C-type natriuretic peptide system in fetal ovine pulmonary vasculature

Satyan Lakshminrusimha1, Christopher A. D'Angelis2, James A. Russell3, Lori C. Nielsen1, Sylvia F. Gugino3, Peter A. Nickerson2, and Robin H. Steinhorn4

Departments of 1 Pediatrics, 2 Pathology, and 3 Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214; and 4 Department of Pediatrics, Northwestern University, Chicago, Illinois 60614


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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C-type natriuretic peptide (CNP) is a recently described endothelium-derived relaxing factor. CNP relaxes vascular smooth muscle and inhibits smooth muscle proliferation by binding to natriuretic peptide receptor (NPR) type B (NPR-B) and producing cGMP. Lung parenchyma and fifth-generation pulmonary arteries (PA) and veins (PV) were isolated from late-gestation fetal lambs. All three types of NPR mRNA were detected in PA and PV by RT-PCR. CNP and NPR-B immunostaining was positive in pulmonary vascular endothelium and medial smooth muscle. CNP concentration-response curves of PA and PV were compared with those of atrial natriuretic peptide (ANP) by use of standard tissue bath techniques. CNP relaxed PV significantly better than PA. ANP relaxed PA and PV equally, but ANP relaxed PA significantly better than CNP. Pretreating PA and PV with natriuretic peptide receptor blocker (HS-142-1) or cGMP-dependent protein kinase inhibitor Rp-beta -phenyl-1- N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate significantly inhibited the CNP relaxation response, indicating that the response was mediated through the NPR-cGMP pathway. We conclude that CNP is important in mediating pulmonary venous tone in the fetus.

natriuretic peptide receptors; protein kinase G; guanosine 3',5'-cyclic monophosphate


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AT BIRTH, SUCCESSFUL TRANSITION from gas exchange by the placenta to gas exchange by the lung depends on a dramatic decrease in pulmonary vascular resistance and an 8- to 10-fold increase in pulmonary blood flow. cGMP is an important second messenger in many biological systems including vascular smooth muscle, where it mediates relaxation. Vasoactive substances such as nitric oxide (NO), produced by the endothelium, modulate pulmonary vascular tone through production of cGMP and thus contribute to the normal fall in pulmonary vascular resistance at birth (1, 7, 8). Guanylate cyclases catalyze the production of cGMP from GTP, leading to the phosphorylation of cGMP-dependent protein kinases (PKG), calcium sequestration, and vascular smooth muscle relaxation (23).

NO is not the only potential agonist for stimulation of cGMP production in the fetal pulmonary vasculature. Natriuretic peptides also have vascular effects mediated by stimulating the production of cGMP. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are produced by cardiac tissue and have potent natriuretic and vasorelaxant properties (19). C-type natriuretic peptide (CNP) was initially identified in the central nervous system (31) and was later shown to be synthesized and released by the endothelial cells (32).

Unlike NO, which stimulates soluble guanylate cyclase (sGC), natriuretic peptides including CNP act by stimulating a membrane-bound particulate guanylate cyclase (pGC) through natriuretic peptide receptors (NPR). There are three known types of NPR. NPR-A and NPR-B are coupled to pGC enzyme activity. ANP and BNP act predominantly through the NPR-A (9), whereas CNP is a specific agonist of the NPR-B (21). Type C receptors (NPR-C) are unique in that they do not possess a kinase (pGC) moiety (33). They mediate endocytosis and subsequent degradation of all the natriuretic peptides and are considered to be clearance receptors (20).

It has been suggested that CNP acts as an endothelium-derived relaxing factor (18) in the control of vascular tone and vascular remodeling (16). Similar to NO, CNP produced in the endothelium stimulates the production of cGMP in adjacent vascular smooth muscle (5). This similarity, and the known importance of the endothelium in the pulmonary vascular changes during gestation and at birth, led us to test the hypothesis that CNP and the NPR-B are expressed in fetal lung and produce dilation of the fetal pulmonary vasculature. We have previously reported that NO is a more potent dilator of pulmonary veins (PV) than of pulmonary arteries (PA) in fetal and newborn lambs (29, 30). Therefore, we further hypothesized that, like NO, CNP would relax PV more than PA.

