Vol.
170,
No.
July
16, 1990
1, 1990
BIOCHEMICAL
AND
Mayumi Yujiro
Furuya*, Hayashil,
Institute
1 Suntory 2 National
COMMUNICATIONS Pages
NOVEL NATRIURETIC PEPTIDE, CNP, POTENTLY PRODUCTION IN RAT CULTURED VASCULAR
Suntory
RESEARCH
BIOPHYSICAL
Maki Takehisa, Norio Ohnumal Kenji Kangawa3,
for
Biomedical
Bio-Pharma
Tech
Cardiovascular
Center
STIMULATES CYCLIC GMP SMOOTH MUSCLE CELLS
Yoshiharu Minamitake, Yasuo Kitajima, , Takafumi Ishihara, Naoto Minamino2, and Hisayuki Matsuo2
Research,
Center,
201-208
Shimamoto,
Akaiwa,
Research
Mishima,
Chiyoda,
Institute,
Ohra,
Osaka
618, Japan
Gunma
370-05,
Japan
Suita,
Osaka
565, Japan
Fujishirodai,
3 Department of Biochemistry, Miyazaki Medical College, Kihara, Kiyotake, Miyazaki 889-l 6, Japan Received
May 30,
1990
SUMMAR‘Y : The newly identified peptide C-type natriuretic peptide (CNP) caused only a slight elevation of cGMP in rat renal glomeruli. In contrast, CNP potently increased cGMP levels in cultured rat vascular smooth muscle cells (VSMC) and stimulated guanylate cyclase activity in the particulate fraction of the cells. The extent of maximum activation of the enzyme induced by CNP was 4-fold higher than that by human atria1 natriuretic peptide (a-hANP) while CNP was 4- and 16fold weak’er than a-hANP in binding affinity for the putative receptors on VSMC and vasorelaxant activity for rat aorta, respectively. These results indicate that CNP is a potent stimulator of cGMP formation in VSMC but not in glomeruli and pharmacological feature of CNP is distinct from that of ANP. 0 1990 AcademrcPress, Inc.
Atria1
natriuretic
regulation
of
body
2).
Identification
heart
(3-5),
to those
which
of human
blood
pressure
third
type: natriuretic
identified to both quite
unilque
fluid of
(ANP)
and brain
has been ANP
brain
feature
*To whom correspondence
blood
pressure
natriuretic
N-terminal
should
mammalian effects
the possibility ANP
has amino
amino
acids,
linkage; and
(1,
brain
and
Very
similar
ends
and
recently, peptide)
sequences
the
functions very
natriuretic acid
in
that water-salt
and BNP.
(C-type
by a disulfide
five
role
in
as CNP CNP
important
physiological
(BNP)
by both
flanked
an
pharmacological
6). discloses
Porcine
play
and other
to elicit
designated
(7).
to
peptide
reported
in a ring in
known
are regulated
pcptide
and BNP
is
(a-hANP)(3,
homeostasis
in porcine ANP
peptide
a was
homologous
however,
it has a
at
second
the
be addressed.
Abbreviations: ANP; atria1 natriuretic peptide (A-type natriuretic peptide), BNP; brain natriuretic peptide (B-type natriuretic peptide). CNP; C-type natriuretic SDS; sodium dodecyl sulfate, PAGE; peptide, tx-hANP; human pro-ANP[99-1261, polyacrylamide gel electrophoresis. 0006-291x/90 201
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
cysteine along
residue with
BIOCHEMICAL
1, 1990
with
porcine
no C-terminal
ANP
CNP ,
and BNP
The
are listed
RESEARCH
amino
COMMUNICATIONS
acid sequences
of porcine
CNP
below.
S-L-R-R-S-S-C-F-G-G-R-M-D-R-I-G-A-Q-S-G-L-G-C-N-S-F-R-Y
BNP ,
S-P-K-T-M-R-D-S-G-C-F-G-R-R-L-D-R-I-G-S-L-S-G-L-G-C-N-V-L-R-R-Y
We
found
that
porcine
hypotensive
actions
in
activity
porcine
CNP
of
suggesting
that
and/or
elevation
CNP
BNP.
and the
and cultured which
tail.
BIOPHYSICAL
G-L-S-K-G-C-F-G-L-K-L-D-R-I-G-S-M-S-G-L-G-C
ANP ,
ANP
AND
than
was
plays
unique activity
smooth identical
elicited ANP
much and
which
cells,
to porcine MATERIALS
and
ANP
diuretic/natriuretic
that
potenl might
porcine
chick than
rectum that
be distinct
we have focused
of synthetic
muscle
potent
and
more
funcrions report,
less
BNP,
3 to 4 times
In the present binding
vascular
is structurally
CNP
rats
from
on the potency
CNP
compared
of
in
with
rat renal those
and relaxant ANP
(7).
those
of
for cGMP glomeruli
of a-h ANP
(8).
AND METHODS
Peptides: Alpha-hANP and porcine CNP were synthesized by solid phase method as described previously (7, 9). We also synthesized a CNP derivative which was elongated with a tyrosine residue at the N-terminal, called YO-CNP for radioiodination. Equivalent potency of Y”-CNP to CNP was confirmed in cGMP accumulation and binding activity in VSMC, and vasorelaxant activity in rat aorta. Iodination of a-hANP and Y”-CNP were carried out by the lactoperoxidase method, and the specific activities of both labeled peptides were equally 22.2MBq/pg. Preoaration of rat renal Plomeruli: Kidneys were dissected from male SpragueDawley rats of 8-9 weeks old. Renal glomeruli were isolated by sieving method (10). Purity and the number of glomeruli were evaluated by light microscopy. Culture of rat vascular smooth muscle cells (VSMC): VSMC were derived from explants of Sprague-Dawley rats aorta (1 I), and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco). Cells were used for experiments between 3th to 7th passage. cGMP accumulation in rrlomeruli and VSMC: A. Glomeruli; Isolated renal glomeruli were incubated with peptides in 125~1 Krebs-Henseleit solution containing 1mM lmethyl 3-isobutylxanthine (MIX) at 30°C for 2min. Concentration of cGMP in the glomeruli was quantified by using a cGMP radioimmunoassay kit (Yamasa Shoyu) (12). B. VSMC; Cells were incubated with peptides in 0.25ml DMEM containing 0.1% BSA, 20mM HEPES and OSmM MIX, pH 7.4 at 37°C for 10min. Cellular cGMP concentration was determined by radioimmunoassay (13). Gu an v 1ate cvc I ase activitv in VSMC membranG: Confluent VSMC were scrapped and homogenized in 8 volumes of 50mM Tris-HCI, pH 7.4 containing 0.25M sucrose, ImM EDTA, 1mM dithiothreitol, 0.2mM phenylmethylsulfonyl fluoride (PMSF) and 2Opg/ml aprotinin. The homogenates were centrifuged at 100,OOOxg for 60min. and particulate fraction was washed and resuspended in the same buffer. The activity of guanylate cyclase in particulate fraction was determined at 37°C in 50mM TrisHCl, pH 7.6, containing 1mM GTP, 5mM MgCl2, 7.5mM phosphocreatine, 13units/ml creatine kinase and 0.5mM MIX with or without 0.5mM ATP. Concentration of cGMP generated during 3min incubation was determined by radioimmunoassay. &mtOr bindine assay: A. Glomeruli; Isolated glomeruli were incubated with 5x 10-l”M [1251]a-hANP (ca. 3x105cpm) in the presence of unlabeled peptide in 0.25ml of 1OmM Tris-HCI, pH 7.4 containing 1OmM MgC12, 1mM EDTA, 0.25M sucrose, 0.2% bovine serum albumin (BSA) and 1mM PMSF at 0°C for 30 min. At the end of the incubation, lml of the ice-cold buffer was added and aliquots were centrifuged 202
Vol.
