Background and purpose: Prenatal patency of ductus arteriosus is maintained by prostaglandin (PG) E 2 , possibly along with nitric oxide (NO) and carbon monoxide (CO), and cyclooxygenase (COX) deletion upregulates NO. Here, we have examined enzyme source and action of NO for ductus patency and whether NO and CO are upregulated by deletion of, respectively, heme oxygenase 2 (HO-2) and COX1 or COX2. Experimental approach: Experiments were performed in vitro and in vivo with wild-type and gene-deleted, near-term mouse fetuses. Key results: N G -nitro-L-arginine methyl ester (L-NAME) contracted the isolated ductus and its effect was reduced by eNOS, but not iNOS, deletion. L-NAME contraction was not modified by HO-2 deletion. Zinc protoporphyrin (ZnPP) also contracted the ductus, an action unaffected by deletion of either COX isoform. Bradykinin (BK) relaxed indomethacin-contracted ductus similarly in wild-type and eNOSÀ/À or iNOSÀ/À. BK relaxation was suppressed by either L-NAME or ZnPP. However, it reappeared with combined L-NAME and ZnPP to subside again with K þ increase or K þ channel inhibition. In vivo, the ductus was patent in wild-type and NOS-deleted fetuses. Likewise, no genotype-related difference was noted in postnatal closure. Conclusions and implications: NO, formed mainly via eNOS, regulates ductal tone. NO and CO cooperatively mediate BKinduced relaxation in the absence of PGE 2 . However, in the absence of PGE 2 , NO and CO, BK induces a relaxant substance behaving as an endothelium-derived hyperpolarizing factor. Ductus patency is, therefore, sustained by a cohort of agents with PGE 2 and NO being preferentially coupled for reciprocal compensation.

The fetal ductus arteriosus (DA) contracts to oxygen, and this feature, maturing through gestation, is considered important for its closure at birth. We have previously obtained evidence of the involvement of cytochrome P-450, possibly of the 3A subfamily (CYP3A), in oxygen sensing and have also identified endothelin (ET)-1 as the attendant effector for the contraction. Here, we examined comparatively wild-type (WT) and CYP3A-null (Cyp3a(-/-)) mice for direct validation of this concept. We found that the CYP3A subfamily is represented only by CYP3A13 in the WT DA. CYP3A13 was also detected in the DA by immunofluorescence microscopy, being primarily colocalized with the endoplasmic reticulum in both endothelial and muscle cells. However, a distinct signal was also evident in the plasma membrane. Isolated DAs from term WT animals developed a sustained contraction to oxygen with transient contractions superimposed. Conversely, no tonic response occurred in Cyp3a(-/-) DAs, whereas the phasic response persisted unabated. Oxygen did not contract the preterm WT DA but caused a full-fledged contraction after retinoic acid (RA) treatment. RA also promoted an oxygen contraction in the Cyp3a(-/-) DA. However, responses of RA-treated WT and Cyp3a(-/-) mice differed in that only the former abated with ET-1 suppression. This implies the existence of an alternative target for RA responsible for the oxygen-induced contraction in the absence of CYP3A13. In vivo, the DA was constricted in WT and Cyp3a(-/-) newborns, although with a tendency to be less narrowed in the mutant. We conclude that oxygen acts primarily through the complex CYP3A13 (sensor)/ET-1 (effector) and, in an accessory way, directly onto ET-1. However, even in the absence of CYP3A13, the DA may close postnatally thanks to the contribution of ET-1 and the likely involvement of compensating mechanism(s) identifiable with an alternative oxygen-sensing system and/or the withdrawal of relaxing influence(s) operating prenatally.

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Background and purpose: Prenatal patency of ductus arteriosus is maintained by prostaglandin (PG) E 2 in concert with nitric oxide (NO) and carbon monoxide (CO). Accordingly, we have previously found that NO activity increases upon deletion of either COX. Here, we have examined whether COX inhibition by indomethacin mimics COX deletion in promoting NO. Experimental approach: Experiments were performed in vitro and in vivo with wild-type (WT) and eNOSÀ/À, near-term mouse foetuses. Indomethacin was given p.o. to the mother as single (acute treatment) or repeated (daily for 3 days; chronic treatment) doses within a therapeutic range (2 mg kg À1 ). Key results: Indomethacin promoted eNOS mRNA expression in the WT ductus. Coincidentally, the drug enhanced the contraction of the isolated ductus to the NOS inhibitor, N G -nitro-L-arginine methyl ester, and its effect augmented with the length of treatment. No such enhancement was seen with the eNOSÀ/À ductus. Chronic indomethacin also increased, albeit marginally, the contraction of the WT ductus to the CO synthesis inhibitor, zinc protoporphyrin. Whether given acutely or chronically, indomethacin induced a little narrowing of the ductus antenatally and had no effect on postnatal closure of the vessel. Conclusions and implications:We conclude that activation of NO and, to a much lesser degree, CO mechanisms is an integral part of the indomethacin effect on the ductus. This relaxing influence may oppose the contraction from PGE 2 suppression and could explain the failures of indomethacin therapy in premature infants with persistent duct.

