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Vol. 297, Issue 1, 181-188, April 2001
Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland Baltimore, School of Medicine, Baltimore, Maryland
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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We tested both relaxation and cGMP generation by atrial (ANP), brain
(BNP), and C-type natriuretic peptide (CNP) in oxytocin-stimulated myometrium from near-term pregnant guinea pigs to investigate the
ability and mechanism of natriuretic peptides to inhibit myometrial contractility. Myometrial strips were contracted by 10
8 M
oxytocin, and relaxation to the cumulative addition
(109-106 M) of the natriuretic peptides
measured. Maximal relaxation to BNP was significantly greater than to
ANP (52 versus 32% respectively; p < 0.05),
whereas CNP failed to produce relaxation. However, the increase in cGMP
produced by BNP (107 M) was significantly less than that
produced by ANP (107 M) (4.5 versus 7.0 times basal;
p < 0.05); CNP did not increase myometrial cGMP.
Anantin, a competitive blocker of the guanylate cyclase A receptor,
significantly reduced the increase in cGMP produced by ANP and BNP, but
had no effect on relaxation induced by either peptide. Rp-8-Br-cGMP, an
inhibitor of the cGMP-dependent protein kinase, did not alter
BNP-induced relaxation. The atrial natriuretic peptide-fragment 4-23 amide, a natriuretic peptide clearance receptor agonist, failed
to inhibit oxytocin-stimulated myometrial contraction. We conclude that
natriuretic peptide induced relaxation of oxytocin-stimulated
myometrium from the pregnant guinea pig is not mediated by either
guanylate cyclase A or B activation, is independent of the cGMP
pathway, and does not involve clearance receptor activation. Our
results suggest that natriuretic peptide-induced relaxation of pregnant
myometrium is mediated via a novel mechanism.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A
successful pregnancy requires relaxation of the uterus during more than
95% of gestation, overcoming the inherent tendency of myometrium to
contract under stretch until the appropriate time for labor. This
active and highly regulated process is called myometrial quiescence. It
entails not only the near absence of myometrial contractions but also
its refractoriness to contractile agents. Myometrial quiescence is
mediated by complex, but as yet poorly understood molecular mechanisms
despite much research (Cunningham et al., 1993
).
Several investigators studying rats, guinea pigs, and rabbits have
documented a time-dependent increase in total myometrial cGMP content
during pregnancy, which decreases shortly before the onset of labor
(Weiner et al., 1994; Yallampalli et al., 1994). This temporal profile
and the known smooth muscle-relaxing capability of cGMP (Lincoln et
al., 1994; Vaandrager and de Jonge, 1996; Carvajal et al., 2000) lead
these researchers to propose this second intracellular messenger has a
central role in the maintenance of uterine quiescence, although
disagreeing on the source of the cGMP (Weiner et al., 1994; Yallampalli
et al., 1994).
Nitric oxide (NO) is a potent, endogenous, relaxing agent of vascular
tissues, and its action is mediated predominantly by an increase in
intracellular cGMP (Moncada et al., 1991). Several groups pursued the
possibility that NO, by its activation of soluble guanylate cyclase,
was responsible for the increase in myometrial cGMP during pregnancy
and consequently was central to uterine quiescence (Natuzzi et al.,
1993; Sladek et al., 1993; Bansal et al., 1997). However, we
demonstrated that the increase in myometrial cGMP in the pregnant
guinea pig was independent of NO production and soluble guanylate
cyclase activation, and proposed that the cGMP increase was secondary
to particulate guanylate cyclase activation (Weiner et al., 1994).
The natural activators of particulate guanylate cyclase receptors are
members of a family of natriuretic peptides (Rosenzweig and Seidman,
1991) and their presence has been demonstrated in gestational tissues.
Atrial natriuretic peptide (ANP) is produced by the human placenta
(Graham et al., 1996). Brain natriuretic peptide (BNP) is present in
amniotic fluid (Itoh et al., 1993) and produced by cultured amnion
cells (Itoh et al., 1993). Furthermore, we have found high levels of
BNP mRNA in human chorion taken from women at term before the onset of
labor (K. Aguan, personal communication).
