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Am J Physiol Heart Circ Physiol 277: H1521-H1531, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 4, H1521-H1531, October 1999

Endothelin A receptor is necessary for O2 constriction but not closure of ductus arteriosus

F. Coceani1, Y.-A. Liu1, E. Seidlitz1, L. Kelsey1, T. Kuwaki3, C. Ackerley2, and M. Yanagisawa4

1 Integrative Biology Programme and 2 Division of Pathology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; 3 Department of Physiology, School of Medicine, Chiba University, Chiba, 260-8670 Japan; and 4 Howard Hughes Medical Institute and Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In vitro and in vivo techniques were developed with genetically modified mice to determine whether endothelin-1 (ET-1) functions as an O2 mediator in closure of the ductus arteriosus (DA) at birth. Wild-type CD-1 and 129/SvEv mice with ETA receptor -/-, +/-, and +/+ genotypes were used. Isolated DA from term ETA +/+ fetuses contracted to O2 (5-95%) and a thromboxane A2 analog (ONO-11113, 0.1 µM). Instead, ET-1 elicited a dual response with weak relaxation (0.1 nM) preceding contraction (1-100 nM). Indomethacin (2.8 µM) was also a constrictor. ETA -/- DA, unlike ETA +/+ DA, contracted marginally to O2 and ET-1 but responded to ONO-11113. O2 contraction was also reduced in ETA +/- DA. In vivo, DA constricted equally in tracheotomized ETA -/- and ETA +/+ newborns. Conversely, no DA constriction was seen in hyperoxic ETA -/- fetuses in utero, although it occurred in ETA +/+ and +/- littermates. We conclude that ET-1 mediates the DA constrictor response to O2. Without ET-1, however, the vessel still closes postnatally, conceivably caused by the withdrawal of relaxing influence(s).

oxygen; endothelin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE DUCTUS ARTERIOSUS is a large muscular shunt in the fetus, connecting the pulmonary artery with the aorta and allowing blood to bypass the unexpanded lungs. At birth, as the lungs acquire their ventilatory function and blood PO2 rises to extrauterine values, the ductus constricts and within hours undergoes functional closure. Both prenatal patency and postnatal closure of the ductus are known to be actively induced, but questions remain about the identity of the effector agents (3, 10, 29). Although there is good evidence implicating PGE2 (3, 10, 29), either alone or in combination with nitric oxide (NO) (1, 7) in ductus patency, the mechanism of ductus closure is being debated (29, 33). We have proposed that ductus closure takes place through a multistep process in which O2 is the trigger, a cytochrome P-450-based monooxygenase reaction the signal transducer, and endothelin-1 (ET-1) the effector (4, 8, 9), acting via the ETA receptor subtype. Coincidentally, we have assumed that subsidence of the relaxing influence of PGE2 at birth, secondary to the fall in blood PGs and the reduced sensitivity of ductal muscle to PGE2 (3, 10, 29), is an accessory event. However, recent work in mice lacking the EP4 receptor subtype, which is the putative target for PGE2 in the ductus (22, 30), points to a major role of this PG mechanism in ductus closure (22). Furthermore, a group (15) has questioned our scheme involving ET-1 as O2 messenger in the ductus, whereas other investigators (21, 32) have linked the O2 constriction to inhibition of the K+ channels.

The development during the past few years of mice strains with the targeted deletion of genes encoding distinct components of the ET-1 system (2, 19, 35) provides the means of directly assessing the role of the peptide in ductus closure. Among the available mutations, we opted to use animals lacking the ETA receptor (2) rather than ET-1 itself (19, 35) to avoid any confounding action of the maternally derived peptide on the null mutant fetus (18). However, before addressing this question, certain methodological issues had to be solved. The mouse fetal ductus had never been studied in vitro, and experiments were required to verify the viability of the preparation. To assess the behavior of the ductus in vivo, specifically its closure, steps had to be taken to overcome the occurrence in ETA null mutants of craniofacial anomalies precluding respiratory function and, hence, survival after birth (2). Once these issues had been settled, our specific aim was to ascertain whether deletion of the ETA receptor, with the attendant loss of ET-1 contractile action, interferes with O2 constriction of the fetal ductus in vitro and the postnatal closure of the vessel in vivo.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were carried out in inbred 129/SvEv mice with ETA -/-, +/-, and +/+ genotypes (litter size 3-12, mean 9). Wild-type CD-1 mice (litter size 7-18, mean 13) formed a separate control. Fetuses were used near term, and their gestational age was 18 days in all but a few early experiments in which age was 19 days. However, results were identical at the two ages and were pooled. ETA -/- mice were identified by their characteristic craniofacial anomaly (2), whereas analysis of tail genomic DNA was necessary with the heterozygous and wild-type littermates (2). About 25% of the ETA -/- fetuses could not be used because of gross cardiovascular malformations (2). Surgical procedures and experimental protocols were approved by the Animal Care Committee of our institutions.