We used immunohistochemical techniques to localize CNP and NPR-B in lungs from late-gestation ovine fetuses. Isolated pulmonary arterial and venous segments were evaluated for mRNA expression of all three types of NPR by use of RT-PCR techniques. We further studied the effects of CNP on isolated PA and PV and compared them with the effects of ANP. Finally, the mechanism of the vascular effects of CNP was studied using inhibitors of NPR and cGMP-dependent protein kinase.


    METHODS
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INTRODUCTION
METHODS
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REFERENCES

This study was approved by the State University of New York at Buffalo Laboratory Animal Care Committee. Near-term-gestation pregnant ewes (135-136 days, term being 146-150 days) were anesthetized with thiopental and halothane, and the fetuses were delivered by cesarean section. The fetus was killed before first breath by rapid exsanguination through a cardiac puncture under anesthesia. The heart and lungs were removed en bloc from the thorax.

RT-PCR to detect NPR mRNA. After removal of the lungs from the lamb, segments of fourth- and fifth-generation PA and PV were dissected (n = 5 lambs). The vessel segments were frozen immediately (<= 5 min of death) in liquid nitrogen and stored at -70°C until processed. Total cellular RNA was isolated from these tissues using TRIzol reagent (Life Technologies, Grand Island, NY) according to manufacturer's protocol. Samples were quantified and run on agarose formaldehyde gels to confirm the integrity of RNA. One microgram of sample was subjected to RT-PCR in a single tube in 50 µl of 1× PCR buffer (1.5 mM magnesium chloride, 20 mM Tris · HCl, pH 8.4, and 50 mM KCl) containing 10 mM dATP, dGTP, dCTP, and dTTP, 30 pM forward and reverse primers, 18S internal standards (Ambion, Austin TX), 2.5 U of AMV reverse transcriptase, and 2.5 U of Taq polymerase (both from Roche Molecular Biochemicals, Indianapolis, IN). Each reaction was subjected to denaturation/annealing at 65° for 10 min, reverse transcription at 50°C for 10 min, and denaturation and inactivation of reverse transcriptase at 94°C for 4 min, followed by 25 cycles at 94°C for 1 min, 60°C for 2 min, and 72°C for 2 min, with a final extension at 72°C for 10 min. Negative controls contained no reverse transcriptase. Cycling profiles for each pair were done with 25 cycles being on the linear part of the cycle vs. product curve.

Forward and reverse primers were designed with restriction sites to facilitate subsequent cDNA cloning. NPR-A primers (forward CCT CCT GCA GTC CCC AAA TGT GGC TTT GAC AA, reverse GGG AAT TCA TTC TGC ACA TCC CGC ATA T) yield a cDNA fragment of 440 bp. NPR-B primers (forward GTGCTG CAG EAA CAC AAC CTG AGC TAT GCC T, reverse AAG AAT TCG GGC CGG TCA TCT GTG CGA) yield a 461-bp cDNA fragment. NPR-C primers (forward GTC CTG CAG TTA CGT GAA GTA CTC AGA GCT GG, reverse CCG AAT TCA TCA CCA ATA ACG TCC TGG G) yield a 206-bp cDNA fragment (10, 11), and 18S internal standards yield a 324-bp cDNA.

Products were sequenced using an ABI 373 sequencer (Advanced Biotechnologies, Columbia, MD) and prism dye-deoxyterminator chemistry to confirm their identity. All reactions were resolved on a single 2% Tris-acetate-EDTA gel to eliminate gel-to-gel variation during subsequent analysis. Receptor signal was normalized to 18S mRNA signal to correct for loading and was analyzed on the basis of each sample's signal: 18S ratio with Molecular Analyst software from Bio-Rad (Hercules, CA) (11).

