Human cardiac plasma concentrations of atrial natriuretic peptide quantified by radioreceptor assay

Vol.

133,

No. 3, 1985

December

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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3 1, 1985

Pages

I,4

E. Bkgisser Departments

, A.E.G. Raine 1,3 , P. Eme' , B. Kamber2 ad F.R. Biih1e.r'

of Research

and Medicine, University Rasel, Switzerland

CH-4031 2

Received October

209

aND?IRATIcpJSoFATRIALNATRIUREI'ICPEPTIDE QuAtVrIFIn,BYRADI~ASSAY

HUMANCARDIAC-

1

1201-l

8,

ClBA-Geigy,

CR-4002

Basel,

Hospital,

Switzerland

1985

SUMMARY: The presence of high affinity receptors for atria1 natriuretic peptide in bovine adrenal cortex has enabled the development of a sensitive, specific and rapid radioreceptor assay for this psptide in human plasma. In 18 norm1 subjects, venous plasm atria1 natriuretic peptide concentration ranged fran 6 to 65 pM. This plasma concentration was two-fold higher in right atrium as canpared to venous blood in 12 patients investigated by cardiac catheterisation, confirming that the right atrium is the site of release of atria1 natriuretic peptide into circulation. There was a further step up in plasma atria1 natriuretic peptide concentration between pulmnory arterial and aortic plasma. This finding indicates that released hormone in man may undergo further activation in the lungs, or that there may be direct release frm the left B 1985 Academic Press, Inc. atrium.

There is now conclusive

evidence

natriuretic

factor,

natriuretic

(2) and vasorelaxant

present

in both rat

peptides

fore,

radioreceptor adrenal 3

(4,s)

and physiological

we have developed

cortex

assay

and describe

precursors

of ANP into of its

of the peptides

the circulation,

for ANP in human plasma, utilising

binding

With this

these

determined

end-organ

as atria1 and possess

(1)

has been established,

sensitive

(8).

knm

cardiocytes

The structure

and their

role

previously

here a specific,

membrane receptors

AKGR is a Medical Dept. of Medicine, 4 Dr. E. &rgisser, Switzerland.

atria1

(3) properties.

is known about the release

concentrations

peptides,

in mammlian

(4) and human (5) atria

have been synthesized

man, little plasm

are present

that

(6,7).

In

its

effects.

There-

and rapid of ANP to bovine

assay we have obtained

direct

Research Council Travelling Fellcw; permanent address: John Radcliffe Hospital, Oxford U.K. ANAWA Laboratories, CH-8602 Wangen, current address:

Abbreviations: ANP, atria1 natriuretic peptide.

natriuretic

peptide;

hANP, human atria1 0006-291X/85 $1.50

1201

All

Copyright 0 1985 rights of reproduction

by Academic Press, Inc. in any form reserved.

Vol.

133,

evidence right

BIOCHEMICAL

No. 3, 1985

from cardiac

atrium.

may further

catheter&&ion

In addition, increase

AND

its

left systemic

BIOPHYSICAL

RESEARCH

that ANP is released

atria1

secretion

circulating

into

COMMUNICATIONS

blood from the

or pulmonary activation

of ANP

concentration.

