(Circulation. 1999;100:1751-1756.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Developmental Changes in Prostaglandin E2 Receptor Subtypes in Porcine Ductus Arteriosus
Possible Contribution in Altered Responsiveness to Prostaglandin E2
From the Departments of Pharmacology and Therapeutics, McGill University (M.B., D.R.V., S.C.), and the Departments of Pediatrics and Pharmacology, Université de Montréal (P.A., P.H., A.-M.G., X.H., A.B., J.-C.F., S.C.), Montréal, Quebec, Canada; the Department of Ophthalmology, Wayne State University, Detroit, Mich (H.S.); and the Department of Pediatrics, University of California at San Francisco (R.I.C.).
Correspondence to Sylvain Chemtob, MD, PhD, FRCP(C), Research Center, Ste-Justine Hospital, 3175 Côte Ste-Catherine, Montréal, Québec, Canada, H3T 1C5. E-mail chemtobs{at}ere.umontreal.ca
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Background—Prostaglandin E2 (PGE2) is important in ductus arteriosus (DA) patency, but the types of functional PGE2 receptors (EP) in the developing DA are not known. We postulated that age-dependent alterations in EP and/or their subtypes may possibly contribute to the reduced responsiveness of the newborn DA to PGE2.
Methods and Results—We determined PGE2 receptor
subtypes by competition binding and immunoblot studies on
the DA of fetal (
75% and 90% gestation) and newborn (<45 minutes
old) pigs. We studied the effects of EP receptor stimulation on cAMP
signaling in vitro and on term newborn (<3 hours old) DA patency in
vivo. Fetal pig DA expressed EP2, EP3, and
EP4 receptors equivalently, but not EP1. In
neonatal DA, EP1, EP3, and EP4 were
undetectable, whereas EP2 density was similar in fetus and
newborn. Prostaglandin-induced changes in cAMP mirrored
binding data. 16,16-Dimethyl PGE2 and 11-deoxy
PGE1 (EP2/EP3/EP4
agonist) produced more cAMP in fetus than newborn, but butaprost
(selective EP2 agonist) caused similar cAMP increases in
both; EP3 and EP4 ligands (M&B28767 and
AH23848B, respectively) affected cAMP production only in fetus.
After birth, administration of butaprost alone was as effective as
11-deoxy PGE1 and 16,16-dimethyl PGE2 in
dilating DA in vivo.
Conclusions—The data reveal fewer PGE2 receptors in the DA of the newborn than in that of the fetus; this may contribute to the decreased responsiveness of the DA to PGE2 in newborn. Because EP2 receptors seem to mediate the effects of PGE2 on the newborn DA, one may propose that a selective EP2 agonist may be preferred as a pharmacological agent to maintain DA patency in infants with certain congenital heart diseases.
Key Words: ductus arteriosus • receptors • prostaglandins • pediatrics
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Prostaglandins play a major role in maintaining patency of the fetal ductus arteriosus (DA).1 2 The marked sensitivity of the DA to prostaglandin E2 (PGE2) suggests that this is the most important prostanoid regulating vessel patency.3 PGE2 exerts its effects through a diverse group of receptors, classified as EP1, EP2, EP3, and EP4.4 Activation of EP1 increases inositol 1,4,5-triphosphate (IP3) formation and elicits vasoconstriction,5 stimulation of EP2 and EP4 increases cAMP and leads to vasodilation,4 6 and stimulation of EP3 decreases cAMP and opposes vasodilation.4
At present, the relative types and importance of EP receptors in the DA are uncertain. Pharmacological evidence suggests that the EP4 receptor is the predominant PGE2 receptor in the fetal rabbit DA.7 Conversely, genetic disruption of the EP4 receptor does not diminish DA patency in the fetal and neonatal mouse.8 However, the type of EP receptors expressed in higher species, and especially in the newborn, is not known. This is of particular relevance because responsiveness of the DA to PGE2 in the newborn is significantly less than that in the fetus.9 10 Because PGE2 acts on several distinct receptor subtypes, each coupled to different second messengers, we hypothesized that differences in the relative density and/or proportion of EP receptors could explain, at least in part, the differences in the responsiveness of the fetal and neonatal DA to PGE2.9 10
