Am J Physiol Heart Circ Physiol
282: H273–H280, 2002.
Differential responses of newborn pulmonary arteries
and veins to atrial and C-type natriuretic peptides
JEAN-FRANCOIS TOLSA,1 YUANSHENG GAO,2 FRED C. SANDER,2
ANNE-CHRISTINE SOUICI,1 ADRIEN MOESSINGER,1 AND J. USHA RAJ2
1
Neonatal Research Laboratory, Division of Neonatology, Department of Pediatrics, Centre
Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland; and 2Department of Pediatrics,
Harbor-University of California Los Angeles Medical Center, Torrance, California 90509
Received 12 January 2001; accepted in final form 4 September 2001
smooth muscle; relaxation; natriuretic peptide receptor; perinatal; pulmonary circulation
ATRIAL AND C-TYPE
natriuretic peptides (ANP and CNP)
are 28- and 22-amino acid peptides, respectively. ANP
induces diuresis and natriuresis, whereas CNP has
minimal effects on diuresis and natriuresis. Both peptides induce vasodilation. However, studies suggest
that ANP is more potent in arteries, whereas CNP is
more potent in veins (17, 20, 28, 48, 49). ANP is
secreted primarily by atrial myocytes in response to
local stretch and acts in an endocrine fashion (40). CNP
acts in a paracrine fashion (15, 23) and is expressed
widely in the central nervous system, with the highest
concentrations found in the cerebellum. Studies show
that CNP may act as a neurotransmitter to coordinate
Address for reprint requests and other correspondence: J.-F. Tolsa,
Division of Neonatology, Dept. of Pediatrics, Univ. Hospital CHUV, 1011
Lausanne, Switzerland (E-mail: [email protected]).
http://www.ajpheart.org
central aspects of salt and water balance and blood
pressure (7, 23, 25, 39, 47). Outside the central nervous
system, CNP is produced mainly by the vascular endothelium (18). Studies suggest that CNP may be released from the endothelium in response to agonists
such as ACh and bradykinin (50) and various cytokines
and growth factors such as transforming growth factor-␣ and tumor necrosis factor-␣ (33).
Two distinctive types of natriuretic peptide receptors, designated NPR-A and NPR-B, mediate actions of
ANP and CNP, respectively. NPR-A is activated with
high affinity by ANP, and CNP is a specific agonist of
the NPR-B (23, 24, 27). Both NPR-A and NPR-B possess guanylyl cyclase activity. Upon binding to these
receptors, ANP and CNP elevate the intracellular
cGMP content, which in turn mediates the biological
actions of these peptides (1, 23).
The role of ANP and CNP in the perinatal pulmonary circulation is not well established. ANP is a potent dilator of pulmonary arteries of the fetus and the
newborn (28). In humans, ANP is detectable in fetal
plasma. The plasma ANP level is higher in early postnatal life than in the adult (41, 42). In the pig, ANP
causes relaxation of pulmonary arteries at all ages, but
the relaxation is greater at 6 and 17 days of age (28).
Plasma CNP levels are increased in adult patients with
cor pulmonale (8), raising the possibility that CNP may
be a circulating hormone involved in the regulation of
cardiovascular function. In experimented animals, the
administration of CNP induces vasodilation of both
arteries and veins (48). In the adult rat, CNP is a
slightly less potent dilator of pulmonary arteries than
ANP (20). In the pig at various ages, CNP is a poor
dilator of pulmonary arteries (28).
Previous studies by others and us show that, in
perinatal lungs, pulmonary veins exhibit considerable
vasoreactivity in response to various stimuli (2, 11, 12,
36, 37, 43, 52). Veins contribute substantially to total
pulmonary vascular resistance (37, 38). More recently,
it has been shown that CNP relaxed fetal ovine pulmonary veins better than pulmonary arteries (26). ReThe costs of publication of this article were defrayed in part by the
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0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society
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Tolsa, Jean-Francois, Yuansheng Gao, Fred C.
Sander, Anne-Christine Souici, Adrien Moessinger,
and J. Usha Raj. Differential responses of newborn pulmonary arteries and veins to atrial and C-type natriuretic peptides. Am J Physiol Heart Circ Physiol 282: H273–H280,
2002.—Atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) are important dilators of the pulmonary
circulation during the perinatal period. We compared the
responses of pulmonary arteries (PA) and veins (PV) of newborn lambs to these peptides. ANP caused a greater relaxation of PA than of PV, and CNP caused a greater relaxation
of PV than of PA. RIA showed that ANP induced a greater
increase in cGMP content of PA than CNP. In PV, ANP and
CNP caused a similar moderate increase in cGMP content.
