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1 Department of Physiology, Institute for Medical Sciences, Jeonbug National University Medical School, Jeonju 561-180; and 2 Department of Physiology, College of Oriental Medicine, Wonkwang University Medicinal Resources Research Center, Iksan 570-749, Korea
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Regulation of atrial release of
atrial natriuretic peptide (ANP) is coupled to changes in atrial
dynamics. However, the mechanism by which mechanical stretch controls
myocytic ANP release must be defined. The purpose of this study was to
define the mechanism by which cAMP controls myocytic ANP release in
perfused, beating rabbit atria. The cAMP-elevating agents forskolin and
3-isobutyl-1-methylxanthine (IBMX) inhibited myocytic ANP release. The
activation of adenylyl cyclase with forskolin inhibited ANP release,
which was a function of an increase in cAMP production. Inhibitors for
L-type Ca2+ channels and protein kinase A (PKA) attenuated
a minor portion of the forskolin-induced inhibition of ANP release.
Gö-6976 and KN-62, which are specific inhibitors for protein
kinase C-
and Ca2+/calmodulin kinase, respectively,
failed to modulate forskolin-induced inhibition of ANP release. The
nonspecific protein kinase inhibitor staurosporine blocked
forskolin-induced inhibition of ANP release in a dose-dependent manner.
Staurosporine but not nifedipine shifted the relationship between cAMP
and ANP release. Inhibitors for L-type Ca2+ channels and
PKA and staurosporine blocked forskolin-induced accentuation of atrial
dynamics. These results suggest that cAMP inhibits atrial myocytic
release of ANP via protein kinase-dependent and L-type
Ca2+-channel-dependent and -independent signaling pathways.
atrial natriuretic peptide; forskolin; phosphodiesterase; channels
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ENDOCRINE ATRIUM SYNTHESIZES and releases a family of natriuretic peptides including atrial natriuretic peptide (ANP) and brain natriuretic peptide (7, 19). C-type natriuretic peptide, a third member of the natriuretic peptide family (32) that is synthesized in the atrium, may have paracrine/autocrine function for the regulation of ANP release (17).
There have been reports on the variable modulators for the control of ANP release (23). However, the specific control mechanism for ANP release must be defined. The most prominent activator for atrial secretion of ANP has been shown to be the stretch and/or release of atrial wall (2, 10, 16). However, the intracellular mechanism responsible for the activation of ANP release by mechanical stimulation is unknown.
The potential roles of cyclic nucleotides and Ca2+ in the regulation of ANP release have been subjects of interest. Recently we found that both cGMP and Ca2+ are negative regulators for atrial myocytic release of ANP (4, 14, 17). There are diverse reports on the effects of cAMP in the regulation of ANP secretion. Forskolin, an activator of adenylyl cyclase (AC), has been shown to decrease ANP release from cultured atrial myocytes (12, 20, 30) and perfused rat heart (25); 3-isobutyl-1-methylxanthine (IBMX), a nonselective inhibitor of cyclic nucleotide phosphodiesterase (PDE) (12), and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) (12, 30), have also been shown to inhibit ANP secretion. In contrast, it has also been shown that cAMP-elevating agents (1, 5, 24) and cell membrane-permeant cAMP analogs (1, 5, 27, 28) increase ANP secretion in cultured cardiac myocytes (5), sliced atria (1), isolated atria (27, 28), and perfused rat heart (24). Hence, the current understanding of cAMP-dependent regulation of ANP secretion is controversial. Further, the mechanism has to be defined.
Diverse effects of Ca2+ on ANP secretion have also been reported. Increases in Ca2+ influx via L-type Ca2+-channel activation by BAY K 8644 increased ANP secretion in perfused heart (24, 26), isolated beating atria (28, 29), and cultured atrial myocytes (18). Similarly, L-type Ca2+-channel blockers inhibit ANP secretion in perfused heart (26) and isolate beating atria (28, 29) and atrial myocytes (5, 18). Increased extracellular Ca2+ also results in an increase in ANP secretion in beating atria (15). In contrast, Ca2+ has also been reported to be a negative regulator for ANP secretion in perfused heart (13, 25) and isolated beating (8, 17, 33) and nonbeating atria (4, 9, 14). Recently (33) it was shown that L- and T-type Ca2+ channels are involved in the opposite regulation of myocytic ANP release.
