Developmental responses to oxygen,arachidonic acid,and indomethacin in the fetal lamb ductus arteriosus in vitro

It has been suggested that ineffective constriction in response to an increase in P0₂ is the primary cause for delayed closure of the ductus arteriosus in preterm infants. The isometric contractile effects of increased P0₂ and prostaglandin synthetase inhibitors (indomethacin and tranylcypromine) were studied on isolated rings of lamb ductus arteriosus from animals of two gestational ages (87–110 days and 135–150 days, term is 150 days). Rings from animals less than 110 days have a significantly smaller oxygen-induced contraction (2.5 ± .3 g/mm², n=16) when compared with rings from animals near term (4.6 ± .7 g/mm², n=9).Oxygen-contracted rings from both gestational age groups contract further upon addition of either prostaglandin synthetase inhibitor. Rings from animals less than 110 days have a significantly larger indomethacin-induced contraction (1.10 ± 1.7 g/mm², n=16) than vessels near term (0.52 ± .12 g/mmm², n=9). In addition, arachidonic acid produces a greater relaxation in the immature oxygen contracted ring (42 ± 9%, n=10) than in the vessel near term (6 ± 2%, n=4). This is consistent with the hypothesis that, early during gestation, endogenous prostaglandins inhibit the vessel's ability to contract in response to oxygen. These observations are also consistent with the ability of indomethacin to constrict the patent ductus arteriosus in preterm infants.     

Endothelin is a potent constrictor of the lamb ductus arteriosus

Endothelin was tested on isolated ductus arteriosus preparations from mature fetal lambs. At low PO2 (18-24 Torr; 1 Torr = 133.3 Pa), the compound constricted the vessel dose-dependently over the range from about 10(-10) to 10(-7) M. The contraction was sustained and did not subside even after an extended period of washing. Endothelin was also effective on tissues (PO2,217-231 Torr; indomethacin, 2.8 X 10(-6) M) that had been completely relaxed with CO (CO/O2 ratio, 0.28). CO treatment interferes with a cytochrome P-450 mechanism, which is considered crucial for the contractile response of the vessel to oxygen. These findings are consistent with a role of endothelin in the closure of the ductus arteriosus at birth.     

Does oxygen regulate prostaglandin-induced relaxation in the lamb ductus arteriosus?

It has been speculated that hypoxia might cause vasodilation of the ductus arteriosus by enhancing the relaxing action of endogenous prostaglandins. Using isolated rings of lamb ductus arteriosus, we measured immunoreactive PGE₂ released into the bath solution. We found that after a period of stabilization following suspension of the rings in low PO₂, only a negligible amount of PGE₂ was released by the rings (1.15 ± 0.52 pg PGE₂/mg wet weight per 45 min, n14, ±SEM). When rings were exposed to a high PO₂, significant amounts of PGE₂ were released (32.3 ± 12.6 pg PGE₂/mg wet weight per 45 min). These observations were supported by our findings that indomethacin had a negligible contractile effect (0.11 ± 0.09 g/mm², n=11) on rings equilibrated in a low PO₂, but caused a significant contraction (0.55 ± 0.12 g/mm², n=11) in rings incubated in a high PO₂. These findings do not support the hypothesis that low PO₂ increases PGE₂ production by the lamb ductus arteriosus. They are consistent with the hypothesis that endogenous PGE₂ inhibits the ability of the vessel to contract in response to oxygen. In addition (if these results can be extrapolated to the situation), the demonstration that the ductus arteriosus needs an oxygen tension greater than that present to produce effective amounts of PGE₂, strengthens the hypothesis that circulating levels of PGE₂ may be important in the prenatal maintenance of ductal patency.     

Responsiveness of the lamb ductus arteriosus to prostaglandins and their metabolites

The relative potencies of the prostaglandins A1, A2, E1, E2, F2alpha and their 15-keto-, 15-keto-13,14-dihydro-, and 13,14-dihydro-metabolites were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO2. All the prostaglandins (except PGF2alpha and its 15-keto-metabolites) relaxed the tissue. However, only PGE1, E2, and their 13,14-dihydro-metabolites, were effective at concentrations below 10(-8) M. Therefore, events that alter metabolism of circulating PGs in the perinatal period may have significant effects on the relative patency or closure of the ductus arteriosus.     

