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The presence of brain natriuretic peptide of 12,000 daltons in porcine heart
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 740-746
September 15, 1988
T H E PRESENCE OF B R A I N NATRIURETIC
Naoto MINAMINO*,
Departments
Masahito ABURAYA,
PEPTIDE OF 12,000 D~LTONS
IN PORCINE HEART
Shuzo UEDA, Kenji KANGAWA & Bisayuki MATSU0
of Biochemistry and Anesthesiology*, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-16, Japan
Received July 28, 1988
SUMMARY: Brain natriuretic peptide (BNP) and its N-terminally six amino acid extended form (BNP-32) have been identified in porcine brain. These peptides exert diuretic-natriuretic and hypotensive effects, and have remarkably high sequence homology to atrial natriuretic peptide (ANP). We have set up a radioimmunoassay system specific to BNP and surveyed immunoreactive (ir-) BNP in peripheral tissue. In porcine cardiac atrium, we found the highest concentration of ir-BNP. By using gel filtration and reverse phase high performance liquid chromatography, ir-BNP was characterized. Most of ir-BNP in the atrium was found to exist as a high molecular weight form of 12,000 daltons; less than 15% of the total ir-BNP exist as low molecular weight forms such as BNP and BNP-32. These results suggest that BNP functions as a circulating hormone in addition to the neuropeptide function in brain. © 1988 Academic Press, Inc.
Atrial atrium
natriuretic
into
fluid and
(1-3).
of
a
natriuretic (4).
blood
through
regulates
as a hormone
the
homeostatic
its vasorelaxant
from
cardiac
balance
of body
and diuretic-natriuretic
We have recently succeeded in the identification
peptide
(BNP)",
having a remarkably high sequence homology to ANP
an N-terminally
identified a
26-amino
in porcine
pharmacological
including
diuretic-natriuretic
bioactive
peptides
originally
been found in the peripheral
and
acid
residues,
in porcine
of
elicit
also present
and
is secreted
peptide
also
peptides
(ANP)
novel
Furthermore,
32) was
stream
blood pressure,
activities brain
the
peptide
"brain
six amino acid extended form of BNP brain
(to be reported
spectrum
very
hypotensive
identified
in the
similar
central
(BNP-
elsewhere). to
activities.
organs and vice versa,
in other organs,
designated
These
that
of
a
number
As
nervous
it is probable
system
ANP, of
have
that BNP is
such as heart, where it may function in concert
with ANP for the control of blood pressure and salt-water balance. By utilizing a recently developed radioimmunoassay screened heart,
ir-BNP
in porcine
especially
characterization
in
the
peripheral atrium.
organs
Here
we
of ir-BNP in cardiac atrium,
of ANP.
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
740
and report
have
(RIA) for BNP, we have found
tissue
ir-BNP
in
the
concentration
and
and we compared these with those
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Peptides: Porcine ~-ANP (identical to human e-ANP (5)), ~-ANP[4-28], ~-ANP[528], BNP and BNP-32 were synthesized in our laboratory by the described method (4,6). BNP-29 was prepared by digesting BNP-32 with lysyl endopeptidase (Wako Pure Chemicals), followed by reverse phase high performance liquid chromatography (HPLC) purification. RIAs for BNP and ANP: Details on RIA for BNP will be reported elsewhere. In brief, antiserum #158-4 primarily recognized the ring portion ~ n k e d by a disulfide linkage; it had equal affinity for BNP and BNP-32. ÷ ~ I - B N P was prepared by the lactoperoxidase method and purified by reverse phase HPLC. Half-maximum inhibition of binding was observed at 9 fmol/tube of BNP, and peptides were measurable in a range of I-i00 fmol/tube. In the RIA system for BNP, ~-ANP showed 0.02% of crossreactivity. RIA for ANP was performed as reported previously (7) by using antiserum #125-8 raised against human ~-ANP. This antiserum had less than 0.003% of crossreactivity with BNP. Extraction methods: Porcine hearts were collected in a local slaughter house soon after killing and kept on ice before dissection and extraction. Method A: Worked up as described (8) with slight modifications. Tissue was boiled in water, and then acetic acid was added to make up a final concentration of 1 M before homogenization. The extracts were stored at -20°C. For precise measurement of tissue concentration of ir-BNP and ir-ANP in the atrium, two other extraction methods were examined as follows. Method B: Diced tissue was heated and then extracted in 5 volumes of 0.1M acetic acid containing 1% Triton X-100 (7). other procedures