Peripheral excitatory effects of two enkephalinase inhibitors, acetorphan and thiorphan, and an enkephalin analogue, [d-Ala2\p=n-\Met5]-enkephalinamide, on uterine motility in periparturient rats in vivo and in vitro O. Adjroud Laboratory of Feto—Maternal Physiology, University of Rouen, 76 30 Mont Saint Aignan, France

The effects of two enkephalinase inhibitors, acetorphan and thiorphan, and the enkephalin analogue [d-Ala2\p=n-\Met5]-enkephalinamide (DAMEA), on spontaneous uterine contractions were studied at day 21 of pregnancy in rats following treatment in vivo or in vitro. Acetorphan (10 mg kg\m=-\1)and thiorphan (1 mg kg\m=-\1),immediately after their i.v. administration, increased the duration of spontaneous contractions 3.4- and 4.6-fold, respectively, but did not modify the maximum amplitude. Similarly, thiorphan (40 \g=m\moll\m=-\1) increased the duration of contractions when administered in vitro. Thiorphan was ineffective during the first 30 min when given into the cerebral ventricles (50 \g=m\gper rat). These results suggest that the enkephalinase inhibitors are acting via a peripheral opioid pathway; and this conclusion is supported by the observation that thiorphan potentiated the stimulatory effect of a submaximal dose of DAMEA administered in vitro. The excitatory effects of DAMEA and the enkephalinase inhibitors were blocked by naloxone. This antagonistic effect of naloxone on uterine motility in the periparturient rat uterus, induced by either acetorphan and thiorphan or DAMEA, seems to be regulated by peripheral opiate receptors. Naloxone (10 mg kg\m=-\1 s.c.) increased both the amplitude and duration of uterine motility in vivo; however, naloxone (26 \g=m\moll\m=-\1 and 52 \g=m\moll\m=-\1) produced a paradoxical dose-dependent biphasic effect in vitro.

Introduction in the regulation of uterine motility. However, little informa¬ tion is available concerning the action of enkephalin on the Opioid peptides are involved in maternal adaptation to preg¬ uterine muscle in the last stage of pregnancy. The present nancy and in uterine motility. In pregnant women, plasma study was therefore undertaken to investigate the response of concentrations of ß-endorphin rise throughout pregnancy the rat myometrium to met-enkephalin on day 21 of pregnancy (Genazzani et al, 1981), and reach peak values during labour and the possible involvement of enkephalinase in this response, (Facchinetti et al, 1982; Genazzani et al, 1985). This increase in using a long lasting enkephalin analogue [D-Ala2—Met' ]- opioid peptide concentrations during labour correlates in a enkephalinamide (DAMEA) and two enkephalinase inhibitors, linear fashion with the number and density of uterine con¬ acetorphan and thiorphan. Acetorphan, a lipophilic derivative tractions (Facchinetti et al, 1982) and with response to the of thiorphan enters into the brain, where it generates thiorphan painful stimuli typical of this condition (Genazzani et al, 1985). (Roques et al, 1980, 1981). When administered in low parental Furthermore, met-enkephalin concentrations increase during doses to rats, it inhibits the cerebral enkephalinase, producing pregnancy and delivery in several brain regions of pregnant more effects than does intracerebral thiorphan (Lecomte et al, rats (Petraglia et al, 1985) and in the human placenta during 1986). Thiorphan penetrates poorly into the brain and elicits labour (Sastry et al, 1980). Similarly, opiate receptors and mainly peripheral effects. Its nanomolar potency in vitro con¬ enkephalinase, an enkephalin-degrading-enzyme at the Gly trasts with the large parental doses required to inhibit the rat - Phe bond (Malfroy et al, 1978) are present in the pregnant rat and mouse cerebral enzyme (Roques et al, 1980; Chipkin et al, uterus (Baraldi et al, 1985; Ottlecz et al, 1991) and in the 1982; Hachisu et al, 1985). In addition to their analgesie human placenta (Valette et al, 1980; Johnson et al, 1984). properties, these inhibitors partially protect the endogenous These data suggest that met-enkephalin could be involved not met-enkephalin release from brain slices and from being exten¬ only in the modulation of analgesia during pregnancy but also sively hydrolysed by enkephalinase (Patey et al, 1981; De La Baume et al, 1983) and increase the half-life of [3H]enkephalin et Central and effects of these *Present address for correspondence: Laboratory of Animal Physiology, (Chaillet al, 1983). peripheral University of Batna 0.5000, Algeria. inhibitors were assessed by comparing the stimulatory actions Revised manuscript received 9 January 1995. of i.V. acetorphan and thiorphan, or thiorphan given i.V., i.e.v.

