Vol. 284, Issue 1, R31-R40, January 2003
Regulation of ANP secretion by cardiac
Na+/Ca2+ exchanger using a new controlled
atrial model
Kyung Hwan
Seul2,
Jeong Hee
Han1,
Keum Yee
Kang2,
Sung Zoo
Kim1, and
Suhn Hee
Kim1
1 Department of Physiology, Medical School,
Institute for Medical Sciences, Chonbuk National University, Jeonju
561-180, Republic of Korea; and
2 Section of Pediatric Hematology/Oncology,
University of Chicago, Chicago, Illinois 60637-1470
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ABSTRACT |
The myocardial
interstitium is important in regulating cardiac function. Between the
atrial lumen and the pericardial space are transmural pathways, and
movement of interstitial fluid (ISF) through these pathways is one of
the main driving forces regulating translocation of substances from the
interstitium into the blood. To define how ISF translocation from the
interstitial space into the luminal space is regulated by each
component of atrial hemodynamics, we devised a new rabbit atrial model
in which each physical parameter could be controlled independently.
Using this system, we also defined the physiological role of the
cardiac Na+/Ca2+ exchanger on secretion of
atrial natriuretic peptide (ANP) by depletion of extracellular
Na+ ([Na+]o). Increases in stroke
volume and atrial end-systolic volume increased ISF translocation and
ANP secretion. However, an increase in atrial rate did not influence
ISF translocation but, rather, increased ANP secretion. Gradual
depletion of [Na+]o caused gradual increases
in ANP secretion and intracellular Ca2+
([Ca2+]i), which were blocked in the presence
of Ca2+-free buffer and Ni2+, but not in the
presence of KB-R7943, diltiazem, mibefradil, caffeine, or monensin.
Amiloride and its analog blocked an increase in ANP secretion but not
an increase in [Ca2+]i by
[Na+]o depletion. Therefore, we suggest that
ANP secretion and ISF translocation may be differently controlled by
each physical factor. These results also suggest that the increase in
ANP secretion in response to [Na+]o depletion
may involve inhibition of Na+/Ca2+ and
Na+/H+ exchangers but not an increase in
[Ca2+]i.
sodium-hydrogen exchanger; interstitial fluid; translocation; atrium; sodium; calcium
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INTRODUCTION |
THE MYOCARDIAL
INTERSTITIUM plays an important role in the regulation of cardiac
function. In the intermediate space, nutrients, bioactive substances,
and metabolites are exchanged between cardiac myocytes and the
bloodstream. Fluid exchange between the interstitial space and blood
depends on the balance of hydraulic and oncotic pressures between these
compartments. The hydraulic pressure caused by the continuous beating
of the heart exerts a dynamic pressure on the interstitial space, and
this pressure is dependent on myocardial hemodynamics. It is well known
that the heart maintains its water content within a narrow critical
range. The heart is supplied by a rich lymphatic network, and the
interstitial fluid (ISF) of the heart is washed down directly into the
atrial lumen. Therefore, the interstitial space is an important route
for the regulation of interstitium, venous flow, and lymphatics. We
have reported the existence of transmural pathways between the atrial
lumen and the pericardial space (5). ISF movement through
these pathways is one of the main driving forces in the secretion of
atrial natriuretic peptide (ANP) from the interstitial space into the
atrial lumen (5). Various models have been proposed for
fluid transport in the myocardium, but some questions remain unanswered
(5). It is uncertain how ISF translocation from the
interstitial space into the luminal space is regulated by each
component of atrial hemodynamics. Using isolated perfused rabbit atria,
we developed a new method whereby specific physical parameters, such as
atrial rate, stroke volume (SV), and atrial end-systolic volume (AESV), can be controlled. Using this perfusion model, we attempted to elucidate the role of each physical parameter on the translocation of
ISF and ANP.
In cardiac muscle cells, three primary transport systems are
responsible for intracellular Ca2+ homeostasis (2,
11, 24). In particular, the Na+/Ca2+
exchanger (NCX) is the primary mechanism responsible for removing Ca2+ from the cells (17). The NCX can function
in "forward mode" or "reverse mode" (15). The
direction of this transport is governed by the magnitude and
orientation of the transmembrane electrical and chemical gradients for
Na+ and Ca2+ (17). NCX activity
directly influences intracellular Ca2+ concentration
([Ca2+]i), which causes cardiac hemodynamic
change followed by the secretion of ANP. However, the effect of
[Ca2+]i on ANP secretion is controversial,
despite extensive studies (6, 9, 14, 19-21).
