J Am Soc Nephrol 9: 1777-1786. 1998. Immunolocalization ... Canada. Abstract. The natnuretic peptide receptor. (NPR) family con- sists of three receptor.
J Am Soc Nephrol
Immunolocalization Rat Kidney
of Natriuretic
MARIE-CHANTAL BONHOMME,* CAROLINE T. CRILLEY,* GAETAN *Lboratory
Abstract. sists
The
of three
contain NPR-B),
of Experimental
Vasoactive
Peptides,
Montreal,
Montreal,
natnuretic receptor
a guanylyl cyclase and one truncated
agonists and to the difficulty
Laboratory
Quebec,
The
(NPR)
family
transmenibrane
forms
brain
natnuretic
(CNP)
(NP),
peptide
are considered
homeostasis
through
atrial
(BNP),
and C-type
important
regulators
their
various
peptide
and NPR-C).
All
sent cell surface
receptor-A,
subtypes
receptors
with
Two of them, NPR-A of approximately 120
weight guanylyl
cyclase
second
messenger
transfected
recognized
a second
domain
the
vasculature,
cloned
NPR-B, and repre-
transrnembrane
domain
that generates, to
from
hormone
GTP,
cGMP
stimulation
as (4).
cGMP is believed to be responsible for meditating most of the biological actions of NP. The third receptor in this family, NPR-C, is a homodimer of 120 kD. Each subunit has a short 37-amino
nylyl
acid
cyclase
clearance
activity.
receptor
transduction Pharmacologic
NPR
cytoplasmic
for their
domain
This
(5),
as well
receptor
although
is devoid
is likel’,
it may
of
any gua-
to function
be involved
as a
in signal
(6).
studies
native
that
have
ligands
shown
some
and synthetic
selectivity
analogs
between
(1,7).
How-
March 25. 1998. IRCM, 1 10 Pine Ave. West Montreal.
1046-6673/090101777$03.O0/0 Journal of the American Society of Nephrology Copyright © 1998 by the American Society of Nephro(ogy
NPR.
rat NPR-B
cDNA.
of
PQ,
The
approximately
antibody
5 to
10 kD
Western
blotting
analysis
with
anti-NPR-B
also identified a protein of appropriate size in membranes. These results were supported by findings
the rat kidney,
that
demonstrated
and medullary study concludes
although
it was
staining
for
capillaries, glomeruli, and that NPR-B is present in
only
detected
in vascular
that
no single
struc-
tures.
they
have
also
revealed
for a particular
binds
ANP
(7-9).
This
ANP
receptor
and BNP, order
(and
NPR-A
or
subtype.
whereas
of
BNP)
potency
reflected
the
respectively. to NPR-A of NPR-C
that is less
conserved,
and
affinity selective
for all NP. for NPR-C
However,
the
this
CNP
ability
of
cyclase
of
HS-l42-l
receptor
has
analog
an equal C-ANF
is
(5).
physiologic
effects
of their
receptors,
but the study
of expression
of NPR-A
onists.
antagonist
synthetic
The
hampered
binds
in the
and NPR-B (10,1 1). The has an amino acid se-
tribution
and regulation
preferentially
guanylyl
The
quence
is absolutely
selectively
to activate
similar affinity binding site
NP
NPR-A
NPR-B is also
or CNP
NPR-B,
binds with extracellular
by the lack Furthermore,
of NP
depend,
been
on the disdistribution
and NPR-B
has been
receptor
agonists
or antag-
demonstrated
that
of selective it has
in part, of tissue
Tyr#{176}-CNP,
which is often used in NPR-B receptor characterization and localization, undergoes a significant reduction in affinity upon iodination (12) and, thus, is not an ideal tool for radioligand binding
assays.
kidneys, detected reverse
because the expression of NPR-B by radioligand binding assays transcniption-PCR reveals the
mRNA Received May 28, 1997. Accepted Correspondence to Dr. Raul Garcia, Canada H2W 1R7.