Natriuretic peptide receptors have been demonstrated in rat (Dos Reis
et al., 1995; Vaillancourt et al., 1998), guinea pig (Aguan at al.,
1998), and human myometrium (Itoh et al., 1994; Aguan et al., 1999). In
the rat uterus, the protein levels of the guanylate cyclase type B
receptor (GC-B) were greater than those of guanylate cyclase type A
(GC-A) (Vaillancourt et al., 1998), whereas in human myometrium, it was
suggested that GC-A is the dominant active receptor based on the
measurement of guanylate cyclase activity (Itoh et al., 1994).
Furthermore, it has been observed in all species studied to date that
GC-B, but not GC-A, is down-regulated during pregnancy (Aguan et al.,
1998, 1999; Vaillancourt et al., 1998).
Thus, we hypothesized that a locally produced natriuretic peptide, by
activation of a particulate guanylate cyclase, is the stimulus for the
increased myometrial cGMP content during pregnancy (Weiner et al.,
1994). We further hypothesized that this natriuretic peptide is central
to the maintenance of myometrial quiescence during pregnancy. The
objective of the present study is to demonstrate the ability of
natriuretic peptides to inhibit oxytocin-induced myometrial
contractility during pregnancy and characterize its mechanism of action.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animal Model. Timed pregnant, Duncan-Hartley guinea pigs were purchased from a commercial breeder (Harlan Sprague-Dawley, Indianapolis, IN). Myometrium was obtained from near-term (60 ± 2 days of gestation), anesthetized (xylazine, 4 mg/kg i.m.; ketamine hydrochloride, 100-200 mg/kg i.p.) guinea pigs respiring spontaneously. Full thickness strips opposite to the placental implantation site were excised, including decidua but excluding fetal membranes. The myometrial strips were placed in Krebs' buffer and stored on wet ice until used within 1 h for isometric tension recording. The Committee on Animal Care at the University of Maryland Baltimore approved the protocol.
The guinea pig was selected as a model for several reasons: 1) its hemomonochorial placentation, which is the most similar to human placenta among all nonprimate mammals (Pijnenborg et al., 1981); 2) the
similarity to humans of its sex hormone profile during the estrus cycle
and pregnancy; and 3) the relatively long and stable gestational period
(65 ± 2 days from copulation), making easy comparisons between
different gestational ages.
Isometric Tension Studies.
Longitudinal strips (~10 mm in
length × 2 mm in width) of full thickness myometrium were placed
in organ chambers and attached to a force transducer (Grass
Instruments, Quincy, MA) for isometric tension recording. The chambers
contained Krebs' buffer composed of 118 mM NaCl, 4.7 mM KCl, 1.18 mM
MgSO4, 1.18 mM
KH2PO4, 11.1 mM
D-glucose, 0.016 mM EDTA, 2.2 mM
CaCl2, 15.8 mM NaHCO3,
pH = 7.35 to 7.45, maintained at 37°C, and continuously bubbled
with 95% O2, 5% CO2. The
myometrial strips were equilibrated under 1-g passive tension until a
stable baseline, typically around 0.5-g basal tension (range 0.4-0.6
g) was achieved (approximately 30 min). The bath buffer solution was
changed every 5 min during equilibration. After the stabilization
period, contractions were produced by a submaximal concentration of
oxytocin (108 M), which approximated the
EC50 obtained from concentration-response curves
previously determined. The experiment was begun after a regular pattern
of contractions was achieved, typically 15 to 20 min after the addition
of the oxytocin.
80% of the oxytocin-stimulated
basal activity (p = N.S.). In preliminary studies, we
determined that 108 M oxytocin was the optimal
concentration to produce this regular pattern of contractions.
Contractions were recorded and analyzed using PowerLab/800 hardware and
Chart v3.4 software (AD Instruments, Mountain View, CA). To quantify
contractile activity, the integral area under the curve over 5-min
intervals was measured and normalized for the cross-sectional area of
the strip. The cross-sectional area was calculated as W/(L × D),
where W is weight (g), L is length (cm), and D (density) is 1.05 g/cm3. Basal activity was designated as the
stabilized contractile response to oxytocin during the 5-min period
before the addition of peptides. The effect of natriuretic peptides on
myometrial contractility was measured as the activity during the 5-min
period after their addition to the organ bath. The difference between the basal activity and that after the addition of natriuretic peptide
was calculated and expressed as a percentage of the basal activity, and
identified as the percentage of relaxation.
Effect of Natriuretic Peptides on Oxytocin-Induced
Contractility.
