Vol.
172,
October
No. 30,
BIOCHEMICAL
2, 1990
AND
RESEARCH
BIOPHYSICAL
COMMUNICATIONS Pages
1990
SEQUENTIAL
MECHANISM OF IN ISOLATED
Kyung Kyung
Kyung Woo Cho, Gou Young Koh,
Department
Received
of Medical
September
ATRIAL NATRIURETIC PERFUSED RABBIT
Hwan Seul, Mee Seul and
Jeonbug Jeonju
Physiology, School,
10,
PEPTIDE ATRIA
Suhn Yun
National 560-180,
423-431
SECRETION
Hee Kim, Ha Hwang
University Korea
1990
well known that the secretion of atria1 natriuretic It is peptide (ANP) is dependent on the atria1 stretch. It has been claimed in this laboratory that the secretion of ANP occurs with in atria1 distension. It was shown in the present a reduction occurs experiment that the secretion of immunoreactive (ir) ANP marker coincidently with a translocation of extracellular space (3-H)-inulin in the isolated perfused rabbit atria. Translocation reduction in of extracellular space fluid was observed with a atria1 distension. The secretion of irANP into the atria1 lumen occurs less than 15 set of the reduction in atria1 distension. It suggested that the incremental response of is therefore irANP secretion to the reduction in atria1 distension is a sequential mechanism of ANP secretion, in which first is the release of ANP from the atria1 myocytes into the extracellular space and then second is the translocation of ANP with extracellular space fluid into the atria1 lumen with a reduction in atria1 distension. ‘D 199”
Rcademlc
Press,
The family
heart of
atria1
the
the
regulation
certainty.
gland.
active
vascular of of
of
(6-10)
been
Among of
ANP, The
exact
have heart
atria1 nature
rate as
suggested distension of
the
423
ANP,
signal
the
heart
Changes
or
contraction
atria1
important
been
is
factors
suggested.
transduction
All
Even
Many
factors has
termed
activities. of
been
a
natriuretic,
system.
considered the
peptides,
relaxant
endocrine
and
have
secretion.
new
contain
potent
source
secretion
(l-5)
pressure
have
muscle
extra-atria1
nyocytes
These
(ANPs),
the
ANP
Atria1
peptides.
smooth
presence organ
frequency
secretion
endocrine
peptides
and
central
ANP
an
natriuretic
the
atria1
is biologically
diuretic though
1°C
for in
stimuli
for
regulating
the
established
with by
atria1
0006-291x/90 $1.50 Copyright 0 1990 by Academic Press. Inc. rights of reproduction in any form reserved.
Vol.
172,
No.
2,
1990
distension been
to
induce
claimed
response
to
atria1
sequence atria1
lumen.
sequential
To
into
translocation
atria
test
by
a
atria1
the
extracellular
reduction
in
atria1
distension-reduction
release
with
ECS
of
fluid
the
isolated
The
into
the has
ANP and
into we
atria1
secretion
(ECS)
distension, in
ANP
studied perfused
in
the
myocytes
space
molecules
in
investigate
the
has
ANP
(11-13).
to
first
It of
stretch
that
the
clarified.
reduction
atria1
hypothesis
i.e.,
ANP
a
atria1
from the
the of
with
an
COMMUNICATIONS
secretion
designed
secretion
mechanism,
myocytes
with
yet
the
occurs
therefore,
ANP
not
that
distension
was,
of
is
laboratory
than
study
BIOPHYSICALRESEARCH
secretion
this
rather
present
of
ANP
in
distension
lumen
BIOCHEMICALAND
from
the
then
the
the the
atria1 effect rabbit
(11-13).
MATERIALS
AND
METHODS
erfused atria1 preparation: New Zealand white rabbits, Isolated weighing 1.%zix-lTg,were sacrificed. Isolated perfused atria1 preparation was made by the method described previously (11,12) with some modifications which permit direct measurement of atria1 volume changes (13). Briefly, the hearts were rapidly removed and placed in oxygenated warm saline. The left atria dissected. A cannula (4.0 mm outer diameter), made of three small Tyson sealed water-tightly with silicone glue inside catheters inserted into the left atrium through the atrioventr;cu;zF orifice by about 2.5 mm. The atria1 cannula was tied by 3-4 One of the three catheters located in the atrium, the tip times. of which was adjacent to the auricular apex, was for the inflow, another was for the outflow from the atrium. The remaining and catheter was located for tracing of pressure changes in the All these procedures were performed in the oxygenated atrium. The cannulated atrium was transferred into the warm saline. constant-temperature (36.5 C) organ chamber. buffer-containing fixed The cannulated atrium was fitted in the organ chamber and silicone rubber cap. The atrium was with a water-tight bicarbonate immediately perfused with Krebs-Henselite buffer The composition of the Krebssolution to the atria1 lumen. bicarbonate buffer in mM was as follows: NaCl, 118; Henseleit 1.2; NaHC03, 25; KCl, 4.7; CaC12, 2.5; KH2P04, 1.2; MgS04, 0.1%. The atria1 lumen glucose, 10.0 and bovine serum albumin, perfused through the exteriorized catheter of which tip was was located in the auricle. The pericardial medium was oxygenated by silicone tubing coils located inside the organ chamber, which was connected to the gas chamber. The pericardial space of the organ chamber was sealed water-tightly with outfit one which was with a calibrated microcapillary tube. connected The calibrated microtube was located in a horizontal position at the level of Atria1 atrium. volume changes were monitored by measuring the of the calibrated microtube occupied by buffer length solution. All these procedures were done within 3 min after atria1 removal. 424
Vol.
