Modulation of opioid system in C57 mice after repeated treatment with morphine and naloxone: biochemical and behavioral correlates

C57 BL/6J (C57) mice display a particular pattern of responses following morphine administration, such as a rapid development of tolerance to the pharmacological action of the opiate and an increase in locomotor activity after a single injection of the drug. We have measured met-enkephalin content and the responsiveness of different opiate receptors after repeated administration of morphine and naloxone. Prolonged morphine administration changes neither met-enkephalin levels, nor the density of the opiate receptors in mice brain. In contrast repeated administration of the opiate antagonist naloxone, produced a marked increase in the number of 3H- DHM and 3H- DADLE binding sites in striatum and brainstem without modifying met-enkephalin concentrations. Behavioral studies have indicated that the morphine-induced increase in locomotor activity is enhanced in naloxone pretreated mice, thus suggesting a possible correlation between the behavioral response to morphine in C57 mice and the higher number of opiate receptors in the striatum.     

Evidence for intrinsic regulation of met-enkephalin-immunoreactivity in gastroenteropancreatic tissues of the rat

In a study whether gastrointestinal endogenous opioids can be modified by vagal denervation or by pharmacological application of an opiate, we examined met-enkephalin-immunoreactivity in gastrointestinal tissue in rats with and without truncal vagotomy and with and without subcutaneously implanted morphine pellets. The immunoreactivity of the tissue extracts gave dose-response lines in the radioimmunoassay for met-enkephalin which were near parallel to that for the standard. On Sephadex chromatography the met-enkephalin immunoreactivity eluted at a position similar to synthetic met-enkephalin. Tissue concentration of met-enkephalin immunoreactivity was not significantly different from the respective control after vagotomy and after morphine treatment. Total gastric met-enkephalin immunoreactive content increased significantly after vagotomy in line with gastric hypertrophy occurring after vagotomy without a drainage procedure. From these results it is concluded that met-enkephalin immunoreactivity in the rat gastrointestinal tract is regulated intrinsically, it is neither altered by vagal denervation nor by exogenous opiate administration.     

Potencies of normorphine and met-enkephalin in the vas deferens of different strains of mice.

The concentration of normorphine causing a 50 per cent inhibition (IC₅₀) of electrically induced twitches in the vas deferens from seven strains of mice varied over a 13-fold range, BALB/cKB being the most, C57BL/6J the least sensitive. There was no significant correlation between the IC₅₀'s of normorphine and met-enkephalin. In the sensitive BALB/cKB mice, both normorphine and met-enkephalin were more effective inhibiting contractions evoked by 0.1 Hz than by 0.01 or 1.2 Hz stimulation. This difference was not observed in the insensitive C57BL/6J mice. Naloxone was purely an antagonist against both normorphine and met-enkephalin in BALB/cKB mice but in low concentration it potentiated the inhibitory effect of both normorphine and met-enkephalin in C57BL/6J mice. Results suggest that qualitative differences in opiate receptors and differences in transmitter release mechanism contribute to the variable sensitivity to morphine of the vas deferens from different strains of mice.     

Intracerebral beta-endorphin, met-enkephalin and morphine: kindling of seizures and handling-induced potentiation of epileptiform effects

The effects of repeated infusion of small, initially subconvulsive amounts of beta-endorphin, met-enkephalin or morphine sulfate into the amygdala and hippocampus were investigated. beta-endorphin and met-enkephalin evoked epileptiform spiking when infused into the posterior amygdala or ventral hippocampus. Morphine evoked epileptiform spiking when infused into the anterior amygdala. Naloxone blocked or terminated the spiking. Repetition of the infusions led to the gradual development of bilateral generalized convulsions by beta-endorphin and met-enkephalin and to the development of tolerance to morphine. An unexpected observation was that handling, immobilization or conspecific threat potentiated the epileptiform effects of beta-endorphin and morphine in many cases. These results suggest that endogenous opiate mechanisms might play a role in convulsive seizures and that stressful stimuli can exacerbate opiate seizures.     

Isolation of human met-enkephalin and two groups of putative precursors (2K-pro-met-enkephalin) from an adrenal medullary tumour

Human met-enkephalin from an adrenal medullary tumour has been purified and characterised. Two groups of putative human met-enkephalin precursors (MW ～ 2000) were also isolated and were only opiate active after trypsinisation and yielded met-enkephalin on further treatment with carboxypeptidase B. Amino acid analyses of these peptides show they are not related to β-LPH nor any other reported putative enkephalin precursors. Their amino acid compositions suggest they are structurally closely related and may represent two groups of heterologous peptides.     