MATERIALsAND-

Materials: Alpha-hANP was purchased frcmn Novabiochem, Switzerland and the synthetic Ciba-Geigy peptide CGP-34089 refers to atriopeptin II (12) to which an extra tyrosine at the N-terminus was synthesized. All other chemicals were ccmnnercially availabel and of highest grade. Icdination of alpha-hANP and CGP-34089: The chloramine-T iodination (22) of at roan alpha-NLNP and CGP-34089 was carried out in 1.5 ml l+propylene-vials temperature. For both peptides, 2.5 mCi (5~1) of Na I wasaddedto 10 ul of 0.25 M sodium phosphate buffer (pH 7.4). The amount of the peptides in the reaction was 15 ug. In order to start the reaction, 5 ul of freshly prepared chloramine-T solution (1 mg/ml in 0.05 M sodium phosphate buffer, pH 7.4) was added. An additional 5 ul was added after 20 and 40 seconds and the reaction was terminated after 60 seconds with 600 ul of a stopsolution, consisting of 0.25% human albumin, 0.1% mercaptoethanol in 0.05 M sodium phosphate buffer, pH 7.4. For separation of unreacted material, the r-action mixture was passed through a reversed-phase minicolumn (300 ul of Spherisorb IO u ODS in a 1 ml plastic syringe). After washing of the column with 1.2 ml of 0.25 M sodium phosphate buffer, a methanol/water step-gradient containing 1% trifluoroacetic acid (TFA) was applied and 600 ul of the radioligands eluted mainly in the 52% methanol fraction. These were then stored at -20 C until use. Under these conditions, the radioligands could be used for one month without significant degradation as checked by HPLC-analysis (data not shown). Reversed-phase HPIC analysis was carried out on a 5 u ODS-Hypersil column (o 4 x 250 sun). The flawrate was 1.2 ml/min and an acetonitrile/water gradient, containing 0.1% TFA, fran 14 to 56% of acetonitrile was applied. One minute fractions were collected and the radioactivity was counted in a gamma scintillation spectrcmeter. For HPIC of non-radioactive peptides (2 - 10 ug), the same conditions were used, excepf2$he optical density ~5 monitored at 210 nm. Typically, the monoicdinated I -alpha-hANP and I -CGP-34089 eluted at 35 and 32% acetonitrile, respectively. Preparation of adrenal cortex membranes: Fresh bovine adrenals were obtained fran the slaughter house and carried on ice. After the medulla was cut out, the cortex was dissected with scissors in the presence of a small volume of icecold Buffer A (5 IIM TRIS, 1 mM MgC12, 250 mM sucrose, pH 7.4 at OOC). Hcmogenization with a Polytron was carried out in Buffer A and larger particles were removed by passing the hcmogenate through a single layer of cheese-cloth, followed by twenty strokes with a Potter harogenizer. In order to remove heavy cell particles; the harcgenate was centrifuged at 3,000 x g for 10 minutes at 4OC and the pellet was discarded. The plasma membrane containing supernatant was spun at 39,000 x g for 10 minutes and the pellet was resuspended in Buffer B (75 mM TRIS, 25 mM MgC12, pH 7.4 at 4OC). After repeating this wash-step twice, the pellet was resuspended in Buffer B, including 1 mM dithiothreitol (DIT) and 250 mM sucrose. Aliguots, containing 1 g of original cortex tissue per ml, were frozen in liquid nitrogen and kept at -7OOC for several months without significant loss of binding activity. Radioligand/receptor binding: Prior to use for binding experiments, the thawed membranes were washed via centrifugation at 39,000 x g for 10 minutes and the final pellet was resuspended in Buffer C (50 mM IRIS, 500 n-&lMgC12, 1 mM EDNA, 0.5% bovine serum albumin (BSA), pH 7.4 at 25OC. The high MgC12 concentration was used and BSA was included to reduce nonspecific binding, due to filter blank. In order to prevent degradation of the radio1202

Vol.

