Br. J. Pharmac. (1985), 86, 899-904 Determination ofthe receptor selectivity of opioid agonists in the guinea-pig ileum and mouse vas deferens by use of 3-funaltrexamine A.G. Hayes, M.J. Sheehan & M.B. Tyers Department ofNeuropharmacology, Glaxo Group Research Ltd, Ware, Herts 1 The irreversible inhibitor of p-opioid receptor-mediated effects, P-funaltrexamine (P-FNA), was used to investigate the selectivity of various opioid agonists at gi-opioid receptors in the electrically stimulated guinea-pig ileum and mouse vas deferens preparations in vitro. 2 In the guinea-pig ileum, pretreatment with P-FNA (3 x 10-8-3 x 10-6M) produced a concentra- tion-dependent antagonism of the inhibitory effect produced by the pt-opioid receptor agonist [D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAGO). High concentrations of P-FNA (3 x 10-6_ X 10-5M) also antagonized the inhibitory effects of the c-opioid agonist U50488. 3 Pretreatment of guinea-pig ileum with P-FNA at I x 10-6M resulted in blockade of the effect of some opioid agonists. The compounds which showed the largest rightward shifts in their concentra- tion-response curves, and hence the greatest i/K opioid receptor selectivity, were nalbuphine, [D-Ser2, Leu5]enkephalinyl-Thr6(DSLET), morphine, DAGO and normophine. Responses to tifluadom, Mr 2034, ethylketocyclazocine, butorphanol, nalorphine, proxorphan and U50488 were not inhibited by P-FNA. 4 In the mouse vas deferens, pre-treatment with P-FNA (I x 10-6M) produced a similar shift in the dose-response curves for normophine as in the guinea-pig ileum. The concentration-response curves for the 6-receptor agonists [D-Ala2, D-Leu'] enkephalin (DADLE) and DSLET were, however, also shifted, indicating that P-FNA will also block 6-opioid receptors. 5 Since P-FNA does not block K-opioid receptor-mediated effects, it can be used in the guinea-pig ileum preparation as a selective is-receptor inhibitor. However, its lack of selectivity between JL- and 6- opioid receptors should be taken into account in many other isolated tissues and experiments in vivo. Introduction The alkylating analogue of naltrexone, P-funaltrex- possible to produce a guinea-pig ileum preparation in amine (f-FNA), was first synthesized by Portoghese et vitro that may be devoid of functional ti-opioid al. (1980). In isolated tissue preparations and in vivo, receptors. Furthermore, by measuring the change in P-FNA initially causes a reversible opioid agonist potency of a series of opioid agonists that occurs after effect, but on prolonged contact a selective irreversible P-FNA pretreatment, we have made an estimate of blockade of responses mediated by p-opioid receptors their relative ;./x-receptor selectivity ratio. develops (Ward et al., 1982a,b); the precise site and The mouse vas deferens is a tissue that possesses 6- mechanism ofaction ofP-FNA are uncertain. Ward et opioid receptors in addition to p- and K-opioid al. (1982a) suggested that P-FNA could thus be used to receptors (Lord et al., 1977). Similar experiments in characterize the receptor selectivity of opioid agonists this tissue were carried out to study the effect of P- by measuring the reduction in potency that occurs FNA on 6-receptors. after inhibition of the r-receptor component of their action. The guinea-pig ileum contains opioid receptors only Methods ofthe pt- and K-types (Ward & Takemori, 1976; Lord et al., 1977) and so treatment with P-FNA should leave it Guinea-pig ileum responsive only to K-opioid agonists. We have exten- ded the findings ofWard et al. (1982a) to show that it is The terminal ileum was removed from male Dunkin- ) The Macmillan Press Ltd 1985 900 A.G.HAYES et al. Hartley guinea-pigs (225-275 g), and the 15 cm sec- Laboratories; clonidine hydrochloride, Boehringer tion closest to the caecum discarded. After gently Ingelheim; dihydrocodeine bitartate, Duncan Flock- flushing out the contents, 1.5 cm sections were suspen- hart; ethylketocyclazocine methane sulphonate, Ster- ded in 5 ml organ baths containing a modified Krebs- ling Winthrop; [D-Ala2, N-MePhe4, Met-(0)-ol5]enke- Henseleit solution ofthe following composition (mM): phalin (FK 33824), Sandoz; fentanyl citrate, Janssen; NaCI 118, NaHCO3 29, glucose I 1.1, KCl 4.7, CaCI2 ketocyclazocine, Sterling Winthrop; morphine 2.6, KH2PO4 1.18 and MgSO4 4, gassed with 95% 02 hydrochloride, Macfarlan Smith; Mr 2034 ((- and 5% CO2 and maintained at 37 ± 0.5°C. Each (1 R,5R,9R)-5,9-dimethyl-2-(L-tetrahydrofuryl)-2'- section of ileum was field-stimulated by two coaxial hydroxy-6,7-benzomorphan), Boehringer Ingelheim; ring electrodes using rectangular pulses of 0.5 ms nalbuphine hydrochloride, Endo; nalorphine hy- duration at IV less than maximal voltage at a drobromide, Wellcome; naloxone hydrochloride, frequency of 0.1 Hz. Drugs were added