Mouse aortic muscle cells respond to oxygen following cytochrome P450 3A13 gene transfer.

We have previously shown that a cytochrome P450 (CYP450) hemoprotein from the 3A subfamily CYP3A13 for the mouse, serves as the sensor in the contraction of the ductus arteriosus in response to increased oxygen tension. In addition, we have identified endothelin-1 (ET-1) as the effector for this response. Here, we examined whether Cyp3a13 gene transfer confers oxygen sensitivity to cultured muscle cells from mouse aorta. Coincidentally, we determined whether the same hemoprotein is normally present in the vessel. Cyp3a13-transfected aortic cells responded to oxygen, whereas no significant response was seen in native cells or in cells transfected with an empty vector. Furthermore, this oxygen effect was curtailed by the ET-1/ETA receptor antagonist BQ-123. We also found that CYP3A13 occurs naturally in aortic tissue and its isolated muscle cells in culture. We conclude that CYP3A13 is involved in oxygen sensing, and its action in the transfected muscle cells of the aorta, as in the native cells of the ductus, takes place through a linkage to ET-1. However, the response of aortic muscle to oxygen, conceivably entailing the presence of CYP3A13 at some special site, is not seen in the native situation, and may instead unfold upon transfection of the parent gene.

Hydrogen sulfide in the mouse ductus arteriosus: a naturally occurring relaxant with potential EDHF function.

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-ß-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.

Dual, constrictor-to-dilator, response of the mouse ductus arteriosus to the microsomal prostaglandin E synthase-1 inhibitor, 2-(6-chloro-1H-phenanthro[9,10d]imidazole- 2-yl)isophthalonitrile.

BACKGROUND: Microsomal prostaglandin E synthase-1 (mPGES1) is critical for prostaglandin E(2) 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.

Cytochrome P-450 3A13 and endothelin jointly mediate ductus arteriosus constriction to oxygen in mice.

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.

EDHF function in the ductus arteriosus: evidence against involvement of epoxyeicosatrienoic acids and 12S-hydroxyeicosatetraenoic acid.

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.

Role of microsomal prostaglandin E synthase-1 (mPGES1)-derived PGE2 in patency of the ductus arteriosus in the mouse.

Prostaglandin E2 (PGE2) 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 (mPGES1-/-), we have examined the importance of this enzyme for PGE2 formation and function. mPGES1-/- ductus, unlike WT ductus, contracted little, or not all, to indomethacin in vitro. Coincidentally, as evident from responses to NG-nitro-L-arginine 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 mPGES1-/- fetuses. Conversely, postnatal closure progressed regularly in mPGES1-/- animals thanks to residual PGE2 originating via mPGES2. We conclude that mPGES1 is critical for PGE2 formation in the ductus but its loss does not entail compensatory up-regulation 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.

Expression, microsomal and mitochondrial activities of cytochrome P450 enzymes in brain regions from control and phenobarbital-treated rabbits.

Expression and monooxygenase activity of various cytochrome P450 (CYP) enzymes along with constitutive androstane (CAR) and the pregnane X (PXR) receptors were investigated in the brain of control and phenobarbital-treated rabbits (80 mg/kg for 4 days). RT-PCR analysis, using specific primers, demonstrated that in control rabbits mRNAs of CYP 2A10, 2B4/5 and 3A6 were expressed, though to a different extent, in the liver, as well as in brain cortex, midbrain, cerebellum, striatum, hippocampus and hypothalamus, whilst CYP2A11 and 4B1 were not expressed in the hypothalamus. CAR was expressed in liver and all the brain regions examined, whereas the PXR was expressed only in liver and cortex. Real time RT-PCR analysis demonstrated that in vivo treatment with phenobarbital, in contrast with what happened in liver, did not induce the expression of CYP 2B4/5 mRNA in cortex, midbrain and cerebellum. NADPH cytochrome c reductase and some other enzymatic activities markers of CYP 2A, 2B, 3A and 4B activities were studied in liver microsomes as well as in microsomes and mitochondria of brain cortex, midbrain and cerebellum of control and phenobarbital-treated rabbits. In contrast to what was observed in liver, phenobarbital treatment did not induce the aforementioned monooxygenase activities in brain. However, we cannot exclude that a longer phenobarbital treatment may lead to a significant induction of CYP activities in brain. These findings indicated that brain CYPs, despite the presence of CAR, were resistant to phenobarbital induction, indicating a possible different regulation of these enzymes between brain and liver.