Vol.
167.
February
No.
BIOCHEMICAL
1, 1990
28,
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
161-167
VASOACTIVRINTESTINAL CONTRACTOR, A NOVRLPEJ'TIDE, SHARESA COMHON RECEPTOR WITB RNDOTRRLIN-1AND STIMULATESCa MOBILIZATION AND DNA SYNTRRSISIN SWISS3T3 CELLS Isabel
Fabregat
Imperial Lincoln’s
Received
December
and Enrique
Rozengurt
Cancer Research Fund, P 0 Box 123, Inn Fields, London WCZA 3PX, U.K.
27,
1989
SUHHARY : Vasoactive intestinal contractor peptide (VIC), a novel member of the en&thelin family, stimulated a rapid increase in the intracellular Ca concentration in fura-Z-loaded Swiss 3T3 cells. Sequential addition of VIC and endothelin-1 (ETl) demonstrated the induction of both homologous and heterologous deser&;‘;.ti;z, i;IC was as potent as ET1 in displacing the binding of stimulating mitogenesis in Swiss 3T3 cells. These findit& suggest that VIC and ET1 share a common receptor in Swiss 3T3 cells. @1990 Academic Press, Inc.
Vasoactive identified
intestinal
as a new member of VIC differs
(1,2). Zl-residue
4, 6, 7).
other
,tissues
inducing
function
from
The
peptide
is
contraction
least
three
distinct
known
whether
these
receptors
the
(3),
expressed
of mouse
and chemical
(1) ileum,
or by a distinct
the endothelin
effects
the
intestine
in
a
acid
residues but
more effective
suggesting
that
Binding
subtypes
of VIC are
receptor
family
(ETl), amino
studies
receptor
been
three
(2).
cross-linking
biological
in
and is
hormone
endothelin
of
or human endothelin
cells
as a gastrointestinal
has recently
peptides
peptide
or endothelial
measurements
(VIC)
the
porcine
vasoconstrictor
(nos
in
contractor
not
in
than
ET1
VIC may
competition
have
identified
(4-8). mediated
It
at is
not
by any of
subtype.
Abbreviations: ET , porciyy or human endothelin. VIC, vasoactive intest.inalntrac∨ [Ca Ii, intracellular Ca2+ concentration; epidermal growth factor; PBt2, phorbol 12,13-dibutyrate; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin.
EGF,
0006-291XN
161
$1.50
Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form resewed.
Vol.
167,
No.
1, 1990
BIOCHEMICAL
A recent in
the
report
intracellular
NG-108-15
and ET1 act binding
(9). not
but
via
to elucidate
growth
factors
cells act
receptor
the
with
for
stimulates growth
factor
we report
Swiss
ET1 and induces
more direct
have
induced that
evidence
a rapid
provided
pathways
(10-12).
VIC
(e.g.
in
model
the action
of
ET1 stimulates
that
the novel VIC shares
increase in
a useful
and DNA synthesis
3T3 cells.
DNA synthesis (EGF)
peptides suggesting
Ca 2+ mobilization
Here
increase
in neuroblastoma
these
transduction peptides
breakdown, (4,13,14).
but
a rapid
([Ca2+li) of
3T3 cells
signal
and mitogenic
caused
COMMUNICATIONS
presented.
of Swiss
as a mitogen
subsequently, epidermal
was not
ETl,
RESEARCH
desensitization,
receptors,
cultures
system
these
like
addition
heterologous
distinct
phosphoinositide
VIC,
of Ca ‘+
Sequential
competition)
Quiescent
that
BIOPHYSICAL
concentration
cells
homologous
also
showed
AND
the
in
[Ca2+li
presence
peptide
in VIC can
a common and of either
or insulin.
MATERIALS
AND MBTBODS
Swiss 3T3 cells were mainlained in culture and assays of DNA synthesis were performed by [ HI-thymidine incorporation as described (15). Determination of [Ca2+].: Confluent and quiescent Swiss 3T3 cells grown in 90 mm dishes were-:ashed twice with Dulbecco’s modified Eagle’s medium (DMEM) and incubated for 10 min in 5 ml DMEM containing 1 uM furatetraacetoxymethyl ester. After this incubation the cells were washed three times with electrolyte solution which contained 120 mM NaCl, 5 mM KCl, 1.8 mM CaC12, 0.9 mM MgC12, 25 mM glucose, 16 mM Hepes, 6 mM Tris and a mixture of amino acids at the same concentrations as those in DMEM (pH 7.2). The cells were suspended in el?ctrolyte solution (2 ml) by gently scraping and transferred to a 1 cm quartz cuvette. The cell suspension was stirred continuously at 37OC. Fluorescence was monitored in a Perkin-Elmer LS-5 luminescence spectrophotometer with an xcitation wavelenght of 336 nm and emission wavelength of 510 nT25 [Ca’+]. was calculated as described (16). Measurement of 125I-ETl binding: Swiss 3T3 cells were incubated at 4’C with 0.5 nM I-ET1 in phosphate buffered saline containing 25 mM N-2-Hydroxyethylpiperazlne-N’2-ethanesulfonic acid, 0.1% fatty acid free bovine serum albumin (BSA) (pH 7.0). After 3 hours, unbound radioactivity was removed by washing the cells three times with cold (4’C) phosphate buffered saline (pH 7.4) containing 0.1% BSA. The cells were extracted (20 min at 37’C) with 0.5 ml of 2% Na CO 1% .2 2’lvity sodium dodecyl sulfate, O.lN NaOH and cell associated radloac was determined in a LKB y-counter. Under these conditions, non-specific binding, determined as cell-associated radioactivity in the presence of 1 vM unlabelled ET1, was about 5% of the total. Materials: ET and sarafotoxin S6b were purchased from Peninsula Laboratories, Inc t U.K.). VIC was obtained from American Peptide Co. 162
Vol.