Receptor binding study showed more specific binding sites for
ANP than for CNP in PA and more for CNP than for ANP in
PV. Relative quantitative RT-PCR for natriuretic peptide
receptor A (NPR-A) and B (NPR-B) mRNAs show that, in PA,
NPR-A mRNA is more prevalent than NPR-B mRNA,
whereas, in PV, NPR-B mRNA is more prevalent than
NPR-A mRNA. In conclusion, in the pulmonary circulation,
arteries are the major site of action for ANP, and veins are
the major site for CNP. Furthermore, the differences in
receptor abundance and the involvement of a cGMP-independent mechanism may contribute to the heterogeneous effects
of the natriuretic peptides in PA and PV of newborn lambs.
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NATRIURETIC PEPTIDES AND PULMONARY VASOACTIVITY
sponses of newborn pulmonary veins to ANP and CNP
have not been well characterized. We postulate that
ANP and CNP may participate differently in the regulation of pulmonary vascular tone in the neonatal
period. In the present study, we have compared the
responses of pulmonary arteries and veins of newborn
lambs to ANP and CNP. Furthermore, the mechanisms
underlying the different responses were investigated.
MATERIALS AND METHODS
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Twenty-five newborn lambs (7–12 days old, either sex)
from Nebeker Ranch (Lancaster, CA) were used. They were
anesthetized with ketamine hydrochloride (30 mg/kg im) and
killed with an overdose of pentobarbital sodium. The lungs
were immediately removed, and fourth-generation pulmonary arteries and veins (outside diameter 3.0–3.5 and 2.5–
3.0 mm, respectively) were dissected free of parenchyma and
cut into rings (length 3 mm). In some vessels the endothelium
was removed mechanically by inserting the tips of a watchmaker’s forceps in the lumen of the vessel and rolling it back
and forth on saline-loaded filter paper. Removal of endothelium was confirmed for each vessel ring by lack of relaxation
to ACh (3 ⫻ 10⫺5 M; see Ref. 13).
Organ chamber study. Vessel rings were suspended in
organ chambers filled with 10 ml of modified Krebs-Ringer
bicarbonate solution [composition (in mM): 118.3 NaCl, 4.7
KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, and
11.1 glucose] maintained at 37 ⫾ 0.5°C and aerated with 95%
O2-5% CO2 (pH ⫽ 7.4). Two stirrups passed through the
lumen suspended each ring. One stirrup was anchored to the
bottom of the organ chamber, and the other was connected to
a strain gauge (model FT03C; Grass Instrument, Braintree,
MA) to measure the isometric force (45).
At the beginning of the experiment, each vessel ring was
stretched to its optimal resting tension. This was achieved by
step-wise stretching, in 0.1-g increments, until the active
contraction of the vessel ring to 100 mM KCl reached a
plateau. The optimal resting tension of pulmonary arteries
with and without endothelium was 0.29 ⫾ 0.05 and 0.26 ⫾
0.04 g/mm2 cross-section area of smooth muscle (CSASM),
respectively (n ⫽ 7 for each group). The optimal resting
tension of pulmonary veins with and without endothelium
was 0.13 ⫾ 0.04 and 0.15 ⫾ 0.03 g/mm2 CSASM, respectively
(n ⫽ 7 for each group). The resting tension of pulmonary
arteries was significantly different from that of veins (P ⬍
0.05). However, there is no significant difference in the resting tension between vessels with and without endothelium
(P ⬎ 0.05). The vessels were allowed to equilibrate at their
optimal resting tension for 1 h. Next, indomethacin (10⫺5 M;
an inhibitor of cyclooxygenase; see Ref. 46) was administered
to exclude the possible involvement of endogenous prostanoids (12). Our previous study shows that there is a basal
release of PGE2 and PGI2 in newborn ovine pulmonary veins
without endothelium (12). Hence, an inhibition of the production of these dilator prostaglandins may contribute to the
increased tone in endothelium-denuded veins caused by indomethacin. There is also a basal release of PGE2 and PGI2
in pulmonary arteries of newborn lambs. The lack of effect of
indomethacin on arterial tone may be partially due to the fact
that the arteries were less sensitive to PGE2 and PGI2 (12).
Effects of ␣-ANP-(1O28) (10⫺12 to 10⫺7 M), CNP-22 (10⫺12
to 10⫺7 M), and exogenous nitric oxide (10⫺7 M) were determined in pulmonary arteries and veins preconstricted with
endothelin-1 (3 ⫻ 10⫺9 to 2 ⫻ 10⫺8 M). In some experiments,
1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 3 ⫻ 10⫺5
M; an inhibitor of soluble guanylyl cyclase; see Ref. 14) was
added to differentiate between relaxation mediated by particulate guanylyl cyclase vs. soluble guanylyl cyclase (14). To
test whether or not a cGMP-independent mechanism may be
involved, some vessels were pretreated with 8-bromo-cGMP
(8-BrcGMP), a cell membrane-permeable analog of cGMP
(29), at a concentration that causes near maximal relaxation
(3 ⫻ 10⫺4 M; see Ref. 13). The vessels were then constricted
with endothelin-1, and the effects of ANP and CNP were
studied.
