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Nanomolar concentrations of neurotensin caused a dose-dependent contraction of the longitudinal muscle layer of the guinea-pig ileum. The contractile activity of neurotensin was partially blocked by tetrodotoxin or atropine, indicating that a component of the neurotensin-mediated contraction is indirect in nature and likely involves the release of endogenous acetylcholine from nervous terminals in the myenteric plexus. Dynorphin and related peptide fragments also blocked in part the neurotensin contraction; the potency of this opioid peptide was about the same as that of atropine. Other peptides and alkaloids tested for ability to block the neurotensin contractures included the enkephalins, beta-endorphin, normorphine and the ketocyclazocines; all these opioids inhibited in a dose-dependent fashion the neuronal component of the excitatory effect of neurotensin. The potency of these compounds to reduce the contractions of neurotensin showed good correlation with the potency of these agents to depress by 50% the electrically evoked neuromuscular twitches in the same tissue (r = 0.99); in these tests dynorphin was found to be the most potent of the endogenous opioid-like peptides. The dynorphin blockade was selective to the excitatory effect of neurotensin because the opioid peptide did not antagonize the contractile action of acetylcholine, histamine, substance P, angiotensin II, bradykinin, Ba++ or K+ ions. In addition, somatostatin, vasointestinal peptide, gastrin or adenosine did not modify the potency of neurotensin whereas thyrotropin releasing hormone and epinephrine caused a modest doubling of the neurotensin EC50. The inhibitory action of dynorphin was reduced in the presence of naloxone, suggesting that the interaction involved opiate receptors. Morphine tolerance was not extended to the inhibitory action of dynorphin as evidenced by the finding that the potency of dynorphin-(1-13) to block the neurotensin responses was increased after chronic morphine exposure. In contrast, the potency of dynorphin-(1-13) was significantly reduced in tissues rendered tolerant to the action of ketocyclazocine or ethylketocyclazocine, suggesting that the action of dynorphin could be partially mediated via occupation of K-opiate receptors. Thus, a cholinergic-neuronal component activated by neurotensin on the myenteric plexus appears to be under the inhibitory influence of opiate receptors, suggesting that dynorphin may play a role in the modulation of cholinergic synapses on the enteric nervous system.

Four hours after the acute administration of L-tryptophan (75 mg/kg) to either, nontolerant or morphine-tolerant mice, the antinociceptive effect of morphine was partially and significantly antagonized. Daily tryptophan administration to rats and mice during a 3-day morphine pellet implantation period increased the rates of both morphine tolerance development and development of physical dependence. The accelerating effect of tryptophan on tolerance and dependence development in mice was antagonized by pretreatment with the tryptophan hydroxylase inhibitor, p-chlorophenylalanine. Acute tryptophan administration (75 mg/kg) significantly increased mouse brain 5-hydroxytryptamine levels for at least 4 hours. Although chronic tryptophan treatment increased 5-hydroxytryptamine turnover in morphine-treated mice, no effect of chronic morphine or tryptophan treatment on the particulate tryptophan hydroxylase activity of whole mouse brain was observed. Slight increases in tryptophan hydroxylase activity were observed in the caudate-putamen and septal areas of rat brain 3 and 6 days, respectively, after s.c. morphine pellet implantation. These and previous studies from our laboratory indicate that the development of morphine tolerance and dependence can be modified by agents affecting serotonergic mechanisms.

The aim of this investigation was to determine the brain regions which were most sensitive to the inhibitory effects of morphine on the shaking response of pentobarbital-anesthetized rats to ice water. The median inhibitory dose (ID50) of morphine sulfate administered intraventricularly was found to be 0.35 mug/rat. When morphine was bilaterally injected into different regions of the brain, the ID50 values ranged from 0.04 to 17.9 mug/rat. The lowest ID50 values (0.04-0.20 mug) were found in the periaqueductal gray, the medial preoptic area and the locus ceruleus. The ID50 values ranged from 0.65 to 1.6 mug for areas around the nucleus accumbens, the fasciculus retroflexus, the medical thalamus and the septal area; from 5.6 to 7.3 mug for various hypothalamic nuclei; and from 11.0 to 17.9 mug for the basal ganglia, reticular formation substantia nigra and the reticular nucleus of the thalamus. The brain areas with the lowest ID50 values are known to have thermoregulatory functions. The similarity of the shaking response to shivering is discussed. It is concluded that the central inhibitory effects of morphine on shaking are subserved by discrete neuroanatomical substrates located in medial subcortical structures.

