Studies on closure of the ductus arteriosus. X. in vivo effect of prostaglandin

STUDIES ON CLOSURE OF THE DUCTUS APTERIOSUS. X. -IN VIVO EFFECT OF PROSTAGLANDIN

GEORGE L SHARPE *

and

K. SUNE LARSSON

LABORATORY OF TERATOLOGY, KAROLINSKA INSTITUTET S-104 01 STOCKHOLM, SWEDEN

The effect of prostaglandins FZoc,E, and of 7-oxa-13-prostynoic acid on the newborn rat and rabbit ductus can be studied using the whole-body freezing technique. PGF2=

and PGE, were

able to re-open the closing ductus arteriosus in adequately oxygenated animals. PGF,_ administration was accompanied by a strong physical reaction in the rat but less in the rabbit. PGE, had sedative effects in both animals. A prostaglandin antagonist, 7-oxa-l3-prostynoic acid had no effect on normal ductal closure nor did it counteract the effects of PGF2_

and

PGH,. The role of prostaglandins in homeostasis during the fetal and newborn period may be to modify ductal tone.

* Holder of a fellowship from the Queen Elizabeth II Canadian Research Fund.

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PROSTAGLANDINS INTRODUCTION -----------_ Among the factors responsible for closure of the ductus arteriosus‘ ,only the role of increased pO2 is demonstrated convincingly (I_).Great attention has also been paid to the presence of adrenergic receptors (2,3) which might play a role but are not essential for ductal closure (4,5). The role of prostaglandins (PGs) on ductal closure was recently studied in vitro independently by Elliott and Starling (6) and Coceani and Olley (7). It was reported that PGF2p could cause contraction of ductal rings from calf, and the constriction was augmented by the presence of oxygen. The effects of PGF2= could be counteracted by a single dose of the PG antagonist 7-oxa-13-prostynoic acid (6j.A relaxation

by PGE, and PGE2 was found in studies on strips of anoxic lamb ductus and the PGs were supposed to regulate the vessel tone during fetal life (7). After in vivo administration of PGE2 or PGF2= it was not possible to produce an effect on either maternal or fetal circulation in the sheep (8). A rapid pulmonary inactivation of PGE, has been demonstrated in the fetal and newborn lamb (9). Speculation on possible clinical use of PGs to control the closure of the ductus arteriosus has“arisen from these early reports. The in vivo effect of PG on the ductus can be accurately determined by the whole-body freezing technique (IO). The ductal closure pattern has been documented in the rat, mouse, rabbit, guinea pig and the pig using this technique (1,701. Species variability in the rate of closure was reported as well as a delay by the influence of hypoxia, hypothermia (11) and phenoxybenzamine (4,5). MATERIAL ________ AND --- METHODS --__--Experimental Animals Virgin Sprague-Dawley rats were obtained from a commercial supplier (Anticimex, Sollentuna) and housed at the laboratory for at least one week before use. After overnight mating, positive vaginal smears were considered as day zero of gestation. Rats were maintained in clear macrolon cages and fed standard lab chow (Astra Ewes, SBdertBlje) and

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water ad libitum until sacrifice by cervical dislocation on day 22 of gestation. Virgin female Havana rabbits weighing 2,500 - 3,000 g were obtained from a local supplier (L. Sjbberg, Ulricehaam). They were housed in individual cages, fed standard lab chow and offered water ad libitum. On arrival rabbits were given 25 IU of luteinising hormone (Pregnyl(E) N.V. Organon, Oss, The Netherlands) intravenously to induce ovulation. They were artifically inseminated 21 days later to produce a known date of gestation (12). Pregnant rabbits were sacrificed by cervical dislocation 30 days following successful insemination. Treatment of Newborn Pups Both rat and rabbit pups were obtained by rapid cesearean section and to prevent a drop in body temperature they were transferred within 3 min to a stable 37'C enviroment in a macrolon incubator (13). The incubator was heated by an overhead 250 watt drying lamp and the internal

1

temperature was raznitoredby a telethermometer .

pGF2ccand PGE, were dissolved in saline within 30 min of use from crystals. The 7-oxa-l3-prostynoic acid was dissolved in 10 per cent ethanol! Substances were given subcutaneously in the dorsum of the thorax in volumes of 50 ul. Control animals received the vehicle only. Strychnine nitrate dissolved in saline was given subcutaneously in the dose of 2 ug/g to duplicate the general stimulatory effects of PGF2=(14). The injections were given 30 or 60 min after delivery to the rats and at 10 min to the rabbits. The 7-oxa-13-prostynoic acid was given to rats in a molar ratio of 2O:l compared to the PG doses ( as recommended by Professor J. Fried ) to explore for a separate effect or a

1

YSI model 43,Single Channel, Yellow Springs Instrument Co.