with
immunohistochemistry
specific
and NPR-B, have a molecular kD and possess an intracellular
in response
institute
probably represents the unglycosylated recep(413-426)-Tyr did not show crossreactivity
(413-426)-Tyr renal vascular
ever,
effects of NP are subtypes, termed
been
a single
recell
peptide
-C (NPR-A,
and
have
and
Research
polypeptide,
NPR-B on papillary renal arteries. This
of cardiovascular on
smaller, which tor. Anti-NPR-B to any other
(ANP),
natriuretic
actions
-B,
three
of Neurobiology
Clinical
membranes
antibodies a region of
peptide
(
natriuretic
(2,3).
natniuretc
adrenal glands, and brain 1 ). The by a family of at least three receptor
kidneys, mediated
CARON,* F. DESCHEPPER,t
tLaboratory
that
of rat NPR-B that is not homologous and NPR-C were developed. Western
peptides
Peptides,
of Hypertension,
con-
intracellular domain (NPR-A and form (NPR-C). Because of the lack
with affinity-purified anti-NPR-B (413-426)-Tyr a polypeptide of approximately 120 kD on COS-l
natriuretic
Vasoactive Biology
and antagonists for each receptor subtype to detect the presence of small quantities
the extracellular domain to sequences in NPR-A
B in the
Canada.
receptor two
and
of Cell
of NPR-B by ligand binding studies, polyclonal against a peptide whose sequence was chosert from
blotting vealed
KEVIN L. GROVE,t SYLVIE THIBAULT,t CHRISTIAN
1-lypertension
and
peptide subtypes:
of specific
Receptor
1998
GARCIA*
RAUL
AND
Peptide
9: 1777-1786.
It is unclear
in different
ANP and BNP, immunoreactive been
found
To further
the regulation
parts
CNP CNP
of the
rat
NPR-B
nephron
is expressed
protein (13-15). presence (16).
in rat
has not been However, of NPR-B In contrast
is undetectable in rat plasma and mRNA coding for CNP
in the kidney understand
whether
to
(17), but have both
(16,18). the renal
of NP receptors,
actions
of CNP
we have
developed
in relation a sensitive
to
1778
Journal
method present antibody
of the American
Society
of Nephrology
for the detection of NPR-B protein expression. In the study, we describe the characterization of a polyclonal recognizing rat NPR-B and its use to determine the
distribution
of NPR-B
Materials
in the rat kidney.
used
were
Triton X-l00, Tween 3,3’-diaminobenzidine phenylmethylsulfonyl Chemical Co. (St.
of the highest
acetylrnuramyl-L-alanyl-D-isoglutamine
and
ethyl]sulfone
were
IL); ECL Western
anti-rabbit
lgG,
and
streptavidin
cured
Arnersham
(Oakville,
from
and CNP
IgG
(1-22)
were
horseradish assay
from
blotting
4B was
MI).
Sepharose
and Freund’s Sprague
were purchased bee, Canada).
from Charles
Preparation
of Anti-NPR
The
peptide
used
goat
from
adjuvant
rats and New
was from
Difco
River Laboratories
Polyclonal
White
with
mem-
5 ml of cold
as
described
vessels,
earlier
glomeruli,
( 13,20).
Isolated
0.25 M sucrose solution Lucerne, Switzerland)
and
kept
membrane
BSA
(200
0.7%
Triton
continuous
used.
Some
experiments
in Triton
X- 100
to 500 j.g) were incubated X-IO0,
and 0.1 mM
agitation,
then
PMSF,
centrifuged
as
in 500 pA pH 7.4,
at 15,000
Protein concentration was measured by
X
in homogethe Bradford
as standard.
