Characterisation of opioid receptors involved in modulating circular and longitudinal muscle contraction in the rat ileum

Abstract

1. The aim of the present investigation was to characterise the opioid receptor subtypes present in the rat ileum using a method that detects drug action on the enteric nerves innervating the circular and longitudinal muscles. 2. Neurogenic contractions were reversibly inhibited by morphine (circular muscle pEC50, 6.43+/-0.17, Emax 81.7+/-5.0%; longitudinal muscle pEC50, 6.65+/-0.27, Emax 59.7+/-7.8%), the mu-opioid receptor-selective agonist, DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin acetate) (circular pEC50, 7.85+/-0.04, Emax 97.8+/-3.6%; longitudinal pEC50, 7.35+/-0.09, Emax 56.0+/-6.1%), the delta-selective agonist DADLE ([D-Ala2,D-Leu5]enkephalin acetate) (circular pEC50, 7.41+/-0.17, Emax, 93.3+/-8.4%; longitudinal pEC50, 6.31+/-0.07, Emax 66.5+/-5.2%) and the kappa-selective agonist U 50488H (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulphonate) (circular pEC50, 5.91+/-0.41, Emax, 83.5+/-26.8%; longitudinal pEC50, 5.60+/-0.08, Emax 74.3+/-7.2%). Agonist potencies were generally within expected ranges for activity at the subtype for which they are selective, except for U 50488H, which was less potent than expected. 3. The mu and delta receptor-selective antagonists, CTAP (H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) and naltrindole, caused progressive, parallel rightward shifts in the DAMGO and DADLE curves, respectively. Analysis indicated conformity to theoretical simple competitive antagonist behaviour. U 50488H effects were insensitive to the kappa-selective antagonist, n-BNI. A high concentration (1 microM) of naltrexone caused apparent potentiation of U 50488H effects. 4. CTAP pK(B) estimates were consistent with previously reported values for mu receptor antagonism (circular 7.84+/-0.17, longitudinal 7.64+/-0.35). However, the naltrindole pK(B) estimates indicated lower antagonist potency than expected (circular 8.22+/-0.23, longitudinal 8.53+/-0.35). 5. It is concluded that mu and possibly atypical delta receptors (but not kappa receptors) mediate inhibition of contraction in this model. Nonopioid actions of U 50488H are probably responsible for the inhibitory effects seen with this compound.

Figure 1

(a) Representative trace of the electrically evoked contractions of the rat ileum, longitudinal muscle (top) and circular muscle (bottom). This example shows the graded inhibition of contractile responses to transmural stimulation (8 s trains of 1 ms pulses at 10 Hz delivered every 3 min) caused by increasing concentrations of DAMGO.

Figure 2

The concentration–response relationship of DAMGO, and DAMGO in the presence of various concentrations of CTAP in circular (a) and longitudinal (b) smooth muscle of the rat ileum. Nonlinear curve fits to pooled data are shown, with common height and slope constraints. The Clark plot (c) indicates the effect of increasing antagonist (CTAP) concentrations on the potency (log EC₅₀) of DAMGO in the different muscle layers. Error bars represent the mean±s.e.m. of four to 12 determinations per point. The lines are plots of DAMGO log EC₅₀=log([CTAP]+K_B)+log c, for circular and longitudinal muscle, representing the ‘theoretical lines' for simple competitive antagonism. The proximity of the lines reflects the similarity of the differences between the antagonist pK_B and agonist control curve pEC₅₀, for the different muscle layers.

Figure 3

The concentration–response relationship of DADLE, and DADLE in the presence of various concentrations of naltrindole in circular (a) and longitudinal (b) smooth muscle of the rat ileum. Nonlinear curve fits to pooled data are shown, with common height and slope constraints. The Clark plot (c) indicates the effect of increasing antagonist (CTAP) concentrations on the potency (log EC₅₀) of DADLE in the different muscle layers. Error bars represent the mean±s.e.m. of four to 12 determinations per point. The lines are plots of DADLE log EC₅₀=log([naltrindole]+K_B)+log c, for circular and longitudinal muscle, representing the ‘theoretical lines' for simple competitive antagonism. The proximity of the lines reflects the similarity of the differences between the antagonist pK_B and agonist control curve pEC₅₀, for the different muscle layers.

Figure 4

The concentration–response relationship of U 50488H and in the presence of various concentrations of n-BNI in circular (a) and longitudinal (b) smooth muscle of the rat ileum. Nonlinear curve fits to pooled data are shown, with common height and slope constraints. The lack of effect exerted by n-BNI on the control curve suggests a nonopioid mechanism of U 50488H action.