We therefore studied the expression of EP receptor subtypes and EP receptor signaling factors in the DA of fetal and newborn pigs. We also assessed the role of these receptors in the newborn in vivo. Our findings reveal that the DA of the fetal pig expresses negligible amounts of the EP1 receptor but does express the other 3 EP subtypes (EP2, EP3, EP4) in equivalent proportions. In the DA of the newborn pig, the density of PGE2 receptors is significantly reduced compared with that in the fetus because of complete loss of EP3 and EP4 receptors; however, the number of EP2 receptors remains unchanged, such that EP2 seems to mediate all PGE2-dependent relaxation in the newborn DA.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Animals
DAs were removed from fetal pigs (78 to 90 and 100 to 105 days of gestation [term, 114 days]) and term newborn piglets (1.25 to 1.7 kg, killed within 45 minutes of vaginal birth with pentobarbital [120 mg/kg intracardiac] under halothane anesthesia). Tissues were immediately rinsed in ice-cold Krebs buffer (pH 7.4) of the following composition (mmol/L): NaCl 120, KCl 4.5, CaCl2 2.5, MgSO4 1.0, NaHCO3 27, KH2PO4 1.0, and glucose 10 and then frozen in liquid N2; we have shown that freezing does not affect PGE2 binding.11 A group of newborn piglets (<3 hours old) was used to study the effects of prostaglandin analogues on DA diameter in vivo.
EP Receptor Characterization
Tissues were prepared for prostaglandin binding as
described.11 12 13 Aliquots of DA homogenate
(200 µg protein) were incubated at 37°C for 30 minutes with various
concentrations of
[3H]PGE2 (Amersham) in
the presence or absence of 25 µmol/L 16,16-dimethyl
PGE2. The reaction was terminated with ice-cold
5 mmol/L Tris-HCl buffer (pH 7.4), the
homogenate was filtered through Whatman GF/C glass
filter disks, and the radioactivity was counted with a ß-counter
(Beckman LS 7500). Subtypes of PGE2 receptors
were studied by displacement of bound
[3H]PGE2 with
16,16-dimethyl PGE2, AH6809 (Glaxo-Wellcome),
butaprost (Bayer), M&B28,767 (Rhone-Poulenc Rorer), and sulprostone,
11-deoxy PGE1, and AH23848B
(Glaxo-Wellcome).4 Receptor densities
(Bmax), affinity constants
(Kd), and concentrations of ligands that
displace 50% of bound
[3H]PGE2
(IC50) were determined from Scatchard plots and
displacement curves with the computer programs Prism (GraphPad) and
Ligand, respectively.14
Immunoblotting of EP Receptors
Western blotting of EP1,
EP3
, and EP4 receptors
was conducted15 on DA membranes prepared as
described13 and on cell lysates from human embryonic
kidney (HEK) 293 cells (Invitrogen), which overexpress each of these
receptors16 and hence were used as positive controls.
After immunoblotting with EP1,
EP3, or EP4 specific
polyclonal rabbit antibodies17 (1:1000;
EP2 antibodies are not available), immunoreactive
bands were visualized by chemiluminescence (Amersham) according to the
manufacturer's instructions.
cAMP Assay
The effects of PGE2 analogues on cAMP were
determined.12 13 Briefly, DA homogenates (100
µg protein) were incubated in an assay mixture (100 µL) containing
10 mmol/L Tris-HCl buffer (pH 8.0), 1 mmol/L ATP, 7.5
mmol/L MgCl2, 15 mmol/L creatine phosphate,
185 U/mL creatine phosphokinase, 200 µg/mL aspirin, 0.5 mmol/L
EGTA, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 1 mmol/L
dithiothreitol, 1 mmol/L benzamidine, 0.1 mmol/L
phenylmethylsulfonyl fluoride, and 100 µg/mL soybean trypsin
inhibitor in the presence or absence of test agents at
37°C for 10 minutes. The reaction was terminated with 200 µL acidic
ethanol. After centrifugation, cAMP was measured by
radioimmunoassay (Diagnostic Products).