to the bath in Sterling Winthrop; normorphine base, Wellcome; volumes of 10-50 pl. Non-cumulative concentration- pentazocine hydrochloride, Sterling Winthrop; prox- response curves were constructed for an agonist and orphan tartrate, Bristol Laboratories; [D-Ser2, the reference drug, normorphine, and an IC50 concen- Leu5]enkephalinyl-Thr6 (DSLET), Universal tration for 50% inhibition of twitch height was Biologicals; tifluadom hydrochloride, Sandoz; determined. U50488 (trans-3,4-dichloro -N-methyl-N-[2-(1-pyrro- Tissues which were to be pretreated with P-FNA llidinyl)cyclohexyl]benzeneacetamide methane sul- were incubated with it for 30 min. During this time the phonate), Upjohn; Win 42610 (D-4-(cyclopropyl- twitch height was depressed due to the agonist action methyl) - 3,4,5,6 - tetrahydro - 1,5 - methano - 1,4- ben- of f3-FNA. The tissues were then extensively washed zodiazocine-9-amine), Sterling Winthrop. with drug-free bathing medium for at least 60 min until the twitch height recovered and all non-covalent- ly bound P-FNA was removed. Agonist concentra- Results tion-response curves were then re-determined, and dose-ratios were calculated as (IC50 after P-FNA/IC50 As reported by Ward et al. (1982a), P-FNA produced a before P-FNA). reversible inhibition of evoked contractions of the For pA2 determinations, four sections of ileum guinea-pig ileum. The IC50 for this opioid agonist (normal or P-FNA-treated) from the same animal effect was 9 x 10-9M. More prolonged pretreatment were incubated with naloxone for 30 min at a range of of the ileum with ,-FNA (3 x 10-8-3 x 10-6M, concentrations, each preparation being exposed to one 30 min, followed by a 60 min period of frequent concentration only. From the dose-ratio for agonist intermittent washout) produced concentration-depen- before and after 13-FNA treatment, the pA2 was dent parallel rightward shifts of the concentration- calculated according to the method of Arunlakshana response curve for the inhibitory effect ofthe pt-opioid & Schild (1959). The statistical significance of the receptor selective agonist, DAGO, ranging from 4.7 difference between the pA2 values obtained in control fold to 42 fold (Figure 1). The response to the selective and P-FNA-pretreated tissues was determined by use K-receptor agonist, U50488, was unaffected by of Student's t test for unpaired samples. pretreatment of the ileum with concentrations of P- FNA up to I x 10-6M, but concentrations greater Mouse vas deferens than 1 x 10-6M did cause a parallel rightward shift of the dose-response curve (Figure 1). Such tissues took Vasa deferentia were taken from male Glaxo-bred up to 2 h to recover their original twitch height. CRH mice (50-100g) and suspended in Sml organ Control tissues not exposed to P-FNA showed only baths containing modified Krebs-Henseleit solution, shifts of 2 fold or less in the IC50 to either agonist over but lacking magnesium sulphate. Each vas deferens the same period. Pretreatment with P-FNA was stimulated at I V supramaximal voltage with (I X 10-6M or 1 x 10-SM) also failed to shift the trains ofthree 0.5 ms pulses 20 ms apart at a frequency concentration-response curve to the a-adrenoceptor of 0.1 Hz. Concentration-response curves were deter- agonist, clonidine. mined by cumulative dosing, and P-FNA was applied In both control and P-FNA-treated tissues, nalox- as described above for the guinea-pig ileum. one produced parallel shifts ofthe dose-response curve for DAGO. Table I shows the pA2 values for antagon- Sources of drugs ism of DAGO by naloxone in control segments of ileum and those treated with P-FNA (I x 10-6M). In [D-Ala2, D-Leu5]enkephalin (DADLE), Peninsula; [D- control tissues, the pA2 value of 8.58 was within the Ala2, MePhe4, Gly(ol)5]enkephalin (DAGO), Cam- range for antagonism of g-opioid receptors but after bridge Research Biochemicals; bremazocine hydro- treatment of the preparations with 10-6M ,B-FNA, the chloride, Sandoz; butorphanol tartrate, Bristol pA2 fell to 7.78, a value consistent with an action of RECEPTOR SELECTIVITY OF OPIOID AGONISTS 901 However, for the partial agonists DSLET, nalorphine, 40 F proxorphan and butorphanol, which did not usually cause a 50% inhibition of the twitch, the dose-ratios were calculated from parallel sections of the concen- tration-response curves. In the case of morphine and 0 30 - nalbuphine, the concentration-response curves after P- 4- FNA often had significantly shallower slopes than in () untreated tissues. The shift for nalbuphine tended to 0 be smaller in tissues in which the maximum Ua response to 20 - nalbuphine was low. The dose-ratios given in Table 2 CA are for the occasions when parallel shifts were 0 0, obtained. The effects of P-FNA were also investigated in the 10 field-stimulated mouse vas deferens preparation.