Additionally, there are few reports about the relationship between NCX
and ANP secretion, possibly because of difficulties in
dissociating ANP secretion from hemodynamic changes. Therefore,
the aim of the present study was to characterize the physiological role
of cardiac NCX in ANP secretion by depleting the concentration of
extracellular Na+ ([Na+]o). The
effects of gradual decreases in [Na+]o on ANP
secretion and [Ca2+]i were studied with and
without KB-R7943, diltiazem, mibefradil, caffeine, Ni2+,
amiloride, 5-(N-methyl-N-isobutyl)-amiloride
(MIA), and monensin.
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MATERIALS AND METHODS |
Animals
New Zealand White rabbits, weighing ~1.5 kg, were used. All
animal experimentation was conducted in accord with the American Association for the Accreditation of Laboratory Animal Care
(1).
Isolated Perfused Atrial Preparation
New Zealand White rabbits were anesthetized with thiopental
sodium and killed by exsanguination. A perfusion catheter was inserted
into the atrium by a method described previously, with some
modifications (5, 7, 23). Briefly, the heart was rapidly
removed and placed in oxygenated saline. The right atrium was dissected
along the tricuspid valve annulus, and then the area above the
sinoatrial node was cut away. A double-barreled cannula (5 mm OD, 4 mm
ID) was inserted into the atrium and secured with ligatures. The inner
barrel (1.5 mm ID) was used for outflow, and the outer barrel was used
for inflow of perfusate. The cannulated atrium was immediately
transferred and fitted into a constant-temperature (36°C) organ
chamber containing HEPES buffer (118 mM NaCl, 4.7 mM KCl, 2.5 mM
CaCl2, 1.2 mM KH2PO4, 1.2 mM
MgSO4, 10 mM HEPES, 10 mM glucose, and 0.1% BSA). Air
bubbles trapped in the pericardial space were removed through
polyethylene tubing located on the water-tight silicone rubber cap of
the organ chamber. Atrial luminal and pericardial pressures were
recorded on a physiograph (model MK IV, Narco Bio-Systems, Houston, TX)
via a pressure transducer throughout the experiment. Perfusate was
continuously oxygenated and infused into the atrial lumen with a
peristaltic pump. The chamber space was oxygenated through silicone
tubing coils located inside the organ chamber.
Control of Physical Parameters
The experimental system was made by some modifications of our
nonbeating model (8, 23) and consisted of three major
components: a perfusion system, a rate control unit, and an SV control
unit (Fig. 1). The circular movement of
the motor (Con-Torque, Eberbach, Ann Arbor, MI) was converted to the
linear movement of the SV syringe through a ball joint. The chamber
space was completely closed, except for the connection to the SV
control unit. Thus the repetitive, push-and-pull motion of chamber
fluid generated a positive-and-negative transmural pressure, which
caused passive, cyclic changes in atrial volume. The amount of SV was
regulated by adjusting distance. One rotation of the adjustment knob of the SV control motor was 15 µl of SV. Flow through the atrial lumen
was maintained only in the forward direction by placing one-way check
balls on inflow and outflow lines. Basal distension of the atrium
(AESV) was adjusted by pull-and-fix of chamber fluid by means of an
AESV syringe.
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Fig. 1.
Schematic illustration of atrial chamber (top)
and pressure trace on atrial and chamber sides in isolated perfused
controlled atria (bottom). Phase A, control of
stroke volume (SV, hatched area) by cyclic movement of SV syringe at
atrial end-systolic volume (AESV) of 0 µl (x). Phase
B, control of SV at basal distension (black area) by adjustment of
AESV syringe from x to y. Bottom:
pressure changes in atrial and chamber sides by cyclic movement of SV
syringe in phases A and B. Arrow, direction of
flow of perfusion buffer.
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Experimental Protocols
The atria were perfused for 30 min to stabilize ANP secretion
and equilibrate the extracellular space with a marker at a steady state. 51Cr-EDTA (10 µCi/ml) was introduced into the
pericardial fluid.
Protocol 1: effect of SV on ISF translocation and ANP secretion.
SV was changed from 65 to 110 and 150 µl/cycle for 10 min in a
stepwise pattern and returned to 65 µl/cycle. AESV was fixed at 0 µl for all atria used in this protocol. Thus the ejection fraction
(EF) was always 100%, and the atrial end-diastolic volume (AEDV) was
the same as SV. Atrial rate was fixed at 30 cycles/min (AR30).
Protocol 2: effect of basal volume (AESV) on ISF translocation
and ANP secretion.