Although cAMP-elevating agents have been known to regulate ANP secretion, direct evidence for the involvement of cAMP in the regulation of ANP secretion must be defined. Because cAMP is known to be involved in the regulation of L-type Ca2+-channel activity (11, 22), the purpose of the present study was twofold; namely, to determine whether 1) cAMP production by cAMP-elevating agents is directly related to the regulation of ANP secretion, and 2) cAMP-induced changes in ANP secretion are related to L-type Ca2+-channel activation or other cAMP signaling.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Beating, Perfused Rabbit Atrial Preparation
New Zealand White rabbits were used. An isolated perfused atrial preparation was prepared using methods described previously (3, 6) that allow atrial pacing and measurements of changes in atrial volume during contraction (stroke volume), pulse pressure, transmural extracellular fluid (ECF) translocation, cAMP efflux, and ANP secretion. The atrium was perfused with HEPES buffer solution by means of a peristaltic pump (1 ml/min).Experimental Protocols
The atria were perfused for 60 min to stabilize ANP secretion. [3H]inulin was introduced to the pericardial fluid 20 min before the start of the sample collection (3). The perfusate was collected at 2-min intervals at 4°C for analyses. In one series of experiments, atrial pacing at 0.8, 1, 1.3, 1.6, and 2 Hz was performed consecutively for 2 min at each frequency with repetitive frequency changes, which were spaced by 2 min of 0.8-Hz pacing. Experiments were carried out using three groups of atria. The control cycle (a 12-min period) was followed by infusion of the cAMP-elevating agents forskolin (1 µM; group 1, n = 6; Fig. 1) or IBMX (1 mM; group 2, n = 6; Fig. 2). The effects were evaluated after two cycles (24 min) of administration of the agent. For the time control, vehicle was introduced, and values obtained during the periods corresponding to the control and experimental observations were compared (group 3, n = 6).
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In another series of experiments, the atria were paced at 1.3 Hz.
Experiments were carried out using 22 groups of atria. The control
cycle (a 12-min period) was followed by an infusion of the
cAMP-elevating agents forskolin or IBMX for 36 min (forskolin, 1 µM,
group 1, n = 14; Figs.
3,
4, and 10; IBMX, 1 mM, group 2, n = 4).
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In other groups of experiments, to define the involvement of L-type
Ca2+ channels or protein kinases, 36 min of infusion of
inhibitors was followed by an infusion of forskolin or vehicle in the
presence of the prior agent. The following inhibitors were used:
1) L-type Ca2+-channel inhibitors including
nifedipine (1µM, group 3, n = 9, Fig.
5A and group 4,
n = 7, Fig. 5B, and Figs.
6 and 10) and diltiazem (10 µM,
group 5, n = 12 and group 6,
n = 6, Fig. 10B); 2) protein kinase A (PKA) inhibitors including KT-5720 (3-6 µM, group
7, n = 6, Fig. 10B) and Rp-adenosine
3',5'-cyclic monophosphorothioate (Rp-cAMPS; 62.5-12.5 µM,
group 8, n = 5, Fig. 10); 3) a specific protein kinase C- (PKC-) inhibitor, Gö-6976 (1 µM,
group 9, n = 5, Fig. 10, and group 10,
n = 3, Fig. 10B); 4) the
Ca2+/calmodulin kinase inhibitor KN-62 (10 µM,
group 11, n = 3, Fig. 10B);
5) the nonspecific protein kinase inhibitor staurosporine at
0.01 µM (group 12, n = 5, and group 13, n = 3, Fig. 10B), 0.1 µM (group 14, n = 9, and group 15, n = 4, Fig.
10B), and 1 µM (group 16, n = 7, Fig.
8A; group 17, n = 6, Figs. 8B, 9,
10); and 6) PKC activator phorbol 12-myristate 13-acetate
(PMA; 60 nM, group 18, n = 5; Fig. 10B) and
its specific inhibitor Gö-6976 (1 µM, group 19, n = 4, and group 20, n = 3; Fig.
10B) and nonspecific inhibitor staurosporine (0.1 µM,
group 21, n = 3, Fig. 10B) were also used.
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In another group of experiments for the control (Figs.
