Immunolocalization of Natriuretic Peptide Receptor B in the Rat Kidney

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