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Department of Obstetrics and Gynecology, University of Glasgow (J.F.T., A.J.T., J.E.N.), Glasgow, Scotland G31 2ER; Departments of Obstetrics and Gynecology, Pediatrics, and Animal Sciences, University of Wisconsin, Perinatal Research Laboratories (H.I., R.R.M.), Madison, Wisconsin 53715; Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine (K.N.), Kyoto 606-01, Japan; and Maternal and Fetal Research Unit, Department of Obstetrics and Gynecology, GKT School of Medicine, St. Thomas’ Hospital (J.S.C., L.P., R.M.T.), London, United Kingdom SE1 7EH
Address all correspondence and requests for reprints to: Ronald R. Magness, Ph.D., Perinatal Research Laboratories, Department of Obstetrics and Gynecology, University of Wisconsin, 7E Meriter Hospital/Park, 202 South Park Street, Madison, Wisconsin 53715. E-mail: rmagness{at}facstaff.wisc.edu
Abstract
The role of cGMP in the regulation of human myometrial smooth muscle contractility is at present unclear. cGMP can be synthesized by a cytoplasmic, soluble guanylate cyclase (sGC), which is stimulated by nitric oxide and carbon monoxide, and by particulate membrane-bound GC, which are activated by natriuretic peptides. The aim of this study was to determine whether sGC or pGC are present in nonpregnant and pregnant human myometrium, and whether the activity and expression of these enzymes and the cGMP content change during pregnancy and with labor. Myometrium was obtained from nonpregnant women (n = 12) and pregnant women who were preterm (25–34 wk gestation; n = 12), term (>38 wk) not in labor (n = 14), or term in active labor (n = 12). The cGMP content in myometrium obtained from preterm deliveries was significantly higher than that in tissue obtained from nonpregnant women and decreased at term, especially in laboring groups. Protein and mRNA for sGC, particulate GC-A, GC-B, and the clearance receptor were detected in human myometrium. cGMP in pregnant human myometrium, however, appears to be produced predominately by sGC and possibly by GC-B, as GC-A was only weakly expressed. sGC activity was greater in myometrium from preterm (nonlabor) deliveries compared those taken at term (in labor), but was down-regulated compared with activity in nonpregnant myometrium. Neither atrial natriuretic peptide nor C-type natriuretic peptide (agonists for GC-A and GC-B, respectively) altered contractility in vitro of myometrium from women at term (not in labor). We conclude that the cGMP/guanylate cyclase system in human myometrium is gestationally regulated and potentially plays an important role in mediating quiescence during early pregnancy. A reduction in cGMP availability may contribute to the switch to contractile activity at term.
THE PRECISE MECHANISMS that maintain uterine quiescence during pregnancy and initiate uterine contractions during labor are unknown. It is hypothesized that agents that promote myometrial relaxation, such as cGMP (1, 2, 3, 4), are highly expressed during early pregnancy but fall before labor onset and so contribute to the maintenance of quiescence during pregnancy and the onset of labor. Indeed, studies of animal pregnancy have reported an increase in endogenous myometrial cGMP content substantially above the nonpregnant level at midgestation before a reduction at term (5, 6, 7). A rise in myometrial cGMP content can be mediated by an increase in the activity of guanylate cyclases, which exist as cytosolic (soluble) and membrane-bound (particulate forms), and/or by decreased phosphodiesterase activity.
Soluble guanylate cyclase (sGC) is a heterodimer of 
- and
ß-subunits (8) and is activated when nitric oxide (NO)
or carbon monoxide binds to the heme site of the molecule (9, 10). Particulate guanylate cyclases (pGC) consist of a single
polypeptide chain, membrane-spanning protein with an intracellular GC
domain and are activated via membrane receptor interaction with
natriuretic peptides (11, 12). The pGC that predominant in
smooth muscle are GC-A and GC-B. Other isoforms have recently been
described in a variety of cell types (GC-C, GC-D, GC-E, GC-F, and GC-F)
(13), but ligands have only been identified for three of
these. The three major natriuretic peptides, atrial natriuretic peptide
(ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide
(CNP) (14, 15). The ligand selectivity for GC-A is
ANP
BNP>>CNP, and that for GC-B is CNP>>ANPBNP (14).
