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. 2005 Jan 24;144(5):687–694. doi: 10.1038/sj.bjp.0706107

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

Andrew C Gray 1, Paul J White 1, Ian M Coupar 1,*
PMCID: PMC1576049  PMID: 15678085

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 μ-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 δ-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 κ-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 μ and δ 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 κ-selective antagonist, n-BNI. A high concentration (1 μM) of naltrexone caused apparent potentiation of U 50488H effects.

  4. CTAP pKB estimates were consistent with previously reported values for μ receptor antagonism (circular 7.84±0.17, longitudinal 7.64±0.35). However, the naltrindole pKB estimates indicated lower antagonist potency than expected (circular 8.22±0.23, longitudinal 8.53±0.35).

  5. It is concluded that μ and possibly atypical δ receptors (but not κ receptors) mediate inhibition of contraction in this model. Nonopioid actions of U 50488H are probably responsible for the inhibitory effects seen with this compound.

Keywords: μ-Opioid receptor, δ-opioid receptor, κ-opioid receptor, rat small intestine, circular smooth muscle, longitudinal smooth muscle

Introduction

The inhibitory effects of opioid drugs on gastrointestinal transit in the rat have been the subject of extensive research. However, only a limited number of reports have been made regarding the muscle contractility changes that give rise to these effects. In particular, effects on circular muscle have received very little attention. Circular muscle contractility plays a dominant role in segmentation and peristaltic propulsion in the gut (Kosterlitz & Lees, 1964), and its sensitivity to many drugs has been shown to differ from that of longitudinal muscle contractility (Brownlee & Harry, 1963; Coupar, 1999).

The antitransit effects of opioid drugs arise from changes in both motility and secretion resulting from their activation of opioid receptors located in the gut wall (Bianchi et al., 1982; 1983). Immunohistochemical studies have revealed that the opioid receptor subtypes, μ, δ and κ, are present in neural tissue of the rat enteric nervous system (ENS), but not in smooth muscle cells (Sternini et al., 1995; Bagnol et al., 1997; Gray et al., unpublished observations). Although transit arrest is a common effect of opioids in mammals, underlying secretomotor changes appear to vary between species (Miller & Hirning, 1989). In the rat, the issue has been complicated by reports of differing tissue responses under different experimental conditions (Burks, 1976; Coupar & De Luca, 1994); studies have examined drug effects on unstimulated tissues, as well as tissues stimulated exogenously with electrical pulses. Offsetting this, efforts to study opioid and opioid receptor pharmacology have capitalised on the availability of highly subtype-selective agonists and antagonists.

With respect to transit in rats, in vivo studies employing the charcoal meal method have indicated that μ and δ receptor activation causes transit slowing, but κ receptor activation has little or no effect (Tavani et al., 1984; 1990; La Regina et al., 1988). With regard to contractility, an in vivo study using anaesthetised rats indicated that morphine caused transient tonic and phasic increases in small intestine intraluminal pressure, which was presumably due to circular and/or longitudinal muscle contraction (Burks, 1976). In apparent contrast, an in vitro study indicated that both μ and δ receptor activation had an inhibitory influence on the peristaltic reflex of the rat ileum (Coupar, 1995). In vitro studies using electrical stimulation have similarly identified an inhibitory influence of δ receptors (but not of μ receptors), on longitudinal muscle contractions in the rat jejunum (Coupar & De Luca, 1994; Hancock & Coupar, 1994).

The primary aim of this study was to examine the effects mediated by μ, δ and κ opioid receptor subtypes on electrically stimulated neurogenic circular muscle contractions in the rat ileum. The in vitro method used in this study also allows for simultaneous measurement of drug effects on the longitudinal muscle (Coupar & Liu, 1996). Therefore, we have taken this opportunity to reinvestigate whether functional μ or κ opioid receptors influence longitudinal muscle contractility, using agonists and antagonists with greater subtype-selectivity than have been used previously. A basic account of these findings has been published as a conference abstract (Gray et al., 2004).

Methods

Animals

Tissues were obtained from male and female Hooded Wistar rats (200–500 g) and adult male tricolour guinea pigs (approx. 400 g). The animals were maintained under a 12-h light/dark cycle, and allowed access to standard chow and water ad libitum prior to use. Ethical approval was obtained from the Monash University Victorian College of Pharmacy Animal Experimentation Ethics Committee.