172,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A peristaltic pump was used to maintain perfusion flow constant The perfusate was equilibrated with an 0.25 or 1.25 ml/min. at mixing by passing silicone tubing into the 02-CO2 mixture gas perfusate To maintain constantly perfusate at pH 7.4, chamber. gases and pH were monitored via periodic sampling measured with a and 175 automatic pH/blood gas system (Corning Medical Corning The solution was prewarmed Scientific, Medfield, MA, U. S. A.). constant temperature water at 36.5 C by passage through a chamber. Protocols: Pericardial medium was introduced by (3-H)-inulin or The atria were (51-Cr)-tDTA at the start of atria1 perfusion. 30 min to stabilize irANP secretion rate and perfused for equilibrate the ECS with a marker in baseline distension, during which distension and reduction were repeated several times. The collected in siliconized tubes at 4 C. In perfusate was one series of experiments, after three collection periods at the baseline distension, atria1 distension was induced by changing elevation of the out-flow catheter tip by 2, 4, 6 and 10 the cmH20 above the atrium. Every 2 rnin of atria1 distension was followed by a reduction in atria1 volume for 10 min. Reduction in atria1 volume was conducted by lowering the elevation of the outflow catheter tip. Atria1 perfusion rate was maintained at 0.25 ml/min. In another series of experiments, rapid sample collections were performed at the higher perfusion rate of 1.25 ml/min. After three collection periods at the baseline distension, atria1 distension wa s induced by changing the the elevation of out-flow catheter tip by 6 cmH20 above the atrium. Two or 10 min of atria1 distension was followed by a reduction in atria1 volume. Atria1 perfusates were collected at 15 set intervals for 2 min (serial 8 samples for 2 min) at the reduction in atria1 distension. In the third series of experiments atria1 tissues were processed to measure the ECS after 60 min equilibration with ECS marker (51-Crj-EOTA. Radioimmunoassay of The irANP in the perfusate was --irANP: measured by radioimmunoassay as described previously (11-13). The pH radioimmunoassay was performed in Tris-acetate buffer (0.1 M containing 0.2% neomycin, 1.0 mM EDTA, 50 benzoyl arginine 7.40), ethyl ester units/ml soybean trypsin inhibitor, 200 KIU/rnl aprotinin, 0.4 mg% phenylmethylsulfonyl fluoride, 0.02% sodium azide and 1% bovine serum albumin. Radioimmunoassay for irANP was done on the day of experiments and all samples in an experiment were analyzed in a single assay. Non-specific binding was less than 3%. The 50% intercept was at 26.6 + 2.9 pg/tube (mean t SE, n=7). The intraand inter-assay coefficients of variation-were 6.3 (n=9) and 7.8% (n=ll), respectively. Reverse-phase high-performance Atria1 perfusates durinq and extracted separately. with Sep-Pak Cl8 cartridges S. A.) and was subjected chromatography (HPLC) Associates) as described with linear gradient trifluoroacetic acid for fractions were collected.
liquid chromatography profiles: basal and stimulated periods were oooled extracted The irANP in perfusates was (Waters Associates, Milford, MA, U. to reverse-phase high-performance liquid on a C-18 ul3ondapak column (Waters previously (14). Elution was performed of 20 to 60% acetonitrile in 0.1% 40 min at a rate of 1 ml/min. One-minute
s ace Extracellular measurement: - r)-tDTA at introduced by d-n Atria1 tissues were processed after 60 min of perfusion. Atria1 the filter paper. The ECS of H20/100 g tissue wet weight.
Pericardial start of measure the tissues were the atrium was
the to
425
medium was atria1 oerfusion. ECS fluid volume gently blotted on expressed as ml
Vol.
172,
No.
2,
1990
BIOCHEMICALAND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS A
reduction
irANP
in
secretion
in
representative
A with
isolated
perfused
induced
resulted 1
A).
by
ECS
in
Very
accompanied
irANP
resulted
the
distension
changes
an
distension
experiment
atria1
(Fig
atria1
an
a
by
2,
The
reduction
in
increase
the
and
and
irANP
into
the
in
secretion
irANP
secretion
of
was
(3-H)-inulin, as
during
the
atria1
well
as
experiments.
lumen
distension.
A
pressure
(3-H)-inulin
constant
(3-H)-inulin atria1
in
of
atria.
cmH20
in
in
Reduction
10
concentration
levels
increase
1.
increase
baseline
in
6
an rabbit
Figure
4,
an
stable of
in
proportional
increase
relatively
translocation
shown
interestingly
marker. were
a
is
in
Increase
occurred in
(A)
irANP
-
a0
-
40
-
3og
z 1. 2 n
-
20%
: z-
-
1OG -
-0
5
10
15
20
25
c:
2
Fr. No.
Coincident increase in the concentration of irANP with Fi ure 1. the of(3-H)-inulin by a reduction in atria1 distension in The changes in the concentration of perfused rabbit atria (A). that of the (3-H)-inulin correlated uell with irANP are The concentration of irANP in terms of ECS ;;;;;located (inset): translocated Increased by a reduction in atria1 distension (6). Each point represents a P-min period. Atria1 perfusion rate was maintained at 0.25 ml/min. ECS; extracellular space. Fr. No.; fraction number.