SV was fixed at 60 µl for all atria used in this protocol, and AESV
was increased from 0 µl to 55 and 110 µl by withdrawal of
pericardial fluid using the AESV syringe. AESV/AEDV changed from 0/60
µl (EF = 100%), 55/115 µl (EF = 52.2%), and 110/170 µl (EF = 35.3%) for 10 min in a stepwise pattern and returned to 0/60 µl. Atrial output was constant, because atrial rate (AR30) and SV (60 µl) were fixed.
Protocol 3: effect of atrial rate on ISF translocation and ANP
secretion.
Atrial rate was changed from 30 to 60, 120, 200, and 280 cycles/min for
10 min in a stepwise pattern and returned to AR30 to check the effect
of the time sequence of the experiments. In some experiments, the order
of changing atrial rate was performed downward to rule out changes due
to the order of changes on the atrial rate. The other parameters were
fixed as follows: SV = 65 µl, AESV = 0 µl, and AEDV = 65 µl.
Protocol 4: effect of Na+ depletion
on ANP secretion.
Atrial rate and AESV were fixed at 23 cycles/min and 0 µl,
respectively. SV was gradually increased from 50 to 100 and 200 µl,
respectively. In another protocol, SV was also fixed at 100 µl. To
determine the effect of NCX on ANP secretion, the Na+
gradient between the extracellular and intracellular space was gradually decreased by extracellular Na+ depletion.
N-methyl-D-glucamine (NMG) or LiCl was used
instead of NaCl. The atrium was perfused with HEPES buffer containing 140 mM [Na+]o and stabilized for 30 min. A
sample was collected every 2 min for 10 min as a control, and then the
buffer on the luminal and pericardial sides was changed to HEPES buffer
containing 70 mM [Na+]o. After 10 min of
stabilization, samples were collected for 10 min, and then buffer was
continuously changed to HEPES buffer containing 28, 14, and 0 mM
[Na+]o, continuing the same protocol as
above. To test the effect on Na+ depletion-induced ANP
secretion, samples were pretreated with KR-B7943 (10
5 M),
diltiazem (106 M), mibefradil (105 M),
caffeine (103 M), Ni2+ (103 M),
amiloride (104 M), MIA (106 M), or monensin
(107 M).
Measurement of ISF Translocation
To estimate ISF translocation, the transmural atrial clearance
of 51Cr-EDTA was measured with a gamma counting system
(Auto Gamma 500 C, Packard, Downers Grove, IL), as described previously
(5). Radioactivity in the atrial perfusate and pericardial
buffer solution was measured, and the amount of ISF translocated
through the atrial wall was calculated as follows
Total ISF translocation was expressed as microliters of ISF per
gram of tissue per minute after calculation of ISF translocated by
measuring 51Cr-EDTA in perfusate for every 2-min fraction.
Radioimmunoassay of Immunoreactive ANP
Immunoreactive ANP in the perfusate was directly measured by
radioimmunoassay, as described previously (5). The
radioimmunoassay was performed on the same day of the experiment, and
all samples in an experiment were analyzed in a single assay.
Nonspecific binding was <3%. The 50% intercept was at 26.6 ± 2.9 pg/tube. The molar concentrations of immunoreactive ANP released
were calculated as follows
Measurement of
[Ca2+]i in Single Atrial
Myocytes
Single atrial myocytes were isolated according to a previously
described technique with minor modifications (18). Changes in the [Ca2+]i of single atrial myocytes were
measured with a fluorescence digital imaging microscopy system (Atto
Instruments, Rockville, MD), as described elsewhere (18).
Isolated myocytes were incubated in HEPES buffer containing 2 µM fura
2-AM (Molecular Probes, Eugene, OR) and 0.02% Pluronic F-127
(Molecular Probes) for 20 min at room temperature and then washed with
fresh HEPES buffer to remove extracellular dye. Cells were attached on
a perfusion chamber coated with matrix gel and placed on the stage of
an inverted fluorescence microscope (Axiovert 135, Karl Zeiss, Jena,
Germany) attached to an Attofluor digital fluorescence microscopy
system. Cells were stimulated at 1 Hz (60 V for 0.6 ms) and perfused
with HEPES buffer containing 1 mM Ca2+ for 5 min at 0.7 ml/min. Buffer was changed to Na+-depleted HEPES buffer.
