Modulating effects of opioids,purine compounds, 5-hydroxytryptamine and prostaglandin E2 on cholinergic neurotransmission in a guinea-pig oesophagus preparation

The submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus was used to study the actions of morphine, opioid peptides, purine compounds, 5-hydroxytryptamine (5-HT) and prostaglandin E₂ (PGE₂) on electrically-induced twitch contractions which are probably mediated by cholinergic nerve stimulation. The twitch contractions were inhibited by morphine (1–100 μM), methionine-enkephalin (1–100 μM) and β-endorphin (0.1–1 μM), but increased by adenosine (1–30 μM), adenosine 5′-triphosphate (1–30 μM), 5-HT (0.01–3 μM) and PGE² (1–10 nM). The submaximal contraction induced by acetylcholine (12 or 20 nM) which is nearly equivalent to the twitch contractions was unaffected by morphine, methionine-enkephalin, β-endorphin and 5-HT, but augmented by purine compounds and PGE₂. It is concluded that cholinergic neurotransmission in the submucous plexus-longitudinal muscularis mucosae of the guinea-pig oesophagus is inhibited by morphine and opioid peptides acting at prejunctional opiate receptors, and facilitated by 5-HT, purine compounds and PGE₂ via prejunctional or postjunctional mechanisms.     

Opioid effects of short enkephalin fragments containing the Gly-Phe sequence on contractile responses of guinea pig ileum

1. Effects of the fragments H-Gly-Phe-OH, H-Gly-Phe-NH₂ or H-Gly-Phe-OMe on the electrically stimulated cholinergic contractions of the longitudinal layer in isolated guinea pig ileum and on the Morphine-, Met-enkephalin- or Leu-enkephalin-induced inhibition of these contractions were analyzed for opioid activity in respect to Gly-Phe sequence.2. H-Gly-Phe-OH or H-Gly-Phe-NH₂ exerted no effects, while H-Gly-Phe-OMe applied cumulatively (1 pM-1 mM), concentration-dependently reduced the contractions to electrical stimulation, the IC₅₀ value being 1.96 ± 0.06 μM. Naloxone (1–5 μM) did not reverse the H-Gly-Phe-OMe effects.3. H-Gly-Phe-OMe at single concentrations (1–10 μM) significantly decreased the maximum inhibition produced by cumulatively added (0.1 nM-100 μM) morphine, Met-enkephalin or Leu-enkephalin. The regression lines for the opioids were shifted to the right but not always in a parallel fashion; the IC₅₀ values were higher as compared to the controls and lower as compared to the IC₅₀ values after naloxone.4. The pA₂ value for H-Gly-Phe-OMe with respect to morphine (6.43 ± 0.14) did not differ from that to Met-enkephalin (6.68 ± 0.35) or Leu-enkephalin (9.06 ± 0.98); the slope of the pA₂ plot to morphine was near unity.5. These data indicated that H-Gly-Phe-OMe exerted predominantly a potent non-competitive opioid antagonistic effect suggesting that short enkephalin fragments containing the Gly-Phe sequence might possess an opioid activity.     

Effects of chronic inflammation and morphine tolerance on the expression of phospho-ERK 1/2 and phospho-P38 in the injured tissue

Opioids are used in humans in the treatment of chronic osteoarticular pain, but the development of tolerance to the analgesic effects after continuous administration is still not well understood. The aim of the present study was to evaluate the expression of phospho-ERK 1/2 and phospho-p38 in mice with monoarthritis chronically exposed to morphine as a possible explanation for the development of tolerance. Inflammation was induced by intraplantar injection of complete Freund’s adjuvant (CFA) and the tolerance by implantation of 75 mg morphine pellets. The results of the present study show that ERKs phosphorylation is unaltered by inflammation or morphine tolerance, each one individually, in the plantar tissue. In contrast, phospho-p38 is similarly decreased by inflammation or morphine tolerance. In naïve but not in tolerant animals, acute injection of morphine induces significant increase in phospho-p38 without any changes in phospho-ERK 1/2 expression. During inflammation, the acute injection of morphine induces a significant increase in the expression of ERK 1/2, but not in phospho-p38, in naïve animals. Phospho-ERK 1/2 expression was significantly decreased in the presence of inflammation plus tolerance. In contrast, no significant differences in phospho-p38 expression were observed between naïve and tolerant animals acutely injected with saline or morphine in presence of CFA inflammation. These results suggest that ERK but not p38 could be implicated in the development of morphine tolerance during peripheral inflammation. These experiments could contribute to establish the mechanisms implicated in the development of morphine tolerance in presence of inflammatory pain.     

