I. Introduction

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The ductus arteriosus is a shunt blood vessel of fetal life; it extends between the pulmonary artery and the aorta. It shunts deoxygenated blood from the main pulmonary artery to the descending aorta. Over half of the blood flow in the descending aorta is diverted to the umbilico-placental circulation (Heymann and Rudolph, 1975), where gaseous exchange takes place. The timing of closure of the ductus after birth varies between species (Heymann and Rudolph, 1975), but it is usually complete within 48 h in humans (Drayton and Skidmore, 1987). Ductal patency in utero is an active state principally maintained by the potent dilator effect of prostaglandins (PGs)^b (Coceani and Olley, 1988). Closure at birth is because of contraction of its smooth muscle. This is secondary to withdrawal of dilation and active stimulation of contraction, particularly by increased oxygen tension (Coceani and Olley, 1988).

The control of the ductus is clinically important in a number of areas. Contraction of the ductus, with or without fetal heart failure, is a recognized side effect of administration of prostaglandin H synthase (PGHS) inhibitors to the mother (Van den Veyver and Moise, 1993). Failure of ductal closure after birth is a common complication of premature delivery, and, conversely, in some forms of congenital heart disease, survival of the neonate is dependent on persistent patency of the ductus (see Gersony, 1986 for review). Understanding the role of PGs in the control of the ductus led directly to therapies in the human neonate, specifically, indomethacin to close the ductus and E series PGs to maintain its patency.

This review seeks to summarize the current state of knowledge of the factors that maintain ductal patency in utero and promote ductal contraction after birth. It also seeks to identify potential novel therapeutic strategies for avoiding ductal contraction as a side effect of maternal anti-PG therapy and for the safer and more effective manipulation of ductal patency in the human neonate.

	II. Physiological Roles of the Ductus Arteriosus

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Flow across the ductus is determined by the difference in pressure between the two vessels it connects, the pulmonary artery and the aorta. In fetal life, pulmonary artery pressure is high and aortic pressure is low (the latter because of the presence of the low resistance umbilico-placental circulation), therefore the flow is right to left (Anderson et al., 1981). The right ventricle pumps approximately two-thirds of the combined ventricular output in the fetal lamb, and 90% of this is shunted from the main pulmonary artery to the descending aorta through the ductus arteriosus (see Heymann and Rudolph, 1975). There is no left to right shunt in the normal lamb fetus (Teitel et al., 1987).

After birth, the pressure gradient across the ductus is reversed. Pulmonary arterial pressure falls after ventilation of the lungs with air and aortic pressure rises because of the loss of the low resistance umbilico-placental circulation (Teitel et al., 1987). In the neonate, flow across the ductus is reversed within minutes after birth (Dawes et al., 1955; Drayton and Skidmore, 1987): i.e., from the aorta to the pulmonary artery. It is a common misconception that closure of the ductus is one of the factors that increases pulmonary blood flow in the neonate. Because the shunt across the ductus in the neonate is left to right, ductal patency increases pulmonary blood flow; ligation of the ductus of the term neonatal lamb decreases pulmonary blood flow (Dawes et al., 1955).

It has been proposed that the left to right shunt of the ductus in the neonate may have an important physiological role in adaptation after birth (Dawes et al., 1955). The magnitude of the shunt in the neonatal preterm lamb varies directly with arterial oxygen tension (Clyman et al., 1987), and ligation of the ductus in the term neonatal lamb decreases arterial oxygen tension (Dawes, et al., 1955). A similar improvement in oxygenation can be demonstrated by creating an artificial ductus arteriosus (with left to right shunt) in adult animals; patency of this artificial vessel increases arterial oxygen tension in the face of an experimentally induced pulmonary arterio-venous shunt (Born et al., 1955). Therefore, the physiological patency of the ductus in the term neonate with a left to right shunt acts to improve arterial oxygen tension in the immediate neonatal period when the lungs are not fully expanded (Dawes et al., 1955; Born et al., 1955).

Initial closure of the ductus is mediated by contraction of its thick muscular wall. In some species, closure is complete in the first few hours after birth, e.g. mice (Tada and Kishimoto, 1990), rats (Jarkovska et al., 1992), and rabbits (Momma et al., 1980); in others, however, it occurs in the first 1 to 2 days of life, e.g. lambs (Dawes et al., 1955), guinea pigs (Fay and Cooke, 1972), and humans (Drayton and Skidmore, 1987). After functional closure, the ductus remodels and closure is permanent. A remnant of the ductus is evident in the adult, the ligamentum arteriosum, which is formed by fibrosis of the closed neonatal vessel.

