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Department of Obstetrics and Gynecology, Perinatal Research Laboratories (H.I., I.M.B., R.R.M.), and Department of Meat/Animal Science (R.R.M.), University of Wisconsin, Madison, Wisconsin 53715; and the Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine (K.N.), Kyoto 606–01, Japan
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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sGC consists of 
- and ß-subunits (7) and is postulated to be a
cytosolic enzyme of vascular smooth muscle (VSM) rather than the
endothelium (3). sGC is stimulated directly by nitric oxide (NO), an
endothelium-derived vasodilator, produced by the calcium-sensitive
constitutive isoform endothelial NO synthase (eNOS) (8, 9). Recently,
we reported that expressions of eNOS protein (9) and messenger RNA
(mRNA; our unpublished data) in endothelial cells of the ovine uterine
artery (UA) are up-regulated during pregnancy and that endogenous and
exogenous NO increase the in vitro production of cGMP by
ovine UA (8).
In contrast to sGC, cGMP production by the enzyme pGC is mediated by specific natriuretic peptide membrane receptors (5) interacting with atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (10). Both ANP and BNP are secreted in an endocrine fashion from the heart (11) and are elevated during normal and hypertensive pregnancies (12, 13) and in distressed newborns (14). BNP also is secreted from human amnion, especially in the first and second trimesters (15, 16). CNP is produced by endothelial cells (10, 17) and may function in a paracrine fashion in addition to NO and prostacyclin, as an endothelium-derived vasodilator (18, 19). It is not known whether CNP is produced by ovine UA or systemic artery (SA) endothelium or whether it is augmented in pregnancy. Particulate guanylate cyclase A (pGC-A; i.e. ANP-A receptor) is the specific receptor for ANP and BNP, whereas particulate guanylate cyclase B (pGC-B; i.e. ANP-B receptor) is the specific receptor for CNP (5, 20, 21). Because the clearance receptor of natriuretic peptide (CR) has no GC domain (5, 7), it regulates local natriuretic peptide concentrations in the vasculature, which modifies exposure of pGC-A/B to their ligands (22, 23, 24).
Thus, both sGC and pGC pathways may have distinct cGMP production systems in the same vasculature. Moreover, as the production of their stimulants, i.e. NO via eNOS protein expression in ovine UA endothelium (9) and plasma ANP levels (12, 13, 25), are only elevated 2- to 5-fold in pregnancy, it is plausible that the sensitivity of the uterine vasculature to these substances is also elevated to explain the local 38-fold substantial elevation of uterine cGMP production (4). Accordingly, we hypothesize that sGC, pGC, and local CNP production are up-regulated in the UA during pregnancy. Our objectives were to evaluate 1) whether activities and/or protein expressions of sGC, pGC-A, and pGC-B as well as CNP production in UA are up-regulated during normal pregnancy; 2) whether CR protein expression in UA is down-regulated by pregnancy; 3) whether changes in pGC-A, pGC-B, and CR mRNA expression are also observed; 4) whether changes in GC and CR are specific to UA and the SA [renal (RA) and/or omental (OmA)]; and 5) whether alterations in GC pathways occur in both endothelium and VSM.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Guanylate cyclase activities in UA
Studies were performed on quadruplicate UA segments obtained
from five nonpregnant and four pregnant (120 ± 2 days’
gestation) ewes treated with pentobarbital sodium (40–50 mg/kg, iv).