Immunohistochemistry. Right upper and lower lobes from six lambs were removed, and the vascular tree was flushed first with normal saline followed by 4% buffered formalin. A 50-ml syringe was inserted into the main stem bronchus, and lung airspaces were infused with 4% buffered formalin until the lobe was firm. The airway was then sutured closed, and the entire lobe was submerged in a formalin-filled container for 24 h. Five to six tissue blocks were cut (~2 cm3) from each of six animals. Tissue blocks were washed, dehydrated in ethanol, and embedded in paraffin. Sections were cut at 5 µm and mounted on poly-L-lysine (Sigma Chemical, St. Louis, MO)-coated slides.

The primary antibodies utilized were as follows: rabbit anti-CNP (human, porcine, rat) (22, 53), which has been shown by the manufacturer not to cross-react with either ANP or BNP, was purchased from Peninsula Laboratories (Belmont, CA); rabbit anti-NPR-B (Z657) was obtained as a gift from Dr. David Garbers (University of Texas Southwestern Medical Center). The anti-NPR-B antibody is directed against the carboxy terminus of the peptide and does not cross-react with NPR-A. For negative controls, sections were incubated with normal rabbit serum in place of the primary antibody. Sections were deparaffinized in three changes of xylene, hydrated in a graded ethanol series, and washed in tap water. Endogenous peroxidase activity was blocked by immersing slides in 0.3% H2O2 for 30 min. After being washed in PBS, slides were incubated for 1 h with 6% horse serum to block nonspecific binding of the primary antibody. The blocking serum was removed by gentle tapping, and slides were incubated overnight at room temperature in a humidified container with either anti-CNP (1:600) or anti-NPR-B (1:3,000). Detection of primary antibody binding was accomplished using the Vectastain Elite horse anti-rabbit Avidin Biotin Complex kit (Vector Laboratories, Burlingame, CA). The following day, slides were washed thoroughly in PBS, and the biotinylated secondary antibody was applied for 45 min. After being washed in PBS, the peroxidase-labeled complex reagent was added, and the slides were incubated for 30 min. Antibody binding was visualized using 3,3'-diaminobenzidine tetrahydrochloride and H2O2 and Sigma Fast DAB (Sigma). Slides were washed in running tap water, counterstained lightly with Mayer's hematoxylin, and mounted in Permount (Fisher Scientific, Fair Lawn, NJ).

Isolated vessel study. Fifth-generation PA and PV were dissected, isolated, and cut into rings (n = 20 lambs) as described previously (29). Rings were suspended in water-jacketed chambers filled with aerated (94% O2-6% CO2) modified Krebs-Ringer solution (in mM: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.5 NaHCO3, and 5.6 glucose) at 37°C. A continuous recording of isometric force generation was obtained by tying each vessel ring to a force-displacement transducer (model UC2, Statham Instruments, Hato Rey, PR) that was connected to a recorder (Gould Instrument Systems, Valley View, OH). After the vessel rings were mounted, they were allowed to equilibrate for 20 min in the bathing solution. A micrometer was used to stretch the tissues repeatedly in small increments over the following 45 min until resting tone remained stable at a passive tension of 0.8 g for arteries and 0.6 g for veins. Preliminary experiments determined that this procedure provided optimal length for generation of active tone to exogenous norepinephrine (NE). The maximal contractile tension of each ring was determined by exposure to KCl (118 mM). Wet tissue weights were obtained at the end of each experiment, and responses were normalized by tissue weight.