Yellow Springs, Ohio, U.S.A. 2 PGF2ccwas kindly supplied by Professor Bengt Samuelsson, Karolinska Institutet, Stockholm, Sweden. PGEl lot No. 10315-VDV-115 was kindly donated by Astra, Sbdertalje, Sweden. The 7-oxa-Y3-prostynoic acid was the kind gift of Professor Josef Fried, The University of Chicago, Chicago, Ill. U.S.A.

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blocking effect. This injection was given both at delivery prior to PG injection or 5 min following injection of PG. For study of the effect on ductal closure the pups were sacrificed at various intervals after injection by immersion in liquid nitrogen (rabbits) or in ethanol cooled to -76'~~by dry ice (rats). Following sacrifice the material was stored at -16OC until sectioning in a cryostat (10). Internal diameter measurements of the ductus at its most narrow point were made with a Zeiss microscope micrometer lens. Pups were assessed for changes in general behaviour up to and at the time of sacrifice (30 or 60 mln after injection). Notations were made of colour change and duration, respiratory pattern, presence of retractions, general activity, amount of spontaneous crying and the crying response to induced head flexion. These observations were mainly made on animals given 5 ug/g PGF2= and studied from 3 to 60 min after injection. Blood gases and pH at the time of the peak effect following 1 ug/g PGF2oc (30 min after administration) were determined in a separate study on rat pups. Mixed arteriovenous blood was collected in heparinised capillary tubes following a short deep cut into the cervical vessels. The blood samples were kept on ice until determination of pC02, pQ2 and pH within 60 min of collection in a Radiometer Microsystem HNS NIC2 coupled with a digital acid-base analyser PHM 72. Student's 't' test was used to compare inner ductal diameter size and blood gas values between the experimental and control groups. RESULTS --_-___ 1. The influence of PGPzocin rats at 30 min of age on ductal closure and general behaviour. The response range of the ductus from 3 to 60 min after injection of 5 ug/g PGPzo:is shown in Fig. 1. A maximal dilatation was seen 30 min after injection. This time was chosen to further spot-check the effect of other doses of PGs. Roth 1 and 5 ug/g PGF2= had an equal dilatory effect on the ductus while 0.2 ug/g had a negligable effect (fig.2). This was also seen in rat pups injected at 60 min of age with the same doses (fig.3).

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1200

A

E 1000

0,., 800

E ,_

Q.1

!

600

$ 400 ,-J

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$

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o% O

Fig.

I.

O

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15

30

45

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Rat pups were injected at 30 min PGF2=

®

( || } or 50 pl saline

rain

( ~ ) with 5 ~g/g

( OO ). Influence on

ductal size is shown up to 60 min after injection.

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1200

z

1000

: 0 0

0

0 00

00°

0 0

C Fig. 2.

$ : 012

i

6 pg/gPGF2aS.C.

Rat pups were injected at 30 min with 0.2, 1 or 5 pg/g PGF2_. Controls

(C )

received 50 1.11

saline. The points plotted represent ductal size 30 min after injection.

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1200

-

E a 2

1000 0

800

0 0

.-E ;

0

600

0

.-E m 400 +-, u tz 200

0

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0

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c Fig. 3.

0.2

PG F20cs.c.

Rat pups were injected at 60 min with 0.2, 1 or 5 pg/g PGF*=..Controls

(C )

received 50 ~1

saline. The points plotted represent ductal size 30 min after injection.

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In the rat pups injected with PGF2= , a marked change in activity occurred within 5 min. Hyperactivity, crying, increased reactivity

CO

stimulation and tonic extensor posturing were present in those injected at 30 min of age with 3 and 5 ug/g PGF2: . A slight colour change from pink to bluish pink was present at 15 min and lasted 5 min. All extra activity had subsided 60 min after injection. In those injected at 60 min of age the physical reaction was faster in onset, usually within 2 min and more sustained with a 7 min period of cyanosis at 15 min. Gasping respirations were noted at 30 min. Adtivity returned to normal 60 min following injection. The extreme physical reaction and change in colour were successfully duplicated with 2 vg/g strychnine nitrate but no effect on the size of the ductus was seen compared to controls (table I). Determinations of pH, pc02 and pG2

performed at the height of the

response in PGFZIE injected animals showed no significant difference when compared to controls (table II).

