Vol.
174,
January
No. 15,
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1991
Pages
Endothelin-3
Stimulates
Nitric Toshiaki Satoru
Oxide
Production via
of
Phosphoinositide
228-235
Endothelium-Derived Breakdown
Yukio Hirata*, Kazuo Kanno, Kazuki Eguchi, Taihei Imai, Masayoshi Shichiri and Fumiaki Marumo
Ohta,
Emori,
Second Department of Internal Medicine, Tokyo Medical and Dental University, l-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan Received
November
26,
1990
Summary: Cultured bovine endothelial cells (EC) have specific receptors for endothelin (ET)-3 functionally coupled to phosphoinositide breakdown. We studied whether ET-3 stimulates synthesis of nitric oxide (NO), an endothelium-derived relaxing factor that activates soluble guanylate cyclase in EC, and whether the ET-3-induced NO formation involves G-proteins. ET-3 dose-dependently stimulated production of intracellular cGMP in EC, of which effects were abolished by pretreatment with No-monomethyl =-arginine, an inhibitor of NO synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The stimulatory effects of ET-3 on cGMP production, inositol trisphosphate formation and increase in cytosolic free Caz+ concentration were similarly These data blocked by pretreatment with pertussis toxin (PTX). suggest that ET-3 induces synthesis of NO mediated by phosphoinositide breakdown via PTX-sensitive G-protein in EC. 0 1‘391 Academic Press,Inc.
There important
is role
elaborating
a growing in
several
awareness
intrinsic potent
that
modulation vasoactive
endothelium
plays
of
tone
vascular
substances
(1),
an by
including
* All correspondence should be addressed to Yukio Hirata, M.D., Second Department of Internal Medicine, Tokyo Medical and Dental University, l-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan. Abbreviations: EC, endothelial cells; ET, endothelin; NO, nitric oxide; =-NMMA, NQ-monomethyl ,-arginine; IP3, inositol-1,4,5trisphosphate; [CaZ+ ]i, cytosolic free Ca2+ concentration; PTX, pertussis toxin, EDRF; endothelium-derived relaxing factor; SNP, sodium nitroprusside; VSMC, vascular smooth muscle cells. 0006-291X/91 Copyrighi All rights
$1.50
0 1991 by Academic Press. Inc. oj. reproduction in any form reserved.
228
Vol.
174,
No.
BIOCHEMICAL
1, 1991
such
vasodilators,
as
endothelin
mainly from
cells
(3)
,-arginine
(,-NMMA), to
is
as
the
a novel
characterized
genomic
cyclase in
DNA cloning
of
of
porcine human
ET-l,
three
ET isopeptides,
ET-3
the
ET-3
response
receptors
coupled
to
free
Caz+
study
was
synthesis
through
recently in
cultured
bovine
EC,
of
cultured
bovine
NO formation
(5).
the and
origi-
Subsequent
(6).
presence
ET-3
potent
(7).
of Among
initial mechanism of
specific
which
are
functionally
and
increase
in
Therefore,
the
whether
ET-3
EC,
and whether
involves
has
,-arginine
presence
(9).
investigate
muscle
No-monomethyl
endothelium-dependent
([Ca"+]i) to
and
peptide,
ET-2
breakdown
designed
synthesized
analogue,
revealed
the
be
smooth
Recently,
NO from
to
AZ
,-arginine,
vascular
most
demonstrated
concentration
receptor-mediated
shows the
phosphoinositide
NO in
of
ET genes
designated
We have
atom
endothelium
ET isopeptides,
(8).
is
vasoconstrictor
three
depressor
which
in
(EDRF)
thromboxane
(2),
arginine
21-residue from
as
shown
EC (4).
formation
factors
been
a stereospecific
antagonize
ET-l nally
as well
(NO)
nitrogen
guanylate
COMMUNICATIONS
relaxing
recently
oxide
guanidido
RESEARCH
such
EDRF has
nitric
soluble (VSMC)
shown
to
terminal
stimulates
endothelium-derived
(ET)-1.
ascribed the
BIOPHYSICAL
and vasoconstrictors,
and prostacyclin, and
AND
cytosolic present
stimulates
GTP-binding
the proteins
(G-proteins). MATERIALS
AND METHODS
Drugs: ET-3 was obtained from Peptide Institute Inc. (Osaka, =-NMMA from Calbiochem Co., Ltd. (La Jolla, CA, USA), Japan), sodium nitroprusside (SNP) and 3-isobutyl-l-methyl=-arginine, xanthine from Sigma Chemical (St. Louis, MO, USA), methylene blue from Schmid GMBH & Co.(Stuttgart-Unterturkheim, FRG), pertussis toxin (PTX) from Kaken Pharmaceutical Co., Ltd. (Tokyo, Japan) and furaacetoxymethyl ester (AM) from Dojin Chemicals (Kumamoto, Japan). Cell culture: Bovine ECs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal calf serum as 229
Vol.
