Distribution Pattern of Metorphamide Compared with Other Opioid Peptides from Proenkephalin and Prodynorphin in the Bovine Brain

Metorphamide is a [Met]-enkephalin-containing opioid octapeptide with a C-terminal alpha-amide group. It is derived from proenkephalin and is, so far, the only endogenous opioid peptide with a particularly high affinity for mu opioid (morphine) receptors, a somewhat lesser affinity for kappa opioid receptors, and a relatively low affinity for delta opioid receptors. The concentrations of metorphamide in the bovine caudate nucleus, the hypothalamus, the spinal cord, and the neurointermediate pituitary were determined by radioimmunoassay and chromatography separation procedures. Metorphamide concentrations were compared with the concentrations of eight other opioid peptides from proenkephalin and prodynorphin in identical extracts. The other opioid peptides were [Met]-enkephalyl-Arg6-Phe7 and [Met]-enkephalyl-Arg6-Gly7-Leu8 from proenkephalin; alpha-neoendorphin, beta-neoendorphin, dynorphin A(1-8), dynorphin A(1-17), and dynorphin B from prodynorphin; and [Leu]-enkephalin, which can be derived from either precursor. All opioid peptides were present in all four bovine neural tissues investigated. Metorphamide concentrations were lower than the concentrations of the other proenkephalin-derived opioid peptides. They were, however, similar to the concentrations of the prodynorphin-derived opioid peptides in the same tissues. Marked differences in the relative ratios of the opioids derived from prodynorphin across brain regions were observed, a finding suggesting differential posttranslational processing. Differences in the ratios of the proenkephalin-derived opioids across brain regions were less pronounced. The results from this study together with previous findings on metorphamide's mu opioid receptor binding and bioactivities suggest that the amounts of metorphamide in the bovine brain are sufficient to make this peptide a candidate for a physiologically significant endogenous mu opioid receptor ligand.     

A novel soluble protein factor with non-opioid dynorphin A-binding activity

A novel soluble non-opioid dynorphin A-binding factor (DABF) was identified and characterized in neuronal cell lines, rat spinal cord, and brain. DABF binds dynorphin A(1-17), dynorphin A(2-17), and the 32 amino acid prodynorphin fragment big dynorphin consisting of dynorphin A and B, but not other opioid and non-opioid peptides, opiates, and benzomorphans. The IC50 for dynorphin A(1-17), dynorphin A(2-17), and big dynorphin is in the 5-10 nM range. Using dynorphin A and big dynorphin fragments a binding epitope was mapped to dynorphin A(6-13). DABF has a molecular mass of about 70 kDa. SH-groups are apparently involved in the binding of dynorphin A since p-hydroxy-mercuribenzoic acid inhibited this process. Upon interaction with DABF dynorphin A was converted into Leu-enkephalin, which remained bound to the protein. These data suggest that DABF functions as an oligopeptidase that forms stable and specific complexes with dynorphin A. The presence of DABF in brain structures and other tissues with low level of prodynorphin expression suggests that DABF as an oligopeptidase may degrade other peptides. Dynorphin A at the sites of its release in the CNS may attenuate this degradation as a competitor when it specifically binds to the enzyme.     

Dynorphin immunocytochemistry in the rat central nervous system

The distribution of dynorphin in the central nervous system was investigated in rats pretreated with relatively high doses (300–400 μg) of colchicine administered intracerebroventricularly. To circumvent the problems of antibody cross-reactivity, antisera were generated against different portions as well as the full dynorphin molecule (i.e., residues 1–13, 7–17, or 1–17). For comparison, antisera to [Leu]enkephalin (residues 1–5) were also utilized. Dynorphin was found to be widely distributed throughout the neuraxis. Immunoreactive neuronal perikarya exist in hypothalamic magnocellular nuclei, periaqueductal gray, scattered reticular formation sites, and other brain stem nuclei, as well as in spinal cord. Additionally, dynorphin-positive fibers or terminals occur in the cerebral cortex, olfactory bulb, nucleus accumbens, caudate-putamen, globus pallidus, hypothalamus, substantia nigra, periaqueductal gray, many brain stem sties, and the spinal cord. In many areas studied, dynorphin and enkephalin appeared to form parallel but probably separate anatomical systems. The results suggest that dynorphin occurs in neuronal systems that are immunocytochemically distinct from those containing other opioid peptides.     