-l+i--
426
Vol.
172,
No.
2,
BIOCHEMICAL
1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(A) 80
t P
I -2
(7
R
min
t
-I
2 min
2 min
0 0
02
200
Distension-
400 Reduction
Volume,
pl
we. &. re
ucfion
in
Fr. No
Dependence atria1 Rapid
perfusates samples volume concentration increased perfusion
were for 2 contained by rate
secretion
was of was
(Fig
1
irANP
in
in
As of
lumen
in increases
2).
in
ECS
fluid from
on
the 1.
Figure
figure
1
the
unexpectedly
concentration
experiments. of
the
the of
concentration by
ECS
reduction
fluid
secretion
and
of
a
into
changes
As irANP
in
translocated
volume
rapid
427
the
B
of
to
concentration
increase
increased
translocation
of
an
(3-H)-inulin
translocated
series the
in of
in
proportion An
another
that
shown
fluid
in
Increase
with
The
was (Fig
observed
ECS
distension.
distension
A,
well
terms
of derived
with
marker.
inset).
atria1
atria1
coincidently
ECS
correlated A,
translocation Data were
increases in the concentration of irANP and (3by a reduction in atria1 distension (A) Atria1 collected at 15 set intervals for 2 min (serial 8 min) at the reduction in atria1 distension. The in the dead space was subtracted. The of irANP in terms of ECS fluid translocated a reduction in atria1 distension (B). Atria1 was maintained at 1.25 ml/min.
observed
translocation irANP
of the distension.
in of
shown the
the atria1
irANP in
Figure
translocation
was 3
Vol.
172,
No.
2, 1990
BIOCHEMICALANDBIOPHYSICALRESEARCH
Pro-ANP
AP III
(A)
COMMUNICATIONS
i
1 /-
_&
.2F1.0 /-
/-
---
1.5
1
_/---
Fr. NO.
40
Reverse-phase HPLC basal (A) and were collected of atriopeptin
4. -aTFs bisten peak respec
of
in
tively.
ECS
less
fluid
than irANP
caused 15
volume
ECS
the of
concentration increased
coincidently
reduction
in
the
ECS
of
irANP
from
it
at
the
confirmed
fluid
was
translocated in the
of
(Fig
perfusate
obtained
mainly
low
of
baseline
3
The at
molecular
well
the
perfusate
The
translocated secretion
by
molecular in
irANP (Fig
a
profiles
reduction
weight
with
inset).
A,
irANP
6).
the
distension.
fluid
in 3
in
correlated
ECS
increase
increase
atria1
(Fig
terms
increase
the
the in
irANP
occurred
that
reduction
of
Atria1 in atria1 icate the pro-ANP,
5.
distension
with
distension
those
atria1
coincidently
with
atria1
in was
concentration
showed
different
again
fluid
15-set-collected
distension
reduction
occurred of
in
a
Here
secretion
Increase
of
by
sec.
translocation
the
profiles of the perfusate 15 set after reduction separately. Arrows ind III (AP III) and
and
atria1 were
not
4).
DISCUSSION To secretion
the
best of
of irANP
our with
this
knowledge, the
coincident
428
is
the translocation
first
to
observe of
ECS
the fluid
Vol.
172,
No.
2, 1990
the
and
very
protocol
BIOCHEMICALANDBIOPHYSICALRESEARCH
rapid
to
equilibrate
technique
is
+- 1.97
ml
EDTA
space
as
present
irANP
is
secretion
of
(11-13).
A
with
a
consistent point
clear
at
a
in
with
time
of
at
the
concentration (
with
Taken is
a
atria1
in in
increased.
the
the in
the
increase
ECS
fluid
not the noteworthy
the
increase
of
the
atria1
lumen. was
in
the
concentration
ECS
by
irANP an
by
but In
also any
accompanied
by case,
by In
an some
concentration was
of not
of
a
increase
(3-H)-inulin.
translocated
429
in
distension,
secretion
myocytes
the
ECS
translocated
atria1
atria1
as
of
of
only
not
lumen
translocation
the
that
exact is
an
of
fluid
the
distension.
not
however,
are
atria1
in
the
of
observed
atria1
with
the
irANP
was
fluid
in
the
distension
step
caused
shown, ECS
ECS
coincident
of
that
though
is
into
into
translocation
volume
concentration
of is
the
fluid
Increase
the
in
of
results
the
a
that
ECS
first
into
reduction
concentration
data
terms It
reduction
ECS
the
experiments
the
into
of was
distension
of
increase
finding
a
in
secretion
atria
distension.
the
the on
release
increase
at
irANP
shown
present
as
with
)
irANP
that
translocation
irANP
terms
with
in irANP
the
in
irANP
clearly
Even
ECS
of
atria1
suggested
reduction
secretion
in
the
values (51-Cr)-
atria1
The
the
the
laboratory
of
was
the
of
(11-13).
into
reduction
dependent
is
data
and
the
in
fluid
atrium
than
of
reduction
The
repetitive
the
was
this
distension.
coincidently
of
in
a
occurs
together
fluid
in
release
the
translocation
ECS
(3-H)-inulin
secretion
it
ANP
of
measured
the
previous
the
step
fluid
atria1
the
present,
final
with of
for
less
step
claimed
translocation
a
SE)
new
a
marker.
with
+
It
final
the
been
occurs
reduction
but (15).
the
presented
of
mean
(n=6, space
with
has ANP
atria
suitable
previously
related It
wt
that
have
space)
inulin
experiment
atrium.
the
wet
the
reported
closely
the
and
g
with
of
We
((51-Cr)-EDTA
HZO/lOO
comparable
the
ECS
irANP.