The analgesic effect of morphine was antagonized in mice by intracerebroventricular injection of Ca++, Mg++ and Mn++ and was potentiated by ethylene glycol tetraacetic acid but was not altered by Sr++, Ba++, Ni++, Hg++, Cd++ or ethylenediamine tetraacetic acid. The antagonistic effect of Ca++ was not altered by pretreatment with pargyline or 6-hydroxydopamine indicating that altered release of catecholamines or serotonin was not involved in this action of Ca++. Induction of morphine tolerance by pellet implantation also did not alter the antagonistic effect of Ca++. The antagonistic effects of Ca++ and naloxone were additive in both nontolerant and tolerant animals and the apparent affinity of naloxone for its receptors, as estimated by in vivo pA2 determinations, was not altered by Ca++. However, the ionophore X537A was found to increase greatly the narcotic antagonist effect of a low dose of Ca++ although the ionophore alone did not alter the effects of morphine. This indicates that Ca"++ must penetrate cell membranes in order to reduce the analgesic effects of morphine. These findings indicate the importance of Ca++ localization in the actions of narcotic agonists and antagonists.

Naloxone hydrochloride, an opiate antagonist, administered via the intracranial or parenteral route precipitates shaking behavior in the morphine-dependent rat. We made localized bilateral injections of naloxone HCl, 1.5 mug/rat, into 60 subcortical sites of the pentobarbital-anesthetized, morphine-dependent rat and found that two circumscribed areas of the brain, the medial hypothalamus and the periaqueductal-4th ventricular spaces, were selectively sensitive to naloxone-precipitated shaking. In the nondependent rat, morphine injections into the anterior diencephalon inhibited the shaking response to ice water; injections of morphine into the medial diencephalon were less effective. However, naloxone antagonized the morphine-inhibited shaking more effectively when injections of naloxone were made in the medial diencephalon than when injections were made in the anterior diencephalon. These results suggest that the reciprocal relationship of morphine and morphine-naloxone effects on shaking behavior may be regulated by topographically different structures in the diencephalon.

In mice implanted with a morphine pellet for 3 days and subsequently withdrawn for various periods of time, a single injection of morphine results in a rapid and marked sensitization of abstinent mice to naloxone-induced jumping behavior when compared to abstinent mice pretreated with saline or to placebo-withdrawn mice given a single morphine injection. Maximum sensitization of abstinent mice occurs at morphine doses of about 10 to 30 mg/kg and peaks at 0.5 to 2 hours after morphine administration. The ability of morphine to sensitize abstinent mice to naloxone declines with time after pellet removal and appears to consist of two components, one with a short T1/2 and one with a much longer T1/2. Abstinent mice are also sensitized to naloxone by levorphanol and methadone, but not by dextrorphan. It is proposed that the administration of a narcotic drug to abstinent mice uncovers a latent, pre-existing state of physical dependence and restores the responsiveness of this state to naloxone.

Intracerebroventricularly (i.vt.) administered lanthanum chloride was found to produce antinociceptive effects as measured by the mouse tail-flick and hot plate tests and the rat tail-flick test. These antinociceptive effects were reduced by peripheral administration of the narcotic antagonist naloxone or by i.vt. administration of calcium chloride. In addition, animals made tolerant to morphine were also tolerant to the effects of La+++. In morphine-dependent mice, the incidence of both abrupt and naloxone-precipitated withdrawal jumping was reduced by La+++ administration. In view of the known inhibitory effects of La+++ on Ca++ binding and movement, these findings suggest that alterations in Ca++ localization may be involved in the analgetic action of narcotic drugs as well as the development of narcotic tolerance and dependence.

