Evidence for the presence of anp-precursor material in the rat thymus

Vol. t55, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 700-708

September 15, 1988

EVIDENCE

FOR

THE

PRESENCE OF ANP-PRECURSOR IN THE RAT THYMUS

MATERIAL

Angelika M. Vollmar and R~diger Schulz Institut fur Pharmakologie, Toxikologie Universit~t M~nchen, K6niginstr. 16, D-8000

und Pharmazie, M ~ n c h e n 22, FRG

Received August 3, 1988

Acidic extraction of the thymus from two day old rats followed by purification on Sephadex G-50 gel filtration revealed the presence of atrial natriuretic peptide-like material (IR-ANP) by radioimmunoassay. Verification of the obtained immunoreactivity has been achieved by the use of two different types of antisera, i.e. two antisera directed against ANP (99-126), the other antiserum recognizing the Nterminal fragment (11-37) of the precursor ANP (1-126) molecule. In addition, reverse phase high performance liquid chromatography (RP-HPLC) and high performance gel permeation chromatography (HP-GPC) monitored by the three antisera have been employed for analysis of the extracted IR-ANP. In both systems the IR-ANP corresponded to the 15 kDa-ANP molecule (i-126) . Furthermore, by using immunohistochemical techniques a distinct localization of the IR-ANP material could be demonstrated. The outer cortical area of the thymus, containing mostly lymphoid cells, showed extensive immunostaining with the three different antisera. The data reported here indicate that the rat thymus is a source of ANP. © 1988 A c a d e m i c Press, Inc.

Mammalian

myocytes

synthesis review ANP

of

see

(99-126) in

glands

(4,5).

functional ANP tide.

natriuretic

influences

kidney,

on

ANP receptors, suggesting report

be

the

the

peptide

agreed salt

blood some

and

the

water and

these

site

(ANP)

that

vessels of

major

of (for

released

balance

by

the

adrenal

organs,

bearing

also seem to be able to synthetize additional

functions

for

of specific ANP receptors

this

pep-

on rat thy-

(9) implies that the thymus may be added to the list

of organs

ANP

to

is generally

Apparently,

A recent

on which

finding

tissue.

considered

atrial It

(3)

the

(6,7,8),

mocytes this

the

1,2).

acting

are

may

Indeed,

precursor

ANP

even

acts.

In analogy

suggest

the

the data communicated (1-126)

is

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

present

700

with

presence in

other

of ANP

here propose the

rat

organs, in

this

that the

thymus,

and

Vol. 155, No. 2, 1988

might therefore

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

indicate that this peptide has a role in the

immunsystem.

MATERIALS AND METHODS Tissue extraction The thymus was removed from 20 two day old male Sprague Dawley rats under a dissection microscope immediately after decapitation and transfered into ice cold 0.1N HCI (2.5 ml). The tissue was homogenized by use of a ground glass homogenizer (10--20 strokes) at 4°C, and protein concentration was d e t e r m i n e d by the Bradford method (i0) (Bio-Rad kit, Munich, FRG). After boiling the homogenate for 60 sec in a m i c r o w a v e oven, it was centrifuged at 20000 x g for 20 min. The clear supernatant (2 ml) was extracted by adsorption to activated Amberlite XAD-4 adsorbens resin (250 mg per column) (Serva, Heidelberg, FRG). Elution was carried out with 2.5 ml 80% acetonitrile in 0.1% trifluoroacetic acid and lyophilized.

Chromatoqraphic analysis of the thymus extracts i.

Gelfiltration (GF): For further purification, the lyophilized tissue extract was dissolved in 200 ~i 0.1M acetic acid, centrifuged and the supernatant applied to a Sephadex G-50 (Pharmacia, Uppsala, Sweden) column (9 mm x i000 mm, Abimed, DUsseldorf, FRG). The flowrate was i0 ml 0.1N acetic acid per h. Calibration was carried out with bovine serum albumin (Vo) , vitamin BI2 (Vt) , rat-proANP (2-126) and rat-ANP (99-126). IR-ANP fractions (2.5 ml), detected by RIA, were pooled and lyophilized.

2.

Hiqh performance gel permeation chromatoqraphy (HPGPC):. An aliquot of the pooled IR-ANP fractions from t h e GF run was applied to a Spherogel TM TSK, 2000 SW column (i0 ~m, 7.5 mm x 300 mm; Beckmann Instruments, San Ramon, CA, USA), and eluted with 0.09% TFA containing 0.005M Na2SO4, 0.002M NaH2PO 4 and 30% acetonitrile (flowrate: 0.3 ml/min). Cal~bration was performed with the standards described above. Fractions (0.3 ml) were lyophilized and tested for IR-ANP by three different antisera (see RIA procedure).