Lack of correlation between sigma binding potency and inhibition of contractions in the mouse vas deferens preparation.

The existence of sigma receptors in the mouse, rat and guinea pig vasa deferentia has previously been proposed, although drug effects are inconsistent and generally occur only at high concentrations. The purpose of the present study was to evaluate lower, physiologically relevant concentrations of ligands for possible sigma effects on electrically stimulated twitch contractions in the mouse vas deferens (MVD). Putative sigma agonists and antagonists all inhibited 0.1 Hz electrically stimulated twitch contractions in nM concentrations. Inhibitory activity plateaued between 20 and 60% for all compounds except 1,3-di(2-tolyl)guanidine (DTG), which had a shallow concentration-effect curve. Subsequent to the plateau, higher concentrations (30 microM) of rimcazole and haloperidol fully inhibited electrically stimulated twitch contractions. There was no correlation between inhibitory potency or maximal effect in the MVD and binding potency at sigma sites in either MVD or guinea pig brain. The inhibitory effects of R(+)-3-(3-hydroxyphenyl)-N-1-propylpiperidine ((+)3-PPP) or DTG on electrically stimulated twitch contractions were not antagonized by the putative sigma antagonists DTG, haloperidol, rimcazole or BMY-14802, nor by alpha 2-adrenoceptor, dopamine D1, dopamine D2 or opiate antagonists. Although the mechanism of sigma ligand effects in the MVD has not been established, the data caution against a presumption that effects of sigma ligands on electrically stimulated twitch contractions in this preparation are mediated by sigma receptors.     

Centrally and peripherally mediated inhibition of intestinal motility by opioids

Summary Intracerebroventricularly injected morphine is 50-fold more potent in arresting intestinal peristalsis in rats, mice or guinea pigs than morphine administered systemically. Using quaternary naloxone as narcotic antagonist, it has been demonstrated that the peripheral pathway of the centrally mediated constipatory effect of morphine does not involve opioid peptidergic mechanisms. Further, this effect is not due to the release of opioid peptides from the pituitary, since hypophysectomy fails to affect the antipropulsive activity of morphine. On the other hand, the intestinal motility can be affected directly by activation of opiate receptors located in the gut. This was best demonstrated with loperamide, which exhibits predominantly a peripheral site of action. Thus, two mechanisms of the action of morphine on gastrointestinal propulsive activity have been demonstrated. One arises in the central nervous system (CNS) and is mediated peripherally not by opioid peptidergic pathways, whereas the other is due to a direct action of morphine on the gut.     

Long-term sensitization to the excitatory effects of morphine: A motility study in post-dependent rats

Motility effects of excitatory and depressant doses of morphine were studied in naïve, dependent (20 mg/kg daily) and post-dependent (about 40 days after withdrawal) rats.Doses of 0.6 and 1.25 mg/kg of morphine produced a greater increase in activity in post-dependent than in naïve rats. A 20-mg/kg dose exerted an excitatory effect in dependent rats instead of the depressant one induced in naïve animals. This excitatory effect was still evident but less pronounced in post-dependent rats.The dose of 1.25 mg/kg was repeatedly challenged in post-dependent (80 mg/kg daily) rats. It was found that the increased excitatory effect of this dose persisted unchanged 160 days after ceasing treatment.The present results demonstrate that several months after the withdrawal of morphine, sensitization still exists to the excitatory effect of the drug while tolerance to the depressant action is slight or no longer present shortly after ceasing treatment.     