Solutions and Drugs

Krebs medium had the following composition (in mM): 118 NaCl, 4.7 KCl, 1 KH2PO4, 0.9 MgSO4, 2.5 CaCl2, 11.1 glucose, and 25 NaHCO3. Potassium-Krebs solution (55 mM) was prepared by substituting NaCl with an equimolar amount of KCl. Solutions were bubbled with gas mixtures containing either no O2 or O2 in various concentrations (2.5, 5, 12.5, 30, and 95%) plus 5% CO2 and, when required, balance N2. In the actual experiment, a single mixture (2.5% O2) was chosen to mimic the fetal condition, whereas several mixtures (5, 12.5, 30, and 95%) were used to ascertain the susceptibility of the vessel to O2 with the aim of duplicating the neonatal condition (optimally at 12.5% O2). PO2 was measured with an Instrumentation Laboratory gas analyzer (model 1610, Lexington, MA) and was 1.17 ± 0.05, 2.35 ± 0.04, 3.1 ± 0.07, 7.51 ± 0.06, 26.6 ± 0.2, and 92.7 ± 0.5 kPa (pH 7.4 ± 0.003) when gas mixtures were used with 0, 2.5, 5, 12.5, 30 and 95% O2, respectively,.

ET-1 (human and porcine type; Peninsula, Belmont, CA) was dissolved under aseptic conditions in sterile water containing 0.05% human serum albumin. Aliquots of this stock solution (0.25 mg/ml) were stored at -20°C and, on the day of the experiment, were diluted with bovine serum albumin-supplemented saline (0.05%). The thromboxane A2 (TxA2) analog 9,11-epithio-11,12-methano-TxA2 (ONO-11113, courtesy of ONO Pharmaceutical, Osaka, Japan) was dissolved in distilled ethanol (5 mg/ml), and aliquots (stored at -70°C) were diluted with Tris buffer (pH 7.4). The cyclooxygenase inhibitor indomethacin (Sigma, St. Louis, MO) was also dissolved in ethanol (10 mg/ml) before preparation of the final solution in Krebs medium. Ethanol in the fluid bathing isolated ductus preparations did not exceed 0.01% (indomethacin) or 0.001% (ONO-11113). BQ-788 (courtesy of Merck Frosst, Montreal, Canada), a selective antagonist for the ETB subtype of endothelin receptors (17), and sodium nitroprusside (SNP) (Sigma) were dissolved directly in saline. Precautions were taken to protect the SNP solution from light. Doses of all compounds are given in molar concentrations and refer to their final concentration in the bath.

In Vitro Studies

Fetal mice were delivered by cesarean section under halothane anesthesia and were killed by cervical dislocation. The procedure for dissection, normalization of internal circumference, and mechanical recording was adapted to the ductus from another protocol (34). Briefly, the animal was secured with its left side up in a dissection chamber containing ice-cold Krebs solution gassed with the zero-O2 mixture. A thoracotomy exposed the ductus and the adjoining large blood vessels, and its length was measured in situ. Any animal with a ductus <300 µm in length could not be used. Afterward, the pulmonary artery trunk was split partially open at the junction with the ductus, and a 25-µm tungsten wire (Cooner wire, Chatsworth, CA) was advanced through the lumen up to the descending aorta. The ductus was then freed by cutting its distal end at the junction with the aorta and completing the cut at the proximal end. The tungsten wire bearing the vessel was secured to a specially designed holder (34), and a second wire was passed through the lumen. The preparation was transferred to an organ bath (capacity, 6.5 ml) containing ice-cold Krebs solution gassed with the 2.5% O2 mixture and was connected by the wires to two, independently controlled micromanipulators. The organ bath had been mounted over the stage of a microscope and was supplied from different reservoirs. Both reservoir and organ bath were continuously bubbled with the required gas mixture, and the same mixture was flushed through a hood covering the bath. Perfusion rate was 5 ml/min. The procedure was completed in about 1 h, and a single preparation was studied on each experimental day.

Equilibration. With the preparation set up in the bath, the temperature was increased to 37°C. The dimensions of the vessel were then measured while a minimal stretch was applied [CD-1 strain, 0.07 ± 0.006 mN/mm (n = 33); 129/SvEv strain, 0.04 ± 0.005 mN/mm (n = 21)], and this measurement (Table 1) was used as a reference for selecting an appropriate operating load and the attendant internal circumference. On the basis of separate experiments that are reported below, the internal circumference of the ductus was set at a value corresponding to a transmural pressure of 50 mmHg in vivo (C50) [CD-1 strain, 0.5 ± 0.01 mN/mm (n = 28); 129/SvEv strain, 0.46 ± 0.01 mN/mm (n = 21)]. For this purpose, vessels were stretched in increments of 10-15 µm/min and, after reaching the desired circumference, were allowed to equilibrate for 40 to 90 min (mean ~60 min).