The effect of natriuretic peptides on myometrial
contractility was measured by generating concentration-response curves
to the cumulative addition of each peptide. Contractility was induced by oxytocin (108 M) and relaxation to ANP, BNP,
and C-type natriuretic peptide (CNP) determined over a concentration
range of 109 to 106 M
(final concentration in the organ bath). The relaxation response was
measured during the 5-min interval after addition of the natriuretic peptide and compared with the basal contractile activity. The 5-min
interval was selected for analysis of the dose response because the
temporal profile of natriuretic peptide-induced relaxation showed an
immediate effect of the peptides that began to decrease after 5 min.
7 M) was used to determine the temporal
course of relaxation for each natriuretic peptide by recording
myometrial contractility for 30 min after peptide addition. The effect
was measured in 5-min intervals, represented in the text and figures by
their mid-time point (e.g., 2.5 min, 7.5 min, etc.)
Effect of 8-Br-cGMP on Oxytocin-Stimulated Myometrial Contractility. To test the ability of cGMP to modulate oxytocin-induced contractile activity of myometrium from pregnant guinea pig, a concentration-response curve was prepared to 8-Br-cGMP, a membrane-permeable cGMP analog.
After the presence of regular and stable contractions was documented, the test drug was added directly to the organ chamber in 0.5 log-unit increments (in volumes of 20-40 µl as appropriate). A new dose was added every 10 min. Vehicle controls were run in parallel. The amount of drug used was expressed as its final concentration in the organ bath. The effect of 8-Br-cGMP was expressed as the contractile activity during the 10-min interval after the addition of the drug, and compared with its basal activity.Mechanism of Natriuretic Peptide-Induced Relaxation.
To
determine whether natriuretic peptide-induced relaxation occurred by
activation of particulate GC-A, myometrial strips were preincubated for
5 min with anantin (106 M), a competitive
antagonist of the GC-A receptor, before the addition of a single dose
of natriuretic peptide. Because the single-dose studies revealed that
the relaxation induced by natriuretic peptides was maximal during the
first 5 min, we selected this interval to compare the effect of anantin
alone, and the effect of anantin on natriuretic peptide-induced relaxation.
). We, too, tested whether this incubation period and drug
concentration were adequate to inhibit PKG by measuring the PKG
activity in three different experiments performed in duplicate. PKG
activity was determined as described by measuring the incorporation of
32Pi into a PKG-specific
substrate (Diwan et al., 1994; Patel and Diamond, 1997). Basal
myometrial PKG activity was 16% of the maximal activity induced by 5 µM cGMP. Five minutes after the addition of a single dose
(107 M) of BNP, PKG activity increased to 40%
of maximum. This effect of BNP was almost completely blocked by the
30-min incubation with 30 µM Rp-8-Br-cGMP; PKG activity was only 10%
of maximum under this condition.
Some of the biological actions of natriuretic peptides are mediated by
activation of the natriuretic peptide clearance receptor (Anand-Srivastava and Trachte, 1993; Hempel et al., 1998). To determine
whether clearance receptor activation is part of the biological action
of the natriuretic peptides on myometrial contractility, the effect of
a single dose of atrial natriuretic peptide
des-(Gln18, Ser19,
Gly20, Leu21,
Gly22)-fragment 4-23 amide (cANP), a specific
agonist of the natriuretic peptide clearance receptor, on
oxytocin-induced contractility was tested. It has been reported that
natriuretic peptide action via the clearance receptor involves a
heterotrimeric G protein-coupled mechanism (Anand-Srivastava and
Trachte, 1993; Murthy and Makhlouf, 1999) and results in an increase in
K+ outward conductance (Anand-Srivastava and
Trachte, 1993; Kanwal and Trachte, 1994). To determine whether the
mechanism of natriuretic peptide relaxation involved a pertussis toxin
(PTX)-sensitive G protein, myometrial strips were preincubated for
3 h with PTX (5 µg/ml), a specific blocker of heterotrimeric G
protein 
I-subunit, before the addition of BNP
to oxytocin-stimulated myometrium. This concentration and incubation
time had been shown to effectively block PTX-sensitive G protein in
porcine myometrial strips (Kitazawa et al., 2000). The effect of 2 mM
tetraethylammonium (TEA), a nonspecific K+
channel blocker, on BNP-induced relaxation was studied by adding the
drug to the organ bath 5 min before the addition of BNP. At this
concentration and incubation time, TEA effectively blocks myometrial
K+ channels (Perez et al., 1993; Khan et al.,
1997).
cGMP Measurement by Radioimmunoassay.