Antianalgesic action of dynorphin A mediated by spinal cholecystokinin

Previous work indicates that the antianalgesic action of pentobarbital and neurotensin administered intracerebroventricularly in mice arises from activation of a descending system to release cholecystokinin (CCK) in the spinal cord where CCK is known to antagonize morphine analgesia. Spinal dynorphin, like CCK, has an antianalgesic action against intrathecally administered morphine. This dynorphin action is indirect; even though it is initiated in the spinal cord, it requires the involvement of an ascending pathway to the brain and a descending pathway to the spinal cord where an antianalgesic mediator works. The aim of the present investigation was to determine if the antianalgesic action of intrathecal dynorphin A involved spinal CCK. All drugs were administered intrathecally to mice in the tail flick test. Morphine analgesia was inhibited by dynorphin as shown by a rightward shift of the morphine dose-response curve. The effect of dynorphin was eliminated by administration of the CCK receptor antagonists lorglumide and PD135 158. One hour pretreatment with CCK antiserum also eliminated the action of dynorphin. On the other hand, the antianalgesic action of CCK was not affected by dynorphin antiserum. Thus, CCK did not release dynorphin. Both CCK and dynorphin were antianalgesic against DSLET but not DPDPE, delta 2 and delta 1 opioid receptor peptide agonists, respectively. The results suggest that the antianalgesic action of dynorphin occurred through an indirect mechanism ultimately dependent on the action of spinal CCK.     

Characterization of dynorphin A-converting enzyme in human spinal cord. An endoprotease related to a distinct conversion pathway for the opioid heptadecapeptide?

A highly specific proteinase, converting dynorphin A (1-17) to enkephalins, was isolated from the human spinal cord and subjected to further characterization. The enzyme was found to be a thiol-dependent protein with a relative molecular mass of 50 kDa and a pH optimum between 5.0 and 5.5. This proteinase appears to exclusively convert dynorphin A (1-17) to Leu-enkephalin and its COOH-terminal extensions Leu-enkephalin-Arg6 (which was a major conversion product) and Leu-enkephalin-Arg6-Arg7 but not the other prodynorphin- or proenkephalin-derived peptides. This high specificity toward a single structure is suggested to be involved in a distinct processing pathway associated with the generation of the opioid peptides with selectivity for delta-opioid receptors.     

Dynorphin converting enzyme in the rat spinal cord. Decreased activities during acute phase of adjuvant induced arthritis.

This paper describes a study on a dynorphin converting enzyme in spinal cord homogenates from rats with experimental arthritis after adjuvant injection into one hindpaw. The enzyme resembles a neutral cysteine endopeptidase which cleaves the opioid peptide dynorphin B and generates its N-terminal fragment, Leu-enkephalin-Arg6 with opioid activity. It exhibits considerably lower activity against dynorphin A and alpha-neoendorphin, the two other prodynorphin derived peptides. The enzyme showed significantly higher activity in the dorsal part than in the ventral part of the spinal cord. A significant decrease in enzyme activity was observed in the dorsal spinal cord during inflammation as compared to vehicle-injected controls. This decrease paralleled a decrease in the tissue level of Leu-enkephalin-Arg6. These data thus indicate that adjuvant-induced arthritis may generate an important change in a converting enzyme acting on peptide structures, which may be involved in pain modulation. Therefore, a functional role of the present enzyme in the regulation of pain-related peptides is suggested.     

Characterization of opioid peptides in the rat stomach

A combination of several chromatographic and assay systems was used to characterize the opioid peptides in rat stomach extracts. Partial purification of opioid material in acetic acid extracts of the corpus plus antrum regions of the rat stomach was carried out by gel filtration chromatography on Sephadex G-50, followed by adsorption onto Amberlite XAD-2 resin. A single peak in opioid activity was determined by both radioreceptor assay (RRA) and bioassay. By high performance liquid chromatography, this peak was resolved into five distinct components, characterized by RRA and (or) radioimmunoassay, with retention times corresponding to methionine enkephalin (met-enk), leucine enkephalin, met-enk-arg6-gly7-leu8, met-enk-arg6-phe7, and dynorphin 1-13. Closer examination of the dynorphin component revealed the presence of dynorphins 1-17, 1-13, and 1-8. Trypsin digestion of the partially purified (Sephadex G-50 and Amberlite XAD-2 chromatographed) extract resulted in an overall increase in opioid activity, suggesting the presence of larger, possibly precursor forms.     

