BIOCHEMICAL
Vol. 166, No. 3, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1080-1087
February 14, 1990
CEARACTERIZATION
OF IMMDNOREACTIVE
BRAIN NATRIDRETIC
PEPTIDE
IN HUNAN CARDIAC ATRIUM TATEYAMA 1.3 , Jun HINOl,
Hitone Kenji 1
Toshio
KANGANA',
Department
of Biochemistry,
'National
of Geriatric
December
18,
MINAMINO',
565,
College,
Japan
Research
Osaka
MATSUO'
Medical
889-16,
Center Suita,
Institute, Japan
Osaka University
Medicine,
Fukushima, Received
Miyazaki
Cardiovascular Fujishirodai,
3 Department
Miyazaki
Kiyotake,
Kihara,
Naoto
OGIHARA3 and Hisayuki
Osaka
553,
Medical
School,
Japan
1989
SUMMARY: Based on CDNA sequence data for human brain natriuretic peptide (BNP) precursor (II, a radioimmunoassay (RIA) system highly specific to human BNP (hBNP1 was developed and used to characterize immunoreactive (ir-1 hBNP in atrium indicated that ir-hBNP cardiac atrium. Gel filtration of ir-hBNP in was mainly comprised of two molecular forms of 13-15K and 4K. In reverse phase high performance liquid chromatography (HPLC), the low molecular weight (MN) ir-hBNP emerged as a single peak at an elution time identical to that of synthetic hBNP-32. The high NN ir-hBNP was also eluted as a single peak. On the other hand, tissue concentrations of ir-hBNP in cardiac atria were found to be 9.98-593.22 pmol/g in 13 specimens, being about l/150 the concentration of ir-human atria1 natriuretic peptide (hANPI. These results demonstrate that hBNP is present as a peptide in human heart, suggesting that hBNP is secreted from heart and functions together with hANP as a hormone. 01990 Academic Press, Inc. Our recent of the
presence
homeostatic neuronal probe
of
(3),
to highly
of
control
body
cloned
(1,4).
fluid
By using
(2,.
we have
mammalian
of BNP in porcine
a dual
balance pathways
precursors that
identification
BNPs exhibit
conserved differences
pathways
giving
rise
data large
sequences of
cDNA for
and sequenced
Structural
species
system
and blood
to different
by
hBNP,
of mammalian BNPs may endogenous
has lead
BNP
and
pressure porcine
cDNAs encoding
for
structural
mammalian
brain
to recognition
ANP
in
through
BNP (pBNPI
precursor
hBNP and rat
rBNP and pBNP have species
differences,
ANPs (5). result molecular
in
regulating hormonal
and as a
BNP (rBNP) demonstrated in contrast
Such large
structural
different
processing
forms
in each
species.
Abbreviations: BNP, brain natriuretic peptide; ANP, atria1 natriuretic p, porcine; rat; y-hBNP, pro-hBNP; hBNP-32, propeptide; h, human; r, hBNP-26, pro-hBNP(83-108); radioimmunoassay; ir-, hBNP(77-108); RIA, inununoreactive; MN, molecular weight; TFA, trifluoroacetic acid; HPLC, high performance liquid chromatography. 0006-291x/98 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
1080
Vol.
166, No. 3, 1990
The proteolytic
BIOCHEMICAL
processing
as indicated
PBNP, (6-8).
by the
In order
tissues,
it
tissue
is
first
RIA for
of rBNP in heart
presence
to determine
of rBNP-45
the
necessary Ir-hBNP
concentration.
established
pattern
AND BIOPHYSICAL
characterize in this
MATERIALS
distinct
from
storage
function was
that
form
of hBNP
ir-hBNP,
in human atrium
hBNP, as reported
is
as a major
physiological to
RESEARCH COMMUNICATIONS
then
characterized
of
of rBNP
in various measure by
its
a newly
paper.
