Mechanism of inhibitory action of 5-hydroxytryptamine on placental function

Mechanism of inhibitory action of 5-hydroxytryptamine on placental function D. P. HONEY, M.B., B.S. J.M. ROBSON, M.D., D.Sc.

F. M. SULLIVAN, B.Sc.* London, England The mechanism by which 5-hydroxytryptamine (5-HT) produces its lethal effect on the fetus has been investigated in mice. The 5-HT was found to markedly reduce placental transfer, as measured by the passage of radiosodium from the maternal blood into the placenta and fetus, and also to reduce the blood flow to the placenta. In general, the duration of the effects on blood flow and placental transfer was the same, but in some animals there was evidence that one or another of these effects could be present in the absence of the other. Therefore, it is suggested that 5-HT may have two partially independent effects-to reduce placental blood flow and to reduce the permeability of the membranes separating the maternal and fetal circulations.

(5-HT) has been shown to interrupt pregnancy in mice at all stages of gestation but it is particularly effective in the second half of pregnancy when a single dose of 5 mg. per kilogram of the drug given to the mother may cause death of the majority of the fetuses within one hour. It has also been shown that 5-HT given at crucial stages of pregnancy (days 6 to 11) will produce congenital abnormalities in surviving fetuses. 5 These effects are probably due to an action of the drug on placental function, since it has been shown that 5-HT interferes with the blood supply to the placenta and also markedly decreases the passage of sodium (2 2 Na) from the maternal circulation into the placenta and fetus. 2 • 6 • 7 The purpose of the present investigation was to elucidate the mechanism by which 5-HT interferes with placental function by

studying the effect of the drug on ( 1) placental blood flow and (2) the passage of radiosodium from the maternal circulation into the placenta and fetus. This was done in order to determine whether the deleterious effects of the drug on the fetus are merely due to an interference with placental blood supply or whether there is also a more specific action on placental permeability. To do this it was decided to use a series of decreasing doses of 5-HT and to measure the duration of the effects on blood flow and on the passage of sodium into the placenta and fetus. If the two phenomena were not interdependent, then it might be expected that at low dose levels one effect would disappear before the other, or alternatively that the duration of the effects would vary independently and so demonstrate a lack of relationship between the effects. On the other hand, a failure to dissociate the effects would not necessarily mean that they were interdependent.

5-HYDROXYTRYPTAMINE

From the Pharmacology Department, Guy's Hospital Medical School Supported by a personal grant and an expense grant from the Population Council, New York. *With honors.

Methods Albino mice bred in the animal house at Guy's Hospital Medical School were used.

250

Inhibitory action of 5-HT on placental function

Volume 99 "'umber 2

rhey were examined daily for vaginal plugs

251

This is an index of the blood flow to the

:tnd day 1 of pregnancy \vas designated as

placenta. !n the norrnal rnouse, full mixing

:he day on which the vaginal plug was :ound. Animals 15 to 16 days pregnant were 1sed, the majority of experiments being car·ied out on the fifteenth day of pregnancy. 5-Hydroxytryptamine creatmme sulfate :May & Baker) was used and all doses are ~xpressed in terms of the salt. This was adninistered subcutaneously in aqueous soluIon. Measurement of placental blood flow. The echnique used has been described in detail ~lsewhere. It consists essentially of labeling nouse erythrocytes with radiochromium. ['hese are then injected intravenously into )regnant mice and the rate of entry of abeled cells into the placenta during the irst few minutes after injection measured.

of the blood in the placenta takes about .1 to 10 minutes, and after this time the radioactivity of the placenta remains constant. lf this is expressed as a percentage of the activity in a mixed venous blood sample then it gives the maternal blood volume of the placenta. If a drug reduces placental blood flow by constricting the uterine arteries or veins then the time taken for full mixing, i.e., for the radioactivity to reach a maximum will be increased, though the final amount of activity will not be different from the controls, unless there has also been constriction of the vessels within the placenta. The duration of the vasoconstriction produced by 5HT was measured as follows: At zero time the mice were injected sub-

7

(a) RadiQ:Ji9dip -

JJperi•ms --

i

~.~

5-B.T

s .c.