	III. Factors Maintaining Ductal Patency In Utero

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The original view of the ductus was that it represented a relatively passive structure in utero that was actively stimulated to contract after delivery (Kennedy and Clark, 1942). It has become clear, however, that patency of the ductus in utero is an active state. That is, it has intrinsic tone, or is tonically stimulated to contract, and these procontractile mechanisms are tonically inhibited by vasodilators. Probably the most important dilator system identified so far is prostaglandin E₂ (PGE₂), which has a profound inhibitory effect on ductal smooth muscle. However, several accessory dilator systems have also been identified.

Increasing oxygen tension has a profound effect on the ductus to promote contraction, both directly and by modulating its response to vasodilators and vasoconstrictors (Smith and McGrath, 1991, 1993). Although the relatively low oxygen tension to which the fetal ductus is exposed helps maintain patency, the effects of oxygen are discussed in section IV in the context of rising oxygen tension promoting contraction.

A. Prostaglandins

1. Dilator effects of prostaglandins. In 1973, Coceani and Olley demonstrated that prostaglandin E₁ (PGE₁) and PGE₂ relaxed the isolated lamb ductus arteriosus. Several studies have compared the relative potencies of PGs in dilating the ductus in a range of species (rats, rabbits, pigs, and sheep), and PGE₁ and PGE₂ have been found, uniformly, to be the most potent, causing ductal relaxation in the picomolar and low nanomolar range (Coceani et al., 1975, 1978b, 1980; Sharpe and Larsson, 1975; Starling et al., 1976; Clyman et al., 1977, 1978c,d; Momma et al., 1980; Friedman et al., 1983; Sideris et al., 1985; Smith et al., 1994). 6-keto-PGE₁ is almost as potent as PGE₂ and, theoretically, could be formed from PGI₂; however, the enzymes required for this have not been demonstrated in the lamb ductus (Coceani et al., 1980). The only mammalian species studied where PGs have not been shown to exert a significant dilator effect is the guinea pig (Bodach et al., 1980).

2. Prostanoid receptors and signal transduction. The pharmacological classification of prostanoid (P) receptors has been reviewed (Coleman et al., 1994b). Each receptor is named after the native PG that is its most potent agonist [i.e., EP for PGE₁ and PGE₂, IP for PGI₂, DP for PGD₂, FP for PGF₂ and TP for thromboxane A₂(TxA₂)]. There are at least four subtypes of EP receptors encoded by separate genes, and there are variable numbers of isoforms (depending on the species) of EP₃ receptors formed by alternative messenger ribonucleic acid (mRNA) splicing from a single gene. The dilator effect of PGE₂ on the rabbit ductus was mediated by the prostanoid EP₄ receptor subtype (Smith et al., 1994); this receptor also mediated dilation of porcine venous smooth muscle by PGE₂ in the picomolar and low nanomolar range of concentrations (Coleman et al., 1994a). The gene encoding the EP₄ receptor has been cloned and sequenced, and, like the other prostanoid receptors, is a member of the superfamily of G-protein coupled receptors with seven transmembrane domains (Pierce et al., 1995). Normal ductal closure fails to take place in mice lacking the EP₄ receptor gene, confirming that this receptor mediates the physiological effects of PGE₂ on the mouse ductus (Nguyen et al, 1997). When the cloned human and murine genes were transfected into mammalian cells, EP₄ receptors were found to be coupled positively to adenylate cyclase (AC) (Honda et al., 1993; Bastien et al., 1994; Nishigaki et al., 1995). Consistent with this (although the EP receptor subtype mediating the dilator effect of PGE₂ on the lamb ductus has not been confirmed), PGE₂ increased the intracellular concentration of cyclic adenosine monophosphate (cAMP) in the lamb ductus arteriosus (Walsh and Mentzer, 1987). Similarly, the effects of PGE₂ on the staphylococcal -toxin permeabilized rabbit ductus were potentiated by a phosphodiesterase inhibitor and were identical with forskolin (a direct activator of AC) and exogenous cAMP (Crichton et al., 1997). The cellular mechanism of action of PGE₂ (presumably acting via cAMP) has been at least partially elucidated in the -toxin permeabilized rabbit ductus, where it was found to inhibit the sensitivity of the contractile proteins to calcium (Crichton et al., 1997).

3. Local sources of prostaglandins. The ductus is exposed to both locally released and circulating PGs (Clyman, 1987). The isolated ductus synthesizes a range of PGs. PGI₂ was the main product of arachidonic acid (AA) in both the ovine and bovine ductus, but it also formed small amounts of PGE₂, PGF₂, and PGD₂all about 10% the level of PGI₂ synthesis (Terragno et al., 1977; Pace-Asciak and Rangaraj, 1977, 1978 and 1983; Skidgell et al., 1984); this adds further support for a physiological role for PGI₂ in the control of the ductus. It has been suggested that PGE₂ is formed by the degradation of PGH₂ in the lamb ductus and not through an enzymatic pathway (Needleman et al., 1981; Skidgell et al., 1984). However, the stimulatory effect of reduced glutathione on PGE₂ release has been interpreted as indicating the presence of PGE₂ isomerase (Coceani et al., 1986). The control of ductal PG synthesis in relation to birth is discussed in section IV and the effect of gestational age is discussed in section V.A.