Downloaded from Bioscientifica.com at 09/27/2021 03:19:58PM via free access or in vitro. The possible involvement of enkephalinase in the equilibration for about 60 min. A 15 min recording period with modulation of uterine was studied motility by adding DAMEA sterile saline was used as a control for subsequent 15 min to the isolated tissue and attempting to protect this peptide exposures to active agents. In the bathing medium, the strip with thiorphan. Naloxone, an opiate receptor antagonist, was was washed between the addition of different drugs for at least used to block the effects induced by both inhibitors and 15 min and allowed to return to a stable baseline. DAMEA. Treatment regimens Materials and Methods Acetorphan and thiorphan, synthesized as described by Roques et al (1980, 1981), were obtained from Bioprojet Animals Labs (Paris). Naloxone and [D-Ala -Met5]-enkephalinamide (DAMEA) were purchased from Chemical Adult Wistar rats (Charles River Labs, St Aubin-les-Elbeuf) Sigma Company (St Louis, MO). Thiorphan, naloxone and DAMEA were were kept in a lighting schedule of 12 h h dark at light:12 dissolved in sterile saline and the pH was adjusted, when 23 ± 1°C with free access to food (U.A.R.103, Villemoison sur necessary, to 7.4. Acetorphan was dissolved in a vehicle and water. Females were Orge) (250-350 g body mass) caged composed of 10% ethanol, 10% Cremophor (Bioprojet Labs), with males and the smear examined for the overnight vaginal and 80% water, pH 7.4. presence of spermatozoa. The day on which spermatozoa were found in the smear was designated day 1 of pregnancy and Experiments in vivo. Thiorphan was given i.v. at 0.15, 1 animals were used at day 21. The average duration of gestation ~ ' and 5 mg kg body mass in 0.3 ml or i.c.v. at 50 µg in 20 µ . in the breeding colony was 22 days. was i.v. at 5 or 10 in ml. Acetorphan given mg kg ~ 0.3 Naloxone was used to block the effects of the enkephalinase inhibitors. Naloxone was s.c. or 10 Preparation in vivo given at 5 mg kg" body mass in 0.3 ml, 10 min before i.v. thiorphan or acetorphan. This Each animal was anaesthetized with sodium route and these doses of naloxone block the analgesic effects of ~ ' pentobarbital (45 mg kg body mass, i.p.; Abbott Laboratories, St Remy sur acetorphan and thiorphan i.v. in rat and mice nociceptive tests. Avre) and the trachea was cannulated to assist respiration. The. (Chaillet et al, 1983; Costentin et al, 1986; Lecomte et al, 1986; abdomen was opened using a midline incision and the medial Hachisu et al, 1985). part of one uterine horn was linked to an isometric strain gauge connected to an ink recording polygraph. The uterus was Experiments in vitro. The met-enkephalin analogue, secured to the strain gauge by a cotton thread passed between DAMEA, was used to reveal the presence of enkephalinase in the myometrium and the blood vessels of the mesometrial the rat uterus. DAMEA was used at i.7, 7.5 and 14.8 1 pmol " 1. end membrane. The cervical of the of the uterus that was at 10, 20 and 40 1 , was or part Thiorphan µ ~ used alone in used for recording was secured to a vertical fixed metal rod to combination with DAMEA (13 µ 1" :). Naloxone at 13, 26 : maintain isometric conditions. A standard tension of 1 was and 52 was or g µ 1 ~ used alone in combination with applied to each uterine horn and care was taken to maintain thiorphan (40 µ l-1) plus DAMEA (13 µ I"1). irrigation by dripping Tyrode's solution (pH 7.4; 36°C) down the outside of the horn. This arrangement records isometric contractions primarily of the longitudinal muscle layer. The Measurement of the spontaneous uterine contractions system was allowed to stabilize for 60 min before recordings Variations in myometrial mechanical activity were evaluated were taken for 30 min to a control value for provide compari¬ in terms of the duration and amplitude of contractions. The son with treatments. The vehicles in which the active agents maximal amplitude (mm) of each contraction was measured and were dissolved were administered the route and by appropriate the duration (s) was taken as the interval between successive in the volume at the start of the 30 min control appropriate relaxations. The amplitude and duration of each contraction Animals were then treated with the active and period. agents during the control were calculated. of 6-13 taken for 30-60 min. period Groups recordings animals were assigned to each treatment in vivo, and each treatment in vitro was performed on at least 6-7 separate preparations. Each recording of the treatment in vivo lasted Isolated preparation in vitro 30-60 min. This time was divided into 10 min periods. The means sem the Each animal was killed with an overdose of pentobarbital ± of amplitude and duration of contractions of each and the uterine horn was split longitudinally. A 40 mm group of treated animals were calculated at 10 min 5 mm uterine strip was removed, as described by Singh and periods during 30-60 min of recording. In the recording Sharma (1984). The endometrium was removed by gentle but in vitro, these parameters were calculated during separate thorough scraping. The uterine strip was then washed several 15 min experiments. times with Krebs' solution (pH 7.4) at 4°C and mounted in a tissue chamber filled with 10 ml of Krebs' solution maintained Statistical at 36°C and gassed with 95% 02:5% C02. Spontaneous analysis isometric contractions (0.25 g resting tension) were recorded Data for each group of 6—13 rats were analysed by analysis with a Gilson transducer (ICT 2H, Villiers-le-Bel) after of variance and expressed as means ± sem and are represented Downloaded from Bioscientifica.com at 09/27/2021 03:19:58PM via free access in Tables 1 and 2. Data from the treated group mean and its cantly increased the duration of contractions by 73 and 220%, own control were examined by Student's t test and differences respectively. were considered to be if P< 0.05. no on or significant Naloxone, 13 µ 1 ~ \ had effect the amplitude duration of uterine but 26 1 contractions, naloxone, µ ~ \ Results significantly increased both the amplitude (44%) and duration (53%) of contractions, whereas 52 µ naloxone I-1 signifi¬ Experiments in vivo cantly decreased both the amplitude {56%) and duration (47%) of contractions with the ~ 1 compared pretreatment period. naloxone, 5 Effect of Naloxone, mg kg body mass (s.c), Treatment with the met-enkephalin agonist, DAMEA, at the did not induce in the or lowest 3.7 any significant change amplitude concentration studied, µ 1~ , which had no duration of uterine contractions over 30 Nalox¬ min (Table 1). significant effect on the duration of uterine contractions when 10 induced an one, mg kg ~ body mass, immediate, significant given alone, augmented the increase in the duration of uterine which increase (about 25%) in the amplitude of contractions, contractions seen with the enkephalinase inhibitor thiorphan at was maintained over 60 min and a increase (40- 20 and 40 1 did not alter of significant 10, µ ~ \ but the effect in the duration of contractions from 10—50 min 50%) after thiorphan on the amplitude of contractions. DAMEA was administration. added to the bathing medium 3 min after the addition of thiorphan. alone or in combination with naloxone. The addition of the Effects of acetorphan I enkephalin antagonist naloxone, mass no 5 _ mg (i.V.), had 26 1 , Acetorphan, kg body significant pmol ~ 5 min before the enkephalinase inhibitor thior¬ effect on the or uterine contractions over duration of at the dose 1 , inhibited amplitude phan highest studied, 40 µ ~ the 30 min. The dose no higher (10 mg kg-1 body mass) had increase in the duration of contractions seen with thiorphan effect on amplitude but induced a significant increase (344%) in alone. Treatment with naloxone also inhibited the increase in the duration of contractions during the first 10 min after the duration of contractions seen with the combination of administration. The duration of contractions had returned to thiorphan and DAMEA, although the highest concentration of the min This values 20—30 1 , was this pretreatment by naloxone, 52 ~ to exert samplingJ period. µ required inhibitory effect was not seen when 5 mass was mg naloxone kg ~ body effect and this dose of naloxone was inhibitory when given given 10 min before acetorphan. The vehicle in which acetor¬ alone. phan was dissolved had no effect on the amplitude or duration of uterine contractions over 30 min (data not shown). Discussion Effects of thiorphan alone or in combination with naloxone. It has been established that the control of myometrial activity and 1 Intravenous injection of thiorphan, 0.15 mg kg-1 body during pregnancy is largely humoral and depends on intrinsic no on the mass, had significant effect amplitude of uterine myogenic factors. Thus, some components are designed to contractions 5 a but mg thiorphan kg ~ induced significant keep the myometrium at rest and others to stimulate motility the increase (about 30%) in amplitude of contractions for the (Fuchs and Fuchs, 1984). Studies of the actions of opioids on first 30 min of the 60 min observation period. All doses of the regulation of myometrial motility have been contradictory. thiorphan significantly increased the duration of uterine con¬ Some investigators have shown that morphine inhibits the an immediate tractions; 5 mg thiorphan kg produced tetanic contractions of nonpregnant rat and mouse uteri (Widy- contraction which lasted the entire 60 min observation period. Tyszkiewicz et al, 1978; Contreras et al, 1982; Acevedo and Pretreatment with 10 mg naloxone kg ~ reduced the utero- Contreras, 1984). Others found that this opioid had no effect