We have previously reported that bradykinin relaxes the fetal ductus arteriosus via endothelium-derived hyperpolarizing factor (EDHF) when other naturally occurring relaxants (prostaglandin E2, nitric oxide, and carbon monoxide) are suppressed, but the identity of the agent could not be ascertained. Here, we have examined in the mouse whether hydrogen sulfide (H2S) is a relaxant of the ductus and, if so, whether it may also function as an EDHF. We found in the vessel transcripts for the H2S synthetic enzymes, cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS), and the presence of these enzymes was confirmed by immunofluorescence microscopy. CSE and CBS were distributed across the vessel wall with the former prevailing in the intimal layer. Both enzymes occurred within the endoplasmic reticulum of endothelial and muscle cells, whereas only CSE was located also in the plasma membrane. The isolated ductus contracted to inhibitors of CSE (d,l-propargylglycine, PPG) and CBS (amino-oxyacetic acid), and PPG contraction was attenuated by removal of the endothelium. EDHF-mediated bradykinin relaxation was curtailed by both PPG and amino-oxyacetic acid, whereas the relaxation to sodium nitroprusside was not affected by either treatment. The H2S donor sodium hydrogen sulfide (NaHS) was also a potent, concentration-dependent relaxant. We conclude that the ductus is endowed with a H2S system exerting a tonic relaxation. In addition, H2S, possibly via an overriding CSE source, qualifies as an EDHF. These findings introduce a novel vasoregulatory mechanism into the ductus, with implications for antenatal patency of the vessel and its transitional adjustments at birth.

Prostaglandin E 2 (PGE 2 ) plays a key role in the ductus arteriosus, prenatally by maintaining patency and postnatally by promoting tissue remodeling for closure. Here, by using near-term mouse fetuses with (wild-type, WT) and without microsomal PGE synthase-1 (mPGES1Ϫ/Ϫ), we have examined the importance of this enzyme for PGE 2 formation and function. mPGES1Ϫ/Ϫ ductus, unlike WT ductus, contracted little, or not all, to indomethacin in vitro. Coincidentally, as evident from responses to N G -nitro-Larginine methyl ester and zinc photoporphyrin, the mutant showed no significant enhancement of nitric oxide (NO)-and carbon monoxide (CO)-based relaxation. mPGES1 suppression differs, therefore, from cyclooxygenase (COX) suppression, whether genetically or pharmacologically induced, where NO is markedly up-regulated. In vivo, the ductus was patent, albeit occasionally with a narrowed lumen, in all mPGES1Ϫ/Ϫ fetuses. Conversely, postnatal closure progressed regularly in mPGES1Ϫ/Ϫ animals thanks to residual PGE 2 originating via mPGES2. We conclude that mPGES1 is critical for PGE 2 formation in the ductus but its loss does not entail compensatory upregulation of other relaxing mechanisms. Accordingly, an mPGES1 inhibitor stands out as a prospective better tool, compared with the currently used COX inhibitors, for the management of premature infants with persistent ductus. (Pediatr Res 64: 523-527, 2008) P rostaglandin (PG) E 2 is viewed as the prime agent for prenatal patency of the ductus arteriosus (1). Conversely, PGI 2 is without a role, notwithstanding its occurrence in the vessel and its known importance for vasoregulation elsewhere (2,3). Ductal PGE 2 is formed primarily through the cyclooxygenase-2 (COX2)/microsomal PGE synthase-1 (mPGES1) complex, while little of the compound originates from the COX1/mPGES1 complex and none at all from cytosolic PGES (cPGES). No information is available on microsomal PGE synthase-2 (mPGES2), but evidence from other vascular districts points to its minor function (4). Several findings support this arrangement in the ductus, namely, immunocytochemical data showing a predominant colocalization of COX2 with mPGES1 (5); the increase in COX2 and mPGES1 expression occurring selectively through development (6); the constrictor action of COX2 inhibitors on the ductus, being close or even equal in magnitude to that of a dual COX1/ COX2 inhibitor (7-9); the greater impairment of PGE 2 -based relaxation upon COX2 than COX1 deletion (5) along with the marked curtailment of PGE 2 formation after inhibition of either COX2 or mPGES1 (6,7). Consistent with the preferential operation of the COX2/mPGES1 route for PGE 2 synthesis is also evidence from adult blood vessels (10) and the notion that mPGES1 is catalytically most active among the PGES (11).Despite these facts, previous attempts to prove the actual importance of the COX2/mPGES1 pathway for ductus patency have failed. As shown by others and ourselves (5,12,13), COX2 removal does not result in loss of patency, likely thanks to ...