Cells were imaged with excitation wavelengths of 338 and 380 nm and an
emission wavelength of 520 nm. The fluorescence images were captured
with an intensified charge-coupled device camera and analyzed with
Attofluor image processing software. Changes in
[Ca2+]i are presented as a ratio of
fluorescence at 340 nm to fluorescence at 380 nm. Background was
subtracted. Experiments were performed at room temperature.
Statistical Analysis
Values are means ± SE. Statistical assessment of the data
was made by Student's t-test or ANOVA followed by Duncan's
multiple range test when appropriate. Linear regressions were
calculated by the least-squares method. P < 0.05 was
taken as significant.
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RESULTS |
Effect of SV on ISF Translocation and ANP Secretion
To evaluate the effect of SV on ISF translocation and ANP
secretion, SV was changed from 65 to 110 and 150 µl/cycle at fixed atrial rate (AR30) and AESV (0 µl/cycle; Fig.
2A; n = 6).
When SV was increased from 65 to 150 µl/cycle and returned to 65 µl/cycle, ANP secretion and ISF translocation were proportionately
increased and returned to basal level. Figure 2B
(top) shows the average of each parameter in terms of SV.
The ISF translocation and ANP secretion increased in proportion to SV.
Therefore, ISF ANP concentration, which represents the release rate of
ANP from atrial myocytes, was increased by increasing SV from 65 to 110 µl/cycle.
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Fig. 2.
Effect of SV on interstitial fluid (ISF) translocation and atrial
natriuretic peptide (ANP) release. A: by increasing SV at
fixed atrial rate [30 cycles/min (AR30)] and AESV (0 µl), ISF
translocation and ANP secretion increased. B: average of ANP
secretion and ISF translocation in terms of SV at fixed atrial rate
(AR30; top) and ANP secretion and ISF translocation in terms
of SV at AR30 and 180 cycles/min (AR180; bottom). Increases
in SV caused proportional increases in ISF translocation and ANP
secretion. Increase in ANP secretion in terms of SV was significantly
attenuated at higher atrial rates. Significantly different from other
group: # P < 0.05 and ## P < 0.01. Significantly different from corresponding value:
** P < 0.01 and *** P < 0.005.
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To evaluate possible differences of ISF response to SV at different
atrial rates, we performed two additional series of experiments at AR30
and 180 cycles/min (AR180). There were no significant differences in
ISF translocation between AR30 and AR180 (45.18 ± 6.98 vs.
39.11 ± 14.24 µl/min at 65 µl/cycle, n = 6).
However, ANP secretion was significantly higher at AR180 than that at
AR30 (179.09 ± 29.17 vs. 54.08 ± 7.74 pg/min at 65 µl/cycle, P < 0.001). Figure 2B
(bottom) shows the comparison of responsiveness of ISF translocation and ANP secretion in terms of SV between AR30 and AR180. The response of ISF translocation to low SV was similar in both
groups, but the response to higher SV was accentuated at AR180.
However, the response of ANP secretion to SV was markedly attenuated at AR180.
Effect of AESV on ISF Translocation and ANP Secretion
In these experiments, AESV/AEDV was changed from 0/60 µl
(EF = 100%), 55/115 µl (EF = 52.2%), and 110/170 µl
(EF = 35.3%) at fixed SV (65 µl/cycle) and atrial rate (AR30,
n = 6). Therefore, atrial output was maintained
constantly. When AESV was increased from 0 to 55 µl, neither ISF
translocation nor ANP secretion changed (Fig.
3). By increasing AESV from 55 to 110 µl, ISF translocation and ANP secretion increased significantly. When
AESV was decreased from 110 to 0 µl, ISF translocation and ANP
secretion increased markedly, but ANP concentration did not change
(fraction 16 in Fig. 3A). The response of ISF
translocation and ANP secretion to AESV at AR180 (n = 6) was similar to that at AR30 (Fig. 3B, bottom).
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Fig. 3.
Effect of AESV on ISF translocation and ANP release. A:
by increasing AESV to 110 µl at fixed SV and atrial rate, ISF
translocation and ANP secretion increased. B: average of ANP
secretion and ISF translocation in terms of AESV at fixed atrial rate
(AR30; top) and ANP secretion and ISF translocation in terms
of AESV at AR30 and AR180 (bottom). AESV was adjusted to 0, 55, and 110 µl, and then ejection fraction (ratio of AESV to atrial
end-diastolic volume) became 100%, 52.2%, and 35.3%, respectively.
ISF translocation and ANP secretion were increased by increasing AESV
to 110 µl. Response to ISF translocation and ANP secretion by
changing AESV was similar for AR30 and AR180. ** Significantly
different from corresponding value, P < 0.01.