Unilateral lesions of the substantia nigra zona compacta (SNC) in rats were produced by electrolytic coagulation or by an injection of 6-hydroxydopamine. Two to 5 weeks later, after being preselected for amphetamine-induced ipsilateral circling behavior, the animals were administered narcotic agonists or antagonists and their circling behavior was observed. Morphine, methadone, levorphanol, nalorphine or pentazocine induced ipsilateral circling movements; both naloxone and dextrorphan were without effect. Ipsilateral circling was also observed in rats with unilateral electrolytic lesions after administration of agents that are thought to enhance central dopaminergic activities: d-amphetamine, l-dopa and apomorphine. In rats with unilateral electrolytic or 6-hydroxydopamine SNC lesions that were rendered highly morphine-dependent by multiple-morphine pellet implantation, contralateral (C) circling behavior was observed within 1 to 2 minutes after a naloxone challenge; the onset and duration of C circling behavior coincided with the initial appearance and duration of precipitated-morphine withdrawal signs. C circling was also observed after administration of putative dopamine receptor blockers, haloperidol and pimozide in rats with either unilateral electrolytic or 6-hydroxydopamine SNC lesions. Morphine pretreatment diminished both the C circling intensity and the appearance of withdrawal signs observed after a naloxone challenge in morphine-dependent, SNC-lesioned rats. The naloxone-precipitated withdrawal in unilaterally lesioned morphine-dependent rats was accompanied by a 20% elevation of neostriatal dopamine in the intact side. In contrast to the effects of a chronic SNC lesion in decreasing neostriatal dopamine, a 77% increase was observed in the lesioned side 30 minutes after electrolytic coagulation. Thus, narcotic agonists and partial agonists may enhance central dopaminergic activities and naloxone-precipitated withdrawal may involve a diminution in central dopaminergic activities of the nigroneostriatal pathway.

In mice rendered morphine-dependent by pellet implantation for 3 days, the administration of pargyline 6 hours after pellet removal intensified narcotic abstinence behavior, particularly the narcotic withdrawal jumping response. Pargyline, 75 mg/kg i.p., caused a 6- to 9-fold increase in the incidence of jumping in mice withdrawing from morphine 6 hours after removal of the pellet, whereas this effect was not observed: 1) 1 hour after the injection of pargyline or 2) in animals still implanted with the morphine pellet. The median effective dose (ED50) of pargyline required to elicit withdrawal jumping in mice implanted with morphine decreased with increasing physical dependence. The ED50 for 72 hours was about one-sixth that after 24 hours of implantation. Additionally, pargyline potentiated naloxone-precipitated withdrawal jumping as evidenced by a reduction of the naloxone ED50 by approximately one-half. Administration of other monoamine oxidase inhibitors such as pheniprazine, iproiazid or tranylcypromine failed to alter the indicence of jumping in dependent mice undergoind abrupt morphine with drawal. Further, dopamine receptor stimulation by amphetamine, pheniprazine or amantadine antagonized the pargyline-induced jumping response. These data suggest that the increased incidence of withdrawal jumping observed after pargyline in morphine-dependent mice is not related to monoamine oxidase inhibition but rather to a possible pargyline-induced decrease in dopaminergic activity.

