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

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

PROSTAGLANDINS RELAXATIONIN THE DOES OXYGEN REGULATE PROSTAGLANDIN-INDUCED LAMB DUCTUS ARTERIOSUS? Ronald I. Clyman, FranGoiseMauray, LaurenceM. Deme...

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PROSTAGLANDINS

RELAXATIONIN THE DOES OXYGEN REGULATE PROSTAGLANDIN-INDUCED LAMB DUCTUS ARTERIOSUS? Ronald I. Clyman, FranGoiseMauray, LaurenceM. Demers, Abraham M. Rudolph,and ChristineRoman The CardiovascularResearch Instituteand the Departmentof Pediatrics,Universityof California,San Francisco,California94143 The Departmentof Pediatrics,Mount Zion Medical Center, San Francisco,California;and the Departmentof Pathology, The Milton S. Hershey Medical Center of the PennsylvaniaState University,Hershey, Pennsylvania

ABSTRACT It has been speculated that hypoxia might cause vasodilationof the ductus arteriosus by enhancing the relaxing action of endogenous prostaglandins. Using isolated rings of lamb ductus arteriosus,we measured immunoreactivePGE2 released into the bath solution. We found that after a period of stabilizationfollowing suspension of the rings in low P02, only a negligible amount of PGE2 was released by the rings (1.15 2 0.52 pg PGE2/mg wet weight per 45 min, n=l4, +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 observationswere supported by our findings that indomethacin had a negligible contractile effect (0.11 + 0.09 g/mm2, n=ll) on rings equilibrated in a low PO2, but caused a significant contraction (0.55 + 0.12 g/mm2, n=ll) in rings incubated in a high P02. 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 in vitro results can be extrapolated to the in vivo situation), the demonstrationthat the ductus arteriosus needs an oxygen tension greater than that present in utero to produce effective amounts of PGE2, strengthens the hypothesis that circulating levels of PGE2 may be important in the prenatalmaintenanceof ductal patency. INTRODUCTION Numerous observationshave drawn attention to the importanceof the postnatal increase in arterial oxygen tension for muscular closure of the ductus arteriosusin newborn infants (1,2). There is increasingevidence that prostaglandinsmaintain the patency of the vessel in the fetus and preterm neonate. Accordingto current views, prostaglandinsare formed intramurallyand exert their action on the muscle cells (3). The marked sensitivityof the tissues to PGE2 makes it the most significantendogenousprostaglandinin the regulation of patency of the vessel (4,5).

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Hypoxia has been shown to increase prostaglandin production in several isolated organ systems (6). Since prostaglandins are not stored intracellularly and are made de novo (7), hypoxia might relax the ductus arteriosus by increasing endogenous PGE2 production and/or decreasing its degradation. PGE2 formation from the endoperoxide precursors, PGG2 and PGH2, is subject to the state of oxidation of the system (8). The increase in reduction potential that accompanies hypoxia may increase the levels of reduced glutathione (which is known to enhance vasodilatory-prostaglandin synthesis in vitro) (9,101, as well as decrease the rate of degradation of PGE2 by inhibiting prostaglandin dehydrogenase (11). In the experiments reported herein, in which we measured PGE2 released by isolated rings of lamb ductus arteriosus at high and low oxygen tensions, we found that hypoxia did not enhance endogenous PGE2 production. METHODS Preparation of Rings of Ductus Arteriosus Time-dated fetal lambs between 100 and 146 days gestational age (term is 150 days) were delivered by caesarean section and rapidly killed by exsanguination. The ductus arteriosus was dissected free from adventitial tissue and divided into 1 mm thick rings that were placed in separate glass organ baths (fluid removed by draining) kept in the dark by an enclosed box (12). The rings were suspended between two stainless steel hooks in 5 ml of bath solution: l27mM NaCl, 5mM KCl, 2.5mM CaC12, 1.27mM MgC12a 6H20, 5.5mM glucose, and 5OmM.Tris HCl, pH 7.39 at 37OC. Isometric responses of circumferential tension were measured on a Grass polygraph. Small samples of the bathing solution were withdrawn and pH and PO2 were measured with Radiometer electrodes and blood gas meter. Each of the rings was stretched to an initial length that we had previously found would result in a maximal contractile response to increases in oxygen tension for rings in that age group (13). Rings from animals between 100 and 108 days were stretched to initial lengths of 6 or 7 mm; rings from animals between 120 and 130 days were stretched to 8 or 9 mm; rings from animals between 136 and 144 days were stretched to 9 or 10 mm. The 5 ml bath solution was changed and collected every 45 min for PGE2 analysis. Initially the bathing solution in the bath was bubbled with 100% N2 (to a PO2 of 24 to 32 torr) and the ring was allowed to equilibrate until a steady state tension developed. The ring was incubated in 100% N2 gassed solution for 135 min. The ring was then exposed to 100% 02 (to a PO2 of 600 to 700 torr) for 90 min allowing the tension to achieve a new plateau. After the 90 min incubation in 100% 02, the bath solution was changed to one containing 1 pg/ml indomethacin (2.8 x 10-6 M) and the ring was allowed to achieve a new increase in tension over the next 90 min while it incubated in the indomethacin-containing solution bubbled with 100% 02. Following the experiment, the ring was removed from the bath and blotted dry before it was weighed (wet weight).

