Unexpected reversal effects of naloxone on the guinea pig ileum.

Distension of the guinea pig ileum segment elicits peristaltic activity. If the distension is maintained the peristaltic activity disappears gradually; naloxone restores normal activity in such “fatigued” preparations. The bath solution surrounding a fatigued preparation inhibits peristaltic reflex activity in non-fatigued segments; this inhibitory effect is reversed by naloxone. The latter also antagonizes the inhibitory effects of adenine-nucleotides. These results indicate that during fatigue a substance is liberated which blocks peristalsis. They further suggest that naloxone-induced reversal of inhibition in the guinea pig ileum does not necessarily demonstrate that the inhibition is caused by a direct action on morphine receptors.     

Morphine dependence in the isolated guinea-pig ileum and its modification by p-chlorophenylalanine.

Experiments were performed to quantitatively determine morphine physical dependence in the isolated guinea-pig ileum and to assess the influence of p-chlorophenylalanine (PCPA) on its development. Ileum segments taken from animals treated with 10 s.c. injections of 100 mg/kg of morphine, given at intervals of 8 hr without interruption, responded with intense, prolonged, dose-dependent contractions to the $\text{in}$$\text{vitro}$ administration of naloxone, although contractions guinea-pigs also responded to naloxone, although contractions were smaller and of short duration. The sensitivity to naloxone on segments isolated from morphinized animals was compared to that of controls. Ilea from morphine-treated guinea-pigs were 8 to 32 times more sensitive to naloxone, as determined by a shift in the naloxone concentration-response curve to the left. There was also a three-fold increase in the maximum response. This phenomenon was taken as evidence of narcotic dependence. PCPA, given before morphine administration, at doses producing only a slight (11%) decrease in intestinal serotonin (5-HT) levels, partially reduced the sensitivity of the morphine-treated ileum to naloxone. However, high doses of PCPA, decreasing intestinal 5-HT by 40%, enhanced the abstinence-like effects of naloxone in the morphine pretreated ileum. PCPA by itself changed the responsiveness of the non-morphinized ileum to naloxone. The direction and magnitude of the change produced by PCPA alone was roughly equivalent to that produced by the serotonin depletor in the morphinized ileum. This finding indicates that PCPA has no effect upon the development of physical dependence in the isolated ileum. It remains to be determined whether or not the increased sensitivity to naloxone induced by high doses of PCPA has something in common with the changes in responsiveness to the antagonist induced by narcotics.     

Effects of chronic treatment with atypical neuroleptics on the biosynthesis and release of opioid peptides in guinea-pig ileum

The guinea-pig ileum myenteric plexus is known to contain opioid peptides, which can be released by electric stimulation at high frequency. Haloperidol, a classic neuroleptic drug, increases the biosynthesis and release of endogenous opioid peptides from the myenteric plexus. In the present work we have examined the effects of chronic treatment with sulpiride and clozapine, two atypical neuroleptics, on the release of these peptides in the myenteric plexus of guinea-pig ileum. Both neuroleptics, administered over a period of 7 days, produced an increase of the inhibitory response obtained by electrical stimulation at 10 Hz of the ileum myenteric plexuslongitudinal muscle preparation. The inhibitory response was reversed by the specific opioid antagonist naloxone, which suggests that the increase in the inhibitory response produced by blocking the dopaminergic receptors is mediated by an increase in the release of opioid peptides. When sulpiride- or clozapine-treated guinea-pigs received cycloheximide (an inhibitor of peptide biosynthesis) there was a significant decrease of the inhibitory response, which indicates that neuroleptics produced an increase of the synthesis of opioid peptides in the ileum myenteric plexus. These results reveal a possible influence of the dopaminergic system on the biological turnover of these peptides.     

Stereospecific increase by narcotic antagonists of evoked acetylcholine output in guinea-pig ileum.

In the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum, naloxone (30–100 nM) increases the output of acetylcholine evoked by electrical field stimulation at 0.017 Hz and to a lesser extent also at 10 Hz. The stereospecific requirements for this effect were studied with three pairs of optical isomers of antagonists of the benzomorphan series. The (−)-isomer of β-9-methyl-5-phenyl-2-allyl-2′-hydroxy-6,7-benzomorphan (GPA 1843) which had no agonist activity, had an effect similar to naloxone whereas the (+)-isomer was inactive in this respect. The (−)-isomer of antagonists with even weak agonist activity gave variable results. It is assumed that naloxone antagonises the action of enkephalin which has been shown to be present in the guinea-pig ileum. It is recommended to establish the stereospecificity of an antagonist action in order to exclude pharmacological effects not due to interaction with opiate receptors.     