in Method B were identical to those used for Method A. Method C: Diced tissue was homogenized in 5 volumes of IM acetic acid/20mM HCI followed by addition of i0 volumes of acetone. No heat treatment was used in Method C. The extract prepared by this method was evaporated to remove acetone and stored at -20°C. Aliquots of the extracts obtained by these three methods were each neutralized with 1.3M Tris solution, diluted with RIA buffer, and submitted to RIAs for BNP and ANP. Characterization of ir-BNP in porcine atrium: Ten gram wet weight tissue equivalent of the extract prepared by Method A was loaded onto a reverse phase C-18 column and then eluted with 60% CH3CN containing 0.1% trifluoroacetic acid (TFA). The eluate was evaporated, lyophilized, and subjected to gel filtration on a Sephadex G-75 fine column (1.8 x 137 cm, Pharmacia). The fraction containing ir-BNP was further separated by reverse phase HPLC on a Hi-Pore RP-318 column (4.6 x 250 mm, Bio-Rad) with a linear gradient elution of CH_CN from 10% to 60% in 0.1% TFA for 80 min at a flow rate 1.5 ml/min. Aliqu~ts of all fractions in gel filtration and reverse phase HPLC were submitted to RIAs for BNP and ANP. Enzymatic digestion of high MW ir-BNP and characterization of ir-BNP in the digests: Two pooled aliquots (i00 ~I/fraction) of high molecular weight (MW) ir-BNP fractions (fractions #39-45 in Sephadex G-75 gel filtration) were lyophilized. The lyophilizates were each dissolved in 200 ~i of 50mM Hepes buffer (pH 7.5) containing 0.1% Triton X-100. Lysyl endopeptidase (20 ~g) was added to each tube and kept at 37°C for 2 hr (9). After addition of 0.1% TFA, one of the resulting digests was separated by reverse phase HPLC under the identical conditions described above. Another digest was lyophilized and then subjected to (IM ion exchange HPLC on a TSK CM-2SW column (4.6 x 250 ram, Tosoh) with a linear gradient elution from 10mM HCOONH 4 (pH 6.6) to 0.5M HCOONH_4 (pH 6.6) each containing 10% CH3CN for 60 min at a flow rate of 1.0 m l / m i n / An aliquot of each fraction was submitted to RIA for BNP.
RESULTS AND DISCUSSION By
the
RIA
specific
to
peripheral organs
of the pig,
gland and heart,
and have
BNP,
we
have
including
found the
preliminarily
the gastrointestinal
highest
741
screened
concentration
ir-BNP
in
tract,
adrenal
of ir-BNP
in the
Vol. 155, No. 2, 1988
Table I.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Tissue concentration of ir-BNP and ir-ANP in porcine cardiac atrium ir-BNP (pmol/g)
method
ir-ANP (pmol/g) ir-BNP/ir-ANP (%)
Method A (boiled and extracted with IM AcOH*)
97.1
4430
2.19%
Method B (boiled and extracted with IM AcOH/I% Triton)
46.2
4450
1.04%
Method C (extracted with 1M AcOH/ 20mM HCI/66% acetone)
51.9
8850
0.59%
65.1
5910
1.10%
mean *AcOH: acetic acid.
heart
(data not shown). In the porcine heart,
the atrium
(65 pmol/g wet wt),
observed in the ventricle. ir-BNP
from
cardiac
In our preliminary experiment,
atrium
were
examined
methods and compared to those of ir-ANP. among the three extraction methods large
(Table i).
ir-BNP was much concentrated
in
while less than 1 pmol/g wet wt of ir-BNP was
By calculating
using
three
However,
recovery yields of different
extraction
differences in the yields
employed in the present
study were not so
a mean value from the three methods,
atrial
concentration of ir-BNP was determined to be 65.1 pmol/g wet wt, while that of ir-ANP was 5,910 pmol/g wet wt. Thus, it was concluded that ir-BNP was present in porcine atrium but only at about 1% the concentration of ir-ANP. For
characterizing
immunoreactivity
found
reverse phase HPLC. 75
column
atrium was BNP
in
to
the
standard
also observed.
the
atrium
molecular
size
and
atrium was
form
of
analyzed by
gel
bulk
of
human
ir-BNP
y-ANP,
exists
as
a high
appeared
where
This indicates
manner similar to the case of ANP.
most
the
low
MW
corresponding
BNP and
that more
MW form
ir-BNP
to ~-ANP of about
#55-63)
3,000 daltons.
the
ir-ANP
position
from porcine
than 70% of the total ir-
of about
Additionally,
(fractions
around
of
12,000
daltons,
in a
two other minor peaks of ir-
BNP were observed around fractions #28-34 and #55-63, of
ir-BNP, filtration
In gel filtration of the atrial extract on a Sephadex G-
(Fig. i),
corresponding
the
in cardiac
respectively.
emerged
around
The peak
the
position
On the other hand,
another
minor peak of ir-BNP eluted in fractions #28-34 may be due to the presence of aggregates
of
displacement
ir-BNP
curve
of
than
12,000 standard
daltons and 3,000 daltons were Pore C-18
column.