AND METHODS
Peptides: Human BNP-32, hBNP-26 and N-Tyr-hBNP-26 were synthesized by solid technique conducted on a phenylacetamidomethyl resin with Applied phase Purification was performed by reverse Biosystems 430A peptide synthesizer. phase and CM ion exchange HPLCs, and correct synthesis and purity of the peptide were confirmed by amino acid analysis and sequencing. Human BNP-26 for immunization was donated from Daiichi Pure Chemicals Co. Ltd. Preparation of antisera: Human BNP-26 (7 mg) was conjugated with bovine thyroglobulin (15 mg, Sigma) by the action of water soluble carbodiimide (9). The conjugates were emulsified with Freund's complete adjuvant and used for immunizing rabbits by the described method (91. by the lactoperoxidase RIA for hBNP: N-Tyr-hBNP-26 was radioiodinated The resulting reaction mixture was immediately (Calbiochemj method (9). subjected to reverse phase HPLC to isolate monoiodo-N-Tyr-hBNP-26, which was stored at -8OV until use. RIA for hBNP was performed by the method used for RIA for pBNP, and N-ethylmaleimide-treated bovine serum albumin (BSA1 was used Peptide standard (hBNP-32) or unknown sample (100 l.11) was pre-incubated (10). 100 ~1 of antiserum (#189-61 for 24 hr. Then, 100 l.11 of tracer solution ;i% I-N-Tyr-hBNP-26) was added and incubated for additional 48 hr. Free and bound tracer were separated by the double antibody method. All procedures were carried out at 4'C, and samples were assayed in duplicate. RIA for hANP: RIA for a-hANP was carried out as reported previously (91. Tissue extraction: Human atria1 tissue was obtained at autopsy from patients within 4 hours post mortem. After weighing, atria mainly including auricles were diced and boiled for 10 min in 10 volumes of water to inactivate and 1M HCl were intrinsic proteases. Glacial acetic acid added (final concentration = 1 M and 20 mM, respectively) after cooling to 5'C, and boiled tissue was homogenized with a Polytron mixer for 4 min. Aliquots of the supernatants, obtained by centrifugation at 18,000 x g for 25 min, were lyophilized, dissolved in the RIA buffer, and then submitted to RIAs for hBNP and hANP. Characterization of ir-hBNP and ir-hANP in human cardiac atrium: The acid extracts of human cardiac atria were each diluted two-fold with water and then loaded onto cartridges (2.5 ml, Waters). The cartridges were Sep-pak C-18 were eluted with 60% washed with 0.5M acetic acid and the adsorbed materials CH3CN containing 0.1% trifluoroacetic acid (TFA). After vacuum evaporation, each extract was subjected to Sephadex G-50 gel filtration (fine, 1.8 x 134 using 1M acetic acid as a solvent. An aliquot of each fraction cm, Pharmacia) was submitted to RIAs for hBNP and hANP. fractions exhibiting hBNP and The hANP immunoreactivity were further analyzed by reverse phase HPLC on a Hi-Pore RP-318 column (4.6 x 250 mm, Bio-Rad) with a linear gradient elution of CH3CN from 10% to 60% in 0.1% TFA solution at a flow rate of 1.0 ml/min. Aliquots of all fractions were submitted to RIAs for hBNP and hANP. RESULTS AND DISCUSSION RIAs
for
hBNP and hANP:
and was usable maximum
inhibition
at a final
Anti-hBNP dilution
of binding
serum
#189-6
showed
was observed 1081
at
high
As shown
of 1:420,000. 2.6
fmol/tube
avidity in
Fig. and
for 1, the
hBNP half-
peptide
Vol.
166, No. 3, 1990
BIOCHEMICAL
O-
10-r
Figure
in by
estimated
peptides.
Since this
crossreact
RIA system
hand,
recognized
by for
hBNP-32
this
were Four
in
ANP-29
the
case
of
since
of ir-hBNP
frog
antiserum
i.e.
ANP-24. than specific
The 0.003%
and hBNP-26
RIA system
for
not pBNP-
highest (Fig.
1). On
to be equally
showed
anti-hANP
a-
to hBNP.
MW hBNP was thought of
did
examined,
was highly
hBNP-32
and the
concentration
rBNP was less
high
antiserum
natriuretic with
peptides
Crossreactivity
to be 0.04%,
known
The
and
were
any crossreactivity
tissue
#189-6
extended
antiserum,
examined
filtration were
exhibit
pBNP-26.
of the
with
natriuretic
antiserum
antiserum.
characterized gel
other
chicken
of ir-hBNP
human cardiac ir-hBNP
I
ld
comparable
serum
#125-8
hANP was also
with found
to hANP.
Characterization atria
not
to measure
that
was determined
to be specific cardiac
I
lo4
Specificity
immunoreactivity.
N-terminally
the
did
hANP
with
verified
fmol/tube.
was able
observed
results
affinity
I
10'
crossreactivity
#189-6
CL-rANP,
crossreactivity
its
of
significantly
other
of 0.2-30
measuring antiserum
rBNP-32.
These the
a range
interference
26,
I 10’
1. Inhibition of binding #189-6. Final dilution: 1:420,000. (M): hBNP-32 and hBNP-26. (-): rBNP-32. (-1: (-U-lJ-): a-hANP, CL-rANP, chicken ANP-29, frog ANP-24. Vasopressin, somatostatin, enkephalins and other neuropeptides examined, but no cross-reactivity was observed in concentrations less than 1 ug/tube.
was measurable
hANP,
I
10'
RESEARCH COMMUNICATIONS
Peptide (fmol/tube) of 125 I-hBNP to antiserum
was
without
,
loo
AND BIOPHYSICAL
and ir-hANP
(Table
by gel atria
ir-hBNP
filtration
were
first
profiles observed
l),
are in
in
and ir-hANP
and subjected shown
fractions
human cardiac reverse
atrium: of five
phase
to Sephadex
in Fig. #27-31 1082
2. and
atria1
HPLC. G-50
In all #40-48
Among 13
gel
cases,
extracts Extracts
of
filtration. two peaks
corresponding
of to
Vol.
BIOCHEMICAL
166, No. 3, 1990
Table
1. Tissue
no.
concentration
sex
1 2 3 4 5 6 7 8 9 10 11 12 13
65 a3 70 a4 67 55 70 19 77 58 75 47 75
F F M M M M F M M F M F M
molecular
weight
form
were
2:3 to 1:5
only
1:lO
in Fig.
AND BIOPHYSICAL
of ir- BNP
and ir- ANP
RESEARCH COMMUNICATIONS
in human
cardiac
atria
cause of death
ir- BNP @mow
ir- ANP (nmoUs)
ir- BNP / ir- ANP (%I
lung cancer rectal cancer lung cancer bladder cancer pleural mesothelioma hepatoma multiple myeloma germinoma of the testis cholangiocarcinoma carcinoma of the uterus myocardial infarction adult T- cell leukemia esophageal cancer
38.86 30.54 18.61 9.98 58.69 19.65 593.22 114.20 64.09 42.32 120.27 66.40 121.82
17.69 5.57 9.16 1.63 18.31 2.66 20.92 3.16 10.56 3.54 35.56 20.05 17.08
0.22 0.55 0.20 0.61 0.32 0.74 2.84 3.61 0.61
13-15K
and 4K.
in
Fig.