4. Circulating prostaglandins. The ductus is also exposed to circulating PGE₂, and it has been suggested that circulating PGE₂ is more important in the control of the vessel than locally released PGE₂ (Clyman, 1987). Circulating concentrations of PGE₂ increased toward term and were approximately 1 to 2 nM in the late gestation fetal lamb (Clyman et al., 1980b) which is close to causing maximal relaxation of the isolated ductus (Coceani et al., 1975; Smith et al., 1994). The placenta is thought to be the major source of circulating PGE₂ in the lamb fetus (see Thorburn, 1992). Because the lungs are the major site of PG catabolism (Tsai and Brown, 1987) and pulmonary blood flow is only 7% of combined ventricular output in the lamb fetus (Heymann and Rudolph, 1975), the high circulating concentrations of PGE₂ are probably also related to reduced catabolism.

5. Effects of prostaglandin H synthase inhibition in vivo and in vitro. Soon after the initial description of the dilator effect of PGE₂, it was demonstrated that indomethacin, a PGHS inhibitor, contracted the rat ductus in vivo (Sharpe et al., 1974) and the lamb ductus in vitro (Coceani et al., 1975), which suggested that (a) the net effect of endogenous PGs on the ductus was dilator and (b) that the ductus had intrinsic tone in utero that was being tonically inhibited by PGs. The effect of indomethacin is likely to have been because of its inhibitory effect on PGHS because a range of structurally diverse PGHS inhibitors contracted the rat ductus (Momma et al., 1984). Indomethacin did not affect the ductus arteriosus of fetal mice lacking the prostanoid EP₄ receptor gene, which indicates that the primary mechanism by which indomethacin contracts the vessel in vivo is eliminating the dilator effect of PGE₂ (Nguyen et al, 1997).

6. Relative roles of local and circulating prostaglandin in fetal life. Early work on the effects of PGs and PGHS inhibitors led to the conclusion that circulating PGs were primarily responsible for maintaining ductal patency in utero (Clyman, 1987). The findings that led to this conclusion were made in the isolated lamb ductus exposed to fetal oxygen tension, where (a) indomethacin had no contractile effect and (b) there was minimal release of PGE₂ (Clyman et al., 1980a). However, the contractile response to indomethacin is not purely an index of the dilator effect of locally released PGs (Smith, 1997). The contraction elicited by indomethacin is also determined by the degree of spontaneous tone in the ductus. The ductus may be profoundly inhibited by locally released PGs, but when there is no spontaneous tone present, it will not contract in response to indomethacin. The lack of a contractile response to indomethacin of the isolated ductus exposed to fetal oxygen tension may simply reflect low levels of spontaneous tone (Smith, 1997).

7. Prostaglandin H synthase isoforms. There are two isoforms of the enzyme PGHS: a constitutive isoform (PGHS-1) and an inducible isoform (PGHS-2) (see Frolich, 1997). There is a recent preliminary report that the fetal ductus primarily expresses PGHS-1, whereas the neonatal vessel primarily expresses PGHS-2 (Guerguerian et al., 1997). The isoforms present in the placenta, the major source of circulating PGE₂ in the fetus (see Section III.A.4.), vary with gestational age. In the preterm sheep, PGHS-1 predominates, whereas with advancing gestation there is induction of PGHS-2, which is correlated with increased placental PG synthesis (Wimsatt et al., 1993). In the preterm fetal ductus, therefore, both locally released and circulating PGE₂ may be produced by PGHS-1. In the term fetal ductus, locally produced PGE₂ is still formed by PGHS-1 whereas PGHS-2 may be the predominant source of circulating PGE_2.