170,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
at 10,OOOxg for Smin. Radioactivity within the pellet was counted by a yspectrophotometer (12). B. VSMC; Cells were incubated with indicated concentrations of [1251]a-hANP or [ 1251]YO-CNP in the presence or absence of the unlabeled peptide in 0.25ml DMEM containing 0.1% BSA and 20mM HEPES, pH 7.4 for 45min at 25°C. Following wash with the same medium, the cells were solubilized with 0.5M NaOH and radioactivity was measured (13). Affinity-crosslinking of labeled oentide with VSMC: VSMC were incubated with 5x10-lOM [1251]a-hANP or [125 I]Y”-CNP in the presence or absence of 5x10T7M unlabeled peptide in the same medium described above at 25°C for 45min. After washing for 3 times with lml of phosphate-buffered saline (PBS), cells were then added with 0.25ml of O.lmM disuccinimidyl suberate (DSS) and incubated on ice for 30min. ‘The DSS solution was aspirated and cells were scraped from each well in 0.5ml of PBS, followed by centrifugation. Cell pellets were resuspended in O.lml SDS sample buffer (60mM Tris-HCI, pH 6.8, 27% glycerol, 0.3% SDS) with 5% 2mercaptoethanol. After boiling for Smin, samples were run on 7.5% polyacryla:mide gels (14). Dried gels were exposed to Kodak X-Omat AR film at -80°C for 7 days. Vasorelaxant assav: Aortic strips were prepared from thoracic aorta of SpragueDawley rats and mounted in a 7ml chamber containing Krebs-Henseleit solution at 37°C. ‘The strips were contracted with 2xlO-7M norepinephrine and once the contractile: response stabilized, they were exposed to peptide cumulatively. RESULTS The renal
effect
of CNP
glomeruli
and
on accumulation cultured
glomeruli,
1.6-fold
increase
of 10e5M
of CNP.
Whereas,
EC50 of
of 3.6x10-7M
cGMP
was
(Fig.
evoked
rat of
cGMP
a-hANP
1A).
by
concentration
up to 160-fold
Alpha-hANP
was less effective
of cGMP
vascular
muscle
concentration
was
increased
In contrast
CNP
in
of basal
rat
cGMP
to renal
VSMC
level
than CNP,
was investigated
smooth
(VSMC).
induced
by
contents glomeruli,
(Fig.
at 10m6M
and the EC50
1B). with
using
cells
rat isolated In
an application
up to 5.4-fold a marked CNP
renal
with
increased
cGMP
an EC5 0 of 7.7x10b8M
was 3.7x10d7M
and maximum
IJ/ -6
-7
-6
Augmentation of cGMP concentrations induced by CNP (closed circles) or Figure 1. ANP (open circles) in rat renal glomeruli (A) and rat vascular smooth muscle cells Renal glomeruli or VSMC were incubated with the peptide in the (VSMC) (B). Data represent mean presence of MIX as described in “Materials and Methods”. of triplicate determinations from two or three separate experiments in values glomeruli or VSMC, respectively. 203
an
accumulation
-5
.
Vol.
170,
No.
elevation
BIOCHEMICAL
1, 1990
was 50-fold
10m5M
at 10e5M.
of a-hANP,
(none,
partial
0.04+0.02;
a-hANP,
pmole/l05celIs,
mean+S.E.
Membrane particulate
fraction
When
BIOPHYSICAL
RESEARCH
the cells were exposed
reduction
of the cGMP
1.84rtO.10;
CNP,
to lo-6M
response
6.98kO.55;
COMMUNICATIONS
of CNP with
to CNP
was
CNP plus a-hANP,
VSMC
cyclase
was
prepared,
activity.
CNP
and
subjected
activated
the
to
by an addition In both with
The
were
VSMC,
ATP
glomet-uli
a less potent
hANP
stimulation
of 0.5mM
renal
without
than a-hANP
8.3~1O-~M
enzyme
by CNP and a-hANP
was
assay
more
of
potently
4-fold greater and 8.0x10-7
both
potentiated
a shift in the EC50. CNP
inhibited
the binding
in
glomeruli,
of [l 251]a-
h ANP
The IC50 values for CNP and a-
(Fig. 3A, B).
and 1.0x10-9M
and
3.5x10mgM
and
1.1x10-9M
in
respectively.
Fig. 4A demonstrates Analysis
of
dissociation
a specific
the saturation
suggesting
plot,
induced
and VSMC,
affinity
4.51kO.01
the
than a-hANP (Fig. 2). Maximum activation induced by CNP was about than that of a-hANP, and the EC50 for CNP and a-hANP was l.l~lO-~M M, respectively.
observed
of 3 determinations).
of
guanylate
AND
the
binding
presence
constant
fmole/105cclls
(Kd)
(Fig. 4B).
a Kd of 0.9x10-gM
and saturable
and
of
data of
according
a
single
3.Xx10-9M
almost
of [l 251]Yo-CNP
to Scatchard class
and
[1251]a-hANP an
binding
comparable
a maximum Bmax
resulted
receptor
of
with
33
in
with
VSMC.
a linear apparent
an
binding
to the cells
bound
in
(Bmax)
a higher
of 42
affinity
fmole/l05cclls
(data
of not
shown).