30,60 or.120---il mins.

22Na

r.v.

examination

25,55or~ m:~.n;.115 W$. I P.M. .

5-HT

~.

a.c.

(c) Dgy.Qle· ~-!··liM

rr ta I

L~ir----0,25,55 or r;~a. 'r mina. 2

5-HT

s.c.

514

... ~r exaltination y. . ··-

2ifa

I.V.

Fig. 1. Scheme of various types of experiments.

September 15, 1967 Am.]. Obst. & Gynec.

252 Honey, Robson, and Sullivan

cutaneously with 5-HT. At 25, 55, and 115 minutes after this, they were injected intravenously with 0.2 ml. (20 ~-tc) of 51 Crlabeled red cells and 5 minutes later the mice were rapidly killed with chloroform, and samples of blood, placentas, fetuses, the whole uterus, and one kidney were removed. These were counted in a well-crystal scintillation counter and weighed and the results calculated as counts per gram per unit time and expressed as a percentage of the activity per milliliter of maternal blood. This percentage was then compared with the corresponding 5 minute values from control mice not injected with 5-HT, and if the values were smaller, they demonstrated that vasoconstriction was still present at that particular time. Measurement of placental transfer function. The passage of radiosodium into the placenta and fetus following intravenous injection into the mother was used as a criterion of placental function. The method is described in detail elsewhere. 7 At zero time the mice were injected subcutaneously with 5-HT and 5 minutes later injected intravenously with 0.2 mi. (3 JhC) of 22NaCl in isotonic saline. Thirty, 60, and 120 minutes after the sodium injection, the mice were rapidly killed with chloroform, and samples of blood, placentas, fetuses, uterus, and kidney were removed and counted as above. The results were again expressed as a percentage of the activity present in the maternal blood. Control animals were similarly injected with radiosodium but without prior 5-HT treatment. Double tracer experiments. In the latter part of the investigation it was decided that additional information might be obtained by injecting both 22 NaCI and 51 Cr-Iabeled cells into each animal. By this means the radiosodium content of each placenta could be correlated with the blood flow of that placenta at the time of examination. The time sequence of the injections of the isotopes and 5-HT was similar to that in the separate experiments described above. The two isotopes were then counted using a singlechannel Dynatron/Ekco scintillation counter

with pulse height analyzer, and the amounts of each were calculated as described by Veall and Vetter. 8 A scheme of the various types of investigation is shown in Fig. 1. Observation of cardiac contraction in the fetus. This was done by transillumination as described by Robson and Sullivan. 7 In the present experiments, if it could be clearly observed that the heart did not beat for a period of 20 to 30 seconds, the fetus was called "dead." This may be semantically incorrect, since evidence is presented that in some fetuses the heart subsequently started beating again. Results

Effect of 5-hydroxytryptamine on the passage of radiosodium into the placenta and fetus. The results are shown in Tables I and II, and are given for the three periods at which measurements were made, i.e., 30, 60, and 120 minutes after the injection of 22 Na into the pregnant mice. It will be seen that in the control animals (i.e., untreated with 5-HT) the passage (uptake) of sodium into the placenta occurred very rapidly during the first half hour and more slowly later on. Thus, at 30 and 60 minutes after the injection of 22 Na, the placental content was, respectively, 62.4 and and 69.0 per cent of the amount of radiosodium found in the maternal blood. These two values are not significantly different from one another ( P > 0.2). At 2 hours the uptake had risen to 84.6 per cent which is significantly greater than the uptake at one hour (P < 0.05). Follovving treatment with 5-HT there were striking changes in the uptake of radiosodium which depended on two factors, namely (a) the dose of 5-HT given and (b) the time after the injection at \Vhich the effect was measured. Thus, at 30 minutes after injection, significantly smaller uptake than controls was noted with doses of 5-HT of 2.0, 1.0, 0.5, and 0.25 mg. At one hour after the injection of 5-HT, a significant decrease was observed only with doses of 2.0 and 1.0 mg., whereas at 2 hours none of the doses used produced