                              
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  		Table 1.   Resting dimensions of isolated ductus arteriosus from CD-1 and 129/SvEv (ETA-deficient) full-term fetal mice

Normalization of internal circumference. Experiments were carried out in CD-1 fetuses using the Laplace equation to extrapolate a value for the applied tension in vitro from the expected transmural pressure in vivo. This tension provided, in turn, a matching internal circumference (C). To assess the optimal load for the isolated ductus, the internal circumference was increased stepwise, starting at C0 with the vessel subjected to a barely detectable stretch (tension 0.09 ± 0.01 mN/mm, n = 6) and progressing up to a value corresponding to a transmural pressure of 60 mmHg in vivo (C60). Resting tension in normal Krebs medium and the tension generated during exposure to potassium-Krebs were measured at each step (Fig. 1), taking care to change the order of tests among experiments. On the basis of the shape of the resulting curves (Fig. 1), the operating circumference was set at C50. When this applied load was used, the isolated ductus did not shorten to any appreciable degree. In addition, the value being calculated for fetal blood pressure accorded with the prediction from pressure values in the adult (27).


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  		Fig. 1.   Relationship between wall tension and internal circumference (C) in isolated ductus arteriosus from CD-1 full-term fetus. Tension output under resting conditions (open circle ) and during exposure to potassium-Krebs (). Both measurements were obtained in same vessel (n = 6) and represent increments over tension at C0. Resting tension includes passive tension and spontaneous tension generated by vessel. Active tension in response to potassium, i.e., total tension minus resting tension, reached peak at about C50.

Experimental design. In addressing the question of the role of ET-1 in the ductus, we assessed first the competence of the preparation. This was done in the CD-1 mouse since this strain can be bred more easily. Subsequent experiments in the 129/SvEv mouse dealt specifically with the involvement of the ETA receptor in the O2 response. Preliminary trials in CD-1 mice showed that the magnitude of the O2 contraction in the isolated ductus was related to body weight. Hence, in vitro data for this particular strain apply to fetuses >= 1.2 g. No such correlation was evident with ETA +/+ fetuses (0.9-1.35 g body wt, mean wt 1.1).

Only one protocol was used with each vessel. In addition, to avoid possible interference caused by tachyphylaxis, ET-1 was tested once, and any change in its effect due to BQ-788 (see EXPERIMENTS IN CD-1 MICE, protocol 3) was ascertained by comparing results in different vessels. Likewise, a single sequence of increasing O2 concentrations was carried out in each experiment. Our study comprised two main experimental series, respectively, in CD-1 (ETA +/+ genotype) and 129/SvEv (ETA -/-, +/-, +/+ genotypes) mice, and distinct protocols. ONO-11113 (0.1 µM) provided a reference contraction. Unless otherwise specified, experiments were carried out at 2.5% O2.

EXPERIMENTS IN CD-1 MICE. In protocol 1, indomethacin (2.8 µM) was added to the perfusion fluid, and its effect was ascertained on the basal tone. An equivalent experiment was carried out with BQ-788 (1 µM) in protocol 2. In protocol 3, ET-1 was tested either on the untreated vessel at rest or the vessel treated with BQ-788 (1 µM). The agonist was added to the organ bath in cumulative doses (0.1-100 nM), using 3- to 10-fold increments. When necessary, BQ-788 was included in the medium 60 min before ET-1. In protocol 4, the capability of the vessel to contract to O2 was assessed by equilibrating the medium with progressively higher concentrations of the gas (5, 12.5, 30, 95% O2). Individual concentrations were tested sequentially, and Krebs medium gassed with 2.5% O2 was passed through the bath between tests. In certain cases, the contraction to O2 did not subside, or subsided only partially, after the tissue was returned to the control medium. This persistent response was then reversed with a 5- to 9-min application of SNP (10 µM).

EXPERIMENTS IN 129SVEV MICE. Only one protocol was used in these preparations and it combined two tests, O2 in increasing concentrations (see EXPERIMENTS IN CD-1 MICE, above) and a maximally effective dose of ET-1 (100 nM).

All compounds were injected into the organ bath in 65-µl volumes, and none of the vehicles had an effect on vessel tone.

In Vivo Studies

Experiments were carried out in fetuses or newborns depending on the protocol. In the former case, animals were delivered by caesarean section under halothane anesthesia unless otherwise specified, whereas in the latter case they were used (under halothane or after cervical dislocation, see EXPERIMENTS IN CD-1 MICE, below) at different intervals after a vaginal delivery. Any animal surviving after birth was placed on a warm metal plate (~37°C) and was also heated with a lamp.

Experimental design. Changes in ductus caliber occurring in vivo during the transition from the pre- to postnatal condition were assessed by fixing the vessel in situ with the whole body freezing technique (16). Because the ETA null mutant does not survive postnatally because of mechanical obstruction in the upper airway (2), separate experiments were performed in CD-1 mice to verify whether the neonatal condition could be reproduced in utero by making the dam hyperoxic, and, whether closure of the ductus progresses normally in fetuses tracheotomized immediately after caesarean delivery. The latter procedure has been used to maintain living mice lacking the ET-1 gene (20).