tonic effect of 1 . Pretreatment with mg ~ 5 mg on rat uteri at et 1979) and thiorphan kg parturition (Yaksh al, ß-endorphin naloxone was ineffective not kg ~ (data shown). caused only a small contraction of the postpartum rat uterus Thiorphan, 50 µ% per rat Lev., induced a significant increase (Leng et al, 1985). The results reported here present the effects in from 20 min (25—35%) the amplitude of uterine contractions of enkephalinase inhibitors and a met-enkephalin analogue after administration to the end of the 60 min observation upon spontaneous uterine mechanical activity of Wistar rats period and a significant increase (33-60%) in the duration of at day 21 of pregnancy (one day before parturition). contractions from 30 to 60 min. Met-enkephalin and enkephalinase inhibitors, that is, acetor¬ phan and thiorphan, stimulated the duration and, to a lesser Experiments in vitro extent, the amplitude of spontaneous uterine contractions. This result is in accordance with the action of enkephalin upon Effects of thiorphan, DAMEA and naloxone. Thiorphan, contractions of rat uteri at metoestrus (Ohia and Laniyonu, ~ 10 µ 1 \ had no significant effect on the amplitude of 1989). When enkephalinase inhibitor was given i.c.v. to the uterine contractions the 15 min observation, whereas 20 in vivo its effects were delayed with ~1 during preparation, compared and 40 µ 1 significantly increased the amplitude of the effects following i.v. administration and its potentiation uterine contractions by 33 and 47%, respectively (Table 2). The of the effects of DAMEA in the in vitro preparation. This highest concentration, 40 µ 1 , also significantly increased finding suggests that the inhibitor was acting only when it had the duration of contractions (262%). reached the periphery via the enkephalinase system. The DAMEA at concentrations of 3.7, 7.4 and 14.8 µ 1_1 pregnant rat uterus is known to contain higher concentrations had no significant effect on the amplitude of contractions but of enkephalinase than does the nonpregnant uterus (Ottlecz