174,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
described (9); the subcultured EC (9-14th passage) was used in the experiments. Determination of intracellular cGMP: Confluent ECs were incubated at 37-C for 10 min in Hanks' medium containing 0.5 mM 3-isobutyl-l-methylxanthine as described (10). After extraction with 6 % trichloroacetic acid, concentrations of intracellular cGMP were determined by cGMP radioimmunoassay kit (New England Nuclear, Boston, MA). Measurement of [Ca"] : Suspended ECs were incubated with 4 ,U M furaAM at 37-C fori min , and the fluorescence of Caz+furaof the suspended cells ( - 5~10~ cells/ml) was measured by a spectrofluorimeter (CAF-100; Jasco Co., Ltd., Tokyo, Japan) as decribed (9). Measurement of inositol-1,4,5-trisphosphate (IP,): Confluent ECs were incubated at 37-C for 30 set in Hanks' medium containing 10 mM LiCl, and IP, levels were determined by a competitive protein binding assay kit (Amersham Japan, Tokyo), as described (9). RESULTS ET-3
dose-dependently
intracellular maximal Table
of
cGMP in bovine
dose to 1, the
production
(1O-9-1O-7
induce
effect
was completely M),
EC (Fig.
1);
cGMP formation
stimulatory
L-NMMA (2~10-~
M) stimulated
of
abolished
of which
Ok?
I'
the
synthesis
approximate
was 5~10~~ M. ET-3
(lode
by the
effect
half-
As shown in
M) on cGMP
simultaneous
was reversed
addition
when
lo-10 IO-Q 10-8 10-7 ET-3
(M)
Fiq. 1. Dose-responsive effect of ET-3 on cGMP production in cultured bovine EC. Confluent cells were incubated at 37-C for 10 min with various doses (10-10-10-7 M ) of ET-3 in the presence of isobutylmethylxanthine. Each point is the mean of 6 dishes; bars shows SE. *P
174,
January
No. 15,
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1991
Pages
Endothelin-3
Stimulates
Nitric Toshiaki Satoru
Oxide
Production via
of
Phosphoinositide
228-235
Endothelium-Derived Breakdown
Yukio Hirata*, Kazuo Kanno, Kazuki Eguchi, Taihei Imai, Masayoshi Shichiri and Fumiaki Marumo
Ohta,
Emori,
Second Department of Internal Medicine, Tokyo Medical and Dental University, l-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan Received
November
26,
1990
Summary: Cultured bovine endothelial cells (EC) have specific receptors for endothelin (ET)-3 functionally coupled to phosphoinositide breakdown. We studied whether ET-3 stimulates synthesis of nitric oxide (NO), an endothelium-derived relaxing factor that activates soluble guanylate cyclase in EC, and whether the ET-3-induced NO formation involves G-proteins. ET-3 dose-dependently stimulated production of intracellular cGMP in EC, of which effects were abolished by pretreatment with No-monomethyl =-arginine, an inhibitor of NO synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The stimulatory effects of ET-3 on cGMP production, inositol trisphosphate formation and increase in cytosolic free Caz+ concentration were similarly These data blocked by pretreatment with pertussis toxin (PTX). suggest that ET-3 induces synthesis of NO mediated by phosphoinositide breakdown via PTX-sensitive G-protein in EC. 0 1‘391 Academic Press,Inc.
There important
is role
elaborating
a growing in
several
awareness
intrinsic potent
that
modulation vasoactive
endothelium
plays
of
tone
vascular
substances
(1),
an by
including
* All correspondence should be addressed to Yukio Hirata, M.D., Second Department of Internal Medicine, Tokyo Medical and Dental University, l-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan. Abbreviations: EC, endothelial cells; ET, endothelin; NO, nitric oxide; =-NMMA, NQ-monomethyl ,-arginine; IP3, inositol-1,4,5trisphosphate; [CaZ+ ]i, cytosolic free Ca2+ concentration; PTX, pertussis toxin, EDRF; endothelium-derived relaxing factor; SNP, sodium nitroprusside; VSMC, vascular smooth muscle cells. 0006-291X/91 Copyrighi All rights
$1.50
0 1991 by Academic Press. Inc. oj. reproduction in any form reserved.