EXPERIMENTS IN CD-1 MICE. Ductus caliber was measured in fetuses, whether delivered from a normoxic or hyperoxic dam, in newborns at different ages (1- to 12-h old), and in fetuses that had been subjected to tracheotomy and survived for 3 h.

Pregnant mice were made hyperoxic by breathing 100% O2 inside a box for 3 h. Throughout this procedure, animals were anesthetized with chloral hydrate (35-70 mg/100 g ip, supplemented as required) and kept warm with water-filled bags at 39°C. Some fetuses were frozen for morphological analysis, whereas others were used for measurement of blood PO2. Fetuses from normoxic dams served as reference. In either case, because of the small size of animals, blood was collected through a cut made at the apex of the heart. Hence, samples were a mixture of placental and venous blood. Blood PO2 was also measured in the mother, using the descending aorta as the sampling site.

Fetuses were tracheotomized as previously described (20) and their respiratory rate was assessed at regular intervals throughout the 3-h observation period. Afterward, they were divided in two groups for morphological analysis and blood PO2 measurement, respectively. Whereas animals in the first group were killed by cervical dislocation, those in the second group were anesthetized with halothane, and blood was collected by inserting a sharp-tipped glass micropipette into the abdominal aorta. Blood PO2 was measured with an Instrumentation Laboratory gas analyzer (see Solutions and Drugs) or, in the case of small-volume samples (20-30 µl), with a Ciba-Corning gas analyzer (model 178, Tokyo, Japan).

EXPERIMENTS IN 129SVEV MICE. Protocols were the same as in CD-1 mice (see EXPERIMENTS IN CD-1 MICE, In Vivo Studies). ETA -/- fetuses, however, could be kept alive only through a tracheotomy. Success rate was about 43%, failures were caused primarily by an inability to breathe. Less frequently, animals stopped breathing for no apparent reason before reaching the 3-h mark. In contrast, all 129/SvEv fetuses, heterozygous for the ETA gene mutation, and the CD-1 fetuses remained alive with or without a tracheotomy.

Processing of specimens. Mice, whether fetal or newborn, were processed according to Hörnblad and Larsson (16) with few modifications. Briefly, animals were placed with their right side up in a Petri dish and were covered with an embedding medium [Tissue-Tek optimum cutting temperature compound (OCT); Sakura Finetek, Torrance, CA]. The dish with the animal was quickly wrapped with aluminum foil and immersed in liquid nitrogen. Once frozen, specimens were stored at -20°C for at least 24 h before further workup. To prepare a block of tissue with the ductus, the carcass was freed in the frozen state of its OCT embedding. With a razor blade, soft tissues covering the dorsum were removed, making certain that the exposed surface would be approximately parallel to the underlying descending aorta and hence perpendicular to the expected orientation of the ductus. With the use of this cut as a reference, a block of tissue was prepared and placed, with its dorsal surface down, onto a vinyl specimen mold (Tissue-Tek II Cryomold; Miles Laboratory, Elkhart, IN) to be embedded in OCT and frozen at -20°C for storage. Afterward, the OCT mold was removed and the tissue block was mounted in a Leitz freezing microtome (model 1720, Leitz, Germany). Serial sections (10-µm thick) were obtained along a plane perpendicular to the main axis of the ductus and were stained with 1% methylene blue.

Morphometric analysis. Images were digitized with Media Grabber v2.2 software (RasterOps, Waco, TX) and stored on a Macintosh IIcx computer. Lumen area of the ductus was measured on the stored images with NIH Image 1.60 software (National Institutes of Health, Bethesda, MD), and the section with the smallest area was selected for computation.

Light and Electron Microscopy

Morphological examination was carried out in ductus specimens that had been prepared in the usual manner and pinned for fixation to a wax board.

For routine histology, the vessel was fixed in neutral buffered Formalin and embedded in paraffin. Transverse sections were examined after staining with Movat pentachrome stain.

For transmission electron microscopy, specimens were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, postfixed in 1% OsO4, and embedded in Epon Araldite. Ultrathin sections were contrasted with uranyl acetate and lead citrate before analysis in a Phillips instrument (model 201, Eindhoven, The Netherlands).

Analysis of Responses

Effects of contractile agents in vitro were measured by the fractional rise in tension over basal tension and were expressed in absolute values (mN/mm).

Data are means ± SE. Comparisons were made with the use of a Student's t-test or ANOVA, followed when required by the Dunnett's test. Differences were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In Vitro Studies

CD-1 mice. The isolated ductus arteriosus from wild-type CD-1 mice was consistent in size among experiments (Table 1) and, in responding to physiological and pharmacological stimuli, showed no major differences compared with other species (29). The ductus developed a variable degree of tension during equilibration at 2.5% O2 (0.14 ± 0.03 mN/mm in 60-90 min, n = 27) and, once stabilized, contracted to O2 in a concentration-dependent manner (Fig. 2). However, whereas four of these tissues showed a maximal, or near-maximal, response with O2 concentrations between 12.5 and 30%, the remainder required the 95% concentration to attain a peak. Regardless of its magnitude, the O2 contraction was sustained and often exhibited rapid, low-amplitude fluctuations (Fig. 2, inset). In no instance, on the other hand, did O2 cause a relaxation.