Affinity-crosslinking in a detection
of either
of only
one radioactive
condition.
Labeling
and a-hANP
at 5x10m7M
Figure 2. Stimulation
[l 251]a-hANP
of the
protein
band
approximately
of
W;IS
inhibited
-9 Cont.
-6 (log M)
almost
with
70kDa
VSMC
under
complctcly
by
resulted
a reduced both
(Fig. 5)
of particulate
(closed symbols) in comparison the reaction mixture for the incubated for 3 min at 37°C
0.5mM ATP.
or [ 1 25 I]Y”-CNP
-7
-6
guanylate cyclase activity
-5
of rat VSMC
by CNP
with ANP (open symbols). Peptides were added to enzyme assay at the indicated concentrations and in the absence (triangles) or presence (circles) of
Each value represents mean*S.E. 204
of triplicate
determinations.
CNP
Vol.
A.
170,
No.
BIOCHEMICAL
1, 1990
AND
120
100
80 -E : m
RESEARCH
COMMUNICATIONS
Bm 120 t
1
100
BIOPHYSICAL
a
80 -2 2 m
60
z
60
ae
40
40
20
20 I_
0
-'a. -71
-es
0 L;;:;c-;; -10 Cont.
-9
-8
-7
-6
.e3
-11
(log M)
-10
Cont.
-9
-8
-7
-6
(log M)
Figure 3. Displacement of 1125 I]a-hANP from binding sites by unlabeled CNP (open circles) or ANP (closed circles) in renal glomeruli (A) and VSMC (B). Renal glomendi or VSMC were incubated with 5x10- l”M [12511a-hANP in the presence of Data represent means of triplicate determinations from two or unlabeled peptides. four separate experiments in glomeruli or VSMC, respectively.
CNP
relaxed
aortic
strips
precontracted
by
norepinephrine
with
an
EC50
71.7k1.8 x10w9M (meankS.E. of 5 preparations), being approximately 16-fold than that of a-hANP (EC50=4.6fO.l x10-oh/l, mean+S.E. of 5 preparations). aortic
tissues
vasorelaxant
were
subjected
responses
to
to the both
assay
CNP
and
after
rubbing
a-hANP
out
were
higher When
the endothelium,
not
affected
of
the
(data
not
whereas,
the
shown). DISCUSSION In renal increment
glomeruli,
CNP competed
of the cGMP
concentration
for the binding
of [’ 251]a-hANP,
was very limited.
A.
This
low
activity
in cGMP
B. 0.5
0.4 -
0
1
2
Labeled
3
Peptide
4
Cl.0
5
0.1
0.2
0.3
Bound
(nM)
0.4
0.5
(nM)
binding t0 rat vs~c. VSMC were Figure 4. (A) Saturation curve of [ ~~~IIY~-CNP incubated at 25°C for 45min with increasing concentrations of [1251]Yo-CNP, and circles) and specific binding (open total (open circles), nonspecific (closed triangles) were measured. Nonspecific binding was determined by incubating the cells with [1251]YO-CNP in the presence of 10.6M unlabeled CNP. (B) Scatchard plot of the specific binding. Each point represents mean of quadruplicate determinations.
205
( 6
Vol.
170,
No.
BIOCHEMICAL
1, 1990
12
AND
34
BIOPHYSICAL
RESEARCH
Mr
56 , * i
COMMUNICATIONS
x lo- 3
-Origin
r-M
-
205
-97
-67 -45 Fi ure 5. Autoradiography of SDS-PAGE analysis under reduced condition of -+---I[I 5I]a hANP and [1251]Yo-CNP covalently coupled to the surface of intact cultured rat VSMC. Confluent cells were incubated with SXIO~~~M 11251]a-hANP or [12511YoCNP in the absence (lane 1 and 4), or prcscnce of ANP (lane 2 and 5) or CNP (lane 3 and 6) at each 5x10e7M, respectively. The migration of known molecular weight markers are shown to the right.
accumulation activity
in
On the Since
other
hand,
the simultaneous
resulted
peptides
mechanism. peptides
would
both
an inhibitor
kinetic
was 4-fold demonstrated a 7OkDa
that
protein,
production
of
cyclase,
did
soluble
guanylate
in VSMC
for cGMP
study
it elicits ANP.
diuretic
at a maximum
effective
at
least
partly,
that CNP as well and
ATP. not
rat VSMC. dose
it can be suggested
VSMC,
formation
revealed
that
of a-hANP.
the detectable
than
low
the
a
Furthermore, affect
the
same
as a-hA effects
that NP
of the
methylene
cGMP
responses
was greater
than that
(data not shown).
of CNP
would
the
in
CNP,
by the findings presence
to
concentrations
through,
in the
than that
and
by
accelerated
for
transduction
CNP
induced
in
does
in VSMC
with
elevation
activity
which
signal
cGMP
cyclase
One interpretation (15). and the cGMP elevation ANP
corresponded
of a-hANP
cGMP
binding
weaker
be
increased
be supported
the potency
of a-hANP,
potently
to
guanylate of
to these two peptides Although
CNP
of cGMP
particulate were
assumed
(7).
stimulate This
stimulated
is
in vivo addition
in a reduction
both
blue,
glomeruli
of the peptide
apparent
Crosslinking
natriuretic not involve
receptor
guanylate
cyclase
between
be that CNP shares Hirose
cyclase et al. 206
binding of
peptide
the discrepancy guanylate
in VSMC
the
on
VSMC
of to
CNP VSMC
was
mostly
domain
in the molecule
binding
to
a common,
activation have
affinity
the peptides
70kDa
through
demonstrated
the receptor receptor
a more the
with
efficient
presence
of
Vol.
170,
No.
