Vol. 275, Issue 5, L931-L941, November 1998
Chronic pulmonary hypertension increases fetal lung cGMP
phosphodiesterase activity
Kimberly A.
Hanson1,
James W.
Ziegler2,
Sergei D.
Rybalkin1,
Jim W.
Miller1,
Steven H.
Abman3, and
William
R.
Clarke1
1 Departments of Pediatrics,
Anesthesiology, and Pharmacology, University of Washington School
of Medicine, Seattle, Washington 98105-00371;
3 Pediatric Heart-Lung Center, Department
of Pediatrics, University of Colorado School of Medicine, Denver,
Colorado 80218; and 2 Department of
Pediatrics, Brown University School of Medicine, Providence, Rhode
Island 02903
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ABSTRACT |
An experimental ovine fetal model for perinatal
pulmonary hypertension of the neonate (PPHN) was characterized by
altered pulmonary vasoreactivity and structure. Because past studies
had suggested impaired nitric oxide-cGMP cascade in this experimental model, we hypothesized that elevated phosphodiesterase (PDE) activity may contribute to altered vascular reactivity and structure in experimental PPHN. Therefore, we studied the effects of the PDE inhibitors zaprinast and dipyridamole on fetal pulmonary vascular resistance and PDE5 activity, protein, mRNA, and localization in normal
and pulmonary hypertensive fetal lambs. Infusion of dipyridamole and
zaprinast lowered pulmonary vascular resistance by 55 and 35%,
respectively, in hypertensive animals. In comparison with control
animals, lung cGMP PDE activity was elevated in hypertensive fetal
lambs (150%). Increased PDE5 activity was not associated with either
an increased PDE5 protein or mRNA level. Immunocytochemistry demonstrated that PDE5 was localized to vascular smooth muscle. We
concluded that PDE5 activity was increased in experimental PPHN,
possibly by posttranslational phosphorylation. We speculated that these
increases in cGMP PDE activity contributed to altered pulmonary
vasoreactivity in experimental perinatal pulmonary hypertension.
persistent pulmonary hypertension of the newborn; vasoregulation; dipyridamole; guanosine 3',5'-cyclic
monophosphate
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INTRODUCTION |
AT BIRTH, THE PULMONARY CIRCULATION must rapidly dilate
to accommodate an 8- to 10-fold increase in pulmonary blood flow to perform its postnatal role of gas exchange (18). The mechanisms that
maintain high basal pulmonary vascular resistance (PVR) in the normal
fetus and cause the dramatic fall in PVR after birth are incompletely
understood. Establishment of an air-liquid interface, shear stress,
changes in oxygen tension, rhythmic distension of the lung, and altered
release of vasoactive mediators contribute to the transition of the
lung circulation (14, 19, 20). Release of nitric oxide (NO) contributes
in part to the normal transition of the pulmonary circulation in fetal
lambs (2, 3, 25, 40). NO is released at birth by increased shear stress, increased oxygen tension, and ventilation of the lung. Pulmonary vasodilation is achieved in part by stimulation of soluble guanylate cyclase, which elevates cGMP content in vascular smooth muscle (1, 16, 43).
Mechanisms that modulate smooth muscle cGMP levels are critical in
determining vascular tone and reactivity. Smooth muscle cGMP content is
regulated, in part, by the hydrolysis of cGMP, thereby lowering
intracellular cGMP, favoring an increase in vascular tone. The majority
of cGMP hydrolysis is mediated by cGMP-specific phosphodiesterases
(PDEs). In the normal fetal circulation, PDE inhibition with
dipyridamole and zaprinast, predominant PDE5 inhibitors, causes potent
and sustained pulmonary vasodilation, suggesting that PDE5 plays a
major role in vasoregulation (9, 44). In addition, PDE5 is present in
high concentrations in lung tissue (38, 41). These physiological and
biochemical studies suggested that an increase in PDE5 activity could
be involved in maintaining high PVR in the fetus, thereby modulating
vasoreactivity in the normal fetal pulmonary circulation.
Persistent pulmonary hypertension of the newborn (PPHN) is the failure
of postnatal adaptation of the pulmonary circulation at birth. PPHN is
characterized by sustained elevations of pulmonary arterial pressure
(PAP) and abnormal pulmonary vasoreactivity, leading to right-to-left
shunting of blood across the ductus arteriosus (DA) and foramen ovale,
causing severe hypoxemia (21). Clinical observations have led to the
hypothesis that intrauterine stimuli may alter the pulmonary
circulation before birth, leading to an inability to achieve or sustain
the normal decrease in PVR at delivery (33). On the basis of
observations that fetal hypertension can alter pulmonary vascular
structure in lambs, an animal model of chronic fetal pulmonary
hypertension involving compression or ligation of the DA in utero was
developed to better understand the etiology and pathophysiology of PPHN
(4, 29). This model was characterized by progressive pulmonary
hypertension and altered vasoreactivity, characterized by the loss of
vasodilation to some pharmacological (ACh) and physiological stimuli
(such as increased oxygen and shear stress) (13, 36). In addition,
these animals had right ventricular hypertrophy and sustained pulmonary
hypertension after delivery, requiring ventilation with increasing
oxygen concentrations. Recent studies (26, 42) demonstrated a decrease
in endogenous endothelial NO synthase (eNOS) mRNA, protein, and
activity in this chronic pulmonary hypertensive model; thus it may have
contributed to sustained pulmonary hypertension in this model. In
addition to decreasing eNOS, a pharmacological study (37) also
demonstrated decreased guanylate cyclase activity in this model. Thus
multiple abnormalities in the NO-cGMP cascade contributed to increasing PVR in this perinatal model.