Response to opioids of isolated small intestine in the house musk shrew, Suncus murinus.

1. Response to the opioids of isolated small intestine of Suncus murinus, an insectivore, was examined and compared with that of guinea-pig ileum. 2. The mechanical response to morphine and U50,488H, preferential mu- and kappa-agonist respectively, was relaxation which was antagonized by tetrodotoxin and naloxone in the Suncus small intestine. 3. Methionine5-enkephalin and D-alanine2, D-leucine5-enkephalin, both preferential delta-agonists, elicited contraction or relaxation in the Suncus small intestine, while enkephalins elicited sustained contraction in the tetrodotoxin-pretreated intestine. 4. In the guinea-pig ileum, the response to morphine and enkephalins was consistently relaxation which was antagonized by tetrodotoxin and naloxone. 5. Morphine, D-alanine2,D-leucine5-enkephalin and U50,488H inhibited electrically-evoked twitch responses of intestine in a concentration-dependent manner in both animals. 6. The relative potencies calculated from IC50 values of opioids for twitches followed the order, D-alanine2,D-leucine5-enkephalin greater than U50,488H greater than morphine in the Suncus small intestine, whereas U50,488 greater than D-alanine2,D-leucine5-enkephalin greater than morphine in the guinea-pig ileum. 7. Suncus small intestine has unique features such that D-alanine2,D-leucine5-enkephalin was most potent in inhibiting electrically-evoked twitches and that enkephalins induced contraction following tetrodotoxin pretreatment.     

The development of opiate tolerance may dissociate from dependence

Experiments on opiate sensitive peripheral tissue preparations such as the mouse vas deferens and the guinea-pig ileum have demonstrated the ability to induce very high degrees of selective tolerance towards particular opiate agonists. Interestingly, the highly tolerant mouse vas deferens failed to display any sign of dependence as judged by the inability of naloxone to precipitate a withdrawal sign. In analogy, the present studies on the guinea-pig ileum revealed a striking dissociation in the degree of tolerance and dependence developed. Opiate receptor binding studies on both tissues point to distinct differences in the opiate-induced effector mechanisms. It is concluded that adaptational changes upon chronic opiate receptor activation may occur at multiple sites within the effector system of the opiate receptor.     

Inhibition of peristaltic activity in the guinea-pig ileum by specific stress stimulus; its reversal by naloxone and indomethacin

During continuous peristaltic reflex activity of the isolated guinea-pig ileum a model stress stimulus, elevated intraluminal pressure (120 mm H₂O) plus increased longitudinal tension (3 g) was applied for 2 min. The resulting inhibition of peristalsis outlasted the initial stimulus by several min. The inhibitory interval was shortened or abolished in the presence of naloxone (0.5 μM), an opiate receptor antagonist, or in the preparations made acutely tolerant to morphine. This seems to suggest an involvement of endorphins. An inhibition of endogenous prostaglandin synthesis by indomethacin (5 μM) decreased the amplitude of peristaltic longitudinal muscle contractions, and these contractions were increased in response to the stress stimulus in the presence of naloxone. Thus the response of the guinea-pig ileum to stress stimulation could be profoundly modified by an interference with endorphin and prostaglandin systems.     

Yohimbine reduces morphine tolerance in guinea-pig ileum

Opiates are known to inhibit electrically-evoked twitches of longitudinal muscle-myenteric plexus strips from guinea-pig ileum. When this preparation was incubated with morphine for 1 h tolerance developed to the inhibitory effect, since dose-response curves were shifted to the right. In the present study, the effects of alpha-2 adrenergic agents on the tolerance induced by morphine in this preparation was investigated. Addition of yohimbine 10 microM (but not 0.1 or 1 microM) to the incubating medium reduced the magnitude of opiate tolerance. This effect did not appear in the presence of the alpha-2 agonists clonidine or guanfacine (10 microM). Our results provide evidence of the longitudinal muscle-myenteric plexus as a useful model for the study of the relationship between morphine tolerance and alpha-2 adrenergic mechanisms.     