7 and 10), vehicle
only was infused for 72 min. The effects of forskolin in the presence
of modulating agent or vehicle were compared with the period before and
the third 12-min period after forskolin infusion. For the
modulating-agent control, values obtained during the periods
corresponding to the control and experimental observations were
compared.
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Radioimmunoassay of cAMP
Production of cAMP was measured by equilibrated radioimmunoassay (6). Briefly, standards or samples were taken up in a final volume of 100 µl of 50 mM sodium acetate buffer (pH 4.8) containing theophylline (8 mM), and then 100 µl of diluted cAMP antiserum (Calbiochem-Novabiochem, San Diego, CA) and iodinated 2'-O-monosuccinyl-adenosine 3',5'-cyclic monophosphate tyrosyl methyl ester (125I-ScAMP-TME, 10,000 counts/min (cpm) per 100 µl) were added and incubated for 24 h at 4°C. For the acetylation reaction, 5 µl of a mixture of acetic anhydride and triethylamine (1:2 dilution) were added to the assay tube before antiserum and tracer were also added. The bound form was separated from the free form by charcoal suspension. 125I-ScAMP-TME was prepared as described previously (31). Briefly, 2 µg of ScAMP-TME (Sigma, St. Louis, MO) was introduced into a vial containing 100 mM phosphate buffer (pH 7.4), and 1 mCi of 125I-Na (Amersham International, Buckinghamshire, UK) was added. Chloramine-T (0.4 mg/ml) was added to the reaction vial (total reaction volume = 50 µl) and mixed gently, and 1 min later the reaction was terminated with sodium metabisulfite (0.2 mg/ml) and NaI (5 mM). The reaction mixture was immediately applied to a Sephadex G-10 column (1 × 20 cm) previously washed with 10 mM phosphate buffer. 125I-ScAMP-TME was eluted with 10 mM phosphate buffer containing 150 mM NaCl (pH 7.4) and stored at
20°C until use.
Immediately before it was used, 125I-ScAMP-TME was
repurified by high-performance liquid chromatography on a
reversed-phase µBondapak column (Waters Associates, Milford, MA) with
a linear gradient (0-60% acetonitrile in 0.1% trifluoroacetic acid) elution. Radioimmunoassay for cAMP was done on the day of experiments, and all samples from one experiment were analyzed in a
single assay. Nonspecific binding was <2.0%. The 50% intercept was
at 16.50 ± 0.79 fmol/tube (n = 10). The intra-
and interassay coefficients of variation were 5.0% (n = 10) and 9.6% (n = 10), respectively. The amount of
cAMP efflux was expressed as picomoles cAMP per minute per gram atrial
tissue. The molar concentration of cAMP efflux in terms of ECF
translocation, which may reflect the concentration of cAMP in the
interstitial space fluid (2, 3, 6), was calculated as cAMP
efflux (µM) = cAMP (in
pmol · min
1 · g1)/ECF
translocated (in
µl · min1 · g1).
Preparation of Samples for cAMP Assay
For the preparation of perfusates, 100 µl of the perfusate was treated with trichloroacetic acid (900 µl) to be a final concentration of 6% for 15 min at room temperature and was centrifuged at 4°C. The supernatant (500 µl) was transferred to a polypropylene tube, extracted with water-saturated ether (1 ml) three times, and then dried using a SpeedVac concentrator (Savant, Farmingdale, NY). The dried samples were resuspended with sodium acetate buffer.Radioimmunoassay of ANP
Immunoreactive ANP in the perfusate was measured by a specific radioimmunoassay as described previously (3). The secreted amount of immunoreactive ANP was expressed as nanograms of ANP per minute per gram of atrial tissue. The molar concentration of immunoreactive ANP in terms of the ECF translocation reflects the concentration of ANP in the interstitial space of the atrium and, therefore, indicates the rate of myocytic release of ANP into the surrounding paracellular space (2, 3). It was calculated as ANP released (µM) = immunoreactive ANP (in pg · min1 · g1)/ECF
translocated (in
µl · min1 · g1) · 3,063 [mol wt of ANP (1-28)]. Most of the ANP secreted is processed ANP (3).