Measurement of smooth muscle contractility

The organ bath method used was described in Coupar & Liu (1996), and enables simultaneous measurement of circular and longitudinal muscle contractions. Rats were stunned with a blow to the head and killed by exsanguination. Up to six 8 cm sections of ileum (minimum 10 cm oral to the caecum) were excised and rinsed with 10 ml Krebs -Henseleit solution, containing (mM): NaCl, 118; KCl, 4.7; NaHCO3, 25; KH2PO4, 1.2; CaCl2, 2.5; MgSO4, 1.2; D-(+)-glucose, 11. Each segment was sheathed over and tied to a tissue hook assembly, securely occluding the lower luminal opening. At the unattached end of the tissue segment, a cannula made from polyethylene tubing, filled with Krebs–Henseleit solution, was inserted and tied in position. The tissue/cannula lumen communicated with a pressure transducer (Gould P23ID), producing a closed-lumen set-up. The assembly was then immersed in Krebs–Henseleit solution bubbled with 95% O2–5% CO2 in a 30 ml organ bath and maintained at 37°C. The cannula was connected to an isometric force transducer (Grass FT03C) with a thread of cotton, and the tissue tensioned to 1 × g. After 30-min equilibration, the lumen was filled with 0.15 ml Krebs–Henseleit solution, and the tissue allowed a further 10-min equilibration. The tissue hook included platinum electrodes for transmural field stimulation, through which pulse trains were delivered. A Grass S48 stimulator supplied (supra-maximal) 40 V square wave pulses of 1 ms duration at a rate of 10 Hz, in 8 s trains to induce neurogenic circular and longitudinal muscle contractions. Pulse trains were applied at intervals of 3 min. Longitudinal and circular muscle contractions were measured by the isometric and pressure transducers, respectively. These dual responses were plotted using a Grass model 79D polygraph.

Complete cumulative concentration–response curves were established to the μ-, δ- and κ-opioid receptor-selective agonists, DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin acetate), DADLE ([D-Ala2,D-Leu5]enkephalin acetate) and U 50488H (trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulphonate), in the presence of various concentrations of their corresponding receptor-selective competitive antagonist, CTAP (H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), naltrindole or n-BNI (nor-binaltorphimine dihydrochloride). The observed lack of effect of n-BNI on the U 50488H concentration–response curve prompted the use of the broad-spectrum opioid receptor antagonist, naltrexone, with this agonist. A concentration–response curve was also established to the prototype opioid compound, morphine. Antagonists were added to the bath 10–20 min prior to the first agonist addition, and agonists were added 90 s prior to the stimulation train at which the response was measured. At least three different antagonist concentrations were used to establish the linearity of antagonist effects, in addition to a control (no antagonist) curve. At least four replicate responses were developed for each antagonist concentration. At the end of the concentration–response determination, stimulation after bath washout verified reversibility of agonist effects and confirmed tissue viability. Rat tissues were discarded after a single concentration–response experiment.

Since experiments indicated low U 50488H potency values and a lack of n-BNI antagonist activity, the chemical integrity of our stocks of these κ ligands was verified by bioassay, using the method of Birch et al. (1987). Segments of guinea-pig ileum approximately 3 cm in length were set up in organ baths containing Krebs–Henseleit solution at 37°C and bubbled with 95% O2–5% CO2. Enteric nerves were stimulated with single pulses (1 ms duration at 0.1 Hz, with supramaximal voltage) from platinum transmural electrodes.

Drugs

Morphine hydrochloride, manufactured by Macfarlan Smith Ltd (London, U.K.); DAMGO, DADLE, CTAP, naltrindole and n-BNI (nor-binaltorphimine dihydrochloride), purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.); U 50488H, a gift from Upjohn, naltrexone, purchased from DuPont Pharmaceuticals (Wilmington, DE, U.S.A.). All compounds were dissolved in distilled water prior to addition to the organ bath.

Data analysis

Experimental data were analysed to obtain pKB values using the global nonlinear regression method described by Lew & Angus (1995). Advantages offered by this method over the traditional Schild method have been discussed previously (Lew & Angus, 1995). Agonist effects on contraction were expressed as a percentage of complete inhibition of contraction, with preagonist contraction strengths serving as the baseline level. Sigmoidal curves were fitted to individual concentration-responses by nonlinear regression, using the four-parameter logistic equation to obtain location and slope parameters. The equation is

graphic file with name 144-0706107e1.gif

where A is the agonist concentration, a is the basal value, b is the vertical range, and d is the mid-point slope.

A curve was fitted by nonlinear regression to the pEC50 values outputted from Eq. (1) regressions, against the corresponding antagonist concentrations, according to the following equation:

graphic file with name 144-0706107e2.gif

where [B] denotes antagonist concentration, pK, log c and s are fitting parameters, with s being a measure of the molecularity of the antagonist–receptor interaction. s is directly equivalent to the Schild slope factor (Arunlakshana & Schild, 1959), and simple competitive antagonism theoretically produces an s value of 1. The F test comparison method was used to indicate the acceptability of constraining the s value to 1. P<0.05 was taken to be significant. When the constraint was permitted and applied, the pK value was taken to be equivalent to the pKB.