ECS
simple
The
25.80
of the
very
stimulations.
are
secretion
COMMUNICATIONS
always irANP
in
Vol.
172,
No.
terms
of
Even
though
A,
BIOCHEMICALAND
ECS
fluid
the vs
the
above
less
concentration translocation
irANP
the
in
concentration. of the
released it into
passes is the
of fluid
it
(16),
with lumen ANP
into
atria1
the
hypothesis
ECS
atria1
by
by
irANP
a
fluid
translocated.
dilute
of
In
a it
to
atria1
a
very
has the
postulate
irANP
micromolar
observe
rapid
been
known
molecules
that
sequential
the
of
range
the A
concentration
by an
and
ECS
the
non-
myocyte-related
may
that
one
is
pathways.
followed
lumen
other
fluid
Since
The
is,
ECS
to
to
extracellular
the
the
permeable
possible
caused
That
translocated.
lumen.
the
is
into the
is
B). channels
fluid
the
is
2
ECS
enough
the
and
of
the in
interesting
through
atria1
be
endothelium
consistent
release ECS
Da
the
that
may
into
endocardial
40,000
proposed
is
irANP
of
6
of
the
1
translocated
spaces. and
(Fig
distension,
functional
secretion
of
terms
ECS
It
secretion
terms
in is
it
released
the
40
rig/ml)-fold
fluid
translocation
both
1.2
1
two
space
in
ECS
endocardial
the
increased.
atria1
(Fig
non-myocyte-related
of context,
the
is
be by
vs in
of
there
Only
not
58.2
increase
and
irANP of
concentration
terms
that
increase of
A,
COMMUNICATIONS
increased
reduction
myocyte-related
may
may
was
2 a
lo-fold
space.
fluid
(Fig
in
pericardial
myocyte-related
to
than
the
through
that
irANP
or
irANP
by
suggests the
this
49
secretion
situation
ECS
to
of
connecting
of
rig/ml)
concentration in
may
concentration
I.8
BIOPHYSICALRESEARCH
translocated
baseline
resulted
is
1990
the
72.0
above the
2,
up
the In
irANP summary,
secretion
of
ANP
mechanism
of
the
rapid
translocation
of
contraction.
ACKNOWLEDGMENTS Yonsei thank Prof. Doo Hee Kang, We for his critical suggestions Medicine, Seoul, was supported by grants from This work Engineering Foundation and the Ministry of
University for the Korea Education,
the
College experiment. Science Korea.
REFERENCES 1 . 2.
Dietz, Lang, tt.,
and
J.R. R.E., Unger,
(1984) Thoelken, T.H.
Am.
J. H.,
(19U5)
Ptrysiol. Ganten, Nature
430
247, D., 314,
R1093-R1096. Luft, F.C., 264-266.
Ruskoaho,
of and
Vol.
3. 4. 5. 6. 7.
a. 9.
10. 11. 12. 13. 14. 15. 16.
172,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Ruskoaho, H., Thelken, H., and Lang, R.E. (1986) Pflugers Arch. 407, 170-174. Katsube, N., Schwartz, O., and Needleman, P. (1985) Biochem. Biophys. Res. Commun. 133, 937-944. Ledsome, J.R., Wilson, N., Courneya, C.A., and Rankin, A.J. 1985) Can. J. Physiol. Pharmacol. 63, 739-742. E.L., Gutkowska, J., Kuchel, O., Cantin, M., and i chiffrin, Genest, J. (1985) N Engl. J. Med. 312, 1196-1197. Gutkowska J Bourassa, M., Roy, O., Thibault, G., Garcia, R Cantin &I, and Genest, J. (1985) Biochem. Biophys. Res. Co,imun. 128: 1350-1357. Schiebinger, R.J., and Linden, J. (1986) Am. J. Physiol. 251, H1095-H1099. Rankin, A.J., Courneya, C.A., Wilson, N., and Ledsome, J.R. (1986) Life Sci. 38, 1951-1957. Bilder, G.E., Siegl, P.K.S., Schofield, T.L., and Friedman, P.A. (1989) Circ. Res. 64, 799-805. Cho, ‘K-W.; Seul, K.H.,. Ryu, H., Kim, S.H., and Koh, G.Y. (1988) Regul. Peptides 22, 333-345. Cho, K.W., Seul, K.H., Kim, S.H., Ryu, H., Seul, K.M., and Koh, G.Y. (1988) Biochem. Biophys. Res. Commun. 153, 811-817. Seul, K.H., Kim, S.H., Seul, K.M., Ryu, H,, and Cho, K.W., Koh, G.Y. (1989) J. Hypertens. 7, 371-375. Cho, K.W., Kim, S.H., Koh, G.Y., and Seul, K.H. (1988) J. Exp. Zool. 247, 139-145. Poole-Wilson, P.A., and Cameron, I.R. (1975) Am. J. Physiol. 229, 1299-1304. Anversa, P., Giacomelli, F., Wiener, J., and Spiro, 0. (1973) Lab. Invest. 28, 728-734.