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  		Fig. 2.   Effect of O2 on isolated ductus arteriosus from CD-1 full-term fetus. Contractile response increased with O2 concentration (n = 10). ONO-11113 (0.1 µM) was tested as reference in same preparations at 2.5% O2 and caused a contraction of 0.76 ± 0.07 mN/mm. Inset: contractile response to 95% O2 (between arrows; baseline, 2.5% O2); scale bars, 0.5 mN/mm for 10 min. * P < 0.01 relative to baseline at 2.5% O2.

Indomethacin (2.8 µM) at 2.5% O2 also contracted the ductus, and the contraction equalled or even exceeded the response to O2 (maximum 0.8 ± 0.17 mN/mm, n = 4). This response had quick onset and a biphasic course, with a first peak at 10-20 min and a second peak at ~60 min. Conversely, the ETB antagonist BQ-788 (1 µM) had a marginal and often transient contractile effect (maximum 0.1 ± 0.07 mN/mm, n = 9).

ET-1 elicited a dual response, consisting of a modest relaxation at 0.1 nM and a progressively greater contraction with increasing concentrations up to a maximum at about 10 nM (Fig. 3A). No ET-1-induced relaxation was seen in ductus preparations pretreated with BQ-788, and the contraction developed unabated at all concentrations (Fig. 3B). This contraction, as evident from preliminary observations, could be inhibited by the ETA antagonist BQ-123 (1 µM) (Y.-A. Liu and F. Coceani, unpublished observations). The TxA2 analog ONO-11113 (0.1 µM) was also a constrictor agent and, at the chosen concentration, produced a greater contraction (0.84 ± 0.06 mN/mm, n = 29) than ET-1 (10 or 100 nM). With either spasmogen, responses were immediate in onset and developed rapidly to a plateau.


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  		Fig. 3.   Effect of ET-1 on isolated ductus arteriosus from CD-1 full-term fetus. Concentration-response curves before and during treatment with BQ-788 (1 µM) at 2.5% O2. A: control (n = 5). B: treatment (n = 5). Experimental groups apply to different tissues, and ET-1 was added cumulatively to bath. Reference contraction to ONO-11113 (0.1 µM) was 1.01 ± 0.1 and 1.04 ± 0.18 mN/mm for A and B, respectively. Two curves are not significantly different by 2-way ANOVA.

129/SvEv mice. The ductus of ETA -/- fetal mice did not differ in size from either that of the littermates, whether heterozygous or wild-type, or from that of the CD-1 mice (Table 1). Despite the similarity, however, disruption of the ETA gene caused marked changes in the contractile behavior of the vessel. ETA -/- preparations did not generate any tone during the initial equilibration period and, in that respect, behaved differently from both the ETA +/- (tension 0.14 ± 0.03 mN/mm, n = 9) and ETA +/+ (tension 0.17 ± 0.03 mN/mm, n = 6) preparations. In addition, as expected, the ETA -/- ductus, unlike the ductus from heterozygous and wild-type littermates, contracted marginally to ET-1 (100 nM) (Fig. 4A). All three preparations developed instead a contraction to ONO-11113 (0.1 µM) whose amplitude was relatively smaller with ETA -/- (Fig. 4B). Most significant, however, was the fact that the tonic contraction to O2, although weaker in ETA +/+ 129/SvEv mice than wild-type CD-1 mice (compare Fig. 5C with Fig. 2), was nearly absent in the ETA null mutant (Fig. 5A). Also missing in the ETA -/- ductus were phasic contractions to O2, which in the ETA +/+ ductus were characteristically superimposed over the tonic contraction (Fig. 5D). These contractions were irregular or rhythmic, but in both cases their magnitude increased with gas concentration (0.02 ± 0.01, 0.05 ± 0.02, 0.11 ± 0.07, and 0.28 ± 0.05 mN/mm with, respectively, 5, 12.5, 30, and 95% O2; n = 6 for all values). Not only were contractile events, whether tonic or phasic, curtailed in the ETA -/- ductus, but also a relaxation occurred in two of six preparations with each O2 concentration. The latter response, which in the ETA +/+ ductus was also seen twice but only at 5% O2 (mean loss of tension, 0.08 mN/mm), had variable magnitude (0.04 to 0.12 mN/mm) and subsided, in part or in full, with time.


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  		Fig. 4.   Comparison of contractile responses to ET-1 and ONO-11113 in isolated ductus arteriosus from full-term fetus. ET-1 (100 nM; A) and ONO-11113 (0.1 µM; B) were tested on ETA-deficient and wild-type 129/SvEv mice. Both compounds were tested on same vessel, and no. of experiments are given above each column. * P < 0.05 relative to ETA -/- genotype. ** P < 0.01 relative to ETA -/- genotype.



