Elevated atrial natriuretic polypeptide in plasma of hypertensive Dahl salt-sensitive rats

Vol. 137. No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June13,1986

Pages 876-883

ELEVATED ATRIAL NATRIURETIC POLYPEPTIDE HYPERTENSIVE DAHL SALT-SENSITIVE Rudolf

M. Snajdar

and John

IN PLASMA OF RATS

P. Rapp"

Departments of Medicine and Pathology Medical College of Ohio Toledo, OH 43699 Received

May 2, 1986

Summary: The relationship between circulating atria1 natriuretic polypeptide (ANP) and blood pressure was studied in inbred Dahl salt-sensitive (S) and inbred Dahl salt-resistant (R) rats. Two month old S and R rats raised on normal rat chow had only small differences in blood pressure and no difference in plasma ANP levels. In contrast, when 6-month-old rats also raised on normal chow were studied, S had markedly elevated blood pressure and a 4 fold increase in plasma ANP compared to R. Similar strain differences in blood pressure and plasma ANP could be induced in young rats by feeding them diets high in salt. In six week old S and R rats which had been fed high salt diet for 3 weeks the S rats showed higher blood pressure and plasma ANP than R rats. The high plasma ANP levels seen in the hypertensive S rats were interpreted to be a response to hypertension and not a cause of hypertension. There was no qualitative strain difference in the plasma ANP molecule as assessed by reverse phase high pressure liquid chromatography. D 1986 Academic Press,

Inc.

It both

has been shown

natriuretic

ANP appears

that

and diuretic to regulate

in the development

known that

Dahl

a

rats,

its

relationship

causing but,

as

diet,

therefore,

low plasma will

while

the

there

high could

Inbred Dahl salt-sensitive rats were used; the animals

0006-291 X/86 $ I .50 Copyr~&hr Cj I986 by Academic Press, All rights IJ~ reproduction in any form

should

Inc. reserved.

(2)

activity.

what

role,

blood

for

It

fulminant

in

which

Since if

any,

is also

well

to

when

do not study

(3,4). ANP and

A genetic

pressure. of salt

possesses

hypertension (R) rats

model

be a cause

was no evidence

(ANP)

hypertension.

salt-resistant

to be an ideal

salt-induced

* To whom correspondence

we wondered

develop

Dahl

MATERIALS (4)

as vasorelaxant

balance

(S) rats

ANP activity

be seen,

as well

polypeptide

of salt-dependent

appear to

natriuretic

and water

salt-sensitive

high-salt

Dahl

(1)

salt

ANP plays

fed

atria1

induced

such a defect

defect

hypertension, in S rats.

AND METHODS

(%/Jr) and inbred Dahl salt-resistant (SR/Jr) 35 generations had been inbred for more than be addressed,

876

Vol.

137,

No.