Evidence for a role of prostaglandin I2 and thromboxane A2 in the ductus venosus of the lamb

The prostaglandin (PG) endoperoxide, PGH2, and the thromboxane (TX) A2 analog, 9,11-epithio-11,12-methano-TXA2, were tested in vitro on the ductus venosus sphincter from fetal (premature and mature) and neonatal (1-day-old) lambs. PGH2 relaxed the indomethacin-contracted fetal ductus in a dose-dependent manner and its action was reduced after treatment with 15-hydroperoxyarachidonic acid. In contrast, reduced glutathione did not affect the PGH2 relaxation in the indomethacin-treated ductus, nor did it modify the response of the untreated ductus to constrictor stimuli. Unlike PGH2, the stable 9 alpha,11 alpha-epoxymethano-PGH2 analog contracted the vessel. Similarly, the TXA2 analog was a contractile agent, its action exceeding that of the PGH2 analog in potency and efficacy. The TXA2 analog was active on preparations from both premature (minimum 117 days gestation) and mature lambs, but a maximal effect was attained during the perinatal period. These results confirm the existence of a PG-mediated relaxing mechanism in the ductus venosus and suggest that the active compound is PGI2. This mechanism is likely responsible for keeping the ductus patent in the fetus. TXA2, formed within the liver parenchyma, is well suited for playing a role in postnatal closure of the vessel.     

Responsiveness of the lamb ductus arteriosus to prostaglandins and their metabolites

The relative potencies of the prostaglandins A₁, A₂, E₁, E₂, F_2α and their 15-keto-, 15-keto-13,14-dihydro-, and 13,14-dihydro-metabolites were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO₂. All the prostaglandins (except PGF_2α and its 15-keto-metabolites) relaxed the tissue. However, only PGE₁, E₂, and their 13,14-dihydro-metabolites, were effective at concentrations below 10⁻⁸ M. Therefore, events that alter metabolism of circulating PGs in the perinatal period may have significant effects on the relative patency or closure of the ductus arteriosus.     

Evidence for an effector role of endothelin in closure of the ductus arteriosus at birth.

The ductus arteriosus is a special muscular shunt that in the fetus allows blood to bypass the unexpanded lungs. It closes rapidly after birth and this event is initiated by the physiologic rise in blood oxygen tension. Endothelin-1 has been proposed by us as a local mediator for oxygen after demonstrating that it is formed within the ductus and is a potent ductus constrictor. To confirm this possibility, we have now measured the release of endothelin-1 from the isolated ductus of near-term fetal lambs at different oxygen concentrations of the medium. In addition, using the same preparation, we have examined the effect on contractile tone of compounds interfering with the synthesis (phosphoramidon, 50 microM) and action (BQ123, 1 microM) of endothelin-1. We report that release of endothelin-1 from the ductus tends to increase with the oxygen concentration up to a value mimicking the neonatal condition. Phosphoramidon and, to a greater degree, BQ123 inhibit the contraction of the vessel to oxygen. These results implicate endothelin-1 as the effector agent for oxygen in the ductus and, by extension, assign to this peptide a critical role in the closure of the vessel at birth.     

Characterization of PGE2 receptors in fetal and newborn lamb ductus arteriosus

Although the role of PGE2 in maintaining ductus arteriosus (DA) patency is well established, the specific PGE2 receptor subtype(s) (EP) involved have not been clearly identified. We used late gestation fetal and neonatal lambs to study developmental regulation of EP receptors. In the fetal DA, radioligand binding and RT-PCR assays virtually failed to detect EP1 but detected EP2, EP3D, and EP4 receptors in equivalent proportions. In the newborn lamb, DA total density was one-third of that found in the fetus and only EP2 was detected. Stimulation of EP2 and EP4 increased cAMP formation and was associated with DA relaxation. Though stimulation of EP3 inhibited cAMP formation, it surprisingly relaxed the fetal DA both in vitro and in vivo. This EP3-induced relaxation was specifically diminished by the ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide. In conclusion, PGE2 dilates the late gestation fetal DA through pathways that involve either cAMP (EP2 and EP4) or K(ATP) channels (EP3). The loss of EP3 and EP4 receptors in the newborn DA is consistent with its decreased responsiveness to PGE2.     