As
daltons, BNP.
since BNP
it
showed
a
immunoreactivities
shallower of
12,000
further separated by reverse phase HPLC on Hi-
shown in Fig. 2,
high M W ir-BNP
(fractions
#39-45
in
Fig. I) emerged as a single peak just after porcine y-ANP. This fact indicates that a high M W
form of
ir-BNP
is
similar
742
to porcine
y-ANP
with
respect
to
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
2
3
4
I
I
I
I
i~- 0.i0
0.05
50 ~G
o"
!, o
0
i0
20
30
~0 50 FrQctlon number
60
70
80
Figure i. Sephadex G-75 gel filtration of the acid extract of porcine cardiac atrium. Sample: Porcine cardiac atrium extract (10 g wet wt equivalent) prepared by Method A. Details of the extraction method are described in Methods. Column: Sephadex G-75 fine (1.8 x 137 cm, Pharmacia). Flow rate: 8 m 1/hr. Fraction size: 5 ml/tube. Solvent: IM acetic acid. The arrows indicate the elution positions of i) Vo, 2) y-hANP, 3) ~-hANP and 4) Vt, respectively.
hydropathy
and molecular
#55-63
Fig.
in
corresponding that B N P controlled
i)
weight.
appeared
in
On the the
to BNP and BNP-32,
and BNP-32
are g e n e r a t e d
proteolytic
processing
other hand,
reverse
as seen
phase
in Fig.
from a high MW system
low M W
HPLC 3.
at
ir-BNP
the
retention
Accordingly, B N P of 1 2 , 0 0 0
in a m a n n e r
similar
(fraction times
it is l i k e l y daltons
to t h a t
for ANP.
6O 0.2 40
z
20 8
o
o.z
i
800
400
.~ 6o = o
O~
30 o
Tlme (mln)
Figure 2. Reverse phase H P L C o f high MW J/m~unoreactive BNP and ANP. Sample: Fractions #39-45 (i00 ~l/tube), shown by arrow A in Fig. i. Colu/m~: Hi-Pore RP-318 (4.6 x 250 ~ , Bio-Rad). Flow rate: 1.5 ml/min. Solvent system: H_O:CH3CN:I0%TFA = (A) 90:10:i, (B) 40:60:1 (v/v). Linear gradient e~ution from (A) to (B) for 80 min. Temperature: ambient.
743
by a
V o l . 155, N o . 2, 1 9 8 8
B I O C H E M I C A L A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
0.f08 0,04
6O 41'
'IF
............
~o 20
.
o
0
4oo~
i.
6
L
o 3 0 10
20
30
200
L O~
40
50
60
70
80
T I m e (mln)
Figure 3. Reverse phase HPLC of low MW Jum~unoreactive BNP and ANP. Sample: Fractions #55-63 (200 ~i/tube), shown by arrow B in Fig. 1. chromatographic conditions were identical to those for Fig. 2. The arrows indicate the elution times of i) e-ANP and 2) BNP or BNP-32, respectively.
In order
to obtain
further
information
about a 12,000-dalton
high M W ir-BNP was digested with lysyl endopeptidase, in the digests was analyzed,
BNP,
the
and the resulting ir-BNP
since BNP-32, having a lysine residue at position
3, undergoes lysyl endopeptidase digestion to yield BNP-29 by removal of three amino acid fractions
residues
at
#39-45 were
the N-terminus lyophilized,
(see Fig.
As shown in Fig. 4a,
Pooled
conditions
BNP immunoreactivity generated
the lysyl endopeptidase to BNP-29
(data not
the digests, BNP-29. of
12,000
on CM
shown).
The peptide
was
exhibiting BNP immunoreactivity
chromatographically
identified to
in
be authentic
BNP is verified to exist also in the heart as a high M W form
daltons,
evidence implies
Furthermore,
from the high M W ir-BNP by
digestion also appeared at the elution time identical
therefore,
Hence,
and then
to those for high
which emerged at 36-37
min identical to authentic BNP-29 on reverse phase HPLC. HPLC,
of
the high MW ir-BNP was found to be converted
into a single component carrying BNP immunoreactivity,
ion exchange
aliquots
digested by lysyl endopeptidase
subjected to reverse phase HPLC under identical M W ir-BNP.
4b).
which
carries
the hormonal
the
BNP
unit
at
its
C-terminus.