The obtained pKB value was then used to represent experimental data in a Clark plot, a graph of agonist log EC50 vs log([B]+KB). Analogous to the Schild plot, the Clark plot indicates the graded effect of the antagonist on agonist potency. By convention, the Clark plot is used to illustrate the deviation of experimental data from theoretical antagonism behaviour. In simple competitive antagonism, data theoretically follow a line with a gradient of 1 (the value of s), offset laterally from the origin by the value of log c. pKB values are read from a Clark plot at the abscissa where [B]=0.

Additional curves were generated from pooled agonist response data to verify curve parallelism over the tested antagonist concentrations. Again, this was carried out using the F test to compare the goodness-of-fit of constrained and unconstrained regression curves to the experimental data. P<0.05 was taken to be significant. Tested constraints to Emax and slope parameters were based on best-fit values provided by regressions set to yield common values between data sets. The minimum response parameter was constrained to 0%. These curves were also used to provide EC50 agonist potency values and a graphical indication of curve positions and shape. The regression curves were not used in subsequent pKB calculations, however.

Concentration–response and Clark plot data are shown as mean±s.e.m. Prism version 4.00 software (GraphPad Software, San Diego, CA, U.S.A.) was used for all computations and graphics.

Results

Effects of agonists

Tissue responses to stimulation were as described previously (Coupar & Liu, 1996), and all data readings were performed within the established ‘stability window' of the tissue preparation.

Circular and longitudinal smooth muscle contractions were inhibited by all agonists tested, that is, morphine, DAMGO, DADLE and U 50488H. Agonist potency and Emax values are summarised in Table 1. Representative traces showing the effects of DAMGO on circular and longitudinal muscle contractions are shown in Figure 1. Potency values did not significantly differ between circular and longitudinal muscle layers for DAMGO or DADLE, although the longitudinal muscle potency of U 50488H was significantly lower than in circular muscle. However, the Emax values of these agonists were all lower for the longitudinal muscle responses than the circular muscle responses. The order of agonist potencies was consistent between the muscle layers, with DAMGO the most potent, followed by DADLE, morphine and U 50488H. Recovery of contractions was apparent after drug washout for each agonist.

Table 1.

Potencies of subtype-selective opioid receptor agonists as inhibitors of transmural stimulation-induced contractions of the rat ileum circular and longitudinal muscle

  pEC50 (−log M) Emax (%)   pKB
Agonist circ. long. circ. long. Antagonist circ. long.
Morphine 6.43±0.17 6.65±0.27 81.7±5.0 59.7±7.8 a a a
DAMGO 7.85±0.04 7.35±0.09 97.8±3.6 56.0±6.1 CTAP 7.84±0.17 7.64±0.35
DADLE 7.41±0.17 6.31±0.07 93.3±8.4 66.5±5.2 Naltrindole 8.22±0.23 8.53±0.35
U 50488H 5.91±0.41 5.60±0.08 83.5±26.8 74.3±7.2 n-BNI b b
U 50488H         Naltrexone b b
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a

Not tested.

b

Antagonism not observed.

Affinity values of subtype-selective antagonists are also shown.

Figure 1.

Figure 1

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(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.

Effects of antagonists

Antagonist results are summarised in Table 1. CTAP and naltrindole produced progressive, parallel rightward shifts of the respective DAMGO and DADLE concentration–response curves in circular and longitudinal muscle (Figure 2a and b; Figure 3a and b). Analysis yielded molecularity values (s) of 1.04±0.25 (circular muscle) and 0.87±0.31 (longitudinal muscle) for CTAP, and 1.10±0.21 (circular muscle) and 0.87±0.31 (longitudinal muscle) for naltrindole. These molecularity values were not significantly different from unity.

Figure 2.

Figure 2

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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]+KB)+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 pKB and agonist control curve pEC50, for the different muscle layers.

Figure 3.

Figure 3

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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]+KB)+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 pKB and agonist control curve pEC50, for the different muscle layers.