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Measurement of Isometric Tension Rings of ductus arteriosus were prepared as described above and placed in separate 150 ml organ baths. The tension developed in the ring was expressed as force/unit area (g/mm2). This area was the surface of the tissue that was perpendicular to the direction of the Since the ring samples assume a contractile-developed force. flattened shape when stretched to their initial length, the cross sectional area was computed as the area of a rectangle the length of which was the initial length of the stretched tissue. We estimated this by dividing the wet weight by the length, assuming a tissue specific gravity of 1. Extraction, %

Chromatographic

Separation,

and

Radioimmunoassay

of

Samples of 5 ml bath solution incubations were extracted in glass tubes. To the 5 ml of bath solution we added 1500 dpm of 3H-PGE2 (130 Ci/mmol, New England Nuclear) for calculation of The sample was then acidified to pH 3.5 with citric recovery. acid. The prostaglandins were extracted twice with 15 ml of a mixture of cyclohexane:ethyl acetate (1:l) with vigorous shaking for 10 min. After centrifugation at 300g for 10 min, the organic phase was separated and subsequently evaporated under a stream of nitrogen Samples were extracted on the same day they were at 3OOC. collected, and residues were stored at -2OOC. We used microcolumns (0.6 x 10 cm) of 0.5g heat-activated silicic acid (Bio-Sil A, 100-200 Mesh, BioRad Laboratories) equilibrated in 2 ml of solvent 1 (benzene:ethyl acetate, 60:40). Each column was washed with 5 ml of solvent 2 (benzene:ethyl acetate: methanol, 60:40:20) and 3 ml of solvent 1. Sample residues were vortexed in 0.2 ml of solvent 3 (benzene:ethyl acetate:methanol, 60:40:10) then in 0.8 ml of solvent 1 and successively applied to the columns. Prostaglandins of the A and B series were eluted with 13 ml of solvent 4 (benzene:ethyl acetate:methanol, 60:40:3) and collected in glass vials (14). This eluate was evaporated under a stream of N2 at 35OC and redissolved in 0.3 ml ethanol. Samples were taken for determination of recovery of 3H-PGE2, which ranged from 50 to 708, and for radioimmunoassay of PGE2. We determined PGE2 by radioimmunoassay, using a specific antiserum against an albumin-conjugated PGE preparation produced in rabbits (15). For each concentration point on the standard curve, we carried a sample of incubation medium plus citric acid through the purification process used for bath solution samples. These evaporated residues were .dissolved in 0.3 ml ethanol, and an appropriate standard of PGE2 was added to each. Samples of unknowns and of these PGE2 standards were added to assay tubes and dried under N2. 0.1 ml of appropriately diluted antiserum (1:2000 diluted with phosphate buffer containing 0.1 M phosphate, pH 7.4, with 0.9% NaCl and 0.1% sodium azide) was added to the dried down assay samples. These were