Table I Effect Of DuplicatingPGF2a InducedActivity With Strychnine

Treatment

No. of animals

Inner ductal diameter (Pm)

Strychninenitrate

9

165 + 38.7

Saline

9

166 + 27.3

Ductal response to 2 pg/g strychninenitrate in 30 min old rats as used to simulate PGF2a induced hyperactivity.Ductal diameter measured 30 min after S.C. injectionof either strychnineor 50 ~1 saline.

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Table II Blood Gases During Ductal Dilatation

Treatment

No. of animals

pH

PC02

p"2

pGF2(L

9

7.40 + 0.03

46.9 + 6.1

52.6 + 5.2

Saline

9

7.37 + 0.04

43.9 t 2.3

46.2 + 9.0

Blood gases and pH (x + S.D.) determined on mixed arteriovenous blood in 30 min old rats given lug/g PGF2(Land measured 30 min later.

2. The influence of PGE, in rats at 30 min of age on ductal closure and general behaviour. The ductal size 30 min after injection of 0.2 and lug/g PGE, is shown in Fig.4. A larger effect was seen compared to the same dose levels of PGFZp. PGE, did not cause any increase in activity. Definite cyanosis was present at 10 min after injection (lug/g) but had subsided at 15 min. A very transient slight colour change occurred with 0.2 ug/g PGE, at 10 min and subsided within 2-3 min. 3. The influence of PGF2= and PGE, in rabbits at IO min of age on ductal closure and general behaviour. Dilatation of the closing rabbit ductus was demonstrated for both PGF2.xand PGE , as seen in Fig. 5. Pups had an increase in active crawling and searching mvements

2

min following injection with PGF2=. At 7 min after injection, tachypnea and chest wall retractions appeared. By 12 min all extra movement had disappeared but chest wall retractions persisted up to the time of sacrifice. No colour change was noted. Rabbits injected with 1 Rg/g PGE, at 10 min of age did not show any increase in activity. These developed inactivity 2 min after and chest

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1200

ii

z

800

0 0

.-co u L- 600 E .-c %

0

0 00

0

0

0 0

400

0

z

00

200

0

“8 L

r

C

Fig.

4.

I

0.2

1 pg/g PG E, S.C.

Rat pups were injected at 30 min with 0.2 pg/g PGE,. Controls

(C )

or 1

received 50 ~1 saline.

The points plotted represent ductal size 30 min after injection.

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1200 0

2

1000

0.0

a $

800

E m e ~ 600 g .-8 m c s 0

400

0 0 0

8 200

0 0

0

C Fig. 5.

PG

F2cd

PGE,

1/@I s-c.

Rabbit pups were injected with I ug/g PGF2= or PGEI at 10 min of age. The points plotted represent ductal size 20 min later compared to controls ( C 1.

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wall retraction 30 min after injectionwhich was sustained

up to the

time of sacrifice. 4. Attempts to block the action of prostaglandinwith 7-oxa-13-prosty-

noic acid. In rat pups injectedwith 7-oxa-33-prostynoic acid, there was no change in general behaviour.It did not alter the ductal response to PGFzGzor PGE1 either when given immediatelyat delivery or after the injectedprostaglandindose (table III). Table III No. of

Treatment

animals 1. PA' Control 2. PA Control 3. PA plus 1 vgh PGFza 1 a/g

PGFza

Inner ductal diameter Age (minutes) injected sacrifice X + S.D. (pm)