167,
No.
BIOCHEMICAL
1, 1990
AND
BIOPHYSICAL
RESEARCH
(U.S.A.). Bovine insulin, EGF, bombesin, vasopressin, lZ,l#ibutyrate (PBt ), BSA were from Sigma Chemical MO. I-ET1 was pure i ased from Amersham International,
COMMUNICATIONS
phorbol Co., St. Louis, Amersham, U.K.
RESULTSAND DISCUSSION Addition fluorescent measurable subsequently
of 10 nM VIC to quiescent Ca 2+ -indicator delay
(Fig.
declined
fura1). towards
[Ca’+]
Swiss
3T3 cells
increased
[Ca2+li
i reached
peak
the basal
level.
loaded
without values
Subsequent
at
with any
15 s and addition
VIC 4nM
VP
Figure 1. Effect of repeated additions of VIC and ET on [Ca*+].. asi Quiescent Swiss 3T3 cells in 90 mm dishes were loaded wit A furadelscribed in Materials and Methods. Fluorescence was recorded continuously while additions were made at the points and co,ncentrations indicated by the arrows: VIC, vasoactive intestinal contractor peptide; ETl, endothelin-1; VP, 10 nM vasopressin; BOM, 6 After a suitable control period, the peptides were added nM bombesin. and fluorescence followed until it returned to near base line levels. After this, additioft+of the same or a different peptide was performed. Measurements of ]Ca 1. were performed after sequential addition of Triton X-100 and ethyl&e glycol-bis(&aminoethyl ether) as previously described (16).
163
the
of
Vol.
167,
No.
1, 1990
either
BIOCHEMICAL
AND
10 nM VIC or 10 nM ET1 did
[C2+li.
Bombesin,
below)
caused
which
a marked
acts
exposed
sequential
responses
nM instead
of 10 nM and when
vasopressin desensitization Identical
completely
in
were
finally not
VIC
(17,18
and
Similar
i.e.
responses
of VIC was 4 with
caused
homologous
response could
by either
in
challenged only
subsequent
was reversed
[Ca2+] i caused
receptor
1, upper).
responsiveness
subsequent
rise
the concentration
Thus, the
COMMUNICATIONS
[Ca 2s ] i in cells
in
cells
blocked
cellular
the
a distinct
when
the
RESEARCH
any further
increase
elicited
addition
blocked
increase
also
in
of peptide
through
of bombesin.
but
elicit
VIC and ET1 (Fig.
were
changes
order
the
to both
instead
not
and rapid
previously
BIOPHYSICAL
to ET1.
be induced
when
the
ET1 either
at 10 or 4 nM
to VIC but
did
bombesin
not
prevent
or vasopressin
(Fig.
1, middle). A possible responses
interpretation
can be blocked
and activation (lower)
Treatment
10 nM vasopressin equivalent
to that
attenuated
the
Crucially,
exposure
[Ca2+li
Ca
caused in Fig.
presence
response
results
from
of a single
(Fig. class
in
not
in
Fig.
1
this fura-
with
[Ca2+li
addition the
1, lower). recently
and raised in Swiss a study
increase
in
The results reported
with
the possibility
that
ET1
3T3 cells. of the
binding
of these
cell-associated shown).
of vasopressin.
abolish
When cultures
at 4’C,
3 h (results
was saturable
not
those
prompted
3T3 cells.
mobilization
shown
with
increase
did
(9)
2+
to test
loaded
VIC or ET1 (Fig.
cells
the ET1 and VIC
Ca
The experiment
to a subsequent
different
125 I-ET1
causes
that
VIC or ET1 and markedly
to vasopressin
by either
a maximum after cells
by either
to a common receptor
with
that
3T3 cells
induced
of Swiss
is
was designed
a transient
2+
The preceding
incubated
(19,20) of Swiss
NG108-15
and VIC bind
C.
promoted
1 are
neuroblastoma
to cultures
kinase
vasopressin
possibility.
results
by any agonist
of protein
using
shown
of these
of cells
radioactivity 125 I-ETI
binding
1251-ET1 were reached
to 3T3
2) and Scatchard
analysis
indicated
the
of high-affinity
sites
(Kd = 0.96
f 0.23
164
Vol.
