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J. Physiol. (1976), 260, pp. 335-349 With 9 text-figures Printed in Great Britain

EFFECTS OF PROSTAGLANDIN E2 AND OXYTOCIN ON THE ELECTRICAL ACTIVITY OF HORMONE-TREATED AND PREGNANT RAT MYOMETRIA BY H. KURIYAMA AND H. SUZUKI the Department of Physiology, Faculty of Dentistry, From Kyushu University, Fukuoka 812, Japan

(Received 2 September 1975) SUMMARY

The effects of prostaglandin E2 (PGE2) and oxytocin on the electrical activity of rat myometrium at various stages of gestation and following hormonal treatment have been investigated. 1. PGE2 and oxytocin produce an excitation of the myometrial membrane under all experimental conditions. The sensitivity of the myometrium markedly increases during the last stage of gestation, and at parturition is more than one thousand times greater than in the midpregnant rat. The sensitivity of the myometrium to oxytocin increases rapidly during the last stage of gestation but to PGE2 the increase is gradual, beginning in the late middle stage of gestation. 2. During the early middle stage of gestation, the sensitivity of the myometrium to PGE2 and oxytocin is lower than in the non-pregnant myometrium. 3. After oestradiol treatment, the sensitivity to PGE2 and oxytocin increases but the sensitivity is much weaker than that during the parturition. On the other hand, after progesterone treatment, the sensitivity is reduced below that of the castrated rat. 4. Differences in the sensitivity of progesterone-treated and oestradioltreated myometria to PGE2 and oxytocin are compared to those of the pregnant and post-partum myometrium. The results show that the sensitivity of the myometrium to PGE2 and oxytocin during the early and early-middle stages of gestation can be simulated by progesterone treatment, but that the sensitivity during the last stage of gestation and during parturition cannot be simulated by oestradiol and progesterone treatment. INTRODUCTION

The various prostaglandins (PGs) have different actions on different reproductive tissues and also act in different ways depending upon the

H. KURIYAMA AND H. SUZUKI 336 hormonal state of a given tissue. For example in human myometrium, the frequency and amplitude of spontaneous contractions are usually depressed by PGE2, but are increased by PGE1 (Sullivan, 1966; Bergstr6m, Carlson & Weeks, 1968). The myometrium of guinea-pig, rat and rabbit, however, contracts in response to prostaglandin E (PGE) and prostaglandin F (PGF). The in vitro application of progesterone depresses the sensitivity of the guinea-pig and rat myometrium to the PGE and PGF series (Bergstr6m, Duner, Euler, Pernow & Sj6val, 1959; Eliasson, 1959; Horton & Main, 1963; Paton & Daniel, 1967; Horton, 1969; Weeks, 1972; Pharris & Shaw, 1974). Suzuki & Kuriyama (1975a, b) reported that the sensitivity of pregnant mouse myometrium to oxytocin was increased only during the last stage of gestation. The sensitivity to PGE2 however, gradually increased from the middle stage of gestation. These results suggest that hormones influence the sensitivity of the myometrium to PGE2 and oxytocin. The present experiments examine the effects of PGE2 and oxytocin on the spayed, hormone-treated and on the pregnant rat myometria. As an indicator of sensitivity to the above agents, changes in the electrical activity of the myometrium were observed. The results show that the sensitivity of the rat myometrium to PGE2 and oxytocin during pregnancy are similar to those previously reported for the mouse myometrium (Suzuki & Kuriyama, 1975a, b). Oestradiol-treated myometria are more sensitive to PGE2 and oxytocin than are progesterone-treated myometria. However, a marked increase in sensitivity to PGE2 and oxytocin during parturition could not be simulated by hormone treatments. METEODS The preparation of uterine muscle strips and the experimental details have been described in the preceding paper (Kuriyama & Suzuki, 1976). The same conditions and the same symbols of hormone treatment have been used in the present paper. The drugs used were oxytocin (synthetic oxytocin, atonin-S, Teikokuzoki Pharm. Co. Ltd), prostaglandin E2 (PG 502, PGE2; Ono Pharm. Co. Ltd), Nembutal (sodium pentobarbitone, Abbott, Lab.), Picrotozin (Wako Pure Chem. Ind. Ltd), progesterone (synthetic progesterone, Mochida Pharm. Co. Ltd) and oestradiol (oestradiol-17,fbenzoate, Teikokuzoki, Pharm. Co. Ltd). Concentrations of the drugs are expressed in g/ml., except for oxytocin (oxytocin units; u./ml.). The PGE2- and oxytocincontaining solutions were prepared fresh daily. RESULTS