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Effect of Atrial Rate on ISF Translocation and ANP Secretion
Surprisingly, total ISF translocation remained relatively
constant over the range of atrial rates. ANP secretion was markedly increased from AR30 to 120 cycles/min and then increased slowly (Fig.
4; n = 6). Rate
dependency is more prominent in ANP secretion than in ISF
translocation. Therefore, ISF ANP concentration gradually increased
with increasing atrial rate and reached a peak at 120 cycles/min.
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Fig. 4.
Effect of atrial rate on ISF translocation and ANP secretion. SV
(55 µl) and AESV (0 µl) were fixed. ANP secretion was markedly
increased by increasing atrial rate to 120 cycles/min. At higher atrial
rates, ANP increased slowly. ISF translocation did not change
significantly. Significantly different from corresponding value:
* P < 0.05 and ** P < 0.01.
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Figure 5 shows the relationship between
atrial output, ANP secretion, and ISF translocation in groups
with changing SV and atrial rate. Atrial output was obtained by
multiplying SV by atrial rate. Atrial output is positively correlated
with ANP secretion in the SV-changed group (Fig. 5A; y = 4.76x + 235, r = 0.55, P < 0.001) and the atrial rate-changed group
(y = 7.34x + 126.12, r = 0.79, P < 0.001). A positive correlation between
atrial rate and ISF translocation was observed in the SV-changed group
(Fig. 5B; y = 1.72x + 35.5, r = 0.49, P < 0.05) but not in the
atrial rate-changed group (y = 1.25x + 60.9, r = 0.3, P = 0.2). There were also close relationships between
extracellular fluid translocation and ANP secretion in both groups
(Fig. 5C).
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Fig. 5.
Relationship between atrial output and other parameters.
AR, atrial rate. A: atrial output and ANP secretion.
B: atrial output and ISF translocation. C: ISF
translocation and ANP secretion.
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Effect of Gradual Decreases in
[Na+]o on ANP Secretion
To determine the effect of NCX on ANP secretion using this model,
[Na+]o was decreased from 140 to 0 mM at
fixed atrial rate (23 cycles/min) and AESV (0 µl/cycle), while SV was
gradually increased from 50 to 100 and 200 µl (Fig.
6A). ANP secretion was
markedly increased by the replacement of extracellular Na+
with NMG; ANP was particularly accentuated at higher SV
(n = 9). An increase in ANP secretion by
Na+ replacement with NMG was similar to that by
Na+ replacement with Li+ (Fig. 6B;
n = 10). This effect was blocked by
Ca2+-free conditions (Fig. 6B; n = 5), but not by pretreatment with mibefradil (105 M,
n = 8) or diltiazem (106 M,
n = 5; Fig. 6C).
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Fig. 6.
Effect of extracellular Na+ depletion on ANP
secretion at fixed atrial rate (AR30) and AESV (0 µl). A:
SV was changed from 50 µl to 100 and 200 µl before (stippled bars)
and after (open bars) exposure to Na+-free buffer.
B: ANP secretion was increased in response to
Na+ replacement by
N-methyl-D-glucamine or Li+, which
was blocked in Ca2+-free buffer (NMG/Ca free).
C: Na+ depletion-induced accentuation of ANP
secretion was not changed by 105 M mibefradil (Mibe) or
106 M diltiazem (Dilt). Dotted line, perfusion of atria
with Na+-free buffer. Cont, control.
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[Na+]o was gradually decreased from 140 to
70, 28, 14, and 0 mM at fixed atrial rate (23 cycles/min), SV (100 µl/cycle), and AESV (0 µl/cycle), and ANP secretion was measured
(Fig. 7A).
We also measured the ratio of increase in ANP
secretion by Na+ depletion to 140 mM
[Na+]o (Fig. 7B). The secretion of
ANP was markedly increased by decreasing [Na+]o to 70 mM and then gradually increased
further as [Na+]o decreased to 0 mM.
Increases in ANP secretion by Na+ depletion from 140 mM to
70, 28, 14, and 0 mM were 1.41 ± 0.20-, 1.71 ± 0.25-, 2.29 ± 0.46-, and 3.04 ± 0.51-fold, respectively (Fig.
7B).
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Fig. 7.