Determination of CSASM. The isometric tension of pulmonary arteries and veins of newborn lambs was standardized
to CSASM, as described earlier (11). Briefly, the total crosssection area of vessels (CSAT) was obtained first using the
following formula: CSAT ⫽ wet weight of tissue (mg) ⫼
density of tissue (mg/mm3) ⫼ optimal length of the tissue
(mm). The tissue density was measured by dividing the
blotted wet weight of the tissue by its volume. The volume
was determined by measuring the volume of Krebs-Ringerbicarbonate solution displaced by the vessels after placing
them in a 1-ml graduated cylinder with an accuracy of 0.01
ml. The optimal length was determined with the aid of a
magnifying eyepiece and a micrometer with an accuracy of
0.01 mm by measuring the distance between two stirrups
passed through the lumen of the vessel ring under their
optimal resting tension.
After CSAT was determined, the ratio of CSASM to CSAT
(CSASM/CSAT) was obtained by counting the total area and
the area occupied by smooth muscle cells on a histological
transverse section of the pulmonary arteries (5 ␮m thick)
under a microscope. The histological sections were treated
with either hematoxilin and eosin stain or Van Giesson stain
to differentiate between smooth muscle cells and other components. The CSASM/CSAT obtained from hematoxylin and
eosin stains and those from Van Giesson staining were averaged. The mean values were multiplied by the CSAT to obtain
the smooth muscle cross-sectional area of the tissue.
RIA of the intracellular content of cGMP. Rings of ovine
pulmonary arteries and veins (without endothelium) were
incubated in 10 ml of modified Krebs-Ringer bicarbonate
solution (37°C, 95% O2-5% CO2) containing indomethacin
(10⫺5 M) and isobutyl methylxanthine (10⫺3 M). These inhibitors were used to eliminate the involvement of cyclooxygenase products and phosphodiesterases, respectively (12,
34). In some experiments, ODQ (3 ⫻ 10⫺5 M) was used to
determine whether or not soluble guanylyl cyclase was involved (14).
After 45 min of equilibration, ANP (10⫺7 M), CNP (10⫺7 M)
or nitric oxide (10⫺7 M) was added to the incubation vials.
Two minutes after the administration of ANP or CNP and 1
min after nitric oxide was added, the tissues were freezeclamped rapidly and thawed in TCA (6%). Preliminary studies showed that the maximal accumulation of cGMP occurred
at 2 min in response to ANP and CNP and at 1 min in
response to nitric oxide. The tissues were then homogenized
in glass with a motor-driven Teflon pestle, sonicated for 5 s,
and centrifuged at 13,000 g for 15 min. The supernatant was
extracted with 4 vol of water-saturated diethyl ether and
lyophilized; the pellets were weighed. The lyophilized samples were resuspended in 0.5 ml of sodium acetate buffer
(0.05 M, pH 6.2), and their intracellular content of cGMP was
determined using a cGMP kit (Biomedical Technologies,
Stoughton, MA). The content of cGMP is expressed as picomoles per milligram protein. The protein concentrations of
the vessels were determined by the Bradford method using
BSA as the standard (6).
Receptor binding. Receptor bindings for ANP and CNP
were determined using a method similar to that described by
NATRIURETIC PEPTIDES AND PULMONARY VASOACTIVITY
AJP-Heart Circ Physiol • VOL
94°C and then 31 cycles of 30 s at 94°C, 45 s at 55°C, and 60 s
at 72°C. Preliminary studies showed that 31 cycles were in
the linear range of the PCR reaction and that the ratio for
18S primer to 18S competimer produced similar yields for the
18S internal standard, NPR-A mRNA from pulmonary arteries, and NPR-B mRNA from pulmonary veins. PCR products
were separated on 1% agarose gels (containing 0.08%
ethidium bromide) and quantified by densitometry using an
Eagle Eye II Still Video System (Stratagene). The quantities
of mRNA for NPR-A and NPR-B were expressed as relative
units to the 18S used.
Preparation of nitric oxide. A gas bulb sealed with a silicone rubber injection septum was filled with nitric oxide from
a cylinder (Union Carbide, Chicago, IL). An appropriate
volume (2.5 ml) was removed with a syringe and injected in
another gas bulb filled with 250 ml of distilled water, which
had been gassed with helium for over 3 h, giving stock
solutions of nitric oxide of 4.2 ⫻ 10⫺4 M. The concentrations
of nitric oxide in the stock solutions were determined by
indirectly measuring the nitrate, which was converted from
nitric oxide using an assay kit (Cayman Chemical, Ann
Arbor, MI; see Ref. 22). The final concentrations of nitric
oxide in the organ chambers were calculated based on the
ratio of the dilution.
Drugs. The following drugs were used (unless otherwise
specified, all were obtained from Sigma, St. Louis, MO):
␣-ANP-(1O28) (Peninsula Laboratories), 8-BrcGMP, CNP-22
(Peninsula Laboratories), endothelin-1 (American Peptide,
Sunnyvale, CA), indomethacin, isobutyl methylxanthine, and
ODQ. ODQ was dissolved in DMSO (final concentrations
⬍0.2%). Preliminary experiments showed that DMSO at the
concentration used had no effect on contraction to endothelin-1 and had no effect on relaxation induced by ANP and
CNP in pulmonary vessels of newborn lambs. Indomethacin
(10⫺5 M) was prepared in equal molar Na2CO3. This concentration of Na2CO3 did not significantly affect the pH of the
solution in the organ chamber. The other drugs were prepared using distilled water.
