Pharmacological characterization in vitro of prostanoid receptors in the myometrium of nonpregnant ewes D. J. Crankshaw and V. Gaspar Department of Obstetrics and Gynecology, McMaster University Health Sciences Centre, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5 Prostanoid receptors regulating the contractility of strips of myometrium obtained from nonpregnant ewes during the breeding season were classified pharmacologically. Natural prostanoids, receptor-type selective synthetic analogues, and selective antagonists were used where available. The natural prostanoids PGD2, PGE2, and PGF2\g=a\were equipotent in causing contractions ( pD2 values of 6.9, 6.7, and 6.9, respectively) but were 100 times less potent than oxytocin ( pD2 = 9.2). The synthetic prostanoids iloprost (pD2 = 8.3), GR63799x ( pD2 = 7.0), cloprostenol ( pD2 = 6.8), and U46619 ( pD2 = 6.2) also caused contractions. The effects of iloprost, but not of GR63799x, were blocked by the selective EP1 antagonist AH6809. This suggests the presence of both EP1 and EP3 receptors. The similar potencies of cloprostenol and PGF2\g=a\suggest the presence of FP receptors. Although the potency of the TP agonist U46619 was relatively low, its effects were blocked by the selective TP antagonist L670596 ( pKB = 8.4), an observation consistent with the presence of TP receptors. Thus, all currently recognized excitatory prostanoid receptors (EP1( EP3, FP and TP) appeared to be present. Contractions induced by cloprostenol and KC1 could be inhibited by the ß-adrenoceptor agonist isoprenaline ( 2 = 8.S against cloprostenol) and the Ca2+-channel blocker, D600 ( pD2 = 6.3 against cloprostenol), but a number of relaxant prostanoids, BW245c, ZK110841, AH13205 and cicaprost, could not produce inhibition. These results suggest that DP, EP2 and IP receptors do not regulate contractility under these conditions. Introduction between 15 September and 15 December over three successive seasons, from crossbred yearling ewes or ewe lambs weighing and that a Novy Liggins (1980) suggest prostanoids play more than 40 kg. The tracts were placed in ice cold physiologi¬ role in the of uterine activity in a cal salt solution physiological regulation (PSS) of the following composition (mmol 1 ~ ): number of species. In ewes, work aimed at delineating this role potassium chloride 4.6, magnesium sulfate 1.16, sodium dihy- has focused, almost exclusively, on alterations in prostanoid drogen phosphate 1.16, calcium chloride 2.5, sodium chloride concentrations in various compartments during different phases 115.5, sodium bicarbonate 21.9 and glucose 11.1 with of the (Olson et al, 1986). Scant attention reproductive cycle indomethacin at 10 pmol \~ , and transported to the labora¬ has been to the actions of these at the paid compounds tory. In the laboratory, the ovaries were inspected for indi¬ putative end-organ. cations of reproductive status. Only uteri from animals with an In humans et the direct (Senior al, 1991, 1992, 1993), effects active corpus luteum, or large fluid-filled follicles accompanied of on uterine are mediated prostanoids contractility through by a recently regressed corpus luteum in at least one ovary, actions at seven different some of which receptors, are excit¬ were included in the study. Results from uteri from 45 animals others In the atory, inhibitory. ewes, however, prostanoid that met these criteria are reported. None of these reproductive status of the is not known. receptor myometrium tracts showed any indication of trophoblastic activation. The In the the status of present study, prostanoid receptor the uteri were washed with oxygenated (95% 02:5% C02) PSS at myometrium of nonpregnant ewes during the breeding season room temperature, cleared of any adhering fat and connective was determined pharmacologically by studying the effects of tissue, and eight longitudinal strips of myometrium approxi¬ prostanoid receptor-selective analogues on the contractility of mately 1.5 cm in length and 0.04 cm2 in cross-sectional area the myometrium in vitro. were cut from the body of the uterus. Materials and Methods Recording of isometric contractions Collection of myometrium General Tissue strips were tied at each end with tracts were obtained from two abattoirs in the protocol. Reproductive silk thread and mounted longitudinally in individual 10 ml of Hamilton, Ontario (43°15'N). They were collected vicinity jacketed muscle baths containing oxygenated PSS at 37°C. One Received 25 July 1994. end of each strip was anchored in the bath; the other was Downloaded from Bioscientifica.com at 09/23/2021 06:14:38PM via free access Table 1. Composition, concentration and sources of compounds used Compound Stock solution Source 3 17-phenyl trinor PGE2 10 g ml 'in absolute ethanol Cayman Chemical, Ann Arbor, MI 3 ' AH6809 2.5 10 ml" in 1% (w/v) Glaxo Ware " g NaHC03 Group Research, AH13205 10 2 ml