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No. 3, 1985

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ligand during incubation with plasma membranes, before starting the reaction, 1 mu final concentration of the phenanthroline (Merck, Dannstadt), which acts in this system as a peptidase inhibitor, was added to the membrane suspension. As checked by,gX-analysis after incubation , no significant degradation of the radioligand I -CEP-34089 was detected. For equilibrium binding experiments, the incubation took place at 25OC for 60 minutes in polypropylene tubes. The reaction was started by the addition of the membrane suspension to the tubes. The final assay volume was 200 ul and typically a binding capacity of 7.7 fmoles corresponding to 25 rrg original adrenal cortex tissue per tubs was used. Pound and free radioligand were separated by rapid filtration assay using Whatman GF/C glass fibre filters (o 25 ma). In order to reduce nonspecific binding, prior to filtration, the filters were wetted with 1 ml of Buffer C. After pouring th sample on the filter, they were washed twice with 4 ml of ice-cold wash buffer (IO mM 'IRIS, 10 mM KgCl 0.1% BSA, pH 7.8 at OOC). The filters were collected and the radioactive 2' y was measured in a gannna scintillaton counter. The nonspecific binding was defined as the binding in the presence of 0.5 us alpha-hAWP, which is sufficient for complete canpetition as shown in Fig.2, panel b. Data analysis: The saturation binding curve was analysed according to a single Mass Action Law model, using weighted, nonlinear regression analysis a described elsewhere (23). The ccmpetition binding curve was also analyzed by computerized nonlinear regression according to a four-parametric logistic function as described elsewhere (24). Collection of blood frcan patients: Blood samples were taken fran 12 patients (8 men, 4 wanen) aged 20-70 years (mean 51 years) during diagnostic cardiac catheterisation. Mean right atria1 pressure was normal (less than 7 n Hg) in seven patients and slightly elevated in five (less than 11 mn Hg). No patients had clinical or radiological evidence of left ventricular failure. 6-8 ml blood was taken into chilled heparinised tubes fran a peripheral vein, the right atrium, main pulmonary artery, and, in seven patients, fran the aorta or left ventricle. Plasma samples werg assayed in triplicate. After centrifugation at 1,600 x g for 10 minutes at 4 C, with addition of 0.1 q/ml aprotinin (SIGMA), the fresh plasma was subjected to Sep-Pak extraction as described below. Using these conditions, exogenously added alpha-hANP was recovered in the radioreceptor assay without significant degradation demonstrable by HpLc analysis, whereas freezing and thawing caused a IO-20% loss of ANP activity (data not shown). In order to increase the sensitivity of the assay and to exclude nonspecific interference of plasma with the bind+% assay, 1 ml of plasma, which contained approximately 600 cpn of added I -alpha-hANP as internal standard for recovery, was slowly passed through a reversed-phase minicolumn (Sep-Pak C-18 cartridge, Waters), mounted on a 2 ml plastic syringe. After three washes with 2 ml of 0.1% TFA/water and two wash-steps with 40% methanol in 0.1% WA/water, the peptides were directly eluted with 0.1% TFA in 100% methanolo into incubation tubes. After evaporation of the solvent under vacuum at 30 C, the radioactivity in each tube was measured, yielding a recovery rate for alpha-hANP fran 60 to 85%. All plasma samples were extracted and assayed in triplicate as described below. The binding reaction occurred Radioreceptor assay for endqenous alpha hANP: as described for the canpetition experiment as shawn in Fig.2. In analogy to curve with known concentrations of alpha-& in radio-=yf2a s* the range of IO to 10 M was constructed. The binding assay with samples containing unknown concentrations of endcgenous ANP was run simultanously. Since the total radioactivity of the radioligand was approximately the small contri70,000 cpn/tube (equivalent to 111 pM assay concentratiofJ5 I-alpha-ANP was bution of radioactivity caused by the internal standard negligible. For guantitation, the binding of the unknown plasma sample was cxmpared with a standard curve as depicted in Fig. 2, by using the four-parametric logistic function. The detection limit for alpha-hANP as defined by 95% of the upper plateau of the standard curve, was 2 fmoles/tube, corresponding to 1203

Vol.

133,

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No. 3. 1985

AND

BIOPHYSICAL

approximately 2.5 @I plasma concentration. interassay C.V. was 10.1% (n=7).

RFSLILTS

The principle

RESEARCH

Intraassay

AND

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C.V. was 8.2% (n=8) and

DISCUSSICN

of the radioreceptor

assay

involves

endcgenous ANP in plasma and a synthetic-24-amino

canpetition

acid

between

radioligand

125

I-m-34089

(for

in bovine adrenal

structure cortex

see Materials

by HEW as described

usedasstandard, concentrations

of alpha-human

unsatisfactory

as a receptor W-34089.

(5),

were iodinated, Unlabelled

in Fig-l.

and assay values

radiolabelled

for AtW receptors

membranes. For use, both CGP-34089 and synthetic

amino acid alpha human ANP (alpha-hANP) characterized

and Methcds)

are expressed

ANP. Iadinated radioligand

used in the assay possess

been

in terms of the equivalent

and for

high affinity

and

alpha-hANPhas

alpha-hANP

As shcwn in Figure

purified

28

this

itself

proved

reason we have used

2, theadrenalcortexmembranes

binding

sites

for ANP (K

=116

@l),

D

this

provides

femtanoles assays

the basis

per tubs ccmpares

atriopeptin

binding

II,

there

reaction angiotensin

and diuretic the following

II,

aldosterone,

LH-RH and scmatastatin. of alpha-m

alpha-hANP concentration.

investigate

possessing

m-34089 full

a large

there

renin,

angiotensinogen,

arginine

vasopressin,

insulin,

a value

The radioreceptor (approx.