Because vasodilation at birth is partly dependent on NO release and
sustained elevation of cGMP in smooth muscle, we hypothesized that
persistent or increased PDE5 may have contributed to the persistently
elevated PVR in PPHN. To test this hypothesis, we studied the
physiological effects of the PDE5 antagonists zaprinast and
dipyridamole in fetal lambs with intrauterine pulmonary hypertension and in age-matched control lambs. To determine whether the effects of
PDE5 were localized to pulmonary vascular tissue, we determined PDE5
protein in fetal ovine pulmonary tissue by immunocytochemistry (ICC).
To determine whether pulmonary hypertension altered lung PDE activity,
we also measured PDE5 activity, protein, and mRNA levels in fetal lambs
with pulmonary hypertension and in age-matched control lambs.
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METHODS |
Physiological Studies
Surgical preparation. All procedures
and protocols were reviewed and approved by the Animal Care and Use
Committee at the University of Colorado Health Sciences Center
(Denver). Mixed-breed (Columbia-Rambouillet) pregnant ewes were fasted
for 24 h before surgery at 125-130 days of gestation (term, 147 days). Ewes were sedated with pentobarbital sodium (2-4 g iv
infusion) and anesthetized with 1% tetracaine hydrochloride (3 mg) by
lumbar puncture. Penicillin (500 mg) and streptomycin (1 g) were
administered to the ewe immediately before surgery. Under sterile
conditions, an abdominal incision was performed to expose the uterus.
The umbilical placental circulation was kept intact throughout fetal
surgery. The fetal left forelimb was delivered through a small uterine
incision to provide access to the left thorax. A skin incision was made
above the fifth rib after local infiltration with lidocaine (2 ml; 1%
solution). Polyvinyl catheters were advanced into the ascending aorta
and superior vena cava after they were inserted into the axillary
arteries and veins. The heart and great vessels were exposed through a left thoracotomy. Catheters were inserted into the left and main pulmonary arteries under direct visualization. Catheters were guided
into position with a 14- or 16-gauge intravenous catheter. The left
pulmonary artery (LPA) catheter was inserted at a site distal to the
branching of the DA from the main pulmonary artery (PA) and guided
through the common PA into the LPA. This catheter was placed to allow
the selective infusion of drugs into the left lung while minimizing
systemic effects. For measurement of PAP, a main PA catheter was
inserted between the DA and the pulmonic valve. A 6-mm ultrasonic flow
transducer (Transonics, Ithaca, NY) was placed around the left lung to
measure blood flow. In hypertensive animals, an umbilical tape was
placed around the DA. This ligature was progressively tightened and
tied to occlude the DA. The thoracotomy, skin, and uterine incisions
were tightly closed with continuous sutures, providing for sufficient
space for the exit of the catheters and flow transducer cables.
Ampicillin (500 mg) was infused into the amniotic cavity after surgery.
Catheters and the flow cable were tunneled for externalization through
a small incision on the maternal flank and were placed in a small pouch. Ampicillin (250 mg) was infused daily in the fetus and amniotic
cavity for 3 days after surgery. All hemodynamic studies were performed
8 days after surgery. Animals were killed after study with high doses
of pentobarbital sodium. Fetal weights were obtained, and
the degree of right ventricular hypertrophy was determined from the
ratio of right ventricle to left ventricle+septum weights.
Physiological measurements. Flow
transducer cables were attached to an internally calibrated flowmeter
(Transonics) for measurement of LPA flow. The main PA, aortic, and
amniotic cavity catheters were connected to a Gould-Statham P23 ID
pressure transducer. Pressures were referenced to the amniotic cavity
pressure and recorded on a Gould chart recorder. Placement of a flow
probe in the LPA measured blood flow continuously; knowing the driving pressure across the main PA-LPA allowed us to know the PVR of the left
lung. Total pulmonary resistance (TPR) was calculated as mean PAP
divided by left pulmonary arterial flow. Blood samples were drawn
through aortic catheters for measurement of pH and arterial
PCO2 and PO2 with a
Radiometer OSM-3 blood gas analyzer (Radiometer, Copenhagen, Denmark).
Study design. Eight days after
surgery, the pulmonary hemodynamic effects of dipyridamole and
zaprinast were studied in control (n = 10) and hypertensive (n = 10) animals.
The doses selected for use in these studies were determined from
previous experience with these agents in which equimolar doses caused a
twofold increase in pulmonary blood flow in normal late-gestation fetal
lambs (44). After at least 30 min of stable baseline measurements,
dipyridamole (0.4 mg/min) or zaprinast (0.22 mg/min) was alternately
infused into the LPA for 30 min. Hemodynamic measurements
were recorded at 10-min intervals throughout the baseline (30-min),
infusion (30-min), and recovery (30-min) periods. Arterial blood gas
tensions and pH were measured during each period.