The relationship between gastrointestinal transit and motility in dogs with postoperative ileus

We investigated the relationship between gastrointestinal (GI) transit and motility during postoperative ileus in dogs undergoing a single laparotomy. We combined X-ray radiography for a GI transit study with chronically implanted force transducers (FTs) for a GI motility study. Radio-opaque markers made of polyethylene and steel wires or barium sulfate were used to examine solid substance transit or liquid substance transit. For a while after the end of the operation, postoperative ileus was observed, with weak irregular contractions of the GI tract. Transmission of the contractions to the lower GI tract was then observed. The start point of interdigestive migrating contraction (IMC)-like motility was observed in the order of small intestine (I-IMC), duodenum (D-IMC), and stomach (G-IMC), and IMC proceeded gradually after the operation. The gastric emptying time of a solid marker was 73.6 +/- 2.3 h (n = 5), and depended on the time of first occurrence of G-IMC (r = 0.674, p = 0.006). The gastric emptying of the liquid marker was finished before the time of the first occurrence of G-IMC, and its small intestinal transit time correlated with the time of the first occurrence of G-IMC (r = 0.888, p = 0.018). Using combined X-ray radiography and FTs we found that recovery from postoperative ileus was aided by GI motility in which contractions were transmitted from the stomach to the lower GI tract, like IMC.     

Substance P and opioid interaction on stimulated and non-stimulated guinea pig ileum

In the past, substance P (SP) has been suggested to be both an opiate agonist and an antagonist. It therefore seemed appropriate to examine potential interactions of SP and opioids on guinea pig ileum. On non-stimulated ileal strips SP caused a dose responsive increase in contraction. Pretreatment of the tissue with morphine (3, 30, 300, 3000 nM), enkephalin (1.42, 14.2, 142, 1420 nM), naloxone (5 nM), or atropine (0.144 μM) did not significantly alter the spasmogenic effect of SP. On stimulated guinea pig ileum, whereas morphine and enkephalin inhibited the electrically induced twitch, SP administration resulted in contraction of the tissue. Additionally, neither strongly effective nor sub-threshold doses of SP antagonized the effects of the narcotics. These data are discussed in terms of separate receptors mediating the effects of the opiates and SP on guinea pig ileum.     

The opiate-like action of tilidine is mediated by metabolites

Summary The analgesic effect of tilidine in rats is completely antagonized by the narcotic antagonist naloxone. Radioreceptor assays revealed, however, that the main metabolites of tilidine, nortilidine and bisnortilidine, rather than tilidine exhibit affinity to opiate receptors. These findings were confirmed in studies using the electrically stimulated guinea pig ileum and the mouse vas deferens. Chronic tilidine administration to rats caused a considerable degree of physical dependence, which was expected from the ability of the intact animal to metabolize tilidine. In the isolated ileum from chronically morphinized guinea pigs, both nortilidine and bisnortilidine fully substituted for morphine in preventing induction of withdrawal, indicating dependence liability of these metabolites.     

Regulation of contractile activity by magnolol in the rat isolated gastrointestinal tracts

This study examined the pharmacological property of magnolol, a phenolic compound purified from Magnolia officinalis, on the GI motility using the rat isolated gastrointestinal (GI) strips. Magnolol (0.3–30 μM) dose-dependently stimulated the tone and amplitude of spontaneous contractions in ileum longitudinal muscles. Magnolol at 3 μM significantly increased the contractions of jejunum longitudinal and colon circular muscles, but not the longitudinal muscle contractions in fundus, antrum and colon. Pretreatment of ileum strips with either atropine (0.5 μM) or 4-diphenyllacetoxy-N(2-chloriethyl)-piperidine (4-DAMP, 0.5 μM) dramatically inhibited the acetylcholine (ACh, 0.1 μM)- and magnolol (3 μM)-induced longitudinal muscle contractions, but they were not affected by methoctramine (0.5 μM) and hexamethonium (0.5 μM). Ondansetron (0.1 μM) and GR113808 (2 μM) significantly reduced the tone of ileum longitudinal muscle contractions stimulated by 5-HT (10 μM), but not the amplitude. Magnolol (3 μM)-induced ileum longitudinal muscle contractions, both tone and amplitude, were significantly blocked by GR113808, but not by ondansetron. Taken together, magnolol differently regulates the spontaneous GI motility according to the region of GI tracts and orientation of smooth muscles, and magnolol-induced regulation of smooth muscle contractions in rat GI strips is likely to be mediated, at least in part, by activation of ACh and 5-HT receptors, possibly the M₃ and/or 5-HT₄ receptors.     