This
function of BNP in the heart in addition to the
neural function in the brain Although ANP was first
identified
in the atrium,
have been shown to exist also in the brain,
ANP and its receptors
especially in the regions thought
to be involved in the control of body fluid homeostasis
(10-12). This strongly
suggests
the neural
that ANP also functions
as a
water and electrolyte balance.
744
neuropeptide
in
control of
VoI. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1 ~,
0.2
2 ~
6O
........
_._..----'-
40 ~5
_.._----
20 ~ 0.i
1'
0
I
o
r
1%
b
20
J.
i "1'
30
800
~00
.11_
~o
~o
T tree (rain)
L 0~
6~
/o
LYS endoDeptldase
I ~5 I0 15 20 2S 30 W BNP-32: S-P-K-T-M-R-D-S'G-C-F-G-R-R-L'D-R" I-6-S-L-S-G-I-G-C-N-V'L-R-R-Y BNP
:
D-S-G-C-F-S-R-R-L-D-RI-G-S-L-S-G'L-G-C-N-V'L-R-R-Y
Figure 4. (a) Reverse phase HPLC of lysyl endopeptidase digests of high MW immunoreactive BNP. Sample: Aliquots (i00 ~l/tube) of fractions #39-45 were lyophilized alad digested with lysyl endopeptidase (20 Bg) at 37°C for 2 hr. Chromatographic conditions were identical to those for Fig. 2. The arrows indicate the elution times of i) BNP-29 and 2) high MW form of i~-BNP. (b) Amino acid sequences of BNP and BNP-32. The arrow indicates the position which lysyl endopeptidase is thought to cleave.
In our recent study using the RIA system for BNP, tissue concentration of ir-BNP in porcine whole brain was estimated
to be 0.63 pmol/g wet wt,
about 13 times higher than that of ir-ANP in brain suggests
that
BNP
together
with
ANP
in
brain
being
(0.05 Imaol/g wet wt).
may
function
in
the
This neural
control of body fluid homeostasis.
In the present study, we demonstrated that
BNP is also present
and that in the cardiac atrium the tissue
concentration
in the heart,
of ir-BNP
(65.1 pmol/g wet wt)
that in the whole brain. BNP
in
the
speculate
heart
Furthermore,
is very similar
that: BNP in
I00 times
higher
than
the molecular distribution pattern of
to that
the atrium,
is about
of ANP.
These
facts
even though its concentration
lead
us to
is only 1%
that of ir-ANP, is highly likely a hormone to be secreted for regulating blood pressure and water-salt balance, working in concert with ANP. The
existence
these two hypotensive
in
two
highly homologous activities,
different
organs
peptides,
such
as
the
heart
and
brain
both sharing natriuretic-diuretic
of and
suggests that body fluid and electrolyte homeostasis
may be controlled by two ways through the neural and hormonal systems, ANP and BNP may function in the concerted manner.
745
where
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS: This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.
REFERENCES i. Flynn, T.G., de Bold, M.L. & de Bold, A.J. (1983) Biochem. Biophys. Res. Commun., 117, 859-865. 2. Kangawa~ K. & Matsuo, H. (1984) Biochem. Biophys. Res. Commun., 118, 131-139. 3. de Bold, A.J. (1985) Science, 230, 767-770. 4. Sudoh, T., Kangawa, K., Minamino, N. & Matsuo, H. (1988) Nature, 332, 78-81. 5. Forssmann, W.G., Birr, C., Carlquist, M., Christmann, M., Finkle, R. Henschen, A., Hock, D., Kirchheim, H., Kreeye, V., Lottspeich, F., Mutt, V. & Reinecke, M. (1984) Cell Tissue Res., 238, 425-430. 6. Ueda, S., Sudoh, T. Fukuda, K., Kangawa, K., Minamino, N. & Matsuo, H. (1987) Biochem. Biophys. Res. Commun., 149, 1055-1062. 7. Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T. & Matsuo, H. (1985) Biochem. Biophys. Res. Commun., 129, 248-255. 8. Kitamura, K., Minamino, N., HayaShi, Y., Kangawa, K. & Matsuo, H. (1982) Biochem. Biophys. Res. Commun., 109, 966-974. 9. Masaki, T., Tanabe, M., Nakamura, K. & Soejima, M. (1981) Biochim. Biophys. Acta, 660, 44-55. i0. Quirion, R., Daple, M., de Lean, A., Gutkowska, J., cantin, M. & Genest, J. (1984) Peptides, 5, i167-1172. ii. Saper, C.B., Standaert, D.G., Currie, M.G., Schwartz, D., Geller, D.M. & Needleman, P. (1985) Science, 227, 1047-1049. 12. Kawata, M., Nakao, K., Morii, N., Kiso, Y., Yamashita, H., Imura, H. & Sano, Y. (1985) Neuroscience, 16, 521-546.