167,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
125 I-ET-l,
RESEARCH
COMMUNICATIONS
nM
Figure 2. Concentration dependence of 1251-ET1 bi’t$;“;T’obf;;a;; quiescent Swiss 3T3 cells. Specific cell-associated . Gas measured in quiescent cells incubated for 3 hour f2ft 4Tlin binding medium containing various concentrations of I-ET . Each point represents the mean of two determinations. Specific B,inding is expressed as pmol/mg of protein. Inset: Scatchard plot of the same datal. Bound (B) radiolabelledlJfgand is expressed in pmol/mg of protein; the concentration of I-ET1 in the medium (F) is in nM. All other experimental details were as described in Materials and Methods .
n=3)
nM;
(Fig. by
and a maximum
2, inset).
unlabelled
Binding
manner
vasopressin
(up
The
feature
saJient
demonstrate
for that
apparent
affinities.
(4,13)
depicted
DNA
synthesis
the
absence
synergistically
in or
other
in
Figs.
growth
ET1
cell
EGF. in
Fig.
eliciting
I-ET1
ET1
a common
share
virtually
can
stimulate
DNA
(21,22)
Swiss
4 shows
that
synthesis
synthesis
in
acting In
whether 3T3
findings
identical
factors.
DNA 165
125
displaced
with
nor
of 1251-ET1.
These
types
of
S6b in a
= 2 nM).
promoting
cultures of
EGF
VIC
and
competed
was
bombesin
binding
1 and 3, we tested
quiescent presence
VIC
that
and other
with
with
that peptides
shown
neither
that
protein
sarafotoxin
specific
3 is
of
3T3 cells
peptide
ET1 (IC50
time
have
to Swiss
the
Fig.
both
pmol/mg
In contrast,
as unlabelled
reports
synergistically
3).
first
of 0.5
cardiotoxic
in
recognises
3T3 cells
results
(Fig.
shown
the
receptor
Swiss
of lz5 I-ET1
to 10 uM) reduced
as potently
Recent
capacity
ET1 and by the
dose-dependent
binding
binding
view
of
the
VIC can stimulate
cells
incubated
in
VIC acted in
Swiss
3T3
Vol.
167,
No.
1, 1990
BIOCHEMICAL
AND
BJOPHYSICAL
RESEARCH
COMMUNICATIONS
: 5;[5,, ,,y\ 1o-2
Id'
0
3
1
ld
lo*
lo3
lo4
0
04
Peptide,nM
1 nM
0.1
Peptide,
10
125 I-ET1 binding in intact, Figure 3. Inhibition of specific quiescent Swiss 3T3 cells by ET (o), VIC (o), sarafotoxin S6b (A), Confluent and quiescenf%S;l;;res bombesin (A) and vasopressin ( b). of 3T3 cells were incubated at 4OC for 3 hours with 0.5 nM either in the presence or in the absence of the related or unrelatkd peptides at the concentrations indicated. Each py@t represents the Binding of I-ET is expressed mean of at least two determinations. as a percentage of the specific binding in the absence o t any unlabelled peptide (100% = 0.147 + 0.014 pmol/mg of protein). Figure 4. Stimulation of DNA synthesis in intact quiescent Swiss for DNA synthesis was performed 3T3 cells by VIC and ET . The assay n , ET ) or in the presence of 5 rig/ml EGF in the absence ( 0 , VIC; or 1 ug/ml insulin (inset) (0, VICf l , ETl). Confluent and quiescent cultures of Swiss 3T3 cells were wafhed and incubated in 2 ml DMEM/Waymouth’s medium containing [ HI-thymidine, with or without insulin or EGF and various concentrations of VIC or ETl. The incorporation of radioactivity into acid-precipitable material was measured after 40 h. Each point represents the mean of 2 determinations. In this experiment, values obtained in the presence of either 10% fgtal bovine seruy or 5 rig/ml EGF plus 1 ug/ml insulin cpm culture, respectively. were 8.65 x loand 7.77 x 10
Maximum
cells.
and
respectively.
nM,
An
obtained
when
ET1
observed
that
VIC,
combination
with
activate
protein
cellular
level VIC
in
the
is
of
neuroblastoma
instead
ET,,,
insulin
(Fig.
kinase
C (e.g.