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  		Fig. 5.   Effect of O2 on isolated ductus arteriosus from 129/SvEv (ETA-deficient) full-term fetus. A: ETA -/- ductus (n = 6); note 1 preparation showed an exceptionally strong contraction at 95% O2 (0.5 mN/mm), thus explaining wide scatter. B: ETA +/- ductus (n = 8); not included in group is a preparation with a large relaxation to O2 (loss of tension: 0.16, 0.21, and 0.42 mN/mm with 12.5, 30, and 95% O2, respectively). C: ETA +/+ ductus (n = 6). D: tracings with response of ETA -/- ductus (top) and ETA +/+ ductus (bottom) to 30% O2 (between arrows; baseline at 2.5% O2); scale bar, 10 min. Note that tonic contraction of wild-type ductus to O2 had consistently phasic contractions superimposed whose amplitude increased with O2 concentration (for details, see In Vitro Studies). Transient contractions were not included in computation of response, hence values in C are an underestimate of actual tension generated by preparation. * P < 0.05 relative to baseline at 2.5% O2. ** P < 0.01 relative to baseline at 2.5% O2.

In responding to O2, the ductus from mice heterozygous for the ETA mutation combined traits of the homozygous -/- and the wild-type +/+ genotypes. This points to an ETA gene-dosage effect. As shown in Fig. 5B, O2 had a contractile effect, but contrary to findings in the wild-type (Fig. 5C), it was barely visible and did not increase linearly with the O2 concentration. Coincidentally, phasic contractions were absent in half the preparations and, when present, had smaller amplitude (0.16 ± 0.04 mN/mm at 95% O2, n = 5). In addition, O2 caused occasionally a transient relaxation rather than a contraction.

In Vivo Studies

The ductus was patent in all fetuses, regardless of strain and ETA genotype (Figs. 6 and 7). The genotype, however, influenced the response of the vessel to O2.


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  		Fig. 6.   Closure of ductus arteriosus in CD-1 mice in vivo. open circle , Time course of postnatal closure of ductus. Zero point refers to full-term fetus in utero (n = 4-14). , Ductal constriction in utero after 3 h of maternal hyperoxia (value at time 0) (n = 10) and in tracheotomized newborn (value at 3 h after cesarean delivery) (n = 4). Where necessary, points have been offset to improve visibility.



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  		Fig. 7.   Postnatal closure of ductus arteriosus in 129/SvEv (ETA- deficient) mice. A: full-term fetus. B: newborn 3 h after birth. In both groups, no. of animals is given above each column. ETA -/- newborns were survived by making a tracheotomy (trach) immediately after cesarean delivery. Part of ETA +/- newborns were tracheotomized and served as control. * P < 0.01 compared with fetus of same genotype.

CD-1 mice. In intact, vaginally delivered mice, the ductus constricted rapidly during the first 3 h after birth and more slowly afterwards until complete closure was attained by 10-12 h (Fig. 6). No difference was noted between these animals and animals tracheotomized after cesarean delivery, at least when comparing vessels at the 3-h mark (Fig. 6). Equally effective was O2 in utero, as documented by the significant constriction of the ductus in the course of maternal hyperoxia (Fig. 6).

129/SvEv mice. ETA deletion did not modify in any obvious manner the behavior of the ductus after birth. As shown in Fig. 7, the ductus of tracheotomized ETA -/- newborns constricted as markedly as that of heterozygous, both tracheotomized and nontracheotomized, and wild-type littermates. Conversely, in utero the vessel was unevenly affected by maternal hyperoxia depending on the genotype (Fig. 8). Its lumen was in the aggregate significantly smaller in ETA +/+ than ETA -/- mice, although not as small as in wild-type CD-1 mice under similar conditions (see Fig. 6). The latter finding accords with data in vitro and points to a relatively weaker responsiveness to O2 of the 129/SvEv +/+ ductus compared with the CD-1 +/+ ductus. Furthermore, mice heterozygous for the ETA mutation gave results spanning over a broad range, from full patency to overt constriction. Regardless of the sign of the response, however, blood PO2 was consistently elevated in both the fetuses from hyperoxic dams and the newborns, its values being within an effective range for constriction of the vessel (Table 2).


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  		Fig. 8.   Effect of maternal hyperoxia on ductus arteriosus in 129/SvEv (ETA-deficient) full-term fetal mice. Dams were made hyperoxic for 3 h, and each point applies to different fetus. Note that lumen size of ETA -/- ductus is similar in hyperoxic and normoxic group (compare with Fig. 7). * P < 0.05 compared with ETA +/+ mice. **P < 0.02 compared with ETA +/+ mice.