Involvement of intramural prostaglandin E2 in prenatal patency of the lamb ductus arteriosus

Release of prostaglandin E2 (PGE2) was studied in isolated ductus arteriosus preparations from immature (103 or 104 days gestation; term, 147 days) and near-term fetal lambs. Mature preparations produced measurable amounts of the compound in most cases and the release rate was 19 +/- 2 pg/(100 mg wet weight X min) at a PO2 of 3-8 Torr (1 Torr = 133.3 Pa). PGE2 release increased with the PO2 of the medium, peak values (about 125 pg/(100 mg X min)) being attained at 106-276 Torr when the oxygen-induced contraction was still submaximal. Experiments in which tissues were either contracted with excess potassium or relaxed with CO proved that PGE2 formation is independent from the contractile state. PGE2 was also released from ductus preparations lacking the adventitia, the intima, or both; however, release values were maximal when the adventitia was preserved. The magnitude of the intrinsic tone in these stripped preparations was inversely related to the rate of PGE2 formation. Reduced glutathione increased PGE2 release from the mature ductus, whole or stripped, and also relaxed hypoxic preparations; both effects were reversed by concomitant treatment with indomethacin. PGE2 synthesis tended to be greater in the immature than the mature ductus, maximal values (115 +/- 27 pg/(100 mg X min)) being observed at 6-8 Torr. We conclude that the ductus arteriosus is endowed with an enzyme system for the synthesis of PGE2 whose function accords with an effector role of the compound in the regulation of tone. These findings, together with the potent relaxation exerted by PGE2 at low PO2, indicate that the locally generated prostaglandin is well suited for keeping the ductus patent in the fetus.     

Age-dependent changes in the response of the lamb ductus arteriosus to oxygen and ibuprofen.

Circular strips of ductus arteriosus from lambs of gestational age between 90 and 144 days (term 147 days) were studied in vitro at low (8--16 torr (1 torr = 133.322 Pa)) and high (426--622 torr) PO2. Potassium- and oxygen-induced contractions increased with the gestational age and attained a maximum at term. At low PO2, ibuprofen, a blocker of prostaglandin synthesis, produced a dose-dependent contraction of the ductus at all ages and enhanced the potassium-induced contraction of the immature ductus (90--124 days). Both effects were relatively greater in the 103- to 107-day gestational group. At that age, ibuprofen also potentiated the oxygen-induced contraction. These findings, while confirming that a prostaglandin is involved in ductus patency, indicate that the prostaglandin-relaxing mechanism becomes functional at an early stage of gestation and reaches maximal activity before term. The existence of an active, prostaglandin-mediated relaxation in the preterm ductus may account, in part, for the reduced responsiveness of the vessel to oxygen. It is confirmed that ibuprofen and other nonsteroidal antiinflammatory drugs are well suited for the management of the premature infant with patent ductus arteriosus.     

Cyclooxygenase-2 inhibitors constrict the fetal lamb ductus arteriosus both in vitro and in vivo

Nonselective cyclooxygenase (COX) inhibitors are potent tocolytic agents; however, they also have adverse fetal effects such as constriction of the fetal ductus arteriosus. Recently, selective COX-2 inhibitors have been used in the management of preterm labor in the hope of avoiding fetal complications. However, both COX-1 and -2 are expressed by cells of the ductus arteriosus. We used fetal lambs (0.88 gestation) to assess the ability of selective COX-2 inhibitors celecoxib and NS398 to affect the ductus arteriosus. Both selective COX-2 inhibitors decreased PGE(2) and 6ketoPGF(1alpha) production in vitro; both inhibitors constricted the isolated ductus in vitro. The nonselective COX-1/COX-2 inhibitor indomethacin produced a further reduction in PG release and an additional increase in ductus tension in vitro. We used a prodrug of celecoxib to achieve 1.4 +/- 0.6 microg/ml, mean +/- standard deviation, of the active drug in vivo. This concentration of celecoxib produced both an increase in pressure gradient and resistance across the ductus; celecoxib also decreased fetal plasma concentrations of PGE(2) and 6ketoPGF(1alpha). Indomethacin (0.7 +/- 0.2 microg/ml) produced a significantly greater fall in ductus blood flow than celecoxib and tended to have a greater effect on ductus resistence in vivo. We conclude that caution should be used when recommending COX-2 inhibitors for use in pregnant women, because COX-2 appears to play a significant role in maintaining patency of the fetal ductus arteriosus.     

Prostaglandin biosynthesis and catabolism in the lamb ductus arteriosus, aorta and pulmonary artery.

Homogenates of tissues from fetal and neonatal lamb ductus arteriosus, aorta and pulmonary artery have the capacity to convert arachidonic acid as well as the intermediate prostaglandin endoperoxide, prostaglandin H2, into three products: prostaglandins E2, F2alpha and a major product 6-ketoprostaglandin F1alpha. The three tissues also displayed prostaglandin 15-hydroxydehydrogenase and 13-reductase catabolic activities. The catabolishing system showed considerable substrate specificity: prostaglandin E1 was a good substrate whereas prostaglandins F1alpha and F2alpha were completely devoid of catabolism. The complete system was observed in immature as well as mature arterial vessels, in the fetus as well as the neonate (up to 7 days old). These experiments demonstrate the presence of several components of the prostaglandin system in these tissues and offer biochemical evidence for the implication of prostaglandins E2 and I2 in the maintenance of the ductus and neighboring vessels in a relaxed state in the fetus.     