3.

Reverse phase hiqh performance liquid c h r o m a t o q r a p h y (RP-HPLC): An aliquot of the lyophilized IR-ANP fractions from the GF ~in was redissolved in 25 ~i 0.1% TFA and loaded onto a HPLC C18 ODS Ultrasphere TM column (5 ~m, 4.6 mm x 2500 mm, Beckman Instruments, San Ramon, USA). Calibration of the column was performed with rat-ANP (103-.123), rat-ANP (103-126), rat-ANP (99-126) and ratpro-ANP (2-126). Elution was carried out with a linear gradient of acetonitrile (20-55%, 55 min) in 0.1% TFA (flowrate 1.5 ml/min). Fractions (1.5 ml) were again assayed with three different antisera by RIA. 701

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Antisera and radioimmunoassay procedures Different types of rabbit polyclonal employed in measuring ANP-like material:

antibodies

were

i.

Antisera raised against the carboxy-terminal sequence of ANP: a) antiserum "Toni III", previously described (ii), was raised against human-ANP (99-126), and b) antiserum "Loisl" was raised against rat-ANP (99-126). Antiserum "Toni III" recognizes the carboxy-terminus of ANP (99-126), showing crossreactivity with ANP (103126), but almost none with ANP (103-125) and ANP (103123). It crossreacts with rat-ANP (99-126) to 82%. A n t i s e r u m "Loisl" is directed against the ring epitope of rat-ANP (99-126), not reacting with the human-ANP (99-126) (1%). Rat and human material differ in only one amino acid (position ii0) situated in the ring structure. Both antisera ("Toni III" and "Loisl") do not detect the N-terminal fragment ANP (1-30) (unpublished results), but crossreact with rat-pro-ANP (2-126) to 30% and 48%, respectively.

2.

A n t i s e r u m "GT-23" has been characterized (12). Briefly, it was generated against rat-ANP (11-37), a fragment of the N-terminal sequence of the ANP molecule (1-126). This antiserum does not recognize ANP (99-126), but shows 100% crossreactivities to N-terminal sequence fragments like human-ANP (1-30), human-ANP (11-37), rat-ANP (1-98) and rat-ANP (1-126).

The radioimmunoassays, employing antiserum "Toni III" and "~Q~sl", were performed as described (ii), except using [izDI]-rat-ANP (99-126) and cold rat-ANP (99-126) instead of the human-ANP (99-126). The RIA procedure for the N-terminal fragments of rat-proANP has been reported el~w~here (13). Human-ANP (1-30) was used as standard and the[ z I]-ANP (1-30) as tracer.

Recovery experiments Duplicates of 20 thymus glands were homogenized in 2.5 ml PBS, using a glass potter and incubated for 2 h at 37°C in order to digest endogenous IR-ANP material. IN HCl was then added to adjust the pH to i. 1 pmol synthetic rat-pro-ANP (2-126) was added to one of the acidic tissue homogenates and the extraction and chromatographic procedures were performed as described. The concentration of IR-ANP was measured by the two RIA systems in the homogenate with or without added pro-ANP, and the values obtained for the amount of pro-ANP (2-126) were corrected for the corresponding crossreactivity of the antiserum ("Toni III" and "GT-23", respectively) used.

Immunohistochemistr7 3-5 mm thick sections of the thymus were fixed in Bouin's fluid for 3 h at 4°C. The tissues were dehydrated in graded alcohols, cleared in xylene and embedded in Paraplast. 702

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Slices (5 ~m) were cut (Leitz microtome, Munich, FRG) and mounted on acid-cleaned, gelatin chrome alumn coated slides. Endogenous peroxidase was inhibited by preincubation of sections with methanol, containing 0.3% H202. Sections were then processed for immunohistochemis~ry with either antiserum "Toni III", "Loisl" or "GT-23". Before applying the primary antisera, sections were preincubated with goat serum (1:30) for 20 min at 25°C to reduce unspecific binding. Primary antibodies ("Toni III", "Loisl" and "GT23", respectively) were applied in a i:i00 dilution for 12 h at 4°C followed by an incubation with goat-anti-rabbit IgG (1:50, Sigma, Munich, FRG) for 30 min at 25°C. Finally, a rabbit peroxidase anti-peroxidase (PAP) (1:200, P-L Biochemicals, Inc., Milwaukee, WI, USA) was applied for 1 h at 25°C. All antisera were diluted in 0.i M PBS containing 1% goat serum and 0.1% BSA. After extensive rinsing with PBS, sections were incubated with a fresh solution of ethylcarbazol (0.3 mg/ml) in 0.i M acetate buffer (pH 5.2) containing 0.003% H202. Controls included incubation with rabbit preimmune serum (i:i00), use of primary antisera previously adsorbed with either synthetic rat-ANP (5 ~g/100 ~i PBS), or rat-pro-ANP (2-126) (25 ~g/100 ~i PBS) for 12 h at 4°C, and omission of the primary antisera, goat-antirabbit IgG, or PAP, respectively. Unless otherwise stated, all these controls gave negative results.