BIOCHEMICAL
1, 1990
dissociative
complexes
inactivation
of
analysis,
for the activation
and
guanylate
cyclase
complex
; Rc*GC
(inactive)
guanylate
cyclase).
that
enzyme
peptide ANP
receptors,
natriuretic
peptide
these
has
been
BNP
to
found
give for
ANF’
residues
was l&fold
analogs
in
vasorelaxant
activities possessed of
cultured
cells
may
reported
previously it
hypotension. be present
This in
in various
interpretation
is
subtype
activated
of natriuretic
by CNP
rather
Recently,
(15,
18-20). of
been (20).
than
multiple One
both
of
ANP
suggested
and
that
CNP
might
vasorelaxant
activity
an be a
must
peptides,
or
from
effectively
in
reported
ii)
that
in
with analog,
observations sufficient
peptide
weak Lysl
to promote
smooth
in
the
muscles
as
be noteworthy
that
in glomeruli,
is originally
including
have
functions
concentration
CNP The system
increases
weak
vasorelaxant
observed
in porcine
ANP. nervous
significantly very
never
of
found than
central
CNP
and shows
in ANP
other
than
brain,
studies
and
cGMP activity
and BNP,
diuresis
and
is assumed
on the activities
are now
going
on.
REFERENCES 1. DeBold., 2.
3. 4. 5. 6.
A. J. (1985) Science, 230, 767-770. H. and Nakazato, H. (1987) Endocrinol.
Metab. Clin. North Am., 16, 43-61. Sudoh, T., Kangawa, K., Minamino, N. and Matsuo, H. (1988) Nature, 332, 78-81. Kojima., M., Minamino, N., Kangawa, K and Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 159, 1420-1426. Sudoh, T., Maekawa, K., Kojima, M., Minamino, N., Kangawa, K. and Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 159, 1427-1434. Itoh, H., Nakao, K., Yamada, T., Shirakami, G., Kangawa, K.. Minamino. N., Matsuo, H. and Imura, H. (1988) Eur. J. Pharmacol., 150, 193-197. Matsuo,
*
suggest
receptors
aortic
that
penicilamine
VSMC
factor
intact
by
an ANP
These
natriuretic
those
of the peptide
peptide
tissues
substituted
(22).
in
We have found
were
may not be a sole
physiological
a higher
in VSMC,
24).
properties
unique
property
different
but not
Such
production
elevation
be
residues
It has also been
similar
(23,
in VSMC
suggesting
CNP
(21).
natriuretic
conclusion,
in aorta.
GC:
Another
and it has be present
may
receptor,
the
than that of a-hANP.
cysteine
cGMP
that i) cGMP
vasorelaxation
In
(Rc:
concentrations
production
less potent
which
stimulated
[7-23]ANP
contents
response, receptor
effect of CNP on cGMP
also
possibilities
this
receptor-enzyme to dissociate
and sequenced high
may
be able
sites in VSMC.
cloned
that ANP
ligand.
rat aorta
some
cGMP for
(active)
radiation
be responsible
also
a certain
and
binding
by
putative
might
through
relatively
the
ANP.
occupied
been
require
ligand
the
To the potent isolated
to
have
a remarkable
natural
candidate
be mediated
cyclase
would
+ GC CNP
COMMUNICATIONS
have proposed
of
than
of the total
receptors
They
Rc*ANP
effectively
is preferably
a minority
guanylate
a dissociation +
RESEARCH
receptor
17).
to the model,
might
which
and
70kDa
(16,
+ ANP more
of CNP
and is only
unidentified1
through
complex
the action
that
cyclase
According
BIOPHYSICAL
receptor
suggested
of guanylate
activate
receptor
ANP
AND
207
to of
Vol.
170,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
7. Sudoh, T., Minamino, N. Kangawa, K. and Matsuo, H. (1990) Biochem. Biophys. Res. Commun., 168, 863-870 8. Forsmann, W.G., Birr, C., Carlquist, M., Christman, M., Finkle, R., Henchen, A., Hock, D., Kirchheim, H., Kreeye, V.. Lottspeich, F., Mutt, V. and Reinecke, M. (1984) Cell Tissue Res., 238, 425-430. 9. Kangawa, K. and Matsuo, H. (1984) Biochem. Biophys. Res. Commun., 118, 131-139. 10. Burlington, H. and Kronkite, E.P. (1973) Proc. Sot. Exp. Biol. Med., 142, 143-147. 11. Ross., R. (1971) J. Cell Biol., 50, 172-186. 12. Iwasa, F., Furuya, M., Hayashi, Y. and Ohnuma, N. (1988) Biochem. Pharmacol.,37, 2757-2763. 13. Kitajima, Y., Minamitake, Y., Furuya, M., Takehisa, M., Katayama, T. and Tanaka, S. (1989) Biochem. Biophys. Res. Commun., 164, 1295-1301. 14. Laemli, U.K. (1970) Nature, 227, 680-685. 15. Fuller, F., Porter, J.G., Arfsten, A.E., Miller, J., Schilling, J.W., Scarborough, R.M., Lewicki, J.A. and Shenk, D.B. (1988) J. Biol. Chem., 263, 9395-9401. 16. Ishido, M., Fujita, T., Shimonaka, M., Saheki, T., Ohuchi, S., Kume, T., Ishigaki, I. and Hirose, S. (1989) J. Biol. Chem., 264, 641-645. 17. Ohuchi, S., Hagiwara, H., Ishido, M., Fujita, T., Kume, T., Ishigaki, I. and Hirose, S. (1989) Biochem. Biophys. Res. Commun., 158, 603-609. 18. Chinkers, M., Garbers, D.L., Chang, M.S., Lowe, D.G., Chin, H., Goeddel, D.V. and Schulz, S. (1989) Nature, 338, 78-83. 19. Chang, MS., Lowe, D.G., Lewis, M., Hellmis, R., Chen, E. and Goeddel, D.V. (1989) Nature, 341, 68-72. 20. Schulz, S., Singh, S., Bellet, R.A., Singh, G., Tubb, D.J., Chin, H. and Garbers, D.L. (1989) Cell, 58, 1155-1162. 21. Minamitake, Y., Furuya, M., Kitajima, Y., Takehisa, M., Katayama, T. and Tanaka, S. (1990) Chem. Pharm. Bull., in press. 22. Budzik, G.P., Firestone, S.L., Bush, E.N., Connolly, P.J., Rockway, T.W., Sarin, V.K. and Holleman, W.H. (1987) Biochem. Biophys. Res. Commun., 144, 423-431. 23. Hassman, CF., Pelton, J.T., Buck, S.H., Shea, P., Heminger, E.F., Boersma, R.J. and Berman, J.M. (1988) Biochem. Biophys. Res. Commun., 152, 1070-1075. 24. Uchida, K., Mizuno, T., Shimonaka, M., Sugiura, N.. Hagiwara, H. and Hirose, S. (1989) Am. J. Physiol., 256, H311-H314.
