COMPARISON BETWEEN PROSTAGLANDIN E2 AND OXYTOCIN …

Jap. J. Physiol., 25, 345-356, 1975

COMPARISON BETWEEN PROSTAGLANDIN E2

AND OXYTOCIN ACTIONS ON PREGNANT

MOUSE MYOMETRIUM

Hikaru SUZUKI and Hirosi KURIYAMA

Department of Physiology, Faculty of Dentistry, Kyushu University,Fukuoka 812, Japan

Abstract Effects of prostaglandin E2 (PGE2) and oxytocin on themembrane activity of pregnant mouse myometrium were investigated withthe microelectrode method. Sensitivity of the myometrium to oxytocinincreased only during the last stage of gestation, however sensitivity toPGE2 gradually increased beginning at the middle stage of gestation.Prolonged treatment with PGE2 but not with oxytocin produced desensi-tization of the myometrium. When the membrane potential was electri-cally displaced to the resting level after the membrane had been markedlydepolarized by either PGE2 or oxytocin, spike activity was restored.However with PGE2 there was continuous spike generation and withoxytocin periodic burst discharges with silent periods. In Ca-free Lockesolution, PGE2 and oxytocin still produced depolarization of the mem-brane, however, oxytocin produced larger depolarization. The dif-ferent responses of the membrane to PGE2 and oxytocin are discussed inrelation to the roles of Ca ion and to the ovarian and placental hormonesduring gestation.

The prostaglandins constitute a group of naturally occurring C-20 unsaturated

hydroxy fatty acids (prostanoic acid), and all prostaglandins of the E type contain

1.1 ƒ¿-hydroxy and 9-keto groups on a 5-membered ring. Their occurrence, tissue

distribution, biosynthesis, metabolism, physiology, and pharmacology have been

the subject of many comprehensive reviews (BERGSTROM et al., 1968; HORTON,

1969 ; PHARRISS and SHAW, 1974). In the female reproductive system, physiolo-

gical roles of the prostaglandin have been suggested in menstruation, dysmenor-rhea, oviduct motility, gamete transport, regression of the corpus luteum, bio-synthesis of ovarian steroids and parturition. It has also been suggested that the

prostaglandins exert a powerful oxytocic effect associated with interruption of

Received for publication December 16, 1974

鈴木光,栗山煕

345

346 H. SUZUKI and H. KURIYAMA

pregnancy at different stages of gestation (PHARRiss and SHAW, 1974).Recently, the effects of prostaglandin E2 (PGE2) and oxytocin on the electrical

activities of pregnant mouse myometrium have been investigated by OSA and TAGA

(1973a, b), OSA et al. (1974) and SUZUKI and KURIYAMA (1975). Both agentsshowed potent excitatory actions in a late stage of gestation. However , PGE2showed weaker effects on circular muscle fibres than the longitudinal muscle fibresand produced desensitization. On the other hand, both types of muscle fibreshowed similar sensitivity to oxytocin and there was only very weak desensitiza-tion. The present experiments were carried out to investigate in more detail thePGE2 and oxytocin actions on pregnant mouse myometrium .

METHODS

Pregnant mice (dd system) were stunned and bled. The uteri were excised andlongitudinal strips were prepared. The period of gestation was calculated from theday the male mouse had been put with the female for a day .

To record electrical activity, the partition stimulating method was used (ABEand TOMITA, 1968). The microelectrodes used for this work were glass capillary

electrodes filled with 3M KCl. Changes in the amplitude of electrotonic potentials

recorded during drug application were measured from photographic records and the

measurements of the electrical activity were taken from ink-writing paper oscil-

logram records.

The normal bathing solution was a modified Locke solution containing (mM);

NaCl:153 ; NaHCO3:8; KCl:5.6; CaCl2:2.2 and glucose:5.5 equilibrated with a

mixture of 97% O2 and 3% CO,. Na-deficient Locke solution (16 .2mM Na),

K-deficient Locke solution were as described by SUZUKI and KURIYAMA (1975) .