Data analyses. Data are shown as means ⫾ SE. When
mean values of two groups were compared, Student’s t-test
for unpaired observations was used. When the mean values
of the same group before and after stimulation were compared, Student’s t-test for paired observations was used. The
comparison of the mean values of more than two groups was
made using the one-way ANOVA test and the Student-Newman-Keuls test for post hoc testing of multiple comparisons.
All analyses were performed using a commercially available
statistics package (SigmaStat; Jandel Scientific, San Rafael,
CA). Statistical significance was accepted when the P value
(two tailed) was ⬍0.05. In all experiments, n represents the
number of animals.
RESULTS
Morphological data. Morphological data of pulmonary arteries and veins of newborn lambs are shown in
Table 1. The wet weight and optimal length of the rings
of pulmonary arteries and veins were significantly different. The density of these tissues was comparable.
The CSAT of pulmonary arteries was significantly
greater than that of pulmonary veins. The CSASM-toCSAT ratio was significantly different between pulmonary arteries and veins. However, there was no significant difference in these values between vessels with
and without endothelium.
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Perreault et al. (35). The membrane preparations of pulmonary arteries and veins (without endothelium) were obtained
by homogenizing the tissues in 5 vol of ice-cold buffer containing 50 mM Tris 䡠 HCl (pH 7.4), 2 mM dithiothreitol (DTT),
10 mM EDTA, 10 ␮g/ml leupeptin, 10 ␮g/ml antipain, 10
␮g/ml pepstatin, 10 ␮g/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 500 g for 10 min at 4°C. The supernatant was
centrifuged at 100,000 g for 1 h at 4°C, and the resulting
pellets were resuspended in a buffer similar to that used for
tissue homogenization with the addition of 0.2% Triton
X-100. Protein concentrations of the preparations were assessed by the Bradford method using BSA as the standard (6).
Binding assay was carried out in an assay mixture (total
volume: 100 ␮l) containing 50 mM Tris 䡠 HCl (pH 7.4), 2 mM
DTT, 10 mM EDTA, 10 ␮g/ml leupeptin, 10 ␮g/ml antipain,
10 ␮g/ml pepstatin, 10 ␮g/ml aprotinin, 0.5 mM PMSF, 10
mg/ml BSA, 5 mM MgCl2, 10–500 pM 125I-labeled ␣-ANP(1O28) (sp act 1,189 Ci/mmol; Peninsula Laboratories, Belmont, CA) or 10–500 pM 125I-labeled [Tyro]CNP-22 (sp act
2,226 Ci/mmol; Peninsula Laboratories), and 30 ␮g membrane protein. Triplicate samples were incubated for 60 min
at 30°C. At the end of the incubation period, the bound tracer
was separated from the free tracer by rapid filtration through
1% polyethylenamine-treated Whatman GF/C glass filters.
Filters were washed with 3 ml of cold Tris 䡠 HCl buffer (50
mM). The radioactivity associated with the filters was determined in an automatic ␥-counter (model 1470; Wallac, Gaithersburg, MD). Nonspecific binding for ANP and CNP was
determined in the presence of 1 ␮M of unlabeled ␣-ANP(1O28) or unlabeled [Tyro]CNP-22, respectively. Specific
binding was calculated as total binding minus nonspecific
binding.
Relative quantitative RT-PCR for NPR-A and NPR-B
mRNAs. Total RNA was extracted from pulmonary arteries
and veins using Trizol reagent (Life Technologies, Grand
Island, NY) according to the manufacturer’s protocol. The
mRNA reverse transcription was started by adding 2 ␮l RNA
to a 11.5 ␮l buffer containing 250 ng random hexamers and
10 unit RNase inhibitor (Life Technologies), incubating at
70°C for 10 min, and quick chilling on ice. Next, 4.0 ␮l of 5⫻
first strand buffer (Life Technologies), 2.0 ␮l DTT (0.1 M), 1.0
␮l dNTP (10 mM), 0.5 ␮l RNase inhibitor (20 U/␮l), and 1 ml
Moloney leukemia virus reverse transcriptase (200 U/␮l; Life
Technologies) were added. The reaction mixture was incubated at 42°C for 1 h and at 90°C for 10 min. The yielded
cDNA products were amplified by PCR with sense and antisense primers.