Materials:

[125I]-rat-ANP (99-126) was purchased ~Kom Amersham (Braunschweig, FRG). Human-ANP (1-30) and [~z°I]-human-ANP (1-30) were from Peninsula Labs (Belmont, CA, USA); rat-ANP (103123), rat-ANP (103-126), human- and rat-ANP (99-126) from Novabiochem (L~ufelfingen, Sitzerland). Rat-pro-ANP (2-126) was kindly provided by Drs. Sch6ne, Preibisch and Seipke (Hoechst AG, Frankfurt, FRG). All other reagents were purchased from Sigma (Taufkirchen, FRG).

RESULTS

AND D I S C U S S I O N

Initial

purification

of

the

acidic

rat

thymus

tissue

extract, preextracted by means of XAD-4 adsorbens resin, was carried out by Sephadex G-50 gelfiltration. tive ANP-like material sera) and

amounted eluted

as

obtained

to appoximately a

single

The immunoreac-

(determined by three 50 pg/mg wet

peak

with

a

tissue

molecular

corresponding to that of synthetic rat-pro-ANP

(2-126)

antiweight weight (data

not shown). The recovery of exogenous synthetic rat-ANP 126)

(i

pmol),

using

this

extraction

procedure, was 22%. In comparison, vein,

vena

and

rat atrium, rat pulmonary

cava and rat hypothalamus

contain

150, 0.6, 3 and 0.02 ng IR-ANP/mg wet tissue (14, 8, 15). 703

(2-

purification approximately , respectively

Vol. 155, No. 2, 1988 T77 o o~

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

7

B

z zz

Ill

o i TONI

4O

III

I00

•~ o o . 99

.,oo[

,

< i

tl

i

A

55

./\

50 126

LOISL

1

>- C i

100

®

so 99 ~.. m

126

20

140

~

,200

55 GT-

23

+

70

' Q

2HN~ 1

99

....

/i\.

loo

126

20 15

25

"

35

20

fraction number FIGURE

30

trl i ~40

fraction number

1

Chromatoqraphic analysis of acidic extracts of rat thymus.

A)

RP-HPLC profiles monitored by three different antisera. A r r o w s i n d i c a t e t h e e l u t i o n p o s i t i o n of A N P A N P (i03-126), A N P (99-126) a n d A N P (2-126).

B)

Recognition employed.

c)

HP-GPC profiles obtained by use of the c o r r e s p o n d i n g antisera shown on the panel B. Elution positions of ANP (99-126) and ANP (2-126) are marked by arrows.

A verification

site

of

ANP

and analysis

(1-126)

by

applying

antisera

recognition tivity

2.

two

ANP-immunore-

approaches:

different

to monitor

chromatographic

sites

of

ANP-immunoreac-

separation

(Figure i, panel A) and HP-GPC

Figure

1 (panel A)

IR-ANP

material

antibodies.

distinctly

(1-126)

antisera

(Figure i, panel B).

conducting RP-HPLC

with

for ANP

the

of the endogenous

activity has been obtained by two different i.

(103-123),

shows

from

a GF

As illustrated

nize different

sites

the RP-HPLC run

(panel C).

profiles

detected

by

techniques

of the pooled

three

different

in panel B, these antisera

of the ANP molecule 704

(1-126):

recog-

Antiserum

Vol. 155, No. 2, 1988

"Toni

III"

molecule. However,

recognizes

the

crossreactivity

epitope

carboxy-terminal

It therefore crossreacts

Antiserum ANP

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

"Loisl"

with

(99-126),

the

100% with ANP

ANP

is apparently

of rat ANP

of

(1-126)

directed

was

ANP

(99-126). only

30%.

the

ring

against

but does not recognize human

(99-126). The synthetic precursor ANP

(2-126)

is detec-

ted to 48% by this antibody. In contrast,

antiserum

"GT-23"

does not recognize ANP well

(100%)

(13),

as

fragments

well

as

raised

against ANP

(99-126) at all. in the

the

region

It detects equally

from amino

rat-precursor

(11-37)

ANP

acid

(1-126)

1-98

molecule

(12). Using these three antisera for monitoring a RP-HPLC run of a thymus

extract

resulted

in

virtually

the

same

profile

(Figure i, A). The IR-ANP material eluted mainly in a single peak,

corresponding to synthetic rat-pro-ANP

rating

the

extracted

identified

IR-ANP

immunoreactivity

by

HP-GPC

possesses

weight as the synthetic rat-pro-ANP This

material

again

is

detected

the

same

(2-126)

by

(2-126). Sepa-

revealed

all

that

the

molecular

(Figure i, C). three

antisera.