431
172,
October
No. 30,
BIOCHEMICAL
2, 1990
AND
RESEARCH
BIOPHYSICAL
COMMUNICATIONS Pages
1990
SEQUENTIAL
MECHANISM OF IN ISOLATED
Kyung Kyung
Kyung Woo Cho, Gou Young Koh,
Department
Received
of Medical
September
ATRIAL NATRIURETIC PERFUSED RABBIT
Hwan Seul, Mee Seul and
Jeonbug Jeonju
Physiology, School,
10,
PEPTIDE ATRIA
Suhn Yun
National 560-180,
423-431
SECRETION
Hee Kim, Ha Hwang
University Korea
1990
well known that the secretion of atria1 natriuretic It is peptide (ANP) is dependent on the atria1 stretch. It has been claimed in this laboratory that the secretion of ANP occurs with in atria1 distension. It was shown in the present a reduction occurs experiment that the secretion of immunoreactive (ir) ANP marker coincidently with a translocation of extracellular space (3-H)-inulin in the isolated perfused rabbit atria. Translocation reduction in of extracellular space fluid was observed with a atria1 distension. The secretion of irANP into the atria1 lumen occurs less than 15 set of the reduction in atria1 distension. It suggested that the incremental response of is therefore irANP secretion to the reduction in atria1 distension is a sequential mechanism of ANP secretion, in which first is the release of ANP from the atria1 myocytes into the extracellular space and then second is the translocation of ANP with extracellular space fluid into the atria1 lumen with a reduction in atria1 distension. ‘D 199”
Rcademlc
Press,
The family
heart of
atria1
the
the
regulation
certainty.
gland.
active
vascular of of
of
(6-10)
been
Among of
ANP, The
exact
have heart
atria1 nature
rate as
suggested distension of
the
423
ANP,
signal
the
heart
Changes
or
contraction
atria1
important
been
is
factors
suggested.
transduction
All
Even
Many
factors has
termed
activities. of
been
a
natriuretic,
system.
considered the
peptides,
relaxant
endocrine
and
have
secretion.
new
contain
potent
source
secretion
(l-5)
pressure
have
muscle
extra-atria1
nyocytes
These
(ANPs),
the
ANP
Atria1
peptides.
smooth
presence organ
frequency
secretion
endocrine
peptides
and
central
ANP
an
natriuretic
the
atria1
is biologically
diuretic though
1°C
for in
stimuli
for
regulating
the
established
with by
atria1
0006-291x/90 $1.50 Copyright 0 1990 by Academic Press. Inc. rights of reproduction in any form reserved.
Vol.
172,
No.
2,
1990
distension been
to
induce
claimed
response
to
atria1
sequence atria1
lumen.
sequential
To
into
translocation
atria
test
by
a
atria1
the
extracellular
reduction
in
atria1
distension-reduction
release
with
ECS
of
fluid
the
isolated
The
into
the has
ANP and
into we
atria1
secretion
(ECS)
distension, in
ANP
studied perfused
in
the
myocytes
space
molecules
in
investigate
the
has
ANP
(11-13).
to
first
It of
stretch
that
the
clarified.
reduction
atria1
hypothesis
i.e.,
ANP
a
atria1
from the
the of
with
an
COMMUNICATIONS
secretion
designed
secretion
mechanism,
myocytes
with
yet
the
occurs
therefore,
ANP
not
that
distension
was,
of
is
laboratory
than
study
BIOPHYSICALRESEARCH
secretion
this
rather
present
of
ANP
in
distension
lumen
BIOCHEMICALAND
from
the
then
the
the the
atria1 effect rabbit
(11-13).
MATERIALS
AND
METHODS
erfused atria1 preparation: New Zealand white rabbits, Isolated weighing 1.%zix-lTg,were sacrificed. Isolated perfused atria1 preparation was made by the method described previously (11,12) with some modifications which permit direct measurement of atria1 volume changes (13). Briefly, the hearts were rapidly removed and placed in oxygenated warm saline. The left atria dissected. A cannula (4.0 mm outer diameter), made of three small Tyson sealed water-tightly with silicone glue inside catheters inserted into the left atrium through the atrioventr;cu;zF orifice by about 2.5 mm. The atria1 cannula was tied by 3-4 One of the three catheters located in the atrium, the tip times. of which was adjacent to the auricular apex, was for the inflow, another was for the outflow from the atrium. The remaining and catheter was located for tracing of pressure changes in the All these procedures were performed in the oxygenated atrium. The cannulated atrium was transferred into the warm saline. constant-temperature (36.5 C) organ chamber. buffer-containing fixed The cannulated atrium was fitted in the organ chamber and silicone rubber cap. The atrium was with a water-tight bicarbonate immediately perfused with Krebs-Henselite buffer The composition of the Krebssolution to the atria1 lumen. bicarbonate buffer in mM was as follows: NaCl, 118; Henseleit 1.2; NaHC03, 25; KCl, 4.7; CaC12, 2.5; KH2P04, 1.2; MgS04, 0.1%. The atria1 lumen glucose, 10.0 and bovine serum albumin, perfused through the exteriorized catheter of which tip was was located in the auricle. The pericardial medium was oxygenated by silicone tubing coils located inside the organ chamber, which was connected to the gas chamber. The pericardial space of the organ chamber was sealed water-tightly with outfit one which was with a calibrated microcapillary tube. connected The calibrated microtube was located in a horizontal position at the level of Atria1 atrium. volume changes were monitored by measuring the of the calibrated microtube occupied by buffer length solution. All these procedures were done within 3 min after atria1 removal. 424
Vol.