BIOCHEMICAL

2, 1986

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Although the official inbred strain designations are SS/Jr in our laboratory. and SR/Jr (5), for simplicity the animals will be referred to by their generic designations of S and R respectively. Rats were fed standard laboratory chow diet which contains 1% salt (Wayne IL) unless stated otherwise. In Rodent Blox, Continental Grain Co., Chicago, In study I rats two separate studies the amount of dietary salt was varied. ingredients containing essentially 0% NaCl were fed diets made from purified (sodium deficient) or the same diet with 8% NaCl added (Teklad, Madison, WI). based diets (Teklad) containing either In study II rats were fed whole grain 0.15% NaCl (low-salt) or 8% NaCl (high-salt). Systolic blood pressures were before animals were measured by the tail-cuff method (6) at least three days killed for blood collection. Blood was collected from the right ventricle of pentobarbital containing inhibitors; expressed as anesthetized rats into a plastic syringe per ml of blood: heparin, 50 U; aprotinin, 500 KIU; EDTA, lmg; soybean trypsin inhibitor, 50 BAEE U. Immediately after centrifugation 1 to 2 ml of plasma was applied to octadecylsilane cartridges (Cl8 SepPak; Waters, Milford, MA) washed with 10 ml of distilled water, eluted with 7 ml of 80% acetonitrile in 0.5 M acetic acid, and lyophilized. On the day of assay the dried residue was resuspended in RIA buffer and aliquotes assayed for ANP by RIA. Atria were removed, frozen on dry ice and weighed. Tissue was then minced, boiled for 10 minutes and homogenized in 1 ml of 1.0 M acetic acid. After centrifugation at 15,000 xg for 20 minutes at 4"C, the supernatant was recovered and stored at -2O"C, until fractionated by high-pressure liquid chromatography. Radioimmunoassay protocol: The polyclonal antisera used in this RIA was raised by us (7) in a New Zealand rabbit immunized with atrioneotin II (Peninsula Laboratories, Belmont, CA) coupled to bovine serum albumin'(i). The RIA incubation buffer consisted of 50mM sodium phosphate buffer, pH 7.2, containing 100 mM sodium chloride, 0.1% bovine serum albumin, 0.01% Triton x-100, and 0.1% sodium azide (RIA buffer). Synthetic atriopeptin III (Peninsula) or unknown sample in 100 ul of RIA buffer was incubated overnight with lOOn of our antisera (final dilution of 1:75,000), at 4°C. Twenty-four hours later iodinated atriopeptin III was added (10,000 CPM in 100 ul) and the tubes were incubated again overnight at 4°C. Separation of antibody-bound and free tracer was accomplished by precipitation with 1% bovine gamma globulin and 25% polyethylene glycol (9) and centrifugation at 2500 rpm for 30 min. at 4°C. The supernatant was aspirated and radioactivity of the antibody-bound fraction was counted in a gamma counter. Reverse-Phase High Pressure Liquid Chromatography (HPLC): Plasma and atria1 extracts were prepared as described above and rehydrated or diluted respectively in 0.1% trifluroacetic acid (TFA). All samples were passed through a 0.45 micron filter (Acre LC13; Gelman Sciences, Ann Arbor, MI) before being injected onto a high-pressure liquid chromatograph (Waters) fitted with a 4.6mm x 25cm C8 Aquapore RP-300 column (Rainin Instrument Co., Woburn, The column was run at 1 ml/min either under isocratic conditions (20% B) MA). or gradient conditions (0% to 15% B in 10 minutes then 15 to 60% B in 60 minutes where A = 0.1% TFA and B = acetonitrile with 0.1% TFA). HPLC fractions were dried in a Speed-Vat concentrator (Savant Instruments Inc., Hicksville, was rehydrated with RIA buffer and assayed for ANP NY), and the dried material by RIA. Statistical Analysis: Unpaired Student's t-test as well as two-way ANOVA were done on Olivetti microcomputer with software provided by the manufacturer (Olivetti, Dallas, TX). A p value less than 0.05 was considered significant.

RESULTS Radioimmunoassay: sensitivity

of

the

By following radioimmunoassay

a two day protocol was 877

dramatically

as described improved

above

the

over

our

Vol. 137, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL

previous one day incubation protocol from 3.1

pg to

400 pg of

displacement point

on the

(7).

10) with

extracted

atriopeptin

I,

was seen with

The within-

the

with

Table

Pressure:

1.

t-test),

Table

plasma and

rat

of variation

were 8% and

gave displacement curves III.

chow diet (containing

1%NaCl)

in terms of ANP levels (p > 0.05, t-test).

of S rats were slightly

Strain

Plasma ANP of

4-fold

differences

At

higher than R rats but the

(p = 0.086, t-test).

relative

in

5- to

to age-matched R

blood pressure

higher than R rats,

were also studied on diets containing

In study I, six-week-old

were also (p < 0.001,

1.

Plasma ANP concentration R female

rats

85 Plasma ANP,pglml nun&

Blood pressures were All values represent

222 119

taken mean

t 22.2 2 3.6

fed

and blood standard

several f SEX.

5-6

8R 251 111

varying amounts of

male S and R rats were placed for two weeks on

2-MONTH OLD

pressure,

added to

arginine-

Table 1.

S and

Blood

III

or

Plasma ANP and blood pressure of Dahl S

this age with S being significantly

S and R rats NaCl.

Only 17%

Plasma values were not corrected for

was however, elevated

rats (p < 0.02, t-test). marked at

bradykinin

atriopeptin

laboratory

were not significant

6-month-old S rats,

(as defined

Plasma from two-month-old S and R rats showed no

difference

this age blood pressures differences

II,

to the standard curve of atriopeptin

strain

Total binding was

human 28 amino acid ANP. The antisera

angiotensin

and R rats maintained on standard

significant

The 50 percent

(Peninsula).

The S and R plasma extracts

Plasma ANP and Blood

shown in

and III

and between-assay coefficients

12.2%, respectively.

are

II

Recovery using synthetic

which were parallel

tube.

showed 100% crossreactivity

as described above was 73%.

recovery.

per

standard curve was 50 + 1.6 pg.

showed no crossreactivity vasopressin.