5

0

30

206 f

32

4

0

30

235 f

13

5

30

60

202 +

50

5

30

60

254 +

36

60

568 f

46

60

532 +

70

60

550 f

72

5 5

30

1 @/g PGFza plus PA

30

5

35

1 M/9 PEl

5

30

60

$60 f

108

1 vg/'gPGEl

5

30

60

580 f

57

60

610 +

96

4. PA plus

0

1 m/g PGEl plus PA

30 5

35

1 PA= 7-oxa-13-prostynoic acid

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DISCUSSION ------^_-In these experiments the administration of PGs effectively reopened the closing ductus arteriosus in both newborn rats and rabbits in vim. It is interesting to note that an equal effect was obtained in both a slow closing species (rat) and a rapid closing species (rabbit) (15). The whole-body freezing method permits an accurate measurement of the in vivo state of the ductus and minute changes in closure rate can be demonstrated (11, since the experimental animals have been shown to freeze completely within 20 seconds. In the rat the width of the ductus can even exceed that seen immediately at birth after treatment with PGEl. Such an influence on the aorta or main pulmonary vessels was not present and,indicates a special effect on the ductus. Even though Elliott and Starling (6) obtained an in vitro constrictor effect with PGFZOc,this may be due to an in viva/ in vitro difference or to the high concentrations (40ug/ml) used in that experiment. The ineffectiveness of 7-oxa-l3-prostynoic acid might be due to excessive protein binding in in vivo systems (personal conrnunication, J.Fried). The effect appears to be due to a direct action of PGs, since a strong physical reaction and colour change in rats to PGF2= did not have any specific effect on the ductus when duplicated by strychnine nitrate. Hypoxia has been shown to delay ductal closure (1) but PGFZoc did not cause hypoxia or acidosis in the present study. It is less likely therefore that the PGs could have caused the dilatation of the closing ductus via hypoxia. The ductus is rich in sympathetic nerve supply and substantial amounts of norepinephrine exist in increasing concentration from the pulmonary to the aortic end of the ductus (

16 ). PGE, has been shown

to inhibit the norepinephrine induced pressor response (17) and may act in the ductus artefiosus. A separate effect of the PGs on the vascular smooth muscle remains a possibility. It is not yet clear if PGs are associated with the oxygen receptors postulated for the ductal smooth muscle (18). However, the presence of very high amounts of PG degrading enzymes in the newborn rat lung (19) may indicate an inhibitory role by endogenous ductal PG

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on the ductal response to oxygen. The closure of

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the ductus in response to oxygen In the newborn period may represent threshold escape by the receptors or a lowering of endogenous PGs by degrading enzymes released by the lung. Exposure in utero to PG synthetase inhibitors may have a potential effect on the ductal response to oxygen if endogenous PGs help control this response. Roth indomethacin and acetylsalicylic acid have been used to delay partuition (20,21,22) and reduce PG synthesis in vivo (23). Indomethacin crosses over the placenta and achieves fetal concentrations 1.6 times the maternal level within 10 hours of exposure (24). If endogenous PGs within the ductal wall decrease to a low level, in utero contraction of the ductus might be possible even at the low pC2 of in utero conditions. Results of recently published studies support this hypothesis (25). The clinical implications of the present'observations can only be speculative. The possibility of using exogenous PGs to modify ductal contraction finds limited use in certain congenital heart defects where ductal contraction is life threatening. Even before this use is contemplated it is necessary to study other systemic side effects of PGs. PGs are being investigated for their use in abortion during early pregnancy and for control of term labour. Exposure at term by the fetus to PGs may have a potential for affecting the ductal closure rate as demonstrated here in the newborn rat and rabbit.

This work was supported by grants No.l4X-993-09,10

from the

Swedish Medical Research Council and Stiftelsen Sigurd and Elsa Goljes Minne.

We wish to acknowledge and thank Marianne Xjellberg and Inger Unnerstad for their valuable technical assistance and Diana Sharpe for preparation of the manuscript and Ulla Kudryk for secretarial assistance.

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REFERENCES _____----1. Hdrnblad,P.Y.: Experimental studies on closure of the ductus arter-

iosus utilizing whole-body freezing. Acta Paediat.Scand. Supp 190, 1969 2. Bof&s,L.O.,Malmfors,T.,McMurphy,D.M.

and L.Olson.: Demonstration of

adrenergic receptor function and innervation in the ductua arteriosus of the human fetus. Acta Physiol.Scand. 77:316, 1969 3. McMurphy,D.M. and L.O.Borius.: Studies on the pharmacology of the perfused human fetal ductus arteriosus. Amer.J.Obstet.Gynec. 109:937 1971