CAMP
(e.g.
peptide
VIC and ET1,
(9)
this
achieved
in
question
In
4,
or
agents
rig/ml
PBt2)
inset)
and
the
remained 166
other
and
0.4
was experiments,
synthesis
in that or
family cells
responses
2.5
we
synergistic either
increase
the
IBMX).
endothelin
endothelial
suggested
VIC. DNA
lo-100
at
relationship
stimulated
the
contractile cells
of
forskolin of
not
were
dose-response
added
like
but
concerning
effects
identical
was
a novel
intestine
studies
for
half-maximal
(2) existence
which
(1,Z).
is
expressed
Although
recent
and Ca 2+ mobilization of
separate
to be elucidated.
in
receptors
Here
we
Vol.
167,
No.
show,
BIOCHEMICAL
1, 1990
for
the
receptor
in
first
Swiss
time,
that
VIC,
like
synergistically preferentially the target
possibility cells
that
3T3 cells,
and cross-desensitization ET1, with
is
other
in the
2+
growth in
RESEARCH
the
novel
signals.
mitogen
for
COMMUNICATIONS
a common
binding
Our results
promoting
competition demonstrate
3T3 cells
acting
factors.
Since
intestine
(l),
peptide
may act
gastrointestinal
with
by 125 I-ET1
as judged
a potent
this
BIOPHYSICAL
VIC and ET1 interact
of Ca
expressed that
AND
these
findings
as a growth
VIC is raise factor
for
tract.
REFERENCES 1.
2. 3. 4. 5. 6. 7. 0. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Saida, K., Mitsui, Y. and Ishida, N. (1989) J. Biol. Chem. 264, 14613-14616. Ishida, N., Tsujioka, K., Tomoi, M., Saida, K. and Mitsui, Y. (1989) FEBS Lett. 247, 337-340. 'Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masakf, T. (1988) Nature 332, 411-415. 'Takuwa, N., Takuwa, Y., Yanagisawa, M., Yamashita, K. and Masaki, T. (1989) J. Biol. Chem. 2, 7856-7861. Watanabe, H., Miyazaki, H., Kondoh, M., Masuda, J., Kimura, S., Yanagisawa, H., Masaki, T. and Murakami, K. (1989) Biochem. Biophys. Res. Commun. 161, 1252-1259. Kloog, Y., Bousso-Mittler, D., Bdolah, A. and Sokolovsky, M. (1989) FEBS Lett. 253, 199-202. Sugiura, M., Snajdar, R.M., Schwartzberg, M., Badr, K.F. and Inagami, T. (1989) Biochem. Biophys. Res. Commun. 162, 1396-1401. Masuda, Y., Miyazaki, H., Kondoh, M., Watanabe, H., Yanagisawa, M., Masaki, T. and Murakami, K. (1989) FEBS Lett. 257, 208-210. Fu, T., Chang, W., Ishida, N., Saida, K., Hitsui, Y., Okano, Y. and Nozawa, Y. (1989) FEBS Lett. 257, 351-353. Rozengurt, E. (1986) Science 234, 161-166. Rozengurt, E., Erusalimsky, J., Mehmet, H., Morris, C., N&berg, E. and Sinnett-Smith, J. (1988) Cold Spring Harbor Symp. Quant. Biol. 53, 945-954. Zachary, I., Wall, P.J. and Rozengurt, E. (1987) Dev. Biol. 124, 295-308. Brown, K.D. and Littlewood, C.J. (1989) Biochem. J. 263, 977-980. Ohnishi, A., Yamaguchi, K., Kusuhara, M., Abe, K. andimura, S. (1989) Biochem. Biophys. Res. Commun. 161, 489-495. Rozengurt, E. and Sinnett-Smith, J. (1983) Proc. Natl. Acad. Sci. USA. 80, 2936-2940. Mendoza, S.A., Schneider, J.A., Lopez-Rivas, A., Sinnett-Smith, J.W. and Rozengurt, E. (1986) J. Cell Biol. 102, 2223-2233. Zachary, I. and Rozengurt, E. (1985) Proc. Natl. Acad. Sci. USA. g, 7616-7620. Zachary, I. and Rozengurt, E. (1987) J. Biol. Chem. 262, 3947-3950. Lopez-Rivas, A., Mendoza, S.A., Nlnberg, E., Sinnett-Smith, J. and Rozengurt, E. (1987) Proc. Natl. Acad. Sci. USA. 84, 5768-5772. Erusalimsky, J.D. and Rozengurt, E. (1989) J. Cell. Physiol. 141, 253-261. Simonson, M.S., Wann, S., Mene, P., Dubyak, G.R., Kester, M., Nakarato, Y., Sedor, J.R. and Dunn, M.J. (1989) J. Clin. Invest. 83,
22.
708-712.
Komuro, I., Kurihara, (1988) FEBS Lett.