172,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A peristaltic pump was used to maintain perfusion flow constant The perfusate was equilibrated with an 0.25 or 1.25 ml/min. at mixing by passing silicone tubing into the 02-CO2 mixture gas perfusate To maintain constantly perfusate at pH 7.4, chamber. gases and pH were monitored via periodic sampling measured with a and 175 automatic pH/blood gas system (Corning Medical Corning The solution was prewarmed Scientific, Medfield, MA, U. S. A.). constant temperature water at 36.5 C by passage through a chamber. Protocols: Pericardial medium was introduced by (3-H)-inulin or The atria were (51-Cr)-tDTA at the start of atria1 perfusion. 30 min to stabilize irANP secretion rate and perfused for equilibrate the ECS with a marker in baseline distension, during which distension and reduction were repeated several times. The collected in siliconized tubes at 4 C. In perfusate was one series of experiments, after three collection periods at the baseline distension, atria1 distension was induced by changing elevation of the out-flow catheter tip by 2, 4, 6 and 10 the cmH20 above the atrium. Every 2 rnin of atria1 distension was followed by a reduction in atria1 volume for 10 min. Reduction in atria1 volume was conducted by lowering the elevation of the outflow catheter tip. Atria1 perfusion rate was maintained at 0.25 ml/min. In another series of experiments, rapid sample collections were performed at the higher perfusion rate of 1.25 ml/min. After three collection periods at the baseline distension, atria1 distension wa s induced by changing the the elevation of out-flow catheter tip by 6 cmH20 above the atrium. Two or 10 min of atria1 distension was followed by a reduction in atria1 volume. Atria1 perfusates were collected at 15 set intervals for 2 min (serial 8 samples for 2 min) at the reduction in atria1 distension. In the third series of experiments atria1 tissues were processed to measure the ECS after 60 min equilibration with ECS marker (51-Crj-EOTA. Radioimmunoassay of The irANP in the perfusate was --irANP: measured by radioimmunoassay as described previously (11-13). The pH radioimmunoassay was performed in Tris-acetate buffer (0.1 M containing 0.2% neomycin, 1.0 mM EDTA, 50 benzoyl arginine 7.40), ethyl ester units/ml soybean trypsin inhibitor, 200 KIU/rnl aprotinin, 0.4 mg% phenylmethylsulfonyl fluoride, 0.02% sodium azide and 1% bovine serum albumin. Radioimmunoassay for irANP was done on the day of experiments and all samples in an experiment were analyzed in a single assay. Non-specific binding was less than 3%. The 50% intercept was at 26.6 + 2.9 pg/tube (mean t SE, n=7). The intraand inter-assay coefficients of variation-were 6.3 (n=9) and 7.8% (n=ll), respectively. Reverse-phase high-performance Atria1 perfusates durinq and extracted separately. with Sep-Pak Cl8 cartridges S. A.) and was subjected chromatography (HPLC) Associates) as described with linear gradient trifluoroacetic acid for fractions were collected.
liquid chromatography profiles: basal and stimulated periods were oooled extracted The irANP in perfusates was (Waters Associates, Milford, MA, U. to reverse-phase high-performance liquid on a C-18 ul3ondapak column (Waters previously (14). Elution was performed of 20 to 60% acetonitrile in 0.1% 40 min at a rate of 1 ml/min. One-minute
s ace Extracellular measurement: - r)-tDTA at introduced by d-n Atria1 tissues were processed after 60 min of perfusion. Atria1 the filter paper. The ECS of H20/100 g tissue wet weight.
Pericardial start of measure the tissues were the atrium was
the to
425
medium was atria1 oerfusion. ECS fluid volume gently blotted on expressed as ml
Vol.
172,
No.
2,
1990
BIOCHEMICALAND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS A
reduction
irANP
in
secretion
in
representative
A with
isolated
perfused
induced
resulted 1
A).
by
ECS
in
Very
accompanied
irANP
resulted
the
distension
changes
an
distension
experiment
atria1
(Fig
atria1
an
a
by
2,
The
reduction
in
increase
the
and
and
irANP
into
the
in
secretion
irANP
secretion
of
was
(3-H)-inulin, as
during
the
atria1
well
as
experiments.
lumen
distension.
A
pressure
(3-H)-inulin
constant
(3-H)-inulin atria1
in
of
atria.
cmH20
in
in
Reduction
10
concentration
levels
increase
1.
increase
baseline
in
6
an rabbit
Figure
4,
an
stable of
in
proportional
increase
relatively
translocation
shown
interestingly
marker. were
a
is
in
Increase
occurred in
(A)
irANP
-
a0
-
40
-
3og
z 1. 2 n
-
20%
: z-
-
1OG -
-0
5
10
15
20
25
c:
2
Fr. No.
Coincident increase in the concentration of irANP with Fi ure 1. the of(3-H)-inulin by a reduction in atria1 distension in The changes in the concentration of perfused rabbit atria (A). that of the (3-H)-inulin correlated uell with irANP are The concentration of irANP in terms of ECS ;;;;;located (inset): translocated Increased by a reduction in atria1 distension (6). Each point represents a P-min period. Atria1 perfusion rate was maintained at 0.25 ml/min. ECS; extracellular space. Fr. No.; fraction number.