of cell
NPR-B
cell membranes were pH 7.4, aliquotted in
membranes
were kept. homogenates
Binding
Aliquots
plasma
proteins
M Tris-HC1,
using
at -40#{176}Cuntil of
for at
Saturation
membrane
(containing
Experiments
suspensions
20 g
of COS-
of protein
1 cells
transfected
in 50 mM Tnis-HC1,
pH 7.4,
5 mM MgC12, 0.5% BSA, 0.1 mM PMSF, and 0.1% bacitracin) were incubated for 90 mm at 22#{176}C in the presence of increasing concentrations of ‘25I-Tyr#{176}-CNP (10 pM to 1300 pM) with or without lO_6
Que-
M unlabelled Tyr#{176}-CNPin a final assay volume of 250 pA. Membranebound radioactivity was separated from free hormone by filtration on
Antibodies was
( 19). For cell
twice
in fresh ice-cold 8, 1 mm; Kinematica,
solubilization
Radioligand with
rabbits
(St. Constant,
volumes
assay,
D’Urf#{233},
washed
Pellets of tissue and M Tnis-HC1, 0. 1 mM PMSF,
g, and the supernatants nates on solubilized
(Baie
Zealand
prepared
for 1 h at 4#{176}C with
Technologies Chemical Co.
Pharmacia
were
in 0.05
of0.05
Bio-Rad
previously
was
x g for 30 mm.
follows:
Canada);
plate
and centrifuged at 1000 X g for 10 mm. This process was repeated the pellet fraction, and both supernatants were then centrifuged
involved
anti-rabbit
of the
as described
each
x g for 20 mm. Rat renal preglomerular papillae
small Chemi-
Ontario,
was from Life was from Aldrich
for immunization
purified by the Institut City, Quebec, Canada).
CA);
reagent
incomplete
Dawley
Pierce
prepared
Soc Nephrol 9: 1777-1786, 1998
0.05 M Tris-HC1, pH 7.4, containing 0. 1 mM PMSF, and then scraped in the same buffer. The cells were homogenized with 10 strokes of a Teflon Potter-Elvejhem tissue grinder at 750 rpm, and centrifuged at
100,000 dispersed
Tyr#{176}-CNP(1-22)
(Mississauga,
(Milwaukee, (Detroit,
Canada);
and the dye BioRad medium
WI);
available.
from Sigma (1-4)-N-
from
(Belmont,
Dulbecco’s modified Eagle BRL; 3-isobutyl-l-rnethyl-xanthine
Canada)
grade
kit, biotinylated donkey peroxidase were pro-
horseradish
Peninsula
from
were preparations,
tissues were homogenized with a Polytron (setting
bis[2-(sulfosuccinimi-
obtained
Ontario,
peroxidase were
Quebec,
reagent
fluoride (PMSF) were purchased Louis, MO); N-acetyl-o-glucosaminyl-
cal Co. (Rockford,
brane
and
20, bacitracin, bovine serum albumin (BSA), tetrahydrochlonide, bovine thyroglobulin, and
dooxycarbonyloxy)
astrocytes
30,000
and Methods
Materials All materials
protein
J Am
synthesized
and
HPLC-
national de la recherche scientifique (Quebec This 13-amino acid peptide (GAEKQIWWT-
Whatman
GF/C
Canada). Tnis-HC1
The filters were washed three times with 3 ml of 50 mM at pH 7.4, and the radioactivity retained on the filters was
filters
in a cell
harvester
system
(Xymotech,
Quebec,
GRPI) corresponds to a portion of the amino acid sequence (3) deduced from the extracellular domain of rat NPR-B (413-426). It was selected by the criteria of hydrophilicity and location in a region showing no homology with sequences present in the extracellular
measured with a gamma counter. Binding data were analyzed by processing raw data with the computer-based EBDA program (Elsevier-Biosoft, Cambridge, UK). The binding capacity and
domain residue
of NPR-A was added
computer-based
peptide
was
glutaraldehyde. injected of
and NPR-C. An extra carboxy-tenminal tyrosine to permit iodination of the antigenic peptide. The
covalently
coupled
Female
intramuscularly
to bovine
New with
Zealand a total
N-acetyl-D-glucosaminyl-f3
isoglutamine NaCl
Blood
followed
and 500
samples
the immunizing
incomplete
by monthly
booster
pl of Freund’s
were collected
by immunoprecipitation antisera. Antibodies
of 500 g
by means
rabbits
(2 kg)
of conjugate
of
were
incomplete
every
month,
adjuvant
and 75 .tg
injections adjuvant
mixed
peptide
coupled
to Sepharose
3: 1 with
of 250
,.tg of
in NaCl
0.9%.
and titers were evaluated
of the ‘251-labeled antigenic peptide were purified by affinity chromatography
affinity
Generation
and
by the using
4B.
into
12-well
was
quantified
rinsed
(Kd)
plates
but
of Recombinant
Receptors
in COS-]
detail generous
elsewhere gift
( I 2). The from
expression
Dr. D.G.