Concentrations of n-BNI up to 1 μM failed to alter the concentration–response curve of U 50488H in either circular or longitudinal muscle (Figure 4a and b). At the highest n-BNI concentration tested, a small leftward shift was produced in the circular muscle curve. Bioassay of the U 50488H and n-BNI drug stocks in the guinea-pig ileum longitudinal muscle preparation produced a pEC50 of 8.01±0.08 (Emax, 84.2±5.2%) for U 50488H and an n-BNI pKB of 10.3±0.04, values virtually identical to those reported in the literature for activity at κ opioid receptors (Birch et al., 1987; Hunter et al., 1990). The possibility of agonist activity at μ and δ receptors was then considered. Although the tested n-BNI concentrations had reached levels approaching 100-fold its reported pA2 at μ and δ receptors (Birch et al., 1987), suggesting these receptors were not involved, affirmation was sought with the use of the nonselective opioid receptor antagonist, naltrexone. Naltrexone (1 μM) failed to produce antagonism; in fact, potentiation was observed. Concentration ratios of 0.17 and 0.45 were obtained for circular and longitudinal muscle, respectively.

Figure 4.

Figure 4

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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.

Discussion

To our knowledge, this is the first in vitro study into the effects of opioids on the rat ileum circular muscle. The method used in this study measures the effect of drugs on neurogenic contractile responses of both circular and longitudinal muscle layers (Coupar & Liu, 1996). It has been used previously to show that α2D-adrenoceptors and adenosine A1 receptors mediate inhibitory effects on neurogenic circular, but not longitudinal muscle contractions in the rat ileum (Liu & Coupar, 1997; Coupar, 1999). The muscular contractions in these studies have appeared to be largely due to neuronal ACh release (as evidenced by sensitivity to atropine), the attenuation of which has been very strongly implicated as a pathway through which the tested agonists caused inhibition of contraction. Similarly, the inhibition of ACh release in opioid-induced inhibition of longitudinal muscle contraction has also been implicated in the stimulated rat small intestine in vitro (Hancock & Coupar, 1994). Our present findings show that certain opioid agonists inhibit electrically induced neurogenic circular, as well as longitudinal muscle contractions. The identities of the active sites of these compounds are discussed below.

μ-Opioid receptor

The observed potencies of DAMGO are consistent with values reported in standard bioassay preparations for μ-opioid receptors; the guinea-pig ileum (Handa et al., 1981; Smith et al., 1988) and mouse vas deferens (Handa et al., 1981; Smith et al., 1988). However, the observed sensitivity of the rat ileum to DAMGO found in our study contrasts with previous findings in the transmurally stimulated longitudinal muscle preparation of the rat jejunum (Coupar & De Luca, 1994; Hancock & Coupar, 1994). The parallel displacement of the DAMGO concentration–effect curve by CTAP and its affinity value derived from this data also indicates μ receptor activity. The CTAP pKB values calculated from the circular and longitudinal muscle data are consistent with findings in the guinea-pig ileum (Kramer et al., 1989).

δ-Opioid receptor

Literature pEC50 values for DADLE cover the range 7.3-9.4 in the standard functional assays of δ receptors. The present values fall towards the lower end of this range, and are very similar to the hamster vas deferens value of 7.3 (Miller & Shaw, 1985). In addition, the values overlie the reported potency in rat jejunum, an intestinal region anatomically contiguous with the ileum, and in which functional δ receptors have been well established (Hancock & Coupar, 1994). Greater DADLE potency has been reported in the guinea-pig ileum (Corbett et al., 1984) and mouse vas deferens (Smith et al., 1988). The observed naltrindole affinity values (KB) for circular and longitudinal muscle responses were approximately 10-fold less than what would be predicted from previous reports, in rat jejunum (Hancock & Coupar, 1994) and mouse vas deferens (Portoghese et al., 1988; Rogers et al., 1990). These reports place the pKB of naltrindole in these δ receptor preparations in the range of 9.6–10.2. The observed affinities were still approximately 10-fold greater than the expected value for antagonism at μ receptors, however (Portoghese et al., 1988; Rogers et al., 1990). The reasons for this deviation are not clear. Variations in δ antagonist affinity have been reported in brain homogenate assays of adenylyl cyclase activity (Buzas et al., 1994; Noble & Cox, 1995). This has been interpreted as being suggestive of δ receptor heterogeneity, although a genetic basis for this conclusion is not currently established. A possible explanation might be modified receptor properties arising from receptor oligomerisation. δ receptor oligomerisation has been demonstrated with μ and κ receptors (Jordan & Devi, 1999; Gomes et al., 2000), and is associated with modified ligand binding affinities and efficacy. In the case of ENS tissue, the potential for such oligomerisation appears to exist; cellular co-expression of μ and δ receptors has been shown electrophysiologically in guinea-pig myenteric plexus neurons (Egan & North, 1981). In the rat ileum, double-labelling immunohistochemistry has indicated μ/δ co-expression is widespread in myenteric and submucous plexus neurons (Gray et al., unpublished observations).