Biochemical Studies
Tissue preparation. Frozen lung
samples were weighed and immediately homogenized on ice in precooled
(4°C) homogenization buffer in a preparative Polytron blender for 1 min. Tissue homogenization was at a ratio (wt/vol) of l g of tissue
to 4 ml of homogenization buffer. Homogenization buffer contained 40 mM
Tris · HCl, pH 7.5, 15 mM benzamidine, 15 mM
2-mercaptoethanol, 1 µg/ml of pepstatin A, 1 µg/ml of leupeptin, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 20 µg/ml of
antipain. After homogenization, samples were centrifuged at 250 g for 10 min at 4°C to remove any
large unhomogenized material. The particulate fraction was discarded, and the supernatant was centrifuged at 100,000 g for 1 h at 4°C. The supernatants
or the soluble fraction from the 100,000 g centrifugation was diluted in a
final concentration of 20% glycerol (vol/vol), divided into 1.0-ml
samples, and either stored in capped cryogenic tubes at 
70°C
or mixed with 2× sample buffer as described in Immunoblot
methodology. The pellets were
placed in 50% of initial homogenization buffer volume containing a
final concentration of 20% glycerol and resuspended with three strokes
of a 15-ml Wheaton glass pestle homogenizer. These pellet
or particulate fractions were divided into 1.0-ml samples and stored at
70°C. Preliminary studies indicated that using either fresh
or frozen lung or freezing the supernatant or pellet fractions did not
alter PDE activity and protein.
Measurement of PDE activity. PDE
activity was measured with a modification of the method of Mumby et al.
(30). The PDE reaction buffer contained a final concentration of 20 mM
Tris · HCl (pH 7.5), 20 mM imidazole (pH 7.5), 3 mM
MgCl2, 15 mM magnesium acetate, 0.2 mg/ml of BSA, 1 µM cold cGMP, and
[3H]cGMP
(105 counts/min). The PDE5
inhibitor zaprinast was added at 4 µM. PDE1 activity was suppressed
in the presence of EGTA (10 µM). To ensure appropriate linear
kinetics of cGMP PDE activity, we initially assayed samples to
determine the volume required for 20-30% hydrolysis of cGMP. All
samples were assayed in triplicate. Protein concentrations were
determined by the method of Bradford (8). cGMP PDE activity is
expressed as picomoles of cGMP hydrolyzed per minute per milligram of
measured protein.
Immunoblot analysis.
PDE5 ANTIBODY PRODUCTION.
Rabbits were used for the production of all the PDE antibodies. Based
on the published sequence of bovine PDE5 (24), a peptide of 16 amino
acids from the COOH-terminal region with the sequence C-R-K-N-R-Q-K-W-Q-A-L-A-E-Q-Q-E-K-OH was selected. The peptide was
conjugated to keyhole limpet hemocyanin (KLH) by the method of Green et
al. (15).
The peptide-KLH conjugate (600 µg) was injected subcutaneously in
rabbits; 3 wk later, a booster injection of peptide-KLH (300 µg)
conjugate was given. The antibody specificity was determined by
immunoblot analysis with partially purified bovine PDE5 and PDE5 fusion
proteins. Partially purified bovine lung PDE5 was obtained by the
method of Thomas et al. (38). As anticipated from the conservation of
homology among PDEs in various species, the PDE5 antibody also
recognized mouse and rat PDE5. An immunoblot with preimmune serum and
the COOH-terminal peptide (100 µg) mixed with PDE5 antibody was
performed. These studies showed no reactivity, confirming that the
antibody is specific for sheep PDE5.
PHOSPHORYLATED PDE5 ANTIBODY PRODUCTION.
With the use of a peptide of 18 amino acids from the cGMP binding
region, the sequence H-R-D-F-E-S-A-S-I-K-R-P-D-T-G-C-OH was synthesized
with phospho- or dephosphoserine at position 92 (Quality Controlled
Biochemicals). Antibodies were produced that were selective for
phosphorylated and unphosphorylated PDE5. With the use of the method of
Thomas et al. (39), partially purified bovine PDE5 was phosphorylated
with protein kinase A for 60 min. The phosphorylated PDE5-selective
antibody showed increased affinity for phosphorylated PDE5, whereas the
unphosphorylated PDE5-selective antibody showed decreased affinity for
phosphorylated PDE5 as assessed by both
32P incorporation
and immunoblot analysis.
ACTIN ANTIBODY.
-Actin vascular smooth muscle antibody was obtained from Sigma.
Immunoblot methodology. Protein
samples were prepared in 2× sample buffer containing 0.12 M
Tris · HCl (pH 6.8), 20% glycerol, 5% SDS, 10%
2-mercaptoethanol, and 0.01% bromphenol blue at both 1 and 2 mg/ml for
immunoblot analysis. The immunoblot samples were boiled for 5 min and
stored at 20°C. To determine the
concentration of PDE5 protein in supernatant from DA-ligated and
control animals and to account for variation in enhanced
chemiluminescence (ECL) exposure, we developed standard curves of
bovine PDE5 (concentrations ranging from 0.0625 to 0.5 µg) at
1-10 s. These experiments demonstrated that ECL was linear at 3 and 5 s over the range of added bovine PDE5 protein. Thus Western blots
were subsequently developed at 3 and 5 s. These immunoblots were
scanned by DATASCAN II and analyzed by National Institutes of Health
(NIH) Image. With the use of NIH Image, each lane containing bovine PDE
or sheep lung protein was analyzed vertically with the background
subtracted, and the number of pixels under the curves was determined.