208
170,
No.
July
16, 1990
1, 1990
BIOCHEMICAL
AND
Mayumi Yujiro
Furuya*, Hayashil,
Institute
1 Suntory 2 National
COMMUNICATIONS Pages
NOVEL NATRIURETIC PEPTIDE, CNP, POTENTLY PRODUCTION IN RAT CULTURED VASCULAR
Suntory
RESEARCH
BIOPHYSICAL
Maki Takehisa, Norio Ohnumal Kenji Kangawa3,
for
Biomedical
Bio-Pharma
Tech
Cardiovascular
Center
STIMULATES CYCLIC GMP SMOOTH MUSCLE CELLS
Yoshiharu Minamitake, Yasuo Kitajima, , Takafumi Ishihara, Naoto Minamino2, and Hisayuki Matsuo2
Research,
Center,
201-208
Shimamoto,
Akaiwa,
Research
Mishima,
Chiyoda,
Institute,
Ohra,
Osaka
618, Japan
Gunma
370-05,
Japan
Suita,
Osaka
565, Japan
Fujishirodai,
3 Department of Biochemistry, Miyazaki Medical College, Kihara, Kiyotake, Miyazaki 889-l 6, Japan Received
May 30,
1990
SUMMAR‘Y : The newly identified peptide C-type natriuretic peptide (CNP) caused only a slight elevation of cGMP in rat renal glomeruli. In contrast, CNP potently increased cGMP levels in cultured rat vascular smooth muscle cells (VSMC) and stimulated guanylate cyclase activity in the particulate fraction of the cells. The extent of maximum activation of the enzyme induced by CNP was 4-fold higher than that by human atria1 natriuretic peptide (a-hANP) while CNP was 4- and 16fold weak’er than a-hANP in binding affinity for the putative receptors on VSMC and vasorelaxant activity for rat aorta, respectively. These results indicate that CNP is a potent stimulator of cGMP formation in VSMC but not in glomeruli and pharmacological feature of CNP is distinct from that of ANP. 0 1990 AcademrcPress, Inc.
Atria1
natriuretic
regulation
of
body
2).
Identification
heart
(3-5),
to those
which
of human
blood
pressure
third
type: natriuretic
identified to both quite
unilque
fluid of
(ANP)
and brain
has been ANP
brain
feature
*To whom correspondence
blood
pressure
natriuretic
N-terminal
should
mammalian effects
the possibility ANP
has amino
amino
acids,
linkage; and
(1,
brain
and
Very
similar
ends
and
recently, peptide)
sequences
the
functions very
natriuretic acid
in
that water-salt
and BNP.
(C-type
by a disulfide
five
role
in
as CNP CNP
important
physiological
(BNP)
by both
flanked
an
pharmacological
6). discloses
Porcine
play
and other
to elicit
designated
(7).
to
peptide
reported
in a ring in
known
are regulated
pcptide
and BNP
is
(a-hANP)(3,
homeostasis
in porcine ANP
peptide
a was
homologous
however,
it has a
at
second
the
be addressed.
Abbreviations: ANP; atria1 natriuretic peptide (A-type natriuretic peptide), BNP; brain natriuretic peptide (B-type natriuretic peptide). CNP; C-type natriuretic SDS; sodium dodecyl sulfate, PAGE; peptide, tx-hANP; human pro-ANP[99-1261, polyacrylamide gel electrophoresis. 0006-291x/90 201
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
cysteine along
residue with
BIOCHEMICAL
1, 1990
with
porcine
no C-terminal
ANP
CNP ,
and BNP
The
are listed
RESEARCH
amino
COMMUNICATIONS
acid sequences
of porcine
CNP
below.
S-L-R-R-S-S-C-F-G-G-R-M-D-R-I-G-A-Q-S-G-L-G-C-N-S-F-R-Y
BNP ,
S-P-K-T-M-R-D-S-G-C-F-G-R-R-L-D-R-I-G-S-L-S-G-L-G-C-N-V-L-R-R-Y
We
found
that
porcine
hypotensive
actions
in
activity
porcine
CNP
of
suggesting
that
and/or
elevation
CNP
BNP.
and the
and cultured which
tail.
BIOPHYSICAL
G-L-S-K-G-C-F-G-L-K-L-D-R-I-G-S-M-S-G-L-G-C
ANP ,
ANP
AND
than
was
plays
unique activity
smooth identical
elicited ANP
much and
which
cells,
to porcine MATERIALS
and
ANP
diuretic/natriuretic
that
potenl might
porcine
chick than
rectum that
be distinct
we have focused
of synthetic
muscle
potent
and
more
funcrions report,
less
BNP,
3 to 4 times
In the present binding
vascular
is structurally
CNP
rats
from
on the potency
CNP
compared
of
in
with
rat renal those
and relaxant ANP
(7).
those
of
for cGMP glomeruli
of a-h ANP
(8).
AND METHODS
Peptides: Alpha-hANP and porcine CNP were synthesized by solid phase method as described previously (7, 9). We also synthesized a CNP derivative which was elongated with a tyrosine residue at the N-terminal, called YO-CNP for radioiodination. Equivalent potency of Y”-CNP to CNP was confirmed in cGMP accumulation and binding activity in VSMC, and vasorelaxant activity in rat aorta. Iodination of a-hANP and Y”-CNP were carried out by the lactoperoxidase method, and the specific activities of both labeled peptides were equally 22.2MBq/pg. Preoaration of rat renal Plomeruli: Kidneys were dissected from male SpragueDawley rats of 8-9 weeks old. Renal glomeruli were isolated by sieving method (10). Purity and the number of glomeruli were evaluated by light microscopy. Culture of rat vascular smooth muscle cells (VSMC): VSMC were derived from explants of Sprague-Dawley rats aorta (1 I), and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco). Cells were used for experiments between 3th to 7th passage. cGMP accumulation in rrlomeruli and VSMC: A. Glomeruli; Isolated renal glomeruli were incubated with peptides in 125~1 Krebs-Henseleit solution containing 1mM lmethyl 3-isobutylxanthine (MIX) at 30°C for 2min. Concentration of cGMP in the glomeruli was quantified by using a cGMP radioimmunoassay kit (Yamasa Shoyu) (12). B. VSMC; Cells were incubated with peptides in 0.25ml DMEM containing 0.1% BSA, 20mM HEPES and OSmM MIX, pH 7.4 at 37°C for 10min. Cellular cGMP concentration was determined by radioimmunoassay (13). Gu an v 1ate cvc I ase activitv in VSMC membranG: Confluent VSMC were scrapped and homogenized in 8 volumes of 50mM Tris-HCI, pH 7.4 containing 0.25M sucrose, ImM EDTA, 1mM dithiothreitol, 0.2mM phenylmethylsulfonyl fluoride (PMSF) and 2Opg/ml aprotinin. The homogenates were centrifuged at 100,OOOxg for 60min. and particulate fraction was washed and resuspended in the same buffer. The activity of guanylate cyclase in particulate fraction was determined at 37°C in 50mM TrisHCl, pH 7.6, containing 1mM GTP, 5mM MgCl2, 7.5mM phosphocreatine, 13units/ml creatine kinase and 0.5mM MIX with or without 0.5mM ATP. Concentration of cGMP generated during 3min incubation was determined by radioimmunoassay. &mtOr bindine assay: A. Glomeruli; Isolated glomeruli were incubated with 5x 10-l”M [1251]a-hANP (ca. 3x105cpm) in the presence of unlabeled peptide in 0.25ml of 1OmM Tris-HCI, pH 7.4 containing 1OmM MgC12, 1mM EDTA, 0.25M sucrose, 0.2% bovine serum albumin (BSA) and 1mM PMSF at 0°C for 30 min. At the end of the incubation, lml of the ice-cold buffer was added and aliquots were centrifuged 202
Vol.