                              
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  		Table 2.   Blood PO2 in fetal and neonatal 129/SvEv mice

Morphological Analysis

On visual examination, the ductus of ETA -/- fetal mice (129/SvEv strain) could not be distinguished from that of littermates, whether wild or heterozygous, and the CD-1 mice. Likewise, no difference was noted among these vessels on both light and electron microscopy. As evident from a representative experiment (Fig. 9A), the wall of the ETA -/- ductus presented a continuous endothelial lining and a well-developed media consisting of layers of smooth muscle cells with elastic laminae interposed. Ultrastructurally, endothelial cells appeared intact and were separated from the underlying muscle by an elastic lamina (Fig. 10). Equally consistent regardless of genotype were ductal changes in the newborn. As shown in Fig. 9B, pertaining again to data from the ETA -/- group, the vessel was clearly constricted and its narrow lumen was occupied, in part, by heaped up endothelial cells.



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  		Fig. 9.   Ductus arteriosus morphology in fetal and newborn ETA null mutant mice. Ductus from ETA -/- full-term fetus (A) and newborn 3 h after cesarean delivery and tracheal intubation (B). Tracheotomized animal breathed room air. a, Photograph. b, Schematic drawing (ductus shown by arrowhead). c, Transverse section of ductus stained with Movat pentachrome stain. ao, Aorta; pt, pulmonary trunk; and pa, pulmonary artery. Scale bars: a and b, 1 mm; c, 100 µm.



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  		Fig. 10.   Electron micrographs of ductus arteriosus from CD-1 and 129/SvEv (ETA-deficient) full-term fetal mice. A: ETA -/- ductus. B: ETA +/- ductus. C: ETA +/+ ductus. D: CD-1 ductus. en, Endothelium; el, elastic lamina; sm, smooth muscle cell. Scale bar, 1 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our results prove that it is feasible to set up an isolated preparation of fetal mouse ductus arteriosus. With this preparation and appropriate interventions to survive the ETA -/- mouse postnatally and expose the same mutant antenatally to O2, we have introduced a new approach to study ductus regulation. The isolated ductus from wild-type mice appears functional in all respects. It is contracted strongly by indomethacin. Hence, from this finding and the reported effectiveness of the drug in vivo (22), we may assume that, in the mouse as in other species (3, 10, 29), prenatal patency is sustained by a vasodilator PG, conceivably PGE2. The ductus appears equally competent in responding to stimuli relevant to the process of closure, with O2 exerting a constrictor effect and ET-1 qualifying as its possible messenger on smooth muscle cells. It is surprising, however, that in a blood vessel as thin as the mouse ductus (see Table 1), hence a preparation with an expectedly modest transmural gradient for O2, the action of O2 does not develop in full over a physiological range of concentrations. Another peculiarity with the mouse ductus, at least in comparison with the sheep and guinea pig ductus (Ref. 5; F. Coceani, unpublished data), is the effectiveness of ONO-11113 as a constrictor agent. In this respect, the mouse behaves as the rabbit (31), implying that there is a dichotomy across species over the susceptibility of ductal muscle to TxA2. It remains to be ascertained whether this observation has any bearing on the normal operation of the vessel.

A most novel finding in our study is that, both in vitro and in vivo, the constrictor effect of O2 on the fetal ductus is curtailed in the absence of the ETA receptor. Significantly, the loss of this response takes place without any obvious modification in the structure of the vessel. This validates a scheme implicating ET-1 as a critical link in the sequence of events being triggered by O2 and leading to contraction of smooth muscle cells (4, 8, 9). Coincidentally with this validation, the use of the ETA -/- ductus unmasks the complexity of O2 action by showing a relaxant component under the normally overriding contraction. This transient relaxation denotes either stimulation of enzyme systems, such as those yielding PGE2 and NO, for which O2 is rate limiting or, in view of the accelerated formation of ET-1, activation of the ETB receptor. Equally important is the realization that in vivo the ETA -/-, but not the ETA +/+, ductus is differentially affected by O2 before and after birth. The lack of prenatal constriction vis-à-vis normal postnatal closure in the mutant, although documenting the impact of ETA deletion under certain conditions, points to some other factor conditioning the effectiveness of O2. On the basis of available data and assuming that such data are applicable to the mouse, this factor is identified with PGE2. It has been known for a long time that the influence of PGE2 on the ductus abates at birth as blood levels of PGs fall and muscle cells become less responsive to this compound (3, 10, 29). The importance of the latter event to closure of the ductus has recently been highlighted by showing that the vessel closes only partially in newborn mice lacking the relevant PGE2 receptor subtype (i.e., EP4) (22). Significantly, partial constriction of the ductus in this EP4 -/- mutant has been ascribed to ET-1 (22). When considering all these facts, it is reasonable to conclude that postnatal closure of the ductus relies on two overlapping and potentially interchangeable processes, i.e., withdrawal of PGE2 relaxation and promotion of ET-1 contraction. This particular arrangement explains, in turn, how the ETA -/- ductus may not respond to O2 in utero and, yet, may close normally after birth. A functional PGE2 mechanism would sustain patency in the former case, whereas its subsidence in the latter case would compensate for the lack of ET-1 and account for closure.