These nonsurvival surgical procedures were approved by the University
of Wisconsin-Madison animal care and use committee. UA were placed in
oxygenated (95% O2-5% CO2) Krebs-Henseleit
buffer and divided into segments of similar weight (10–30 mg) and size
(1–3 mm) (9). The activities of sGC and pGC were estimated as the
production of cGMP (8). After 20- to 30-min preincubation (37 C),
segments of UA were incubated for 10 min (based on triplicate time
courses), either alone (control) or in the presence of maximal
stimulating doses of 100 µM sodium nitroprusside (SNP;
the NO donor used as a sGC stimulant), 1 µM human
ANP-(1–28) (the specific ligand of pGC-A), or 1 µM human
CNP-(1–22) (Peptide Institute, Minoh, Japan; the specific ligand of
pGC-B). Incubation times and maximal doses of each treatment were based
on our previous reports (8, 20, 21, 43). To clarify the effect of
simultaneous stimulation of sGC and pGC-A and to test for possible
synergy, cotreatment with 100 µM SNP and 1
µM ANP also was performed. Parallel experiments were
performed in the presence of 10 µM methylene blue (MB;
20- to 30-min preincubation) to specifically inhibit sGC (8). The
oxygenated Krebs-Henseleit buffer containing 100 µM
isobutylmethylxanthine for phosphodiesterase inhibition and 0.5% BSA
(crystallized) for natriuretic peptide delivery to the tissues. After
10-min incubation, the reaction was terminated by adding an equal
volume (0.5 ml) of ice-cold 10% trichloroacetic acid (TCA) and
freezing (-20 C). Segments of UA were homogenized and centrifuged
(1300 x g, 10 min), and pellets were dissolved in 2
N NaOH for protein determination. TCA was extracted from
the supernatant as previously described (9). Total cGMP content in the
medium plus ground tissue, divided by total tissue protein content, was
regarded as sGC and/or pGC activities after subtracting the time zero
content of cGMP (0.2–0.7 pmol/mg protein).
Tissue preparation for Western immunoblot analysis
Endothelium-derived protein and intact and denuded arteries
(VSM) were obtained and processed as previously described (9). They
were homogenized in a solubilizing buffer containing 150 mM
NaCl, 50 mM Tris-HCl, 10 mM EDTA (pH 7.4),
0.1% Tween-20, 0.1% ß-mercaptoethanol, 0.1 mM
phenylmethylsulfonylfluoride, 5 µg/ml leupeptin, and 5 µg/ml
aprotinin.
Western immunoblot analysis of sGC ß1-subunit
UA proteins from four nonpregnant and four pregnant (121 ±
3 days’ gestation) ewes were fractionated by 7.5% SDS-PAGE (50
µg/lane, 100 V, 2 h), along with positive controls (50 µg/lane
of ovine kidney protein) and Rainbow mol wt markers (Bio-Rad
Laboratories, Richmond, CA) as previously described (9).
Immunodetection was achieved using rabbit antisera raised against sGC
ß1-subunit (Cayman Chemical Co., Ann Arbor, MI; 1:1000, 16 h, 4
C) followed by the enhanced chemiluminescence (ECL) reagent detection
system with exposure to Hyperfilm (Amersham, Arlington Heights,
IL).
Western immunoblot analysis of pGC-A after
immunoprecipitation
Rabbit antirat pGC-A and pGC-B and C-terminal synthetic peptides
were donated by D. L. Garbers, Ph.D., University of Texas
Southwestern Medical Center, and were used as previously described
(26). Both intact and denuded UA from five nonpregnant and five
pregnant (126 ± 2 days’ gestation) ewes were studied. Intact RA
from four nonpregnant and four pregnant (125 ± 3 days’
gestation) ewes were studied as nonuterine SA. Arteries were
homogenized in solubilizing buffer (9), gently vortexed (16 h, 4 C) in
0.5% Triton X-100, and centrifuged (12,250 x g, 20
min, 4 C), and pGC-A antiserum (1:400) was added to 0.5 ml of this
fraction containing 5 mg total protein (16 h, 4 C). Protein A-Sepharose
suspension (16%) was added (30 µl) and vortexed (2 h, 4 C), followed
by centrifugation (600 x g). The pellet was washed
(three times) and fractionated by 6.5% SDS-PAGE (100 V, 2 h) with
positive controls (immunoprecipitant from ovine kidney) before transfer
to Immobilon-P membrane (30 V, 16 h). The same primary pGC-A
antiserum was used (1:2000, 16 h, 4 C), and ECL detection was
performed. The pGC-A C-terminal synthetic peptide (25 µg/ml) used to
raise the antiserum was used as a competitive negative control to show
the specificity of the antiserum (data not shown).