Dynorphin-A-(1–13) antagonizes morphine analgesia in the brain and potentiates morphine analgesia in the spinal cord

Intrathecal injection of subanalgesic doses of morphine (7.5 nmol) and dynorphin-A-(1–13) (1.25 nmol) in combination resulted in a marked analgesic effect as assessed by tail flick latency in the rat. The analgesic effect of the composite dynorphin/morphine was dose-dependent in serial dilutions so that a composition of 1/8 of the analgesic dose of dynorphin and 1/3 that of morphine produced an analgesic effect equipotent to full dose of either drug applied separately. The analgesic effect induced by dynorphin/morphine mixture was not accompanied by motor dysfunction and was easily reversed by a small dose (0.5 mg/kg) of naloxone. Contrary to the augmentatory effect of dynorphin on morphine analgesia in the spinal cord, intracerevroventricular (ICV) injection of 20 nmol of dynorphin-A-(1–13) exhibited a marked antagonistic effect on the analgesia produced by morphine (120 nmol, ICV). The theoretical considerations and practical implications of the differential interactions between dynorphin-A-(1–13) and morphine in the brain versus spinal cord are discussed.     

Analgesia induced by intrathecal injection of dynorphin B in the rat

A dose-dependent analgesic effect of intrathecally injected dynorphin B was observed in rats using the tail flick as nociceptive test. Intrathecal injection of 20 nmol of dynorphin B increased the tail flick latency by 90 +/- 23%, an effect that lasted about 90 min. For the same degree of analgesia, dynorphin B was 50% more potent than morphine on a molar basis. The analgesic effect of this dose of dynorphin B was partially blocked by 10 mg/kg, but not by 1 mg/kg, of subcutaneous naloxone, showing a relative resistance to naloxone reversal as compared with morphine analgesia. The analgesia produced by dynorphin B was unchanged in morphine-tolerant rats but was significantly decreased in rats tolerant to ethylketazocine. These results suggest that dynorphin B produces its potent analgesic effect by activation of kappa rather than mu opioid receptors in the rat spinal cord.     

Involvement of spinal kappa opioid receptors in the antagonistic effect of dynorphins on morphine antinociception.

The modulatory effects of intrathecally (i.t.) administered dynorphin A(1-17) and dynorphin A(1-13) on morphine antinociception have been studied previously in rats by other investigators. However, both potentiating and attenuating effects have been reported. In this study, the modulatory effects of i.t. administered dynorphin A(1-17) as well as the smaller fragment, dynorphin A(1-8), were studied in mice. In addition, nor-binaltorphimine (nor-BNI), a highly selective kappa opioid receptor antagonist, and naltrindole (NTI), a highly selective delta opioid receptor antagonist, were used to characterize the possible involvement of spinal kappa and delta opioid receptors in the modulatory effects of the dynorphins. Dynorphin A(1-17) and dynorphin A(1-8) administered i.t. at doses that did not alter tail-flick latencies, were both able to antagonize in a dose-dependent manner, the antinociceptive action of s.c. administered morphine sulfate. The antinociceptive ED50 of morphine sulfate was increased 3.9- and 5.3-fold by 0.4 nmol/mouse of dynorphin A(1-17) and dynorphin A(1-8), respectively. Injections of 0.4 and 0.8 nmol/mouse of nor-BNI i.t., but not its inactive enantiomer (+)-1-nor-BNI, inhibited dose-dependently the antagonistic effects of the dynorphins. These doses of nor-BNI alone did not affect the antinociceptive action of morphine sulfate. Intrathecal administration of 5 nmol/mouse of NTI also did not affect the modulatory effects of dynorphins. These observations that dynorphins exert their antagonistic effects on morphine-induced antinociception stereoselectively through spinal kappa opioid receptors may suggest a coupling between spinal kappa and mu opioid receptors.     

Functional blockage of the cannabinoid receptor type 1 evokes a kappa-opiate-dependent analgesia

Progress in the control and treatment of pain may be facilitated by a better understanding of mechanisms underlying nociceptive processing. Cannabinoids and opioids are endogenous modulator of pain sensation, but therapies based in these compounds are not completely exploited because of their side effects. To test the role of cannabinoid receptor type 1 (CB1-R) inhibition in nociception, we performed a subchronic administration of the CB1-R antagonist N -(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM281) in mice. This treatment provoked analgesia in peripheral thermal and visceral models of pain. Analysis of genes encoded for the opioid system in the spinal cord showed an increase in the expression of genes encoded for the κ-opioid system in AM281-injected mice compared with vehicle-injected ones. Furthermore, systemic administration of nor-binaltorphimine, a κ-opioid receptor antagonist, blocked AM281-induced analgesia. Finally, c-fos expression in the dorsal spinal cord and higher centers of pain processing after noxious stimulation were significantly lower in AM281-injected mice than in vehicle-injected animals, indicating that dynorphin could block nociceptive information transmission at the spinal cord level. These results indicate the existence of a cross-talk between opioid and cannabinoid systems in nociception. Furthermore, the results suggest that CB1-R antagonists could be useful as a new therapeutic approach for pain relief.     