the two 1 , this of higher concentrations, 7.4 and 14.8 µ ~ signifi- et al, 1991). However, high concentration enkephalinase Downloaded from Bioscientifica.com at 09/27/2021 03:19:58PM via free access o 00 O O O in -o o in IN 3 o - G +1 +! +1 +1 +1 +1 +1 +1 +1 +1 +1 O O oo t>. o o o o o O q in! ö uS IN LTl IS 00

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O a· O in re . a. — -C cu Cu J » 60 c re cu pL SP o -C 60 J= 60 2 oo j^ , O 2 Oh O M X cu fr 60 > 00 o m S « S 3- "re_0 li5 g m 9 V te re ° § , <2 2 m z2 Ho Hh Downloaded from Bioscientifica.com at 09/27/2021 03:19:58PM via free access Table 2. Effects of thiorphan, [D-Ala —Met ]-enkephalinamide (DAMEA) and naloxone administered alone or in combination on maximal amplitude (A; mm) or duration (D; s) on spontaneous uterine contractions in periparturient rats in vitro

Control Treatment Number of Agent used experiments 15-0 min

Thiorphan A 26.6 ± 2.3 28.2 ± 1.0 35.5 ± 3.7* 39.2 ± 4.6* D 49.0 + 16.4 54.2 ± 9.0 52.0 ± 2.5 177.5 ± 2.5** DAMEA A 27.0 ± 0.4 25.2: 3.3 28.1 ± 1.4 27.1 ± 3.5 D 72.8 ±27.0 91.8: 12.0 125.9 ± 12.0* 232.8 ± 54.0** Naloxone A 34.1 ± 2.5 35.5 ± 3.4 49.1 ± 4.1* 15.0: 3.5* D 49.0+ 5.9 50.0 ± 2.5 75.0 ± 3.5* 25.8: 2.1" Thiorphan (1, 2, 4) A 22.3 ± 2.7 25.0 ± 2.0 24.7 ± 2.9 30.9 ± 1.6 + DAMEA (1) D 64.5 ± 16.0 162.6 ± 35.0 380.3 ± 94.1 848.0 ±172.7 Naloxone (2) A 29.3 ± 4.4 39.0 ± 10.0 + thiorphan (4) D 48.9 + 16.0 72.4 ± 18.0 Naloxone (2) A 22.3 ± 2.7 17.0: 2.6 + thiorphan (4) D 64.6 ±16.0 27.3: 6.0 + DAMEA (I)