The bathing solutions were kept at 35•Ž and perfused at a constant rate (1ml/sec)

The drugs used were synthetic prostaglandin E2 (G 502, Ono Pharm . Co. Ltd.)

and oxytocin (Atonin-O, Teikokuzoki Pharm. Co. Ltd.).

The concentrations of PGE2 are give in g/ml and those of oxytocin in U/ml.

The drug solutions were prepared fresh every day.

RESULTS

Effects of oxytocin on the membrane activity at various stages of gestationOxytocin showed excitatory action on pregnant mouse myometrium . How-

ever the actual concentration of oxytocin necessary to produce excitation variedduring the progress of gestation. Membrane activity induced by oxytocin wasclassified tentatively into three grades, as was done for the action of PGE2 (SUZUKIand KURIYAMA, 1975). There was the first grade which increased only spike fre-

quency, the number of spikes in a train discharge and the frequency of traindischarges without any marked change of the membrane potential; a second grade

PGE2 AND OXYTOCIN ACTIONS ON MYOMETRIUM 347

which depolarized the membrane and induced continuous generation of spikes

eliminating the quiescent periods between train discharges. The largest effect

was depolarization block of spike generation and this was called the third grade

response.

Figure 1 shows the effects of oxytocin on the membrane activity of mouse

myometrium (16th day of gestation). The three different grades of oxytocin effect

are illustrated, and are nearly the same as those observed with PGE2. On the 16th

day of gestation, 10-4U/ml oxytocin produced the 1st grade effect on the myo-

metrium, and 1•~10-3 and 3•~10-3U/ml oxytocin produced 2nd and 3rd grade ef-

fects, respectively. The increased membrane activity produced by oxytocin was not

mediated by the activation of the nervous elements, since pretreatment with tetro-

dotoxin (10-3g/ml) had no effect on the oxytocin action.

Longitudinal Muscle (16th day)

a

b

c

Fig. 1. Effects of oxytocin on the electrical activity of the pregnant mouse myometrium.

(16th day of gestation). a; 1•~10-4U/ml (1st grade effect), b; 1•~10-3U/ml (2nd grade

effect), c; 3•~10-3U/ml (3rd grade effect). Dots indicate application and removal of

oxytocin.

Figure 2 shows the effects of oxytocin on the membrane activity at variousstages of the gestation. During the late middle stage of gestation (14-16th days),the very high concentration of oxytocin (10-3U/ml) only produced the 1st gradeeffect on the myometrium but during the last stage of gestation (20th days), the 1st

grade effect of oxytocin appeared at 10-5U/ml.In the non-pregnant myometrium, 10-5g/ml PGE2 or 10-2U/ml oxytocin was

required to produce a 1st grade effect, whereas at 14 days of gestation, 10-3U/mloxytocin or 10-8g/ml PGE2 was required to produce the 1st grade effect.


Effects of prolonged treatment with PGE2 and oxytocin on the myometrium

Figure 3c shows the effect of simultaneous treatment with low concentrations

of PGE2 and oxytocin on the electrical activity of the myometrium (14th day of

gestation). To produce the 1st grade effect, 10-3U/ml oxytocin (a) or 2•~10-8g/ml

PGE2 (b) were required. However when the above concentrations of oxytocin and

PGE2 were simultaneously applied to the myometrium the 2nd grade effect on the

myometrium was observed, i.e. the membrane was depolarized and continuous spike

discharge appeared.

Fig. 2. Effects of oxytocin on the membrane activity during various stages of gestation of

mouse myometrium.•› 1st grade effect, 2nd grade effect,•œ 3rd grade effect. More

details are given in the text.