Effects of morphine and methionine-enkephalin on the smooth muscle tonus and the contraction induced by transmural stimulation in the carp (Cyprinus carpio) intestinal bulb

The effects of morphine and methionine-enkephalin (met-enkephalin) on the smooth muscle tonus and the contraction induced by transmural stimulation were investigated in the isolated intestinal bulb of carp in vitro. Morphine (30 nM-3 microM) and met-enkephalin (3 nM-5 microM) caused dose-dependent non-sustained contraction. Naloxone (10 nM) inhibited the contraction induced by morphine or met-enkephalin in a competitive manner. Tetrodotoxin (400 nM) or atropine (500 nM) did not inhibit the contraction induced by morphine or met-enkephalin. Cooling of the bath fluid from 20 to 10 degrees C decreased nicotine- and transmural stimulation-induced contraction. But met-enkephalin-induced contraction was not affected. Transmural stimulation-induced contraction (3 Hz) was not affected by pretreatment with morphine, met-enkephalin or naloxone. The results demonstrated that morphine or met-enkephalin caused contraction of the smooth muscle directly through the activation of opiate receptors on the smooth muscle cells and neither morphine nor met-enkephalin regulated the cholinergic neurotransmission presynaptically.     

Monoclonal antibody to enkephalins with binding characteristics similar to opiate receptor

Monoclonal antibodies to enkephalins were established by immunization of mice with met-enkephalin, leu-enkephalin or both. Twenty-three clones with a high titer were classified into 6 types according to the binding properties to enkephalins and their derivatives. Antibody LM 239 showed binding characteristics similar to opiate receptor. It has a very high affinity to enkephalins and their derivatives which have a potent opioid activity, but a low affinity to enkephalin derivatives which devoid of opioid activity. The binding of 3H-met-enkephalin to the antibody was inhibited by naloxone and morphine, although the ID50 values were considerably higher than the Ka values of the alkaloids to opiate receptor.     

Endogenous ligands for kappa-opiate receptors

The receptor preferences of opioids in the mouse vas deferens was tested by means of tolerance and cross-tolerance studies. The preparations were rendered tolerant in situ by superfusion with the kappa-receptor agonist dynorphin and with alpha-neoendorphin, respectively, and set up in vitro in the presence of the respective peptide to maintain tolerance. The investigations revealed strong kappa-agonistic activities both of alpha-neoendorphin and of dynorphin and its fragments 1-13 and 1-11. As the dynorphin chain shortened, the kappa-receptor activity declined and delta-receptor activity became progressively apparent. Interestingly, the octapeptide met-enkephalin[Arg6,Gly7,Leu8], a fragment of the adrenal medulla proenkephalin, also displayed considerable kappa-agonistic properties under the experimental conditions employed. Presumably, the decapeptide alpha-neoendorphin and the octapeptide met-enkephalin[Arg6,Gly7,Leu8] cover in addition to the kappa-receptor population in the MVD further opiate receptors, most probably delta-receptors.     

Interaction of various types of opiate receptors of cortical neurons in the turtle during activation by specific agonists

The influence of polycomponent solutions of agonists of opiate mu-, delta, chi- and sigma-receptors (morphine, D-Ala2, D- Ley5 -enkephalin, bremazocine, SKF 10,047) and of met-enkephalin on the habituation of orthodromic evoked potential in the visual cortical area was studied in turtles. Interaction between the different types of opiate receptors was observed at their combined activation. The interaction resulted in an enhancement or attenuation of modulation of separate phases of evoked potential habituation which differed from simple sum of effects during isolated activation of each type of receptors.     

Binding of the opiate-like pentapeptide methionine-enkephalin to a particulate fraction from rat brain

The characteristics of stereospecific binding of [3H] met-enkephalin (15 Ci/mmole) were studied in a particulate fraction from rat brain. The binding assay was performed for 70 min at degrees C and the bound radioactivity separated by filtration through glass fiber filters (Whatman, GF/C). In the absence of sodium, binding of [3H] met-enkephalin could be described on the basis of two independent binding sites with apparent KDs of 2.1 and 53 nM, respectively. The data are also consistent with one class of binding sites showing negative cooperativity. In the presence of 100 mM NaCl, binding of [3H] met-enkephalin was 90-95% reduced, thus indicating the agonist properties of the peptide. The highly stereospecific binding of [3H] met-enkephalin was evidenced by the 10,000-fold greater potency of levorphanol than its analgesically inactive enantiomer dextrorphan to compete for [3H] met-enkephalin binding. Similar conclusions could be reached using levallorphan, (+)-3-hydro-N-allyl-morphinan, (-) methadone and (+) methadone. The apparent affinity of various opiate agonists and antagonists for the binding sites was closely correlated with their known pharmacological activity.     