-l+i--
426
Vol.
172,
No.
2,
BIOCHEMICAL
1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(A) 80
t P
I -2
(7
R
min
t
-I
2 min
2 min
0 0
02
200
Distension-
400 Reduction
Volume,
pl
we. &. re
ucfion
in
Fr. No
Dependence atria1 Rapid
perfusates samples volume concentration increased perfusion
were for 2 contained by rate
secretion
was of was
(Fig
1
irANP
in
in
As of
lumen
in increases
2).
in
ECS
fluid from
on
the 1.
Figure
figure
1
the
unexpectedly
concentration
experiments. of
the
the of
concentration by
ECS
reduction
fluid
secretion
and
of
a
into
changes
As irANP
in
translocated
volume
rapid
427
the
B
of
to
concentration
increase
increased
translocation
of
an
(3-H)-inulin
translocated
series the
in of
in
proportion An
another
that
shown
fluid
in
Increase
with
The
was (Fig
observed
ECS
distension.
distension
A,
well
terms
of derived
with
marker.
inset).
atria1
atria1
coincidently
ECS
correlated A,
translocation Data were
increases in the concentration of irANP and (3by a reduction in atria1 distension (A) Atria1 collected at 15 set intervals for 2 min (serial 8 min) at the reduction in atria1 distension. The in the dead space was subtracted. The of irANP in terms of ECS fluid translocated a reduction in atria1 distension (B). Atria1 was maintained at 1.25 ml/min.
observed
translocation irANP
of the distension.
in of
shown the
the atria1
irANP in
Figure
translocation
was 3
Vol.
172,
No.
2, 1990
BIOCHEMICALANDBIOPHYSICALRESEARCH
Pro-ANP
AP III
(A)
COMMUNICATIONS
i
1 /-
_&
.2F1.0 /-
/-
---
1.5
1
_/---
Fr. NO.
40
Reverse-phase HPLC basal (A) and were collected of atriopeptin
4. -aTFs bisten peak respec
of
in
tively.
ECS
less
fluid
than irANP
caused 15
volume
ECS
the of
concentration increased
coincidently
reduction
in
the
ECS
of
irANP
from
it
at
the
confirmed
fluid
was
translocated in the
of
(Fig
perfusate
obtained
mainly
low
of
baseline
3
The at
molecular
well
the
perfusate
The
translocated secretion
by
molecular in
irANP (Fig
a
profiles
reduction
weight
with
inset).
A,
irANP
6).
the
distension.
fluid
in 3
in
correlated
ECS
increase
increase
atria1
(Fig
terms
increase
the
the in
irANP
occurred
that
reduction
of
Atria1 in atria1 icate the pro-ANP,
5.
distension
with
distension
those
atria1
coincidently
with
atria1
in was
concentration
showed
different
again
fluid
15-set-collected
distension
reduction
occurred of
in
a
Here
secretion
Increase
of
by
sec.
translocation
the
profiles of the perfusate 15 set after reduction separately. Arrows ind III (AP III) and
and
atria1 were
not
4).
DISCUSSION To secretion
the
best of
of irANP
our with
this
knowledge, the
coincident
428
is
the translocation
first
to
observe of
ECS
the fluid
Vol.
172,
No.
2, 1990
the
and
very
protocol
BIOCHEMICALANDBIOPHYSICALRESEARCH
rapid
to
equilibrate
technique
is
+- 1.97
ml
EDTA
space
as
present
irANP
is
secretion
of
(11-13).
A
with
a
consistent point
clear
at
a
in
with
time
of
at
the
concentration (
with
Taken is
a
atria1
in in
increased.
the
the in
the
increase
ECS
fluid
not the noteworthy
the
increase
of
the
atria1
lumen. was
in
the
concentration
ECS
by
irANP an
by
but In
also any
accompanied
by case,
by In
an some
concentration was
of not
of
a
increase
(3-H)-inulin.
translocated
429
in
distension,
secretion
myocytes
the
ECS
translocated
atria1
atria1
as
of
of
only
not
lumen
translocation
the
that
exact is
an
of
fluid
the
distension.
not
however,
are
atria1
in
the
of
observed
atria1
with
the
irANP
was
fluid
in
the
distension
step
caused
shown, ECS
ECS
coincident
of
that
though
is
into
into
translocation
volume
concentration
of is
the
fluid
Increase
the
in
of
results
the
a
that
ECS
first
into
reduction
concentration
data
terms It
reduction
ECS
the
experiments
the
into
of was
distension
of
increase
finding
a
in
secretion
atria
distension.
the
the on
release
increase
at
irANP
shown
present
as
with
)
irANP
that
translocation
irANP
terms
with
in irANP
the
in
irANP
clearly
Even
ECS
of
atria1
suggested
reduction
secretion
in
the
values (51-Cr)-
atria1
The
the
the
laboratory
of
was
the
of
(11-13).
into
reduction
dependent
is
data
and
the
in
fluid
atrium
than
of
reduction
The
repetitive
the
was
this
distension.
coincidently
of
in
a
occurs
together
fluid
in
release
the
translocation
ECS
(3-H)-inulin
secretion
it
ANP
of
measured
the
previous
the
step
fluid
atria1
the
present,
final
with of
for
less
step
claimed
translocation
a
SE)
new
a
marker.
with
+
It
final
the
been
occurs
reduction
but (15).
the
presented
of
mean
(n=6, space
with
has ANP
atria
suitable
previously
related It
wt
that
have
space)
inulin
experiment
atrium.
the
wet
the
reported
closely
the
and
g
with
of
We
((51-Cr)-EDTA
HZO/lOO
comparable
the
ECS
irANP.
