Prostaglandins

and

Medicine

1:

167-174,

1978.

DEVELOPMENTAL RESPONSES TO OXYGEN, ARACHIDONIC ACID, AND INDOMETHACIN IN THE FETAL LAMB DUCTUS ARTERIOSUS -IN VITRO R.I. Clyman. Cardiovascular Research Institute and the Department of Pediatrics, University of California, San Francisco, California 94143 and the Department of Pediatrics, Mt. Zion Medical Center, San Francisco, California 94115. (reprint requests to RIC) ABSTRACT It has been suggested that ineffective constriction in response to an increase in PO2 is the primary cause for delayed closure of the ductus arteriosus in preterm infants. The isometric contractile effects of increased Pop and prostaglandin synthetase inhibitors (indomethacin and tranylcypromine) were studied on isolated rings of lamb ductus arteriosus from animals of two gestationRings from animals days, term is 150 days). al ages (87-110 days and 135-150 less than 110 days have a significantly smaller oxygen-induced contraction (2.5 + .3 g/ntm2, 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 indomethatin-induced contraction (1.10 + 1.7 g/mm2, n=16) than vessels near term (0.52 i: .12 g/mm2, n-9). In addition, arachidonic acid produces a greater relaxation in the immature oxygen contracted ring (42 + 9%, n=lO) 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. INTRODUCTION In contrast to full term infants in whom functional closure of the ductus arteriosus occurs within the first 24 hours after birth, preterm infants frequently have delayed spontaneous closure (1). The exact mechanism responsible for the constriction of the ductus arteriosus at birth and for its delay in closure in preterm infants is as yet unknown. Numerous observations have drawn attention to the importance of the postnatal increase in arterial oxygen tension (PO2) for muscular closure of the ductus arteriosus (2-5). There is increasing evidence that prostaglandins maintain the patency of the vessel in the fetus near term. Prostaglandins El (PGE1) and E7_ (PGE2) relax the iso-

167

lated calf and lamb ductus arteriosus in a low oxygen environment (6,7). Pretreatment of isolated lamb ductus arteriosus with prostaglandin synthesis inhibitors also contracts the hypoxic vessel (8). Blockers of prostaglandin synthesis, when given to pregnant mothers near term, constrict the ductus arteriosus of fetal rats, rabbits, and lambs in vivo (9,lO). An increasing sensitivity to indomethacin has been demonstrated as fetal rats approach term gestation (10). Recently, we have observed that the isolated lamb ductus arteriosus exposed to high oxygen tension is as sensitive to prostaglandins and their metabolites as it is in a low oxygen environment (11,12). In the exper&ments reported herein, the contracting effects of indomethacin on the isolated lamb ductus arteriosus from two gestational age groups were examined at high oxygen tensions. Indomethacin caused further contraction of the ductus arteriosus already constricted by elevated P02. This is consistent with the hypothesis that endogenous prostaglandins inhibit the tendency of the vessel to contract in response to oxygen. In contrast with studies in the rat, we found a more intense response to indomethacin in the youngest fetuses, indicating that the prostaglandin mechanism develops early during gestation. METHODS AND MATERIALS Time-dated fetal lambs, between 87 and 150 days gestational age (term is 150 days), were delivered by cesarean section and rapidly killed by exsanguination. The ductus arteriosus was dissected free from adventitial tissue and divided into three to four 1 mm thick rings that were placed into separate 150 ml lucite plastic organ baths (fluid removed by draining) enclosed in a box excluding light. The rings were suspended between two stainless steel hooks in a solution containing 127 mM NaCl, 5 mM KCl, 2.5 mM CaC12, 1.27 mM MgC12*6H20r 5.5 mM glucose, and 50 mM Tris*HCl, pH 7.39 at 37'C. Isometric responses of circumferential tension were measured by a Grass F'TO3Cforce transducer and recorded 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 from a single vessel was stretched to an initial length that would result in a maximal contractile response to increases in oxygen tension for rings in that age group. Rings from animals between 87 and 110 days were stretched to an initial length of 7 mm, whereas rings from animals between 136 and 147 days were stretched to 9 or 10 mm (unpublished observations). The bathing solution in each bath was bubbled with 100% nitrogen (to a PO2 of 16 to 20 torr) and the tissues were allowed to equilibrate for 45 min until a steady tension was developed. The tissues then were exposed to 100% oxygen (to a PO2 of 680 to 720 torr) for 1 hour to allow the tension to achieve a new plateau. The oxygen-induced contraction was considered as the difference in the tensions at high and low PO2. After the rings reached a new steady tension in the high PO2 environment, indomethacin was added to the bath in a final concentration of 1 ug/ml (2.8 x 10-6 M) and the rings were allowed to achieve a new increase in tension over the next hour. Additional doses of indomethac3.nto a final concentration of 5 ug/ml or 10 ug/ml produced no further increase in tension. The indomethacin-induced contraction was considered as the difference in the steady tensions at high PO2 and high PO2plus-indomethacin (1 ug/ml). 168

