J. Bläsig

Development of physical dependence on morphine in respect to time and dosage and quantification of the precipitated withdrawal syndrome in rats

In rats implanted subcutaneously with morphine containing pellets different degrees of dependence were induced by varying the dosage, frequency of implantation and duration of exposure to morphine. Withdrawal was precipitated by intraperitoneal injection of morphine antagonists, mostly levallorphan. The absorption of morphine from the subcutaneous depots was estimated chemically.When withdrawal was precipitated with a constant dose of antagonist the frequency of occurrence of various counted signs and the presence of some checked signs were studied in respect to varying degrees of dependence. The results were compared to those obtained after administration of increasing doses of antagonist in groups of animals that had developed a constant degree of dependence.In both types of experiments the results were rather similar. Some signs became progressively more pronounced when dependence got stronger or the dose of the antagonist was increased. In contrast, other signs showed a maximal frequency at the lower degrees of dependence or after administration of the lower doses of antagonist and decreased or even disappeared when the degree of dependence was higher or the dose of antagonist further increased. Obviously, in withdrawal the intensity of “recessive” signs like writhing and wet dog shaking declines when “dominant” signs like jumping, flying (a vigorous kind of jumping) and teeth chattering increase. An inverse relationship between the occurrence of various signs could also be shown within the 30 min observation period. Changes in the integrative mechanisms controlling behaviour during withdrawal are supposed to be the reason for this shift of signs.In other experiments in which the interval between each morphine implantation was prolonged the frequency of some signs like jumping and teeth chattering tented to plateau. This finding seems to be correlated to some kind of steady state on resorption of morphine from the subcutaneous depots, as was found in chemical analysis.

Opioid activities of β-casomorphins

Abstract β-Casomorphin-7 (H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH) and its analogues: β-casomorphin-6, (-5) and (-4) (derived by sequential removal of respectively one, two or three amino acid residues from the C-terminus), were tested for their opioid activities in a variety of assay systems. Each of the four peptides displayed opioid activity in an opiate receptor binding assay, the isolated mouse vas deferens (MVD), the guinea-pig ileum longitudinal muscle myenteric plexus preparation (GPI) and produced naloxone-reversible analgesia after intracerebroventricular injection into rats. In contrast, none of the peptides displayed opioid activity in the isolated rat vas deferens preparation (RVD). β-Casomorphin-5 was the most potent compound in all the assays employed. Each β-casomorphin was more potent on the GPI than on the MVD. In view of the fact that the GPI, MVD and RVD are populated predominantly by μ-, δ- and e-receptors, respectively, the β-casomorphins probably represent μ-type opiate receptor agonists.

Involvement of endorphins in emotional hyperthermia of rats.

Abstract When rats were confronted with the novel experience of a new environment and stressful handling procedure their body temperature increased within minutes. At the same time β-endorphin-like immunoreactivity in the plasma increased dramatically. The stress-induced hyperthermia could be antagonized or reversed by the active, not however, by the inactive enantiomer of naloxone. The data provide evidence for a physiological role of endorphins.

Comparison of in vivo and in vitro parameters of opiate receptor binding in naive and tolerant/dependent rodents

Abstract In vivo and in vitro approaches were used to investigate a possible change in the opiate receptors during the development of tolerance/ depende. With the pAx method no significant change in the apparent pA 2 of naloxone in tolerant rats in vivo could be found, indicating that no substantial change in the affinity for the receptors takes place. Comparison of receptor binding of 3 H-etorphine and 3 H-naloxone to rat brain homogenate in vitro showed no difference in binding between naive and tolerant rats. The displacement of small amounts of high labeled antagonist or agonist by increasing amounts of unlabeled antagonist in mouse brain in vivo offered the possibility of characterizing properties of receptors in the intact animal. This technique revealed no indication of a change in the number of receptor sites in tolerant animals. An apparently lower affinity in the tolerant animals could be explained by the morphine present in these animals. Displacement of 3 H-etorphine from receptors by a high amount of unlabeled naltrexone in vivo could also be demonstrated by autoradiography.

Effect of 6-hydroxydopamine, 5,6-dihydroxytryptamine and raphe lesions on the antinociceptive actions of morphine in rats

The antinociceptive effect of morphine was studied in rats in which degeneration of catecholamine- or serotonin-containing nerve terminals had been induced. Vocalisation during electric stimulation of the tail was used as a test for nociception. Brain catecholamines were estimated fluorimetrically.Intraventricular injection of 6-hydroxydopamine markedly reduced brain catecholamines without affecting 5-HT levels; this change was accompanied by a significant reduction of the stimulation threshold both before and after application of morphine.Intraventricular injection of 5,6-dihydroxytryptamine significantly depleted central 5-HT content; the stimulation threshold before and after morphine injection was not affected.Lesions of the midbrain raphe system induced a great reduction of forebrain 5-HT; the morphine effect was not significantly changed.The role of catecholamines and 5-HT in the antinociceptive effect of morphine is discussed in respect to these and other results recently published by this laboratory.