Relative
2a,
2b (patient
ratios
of
high
2c and 2d (patients
#71.
On the
other
MW form
#5,
hand,
1.20 0.34 0.33 0.71
#ll
ir-ANP
to
low MW
and #12), emerged
but as two
n
z
3.0 -
.k 8 :s 5
15-*0 ;L
P4
20
30
40
Fraction
Figure
50
60
70
,1
20
30
40
Fraction
number
50
60
70
number
2. Sephadex G-50 gel filtration of acid extracts of human atrium. Sample: Atria1 extracts (450 mg wet wt equivalent) of (a) patient #5, #ll, and (dl patient #12. (bl patient #7, (c, patient Column: Sephadex G-50 (fine, 1.8 x 134 cm, Pharmacia). Flow rate: 10 ml/hr. Fraction size: 5 ml/tube. Solvent: 1M acetic acid. Arrows indicate 4) whANP and
5)
elution Vt.
positions
of
1083
II
Vo,
21
y-hANP,
31
B-hANP,
Vol.
166, No. 3, 1990
peaks Fig.
of MW 13K and 3K in Fig.
phase
#40-46
2a-2c
and #48-52
HPLC on a C-18
eluted
as
while
The
hANP (Fig.
and as three
RESEARCH COMMUNICATIONS
peaks
of
13K,
5K and 3K in
of hBNP-32
to
consist
hand,
found
in patient
subjected of high
subjected
to reverse
identical
phase
the
low
not
component
was mainly
MW ir-hBNP
was
to that
of synthetic
MW emerged
at the
elution
time
to that
of o-
identical
shown
HPLC and emerged
(data
of a single
reverse
phase
shown).
at the
Thus,
corresponding
comprised
in Fig.
elution
2atimes
low MW ir-BNP
to hBNP-32.
of cc-hANP,
emerged
30
20
Time
obtained
4 shows from
as single
peaks,
of y-hANP.
High
was much earlier
time
fractions
Figure
HPLC.
and ir-hANP
to that
elution
3a, identical
at a time
MW hBNP- and hANP-immunoreactive
hBNP and ir-hANP
Figure
time
each
with
was
On the
B-hANP being
#12.
MW ir-hBNP
its
in Fig.
was eluted
respectively
low MW ir-hANP
to
were
of an intermediate
to reverse
and cc-hANP,
concluded
2d
an elution
low MW ir-hANP
subjected
other
As shown
at
ir-hANP
of Fig.
The low MW hBNP- and hANP-immunoreactivity
3b). also
High
peak
the
of B-hANP. 2c were
column.
a single
hBNP-32,
but
AND BIOPHYSICAL
2d. Fractions
time
BIOCHEMICAL
of Fig. one of the
patient
#12
and ir-hANP MW ir-hBNP than
that
2a-2d elution
(Fig.
2dl.
was eluted appeared
were
profiles Both
ir-
at an elution
as a single
of y-hANP.
also
Since
peak, high
40 Time (min)
(mln)
3. Reverse phase HPLC of (a) low MW ir-hBNP and (b) low MW ir-hANP of human cardiac atrium. Sample: (a1 Fractions #40-46 (190 ul/tubel and (b1 fractions #48-52 (1.2 ul/tube) in Fig. 2d. Temperature: ambient. Column: Hi-Pore RP-318 (4.6 x 250 mm, Bio-Rad). Flow rate: 1.0 ml/min. Solvent system: H20:CH3CN:10%TFA = (Al 90:10:1, (BI 40:60:1 (vol/vol). Linear gradlent elution from (Al to (BI for 60 min. Arrows indicate elution times of II hBNP-32, 2) 8-hANP and 3) a-hANP. 1084
MW
Vol.
BIOCHEMICAL
166, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I --
Time
Figure
ir-hBNP
in
in Fig.
2d,
four
other
the
cases
to be derived
these
high
facts, Very
were
recently,
as hBNP-32
characterization
of
HPLC,
atria
was
showed
tissue
serial
Table
patient's
profile. ,
of
in human cardiac to that However,
atrium
in porcine differences
(99.9
cardiac
hBNP-32
and y-hBNP
in
present
isolating
in human atria the
of ir-hBNP in tissue concentration
pmol/g)
of tissue
on
y-hBNP. the
A
mean
from study
concentration 1085
atrium:
filtration
human cardiac
of human caridic
of synthetic
hBNP-32
of ir-hBNP was in
a range
varied tissue
(data
among specimens.
widely Table
l/6
observed
not with
of 9.98-593.22 with
concentration
from
atria
and ir-hANP
concentration
about
After and reverse
in 13
extracts
calculated and was
cardiac
by gel and ir-hANP
to that
atrium
HPLC
Based
in
concentration
nmol/g.
phase
from
parallel
tissue
1.63-35.56
reverse
property.
to be derived
tissue
differences
in
as that
time
(to be published).