In other experiments, tranylcypromine hydrochloride (a monoamine oxidase inhibitor and inhibitor of prostaglandin biosynthesis [13]) was added to the bath (instead of indomethacin) after the rings reached a steady tension in high P02. Concentrations of 500 ug/ml produced a maximal tranylcypromineinduced contraction (the difference in tensions at high PO2 and high PO,-plustranylcypromine). In some experiments rings of ductus arteriosus were contracted with high PO2 or high P02-plus-indomethacin (1 ug/ml) following which arachidonic acid (a precursor,of prostaglandin biosynthesis) was added to a final concentration of 0.2 pg/ml. Followlng the experiment, the tissues were removed from the baths and blotted dry before they were weighed (wet weight). The tension developed in the tissue was expressed as force/unit area (g/mm2>. This area was the surface of the tissue that was perpendicular to the direction of the contractile-developed force. Since the ring samples assume a 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. This was estimated by dividing the wet weight by the length, assuming a tissue specific gravity of 1. Stock solutions of indomethacin (16 mg/ml> and arachidonic acid (33 mg/dl) were prepared in ethanol each day and aliquots were added to the bath solution. The maximal concentration of ethanol in the bath (0.06%) had no effect on tissue contractility. Tranylcypromine hydrochloride was dissolved in 5 ml of bath solution and added to the bath. Chemicals were obtained from Sigma Chemical Co. RESULTS All the tissues of ductus arteriosus contracted when exposed to high PO,. Figure 1 shows the mean oxygen-induced contraction in rings (from two different gestational age groups) that were stretched to initial lengths that result in maximal contractile responses for rings in that age group (unpublished observations). Vessels from animals less than 110 days gestation have a significantly smaller oxygen-induced contraction (2.5 +. .3 g/mm2, ?SEM, n=16) when compared with vessels near term (4.6 + .7 g/mm2, n=9, p c.005 unpaired t-test). The oxygen-induced response of animals less than 110 days is about 55% of the response in animals near term. As can be seen from Figure 1, oxygen contracted rings contract further upon addition of indomethacin (1 pg/ml) or tranylcypromine (500 ng/ml). The tranylcypromine-induced contraction was not inhibited by phentolamine (data not shown). Rings from animals less than 110 days gestation have a significantly larger indomethacin-induced contraction (1.6 + .17 g/mm2, n=16, p c.025 unpaired t-test) than do vessels near term (0.52 ;f: .12 g/mm2, n=9). In addition, the indomethacin-induced contraction in animals less than 110 days gestation represents a significantly larger proportion of the combined oxygen-plus-indomethacin-induced contraction than it does in animals near term. 169

n

I

Tension (g/mm2) 4

cl 02 ??lndomethoch ?? Trony/cyprom/ne :.:.:.:.:.:. :::::::::::: :::::::::::: .:.:.:.:.:.: y::::::::: :.:.:.:.:.:. :::::::::::: .:.:.:.:.:.: :.:.:.:.:.:. :::::::::::: .:.:.:.:.:.: :.:.:.:.:.:.