On the balance of the available evidence, we consider it likely that DADLE acts via δ-opioid receptors to inhibit electrically induced neurogenic contractions. These receptors may be atypical in nature, possibly owing to the reasons outlined above.

κ-Opioid receptor

The observed inhibitory potencies of U 50488H were 100- to 1000-fold lower than previously reported in the guinea-pig ileum (Birch et al., 1987; Hunter et al., 1990; Choi et al., 1992). This brings into question the role for the κ receptor as an inhibitory influence on neurogenic excitation in the rat ileum. Moreover, U 50488H effects were not sensitive to n-BNI or naltrexone. n-BNI has been consistently reported as possessing subnanomolar antagonist activity at κ receptors, yet concentrations up to 1 μM failed to produce any detectable change in the agonist concentration–response curve in the present experiments. Similarly, the concentration of naltrexone used would be expected to cause large rightwards shifts to opioid receptor-mediated responses (Takemori & Portoghese, 1984). It therefore appears highly unlikely that the observed responses to U 50488H arise from the activation of opioid receptors.

The present in vitro results correlate with those obtained in vivo. For example, U 50488H did not affect gastrointestinal transit in the rat, even at doses causing profound CNS effects (Tavani et al., 1984). A number of other studies have indicated that the role for κ receptors is minor (at best) in opioid-induced constipation in rats (La Regina et al., 1988; Tavani et al., 1990). Nonetheless, κ receptors were considered a reasonable subject for examination. This is partly because they mediate strong inhibitory effects on contractility and transit in several mammals, including the guinea-pig (Culpepper-Morgan et al., 1995) and humans (De Schepper et al., 2004). Secondly, κ receptors are located in close proximity to μ and δ receptors in the rat ENS. In addition, the κ-selective agonist U 69593 ((+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide) has been shown to antagonise morphine constipation, and cause minor reduction in gastrointestinal transit in rats, with uncertainty surrounding the involvement of κ receptors (La Regina et al., 1988).

Nonopioid actions have previously been attributed to U 50488H and related κ opioid arylacetamides in a variety of settings (Alzheimer & ten Bruggencate, 1990; Joshi et al., 2000; Su et al., 2002). As in the present study, these effects have been characterised by low (μM) potency and insensitivity to n-BNI and nonselective opioid antagonists. The study of these effects has been pursued in a noxious colonic distension model in rats, with observation of a variety of end points (Joshi et al., 2000; Su et al., 2002). A local-anaesthetic-like blockade of sodium currents in local sensory neurons is considered the likely mechanism, and is supported by electrophysiological findings (Joshi et al., 2003).

There is insufficient information to determine the basis for the apparent potentiation of U 50488H by naltrexone. Potentiation of nonopioid U 50488H effects has also been reported with the pharmacologically analogous compound, naloxone, in an electrophysiological study of hippocampal CA3 neurons of the guinea-pig (Alzheimer & ten Bruggencate, 1990).

Morphine

The stimulatory effects of morphine reported previously in vivo (Burks, 1976) appear to be at odds with opioid agonist-induced inhibition of contraction observed here. However, critical differences in experimental conditions likely account for this. In particular, the use of electrical stimulation in the present study would have created an altered neuronal state. Indeed, the neurotransmitters at play in stimulated and unstimulated conditions appear to differ. In the in vivo study, morphine effects appeared to arise from induction of 5-HT release, whereas in the stimulated tissue, inhibition of ACh release appears to be the major mechanism responsible, at least for longitudinal muscle (for a review, see De Luca & Coupar, 1996). The validity and relevance of the electrically stimulated rat intestine is supported by correlations with drug effects on gastrointestinal transit in vivo. Significantly, our finding that μ and δ (but not κ) receptors are involved in attenuating neurogenic contractions correlates with in vivo antitransit findings reported previously (Tavani et al., 1984; 1990; La Regina et al., 1988; Tavani et al.).

It is interesting that morphine, considered a full agonist at all opioid receptor subtypes, produced Emax values that were exceeded by both DAMGO and DADLE. An explanation for this might be functional self-antagonism via activation of κ receptors. Alternatively, contractile effects, as previously reported (Burks, 1976), may have mitigated the drug's inhibitory efficacy on responses to stimulation.

In conclusion, this study provides evidence that neurogenic circular and longitudinal smooth muscle contractions are inhibited by activation of μ opioid receptors and δ opioid receptors (which are possibly of an atypical form) in the ileum of the rat. These effects are probably mediated in the cholinergic neuronal pathway in the ENS. These findings provide a mechanical basis for earlier in vivo functional observations, in which apparent activation of these opioid receptor subtypes resulted in arrest of gastrointestinal transit. The unusual properties exhibited by U 50488H in this preparation call for further investigation, and the method used here may provide a convenient tool for the future study of nonopioid properties of arylacetamide κ opioids.