Both sodium nitroprusside (SNP) and glyceryl trinitrate dilated the lamb ductus in vivo (Walsh et al., 1988) and the lamb and rabbit ductus in vitro (Walsh and Mentzer, 1987; Smith and McGrath, 1993). These agents are nitric oxide (NO) donors, and they increased the intracellular concentrations of cAMP and cyclic guanosine monophosphate (cGMP) in the lamb ductus (Walsh and Mentzer, 1987; Coceani et al., 1996a). Inhibitors of nitric oxide synthase (NOS) contracted the ductus both in vitro (Coceani et al., 1994b) and in vivo (Fox et al., 1996), and contraction of the isolated lamb ductus was associated with decreased intracellular concentrations of cGMP (Coceani et al., 1996a). Removing the luminal endothelium of the ductus decreased, but did not abolish, the contractile response to a NOS inhibitor (Coceani et al., 1994b; Clyman et al., 1997b), implying an extraluminal source of NOS. Immunohistochemistry localized endothelial nitric oxide synthase (eNOS) to the luminal endothelium (Fox et al., 1996) and the vasa vasorum endothelium (Clyman et al., 1997b). The presence of mRNA encoding the inducible nitric oxide synthase (iNOS) gene in a homogenate of the heart and great vessels from the fetal rat has been demonstrated (Bustamante et al., 1996), and immunohistochemistry of the fetal lamb ductus has located this to the luminal endothelium (Clyman et al., 1997b). Oral administration of a selective iNOS inhibitor (L-N^G-1[1-aminomethyl] lysine) to pregnant rats caused contraction of the fetal rat aorta, pulmonary artery, and ductus arteriosus in vivo in a dose-dependent manner, and this could be reversed with SNP (Bustamante et al., 1996).

However, in vitro studies have demonstrated that in the indomethacin-treated, endothelium-denuded rabbit ductus exposed to neonatal oxygen tension, the maximal effect of a NO donor (SNP) was only 4% of maximal relaxation compared with 80% for PGE₂ and 87% for cicaprost (Smith and McGrath, 1993). Consistent with this, L^G-nitro-L-arginine, an inhibitor of eNOS and neuronal NOS (Moncada et al, 1997), at a dosage sufficient to increase mean arterial pressure, had a much smaller contractile effect than indomethacin in the fetal lamb ductus in vivo (Fox et al., 1996). These observations suggest that PGE₂ is the most important ductal dilator and that NO may only play an accessory role (Smith and McGrath, 1993; Fox et al., 1996). Given the profound contractile effect of indomethacin on the fetal lamb ductus in vivo (Friedman et al., 1983; Fox et al., 1996), NO is insufficient on its own to oppose the intrinsic tone of the fetal vessel in utero.

A study in the isolated lamb ductus demonstrated that NO donors had a greater effect than E series PGs in vitro (Walsh and Mentzer, 1987). These experiments were conducted in the absence of indomethacin. Endogenous PGE₂ potentiated the sensitivity and maximum response of the rabbit ductus to nonprostanoid vasodilators and inhibited its sensitivity to exogenous PGE₂ (Smith and McGrath, 1994), and the absence of indomethacin (and, therefore, the effects of locally released PGE₂) probably explains greater sensitivity to NO donors compared with PGE₂ (see section X.). The effects of endogenous and exogenous NO on ductal patency have not been studied in the instrumented neonate. This area warrants investigation.

The effects of carbon monoxide (CO) on the ductus have been studied for over a decade in investigations of the mechanism of the oxygen-induced contraction of the ductus (see section IV.). More recently, it has been appreciated that smooth muscle contains an enzyme, heme oxygenase, that can produce CO from heme and that the CO produced may cause vasodilatation through stimulation of cGMP (Morita et al., 1995; Werkstrom et al., 1997) or by effects on potassium channels (Farrugia et al., 1993; Wang and Wu, 1997; Werkstrom et al., 1997).

The expression of the inducible (heme oxygenase-1) and constitutive (heme oxygenase-2) isoforms of the enzyme have been studied by immunohistochemistry in the lamb ductus (Coceani et al., 1997). Heme oxygenase-1 was expressed in both endothelial and smooth muscle cells, whereas heme oxygenase-2 was only found in smooth muscle. The formation of CO from exogenous hemin was blocked by the heme oxygenase inhibitor zinc protoporphyrin IX (ZnPP) (10 µM). However, ZnPP only contracted the ductus exposed to fetal oxygen tension when heme oxygenase-1 had been induced by endotoxin (Coceani et al., 1997). It remains to be established whether CO acts as a dilator of the ductus under physiological conditions in utero.

In the chronically instrumented fetal lamb, adenosine reversed the contraction of the ductus induced by ventilation of the fetal lungs with oxygen (Mentzer et al., 1985). Furthermore, circulating concentrations of endogenous adenosine varied inversely with both fetal arterial oxygen tension and the degree of contraction of the ductus arteriosus (Mentzer et al., 1985). However, adenosine had no effect on the indomethacin-induced contraction of the fetal lamb in vivo (Friedman et al., 1983) and its maximal effect on the indomethacin-treated, endothelium-denuded rabbit ductus exposed to neonatal oxygen tension was 4% of maximal relaxation compared with 80% for PGE₂ (Smith and McGrath, 1993). It is likely that adenosine has only a minor and accessory role in maintaining patency of the ductus in utero.

The ductus also has -adrenoceptors that mediate relaxation (Bodach et al., 1980). The contractile effect of catecholamines through -adrenoceptor activation is offset by its dilator effect through -adrenoceptors. However, infusion of the -adrenoceptor antagonist, propranolol, had no effect on ductal patency in the in vivo lamb (Friedman et al., 1983).