Effects of PGE2 and oxytocin on the non-pregnant, pregnant and post-partum myometrium The action of oxytocin and PGE2 was excitatory for all stages of pregnancy examined; however the actual drug concentration necessary to

-3:S PGE2 AND OXYTOCIN ON RAT MYOMETRIUM produce excitation varied during the progress of gestation. As described for the mouse myometrium (Suzuki & Kuriyama, 1975a, b) the effects on membrane activity induced by oxytocin and PGE2 were classified into three grades. At the first grade, spike frequency, the number of spikes in a burst and the frequency of bursts increased without any marked change in membrane potential. At the second grade, the membrane was depolarized and spikes were generated continuously, thus eliminating the quiescent periods between bursts. At the third grade, spike generation was blocked during depolarization of the membrane. In the present experiments, the sensitivity of the myometrium to PGE2 or oxytocin was examined for drug concentrations which induced only the first grade response. The maximum concentrations used for these experiments were PGE2, 106 g/ml. and oxytocin 5 x 10-2 u./ml. Fig. 1 shows the effects of PGE2 and oxytocin on the electrical activity of the myometrium from non-pregnant rats. The membrane potential of the non-pregnant myometrium was -56 mV and the burst discharges occurred between silent periods of variable length (2-3 min). The amplitude of the spikes in a burst was irregular, often overshooting. The concentration of PGE2 10-7 g/ml. was threshold. PGE2, 10 6g/mi., produced a first grade response of the myometrium, and oxytocin, 10- u./ml., had nearly the same action as PGE2, 106g/ml., while 10-3 u./ml. oxytocin was more active. However, during the progress of gestation, the sensitivity of the myometrium to PGE2 and oxytocin changed markedly. Fig. 2 shows the effects of PGE2 and oxytocin on the membrane activity during the middle stage of gestation (14 days). Concentrations of PGE2 10-6 g/ml. induced a first-grade response but oxytocin 10-3 u./ml. had little effect. On the other hand, just after the delivery (as shown in Fig. 3) PGE2 10-9 g/ml. and oxytocin l0- u./ml. induced first-grade responses. In this preparation (1 hr post-partum), PGE2 10-9 g/ml. and oxytocin 10- u./ml. oxytocin increased the frequency of the burst discharges (B and D), and ten times higher concentrations (10-8 g/ml. and 10-5 u./ml., respectively) prolonged the duration of the burst discharges (C and E). The minimum concentrations of PGE2 and oxytocin necessary to induce first-grade responses of the myometrium before, during and after gestation, are summarized in Fig. 4. During the first 2 weeks of pregnancy, the sensitivity to both substances was slightly less than that of non-pregnant uterus. From the 15th to 16th day onwards the sensitivity to PGE2 steadily increased. However, the sensitivity to oxytocin increased later and rapidly from the 19th day of gestation. At the end of gestation and during parturition, the sensitivity of the myometrium to PGE2 and oxytocin was 500-1000 times higher than that of non-pregnant myometrium.

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(A-C, D-F). Both PGE2 (104 g/ml.) and oxytocin (10-3 u./ml.) accelerated the membrane activity, and the responses were similar to those of nonovariectomized rat myometria (Fig. 1 C-F). Hormone treatment caused marked differences in the electrical activity. The specific activity patterns have been described by Kuriyama & Suzuki (1976). When progesterone was administered (P.), both PGE2 and oxytocin failed to excite the muscle, i.e. the sensitivity of the myometrium was reduced to less than that of the non-pregnant or castrated uterus (Fig. 6A-C). Oestradiol treatment (E6) increased the sensitivity of the myometrium to more than that of the castrated uterus (Fig. 6D-F). Simultaneous treatment with oestradiol and progesterone (both EP6 and

