The synthesis of PGE2, the major vasodilator prostanoid of the ductus arteriosus (DA), is catalyzed by PGE2 synthases (PGES). The factors implicated in increased PGE2 synthesis in the perinatal DA are not known. We studied the developmental changes of PGES along with that of cyclooxygenase (COX)-2 and cytosolic phospholipase A2 (cPLA2) in the DA of fetal (75-90% gestation) and immediately postnatal newborn (NB) piglets. Levels of microsomal PGES (mPGES), COX-2, and PGE2 in the DA of NB were ∼7-fold higher than in fetus; activities of cytosolic PGES (cPGES) and cPLA2 in DA of the fetus and NB did not differ. Because platelet-activating factor (PAF) could regulate COX-2 expression, the former was measured and found to be more abundant in the DA of the NB than of fetus. PAF elicited an increase in mPGES, COX-2, and PGE2 in fetal DA to levels approaching those of the NB; cPGES, cPLA2, and COX-1 were unaffected. In perinatal NB DA, PAF receptor antagonists BN-52021 and THG-315 reduced mPGES, COX-2, and PGE2 levels and were associated with increased DA tone. It is concluded that PAF contributes in regulating DA tone by governing mPGES, COX-2, and ensuing PGE2 levels in the perinate.
- platelet-activating factor
- cytosolic phospholipase A2
prostaglandin E2 (PGE2) is the major vasodilator prostanoid of ductus arteriosus (DA) (9, 12). Cytosolic phospholipase A2 (cPLA2) causes the release of arachidonic acid, which is converted into PGH2 by cyclooxygenase (COX)-1 and COX-2 (39); PGH2 is then converted to PGE2 by PGE2 synthases (PGES). Two glutathione-dependent PGES isoforms have recently been identified (23, 36, 48). The constitutive PGES, cPGES, is a 26-kDa cytosolic protein (48) that is abundantly expressed in many tissues and cell lines (48). An inducible isoform, microsomal PGES (mPGES), is a 16-kDa microsomal protein that can be expressed and regulated in a cell-specific manner (23, 36). It is highly upregulated by proinflammatory factors and believed to be coupled principally to COX-2 (19, 36, 48, 51).
PGE2 levels increase in the circulation and numerous tissues at the onset of parturition, peak soon before birth (24), and decline rapidly thereafter (24, 28, 29, 34). A cytoprotective role for the perinatal rise in PGE2 has been suggested (38), consistent with maintenance of a patent DA to supply adequate blood flow to the descending aorta especially during recurring hypoxic episodes induced by the strong uterine contractions of the end of labor (17). However, the factors responsible for changes in PGE2 levels in the perinate have not been clearly identified. COX-2 is developmentally regulated (18, 43, 45, 46), but this cannot explain the relatively selective increase in PGE2 during the perinatal period (1, 24). On the other hand, specific ontogenic changes in PGES isoforms may be responsible for this developmental process; if so, selective regulation of PGE2 synthesis may constitute a novel approach for controlling DA tone.
Platelet-activating factor (PAF) is a potent proinflammatory cytokine that induces COX-2 expression (4, 33). PAF levels are reported to be increased in the perinate (22, 30). It is thus possible that PAF contributes to the regulation of COX-2 and coupled mPGES as well as PGE2 levels in DA of the perinate, but this remains to be determined. We therefore investigated the developmental changes in mPGES and COX-2 expression in the DA of the piglet and the possible role of PAF in their regulation. Our findings reveal a developmental increase in mPGES expression along with that of COX-2 in the DA, which is regulated at least in part by PAF, and impacts on DA tone.
MATERIAL AND METHODS
Animals. Fetal pigs (Abattoir Laurentien, Quebec, Canada) at ∼75-90% gestation (term 114 days) and newborn piglets (Fermes Ménard, Quebec, Canada) within 2 h of vaginal birth were used. Tissues of the immediately postnatal newborn pigs were studied to reflect as closely as possible physiological characteristics associated with labor; difficulty in conducting studies in fetuses of sows during labor precluded such experiments. Animals were killed with intracardiac pentobarbital sodium (120 mg/kg); newborns were first anesthetized with halothane. Tissues were rinsed in ice-cold Krebs buffer (pH 7.4) of the following composition (in mM): 120 NaCl, 4.5 KCl, 2.5 CaCl2, 1.0 MgSO4, 27 NaHCO3, 1.0 KH2PO4, and 10 glucose; tissues were then frozen in liquid N2 and stored at -80°C until assayed.