	IV. Factors Mediating Contraction at Birth

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Although the maintenance of patency in utero is an active state and the loss of the dilator effect of PGE₂ is central to the control of the ductus in the neonate (Coceani and Olley, 1988), the trigger to close the vessel after birth is more than just the withdrawal of dilator influences. The major factor actively stimulating contraction is probably the effect of increasing oxygen tension although the isolated ductus is sensitive to a wide range of contractile agonists (see Sections IV.D. and IV.G.). The multiplicity of these contractile systems seems at odds with the relatively simple physiological role of the ductus. This can be explained by the fact that the two main systems that vary at birth, namely oxygen tension and PGE₂, act synergistically to modulate the response of the ductus to vasoconstrictors (Smith and McGrath, 1991, 1993). In the presence of fetal oxygen tension and physiological concentrations of PGE₂, the ductus is virtually unresponsive to even high micromolar concentrations of norepinephrine (Smith and McGrath, 1991). Loss of this profound synergistic inhibition after delivery will uncover the vessel's response to a range of vasoconstrictors.

In fetal life, the ductus is exposed to an oxygen tension that has been estimated as between 18 to 28 mmHg (Heymann and Rudolph, 1975). After birth, the ductus is exposed to arterial blood because of the reversal of the direction of flow (Dawes et al., 1955) and arterial oxygen tension rises rapidly after delivery (Heymann and Rudolph, 1975). Rising oxygen tension profoundly contracts the ductus (Kennedy and Clark, 1942; Kovalcik, 1963; Fay, 1971). With the exception of the pulmonary circulation, most vascular smooth muscles relax in a low oxygen environment and contract in response to increasing oxygen tension. The response of the ductus to oxygen, although qualitatively similar to other blood vessels, is much greater in magnitude (Heymann and Rudolph, 1975; Smith and McGrath, 1988). Several different mechanisms have been proposed to explain the profound contractile effect of physiological increases in oxygen tension on the ductus. None of the models fit all of the experimental observations, and it is likely that there is more than one oxygen sensor in the ductus. Furthermore, oxygen tension has a profound modulatory effect on other vasoactive systems (Smith and McGrath, 1991, 1993). It follows, therefore, that the effects of locally released vasoactive agents that exert a tonic effect on ductal contractility will vary with alterations in oxygen tension.

1. Cytochrome a₃ hypothesis. Fay (1971) demonstrated some fundamental properties of the oxygen-induced contraction of the guinea pig ductus: exposure of the vessel to increasing oxygen tension across the range 0 to 140 mmHg induced incremental contraction; the effect was the same when either the luminal or the adventitial side of the ductus was exposed to elevated oxygen; and local anesthetics and tetrodotoxin (inhibitors of nerve-mediated effects) had no effect on the response. These findings suggested that oxygen acts directly on the smooth muscle cells of the ductus, which was consistent with the lack of effect of a range of antagonists of autonomic nervous system mediators on the oxygen-induced contraction of the lamb and guinea pig ductus (Kovalcik, 1963). In addition, Fay observed that the oxygen-induced contraction was inhibited by cyanide and several other inhibitors of oxidative phosphorylation (Fay, 1971; Fay and Jobsis, 1972). Cyanide had little effect on the acetylcholine-induced contraction, although some of the other inhibitors of oxidative phosphorylation reduced the response to acetylcholine. Fay postulated that cytochrome a₃ was the oxygen sensor and that the contractile effect of oxygen was related to adenosine triphosphate (ATP) levels in ductal smooth muscle cells (Fay and Jobsis, 1972). Subsequently, it was demonstrated that CO had a greater affinity than oxygen for the target hemoprotein in the ductus (Coceani et al., 1984). Because oxygen has a nine-fold greater affinity for cytochrome a₃ than CO (Ball et al., 1951), it was concluded that CO was acting at a different hemoprotein (Coceani et al., 1984).

2. Arachidonate hypothesis. Given that dilation of the ductus in utero is mediated by an AA metabolite (PGE₂), it was an attractive hypothesis that an AA metabolite might also mediate oxygen-induced contraction of the vessel with a shift in AA metabolism occurring at birth. Furthermore, the oxygen-induced contraction of the human umbilical artery was blocked by PGHS inhibitors (McGrath et al., 1986) and was found to be mediated by TxA₂ (Templeton et al., 1991). However, the oxygen-induced contraction of the lamb ductus arteriosus was not blocked by PGHS (Coceani et al., 1979) or lipoxygenase (Coceani et al., 1982) inhibitors. The remaining major enzymatic AA pathway is the formation of epoxides by cytochrome P₄₅₀ (also called the monooxygenase or epoxygenase pathway), which generates contractile agonists in other vascular smooth muscles (see Harder et al., 1995 for review). Although there is some evidence to suggest that inhibiting cytochrome P₄₅₀ relaxes the ductus (see Section IV.A.3.), there are several pieces of evidence to suggest that this is not mediated by preventing synthesis of a constrictor epoxide. First, in the presence of combined PGHS and lipoxygenase inhibition, exogenous AA only relaxed the ductus in elevated oxygen tension (Coceani et al., 1988). Second, a range of AA epoxides were without effect or caused relaxation of the isolated lamb ductus (Coceani et al., 1988). And third, no monooxygenase activity could be demonstrated in the closing lamb ductus (Coceani et al., 1994a).