III

of the standard curve was

28 amino acid rat ANP and with the major biologically

active ANP fragments, crossreactivity

Linearity

atriopeptin

maintained at 35 + 3%. The antisera in reference

RESEARCH COMMUNICATIONS

MONTH

of

before

1079 199

+ 259.3 f 5.3

plasma

878

2 and 5-to chow diet

10 R 242 119

collection.

f r

6-month-old

PROBABILITIES, 2x2ANOVA

OLD

10 s

+ 20.3 f 2.6

days

pressure laboratory

34.4 2.0

Strain 0.010
Age 0.007
Interaction 0.006
Vol.

137,

BIOCHEMICAL

No. 2, 1986

containing

purified

diets

2. There

was no strain

these

two

diets

significantly

systolic than

blood those

in the

R

long

relative

containing

above,

Table

With

2.

plasma

rats,

significant

weeks

Thus,

as is always of

Reverse identify Table

2.

STUDY I 2 weeks of diets

weeks

of 8% NaCl compared

STUDY II 3 weeks of diets

in S rats

interaction Similarly,

blood

the

strain

case

with

and diet

circulating

form

salt

on blood and

Atria1

of atria1

Plasma ANP concentration

also

shown

Extracts:

The increase

after

rats

rose

duration

best

resolve

PROBABILITIES, Strain

Diet

119 + 11.4 128 + 3.9

188 i 37.5 112 r 1.9

307 t 44.5 131 i 3.8

258 C 37.8 112 + 2.8

0.772
(0.001 0.578

PROBABILITIES,

20 s Females

20 R Females

24 S Females

19 R Females

Strain

Diet

227 + 34.2 131 + 3.9

251 + 52.8 101 + 2.5

642 2 117.8 177 _+ 3.9

192 t 50.4 105 + 2.4

0.008 (0.001

0.027 co.oo1

mean

days before

plasma samples were collected.

f SEM.

879

and to use

manipulation

17 R Males

8% NaCl

the

(p < 0.001).

16 S Males

HIGH SALT,

markedly

was necessary

SODIUM DEFICIENT + 8% NaCl

NaCl

ANP was

was unchanged.

enough

diet

in

was observed

on plasma

To it

in

of S rats

17 R Males

taken several

represent

long

diets

are

of R rats

peptide

grain

18 S Males

0.15%

the S

weeks as

was significant

in S and R rats

of

ANP levels

of S

pressure

pressure

not

two

diet

pressure

of

18 mmHg)

than

no such increase

feeding

The

however,

on whole

Results

and salt

blood

of 0% NaCl

pressure

(p < 0.008).

while

however,

(about was,

rather

plasma

rats

of strain

8% NaCl but the

LOW SALT,

Plasma ANP, pg/ml Blood Pressure, mmHg

Blood pressure

to R

given

t-test).

of age.

feeding,

R rats

those

diet

placed

weeks,

at 9 weeks

three-fold

of

three

SODIUM DEFICIENT, 0% N&l

Plasma ANP, pg/ml Blood Pressure, mmHg

values

were

in Table

S and R rats.

blood

female

Phase HPLC of Plasma

the

the

rats

given

were,

higher

six-week-old

(p < 0.00s).

interaction

All

to increase

for

the

8% NaCl

of

are

than

both

(p > 0.05

the

three

in

significantly

weeks

on purpose)

elevated

after

(p < 0.001)

on 0% NaCl diet

three

thus

rats

data

COMMUNICATIONS

ANP levels

S rats

ANP was about

in R

Two

and sacrificed

was significantly

Plasma

0.7).

were

0.15% NaCl or 8% NaCl

discussed

RESEARCH

ANP of the S and

was observed

of S rats

(done

II,

(p >

plasma

8% NaCl fed

rats.

to the

In study

in the

increase

pressures

BIOPHYSICAL

0% NaCl or 8% NaCl;

two weeks

relative

of

sufficiently rats

for

this

either difference

higher

fed group;

AND

2x2 ANOVA Interaction 0.091 0.526 2x2 ANOVA Interaction 0.003 to.001

Vol.