Effect of gradual decrease in extracellular
Na+ concentration ([Na+]o) on ANP
secretion. Atrial rate (AR30), AESV (0 µl), and SV (100 µl) were
fixed. A: ANP secretion was gradually increased by
decreasing [Na+]o from 140 mM to 70, 28, 14, and 0 mM. B-D: modification of accentuation of ANP
secretion by gradual decreases in [Na+]o by
treatment with 106 M
5-(N-methyl-N-isobutyl)-amiloride (MIA),
103 M, Ni2+, 106 M monensin
(MONEN), 104 M amiloride, 104 M caffeine,
or mibefradil + diltiazem. Pretreatment with MIA,
Ni2+, and amiloride markedly attenuated an accentuation of
ANP secretion by Na+ depletion; pretreatment with monensin,
mibefradil + diltiazem, and caffeine did not. Significantly
different from control: * P < 0.05 and
** P < 0.01.
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To define the mechanism involved in [Na+]o
depletion-induced ANP secretion, we used KB-R7943 and Ni2+,
an NCX blocker. KB-R7943, a selective inhibitor of the reverse mode of
NCX, did not cause any significant changes in
[Na+]o depletion-induced accentuation of ANP
secretion (105 M, n = 5). Increases in
ANP secretion by [Na+]o depletion were
completely blocked by pretreatment with a high dose of Ni2+
(103 M, n = 5; Fig. 7B). An
increase in ANP secretion by [Na+]o depletion
was partially or completely blocked by pretreatment with amiloride
(104 M, n = 5) or MIA (106
M, n = 6), which are known
Na+/H+ exchange (NHX) inhibitors (Fig. 7,
B and C). Amiloride is also an Na+
channel blocker. Therefore, we used an Na+ channel
ionophore monensin, which did not show any significant effect on
[Na+]o depletion-induced ANP secretion
(106 M, n = 5; Fig. 7C).
Pretreatment with diltiazem (106 M) and mibefradil
(105 M) together did not significantly change
[Na+]o depletion-induced accentuation of ANP
secretion (Fig. 7D; n = 5). Caffeine
(104 M) tended to attenuate the response of ANP secretion
by [Na+]o depletion, but not to a significant
extent (Fig. 7D; n = 6). KB-R7943,
Ni2+, MIA, amiloride, and monensin alone did not cause any
significant changes in ANP secretion in 140 mM
[Na+]o (13.25 ± 3.45, 15.20 ± 2.37, 11.42 ± 1.97, 15.83 ± 5.90, and 8.17 ± 0.41 vs.
12.12 ± 1.85 ng · min1 · g1).
Effect of Gradual Decreases in
[Na+]o on
[Ca2+]i
Decreasing [Na+]o from 140 to 70 mM did
not cause any significant changes in [Ca2+]i
in single beating atrial myocytes. However, gradual decreases in
[Na+]o from 70 to 42, 28, 14, and 0 mM caused
increases in [Ca2+]i, which were abolished in
Ca2+-free conditions (Fig.
8A) and in the presence of
Ni2+. However, pretreatment with MIA, diltiazem, and
mibefradil or caffeine did not affect [Na+]o
depletion-induced increases in [Ca2+]i (Fig.
8B).
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Fig. 8.
Changes in intracellular Ca2+ concentration
by extracellular Na+ depletion in beating rabbit atrial
myocytes. Extracellular Na+ depletion caused marked
increases in intracellular Ca2+ concentration, which was
completely blocked by pretreatment with 102 M
Ni2+ (A), but not by 106 M
5-(N-methyl-N-isobutyl)-amiloride,
104 M caffeine, or mibefradil + diltiazem
(B). Significantly different from control group:
# P < 0.05 and ## P < 0.01.
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DISCUSSION |
The myocardial interstitium plays an important role in the
regulation of cardiac function under physiological and pathological conditions. It is believed that exchanges of substances between atrial
tissue and blood are dependent on their metabolic rates and the
transport rate of ISF. Using a new experimental model that can control
the physical parameters of atrial hemodynamics, we showed that the
translocation of ISF from atrial tissue is differently regulated by
each physical factor. The translocation of ISF increased more by
increasing SV than by increasing atrial rate or basal volume.
Interestingly, the translocation of ISF was maintained relatively
constant across the range of atrial rates by abruptly decreasing the
amount of ISF translocation in each cycle. The maintenance of total ISF
translocation, regardless of atrial rates, appears to be the ideal way
for the atrium to maintain a constant interstitial environment.
However, ISF translocation in terms of SV was decreased by increasing
SV, similar to ISF translocation in terms of atrial rate by increasing
atrial rate. These decrements caused an attenuation of total ISF
translocation per unit time by changing SV and atrial rate. These
results suggest that total ISF movement across the atrial wall through
paracellular pathways may be more dependent on cardiac output than on
atrial rate or SV.