3. Endothelin/cytochrome P₄₅₀ hypothesis. Inhibition of the oxygen-induced contraction of the ductus by cyanide (Fay, 1971) implied the role of a hemoprotein. It was found that CO relaxed the oxygen contracted ductus and this effect was reversed by monochromatic light at a peak of 450 nM (Coceani et al., 1988). It was concluded that cytochrome P₄₅₀ was the oxygen sensor and its activation promoted contraction. As discussed above (see Section IV.A.2.), if the enzyme has a role, it is unlikely to be acting through formation of a constrictor AA epoxide (Coceani et al., 1988, 1994a). The effect of oxygen on the enzyme is proposed to lead to the release from endothelial and smooth muscle cells of endothelin (ET)-1, which causes contraction of the ductus mediated by the ET_A receptor (Coceani et al., 1992). There is no currently known nonepoxide mechanism linking cytochrome P₄₅₀ to the production of ET-1, although conformational change in the enzyme or the production of a nonepoxide stimulatory metabolite have been proposed (Coceani, 1994).

4. Membrane hypothesis. It was found that the oxygen-induced contraction of the guinea pig ductus arteriosus was associated with smooth muscle cell depolarization (Roulet and Coburn, 1981). Subsequently, it was observed that glibenclamide, a blocker of ATP-sensitive potassium channels, contracted the isolated rabbit ductus exposed to fetal oxygen tension and had little effect on the oxygen-contracted ductus (Nakanishi et al., 1993). Furthermore, cromakalim, an ATP-sensitive potassium channel activator, relaxed the ductus contracted by oxygen, but had much less of an effect on the vessel precontracted with 10 µM norepinephrine (Nakanishi et al., 1993). These authors proposed that oxygen depolarized the ductus by closing ATP-sensitive potassium channels.

5. Characterizing an oxygen sensor. I propose that the definition of "sensor" in this context is a system that is directly altered (e.g. synthesis or release of a vasoactive agent from the ductus, or the state of an ion channel) by increasing oxygen tension in such a way as to promote contraction. Oxygen profoundly influences the response of the ductus to a wide range of vasodilators (Smith et al., 1993) and vasoconstrictors (Ikeda et al., 1973a; Smith and McGrath 1988, 1991). Indeed, these studies failed to identify a vasoactive agent whose effect on the ductus was not affected by oxygen tension. It follows, therefore, that when an agent has a tonic effect on the ductus (dilator or contractile), the magnitude of its effect is likely to vary with oxygen tension, i.e., the change is secondary to altered sensitivity to the effect of the given system rather than an activation (or inhibition) of the system per se. Therefore, an agonist with a tonic effect on ductal tone may contribute to the oxygen-induced contraction but not be an oxygen sensor.

Two early in vitro studies failed to demonstrate significant contractile effects of prostanoids on the ductus. The first looked at the effect of a single high concentration (about 7 µM) of PGF₂ that was found to cause a small contraction of the bovine ductus exposed to either fetal or neonatal oxygen tension (Starling and Eliott, 1974). The other study (Coceani et al., 1978a) incubated PGG₂ or PGH₂ with microsomal fractions of human platelets or guinea pig lungs to generate TxA₂. PGG₂ or PGH₂ on their own relaxed the ductus (presumably by the formation of PGE₂), but when PGG₂ or PGH₂ were incubated in platelet or lung microsomes, they were without effect.

More recently, the effect of stable synthetic agonists of contractile prostanoid receptors have been studied (Smith and McGrath, 1995). It was demonstrated that the fetal rabbit ductus arteriosus has two prostanoid receptors coupled to contractile pathways: namely, a TP receptor and a contractile EP receptor, probably EP₃. The TP receptor agonist, U46619 (5-heptenoic acid, 7-[6(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl-,[1R[1,4,5,(Z),6,(1E, 3S*)]]-), contracted the ductus in the nanomolar range, had a maximal effect similar to norepinephrine, and was antagonized by a TP receptor antagonist. The ductus also contracted in response to nanomolar concentrations of the selective EP₃ agonists GR63799X ([1R-[1a(Z),2b(R*), 3a]]-4-(benzoylamino) phenyl 7-[3-hydroxy- 2 (2-hydroxy- 3- phenoxypropoxy)- 5- oxocyclopentyl]- 4- heptenoate) and sulprostone. The maximum response to these agonists was smaller than the response to U46619. Interestingly, 10 nM sulprostone decreased the sensitivity of the ductus to the dilator effect of PGE₂, whereas 10 nM U46619 did not. Therefore, the ductus arteriosus has both dilator and contractile receptors to PGE₂, and the contractile receptor modulates the effects of the dilator receptor (Smith and McGrath, 1995). The significance of this observation is discussed in section IV.C.2.