Every PDE5 immunoblot contained the same internal standards (bovine PDE
at 0.25 µg). Therefore, because samples were assayed on the linear
portion of the ECL exposure curves and the same internal standard was present on each immunoblot, quantitative comparison of ovine PDE5 could
be made from the immunoblots. Prestained broad molecular-weight and
kaleidoscope molecular-weight standards (Bio-Rad) were used and boiled
for 1.5 min. The samples were loaded onto an 8% SDS resolving gel and
5% stacking gel in a 1-mm-thick slab with the products of Protogel.
The proteins were blotted onto nitrocellulose paper and reacted with
the respective polyclonal antibodies. The specific PDE isoform and
actin signals were detected with the supersignal
chemiluminescent-horseradish peroxidase substrate system (Amersham).
The immunoblots of -actin were scanned by DATASCAN II and analyzed
by NIH Image and are reported as the number of pixels.
Measurement of PDE5 mRNA by RNase protection assay.
TOTAL RNA EXTRACTION AND MRNA EXTRACTION.
Sheep RNA was extracted with a modification of the single-step method
by Chomczynski and Sacchi (11). A purified mRNA preparation was made
from total RNA with reagents and protocols supplied in the Fast Trak
2.0 mRNA Isolation Kit (Clontech). mRNA was then quantitated via
ultraviolet spectrophotometry and used in the preparation of a cDNA
library. Sheep lung cDNA was made with the Marathon kit from
Clontech. This cDNA pool provided amplified sheep lung
PDE5 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene
fragments via PCR as described in GENERATION OF OVINE
PDE5 PCR FRAGMENTS and
PRODUCTION OF GAPDH CLONES,
respectively.
GENERATION OF OVINE PDE5 PCR FRAGMENTS.
Ovine PDE5 gene fragments were made via PCR. PDE5-specific
oligopeptides based on bovine PDE5 sequences (Genetics Computer Group
accession no. L16545) were synthesized in the University of Washington Department of Pharmacology Core Molecular Biology Facility (Seattle). Two sets of PCR primers were
developed that would produce nonoverlapping PCR products of PDE5. The
first set of primers was designed to produce a PCR product
corresponding to base pairs 1687-2399 of bovine PDE5. The primers
used to generate this product were
5'-TGCAGTCCTTAGCGGCTGCT-3' (sense primer) and 5'-CCGTTGTTGAATAGGCCAGG-3' (antisense primer). The product
of this PCR was termed PDE5-1. A second set of primers was designed to
cover base pairs 1-1046 of bovine PDE5. The primers used to generate this ovine PCR product were
5'-GGGAGGGTCTCGAGGCGAGTTC-3' (sense primer) and
5'-CTCCAGCAGTGAAGTCTCATAG-3' (antisense primer). The
product of this PCR reaction was termed PDE5-2. Sense and antisense
primers (20 pmol each) were mixed with 5 µl of the cDNA pool
described above. PCR was done in the following sequence: 94°C for
30 s, 60°C for 30 s, and 68°C for 5 min. This PCR reaction was
performed for a total of 40 cycles, and the purity of PCR products was
determined by running 20 µl of each reaction on a 1%
agarose-1× tricine-KOH gel. Fragments generated
were purified out of low-molecular-weight protein agarose-gel, and
poly(A)+ tails were added to PCR fragments to increase
ligation efficiency with Tailing Reaction Mix (Promega).
Escherichia coli
(Invitrogen INVF') was transformed with the above ligation
reactions with the Original TA Cloning Kit protocol and reagents
(Invitrogen).
Production of probes for internal control of total RNA loading for
analysis of mRNA levels.
PRODUCTION OF GAPDH CLONES.
Consensus sequence (human, mouse, and rat) primers for PCR
amplification of GAPDH were purchased from Clontech. The 5'
primer had the sequence 5'-ACCACAGTCCATGCCATCAC-3'. The
3' primer had the sequence
5'-TCCACCACCCTGTTGCTGTA-3'. With the use of the method described above for the production of PDE5 clones, ovine GAPDH clones
were obtained. These GAPDH clones were confirmed by sequencing.
PRODUCTION OF PROBES FOR RNASE PROTECTION
ASSAY.
Both PDE5-1 and PDE5-2 clones were used to make probes for RNase
protection assay (RPA) analyses. Plasmids were cut with appropriate enzymes, and 32P-labeled
transcripts were made with the MaxiScript system (Ambion). RPAs were
begun by adding 15 µg of sample of sheep lung RNA. This was mixed
with 1 × 106 counts/min of
one or more of the probes described above. Nucleic acid was
precipitated with 0.5 M ammonium acetate and 70% ethanol for 15 min at
70°C and centrifuged (16,000 g, 15 min at 4°C). Pellets were
resuspended in 20 µl of hybridization buffer [80% deionized
formamide, 100 mM sodium citrate (pH 6.4), and 1 mM EDTA] and
denaturated (3 min at 90°C and 10-s vortex) and incubated overnight
at 45°C. The next day, selected samples were digested for 1 h at
37°C with a mixture of RNase A (0.9 U/ml) and RNase T1 (36 U/ml) in
a total volume of 220 µl. The RNA was then precipitated, air-dried,
and resuspended in 4 µl of gel loading buffer, then visualized by SDS-PAGE (5% acrylamide-8 M urea) and autoradiography.