To determine the
effect of natriuretic peptides on particulate guanylate cyclase
activation, we measured the cGMP content of guinea pig myometrial
samples both under basal condition and after the stimulus of a single
dose (107 M) of ANP, BNP, and CNP, either in
the presence or absence of anantin (106
M). To correlate the effect of natriuretic peptides on cGMP
levels with their effect on contractility, three additional experiments were performed (in duplicate) in the presence of
108 M oxytocin, the same concentration used to
induce contraction of the myometrial strips. To determine the temporal
profile of natriuretic peptide-induced myometrial cGMP content, cGMP
was measured in duplicate tissue samples every 5 min beginning 2.5 min
after the addition of the peptide (up to 27.5 min total).
). At the
appropriate time, myometrial samples (about 5 mm2) were placed directly into boiling 50 mM Tris
with 4 mM EDTA at a pH of 7.5 for 10 min. The tissue was then minced,
homogenized, centrifuged at 14,000g (
4°C) for 20 min,
and the supernatant snap frozen until assayed for cGMP content. The
total protein content of the pellet was measured spectrophotometrically
using bicinchoninic acid method (BCA kit; Pierce, Rockford, IL). The frozen supernatants were thawed and diluted (1:75) in 25 mM Tris/4 mM
EDTA, pH 7.5. cGMP content was measured by radioimmunoassay using a
commercially available kit (Amersham, Piscataway, NJ). All cGMP
measurements were corrected for total protein content in the sample.
Duplicate values were averaged and the measurements expressed as
picomoles per milligram protein. To represent the changes in cGMP
content, we compared the cGMP levels induced by natriuretic peptides
with their basal level and expressed the results as times basal. Intra-
and interassay variations were each less than 5%.
Drugs and Solutions. Oxytocin, human ANP, BNP, and CNP, anantin, Rp-8-Br-cGMP, cANP, PTX, and TEA were obtained from Sigma Chemical Co. (St. Louis, MO). All drugs were dissolved in double deionized water.
Statistical Analysis. All data sets (mean ± standard error of the mean) were subject to a test of normalcy (Shapiro-Wilk test) and parametric or nonparametric tests performed where appropriate. Statistical comparisons between two groups were performed using Student's t test. For multiple group comparisons, either one-way ANOVA followed by post hoc Student-Newman-Keuls (parametric) or Kruskall-Wallis one-way ANOVA on ranks followed by Dunnett's multiple comparison test (nonparametric) was used. A two-tailed p < 0.05 was considered indicative of statistical significance.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect of Natriuretic Peptides on Oxytocin-Induced
Contractility.
Both ANP and BNP, but not CNP, produced
dose-dependent relaxation of oxytocin-induced myometrial contractility
(Fig. 1). The effect of BNP was
significantly greater than ANP (p < 0.05), and the
relaxation curve to ANP did not lie to the left of the relaxation curve
to BNP, surprising results considering that ANP, compared with BNP, has
greater potency and affinity for the GC-A receptor. The threshold
concentration for both BNP and ANP was 108 M;
relaxation induced at 106 M (the maximal dose
used), was 52 and 32% respectively (p < 0.05).
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Temporal Course of Natriuretic Peptide-Induced Relaxation.
Figure 2 illustrates the effect of a
single concentration (107 M) of ANP and BNP on
oxytocin-induced contractility. During the first 5 min, the magnitude
of the relaxation induced by BNP was greater than ANP (31 versus 12%
relaxation, respectively; p < 0.05).
|
Effect of 8-Br-cGMP on Oxytocin-Stimulated Myometrial
Contractility.
8-Br-cGMP produced a dose-dependent decrease in
oxytocin-stimulated contractile activity in myometrium of pregnant
guinea pig. Relaxation was apparent at 105 M,
ultimately reaching 63% relaxation from baseline at 3 × 104 M (the maximal concentration used) as
illustrated in Fig. 3.
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Effect of Anantin on Natriuretic Peptide-Induced Relaxation.