All
Concentration large
MW ir-hBNP
of ir-hBNP
curves
elution
characterized
cardiac
precisely.
of ir-hANP,
a range
in
same
hydrophilic
and ir-hANP
1 summarizes
indicating
In the case within
and y-hBNP
ir-hBNP
dilution
shown). pmol/g
forms
concentration
re-measured
of high
succeeded
of ir-hBNP
at the
intrinsic
was deduced
we have
concentration
phase
its
and two molecular
identified
eluted
time
from
MW ir-hBNP
atrium,
Tissue
was also
elution
earlier
was thought
cardiac
(min)
4. Reverse phase HPLC of high MW ir-hBNP of human cardiac atrium. Sample: Fractions #27-31 (630 ul/tube) in Fig. 2d. Chromatographic conditions were identical to those of Fig. 3. Arrow indicates elution time of y-hANP.
1
that
specimens of ir-hBNP
was comparable in
rat
in human
atrium. were
much
Vol.
166, No. 3, 1990
larger
than
BIOCHEMICAL
those
differences
expected
in age,
Tissue
sex,
in part
ir-hBNP
in a
was
measurement
reported
in
be similar
body
varied
between
concentrations
suggests
the
regulated
form the
to low
is
processing
MW form of
8-hANP facts
indicated systems
of
physiological
used
cardiac
that
of
of atrium.
ventricle
ir-hBNP
relative
and no
for
in heart
is
atrium
will
be
found
to
demonstrated
that
hBNP functions
homeostatic concentration
apparent
balance
of
of ir-hBNP
to
correlation
was observed
in 13 human specimens.
and secretion
This
and BNP
of ANP
hBNP and functions
form,
as seen heart
(1).
2d
hBNP-32
and functions
fact may be
similar of
pBNP shown
each
did is
ir-hBNP,
relative not
affect
probably
in
of mammalian
was
of high
specimen.
in
to that
cardiac
MW
Furthermore, predominance
a mature
atrium
form
in
the
indicates
of pBNP (10,121,
rBNP has a distinct
rBNP-45
hBNP-32 ratio
of of hBNP
as a hormone.
and y-hBNP Although
presence
in
in Fig. that
of
although
was different
of hBNP is
the
characterization
molecular
of hBNP-32
paper by
l/100
maintaining
two peptides
suggest
from
pathway
Concentration
1.
about
suggesting
atrium,
production
of ir-hBNP
These
previous
ventricle
mechanisms. atria
The presence our
that
as a major
secreted
patient.
Table
in human cardiac
However,
of these
cardiac
presence
hBNP-32.
in of
a hormone
0.2% to 3.6%,
by separate present
which
hANP as
possibility
In all always
of ir-hBNP
pressure.
from
of each
in human cardiac
in
distribution
in human cardiac
with
and blood
of death
being
concentration
considering
ir-hANP.
present
ir-hANP
summarized pmol/g,
but
characterization is
fluid
paper,
of
in human together
and cause
even
(8,11),
and ir-hANP
patients
of 0.3-3.6
of ir-hBNP
to that
hBNP
of the
a separate
Present
course
of ir-hBNP
range
Detailed
pBNP and rBNP data
clinical
concentration
was measured
that
from
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
heart present
(6-81, paper
that
the
as predicted
processing
system
processing
similar
will
in as
help
explain
BNPs.
Acknowledgments: The authors are grateful to Mr. T. Sudoh (Daiichi Pure Chemicals Co. Ltd.) for his kind donation of hBNP-26. Human heart tissue was presented by courtesy of Profs. A. Sumiyoshi Medical and M. Koono (Miyazaki College). This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Sudoh, T., Maekawa, K., Kojima, M., Minamino, N., Kangawa, K. & Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 159, 1427-1434. 2. Sudoh, T., Kangawa, K., Minamino, N. & Gsuo, H. (1988) Nature, -'332 78-81. 3. Maekawa, K., Sudoh, T., Furusawa, M., Minamino, N., Kangawa, K., Ohkubo, H ., Nakanishi, S. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., 157, 410-416. 1086
Vol.
166,
4. 5.
6.
8. 9. 10. 11. 12.
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Kojima, M., Minamino, N., Kangawa, K. & Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 159, 1420-1426. Matsuo, H. & Nakazato, H. (1987) Endocrinol. Metab. Clin. North Am., 16, 43-61. Aburaya, M., Hino, J., Minamino, N., Kangawa, K. & Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 163, 226-232. Kambayashi, Y., Nakao, K., Itoh,x, Hosoda, K., Saito, Y., Yamada, T., Mukoyama, M., Arai, H., Shirakami, G., Suga, S., Ogawa, Y., Jougasaki, M., Minamino, N., Kangawa, K., Matsuo, H., Inouye, K. & Imura, H. (19891 Biochem. Biophys. Res. Commun., 163, 233-240. M. Aburaya, N. Minamino, J. Hino, K. Kangawa, K. Tanaka & H. Matsuo. Biochem. Biophys. Res. Conunun., in press. Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T. & Matsuo, H. (19851 Biochem. Biophys. Res. Commun., 129, 248-255. Ueda, S., Minamino, N., Sudoh, T., Kangawa, K. & Matsuo, H. (19881 Biochem. Biophys. Res. Commun., 155, 733-739. Aburaya, M., Minamino, N., Kangac K., Tanaka, K. & Matsuo, H. Biochem. Biophys. Res. Commun., in press. Minamino, N., Aburaya, M., Ueda, S., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res Commun., -155, 740-746.