The sense and antisense primers for NPR-A were 5⬘-AAGAGCCTGATAATCCTGAGTACT-3⬘ (1302–1325: accession
no. NM_12613) and 5⬘-TTGCAGGCTGGGTCCTCATTGTCA-3⬘
(1752–1729: accession mo. NM_12613), respectively. The sense
and antisense primers for NPR-B were 5⬘-AACGGGCGCATTGTGTATATCTGCGGC-3⬘ (730–756: accession no. M26896) and
5⬘-TTATCACAGGATGGGTCGTCCAAGTCA-3⬘ (1421–1395:
accession no. M26896), respectively (19, 23).
Relative quantitative (RQ) RT-PCR was performed on a
Stratagene (La Jolla, CA) thermal cycler (RoboCycler Gradient 96) according to a protocol by Ambion (Austin, TX).
Briefly, a 50-␮l reaction mixture contains 5 ␮l 10⫻ ThermalAce Buffer (Invitrogen, Carlsbad, CA), 1.0 ␮l 50⫻ dNTPs (10
mM each; Invitrogen), 20 units ThermalAce DNA Polymerase (Invitrogen), 2.0 ␮l cDNA, 25 pM each for NPR-A sense
and antisense and for NPR-B sense and antisense, 1.0 ␮l
Universal 18S primer, and 1.5 ␮l 18S competimers (Ambion).
The mixture was initially subjected to heating for 2 min at
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NATRIURETIC PEPTIDES AND PULMONARY VASOACTIVITY
Table 1. Morphological data of pulmonary arteries
and veins of newborn lambs
Pulmonary Arteries
With
Without
endothelium endothelium
Tissue weight,
mg
Lo, mm
Density, mg/
mm3
CSAT, mm2
CSASM/CSAT
H&E stain
Van Giesson
stain
Pulmonary Veins
With
endothelium
Without
endothelium
18.1 ⫾ 2.5 19.6 ⫾ 3.1 7.75 ⫾ 0.41* 7.17 ⫾ 0.50*
2.82 ⫾ 0.19 3.07 ⫾ 0.18 2.33 ⫾ 0.07* 2.36 ⫾ 0.11*
1.01 ⫾ 0.03 1.09 ⫾ 0.02 1.06 ⫾ 0.03 1.12 ⫾ 0.03
2.46 ⫾ 0.17 2.02 ⫾ 0.31 0.77 ⫾ 0.04* 0.75 ⫾ 0.06*
0.36 ⫾ 0.02 0.35 ⫾ 0.02 0.25 ⫾ 0.03* 0.28 ⫾ 0.01*
0.37 ⫾ 0.01 0.35 ⫾ 0.02 0.27 ⫾ 0.02* 0.28 ⫾ 0.02*
Organ chamber studies. Experiments were carried
out in the presence of indomethacin to exclude the
involvement of endogenous prostaglandins (13). Under
basal conditions, indomethacin (10⫺5 M) had no effect
on pulmonary arteries but caused a greater increase in
the resting tone of pulmonary veins without endothelium than that of pulmonary veins with endothelium
[0.39 ⫾ 0.07 g/mm2 CSASM (n ⫽ 13) vs. 0.14 ⫾ 0.06
g/mm2 CSASM (n ⫽ 6); P ⬍ 0.05]. Before testing the
effect of various vasodilators, pulmonary arteries and
veins were constricted with endothelin-1 (3 ⫻ 10⫺9 to
2 ⫻ 10⫺8 M) to a similar tension (Table 2). At the
concentrations of endothelin-1 used, the arteries with
and without endothelium contracted to 73.9 ⫾ 8.5 and
72.6 ⫾ 6.1% of their maximal responses, respectively
(n ⫽ 6 for each group); the veins with and without
endothelium contracted to 82.1 ⫾ 6.5 and 75.3 ⫾ 8.1%
of their maximal responses, respectively (n ⫽ 6 for each
group).
ANP and CNP induced concentration-dependent relaxations of pulmonary arteries and veins. ANP-in-
duced relaxation was greater in arteries than in veins,
whereas CNP-induced relaxation was greater in veins
than in arteries, with or without endothelium (Fig. 1).
In vessels without endothelium, nitric oxide (10⫺7 M)
induced a greater relaxation of pulmonary veins than
that of arteries. Relaxation to nitric oxide was inhibited by ODQ (3 ⫻ 10⫺5 M), an inhibitor of soluble
guanylyl cyclase (14). In contrast, relaxation induced
by ANP (10⫺7 M) or CNP (10⫺7 M) was not affected
significantly by ODQ (Fig. 2). In some experiments, the
effects of ANP, CNP, and nitric oxide were examined in
pulmonary vessels without endothelium pretreated
with 8-BrcGMP (3 ⫻ 10⫺4 M). In the presence of
8-BrcGMP, a cell membrane-permeable analog of
cGMP (29), higher concentrations of endothelin-1 (10⫺8
to 2 ⫻ 10⫺8 M) were used to raise the vessel tension to
a level similar to that of control vessels (Table 3). After
treatment with 8-BrcGMP, relaxation of pulmonary
Table 2. Tension in pulmonary arteries and veins
increased by endothelin-1 before determination of
relaxing effects of ANP and CNP
Increase in Tension, g/mm2 CSASM
Pulmonary
arteries
Pulmonary
veins
Vessels for ANP study
With endothelium
Without endothelium
1.57 ⫾ 0.20
1.46 ⫾ 0.27
1.63 ⫾ 0.18
1.55 ⫾ 0.22
Vessel for CNP study
With endothelium
Without endothelium
1.67 ⫾ 0.23
1.57 ⫾ 0.21
1.58 ⫾ 0.21
1.51 ⫾ 0.25
Values are expressed as means ⫾ SE; n ⫽ 7 for each group. ANP,
atrial natriuretic peptide; CNP, C-type natriuretic peptide. The
increase in tension was obtained with endothelin-1 at 3 ⫻ 10⫺9 M for
veins without endothelium and 6 ⫻ 10⫺9 M for the other groups.