H., Sugiyama, 238, 249-252. 167
T.,
Takaku,
F. and Yazaki,
Y.
167.
February
No.
BIOCHEMICAL
1, 1990
28,
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
161-167
VASOACTIVRINTESTINAL CONTRACTOR, A NOVRLPEJ'TIDE, SHARESA COMHON RECEPTOR WITB RNDOTRRLIN-1AND STIMULATESCa MOBILIZATION AND DNA SYNTRRSISIN SWISS3T3 CELLS Isabel
Fabregat
Imperial Lincoln’s
Received
December
and Enrique
Rozengurt
Cancer Research Fund, P 0 Box 123, Inn Fields, London WCZA 3PX, U.K.
27,
1989
SUHHARY : Vasoactive intestinal contractor peptide (VIC), a novel member of the en&thelin family, stimulated a rapid increase in the intracellular Ca concentration in fura-Z-loaded Swiss 3T3 cells. Sequential addition of VIC and endothelin-1 (ETl) demonstrated the induction of both homologous and heterologous deser&;‘;.ti;z, i;IC was as potent as ET1 in displacing the binding of stimulating mitogenesis in Swiss 3T3 cells. These findit& suggest that VIC and ET1 share a common receptor in Swiss 3T3 cells. @1990 Academic Press, Inc.
Vasoactive identified
intestinal
as a new member of VIC differs
(1,2). Zl-residue
4, 6, 7).
other
,tissues
inducing
function
from
The
peptide
is
contraction
least
three
distinct
known
whether
these
receptors
the
(3),
expressed
of mouse
and chemical
(1) ileum,
or by a distinct
the endothelin
effects
the
intestine
in
a
acid
residues but
more effective
suggesting
that
Binding
subtypes
of VIC are
receptor
family
(ETl), amino
studies
receptor
been
three
(2).
cross-linking
biological
in
and is
hormone
endothelin
of
or human endothelin
cells
as a gastrointestinal
has recently
peptides
peptide
or endothelial
measurements
(VIC)
the
porcine
vasoconstrictor
(nos
in
contractor
not
in
than
ET1
VIC may
competition
have
identified
(4-8). mediated
It
at is
not
by any of
subtype.
Abbreviations: ET , porciyy or human endothelin. VIC, vasoactive intest.inalntrac∨ [Ca Ii, intracellular Ca2+ concentration; epidermal growth factor; PBt2, phorbol 12,13-dibutyrate; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin.
EGF,
0006-291XN
161
$1.50
Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form resewed.
Vol.
167,
No.
1, 1990
BIOCHEMICAL
A recent in
the
report
intracellular
NG-108-15
and ET1 act binding
(9). not
but
via
to elucidate
growth
factors
cells act
receptor
the
with
for
stimulates growth
factor
we report
Swiss
ET1 and induces
more direct
have
induced that
evidence
a rapid
provided
pathways
(10-12).
VIC
(e.g.
in
model
the action
of
ET1 stimulates
that
the novel VIC shares
increase in
a useful
and DNA synthesis
3T3 cells.
DNA synthesis (EGF)
peptides suggesting
Ca 2+ mobilization
Here
increase
in neuroblastoma
these
transduction peptides
breakdown, (4,13,14).
but
a rapid
([Ca2+li) of
3T3 cells
signal
and mitogenic
caused
COMMUNICATIONS
presented.
of Swiss
as a mitogen
subsequently, epidermal
was not
ETl,
RESEARCH
desensitization,
receptors,
cultures
system
these
like
addition
heterologous
distinct
phosphoinositide
VIC,
of Ca ‘+
Sequential
competition)
Quiescent
that
BIOPHYSICAL
concentration
cells
homologous
also
showed
AND
the
in
[Ca2+li
presence
peptide
in VIC can
a common and of either
or insulin.