1087
Vol. 166, No. 3, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1080-1087
February 14, 1990
CEARACTERIZATION
OF IMMDNOREACTIVE
BRAIN NATRIDRETIC
PEPTIDE
IN HUNAN CARDIAC ATRIUM TATEYAMA 1.3 , Jun HINOl,
Hitone Kenji 1
Toshio
KANGANA',
Department
of Biochemistry,
'National
of Geriatric
December
18,
MINAMINO',
565,
College,
Japan
Research
Osaka
MATSUO'
Medical
889-16,
Center Suita,
Institute, Japan
Osaka University
Medicine,
Fukushima, Received
Miyazaki
Cardiovascular Fujishirodai,
3 Department
Miyazaki
Kiyotake,
Kihara,
Naoto
OGIHARA3 and Hisayuki
Osaka
553,
Medical
School,
Japan
1989
SUMMARY: Based on CDNA sequence data for human brain natriuretic peptide (BNP) precursor (II, a radioimmunoassay (RIA) system highly specific to human BNP (hBNP1 was developed and used to characterize immunoreactive (ir-1 hBNP in atrium indicated that ir-hBNP cardiac atrium. Gel filtration of ir-hBNP in was mainly comprised of two molecular forms of 13-15K and 4K. In reverse phase high performance liquid chromatography (HPLC), the low molecular weight (MN) ir-hBNP emerged as a single peak at an elution time identical to that of synthetic hBNP-32. The high NN ir-hBNP was also eluted as a single peak. On the other hand, tissue concentrations of ir-hBNP in cardiac atria were found to be 9.98-593.22 pmol/g in 13 specimens, being about l/150 the concentration of ir-human atria1 natriuretic peptide (hANPI. These results demonstrate that hBNP is present as a peptide in human heart, suggesting that hBNP is secreted from heart and functions together with hANP as a hormone. 01990 Academic Press, Inc. Our recent of the
presence
homeostatic neuronal probe
of
(3),
to highly
of
control
body
cloned
(1,4).
fluid
By using
(2,.
we have
mammalian
of BNP in porcine
a dual
balance pathways
precursors that
identification
BNPs exhibit
conserved differences
pathways
giving
rise
data large
sequences of
cDNA for
and sequenced
Structural
species
system
and blood
to different
by
hBNP,
of mammalian BNPs may endogenous
has lead
BNP
and
pressure porcine
cDNAs encoding
for
structural
mammalian
brain
to recognition
ANP
in
through
BNP (pBNPI
precursor
hBNP and rat
rBNP and pBNP have species
differences,
ANPs (5). result molecular
in
regulating hormonal
and as a
BNP (rBNP) demonstrated in contrast
Such large
structural
different
processing
forms
in each
species.
Abbreviations: BNP, brain natriuretic peptide; ANP, atria1 natriuretic p, porcine; rat; y-hBNP, pro-hBNP; hBNP-32, propeptide; h, human; r, hBNP-26, pro-hBNP(83-108); radioimmunoassay; ir-, hBNP(77-108); RIA, inununoreactive; MN, molecular weight; TFA, trifluoroacetic acid; HPLC, high performance liquid chromatography. 0006-291x/98 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
1080
Vol.
166, No. 3, 1990
The proteolytic
BIOCHEMICAL
processing
as indicated
PBNP, (6-8).
by the
In order
tissues,
it
tissue
is
first
RIA for
of rBNP in heart
presence
to determine
of rBNP-45
the
necessary Ir-hBNP
concentration.
established
pattern
AND BIOPHYSICAL
characterize in this
MATERIALS
distinct
from
storage
function was
that
form
of hBNP
ir-hBNP,
in human atrium
hBNP, as reported
is
as a major
physiological to
RESEARCH COMMUNICATIONS
then
characterized
of
of rBNP
in various measure by
its
a newly
paper.