H. KURIYAMA AND H. SUZUKI 342 E3EP3) resulted in the generation of spikes with a sustained depolarization, a plateau potential, whose duration often exceeded 40 sec (Fig. 7). PGE2 slightly increased the plateau duration in EP6-treated myometrium, and it increased both the frequency of discharge and the duration of the plateau in E3EP3-treated myometrium. The effects of oxytocin were Castrated

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similar, but much weaker. Thus, hormone treatment sensitized the uterus more to PGE2 than to oxytocin. The degree of sensitization was of the order E6 > EP6 E3EP3 > P6. This indicates that the sensitization was mainly caused by oestradiol while progesterone had little or no effect or reduced sensitivity. Another series of experiment was carried out to observe the effects of PGE2 and oxytocin further on the myometrium of rats which were treated for more than 9 days with different combinations of oestradiol and

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H. KURIYAMA AND H. SUZUKI '344 progesterone given in different order. Figs. 8 and 9 show examples of such experiments, i.e. E.P6, P7E5, E3EP3E3 and E3EP3P3. The concentrations of PGE2 and oxytocin were kept constant at 106 and 1O-3 u./ml., respectively. When progesterone was administered after pre-treatment with oestradiol (E5?P6), both PGE2 and oxytocin failed to excite the muscle (as observed after progesterone treatment alone (see Fig. 6 (P6)) and resembled-the responses observed during the middle stage of gestation (-cf. Figs. 8B, C and 2). When oestradiol was administered after pre-treatment with progesterone (P7 E5), PGE2 and oxytocin had strong actions (Fig. 8E and F); however, these actions were nearly the same as those observed after oestradiol treatment alone (see Fig. 6 (E6)). Simultaneous hormone treatment followed by 3 days oestradiol alone (E3EP3E3) produced strong responses to PGE2 and oxytocin (Fig. 6A-C), but no response if the last hormone was progesterone (E3EP3P3, Fig. 9D, E). Although the passive membrane properties of the uterus after treatment with E3EP3E3, are similar to those during the last stage of gestation and .during parturition (Kuriyama & Suzuki, 1976), the sensitivity to PGE2 and oxytocin was much weaker. During the last stage of gestation PGE2 10- g/ml. and oxytocin 10-3 u./ml. produced depolarization block of membrane activity (third-grade response). But these concentrations failed to produce even a second grade response of the hormone-treated myometria (defined as 'depolarization -of the membrane and continuous spike generation'). The obtained results could be summarized as follows. (i) Ovarian hormones change the sensitivity of the myometrium to PGE2 and oxytocin, i.e. oestradiol increases and progesterone suppresses. (ii) If the two hormones are administrated one after the other, the sensitivity of the myometrium is determined by the second hormone. (iii) If the two hormones are given simultaneously, the sensitivity to PGE2 and oxytocin resembles more closely that observed after oestradiol treatment than that after progesterone treatment. (iv) The response of the myometrium to.PGE2 and oxytocin during the middle stages of gestation can be simulated by Fig. 7. Effects of prostaglandin E1 (10- g/ml) and oxytocin (10 3 u./ml;) on the electrical activity of the myometria of castrated rats treated with two different combinations of oestradiol (10 14g/day) and progesterone (10-2 g/day). EP.: oestradiol (10-2 g/day) and progesterone (10-2 g/day) were given for 6 days. E3EP3: oestradiol (10-5 g/day) was given for 6 days and from the 4th to 6th day, progesterone (10 mg/day) was given additively. A and D, control for EP,, (A-C) and E3EP3 (D-F), respectively. B and F, effect of PGE2 (104 g/ml.). C and E, effect of oxytocin (103 u./ml.).