Western blots. Frozen tissues were homogenized with lysis buffer consisting of 10 mM Tris·HCl, pH 7.4, 0.1 M NaCl, 2 mM MgCl2, 0.02% acetylsalicylic acid, 0.5 mM EDTA, 0.1 mM PMSF, and 100 μg/ml trypsin inhibitor and centrifuged twice for 15 min at 1,000 g at 4°C. Immunoblots were performed as described (33, 43). Equal amounts of supernatant (30-40 μg protein/lane) in SDS-containing sample buffer were boiled for 5 min (27), and proteins were resolved by 15 and 8% SDS-PAGE for PGES and COX-2/cPLA2, respectively; β-actin served as control and was stable. The proteins were electroblotted onto polyvinylidine difluoride (PVDF transfer) membranes (Perkin Elmer Life Sciences, Boston, MA; Ref. 50). The filters were blocked overnight at 4°C in Tris-buffered saline (TBS) [10 mM Tris·HCl (pH 7.4)-100 mM NaCl] containing 0.1% (vol/vol) Tween 20 (0.1% T-TBS) and 5% (wt/vol) nonfat dried milk. The membrane was subsequently incubated overnight at 4°C in 0.1% T-TBS with 5% (wt/vol) nonfat dried milk with the corresponding IgG primary antibodies: polyclonal rabbit anti-human mPGES (1:500) (Cayman Chemical, Ann Arbor, MI), monoclonal anti-mouse cPGES (1:500) (gift from Dr. David Toft, Mayo Clinic, Rochester, MI), polyclonal rabbit anti-mouse COX-2 (1:250) (Biomol Research, Philadelphia, PA), polyclonal rabbit anti-human cPLA2 (1:500) (Santa Cruz Biotechnologies, Santa Cruz, CA), and polyclonal anti-human β-actin (Abcam, Cambridge, UK). After washing, secondary horseradish peroxidase-linked anti-rabbit antibodies (1:2,500; Pierce, Rockford, IL) were applied for 1 h at 25°C. Detection of signal was enhanced using chemiluminescence (PerkinElmer, Boston, MA), and the resulting bands were analyzed densitometrically. The positive control for mPGES was ram seminal vesicle microsomes (5 mg/ml, Oxford Biomedical Research, Oxford, MI), while positive control for cPGES was purified human protein overexpressed in mouse cells hybridized with cancerous cells kindly provided by Dr. D. O. Toft (Mayo Graduate School, Rochester, MI).
PAF assay. DA homogenates were prepared in 10% dimethyldichlorosilane siliconized glass tubes as previously described (6). PAF extraction was conducted on octadecylsilyl silica columns (41) and quantified with the scintillation proximity assay commercial kit (Amersham Pharmacia Biotech, Piscataway, NJ) (21). Cross-reactivity of the antibody for other structurally related lipids was <0.06% (lyso-PAF <0.01%), and interassay variability was <5%.
Vasomotor responses of DA. Isometric tension of DA was determined as described (8). DA was cut into 2-mm rings and suspended between two stainless steel wires at initial tension of 2 g in 20-ml tissue baths containing Krebs buffer aerated with a mixture of 95% O2-5% CO2. Isometric tension was recorded by means of force transducers (Kent Scientific). Preparations were allowed to equilibrate until the tension became stable before test agents were added. Time-dependent response to C-PAF (stable PAF analog, 0.1 μM) in the absence and the presence of the putative mPGES inhibitor MK-886 (10 μM) (32) was determined over a 6-h period.