Preparation of Cultured After transfection, COS-l
vector
vectors
Cells for
rat
as described
in
for rat NPR-C
(2) was
Lowe.
Cell and Tissue Membranes cells were grown for 48 h in 150-mm
dishes in Dulbecco’s modified Eagle medium supplemented with 5% fetal calf serum before being harvested. Primary cultures of rat type-i
a
COS-
I cells
for an additional previously
by the
day.
(19).
were
cGMP
Briefly,
passaged production
the
cells
modified
were Eagle
containing 0.5 mM 3-isobutyl-l-methylwith increasing doses of NP in the same
supplemented
with
0. 1 % protease-free
BSA.
Twenty
was aspirated and the reaction was stopped 0. 1 N HC1 to each well. The samples were
at -70#{176}C, then
were
determined
15 mm at 37#{176}C in Dulbecco’s
minutes later, the medium by adding 250 p.1 of cold frozen
then
of cGMP
as described for
were
(21).
transfection,
medium and 20 mM Hepes xanthine, before incubation
immediately
COS- 1 cells were transfected with expression NPR-A and NPR-B by the DEAE-dextran method,
after
and grown
and incubated
medium
sites
program
Detection
hours
4#{176}C. The cells Transfection
of binding
LIGAND
Twenty-four
(l-4)-N-acetylmuramyl-L-alanyl-D-
in I ml of Freund’s
0.9%,
conjugate
thyroglobulin
White
apparent
removed
allowed
from
to thaw
the wells
progressively
with
a cell
at
scraper,
transferred to a microcentrifuge tube, and sonicated. After another 48 h of incubation at 4#{176}C, the proteins precipitated were separated by centnifugation. Aliquots tration by radioimmunoassay
ment
was repeated
using
triplicate
Western
twice
wells
acetylated as described
with
for each
different
to measure cGMP concenearlier (22). Each expeni-
batches
of transfected
cells,
sample
were
concentration.
Blotting
Solubilized resolved
were
proteins,
by sodium
mixed
dodecyl
with
Laemmli’s
sulfate-polyacrylamide
buffer,
gel electrophore-
J Am Soc Nephrol
9: 1777-1786.
sis (SDS-PAGE)
and transferred
binding
sites
NPR-B
1998
to nitrocellulose.
on the nitrocellulose
were
blocked
Nonspecific
protein
Statistical
by overnight
incuba-
Data program
tion at 4#{176}C in PBS-T (PBS with 0. 1% Tween-20, skimmed milk powder, 0.3% BSA, and 0.1% Affinity-purified PBS-T
anti-NPR-B
containing
0.3%
(413-426)-Tyr
BSA.
After
pH 7.4) plus 5% bovine y-globulin.
was
diluted
2 h of incubation
1 :250
at room
conjugate
antibody
(1 :30,000).
was visualized
luminescence
Affinity
on Kodak
detection
transfected
XRP-l
washing,
using
of solubilized
were
0.05
incubated
the ECL chemi-
M Tnis-HCI,
NPR-A
and NPR-B
in a total volume pH 7.4,
5 mM
of 0. 1 ml
MgCl,,
0.5%
±
30 mM
bis[2-(sulfosuccinimidooxycarbonyloxy)
little
30 mm
of incubation, heated
transferred above,
the reaction
pH 7.4.
buffer,
Proteins
for
to
were
5 mm
at
exposure
was
quenched
then
diluted
with
After paper
to Kodak
XRP-l
10 pA of 2 M
sample
Laemmli by SDS-PAGE
Western
was washed
After
in
95#{176}C,separated
nitrocellulose.