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condition of the 'progesterone dominated myometrium' can be simulated by hormone treatment, but the condition of the 'oestrogen dominated myometrium' cannot. DISCUSSION

It is known that the hormonal state of the mouse myometrium modifies the membrane properties and the membrane sensitivity to PGE2 and oxytocin (Gsa, Suzuki, Katase & Kuriyama, 1974; Suzuki & Kuriyama, 1975a, b). Almost identical changes in the sensitivity to PGE2 and oxytocin have been observed for the rat myometrium. The only important exception occurs during the middle stage of gestation where the rat myometrium appears to be less sensitive than the mouse myometrium. The results presented confirm the previous observations that the 'oestrogen dominated myometrium' is more -sensitive to oxytocin than the 'progesterone dominated myometrium' (Caldeyro-Barcia & Alvarez, 1952;

Csapo, 1960). It has also been reported that in ovariectomized and adrenalectomized rats, either 10 mg progesterone containing 0-5 /sg oestradiol, or 5 mg progesterone containing 0-25 #sg oestradiol is necessary for implantation and also to maintain gestation (Mayer, 1959). Therefore the injected doses of progesterone and oestradiol in the present experiments may be adequate to reproduce the physiological changes of the myometrium during gestation. In fact., the oestradiol-treated myometrium showed a higher sensitivity to PGE2 and oxytocin than that of the progesterone-treated myometrium. In spite of the increased sensitivity of the 'oestradiol-treated myometrium' to PGE2 and to oxytocin, compared with the non-pregnant myometrium, the sensitivity was only one hundredth of that observed during the last stage of gestation. Therefore, at this stage, other factors may contribute to the changes of the membrane activity (e.g. relaxin and adenocorticosteroids). However, the sensitivities to PGE2 and to oxytocin were nearly the same for the middle stage of pregnancy (progesterone dominated myometrium) and the progesterone-treated myometrium. Thus, it is possible to simulate the 'progesterone-dominated myometrium' by treatment with progesterone, but it is difficult to reproduce the 'oestrogendominated myometrium' by oestradiol treatment. Though combined treatment with oestradiol and progesterone followed by progesterone produced similar passive membrane properties as during parturition, there are big differences in the shape of electrical activity and in the sensitivity to PGE2 and oxytocin between the two conditions. The membrane properties of the myometrium differ greatly among

349 PGE2 AND OXYTOCIN ON RAT MYOMETRIUM animal species, and the present results have been only obtained from rat myometrium. Therefore, it is conceivable that the conclusions derived from the present experiments may not be strictly applicable to other species. We wish to thank Professor Edith Biilbring for reading the manuscript and for helpful comments. This study was supported in part by a research grant from Ministry of Education of Japan (048221). REFERENCES

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EIJAsSoN, R. (1959). Studies on prostaglandin, occurrence, formation and biological actions. Acta phy&iol. 8cand. 46, 1-73. HORTON, E. W. (1969). Hypotheses on physiological roles of prostaglandins. Phyiol. Rev. 49, 122-126. HORTON, E. W. & MEN, I. H. M. (1963). A comparison of the biological activities of four prostaglandins. Br. J. Pharmac. Chemother. 21, 182-189. Kuirov, H. & SUZUKI, H. (1976). Changes in electrical properties of rat myometrium during gestation and following hormonal treatments. J. Phy8iol. 260, 315-333.

MAYER, G. (1959). Recent studies on hormonal control of delayed implantation and superimplantation in the rat. Mem. Soc. Endocr. 6, 76-83. OSA, T., Suzuxi, H., KATASE, T. & KuRiuvA, H. (1974). Excitatory action of synthetic prostaglandin E2 on the electrical activity of pregnant mouse myometrium in relation to temperature changes and external sodium and calcium concentrations. Jap. J. Physiol. 24, 233-248. PATON, D. M. & DANIEL, E. E. (1967). On the contractile response of the isolated rat uterus to prostaglandin E1. Can. J. Phy8iol. Pharnacol. 45, 795-804. PHAmais, B. B. & SHAw, J. E. (1974). Prostaglandins in reproduction. A. Rev. Phyaiol. 26, 391-412. SuLImvAN, T. J. (1966). Response of the mammalian uterus to prostaglandins under differing hormonal conditions. Br. J. Pharmac. Chemother. 26, 678-685. SuzuE, H. & K WIYAMA, H. (1975a). Effects of prostaglandin E2 on the electrical property of the pregnant mouse myometrium. Jap. J. Physiol. 25, 201-215. Suzuxi, H. & KuRiYA, H. (1975b). Comparison between prostaglandin E2 and oxytocin actions on the pregnant mouse myometrium. Jap. J. Physiol. 25,

345-356. WEEKS, J. R. (1972). Prostaglandins. A. Rev. Pharmac. 12, 317-336. 12-2