Loss of the dilator effect of PGE₂ is central to the closure of the ductus, and treatment of the neonatal rat with PGE₂ is sufficient on its own to prevent postnatal closure (Jarkovska et al., 1992). The relative roles of withdrawal of locally released and circulating PGE₂ remain to be determined.

1. Circulating prostaglandin E₂. The high fetal circulating concentrations of PGE₂ fall dramatically after birth, by ten-fold at 1 h and by twenty-fold at 3 h, in the term lamb (Clyman et al., 1980b). Loss of the dilator effect of circulating PGE₂ has been postulated to be fundamental to the closure of the ductus (Clyman, 1987). The fall in circulating concentrations of PGE₂ is because of (a) the increase in lung blood flow that occurs at birth because the lungs are the major site of PG catabolism (Tsai and Brown, 1987) and (b) the loss of the placenta, the major source of circulating PGE₂ in the fetus (see Thorburn, 1992).

2. Locally released prostaglandin. The elimination of the dilator effects of locally released PGs after birth is more complex and less well understood than for circulating PGE₂ (Clyman, 1987). Paradoxically, increasing oxygen tension stimulates PGE₂ release by the lamb ductus (Clyman et al., 1980a; Coceani et al., 1986) i.e., oxygen tension, which contracts the vessel, stimulates the release of PGE₂, which relaxes it. This apparent anomaly has been resolved at least in part with the appreciation of the contractile effects of PGs on the ductus (section IV.)

The ductus arteriosus of all species studied is innervated by catecholamine containing nerves (Boreus et al., 1969; Ikeda, 1970; Ikeda et al., 1972; Bodach et al., 1980). The catecholamine content of the lamb ductus was similar to peripheral arteries that are known to be under autonomic neural control (Ikeda et al., 1972). The guinea pig and lamb ductus contracted in response to transmural stimulation of nerves (Ikeda et al., 1973a; Bodach et al., 1980), and the ductus of several species contracted in response to exogenous norepinephrine (Kovalcik, 1963; Aronson et al., 1970; Smith and McGrath, 1988). The effect of transmural stimulation on the guinea pig ductus was potentiated by raised oxygen tension, as was the response of the vessel to exogenous norepinephrine (Ikeda et al., 1973a). The effect of transmural stimulation was blocked in part by -adrenoceptor blockade (Bodach et al., 1980), and the treatment of the pregnant guinea pig with phenoxybenzamine (a nonselective -adrenoceptor antagonist) delayed closure of the ductus in the offspring (Hornblad and Larsson, 1972). The -adrenoceptor subtype mediating the contractile effect of norepinephrine on the ductus has not been elucidated.

The guinea pig and human ductus also contracted in response to acetylcholine (Kovalcik, 1963; McMurphy and Boreus, 1971; Ikeda et al., 1973a), and the lamb ductus is innervated with acetylcholine-containing nerves (Silva and Ikeda, 1971). Atropine blocked the contractile response of the ductus to exogenous acetylcholine, but had no effect on the contraction induced by transmural stimulation of nerves of the isolated lamb ductus arteriosus (Bodach et al., 1980).

The central control of activity of these pressor nerves is unknown. Interestingly, the ductus of several species has structures in its wall that are similar to the carotid and aortic bodies (Boyd, 1941; Fay, 1971; MacDonald et al., 1983) and send afferent fibers to the left vagus nerve (Boyd, 1941). There is no information on what physiological stimuli might activate this apparent sensory system, but the presence of afferent and efferent neural pathways in the ductus suggests the possibility of a control loop.

The potential role of ET-1 is discussed in section IV.A.3. The adventitia of the guinea pig ductus has many mast cells present, which can release other vasoconstrictors such as histamine and 5-hydroxytryptamine (Fay, 1971), both of which contracted the isolated ductus of several species (Aronson et al., 1970; McMurphy and Boreus, 1971; Smith and McGrath, 1991). The factors that control degranulation of ductal mast cells are not known. Infusion of 5-hydroxytryptamine into the chronically instrumented fetal lamb had no effect on ductal patency (Friedman et al., 1983), and this may reflect the profound synergistic dilator effect of fetal oxygen tension and PGE₂ on the response of the ductus to vasoconstrictors (Smith and McGrath, 1991).