Western immunoblot analysis of CR
Intact UA were studied from nine nonpregnant and five pregnant
(124 ± 3 days’ gestation) ewes. Intact, denuded, and
endothelium-derived protein from UA were also studied from six
nonpregnant and six pregnant (125 ± 5 days’ gestation) ewes.
Intact RA and OmA, used as nonuterine SA, were studied from seven
nonpregnant and six pregnant (125 ± 5 days’ gestation) ewes.
Proteins were fractionated by 7.5% SDS-PAGE (20 µg/lane, 100 V,
2 h) with positive controls (ovine kidney protein) as previously
described (9). Mouse monoclonal antibody raised against bovine CR (23)
was used (1:1000, 1 h, room temperature), and ECL detection was
performed.
Immunohistochemical analysis of sGC ß1-subunit, pGC-B, and
CNP
Intact UA, RA, and OmA from 5 nonpregnant and 5 pregnant
(122 ± 6 days’ gestation) ewes were placed in 4% formaldehyde
(0.1 M sodium cacodylate buffer, pH 7.4), fixed overnight,
and embedded in paraffin (9). Rabbit anti-sGC ß1-subunit antisera
(Cayman Chemical Co.; 800:1), rabbit anti-pGC-B antisera (200:1) (26),
and mouse monoclonal anti-CNP antibody ascites (100:1) (27) were
applied to sections (1 h, room temperature). Staining was detected
using the avidin-biotin-peroxidase method (ELITE ABC, Vector
Laboratories, Burlingame, CA) with 3,3'-diaminobenzidine as previously
described (9). As negative controls for sGC ß1-subunit, normal rabbit
serum (1:800) or sGC ß1-subunit control peptide (Cayman Chemical Co;
100 µg/ml) was used to show antibody specificity. As negative
controls for pGC-B and CNP, the pGC-B C-terminal synthetic peptide (25
µg/ml) or CNP-(1–22) (100 µg/ml) was used with the primary
antibodies to demonstrate antibody specificity. As positive control
sections, ovine kidney (for sGC ß1-subunit and pGC-B) and ovine fetal
brain (for CNP) were used (data not shown). In each study group of
nonpregnant and pregnant ewes, immunostaining was performed
simultaneously. The density of the sGC ß1-subunit and pGC-B
immunostaining in VSM were evaluated with a microvideo system and
computer, using NIH Image version 1.55 software (28). The mean density
of immunostaining (pixel; arbitrary unit calculated by the image
analysis software) was quantified for 10 randomly chosen fields, with
3.6 x 103 µm2 VSM in each section. The
density of background staining in parallel negative control sections
was subtracted from the positive staining.
RT-PCR analysis of pGC-A, pGC-B, and CR mRNA: mass assay
Ovine pGC-A, pGC-B, and CR complementary DNA (cDNA)
partial clones were donated by P. Aldred, Ph.D., University of
Melbourne (Melbourne, Australia). Total RNA was extracted from intact
UA from eight nonpregnant and seven pregnant (125 ± 1 days’
gestation) ewes using a phenol-chloroform-isoamyl alcohol extraction
procedure, and pGC-A, pGC-B, and CR mRNA were quantified by coupled
RT-PCR amplification in a single tube as recently described (29, 30).