We have previously shown (Ref. 2) that endothelium-derived hyperpolarizing factor (EDHF) becomes functional in the fetal ductus arteriosus on removal of nitric oxide and carbon monoxide. From this, it was proposed that EDHF originates from a cytochrome P-450 (CYP450)-catalyzed reaction being inhibited by the two agents. Here, we have examined in the mouse ductus whether EDHF can be identified as an arachidonic acid product of a CYP450 epoxygenase and allied pathways. We did not detect transcripts of the mouse CYP2C subfamily in vessel, while CYP2J subfamily transcripts were expressed with CYP2J6 and CYP2J9. These CYP2J hemoproteins were also detected in the ductus by immunofluorescence microscopy, being colocalized with the endoplasmic reticulum in both endothelial and muscle cells. Distinct CYP450 transcripts were also detected and were responsible for omega-hydroxylation (CYP4A31) and 12R-hydroxylation (CYP4B1). Mass spectrometric analysis showed formation of epoxyeicosatrienoic acids (EETs) in the intact ductus, with 11,12- and 14,15-EETs being more prominent than 5,6- and 8,9-EETs. However, their yield did not increase with nitric oxide/carbon monoxide suppression, nor did it abate with endothelium removal. No evidence was obtained for formation of 12R-hydroxyeicosatrienoic acid and omega-hydroxylation products. 2S-hydroxyeicosatetraenoic acid was instead detected, and, contrary to data implicating this compound as an alternative EDHF, its suppression with baicalein did not modify the EDHF-mediated relaxation to bradykinin. We conclude that none of the more common CYP450-linked arachidonic acid metabolites appears to qualify as EDHF in mouse ductus. We speculate that some novel eicosanoid or a totally unrelated compound requiring CYP450 for its synthesis accounts for EDHF in this vessel.

Background: Microsomal prostaglandin E synthase-1 (mPGES1) is critical for prostaglandin E₂ formation in ductus arteriosus (DA) and, accordingly, in its patency. We previously reported that mPGES1 deletion, unlike cyclo-oxygenase (COX) suppression, is not followed by upregulation of relaxant nitric oxide (NO). Consequently, we proposed that a mPGES1 inhibitor may be better than currently used COX inhibitors in managing premature infants with persistent DA (PDA). Objective: To assess the effect of the mPGES1 inhibitor, 2-(6-chloro-1H-phenanthro[9,10d]imidazole-2-yl)isophthalonitrile (MF63) on DA ex vivo and in vivo (p.o. to the mother). Methods: Experiments were carried out with mice bearing human mPGES1. We utilized isolated, wire-mounted DA for isometric recording and a whole-body freezing technique to assess the DA caliber as it occurs in vivo. Results: MF63 (10 µM) contracted the isolated DA. DA constriction was also seen in vivo after a single 10-mg kg^–1 dose. Conversely, a 30-mg kg^–1 dose gave inconsistent results, combining constriction with no effect. DA dilatation followed instead a repeated lower dose (twice daily for 3 days), and postnatal closure of the vessel was also delayed. Chronic pretreatment had no effect on endothelial NO synthase mRNA expression in fetal DA, nor did it modify the contraction to NO synthase inhibitor N^G-nitro-L-arginine methyl ester (100 µM). Conclusions: MF63 has a dual action on DA, the constriction being associated with accessory dilatation. The latter effect should be explained before considering further a mPGES1 inhibitor for management of PDA.