4. Hbrnblad,P.Y.,Bor&s,L.O.

and K.S.Larsson.: Studies on closure of

the ductus arteriosus. VIII. Reduced closure rate in guinea pigs treated with phenoxybenzamine. Cardiol. 51:237, 1970 5. Hbrnblad,P.Y. and K.S.Larsson.: Studies on closure of the ductus arteriosus. IX. Transitory effect of phenoxybenzamine on ductal closure in the guinea pig. Acta Pharnncol.toxicol. 31:149, 1972 6. Elliott,R.B. and M.B.Starling.: The effect of prostaglandin FZoc in the closure of the ductus arteriosus. Prostaglandins 2:399, 1972 7. Coceani,F. and P.M.Olley.: The response of the ductus arteriosus to prostaglandins. Canad.J.Physiol.Pharmacol. 51:220, 1973 8. Oakes,G.,Mofid,M.,Brinkman 111,C.R. ans N.S.Assali.: Insensitivity of the sheep to prostaglandins. Proc.Soc.Exp.Biol.Med. 142:194 1973 9. Olley,P.'M.,Coceani,F.and G.Kent.: Pulmonary inactivation of prostaglandin El in the fetal and newborn lamb. Pediat.Res. 7:425, 1973 IO. Hbrnblad,P.Y. and K.S.Larsson.: Studies on closure of the ductus arteriosus. I. Whole-body freezing as improvment of fixation procedures. Cardiol. 51:231, 1967 11.

Hbrnblad,P.Y. and K.S.Larsson.: Studies on closure of the ductus arteriosus. II. Closure rate in the rat and its relation to enviromental temperature. Cardiol. 51:242, 1967

12. Tesh,S. and J.M.Gesh.: Artificial. insemination in the.rabbit and its use in routine teratogenic studies. Excerpta Med.Internat.Congr. Series No. 220:332, 1970

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13.

Thalme,B.: Electrolyte and acid-base balance in fetal and maternal blood. An experimental and clinical study. Acta Obstet. Gynecol. Stand. Vol. XLV, Suppl. 8, 1967

14. Pylkkd,0.0.: The effect of maturation on chemically induced seizures in

rats. J.Fharmacol.Exp.Ther. 131:185, 1961

15. Hdrnblad,P.Y.: Studies on closure of the ductus arteriosus. III. Species differences in closure rate and morphology. Cardiol. 51:262, 1967 16. Ikeda,M.,Sonnenschein,R.R. and D.T.Masuoka.: Catecholamine content and uptake of the fetal ductus arteriosus of the fetal lamb. Experentia 28:914, 1972 17. Hedqvist,P. and J.Brundin.: Inhibition by prostaglandin E, of noradrenaline release

and of effector response to nerve stimulation

in the cat spleen. Life Sciences 8:389, 1969 18.

Fay,F.S.

: Guinea pig ductus arteriosus.I.Cellular and metabolic

basis for oxygen sensitivity. Amer.J.Physiol. 221:470, 1971 19. Pace-Asciak,C. and D.Miller.: Prostaglandins during developement. I. Age dependent activity profiles of prostaglandin 15-hydroxy-dehydrogenaee

and 13,14_reductase in lung tissue from late prenatal, early

postnatal and adult rats. Prostaglandins 4:351, 1973 20. Aiken,J.W.: Aspirin and indomethacin prolong parturitionin rats. Evidence that prostaglandins contribute to expulsion of the fetus. Nature 240:21, 1972 21. Chester,R.,Dukes,M.,Slater,S.R. and A.L.Walpole.: Delay of parturition in the rat by anti-inflanrmatiryagents which inhibit the biosynthesis of prostaglandins. Nature 240:37, 1972 22. Nosler,K.H. and W.Dornhbfer.: The inhibition of premature labor by B-adrenergic sympathomimetics and prostaglandin antagonists in managing premature onset of labor. Naunyn-Schmiedebergs Arch. Pharmacol. Suppl. 277 R48, 1973 23. Hamberg,M. and B.Samuelsson.: On the metabolism of prostaglandins El and E2 in the guinea pig. J.Biol.Chem. 247:3495, 1972 24. Traeger,A.,Nbschel,H. and J.Baumseil.: Zur pharmakokinetik von indomethacin bei schwangeren, Kreissenden und deren neugeborenen. Bbl.Gyn&k. 95:635, 1973

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25. Sharpe,G.L., Thalme,B. and K.S.Larsson.: Studies on closure of the ductus arteriosus. XI. Ductal closure in utero by a prostaglandin synthetase inhibitax. Prostaglandins

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