The effect of isoquinoline alkaloids on opiate withdrawal

Our interest has been centered on isoquinoline alkaloids obtained from Argemone mexicana (Papaveraceae), Aristolochia constricta (Aristolochiaceae) and the opium alkaloid, papaverine. In this respect, the effect of these isoquinoline alkaloids was investigated on contractions induced by naloxone of isolated guinea pig ileum acutely exposed to morphine in vitro. The activity of these alkaloids was compared to the control compound, papaverine. Furthermore, the effect of these isoquinoline alkaloids was also determined on naloxone-precipitated withdrawal in isolated guinea pig ileum exposed to DAMGO (highly selective mu opioid receptor agonist) and U50-488H (highly selective kappa opioid receptor agonist) to test whether the possible interaction of isoquinoline alkaloids on opioid withdrawal involves mu- and/or kappa-opioid receptors. Isoquinoline alkaloids from A. mexicana (from 5 x 10(-6) to 1 x 10(-4) M), from A. constricta (1 x 10(-5) x 10(-5)-1 x 10(-4) M) as well as papaverine treatment (1 x 10(-7)-5 x 10(-6)-1 x 10(-6) M) before or after the opioid agonists were able of both preventing and reversing the naloxone-induced contraction after exposure to mu (morphine and DAMGO) or kappa (U50-488H) opiate receptor agonists in a concentration-dependent manner. Both acetylcholine response and electrical stimulation were also reduced by isoquinoline alkaloids and papaverine treatment as well as the final opiate withdrawal was still reduced. The results of the present study indicate that isoquinoline alkaloids as well as papaverine were able to produce significant influence on the opiate withdrawal in vitro and these compounds were able to exert their effects both at mu and kappa opioid agonists.     

The Novel Endomorphin Degradation Blockers Tyr-Pro-DClPhe-Phe-NH2 (EMDB-1) and Tyr-Pro-Ala-NH2 (EMDB-2) Prolong Endomorphin-2 Action in Rat Ileum In Vitro

The endogenous opioid system is involved in the control of gastrointestinal (GI) motility. The potential use of endogenous MOR ligands, endomorphins (EMs), as therapeutics is limited because of their rapid enzymatic degradation and short duration of action. Targeting enzymatic degradation is an approach to prolong EM activity. In the present study, we characterized the effects of novel blockers of EM degradation in GI tissue preparation in vitro. The effects of actinonin, diprotin A (DIP) and the novel peptide EM degradation blockers Tyr-Pro-D ClPhe-Phe-NH₂ (EMDB-1), Tyr-Pro-Ala-NH₂ (EMDB-2) and Tyr-Pro-Ala-OH (EMDB-3) on EM-2-mediated inhibition of electrically induced cholinergic twitch contractions were compared in rat ileum in vitro using an organ bath. EMDB-1 and EMDB-2 significantly prolonged the inhibitory effect of EM-2 on smooth muscle contractility in rat ileum. EMDB-2 extended the EM-2 action for up to 60 min compared to 10 min in controls and was more potent than the conventional peptidase inhibitor DIP. EMDB-1 and EMDB-2 are potent EM degradation blockers, which prolong the inhibitory effects of EM-2 on smooth muscle contractility in rat ileum. These novel compounds may be of future use when targeting the endogenous opioid system in the treatment of GI motility disorders such as diarrhea.     

Morphine withdrawal in cortical slices: suppression by Ca2+-channel inhibitors of abstinence-induced [3H]-noradrenaline release.

1. The effects of morphine withdrawal were evaluated in vitro by monitoring the actions of naloxone on the depolarization-induced release of [3H]-noradrenaline (NA) in cortical slices taken from naïve or dependent rats. The effects of dihydropyridine molecules acting on Ca2+-channels (nimodipine and Bay K 8644) were also studied in this model. 2. Naloxone (10(-8)-10(-5) M) dose-dependently enhanced the K+ induced release of [3H]-NA in slices taken from dependent rats, but failed to modify the [3H]-NA release from ''naïve'' slices. 3. The naloxone-induced potentiation of release was significantly reversed by nimodipine (10(-8)-10(-6) M). These doses of nimodipine did not change [3H]-NA release (both basal and K+ induced) in preparations obtained from naïve rats. 4. Bay K 8644 potentiated the K+-induced [3H]-NA release from cortical slices taken from naïve rats to a similar extent as that of naloxone in dependent rats. 5. These results suggest that the naloxone potentiation of the depolarization-induced [3H]-NA release in slices taken from dependent rats may be considered a model of morphine withdrawal in vitro. In this model dihydropyridine Ca2+-channel antagonists suppress morphine-withdrawal effects in a similar manner to observations made in vivo.     