For mRNA quantification, total RNA (1 µg/tube) was incubated in a
50-µl final volume containing 1 x PCR buffer [2 mM
MgCl2; 10 nmol of each deoxy (d)-ATP, dCTP, dTTP, and dGTP;
30 pmol forward and reverse temperature-matched primers; 1 µl (2.5 U)
AMV reverse transcriptase; and 1 µl (5 U) Taq polymerase
transcriptase; all from Life Technologies, Grand Island, NY); RT
controls only contained Taq polymerase. Forward and reverse
primers, used for targeting amplification from part of the ovine pGC-A,
pGC-B, and CR protein-coding regions (31), were: pGC-A: forward,
5'-CCTGCAACCAAGACCA-3'; and reverse, 5'-CACAGTCGAGTTACGCAA-3'; pGC-B:
forward, 5'-AACACAACCTGAGCTATGC-3'; reverse, 5'-CATCTGTGCGAGCA TCC-3';
CR: forward, 5'-TACGTGAAGTACTCAGAG CTG-3'; reverse,
5'-AGTAATCACCAATAACCTCCTG-3'. The expected final products from ovine
pGC-A, pGC-B, and CR mRNA were 345, 432, and 192 bases, respectively.
The program used was annealing at 62 C for 10 min, reverse
transcription at 50 C for 10 min, denaturing at 94 C for 2 min, and
amplification for 28 cycles using 94 C for 30 sec, 55 C for 30 sec, and
72 C for 30 sec. Final products were extended to full length by
incubation at 72 C for 30 sec. Controls for each assay included total
RNA extracted from ovine kidney and a standard curve containing known
copy numbers of ovine pGC-A, pGC-B, and CR cDNA target sequences,
respectively. At the end of the assay, 10 µl of products were
separated on a 2% agarose Tris-acetate-cDNA gel and transferred to
MagnaGraph hybridization membrane (Molecular Separations, Westborough,
MA) for Southern blotting against probes (generated against pOpGC-A,
pOpGC-B, and pOCR using asymmetric PCR) (30, 31) encoding the
protein-coding sequences. After hybridization, membranes were washed
once in 2 x SSC (standard saline citrate)-0.1% SDS for 15 min
and twice in 0.1 x SSC-0.1% SDS (twice, 30 min each time) before
drying and direct exposure to a phosphorimager (BI screen, Bio-Rad
Laboratories, Hercules, CA; 15–30 min) for direct quantification
(Molecular Analysis version 1.4, Bio-Rad Laboratories). Data were
normalized to 28S ribosomal RNA and expressed as copy number per µg
RNA.
Statistical analysis
Values were expressed as the mean ± SE.
Mann-Whitney U test was used for RT-PCR data. Other statistical
analyses performed were Student’s t test (comparing two
means) or ANOVA, followed by Fisher’s protected least significant
difference test (comparing three or more means). P <
0.05 was regarded as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). sGC activity in pregnant UA treated
with 100 µM SNP was 61.4 ± 20.3 pmol/mg·10 min,
which was 47% higher (P < 0.05) than that in UA from
nonpregnant ewes (41.7 ± 8.2 pmol/mg·10 min). Activities of
pGC-A and pGC-B in UA from pregnant ewes, estimated by treatment with 1
µM ANP and 1 µM CNP, were 10.5 ± 3.1
and 8.1 ± 1.9 pmol/mg·10 min, which were 62% and 65% higher
(P < 0.05) than those in UA from nonpregnant ewes
(6.5 ± 1.7 and 4.9 ± 2.7 pmol/mg·10 min, respectively;
Fig. 1A). Thus, the potencies of GC activities in UA from pregnant ewes
were sGC >> pGC-A > pGC-B, and the sGC activity was 5.8- to
7.6-fold higher (P < 0.05) than that of either pGC-A
or pGC-B. Moreover, after treatment with 10 µM MB, an
inhibitor of sGC, the sGC activities in UA from pregnant and
nonpregnant ewes were suppressed from 61.4 ± 20.3 to 5.6 ±
4.4 (-91%) and 41.7 ± 8.2 to 18.8 ± 4.4 (-55%)
pmol/mg·10 min, respectively. The suppressive effect of MB in
pregnant ewes was stronger that that in nonpregnant ewes; we have no
explanation for this. MB treatment did not alter the activities of
pGC-A and pGC-B in pregnant or nonpregnant ewes (Fig. 1B). Production
of cGMP by UA from pregnant ewes after cotreatment with SNP (100
µM) and ANP (1 µM) was 81.7 ± 25.7
pmol/mg·10 min, which was slightly (18%), but not significantly,
higher (P > 0.05) than that of the summation of sGC
and pGC-A activities (69.4 pmol/mg·10 min, estimated individually;
black bar inset in Fig. 1C). Similarly, cGMP production by
UA from nonpregnant ewes with the same combination treatment was
42.2 ± 13.1 pmol/mg·10 min, which was also slightly (17%)
higher (P > 0.05) than that of the summation of sGC
and pGC-A activities (36.0 pmol/mg·10 min; black bar inset
in Fig. 1C). These data suggest no clear synergistic effect of sGC and
pGC-A in UA treated with ANP; 10 µM MB could only inhibit
34% and 58% of the response to SNP in nonpregnant and pregnant UA. We
have no explanation for these findings.