The role of cerebroside sulfate in opiate action and binding was studied by examining the effect of decreasing the availability of the glycolipid in vivo on morphine analgetic activity and receptor affinity. Available cerebroside sites were decreased either by injecting Azure A at a dose with selective high affinity for sulfo-lipids, or by using "jimpy" mice, a genetic leukodystropic mutant mouse with a deficiency in brain sulfatides. Injections of Azure A (4.4 MG/KG) intracerebroventricularly produced a 2-fold increase in the AD50 of morphine (tail-flick test) within 2 hr. The genetic mutation also resulted in a decreased sensitivity to morphine; the morphine AD50 was 6- to 11-fold higher in jimpy mice than in their normal littermates. Azure A produced a dose-dependent inhibition of opiate binding to synaptosomal plasma membranes. There was 85% inhibition of [3H]morphine binding with 0.2 micrometer Azure A, and 40% inhibition of [3H]naloxone binding with 5 micrometer Azure A. The inhibition of morphine binding was competitive and was demonstrated to be different from that elicited by Na+. Synaptic membranes from jimpy mice exhibited a decrease in number of binding sites for morphine. Binding of Azure A to cerebroside sulfate, associated with or in the proximity of the receptor sites, was suggested by the fact that in the presence of 0.2 micrometer Azure A, no inhibition of [3H]morphine binding to synaptosomal plasma membranes of jimpy mice could be demonstrated whereas 31% inh-bition was observed in membranes from control littermates. Based on the findings, it is concluded that cerebroside sulfate strategically located may have a role in binding morphine and mediating its effects.

The effect of morphine on the uptake of 45Ca++ was studied in synaptosomes from mouse brain using two procedures, centrifugation and filtration. The addition of morphine (1.7 x 10(-7) or 3.4 x 10(-7) M) reduced 45CA++ uptake by either technique, although the basal 45Ca++ uptake by the filtration method was approximately 7-fold higher than that by the centrifugation procedure. Similar effects were obtained after acute morphine treatment with 10 mg/kg s.c. Previous naloxone in vitro treatment (1.9 x 10(-8) M) or in vivo administration (2 mg/kg s.c.) reversed the morphine inhibition of the 45Ca++ uptake. On the other hand, after the animal was rendered tolerant and dependent by morphine pellet implantation, an enhancement of the synaptosomal 45Ca++ uptake was observed. It is concluded that changes in Ca++ fluxes in synaptosomes observed after acute and chronic morphine treatment may be involved with morphine pharmacological action related with analgesia, tolerance and physical dependence.

Levels of brain acetylcholine and choline were measured with a gas chromatograph in Swiss-Webster or ICR mice and Sprague-Dawley rats: a) 30 minutes after various single doses of morphine sulfate s.c.; b) rendered highly tolerant to and dependent on morphine by pellet implantation for 3 days; c) at various times after abrupt withdrawal (pellet removal); or d) during abstinence precipitated by the narcotic antagonist naloxone. The specific activity of brain acetylcholinsterase was determined in treatment d. Brain choline levels generally remained unaffected by the above manipulations. It was found that analgetic doses of morphine did not alter the steady-state levels of brain acetylcholine, but slight increases were observed after high doses and in morphine-tolerant animals. Abrupt withdrawal of morphine in the mouse caused a significant increase in brain acetylcholine levels, which was observed at 6 hours but not at 12 and 24 hours. In rats, abrupt withdrawal had no effect on acetylcholine levels at 6 and 18 hours. Nalonone-precipitated withdrawal significantly lowered the brain acetylcholine in both mice and rats without affecting acetylcholinesterase activity. This lowering was observed in animals that jumped after naloxone, but not in those failed to jump. It is concluded that this decrease in brain acetylcholine may be related to an increased neuronal release.

Mice of the DBA/2J, A/J, C3H, C57BL/L, ICR and Swiss strains were studied with respect to acute and chronic morphine administration. The acute administration of morphine resulted in a dose-dependent running response in C57BL/6, C3H, ICR and Swiss strains, but no running response in DBA/2J and A/J strains. Strain differences in sensitivity to morphine-induced running activity did not parallel differences in sensitivity to morphine-induced running activity did not parallel differences in sensitivity to antinociception in the abdominal constriction test (r = 0.404), but significantly correlated with differences in the expression of physical dependence as measured by either precipitated (r = 0.957) or abrupt (r = 0.927) withdrawal jumping behavior in mice made dependent by morphine pellent implantation. Mortality for 3 days of pellet implantation ranged from 5% in ICR mice to 84% in A/J mice. Strain differences in degree of initial physical dependence. These results suggest the possibility that the running response and withdrawal jumping may involve at least part of the same neuronal pathway. This pathway may include dopamine-containing neurons which terminate in the neostriatum.