Degrees of contraction (AESV) and distension (AEDV) of atrial muscles
are different in physiological and pathological conditions of the
heart. The contraction of cardiac muscle is severely disturbed in the
distended atrium of congestive heart failure. There was no accumulation
of atrial ISF when atria were working with 100% EF, but significant
amounts of ISF accumulated in the interstitial space when EF was
severely disturbed. This stagnated ISF was translocated into the atrial
lumen by the release of basal volume distension (shortening of muscle
length), as clearly shown in fraction 16 of Fig.
3A. The accumulation of ISF by atrial distension was
dependent on the degree of distension volume (2, 24), but
not on the duration of distension. These data suggest that significant
amounts of metabolites may accumulate in the ISF during atrial
fibrillation or congestive heart failure, where atria are severely
distended and SV is decreased SV (decreased EF). These metabolites may
further aggravate atrial function. Our data clearly show that changes of muscle length and frequency play a unique role in ISF translocation during atrial cycles. It is not clear whether paracellular pathways in
cardiac atria are important for the exchange of molecules through ISF translocation.
The patterns of ANP release observed in this model provided interesting
facts about the exchange patterns of molecules between atrial tissue
and blood. First, ANP molecules are released from myocytes into the
interstitial space in a unique pattern by each physical factor, and
then these molecules are transported into the atrial lumen
concomitantly with unique patterns of ISF translocation for each
physical factor (5). One example is the relationship between heart rate and plasma ANP concentration. Tachycardia generally raises plasma ANP concentration (18, 22), but this
relationship was not linear over all ranges of heart rate and showed a
significantly blunted response at higher heart rate. There are many
hypotheses related to blunted response of increased plasma ANP to heart
rate, such as depressed secretion from myocytes, increased metabolism, and concomitant changes of other physical parameters that can inhibit
ANP secretion. Our study clearly showed that a lesser increase in
plasma ANP in tachycardia resulted from the function of unique
characteristics of ANP release from myocytes and of ISF regulation by
atrial rate. Patterns of decreasing unit ISF translocation caused by
atrial rate and SV resulted in a lower release of total ANP into the
atrial lumen. The amounts of ANP secreted into the atrial lumen may
show linear patterns if unit ISF translocation was not decreased. This
means that plasma ANP concentration is under fine control by each
hemodynamic factor. This fine control provides maximum response when
atrial rate and SV are increased (e.g., exercise), moderate response
when only one factor is increased (e.g., palpitation), and minimal
response when one factor is increased but the other is decreased (e.g., hemorrhagic hypotension). Therefore, we believe that this kind of ANP
regulation may help atria differentiate various stimuli and may help
properly regulate the body fluid through the blood vessels and kidneys.
Using this model to characterize the physiological role of cardiac NCX
on ANP secretion, we studied the effects of gradual decreases in
[Na+]o on ANP secretion under conditions of
fixed atrial rate, SV, and AESV. A decrease in
[Na+]o to 70 mM caused a twofold increase in
ANP secretion without a change in [Ca2+]i.
Further decreases in [Na+]o to 28, 14, and 0 mM caused gradual increases in [Ca2+]i and
ANP secretion, which were blocked under Ca2+-free
conditions. These data suggest that [Na+]o
depletion-induced increases in ANP secretion may be a direct effect,
rather than due to changes in SV and AESV. To determine the mechanism
involved in [Na+]o depletion-induced increase
in ANP secretion, Ca2+ channel modulators such as diltiazem
and/or mibefradil or caffeine were used. However, they did not show any
significant effects on [Na+]o
depletion-induced increase in ANP secretion or on
[Ca2+]i. Therefore, we used KB-R7943, a
selective inhibitor of the reverse mode of NCX. KB-R7943 had no effect
on basal ANP secretion and accentuation of ANP secretion by
[Na+]o depletion, which caused an inhibition
of the forward mode of NCX. The reverse mode of NCX seems at least to
be not operational in the isolated controlled atria. Heavy metal ions
inhibit NCX current when externally applied (13, 15). In
particular, Ni2+ has been widely used in investigations of
NCX. Ni2+ inhibits nonspecific cation channels
(4). Hinde et al. (12) reported that a high
concentration of Ni2+ was sufficient to produce maximal
inhibition of NCX current in ventricular myocytes. Therefore, we used a
high dose of Ni2+ as an NCX inhibitor to block the effect
of [Na+]o depletion on ANP secretion.