746
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 740-746
September 15, 1988
T H E PRESENCE OF B R A I N NATRIURETIC
Naoto MINAMINO*,
Departments
Masahito ABURAYA,
PEPTIDE OF 12,000 D~LTONS
IN PORCINE HEART
Shuzo UEDA, Kenji KANGAWA & Bisayuki MATSU0
of Biochemistry and Anesthesiology*, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-16, Japan
Received July 28, 1988
SUMMARY: Brain natriuretic peptide (BNP) and its N-terminally six amino acid extended form (BNP-32) have been identified in porcine brain. These peptides exert diuretic-natriuretic and hypotensive effects, and have remarkably high sequence homology to atrial natriuretic peptide (ANP). We have set up a radioimmunoassay system specific to BNP and surveyed immunoreactive (ir-) BNP in peripheral tissue. In porcine cardiac atrium, we found the highest concentration of ir-BNP. By using gel filtration and reverse phase high performance liquid chromatography, ir-BNP was characterized. Most of ir-BNP in the atrium was found to exist as a high molecular weight form of 12,000 daltons; less than 15% of the total ir-BNP exist as low molecular weight forms such as BNP and BNP-32. These results suggest that BNP functions as a circulating hormone in addition to the neuropeptide function in brain. © 1988 Academic Press, Inc.
Atrial atrium
natriuretic
into
fluid and
(1-3).
of
a
natriuretic (4).
blood
through
regulates
as a hormone
the
homeostatic
its vasorelaxant
from
cardiac
balance
of body
and diuretic-natriuretic
We have recently succeeded in the identification
peptide
(BNP)",
having a remarkably high sequence homology to ANP
an N-terminally
identified a
26-amino
in porcine
pharmacological
including
diuretic-natriuretic
bioactive
peptides
originally
been found in the peripheral
and
acid
residues,
in porcine
of
elicit
also present
and
is secreted
peptide
also
peptides
(ANP)
novel
Furthermore,
32) was
stream
blood pressure,
activities brain
the
peptide
"brain
six amino acid extended form of BNP brain
(to be reported
spectrum
very
hypotensive
identified
in the
similar
central
(BNP-
elsewhere). to
activities.
organs and vice versa,
in other organs,
designated
These
that
of
a
number
As
nervous
it is probable
system
ANP, of
have
that BNP is
such as heart, where it may function in concert
with ANP for the control of blood pressure and salt-water balance. By utilizing a recently developed radioimmunoassay screened heart,
ir-BNP
in porcine
especially
characterization
in
the
peripheral atrium.
organs
Here
we
of ir-BNP in cardiac atrium,
of ANP.
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
740
and report
have
(RIA) for BNP, we have found
tissue
ir-BNP
in
the
concentration
and
and we compared these with those
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Peptides: Porcine ~-ANP (identical to human e-ANP (5)), ~-ANP[4-28], ~-ANP[528], BNP and BNP-32 were synthesized in our laboratory by the described method (4,6). BNP-29 was prepared by digesting BNP-32 with lysyl endopeptidase (Wako Pure Chemicals), followed by reverse phase high performance liquid chromatography (HPLC) purification. RIAs for BNP and ANP: Details on RIA for BNP will be reported elsewhere. In brief, antiserum #158-4 primarily recognized the ring portion ~ n k e d by a disulfide linkage; it had equal affinity for BNP and BNP-32. ÷ ~ I - B N P was prepared by the lactoperoxidase method and purified by reverse phase HPLC. Half-maximum inhibition of binding was observed at 9 fmol/tube of BNP, and peptides were measurable in a range of I-i00 fmol/tube. In the RIA system for BNP, ~-ANP showed 0.02% of crossreactivity. RIA for ANP was performed as reported previously (7) by using antiserum #125-8 raised against human ~-ANP. This antiserum had less than 0.003% of crossreactivity with BNP. Extraction methods: Porcine hearts were collected in a local slaughter house soon after killing and kept on ice before dissection and extraction. Method A: Worked up as described (8) with slight modifications. Tissue was boiled in water, and then acetic acid was added to make up a final concentration of 1 M before homogenization. The extracts were stored at -20°C. For precise measurement of tissue concentration of ir-BNP and ir-ANP in the atrium, two other extraction methods were examined as follows. Method B: Diced tissue was heated and then extracted in 5 volumes of 0.1M acetic acid containing 1% Triton X-100 (7). other procedures in Method B were identical to those used for Method A. Method C: Diced tissue was homogenized in 5 volumes of IM acetic acid/20mM HCI followed by addition of i0 volumes of acetone. No heat treatment was used in Method C. The extract prepared by this method was evaporated to remove acetone and stored at -20°C. Aliquots of the extracts obtained by these three methods were each neutralized with 1.3M Tris solution, diluted with RIA buffer, and submitted to RIAs for BNP and ANP. Characterization of ir-BNP in porcine atrium: Ten gram wet weight tissue equivalent of the extract prepared by Method A was loaded onto a reverse phase C-18 column and then eluted with 60% CH3CN containing 0.1% trifluoroacetic acid (TFA). The eluate was evaporated, lyophilized, and subjected to gel filtration on a Sephadex G-75 fine column (1.8 x 137 cm, Pharmacia). The fraction containing ir-BNP was further separated by reverse phase HPLC on a Hi-Pore RP-318 column (4.6 x 250 mm, Bio-Rad) with a linear gradient elution of CH_CN from 10% to 60% in 0.1% TFA for 80 min at a flow rate 1.5 ml/min. Aliqu~ts of all fractions in gel filtration and reverse phase HPLC were submitted to RIAs for BNP and ANP. Enzymatic digestion of high MW ir-BNP and characterization of ir-BNP in the digests: Two pooled aliquots (i00 ~I/fraction) of high molecular weight (MW) ir-BNP fractions (fractions #39-45 in Sephadex G-75 gel filtration) were lyophilized. The lyophilizates were each dissolved in 200 ~i of 50mM Hepes buffer (pH 7.5) containing 0.1% Triton X-100. Lysyl endopeptidase (20 ~g) was added to each tube and kept at 37°C for 2 hr (9). After addition of 0.1% TFA, one of the resulting digests was separated by reverse phase HPLC under the identical conditions described above. Another digest was lyophilized and then subjected to (IM ion exchange HPLC on a TSK CM-2SW column (4.6 x 250 ram, Tosoh) with a linear gradient elution from 10mM HCOONH 4 (pH 6.6) to 0.5M HCOONH_4 (pH 6.6) each containing 10% CH3CN for 60 min at a flow rate of 1.0 m l / m i n / An aliquot of each fraction was submitted to RIA for BNP.
RESULTS AND DISCUSSION By
the
RIA
specific
to
peripheral organs
of the pig,
gland and heart,
and have
BNP,
we
have
including
found the
preliminarily
the gastrointestinal
highest
741
screened
concentration
ir-BNP
in
tract,
adrenal
of ir-BNP
in the
Vol. 155, No. 2, 1988
Table I.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Tissue concentration of ir-BNP and ir-ANP in porcine cardiac atrium ir-BNP (pmol/g)
method
ir-ANP (pmol/g) ir-BNP/ir-ANP (%)
Method A (boiled and extracted with IM AcOH*)
97.1
4430
2.19%
Method B (boiled and extracted with IM AcOH/I% Triton)
46.2
4450
1.04%
Method C (extracted with 1M AcOH/ 20mM HCI/66% acetone)
51.9
8850
0.59%
65.1
5910
1.10%
mean *AcOH: acetic acid.
heart
(data not shown). In the porcine heart,
the atrium
(65 pmol/g wet wt),
observed in the ventricle. ir-BNP
from
cardiac
In our preliminary experiment,
atrium
were
examined
methods and compared to those of ir-ANP. among the three extraction methods large
(Table i).
ir-BNP was much concentrated
in
while less than 1 pmol/g wet wt of ir-BNP was
By calculating
using
three
However,
recovery yields of different
extraction
differences in the yields
employed in the present
study were not so
a mean value from the three methods,
atrial
concentration of ir-BNP was determined to be 65.1 pmol/g wet wt, while that of ir-ANP was 5,910 pmol/g wet wt. Thus, it was concluded that ir-BNP was present in porcine atrium but only at about 1% the concentration of ir-ANP. For
characterizing
immunoreactivity
found
reverse phase HPLC. 75
column
atrium was BNP
in
to
the
standard
also observed.
the
atrium
molecular
size
and
atrium was
form
of
analyzed by
gel
bulk
of
human
ir-BNP
y-ANP,
exists
as
a high
appeared
where
This indicates
manner similar to the case of ANP.
most
the
low
MW
corresponding
BNP and
that more
MW form
ir-BNP
to ~-ANP of about
#55-63)
3,000 daltons.
the
ir-ANP
position
from porcine
than 70% of the total ir-
of about
Additionally,
(fractions
around
of
12,000
daltons,
in a
two other minor peaks of ir-
BNP were observed around fractions #28-34 and #55-63, of
ir-BNP, filtration
In gel filtration of the atrial extract on a Sephadex G-
(Fig. i),
corresponding
the
in cardiac
respectively.
emerged
around
The peak
the
position
On the other hand,
another
minor peak of ir-BNP eluted in fractions #28-34 may be due to the presence of aggregates
of
displacement
ir-BNP
curve
of
than
12,000 standard
daltons and 3,000 daltons were Pore C-18
column.