170,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
at 10,OOOxg for Smin. Radioactivity within the pellet was counted by a yspectrophotometer (12). B. VSMC; Cells were incubated with indicated concentrations of [1251]a-hANP or [ 1251]YO-CNP in the presence or absence of the unlabeled peptide in 0.25ml DMEM containing 0.1% BSA and 20mM HEPES, pH 7.4 for 45min at 25°C. Following wash with the same medium, the cells were solubilized with 0.5M NaOH and radioactivity was measured (13). Affinity-crosslinking of labeled oentide with VSMC: VSMC were incubated with 5x10-lOM [1251]a-hANP or [125 I]Y”-CNP in the presence or absence of 5x10T7M unlabeled peptide in the same medium described above at 25°C for 45min. After washing for 3 times with lml of phosphate-buffered saline (PBS), cells were then added with 0.25ml of O.lmM disuccinimidyl suberate (DSS) and incubated on ice for 30min. ‘The DSS solution was aspirated and cells were scraped from each well in 0.5ml of PBS, followed by centrifugation. Cell pellets were resuspended in O.lml SDS sample buffer (60mM Tris-HCI, pH 6.8, 27% glycerol, 0.3% SDS) with 5% 2mercaptoethanol. After boiling for Smin, samples were run on 7.5% polyacryla:mide gels (14). Dried gels were exposed to Kodak X-Omat AR film at -80°C for 7 days. Vasorelaxant assav: Aortic strips were prepared from thoracic aorta of SpragueDawley rats and mounted in a 7ml chamber containing Krebs-Henseleit solution at 37°C. ‘The strips were contracted with 2xlO-7M norepinephrine and once the contractile: response stabilized, they were exposed to peptide cumulatively. RESULTS The renal
effect
of CNP
glomeruli
and
on accumulation cultured
glomeruli,
1.6-fold
increase
of 10e5M
of CNP.
Whereas,
EC50 of
of 3.6x10-7M
cGMP
was
(Fig.
evoked
rat of
cGMP
a-hANP
1A).
by
concentration
up to 160-fold
Alpha-hANP
was less effective
of cGMP
vascular
muscle
concentration
was
increased
In contrast
CNP
in
of basal
rat
cGMP
to renal
VSMC
level
than CNP,
was investigated
smooth
(VSMC).
induced
by
contents glomeruli,
(Fig.
at 10m6M
and the EC50
1B). with
using
cells
rat isolated In
an application
up to 5.4-fold a marked CNP
renal
with
increased
cGMP
an EC5 0 of 7.7x10b8M
was 3.7x10d7M
and maximum
IJ/ -6
-7
-6
Augmentation of cGMP concentrations induced by CNP (closed circles) or Figure 1. ANP (open circles) in rat renal glomeruli (A) and rat vascular smooth muscle cells Renal glomeruli or VSMC were incubated with the peptide in the (VSMC) (B). Data represent mean presence of MIX as described in “Materials and Methods”. of triplicate determinations from two or three separate experiments in values glomeruli or VSMC, respectively. 203
an
accumulation
-5
.
Vol.
170,
No.
elevation
BIOCHEMICAL
1, 1990
was 50-fold
10m5M
at 10e5M.
of a-hANP,
(none,
partial
0.04+0.02;
a-hANP,
pmole/l05celIs,
mean+S.E.
Membrane particulate
fraction
When
BIOPHYSICAL
RESEARCH
the cells were exposed
reduction
of the cGMP
1.84rtO.10;
CNP,
to lo-6M
response
6.98kO.55;
COMMUNICATIONS
of CNP with
to CNP
was
CNP plus a-hANP,
VSMC
cyclase
was
prepared,
activity.
CNP
and
subjected
activated
the
to
by an addition In both with
The
were
VSMC,
ATP
glomet-uli
a less potent
hANP
stimulation
of 0.5mM
renal
without
than a-hANP
8.3~1O-~M
enzyme
by CNP and a-hANP
was
assay
more
of
potently
4-fold greater and 8.0x10-7
both
potentiated
a shift in the EC50. CNP
inhibited
the binding
in
glomeruli,
of [l 251]a-
h ANP
The IC50 values for CNP and a-
(Fig. 3A, B).
and 1.0x10-9M
and
3.5x10mgM
and
1.1x10-9M
in
respectively.
Fig. 4A demonstrates Analysis
of
dissociation
a specific
the saturation
suggesting
plot,
induced
and VSMC,
affinity
4.51kO.01
the
than a-hANP (Fig. 2). Maximum activation induced by CNP was about than that of a-hANP, and the EC50 for CNP and a-hANP was l.l~lO-~M M, respectively.
observed
of 3 determinations).
of
guanylate
AND
the
binding
presence
constant
fmole/105cclls
(Kd)
(Fig. 4B).
a Kd of 0.9x10-gM
and saturable
and
of
data of
according
a
single
3.Xx10-9M
almost
of [l 251]Yo-CNP
to Scatchard class
and
[1251]a-hANP an
binding
comparable
a maximum Bmax
resulted
receptor
of
with
33
in
with
VSMC.
a linear apparent
an
binding
to the cells
bound
in
(Bmax)
a higher
of 42
affinity
fmole/l05cclls
(data
of not
shown).