ICC.
Portions of left and right fetal lung were prepared in 1%
paraformaldehyde-PBS (pH 7.4) as previously described (17). The frozen
tissue sections were blocked with a solution containing 5% goat serum
to prevent nonspecific binding. The sections were incubated with
anti-PDE5 COOH-terminal antibody at 1:1,000 dilution overnight. As
controls, other sections were incubated overnight with preimmune serum
at a 1:1,000 dilution or anti-PDE5 COOH-terminal antibody at a 1:1,000
dilution and 100 µg/ml of COOH-terminal peptide. The sections were
washed and incubated with a biotinylated horse anti-rabbit antibody. In
the tissue sections, intrinsic peroxidase activity was quenched by
hydrogen peroxide (0.3%). The slides were covered with a streptavidin
solution stained by the avidin-biotin-peroxidase complex method with
ABC Vecastain Kit from Vector Laboratories.
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RESULTS |
Physiological Studies
Table 1 summarizes the gestational ages and
baseline hemodynamic measurements in control and DA-ligated study
groups. Gestational age was similar in each group. The DA-ligated group
developed increased mean PAP and decreased LPA flow without changing
aortic pressure. TPR (mean PAP divided by flow) was increased nearly threefold 8 days after DA ligation. At autopsy, fetal weights in
control (3,135 ± 310 g) and hypertensive lambs (3,175 ± 85 g)
were not different. Right ventricular hypertrophy, expressed as the
ratio of right ventricular to left ventricular+septal weight was
markedly elevated in the DA-ligated group (control 0.53 ± 0.03 vs.
hypertension 0.82 ± 0.05; P < 0.05).
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Table 1.
Gestational age and baseline hemodynamic measurements in normal
(control) and hypertensive (DA-ligated) fetal lambs before dipyridamole
or zaprinast treatment
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Brief infusions of zaprinast and dipyridamole decreased TPR in both
control late-gestation fetal lambs and DA-ligated animals (hypertensive). Figures 1 and
2 demonstrate the pulmonary effects of
zaprinast and dipyridamole, respectively, in hypertensive vs. control
fetal sheep. As shown in Fig. 1, zaprinast lowered TPR in
both study groups. As shown in Fig. 2, dipyridamole also lowered TPR in
both study groups.
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Fig. 1.
Pulmonary hemodynamic effects of zaprinast (ZAP) in control (CTL) and
ductus arteriosus-ligated [hypertensive (HTN)] fetal lambs.
ZAP lowered total pulmonary resistance (TPR; mean pulmonary arterial
pressure divided by flow) in both study groups
(n = 5 animals/group)
expressed as absolute change in TPR
(A) and %change from baseline (BL;
B).
* P < 0.05 vs. BL.
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Fig. 2.
Pulmonary hemodynamic effects of dipyridamole (DIP) in CTL and HTN
fetal lambs. DIP lowered TPR in both study groups
(n = 5 animals/group)
expressed as absolute changes in TPR
(A) and %change from BL
(B).
* P < 0.05 vs. BL.
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Figure 3 is a summary of the maximum
reduction in TPR in control and DA-ligated animals receiving either
zaprinast or dipyridamole. These results demonstrate that the maximal
reduction in TPR in the DA-ligated group was 35%, whereas in the
control group, the maximal reduction in TPR was 46%. With the
administration of dipyridamole, the maximal reduction in TPR was
similar in both the control (63%) and DA-ligated groups (63%). In
both groups (control and DA ligated), dipyridamole caused a slightly
greater reduction in TPR than zaprinast.
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Fig. 3.
Effects of DIP and ZAP on TPR in CTL and chronic HTN lambs.
Responsiveness to phosphodiesterase (PDE) inhibitors was similar in
both study groups.
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Biochemical Studies
cGMP-dependent PDE activity in hypertensive animals was significantly
elevated compared with control animals
(P < 0.0001) (Fig.
4). Inhibition of
cGMP-dependent PDE activity with zaprinast demonstrated that the
increase in cGMP PDE activity was secondary to PDE5-specific activity.
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Fig. 4.
Effects of chronic pulmonary hypertension on average cGMP PDE activity
at 1 µM cGMP in presence and absence of ZAP in CTL
(n = 6) and HTN
(n = 6) animals. Total cGMP PDE
activity is significantly elevated in HTN fetal lung compared with CTL.
Presence of ZAP demonstrates that majority of cGMP PDE activity in
presence of EGTA is PDE5. Significantly different from CTL:
* P < 0.0001;
** P < 0.001.
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Figures 5-8 show the immunoblot data for analysis of -actin,
PDE5, and phosphorylated PDE5 in hypertensive vs. control fetal sheep.
Figure 5 shows no significant difference in
vascular smooth muscle -actin levels in hypertensive vs. control
animals. Therefore, increases in PDE5 activity were not attributed to
changes in vascular smooth muscle content.
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Fig. 5.
A: representative immunoblot of
-actin vascular smooth muscle (VSM) protein in fetal HTN
(n = 3) and CTL
(n = 3) animals. Nos. at
left, molecular-weight markers.