Each value of amplitude and duration represents the mean ± sem of 6-7 animals per group. Each drug was given in increasing concentrations and each concentration was a factor of 2. 20 and 40 µ 1 DAMEA 7.4 and 14.8 mol 1 ') and naloxone (13, 26 and 52 mol 1 '). (3.7, ~ (10, '), ~ successively multiplied by Thiorphan " *P< 0.05, **P< 0.01 compared with the control value (Student's f test). declined 1 day before parturition (Ottlecz et al, 1991), suggest¬ scious sheep (Maas and Leek, 1985). The mechanism by which ing that during pregnancy, it may contribute to the prevention naloxone stimulates uterine motility is not clear. Its excitatory of parturition by regulating the activity of uterine motility by effect could be explained by the fact that it is an opioid inactivating enkephalin and other circulatory regulatory pep- antagonist that crosses the blood—brain barrier easily (Calligan tides that have an important role in uterine motility, such as and Burks, 1983) and acts on other components involved in the oxytocin (Adjroud, 1988; Ottlecz et al, 1991), which is also a excitation of periparturient rat uterus such as oxytocin. This substrate for enkephalinase (Johnson et al, 1984). The pregnant hypothesis is supported by the finding of Hartman et al (1984) rat uterus also contains large amounts of mRNA encoding that plasma concentrations of oxytocin, measured during proenkephalin (Jin et al, 1988), which appears to increase in the parturition, are raised in rats treated chronically with naloxone, mouse uterus during pregnancy (Rosen et al, 1990), but the and the observation that the uterotonic effect of oxytocin is mechanism by which enkephalins act on uterine muscle is not potentiated in the presence of naloxone (Adjroud, 1988). In known. Enkephalin stimulates the amplitude of contractions contrast to experiments in vivo, naloxone showed a biphasic in cultured chick embryo heart muscle cells by stimulating effect on preparations in vitro. At a lower dose it exhibited a adenylate cyclase and increasing cAMP concentrations stimulatory effect and at a higher dose it was inhibitory. Similar (Laurent et al, 1986), and exerts a positive inotropic effect by paradoxical effects of naloxone are reported from analgesic augmenting Ca2+ influx (Laurent et al, 1986). Enkephalin has tests in mice (Noble et al, 1994). been shown to reduce adenylate cyclase activity in rat vas The results of present study suggest that the uterotonic deferens (Bhoola and Pay, 1986). Naloxone acts as an antago¬ effects of enkephalinase inhibitors and met-enkephalin in the nist of enkephalin in the brain (Otero et al, 1993; Coffield and periparturient uterus are due to the presence of specific local Miletic, 1993) and the periphery (Maas and Leek, 1985). In the enkephalin receptors that modulate myometrial activity. These present study, naloxone reduced the effects of enkephalinase local enkephalinergic receptors can be blocked by naloxone and inhibitors and the met-enkephalin analogue on the motility of by part of a local enkephalinergic system, which remains to be the periparturient uterus in vivo and in vitro, suggesting that defined. opioid peptides act locally in the uterus via opioid receptor systems, which have been identified in the pregnant rat uterus This work was carried out in the Laboratory of Feto-Maternal of The author thanks (Baraldi et al, 1985). This antagonistic effect of naloxone is Physiology, University Rouen, France. M. Clabaut for provision of laboratory facilities. in agreement with other results obtained on the motility of isolated rat and mouse vas deferens (Aoki et al, 1986; Lingyuan et al, 1986), isolated dog intestine (Burks et al, 1982) and References in vivo sheep reticulo-rumen (Maas and Leek, 1985). However, naloxone ~ alone increased the duration (10 mg kg ) markedly Acevedo CG and Contreras E (1984) Effects of morphine on the responses of and amplitude of uterine contractions in rats. Similar excitation acetylcholine and adrenaline in the mouse uterus Cellular and Molecular by naloxone is observed in reticulo-ruminal motility in con- Biology 30 239-242

Downloaded from Bioscientifica.com at 09/27/2021 03:19:58PM via free access Adjroud 0 (1988) Identißcation d'une Réactivité Enképhalinergique du Myomètre Laurent S, Marsh JD and Smith TW (1986) Enkephalins increase cyclic adenosine chez la Rate Préparturiente. interaction des Enképhalines avec Quelques Facteurs de monophosphate content, calcium uptake, and contractile state in cultured Régulation de la Motricité Utérine. 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