The ligands for the third receptor GC-C are not natriuretic peptides,
but include guanylin and uroguanylin (16, 17). There is
also a natriuretic peptide clearance receptor (CR) that does not have a
guanylate cyclase domain and is considered to modulate the natriuretic
peptide concentration through local metabolism (15).
Human amnion cells secrete BNP (18), and during pregnancy the concentration of BNP in amniotic fluid is approximately 50 times that in the systemic circulation (18, 19, 20) until the third trimester of human pregnancy when the concentration in amniotic fluid dramatically declines (18). In human myometrium obtained during the second trimester both GC-A and GC-B mRNA have been detected, and cGMP generation has been shown to be augmented by ANP and BNP (21). This suggests that natriuretic peptides may contribute to myometrial quiescence in pregnancy. In the pregnant guinea pig, the midgestation rise in cGMP content has been attributed to increased activity of GC-A (17). However, in human myometrium, there has been no systematic study of the pGC or sGC enzyme systems in relation to cGMP production in pregnancy, although some studies have focused on the assumption that NO is the primary modulator of myometrial cGMP (22, 23, 24, 25, 26) and hence quiescence.
The aims of this study were to determine 1) whether sGC, GC-A, GC-B, and CR are present in myometrium from nonpregnant and pregnant women; 2) whether the expression or activity of sGC and pGC, and hence cGMP production, are increased during pregnancy but down-regulated before or during labor; and 3) whether natriuretic peptides modulate myometrial contractility of pregnant human myometrium in vitro.
Subjects and Methods
Subjects and collection of tissue
A total of 50 women were recruited to this study. Biopsies of myometrium (taken from the lower third of uterus) were obtained from nonpregnant premenopausal women undergoing hysterectomy for benign disease (n = 12), and biopsies were taken during elective cesarean section from the upper margin of the lower uterine segment incision from women undergoing preterm delivery who were not in labor (25–34 wk; n = 12) and women at term but not in labor (>38 wk gestation; n = 14). Lower segment myometrium was obtained from 12 additional women who were delivered by emergency cesarean section during active labor. Preterm myometrium was obtained from women who required elective cesarean section for maternal disease, placenta praevia, or fetal distress of unknown etiology. Women were excluded from the study if they had multiple pregnancy, evidence of active infection, or had had induction of labor. Biopsies for protein and mRNA studies were divided; one half was fixed in 10% neutral buffered formalin and embedded in paraffin, and the other half was snap-frozen in liquid nitrogen and stored at -70 C. Samples to be used for the evaluation of tension were placed immediately into ice-cold physiological salt solution (PSS). Informed consent was obtained at the time of recruitment, and the study was approved by the ethical committees of Glasgow Royal Infirmary National Health Service Trust and of the Guys and St. Thomas’ Hospital Trust, United Kingdom.
Assay for myometrial cGMP content
Frozen tissue was weighed, cut into small pieces, and homogenized in 5 vol ice-cold 5% (vol/vol) trichloroacetic acid (TCA). The tube used for homogenization was washed in 0.5 ml 5% TCA to remove any residual cGMP. The homogenates were centrifuged at 600 x g for 5 min at 4 C. TCA was extracted from the reaction mixture using a 1:1 mixture of trioctylamine (27, 28, 29) (Sigma, St. Louis, MO) and 1,1,2-trichlorofluoroethane (Sigma). The cGMP content (expressed as picomoles per frozen weight of tissue in grams) of the aqueous phase was determined using a commercially available cGMP enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI). The intra- and interassay coefficients of variation of this assay, as determined by the manufacturer, are 10–15%.