Acknowledgments

We are grateful to Upjohn for the gift of U 50488H. Andrew Gray is grateful for the support for this work provided by a Monash Graduate Scholarship.

Abbreviations

CTAP

H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (disulphide bridge 2–7)

DADLE

[D-Ala2,D-Leu5]enkephalin acetate

DAMGO

[D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin acetate

n-BNI

nor-binaltorphimine dihydrochloride

U 50488H

trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulphonate

U 69593

(+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide

References

  1. ALZHEIMER C., TEN BRUGGENCATE G. Nonopioid actions of the kappa-opioid receptor agonists, U 50488H and U 69593 on electrophysiologic properties of hippocampal CA3 neurons in vitro. J. Pharmacol. Exp. Ther. 1990;255:900–905. [PubMed] [Google Scholar]
  2. ARUNLAKSHANA O., SCHILD H.O. Some quantitative uses of drug antagonists. Br. J. Pharmacol. 1959;14:48–58. doi: 10.1111/j.1476-5381.1959.tb00928.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BAGNOL D., MANSOUR A., AKIL H., WATSON S.J. Cellular localization and distribution of the cloned mu and kappa opioid receptors in rat gastrointestinal tract. Neuroscience. 1997;81:579–591. doi: 10.1016/s0306-4522(97)00227-3. [DOI] [PubMed] [Google Scholar]
  4. BIANCHI G., FERRETTI P., RECCHIA M., ROCCHETTI M., TAVANI A., MANARA L. Morphine tissue levels and reduction of gastrointestinal transit in rats. Correlation supports primary action site in the gut. Gastroenterology. 1983;85:852–858. [PubMed] [Google Scholar]
  5. BIANCHI G., FIOCCHI R., TAVANI A., MANARA L. Quaternary narcotic antagonists' relative ability to prevent antinociception and gastrointestinal transit inhibition in morphine-treated rats as an index of peripheral selectivity. Life Sci. 1982;30:1875–1883. doi: 10.1016/0024-3205(82)90467-2. [DOI] [PubMed] [Google Scholar]
  6. BIRCH P.J., HAYES A.G., SHEEHAN M.J., TYERS M.B. Norbinaltorphimine: antagonist profile at kappa opioid receptors. Eur. J. Pharmacol. 1987;144:405–408. doi: 10.1016/0014-2999(87)90397-9. [DOI] [PubMed] [Google Scholar]
  7. BROWNLEE G., HARRY J. Some pharmacological properties of the circular and longitudinal muscle strips from the guinea-pig isolated ileum. Br. J. Pharmacol. 1963;21:544–554. doi: 10.1111/j.1476-5381.1963.tb02022.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. BURKS T.F. Acute effects of morphine on rat intestinal motility. Eur. J. Pharmacol. 1976;40:279–283. doi: 10.1016/0014-2999(76)90063-7. [DOI] [PubMed] [Google Scholar]
  9. BUZAS B., IZENWASSER S., PORTOGHESE P.S., COX B.M. Evidence for delta opioid receptor subtypes regulating adenylyl cyclase activity in rat brain. Life Sci. 1994;54:PL101–PL106. doi: 10.1016/0024-3205(94)00412-9. [DOI] [PubMed] [Google Scholar]
  10. CHOI H., MURRAY T.F., DELANDER G.E., CALDWELL V., ALDRICH J.V. N-terminal alkylated derivatives of [D-Pro10]dynorphin A-(1-11) are highly selective for kappa-opioid receptors. J. Med. Chem. 1992;35:4638–4639. doi: 10.1021/jm00102a019. [DOI] [PubMed] [Google Scholar]
  11. CORBETT A.D., GILLAN M.G., KOSTERLITZ H.W., MCKNIGHT A.T., PATERSON S.J., ROBSON L.E. Selectivities of opioid peptide analogues as agonists and antagonists at the delta-receptor. Br. J. Pharmacol. 1984;83:271–279. doi: 10.1111/j.1476-5381.1984.tb10143.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. COUPAR I.M. The peristaltic reflex in the rat ileum: evidence for functional mu- and delta-opiate receptors. J. Pharm. Pharmacol. 1995;47:643–646. doi: 10.1111/j.2042-7158.1995.tb05851.x. [DOI] [PubMed] [Google Scholar]