Statistical Analysis
Significant difference was compared using a two-way ANOVA analysis for repeated measures (Figs. 1 and 2, E-G, and Fig. 10A). Significant differences between paired data for a given pacing frequency (Figs. 1 and 2, A-D) and also the data of Figs. 3, 5, 7, and 8 were analyzed by repeated-measures ANOVA followed by Bonferroni's multiple-comparison test. Student's t-test for unpaired data (Fig. 10B) was also applied. Correlation coefficients were determined with the use of linear regression analysis. Statistical significance was defined as P < 0.05. The results are given as means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Inhibition by cAMP-Elevating Agents of Myocytic ANP Release
Activation of adenylyl cyclase with forskolin inhibits ANP secretion. An increase in pacing frequency resulted in an increase in secretion of ANP concomitantly with translocation of the ECF, which is coincident with an increase in atrial stroke volume (see Fig. 1, A, B, and D). The concentration of ANP in perfusate in terms of the ECF translocation was 0.4-0.6 µM (see Fig. 1C). Because the secretion of ANP is related to the atrial work in beating atria (Ref. 3 and present data), the response of ANP secretion was analyzed as a function of atrial stroke volume (see Fig. 1E). Both secretion of ANP and translocation of the ECF were functions of atrial stroke volume (see Fig. 1, E and F). At higher atrial stroke volumes, the incremental changes in the secretion of ANP and translocation of the ECF showed a peak and fall (see Fig. 1, A and B). The change in ANP secretion was well correlated with translocation of the ECF (see Fig. 1G).
Forskolin inhibited the secretion of ANP (all P < 0.01 at 1.3-2.0 Hz; see Fig. 1A). The concentration of ANP in perfusate in terms of the ECF translocation was significantly decreased by forskolin (all P < 0.01; see Fig. 1C). Translocation of the ECF was increased at low (both P < 0.01 at 0.8 and 1.0 Hz) and decreased at higher (P < 0.01 at 1.6 Hz) atrial rates, respectively, by forskolin (see Fig. 1B). Atrial stroke volume was increased by forskolin (all P < 0.01 at 0.8-1.6 Hz; see Fig. 1D). Forskolin shifted relationships between the secretion of ANP and atrial stroke volume or translocation of the ECF downward and rightward (both P < 0.01; see Fig. 1, E and G). This means that forskolin inhibits myocytic release of ANP. This is related to the decrease in the concentration of ANP shown in Fig. 1C. Forskolin shifted the relationship between translocation of the ECF and atrial stroke volume downward and rightward (P < 0.01; see Fig. 1F). For the time control, changes in secretion of ANP and translocation of the ECF in response to repetitive changes in pacing frequency were constant and stable. The responses of the parameters were reproducible during the periods corresponding to the control and experimental observations (differences between periods were not significant; n = 6).Inhibitor of phosphodiesterase inhibits ANP secretion. IBMX inhibited the secretion of ANP (both P < 0.01 at 1.6 and 2.0 Hz; see Fig. 2A). The concentration of ANP was significantly decreased by IBMX (all P < 0.01 at 0.8-2.0 Hz; see Fig. 2C). Translocation of the ECF was increased at low and decreased at higher atrial rates without significance, respectively, by IBMX (see Fig. 2B). Atrial stroke volume was increased by IBMX (all P < 0.01 at 0.8-1.3 Hz; see Fig. 2D). IBMX shifted relationships between the secretion of ANP and atrial stroke volume or translocation of the ECF downward and rightward (both P < 0.05; see Fig. 2, E and G). This means that IBMX inhibits myocytic release of ANP. IBMX shifted the relationship between translocation of the ECF and atrial stroke volume downward and rightward (P < 0.05; see Fig. 2F).
Protein Kinase-Dependent Inhibition by cAMP of Myocytic ANP Release
Relationship between changes in cAMP production and atrial release of ANP. To define more clearly the relationship between changes in cAMP production and release of ANP, another series of experiments have been done in atria paced at a fixed frequency. Forskolin decreased ANP secretion and myocytic ANP release (ANP concentration) concomitantly with increases in atrial cAMP efflux and dynamics in a time-dependent manner (see Fig. 3). IBMX revealed similar effects (n = 4; data not shown). The effects observed with the atria paced at a fixed frequency of forskolin on ANP secretion and atrial dynamics were similar to those observed with the atria paced at repetitive frequency changes. The responses were stable during the third cycle of the administration of forskolin. As shown in Fig. 4, inhibition by forskolin of myocytic ANP release was a function of elevation of cAMP production. Inhibition in atrial myocytic ANP release by forskolin was coincident with an increase in cAMP production (see Fig. 4A). Figure 4B shows an inverse relationship between the changes in cAMP production and ANP concentration.