MATERIALS
AND MBTBODS
Swiss 3T3 cells were mainlained in culture and assays of DNA synthesis were performed by [ HI-thymidine incorporation as described (15). Determination of [Ca2+].: Confluent and quiescent Swiss 3T3 cells grown in 90 mm dishes were-:ashed twice with Dulbecco’s modified Eagle’s medium (DMEM) and incubated for 10 min in 5 ml DMEM containing 1 uM furatetraacetoxymethyl ester. After this incubation the cells were washed three times with electrolyte solution which contained 120 mM NaCl, 5 mM KCl, 1.8 mM CaC12, 0.9 mM MgC12, 25 mM glucose, 16 mM Hepes, 6 mM Tris and a mixture of amino acids at the same concentrations as those in DMEM (pH 7.2). The cells were suspended in el?ctrolyte solution (2 ml) by gently scraping and transferred to a 1 cm quartz cuvette. The cell suspension was stirred continuously at 37OC. Fluorescence was monitored in a Perkin-Elmer LS-5 luminescence spectrophotometer with an xcitation wavelenght of 336 nm and emission wavelength of 510 nT25 [Ca’+]. was calculated as described (16). Measurement of 125I-ETl binding: Swiss 3T3 cells were incubated at 4’C with 0.5 nM I-ET1 in phosphate buffered saline containing 25 mM N-2-Hydroxyethylpiperazlne-N’2-ethanesulfonic acid, 0.1% fatty acid free bovine serum albumin (BSA) (pH 7.0). After 3 hours, unbound radioactivity was removed by washing the cells three times with cold (4’C) phosphate buffered saline (pH 7.4) containing 0.1% BSA. The cells were extracted (20 min at 37’C) with 0.5 ml of 2% Na CO 1% .2 2’lvity sodium dodecyl sulfate, O.lN NaOH and cell associated radloac was determined in a LKB y-counter. Under these conditions, non-specific binding, determined as cell-associated radioactivity in the presence of 1 vM unlabelled ET1, was about 5% of the total. Materials: ET and sarafotoxin S6b were purchased from Peninsula Laboratories, Inc t U.K.). VIC was obtained from American Peptide Co. 162
Vol.
167,
No.
BIOCHEMICAL
1, 1990
AND
BIOPHYSICAL
RESEARCH
(U.S.A.). Bovine insulin, EGF, bombesin, vasopressin, lZ,l#ibutyrate (PBt ), BSA were from Sigma Chemical MO. I-ET1 was pure i ased from Amersham International,
COMMUNICATIONS
phorbol Co., St. Louis, Amersham, U.K.
RESULTSAND DISCUSSION Addition fluorescent measurable subsequently
of 10 nM VIC to quiescent Ca 2+ -indicator delay
(Fig.
declined
fura1). towards
[Ca’+]
Swiss
3T3 cells
increased
[Ca2+li
i reached
peak
the basal
level.
loaded
without values
Subsequent
at
with any
15 s and addition
VIC 4nM
VP
Figure 1. Effect of repeated additions of VIC and ET on [Ca*+].. asi Quiescent Swiss 3T3 cells in 90 mm dishes were loaded wit A furadelscribed in Materials and Methods. Fluorescence was recorded continuously while additions were made at the points and co,ncentrations indicated by the arrows: VIC, vasoactive intestinal contractor peptide; ETl, endothelin-1; VP, 10 nM vasopressin; BOM, 6 After a suitable control period, the peptides were added nM bombesin. and fluorescence followed until it returned to near base line levels. After this, additioft+of the same or a different peptide was performed. Measurements of ]Ca 1. were performed after sequential addition of Triton X-100 and ethyl&e glycol-bis(&aminoethyl ether) as previously described (16).
163
the
of
Vol.
167,
No.
1, 1990
either
BIOCHEMICAL
AND
10 nM VIC or 10 nM ET1 did
[C2+li.
Bombesin,
below)
caused
which
a marked
acts
exposed
sequential
responses
nM instead
of 10 nM and when
vasopressin desensitization Identical
completely
in
were
finally not
VIC
(17,18
and
Similar
i.e.
responses
of VIC was 4 with
caused
homologous
response could
by either
in
challenged only
subsequent
was reversed
[Ca2+] i caused
receptor
1, upper).
responsiveness
subsequent
rise
the concentration
Thus, the
COMMUNICATIONS
[Ca 2s ] i in cells
in
cells
blocked
cellular
the
a distinct
when
the
RESEARCH
any further
increase
elicited
addition
blocked
increase
also
in
of peptide
through
of bombesin.
but
elicit
VIC and ET1 (Fig.
were
changes
order
the
to both
instead
not
and rapid
previously
BIOPHYSICAL
to ET1.
be induced
when
the
ET1 either
at 10 or 4 nM
to VIC but
did
bombesin
not
prevent
or vasopressin
(Fig.
1, middle). A possible responses
interpretation
can be blocked
and activation (lower)
Treatment
10 nM vasopressin equivalent
to that
attenuated
the
Crucially,
exposure
[Ca2+li
Ca
caused in Fig.
presence
response
results
from
of a single
(Fig. class
in
not
in
Fig.