The following pharmacological agents were used: indomethacin, DL-propranolol, N-nitro-L-arginine (L-NNA), L-norepinephrine hydrochloride, ANP (rat ANP 1-28), and CNP (human CNP 22; Peptides International, Louisville, KY). All other drugs were purchased from Sigma-Aldrich. The NPR antagonist, HS-142-1, was a gift from Dr. S. Nakanishi (Kyowa Hakko, Tokyo Research Laboratories, Tokyo, Japan). This compound selectively blocks NPR-A and -B but not the C receptor (25). In the canine coronary circulation, this agent specifically attenuates CNP-mediated vasodilation and does not alter vasodilation induced by acetylcholine (36). The specific PKG inhibitor, Rp-beta -phenyl-1-N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate (Rp-8-Br-PET-cGMPS) was purchased from Biolog Life Science Institute (Bremen, Germany, via Ruth Langhorst International Marketing, La Jolla, CA). This agent is a competitive inhibitor of cGMP-dependent kinase, with an inhibition constant (Ki) of 0.03 µM, at which no effect on protein kinase A (PKA) was observed. This drug was chosen because of its selectivity for PKG and excellent membrane permeability (3). It effectively inhibits both PKG isoforms (Ialpha and Ibeta ) found in vascular smooth muscle (24). Preliminary experiments demonstrated that Rp-8-Br-PET-cGMPS significantly inhibited relaxations to NO and 8-bromo-cGMP in both fetal and neonatal pulmonary vessels (data not shown). This finding has been reported by others (13). Rp-8-Br-PET-cGMPS was dissolved first in a small quantity of DMSO and then diluted in distilled water. L-NNA was dissolved directly in Krebs-Ringer solution, and indomethacin was dissolved in ethanol. All other drugs were dissolved in distilled water. Ethanol and DMSO, at the concentrations used in these experiments, did not alter the preexisting tone of PA or PV. Vessels were pretreated with indomethacin (10-5 M) to prevent the formation of vasoactive prostaglandins, propranolol (10-6 M) to block the beta -adrenergic receptors, and L-NNA (10-3 M) to inhibit NO synthase. All experiments were performed in a darkened room because L-NNA is sensitive to light.

The vessels were first constricted with NE to produce half-maximal constriction. This EC50 of NE was determined from preliminary studies in which cumulative concentration-response curves for NE (10-8 to 10-5 M) were developed in PA and PV. Once the response to NE had reached a steady level, cumulative concentration-response curves to natriuretic peptides were obtained by increasing the bath concentration of these agents in successive steps. The next concentration was added only when the response to the previous concentration had reached a plateau. Vessel rings were used for one experimental protocol and then discarded. In all experiments, n is the number of animals from which vessel rings were studied.

The inhibitors HS-142-1 (2 × 10-5 M) and Rp-8-Br-PET-cGMPS (3 × 10-5 M) were added at the plateau of NE constriction and incubated for a period of 5 min before the addition of CNP. The relaxation responses in these pretreated vessels were compared with control vessels from the same animal.

The statistical comparison of the curves was performed with ANOVA factorial for repeated measures. When appropriate, individual data points between curves were compared using Student's unpaired t-test. All statistical analysis was performed with StatView 4.5 software (Abacus Concepts, Berkley, CA). Significance was accepted at P < 0.05.


    RESULTS
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INTRODUCTION
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RT-PCR. RT-PCR analysis for detection of NPR-A, -B, and -C in isolated fifth-generation PA and PV yielded single, distinct, appropriately sized bands for each cDNA. Subsequent sequencing of the products and comparison with published sequences for NPR-A (22), -B (6), and -C (12) determined that each primer pair yielded the expected product. A representative sample is shown in Fig. 1. NPR-B mRNA content (normalized to the 18S signal) tended to be higher in PV than in PA (signal/18S ratio 1.81 ± 0.34 in PV vs. 0.92 ± 0.28 in PA, P = 0.07). In contrast, NPR-A mRNA content was similar in PV and PA (signal/18S ratio 1.41 ± 0.16 in PV vs. 1.31 ± 0.31 in PA).


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Fig. 1.   Representative sample of RT-PCR analysis for detection of natriuretic peptide receptors (NPR)-A, -B and -C in isolated fifth-generation pulmonary arteries (PA) and pulmonary veins (PV). 18S rRNA (18S) is shown as an internal standard.

Immunohistochemistry. Staining was absent within all structures in all sections incubated with normal rabbit serum in place of primary antibody (Fig. 2). The intensity and pattern of staining for both CNP and NPR-B were consistent for all six animals examined.