137,

No. 2, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

8” *w 60-

S

S PLASMA 50-

PLISMA

i

c

oJ, ,, ,; 1, j 20 25 30 35 40 45 50 55 60 FractionNumber Figure 1. Immunoreactive ANP-like material (ir-ANP) after reverse phase HPLC. Elution position of synthetic atria1 peptides is indicated by bars at the top of each graph, where I = atriopeptin I, II = atriopeptin II, III = atriopeptin III and ANP = the 28 amino acid form of rat ANP. Panels A and B were obtained from S and R plasma extracts, respectively, using isocratic elution. Panel C and D were obtained from S plasma and S atria1 extracts, respectively using gradient elution as described in the methods section. R plasma and R atria1 extracts run with gradient elution gave identical patterns (not shown) to the corresponding S samples shown in panels C and D.

isocratic

conditions.

fractionated

Figure

using

20% acetonitrile

elution

position

for

shown

in Figure

1.

shown

in Figure

1A and lB,

immunoreactivity rat also

of other 95% of component made by

on HPLC forms total) (about boiling

immunoreactive

synthetic Both

than

at the

As shown

III.

lC,

eluting acid

in each

give peak

the

S

plasma

in fraction

880

reversed,

28 as

acid

plasmas

were

for gave

the presence a major

30 and a very

47.

same two peaks

was

are

28 amino

and R

to look

S rat

in fraction

III

in fraction

synthetic

in order

in Figure

and

The

95% of the total

as the

ANP peak eluting

2% of total)

material

conditions

II

eluted

In addition

fractions.

ml

I,

ANP peaks

atriopeptin

as

of 0.5

and atriopeptins

same position

gradient

immunoreactive

in acetic

ANP

respectively.

in plasma.

HPLC of S and R plasma

and collection rat

5% eluted

under

reverse-phase

the S and R plasma

was found

ANP, and less run

1 shows the

Extracts but

Figure

of

minor S atria

the proportions 1D.

Under

(>

of these

Vol.

137,

same

No

gradient

identical

BIOCHEMICAL

2. 1986

conditions,

profiles

AND

plasma

(data

not

BIOPHYSICAL

and

shown)

atria1

as those

RESEARCH

extracts

COMMUNICATIONS

of R rats

also

gave

of S rats.

DISCUSSION Our data

show that

R rats

in situations

levels

diverge

exist

where

between the

between Two possible

levels

in the

2) a direct that

plasma

S

in

S rats;

of hypertension

per

two-month

one would

levels

under

plasma

ANP levels irrespective

studies

involving

a heart

(16)

and posture

immersion primary study

stimulus

that

atria1

with

in

hypertension

show a single

form and R

acid

increased

that

changes

intake.

Alternatively,

Our data

plasma

loading

(14.151,

plasma

SHR

(191.

a recent ANP levels

there

is evidence

Therefore

distention plasma

water

may be the

In addition

also

on

Previous

stretch

(lg),

ANP

consequence

pressure.

that

the pressure-induced

end-organ

in

stores.

in hypertensive

ANP levels.

With

was

increase

atria1

(SHR) showed

pressure

have shown

it

of atria1

ANP

are

is wall

compatible

interpretation.

Fractionation

amino

blood

rats

(12)

to R.

blood

volume

atria1

to ANP or

may be a direct

of high (131,

of ANP from

with

plasma

cause

have suggested

hypertensive

salt

S rats

preparation

(17)

is

to increased this

lung

pressure

that

compared

ANP

hyporesponsiveness

a compensatory

the

diet.

circulating

and others

S rats

ANP

differences

hyporesponsiveness

Renal

S and

Plasma

pressure

the elevated

of increased

of

Dahl

fed high-salt

to ANP.

in hypertensive

release

correlated

likely leads

for

on spontaneously

positively

anticipate

blood

in rats for

between

are minimal.

We (11)

se.

old

conditions

of hypertension,

or

1) renal

is hyporesponsive and

different

when large

can be offered

hyporesponsiveness

increased

not

differences

rats

Dahl

one-

especially

pressure

as in old

explanations

kidney

are

and R strains

strains,

result

demonstrated

blood

hypertensive

the S-rat

ANP levels

of major

rat

of atria1 plasma

Dahl

S

and R

immunoreactive

ANP, which peptide ANP peaks

plasma

plasma

eluted

rat

(20).

in the

It

reverse

phase

with

the

as the most probable is

same position 881

the

peak coeluting

has been identified

in the

on

important

to note

in HPLC.