1
this fura-
with
[Ca2+li
addition the
1, lower). recently
and raised in Swiss a study
increase
in
The results reported
with
the possibility
that
ET1
3T3 cells. of the
binding
of these
cell-associated shown).
of vasopressin.
abolish
When cultures
at 4’C,
3 h (results
was saturable
not
those
prompted
3T3 cells.
mobilization
shown
with
increase
did
(9)
2+
to test
loaded
VIC or ET1 (Fig.
cells
the ET1 and VIC
Ca
The experiment
to a subsequent
different
125 I-ET1
causes
that
VIC or ET1 and markedly
to vasopressin
by either
a maximum after cells
by either
to a common receptor
with
that
3T3 cells
induced
of Swiss
is
was designed
a transient
2+
The preceding
incubated
(19,20) of Swiss
NG108-15
and VIC bind
C.
promoted
1 are
neuroblastoma
to cultures
kinase
vasopressin
possibility.
results
by any agonist
of protein
using
shown
of these
of cells
radioactivity 125 I-ETI
binding
1251-ET1 were reached
to 3T3
2) and Scatchard
analysis
indicated
the
of high-affinity
sites
(Kd = 0.96
f 0.23
164
Vol.
167,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
125 I-ET-l,
RESEARCH
COMMUNICATIONS
nM
Figure 2. Concentration dependence of 1251-ET1 bi’t$;“;T’obf;;a;; quiescent Swiss 3T3 cells. Specific cell-associated . Gas measured in quiescent cells incubated for 3 hour f2ft 4Tlin binding medium containing various concentrations of I-ET . Each point represents the mean of two determinations. Specific B,inding is expressed as pmol/mg of protein. Inset: Scatchard plot of the same datal. Bound (B) radiolabelledlJfgand is expressed in pmol/mg of protein; the concentration of I-ET1 in the medium (F) is in nM. All other experimental details were as described in Materials and Methods .
n=3)
nM;
(Fig. by
and a maximum
2, inset).
unlabelled
Binding
manner
vasopressin
(up
The
feature
saJient
demonstrate
for that
apparent
affinities.
(4,13)
depicted
DNA
synthesis
the
absence
synergistically
in or
other
in
Figs.
growth
ET1
cell
EGF. in
Fig.
eliciting
I-ET1
ET1
a common
share
virtually
can
stimulate
DNA
(21,22)
Swiss
4 shows
that
synthesis
synthesis
in
acting In
whether 3T3
findings
identical
factors.
DNA 165
125
displaced
with
nor
of 1251-ET1.
These
types
of
S6b in a
= 2 nM).
promoting
cultures of
EGF
VIC
and
competed
was
bombesin
binding
1 and 3, we tested
quiescent presence
VIC
that
and other
with
with
that peptides
shown
neither
that
protein
sarafotoxin
specific
3 is
of
3T3 cells
peptide
ET1 (IC50
time
have
to Swiss
the
Fig.
both
pmol/mg
In contrast,
as unlabelled
reports
synergistically
3).
first
of 0.5
cardiotoxic
in
recognises
3T3 cells
results
(Fig.
shown
the
receptor
Swiss
of lz5 I-ET1
to 10 uM) reduced
as potently
Recent
capacity
ET1 and by the
dose-dependent
binding
binding
view
of
the
VIC can stimulate
cells
incubated
in
VIC acted in
Swiss
3T3
Vol.
167,
No.
1, 1990
BIOCHEMICAL
AND
BJOPHYSICAL
RESEARCH
COMMUNICATIONS
: 5;[5,, ,,y\ 1o-2
Id'
0
3
1
ld
lo*
lo3
lo4
0
04
Peptide,nM
1 nM
0.1
Peptide,
10
125 I-ET1 binding in intact, Figure 3. Inhibition of specific quiescent Swiss 3T3 cells by ET (o), VIC (o), sarafotoxin S6b (A), Confluent and quiescenf%S;l;;res bombesin (A) and vasopressin ( b). of 3T3 cells were incubated at 4OC for 3 hours with 0.5 nM either in the presence or in the absence of the related or unrelatkd peptides at the concentrations indicated. Each py@t represents the Binding of I-ET is expressed mean of at least two determinations. as a percentage of the specific binding in the absence o t any unlabelled peptide (100% = 0.147 + 0.014 pmol/mg of protein). Figure 4. Stimulation of DNA synthesis in intact quiescent Swiss for DNA synthesis was performed 3T3 cells by VIC and ET . The assay n , ET ) or in the presence of 5 rig/ml EGF in the absence ( 0 , VIC; or 1 ug/ml insulin (inset) (0, VICf l , ETl). Confluent and quiescent cultures of Swiss 3T3 cells were wafhed and incubated in 2 ml DMEM/Waymouth’s medium containing [ HI-thymidine, with or without insulin or EGF and various concentrations of VIC or ETl. The incorporation of radioactivity into acid-precipitable material was measured after 40 h. Each point represents the mean of 2 determinations. In this experiment, values obtained in the presence of either 10% fgtal bovine seruy or 5 rig/ml EGF plus 1 ug/ml insulin cpm culture, respectively. were 8.65 x loand 7.77 x 10
Maximum
cells.
and
respectively.
nM,
An
obtained
when
ET1
observed
that
VIC,
combination
with
activate
protein
cellular
level VIC
in
the
is
of
neuroblastoma
instead
ET,,,
insulin
(Fig.
kinase
C (e.g.