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incubated at room temperature for 60 min. 0.1 ml of 3H-PGE2 (14,000 dpm of 130 Ci/mmol) was then added and the equilibrium reaction was allowed to proceed at 4oC for 18 hr. The free fraction was separated'fromthe fraction bound to the antibody with dextran-charcoal. One ml of dextran-charcoal(Charcoal activated, Sigma: 250 mg, dextran T-70, Pharmacia: 25 mg/lOO ml phosphate buffer) was added to the reaction mixture at 4OC. Ten minutes later, the tubes were centrifuged for 15 min at 2000 g, the supernatantwas decanted into counting vials, and 8 ml of a liquid scintillationcoctail (1 liter triton X-100, Sigma; 2 liters xylenes, Mallinckrodt; 21g PPO, Mallinckrodt; 0.3g POPOP, New England Nuclear) added and counted in a liquid scintillationcounter. This procedure yielded reproduciblestandard curves with linear titration from 5 to 250 pg of PGE2. The cross reactivityof the PGE-antiserum used in these studies with the 6 keto PGFl metaboliteof prostacyclin (calculated from the relative amounts of PGE2 and 6 keto PGFlc required to reduce the initial binding of 3H-PGE2 by 50%) was 0.1%; cross reactivitywith PGF2c was 1.5%. Indomethacin (Sigma) was prepared in ethanol (16 mg/ml) and aliquots were added to the bath solution. Prostaglandins were generously supplied by Dr. J.E. Pike of the Upjohn Company, Kalamazoo, Michigan, USA. Concentrations of prostaglandins are expressed as free acid concentrations. Data were compared with either a paired or unpaired t-test,where appropriate. RESULTS In Figure 1, PGE2 production by rings .of ductus arteriosus from 14 fetal lambs (between 135 and 144 days gestation) is expressed in terms of the amount of immunoreactivePGE2 released by rings during a 45-minute incubation period. There was initially a moderate production of PGE2 following the manipulation of mounting the rings in the organ culture bath (3.69 + 1.11 pg PGE2/mg wet weight per 45 min, $SEM). By 90 min of exposure to low PO2, however, the PGE2 production declined to a low steady state level (1.15 _c 0.52 pg PGE2/mg wet weight per 45 min, n=l4, p c.005 paired t-test). When these rings were exposed to high PO2 there was a marked increase in PGE2 production (32.3 + 12.6 pg/mg wet weight per 45 min) which could be inhibited by indomethacin (2.26 + 1.01 pg/mg wet weight per 45 min, pt .OOl).

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WE2

Released (w/w wet

W/45

min)

Fig. 1. PGE2 productionby rings of lamb ductus arteriosus. Rings of ductus arteriosus from 14 fetal lambs between 135 and 144 days gestation were treated as described in METHODS. The-height of the bars represents the mean (&SEMI immunoreactivePCE2 released into the 5 ml bath solution during the 45 min incubationperiods.

These findings are consistent with the effects of indomethacin the isometric tension of rings of ductus arteriosus. Figure 2 demonstrates that when rings of ductus arteriosus were exposed to indomethacin after incubating in low PO2 for less than 90 qin, there was a significant increase in tension (1.41 2 0.33 g/mm2, n=6, p L 0.025 paired t-test); however, when rings were exposed to indomethacinafter more than 90 qin exposure to low PO2 there was no consistent significant effect of indomethacin on isometric tension (0.14 _C0.11 g/mm2, n=8). on

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Low PO2 f

-

<90

min

Exposure

> 90 min

to Low

PO;!

Fig. 2 Comparison of indomethacin-induced changes in tension in the ductus arteriosus exposed to low PO2 for different lengths of time. Rings of ductus arteriosus from 14 different fetal lambs between 122 and 146 days gestation were treated as described in METHODS. The heights of the bars represent the mean (I) resting tension in the rings when incubated in low PO2 (24 to 32 torr alone) and the (2) increase in tension produced by indomethacin in the low PO2 environment. Rings from 6 animals were incubated for 40 to 70 min in a low PO2 environment ( < 90 min); rings from 8 animals were incubated for 95 to 180 min in a low PO2 environment (790 qin). There was no significant difference in the passive tensions applied to the rings to stretch them to their initial isometric length ( ( 90 min: 4.33 + 0.37 g/mm2 versus ~90 min: 3.86 + 0.72 g/mm2); nor was there any significant difference in the final resting tensions between rings incubated for less than 90 min (4.54 2 0.33 g/mm2) and those incubated for more than 90 min (4.66 -, 1.0 g/mm2) with low P02. Figure 3 demonstrates that, although indomethacin had no significant effect on rings incubated in low PO2 for greater than 90 min, following exposure of the rings to oxygen, indomethacin produced a significant increase in tension. In addition, the final tension achieved in rings exposed to high PO2 and indomethacin was independent of whether the indomethacin was added before or after the increase in oxygen tension.