sGC activity in whole tissue homogenates
Frozen myometrium or ovine kidney, used as a positive control for this assay, was thawed, weighed, and homogenized with a Polytron homogenizer (Brinkmann Instruments, Inc. (Westbury, NY), in 5 vol ice-cold 20 mM potassium phosphate buffer, pH 7.4, containing 5 mM EDTA, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 10 mg/ml pepastatin A (Sigma), and 100 mg/ml phenylmethylsufonylfluoride. Homogenates were centrifuged at 600 x g for 5 min at 4 C, and supernatants were collected for assay. Supernatants were added to 100 µl GC reaction mixture (50 mM Tris-HCl buffer (pH 7.6) containing 0.5 mM isobutylmethylxanthine, 0.1% crystalline BSA, 4 mM MgCl2, 1 mM GTP, 15 mM creatinine phosphate, and 3 U creatinine phosphokinase. All procedures were carried out at 4 C. The sGC activity was estimated from the rate of cGMP production (27, 28, 29, 30). Assays were carried out in triplicate. Assay mixtures were incubated for 10 min at 37 C (based on time courses) either with or without (basal activity) a maximally stimulatory dose (100 µM) of the NO donor sodium nitroprusside, which stimulates sGC (21, 27, 29). The reaction was terminated by adding 10% TCA, which was extracted from the reaction mixture using a 1:1 mixture of trioctylamine and 1,1,2-trichlorofluoroethane (Sigma). The cGMP content of the aqueous phase was determined using the cGMP enzyme immunoassay kit (Cayman Chemical Co.) described above. The protein content of the supernatants was determined using a modified Lowry assay procedure (Bio-Rad Laboratories, Inc., Hercules, CA). sGC activity (femtomoles per 10 min/µg protein) was determined as cGMP content in supernatants at 10 min minus cGMP content in time zero supernatants divided by total protein content. As basal activity in myometrium was similar among all three groups of pregnant women (preterm, n = 6; term not in labor, n = 6; term in labor, n = 6), the mean basal activity was calculated from all samples from the three pregnant groups (n = 18). Basal sGC activity in myometrial from nonpregnant women (n = 5) was also determined. Maximal NO-stimulated sGC activity was compared between nonpregnant and pregnant groups, and with the respective mean basal sGC activity. ANP- and CNP-stimulated cGMP productions were also measured in whole tissue homogenates of nonpregnant human myometrium, and basal and SNP- and ANP-stimulated GC activities were measured in whole tissue homogenates of ovine kidney; the latter was used as a positive control.
pGC activity in myometrial membrane protein preparations
Frozen myometrium or ovine kidney was thawed and homogenized with a Polytron homogenizer in 10 vol ice-cold 20 mM potassium phosphate buffer, pH 7.4, containing 5 mM EDTA, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 10 mg/ml pepasatin A (Sigma), and 100 mg/ml phenylmethylsufonylfluoride. The homogenates were centrifuged at 6,000 x g (5 min) and 100,000 x g (30 min). The resultant particulate fractions were washed in homogenization buffer and spun at 100,000 x g (30 min). The crude membrane fractions obtained (20–50 mg/tube) were added to 100 µl GC assay mixture (21, 29). Assays were carried out in triplicate. Assay mixtures were incubated for 10 min at 37 C (based on time courses) either with or without (basal activity) a maximally stimulatory dose (1 µM) of ANP and CNP (21, 29). The reaction was terminated by the addition of 10% TCA, and the TCA was extracted as before. cGMP measurements were carried out as described above, and ligand-selective stimulation of pGC activity, GC-A (measured in the presence of ANP), and GC-B (measured in the presence of CNP) were calculated (femtomoles per 10 min/µg protein) (21). Basal GC-A and GC-B activities were expressed as an average of all the pregnant myometrium or nonpregnant myometrial samples. Stimulated activity was compared among the three pregnant groups and the nonpregnant group, and with respective mean basal activity values. Basal and ANP-stimulated cGMP productions were also measured in membrane preparations from ovine kidney as a positive control for GC-A activity.