Inhibitory action of 5-HT on placental tunct10n

Volume 99 Number 2

Table I. The placental content of

22

253

Na as percentage of maternal blood activity*

I

I

60 minutes I 20 minutes 30 minutes Dose, of ~-HT -u--_,_-n-...--;~-N-.~-.-o-f,-p-la-.~-en_t_'as INo. of placentas ~~~· of,pla~ent.as (mg.) 1 lYiean.:.. J.LI. 1 1vo. OJ ammats 1 1'vfean ±S.D. 1 lvo. of animals 1 lvfean ±S.D. 1 1vo. OJ ammats

I

62.4 ± 8.5

65 9

69.0 ± 14.4

66 10

84.6 ± 13.9

67 10

2.0

19.6 ± 8.6t

29 5

28.8 ± 15.2t

35 6

58.6 ± 28.4

51 6

1.0

16.5 ± 1.7t

20

39.7 ± 16.5:1:

19

96.8 ± 16.5

18 2

Untreated control

2 21.8:!: 6.8§

0.5

21

2

51.8±19.7

25 3

90.2 ± 27.2

26 3

63.1 ± 10.9

35

78.9 ± 8.4

33 4

2 0.25

22.8 ± 14.0t

36

5

4

0.05

51.0 ± 3.8

8 2

65.4

6

0.02

51.2 ± 16.3

19 4

72.9

7

*5 .. HT creatinine sulfate, when given, was injected subcutaneously 5 minutes before the

2 2Na.

The average value for the

placentas (about 6 to 10) for each mouse was first determined and the figures in the table are the means values.

tP ~p

§P

< <

<

±

S.D. of these

0.001. 0.05. 0.01.

a significant effect on the uptake of radiosodium by the placentas. The passage of radiosodium into the fetuses in control animals was much slower than into the placenta. The values were 26.6, 42.9, and 79.4 per cent at 30, 60, and 120 minutes and are significantly different from each other (P < 0.001). Administration of 5-HT produced a marked inhibition of sodium uptake by the fetus. The results are complicated by the fact that many of the fetuses died as a result of the 5-HT treatment, and the sodium content was usually quite different in the dead and live fetuses; thus, the resuits for the live and dead fetuses are shown separately. From the results in which a sufficient number of live fetuses were available for analysis, it can be seen that there was a significant reduction in sodium transfer to the fetuses up to one hour following doses of 5-HT of from 2.0 to 0.25 mg. Doses of 0.05 and 0.02 mg., however, did not cause :o~nv --o---------siP"niflr:o~nt ---------rPrlnrtion --in soclinm ----; --------- tr:o~nsfpr

-----~---.

By 2 hours following the injections there

was no significant reduction in any of the live fetuses. In the dead fetuses, the content of sodium was always very much less (P < 0.001) than in the control fetuses. Effect of 5-HT on blood flow in the pia· centa. The rate of entry of 51 Cr-labeled erythrocytes into the placenta was used as an index of placental blood flow. The results are shown in Table III, and are given for the three periods at which measurements were made, i.e., 30, 60, and 120 minutes after the injection of 5-HT into the pregnant m1ce. In untreated animals, mixing of maternal systemic blood and placental blood occurred rapidly, so that by 5 minutes after the injection of tagged erythrocytes the placentas already had a count of 13.4 per cent of the mixed maternal blood radioactivity. It has been shown previously that the total maternal blood volume of the placenta at this stage of pregnancy in the mouse, is 17 per cent. 7 Thus, the value of 13.4 per cent reached in 5 minutes corresponds to 79 per cent mixing within the placenta.