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8-

6Tension (q/mtl12)

.

High

High

\

PO* f ’

fndome?hocin POp-

Low PO2 + lndome fhomh

4-

-

Low

PO,

-

2-

O-

Fig. 3 Comparison of indomethacin-inducedchanges in tension in rings of ductus arteriosus exposed to different oxygen tensions. Two parallel rings from each ductus arteriosus were suspended in separate organ baths as described in METHODS. Eleven lambs between 100 and 143 days gestation were used. (a) One ring was incubatedin 100% N-7 for 100 min. Following this, indomethacinwas added and the ring was incubated in 100% N2 plus indomethacin for 90 min. The ring was then incubated in 100% 02 plus indomethacin for 90 min. (b) The other ring, from the same ductus, was exposed to (in consecutive order): 100% N2 for 100 min, followed by 100% O2 for 90 min, followed by 100% 02 plus indomethacin for 90 min. There was no significantdifferencein the passive tensions applied to the rings to stretch them to their initial isometric length (a: 4.17 + 0.79 g/mm2 versus b: 3.57 + 0.79 g/mm2, n=ll); nor was there any significant difference in the final resting tensions in low PO2 (a: 5.11 2 0.84 g/mm2 versus b: 4.58 2 0.50 g/mm2, n=ll) or in the final oxygen-plus-indomethacin-induced tensions (a: 7.30 2 0.99 g/mm2 versus b: 6.74 + 0.83 g/mm2, n=ll) in parallel rings from the same ductus. There was a significant difference in the indomethacin-inducedincrease in tension in rings incubated in low PO2 (0.11 + 0.09 g/mm2) when compared with those in high PO2 (0.55 2 0.12 g/mm2, kll, pd .05).

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DISCUSSION Despite the observations that hypoxia increases prostaglandin production in several isolated organ systems (6), our findings do not support the hypothesis that hypoxia increases PGE2 production by the isolated lamb ductus arteriosus. In fact, 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; it was when rings were exposed to an elevated PO2 that significant amounts of PGE2 were released. These observations were supported by our findings that indomethacin had a negligible effect on rings equilibrated in a low PO2 but caused a significant contraction in rings incubated in a high P02. It is possible that the intratissue PO2 in our in vitro preparation may be lower than in vivo, and inadequate to sustain prostaglandin synthesis. Experiments designed to monitor intratissue PO2 will be necessary to see how well these in vitro results can be extrapolated to the in vivo situation. Preliminary experiments demonstrate that, although the ductus arteriosus produces only a negligible amount of PGE2 when equilibrated in a low-oxygen medium, it produces significant amounts of PGE2 when exogenous arachidonic acid is added to the low PO2 medium; this increased PGE2 production is completely blocked by indomethacin (Clyman: unpublished results). Our findings are consistent with the hypothesis that endogenous PGE2 inhibits the ability of the vessel to contract in response to oxygen. We cannot explain the discrepancy between our observations and those of Coceani et al (in which indomethacin caused a significant increase in tension in rings incubated in a low P02) (16) other than to suggest that the former experiments may have been performed while the tissues still were recovering from the initial manipulation and still were producing significant amounts of PGE2 (c.f., Fig. 2). However, Coceani et al appear to have observed this effect several hours after mounting the preparation in the bath (personal communication). Although the foregoing discussion has assumed that prostaglandins which act on the ductus arteriosus are formed locally, the fetus has high circulating levels of PGE2 that may play a hormonal role, particularly in maintaining the patency of the ductus arteriosus (17). Friedman and coworkers used sonomicrometer dimension crystals, chronically implanted on the fetal lamb ductus arteriosus, to measure the effects of indomethacin doses on ductal constriction (18). They found that a good correlation existed between the magnitude of ductal constriction and the depression in circulating PGE2. Our results demonstrate that the isolated lamb ductus arteriosus needs an oxygen tension greater than that present in utero to produce effective amounts of PGE2. These observations are consistent with the hypothesis that circulating levels of PGE2 may be important in the prenatal maintenance of ductal patency. Whether circulating prostaglandins are important in the etiology of persistent patency of the ductus arteriosus in the newborn still requires further investigation.