Ni2+ at 103 M completely blocked the effect
of [Na+]o depletion, but at 104
M it did not. From these data, we suggest that an inhibition of NCX by
pretreatment with Ni2+ may block the
[Na+]o depletion-induced increase in ANP
secretion. However, it is difficult to explain why Ni2+ did
not show any significant changes in ANP secretion. If Ni2+
inhibits NCX activity, the secretion of ANP as well as
[Ca2+]i may be increased. However, because
Ni2+ also acts as a nonspecific cation channel inhibitor
(4) and Ca2+ channel blocker, its effects may
be due to actions at other sites.
To define whether [Na+]o depletion-induced
accentuation of ANP secretion may be related to NHX activity, we used
MIA. MIA, as well as amiloride, markedly attenuated the effect of
gradually decreased [Na+]o on ANP secretion.
Amiloride is an Na+ channel blocker as well as an NHX
inhibitor. To discriminate between the blocking effects of amiloride,
we used monensin, an Na+ channel ionophore. Monensin did
not cause any significant changes in [Na+]o
depletion-induced increase in ANP secretion. We expected that monensin
would cause a decreased Na+ gradient followed by inhibition
of NCX activity, whereas amiloride may cause an increased
Na+ gradient followed by activation of NCX activity.
However, monensin did not influence NCX activity. It has been reported
that intracellular acidification clearly inhibits outward NCX current
in guinea pig ventricular myocytes (10). Therefore, we
suggest that MIA or amiloride may inhibit NCX activity by intracellular
acidification through the inhibition of NHX activity.
A decrease in [Na+]o from 140 to 70 mM may
cause a decrease in Na+ gradient followed by a decrease in
NCX activity and an increase in [Ca2+]i. In
the present study, decreasing [Na+]o to 70 or
42 mM did not cause any significant changes in
[Ca2+]i in single beating atrial myocytes.
Further decreases in [Na+]o to 28, 14, and 0 mM caused gradual increases in [Ca2+]i. This
result can be explained as follows: another Ca2+ extrusion
mechanism, such as a Ca2+ pump, may be activated when NCX
activity is slightly inhibited by reduction of
[Na+]o to 70 mM, and then
[Ca2+]i may not be changed. However, a marked
inhibition in NCX activity by further decreases in
[Na+]o may cause an increase in
[Ca2+]i, even though the Ca2+
extrusion mechanism may be fully activated. Because there are few
specific blockers of NCX, manipulation of ion concentrations is
essential for identification of this transporter. To define the
mechanisms involved in [Na+]o
depletion-induced increase in ANP secretion and
[Ca2+]i, we used Ni2+ as an NCX
blocker. An increase in [Ca2+]i by
[Na+]o depletion was attenuated in the
presence of Ca2+-free buffer and Ni2+, but not
in the presence of MIA and Ca2+ channel modulators. These
results suggest that extracellular Ca2+ may be essential
for the [Na+]o depletion-induced increase in
[Ca2+]i through an Ni2+-sensitive
nonselective cation channel, but not through L- or T-type
Ca2+ channels. The relationship between increased ANP
secretion and [Ca2+]i by
[Na+]o depletion appears to be independent.
Increases in ANP secretion and [Ca2+]i by
[Na+]o depletion were blocked by
Ca2+-free buffer and Ni2+. MIA and amiloride
inhibited the response of ANP, but not Ca2+, to
[Na+]o depletion. However, diltiazem,
mibefradil, and caffeine did not affect either response.
In conclusion, we believe that this model may provide many benefits in
the study of the mechanism of atrial ANP secretion and ISF
translocation. These results also suggest that the increase in ANP
secretion in response to [Na+]o depletion may
partly involve inhibition of the NCX and NHX but may not involve an
increase in [Ca2+]i.
| |
ACKNOWLEDGEMENTS |
This study was supported by Korea Science and Engineering
Foundation Grant 941-0700-001-1 and Korea Health 21 Research and Development Project HMP-00-B-21400-0055 and HMP-01-PJ1-PG1-01CH06-0003 from the Ministry of Health and Welfare, Republic of Korea.
| |
FOOTNOTES |
Address for reprint requests and other correspondence:
S. H. Kim, Dept. of Physiology, Chonbuk National University
Medical School, 2-20 Keum-Am-Dong San, Jeonju 561-180, Republic of
Korea (E-mail:
shkim{at}moak.chonbuk.ac.kr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
September 5, 2002;10.1152/ajpregu.00408.2002
Received 9 July 2002; accepted in final form 4 September 2002.
| |
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