ECS
simple
The
25.80
of the
very
stimulations.
are
secretion
COMMUNICATIONS
always irANP
in
Vol.
172,
No.
terms
of
Even
though
A,
BIOCHEMICALAND
ECS
fluid
the vs
the
above
less
concentration translocation
irANP
the
in
concentration. of the
released it into
passes is the
of fluid
it
(16),
with lumen ANP
into
atria1
the
hypothesis
ECS
atria1
by
by
irANP
a
fluid
translocated.
dilute
of
In
a it
to
atria1
a
very
has the
postulate
irANP
micromolar
observe
rapid
been
known
molecules
that
sequential
the
of
range
the A
concentration
by an
and
ECS
the
non-
myocyte-related
may
that
one
is
pathways.
followed
lumen
other
fluid
Since
The
is,
ECS
to
to
extracellular
the
the
permeable
possible
caused
That
translocated.
lumen.
the
is
into the
is
B). channels
fluid
the
is
2
ECS
enough
the
and
of
the in
interesting
through
atria1
be
endothelium
consistent
release ECS
Da
the
that
may
into
endocardial
40,000
proposed
is
irANP
of
6
of
the
1
translocated
spaces. and
(Fig
distension,
functional
secretion
of
terms
ECS
It
secretion
terms
in is
it
released
the
40
rig/ml)-fold
fluid
translocation
both
1.2
1
two
space
in
ECS
endocardial
the
increased.
atria1
(Fig
non-myocyte-related
of context,
the
is
be by
vs in
of
there
Only
not
58.2
increase
and
irANP of
concentration
terms
that
increase of
A,
COMMUNICATIONS
increased
reduction
myocyte-related
may
may
was
2 a
lo-fold
space.
fluid
(Fig
in
pericardial
myocyte-related
to
than
the
through
that
irANP
or
irANP
by
suggests the
this
49
secretion
situation
ECS
to
of
connecting
of
rig/ml)
concentration in
may
concentration
I.8
BIOPHYSICALRESEARCH
translocated
baseline
resulted
is
1990
the
72.0
above the
2,
up
the In
irANP summary,
secretion
of
ANP
mechanism
of
the
rapid
translocation
of
contraction.
ACKNOWLEDGMENTS Yonsei thank Prof. Doo Hee Kang, We for his critical suggestions Medicine, Seoul, was supported by grants from This work Engineering Foundation and the Ministry of
University for the Korea Education,
the
College experiment. Science Korea.
REFERENCES 1 . 2.
Dietz, Lang, tt.,
and
J.R. R.E., Unger,
(1984) Thoelken, T.H.
Am.
J. H.,
(19U5)
Ptrysiol. Ganten, Nature
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247, D., 314,
R1093-R1096. Luft, F.C., 264-266.
Ruskoaho,
of and
Vol.
3. 4. 5. 6. 7.
a. 9.
10. 11. 12. 13. 14. 15. 16.
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No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Ruskoaho, H., Thelken, H., and Lang, R.E. (1986) Pflugers Arch. 407, 170-174. Katsube, N., Schwartz, O., and Needleman, P. (1985) Biochem. Biophys. Res. Commun. 133, 937-944. Ledsome, J.R., Wilson, N., Courneya, C.A., and Rankin, A.J. 1985) Can. J. Physiol. Pharmacol. 63, 739-742. E.L., Gutkowska, J., Kuchel, O., Cantin, M., and i chiffrin, Genest, J. (1985) N Engl. J. Med. 312, 1196-1197. Gutkowska J Bourassa, M., Roy, O., Thibault, G., Garcia, R Cantin &I, and Genest, J. (1985) Biochem. Biophys. Res. Co,imun. 128: 1350-1357. Schiebinger, R.J., and Linden, J. (1986) Am. J. Physiol. 251, H1095-H1099. Rankin, A.J., Courneya, C.A., Wilson, N., and Ledsome, J.R. (1986) Life Sci. 38, 1951-1957. Bilder, G.E., Siegl, P.K.S., Schofield, T.L., and Friedman, P.A. (1989) Circ. Res. 64, 799-805. Cho, ‘K-W.; Seul, K.H.,. Ryu, H., Kim, S.H., and Koh, G.Y. (1988) Regul. Peptides 22, 333-345. Cho, K.W., Seul, K.H., Kim, S.H., Ryu, H., Seul, K.M., and Koh, G.Y. (1988) Biochem. Biophys. Res. Commun. 153, 811-817. Seul, K.H., Kim, S.H., Seul, K.M., Ryu, H,, and Cho, K.W., Koh, G.Y. (1989) J. Hypertens. 7, 371-375. Cho, K.W., Kim, S.H., Koh, G.Y., and Seul, K.H. (1988) J. Exp. Zool. 247, 139-145. Poole-Wilson, P.A., and Cameron, I.R. (1975) Am. J. Physiol. 229, 1299-1304. Anversa, P., Giacomelli, F., Wiener, J., and Spiro, 0. (1973) Lab. Invest. 28, 728-734.
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