 dpm/dish (n=25) in granule 1195+105 dpm/dish (n=18) in astrocytes and 1510+150 cells, dpm/dish (n=lO) in C -gliomas. Data in parentheses indicate numbers of independent gxperiments; each performed in triplicate.
TABLE
2. Additivity other receptor
by ‘H-IP agonists
accumulation in cerebellar IP
Agonist
Concentration
None ifib Carbachol ATP Angiotensin
accumulation Control 100
II
10 1 1 100 1
nM mid ml4 UM un
1482+34
635521 181*16 24Ot25
1918+43
induced astrocytes (% of
by ET
and
control)
+ET
(10
nt4)
1677+55 1690+64 2608266
1974+102 1974+15 3373*767
Measurements of PI turnover were performed as described in the Methods in the absence or presence of indicated receptor agonists. The 100% basal values were 1185*50 dpm/dish. Results are mean + S.E.M. from a triplicated experiment which was performed three times with similar results.
516
Vol.
168,
No.
BIOCHEMICAL
2, 1990
TABLE
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
3. Effects of PTX and PDBu on agonist-induced in cerebellar astrocytes and C6-gliomas 4: of response
to agonist
Astrocyte PTX (24hr)
Agonist
PI turnover
alone Cs-gliomas
PDBu (10min)
PDBu (‘24hr)
PTX (24hr)
ET (10 nM) 63*6(3) 64*5(3) 169*17(5) 75+12(4)26+5(3) 180+23(5) ATP (100 ut4) Angiotensin II 86+20(3)50_+13(3)117+24(3) (1 uM) Neurotensin 122&E(3) 47+19(3)122+12(3) (1 uM) NE (1 mM) 44*8(3) 18*6(3) 118+26(3)
PDBu (10min)
PDBu (24hr)
24&6(3) 25*3(3) 92*20(3) 7*1(3)
33+4(3) 2&9(3)
Cells pretreated with vehicle, PTX or PDBu (10 min or 24 hr as indicated) were exposed to various agonists for 45 min. The agonist-induced PI turnover in the PTX or PDBu treatment was expressed as the percentage of the agonist response measured in the absence of PDBu or PTX. The concentrations of PDBu and PTX used were 500 nM and 500 rig/ml, respectively. Basal PI turnover was 66+5(5), 92*4(3), 84&5(6) ( % of control) in astrocytes and 101+3(3), ?9+2(3) in C -gliomas after treatment with 115+9(3), PTX (24hr), PDBu (10min) and PDBb (24hr), respectively. Data in parentheses indicate numbers of independent experiments.
pretreatment to
ET,
of cells ATP
gliomas
with
and NE
(Table
neurotensin
3),
PTX for
whereas
the
in astrocytes
not
significantly
PI
hydrolysis
24
in astrocytes
responses
and
changed.
by ET
PI response response
to angiotensin
the effect
Long-term
induced
hr decreased
and ET-induced
in
II
and
of ATP in gliomas
PTX treatment
and carbachol
did
were
not
alter
in granule
cells
(13,14). DISCUSSION Although of
ET-l
PI
turnover
astrocytes
and
effects
were
differed three gliomas with kinase while gliomas Exposure response
and its in
C6 -gliomas noted.
types
in
with
of
the
to
3) cells ET
being
a robust cells,
major
differences
five
rank
(Fig.1).
same treatment
(Table
the
induced granule
peptides
order a
increase cerebellar in
examined,
of potencies
much weaker
Long-term
their they
in
these
stimulator
treatment
of
in cells
presumably
C, enhanced the
some
their
ET-3
and astrocytes PDBu, which
,
Among
considerably cell
homologues
cerebellar
resulted in depletion of protein to ET and ATP in astrocytes, responses reduced
the responses
and the
ET effect
to
for
PTX
in astrocytes
24
hr markedly
and gliomas 517
to ET and ATP in
in granule (Table
cells
(13).
inhibited 3)
the with
no
BIOCHEMICAL
Vol. 168, No. 2, 1990 effect
in granule
agonists
cells
of
suggest
that
that
cerebellar
granule in
different
coupling PTX
3),
but
classes
or
It
is
3).
This
PLC-coupled
it
may be
distinct astrocytes to that
shown
cells suggests
pools,
cells
in
these
stimulation
with
these
neurons
glial
cells,
Ross,
C.A.
related
are
to ET-induced
are
heterologous cell
of ET-l (data
(10)
subtypes.
shown)
is
2) and C6-gliomas for
(data these
cerebellar
granule
the
the synthesis personal
to determine
if
release
of
and
primary
shown)
agonists
to separate
In C6-glioma
The
not
cultures. triggers
that
in granule
subpopulations
and in
similar
receptors.
PI turnover
linked
in
and suggests of
and receptors
with
and sarafotoxin
not
cells
classes
different
Snyder,S.H.,
required
of others
with
ET promotes and
some
associated ET-1
(15)
protein.
Alternatively,
in different
but
G
cyclic
(ai)
of different
and agonist-evoked
on the same cells or
Gi
to
PLC-coupled
the effects
same population
(Table
in
PTX decreased
copresence
granule the
suggest
involved
the
the same astrocyte.
expressed
ET receptors
either
lipid
studies
ET-
astrocytes that
present
are
the
in cultures
at
(13),
of intracellular of
altering
the
in astrocytes
of ET response of
a rise
2) and C6-gliomas
between (13),
G proteins
of the effects
act
of
astrocytes
cells
astrocytes,
in
in cerebellar
peptides
in granule
without
being
(Table
additivity
in
astrocytes
The nonadditivity
both
of
may reflect
G proteins
ET the
cultures
effects
inactivation
G proteins
that
out
primary
ADP-ribosylation
responses
of
for
and rule
to contaminating
of putative
that,
agonist-induced
results
mechanisms
to PLC. The sensitivity
persistent
of interest
(Table
not
the consequence
following
three
these
types in
receptor
these
preparation.
ET receptors
protein,
cell
is due
may be due to direct
AMP
transduction
by PTX of ET-induced
(Table
of in
together,
of ET shown
cells
inhibition
gliomas
that
effect
the neuronal
The
Taken
in different
the
a variety
PI hydrolysis
1). signal
involved
possibility present
(Table
distinct
are
Moreover,
stimulated
cultures
receptors
and
(13).
differentially
types
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
are
inositol of cells,
glutamate
from
cultures
of
of DNA (Maccumber,N.W., communications). these
events
Further are
causally
PI breakdown. RJWKRENCES
1.
Yanagisawa, Kobayashi, Nature 332,
M., Kurihara, H. M., Mitsui, Y., ,Goto, 411-415. 518
Kimura, S., Tomobe, Y., K. and Masaki, T. (1988)
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168, No. 2, 1990
2.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Inoue, A., Yanagisawa, H., Kimura, S., Kasuya, Y., Miyauchi, T Goto, K. and Masaki, T. (1989) Proc. Natl. Acad. Sci. Usi
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