In apparent disagreement with our present results in the mouse and those reported earlier in the lamb (6) is a recent study (23) in which an ETA antagonist PD-156707 is ineffective on the contraction of the isolated lamb ductus to O2 (15). In our view, such inconsistency has a methodological cause. Fineman et al. (15) used a wire-mounted ring preparation, which because of the thickness of the wall and the consequent absence of a lumen even under stretch, may not equilibrate to O2 changes as efficiently as the circular strip used by us (6). In fact, contrary to our results (6), the contraction of a ductal ring to O2 is relatively small (15). Equally curtailed appears the response of the latter preparation to ET-1 (15). More importantly, we have found that compound PD-156707 inhibits the contraction of the isolated lamb ductus to O2 (Y.-A. Liu and F. Coceani, unpublished observations), albeit not as effectively as BQ-123 (6). In this connection, it should be noted that PD-156707 acquires two configurations in solution, an open form and the lactone (12), sharing the antagonistic action on the ETA receptor but differing in solubility (the lactone is sparingly soluble and may precipitate on lowering pH; S. Haleen, personal communication). Hence, accessibility of this compound to its target within the tissue may not be optimal and may change with the local pH.

Our scheme for ductus regulation has far-reaching consequences. The two processes being implicated in postnatal closure of the vessel may be unevenly expressed across species and, in a given species, may have varying importance depending on physiological and pathophysiological conditions. The PGE2-linked mechanism would appear to be more important than the ET-1-linked mechanism in the mouse ductus. Evidence supporting our view includes the impact of EP4 deletion on ductus closure (22) and the coexistence in the isolated vessel of a brisk contraction to indomethacin with limited efficacy of O2 over a physiological range of concentrations. An opposite situation is expected in the guinea pig ductus in which susceptibility to O2 is greater (14, 24) and the intramural PGE2 mechanism is peculiarly missing (11, 24). This dual control on ductus closure may also become evident under certain conditions. It is known that cyanotic infants are able to close their ductus, albeit more slowly than normally oxygenated infants. On the basis of our scheme, ductus closure in these patients can be ascribed to the removal of the relaxing influence. Indeed, we have experimental evidence that the ET-1 system is not operational in the hypoxic ductus (8).

Two final points deserve a comment and they relate to the residual contraction to ET-1 in the ETA -/- ductus and the apparent conflict between our scheme assigning a mediator function to ET-1 in O2 vasoconstriction and that of investigators (21, 32) implicating blockade of the K+ channels in the same response. It is known that ETB receptors may in certain cases mediate vasoconstriction rather than vasodilatation. In fact, two subsets of the ETB receptor, ETB1 (relaxant) and ETB2 (contractile), have been characterized pharmacologically to account for this dual response (13). Barring an unspecific effect, any ET-1 contraction in the ETA -/- ductus likely reflects the presence of the ETB2 subtype. On the surface, it is more difficult to explain the coexistence of two processes in O2 vasoconstriction, i.e., activation of the ET-1 system and inhibition of the K+ channels, seemingly distant operationally. In actual fact, however, there may not be incongruence in this finding because recent work has identified an inhibitory action of ET-1 on K+ channels (25, 26, 28). Hence, after considering all these facts, one could formulate a scheme with ET-1 acting as a messenger for the O2 constriction both directly and through the inhibition of K+ channels. Further work would be required to verify this possibility.

In conclusion, our study demonstrates that ET-1, acting via the ETA receptor subtype, plays a critical role in the constrictor response of the ductus to O2. However, closure of this vessel in the normal condition in vivo entails not only activation of the ET-1 mechanism, but also removal of the relaxing influence of PGE2. These concepts have practical implications and introduce new possibilities for the management of infants requiring ductus patency for survival. An E-type PG is currently the treatment of choice; however, an ETA antagonist could become a useful adjunct, particularly if persistence of the shunt is necessary over an extended period of time.


    ACKNOWLEDGEMENTS

We thank Dr. E. Cutz and L. Morikawa for morphological analysis, and D. E. Clouthier, D. DeWitt, and S. Dixon for analysis of tail genomic DNA. We also thank Dr. H. Reeve at the University of Minnesota for constructive discussion concerning the endothelin effect on potassium channels.


    FOOTNOTES

This work was supported by the Heart and Stroke Foundation of Ontario, Grant T-3329 (F. Coceani), the W. M. Keck Foundation (M. Yanagisawa), the Perot Family Foundation (M. Yanagisawa), and the Naito Foundation (T. Kuwaki). M. Yanagisawa is an investigator of the Howard Hughes Medical Institute. Y.-A. Liu was supported in part by Parke Davis.

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 and other correspondence: F. Coceani, Scuola Superiore S. Anna, Via Carducci 40, 56127 Pisa, Italy (E-mail: coceani{at}sssup.it).

Received 1 April 1999; accepted in final form 19 May 1999.


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