the nitrocellulose
2 d before
ethyl]sulfone.
analysis, in PBS-T
9 fmol/mg
COS-l
cells
(65
g)
and air-dried
Triton
X-lOO,
and 0.02%
sodium
NPR-B.transfected
N-glycosidase
F. In separate
transfected N-glycosidase
COS-cells were F under the
more,
to assess
sylated,
whether
solubilized
from
COS-l
U/ml
of N-glycosidase
PAGE
cells
under
azide
with
se-coupled
experiments
incubated with 0.5, same experimental
NPR-B
on vascular
membranes
from
isolated
transfected
with
NPR-B
F. The
reducing
samples
conditions,
in 50
membranes
from
merribranes were
were
NPR-B
was glycovessels
incubated
by Western
or
with
then. subjected
followed
ruM
units/ml of Further-
pregEomerular
1
the COS
0.7%
pentobarbital
(65 mg/kg,
ip) and perfused
through
with sodium
the aorta
first with
PBS and then with Bouin’s fixative solution. After perfusion, the kidneys were removed, cut in half, and fixed fcr 24 h in the same fixative, then embedded in paraffin. Tissue sections were cut, collected
on slides,
peroxidases, mm at room and incubated
HC1, 0.9% 4#{176}C with corresponding slides were then diluted
deparaffinated,
they were temperature. with
NaCI, the
To block
I % normal
donkey
serum
affinity-purified
in streptavidin
antibody
in TBS
( 1:50)
(50
with
horseradish
peroxidase
peroxidase
reaction
conjugate
mM
overnight
Tris-
at the
in TBS, the IgG 1:200, 1 :300
was carried
3,3’-diamino benzidine tetrahydrochlonide (0.025% HC1, pH 7.6) along with 0.0003% hydrogen peroxide. dehydrated and mounted for light microscopy.
for 30 water
or without
(lO_6 M). After several washes in biotinylated donkey anti-rabbit
for 2 h. The
endogenous
in methanol with running
pH 7.4) for 60 mm and then incubated
antigen incubated
in TBS)
and hydrated.
incubated with 3% H2O, The slides were washed
in
(n
3).
=
NPR-B-transfected whereas
of type
cells
ANP
had
B guanylyl
(Figure
(both
out with
in 50 mM
Tris-
The slides
were
performed
lB;
cycla-
< 0.05 at all
P
to
that
NPR-B
(Figure
plasma
membrane
Triton
remove
2B).
the
to determine
To improve
optimal
the intensity
fraction
was
anti-NPR-B
nonspecific
serum
antibodies.
affinity-purified
no crossreactivity
Preincubation
of anti-NPR-B
was was
used
affinity-
Immunoblotting
antibody
and showed
of the
solubilized
and the supernatant
X-lOO
In addition,
revealed with
were
(vollvol)
purified
reacted with
strongly
the other
NPR
(413-426)-Tyr
with
the synthetic peptide (NPR-B (4 1 3-426)-Tyr) blocked the labeling of this band (Figure 2A). A doublet of polypeptides with an apparent molecular weight of approximately 1 10 and 120 kD was detected. After treatment with 1 unitlml endoglylower
blotting.
anesthetized
on these
cell
SDS-PAGE.
cosidase
to SDS-
immunocytochemistry to 300 g) were
were
of NPR
± 5 pmol/L
in manner,
for immunoblotting.
F (to
band
remove
could
N-linked
form
membranes centrations
carbohydrate
be recognized,
unglycosylated
glycosylated rats (250
Cells rat NPR
points).
experiments
to the
Sprague-Dawley
generation the presence
receptors
Initial
for
0.1% 2 units of
5, or 40 conditions.
are
Immunoblotting for
COS-l
or without
COS
presence
and a Kd of 208
cGMP
confirming
conditions
cells were incubated for 18 h at 36#{176}Cunder agitation sodium phosphate, pH 7.1 , 50 mM ethylenediaminetetraacetate,
values
and
as described
film for 3 wk.
from
The
in a dose-dependent
effect,
with
membranes
0.05