2.7:!: 3.8t

9.0 ± 7.81f

17.9±0.4

24.9

0.25

0.05

0.02

20

- 1-

0

16

2

-2--~2-

8

9 __ 2 _ _27 _ 4_

20

0

5 24 --5

659_ _ _09_ __

7.7±6.7t

16.6 ± 8.0[[

48.8

39.3

1.8 ± 0.5t

1.8 ± 0.3t

1.6 ± 1.4t

17.8± 1.1[1

7.7

42.9 ± 9.3

0

0

0

6 7 ----

4

22

3

22

33 6

13 3

3

0

2 ---

--1o-- ·-w-66

57.9 ± 21.1

44.8

79.4 ± 6.5

1.5

3.2 :t l.Bt

2.9 ± 0.3t§

2.5 ± 1.5t:):

65

29 4

0

1

0 6

·-~10-

120 minutes

4

26

2

-~-

16§

47:j: 6

0 10

tP < 0.001. tExcludes high value ( 48.4 per cent) of 4 fetuses of I animal. §Excludes high value (36.2 per cent) of 2 fetuses. I!P < 0.01. 1]P < 0.05.

*5-HT creatinine sulfate, when given, was injected subcutaneously 5 minutes before the 22Na. The average values for the foetuses (about 6-10) for each mouse was first determined and the figures in the table are the means ± S.D. of these values. Values for live and dead foetuses are shown separately.

5.8±1.5t

1.6:!: 0.2t

8.8

0.5

1.3 ± 1.3t

0.8 ± 0.07t

5.9± 2.8t

2.0

Dead

1.0

26.6 :±: 6.9

Alive

minutes

Na as percentage of maternal blood activity*

Fetal uptake ± S.D.

Untreated controls

Dose of 5-HT (mg.)

22

30 minutes

Table II, Fetal content of

-

...G'l::; . r" ~

.!':'"'"

[§.

(/>

""'" . "" 0;;

§:: <" c ::1

(1'1

0..

.? c:;:,

~

•

~

"'0

0"

(1)

::s -;:::.

0

::c

~

"->

Inhibitory action of 5-HT on placental function

Volume 99 Numbe1· 2

255

Table III. Placental content of 51 Cr-labeled erythrocytes as percentage of maternal blood activity* Dose of

5-HT (mg.)

Untreated control

2.0

I

30 ":inutes

.. •o.;:;::.J . . . .

N o. of placentas Mean± S.D. No. of animals Mean± S.D.

I

13.4 ± 3.0

110

No. of animals

Mean± S.D.

No. of animals

110

13.4±3.0

110

13.4 ± 3.0

15 4.0 ± 1.9t

15

15 3.6 ± 1.1 t

4.3 ± l.lt

20

3.6 ± 2.2t

15

24 2

2

3.9 ± 0.9t

11.1 ± 5.0

17

15 2

2

2 0.25

15 5.8 ± 4.6t

17

2

1.0

120 ~:·:;:,:.::.=

10.4±4.1

9.6 ± 2.8§

25

4

35 4

5

0.05

13.9

4

17.7

6

0.02

15.9

6

13.9

7

*After subcutaneous injection of various doses of 5-RT creJOtinine $\llfate. The average' vatues for the placentae (about 6 to 10) for each mouse was first determined and the figures. in the table are the means ± S.D. of these values. tP < 0.001. j:P < 0.01. §P < 0.05.

No significant amount of radioactivity was detected in the fetuses showing that 51 Crlabeled erythrocytes did not readily pass the placental barrier. Following treatment with 5-HT there was marked reduction in the rate of entry of labeled cells into the placenta, so that 30 minutes after injection of 5-HT there was a significant reduction (P < 0.001) of placental flow with doses of 5-HT of 2.0, 1.0, and 0.25 mg. By one hour there was still a significant reduction ( P < 0.001 ) with doses of 2.0 and 1.0 mg. and by 2 hours there was oniy a significant reduction ( P < 0.01) with the 2 mg. dose. With doses of 5=HT of 0.05 mg. and 0.02 mg., no detectable reduction in blood flow was present by 30 minutes. Double tracer experiments. Some experiments were carried out using both 22Na and 51Cr-labeled red cells, so that the sodium content and blood flow could be measured simultaneously. Only about 20 animals were used in these experiments so no very significant conclusions could he drawn. In animals treated with 2.0 mg. of 5·HT, occasional placentas (2 out of 10) had good