B: means ± SE of -actin VSM
protein by immunoblot analysis in fetal HTN
(n = 6) and CTL
(n = 6) lambs. After 30-s
exposure, immunoblots were analyzed by DATASCAN II and NIH Image.
Pixels were determined and averaged. Data demonstrate no significant
difference between the 2 groups in -actin VSM protein content and
also illustrate lack of difference between individual samples.
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Although cGMP-dependent PDE5 activity was increased in hypertensive
animals, immunoblot levels of PDE5 were not significantly different
between hypertensive and control lungs (Fig.
6). These results suggest that the increase
in PDE5 activity in the experimental PPHN model may involve a
posttranslational modification of PDE5 without a change in total
protein. A previous study by Burns et al. (10) indicated that PDE5
activity may be increased posttranslationally by phosphorylation.
Therefore, antibodies were developed to both phosphorylated and
unphosphorylated PDE5. As a control, partially purified bovine lung
PDE5 was incubated with protein kinase A in a phosphorylation reaction
for 60 min, with increasing
32P incorporation
observed after 60 min. Figure 7 shows the
selectivity of the dephosphorylated PDE5 antibody for PDE5 before
phosphorylation and lower selectivity after phosphorylation. The
selectivity of the phosphorylated antibody increased after
phosphorylation of PDE5. The phosphorylated antibody demonstrated lower
selectivity for unphosphorylated PDE5. The unphosphorylated and
phosphorylated antibodies recognized both forms of PDE5 but with
greater selectivity for unphosphorylated and phosphorylated PDE5,
respectively.
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Fig. 6.
A,
top: representative immunoblot of PDE5
protein in fetal HTN (n = 3) and CTL
(n = 3) animals. Protein added to each
lane was 50 µg. Standard PDE5 protein at 0.25 µg was added to
lane 1. Nos. at
left: molecular-weight markers. No
significant difference in PDE5 protein was observed between HTN and
normal fetal lamb lungs. A,
bottom: immunoblot analysis of 3 separate sections, each with partially purified bovine PDE5 (PDE5),
adult ovine lung tissue extract (adult), and fetal ovine lung tissue
(fetal). Left, antibody (Ab) specific
for COOH-terminal region of PDE5 at 1:10,000;
middle, preimmune serum at 1:10,000;
right, Ab for COOH-terminal region
mixed with COOH-terminal peptide (100 ng) to which Ab was made.
Specificity of Ab for PDE5 was observed.
B: means ± SE of PDE5 protein by
immunoblot analysis in fetal HTN (n = 6) and CTL (n = 6) lambs. Immunoblots
at 3-min exposure were analyzed by DATASCAN II and NIH Image
and compared with standard PDE5 curves described in
METHODS. No significant difference in PDE5 protein was
observed between HTN and normal fetal lamb lungs.
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Fig. 7.
Immunoblot analysis of 3 separate sections, each with partially
purified lung bovine PDE5 before and after phosphorylation with protein
kinase A for incubation period of 60 min. Protein added to each lane
was 50 µg (wt/vol equivalent between HTN and CTL animals).
Left, Ab specific for dephosphorylated PDE5 (Dephospho Ab)
at 1:1,000; middle, phosphorylated Ab (Phospho Ab) at
1:1,000; right, Ab specific for COOH terminus (COOH-terminal
Ab) at 1:10,000. Ab for unphosphorylated PDE5 was selective for PDE5
before phosphorylation and demonstrated decreased affinity for
phosphorylated PDE5. Ab for phosphorylated PDE5 was selective for PDE5
after phosphorylation and
32P incorporation.
COOH-terminal Ab selectivity did not change with phosphorylation of
PDE5.
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Figure 8A
shows representative immunoblots of three individual hypertensive and
three age-matched control animals with the COOH-terminal antibody
(immunoblot
a; antibody that recognizes both
phosphorylated and unphosphorylated forms of PDE5), dephosphorylated antibody (immunoblot
b), and the phosphorylated antibody
(immunoblot c). Although the
COOH-terminal antibody demonstrated no difference in total PDE5 protein
between hypertensive and control animals, the dephosphorylated PDE5 was
increased in control animals and the phosphorylated PDE5 was increased
in hypertensive animals. Figure 8B
demonstrates the specificity of the dephosphorylated antibody
(immunoblot
d) and the phosphorylated antibody
(immunoblot e). Figure
8C demonstrates the levels of the
unphosphorylated and phosphorylated PDE5 in six hypertensive and six
age-matched control animals. The protein levels of phosphorylated PDE5
were significantly elevated (P < 0.05) in the hypertensive animals compared with control sheep. With the
use of these same animals, the unphosphorylated PDE5 protein levels
were lower in the hypertensive animals and elevated in the control
sheep.
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Fig. 8.
A: representative immunoblots of COOH-terminal
(immunoblot a), unphosphorylated (Dephospho;
immunoblot b) and Phospho (immunoblot c) PDE5
protein in fetal HTN (n = 3) and CTL (n = 3)
animals. Protein added to each lane was 50 µg. Total protein was
similar between HTN and CTL animals. COOH-terminal Ab (immunoblot
a), which recognizes both Phospho and Dephospho, was not
significantly different between HTN and CTL animals. PDE5 was higher in
CTL than in HTN animals (immunoblot b), whereas Phospho
PDE5 was elevated in HTN compared with CTL animals (immunoblot
c). B: immunoblots d and e
demonstrate specificity of Ab for Dephospho and Phospho PDE5,
respectively. Each immunoblot has 3 separate sections: partially
purified bovine PDE5 (Std), fetal HTN ovine lung tissue (HTN), and CTL
ovine lung tissue extract (CTL). Protein added to each lane was 50 µg. Immunoblot d: left, Ab specific for
Dephospho PDE5 at 1:2,000; middle, preimmune serum;
right, Ab for COOH-terminal region with Dephospho peptide
(100 ng) to which Ab was made. Ab was specific for Dephospho PDE5, and
Dephospho PDE5 was elevated in CTL compared with HTN animals.