Statistical analysis. Ontogenic variation of mPGES, COX-2, cPLA2, and cPGES protein expression was assessed by unpaired Student's t-test; effects of various agents on DA tension were determined by two-way ANOVA factoring for time and treatment. Comparison among means tests was performed by the Tukey-Kramer method. Statistical significance was set at P < 0.05. Data are expressed as means ± SE.
PGES, COX-2, and cPLA2 expression and PGE2 and PAF levels in fetal and immediate postnatal newborn DA. The expression of mPGES and COX-2 proteins in the DA of pigs immediately postnatally (within 2 h of birth) was six- to sevenfold higher than in the DA of fetus (Fig. 1, A and B); in adjacent aorta and pulmonary artery there was only a two- to threefold higher expression of these proteins in the newborn. Expression of cPGES and cPLA2 was relatively unchanged between ages in all tissues (Fig. 1, C and D). The higher mPGES and COX-2 protein in newborn DA was paralleled by a developmental rise in PGE2 levels (Fig. 2A); PGE2 levels in aorta and pulmonary artery increased with age to a lesser extent (Fig. 2, B and C, respectively) as seen for corresponding mPGES and COX-2 immunoreactivity.
Effects of PAF on PGES and COX-2 expression and PGE2 levels in fetal DA. Because PAF generation is increased in the perinatal period (22, 30) and is reported to upregulate COX-2 expression (4, 33), we tested if PAF can induce mPGES and COX-2 in the fetal DA. PAF concentrations in the DA of immediate postnatal newborn pigs were indeed significantly greater than in fetal tissues (Fig. 3A). C-PAF caused a dose-dependent increase in mPGES and COX-2 expression in the fetal DA (6 h incubation), which was associated with a time-dependent rise in PGE2 levels (Fig. 3, C-E), which approached the higher values observed in the newborn (Figs. 1 and 2); COX-1 (not shown) as well as cPGES and cPLA2 expressions were not affected by C-PAF (0.1 μM; Fig. 3B). Concordant with these results, C-PAF induced a transient rise in DA tone, which in contrast to the contractant KCl (100 mM) was not sustained but did persist in presence of the mPGES inhibitor MK-886 (32) (see Fig. 5A).
Effects of endogenous PAF on mPGES, COX-2, and PGE2 levels in the DA of the perinate. We determined the role of endogenous PAF on mPGES, COX-2, and PGE2 levels in immediately postnatal newborn DA using distinct PAF receptor antagonists. Treatment of DA from immediate postnatal newborn with PAF receptor antagonists BN-52021 (0.1 μM; Ref. 6) and THG-315 (0.1 μM; Ref. 6) for 6 h (but not <2 h) decreased mPGES expression by 50-85% (Fig. 4A) and COX-2 by 75-80% (Fig. 4B) but not that of cPGES and cPLA2; PAF receptor antagonist-induced changes were associated with a corresponding reduction in DA PGE2 levels (Fig. 4C) and a coincidental delayed rise in DA tone starting 2 h after treatment with the agents (Fig. 5B). Stimulation of newborn DA with PAF induced a smaller rise in mPGES and COX-2 protein density (data not shown), possibly due to inherently augmented expression of these proteins in newborn (Fig. 1).
The mechanisms that govern PGE2 formation in DA during development are not well known. Although COX-2 has been shown to contribute significantly to prostanoid formation in the perinatal DA (18, 47), its expression and activity cannot explain the specific increase in PGE2 in this tissue especially at the end of gestation. Our findings indicate that the PGE2-generating enzyme mPGES (23, 36), like COX-2, is developmentally regulated in the DA by PAF, the levels of which are relatively augmented in the DA of the perinate.
