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.