Immunoblot e: left, Ab specific for Phospho PDE5
at 1:2,000; middle, preimmune serum; right, Ab
for COOH-terminal region mixed with Phospho peptide (100 ng) to which
Ab was made. Ab was specific for Phospho PDE5, and Phospho PDE5 was
elevated in HTN compared with CTL animals.
C: averages of Dephospho and Phospho
PDE5 protein by immunoblot analysis in fetal HTN
(n = 6) and CTL
(n = 6) lambs. After 3-min exposure,
immunoblots were analyzed by DATASCAN II and NIH Image as discussed in
METHODS. Dephospho PDE5 was
significantly elevated in CTL animals compared with HTN animals, and
Phospho PDE5 was significantly elevated in HTN animals compared with
CTL animals. * P < 0.05.
|
|
Figure 9 shows the RPA analysis of PDE5
message and control GAPDH in hypertensive and control fetal sheep
lungs. As with PDE5 COOH-terminal protein levels, there was no
significant difference in the quantitative level of PDE5 mRNA in either
hypertensive or control animals.
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Fig. 9.
A, top: representative RNase protection assay
(RPA) of HTN (n = 3) and CTL (n = 3) fetal sheep
PDE5 mRNAs. PDE5-1 and PDE5-2 are 2 probes from different regions of
PDE5. With each probe, HTN and CTL animals have similar mRNA PDE5
levels. A,
bottom: glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) representative RPA in HTN
(n = 4) and CTL
(n = 3) sheep. GAPDH levels are for
standardizing each sample and indicate similar levels within HTN and
CTL animals. B: means ± SE of PDE5
mRNA levels as assessed by RPA in fetal HTN
(n = 4) and CTL
(n = 4) lambs. RPA was scanned by
DATASCAN II and NIH Image. PDE5 mRNA is expressed as ratio of PDE5 mRNA
pixels to GAPDH pixels. PDE5 mRNA is not significantly different
between HTN and CTL fetal sheep.
|
|
The localization of PDE5 in control fetal ovine sheep in pulmonary
tissue is shown in Fig. 10. PDE5 protein
was localized to vascular smooth muscle in both control fetal lung and
hypertensive fetal lung. ICC also demonstrated a small amount of
staining in bronchial cilia in both control and hypertensive lungs.
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Fig. 10.
Immunocytochemistry analysis of fetal CTL lung with PDE5 Ab
(A), fetal HTN lung with PDE5 Ab
(B), fetal CTL sheep lung cilia with
PDE5 Ab (C), fetal CTL lung with
PDE5 Ab and COOH-terminal peptide (100 µg/ml;
D), and fetal HTN lung with PDE5 Ab
and COOH-terminal peptide (100 µg/ml)
(E). B, bronchiole; A, alveola. PDE5
is localized to pulmonary VSM and cilia. Similar results were obtained
with HTN lung tissue.
|
|
| |
DISCUSSION |
cGMP plays a crucial regulatory role in vascular tone. An elevation in
cGMP induces vasorelaxation. In addition to regulating smooth muscle
tone, cGMP was also able to inhibit smooth muscle cell proliferation
(5, 7). Intracellular cGMP concentrations are regulated by a balance
between synthesis by smooth muscle guanylate cyclases and degradation
by cyclic nucleotide PDEs. Seven PDE gene families have been described
based on different kinetic properties, cyclic nucleotide preferences,
regulatory mechanisms, and sensitivities to pharmacological inhibitors
(6). Recently, it was determined that within each major gene family, there were multiple PDE isoforms and splice variants, each exhibiting different characteristics and tissue expressions (6). PDE1 (calcium/calmodulin-dependent PDE), PDE2 (cGMP-stimulated PDE), PDE3
(cGMP-inhibited PDE), PDE4 (cAMP-specific PDE), and PDE5 (cGMP-specific
PDE) were identified in vascular smooth muscle (23, 27, 35). PDE5
accounts for the majority of cGMP hydrolysis in vascular smooth muscle.
Dipyridamole and zaprinast are potent inhibitors of PDE5 and PDE1C,
with IC50 values in the 1-4
µM and 4-6 µM ranges, respectively (12, 32). A previous study
(44) demonstrated that dipyridamole- and zaprinast-induced vasodilation was dependent on NO-cGMP cascade and not related to other nonspecific effects (such as increases in adenosine activity). Therefore, the
effects of zaprinast and dipyridamole were studied in a chronic hypertensive fetal model and normotensive fetal control model to
determine whether these inhibitors were effective vasodilators in a
pathological model of pulmonary hypertension.