Oxytocin and PGE2 had different actions on the myometrium when applied fora prolonged period. Figure 4 shows the successive application of PGE2 andoxytocin on the myometrium (18th day of gestation). When 10-7g/ml PGE2 wasapplied to the tissue, the 2nd grade effect observed, however the membrane gradual-ly repolarized and the spike amplitude increased even in the presence of PGE2.When 10-3U/ml oxytocin was added, the 3rd grade effect on the myometrium wasobserved and the depolarization persisted without reduction of amplitude (a). Onthe other hand, when 10-3U/ml oxytocin was applied to the tissue in the absenceof PGE2, the 3rd grade effect was observed and the depolarization was sustainedfor more than 10min. On addition of 10-7g/ml PGE2 in the above solution, the



a

b

c

Fig. 3. Effects of oxytocin and PGE2 on the membrane activity of the pregnant mouse myo-metrium (14th day of gestation). Dots indicate application and removal of drugs.Throughout the recording, inward current pulses were applied (botton trace).


a

b

Fig. 4. Effects of PGE2 and oxytocin on the membrane activity of the pregnant mousemyometrium (18th day of gestation). a; Effect of oxytocin (10-3U/ml) in the presenceof PGE2 (10-7g/ml). b; Effect of PGE2 (10-7g/ml) in the presence of oxytocin (10-3U/ml). Long and short bars in the figure indicate the application of the drugs. Theupper trace indicates two different speeds of the continuous records.

membrane was further depolarized but was repolarized within 5min to the levelbefore application of PGE2 to the oxytocin containing solution.

These results might indicate that PGE2 produces desensitization of the myo-metrium and confirmed the previous observations (OSA et al., 1974).


Effects of oxytocin under electrical modulation of the membraneThe effects of oxytocin on the membrane potential and membrane resistance

were observed. To calculate the relative membrane resistance, the ratio (V'/V)2was used, where V and V' are the amplitude of electrotonic potentials measuredbefore and during application of the agent . However if the drug markedly changesthe length constant, the value obtained from this ratio is not reliable , though itwill indicate whether a decrease or increase of the membrane resistance occurs

(SUZUKI and KURIYAMA, 1975). In Locke solution, the length constant was 1.2mmand the microelectrode was impaled into a muscle cell at about 0.3mm from thestimulating partition.

Fig. 5. Current-voltage relationships of the pregnant mouse myometrium (18th day of gesta-

tion) observed in Locke solution (•œ) and in oxytocin (10-4U/ml)-containing Locke solu-

tion (•›) at various membrane potential levels . The membrane potential was displaced

by electrical current of various intensities (expressed as V/cm). RMP shows the resting

membrane potential (mV).

Figure 5 shows an example of current-voltage relationships recorded before and

during the application of oxytocin. The membrane potential was displaced ineither a depolarizing or hyperpolarizing direction in steps and short inward current

pulses of different intensity were applied during the displacement. When the cur-rent-voltage relationships were compared, the amplitudes of the electrotonic poten-tial in the presence of oxytocin were consistently less than in the absence of oxyto-cin at the same membrane potential level. These results agree with those observedon treatment with PGE2 (Suzum and KURIYAMA, 1975). Moreover, when themembrane was slightly depolarized by oxytocin, the membrane resistance was


Longitudinal muscle (19th day)

a

b

c

Fig. 6. Effects of displacement of the membrane potential on the membrane activity duringthe generation of the depolarization block of the spike induced by PGE2 (10-6g/ml).19th day of gestation. a-c; various intensities of short inward current pulse were appliedto the tissue before, during and after application of PGE2. The membrane potentialwas displaced to five different levels in the presence of PGE2. Dots in the figure indicateapplication and removal of PGE2. Lengths of horizontal bar indicate 1min.

greater than that in the resting state. These findings confirmed the previousobservations made on the effects of PGE2 and excess K solutions, that increasedmembrane resistance during the depolarization of the membrane is not a specificaction of the drugs but is due to the anomalous rectifying property of the membranein the mouse myometrium (SuzuKI and KURIYAMA, 1975).

A further difference in the action of PGE2 and oxytocin was seen on repolari-zation of the membrane by application of inward current during the 3rd graderesponse. Repolarization in the presence of PGE2 produced continuous spikedischarges and the spike amplitude was not suppressed (Fig. 6). On the otherhand, repolarization of the membrane in the presence of oxytocin produced the

periodic appearance of burst discharges and the spike amplitude was lower than inthe control (Fig. 7).