A minor effect of L-type Ca2+-channel
blockade on cAMP-induced inhibition in ANP release.
To define involvement of L-type Ca2+ channels in inhibition
of ANP release by forskolin, nifedipine was administered. L-type Ca2+-channel blockade with nifedipine significantly
increased ANP secretion and myocytic ANP release (see Fig. 5,
Aa and Ac). No significant change in cAMP
production was observed by nifedipine (see Fig. 5, Ad and
Ae). Nifedipine decreased atrial dynamics (see Fig. 5,
Af and Ag). In the presence of nifedipine,
forskolin inhibited ANP secretion and myocytic ANP release (see Fig. 5, Aa and Ac) with concomitant increases in cAMP
production (see Fig. 5, Ad and Ae). Nifedipine
attenuated a minor portion of forskolin-induced inhibition of ANP
release (in difference in percent changes over the control,
51.57 ± 4.34% for nifedipine plus forskolin, n = 9, vs. 68.48 ± 2.56% for forskolin alone, n = 14; P < 0.01, Fig. 10B; in percent
changes of the control, P > 0.05; Fig.
10A). In the presence of nifedipine, forskolin significantly
increased cAMP production (see Fig. 5, Ad and
Ae). In the presence of nifedipine, forskolin-induced
accentuation of atrial dynamics was not observed (see Fig. 5,
Af and Ag). Responses in cAMP production and
atrial dynamics by forskolin were dissociated by
Ca2+-channel blockade. Nifedipine-induced changes in ANP
release, cAMP production, and atrial dynamics were maintained stably
during the period corresponding to forskolin infusion (see Fig. 5,
Ba-Bg). As shown in Fig. 6A, in the presence of
nifedipine, forskolin inhibited myocytic release of ANP concomitantly
with an increase in cAMP production. The inverse relationship between
cAMP production and ANP concentration was not changed by the treatment
with nifedipine (Fig. 6B). The slopes of the relationships
were not significantly different between the groups of forskolin and
nifedipine plus forskolin (0.316 ± 0.058 for nifedipine plus
forskolin, n = 9 vs. 0.279 ± 0.050 for
forskolin alone; n = 14; P > 0.05).
Diltiazem (10 µM), another L-type Ca2+-channel inhibitor
attenuated slightly but not significantly forskolin-induced inhibition
of ANP release (in difference in percent changes, 57.64 ± 4.88% for diltiazem plus forskolin, n = 12 vs.
68.48 ± 2.56% for forskolin alone, n = 14;
P = 0.0515; Fig. 10B). For the time control,
changes in secretion of ANP, ECF translocation, cAMP production, and
atrial dynamics were constant and stable (see Fig. 7). The differences
between control observations and periods corresponding to experimental
observations were not significant.
Inhibition of protein kinases blocks cAMP-induced decrease in ANP
release.
To further dissect the pathway of cAMP-induced inhibition in ANP
secretion, the atrium was treated with a nonspecific protein kinase
inhibitor, staurosporine. At a dose of 1 µM but not 0.01 or 0.1 µM,
staurosprine decreased ANP secretion, and the effect was stable and
maintained after the third cycle of staurosporine (P < 0.01, sixth cycle of staurosporine vs. control; P > 0.05, sixth cycle of staurosporine vs. third cycle of staurosporine, see METHODS, Fig. 8). No significant change in cAMP
production was observed by staurosporine alone. Staurosporine
attenuated forskolin-induced inhibition of atrial ANP release in a
concentration-dependent manner (Fig. 10). No significant change in
forskolin-induced inhibition of ANP release was observed by 0.01 µM
of staurosporine. Staurosporine (0.1 µM) significantly attenuated
forskolin-induced inhibition of ANP release (in difference in percent
changes, 45.24 ± 6.09% for staurosporine plus forskolin,
n = 9 vs. 68.48 ± 2.56% for forskolin,
n = 14; P < 0.001). In the presence of
staurosporine (1 µM), forskolin slightly decreased ANP secretion and
the concentration of ANP (see Fig. 8, Aa and Ac).