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). Positive
staining (brown) for sGC ß1 was detected mostly in the tunica media
(VSM) of UA, RA, and OmA in both groups. Negative controls, using
normal rabbit serum, showed greatly reduced staining. Additional
negative controls, using sGC ß1-subunit control peptide (100 µg/ml)
to absorb primary antibody, also showed greatly reduced staining (data
not shown). Positive sGC ß1-subunit staining in UA VSM was much
stronger in pregnant vs. nonpregnant ewes. Density of
positive sGC ß1-subunit immunostaining in UA of pregnant ewes was
27.6 ± 6.6 pixels, 128% higher (P < 0.05) than
that in UA from nonpregnant ewes (12.1 ± 1.9 pixel; Fig. 2).
These changes were specific to UA, as the densities of positive sGC
ß1-subunit immunostaining of RA and OmA from nonpregnant and pregnant
ewes were similar (RA, 12.1 ± 2.4 and 17.6 ± 4.6 pixels;
OmA, 6.0 ± 1.5 and 6.7 ± 1.0 pixels; P >
0.05).
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). As we had to load immunoprecipitant
from a large amount of tissue protein (5 mg), the 130-kDa bands were
somewhat broad and were not sharp. Specificity was demonstrated, as the
negative controls showed greatly reduced 130-kDa bands upon antibody
absorption with 25 µg/ml C-terminal peptides (data not shown).
Protein expression of pGC-A in intact UA from pregnant ewes was
2.4 ± 0.4 arbitrary units, which was 60% higher
(P < 0.05) than that from nonpregnant ewes (1.5
± 0.1 arbitrary units; n = 5). In contrast, the protein
expressions of pGC-A in intact RA from nonpregnant and pregnant ewes
were similar (1.8 ± 0.3 and 1.5 ± 0.2 arbitrary units,
respectively). To investigate the cellular distribution of pGC-A in UA,
Western analysis was performed after immunoprecipitation in intact UA
(UA+) and denuded UA (UA-) from pregnant ewes. Studies had to be
performed in this manner, because antiserum against rat pGC-A did not
work well for immunohistochemistry in ovine tissues, and it was not
possible to obtain enough endothelial cell protein for
immunoprecipitation. Protein expressions of UA+ and
UA- were similar (Fig. 3), suggesting the lack of
endothelial pGC-A receptor.
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). In contrast, no such dramatic
pregnancy-mediated difference in immunostaining was observed in
sections of RA or OmA. Negative controls, using the C-terminal
synthetic peptide (25 µg/ml) to absorb primary antibody, demonstrated
greatly reduced pGC-B staining. The mean density of positive pGC-B
immunostaining in UA VSM of pregnant ewes was 41.2 ± 1.4 pixels,
83% higher (P < 0.05) than that in UA from
nonpregnant ewes (22.5 ± 2.7 pixels). The densities of
positive immunostaining of nonpregnant and pregnant ewes were
similar (P > 0.05) for RA VSM (19.2 ± 3.0 and
20.1 ± 3.4 pixels) and OmA VSM (22.7 ± 4.9 and 20.0 ±
3.7 pixels). Immunohistochemical staining of pGC-B in tunica intima of
UA from pregnant ewes was stronger than that from nonpregnant ewes,
suggesting up-regulation of pGC-B protein in endothelial cells. By
contrast, the immunohistochemical staining of pGC-B in endothelium of
RA and OmA was unaltered by pregnancy.