Immunolocalization of sGC, GC-B, CR, and GC-C
Immunohistochemistry was performed as previously described (14, 18, 20). Myometrial biopsies were fixed in 10% neutral buffered formalin and embedded in paraffin as we have previously described (28, 29, 30, 31). Sections (5 µm thick) of paraffin-embedded tissue were cut and mounted on saline-coated slides, heated to 60 C for 30 min, cleared in xylene, and rehydrated in a graded alcohol series. Sections were incubated with 1) a polyclonal antibody raised against the ß1-subunit of sGC (Cayman Chemical Co.) diluted 1:750; 2) polyclonal antibodies raised against the C-terminal peptides of rat GC-A and GC-B (donated by Prof. D. L. Garbers, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX) diluted 1:500; and 3) a mouse monoclonal antibody against bovine CR diluted 1:500 (32). Antibody binding was detected using an antirabbit IgG or antimouse IgG peroxidase kit (Vectastain Elite ABC kit, Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions.
Negative control slides were incubated without primary antibody (peptide for preabsorbed antibody controls was unavailable). Immunoreactivity was localized using 1 mg/ml diaminobenzidine tetrahydrochloride (Sigma) and 0.02% hydrogen peroxide in 50 mM Tris-HCl, pH 7.6. Sections were washed in distilled water, counterstained with Harris hematoxylin, and mounted in DPX (BDH, Glasgow, Scotland).
Identification of GC-A, GC-B, and CR by RT-PCR
RNA was extracted from snap-frozen myometrial tissue using a commercially available RNA guanidium isothiocyanate extraction kit (Hybaid Ribolyser Green kit; Thermo Hybaid-Ashford, Middlesex, UK). RNA samples (5 µg) were denatured at 70 C for 5 min and cooled to 37 C. RT was carried out at 37 C for 60 min in a final volume of 20 µl using random hexanucleotide primers (0.2 µg), 1 x RT buffer, 10 mM dithiothreitol, 1 mM dNTP, 40 U Moloney murine leukemia virus reverse transcriptase, and 1 U ribonuclease inhibitor. The reaction was terminated by heating to 90 C for 5 min. PCR amplification was carried out using specifically designed human PCR primers for GC-A, GC-B, and CR (33). PCR was performed on the RT products (cDNA) in a reaction mixture containing 1.5 mM MgCl2, 0.2 mM dNTPs, 125 ng of each primer, and 1 U DNA polymerase (AmpliTaq, Perkin-Elmer Corp., Norwalk, CT; final volume, 25 µl). Amplification for ß2-microglobulin was carried out on the samples as a parallel control. The amplification profile used for the detection of GC-A mRNA was 35 cycles at 93 C for 20 sec, 55 C for 30 sec, 73 C for 60 sec, and 1 cycle at 73 C for 10 min. The profile used for the detection of GC-B and CR mRNA was 1 cycle at 94 C for 2 min, 55 C for 1 min, and 72 C for 1 min and 30 sec; 33 cycles at 94 C for 50 sec, 55 C for 1 min, and 72 C for 1 min and 30 sec; and 1 cycle at 94 C for 50 sec, 55 C for 1 min, and 72 C for 5 min. Aliquots (15 µl) of the PCR products were separated by horizontal gel electrophoresis on a 1% agarose-ethidium bromide gel.
Effect of natriuretic peptide on spontaneous and oxytocin-augmented human myometrial contractions in vitro
Tension was measured as we have previously described
(34). Human myometrium taken at the time of term nonlabor
elective cesarean section was dissected longitudinally with fiber
structure into a maximum of four similar strips (
10 x 4
x 2 mm), which were each mounted vertically in a 10-ml organ bath; one
end was tied by a thin cotton thread to a fixed support, and the other
to an isometric transducer (Linton, Norfolk, UK). The strips were
maintained in PSS (119 mM sodium chloride, 25
mM sodium bicarbonate, 5.5 mM glucose, 1.18
mM potassium phosphate, 1.17 mM magnesium
sulfate, 4.7 mM potassium chloride, 2.5 mM
calcium chloride, and 26 µM EDTA, pH 7.4) and were
stretched to achieve 3 g resting tension. During an equilibration
period of 90–120 min, regular spontaneous contractions occurred in
approximately 90% of the strips. Oxytocin (1 nmol/liter;
Sigma, Poole, UK) was then added to all the strips (strips
that did not develop regularly or in some cases any contractions with
oxytocin stimulation were discarded), control contractions were
recorded for 40 min, and recording was continued during a second
experimental period. One strip in each protocol was exposed to PSS
during the experimental period to serve as a time control. The
remaining strips were exposed to different concentrations of either
ANP, which has a high ligand selectivity for GC-A (0.01, 1.0, or 100
nmol/liter), or CNP, which has a high ligand selectivity for GC-B
(0.01, 1.0, or 100 nmol/liter).