228
Vol.
174,
No.
BIOCHEMICAL
1, 1991
such
vasodilators,
as
endothelin
mainly from
cells
(3)
,-arginine
(,-NMMA), to
is
as
the
a novel
characterized
genomic
cyclase in
DNA cloning
of
of
porcine human
ET-l,
three
ET isopeptides,
ET-3
the
ET-3
response
receptors
coupled
to
free
Caz+
study
was
synthesis
through
recently in
cultured
bovine
EC,
of
cultured
bovine
NO formation
(5).
the and
origi-
Subsequent
(6).
presence
ET-3
potent
(7).
of Among
initial mechanism of
specific
which
are
functionally
and
increase
in
Therefore,
the
whether
ET-3
EC,
and whether
involves
has
,-arginine
presence
(9).
investigate
muscle
No-monomethyl
endothelium-dependent
([Ca"+]i) to
and
peptide,
ET-2
breakdown
designed
synthesized
analogue,
revealed
the
be
smooth
Recently,
NO from
to
AZ
,-arginine,
vascular
most
demonstrated
concentration
receptor-mediated
shows the
phosphoinositide
NO in
of
ET genes
designated
We have
atom
endothelium
ET isopeptides,
(8).
is
vasoconstrictor
three
depressor
which
in
(EDRF)
thromboxane
(2),
arginine
21-residue from
as
shown
EC (4).
formation
factors
been
a stereospecific
antagonize
ET-l nally
as well
(NO)
nitrogen
guanylate
COMMUNICATIONS
relaxing
recently
oxide
guanidido
RESEARCH
such
EDRF has
nitric
soluble (VSMC)
shown
to
terminal
stimulates
endothelium-derived
(ET)-1.
ascribed the
BIOPHYSICAL
and vasoconstrictors,
and prostacyclin, and
AND
cytosolic present
stimulates
GTP-binding
the proteins
(G-proteins). MATERIALS
AND METHODS
Drugs: ET-3 was obtained from Peptide Institute Inc. (Osaka, =-NMMA from Calbiochem Co., Ltd. (La Jolla, CA, USA), Japan), sodium nitroprusside (SNP) and 3-isobutyl-l-methyl=-arginine, xanthine from Sigma Chemical (St. Louis, MO, USA), methylene blue from Schmid GMBH & Co.(Stuttgart-Unterturkheim, FRG), pertussis toxin (PTX) from Kaken Pharmaceutical Co., Ltd. (Tokyo, Japan) and furaacetoxymethyl ester (AM) from Dojin Chemicals (Kumamoto, Japan). Cell culture: Bovine ECs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal calf serum as 229
Vol.
174,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
described (9); the subcultured EC (9-14th passage) was used in the experiments. Determination of intracellular cGMP: Confluent ECs were incubated at 37-C for 10 min in Hanks' medium containing 0.5 mM 3-isobutyl-l-methylxanthine as described (10). After extraction with 6 % trichloroacetic acid, concentrations of intracellular cGMP were determined by cGMP radioimmunoassay kit (New England Nuclear, Boston, MA). Measurement of [Ca"] : Suspended ECs were incubated with 4 ,U M furaAM at 37-C fori min , and the fluorescence of Caz+furaof the suspended cells ( - 5~10~ cells/ml) was measured by a spectrofluorimeter (CAF-100; Jasco Co., Ltd., Tokyo, Japan) as decribed (9). Measurement of inositol-1,4,5-trisphosphate (IP,): Confluent ECs were incubated at 37-C for 30 set in Hanks' medium containing 10 mM LiCl, and IP, levels were determined by a competitive protein binding assay kit (Amersham Japan, Tokyo), as described (9). RESULTS ET-3
dose-dependently
intracellular maximal Table
of
cGMP in bovine
dose to 1, the
production
(1O-9-1O-7
induce
effect
was completely M),
EC (Fig.
1);
cGMP formation
stimulatory
L-NMMA (2~10-~
M) stimulated
of
abolished
of which
Ok?
I'
the
synthesis
approximate
was 5~10~~ M. ET-3
(lode
by the
effect
half-
As shown in
M) on cGMP
simultaneous
was reversed
addition
when
lo-10 IO-Q 10-8 10-7 ET-3
(M)
Fiq. 1. Dose-responsive effect of ET-3 on cGMP production in cultured bovine EC. Confluent cells were incubated at 37-C for 10 min with various doses (10-10-10-7 M ) of ET-3 in the presence of isobutylmethylxanthine. Each point is the mean of 6 dishes; bars shows SE. *P