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). Moreover,
positive CNP staining in UA endothelium was substantially stronger in
pregnant vs. nonpregnant ewes. In contrast, no such dramatic
pregnancy-mediated increase in endothelial CNP immunostaining was
observed in the sections of RA (data not shown). Negative controls,
using normal mouse ascites and 100 µg/ml human CNP-(1–22) to absorb
the primary antibody, greatly reduced staining.
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). In
contrast, the results of Western analysis of CR protein levels from
nonpregnant and pregnant ewes were similar (P > 0.05)
for intact RA (10.4 ± 1.9 and 9.5 ± 2.0 arbitrary units)
and intact OmA (7.1 ± 2.4 and 10.9 ± 4.9 arbitrary units).
In Fig. 7, we confirmed the linearity of
immunoblots of CR in UA+ from a nonpregnant ewe (r =
0.925; P < 0.025) and show a representative immunoblot
of CR for UA+, UA-, and UA
endothelium-isolated protein (Endo) from nonpregnant and pregnant ewes.
The CR protein expression in six nonpregnant and pregnant ewes (using
three different Western blots) also was calibrated by the CR protein
expression of ovine kidney standard (20 µg/lane). Expression of CR in
UA+, UA-, and Endo were similar within either
the nonpregnant or pregnant groups. However, CR protein expressions in
UA+ and Endo from pregnant ewes were -72% and -81%
lower (P < 0.05) than those in nonpregnant ewes. CR
protein expression in UA- from pregnant ewes was (-50%)
lower than that in nonpregnant ewes; however, this did not reach
statistical significance (P > 0.05).
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). Coupled RT-PCR followed by Southern
blot analysis of pGC-A, pGC-B, and CR in total RNA of UA and positive
control kidney showed the expected sized bands of 345, 432, and 192
bases, respectively (Fig. 8B). No signals were observed in
RT- (without RT) kidney total RNA. The copy number of
pGC-B mRNA/µg UA total RNA from pregnant ewes was 44% higher
(P < 0.05) than that from nonpregnant ewes (Fig. 8C);
CR mRNA per µg UA total RNA was reduced 34% (P <
0.05) by pregnancy. Consistent with the need to use immunoprecipitation
before performing Western analysis for GC-A protein, we detected too
few copies of pGC-A mRNA per µg UA total RNA to make definitive
comparisons. This low expression of pGC-A mRNA in UA was inconsistent
with the findings that the potency of GC activity in ovine UA was sGC
>> pGC-A > pGC-B and that both pGC-A activity and protein
expression were increased during pregnancy. We cannot eliminate the
possibility that ovine pGC-A mRNA is unstable or that pregnancy
up-regulated pGC-A protein expression at the posttranscriptional
level.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). We report the first direct evidence
in UA that both sGC and pGC-A/B activities/expressions and CNP levels
are increased during pregnancy. The potency of GC activities in UA from
nonpregnant and pregnant ewes was sGC >> pGC-A > pGC-B. This
is the first reported comparison of the potencies and expressions of
sGC with pGC-A/B in any vascular tissue. These mechanisms function in
addition to elevations in UA NO production (8) and eNOS expression (9)
during pregnancy. We also report the novel observation of a 34%
down-regulation of CR expression in UA endothelium and VSM during
pregnancy. These responses in sGC, pGC-A, pGC-B, and CR expressions
were specific to UA and were not observed in RA or OmA, suggesting the
potential involvement of both sGC and pGC-A/B pathways in modulating
the 30- to 50-fold increase in uteroplacental blood flow during
pregnancy (1, 2).