Adrenalectomy‐induced potentiation of morphine action in guinea‐pig ileum: possible decrease in the release of endogenous opioids from opioidergic neurones

1 The effects of adrenalectomy on exogenous and endogenous opioid actions in guinea‐pig isolated myenteric plexus‐longitudinal muscle (MPLM) were investigated. 2 A decrease in serum cortisol level to about 37% of the level in the sham‐operated group was obtained in adrenalectomized animals (sham: 53.5 ± 7.2 μg 100 ml^–1; adrenalectomized: 20.0 ± 3.6 μg 100 ml^–1). 3 The concentration–response curve of twitch inhibition, which was induced by electrical field stimulation (0.1 Hz, 0.5 ms pulse width, maximum intensity), caused by a low concentration of morphine (5 × 10^–9–5 × 10^–7 M ) was not affected, but at high concentration (10^–6–10^–5 M ) there was an upward shift in the adrenalectomized group compared with the sham‐operated control, although the basal twitch contraction was not changed by adrenalectomy. 4 The twitch inhibition induced by a high concentration of morphine (10^–6 M ) in the adrenalectomized group was antagonized to the same level as that in sham‐operated controls by naloxone (NLX) (3 × 10^–7 M ). 5 Post‐tetanic twitch inhibition, an indicator of endogenous opioid release, induced by tetanic stimulation (10 Hz, 0.5 ms pulse width, maximum intensity, for 1 min) was inhibited in the adrenalectomized group compared with the sham‐operated controls. The antagonism of inhibition in both groups was equivalent to that exerted by NLX (10^–7 M ). 6 Acetylcholine‐evoked contraction of the muscle was not influenced by adrenalectomy. 7 These results suggested a possible mechanism for the increase in sensitivity of the opioid receptors to morphine by adrenalectomy, resulting from a decrease in the release of endogenous opioids from the opioidergic neurones in the ileum.     

Modulation of gastrointestinal function by MuDelta,a mixed μ opioid receptor agonist/ μ opioid receptor antagonist

BACKGROUND & PURPOSE

Loperamide is a selective µ opioid receptor agonist acting locally in the gastrointestinal (GI) tract as an effective anti-diarrhoeal but can cause constipation. We tested whether modulating µ opioid receptor agonism with δ opioid receptor antagonism, by combining reference compounds or using a novel compound (‘MuDelta’), could normalize GI motility without constipation.

EXPERIMENTAL APPROACH

MuDelta was characterized in vitro as a potent µ opioid receptor agonist and high-affinity δ opioid receptor antagonist. Reference compounds, MuDelta and loperamide were assessed in the following ex vivo and in vivo experiments: guinea pig intestinal smooth muscle contractility, mouse intestinal epithelial ion transport and upper GI tract transit, entire GI transit or faecal output in novel environment stressed mice, or four weeks after intracolonic mustard oil (post-inflammatory). Colonic δ opioid receptor immunoreactivity was quantified.

KEY RESULTS

δ Opioid receptor antagonism opposed µ opioid receptor agonist inhibition of intestinal contractility and motility. MuDelta reduced intestinal contractility and inhibited neurogenically-mediated secretion. Very low plasma levels of MuDelta were detected after oral administration. Stress up-regulated δ opioid receptor expression in colonic epithelial cells. In stressed mice, MuDelta normalized GI transit and faecal output to control levels over a wide dose range, whereas loperamide had a narrow dose range. MuDelta and loperamide reduced upper GI transit in the post-inflammatory model.