13

release,

activity

was no crossangiotensin calcitonin,

I,

ACXR,

by adding known concentrations and assayed

as described

of 96.4 + 8.0% (n=6) of the added assay described 2 hours

blood was first

total

taken after

males aged 22-52 and 5 females aged 1204

and

biological

substances:

giving

specific.

number of only-used

agents and in particular

was confirmed

of 2

radioimnuno-

is also highly

alpha-hANP,

fran

of detection

described

a normal range of plasma NW concentrations

intracardiac

normal subjects,

with

peptide

advantage of being very rapid To establish

recently

which was then extracted

and Methods,

the limit

The assay

interference

Accuracy

to plasma,

under Materials

futher

was observed

was no detectable

fran

with

(9,10,11).

the minimum length

antihypertensive

sensitivity;

favorably

for ANP in human plasma

While eguipotent

(12),

of the assay's

21-41,

here carries

the

duration). in man and to rest who

from had

18 110

Vol.

133,

No. 3, 1985

BIOCHEMICAL

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a

a04 a 0 a032 2, .-s 002 05

3oowo B T,

2ooooo

.Z 3

ii .Y am E

0 i

100000

,

i

0

0 IIJ

% Methanol

t

0

10

20

M

40

50

64

Fraction

Figure 1. Purification

human atria1

natriuretic

and HPK-+@erization peptide ( I -alpha-W)

of radiT@vely and

labelled

a step-gradient elution over a reversed-phase minicolumn after icdination. For both of the two peptides the main peaks eluted at 52% methanol and contained the radioligands which were used routinely in the radioreceptor aB%y and for I reversed-phase EiPIC chqu@erization as documented in Panels c ( -alpha-hANPI and d, ( I -GP-34089). Under the conditions used, a main peak, representing the monoialinated peptide, at-d a later, minor peak with the diicdinated peptide, was resolved. The unlabelled peptides eluted fran the reversed-phase minicolunm at 40% methanol ard fran the HPIC-column a few fractions earlier as the monoiodinated peptides. Since after the minicolumn separation, the fraction of the monoicdinated peptides was greater than 95% and free of unreacted iodine and peptide,,25specific radioactivity of 2,200 Ci/tMol I -EP-34089 proved to be a radiofor these radioligands was assumeL ligand with excellent birx@g properties for the ANP-receptor in adrenal cortex membranes. In contrast, I -alpha-W proved unsuitable for direct binding, and was used as the internal staru3ard for recovery measurements during plasma extraction of endogenous ANP as described in Fig.3.

significant (range

6-65)

plasma

levels

at the

time

medical history.

Plasm ANP concentration

for men and 30 2 5 pM (range were

sanewhat

of diagnostic

higher cardiac

catheterisation 1205

was

32

+

6

(SD!)

@I

for wanen. Peripheral venous

18-42)

(47 + 8pM)

30 m E

20

in

12

in

patients five

who were of whan right

studied atria1

i t Y SC

0

4: 0

40

lo

Vol.

133,

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No. 3, 1985

AND

BIOPHYSICAL

.

RESEARCH

COMMUNICATIONS

b

I 500 Total

loo0

Ixlo

125I -CGP-34089

-11

-10

-9

alpha-hANP

(PM)

-8

-7

(log M)

Binding of 125 I -CGP-34089 to bovine adrenal cortex membrane. Figure 2. (filled circles) and A typical saturation binding curve, inc)y$iihg specific nonspecific (open circles) binding of I -CGP-34089 to bovine adrenal cortex membrane is shown in panel a. A saturable, high affinity binding site with a dissociation constant of 116 pM and a binding capacity of 45 fmoles/mg of protein was obtained. In panel b a represen&ive standard curve with alpha-hAW as cmpetitor for the radioligand I -a-34089 is shown. An of 582 k 27 p&l and a slope of 0.98 + 0.02 fran iine consey$Yay&l. rime&s was obtained. For saturation and canpetition experiments as depicted in the figure, the conditions were very close to equilibrium (kintics data not shown) ard applicable for quantitative and reproducible determinations of alpha-m in humn plasma. pressure illustrates, concentration providing bloodstream

was slightly blood

increased taken

fran

of alpha-hANP strong fran

support within

100

r

(for the right

than for

the

that

-pp-0.005

see legend

atrium of venous

the concept right

W”O”S

details

that

atrium.