Figure 4 illustrates the effect of
anantin (106 M) on the natriuretic
peptide-induced relaxation during the first 5-min period. There was no
effect of anantin alone on oxytocin-induced contractility. Alone, both
ANP and BNP relaxed oxytocin-stimulated myometrium by 9 and 31%,
respectively (p < 0.05). There was no effect of anantin on the relaxation induced by a single concentration
(107 M) of either ANP or BNP.
|
Effect of Rp-8-Br-cGMP on BNP-Induced Relaxation.
The effect
of Rp-8-Br-cGMP on BNP-induced relaxation of oxytocin-stimulated
myometrium is illustrated in Fig. 5.
Under control conditions, a single dose of 107
M BNP produced a 30% relaxation. The addition of 30 µM Rp-8-Br-cGMP (30-min preincubation) had no effect on BNP-induced relaxation (32%
relaxation, Fig. 5).
|
Effect of cANP on Oxytocin-Induced Contractility.
The atrial
natriuretic peptide-fragment cANP, a specific agonist of the
natriuretic peptide clearance receptor, had no significant effect on
oxytocin-induced myometrial contractions at either
107 or 106 M (Fig.
6).
|
Effect of PTX and TEA on BNP-Induced Relaxation.
The effects
of preincubation with either PTX or TEA on BNP-induced relaxation are
illustrated in Fig. 7. Under control
conditions, BNP at 107 M produced 26%
relaxation; neither PTX (5 µg/ml) nor TEA (2 mM) had any effect on
BNP-induced relaxation of oxytocin-stimulated myometrial contractions
(Fig. 7).
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Natriuretic Peptide-Induced Myometrial cGMP Generation.
Both
ANP and BNP, but not CNP, increased myometrial cGMP content (Fig.
8). Myometrial cGMP content under basal
conditions was 7.41 ± 1.6 pmol/mg of protein (average ± S.E.). After the addition of 107 M ANP or BNP,
cGMP rose to 45.14 ± 9.5 and 28.87 ± 4.58 pmol/mg of
protein, respectively (p < 0.05).
|
7 M) of ANP, compared with BNP, generated a
larger increase in cGMP at 2.5 min (7- versus 4.5-fold increase above
the basal level, respectively; p < 0.05). This finding
is consistent with the reported higher GC-A receptor affinity of ANP
compared with BNP. The results of this experiment were not affected by
the presence of 108 M oxytocin. In this
situation, basal levels of cGMP were increased 6.5-fold by
107 M ANP and 4.5-fold by
107 M BNP (p < 0.05).
The temporal course of the cGMP increase (Fig. 8) paralleled the
relaxation profile produced by both ANP and BNP (Fig. 2). cGMP content
decreased over time, but remained significantly elevated over the basal
level at the end of the experiment (p < 0.05).
Effect of Anantin on Natriuretic Peptide-Induced cGMP
Generation.
Figure 9 illustrates the
effect of anantin (106 M) on myometrial cGMP
content 2.5 min after a single concentration
(107 M) of either ANP or BNP. Anantin alone had
no effect on cGMP content (6.57 ± 2.0 pmol/mg of protein),
compared with the basal myometrial cGMP level of 5.26 ± 1.1 pmol/mg of protein.
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6 M anantin. Under this
condition, ANP increase myometrial cGMP 6.5- and 4.5-fold either in the
absence or presence of anantin, respectively. And BNP increase
myometrial cGMP (in the presence of oxytocin) 4.5- and 2-fold (absence
versus presence of anantin, respectively).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We tested the ability of natriuretic peptides to decrease oxytocin-induced myometrial contractility. Our studies reveal that ANP and BNP, but not CNP, inhibit oxytocin-induced contractions of myometrium obtained from near-term pregnant guinea pigs. Furthermore, although BNP produced greater relaxation than ANP, ANP stimulated a greater increase in cGMP. Anantin had no effect on natriuretic peptide-induced relaxation, but inhibits the increase in cGMP stimulated by both ANP and BNP. The results of these experiments suggest that natriuretic peptide-induced relaxation of myometrium from the pregnant guinea pig is not mediated by receptor guanylate cyclase activation.