Effects of beta-endorphin, met-enkephalin and somatostatin on the neurotensin-induced neurogenic contraction in the guinea-pig ileum

At maximally effective concentrations, the opiate peptides β-endorphin (240 nm) and Met-enkephalin (1400 nM) virtually abolished the contractions induced by a maximally effective concentration of 60 nM neurotensin (NT), either in the longitudinal smooth muscle strip or in the intact segment of guinea-pig ileum. This inhibitory effect was concentration-dependent and was totally blocked by naloxone at 100 nM. In contrast a maximally effective concentration of somatostatin (60 nM) partially inhibited (50–60%) the contraction induced by 60 nM NT in either smooth muscle preparation. Somatostatin inhibition was concentration-dependent and was not blocked by naloxone at 100 nM. Atropine at 100 nM inhibited by 50% the contractions induced by 60 nM NT in the intact segment of guinea-pig ileum. The remaining contraction was abolished by β-endorphin and Met-enkephalin and partially reduced by somatostatin. Our results confirm that NT-induced contractions in the guinea-pig ileum are neurogenic and involve a cholinergic as well as a non-cholinergic component. Furthermore, we show that the release of mediators from both components     

Antinociceptive effect of 7-hydroxymitragynine in mice: Discovery of an orally active opioid analgesic from the Thai medicinal herb Mitragyna speciosa

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa. We previously reported the morphine-like action of mitragynine and its related compounds in the in vitro assays. In the present study, we investigated the opioid effects of 7-hydroxymitragynine, which is isolated as its novel constituent, on contraction of isolated ileum, binding of the specific ligands to opioid receptors and nociceptive stimuli in mice. In guinea-pig ileum, 7-hydroxymitragynine inhibited electrically induced contraction through the opioid receptors. Receptor-binding assays revealed that 7-hydroxymitragynine has a higher affinity for micro-opioid receptors relative to the other opioid receptors. Administration of 7-hydroxymitragynine (2.5-10 mg/kg, s.c.) induced dose-dependent antinociceptive effects in tail-flick and hot-plate tests in mice. Its effect was more potent than that of morphine in both tests. When orally administered, 7-hydroxymitragynine (5-10 mg/kg) showed potent antinociceptive activities in tail-flick and hot-plate tests. In contrast, only weak antinociception was observed in the case of oral administration of morphine at a dose of 20 mg/kg. It was found that 7-hydroxymitragynine is a novel opioid agonist that is structurally different from the other opioid agonists, and has potent analgesic activity when orally administered.     

Tyr-MIF-1 and hemorphin can act as opiate agonists as well as antagonists in the guinea pig ileum.

The brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) was tested for its effects on electrically stimulated contractions in the guinea pig ileum assay. Tyr-MIF-1 acted as an opiate agonist in reducing these contractions. Its IC50 was about 9 microM, and its effects were reversed by naloxone and CTOP. The ability of Tyr-MIF-1 also to antagonize the inhibitory effects of opiates on electrically stimulated contractions was more evident in the ileum removed from a guinea pig tolerant to morphine or after partial inactivation of opiate receptors with beta-CNA. Similar results were observed with hemorphin. The endogenous peptide Tyr-MIF-1 and the blood-derived peptide hemorphin, therefore, can act as agonists as well as antagonists in the guinea pig ileum. The effects as antagonists are best observed in preparations of ileum with reduced receptor reserve (tolerant or beta-CNA treated) and are consistent with the idea that properties of endogenous peptides as opiate antagonists are enhanced in the tolerant state.     

Bremazocine: a potent, long-acting opiate kappa-agonist

The benzomorphan analogue bremazocine is a potent, centrally-acting analgesic with a long duration of action. In animal models it is free of physical and psychological dependence liability, produces no respiratory depression, and has a variety of other properties which justify its classification as a putative opiate kappa-receptor agonist.Binding studies with tritiated (?)-bremazocine on rat brain membrane preparations show that this molecule differs in its binding properties from previously investigated exogenous or endogenous opioids. Studies on isolated guinea-pig ileum and mouse vas deferens indicate a preference for opiate kappa-receptors.In mice (hot plate, tail flick) and rhesus monkeys (shock titration), bremazocine is a potent analgesic with a long duration of action. Here also, the actions of the antagonists naloxone and Mr 2266 suggest a preference for opiate kappa-receptors.Bremazocine differs from morphine in the non-production of mydriasis and the Straub tail phenomenon in mice, in its lack of effects on respiration in rats, in that it is not self-administered by rhesus monkeys, and in that programmed administration in the same species does not lead to a morphine-like withdrawal syndrome upon cessation of drug treatment or upon naloxone challenge. Prolonged treatment of animals with bremazocine leads to tolerance to its analgesic effects; morphine treatment of such tolerant animals causes analgesia. Conversely, treatment of morphine-tolerant animals with bremazocine does not cause analgesia; these findings suggest that morphine and bremazocine interact with different subpopulations of opiate receptors.     