Acute subcutaneous injection of 25 mg/kg of morphine in rats decreased synaptosomal CA++ levels by 29% without altering the Ca++ content of other subcellular fractions. In contrast, chronic morphine treatment of mice or rats by pellet implantation selectively increased synaptosomal Ca++ levels by almost 100%. This increase in Ca++ induced by morphine was blocked by simultaneous chronic administration of naloxone. The binding of low concentrations (10(-7)-10(-5) M) of 45Ca++ to synaptic plasma membranes was increased by acute morphine treatment and decreased by chronic administration. The binding of higher concentrations (10(-3) M) of 45Ca++ to synaptic vesicles was also increased by acute morphine treatment and decreased by chronic treatment. Changes in binding were not observed with other subcellular fractions. It is suggested that these highly selective changes in Ca++ levels and binding may represent mechanisms by which acute morphine treatment interferes with synaptic transmission and by which chronic administration overcomes these effects, resulting in tolerance and dependence.

The effect of morphine on the uptake of 45Ca++ was studied in lysed synaptosomes obtained from homogenates of whole mouse brain. The addition of morphine, 10(-6) M, to the incubation medium or acute administration of 10 or 20 mg/kg s.c. resulted in a decrease in 45Ca++ uptake; this decrease was observed only in the presence of ATP (3 mM). In contrast, after morphine pellet implantation (72 hr) to induce tolerance and physical dependence, an enhancement of lysed synaptosomal 45Ca++ uptake occurred; the increase was obtained in the presence but not in the absence of ATP. The enhancement of Ca++ uptake appears to be related with the degree of tolerance and dependence development since a linear relationship was noted between the time of morphine pellet implantation and the increase in 45Ca++ uptake by lysed synaptosomes. The acute inhibitory action on 45Ca++ uptake by morphine was prevented in vitro by naloxone, 1.9 x 10(-8) M, and in vivo by 2 mg/kg of naloxone s.c. and the chronic enhancing action of morphine by the simultaneous implantation of a naloxone pellet with the morphine pellet. The present findings lend further support to our previous reports in which we suggest that alterations in Ca++ flux may be involved with morphine analgesia, tolerance and physical dependence.

Using a modified tail-flick procedure we have found a highly significant hyperalgesic response in narcotic dependent mice peaking 12 hr following the removal of chronically implanted morphine pellets. Withdrawal-induced hyperalgesia correlated well with other signs of opiate withdrawal behavior. Intracerebroventricular injections of CaCl2, MnCl2 further enhanced the hyperalgesic response in morphine-dependent mice; morphine-dependent mice were more than twice as sensitive to calcium-induced hyperalgesia as placebo-treated controls. On the other hand, i.c.v. injections of the calcium-specific chelator EGTA produced highly significant, dose-dependent antinociceptive responses in both morphine-dependent and control mice, but morphine dependent mice were only half as sensitive to EGTA-induced analgesia as controls. Withdrawal hyperalgesia and EGTA analgesia may be directly related to changes in brain localization of calcium that have been reported previously; it is concluded that morphine and EGTA-induced analgesia may be associated with a calcium depleted state within a relatively small calcium pool of the nerve cell and that opiate withdrawal hyperalgesia is a sensitive measure of narcotic dependency that is likely associated with an increased Ca++ content in the same region.