Surgical Preparation and Echocardiography
Newborn pigs (between 1.5 and 2 hours after delivery; 1.3 to 1.7
kg) were prepared to test the effects of specific EP receptor agonists
on DA patency; in the piglet, the DA closes completely by 4 to 6 hours
of age.18 We were not able to perform in vivo studies on
fetal pigs because facilities were not available to operate on sows.
The newborn piglets were anesthetized with halothane (2%) for
15 minutes during tracheostomy and catheterization of
the umbilical vein and artery, and anesthesia was
discontinued after surgery. Animals were ventilated with air with a
Harvard small-animal respirator, maintained on -chloralose (50 mg/kg
bolus followed by 10 mg · kg-1 ·
h-1 infusion), and paralyzed with pancuronium
(0.1 mg/kg); body temperature was maintained at 38°C. The surgical
procedure was completed within 15 minutes, and piglets were allowed to
stabilize for an additional 30 minutes.
Echocardiographic measurements were performed with an Acuson 128 XP/10C real-time ultrasound imaging system using 7.5- and 5-MHz transducers duplexed with a range-gated Doppler as previously described19 20 ; Doppler signals were filtered by 100-Hz high-pass filter. The DA was visualized through a left second intercostal parasternal approach. Measurements of the DA diameter were repeated 3 times. The smallest diameter of the DA lumen was monitored until it reached 0.6 to 0.8 mm in diameter. Experimental drugs were then injected, and the smallest diameter was measured every minute for the next 10 to 25 minutes.
Animals (3 to 4 per treatment) were randomly assigned to receive 10-minute infusions of saline (1 mL, controls) or 0.083 µg · kg-1 · min-1 of EP receptor agonists; the doses used have been shown previously to be effective in vivo.21 The effect of EP receptor agonists on DA patency was also tested in animals pretreated with indomethacin (3 mg/kg IV for 5 minutes), once the DA diameter reached 0.6 to 0.9 mm (within 20 minutes).
Statistical Analysis
Data were analyzed by Student's t test and
by 2-way ANOVA factoring for time and treatment; means tests were
compared by the Tukey-Kramer method. Statistical significance was set
at P<0.05. Data are expressed as mean±SEM.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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PGE2 Receptors in Fetal and Newborn DA
[3H]PGE2 bound specifically to fetal and newborn DA membranes. Maximum binding of [3H]PGE2 to the fetal DA was similar at both gestational ages studied (78 to 90 days: 34.9±2.5 fmol/mg protein, n=3; 100 to 105 days: 35.1±3.1 fmol/mg protein, n=3). Maximum specific binding of [3H]PGE2 was 3-fold greater in fetal than newborn DA (Figure 1A
and 1B and Table 1); the
Kd did not differ between the fetus and
newborn.
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),
EP1 antagonist AH6809 (
),
EP2 agonist butaprost (•), EP3 agonist
M&B28,767 (
), EP1/EP3 agonist
sulprostone (
), EP4 antagonist
AH23848B (
), and EP2/EP3/EP4
agonist 11-deoxy PGE1 (
). E, Estimated
Bmax of specific EP receptors was calculated as maximum
binding of PGE2xproportion of EP subtypes derived
from indicated ligands.
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In fetal DA membrane preparations (Figure 1C
), butaprost
(EP2 agonist), M&B28,767
(EP3 agonist), sulprostone
(EP1/EP3 receptor agonist),
and AH23848B (EP4 antagonist) caused
an equivalent displacement of
[3H]PGE2 (Figure 1C and 1E and Table 2); 11-deoxy
PGE1
(EP2/EP3/EP4
agonist) displaced virtually all
[3H]PGE2, but the
EP1 antagonist AH6809 was ineffective
(Figure 1C and 1E and Table 2). In contrast to fetal DA,
[3H]PGE2 bound to newborn
DA membranes was no longer displaced by M&B28,767, sulprostone, or
AH23848B (Figure 1D). Butaprost and 11-deoxy
PGE1 were the only PGE2
analogues (along with 16,16-dimethyl PGE2)
capable of fully displacing
[3H]PGE2 from the newborn
DA (Figure 1D and Table 2). IC50
values of ligands tested were comparable to those reported in other
porcine tissues.11 12 Results indicate the presence of
EP2, EP3, and
EP4 in fetal DA.