As
daltons, BNP.
since BNP
it
showed
a
immunoreactivities
shallower of
12,000
further separated by reverse phase HPLC on Hi-
shown in Fig. 2,
high M W ir-BNP
(fractions
#39-45
in
Fig. I) emerged as a single peak just after porcine y-ANP. This fact indicates that a high M W
form of
ir-BNP
is
similar
742
to porcine
y-ANP
with
respect
to
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
2
3
4
I
I
I
I
i~- 0.i0
0.05
50 ~G
o"
!, o
0
i0
20
30
~0 50 FrQctlon number
60
70
80
Figure i. Sephadex G-75 gel filtration of the acid extract of porcine cardiac atrium. Sample: Porcine cardiac atrium extract (10 g wet wt equivalent) prepared by Method A. Details of the extraction method are described in Methods. Column: Sephadex G-75 fine (1.8 x 137 cm, Pharmacia). Flow rate: 8 m 1/hr. Fraction size: 5 ml/tube. Solvent: IM acetic acid. The arrows indicate the elution positions of i) Vo, 2) y-hANP, 3) ~-hANP and 4) Vt, respectively.
hydropathy
and molecular
#55-63
Fig.
in
corresponding that B N P controlled
i)
weight.
appeared
in
On the the
to BNP and BNP-32,
and BNP-32
are g e n e r a t e d
proteolytic
processing
other hand,
reverse
as seen
phase
in Fig.
from a high MW system
low M W
HPLC 3.
at
ir-BNP
the
retention
Accordingly, B N P of 1 2 , 0 0 0
in a m a n n e r
similar
(fraction times
it is l i k e l y daltons
to t h a t
for ANP.
6O 0.2 40
z
20 8
o
o.z
i
800
400
.~ 6o = o
O~
30 o
Tlme (mln)
Figure 2. Reverse phase H P L C o f high MW J/m~unoreactive BNP and ANP. Sample: Fractions #39-45 (i00 ~l/tube), shown by arrow A in Fig. i. Colu/m~: Hi-Pore RP-318 (4.6 x 250 ~ , Bio-Rad). Flow rate: 1.5 ml/min. Solvent system: H_O:CH3CN:I0%TFA = (A) 90:10:i, (B) 40:60:1 (v/v). Linear gradient e~ution from (A) to (B) for 80 min. Temperature: ambient.
743
by a
V o l . 155, N o . 2, 1 9 8 8
B I O C H E M I C A L A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
0.f08 0,04
6O 41'
'IF
............
~o 20
.
o
0
4oo~
i.
6
L
o 3 0 10
20
30
200
L O~
40
50
60
70
80
T I m e (mln)
Figure 3. Reverse phase HPLC of low MW Jum~unoreactive BNP and ANP. Sample: Fractions #55-63 (200 ~i/tube), shown by arrow B in Fig. 1. chromatographic conditions were identical to those for Fig. 2. The arrows indicate the elution times of i) e-ANP and 2) BNP or BNP-32, respectively.
In order
to obtain
further
information
about a 12,000-dalton
high M W ir-BNP was digested with lysyl endopeptidase, in the digests was analyzed,
BNP,
the
and the resulting ir-BNP
since BNP-32, having a lysine residue at position
3, undergoes lysyl endopeptidase digestion to yield BNP-29 by removal of three amino acid fractions
residues
at
#39-45 were
the N-terminus lyophilized,
(see Fig.
As shown in Fig. 4a,
Pooled
conditions
BNP immunoreactivity generated
the lysyl endopeptidase to BNP-29
(data not
the digests, BNP-29. of
12,000
on CM
shown).
The peptide
was
exhibiting BNP immunoreactivity
chromatographically
identified to
in
be authentic
BNP is verified to exist also in the heart as a high M W form
daltons,
evidence implies
Furthermore,
from the high M W ir-BNP by
digestion also appeared at the elution time identical
therefore,
Hence,
and then
to those for high
which emerged at 36-37
min identical to authentic BNP-29 on reverse phase HPLC. HPLC,
of
the high MW ir-BNP was found to be converted
into a single component carrying BNP immunoreactivity,
ion exchange
aliquots
digested by lysyl endopeptidase
subjected to reverse phase HPLC under identical M W ir-BNP.
4b).
which
carries
the hormonal
the
BNP
unit
at
its
C-terminus.
This
function of BNP in the heart in addition to the
neural function in the brain Although ANP was first
identified
in the atrium,
have been shown to exist also in the brain,
ANP and its receptors
especially in the regions thought
to be involved in the control of body fluid homeostasis
(10-12). This strongly
suggests
the neural
that ANP also functions
as a
water and electrolyte balance.