Affinity-crosslinking in a detection
of either
of only
one radioactive
condition.
Labeling
and a-hANP
at 5x10m7M
Figure 2. Stimulation
[l 251]a-hANP
of the
protein
band
approximately
of
W;IS
inhibited
-9 Cont.
-6 (log M)
almost
with
70kDa
VSMC
under
complctcly
by
resulted
a reduced both
(Fig. 5)
of particulate
(closed symbols) in comparison the reaction mixture for the incubated for 3 min at 37°C
0.5mM ATP.
or [ 1 25 I]Y”-CNP
-7
-6
guanylate cyclase activity
-5
of rat VSMC
by CNP
with ANP (open symbols). Peptides were added to enzyme assay at the indicated concentrations and in the absence (triangles) or presence (circles) of
Each value represents mean*S.E. 204
of triplicate
determinations.
CNP
Vol.
A.
170,
No.
BIOCHEMICAL
1, 1990
AND
120
100
80 -E : m
RESEARCH
COMMUNICATIONS
Bm 120 t
1
100
BIOPHYSICAL
a
80 -2 2 m
60
z
60
ae
40
40
20
20 I_
0
-'a. -71
-es
0 L;;:;c-;; -10 Cont.
-9
-8
-7
-6
.e3
-11
(log M)
-10
Cont.
-9
-8
-7
-6
(log M)
Figure 3. Displacement of 1125 I]a-hANP from binding sites by unlabeled CNP (open circles) or ANP (closed circles) in renal glomeruli (A) and VSMC (B). Renal glomendi or VSMC were incubated with 5x10- l”M [12511a-hANP in the presence of Data represent means of triplicate determinations from two or unlabeled peptides. four separate experiments in glomeruli or VSMC, respectively.
CNP
relaxed
aortic
strips
precontracted
by
norepinephrine
with
an
EC50
71.7k1.8 x10w9M (meankS.E. of 5 preparations), being approximately 16-fold than that of a-hANP (EC50=4.6fO.l x10-oh/l, mean+S.E. of 5 preparations). aortic
tissues
vasorelaxant
were
subjected
responses
to
to the both
assay
CNP
and
after
rubbing
a-hANP
out
were
higher When
the endothelium,
not
affected
of
the
(data
not
whereas,
the
shown). DISCUSSION In renal increment
glomeruli,
CNP competed
of the cGMP
concentration
for the binding
of [’ 251]a-hANP,
was very limited.
A.
This
low
activity
in cGMP
B. 0.5
0.4 -
0
1
2
Labeled
3
Peptide
4
Cl.0
5
0.1
0.2
0.3
Bound
(nM)
0.4
0.5
(nM)
binding t0 rat vs~c. VSMC were Figure 4. (A) Saturation curve of [ ~~~IIY~-CNP incubated at 25°C for 45min with increasing concentrations of [1251]Yo-CNP, and circles) and specific binding (open total (open circles), nonspecific (closed triangles) were measured. Nonspecific binding was determined by incubating the cells with [1251]YO-CNP in the presence of 10.6M unlabeled CNP. (B) Scatchard plot of the specific binding. Each point represents mean of quadruplicate determinations.
205
( 6
Vol.
170,
No.
BIOCHEMICAL
1, 1990
12
AND
34
BIOPHYSICAL
RESEARCH
Mr
56 , * i
COMMUNICATIONS
x lo- 3
-Origin
r-M
-
205
-97
-67 -45 Fi ure 5. Autoradiography of SDS-PAGE analysis under reduced condition of -+---I[I 5I]a hANP and [1251]Yo-CNP covalently coupled to the surface of intact cultured rat VSMC. Confluent cells were incubated with SXIO~~~M 11251]a-hANP or [12511YoCNP in the absence (lane 1 and 4), or prcscnce of ANP (lane 2 and 5) or CNP (lane 3 and 6) at each 5x10e7M, respectively. The migration of known molecular weight markers are shown to the right.
accumulation activity
in
On the Since
other
hand,
the simultaneous
resulted
peptides
mechanism. peptides
would
both
an inhibitor
kinetic
was 4-fold demonstrated a 7OkDa
that
protein,
production
of
cyclase,
did
soluble
guanylate
in VSMC
for cGMP
study
it elicits ANP.
diuretic
at a maximum
effective
at
least
partly,
that CNP as well and
ATP. not
rat VSMC. dose
it can be suggested
VSMC,
formation
revealed
that
of a-hANP.
the detectable
than
low
the
a
Furthermore, affect
the
same
as a-hA effects
that NP
of the
methylene
cGMP
responses
was greater
than that
(data not shown).
of CNP
would
the
in
CNP,
by the findings presence
to
concentrations
through,
in the
than that
and
by
accelerated
for
transduction
CNP
induced
in
does
in VSMC
with
elevation
activity
which
signal
cGMP
cyclase
One interpretation (15). and the cGMP elevation ANP
corresponded
of a-hANP
cGMP
binding
weaker
be
increased
be supported
the potency
of a-hANP,
potently
to
guanylate of
to these two peptides Although
CNP
of cGMP
particulate were
assumed
(7).
stimulate This
stimulated
is
in vivo addition
in a reduction
both
blue,
glomeruli
of the peptide
apparent
Crosslinking
natriuretic not involve
receptor
guanylate
cyclase
between
be that CNP shares Hirose
cyclase et al. 206
binding of
peptide
the discrepancy guanylate
in VSMC
the
on
VSMC
of to
CNP VSMC
was
mostly
domain
in the molecule
binding
to
a common,
activation have
affinity
the peptides
70kDa
through
demonstrated
the receptor receptor
a more the
with
efficient
presence
of
Vol.
170,
No.