Antagonism of diazepam-induced feeding in rats by antisera to opioid peptides

Diazepam-induced feeding in rats is antagonized not only by the opiate antagonist naloxone but also intraventricular administration of specific antisera to the endogenous opioid peptides met-enkephalin or beta-endorphin. Pituitary beta-endorphin is probably not implicated in the diazepam effect since blockade with the glucocorticoid dexamethasone of the release of beta-endorphin from the anterior pituitary does not modify the diazepam-induced feeding, which is however prevented by TRH, a suggested physiological antagonist of some of the effects of opioid peptides. The possible central participation of both beta-endorphin and met-enkephalin in the ingestive behavior induced by diazepam gives further support to the postulated physiological role of endogenous opioids in appetite regulation.     

Evidence contradicting the notion that gonadal hormones regulate brain opiate receptors

Castration of male rats has been reported to increase brain opiate receptors by nearly 100%. We assayed brain opiate receptors with both naloxone and met-enkephalin, but found no effect of gonadectomy on the Kd or Bmax for either ligand in male or female mice or in male rats. Experiments were performed with 2 strains of mice and 3 strains of rats; mice were gonadectomized 1–7 weeks and rats were castrated 3 weeks before assay. Both washed and unwashed brain membrane preparations were used. Administration of testosterone or estrogen to intact male or female mice did not alter opiate receptors. Castration did not affect the strain or age and brain-region differences found for naloxone binding in male rats.     

Opioid activity of peptides and wound healing of the skin

The binding of dalargin, its four analogues and FK-33824, DADLE, met-enkephalin and morphine to peripheral mu- and delta-receptors and to brain receptors has been investigated in comparison with their influence on skin wound healing in rats. It has been shown that only substances with opiate activity, including morphine, stimulated wound healing. No correlation between wound healing effect of peptides and their binding to a definite receptor has been found. Naloxone inhibited wound healing and suppressed opiate peptide-mediated healing process. It is suggested that endogenous opiate peptides are involved in the maintenance of structural homeostasis.     

Opiate binding to membrane preparations of neuroblastoma x glioma hybrid cells NG108-15: effects of ions and nucleotides.

Interaction of a number of opiate agonists with the opiate receptors in NG108-15 cell membranes is influenced by ions, as well as certain nucleotides. Steady state binding of [³H]leu-enkephalin is increased by Mg⁺⁺ and decreased by Na⁺, GMP-P(NH)P, GTP, GDP, ITP and IMP-P(NH)P. Half-maximal inhibition produced by GMP-P(NH)P occurred at 4.6 μM. The dissociation of [³H]leu- and [³H]met-enkephalin, as well as [³H]etorphine, from these opiate receptors was also shown to be altered by both ions and nucleotides.     

The regulatory effects of opioid peptides--enkephalins--in controlling the activities of the cardiovascular system

In this review we analyse the experimental and clinical findings demonstrating important regulatory significance of met-enkephalin, leu-enkephalin and their derivatives in the control of cardiovascular system activity. Enkephalin-positive immunoreactivity is revealed in the heart of different species of animals, and their cardiovascular effects are established in numerous investigations. It is determined that cardiac effects of enkephalins are essentially associated with modulatory influence at the presynaptic and postsynaptic levels on the activity of extracardiac neural regulation. Cardiovascular effects of endogenous opioid system are extremely important in developing of myocardial ischemia, cardiac arrhythmias and congestive heart failure. The cellular mechanisms of opioid effects are associated with stimulation of mu- and delta-subtypes of opiate receptors which stimulation of mu- and delta-subtypes of opiate receptors which are coupled with conductivity of ion channels, adenylate cyclase activity, phosphoinositide turnover and calcium-calmodulin-dependent protein kynases.     

Cardiovascular effects of intraventricular injection of FMRFamide, Met-enkephalin and their common analogues in the rat

The molluscan neuropeptide, Phe-Met-Arg-Phe-NH2 (FMRFamide), the mammalian opioid peptide met-enkephalin, and their common analogues, met-enkephalin-Arg6-Phe7 (YGGFMRF) and Tyr-Gly-Gly-Phe-Met-Arg-Phe-amide (YGGFMRFamide), were injected into the lateral ventricle of the rat; the cardiovascular effects were studied. FMRFamide caused a rapid, transient elevation in blood pressure accompanied by a great increase in pulse pressure. These effects were followed by secondary increases in blood and pulse pressures. Met-enkephalin produced an initial reduction in blood pressure which was followed by a gradual increase at the higher of two test doses (300 nmole). Injection of YGGFMRF resulted in a gradual increase in blood pressure. This response resembled that to met-enkephalin. The initial response to YGGFMRFamide was similar to that to FMRFamide: increases in both blood and pulse pressures after injection. However, the secondary effect of YGGFMRFamide, a prolonged reduction in blood pressure, was not produced by FMRFamide. These results suggest that the initial excitatory cardiovascular responses may be due to the presence of the C-terminal amide. All of the cardiovascular effects of injecting these peptides into the lateral ventricle were abolished by pre-treatment with naloxone in a dose that, itself, produced no cardiovascular changes. In conclusion, these peptides seem to act via the naloxone sensitive opiate receptors in the rat brain.     