  13. COUPAR I.M. Characterization and tissue location of the neural adenosine receptor in the rat ileum. Br. J. Pharmacol. 1999;126:1269–1275. doi: 10.1038/sj.bjp.0702411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. COUPAR I.M., DE LUCA A. Opiate and opiate antidiarrhoeal drug action on rat isolated intestine. J. Auton. Pharmacol. 1994;14:69–78. doi: 10.1111/j.1474-8673.1994.tb00591.x. [DOI] [PubMed] [Google Scholar]
  15. COUPAR I.M., LIU L. A simple method for measuring the effects of drugs on intestinal longitudinal and circular muscle. J. Pharmacol. Toxicol. Methods. 1996;36:147–154. doi: 10.1016/s1056-8719(96)00110-4. [DOI] [PubMed] [Google Scholar]
  16. CULPEPPER-MORGAN J.A., HOLT P.R., LAROCHE D., KREEK M.J. Orally administered opioid antagonists reverse both mu and kappa opioid agonist delay of gastrointestinal transit in the guinea pig. Life Sci. 1995;56:1187–1192. doi: 10.1016/0024-3205(95)00057-d. [DOI] [PubMed] [Google Scholar]
  17. DE LUCA A., COUPAR I.M. Insights into opioid action in the intestinal tract. Pharmacol. Ther. 1996;69:103–115. doi: 10.1016/0163-7258(95)02053-5. [DOI] [PubMed] [Google Scholar]
  18. DE SCHEPPER H.U., CREMONINI F., PARK M.I., CAMILLERI M. Opioids and the gut: pharmacology and current clinical experience. Neurogastroenterol. Motil. 2004;16:383–394. doi: 10.1111/j.1365-2982.2004.00513.x. [DOI] [PubMed] [Google Scholar]
  19. EGAN T.M., NORTH R.A. Both mu and delta opiate receptors exist on the same neuron. Science. 1981;214:923–924. doi: 10.1126/science.6272393. [DOI] [PubMed] [Google Scholar]
  20. GOMES I., JORDAN B.A., GUPTA A., TRAPAIDZE N., NAGY V., DEVI L.A. Heterodimerization of mu and delta opioid receptors: a role in opiate synergy. J. Neurosci. 2000;20:RC110. doi: 10.1523/JNEUROSCI.20-22-j0007.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. GRAY A.C., WHITE P.J., COUPAR I.M. Abstracts of the 8th World Congress on Clinical Pharmacology & Therapeutics. Clin. Exp. Pharmacol. Physiol. 2004;31:I–X. doi: 10.1111/j.1440-1681.2004.04042.x. [DOI] [PubMed] [Google Scholar]
  22. HANCOCK D.L., COUPAR I.M. Evidence for functional delta-opiate receptors in the rat intestine. J. Pharm. Pharmacol. 1994;46:805–808. doi: 10.1111/j.2042-7158.1994.tb03734.x. [DOI] [PubMed] [Google Scholar]
  23. HANDA B.K., LAND A.C., LORD J.A., MORGAN B.A., RANCE M.J., SMITH C.F. Analogues of beta-LPH61-64 possessing selective agonist activity at mu-opiate receptors. Eur. J. Pharmacol. 1981;70:531–540. doi: 10.1016/0014-2999(81)90364-2. [DOI] [PubMed] [Google Scholar]
  24. HUNTER J.C., LEIGHTON G.E., MEECHAM K.G., BOYLE S.J., HORWELL D.C., REES D.C., HUGHES J. CI-977, a novel and selective agonist for the kappa-opioid receptor. Br. J. Pharmacol. 1990;101:183–189. doi: 10.1111/j.1476-5381.1990.tb12110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. JORDAN B.A., DEVI L.A. G-protein-coupled receptor heterodimerization modulates receptor function. Nature. 1999;399:697–700. doi: 10.1038/21441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. JOSHI S.K., LAMB K., BIELEFELDT K., GEBHART G.F. Arylacetamide kappa-opioid receptor agonists produce a tonic- and use-dependent block of tetrodotoxin-sensitive and -resistant sodium currents in colon sensory neurons. J. Pharmacol. Exp. Ther. 2003;307:367–372. doi: 10.1124/jpet.103.052829. [DOI] [PubMed] [Google Scholar]
  27. JOSHI S.K., SU X., PORRECA F., GEBHART G.F. kappa -opioid receptor agonists modulate visceral nociception at a novel, peripheral site of action. J. Neurosci. 2000;20:5874–5879. doi: 10.1523/JNEUROSCI.20-15-05874.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. KOSTERLITZ H.W., LEES G.M. Pharmacological analysis of intrinsic intestinal reflexes. Pharmacol. Rev. 1964;16:301–339. [PubMed] [Google Scholar]