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In the immediate postnatal newborn, EP3 and
EP4 are absent, without significant change in the
number of EP2 receptors (Figure 1E); this
is reflected by the reduced
[3H]PGE2 binding in the
newborn DA (Figure 1A and 1B and Table 1).
Expression of EP1, EP3, and
EP4 Immunoreactive Protein in DA of Fetal and Newborn
Pig
The DA of the fetal pig, but not of the newborn, expressed
EP3 (55-kDa band) and
EP4 (63-kDa band) immunoreactive protein
consistent with binding data (Figure 2); EP2 antibodies
are unavailable.
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PGE2 Analogue–Induced Production of
cAMP
PGE2 receptor–coupled changes in cAMP
generation were also consistent with the binding data.
16,16-Dimethyl PGE2 and 11-deoxy
PGE1
(EP2/EP3/EP4
agonist) produced a comparable dose-dependent increase in cAMP
synthesis, which was greater in fetus than in newborn (Table 3).
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In fetal DA, butaprost (EP2 agonist) stimulated
cAMP production in a concentration-dependent manner. The
EP3 agonist M&B28,767 and
EP1/EP3 agonist sulprostone
had no effect on cAMP formation by themselves but reduced
forskolin-induced cAMP synthesis; this suggests that
EP3 is coupled to inhibition of cAMP formation.
Because no selective EP4 agonist is currently
available, we estimated the effects attributed to
EP4, as we previously reported,12 by
subtracting the increase in cAMP produced by the combination of
16,16-dimethyl PGE2 and EP4
antagonist AH23848B (which would stimulate all of the EP
receptors except EP4) from the increase in cAMP
produced by 16,16-dimethyl PGE2 alone (which
stimulates all EP receptors). In the fetal DA, AH23848B decreased the
cAMP production induced by 16,16-dimethyl
PGE2 but not that stimulated by butaprost (Table 3).
In the newborn DA, butaprost increased cAMP generation to values
comparable to those in the fetus (Table 3); M&B28,767,
sulprostone, and AH23848B had no effect on cAMP production.
Effects of EP Receptor Agonists on DA Diameter In Vivo
We examined the effects of EP receptor agonists (17-phenyl-trinor
PGE2 [EP1]; butaprost
[EP2]; M&B28,767 [EP3];
sulprostone [EP1/EP3];
and 11-deoxy PGE1
[EP2/EP3/EP4])
on patency of the newborn DA in vivo (Figure 3). 16,16-Dimethyl
PGE2 dilated both the untreated, spontaneously
closing (Figure 3A) and the
indomethacin-constricted DA (Figure 3B).
Butaprost and 11-deoxy PGE1 dilated the
constricting DA as seen with 16,16-dimethyl PGE2
(Figure 3); cessation of infusion of these
PGE2 analogues resulted in constriction of the
DA. In contrast, 17-phenyl-trinor PGE2,
M&B28,767, and sulprostone did not affect DA diameter; 2- to 3-fold
higher infusion rates of the EP1 and
EP3 agonists also had no effects (data not
shown). Hence, the vasodilatory effects of PGE2
in the newly born seem to be accounted for by action on
EP2.
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),
EP2 agonist butaprost (•), EP3 agonist
M&B28,767 ( |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Previous pharmacological studies have suggested that EP4 is the dominant (relaxant) PGE2 receptor in the fetal rabbit DA.7 In the mouse, however, disruption of the EP4 gene did not affect ductal patency,8 suggesting possible species differences. We therefore set out to characterize the EP receptors in the DA of the fetus and newborn of a higher species, namely the pig.