The inhibitory activity of a variety of opioid peptides was tested on the electrically stimulated rat vas deferens. Human beta-endorphin (beta h-EP) was the most potent of the opoioid-like peptides; it produced half-maximal inhibition of the neuromuscular twitching at a concentration of about 100 nM. The potency of beta h-EP was greater than that of camel, porcine, ovine or leucine5 beta h-EP, alpha-N-acetyl beta-ovine-EP was inactive. In contrast to beta-endorphin (beta-EP), methionine and leucine-enkephalin, dynorphin-(1-13), morphine and other narcotic analgesics were devoid of opioid-like activity. Fragments of beta-EP with amino acid deletions at the carboxy end of the molecule were considerably less potent than the parent compound. The fragment beta h-EP 1-21 was 70 times less potent than beta h-EP, whereas the segments beta h-EP 1-19 and beta h-EP 1-16 were both completely inactive. Deletions at the amino terminal of beta-EP yielded inactive compounds. The potency of beta h-EP was reduced in a dose-related fashion by applications of nanomolar concentrations of naloxone or N-allylnormetazocine and by micromolar concentrations of levorphanol or morphine. In decided contrast, opioid peptides such as methionine enkephalin, dynorphin-(1-13), beta-casomorphan derivatives or short chain beta h-EP segments such as beta-EP 1-16, beta-EP 1-19, beta-EP 6-31, beta-EP (1-5)-(16-31), methionine enkephalin plus beta-EP 6-31 or the N-acetyl beta-bovine-EP did not antagonize the inhibitory action of beta h-EP. The present results demonstrate that the rat vas deferens contains opioid receptors with considerable selectivity for beta-EP. It is concluded from this structure activity relationship study that the activation of the beta-EP receptor involves at least two sites of recognition on the beta-EP structure.

The use of the guinea pig ileum (GPI) and mouse vas deferens (MVD) to quantify opiate tolerance and physical dependence in the same preparation was examined. In the GPI, tolerance to and physical dependence on various opiates were induced by direct incubation with an agonist and tolerance and abstinence were assessed after its rapid removal by extensive washing. Dependence, as evidenced by an increase in twitch height and after removal of the agonist as compared to that before incubation, provided an index of the degree of physical dependence. The extent of the supersensitive response after agonist removal was noted to be related directly to the agonist concentration and the degree of tolerance development. Furthermore, the supersensitive response was found to be stereospecific, naloxone reversible and preferentially mu receptor-mediated. Likewise, tolerance to opiates could be demonstrated in the MVD by direct incubation with an agonist. Unlike the GPI, rapid removal of the agonist by washing did not produce a supersensitive response to electrical stimulation. However, a supersensitive response was demonstrable in the presence of naloxone. Thus, after a tolerant/dependent state was induced by incubation of the vas with a predetermined dose of [D-Ala2, D-Leu5]enkephalin that completely abolished the twitch response, the subsequent addition of naloxone resulted in a supersensitive response to electrical stimulation that was concentration-dependent. The maximum twitch response in the presence of naloxone, compared to that obtained before incubation with the agonist, provided an index of the extent of dependence.(ABSTRACT TRUNCATED AT 250 WORDS)

The rate and degree of tolerance development of morphine, normorphine and methadone were assessed in vitro on the guinea-pig ileum. After the half-maximal concentration to inhibit electrically induced contractions (IC50) for each compound was determined, tolerance to a fixed concentration representing 0.5, 1 or 2 times its IC50 was induced by incubation at 37 degrees C for 1, 2 or 4 hr. The IC50 was then redetermined and the ratio of the IC50 after and before incubation provided a quantitative index of the degree of tolerance development to each agonist. For any given concentration or time, morphine induced the highest, whereas normorphine the intermediate and methadone the lowest degree of tolerance. Tolerance to opiates was associated with some degree of physical dependence as evidenced by the fact that the application of naloxone at the end of each experiment elicited a muscular contraction. Specificity of tolerance development to opiates was demonstrated by several experiments. Coincubation of morphine with naloxone under the same conditions resulted in an inhibition of tolerance development. The stereospecificity of the process was demonstrated by the fact that levorphanol and I-methadone induced a high degree of tolerance, whereas, under the same conditions the less active d-isomers, dextrorphan and d-methadone, did not. Moreover, subsensitivity to acetylcholine, norepinephrine and adenosine monophosphate did not develop in the presence of tolerance to morphine. The validity of this method for investigating mechanisms involved in tolerance and physical dependence was further demonstrated by obtaining data compatible with earlier experiments in vivo that cyclic AMP enhances tolerance development and cycloheximide inhibits this phenomenon. The present method should facilitate studies on the mechanisms involved in opiate tolerance and physical dependence development.