AJP-Heart Circ Physiol • VOL
Fig. 2. Relaxation of PA (A) and PV (B) without endothelium of
newborn lambs induced by ANP (10⫺7 M), CNP (10⫺7 M), and nitric
oxide (10⫺7 M) under control conditions, in the presence of 1H[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 3 ⫻ 10⫺5 M), or in
the presence of 8-bromo-cGMP (8-BrcGMP; 3 ⫻ 10⫺4 M). Vessels
were preconstricted to a similar tension with endothelin-1 (3 ⫻ 10⫺9
to 2 ⫻ 10⫺8 M). Data are shown as means ⫾ SE; n ⫽ 6 for each group.
*Significantly different from control (P ⬍ 0.05).
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Values are expressed as means ⫾ SE; n ⫽ 7 animals for each
group. Lo, optimal length of the vessel ring; CSASM/CSAT, ratio of the
cross-sectional area occupied by smooth muscle to the total crosssectional area determined by transverse histological section treated
with hematoxylin and eosin (H&E) or Van Giesson stain. * Significantly different from pulmonary arteries (P ⬍ 0.05).
Fig. 1. Relaxation of pulmonary arteries (PA) and veins (PV) of
newborn lambs with endothelium (E⫹) or without endothelium (E⫺)
induced by atrial natriuretic peptide (ANP; A) and C-type natriuretic
peptide (CNP; B). Tissues were preconstricted to a similar tension
with endothelin-1 (3 ⫻ 10⫺9 to 6 ⫻ 10⫺9 M). Data are shown as
means ⫾ SE; n ⫽ 7 animals for each group. *Significant difference
between PA and PV (P ⬍ 0.05).
NATRIURETIC PEPTIDES AND PULMONARY VASOACTIVITY
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Table 3. Tension in pulmonary arteries and veins
increased by endothelin-1 before determining the
effects of ODQ and 8-BrcGMP on relaxing
effects induced by ANP, CNP, and nitric oxide
Increase in Tension, g/mm2 CSASM
Pulmonary
arteries
Pulmonary
veins
Vessels for ANP study
Control
ODQ (3 ⫻ 10⫺5 M)
8-BrcGMP (3 ⫻ 10⫺4 M)
1.49 ⫾ 0.25*
1.53 ⫾ 0.21*
1.63 ⫾ 0.13‡
1.56 ⫾ 0.27†
1.64 ⫾ 0.25†
1.60 ⫾ 0.22§
Vessel for CNP study
1.56 ⫾ 0.30*
1.61 ⫾ 0.26*
1.50 ⫾ 0.17‡
1.59 ⫾ 0.20†
1.60 ⫾ 0.14†
1.49 ⫾ 0.31§
Vessels for nitric oxide study
Control
ODQ (3 ⫻ 10⫺5 M)
8-BrcGMP (3 ⫻ 10⫺4 M)
1.58 ⫾ 0.20*
1.71 ⫾ 0.23*
1.65 ⫾ 0.19‡
1.51 ⫾ 0.17†
1.62 ⫾ 0.19†
1.50 ⫾ 0.28§
Values are expressed as means ⫾ SE; n ⫽ 6 for each group. All
vessels used were denuded of the endothelium. Tension increased by
endothelin-1 at 3 ⫻ 10⫺9 (†), 6 ⫻ 10⫺9 (*), 10⫺8 (‡), and 2 ⫻ 10⫺8 (§)
M. ANP, CNP, and nitric oxide were used at 10⫺7 M. 8-BrcGMP,
8-bromo-cGMP; ODQ, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one.
arteries induced by ANP (10⫺7 M), CNP (10⫺7 M), and
nitric oxide (10⫺7 M) was attenuated by 58.7 ⫾ 3.6,
26.3 ⫾ 2.1, and 83.4 ⫾ 5.1%, respectively (n ⫽ 6 for
each group; Fig. 2). In pulmonary veins, treatment
with 8-BrcGMP attenuated relaxation induced by ANP
(10⫺7 M), CNP (10⫺7 M), and nitric oxide (10⫺7 M) by
47.9 ⫾ 3.0, 20.7 ⫾ 2.6, and 68.7 ⫾ 4.3%, respectively
(n ⫽ 6 for each group). In comparison, treatment with
8-Br-cGMP attenuated relaxation induced by nitric
oxide to a greater extent than that induced by ANP and
attenuated the relaxation induced by CNP the least
(P ⬍ 0.05; Fig. 2).