Myometrial force development was recorded and analyzed using MacLab hardware with Chart version 3.2 software [AD Instruments (Europe) Limited, Hastings, UK] (34). Parameters measured included maximum tension, development of each contraction, the contraction integral (total tension developed in each contraction, and contraction interval time from the origin of one contraction to the next). The mean and SEM were calculated for each of these parameters over the control and experimental periods. As a measure of mean myometrial activity per unit time, the mean integral tension (grams per sec) was obtained for each period (the sum of the integrals during the period divided by the duration of the period) and was taken from the onset of the first contraction to the onset of the contraction when the solution was changed or the end of the experiment. The response of the myometrium to the addition of each compound was expressed as a percentage of the mean integral tension during the first 60-min control period for the same strip.
Statistical analysis
Experimental data were analyzed using ANOVA with Fisher’s protected least significant difference Wilcoxon signed-rank analysis or Dunnett’s correction for multiple comparisons (tension protocols). P < 0.05 was taken to be significant.
Results
cGMP content myometrium from nonpregnant and pregnant women
cGMP content was highest in myometrium obtained from preterm (not
in labor) pregnancies (137 ± 45 pmol/g wet wt) and was
significantly greater than in nonpregnant myometrium (33 ± 4
pmol/g) and term in labor (47 ± 10 pmol/g) samples. cGMP content
of myometrium obtained at term not in labor (70 ± 19 pmol/g)
tended to be lower than that measured in myometrium obtained from
preterm deliveries, although this did not reach statistical
significance. The cGMP content of myometrium obtained from term
deliveries during labor was not significantly different from that of
myometrium from term deliveries before labor (Fig. 1
).
|
Total unstimulated production of cGMP (basal GC activity) by
myometrium from nonpregnant subjects (14.7 ± 5.2 fmol/µg·10
min) protein was not significantly different from that of myometrium
obtained from pregnant women (11.9 ± 1.4 fmol/µg·10 min; Fig. 2). However, sGC activity in myometrium
obtained from nonpregnant women estimated by stimulation with 100
µM SNP was significantly higher than that of myometrium
obtained from both preterm and term pregnant women (Fig. 2).
SNP-stimulated myometrial sGC activity was significantly higher than
basal GC activity in myometrium from the four groups of women with the
exception of myometrium from term deliveries before labor
(16.7 ± 4.4 fmol/µg·10 min). Myometrial sGC activity in
preterm pregnant women (142 ± 107 fmol/µg·10 min) was
significantly higher than that in myometrium from term pregnant women
with and without labor.
|
Initially, a comparison of sGC, GC-A, and GC-B activities in whole
tissue homogenates was planned (Fig. 3A).
However, we observed that neither GC-A or GC-B activity was detectable
in nonpregnant human myometrium tissue homogenate, although
ANP-stimulated cGMP production was measurable in whole ovine kidney
homogenate (Fig. 3B). Experiments subsequently performed in
membrane-rich protein preparations, however, clearly demonstrated ANP-
and CNP-stimulated cGMP production (GC-A and GC-B activities,
respectively) in ovine kidney membrane preparations and to a much
lesser degree in nonpregnant human myometrial preparations (Figs. 3 and 4).