CONCLUSIONS AND IMPLICATIONS

MuDelta normalizes, but does not prevent, perturbed GI transit over a wide dose-range in mice. These data support the subsequent assessment of MuDelta in a clinical phase II trial in patients with diarrhoea-predominant irritable bowel syndrome.     

Characterisation of the prostanoid receptor mediating inhibition of smooth muscle contractility in the rat prostate gland

This study characterised the inhibitory actions of prostaglandins on smooth muscle contractility in the rat prostate gland. Immunohistochemical studies were carried out to identify and localise the two isoforms of cyclooxygenase (COX) enzyme and the subtypes of prostanoid receptors present in the rat prostate. Isolated organ bath studies were carried out to pharmacologically characterise the subtype of prostanoid receptor mediating the inhibitory effects of prostanoids on the rat prostate. Immunohistochemical studies confirmed the presence of mainly COX-2 within the prostatic stroma. Isolated organ bath studies showed that prostaglandin E₂ (PGE₂; 10 nM–10 μM) but not prostaglandin D₂ (10 nM–10 μM), $ {\hbox{PG}}{{\hbox{F}}_2}_\alpha $ (10 nM–10 μM), prostacyclin (10 nM–10 μM) or U46619 (10 nM–10 μM) inhibited nerve-mediated contractile responses to electrical field stimulation. Similarly, sulprostone (10 nM–10 μM) had no affect on the magnitude of the electrically evoked contractions. PGE₂ (0.1–10 μM) did not affect contractions elicited by noradrenaline or adenosine 5′-triphosphate. PGE₂-mediated inhibition of electrical field stimulation induced contractions was attenuated by AH 6809 (10 μM) but not SC 19220 (10 μM) or AH 23848 (10 μM). It is concluded that prostaglandins can inhibit contractions of the rat prostate gland through a prostanoid receptor of the EP₂ subtype.     

Naloxone elicits an excitatory response in the morphine-tolerant mouse vas deferens

The effects of naloxone on the electrically stimulated vas deferens from mice implanted with morphine (tolerant) or placebo (naive) pellets were studied. In tolerant vas deferens, naloxone produced an 86.6 % increase of the twitch contractions when the preparations were stimulated with 15 V, while only a 12 % increase was observed with supramaximal voltage (40 V). Naloxone had no effect at either voltage in preparations from naive animals. The effect of morphine in naive vas deferens stimulated with 15 V, was reversed by naloxone without further increase over baseline contractions. The results suggest that opioid receptors other than those located in the neuronal soma and/or coupled to adenylate cyclase may be involved in the development of dependence.     

Acute morphine administration and withdrawal from chronic morphine increase afterdepolarization amplitude in rat supraoptic nucleus neurons in hypothalamic explants

Supraoptic nucleus (SON) neurons secrete either oxytocin or vasopressin into the bloodstream from their axon terminals in the posterior pituitary gland. SON neurons are powerfully inhibited by the classical μ-opioid receptor agonist, morphine. Oxytocin neurons develop morphine dependence when chronically exposed to this opiate, and undergo robust withdrawal excitation when morphine is subsequently acutely antagonized by naloxone. Morphine withdrawal excitation is evident as an increased firing rate and is associated with an increased post-spike excitability that is consistent with the expression of an enhanced post-spike afterdepolarization (ADP) during withdrawal. Here, we used sharp electrode recording from SON neurons in hypothalamic explants from morphine naïve and morphine treated rats to determine the effects of morphine on the ADP, and to test the hypothesis that morphine withdrawal increases ADP amplitude in SON neurons. Acute morphine administration (0.05-5.0 μM) caused a dose-dependent hyperpolarization of SON neurons that was reversed by concomitant administration of 10 μM naloxone, or by washout of morphine; counter-intuitively, acute exposure to 5 μM morphine increased ADP amplitude by 78 ± 11% (mean ± SEM). Naloxone-precipitated morphine withdrawal did not alter baseline membrane potential in SON neurons from morphine treated rats, but increased ADP amplitude by 48 ± 11%; this represents a hyper-activation of ADPs because the basal amplitude of the ADP was similar in SON neurons recorded from explants prepared from morphine naïve and morphine treated rats. Hence, an enhanced ADP might contribute to morphine withdrawal excitation of oxytocin neurons.