AND METHODS
Peptides: Human BNP-32, hBNP-26 and N-Tyr-hBNP-26 were synthesized by solid technique conducted on a phenylacetamidomethyl resin with Applied phase Purification was performed by reverse Biosystems 430A peptide synthesizer. phase and CM ion exchange HPLCs, and correct synthesis and purity of the peptide were confirmed by amino acid analysis and sequencing. Human BNP-26 for immunization was donated from Daiichi Pure Chemicals Co. Ltd. Preparation of antisera: Human BNP-26 (7 mg) was conjugated with bovine thyroglobulin (15 mg, Sigma) by the action of water soluble carbodiimide (9). The conjugates were emulsified with Freund's complete adjuvant and used for immunizing rabbits by the described method (91. by the lactoperoxidase RIA for hBNP: N-Tyr-hBNP-26 was radioiodinated The resulting reaction mixture was immediately (Calbiochemj method (9). subjected to reverse phase HPLC to isolate monoiodo-N-Tyr-hBNP-26, which was stored at -8OV until use. RIA for hBNP was performed by the method used for RIA for pBNP, and N-ethylmaleimide-treated bovine serum albumin (BSA1 was used Peptide standard (hBNP-32) or unknown sample (100 l.11) was pre-incubated (10). 100 ~1 of antiserum (#189-61 for 24 hr. Then, 100 l.11 of tracer solution ;i% I-N-Tyr-hBNP-26) was added and incubated for additional 48 hr. Free and bound tracer were separated by the double antibody method. All procedures were carried out at 4'C, and samples were assayed in duplicate. RIA for hANP: RIA for a-hANP was carried out as reported previously (91. Tissue extraction: Human atria1 tissue was obtained at autopsy from patients within 4 hours post mortem. After weighing, atria mainly including auricles were diced and boiled for 10 min in 10 volumes of water to inactivate and 1M HCl were intrinsic proteases. Glacial acetic acid added (final concentration = 1 M and 20 mM, respectively) after cooling to 5'C, and boiled tissue was homogenized with a Polytron mixer for 4 min. Aliquots of the supernatants, obtained by centrifugation at 18,000 x g for 25 min, were lyophilized, dissolved in the RIA buffer, and then submitted to RIAs for hBNP and hANP. Characterization of ir-hBNP and ir-hANP in human cardiac atrium: The acid extracts of human cardiac atria were each diluted two-fold with water and then loaded onto cartridges (2.5 ml, Waters). The cartridges were Sep-pak C-18 were eluted with 60% washed with 0.5M acetic acid and the adsorbed materials CH3CN containing 0.1% trifluoroacetic acid (TFA). After vacuum evaporation, each extract was subjected to Sephadex G-50 gel filtration (fine, 1.8 x 134 using 1M acetic acid as a solvent. An aliquot of each fraction cm, Pharmacia) was submitted to RIAs for hBNP and hANP. fractions exhibiting hBNP and The hANP immunoreactivity were further analyzed by reverse phase HPLC on a Hi-Pore RP-318 column (4.6 x 250 mm, Bio-Rad) with a linear gradient elution of CH3CN from 10% to 60% in 0.1% TFA solution at a flow rate of 1.0 ml/min. Aliquots of all fractions were submitted to RIAs for hBNP and hANP. RESULTS AND DISCUSSION RIAs
for
hBNP and hANP:
and was usable maximum
inhibition
at a final
Anti-hBNP dilution
of binding
serum
#189-6
showed
was observed 1081
at
high
As shown
of 1:420,000. 2.6
fmol/tube
avidity in
Fig. and
for 1, the
hBNP half-
peptide
Vol.
166, No. 3, 1990
BIOCHEMICAL
O-
10-r
Figure
in by
estimated
peptides.
Since this
crossreact
RIA system
hand,
recognized
by for
hBNP-32
this
were Four
in
ANP-29
the
case
of
since
of ir-hBNP
frog
antiserum
i.e.
ANP-24. than specific
The 0.003%
and hBNP-26
RIA system
for
not pBNP-
highest (Fig.
1). On
to be equally
showed
anti-hANP
a-
to hBNP.
MW hBNP was thought of
did
examined,
was highly
hBNP-32
and the
concentration
rBNP was less
high
antiserum
natriuretic with
peptides
Crossreactivity
to be 0.04%,
known
The
and
were
any crossreactivity
tissue
#189-6
extended
antiserum,
examined
filtration were
exhibit
pBNP-26.
of the
with
natriuretic
antiserum
antiserum.
characterized gel
other
chicken
of ir-hBNP
human cardiac ir-hBNP
I
ld
comparable
serum
#125-8
hANP was also
with found
to hANP.
Characterization atria
not
to measure
that
was determined
to be specific cardiac
I
lo4
Specificity
immunoreactivity.
N-terminally
the
did
hANP
with
verified
fmol/tube.
was able
observed
results
affinity
I
10'
crossreactivity
#189-6
CL-rANP,
crossreactivity
its
of
significantly
other
of 0.2-30
measuring antiserum
rBNP-32.
These the
a range
interference
26,
I 10’
1. Inhibition of binding #189-6. Final dilution: 1:420,000. (M): hBNP-32 and hBNP-26. (-): rBNP-32. (-1: (-U-lJ-): a-hANP, CL-rANP, chicken ANP-29, frog ANP-24. Vasopressin, somatostatin, enkephalins and other neuropeptides examined, but no cross-reactivity was observed in concentrations less than 1 ug/tube.
was measurable
hANP,
I
10'
RESEARCH COMMUNICATIONS
Peptide (fmol/tube) of 125 I-hBNP to antiserum
was
without
,
loo
AND BIOPHYSICAL
and ir-hANP
(Table
by gel atria
ir-hBNP
filtration
were
first
profiles observed
l),
are in
in
and ir-hANP
and subjected shown
fractions
human cardiac reverse
atrium: of five
phase
to Sephadex
in Fig. #27-31 1082
2. and
atria1
HPLC. G-50
In all #40-48
Among 13
gel
cases,
extracts Extracts
of
filtration. two peaks
corresponding
of to
Vol.
BIOCHEMICAL
166, No. 3, 1990
Table
1. Tissue
no.
concentration
sex
1 2 3 4 5 6 7 8 9 10 11 12 13
65 a3 70 a4 67 55 70 19 77 58 75 47 75
F F M M M M F M M F M F M
molecular
weight
form
were
2:3 to 1:5
only
1:lO
in Fig.
AND BIOPHYSICAL
of ir- BNP
and ir- ANP
RESEARCH COMMUNICATIONS
in human
cardiac
atria
cause of death
ir- BNP @mow
ir- ANP (nmoUs)
ir- BNP / ir- ANP (%I
lung cancer rectal cancer lung cancer bladder cancer pleural mesothelioma hepatoma multiple myeloma germinoma of the testis cholangiocarcinoma carcinoma of the uterus myocardial infarction adult T- cell leukemia esophageal cancer
38.86 30.54 18.61 9.98 58.69 19.65 593.22 114.20 64.09 42.32 120.27 66.40 121.82
17.69 5.57 9.16 1.63 18.31 2.66 20.92 3.16 10.56 3.54 35.56 20.05 17.08
0.22 0.55 0.20 0.61 0.32 0.74 2.84 3.61 0.61
13-15K
and 4K.
in
Fig.