Six isoforms of the receptors for natriuretic peptides with guanylate
cyclase activity have been identified, in addition to the nonguanylate
cyclase clearance receptor (Foster et al., 1999). The type A (GC-A) and
type B (GC-B) receptors have been demonstrated in rat (Dos Reis et al.,
1995: Vaillancourt et al., 1998), guinea pig (Aguan et al., 1998), and
human myometrium (Itoh et al., 1994; Aguan et al., 1999). The binding
affinity and the order of potency for GC-A activation is ANP 
BNP >>> CNP, whereas for GC-B it is CNP 
ANP BNP (Suga et al., 1992). The results of our experiments on
natriuretic peptide-induced myometrial relaxation are inconsistent with
the above-described receptor affinity because of the absent left shift
in the relaxation response to ANP compared with BNP.
In the present study, BNP was a more effective inhibitor of
oxytocin-induced myometrial contractions than ANP; CNP had no effect on
contractility. Since CNP does not induce relaxation of guinea pig
myometrium, we conclude that GC-B activation is not involved in
mediating the studied response. Our relaxation-response curve to
natriuretic peptides did not show a larger affinity of ANP compared
with BNP, and anantin, a specific, competitive blocker of GC-A (Weber
et al., 1991), had no effect on the relaxation induced by both
peptides. This supports our conclusion that BNP-induced relaxation is
not mediated by GC-A activation. Based on these results, we suggest
that BNP induces relaxation by a mechanism that does not involve
activation of either GC-A or GC-B.
In contrast to relaxation, natriuretic peptides increase myometrial cGMP by GC-A activation. The functional presence of GC-A in myometrium obtained from pregnant guinea pigs and its higher affinity for ANP were demonstrated by the significantly larger increase in cGMP content induced by ANP compared with BNP. Furthermore, anantin significantly reduced the ANP- and BNP-mediated increase in myometrial cGMP content, suggesting that both peptides activate GC-A to increase cGMP. Additionally, CNP did not produce any significant change in myometrial cGMP content, suggesting that GC-B activation is not related to cGMP production in the pregnant guinea pig.
Although the time course of the natriuretic peptide-induced cGMP increase paralleled the relaxing effect of the peptides on myometrial contractility, anantin had no effect on natriuretic peptide-induced relaxation, while inhibiting the peptide-stimulated increase in cGMP. Additionally, inhibition of cGMP-dependent protein kinase with Rp-8-Br-cGMP also did not alter the relaxation response to BNP. These results suggest that the cGMP pathway does not mediate the relaxation induced by ANP and BNP. The conclusion that neither GC-A activation nor the resulting cGMP increase mediates BNP-induced relaxation is supported by the observation that whereas BNP-induced relaxation was greater than that induced by ANP, the stimulated increase in cGMP content was less. Therefore, we conclude that the relaxation of pregnant guinea pig myometrium to natriuretic peptides is mediated by activation of a receptor distinct from GC-A and GC-B, which lacks guanylate cyclase activity.
This conclusion is central to the support of our hypothesis of a role
for natriuretic peptides in the maintenance of uterine quiescence,
because it has been reported that the cGMP-PKG pathway was uninvolved
in the regulation of myometrial contractility during pregnancy (Word
and Cornwell, 1998). These authors found that cGMP analogs produced
only modest relaxation of myometrial preparations of rat uterus and
concluded that pregnant rat myometrium was insensitive to cGMP-induced
relaxation (Word and Cornwell, 1998). This interpretation is consistent
with our finding that cGMP analogs produced significant relaxation of
oxytocin-induced myometrial contractility only at high concentrations.
Furthermore, myometrial insensitivity to cGMP was explained because of
a pregnancy-induced decrease PKG expression and function (Word and
Cornwell, 1998). This line of evidence could be used to argue against a
role for natriuretic peptides in the maintenance of uterine quiescence
during pregnancy if their action was PKG mediated. However, the
interpretation of our results refute this argument since we document
that natriuretic peptide-induced relaxation occurs via a pathway that
does not involve either cGMP or PKG activation.
There is a minor concern regarding the experiments using Rp-8-Br-cGMP
to inhibit PKG activity. The effectiveness of this cGMP analog in
inhibiting the activity of the cGMP-dependent protein kinase is
supported by previous work (Butt et al., 1994; Patel and Diamond,
1997), and our experiments do demonstrate a decrease in PKG activity
after incubation with 30 µM Rp-8-Br-cGMP. However, we cannot exclude
the chance that the inhibition detected is explained by an effect of
the drug on PKG during the PKG assay after tissue homogenization rather
than its effect on the intact tissue. This possibility, however, does
not argue against our conclusion that natriuretic peptide-induced
relaxation does not occur trough the cGMP-PKG pathway, since the lines
of evidence presented supporting our conclusion are multiple and do not
only rely solely on the use of this cGMP analog.