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Fig. 2.   Negative controls. Fetal lung sections were incubated with a 1:800 dilution of normal rabbit serum to control for both C-type natriuretic peptide (CNP) and NPR-B staining. They were lightly counterstained with hematoxylin to identify the site of positive antibody immunostaining in Fig. 3. Negative staining is demonstrated in a bronchiole-associated artery (A); a conduit artery with associated airway (C) and resistance arteries with associated terminal bronchioles are shown (B and D). Magnification: A and C, ×40; B, ×60; D, ×100.

Immunostaining for CNP was prominent within the endothelium of large arteries and veins (arrows in Fig. 3, A and C, respectively). These vessels have an approximate inner diameter of 0.5-0.75 mm and correspond to the vessels used for RT-PCR and functional studies. Endothelial CNP immunostaining diminished as vessel diameter decreased, becoming absent within the endothelium of both small arteries and small veins (Fig. 3, E and G). Weak diffuse staining for CNP was present in the adventitial surface of the media of large arteries (Fig. 3A) and was seen throughout the media of large veins (Fig. 3C). Within the medial layer of resistance arteries, CNP staining was present in the form of large granules (Fig. 3E). CNP staining was absent within small veins (Fig. 3G).


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Fig. 3.   Immunohistochemical localization of CNP and NPR-B in fetal ovine lung: immunostaining for CNP (A, C, E, and G) and NPR-B (B, D, F, and H). CNP immunostaining is seen within the endothelial cells (arrows) of large arteries (A) and veins (C). Large immunoreactive granules can be seen within the media of smaller resistance arteries (E) accompanying terminal bronchioles (TB) but not septal veins (G). NPR-B immunostaining is present in the media of large arteries (B), veins (D), and resistance arteries (F) but is absent from septal veins (H). Magnification: conduit artery (A and B) ×200; large vein (C and D) ×200; resistance artery (E and F) ×200; septal vein (G and H) ×100.

Staining for NPR-B was present within smooth muscle cells (SMC) comprising the adventitial half of the media of large arteries (Fig. 3B) and could be seen in SMC throughout the entire media of large veins (Fig. 3D). NPR-B immunostaining decreased in smaller resistance arteries but could still be seen within the SMC of many precapillary arteries (Fig. 3F). Staining for NPR-B was absent in small veins (Fig. 3H) and within the endothelium of all vessels.

Isolated vessel studies. The constrictor response to NE (expressed as grams of tension per gram of tissue weight) was 495.7 ± 39.3 g/g in PV and 252.1 ± 25 g/g in PA. However, the level of constriction was similar between the two vessel types when compared as a percentage of their maximal constriction to 118 M KCl (63 ± 8% in veins vs. 72 ± 4% in arteries, P = nonsignificant).

Relaxation responses to increasing concentrations of CNP (10-10 to 3 × 10-7 M) were obtained in PA and PV and compared with responses obtained to similar concentrations of ANP. CNP relaxed pulmonary veins significantly better than arteries (Fig. 4). In contrast, ANP relaxed PA and PV in a similar fashion (Fig. 5). ANP relaxed PA significantly better than CNP (Figs. 5 vs. 4). There was a trend for enhanced relaxations to CNP vs. ANP in PV (P = 0.06), and relaxations to CNP were significantly better than ANP at all concentrations >3 × 10-9 M (Fig. 4 vs. 5).


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Fig. 4.   Cumulative concentration-response curves to CNP in PA and PV isolated from late-gestation fetal lambs. All vessels were pretreated with indomethacin, propranolol, and N-nitro-L-arginine (L-NNA). Relaxations are expressed as percentage of plateau norepinephrine (NE) constriction. n, No. of animals. PV relax significantly better than PA to CNP.



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Fig. 5.   Cumulative concentration-response curves to atrial natriuretic peptide (ANP) in PA and PV isolated from late-gestation fetal lambs. All vessels were pretreated with indomethacin, propranolol, and L-NNA. Relaxations are expressed as percentage of plateau NE constriction. n, No. of animals. PA and PV relax similarly to ANP.