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-nitro-L-arginine methyl ester (an inhibitor of NOS)
directly into the uterine circulation of pregnant sheep decreases the
secretion of cGMP into the uterine venous effluent (4). In the present
study, we not only reveal that sGC activity is increased 48% during
pregnancy, but also that the expression of the sGC ß1-subunit protein
of UA, but not those of RA and OmA, increases 52–128%, suggesting
that both eNOS and sGC enzyme expressions are locally up-regulated in
uterine vasculature by pregnancy.
The enzyme sGC is a heterodimer consisting of - and ß-subunits and
cGMP production occurs upon binding of NO to its prosthetic group (7).
The antibody we used to detect sGC recognizes the ß1-subunit;
however, the isoform distribution of sGC in different vasculature is
not well defined, as at least two different sGCs, 1/ß1 and
2/ß1, are present in the rat renal vasculature, and only slight
ß2 expression was observed using PCR (33). In contrast, others could
not detect positive immunostaining in rat renal vessels using different
antibodies specific for 1-, 2-, ß1-, and ß2-subunits,
suggesting the possible presence of an as yet undefined sGC subunit
(34). Using immunohistochemistry, this increased UA sGC expression was
localized primarily to the VSM, demonstrating that sGC ß1-subunit is
not expressed to a great extent in the endothelium. By contrast, we
also have shown that the endothelium is the main source of eNOS
expression (9), establishing the specific cellular compartmentalization
for the regulation of VSM cGMP by endothelial NO. Therefore, pregnancy
up-regulates NO production by increasing eNOS expression (9) as well as
the sensitivity of the VSM to respond to NO by increasing the
expression of sGC in the UA VSM (Fig. 9
). These conclusions are in
contrast to the those obtained indirectly using in vitro
bioassays in studies of norepinephrine-precontracted UA in which
relaxation responses to SNP were similar in UA obtained from pregnant
vs. nonpregnant rats (35) and women (36). In contrast,
Weiner et al. reported that the relaxation response of the
guinea pig UA to SNP was reduced in pregnancy and suggested that
increased production of NO may down-regulate VSM sGC (37). Ours is the
first report demonstrating that pregnancy specifically increases sGC in
UA, but not SA, using direct measurement of both functional activity
and comparing this to protein expression.
The biologically active natriuretic membrane receptors, pGC-A
(i.e. ANP-A receptor) and pGC-B (i.e. ANP-B
receptor), constitute a major part of GC in the vasculature. ANP and
BNP stimulate pGC-A with a ligand receptor specificity of ANP 
BNP >>> CNP (5, 20). Maternal plasma ANP levels are increased (12, 13, 25) or unchanged (38, 39) during normal pregnancy. There are
several reports that demonstrated that pGC-A is present in the uterine
vasculature and has biological activity. Treatment of pregnant ewes
with ANP was reported to increase uteroplacental blood flow (40), to
antagonize the in vitro uterine vasoconstriction response of
angiotensin II (41), and in UA from nonpregnant women to cause in
vitro vasodilatation (41, 42).
The ligand selectivity for pGC-B (ANP-B receptor) is CNP >>>
ANP BNP (5, 20), and pGC-B has been reported to be present in
VSM (5, 43). The natriuretic peptide CNP was originally thought to be
present mainly in the central nervous system (10); however, CNP was
also recently reported to be secreted from endothelial cells and was
postulated to act in addition to PGI2 and NO as an
endothelium-derived vasodilator (10, 17, 18). Thus, in a fashion
analogous to NO/sGC in the vasculature, CNP and pGC-B were hypothesized
to constitute a local autocrine/paracrine vascular natriuretic peptide
system (5, 19, 44). Although the ovine CNP gene has not yet been
cloned, the structure of CNP is highly conserved among other species
(45). Accordingly, we used human CNP-(1–22) as a stimulant of pCG-B in
ovine UA, which is similar to rat, mouse, and porcine CNP. We report
herein the first observation that pregnancy increases CNP levels in UA,
but not SA, endothelial cells, as demonstrated by immunohistochemistry.