电针引起脊髓P物质释放的频率依赖性

我室以往的研究表明,不同频率的电针可引起脊髓释放不同种类的阿片肽。本工作观察Ｐ物质（ＳＰ）释放的频率依赖性,电针频率选择２,４,８,１５,３０和１００Ｈｚ,分别收集电针期间及电针前后各３０ｍｉｎ的脊髓灌流液,通过放射免疫方法测定大鼠电针有效组和电针无效组Ｐ物质免疫活性（ＳＰ－ｉｒ）．结果如下：（１）电针有效组：２Ｈｚ引起ＳＰ－ｉｒ降低,与电针前相比,Ｐ＜０．０１;４Ｈｚ电针前后ＳＰ－ｉｒ比较,无统计学意义;８,１５,３０,１００Ｈｚ时ＳＰ－ｉｒ均增加（Ｐ＜０．０１）,其中１５Ｈｚ时ＳＰ增加最多（Ｐ＜０．００１）,表明刺激引起ＳＰ释放有频率依赖性。（２）电针无效组：不论应用何种频率,电针前后脊髓灌流液中ＳＰ－ｉｒ变化不大（均Ｐ＞０．０５）。提示,电针时脊髓液中ＳＰ含量变化与镇痛效果有密切关系。     

Dynorphin A(1-17) biotransformation in striatum of freely moving rats using microdialysis and matrix-assisted laser desorption/ionization mass spectrometry

The biotransformation of the opioid peptide dynorphin A(1-17) was investigated in striatum of freely moving Fischer rats, by direct infusion of this peptide, followed by recovery of the resulting biotransformation products via microdialysis and identification using matrix-assisted laser desorption/ionization mass spectrometry. The observed peptides are consistent with enzymatic cleavage at the Arg7-Ile8 position of dynorphin A(1-17), followed by terminal degradation of the resulting dynorphin A(1-7) and dynorphin A(8-17) peptides. Unexpectedly, novel post-translational modifications were found on C-terminal fragments of dynorphin A(1-17). Using tandem mass spectrometry, a covalent modification of mass 172 Da, the nature of which is not understood, was found on the tryptophan residue of C-terminal fragments (Trp14). Additional modifications, of mass 42 and 113 Da, were also found on the N-terminus (Ile8 or Pro10) of these same C-terminal fragments. The role of these modifications of C-terminal fragments has not yet been characterized.     

Alternative Forms of Interaction of Substance P and Opioids in Nociceptive Transmission

Summary Many years preclinical and clinical anatomic, pharmacologic, and physiologic studies suggest that SP- and opioid-expressing neurons produce opposite biological effects at the spinal level, i.e., nociception and antinociception, respectively. However, in certain circumstances intrathecally administered SP is capable of reinforcing of spinal morphine analgesia and may therefore function as an opioid adjuvantin vivo. The SP dose-response curve of spinally administered SP follows a bell-shaped or inverted-U configuration, permitting pharmacological dissociation of opioid-potentiating and analgesic properties of SP from traditional hyperalgesic effects seen at significantly higher concentrations. This analgesic effect is blocked by naloxone but unaffected by transection of the spinal cord, thus demonstrating the lack of supraspinal modulation. The present report briefly describes both reinforcing and opposing interactions between multiple opioid systems and substance P at the spinal level. We propose that a likely mechanism underlying SP-mediated enhancement of opioid analgesia is the ability of SP to release endogenous opioid peptides within the local spinal cord environment.     

Decoy peptides that bind dynorphin noncovalently prevent NMDA receptor-mediated neurotoxicity

Prodynorphin-derived peptides elicit various pathological effects including neurological dysfunction and cell death. These actions are reduced by N-methyl-d-aspartate receptor (NMDAR) but not opioid receptor antagonists suggesting NMDAR-mediation. Here, we show that a conserved epitope (KVNSEEEEEDA) of the NR1 subunit of the NMDAR binds dynorphin peptides (DYNp) noncovalently. Synthetic peptides containing this epitope form stable complexes with DYNp and prevent the potentiation of NMDAR-gated currents produced by DYNp. They attenuate DYNp-evoked cell death in spinal cord and prevent, as well as reverse, DYNp-induced paralysis and allodynia. The data reveal a novel mechanism whereby prodynorphin-derived peptides facilitate NMDAR function and produce neurotoxicity. Furthermore, they suggest that synthetic peptides that bind DYNp, thus preventing their interaction with NMDAR, may be novel therapeutic agents for the treatment of spinal cord injury.     