The effects of PGE2 and oxytocin on the myometrium in various ionic environmentsThe effects of PGE2 in K-deficient, in Na-deficient and in Ca-free Locke solu-

tion were observed by SUZUKI and KURIYAMA (1975). For comparison the effectsof oxytocin in the same ionic environments were observed. In K-deficient Lockesolution and Na-deficient Locke solution, the effects of oxytocin on the membrane


Longitudinal Muscle(16th day)

a

b c

d

e

Fig. 7. Effects of displacement of the membrane potential on the membrane activity duringthe generation of depolarization block of the spike induced by oxytocin (10-2U/ml). 16thday of gestation. a, b and c; displacements of the membrane potential in hyperpolarizing

(b) and depolarizing directions in Locke solution. d and e; the membrane potentialdisplaced to two different levels in the presence of oxytocin. a-e; various intensitiesof short inward current pulse (1sec) were applied to measure the voltage-current relationship. Dots in d and e indicate application and removal of oxytocin.

activity were nearly the same as those of PGE2 (Suzum and KURIYAMA 1975), i.e.in K-deficient solution oxytocin produced the depolarization of the membrane tothe same extent as that observed in Locke solution. However, in Na-deficientsolution the depolarization of the membrane was smaller than that in Lockesolution.

When the tissues were perfused in Ca-free Locke solution (Ca being replacedby 4.4mM Mg), all electrical activity ceased and even strong intensity of outward cur-rent pulse could not evoke a spike. However, on treatment with either PGE2 oroxytocin the membrane was depolarized. Figure 8 shows the effects of oxytocin onthe myometrium in Ca-free Lockes solution. After the tissue was rinsed withCa-free solution, following depolarization of the membrane induced by oxytocin

(10U/ml) burst discharges of small amplitude or periodic appearance of smalldepolarizations (c and d) were observed. The depolarization of membrane in-duced by oxytocin was maintained throughout the presence of the drug, but re-

petitive application of oxytocin reduced the amplitude of depolarization. However,as shown in Fig. 9, when PGE2 was added in Ca-free Locke solution, the membranewas depolarized but the amplitude was gradually lowered in the presence of thedrug. When PGE2 was applied a second time in Ca-free Locke solution, the de-



a

b

c

d

e f

Fig. 8. Effects of oxytocin (10-3U/ml) on the membrane activity of the pregnant mouse myo-metrium in Ca-free Locke solution. a; Effects of oxytocin in Locke solution. b; Careplaced by 4.4mM Mg (Mg-Locke). Inward and outward current pulses (1sec) wereapplied alternately. c; Application of oxytocin after outward current pulses failed togenerate action potential. d; oxytocin was again applied after the membrane had com-pletely repolarized to the level before application of oxytocin in c.e, f and g; recoveryin Locke solution.


a

b

c

d

Fig. 9. Effects of PGE2 (10-6g/ml) on the electrical activity of the pregnant mouse myome-trium (13th day of gestation). Mg-Locke; Ca-free 4.4mM Mg containing Locke solu-tion. Intervals between a and b, and b and c are 6min and 5min respectively.


polarization was suppressed more rapidly than with oxytocin.

DISCUSSION

The main differences between the actions of PGE2 and oxytocin on the mousemyometrium can be summarized as follows; i) sensitivity to PGE2 and to oxytocindiffered markedly during the early and middle stages of gestation and PGE2 wasmore effectively evoked excitation than oxytocin. Presumably progesterone dom-inated myometrium is less sensitive to oxytocin than to PGE2. Therefore whilePGE2 and oxytocin both have excitatory actions on the myometrium , the me-chanism underlying the two drug actions on the myometrium may not be the same.ii) the two agents do not react with the same receptor, because the myometrium wasdesensitized to PGE2 but not to oxytocin and the desensitization induced by PGE2did not influence the action of oxytocin. It is common to produce desensitizationof receptors by prolonged application of neuro-hormones and therefore PGE2behaves more like a neurohormone than oxytocin. iii) sensitivity to PGE2 is lowerin the circular than in the longitudinal muscle layer . However both muscle layerssimilar sensitivity to oxytocin (SUZUKI and KURIYAMA, 1975).