cGMP. The measurement of cGMP was performed in
vessels without endothelium. Under basal conditions,
the intracellular level of cGMP of pulmonary arteries
(0.96 ⫾ 0.33 pmol/mg protein; n ⫽ 7) was not significantly different from that of pulmonary veins (1.02 ⫾
0.35 pmol/mg protein; n ⫽ 7, P ⬎ 0.05). In pulmonary
arteries, ANP (10⫺7 M) caused a greater increase in
cGMP content than CNP (10⫺7 M) or nitric oxide (10⫺7
M). In veins, both ANP and CNP caused a similar,
moderate increase in cGMP content; the increase was
significantly less than that induced by nitric oxide
(10⫺7 M). The effects of ANP and CNP on cGMP content were not significantly affected by ODQ (3 ⫻ 10⫺5
M). The effect of nitric oxide was inhibited by ODQ (3 ⫻
10⫺5 M; Fig. 3).
ANP and CNP receptors. After a 60-min incubation
with 125I-ANP-(1O28) (10–500 pM) or 125I-CNP (10–
500 pM) at 30°C, the membrane preparations of pulmonary arteries showed a greater specific binding to
125
I-ANP than those of veins [maximal binding (Bmax)
116.6 ⫾ 8.9 vs. 69.7 ⫾ 6.3 fmol/mg protein; n ⫽ 7, P ⬍
0.05]. In contrast, the membrane preparations of pulAJP-Heart Circ Physiol • VOL
monary veins showed greater specific binding to 125ICNP than that of arteries (Bmax 102.8 ⫾ 4.2 vs. 57.7 ⫾
3.1 fmol/mg protein; n ⫽ 7, P ⬍ 0.05). The equilibrium
dissociation constant value (KD) for ANP binding receptors was 268.9 ⫾ 53.0 and 242.4 ⫾ 59.0 pM for
pulmonary arteries and veins, respectively. The KD
values for CNP was 211.4 ⫾ 32.2 and 236.5 ⫾ 25.9 pM
for pulmonary arteries and veins, respectively. There is
no significant difference between these KD values (n ⫽
7 for each group, P ⬎ 0.05; Fig. 4).
NPR-A and NPR-B mRNAs. RQ RT-PCR yielded
three distinctive bands corresponding to the predicted
sizes for mRNA fragments of NPR-B, NPR-A, and the
18S internal standards (691, 450, and 315 bp, respectively) according to the base pair ladders. Pulmonary
arteries showed a greater content of NPR-A mRNA
than that of NPR-B mRNA (relative density to the 18S:
1.46 ⫾ 0.10 vs. 0.97 ⫾ 0.11; n ⫽ 6, P ⬍ 0.05), whereas
pulmonary veins showed a greater content of NPR-B
mRNA than that of NPR-A mRNA (relative density to
the 18S: 1.83 ⫾ 0.15 vs. 1.07 ⫾ 0.11; n ⫽ 6, P ⬍ 0.05;
Fig. 5).
Fig. 4. Specific binding of 125I-ANP (A) and 125I-CNP (B) to membrane preparations of PA and PV of newborn lambs. Data are shown
as means ⫾ SE; n ⫽ 7 for each group.
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Control
ODQ (3 ⫻ 10⫺5 M)
8-BrcGMP (3 ⫻ 10⫺4 M)
Fig. 3. Changes in the intracellular cGMP content of PA and PV of
newborn lambs after treatment with ANP (10⫺7 M; n ⫽ 7), CNP
(10⫺7 M; n ⫽ 7), and nitric oxide (10⫺7 M; n ⫽ 8) under control
conditions or in the presence of ODQ (3 ⫻ 10⫺5 M). Data are shown
as means ⫾ SE. *Significantly different from vessels treated with
ANP; †significantly different from vessels treated with ANP or CNP;
‡significantly different from control (P ⬍ 0.05).
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NATRIURETIC PEPTIDES AND PULMONARY VASOACTIVITY
Fig. 5. Detection of natriuretic peptide receptor type A (NPR-A) and
type B (NPR-B) mRNAs by relative quantitative RT-PCR. 18S rRNA
(18S) is shown as an internal standard.
DISCUSSION
AJP-Heart Circ Physiol • VOL
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In the present study, ANP caused greater relaxation
of pulmonary arteries of newborn lambs than CNP.