blood flows with reduced sodium contents. In animals treated with 0.02 mg. 5-HT, there was a marked reduction in blood flow at 5 to 10 minutes following the injection and this was accompanied ·by reduced so~ dium transfer into the placenta and fetus (values about one fifth of controls) , By 30 minutes, however, the blood flow was normal though the sodium transfer values were still reduced. Therefore, from these few experiments it appeared that occasionally it was possible to detect a reduced permeability to sodium without a simultaneous n ream:uon m mooa now. Fetal mortality. The criterion of fetal death was absence of the fetal heart beat for a period of observation of about 2.0 to 30 seconds, Table IV shows an analysis of the . fetal mortality in all e~riment.s de.. scribed above. It <;;an be seen that 5-HT in a dosage of 0.5 to 2 mg. had 'l- marked letb~l effect on the fetus at all tim¢8~-after in· jection of 5-HT. At the dose of 0,?5 rpg. the over~au fetal m(Jrtality was reduCed. of 5~BT Doses Qf 0;05 mg. ~. 0~02 dose rea $ ki:llfld very few f~ There. which 5~HT of doses the lationship between 1

.•

'"

1 1

1

mg.

256

September 1.5, 1967 Am. J. Obst. & Gynec.

Honey, Robson, and Sullivan

Comment

Table IV* Dose of 5-HT

(mg.)

2.0

% Fetal mortality

! 35 minutes I 65 minutes 82.8 (29)

94.3 (35)

1125 minutes

100

(51)

(36)

97.1 (34)

0.5

95.2 (21)

88.0 (25)

96.7 (30)

0.25

76.5 (51)

57.6 (59)

32.1 (81)

1.0

0.05 0.02

100

0

(40)

(8)

1l.l (18)

100

0

(6)

0

(7)

*The fetuses were examined 35, 65 1 and 125 mmutes after the 5-HT injection. Total numbers of fetuses shown in parentheses. In control untreated mice) no- dead fetuses were

found in 196 examined.

lead to death of the fetuses and the doses which produce marked effects on the blood flow and sodium transfer in the placenta. Examination of the data on fetal mortality following the injection of 0.25 mg. 5-HT suggests that the percentage mortality decreases if the animals are examined at 125 minutes rather than at 35 and 65 minutes following the injection. The mortality, in all experiments performed at 35, 65, and 125 minutes was 76.5, 57.6 and 32.1 per cent, respectively; differences which are very significant (P < 0.001). The data suggest that as the action of 5-HT diminishes with time, some fetuses are capable of recovering from the cardiac arrest caused by the temporary reduction in placental function. The effect of 5-HT on the uterus and kidney. The diffusion on radiosodium into the uterus and kidney was measured at 30, 60, and 120 minutes following its injection into normal or 5-HT treated pregnant mice. Uterine sodium values of 60.8, 60.6, and 77.6 per cent and renal sodium values of 76.2, 80.6, and 83.0 per cent, respectively, were found at these times. These values were not significantly reduced by prior 5-HT treatment in any of the doses tested up to 2.0 mg. Uterine blood flow was also measured at intervals up to 2 hours following 5-HT administration, but no significant reduction from the control value of 5.6 per cent was observed in any of the groups.