744
neuropeptide
in
control of
VoI. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1 ~,
0.2
2 ~
6O
........
_._..----'-
40 ~5
_.._----
20 ~ 0.i
1'
0
I
o
r
1%
b
20
J.
i "1'
30
800
~00
.11_
~o
~o
T tree (rain)
L 0~
6~
/o
LYS endoDeptldase
I ~5 I0 15 20 2S 30 W BNP-32: S-P-K-T-M-R-D-S'G-C-F-G-R-R-L'D-R" I-6-S-L-S-G-I-G-C-N-V'L-R-R-Y BNP
:
D-S-G-C-F-S-R-R-L-D-RI-G-S-L-S-G'L-G-C-N-V'L-R-R-Y
Figure 4. (a) Reverse phase HPLC of lysyl endopeptidase digests of high MW immunoreactive BNP. Sample: Aliquots (i00 ~l/tube) of fractions #39-45 were lyophilized alad digested with lysyl endopeptidase (20 Bg) at 37°C for 2 hr. Chromatographic conditions were identical to those for Fig. 2. The arrows indicate the elution times of i) BNP-29 and 2) high MW form of i~-BNP. (b) Amino acid sequences of BNP and BNP-32. The arrow indicates the position which lysyl endopeptidase is thought to cleave.
In our recent study using the RIA system for BNP, tissue concentration of ir-BNP in porcine whole brain was estimated
to be 0.63 pmol/g wet wt,
about 13 times higher than that of ir-ANP in brain suggests
that
BNP
together
with
ANP
in
brain
being
(0.05 Imaol/g wet wt).
may
function
in
the
This neural
control of body fluid homeostasis.
In the present study, we demonstrated that
BNP is also present
and that in the cardiac atrium the tissue
concentration
in the heart,
of ir-BNP
(65.1 pmol/g wet wt)
that in the whole brain. BNP
in
the
speculate
heart
Furthermore,
is very similar
that: BNP in
I00 times
higher
than
the molecular distribution pattern of
to that
the atrium,
is about
of ANP.
These
facts
even though its concentration
lead
us to
is only 1%
that of ir-ANP, is highly likely a hormone to be secreted for regulating blood pressure and water-salt balance, working in concert with ANP. The
existence
these two hypotensive
in
two
highly homologous activities,
different
organs
peptides,
such
as
the
heart
and
brain
both sharing natriuretic-diuretic
of and
suggests that body fluid and electrolyte homeostasis
may be controlled by two ways through the neural and hormonal systems, ANP and BNP may function in the concerted manner.
745
where
Vol. 155, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS: This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.
REFERENCES i. Flynn, T.G., de Bold, M.L. & de Bold, A.J. (1983) Biochem. Biophys. Res. Commun., 117, 859-865. 2. Kangawa~ K. & Matsuo, H. (1984) Biochem. Biophys. Res. Commun., 118, 131-139. 3. de Bold, A.J. (1985) Science, 230, 767-770. 4. Sudoh, T., Kangawa, K., Minamino, N. & Matsuo, H. (1988) Nature, 332, 78-81. 5. Forssmann, W.G., Birr, C., Carlquist, M., Christmann, M., Finkle, R. Henschen, A., Hock, D., Kirchheim, H., Kreeye, V., Lottspeich, F., Mutt, V. & Reinecke, M. (1984) Cell Tissue Res., 238, 425-430. 6. Ueda, S., Sudoh, T. Fukuda, K., Kangawa, K., Minamino, N. & Matsuo, H. (1987) Biochem. Biophys. Res. Commun., 149, 1055-1062. 7. Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T. & Matsuo, H. (1985) Biochem. Biophys. Res. Commun., 129, 248-255. 8. Kitamura, K., Minamino, N., HayaShi, Y., Kangawa, K. & Matsuo, H. (1982) Biochem. Biophys. Res. Commun., 109, 966-974. 9. Masaki, T., Tanabe, M., Nakamura, K. & Soejima, M. (1981) Biochim. Biophys. Acta, 660, 44-55. i0. Quirion, R., Daple, M., de Lean, A., Gutkowska, J., cantin, M. & Genest, J. (1984) Peptides, 5, i167-1172. ii. Saper, C.B., Standaert, D.G., Currie, M.G., Schwartz, D., Geller, D.M. & Needleman, P. (1985) Science, 227, 1047-1049. 12. Kawata, M., Nakao, K., Morii, N., Kiso, Y., Yamashita, H., Imura, H. & Sano, Y. (1985) Neuroscience, 16, 521-546.
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