Buprenorphine: Demonstration of physical dependence liability

This study was designed to assess the dependence-producing capacity of the opiate partial agonist, buprenorphine. Rats chronically treated with buprenorphine for 4 days showed only very weak signs of withdrawal upon cessation of buprenorphine treatment or upon challenge with naloxone, although complete tolerance had developed to the drug at this time. However, more intense withdrawal could be induced when buprenorphine treatment was followed by substitution treatment with morphine. Even one injection of morphine given 12 h after the last buprenorphine treatment enabled the precipitation of withdrawal with naloxone. Naloxone could not precipitate signs of withdrawal in naive rats treated with this dose of morphine. Thus, contrary to some claims in the literature, buprenorphine, like other opiate agonists and partial agonists, induces dependence. The fact that only few signs of withdrawal are seen in direct dependence tests, probably reflects the slow dissociation of the drug from the receptor - which probably limits the intensity of withdrawal by preventing the rapid uncovery of the receptor upon discontinuance of treatment with the drug or upon injection of an antagonist. In addition, the maximum degree of dependence induced by buprenorphine - in comparison to pure agonists is limited, like that of other partial agonists.

Tolerance and dependence induced by morphine-like pituitary peptides in rats

SummaryAn extract from porcine pituitaries containing peptides with opiate-like activity (endorphins) was investigated for possible tolerance/dependence liability in rats. Repeated intraventricular administration of endorphins induced a degree of tolerance similar to that induced by normorphine. Rats made tolerant to morphine by repeated pellet implantation proved cross-tolerant to the pituitary extract as well as to synthetic methionine-enkephalin. Naloxone given to rats, repeatedly pretreated with endorphins, precipitated a withdrawal syndrome strongly resembling that induced in rats treated with normorphine. Withdrawal precipitated in morphine-dependent rats was suppressed by intraventricularly applied endorphins. The results suggest that identical sites and mechanisms are involved in the development of tolerance/dependence induced by opiates and pituitary endorphins.

Changes in brain concentration of biogenic amines and the antinociceptive effect of morphine in rats

SummaryThe antinociceptive activity of morphine was determined in rats using the vocalisation test, whereby vocalisation was elicited by electrical stimulation of the tail. The effects of various drugs were compared with the corresponding changes in brain amine concentration.1.Manipulation of 5-hydroxytryptamine (5-HT) levels with parachlorophenylalanine (PCPA) or 5-hydroxytryptophan (5-HTP) did not modify the effect of morphine.2.Catecholamine (CA)-depleting agents antagonized the effect of morphine. α-methyl-p-tyrosine (α-MT) treatment attenuated the morphine effect and decreased the CA levels, whereas the stimulation threshold before morphine administration was not changed. Inhibition of noradrenaline (NA) synthesis by FLA-63 also reduced the morphine effect.3.Intraventricular injections of 5-HT, NA and dopamine (DA) (without morphine) did not alter the nociceptive threshold. The effect of morphine was not altered by 5-HT or DA.NA attenuated the action of morphine when injected intraventricularly after morphine, but not when injected before morphine. The reduced activity of morphine in rats treated with α-MT could not be restored by intraventricular injection of NA or DA.4.The concentration of morphine in brain was not altered by the α-MT treatment. It is suggested that the antinociceptive activity of morphine as tested by this method depends on the concentration of CA in brain, NA being more important than DA. 5-HT does not seem to be involved. Apparently the antinociceptive effect of morphine does not follow a single mechanism since the putative transmitter substances play a different role depending on the animal species and test methods used.

Endorphin-induced hyperthermia: Characterization of the exogenously and endogenously induced effects

SummaryEndorphins-like opiates-were found to increase temperature at very low doses and to decrease temperature at high doses. Not only the decreasing, but also the increasing effect on temperature, seems to be mediated through opiate receptors, since it proved to be antagonizable by naloxone and to be diminished in morphine-tolerant and-dependent animals. Like opiate-induced hyperthermia, both the pharmacological as well as the supposedly physiological increase in temperature induced by endorphins were strongly diminished in restrained animals. Hyperthermia induced by pyrogens, however, was not antagonizable by either naloxone or restraining of the animals. Maintenance of normal body temperature in animals being exposed to acute cold could not be disturbed by administration of naloxone. As a whole, such findings support the suggestion derived from our previous observation that physiologically induced naloxone-antagonizable hyperthermia may be a sensitive measure for endorphin release. However, endorphins although being potent hyperthermic modulators of temperature, do not seem to be directly involved in the central regulation of temperature. Instead, the physiologically induced increase in temperature by endorphins seems to represent some epiphenomenon of physiological events primarily initiated for physiological requirements other than the support of thermostability.

Non-competitive nature of the antagonistic mechanism responsible for tolerance development to opiate-induced analgesia

Abstract The mechanism responsible for development of tolerance to opiate-induced analgesia was investigated by means of an analysis of a series of dose-response curves in groups of rats which had developed different degrees of tolerance. Increasing tolerance was reflected in the curves by an increasingly large shift to the right and depression of the maximum effect. When the responses produced by morphine were plotted against the concentrations of the drug in the brain, instead of against the doses applied, and the curves analysed with a double reciprocal (Lineweaver-Burk) plot, it became evident that the KD value of morphine was not changed during tolerance development. The KD value obtained with this behavioral method was close to that obtained with receptor binding studies performed under identical conditions in vivo. The data implies that the mechanism(s) responsible for tolerance development to opiate-induced analgesia does not involve a qualitative change in the opiate binding sites. Instead, tolerance seems to be due to non-competitive changes in the transduction process between receptor occupation and response.