The responses were not significantly different from those of the atrium
treated with staurosporine alone (see Figs. 8, Ba-Bg, and
Fig. 10B). Percent changes of the forskolin-induced
inhibition of ANP release were significantly attenuated by the
staurosporine (P < 0.05; Fig. 10A).
Forskolin significantly increased cAMP production in the presence of
staurosporine (see Fig. 8, Ad and Ae).
Forskolin-induced increases in atrial dynamics were not observed in the
presence of staurosporine (see Fig. 8, Af and
Ag). In this condition, forskolin rather decreased atrial
dynamics. In the presence of staurosporine, forskolin-induced changes
in atrial dynamics were dissociated from the increase in cAMP
production. As shown in Fig. 9, in the
presence of staurosporine, forskolin increased cAMP production without
significant changes in ANP release, which indicates a dissociation of
the relationship between cAMP production and ANP release. The slopes of
the relationships were significantly different between the groups of
forskolin and staurosporine plus forskolin (0.029 ± 0.012 for
staurosporine plus forskolin, n = 7 vs. 0.279 ± 0.050 for forskolin, n = 14; P < 0.01). Forskolin-induced decrease in the concentration of ANP was
significantly attenuated in the presence of staurosporine (see Figs. 8,
9, and 10). The decrease in the concentration of ANP in the presence of
staurosporine plus forskolin was not significantly different from that
in the presence of staurosporine alone.
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A minor effect of PKA inhibition on the cAMP-induced decrease in
ANP release.
To define involvement of the PKA activity in inhibition of ANP release
by forskolin, the PKA-specific inhibitor KT-5720 or Rp-cAMP was
administered. KT-5720 blocked forskolin-induced accentuation of atrial
dynamics (in difference in percent changes over the control, 1.71 ± 3.81% for KT-5720 plus forskolin, n = 6 vs.
27.94 ± 3.49% for forskolin alone, n = 14;
P < 0.001). KT-5720 slightly but not significantly
attenuated forskolin-induced decrease in atrial ANP release (in
difference in percent changes, 62.84 ± 4.08% for KT-5720 plus
forskolin, n = 6 vs. 68.48 ± 2.56% for forskolin alone, n = 14; P > 0.05;
Fig. 10B). Another PKA
inhibitor, Rp-cAMPS, slightly but significantly attenuated the
forskolin-induced decrease in ANP release (in percent changes of the
control; P > 0.05, Fig. 10A; in difference
in percent changes over the control, 54.72 ± 3.48%,
n = 5 vs. 68.48 ± 2.56%; P < 0.01, Fig. 10B; see legends for Fig. 10). Rp-cAMPS blocked
forskolin-induced accentuation of atrial dynamics (in difference in
percent changes over the control, 4.05 ± 2.27% for Rp-cAMPS plus
forskolin, n = 5 vs. 27.94 ± 3.49% for forskolin
alone, n = 14; P < 0.01). In the
presence of PKA inhibitors, forskolin significantly increased cAMP
production (data not shown).
Absence of effect of inhibition of PKC- or
Ca2+/calmodulin kinase on cAMP-induced
decrease in ANP release.
To define the involvement of PKC in inhibition of ANP release by
forskolin, Gö-6976, a specific PKC- inhibitor, was
administered. Gö-6976 failed to modulate the forskolin-induced
decrease in ANP release (Fig. 10). KN-62, a specific
Ca2+/calmodulin inhibitor, also failed to modulate the
forskolin-induced decrease in ANP release (Fig. 10B).
An activation of PKC with PMA accentuates ANP release and its specific blockade blocks increase in ANP release. To test the effect of staurosporine and Gö-6976 in relation to an accentuation of ANP release by PKC activation, PMA, an activator of PKC, was administered. PMA accentuated atrial ANP release (Fig. 10B). In the presence of Gö-6976 or staurosporine (0.1 µM), PMA failed to accentuate atrial ANP release.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present data indicate that cAMP inhibits atrial myocytic ANP release via protein kinase-dependent and L-type Ca2+-channel-dependent and -independent signaling pathways in rabbit atria.