7

bad a significantly plasma, alpha-hANP

‘I Pulmonary artery

p-002

As Figure higher

76 2 8 @I (p>O.O05), is released

ANP concentrations

-

Right atrium

to Fig.3).

were

into lcwer

the (64 5

7

L

-

Arterial

Figure 3. Plasma ANP levels in venous bleed, right atrium, pulmonary artery and arterial blood. Results are mean + SEN for 12 subjects, and statistical significance was calculated by paired t-test. The figure demmstrates a step-up of ANP concentration fran peripheral to right atria1 plasma, and fran pulmonary artery to arterial plasm. 1206

3

Vol.

133,

No. 3, 1985

BIOCHEMICAL

11 @l) in the pulmonary Strikingly,

there

concentration

of aortic

circulate

life

in blood

place

recent

from within

release

and degradation.

to left

atria1

the right

atrium.

that

Right

indicate

that,

left

is that

Haiever,

ventricle,

convert

there

and an alternative

and left

effects

relased

clear

atria1

respectively.

cient

stimulus

heart

(16).

fran

the right

of right

and it

(15),

to arise

mainly

will

fran

reflect

ANP

Our results

One explanation

of

fran within

the left

venous drainage

into

atrium

is

in alpha-hANP

is that

atria1

In the rat,

In vitro

what the release

atria1

studies

increased

lung peptidases into

the may

physiologically

on isolated

may cause diuresis

the humoral efferent

mechanism and the roles

to pulmonary right

ANP release,

and vagal modulation

distension

alpha-m

at present

ANP may be in relation

for right

both adrenergic

atria1

as it

atria

through

and systemic pressure

vascular

is a suffi-

the possibility

Either

(17).

the Henry-Gauer weight

right

or left

reflex

(18),

and distinct

of

but

frcrn

(19).

are in a similar

range,

of alpha-m but

slightly 1207

we have measured higher

of

is seen also in the isolated

have raised

of release

agent is of low molecular

The plasma concentrations assay

direct

fast

forms. It is not all

right

place

possibility

imply that

of the myocardium. step-up

takes

little

thus

a preponderance

and the aorta.

ANP release

is relatively

ANp precursors

active

artery

the plasma half-

concentration

is a further

secretion

between rather

is likely

fran most parts

there

state:

to 2.5 fold

(14)

alphahARP

the pulmonary further

1.7

peptides

continous

a balance

plasma alpha-m

sinus

unexpectedly, between

finding

circulating atria1

via the coronary

concentration

that

have reported

fran

that atria1

and our findings

represents

ranging

of mixing.

in alpha-m

(9,10,11)

seems likely

(13),

Animal studies

draining

atrium.

2-3 minutes

ANP content

considered

this

it

plasma concentration

generally

reports

the atria , even in the resting

of ANP is very short,

the alpha-W

step-up

COMMUNICATIONS

@I (pPO.02).

15

initial

in man. Moreover,

RESEARCH

as a consequence

significant

to 76 +

blood

confirm

BIOPHYSICAL

presumably

was then a further

Our results

takes

artery,

AND

by

radioreceptor

than radiotiunoassay-based

Vol.

133,

No. 3, 1985

in

estimates be

released

directed all

man

into

the

bloodstream

a specific

circulating

It is pertinent

receptors

studied

(lo),

it

determinant

ANP petides,

alpha-hANP egualto

AND

BIOPHYSICAL

RESEARCH

Since more than one biologically

(9,10,11).

against

receptor.

BIOCHEMICAL

or greater

active

that

possible

of a peptide

in contrast that

so far

is

COMMUNICATIONS

peptide

my

radioimunoassays

may not fully

recognize

to the naturally-occurring

adrenal

the adrenal

ANP receptor

has an affinity

for

thaneither

thevascular

or renal alpha-hANP

(20).