We reported that dipyridamole and zaprinast caused pulmonary
vasodilation during the development of intrauterine pulmonary hypertension by chronic compression of the DA in late-gestation fetal
lambs. The percent decrease in TPR to either dipyridamole or zaprinast
was similar in both normal fetal lambs and hypertensive fetuses. These
data demonstrated the persistence of the potent pulmonary vasodilator
response to PDE5 antagonists in the hypertensive lambs. A previous
study (44) in normal fetal lambs demonstrated that inhibition of eNOS
activity blocks the vasodilator effects of PDE5 antagonists. Therefore,
dipyridamole- and zaprinast-induced vasodilation was more dependent on
endogenous eNOS activity in the normal fetal lung. However, chronic
pulmonary hypertension decreased endogenous eNOS activity, protein, and
mRNA in perinatal lambs (26, 42). Despite this reduction in eNOS
activity, we reported that the PDE5 antagonists still induce marked
pulmonary vasodilation. These findings suggested that PDE5 activity
regulated basal PVR in the normal fetus and remained critical in the
hypertensive fetal lung circulation.
Several other studies have reported the efficacy of dipyridamole and
zaprinast as vasodilators in animal models of pulmonary hypertension.
Rosenkrantz et al. (31) showed that dipyridamole attenuated the pressor
response to acute hypoxia in pigs, an effect that was attributed to
antiplatelet activity of dipyridamole. Mlczoch et al. (28)
subsequently showed in a canine model of hypoxic pulmonary hypertension
that dipyridamole administration inhibited hypoxic pulmonary
vasoconstriction, even in dogs rendered thrombocytopenic by platelet
antiserum. As noted in fetal lambs (44), these findings suggested a
direct effect of dipyridamole in causing vasodilation in the adult
pulmonary circulation. Similar findings were observed in normal newborn
lambs in response to zaprinast (9).
Our results demonstrated that cGMP PDE activity was elevated in the
hypertensive model compared with normal control lungs, suggesting that
higher PDE activity may have contributed to an elevated PVR after DA
ligation. Although multiple cGMP PDE activities are present in lung
tissue, PDE5 is present in high concentrations in the lung and is the
only PDE known that is zaprinast sensitive in the presence of EGTA
(which will inhibit all PDEs in the PDE1 family, including
PDE1C). Previous studies demonstrated the
IC50 for zaprinast for guinea pig
and bovine PDE5 to be 0.4-8 µM. Therefore, our data suggested
that the majority of the cGMP PDE activity in the presence of EGTA, 1 µM cGMP, and 4 µM zaprinast was PDE5. However, PDE5
protein levels and message levels did not differ between hypertensive
and normal control lungs, suggesting that the increase in PDE5 activity
without a concurrent rise in PDE5 protein in the hypertensive fetal
lungs was secondary to posttranslational control of PDE5. Previous work
suggested that PDE5 activity increased after phosphorylation and that
PDE5 activity decreased with dephosphorylation (10). Our
results with an antibody that recognized only the phosphorylated state
of PDE5 indicated that hypertensive animals had an increase in
phosphorylated PDE5 compared with control animals. Therefore, in the
experimental model of PPHN, increases in PDE5 activity may be due to an
increase in phosphorylation of PDE5. It was also reported that PDE5
activity posttranslationally may be altered by the presence of factors,
referred to as a 
- and 
-factor (22). These factors may inhibit
PDE5 activity similar to - and -factor mechanisms with PDE6 (34).
Our results localized PDE5 to fetal pulmonary vasculature by ICC (Fig.
10). We also observed a small amount of staining in the ciliary bodies
of bronchioles in fetal lung tissue. The alterations in PDE5 in the
hypertensive animals most likely reflected changes to vascular smooth
muscle content.
In summary, physiological studies demonstrated the effectiveness of the
PDE inhibitors zaprinast and dipyridamole in reducing a pathological
elevation in PVR in a model of PPHN. Biochemical studies demonstrated
that PDE5 activity is significantly elevated in PPHN, and this activity
is not associated with an increase in PDE5 protein or mRNA. However,
our results strongly implied that posttranslational modification of
PDE5 by phosphorylation accounts for the increase in PDE5 activity in
this model of PPHN. Therefore, a pathological elevation in PDE5 in
vascular smooth muscle, suggested by ICC localization, that resulted in
an even lower cGMP level may have contributed to the elevated PVR by
vasoconstriction and possibly smooth muscle vascular proliferation. We
speculate that the elevation in cGMP PDEs may also be important in
downregulating the activity of endogenous NO in the pulmonary
hypertensive model. We further speculate that isozyme-specific PDE
inhibitors, alone or in combination with other cGMP-dependent dilators
(inhaled NO), may prove to be potential treatments of severe pulmonary hypertension.
| |
ACKNOWLEDGEMENTS |
We thank Dr. Joe Beavo for the antibody reagents and helpful
discussions in the area of phoshodiesterases.
| |
FOOTNOTES |
This study was supported in part by a grant from the ONO Pharmaceutical
Company (Japan) and National Heart, Lung, and Blood Institute Grant
HL-46481.
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. §1734 solely to indicate this fact.
Address for reprint requests: K. Hanson, Dept. of Anesthesia and
Critical Care, Children's Hospital and Medical Center, Sand Point Way
NE, PO Box 5371, Seattle, WA 98105-0371.
Received 26 January 1998; accepted in final form 1 July 1998.
| |
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Abstract
Introduction