The expression of mPGES, like that of COX-2, is regulated by inflammatory mediators (5, 19, 23, 31, 36, 49). Although PAF levels are increased in tissues of the perinate (22, 30), its role in the physiological regulation of mPGES and COX-2 in the DA is not known. Overall data of this study suggest that PAF increases mPGES and COX-2 expression and PGE2 levels, which impacts on ductal tone. Specifically, 1) PAF levels were threefold higher in the DA of the immediate postnatal newborn than of the fetus (Fig. 3A). One can argue that our experiments on fetus were conducted on tissues obtained from animals over a range of 75-90% gestation. Conceivably a gradual increase in PGE2 production during gestation may alter vasomotor response to PGE2, but by far the most significant rise in PGE2 levels occurs at the time of parturition (10), including in the DA (18). Accordingly, the biggest impact on PGE2 synthesis and tone would occur in the perinate (9, 18) consistent with our principal objective of elucidating mechanisms that govern COX-2 and mPGES expression in the immediate perinate. 2) In fetal DA, PAF stimulated a dose-dependent increase in mPGES and COX-2 expression and an associated time-dependent rise in the levels of PGE2 (Fig. 3, C-E); these findings are consistent with the concentration profile of PAF in inducing the immediate-early gene COX-2 (4) as well as with the documented role for COX-2 in the perinatal DA (12, 18, 47). Furthermore, PAF caused a rapid but transient rise in fetal DA tone; constrictor effects of PAF are mostly independent of cyclooxygenase (3) but likely secondary to calcium mobilization (11, 26). The rapid loss of this tone coincided with and could be explained by an increase in mPGES, COX-2, and the major DA relaxant PGE2 (Fig. 5A), as it was prevented with MK-886, an eicosanoid synthesis blocker that inhibits mPGES (32). 3) In the DA of the immediately postnatal newborn, inhibition of endogenous PAF signaling through PAF receptor blockade caused a striking decrease in mPGES and COX-2 expression as well as PGE2 levels (Fig. 3, C and D). The decrease in PGE2 concentrations coincided with an increase in DA tone starting 2 h after the addition of PAF receptor antagonists (Fig. 5B); observations are consistent with a short half-life for the products of mPGES and COX-2 genes, as reported (19).
mPGES and COX-2 seem to exhibit a similar ontogenic and regulatory profile in DA. Our results from this and previous studies (12, 18, 47) suggest a physiological functional coupling between COX-2 and mPGES that would participate signifi-cantly in the perinatal increase in PGE2 levels. Such a COX-2/mPGES coupling has been proposed in isolated cell systems, namely in transfected HEK-293 cells exposed to proinflammatory agents (19, 36), whereas COX-1 has been reported to couple preferentially to cPGES and was not affected by inflammatory mediators (48).
The mechanisms responsible for the perinatal rise in PAF levels are not known. Possible regulatory factors can be proposed. These include the activity of the catabolic enzyme PAF acetylhydrolase, which varies with development, yielding higher activities in the adult than the fetus (42). Also, a number of mediators known to induce PAF formation such as cytokines (52), endothelin (37), and angiotensin (40) exhibit increased levels in the perinatal period and may contribute to high PAF levels in the perinate.
In summary, the present paper provides a mechanism for the increase in the levels of the important DA autacoid PGE2 at the end of gestation, especially at parturition, specifically through a concerted developmental regulation of mPGES and COX-2 contributed by PAF, which impacts on DA tone. One could speculate that as sensitivity of the DA to PGE2 decreases with advancing gestation (14), the increased expression of COX-2 and mPGES may supply the necessary PGE2 levels to ensure adequate relatively oxygenated blood flow across the DA to the descending aorta-dependent tissues, especially during recurring hypoxic episodes associated with increased uterine contractions at the end of parturition (17). Because all PGE2 receptors in the DA evoke its vasorelaxation (8) and because COX-2 inhibitors may cause serious renal complications especially to the developing subject (44), one may postulate that mPGES, which catalyzes the formation of the ligand common to all PGE2 receptors, could be a preferred therapeutic target in the control of DA tone.
This study was supported by grants from the Canadian Institute of Health Research, Heart and Stroke Foundation of Québec, Fonds de la Recherche en Santé du Québec, and from the National Heart, Lung, and Blood Institute (HL-46691). A. Bouayad is recipient of a Hôpital Ste-Justine Foundation fellowship, M. Beauchamp a recipient of a studentship from the Canadian Institute of Health Research, and C. Quiniou a studentship from the Heart and Stroke Foundation of Canada. S. Chemtob is an awardee of a Canada Research Chair (perinatology).
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