Endothelin-1 release from lamb ductus arteriosus: relevance to postnatal closure of the vessel

Our previous investigations have shown that endothelin-1 (ET-1) is a singularly potent constrictor of the ductus arteriosus and that a cytochrome P-450 system located in the sarcolemma is crucial for the contractile response of the vessel to oxygen. We have now studied the release of ET-1 from isolated ductus arteriosus preparations of near-term fetal lambs. Preparations produced measurable amounts of ET-1 under basal conditions (about 0.04 pg/100 mg wet weight.min) both in the presence and absence of the endothelium. Anisomycin (10(-4) M) reduced this release by 50%, while thrombin (1 U/mL) doubled the release. Treatment with a CO mixture (CO/O2 ratio, 0.27) inhibited ET-1 release from intact and endothelium-denuded preparations. We propose that oxygen triggers closure of the ductus arteriosus at birth by causing a conformational change in a specific cytochrome P-450, which, in turn, provides the signal for the synthesis of the constrictor ET-1.     

Developmental response to indomethacin: a comparison of isometric tension with PGE2 formation in the lamb ductus arteriosus

We studied the effects of oxygen and indomethacin on the isometric contractile response and the production of PGE2 in isolated rings of lamb ductus arteriosus from animals of different gestational ages (100 to 144 days; term is 150 days). Rings of ductus arteriosus from animals less than 110 days released significantly less PGE2 than did rings from animals greater than 120 days. The indomethacin-induced increase in muscle tension in relation to the decrease in endogenous PGE2 production in preparations from animals less than 110 days gestation was greater than in animals older than 120 days. These findings do not support the hypothesis that immature animals have a larger indomethacin-induced contraction due to an increased production of PGE2 earlier in gestation. They are, however, consistent with a decreased sensitivity to PGE2 in the more mature animals; they also support the hypothesis that the decreased effectiveness of indomethacin on the ductus arteriosus from later gestation animals reflects primarily a decrease in the sensitivity of the vessel to PGE2 during development.     

The response of the lamb ductus arteriosus to endothelin: developmental changes and influence of light

Endothelin-1 (ET-1) is a putative messenger of oxygen in the ductus arteriosus. Since the ability of the vessel to contract to oxygen increases with gestation, we wished to ascertain whether ET-1 action is also developmentally regulated. A corollary objective was to assess whether any gestational variation in the ET-1 contraction is due to a change in the ET(A)-mediated action or to a shift in the balance between opposing, contractile (ET(A) - mediated) and relaxant (ET(B)-mediated), actions. Experiments were performed with isolated ductal strips from preterm (0.7 gestation) and near-term fetal lambs. ET-1 contracted the ductus dose-dependently (10(-10)-10(-7) M) at both ages; however, the peak contraction was about double in magnitude at term. Regardless of age, ET-1 contraction was greater with preparations kept in the dark compared to those exposed to light. This effect of light was not seen after removing the endothelium or when treating the intact tissue with the ET(B) antagonist BQ788 (1 microM). In the dark, however, BQ788 did not modify significantly the ET-1 response at either age. We conclude that ET-1 becomes a stronger ductus constrictor with fetal age, conceivably by acting on ET(A) receptors. Hence, the concept of ET-1 mediating the oxygen contraction is further validated. Peculiarly, the ET-1 contraction is curtailed by light through a hitherto undefined ET(B) receptor-linked process.     

Metabolic products of arachidonic acid in rats

The pattern of eicosanoid metabolites appearing in urine and feces following oral administration of radioactive arachidonic acid was investigated using rats deficient in essential fatty acids. About 70-80% of the radioactivity in the urine during the first day after feeding was adsorbed to XAD-2 resin and represented eicosanoid metabolites, whereas the rest of the radioactivity was mainly 3H2O. The eicosanoid metabolites were fractionated into different polarity classes using reverse phase Sep-Pak C18 cartridges. Gas chromatographic analysis of the urinary metabolites following their derivatization into methyl ester-methoxime-tert-butyl-dimethylsilyl ethers revealed that nearly one-half of the metabolites had ECL values less than 22 and represented metabolites more oxidized than commonly described. Only 30% of the metabolites had ECL values between 26 to 32, corresponding to the values for the metabolites that originate from exogenously infused prostaglandins. A large portion of the eicosanoid metabolites was also excreted with the feces. The isotopic patterns from the reverse phase chromatography indicated that many of the fecal metabolites may be similar to those in urine although some metabolites in feces were not present in urine. Based on the specific radioactivity of the administered arachidonic acid, it appeared that at least 6 to 8 mg of eicosanoid metabolites were excreted through urine and feces within 24 hrs following an oral bolus of 60 mg arachidonic acid. The rapid increase and subsequent decrease in eicosanoid metabolite excretion after oral administration of arachidonate indicates that the dietary intake of polyunsaturated fatty acids may have a more rapid effect upon the endogenous production of eicosanoids than is generally recognized.     