Isolated rings of guinea pig ductus arteriosus contracted in response to stretch (Ikeda et al., 1973a), and isolated perfused rabbit ductus contracted in response to increased perfusion pressure (Kriska et al., 1990). Myogenic tone of both isolated rings of rabbit ductus and the isolated perfused vessel was inhibited by both endogenous and exogenous PGs (Kriska et al., 1990; Smith, 1997). However, the role of myogenic tone in the physiological control of the ductus is as yet obscure.

Enalapril, an angiotensin converting enzyme inhibitor, delayed ductal closure when given to fetal rats and could reopen the closed ductus when given at 3 h of life (Takizawa et al., 1994a,c). Surprisingly, the effects of angiotensin II on the isolated ductus have not been studied, but the drug had no effect on the patency of the vessel when infused into the chronically instrumented fetal sheep (Friedman et al., 1983). There have been several reports linking maternal consumption of angiotensin converting enzyme inhibitors with patent ductus arteriosus (PDA) in the neonate, but a systematic review of the literature did not support an association, although the drugs in clinical use can cross the placenta (Hanssens et al., 1991).

The ductus contracts in response to other circulating vasoactive agents, such as epinephrine (Kovalcik, 1963), through -adrenoceptors, and bradykinin (Kovalcik, 1963; Aronson et al., 1970). The latter was released after ventilation of the lungs with oxygen in the lamb (Heymann et al., 1969), and the levels of bradykinin in human cord blood were higher than in the adult (Melmon et al., 1968). The rat ductus also contracted in response to steroid hormones, namely, corticosteroids (Momma et al., 1981) and progesterone (Pulkkinen et al., 1986). The contractile effect of corticosteroids is probably related to modulating the sensitivity of the ductus to PGE₂ (see section V.B.). The mechanism of action of progesterone is unknown, but, unlike corticosteroids (Momma and Takao, 1989a), it does not interact with the effect of indomethacin (Pulkkinen et al., 1986).

	V. Ontogeny of Pharmacological Responses

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As might be expected, the ductus undergoes alterations in its pharmacological responsiveness with advancing gestational age. The procontractile pathways are clearly held in check at term, however, because the ductus is widely patent immediately before and after delivery (e.g. Momma et al., 1980). It may be that the increasing concentrations of PGE₂ in fetal blood with advancing gestational age (Clyman et al., 1980b) provide a parallel increase in inhibition that is then lost at birth by the mechanisms discussed in section IV.C.1.

The contractile effect of indomethacin on the ductus varies with gestational age, but the findings are different when in vitro and in vivo studies are compared. The contractile effect of indomethacin and ibuprofen on the isolated lamb ductus decreased towards term (Clyman et al., 1978a; Coceani et al., 1979), whereas the contractile effect of indomethacin in vivo increased with advancing gestational age in fetal rats, lambs, and humans (Friedman et al., 1983; Momma and Takao, 1987; Moise et al., 1988). The discrepancy between the findings in vivo and in vitro may be because of the additional dilator effect of circulating PGE₂ in vivo. The sensitivity of the lamb ductus to PGE₂ and PGI₂ decreased towards term (Clyman et al., 1980c). The preterm lamb ductus had greater PGI₂ synthase activity than term (Clyman et al., 1978b), but released less PGE₂ than the term ductus (Clyman et al., 1979). The increased activity of PGI₂ synthase in the preterm ductus may result in less accumulation of PGH₂, and, because PGE₂ is formed from PGH₂, it may explain the low levels of PGE₂ released. The main catabolic pathway for circulating PGE₂ is 15(OH)PGDH: the activity of this enzyme in the fetal rat and rabbit lung increased towards term (Simberg, 1983; Tsai and Einzig, 1989).

There is also maturation of non-PG procontractile pathways with advancing gestational age. The contractile effect of transmural stimulation of pressor nerves in the isolated guinea pig ductus increased towards term (Ikeda et al., 1973b), as did the oxygen-induced contraction of the guinea pig and lamb vessels (Noel and Cassin, 1976; Clyman et al., 1979). The reduced contractile response of the preterm lamb ductus to oxygen may reflect greater inhibition by locally released PGs because the combined contractile effect of oxygen and indomethacin did not differ significantly comparing the preterm and term vessels (Clyman et al., 1980c). However, a recent preliminary report of patch clamp studies of smooth muscle cells from the rabbit ductus found that the potassium channels controlling membrane potential change from the oxygen-insensitive Ca-activated channel to the oxygen-sensitive delayed rectifier channel with advancing gestational age (Reeve et al., 1997). This suggests that the fundamental pathway involved in the oxygen-induced contraction changes with advancing gestational age rather than merely a reduced degree of inhibition of the oxygen-induced contraction by locally released PG.