HPLC system synthetic

28

circulating that

Our results

the rule

S

Vol. 137, No. 2, 1986

out

BIOCHEMICAL

the possibility

form

in

S and R rats.

amounts by

of a gross

of another

In addition

HPLC

material

was observed

directly

addressed

is

to

intact

the same position. in

into

whether

processing

shortly

after

of the

indicate

which

probably

may

of atria1

into

acid

seen

in

However, to

that

the

be a

hypertensih

direct

Dahl

consequence

It

to be shown how much each of these

hyporesponsiveness,

was only that

by methods

(22,23),

eluted

and

in

the minor

that

ANP precursor

to

be determined

needs

it

acid

form

occurs

during

S rats

have

increased

or

circulation.

a consequence

renal

made

still

of this

it

extracts

extracts

28 amino

ANP and/or remains

it

presence

indicate

suggests

small

was fractionated

Our data

atria1 This

peak,

but

ANP precursor

identical.

precursor

plasma

(14,20,21)

(20).

circulating

ANP plasma

The

groups

126 amino

circulation.

release

Our data ANP levels

are

of the

conditions.

and coworkers

peak

acid

seen when

by several

the

RESEARCH COMMUNICATIONS

structure

28 amino

preparations

The large

plasma

may be released

to the

gradient

by Schwartz

preserve

component

under

since

in the

peak were

in plasma

the ANP precursor,

known

alteration

immunoreactive

reverse-phase

AND BIOPHYSICAL

of pressure-induced

contributes

of renal

hyporesponsiveness

distention

to the

factors, elevation

plasma

of that

the is

atria1

to wall.

hypertension

of plasma

or

ANP levels.

ACKNOWLEDGEMENTS The work NL20176

was supported

by grants

from

the

National

Institute

of Health,

and HL34394.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

deBold, A.J., Flynn, T.G. (1984) Life Science 33, 297-302. Currie, M.G., Geller, D.M., Cole, B.R., Siegel, N.R., Fok, K.G., Adams, S.P., Eubanks, S.R., Galluppie, G.R., Needleman, P. (1984) Science 233, 67-69. Dahl, L.K., Heine, M., Tassinari, L. (1962) J. Exp. Med. 115, 1173-1190. Rapp, J.P., Dene, H. (1985) Hypertension 7, 340-349. Rapp, J.P. (1986) Rat News Letter 16, 22. Friedman, M., Freed, C. (1949) Proc. Exper. Biol. Med. 70, 670-672. Snajdar, R.M., Dene, H., Rapp, J.P. Endocrinology. Submitted. (1977) Endocrinology 100, Tager, H.S., Hohenboken, M., Markese. J. 367-372. (1971) J. Clin. Endocrinol. Metab. Desbugois, B., Aurbach, G.D. 3,732-738. Chard, T. (1982) An Introduction to Radioimmunoassay and Related Techniques, pp. 188, Elsevier Biochemical Press, Amsterdam. 882

Vol.

11.

12. 13. 14. 15. 16.

17. 18. 19.

20. 21. 22. 23.

137,

No. 2. 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

7. 775-782. Snajdar, R.M., Rapp, J.P. (1985) Hypertension Hirata, Y., Ganguli, M., Tobian, L. (1984) Hypertension 6 (Suppl I), 148-155. Dietz, J.R. (1984) Am. J. Physiol. 247, R1093-R1096. Lang, R.E., Tholken, H., Ganten, D., Luft, F.C., Ruskoaho, H., Unger, Th. (1985) Nature 314, 264-266. Rascher, W., Tulassay, T., Lang, R.E. (1985) Lancet 2, 303-305. Katsube, N., Schwartz, D., Needleman, P. (1985) Biochem. Biophys. Res. comm. 133, 937-944. Hodsman, G.P., Tsunoda, K., Ogawa, K., Johnston, C.I. (1985) Lancet 2, 1427. Imada, T., Takayanagi, R., Inagami, T. (1985) Biochem. Biophys. Res. Comm. 133, 759-765. Noresson, E., Ricksten, S-E,, Thoren, P. (1979) Acta. Physiol. Stand. 107, 9-12. Schwartz, D., Geller, D.M., Manning, P.T., Siegel, N.R., Fok, K.F., Smith, C.E., Needleman, P., (1985) Science 229, 397-400. Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T., Matsuo, H. (1985) Biochem. Biophys. Res. Comm. 129, 248-255. Kangawa, K., Fukuda, A., Minamino, M., Matsuo, H. (1983) Biochem. Biophys. Res. Comm. 119, 933-940. Kangawa, K., Tawaragi, Y., Oskawa, S., Mizuno, A., Sakuragawa, Y., Nakazato, H., Fukada, A., Minamino, M., Matsuo, M. (1984) Nature 312, 152-155.

883