|
|
). ANP-stimulated GC-A activity was undetectable in pregnant
human myometrium, but was present in nonpregnant myometrium (13.7
± 5.4 fmol/µg·10 min). In contrast, GC-B activity (as estimated by
CNP-stimulated cGMP synthesis) was significantly higher in membrane
preparations of myometrium from both nonpregnant and pregnant women
compared with the appropriate basal activities (Fig. 4). GC-B activity
was similar in all three groups of pregnant human myometrium, although
cGMP production tended to be lower in myometrium from women at term in
labor. In addition, although GC-A activity was significantly lower in
pregnant myometrium compared with nonpregnant myometrium, there was
no difference in GC-B activity in nonpregnant vs. pregnant
tissues (Fig. 4). Myometrial tissue expression of the ß1 subunit of soluble guanylate cyclase
Immunostaining for sGC (Fig. 5a) was evident in myometrial
smooth muscle and vascular smooth muscle cells. The intensity of sGC
staining supported the profile of sGC activity described above.
Immunostaining intensity was greatest within myometrial smooth muscle
and vascular smooth muscle of myometrium from nonpregnant women and was
considerably less intense in myometrial smooth muscle cells in tissue
obtained from pregnant women.
|
GC-A and GC-B immunostaining were detected in myometrial smooth
muscle from both nonpregnant and pregnant subjects (Fig. 5, C and D)
compared with negative controls (data not shown). For GC-A, the degree
of staining was weaker in pregnant myometrial tissue compared with
nonpregnant myometrium, and the majority of myometrial smooth muscle
cells lacked any staining, although there were some areas where some
myometrial smooth muscle cells (myocytes) exhibited moderate staining
(Fig. 5C). GC-B was detected in pregnant myometrium at a similar
intensity to that of nonpregnant myometrium, but the staining was
concentrated in discrete regions associated with myometrial smooth
muscle cells (Fig. 5D).
Immunohistochemistry for natriuretic peptide CR
In the nonpregnant group, natriuretic peptide CR immunoreactivity
(Fig. 5B) was present within myometrial myocytes and vascular smooth
muscle cells and was absent from endothelial cells when viewed at a
higher magnification (data not shown). In myocytes of myometrium
obtained from pregnant women there was very little staining compared
with negative controls.
RT-PCR for GC-A, GC-B, and CR mRNA expression
Figure 6 shows the expression of the
three receptors in myometrium from nonpregnant women and pregnant women
at the different stages of gestation studied. The sizes of the PCR
products were confirmed as: GC-A, 209 bp; GC-B, 600 bp; CR, 510 bp; and
ß2-microglobulin, 332 bp. All three receptors
were expressed in myometrial tissue from nonpregnant women;
i.e. all of the biopsies from nonpregnant women expressed
GC-A, whereas expression of GC-B and CR occurred in five of six and
four of six of the samples. It is notable that GC-B was absent from all
preterm pregnant samples, but was observed in term myometrium with and
without active labor (four of six and eight of eight, respectively).
There was widespread presence of GC-A and CR in preterm (four of six),
term nonlaboring (eight of eight), and term laboring (five of six)
myometrial samples.
|
As might be anticipated from the low expression of GC-A and GC-B
and the absent/small increase in cGMP observed in response to ANP and
CNP, respectively, these peptides had no significant effect on any
parameter of oxytocin-induced myometrial tension development in
isolated strips from term, nonlaboring women (Table 1).
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The role of cyclic nucleotides in the maintenance of uterine quiescence during pregnancy is unresolved. Evidence from animal and human studies suggests that the adenylate cyclase/cAMP signaling pathway is gestationally regulated (35, 36, 37), but the importance of cGMP in myometrial smooth muscle relaxation during pregnancy remains to be clarified. Functional studies in the rat and guinea pig report that myometrium is relatively insensitive to cGMP (38, 39, 40), particularly in comparison to vascular smooth muscle (41). Other studies, in contrast, demonstrate that cGMP can modulate myometrial contractility (42) and that ANP inhibits spontaneous contractions in myometrial strips from estrogen-treated (43) and pregnant rats (44). Several reports indicate that exogenous NO (via cGMP) can inhibit spontaneous contractions of pregnant rat and human uteri at term (1, 2, 4, 44, 45, 46), but whether endogenous NO is an important modulator of myometrial tone remains controversial (47).