BIOCHEMICAL
1, 1990
dissociative
complexes
inactivation
of
analysis,
for the activation
and
guanylate
cyclase
complex
; Rc*GC
(inactive)
guanylate
cyclase).
that
enzyme
peptide ANP
receptors,
natriuretic
peptide
these
has
been
BNP
to
found
give for
ANF’
residues
was l&fold
analogs
in
vasorelaxant
activities possessed of
cultured
cells
may
reported
previously it
hypotension. be present
This in
in various
interpretation
is
subtype
activated
of natriuretic
by CNP
rather
Recently,
(15,
18-20). of
been (20).
than
multiple One
both
of
ANP
suggested
and
that
CNP
might
vasorelaxant
activity
an be a
must
peptides,
or
from
effectively
in
reported
ii)
that
in
with analog,
observations sufficient
peptide
weak Lysl
to promote
smooth
in
the
muscles
as
be noteworthy
that
in glomeruli,
is originally
including
have
functions
concentration
CNP The system
increases
weak
vasorelaxant
observed
in porcine
ANP. nervous
significantly very
never
of
found than
central
CNP
and shows
in ANP
other
than
brain,
studies
and
cGMP activity
and BNP,
diuresis
and
is assumed
on the activities
are now
going
on.
REFERENCES 1. DeBold., 2.
3. 4. 5. 6.
A. J. (1985) Science, 230, 767-770. H. and Nakazato, H. (1987) Endocrinol.
Metab. Clin. North Am., 16, 43-61. Sudoh, T., Kangawa, K., Minamino, N. and Matsuo, H. (1988) Nature, 332, 78-81. Kojima., M., Minamino, N., Kangawa, K and Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 159, 1420-1426. Sudoh, T., Maekawa, K., Kojima, M., Minamino, N., Kangawa, K. and Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 159, 1427-1434. Itoh, H., Nakao, K., Yamada, T., Shirakami, G., Kangawa, K.. Minamino. N., Matsuo, H. and Imura, H. (1988) Eur. J. Pharmacol., 150, 193-197. Matsuo,
*
suggest
receptors
aortic
that
penicilamine
VSMC
factor
intact
by
an ANP
These
natriuretic
those
of the peptide
peptide
tissues
substituted
(22).
in
We have found
were
may not be a sole
physiological
a higher
in VSMC,
24).
properties
unique
property
different
but not
Such
production
elevation
be
residues
It has also been
similar
(23,
in VSMC
suggesting
CNP
(21).
natriuretic
conclusion,
in aorta.
GC:
Another
and it has be present
may
receptor,
the
than that of a-hANP.
cysteine
cGMP
that i) cGMP
vasorelaxation
In
(Rc:
concentrations
production
less potent
which
stimulated
[7-23]ANP
contents
response, receptor
effect of CNP on cGMP
also
possibilities
this
receptor-enzyme to dissociate
and sequenced high
may
be able
sites in VSMC.
cloned
that ANP
ligand.
rat aorta
some
cGMP for
(active)
radiation
be responsible
also
a certain
and
binding
by
putative
might
through
relatively
the
ANP.
occupied
been
require
ligand
the
To the potent isolated
to
have
a remarkable
natural
candidate
be mediated
cyclase
would
+ GC CNP
COMMUNICATIONS
have proposed
of
than
of the total
receptors
They
Rc*ANP
effectively
is preferably
a minority
guanylate
a dissociation +
RESEARCH
receptor
17).
to the model,
might
which
and
70kDa
(16,
+ ANP more
of CNP
and is only
unidentified1
through
complex
the action
that
cyclase
According
BIOPHYSICAL
receptor
suggested
of guanylate
activate
receptor
ANP
AND
207
to of
Vol.
170,
No.
1, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
7. Sudoh, T., Minamino, N. Kangawa, K. and Matsuo, H. (1990) Biochem. Biophys. Res. Commun., 168, 863-870 8. Forsmann, W.G., Birr, C., Carlquist, M., Christman, M., Finkle, R., Henchen, A., Hock, D., Kirchheim, H., Kreeye, V.. Lottspeich, F., Mutt, V. and Reinecke, M. (1984) Cell Tissue Res., 238, 425-430. 9. Kangawa, K. and Matsuo, H. (1984) Biochem. Biophys. Res. Commun., 118, 131-139. 10. Burlington, H. and Kronkite, E.P. (1973) Proc. Sot. Exp. Biol. Med., 142, 143-147. 11. Ross., R. (1971) J. Cell Biol., 50, 172-186. 12. Iwasa, F., Furuya, M., Hayashi, Y. and Ohnuma, N. (1988) Biochem. Pharmacol.,37, 2757-2763. 13. Kitajima, Y., Minamitake, Y., Furuya, M., Takehisa, M., Katayama, T. and Tanaka, S. (1989) Biochem. Biophys. Res. Commun., 164, 1295-1301. 14. Laemli, U.K. (1970) Nature, 227, 680-685. 15. Fuller, F., Porter, J.G., Arfsten, A.E., Miller, J., Schilling, J.W., Scarborough, R.M., Lewicki, J.A. and Shenk, D.B. (1988) J. Biol. Chem., 263, 9395-9401. 16. Ishido, M., Fujita, T., Shimonaka, M., Saheki, T., Ohuchi, S., Kume, T., Ishigaki, I. and Hirose, S. (1989) J. Biol. Chem., 264, 641-645. 17. Ohuchi, S., Hagiwara, H., Ishido, M., Fujita, T., Kume, T., Ishigaki, I. and Hirose, S. (1989) Biochem. Biophys. Res. Commun., 158, 603-609. 18. Chinkers, M., Garbers, D.L., Chang, M.S., Lowe, D.G., Chin, H., Goeddel, D.V. and Schulz, S. (1989) Nature, 338, 78-83. 19. Chang, MS., Lowe, D.G., Lewis, M., Hellmis, R., Chen, E. and Goeddel, D.V. (1989) Nature, 341, 68-72. 20. Schulz, S., Singh, S., Bellet, R.A., Singh, G., Tubb, D.J., Chin, H. and Garbers, D.L. (1989) Cell, 58, 1155-1162. 21. Minamitake, Y., Furuya, M., Kitajima, Y., Takehisa, M., Katayama, T. and Tanaka, S. (1990) Chem. Pharm. Bull., in press. 22. Budzik, G.P., Firestone, S.L., Bush, E.N., Connolly, P.J., Rockway, T.W., Sarin, V.K. and Holleman, W.H. (1987) Biochem. Biophys. Res. Commun., 144, 423-431. 23. Hassman, CF., Pelton, J.T., Buck, S.H., Shea, P., Heminger, E.F., Boersma, R.J. and Berman, J.M. (1988) Biochem. Biophys. Res. Commun., 152, 1070-1075. 24. Uchida, K., Mizuno, T., Shimonaka, M., Sugiura, N.. Hagiwara, H. and Hirose, S. (1989) Am. J. Physiol., 256, H311-H314.
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