We have shown previously, in a detailed study on the mechanism of the lethal effect of 5-HT on the fetus/ that the substance does not produce any harmful effect if injected directly into the fetus; it does not produce constriction of the umblical vessels in the mouse; and it does not cause any significant uterine contractions under these conditions in vivo. Furthermore, when injected into the mother, very little 5-HT passes into the fetus. From all of this, it can be assumed that the effects described in the present paper are not secondary to direct effects on the fetus. It can be seen from Tables I through III that the injection of 5-HT into pregnant mice produces a reduction in blood flow through the placenta and that the passage of radiosodium from the maternal blood into the placenta and fetus is also markedly reduced. With doses of 0.25 mg. 5-HT and over, the reduction in placental function lasts for over 30 minutes, and many of the fetuses die. With doses of 0.02 and 0.05 mg., however, there was no clear evidence of impaired placental function, and the fetal mortality in these groups is low. Previously, we have shown that severe anoxia of the fetus will lead to death in 20 to 30 minutes 7 so that the above results are compatible with the hypothesis that 5-HT produces its lethal effect on the fetus by the production of anoxia following reduced placental function. It is interesting, however, that some of the fetuses may still recover following complete cessation of cardiac activity even after periods of up to 2 hours, though whether this is the limit has not been investigated. The question remains as to the cause of the reduced placental function and how is it related to the reduced blood flow? By comparing the results in Tables I and III it is clear that in those groups of animals in which the sodium uptake in the placenta is reduced, the blood flow is also reduced. In general the converse is also true but in two groups receiving respectively 2.0 and 0.25 mg. both at 2 hours, the placental blood flow is still significantly reduced, but the

Volume 99 Number~

content of sodium is within normal lilllits. This, together with tlw few experinH'nts using the double isotope technique in which the converse was found. i.e., that then• could lw reduced sodium content with near nor" mal blood flows, suggest> that the two phenomena of blood flow reduction and reduced sodium transfer may not be Pntirely causall1 related. It seems possible that at least part of the effect of 5-HT may be to reduce the permeability of the vascular membrane> within the placenta. The oxygen consumption of the placeuta is very high, and at some stages it may t'll'lt be greater than that of the fetus itself. t Reduced plact>ntal blood flow would obviously reduce the oxygen available within the placenta, and this in turn could reduce the activity of the active transport mechanisms involved in sodium transport to the fetus. However, it is much more difficult to explain the failure of sodium to diffuse into the extravascular extracellular space within the placenta itself. In Table I, the sodiur11 space at 30 minutes >vith doses of 0.25 to 2.0 mg. 5-HT is of the order of 16 to 2:)

Inhibitory action of 5-HT on placental function

257

per n·nt which ts n:n dose to tlw watPtll:rl \ascular SJldun·d. Tlrf'w j, '" fact SOl! It' ,,tJwr ,., idt:rt< v that ~·-liT ca havt• a11 dlen un cl'il rrwmbtal!l' !'t'l'lltl'abilitY. 1 The failure of sodll!lll lP JWIIt'tnd•· itrt<\ the fetus tn,t'. 1\ell l." St'C0111brv to thv placental dl't:ns. thnugh utfwr llltcchanism,.,, such as inlt·rferew:c \1ith adi\'t' tran~porl. reduction in countercurrent How. Pte .. ura'> also ('OTJtributc to the ff'tal dfcct. In conclminr,, it t·an lw said th tlw1 othvt !actors may lw in\·okcd and tltcsc llllJUin·

furthn im·(•stigation.

REFERENCES

1. Assali, N. S., Rauranw, L, and Peltonen, T. · AM. J. 0BST. & GYNEC. 79: 86, 1960. 2. Craig, J. M.: Arch. Path. 81: 257, 1966. :t Hyman, C., Rapaport, S. I., Saul. A. M., and Morton, M. E.: Am. J. Physiol. 168: 67·1. 1952. ~- Pickles, V.; J. Physiol. 134: 48+, 1956. 5. Poulson, E., Robson. J. M., and Sullivan. F M.: Scien('c 141:717. 1963.

H.obso11. J :-.1 and Suill\ <>II, I' \1: J Eud" ninol. 25: ~5:\, l96~l. H.obsou. J \L and Sulli1 .JIL F. ~1 : .T Phvsiol. 11!4: 717, 1. \'t·all, :-\., .l!id Vetter. ll : Radioisotope Te-ch· niqu<'~ in Clinical R•·.;r·arch ami Din1J:nnsi' London. 1'.~'i~L Butt<·rwr•rth & Com pam·. Ltd

t>.