Dynorphins Modulate DNA Synthesis in Fetal Brain Cell Aggregates

Abstract: Previously, opioid peptide analogues, β-endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7-day fetal rat brain cell aggregates via κ-and μ-opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 µ M , all dynorphins tested and β-endorphin inhibited [³H]thymidine incorporation into DNA by 20–38% in 7-day rat brain cell aggregates. The putative ε-antagonist β-endorphin (1–27) did not prevent the effect of β-endorphin, suggesting that the ε-receptor is not involved in opioid inhibition of DNA synthesis. The κ-selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a κ-opioid receptor. In dose-dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete κ-receptor subtype. The IC₅₀ value of 0.1 n M estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the κ-receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21-day aggregates were treated with dynorphins, a 33–86% enhancement of thymidine incorporation was observed. Because both 7- and 21-day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express κ-receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.     

Immunoreactive peptides related to dynorphin B (=rimorphin) in the rat brain

We have developed a radioimmunoassay for synthetic dynorphin B, a novel opioid tridecapeptide, which shares a common precursor molecule with dynorphin_1–17 (=dynorphin A) and the neo-endorphins. The levels of immunoreactivity towards this peptide in rat brain and pituitary show a pattern quantitatively and qualitatively similar to those found for dynorphin A and -neo-endorphin in earlier studies. The antiserum used was highly specific with only dynorphin-32 and dynorphin B-29, both of which contain the dynorphin B sequence, showing substantial cross-reactivity. Gel filtration of whole rat brain extracts in combination with HPLC analysis provide strong evidence for the existence of these latter two peptides in rat brain.     

A novel bovine spinal cord endoprotease with high specificity for dynorphin B

An endopeptidase that converts the opioid peptide dynorphin B (Tyr-Gly-Gly-Phe-Leu-Arg-aRg-Gln-Phe-Lys-Val-Val-Thr) to its bioactive fragment Leu-enkephalin-Arg6 was isolated from bovine spinal cord. The enzyme was purified about 230-fold from a concentrated spinal cord extract. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it stained as a protein of Mr 55,000. The purified enzyme is optimally active at around pH7 and has essential thiol groups. It appears to be highly specific for dynorphin B (Km = 11 microM) but not for alpha-neoendorphin or dynorphin A, two other opioids included in the prodynorphin precursor. From its specificity, molecular size, and inhibitory spectrum, this enzyme is different from other known dynorphin-converting or -degrading enzymes and appears to be a unique and novel endoprotease.     

Dynorphin: potent analgesic effect in spinal cord of the rat

Evidence is presented to show a strong and long-lasting analgesic effect after injection of dynorphin into the subarachnoid space of the spinal cord of the rat. Calculating on a molar basis dynorphin was 6-10 times more potent than morphine and 65-100 times more potent than morphiceptin, the specific mu receptor agonist. Dynorphin analgesia was completely reversed by intrathecal injection of anti-dynorphin IgG and partially reversed by naloxone. Acute tolerance to morphine analgesia did not affect the occurrence of dynorphin analgesia. Evidence from different lines of approach suggest that dynorphin may bind with kappa receptors in the spinal cord to exert its analgesic effect.     

Hypothalamo-posterior pituitary system in Brattleboro rats: immunoreactive levels of leucine-enkephalin, dynorphin (1-17), dynorphin (1-8) and alpha-neo-endorphin

The levels of immunoreactive leucine-enkephalin, alpha-neo-endorphin, dynorphin (1-17) and dynorphin (1-8) have been determined in the hypothalamus and posterior pituitary from male and female Brattleboro rats homozygous (unable to produce vasopressin) and heterozygous (producing vasopressin) for diabetes insipidus, and from male and female Long Evans rats. In the hypothalamus we found no significant differences in the levels of these peptides while there were great differences in extracts from the posterior pituitary: female homozygous animals have greatly reduced levels in all four peptides compared to the heterozygous controls. In male homozygous animals the differences in the dynorphin (1-17) and leucine-enkephalin levels were small whereas the concentrations of alpha-neo-endorphin and dynorphin (1-8) showed a significant decrease compared to the male heterozygous controls. The results indicate a reduction in opioid peptides linked to the vasopressin deficiency in a partially sex dependent manner.