Our data demonstrate a clear increase in the cGMP content of human myometrium obtained at the time of preterm cesarean section compared with myometrium procured from nonpregnant women. In addition, they indicate that cGMP levels are lower in myometrium obtained at term at the onset of labor. If in the pregnant human, as in the rat (1, 4, 48), myometrium is less sensitive to the relaxant effect of cGMP at term, this would compound the decrease in cGMP content at term and shift the balance further toward enhanced myometrial contractility at labor onset. Therefore, cGMP may play an important role in maintaining myometrial quiescence during pregnancy, and a decrease in cGMP at term may contribute to the increase in uterine contractility during active labor.
We report the first direct evidence that functional sGC protein is present in pregnant and nonpregnant human myometrium. Although no differences in basal activity of the enzyme in myometrial tissue from pregnant and nonpregnant women were observed, significant differences were present in the SNP-mediated, maximally stimulated activity of sGC, as measured by the rate of cGMP production in the presence of a phosphodiesterase inhibitor. Stimulated sGC activity was lower at term and in labor than in preterm uterine muscle and therefore mirrored the gestational changes in cGMP content in pregnant human myometrium. The lower values in the term pregnant compared with nonpregnant uterine tissue are not immediately explicable, but might also suggest an important role for cGMP in nonpregnant myometrium. As the measurement in vitro was carried out in the presence of a phosphodiesterase inhibitor, underlying variability in the activity of endogenous cGMP phosphodiesterases would be masked. Indeed, there is evidence that myometrial cAMP phosphodiestrase 4 is gestationally and hormonally regulated (49, 50, 51). It is plausible, therefore, that in addition to a reduction of sGC activity during pregnancy, there is also a concomitant attenuation of phosphodiesterase activity that would result in a net gain in available cGMP and a higher intracellular cGMP content in the early pregnant state compared with nonpregnant myometrium. More in vivo studies of cGMP phosphodiesterase in myometrium are necessary to clarify this possibility. Assuming that sGC plays an important role in the regulation of myometrial cGMP, the question of the endogenous stimulant arises. Although early evidence (45, 46, 52, 53) was strongly supportive of a role of NO in control of myometrial tone, the importance of this radical to uterine quiescence in human pregnancy remains questionable (26, 47, 48). Moreover, the greater effect of the NO donor, SNP, on cGMP in the nonpregnant myometrial biopsies compared with pregnant samples would suggest reduced sensitivity to this agonist in pregnancy and might argue against a predominant role for NO. Alternatively, carbon monoxide may also regulate human sGC activity (10), although the carbon monoxide/hemoxygenase system has not been extensively studied in pregnancy.
The increase in cGMP content in pregnancy could also be potentially accounted for by enhanced GC-A or GC-B activity; however, activities of these enzymes in the human myometrium were very low (>20-fold less) compared with the ovine kidney preparation studied, which may mitigate against a major role. Recently, GC-A activity has been shown to be significantly raised in myometrium from term pregnant guinea pigs (17), but in complete contrast we found GC-A activity and protein was low or undetectable in preterm or term (with or without labor) pregnant human myometrium, although GC-A mRNA expression was observed by RT-PCR in myometrium from all three groups of pregnant women. Our previously published data from women at earlier gestation than those studied herein showed higher GC-A activity, and taken together these data might suggest a fall in activity very early in pregnancy (21). The discrepancy between GC-A activity and protein expression with the mRNA is likely to reflect poor translation and/or low mRNA expression, as 35 cycles of amplification were required to obtain GC-A PCR product. Overall, our data support a previous study that reported reduced uterine GC-A activity, protein, and mRNA expression in the pregnant rat (54).