  29. KRAMER T.H., SHOOK J.E., KAZMIERSKI W., AYRES E.A., WIRE W.S., HRUBY V.J., BURKS T.F. Novel peptidic mu opioid antagonists: pharmacologic characterization in vitro and in vivo. J. Pharmacol. Exp. Ther. 1989;249:544–551. [PubMed] [Google Scholar]
  30. LA REGINA A., PETRILLO P., SBACCHI M., TAVANI A. Interaction of U-69,593 with mu-, alpha- and kappa-opioid binding sites and its analgesic and intestinal effects in rats. Life Sci. 1988;42:293–301. doi: 10.1016/0024-3205(88)90638-8. [DOI] [PubMed] [Google Scholar]
  31. LEW M.J., ANGUS J.A. Analysis of competitive agonist-antagonist interactions by nonlinear regression. Trends Pharmacol. Sci. 1995;16:328–337. doi: 10.1016/s0165-6147(00)89066-5. [DOI] [PubMed] [Google Scholar]
  32. LIU L., COUPAR I.M. Characterisation of pre- and post-synaptic alpha-adrenoceptors in modulation of the rat ileum longitudinal and circular muscle activities. Naunyn. Schmiedebergs Arch. Pharmacol. 1997;356:248–256. doi: 10.1007/pl00005048. [DOI] [PubMed] [Google Scholar]
  33. MILLER L., SHAW J.S. Characterisation of the delta-opioid receptor on the hamster vas deferens. Neuropeptides. 1985;6:531–536. doi: 10.1016/0143-4179(85)90115-5. [DOI] [PubMed] [Google Scholar]
  34. MILLER R.J., HIRNING L.D.Opioid peptides of the gut Handbook of Physiology, The Gastrointestinal Tract 1989Bethesda, MD: American Physiological Society; 631–660.ed. Shultz, S.T., Makhlouf, G.N. & Rauner, B.B., pp [Google Scholar]
  35. NOBLE F., COX B.M. Differential regulation of D1 dopamine receptor- and of A2a adenosine receptor-stimulated adenylyl cyclase by mu-, delta 1-, and delta 2-opioid agonists in rat caudate putamen. J. Neurochem. 1995;65:125–133. doi: 10.1046/j.1471-4159.1995.65010125.x. [DOI] [PubMed] [Google Scholar]
  36. PORTOGHESE P.S., SULTANA M., TAKEMORI A.E. Naltrindole, a highly selective and potent non-peptide delta opioid receptor antagonist. Eur. J. Pharmacol. 1988;146:185–186. doi: 10.1016/0014-2999(88)90502-x. [DOI] [PubMed] [Google Scholar]
  37. ROGERS H., HAYES A.G., BIRCH P.J., TRAYNOR J.R., LAWRENCE A.J. The selectivity of the opioid antagonist, naltrindole, for delta-opioid receptors. J. Pharm. Pharmacol. 1990;42:358–359. doi: 10.1111/j.2042-7158.1990.tb05428.x. [DOI] [PubMed] [Google Scholar]
  38. SMITH C.F., WALDRON C., BROOK N.A. Opioid receptors in the mouse ileum. Arch. Int. Pharmacodyn. Ther. 1988;291:122–131. [PubMed] [Google Scholar]
  39. STERNINI C., SPANN M., DE GIORGIO R., ANTON B., KEITH D.E., JR, EVANS C., BRECHA N.C. Cellular localization of the mu opioid receptor in the rat and guinea pig enteric nervous system. Analgesia. 1995;1:762–765. [Google Scholar]
  40. SU X., JOSHI S.K., KARDOS S., GEBHART G.F. Sodium channel blocking actions of the kappa-opioid receptor agonist U50,488 contribute to its visceral antinociceptive effects. J. Neurophysiol. 2002;87:1271–1279. doi: 10.1152/jn.00624.2001. [DOI] [PubMed] [Google Scholar]
  41. TAKEMORI A.E., PORTOGHESE P.S. Comparative antagonism by naltrexone and naloxone of mu, kappa, and delta agonists. Eur. J. Pharmacol. 1984;104:101–104. doi: 10.1016/0014-2999(84)90374-1. [DOI] [PubMed] [Google Scholar]
  42. TAVANI A., GAMBINO M.C., PETRILLO P. The opioid kappa-selective compound U-50,488H does not inhibit intestinal propulsion in rats. J. Pharm. Pharmacol. 1984;36:343–344. doi: 10.1111/j.2042-7158.1984.tb04391.x. [DOI] [PubMed] [Google Scholar]
  43. TAVANI A., PETRILLO P., LA REGINA A., SBACCHI M. Role of peripheral mu, delta and kappa opioid receptors in opioid-induced inhibition of gastrointestinal transit in rats. J. Pharmacol. Exp. Ther. 1990;254:91–97. [PubMed] [Google Scholar]

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