CAMP
(e.g.
peptide
VIC and ET1,
(9)
this
achieved
in
question
In
4,
or
agents
rig/ml
PBt2)
inset)
and
the
remained 166
other
and
0.4
was experiments,
synthesis
in that or
family cells
responses
2.5
we
synergistic either
increase
the
IBMX).
endothelin
endothelial
suggested
VIC. DNA
lo-100
at
relationship
stimulated
the
contractile cells
of
forskolin of
not
were
dose-response
added
like
but
concerning
effects
identical
was
a novel
intestine
studies
for
half-maximal
(2) existence
which
(1,Z).
is
expressed
Although
recent
and Ca 2+ mobilization of
separate
to be elucidated.
in
receptors
Here
we
Vol.
167,
No.
show,
BIOCHEMICAL
1, 1990
for
the
receptor
in
first
Swiss
time,
that
VIC,
like
synergistically preferentially the target
possibility cells
that
3T3 cells,
and cross-desensitization ET1, with
is
other
in the
2+
growth in
RESEARCH
the
novel
signals.
mitogen
for
COMMUNICATIONS
a common
binding
Our results
promoting
competition demonstrate
3T3 cells
acting
factors.
Since
intestine
(l),
peptide
may act
gastrointestinal
with
by 125 I-ET1
as judged
a potent
this
BIOPHYSICAL
VIC and ET1 interact
of Ca
expressed that
AND
these
findings
as a growth
VIC is raise factor
for
tract.
REFERENCES 1.
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Saida, K., Mitsui, Y. and Ishida, N. (1989) J. Biol. Chem. 264, 14613-14616. Ishida, N., Tsujioka, K., Tomoi, M., Saida, K. and Mitsui, Y. (1989) FEBS Lett. 247, 337-340. 'Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masakf, T. (1988) Nature 332, 411-415. 'Takuwa, N., Takuwa, Y., Yanagisawa, M., Yamashita, K. and Masaki, T. (1989) J. Biol. Chem. 2, 7856-7861. Watanabe, H., Miyazaki, H., Kondoh, M., Masuda, J., Kimura, S., Yanagisawa, H., Masaki, T. and Murakami, K. (1989) Biochem. Biophys. Res. Commun. 161, 1252-1259. Kloog, Y., Bousso-Mittler, D., Bdolah, A. and Sokolovsky, M. (1989) FEBS Lett. 253, 199-202. Sugiura, M., Snajdar, R.M., Schwartzberg, M., Badr, K.F. and Inagami, T. (1989) Biochem. Biophys. Res. Commun. 162, 1396-1401. Masuda, Y., Miyazaki, H., Kondoh, M., Watanabe, H., Yanagisawa, M., Masaki, T. and Murakami, K. (1989) FEBS Lett. 257, 208-210. Fu, T., Chang, W., Ishida, N., Saida, K., Hitsui, Y., Okano, Y. and Nozawa, Y. (1989) FEBS Lett. 257, 351-353. Rozengurt, E. (1986) Science 234, 161-166. Rozengurt, E., Erusalimsky, J., Mehmet, H., Morris, C., N&berg, E. and Sinnett-Smith, J. (1988) Cold Spring Harbor Symp. Quant. Biol. 53, 945-954. Zachary, I., Wall, P.J. and Rozengurt, E. (1987) Dev. Biol. 124, 295-308. Brown, K.D. and Littlewood, C.J. (1989) Biochem. J. 263, 977-980. Ohnishi, A., Yamaguchi, K., Kusuhara, M., Abe, K. andimura, S. (1989) Biochem. Biophys. Res. Commun. 161, 489-495. Rozengurt, E. and Sinnett-Smith, J. (1983) Proc. Natl. Acad. Sci. USA. 80, 2936-2940. Mendoza, S.A., Schneider, J.A., Lopez-Rivas, A., Sinnett-Smith, J.W. and Rozengurt, E. (1986) J. Cell Biol. 102, 2223-2233. Zachary, I. and Rozengurt, E. (1985) Proc. Natl. Acad. Sci. USA. g, 7616-7620. Zachary, I. and Rozengurt, E. (1987) J. Biol. Chem. 262, 3947-3950. Lopez-Rivas, A., Mendoza, S.A., Nlnberg, E., Sinnett-Smith, J. and Rozengurt, E. (1987) Proc. Natl. Acad. Sci. USA. 84, 5768-5772. Erusalimsky, J.D. and Rozengurt, E. (1989) J. Cell. Physiol. 141, 253-261. Simonson, M.S., Wann, S., Mene, P., Dubyak, G.R., Kester, M., Nakarato, Y., Sedor, J.R. and Dunn, M.J. (1989) J. Clin. Invest. 83,
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