CNP-stimulated GC-B activity (and GC-B protein), however, was present in pregnant human myometrium, as also reported in the rat (54), but this was different from the guinea pig myometrium, where GC-B activity, similarly stimulated by CNP, is reported to be absent. However, there was no indication of any pregnancy-associated increase in the activity of the enzyme, as the degree of CNP-stimulated cGMP production over basal levels in myometrium from pregnant biopsies was similar to that of nonpregnant human myometrium. However, despite the demonstration of CNP-induced cGMP synthesis, GC-B mRNA expression could not be detected in preterm myometrium, and conflicts with the presence of CNP-induced cGMP production, albeit low, in myometrium. We have no definite explanation for this observation, except for the possibility that CNP may not solely activate GC-B in myometrial tissue. Recently, other receptor-linked particulate guanylate cyclases have been reported in nonmyometrial tissues (8, 13, 54, 55). Therefore, we hypothesize that CNP may activate one of these complexes, or alternatively, that there is a hitherto undiscovered GC-associated receptor in human myometrium that has a great affinity for CNP than ANP. The lack of response to ANP compared with the demonstrated CNP-induced cGMP synthesis might also be related to the presence of the CR in pregnant human myometrium, which has a greater affinity for ANP than CNP (14, 32).
ANP or CNP did not affect spontaneous or agonist-augmented contractions in term myometrium in vitro. There is much evidence to support the loss of prorelaxation pathways in human myometrium during late pregnancy and the onset of labor (56), and our data would further support these observations. In term pregnant rat myometrium there is evidence that relaxation to cGMP is greatly reduced compared with vascular smooth muscle (41) and is insensitive to physiological concentrations of ANP (43), although this is in contrast to another study that reported substantial relaxation of myometrium from mid and term gestation rats (44). In future studies it would be of interest to investigate responses to the natriuretic peptides in preterm biopsies. However, the remarkably low activities of the particulate GCs that we observed might suggest that if present at all the relaxation might be only modest.
A potential limitation of our study is the comparison of nonpregnant myometrial and term pregnant myometrial (with or without labor) biopsies, as theoretically these biopsies may have been taken from different sites of the uterus. However, the nonpregnant myometrial biopsies were taken from the lower third of the uterus and should be comparable to the samples taken from the lower segment at term nonlabor. The myometrial biopsies obtained from women in active labor could originate from the upper region of the cervix, but tension studies in vitro clearly demonstrate that term active labor biopsies are indeed contractile, and myometrial smooth muscle, not cervical, in origin (57). All of the pregnant myometrial samples used in this study were taken from the lower segment, and although such samples are contractile in nature, it cannot be assumed that they are the same as fundal biopsies. We suggest, however, that as the fundal myometrium is associated with force generation, any changes in the guanylate cyclase system(s) reported in the lower segment samples will be magnified in fundal myometrium.
In conclusion, the current studies suggest that cGMP content is gestationally regulated in human myometrium and decreases at term at the time of labor onset, and that sGC and possibly CNP-mediated pGC may be involved. It is highly unlikely that ANP or BNP, although present in high concentrations in amniotic fluid (18) and also a stimulator of GC-A, plays a major role. An elevated myometrial cGMP content during early pregnancy may help to maintain the uterus in a quiescent state, and the down-regulation in sGC responsiveness that occurs at term may assist in the preparation of the myometrium for active labor. The lack of any ANP- or CNP-induced relaxation in term human myometrium in vitro also supports the conclusion that cGMP systems are reduced in preparation for the onset of labor.
Acknowledgments
We acknowledge Cindy Goss for her help in preparing this manuscript for publication.
Footnotes
This work was supported in part by a grant from the Medical Research Council (G94 37277); Tommy’s Campaign (Reg. Charity 1060508); NIH Grants-in-Aid HL-49210, HD-33255, HL-57653, and HD-38843; and a Research Fellowship from the Uehara Memorial Foundation of Japan.
Abbreviations: ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide; CNP, C-type natriuretic peptide; CR, clearance receptor; GC, guanylate cyclase; NO, nitric oxide; pGC, particulate membrane-bound guanylate cyclase; PSS, physiological salt solution; sGC, soluble guanylate cyclase; TCA, trichloroacetic acid.
Received February 22, 2001.
Accepted August 31, 2001.
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