Recently, Okahara et al. reported that shear stress
dramatically augmented CNP gene expression in cultured endothelial
cells (46). As uteroplacental blood flow increases 30- to 50-fold
during pregnancy (1, 2), it is possible that shear stress augments CNP
expression in UA endothelium.
In the present study, UA pCG-A and pCG-B activities were increased 60–65% during pregnancy. We also report herein for the first time that UA expressions of pGC-A protein, pGC-B protein, and pGC-B mRNA were up-regulated by 60%, 83%, and 44%, respectively, during pregnancy. Because the pGC-A antibody did not work well for immunohistochemistry in ovine tissues, we could only indirectly determine the cellular distribution of pGC-A in ovine UA. Using immunoblot analysis after immunoprecipitation of pGC-A of UA+vs. UA- from pregnant ewes, we showed no significant difference, suggesting mainly VSM pGC-A expression. Immunohistochemical staining of pGC-B was substantially up-regulated in both endothelial and VSM cells in UA during pregnancy. This pregnancy-associated up-regulation of pGC-B protein expression in UA was paralleled by mRNA levels. By contrast, immunohistochemical staining for pGC-B in endothelium and VSM of the systemic vasculature (RA and OmA) was unaltered by pregnancy. Thus, the local pGC-A/B protein expressions and pGC-A/B activities in ovine UA were specifically up-regulated during pregnancy, strongly suggesting that natriuretic peptides/pGC-A/B pathways in addition to NO/sGC contribute to the increase in uterine production of cGMP during pregnancy.
Another natriuretic peptide receptor, CR, has no GC domain and plays a role in modulating the local concentration of natriuretic peptides, as it functions as a clearance receptor (5, 22, 23, 24), with a ligand selectivity of ANP > CNP > BNP (5, 20). Although we did not observe a statistical difference between endothelial and VSM CR protein expressions, the major amount of CR in UA is in the VSM, because endothelial-derived protein is less than 1% of the total protein from vasculature. CR protein expression was down-regulated by 34% during pregnancy in both endothelium and VSM of UA, but not in RA or OmA; RT-PCR analysis confirmed CR mRNA down-regulation in UA. Brandt et al. reported that infusion of C-ANF, the specific ligand that blocks CR, elevated plasma CNP concentrations, demonstrating that CR does indeed modulate local natriuretic peptide levels (24). Although it is difficult to estimate the exact increase in local natriuretic peptide (ANP, BNP, and CNP) concentrations by this down-regulation of CR in pregnant UA, it is expected that the physiological rate of cGMP production will be dramatically increased, as this CR down-regulation is associated with the up-regulation of pGC-A/B activity/protein as well as local levels of CNP in the endothelium. Kishimoto et al. reported that ß2-adrenergic stimulation (23) or treatment with 8-bromo-cGMP caused down-regulation of VSM CR (22). As we have demonstrated a 38-fold increase in cGMP production from the gravid ovine uterus (4), a 2-fold increase in plasma and urinary cGMP levels (9), and that basal cGMP production by UA is increased 110% during pregnancy, it is plausible that the locally increased cGMP produced by sGC and pGC-A/B may underlie the decrease in CR expression in UA during pregnancy. These findings suggest the possibly important role of the natriuretic peptide-pGC pathway in UA via local pGC up-regulation and CR down-regulation in the local regulation of uteroplacental blood flow during pregnancy.
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2 Visiting scientist. Current address: Department of Obstetrics and
Gynecology, Kyoto University Graduate School of Medicine, 54 Shogoin
Kawahara-cho, Sakyo-ku, Kyoto 606-01, Japan.
Received December 29, 1997.
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pregnant; +, P < 0.05,
methylene blue effect.