NPR blockade with HS-142-1 resulted in significant inhibition of relaxation to CNP in both PA and PV (Fig. 6). PKG blockade with Rp-8-Br-PET-cGMPS resulted in a significant inhibition of the relaxation responses of PA and PV to CNP (Fig. 7, A and B).


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Fig. 6.   Cumulative concentration-response curves to CNP in PA (A) and PV (B) isolated from late-gestation fetal lambs in the presence and absence of NPR-particulate guanylate cyclase inhibitor (HS-142-1; 2 × 10-5 M). All vessels were pretreated with indomethacin, propranolol, and L-NNA. Relaxations are expressed as percentage of plateau NE constriction. n, No. of animals. Pretreatment with HS-142-1 results in significant inhibition of CNP-mediated relaxation in PA and PV.



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Fig. 7.   Cumulative concentration-response curves to CNP in PA (A) and PV (B) isolated from late-gestation fetal lambs in the presence and absence of protein kinase G inhibitor, Rp-beta -phenyl-1-N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate (Rp-8-Br-PET-cGMPS, PET; 3 × 10-5 M). All vessels were pretreated with indomethacin, propranolol, and L-NNA. Relaxations are expressed as percentage of plateau NE constriction. n, No. of animals. Pretreatment with Rp-8-Br-PET-cGMPS results in significant inhibition of CNP-mediated relaxation in PA and PV.


    DISCUSSION
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INTRODUCTION
METHODS
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DISCUSSION
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We studied the CNP-pGC system in pulmonary arteries and veins isolated from normal, late-gestation fetal lambs. Using RT-PCR, we demonstrated the presence of all of the three NPR mRNAs (NPR-A, -B and -C) in pulmonary arteries and veins (Fig. 1). Having detected NPR mRNA in pulmonary arteries and veins, we localized CNP and NPR-B protein within these vessels by immunohistochemistry. CNP was localized primarily to the endothelium and smooth muscle of large arteries and veins (Fig. 2, A and C). NPR-B was localized in the smooth muscle of large arteries and veins and resistance arteries.

Recently, Nakanishi et al. (26) reported the ontogeny and localization of CNP expression in the rat embryo lung. CNP mRNA was present in embryonic lung and increased rapidly during the immediate postnatal period. Similarly, CNP mRNA and immunoreactivity localized to vascular smooth muscle cells just before birth and expression increased with advancing postnatal age. Jankowski et al. (17) reported that rat pulmonary parenchymal NPR-B mRNA content is low on postnatal day 1, increased on day 4, and decreased thereafter. They did not determine localization of NPR-B expression.

Although ontogeny was not the focus of the current study, we examined NPR mRNA expression and localized CNP and NPR-B within the pulmonary vasculature of late-gestation ovine fetuses. We chose the fetal lamb because many physiological and pathological studies of the perinatal pulmonary vasculature have been performed in fetal lambs and because models of pulmonary hypertension can be relatively easily developed in this species (1, 8). It is important to note that the chronology of lung development differs from rats to sheep and humans (2). Rats have immature saccular lungs at birth, whereas lambs and humans have mature alveolar lungs at term gestation (4).

In the present study, we did not examine CNP mRNA. However, using immunohistochemical staining, we found abundant immunoreactive CNP in the endothelium of the large pulmonary arteries and veins, likely representing areas of CNP synthesis. The absence of NPR-B staining in the endothelium and its presence in the smooth muscle indicate that smooth muscle is the likely primary site of action of CNP. The distribution of CNP protein in smooth muscle cells parallels the distribution of NPR-B. This CNP immunoreactivity in the smooth muscle could be secondary to its binding to the NPR-B or may represent active CNP synthesis in the smooth muscle (26).

In the current study, we observed that CNP and NPR-B immunoreactivity in pulmonary arterial smooth muscle was more intense toward the adventitial layer (Fig. 2, A and B), whereas their distribution was more uniform throughout the medial layer in pulmonary veins. The significance of this differential pattern of distribution of CNP and NPR-B protein within the medial layer of large arteries is not clear.