Using binding and displacement, immunoblot, and stimulation
of second-messenger cAMP, our studies revealed that the number and
types of PGE2 receptors differ between fetal and
immediate postnatal newborn DA. There was a 3-fold higher density of
PGE2 receptors and a greater
PGE2-induced increase in cAMP in the fetus than
in the newborn (Table 3); a rise in cAMP is usually associated
with vasorelaxation.6 We identified
EP2, EP3, and
EP4 receptors in the fetal pig DA;
EP1 receptor was undetectable (Figures 1 and 2). In the newborn DA, we observed a decrease in
PGE2 binding due to a loss of
EP3 and EP4 receptors
(Figures 1C through 1E and 2); the number of
EP2 receptors was essentially the same in the
fetus and newborn. As a result, the EP2 receptor
appeared to mediate the vasorelaxant effects of
PGE2 on the full-term neonatal DA (Figure 3).
The mechanism(s) responsible for the birth-related decrease in PGE2 receptors in the DA are currently unknown. We have previously observed that the loss of DA responsiveness to PGE2 is directly related to the degree of postnatal DA constriction.9 However, a role for hypoxia to explain the selective loss of EP3 and EP4 is unlikely. Our data on [3H]PGE2 binding, immunoreactivity, and cAMP generation in newborn were obtained on DA of animals <45 minutes after birth. Although a 50% ductal constriction occurs over this time period,18 the partial pressure of oxygen also rises markedly immediately after birth; as a result, the DA tunica media may not develop significant hypoxia during this time period. In support of this inference, tissues from 15-minute-old newborn animals exhibited [3H]PGE2 binding properties similar to those of piglets 30 to 45 minutes old (data not shown). Another more likely explanation for the changes in EP receptor profile after birth relates to the sharp increase in PGE2 concentrations in both the DA and the circulating plasma during the perinatal period.3 20 22 23 We have previously observed that EP3 and EP4 receptors in cerebral and ocular vessels can be altered by high levels of circulating PGE2 concentrations in the perinatal period.12 13 EP2 receptors, conversely, do not appear to be regulated by PGE2 concentrations.12 We speculate that the EP receptors in the DA also may undergo this same type of homologous downregulation during the perinatal period when plasma PGE2 levels peak at the end of labor.24 Although PGE2 levels decrease immediately after birth to reach fetal values by 1 to 2 hours,24 this time period is insufficient to reverse the downregulation of PGE2 receptors.13
A reduction in PGE2 receptors associated with a decrease in PGE2-induced cAMP formation in the immediately postnatal newborn is consistent with and may contribute to the reduced responsiveness to PGE2 of the newborn DA compared with that of the fetus.9 10 In the present study, we focused on ontogenic changes in PGE2 receptors in the DA. Other mechanisms are also likely to participate in this age-dependent altered responsiveness of the DA to PGE2.9 10 These include developmental changes in the rates of prostaglandin production, uptake, and degradation as well as in prostaglandin-coupled signal transduction and relaxant mechanisms, and the role of increasing oxygen tension on ductal function,25 which has been debated26 ; these various aspects need to be examined separately.
In summary, the present study demonstrates a developmental alteration of EP receptors in the DA. However, changes in EP receptor profile in DA of the prematurely born newborn remain to be determined. The present study also provides a physiological basis for the potential use of more selective EP receptor ligands to control DA patency and potentially diminish the side effects associated with nonselective PGE therapy.27 For example, one could suggest specific therapies for maintaining ductal patency in infants with congenital heart disease, such as selective EP2 agonists, and possibly reduce PGE2-mediated fever due to EP3 stimulation.28 In the future, selective EP receptor antagonists targeted toward inflammation,29 fever,28 and pain21 in the pregnant subject will also need to be evaluated for their effects on the fetal DA.
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Acknowledgments |
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This study was supported by grants from the Medical Research Council of Canada, the Heart and Stroke Foundation of Quebec, and the Fonds de la Recherche en Santé du Québec, and from the US Public Health Service, NIH, NHLBI HL-46691. The authors thank Hendrika Fernandez and Manon Lessard for their technical assistance. M. Bhattacharya and Dr Hardy are recipients of Doctoral Research and Fellowship Awards, respectively, from the Medical Research Council of Canada.
Received March 16, 1999; revision received June 8, 1999; accepted June 14, 1999.
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