Neurotransmitter-related features of the retinal pigment epithelium

Various neurotransmitter-related biochemical features of the separated pigment epithelium and neural retina of the cow have been examined. The pigment epithelium contains high affinity binding sites for several pharmacological agents thought to attach to neurotransmitter receptor sites with a high degree of specificity. Thus, serotonergic, adrenergic and opiate receptors appear to be present in the pigment epithelium. Serotonin has also been detected in this region.Several neuropeptides were found in the pigment epithelium. Relatively large amounts of neurotensin and met-enkephalin were present, but substance P was not detected.     

Endogenous ligands for κ-opiate receptors

The receptor preferences of opioids in the mouse vas deferens was tested by means of tolerance and cross-tolerance studies. The preparations were rendered tolerant in situ by superfusion with the κ-receptor agonist dynorphin and with α-neoendorphin, respectively, and set up in vitro in the presence of the respective peptide to maintain tolerance. The investigations revealed strong κ-agonistic activities both of α-neoendorphin and of dynorphin and its fragments 1–13 and 1–11. As the dynorphin chain shortened, the κ-receptor activity declined and δ-receptor activity became progressively apparent. Interestingly, the octapeptide met-enkephalin[Arg⁶,Gly⁷,Leu⁸], a fragment of the adrenal medulla proenkephalin, also displayed considerable κ-agonistic properties under the experimental conditions employed. Presumably, the decapeptide α-neoendorphin and the octapeptide met-enkephalin[Arg⁶,Gly⁷,Leu⁸] cover in addition to the κ-receptor population in the MVD further opiate receptors, most probably δ-receptors.     

Opiate receptor affinities and behavioral effects of enkephalin: Structure-activity relationship of ten synthetic peptide analogues

Synthetic met- and leu-enkephalin bind to rat brain opiate receptors with $\text{1}\text{2}$ and $\text{1}\text{7}$ the affinity of morphine. The aromatic hydroxyl moiety of the tyrosine residue is critical for receptor binding. Intracranial microinjection of met-enkephalin requires very high doses to produce an evanescent, naloxone reversible analgesia and stuperous immobility, presumably because of its rapid enzymatic degradation. Leu-enkephalin fails to elicit analgesia.     

Immunohistochemical and biochemical evidence for the presence of the pentapeptide met-enkephalin and the heptapeptide met-enkephalin-Arg6-Phe7 but not the octapeptide met-enkephalin-Arg6-Gly7-Leu8 in amphibian chromaffin cells

Using specific antibodies to met-enkephalin, met-enkephalin-Arg⁶-Phe⁷ and met-enkephalin-Arg⁶-Gly⁷-Leu⁸, we have studied the distribution of these opioid peptides in the frog adrenal gland by means of the indirect immunofluorescence technique. Bright staining of all chromaffin cells was observed by application of met-enkephalin and met-enkephalin-Arg⁶-Phe⁷ antisera. No nerve endings could be detected. A few chromaffin cells were weakly stained by met-enkephalin-Arg⁶-Gly⁷-Leu⁸ antiserum. Using a specific radioimmunoassay for met-enkephalin, the dilution curve of frog adrenal extracts was parallel to that of synthetic met-enkephalin. The concentration of met-enkephalin-like material measured in crude acetic extracts of frog adrenals (2.31 ± 0.16 pmol/mg w. wt) was high when compared to those reported for most mammalian species. No leu-enkephalin and virtually no met-enkephalin-Arg⁶-Gly⁷-Leu⁸ were detected by the corresponding radioimmunoassays. Reverse phase HPLC analysis revealed that oxidized met-enkephalin was the main form of met-enkephalin detected in acetic extracts of frog adrenals. HPLC separation showed the presence of a peptide co-eluting with synthetic met-enkephalin-Arg⁶-Phe⁷. Higher molecular weight forms were also separated by HPLC. These results show the presence of both met-enkephalin and the heptapeptide met-enkephalin-Arg⁶-Phe⁷ and the lack of met-enkephalin-Arg⁶-Gly⁷-Leu⁸ in frog chromaffin cells.