Another peak of lower molecular weight is present when monitoring the run with antiserum "Loisl" and "GT-23",

respec-

tively.

product

It

most

likely

represents

a

degradation

lacking the intact carboxy-terminal of ANP

(1-126), because

it is not detected by antiserum "Toni III". Based

on

material

these which

results, seems

rat

thymus

contains

chromatographically

to

IR-ANP-Iike

represent

the

precursor ANP (1-126) molecule. Identifying

the

precursor-ANP molecule

in a tissue

raises

the question of whether this organ might also be the site of synthesis for the peptide, as it has been suggested for several other organs,

including the heart

(7, 8, 16). Ascri-

bing this putative ANP-precursor molecule to an anatomical localization distinct to the thymus would support this hypothesis. Immunohistochemical analysis of rat thymus sections, indeed, revealed ANP-immunopositive staining localized mainly in cells of the outer cortex sera

("Toni

III",

"Loisl"

the same staining pattern

and

(Figure 2). The three anti"GT-23")

showed

essentially

(data not shown). By using the N705

Vol. 155, No. 2, 1988

FIGURE

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2

Immunohistochemical analysis of rat thymus. The outer part of the cortex (c) of two thymus lobules separated by the connective tissue capsule (tc) is shown. Arrows mark representative immunopositive cells, most likely thymocytes. Their characteristic thin layer of cytoplasma is heavily stained by antiserum "GT-23" (x i000). terminally could

directed

exclude

ANP

(99-126)

the

precursor

does

not

antiserum

that

the

molecule

appear

to

experiments

cubated

with

synthetic

staining

has

staining

occured

with

(99-126)

antiserum 126)

(1-126)

circulate

is in

the

for immunopositive

The

prevailing

and

a much

staining

weaker

less the

with

rat

observed.

antiserum

As

"GT-23"

of

because

it

(12).

In

"GT-23"

almost

prein-

no

immuno-

expected,

immuno-

preincubated

This demonstrates

N-terminal

from

uptake

plasma

antiserum

2) we

result An

likely,

(2-126)

(results not shown).

requires

might

the thymus.

rat-ANP been

(shown in Figure

staining

outside

conducted

positive ANP

positive

synthesized

control

"GT-23"

sequence

of

with

that the

pro-ANP

(i-

staining. in the

immunoreaction 706

outer

cortex

in the

of

medulla

the

thymus

(data

not

Vol. 155, No. 2, 1988

shown)

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

suggest that most of the

represent

lymphoid cells

immunopositive cells might

(thymocytes),

being mainly

local-

ized in the cortical area and to a much lower degree in the medulla (17). Since light microscopy does not allow an exact identification

of

the

stained

cells,

electronmicroscopic

analysis is needed. Furthermore, thymocyte maturation occurs on the way

from the

cortex to the medulla

(18),

and thus

especially the outer cortical area contains mainly proliferating

cells

which

originate

from

the

bone

marrow

(19).

Therefore, the actual site of expression of ANP is difficult to ascribe. In

summary,

the

thymus,

representing

organ that generates T-cells ANP

(1-126).

This

finding

(20),

in

the

central

lymphoid

is suggested to express

context

that thymocytes bear receptors for ANP as to whether the atrial natriuretic

with

recent

reports

(9) evokes questions peptide might play a

role in the immune defence mechanisms.

ACKNOWLEDGEMENTS

We would like to thank Drs. M. Cantin and G. Thibault (Montreal, Canada) for providing the antiserum "GT-23" and especially for the support in establishing the RIA, Dr. R.M. Arendt (M~nchen, FRG) for donating the antiserum "Toni III", and Drs. Sch6ne, Preibisch and Seipke (Frankfurt, FRG) for providing the synthetic rat-ANP (2-126), Dr. Achmed Hassan (M~nchen, FRG) for his help performing the immunohistochemistry. The valuable support of Ms. A. Friedrich, G. Hach, D. Misera and U. R~berg and the secreterial assistance of Ms. B. Hilz is gratefully acknowledged. Supported by DFG (R.S.).

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Van Haelst, V.

(1967) Z. Zellforsch. 77, 534-549.

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Reinherz, 821-856.

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Harris, J.E. and Ford, C.E.

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Stutman, O.

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E.L.

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