cAMP Inhibits Myocytic ANP Release
The present study clearly shows that cAMP-elevating agents inhibit release of ANP. Both AC activation with forskolin and PDE inhibition with IBMX inhibited release of ANP. Both agents decreased the concentration of ANP in perfusate in terms of the ECF translocation and shifted the relationship between release of ANP and translocation of the ECF downward and rightward. This means that cAMP-elevating agents inhibit myocytic release of ANP. An inhibition of myocytic ANP release by AC activation is a function of an increase in cAMP production. Because cAMP efflux reflects the intracellular cAMP content, amplitude of cAMP efflux appears to represent atrial cAMP production (6). The present data are in agreement with previous reports showing an inhibition in ANP secretion by forskolin (12, 20, 25, 30) or IBMX (12) in cultured atrial myocytes or perfused rat hearts. However, the present data contrast to the previous reports on the stimulation of ANP release by cAMP-elevating agents (1, 5, 24) in cultured cardiac myocytes, isolated atria, or perfused rat hearts. The reasons for the discrepancy are not clear at present. Differences in methodology and/or basal status of atrial or cellular dynamics may account for the discrepancy. Although intracellular cAMP has been claimed to be a negative or positive regulator for ANP release, the mechanism by which cAMP controls atrial myocytic ANP release must be defined.cAMP Inhibits ANP Release by a Protein Kinase-Dependent and L-Type Ca2+-Channel-Dependent and -Independent Mechanism
The present study clearly shows that cAMP inhibits myocytic ANP release via protein kinase-dependent signaling. The data also suggest that Ca2+ influx via L-type Ca2+ channels may be involved in a minor portion of the cAMP-induced inhibition of ANP release. A major portion of cAMP-induced inhibition of ANP release is related to protein kinase-dependent signaling. Inhibition of PKA attenuated a minor portion of the cAMP-induced inhibition of ANP release, which may be relevant to the inhibition of L-type Ca2+-channel activity. Gö-6976, a specific PKC-
inhibitor, and KN-62, a Ca2+/calmodulin kinase inhibitor,
failed to modulate cAMP-induced inhibition of ANP release. This means
that the mechanism by which staurosporine blocks the cAMP-induced
inhibition of ANP release may be related to protein kinase(s) other
than PKA, PKC-, and Ca2+/calmodulin kinase.
Because the cAMP signaling pathway contains cAMP-related activation of L-type Ca2+ channels (11, 22) and also because an increase in the intracellular Ca2+ concentration inhibits ANP release in beating atria and perfused heart (8, 13, 17, 25, 33), it was expected that cAMP-induced inhibition of ANP release would be related to Ca2+ influx via the L-type channel. Treatment with L-type Ca2+ channel or PKA inhibitors only slightly attenuated the inhibition of ANP release by forskolin. L-type Ca2+-channel inhibitors failed to change the relationship between changes in cAMP production and ANP release. On this occasion, treatment with Ca2+-channel or PKA inhibitors inhibited forskolin-induced increases in atrial dynamics. This suggests that cAMP negatively regulates myocytic ANP release via largely Ca2+-independent pathways. An increase in ANP secretion by L-type Ca2+-channel inhibitors is consistent with the previous report (33).
cAMP-induced inhibition of myocytic ANP release as well as an increase
in atrial dynamics were not observed in the presence of staurosporine,
a nonspecific protein kinase inhibitor. The effect was dose dependent.
Staurosporine significantly shifted the relationship between changes in
cAMP production and ANP release. An accentuation of ANP release by PMA
is consistent with the previous report (23, 24). The
findings that both the specific PKC- inhibitor Gö-6976 and the
nonspecific protein kinase inhibitor staurosporine (0.1 µM)
completely blocked the PMA-induced increase of ANP release and that
Gö-6976 but not staurosporine failed to modulate the
forskolin-induced decrease of ANP release suggest that at least PKC-
is not involved in cAMP-induced inhibition of ANP release. Although the
specific protein kinase responsible for the effect has yet to be
defined, the present data indicate that cAMP-induced inhibition of ANP
release is related to the activation of protein kinase(s) other than
PKA, PKC-, and Ca2+/calmodulin kinase. From these data
it is hypothesized that AC-activated cAMP production has two signaling
pathways in the cardiac atrium: one is to increase Ca2+
influx leading to an increase in atrial dynamics, and the other is to
activate protein kinase(s) leading to inhibition of secretory function.
Therefore, these findings suggest that the mechanisms by which cAMP or
Ca2+ inhibits myocytic release of ANP may not be the same.