It is noteworthy that the half maximal suppression (EC ) of stimula50

ted adrenal aldosterone and cortisol circulating

plasm concentrations

release,

70-120

@I

is close to the

(8),

of alpha-hANP, lower then the EC

for

50

vasorelaxation

(400-600 @I) (20) and considerably

of alpha-hANP required to produce natriuresis

lmer than the concentration

(14,201. The implication

is that

a major role for ANP in normal subjects may be a tonic modulation of adrenal aldosterone and cortisol natriuretic

effects

release. The vasorelaxant

of alpha-m

high plasm concentrations failure

my becane relevant

natriuretic

and left

developed and employed is specific, of measuring physiologically

5. 6. 7. 8.

peptides circulate

atria,

adrenal and possibly vascular function.

4.

only when pathologically

(21).

released fran both right

3.

the

of the hormoneoccur, as my be the case in heart

Weconclude that atria1

1. 2.

and especially

relevant

in concentrations The radioreceptor

sensitive,

in man, and my be sufficient

assay ws have

rapid ard carries

concentrations

to modify

the advantage

of ANP.

Sagnella, G.A. and Mac Gregor, G.A. Nature 309, 666-667 (1984). de Bold, A-J., Borenstein, H.B., Veress, A.T. and Sormekerg, H. Life Sci. 28, 89-94 (1981). Currie, M.G., Geller, D.M., Cole, B.R., Boylan, J.G., YuSheng, W., Holmberg, S.W. and Needleman, P. Science 221,71-73 (1983). Atlas, S.A., Kleinert, H.D., Camargo, M.J., Januszewicz, A., Sealey, J.E., Laragh, J.H., &chilling, J.W., Lewicki, J.A., Johnson, L.K. and Maack, T. Nature 309, 717-719 (1984). Kangawa, K. and mtsuo, H. Biochm.Biophys.Res.Carmun. 118, 131-139 (1984). Maki, M., Takayanagi, R., Misono, K-S., Parley, K-N., Tibbetts, C. and Inagami, T. Nature 309, 722-724 (1984). Oikawa, S., Imai, M., Ueno,A., Tanaka, S., Noguchi, T., Nakazato, H., Kangawa, K., F'ukuda, A., and Matsuo, H. Nature 309, 724-726 (1984). De Lean, A., Racz, K., Gutkowska, J., Nguyen, 'FT., Cantin, M. and Genest, T. Endocrinolcgy 115, 1636-1638 (1984). 1208

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Napier, M.A., Vandlen, R.L., Alters-SchZjnberg, G., Nutt, R-F., Brady, S., Lyle, T., Winguist, R., Faison, E.P., Heinel, L.A. and Blaine, E.H. Proc.Natl.Acad.Sci.USA 81, 5946-5950 (1984). K. and Leidenius, R. Lancet & Tikkanen, I., Fyhrquist, F., Met&&me, 66-69

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Kuribayashi, T., Nakazato, M., Tanaka, M., Nagamine, M., Kurihara, Kangawa, K. ard Matsuo, H. N.Engl.J.Med. 312,1456-1457 (1985). Tanaka, I., Misono, K.S., arid Inagami, T. Biochem.Biophys.Res.Curmun. 124,

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M., Nakaoka, H., Imtako, K., Masao, A., ard Yamaji, T., Ishibashi, Fujii, J. Lancet1, 1211 (1985). Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T. and Matsuo, H. Bicchem.Biophys.Res.(%mnm 129, 248-255 (1985). Sugawara, A., Nakao, K., Morii, N., Sakatmto, M., Suda, M., Shinmkura, M., K., Soneda, J., Ban, T. and KiSO, Y., Kihara, M., Yamori, Y., Nishimura, Imura, H. Bicchem.Biophys.Res.Cutmun. 129, 439-446 (1985). Currie, M.G., Gelfer, D.M., Cole, B.R., Siegel, N.R., Fok, K.F., Adams, S.P., Eubanks, S.R., Galluppi, G.R. and Needleman, P. Science 223, 67-69

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