Adenosine receptors are involved in the control of acute naloxone-precipitated withdrawal: in vitro evidence

The effects exerted by adenosine A1 and A2 receptor agonists and antagonists on the acute opiate withdrawal induced by morphine were investigated in vitro. Following a 4 min in vitro exposure to morphine, the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. The P1 adenosine receptor agonist, adenosine, was able to reduce dose-dependently naloxone-precipitaded withdrawal. The same effect was induced by the adenosine A1 receptor agonist, N6-Cyclopentyladenosine (CPA) whereas the selective adenosine A2A receptor agonist CGS 21680 increased the naloxone-precipitated withdrawal phenomenon. Dipyridamole, a blocker of adenosine reuptake, induced a significant reduction of morphine dependence. Caffeine, an adenosine receptor antagonist, significantly increased the naloxone-precipitated withdrawal effect in a concentration dependent manner. The same effect was observed with 8-phenyltheophylline (8PT), an A1 adenosine receptor antagonist, whereas 3,7-dimethyl-1-propargylxanthine (DMPX), an A2 adenosine receptor antagonist, reduced the naloxone-precipitated withdrawal phenomenon. The results of our experiments indicate that both A1 and A2 adenosine receptor agonists and antagonists are able to influence opiate withdrawal in vitro, suggesting an important functional interaction between the adenosine receptors and opioid withdrawal.     

Purinoreceptors are involved in the control of acute morphine withdrawal.

The effects exerted by P1 and P2 purinoceptor agonists and antagonists on the acute opiate withdrawal induced by morphine were investigated in vitro. Following a 4 min in vitro exposure to morphine, the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. The P1 purinoceptor agonist, adenosine, was able dose-dependently to reduce morphine withdrawal whereas alpha,beta-methylene ATP (APCPP), a P2 purinoceptor agonist, increased morphine withdrawal. Caffeine, a P1 purinoceptor antagonist, was able significantly and in a concentration dependent manner to increase morphine withdrawal whereas quinidine, a P2 receptor antagonist, reduced it. The results of our experiments indicate that both P1 and P2 purinoceptor agonists and antagonists are able to influence opiate withdrawal in vitro, suggesting an important functional interaction between the purinergic system and opioid withdrawal.     

The development and reversal of the tolerance to morphine in the longitudinal smooth muscle-myenteric plexus preparation of the guinea pig

Chronic treatment with morphine results in a reduction in the potency of morphine in the longitudinal smooth muscle-myenteric plexus of the guinea-pig ileum. Implantation of morphine pellets leads to the development of tolerance to the inhibitory effects of morphine upon neurogenic contractions of this preparation. Tolerance develops within 24 hours, peaks between days 4 and 7 and disappears by day 14. A similar time course for the development of tolerance to the inhibitory effects of 2-chloroadenosine is also seen in these same morphine-tolerant preparations. The rate of reversal of morphine tolerance was assessed after the removal of the morphine pellets four days after implantation. In this situation, tolerance to the effects of morphine were maintained for at least 24 hours, were partially reversed at day 2 and were totally reversed by day 4. The delay in the development and reversal of the effect are consistent with the fact that chronic treatment with morphine evokes an adaptive sensitivity change.     

Characterization of nicotine-induced contraction in the canine bronchus

1. The modes of action of nicotine on the dog bronchial smooth muscle preparation was investigated, in order to compare with those on the bronchial preparations from the guinea-pig, rabbit and monkey. 2. Nicotine induced a contraction in the dog bronchial preparation, and this response was abolished by hexamethonium and atropine and potentiated by physostigmine. 3. These findings suggest that the contractile response to nicotine was mediated through an action on the nicotinic receptors and due to the release of acetylcholine. 4. Tetrodotoxin did not inhibit the contractile response to nicotine in the dog bronchial preparation, suggesting that the nicotine-induced response may be produced mainly through a sodium action potential-independent process. 5. The present observations in the dog bronchial preparations coincided with those in the rabbit and monkey bronchi but not with the findings in the guinea-pig bronchus.     