Relative
2a,
2b (patient
ratios
of
high
2c and 2d (patients
#71.
On the
other
MW form
#5,
hand,
1.20 0.34 0.33 0.71
#ll
ir-ANP
to
low MW
and #12), emerged
but as two
n
z
3.0 -
.k 8 :s 5
15-*0 ;L
P4
20
30
40
Fraction
Figure
50
60
70
,1
20
30
40
Fraction
number
50
60
70
number
2. Sephadex G-50 gel filtration of acid extracts of human atrium. Sample: Atria1 extracts (450 mg wet wt equivalent) of (a) patient #5, #ll, and (dl patient #12. (bl patient #7, (c, patient Column: Sephadex G-50 (fine, 1.8 x 134 cm, Pharmacia). Flow rate: 10 ml/hr. Fraction size: 5 ml/tube. Solvent: 1M acetic acid. Arrows indicate 4) whANP and
5)
elution Vt.
positions
of
1083
II
Vo,
21
y-hANP,
31
B-hANP,
Vol.
166, No. 3, 1990
peaks Fig.
of MW 13K and 3K in Fig.
phase
#40-46
2a-2c
and #48-52
HPLC on a C-18
eluted
as
while
The
hANP (Fig.
and as three
RESEARCH COMMUNICATIONS
peaks
of
13K,
5K and 3K in
of hBNP-32
to
consist
hand,
found
in patient
subjected of high
subjected
to reverse
identical
phase
the
low
not
component
was mainly
MW ir-hBNP
was
to that
of synthetic
MW emerged
at the
elution
time
to that
of o-
identical
shown
HPLC and emerged
(data
of a single
reverse
phase
shown).
at the
Thus,
corresponding
comprised
in Fig.
elution
2atimes
low MW ir-BNP
to hBNP-32.
of cc-hANP,
emerged
30
20
Time
obtained
4 shows from
as single
peaks,
of y-hANP.
High
was much earlier
time
fractions
Figure
HPLC.
and ir-hANP
to that
elution
3a, identical
at a time
MW hBNP- and hANP-immunoreactive
hBNP and ir-hANP
Figure
time
each
with
was
On the
B-hANP being
#12.
MW ir-hBNP
its
in Fig.
was eluted
respectively
low MW ir-hANP
to
were
of an intermediate
to reverse
and cc-hANP,
concluded
2d
an elution
low MW ir-hANP
subjected
other
As shown
at
ir-hANP
of Fig.
The low MW hBNP- and hANP-immunoreactivity
3b). also
High
peak
the
of B-hANP. 2c were
column.
a single
hBNP-32,
but
AND BIOPHYSICAL
2d. Fractions
time
BIOCHEMICAL
of Fig. one of the
patient
#12
and ir-hANP MW ir-hBNP than
that
2a-2d elution
(Fig.
2dl.
was eluted appeared
were
profiles Both
ir-
at an elution
as a single
of y-hANP.
also
Since
peak, high
40 Time (min)
(mln)
3. Reverse phase HPLC of (a) low MW ir-hBNP and (b) low MW ir-hANP of human cardiac atrium. Sample: (a1 Fractions #40-46 (190 ul/tubel and (b1 fractions #48-52 (1.2 ul/tube) in Fig. 2d. Temperature: ambient. Column: Hi-Pore RP-318 (4.6 x 250 mm, Bio-Rad). Flow rate: 1.0 ml/min. Solvent system: H20:CH3CN:10%TFA = (Al 90:10:1, (BI 40:60:1 (vol/vol). Linear gradlent elution from (Al to (BI for 60 min. Arrows indicate elution times of II hBNP-32, 2) 8-hANP and 3) a-hANP. 1084
MW
Vol.
BIOCHEMICAL
166, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I --
Time
Figure
ir-hBNP
in
in Fig.
2d,
four
other
the
cases
to be derived
these
high
facts, Very
were
recently,
as hBNP-32
characterization
of
HPLC,
atria
was
showed
tissue
serial
Table
patient's
profile. ,
of
in human cardiac to that However,
atrium
in porcine differences
(99.9
cardiac
hBNP-32
and y-hBNP
in
present
isolating
in human atria the
of ir-hBNP in tissue concentration
pmol/g)
of tissue
on
y-hBNP. the
A
mean
from study
concentration 1085
atrium:
filtration
human cardiac
of human caridic
of synthetic
hBNP-32
of ir-hBNP was in
a range
varied tissue
(data
among specimens.
widely Table
l/6
observed
not with
of 9.98-593.22 with
concentration
from
atria
and ir-hANP
concentration
about
After and reverse
in 13
extracts
calculated and was
cardiac
by gel and ir-hANP
to that
atrium
HPLC
Based
in
concentration
nmol/g.
phase
from
parallel
tissue
1.63-35.56
reverse
property.
to be derived
tissue
differences
in
as that
time
(to be published).