0

~

:g$g; ::::::::::::

87-

110

135- 150

Days Gestation

Figure 1. Comparison of oxygen-, indomethacin-, and tranylcypromine-induced contraction in lamb ductus arteriosus. Rings of ductus arteriosus from two different gestational age groups were prepared as described in METHODS. The bars represent the mean (fSRM) increase in tension produced by the oxygeninduced contraction, the indomethacin (1 ug/ml)-induced contraction, and the tranylcypromine (500 pg/ml)-induced contraction. Tranylcypromine was used with 8 fetuses of gestational age 87-110 days; indomethacin was used with 16 fetuses of 87-110 days and g-fetuses of 135-150 days.

The effect of arachidonic acid on the ductus arteriosus shown in Figure 2 is expressed in terms of its ability to reverse the oxygen or oxygen-plus-indomethacin increase in tension. As can be seen in Figure 2, relaxation associated with arachidonic acid (0.2 us/ml> was completely blocked by pretreatment with 1 pg/ml indomethacin. In addition, the arachidonic acid-induced relaxation in rings less than 110 days gestation was significantly greater (42 f 9%) than it was in rings near term (6 t 2X, p c.05, unpaired t-test).

170

60

40

20

0

87-102

140-142

Days

Gestation

Figure 2. Arachidonic acid-induced relaxation in mature and immature rings from the same ductus arteriosus. Parallel rings from the same ductus arteriosus were prepared as described in METHODS. One ring was contracted by oxygen alone and the other by oxygen and indomethacin. Bars represent the mean (+SEM) % decrease in tension caused by arachidonic acid (0.2 pg/ml) of the oxygen- or oxygen-plus-indomethacin-inducedcontraction. Arachidonic acid was used in 10 fetuses of 87-102 days and 4 fetuses of 140-142 days.

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DISCUSSION Other investigators who have studied the in vitro lamb ductus arteriosus have suggested that the ineffective oxygen-induced constriction in immature vessels was the major cause of the delayed closure of the vessel in preterm infants (5,14). They observed that the immature vessel's response was only 10 to 20% of the response in vessels from near term animals. These studies, however, were all performed with vessels exposed to overhead laboratory illumination which causes a photorelaxation in Immature vessels (15). In addition, former studies were not performed at initial lengths of stretch that would result in maximal contractile responses for the rings in a particular age group. The experiments reported herein found that even when oxygen-induced responses are compared at maximally effective initial lengths in a dark environment, vessels less than 110 days gestation have a significantly reduced response to oxygen in comparison with those from older animals. Inhibition of prostaglandin production in rings less than 110 days, however, resulted in a total combined oxygen and indomethacin-induced tension that was not significantly different from the oxygen-induced or total combined oxygen-and-indomethacininduced tension developed in rings from animals near term. This is consistent with the hypothesis that early in gestation endogenous prostaglandins inhibit the vessel's ability to contract in response to oxygen. In addition, indomethacin proved to be more effective in producing contraction in the more immature vessels. Although the difference in effectiveness may reflect a change in the sensitivity of the prostaglandin biosynthetic system to indomethacin, it may also represent a change in the sensitivity of the vessel to, or a change in the synthesis and degradation of, locally produced prostaglandins during development. The loss of arachidonic acid (a precursor of prostaglandin biosynthesis)-induced relaxation in older tissue would support this latter concept. It is unlikely that the action of indomethacin on the ductus arteriosus, in the above studies, occurs by a nonspecific action of the drug (16). Few of the other enzymes known to be susceptible to indomethacin are inhibited by the concentrations used here (9). Prostaglandin synthetase inhibitors (indomethatin and tranylcypromine) behave similarly on the ductus arteriosus despite their structural diversity (8,13). Furthermore, nonspecific effects of indomethacin (e.g., inhibition of phosphodiesterase and prostaglandin 15-dehydrogenase activity, or uncoupling of oxidative phosphorylation) would have led to relaxation rather than contraction. Recently, Pace-Asciak and Rangaraj (17) have shown that homogenates of lamb ductus arteriosus formed PGEZ, PGF, , 6 keto prostaglandin Flee (and its unstable precursor prostacyclin, PG12y but did not form PGD2 or thromboxanes when incubated in high P02. All the prostaglandins known to be produced by the lamb ductus arteriosus (except PGF,, which has no effect on the tissue) relax the veesel (unpublished results). The current findings support the concept of the continued sensitivity of the ductus arteriosus to prostaglandins in the oxygen environment of postnatal, as well as prenatal, existence. If the tone of the vessel represents a balance between contracting (e.g., oxygen) and relaxing (e.g., prostaglandins) influences, then it is possible that patency of the ductus arteriosus in pre172