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ACKNOWLEDGEMENTS This work was supported by USPHS Program Project Grant HL 06285 from the National Heart, Lung, and Blood Institute and from a grant from the National Foundation-March of Dimes. Dr. Clyman is the recipient of a Young Investigator's Award from the National Heart, Lung, and Blood Institute, HL 21409. We wish to thank Susan Axelrod for her skillful preparation of this manuscript.

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I. Cellular and Guinea .pig ductus arteriosus. Fay, F.S. Am. J. Physiol. metabolic basis for oxygen sensitivity. 221:470, 1971. Kennedy, J.A. and S.L. Clark. Observations on the physiological reactions of the ductus arteriosus. Am. J. Physiol. 136:140, 1942. ductus Prostaglandins and patency of the Geani, F. Ductus Arteriosus, Report of the arteriosus. In: The Seventy-Fifth Ross Conference on Pediatric Research. (M.A. Heymann and A.M. Rudolph, eds.) Columbus, Ohio, 1978, p. 28. Clyman, R.I., F. Mauray, C. Roman, and A.M. Rudolph. PGE2 is a more potent vasodilator of the lamb ductus arteriosus than is either PG12 or 6 keto PGFlcc. Prostaglandins 16:259, 1978. Clyman, R.I. Ontogeny of the ductus artexosus response to prostaglandins and inhibitors of their synthesis. Seminars in Perinatology (In Press). Markelonis and G., J. Garbus. Alterations of intracellular oxidative metabolism as stimuli evoking prostaglandin biosynthesis: A review of prostaglandins in cell injury and an hypothesis. Prostaglandins 10:1087, 1975. Piper, P.J. and J.R. Vane. -The release of prostaglandins from lung and other tissues. Ann. N.Y. Acad. Sci. 180:363, 1971. Hamberg, M., S. Svensson, and B. Samuelsson. Gel transformations of Formation of prostaglandin endoperoxides: thromboxanes. In: Advances in Prostaglandin and Thromboxane Research, Vol. 1 (B. Samuelsson, R. Paoletti, eds.) Raven Prewss, New York, 1976, p. lg. Factors regulating the Lands, W., R. Lee, and W. Smith. biosynthesis of various prostaglandins. Ann. N.Y. Acad. Sci.

180:107, 1971. 10. Takeguchi, C., E. Kohno, and C. Sih. Mechanism of prostaglandin biosynthesis. I. and Characterization assay of bovine prostaglandin synthetase. Biochem. 10:2372, 1971. 11. Hansen, H.S. 15-hydroxyprostaglands dehydrogenase. A review. Prostaglandins 12:647, 1976. 12. Clyman, R.I. andA.M. Rudolph. Patent ductus arteriosus: A new light on an old problem. Pediatr. Res. 12:92, 1978. The developmental 13. Clyman, R.I., F. Mauray, L. Wong, etT1. response of the ductus arteriosus to oxygen. Biol. Neonate 34:

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Valenzuela, G. and R. Antonini. Influences of different batches of silicic acid on the chromatography of PGE. Prostaglandins -11: 769, 1976. Friedman, Z. and L.M. Demers. Essential fatty acids, prostaglandins, and respiratory distress syndrome of the newborn. Pediatrics 61:341, 1978. Coceani, F.7P.M. Olley, I. Bishair, et al. Significance of the prostaglandin system to the control of muscle tone of the ductus arteriosus. In: Advances in Prostaglandin and Thromboxane Raven Research, Vol. 4 (F. Coceani and P.M. Olleys, eds.) Press, New York, 1978, p. 325. Robinson, et al. Challis, Dilley, J.S. J.R.G., S.R. Prostaglandins in circulation of the fetal lamb. the Prostaglandins 11:1041, 1976. Kirkpatrick. Malony, and S.E. Friedman, W.F., D.A. Prostaglandins: Physiological and clinical correlations. In: Advances in Pediatrics, vol. 25 (L. Barness, ed.) Year Book Medical Publishers, Chicago, 1978, p. 151. Editor: F. Coceani Received 9/N/79 Accepted 2/13/80

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