All
Concentration large
MW ir-hBNP
of ir-hBNP
curves
elution
characterized
cardiac
precisely.
of ir-hANP,
a range
in
same
hydrophilic
and ir-hANP
1 summarizes
indicating
In the case within
and y-hBNP
ir-hBNP
dilution
shown). pmol/g
forms
concentration
re-measured
of high
succeeded
of ir-hBNP
at the
intrinsic
was deduced
we have
concentration
phase
its
and two molecular
identified
eluted
time
from
MW ir-hBNP
atrium,
Tissue
was also
elution
earlier
was thought
cardiac
(min)
4. Reverse phase HPLC of high MW ir-hBNP of human cardiac atrium. Sample: Fractions #27-31 (630 ul/tube) in Fig. 2d. Chromatographic conditions were identical to those of Fig. 3. Arrow indicates elution time of y-hANP.
1
that
specimens of ir-hBNP
was comparable in
rat
in human
atrium. were
much
Vol.
166, No. 3, 1990
larger
than
BIOCHEMICAL
those
differences
expected
in age,
Tissue
sex,
in part
ir-hBNP
in a
was
measurement
reported
in
be similar
body
varied
between
concentrations
suggests
the
regulated
form the
to low
is
processing
MW form of
8-hANP facts
indicated systems
of
physiological
used
cardiac
that
of
of atrium.
ventricle
ir-hBNP
relative
and no
for
in heart
is
atrium
will
be
found
to
demonstrated
that
hBNP functions
homeostatic concentration
apparent
balance
of
of ir-hBNP
to
correlation
was observed
in 13 human specimens.
and secretion
This
and BNP
of ANP
hBNP and functions
form,
as seen heart
(1).
2d
hBNP-32
and functions
fact may be
similar of
pBNP shown
each
did is
ir-hBNP,
relative not
affect
probably
in
of mammalian
was
of high
specimen.
in
to that
cardiac
MW
Furthermore, predominance
a mature
atrium
form
in
the
indicates
of pBNP (10,121,
rBNP has a distinct
rBNP-45
hBNP-32 ratio
of of hBNP
as a hormone.
and y-hBNP Although
presence
in
in Fig. that
of
although
was different
of hBNP is
the
characterization
molecular
of hBNP-32
paper by
l/100
maintaining
two peptides
suggest
from
pathway
Concentration
1.
about
suggesting
atrium,
production
of ir-hBNP
These
previous
ventricle
mechanisms. atria
The presence our
that
as a major
secreted
patient.
Table
in human cardiac
However,
of these
cardiac
presence
hBNP-32.
in of
a hormone
0.2% to 3.6%,
by separate present
which
hANP as
possibility
In all always
of ir-hBNP
pressure.
from
of each
in human cardiac
in
distribution
in human cardiac
with
and blood
of death
being
concentration
considering
ir-hANP.
present
ir-hANP
summarized pmol/g,
but
characterization is
fluid
paper,
of
in human together
and cause
even
(8,11),
and ir-hANP
patients
of 0.3-3.6
of ir-hBNP
to that
hBNP
of the
a separate
Present
course
of ir-hBNP
range
Detailed
pBNP and rBNP data
clinical
concentration
was measured
that
from
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
heart present
(6-81, paper
that
the
as predicted
processing
system
processing
similar
will
in as
help
explain
BNPs.
Acknowledgments: The authors are grateful to Mr. T. Sudoh (Daiichi Pure Chemicals Co. Ltd.) for his kind donation of hBNP-26. Human heart tissue was presented by courtesy of Profs. A. Sumiyoshi Medical and M. Koono (Miyazaki College). This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Sudoh, T., Maekawa, K., Kojima, M., Minamino, N., Kangawa, K. & Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 159, 1427-1434. 2. Sudoh, T., Kangawa, K., Minamino, N. & Gsuo, H. (1988) Nature, -'332 78-81. 3. Maekawa, K., Sudoh, T., Furusawa, M., Minamino, N., Kangawa, K., Ohkubo, H ., Nakanishi, S. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., 157, 410-416. 1086
Vol.
166,
4. 5.
6.
8. 9. 10. 11. 12.
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Kojima, M., Minamino, N., Kangawa, K. & Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 159, 1420-1426. Matsuo, H. & Nakazato, H. (1987) Endocrinol. Metab. Clin. North Am., 16, 43-61. Aburaya, M., Hino, J., Minamino, N., Kangawa, K. & Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 163, 226-232. Kambayashi, Y., Nakao, K., Itoh,x, Hosoda, K., Saito, Y., Yamada, T., Mukoyama, M., Arai, H., Shirakami, G., Suga, S., Ogawa, Y., Jougasaki, M., Minamino, N., Kangawa, K., Matsuo, H., Inouye, K. & Imura, H. (19891 Biochem. Biophys. Res. Commun., 163, 233-240. M. Aburaya, N. Minamino, J. Hino, K. Kangawa, K. Tanaka & H. Matsuo. Biochem. Biophys. Res. Conunun., in press. Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T. & Matsuo, H. (19851 Biochem. Biophys. Res. Commun., 129, 248-255. Ueda, S., Minamino, N., Sudoh, T., Kangawa, K. & Matsuo, H. (19881 Biochem. Biophys. Res. Commun., 155, 733-739. Aburaya, M., Minamino, N., Kangac K., Tanaka, K. & Matsuo, H. Biochem. Biophys. Res. Commun., in press. Minamino, N., Aburaya, M., Ueda, S., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res Commun., -155, 740-746.
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