mature infants is due to an alteration in the metabolism of endogenous prostaglandins, and not to a lack of responsiveness to oxygen. ACKNOWLEDGEMENTS The author wishes to thank Ms. Francoise Mauray for her skillful assistance and Ms. Anne Marsh for her help in the preparation of this manuscript. This work was supported by USPHS Program Project Grant HL 06285 and HL 21409-01 from the National Heart, Lung, and Blood Institute. REFERENCES 1.

Danilowicz D, Rudolph AM, Hoffman JIE. Delayed closure of the ductus arteriosus in premature infants. Pediatrics 37: 74, 1966.

2.

Fay FS. Guinea pig ductus arteriosus. I. Cellular and metabolic basis for oxygen sensitivity. Am. J. Physiol. 221: 470, 1971.

3.

Kennedy JA, Clark SL. Observations on the physiological reactions of the ductus arteriosus. Am. J. Physiol. 136: 140, 1942.

4.

Kovalcik V. The response of the isolated ductus arteriosus to oxygen and anoxia. J. Physiol. (Lond.) 169: 185, 1963.

5.

McMurphy DM, Heymann MA, Rudolph AM, Melmon KL. Developmental changes in constriction of the ductus arteriosus: Responses to oxygen and vasoactive agents in the isolated ductus arteriosus of the fetal lamb. Pediatr, Res. 6: 231, 1972.

6.

Starling MB, Elliott RB. The effects of prostaglandins, prostaglandin inhibitors, and oxygen on the closure of the ductus arteriosus, pulmonary arteries, and umbilical vessels in vitro. Prostaglandins 8: 187, 1974.

7.

Coceani F, Olley PM. The response of the ductus arteriosus to prostaglandins. Can. J. Physiol. Pharmacol. 51: 220, 1973.

8.

Coceani F, Olley PM, Bodach E. Lamb ductus arteriosus: Effect of prostaglandin synthesis inhibitors on the muscle tone and the response to prostaglandin E2. Prostaglandins 9: 299, 1975.

9.

Olley PM, Bodach E, Heaton J, Coceani F. Further evidence implicating E-type prostaglandins in the patency of the lamb ductus arteriosus. Eur. J. Pharmacol. 34: 247, 1975.

10.

Sharpe GL, Larsson KS, Thalme B. Studies on closure of the ductus arteriosus. XII. Prostaglandins 9: 585, 1975.

11.

Clyman RI, Heymann MA, Rudolph AM. Ductus arteriosus responses to prostaglandin El at high and low oxygen concentration. Prostaglandins 12: 219, 1977.

12.

Clyman RI, Wong L, Heymann MA, Rudolph AM. Responsiveness of the lamb ductus arteriosus to prostaglandins and their metabolites. Prostaglandins 15: 325, 1978.

13.

Lee RE. The influence of psychotropic drugs on prostaglandin biosynthesis. Prostaglandins 5: 63, 1974.

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14.

Oberhansli-Weiss I, Heymann HA, Rudolph AM, Melmon IU. The pattern and mechanism of response to oxygen by the ductus arteriosus and umbilical artery. Pediatr. Res. 6: 693, 1972.

15.

Clyman RI, Rudolph AM. Patent ductus arteriosus: A new light on an old problem. Pediatr. Res. 12: 92, 1978.

16.

Flower RJ, Vane JR. Inhibition of prostaglandin biosynthesis. Biochem. Pharmacol. 23: 1439, 1974.

17.

Pace-Asciak CR, Rangaraj G. The 6-lcetoprostaglandin Flclpathway in the lamb ductus arteriosus. Biochim. Biophys. Acta 486: 583, 1977.

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

Prostaglandins and Medicine 1: 167-174, 1978. DEVELOPMENTAL RESPONSES TO OXYGEN, ARACHIDONIC ACID, AND INDOMETHACIN IN THE FETAL LAMB DUCTUS ART...
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