Functional responses to exogenous CNP were studied in isolated pulmonary arteries and veins. These effects were compared with the known effects of ANP (30) on fetal pulmonary vessels. CNP and ANP were used for comparison because of their respective specificity for NPR-B and -A, respectively (14, 21). Although both peptides relaxed each type of vessel tested, distinct differences were observed. CNP was a significantly more potent vasodilator in pulmonary veins than in arteries. This pattern is similar to what we have previously reported for NO (29, 30). In contrast, ANP produced equivalent relaxation in arteries and veins. A differential distribution of NPR-A receptors to pulmonary arteries and NPR-B to pulmonary veins is the most likely explanation for these findings. This pattern of arterial and venous responses to ANP and CNP is similar to that reported in systemic vessels isolated from dogs (35) and humans (37). Perreault et al. (27) have reported that pulmonary veins from neonatal piglets have fewer NPR-A than pulmonary arteries.

The constrictor response to NE was greater in pulmonary veins than arteries, perhaps in part due to pretreatment with L-NNA, which we previously reported to constrict pulmonary veins more than arteries in lambs (29). Although it is possible that these differences in constrictor response contributed to the enhanced venous relaxations to CNP, we found relaxations to ANP were very similar in arteries and veins (Fig. 5). Furthermore, the constrictor response to NE was equivalent in veins and arteries when expressed as a percentage of maximal constriction to 118 M KCl. Therefore, we believe that venous relaxations to CNP are greater because of differences in NPR-B expression.

We used HS-142-1, which inhibits both NPR-A and NPR-B (25, 28), to determine whether CNP relaxed pulmonary vessels through receptor-mediated activation of pGC. HS-142-1 has been shown to inhibit CNP-mediated relaxation of canine coronary arteries (36) and ANP-mediated relaxation of rabbit thoracic aorta. We were able to conduct experiments with HS-142-1 in only a limited number of animals because it is no longer manufactured (Dr. S. Nakanishi, personal communication), but we found a significant inhibition of CNP-mediated relaxation in both pulmonary arteries and veins. The degree of inhibition observed in the current study is similar to that previously reported with ANP-mediated relaxation of isolated rabbit thoracic aorta (15).

We used Rp-8-Br-PET-cGMPS, a potent inhibitor of cGMP-dependent PKG, to determine whether CNP relaxations were mediated by cGMP and activation of PKG. Rp-8-Br-PET-cGMPS has been reported to inhibit relaxations to NO donors (3) and 8-Br-cGMP (13). Significant attenuation of CNP-mediated relaxation by Rp-8-Br-PET-cGMPS pretreatment in both pulmonary arteries and veins demonstrates that CNP relaxes these vessels, at least in part, by a cGMP-PKG-dependent mechanism. To our knowledge, this is the first reported use of this compound to inhibit NPR-mediated relaxation.

In conclusion, we found that CNP and its receptors are present in the pulmonary circulation of late-gestation fetal lambs. We also found that exogenous CNP relaxes pulmonary veins and arteries and that this relaxation is mediated by the NPR-pGC-cGMP-PKG pathway. Similar to NO, CNP is a more potent dilator of pulmonary veins than of pulmonary arteries. We speculate that CNP may act in a paracrine fashion to modulate vasomotor tone and smooth muscle cell proliferation in pulmonary vessels. Further experiments will be necessary to elucidate the exact role of CNP in physiological transition at birth and in pathological states associated with pulmonary hypertension.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Daniel D. Swartz and Huamei Wang for expert technical assistance in performing these experiments.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-54705 (R. H. Steinhorn) and American Heart Association Grant 9740024 (R. H. Steinhorn).

Address for reprint requests and other correspondence: R. H. Steinhorn, Div. of Neonatology, Children's Memorial Hospital, 2300 Children's Plaza no. 45, Chicago, IL 60614 (E-mail: r-steinhorn{at}northwestern.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 20 November 2000; accepted in final form 13 March 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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