On the rabbit myometrium, KLEINHAUS and KAO (1969) reported that low con-centration of oxytocin did not cause depolarization, although the amplitude andthe rate of rise of the spike potential increased . They, therefore, postulated thatoxytocin accelerated the activation of Na influx. On the other hand , OSA andTAGA (1973a) concluded that in the mouse myometrium low concentrations ofoxytocin neither depolarized the membrane nor increased the amplitude of theaction potential, but facilitated the conduction of excitation and increased thefrequency and number of spikes in a train. They found no acceleration of therate of rise of the spike by treatment with oxytocin. The present experimentsagreed the observations made by OSA and TAGA (1973a).

OSA et al.(1974) thought that in the mouse myometrium an important factorin the excitatory action induced by PGE2 was the ratio of [Na]0/[Ca]20. SUZUKI

and KURIYAMA (1975) confirmed this. Moreover, both PGE2 and oxytocin pro-duced depolarization of the membrane in Ca-free solution. The PGE2 actions onthe myometrium might be related to mobilization of Ca from the sequestered sitesas observed in microsomes excised from the human and bovine myometrium

(CARSTEN, 1973a, b, 1974). However, effect of oxytocin on the above mechanismhas not been examined.

Differences observed between the actions of PGE2 and oxytocin during the pro-

gress of gestation and the selectivity of longitudinal and circular muscle layers mightbe related to the hormonal state of the mouse myometrium. Concerning the rolesof progesterone during gestation, CSAPO (1963) proposed that sensitivity to oxyto-cin of pregnant myometrium increases when the concentration of oestradiol in theblood is high and is reduced when the concentrations of progesterone is high. This


was called 'Progesterone block' and could maintain pregnancy. Progesteronemight modify the actions of PGE2 and oxytocin on the myometrium, since the sensi-tivity of the myometrium is closely related to the progress of gestation (CSAPO et al.,1972, 1973). However it has also been reported in the human myometrium bySPEROFF et al.(1972) that a significant decrease in oestriol levels preceded the de-crease in oestradiol during PG infusion and no significant change in progesteroneconcentration in the blood is seen prior to abortion. On the other hand, HARBONand CLAUSER (1971) have demonstrated that PGE2 stimulates adenyl cyclaseactivity in rat uteri in vitro, indicating that PGE2 would increase CAMP in thistissue, although ROBISON (1968) reported that extracellularly applied CAMPrelaxes uterine smooth muscle rather than exciting it. Therefore, it is not yet clearwhether the action of PGE2 is mediated by CAMP.

Several differences between PGE2 and oxytocin on the pregnant mouse myo-metrium have been revealed using the electrophysiological method. However, tofully understand the actions of PGE2 and oxytocin on the myometrium, the role ofovarian and placental hormones which modify the membrane, must be clarified.

REFERENCES

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biologically active lipids. Pharmacol. Rev., 20:1-48.CARSTEN, M.E.(1973a) Prostaglandins and cellular calcium transport in the pregnant human

uterus. Am.J. Obst. Gynecol., 117:824-832.CARSTEN, M.E.(1973b) Sarcoplasmic reticulum from pregnant bovine uterus. Gynec. Invest.,

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Prostaglandins, 5:33-40.CSAPO, A.I.(1963) Model experiments and clinical trials in the control of pregnancy and parturi-

tion. Am.J. Obst. Gynecol., 85:359-379.CSAPO, A.I., MOCSARY, P., NAGY, T., and KAIHOLA, H.L.(1973) The efficacy and acceptability

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COMPARISON BETWEEN PROSTAGLANDIN E2 AND OXYTOCIN …

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