This is consistent with previous results reported in
pulmonary arteries of piglets (28) and pulmonary arteries of adult rats (20). In bovine, atriopeptin II, a
synthetic fragment of ANP, causes relaxation of pulmonary arteries but not of veins (17). Our present
study showed that ANP caused less relaxation of pulmonary veins than that of arteries. Thus pulmonary
arteries may be the major site of action of ANP in the
lung. It is interesting to note that ANP caused greater
relaxation of the pulmonary arteries of newborn lambs
than nitric oxide at an equal concentration. Considering that nitric oxide or nitric oxide donors are less
potent dilators of pulmonary arteries than those of
veins in perinatal lambs, adult pigs, and cows (5, 10,
13, 17, 43), it is possible that ANP may play a more
important role in modulating pulmonary arterial activity than previously thought, at least in certain species.
Our present study demonstrates for the first time
that CNP is a potent dilator of newborn pulmonary
veins, greater than that of arteries. CNP caused pulmonary veins to relax at concentrations as low as 3 ⫻
10⫺11 M. In fetal ovine pulmonary veins, CNP has
recently been shown to be a more potent dilator than
ANP (26). However, ANP and CNP were equally potent
in relaxing fetal pulmonary arteries (26). This is different from our results obtained in newborn lambs and
from results obtained in rat pulmonary arteries (20)
and in newborn porcine pulmonary arteries (28). In
porcine pulmonary arteries, there is a maturational
increase in ANP-induced relaxation when comparing
vessels of fetus vs. vessels of newborns of 6 and 17
days. However, in vessels of all these groups, relaxation to CNP was ⬍5% (28). Thus a difference in
maturational change between ANP and CNP may contribute to the different findings between our results
from pulmonary arteries of the newborn lambs vs.
those from the fetal lambs. This suggests also that
CNP may be of physiological importance in the modulation of pulmonary venous tone in the fetus and in the
newborn.
In contrast to ANP, which is a circulating hormone
released mainly from the heart in response to stretching and other stimuli, CNP is released locally in various tissues, including vascular endothelial cells (1, 23,
31, 44). In bovine aortic endothelial cells, the synthesis
and release of CNP is stimulated by ANP and brain
natriuretic peptides (32). If this is the case in lungs,
ANP may cause endothelium-dependent relaxation of
pulmonary vessels through the release of CNP. Our
data show that relaxations of pulmonary vessels with
endothelium induced by ANP and CNP are not significantly different from those of vessels without endothelium (Fig. 1). Likewise, in bovine and porcine pulmonary arteries (17, 28), rabbit aorta (51), and canine
renal arteries (48), relaxations induced by ANP or ANP
fragments are comparable between vessels with or
without endothelium. CNP causes relaxation of intact
canine saphenous arteries to a similar extent as that of
endothelium-denuded vessels (48). Our present results
are in line with these studies. In canine renal, saphenous, and femoral veins, CNP causes greater relaxation in vessels without endothelium than that of vessels with endothelium (48). Hence, the possibility that
the endothelium is a source of ANP or CNP in response
to these natriuretic peptides and affects vascular tone
may not be a general phenomenon. In porcine coronary
arteries, CNP induces endothelium- and nitric oxideindependent hyperpolarization and relaxation (4). In
bovine aortic endothelial cells, shear stress induces the
release of CNP (53). The underlying mechanism of
CNP-induced relaxation of pulmonary vessels is not
clear and remains to be determined.
Natriuretic peptides and nitric oxide cause vasodilation by elevating the intracellular content of cGMP
after stimulation of particulate and soluble guanylyl
cyclase, respectively (23). In the present study, the
relaxation and increase in cGMP of pulmonary vessels
induced by nitric oxide, but not that induced by ANP
and CNP, was inhibited by ODQ, an inhibitor of soluble
guanylyl cyclase (14). Thus particulate but not soluble
guanylyl cyclase is involved in the responses of pulmonary vessels induced by ANP and CNP. In pulmonary
arteries of the newborn lambs, the greater relaxation
induced by ANP compared with that induced by CNP
was accompanied by a greater increase in cGMP content. In veins, although CNP induced a markedly
larger relaxation than ANP, the increase in cGMP
induced by CNP was moderate and similar to that
induced by ANP. This would suggest that relaxation
induced by CNP is less dependent on cGMP than that
induced by ANP. In our study, relaxation induced by
ANP and CNP was also tested in vessels pretreated
with 8-BrcGMP at a concentration that we have previously shown to induce near maximal relaxation (3 ⫻
10⫺4 M; see Ref. 13). This was done so that any further
increase in cGMP content induced by ANP or CNP
would not cause any further relaxation. As expected,
under these conditions, relaxation to nitric oxide was
largely attenuated. However, relaxation to CNP was
less attenuated than that induced by ANP. These results suggest that a cGMP-independent mechanism
plays a larger role in mediating relaxation to CNP than
to ANP. In human kidney epithelial cells, it has been
reported that CNP depolarizes the membrane potential by increasing K⫹ conductance via a cGMP-inde-
NATRIURETIC PEPTIDES AND PULMONARY VASOACTIVITY
AJP-Heart Circ Physiol • VOL
We thank Marry Lee Ryba for secretarial assistance.
This study was supported by a grant from the Emma Mushamp
Foundation, Switzerland, and National Heart, Lung, and Blood
Institute Grant HL-59435.
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