Action of morphine on guinea-pig myenteric plexus and mouse vas deferens studied by intracellular recording.

Morphine reduces the output of transmitter from the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum and from the mouse vas deferens. Intracellular recordings were made from ganglion cells of the myenteric plexus and smooth muscle cells of the vas deferens. Synaptic transmission within the myenteric plexus was blocked by hexamethonium. Morphine did not change the properties of the ganglion cells, nor did it affect synaptic potentials. 5-Hydroxytryptamine inhibited acetylcholine release at intraganglionic synapses by an action which was unaffected by morphine. In the vas deferens, excitatory junction potentials were elicited by stimulation of postganglionic adrenergic nerve fibres. The junction potentials were depressed by morphine and levorphanol but not by dextrorphan. This depression was reversed by naloxone. The results indicate that morphine acts directly to reduce transmitter release at the neuro-effector junctions in the myenteric plexus-longitudinal muscle preparation and in the vas deferens in these species.     

Further evidence that naloxone acts as an inverse opiate agonist: Implications for drug dependence and withdrawaL

To test if naloxone behaved as an inverse agonist rather than as an antagonist we evaluated its responses in guinea-pig ilea with and without morphine (480 nM, 24 h). In control ilea, naloxone (100 nM) had no effect. In morphine-treated ilea, naloxone as a bolus, but not as an infusion, elicited an abstinence response. Preadministration of naloxone blocked the response to subsequent administrations. Similarly, naloxone failed to produce an abstinence response in ilea pretreated with kappa compounds (bremazocine, U50488 or xorphanol 100 nM) or with kinase inhibitors (H7 or H8 30 μM). These findings can be interpreted in the light of the two-state receptor model if naloxone behaves as an inverse agonist: Incubation with morphine increased the active state of receptors making them susceptible to the inverse agonist (naloxone); exposure to naloxone favored the inactive conformation making them insensitive to further administration of naloxone; kappa compounds behaved as antagonists preventing the response to naloxone; and kinase inhibitors interfered with the active conformation making the system insensitive to naloxone. According to this model, dependence can be viewed as an overexpression of the active receptors and withdrawal as an abrupt change from the active to the inactive state.     

Interaction between morphine and putative excitatory neurotransmitters in cortical neurons in naive and tolerant rats.

Microelectrophoretically applied morphine depressed spontaneously discharging cortical neurones of rats and blocked excitation induced by electrophoretic administrations of either acetylcholine or l-glutamate. This depressant effect and both the anti-acetylcholine and the anti-glutamate effect were naloxone antagonizable and therefore regarded as specific morphine actions. The excitatory effects of morphine were not affected by naloxone application and were classified as non-specific.In chronically morphinized rats the depressant effect of morphine on spontaneous discharge activity and also its blocking action upon acetylcholine and l-glutamate-induced excitation were almost completely abolished. The predominant response in such pre-treated animals was non-specific excitation. Acetylcholine and l-glutamate were found to be more effective in tolerant rats (supersensitivity).     

Naloxone potentiation of apomorphine-induced stereotypic climbing in mice and interaction with mu-, sigma- and kappa-opiate drugs

Pretreatment with the narcotic antagonist naloxone produced a dose-related potentiation of mouse stereotypic climbing behavior induced by the dopaminergic agonist apomorphine. In further experiments, mice were also pretreated with various drugs specific for mu-opiate receptors (morphine), sigma-opiate receptors (N-allylnormetazocine) and kappa-opiate receptors (ketocyclazocine). Doses of morphine that alone did not affect apomorphine-induced climbing antagonized naloxone potentiation of apomorphine. Doses of N-allylnormetazocine that did not influence apomorphine stereotypy also reversed naloxone potentiation of apomorphine. On the other hand, ketocyclazocine alone exerted a behavioral suppressant effect upon apomorphine- induced stereotypic climbing, however, these same doses failed to prevent naloxone potentiation of apomorphine.