These data for the first time indicate that intracellular cAMP inhibits
ANP release independently from the activation of L-type
Ca2+ channels. These data suggest that a cAMP signaling
pathway other than the activation of the L-type Ca2+
channel is very potent and distinct in the regulation of ANP secretion.
Alternatively, activated cAMP signaling may sensitize the
Ca2+-related inhibitory mechanism so that only a minor
fraction of Ca2+ is enough for the inhibition. Or, it seems
likely that cAMP signaling is distal to the role of Ca2+ in
the regulation of ANP release.
Previously it was shown that forskolin-induced inhibition of ANP secretion was not observed in the cultured noncontracting rat atrial myocytes treated with ryanodine and Ca2+ depletion (12), which suggested that cAMP-induced inhibition of ANP secretion was related to the increases in Ca2+ influx and intracellular Ca2+ release. The present data contrast with that report. The reasons for the discrepancy are not clear at present. Both differences in species and methodology may account for the discrepancy.
The blockade of the forskolin-induced accentuation of atrial dynamics by L-type Ca2+ channels, PKA inhibitors, or staurosporine that was observed in the present study is consistent with the notion (11, 22) that cAMP activates L-type Ca2+ channels via PKA. In the presence of staurosporine, forskolin actually decreased the atrial dynamics. The mechanism responsible for this phenomenon is unclear at present.
Taken together with previous reports showing inhibition of ANP release by cGMP (12, 17, 21, 24), it is clear that cyclic nucleotides are inhibitory regulators for the ANP secretion.
In conclusion, the present study shows that cAMP inhibits atrial myocytic ANP release via protein kinase-dependent signaling. The present study also suggests that an accentuation of Ca2+ influx via L-type Ca2+ channels by AC-activated cAMP production is related to the control of dynamics with only a minor contribution to the control of secretory function of the cardiac atrium.
Perspectives
It has been shown that ANP secretion is controlled by a "two-step sequential mechanism" (2, 3). The first step is atrial myocytic ANP release into the interstitial space, and the second is convective translocation of the ECF with the released ANP into the atrial lumenal bloodstream. Atrial myocytic ANP release is under the control of variable second messengers (23). The second step, convective ECF translocation, is controlled by changes in atrial dynamics and the size of the extracellular space; therefore it is expected that factors affecting atrial dynamics such as cyclic nucleotide and Ca2+ modulating agents may influence atrial myocytic ANP release in a complex way. However, the exact nature of the roles for second messengers in the regulation of myocytic ANP release is largely unknown. Ca2+ is involved in both negative and positive regulation of atrial ANP release (33). As shown in the present study, cAMP is involved in the negative regulation of ANP release. This pathway is distinct from L-type Ca2+ channels. In the cardiac atrium, cAMP as an inhibitory factor for the regulation of ANP release may work through protein kinase signaling. However, it looks as though the signaling pathway is unusual. This suggests that within the cardiac atrium, roles for cAMP in the regulation of secretory function are complex. The signaling pathway for the AC-cAMP system in the endocrine atrium has to be defined.| |
ACKNOWLEDGEMENTS |
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The authors thank Kyong Sook Kim for secretarial work.
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FOOTNOTES |
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This work was supported by research grants from the Korea Science and Engineering Foundation (98-0403-10-01-5), Korea Research Foundation (1998-001-F00112, 2000-015-FP0023), and Ministry of Health and Welfare (HMP-00-CO-03-0003).
Address for reprint requests and other correspondence: Send all correspondence to: K. W. Cho, Dept. of Physiology, Jeonbug National Univ. Medical School, 2-20, Keum-Am-Dong-San, Jeonju 561-180, Republic of Korea (E-Mail: kwcho{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.
10.1152/ajpregu.00316.2001
Received 5 June 2001; accepted in final form 22 January 2002.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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% changes over the mean value of 2 periods before
administration of forskolin. Fraction no, serial 2-min sample
collections after the administration of forskolin.
P < 0.01 vs.
nifedipine (values of the one cycle before the administration of
forskolin or vehicle).
)
coincident with an increase in cAMP production (
) in
the presence of nifedipine (1.0 µM). Effects of nifedipine alone on
changes in ANP concentration (
) and cAMP production
(
). Data were derived from Fig. 