in 1% Glaxo Research - (w/v) g - NaHC03 Group BW245C 10 2 mol 1 1 in absolute ethanol Wellcome Research Beckenham ~ ~ Laboratories, BW A868c 10 2 mol 1 in absolute ethanol Wellcome Research Laboratories ~~ ~ Carbachol 10 1 mol 1 ' in 0.9% saline Chemical Co., St Louis, MO " (w/v) " Sigma 10 3 ml l in 0.9% saline ICI Park Cloprostenol ~ g ~ (w/v) Pharmaceuticals, Alderley 5 2 5 10 ml in solution AG, Berlin Cicaprost ~ g ~ aqueous Schering D600 10 3 mol 1 in 0.9% saline Chemical Co. " " (w/v) Sigma GR63799X 10 2 ml ' in absolute ethanol Glaxo Research ~ ~ g Group 10 5 ml ' in solution AG Iloprost ~ g ~ aqueous Schering 3 : Indomethacin 5 10 mol " Na2C03 Sigma 10 2 mol 1 1 in 0.9% saline and Isoprenaline ~ ~ (w/v) Sigma ascorbate L670596 10 3 ml l in DMSO Merck Frosst Centre for " g " Therapeutic Research, Pointe Clare, PQ 10 'gmr1 in absolute ethanol G. D. Searle, Skokie, IL Misoprostol 1 10 mol 1 l in 0.9% saline Tucson, AZ Oxytocin " (w/v) Vega, 10 1 mol 1 in absolute ethanol Chemical PGD2 ~ Cayman 10 1 mol 1 in absolute ethanol Chemical PGE2 ~ Cayman PGF2a tromethamine salt 10" 1 mol 1_ 1 in 0.9% (w/v) saline Cayman Chemical [0 ' ml ' in solution AG Sulprostone g ~ aqueous Schering U46619 10 ml J in acetate Chemical g ~ methyl ' Cayman 3 : ZK110841 1.25 10 g ml in absolute ethanol Schering AG attached to an FT-03 force displacement transducer writing to force developed in response to that concentration of agonist. a 7D polygraph (Grass Instruments, Quincy, MA). An initial Additions were continued until further addition produced no resting force of 40 mN was applied to each tissue. This usually further increase in developed force. Concentration—effect declined rapidly and was readjusted throughout the next hour curves (response versus log molar agonist concentration) were to obtain a steady baseline force of 25 mN. Throughout this then constructed from these data by fitting the equation period, the bath solution was changed regularly. Tissues were E = E + Œ E Vi + e k('08C "logD) " then with chloride mmol 1 - challenged potassium (90 ~ ). Any strip that failed to develop force in response to this challenge where E is the effect of the agonist, C is the molar concen¬ was discarded. Potassium chloride was washed from the bath tration of the agonist and D is the molar concentration of and were allowed for a further hour. The tissues to equilibrate the agonist that produces a half maximal response (£C50). mean force developed by the individual muscle strips during The value of logD is equivalent to the negative logI0 of 10 min was used as a measure of their contractility. This the molar concentration— of the agonist that produces 50% of technique has been used routinely in this laboratory (Dyal and the maximal response, pD2 values. Wainman and others Crankshaw, 1988; el al, 1988) (Cheuk Normally the same agonist was tested on two muscle strips el al, 1993), since it can be used to quantify drug effects in from the same animal and the average of the two pD2 values tissues that develop significant spontaneous activity and obtained was used. Thus values represent the number of respond to stimulation by changes in both tonic and phasic animals on which each drug was tested. activity (Crankshaw, 1990). Mean force was determined exactly as described by Wainman et al (1988). Concentration-effect curves to inhibitory agonists most tissues contractile Concentration-effect curves to excitatory agonists Although developed spontaneous activity during the equilibration period, this activity decayed At the end of the equilibration period, the mean force with a time course that was highly variable, particularly developed during a 10 min control period was determined. between animals, but also between strips from the same animal. Drugs were then added to the baths, in a cumulative fashion, This made it difficult to obtain concentration-effect data for by increments that would produce approximately one half log inhibitors. To remedy this problem, strips were stimulated in unit changes in the concentration in the bath. After each two different ways, and the effect of drugs on this stimulated addition, the mean contractile force was determined for 10 min. activity was examined. At the end of the equilibration period, The mean force recorded in 10 min immediately after agonist the PSS was changed to a solution that contained either 1 addition minus the mean control force was considered to be the 40 mmol potassium chloride 1~ or 2 pmol cloprostenol I-1. Downloaded from Bioscientifica.com at 09/23/2021 06:14:38PM via free access Effects of antagonists 50 mN In attempts to delineate the site of action of a number of excitatory agonists, experiments were performed to investigate the effects of selective receptor antagonists on their action. The as described above was used, that four —10 min general protocol except tissue strips from each animal were used.