The observation that cGMP analogs such as 8-Br-cGMP require a high concentration to produce relaxation in the myometrial strips should be carefully considered. It is difficult to interpret whether this is due to poor tissue permeability, or the minimal role of cGMP generated by the myometrium in relaxation. Furthermore, this may also indicate that although exogenous administration of cGMP causes relaxation, agonist-stimulation of endogenous cGMP is either not sufficient or is compartmentalized in the cell and is not a significant second messenger.
It has been reported that natriuretic peptides can have biological
actions not mediated by guanylate cyclase receptor activation and cGMP
production, but by a signaling activity via the natriuretic peptide
clearance receptor (Johnson et al., 1991; Hempel et al., 1998). This
signaling pathway does not lead to cGMP generation (Drewett et al.,
1992), and involves a pertussis toxin-sensitive G protein
(Anand-Srivastava et al., 1996; Murthy et al., 1998; Murthy and
Makhlouf, 1999). Activation of the clearance receptor inhibits
adenylate cyclase (Drewett et al., 1992), increases phospholipase C
activity (Anand-Srivastava and Trachte, 1993), and increases K+ outward conductance (Anand-Srivastava and
Trachte, 1993; Kanwal and Trachte, 1994). Although a relationship
between clearance receptor activation and myometrial
contraction/relaxation has not been shown, the presence of the
natriuretic peptide clearance receptor mRNA has been identified in the
rat myometrium (Vaillancourt et al., 1998). In the present study, we
report that cANP, a specific agonist of the clearance receptor (Maack
et al., 1987), does not inhibit oxytocin-induced contractility.
Although the presence of the clearance receptor in the myometrium of
the pregnant guinea pig has yet to be demonstrated, our studies do not
support a role for it (if present) in the inhibition of
oxytocin-induced contraction by natriuretic peptides. In addition,
neither PTX (a trimeric G protein inhibitor) nor TEA (a
K+ channel blocker) interfered with BNP-induced
relaxation of oxytocin-induced contractions, further supporting our
conclusion that natriuretic peptides do not activate the clearance
receptor to cause relaxation.
In conclusion, both BNP and ANP, but not CNP, inhibit oxytocin-induced contractions of the pregnant guinea pig myometrium. We propose that natriuretic peptide-induced relaxation is mediated by activation of a previously undescribed receptor molecule that lacks guanylate cyclase activity, and therefore does not involve the cGMP pathway. Our findings suggest that BNP has a greater efficacy in activating this receptor molecule than either ANP or CNP. We suggest that this pathway of relaxation is via a novel mechanism, independent of GC-A or CG-B activation, cGMP generation, or clearance receptor activation. Further studies are necessary to elucidate the molecular mechanism of natriuretic peptide-induced myometrial relaxation of the pregnant guinea pig myometrium.
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Footnotes |
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Accepted for publication December 21, 2000.
Received for publication August 21, 2000.
This study was supported by grants from the National Institutes of Health: Fogarty International Center 5 F05 TW05442-02 (to J.A.C.); HL49999 (to L.P.T.); and HL49041, HD24492, and HL51735 (to C.P.W.). This work was presented in part at the 47th Annual Meeting of the Society for Gynecological Investigation in Atlanta, GA, March 2000.
Send reprint requests to: Carl P. Weiner, M.D., Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Bressler Research Bldg., Room 11-033, 655 W. Baltimore St., Baltimore, MD 21201. E-mail: cweiner{at}umm.edu
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Abbreviations |
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NO, nitric oxide; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; GC-B, guanylate cyclase B; GC-A, guanylate cyclase A; CNP, C-type natriuretic peptide; 8-Br-cGMP, 8-bromoadenosine cGMP: PKG, cGMP-dependent protein kinase; cANP, atrial natriuretic peptide des-(Gln18, Ser19, Gly20, Leu21, Gly22)-fragment 4-23 amide; PTX, pertussis toxin; TEA, tetraethylammonium.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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a peptide antagonist of the atrial natriuretic factor (ANF). I. Producing organism, fermentation, isolation and biological activity.
J Antibiot
44:
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