Prostaglandin I2 is less relaxant than prostaglandin E2 on the lamb ductus arteriosus.

Prostaglandin (PG) I2 and its stable metabolite, 6-keto-PGF1alpha, were tested on the isolated ductus arteriosus from mature fetal lambs. PGI2 relaxed the ductus in high doses (threshold 10(-6)M) and its activity disappeared on standing at room temperature for 30 minutes. 6-keto-PGF1alpha was inactive at all doses. By contrast, PGE2 produced a dose-dependent relaxation over a range between 10(-10) and 10(-6)M. These findings confirm that PGE2 is the most potent ductal relaxant among the known derivatives of arachidonic acid. PGE2 probably maintains ductus patency in the fetus and, together with PGE1, remains the compound of choice in the management of newborns requiring a viable ductus for survival.     

Metabolic products of arachidonic acid in rats

The pattern of eicosanoid metabolites appearing in urine and feces following oral administration of radioactive arachidonic acid was investigated using rats deficient in essential fatty acids. About 70–80% of the radioactivity in the urine during the first day after feeding was adsorbed to XAD-2 resin and he represented eicosanoid metabolites, whereas the rest of the radioactivity was mainly ³H₂O. The eicosanoid metabolites were fractioned into different polarity classes using reverse phase Sep-Pak C₁₈ cartridges. Gas chromatographic analysis of the urinary metabolites following their derivatization into methyl ester-methoxime- -butyl-dimethylsilyl ethers revealed that nearly one-half of the metabolites had ECL values less than 22 and represented metabolites more oxidized than commonly described. Only 30% of the metabolites had ECL values between 26 to 32, corresponding to the values for the metabolites that originate from exogenously infused prostaglandins. A large portion of the eicosanoid metabolites was also excreted with the feces. The isotropic patterns from the reverse phase chromatography indicated that many of the fecal metabolites may be similar to those in urine although some metabolites in feces were not present in urine. Based on the specific radioactivity of the administered arachidonic acid, it appeared that at least 6 to 8 mg of eicosanoid metabolites were excreted through urine and feces within 24 hrs following an oral bolus of 60 mg arachidonic acid. The rapid increase and subsequent decrease in eicosanoid metabolite excretion after oral administration of arachidonate indicates that the dietary intake of polyunsaturated fatty acids may have a more rapid effect upon the endogenous production of eicosanoids than is generally recognized.     

Smooth muscle contraction as a model to study the mediator role of endogenous lipoxygenase products of arachidonic acid

In the lung, the contraction of smooth muscle, or bronchospasm, is generally caused by an immunologic insult resulting in mast cell degranulation and the release of histamine, slow reacting substances, and other mediators of inflammation (1). Although the immediate response is bronchospasm, continued activation of this sequence of events results in a chronic inflammatory disease. In the uterus, numerous conditions can result in smooth muscle contraction. One major pathophysiological syndrome associated with increased uterine tone and severe rhythmic contraction is primary dysmenorrhea (2). In this disease state, prostaglandins have been shown to play a major role in these contractions (3,4), and inhibitors of cyclooxygenase have proven beneficial in clinical practice (5). Both dysmenorrhea and cervical ripening have been likened to inflammatory reactions due to varying degrees of vasodilation, invasion by inflammatory cells, proliferation of fibroblasts and smooth muscle contraction (6,7). Metabolism of arachidonic acid (AA) via cyclooxygenase to prostaglandins and thromboxanes and via lipoxygenase to hydroxyeicosatetraenoic acids (HETEs) and leukotrienes is an integral part of both the acute and chronic inflammatory reaction in the lung or uterus. The material reviewed here examines the effect of endogenous leukotrienes on both the lung and uterus and suggests that other smooth muscles and pathophysiological states may be more involved with the lipoxygenase pathway of AA metabolism than previously believed.