Action Of Opioid Peptides Research Articles

Activation of central opioid receptors may induce gastric mucosal defence in the rat

The effect of different opioid peptides on acidified ethanol- and indomethacin-induced gastric mucosal lesions was studied following intracerebroventricular (i.c.v.) administration. It was found that both the selective δ opioid receptor agonists—deltorphin II, [D-Ala 2, D-Leu 5]-enkephalin (DADLE), [D-Pen 2, D-Pen 5]-enkephalin (DPDPE)-, μ-opioid receptor agonist — [D-Ala 2, Phe 4, GlyT-ol]-enkephalin (DAGO) — as well as β-endorphin inhibited the mucosal damage induced by both ethanol and indomethacin in pmolar dose range. In contrast, the gastric acid secretion was not influenced by DADLE in the dose of 16 nmol/rat and only a slight reduction (40%) was induced by DAGO in the dose of 1.9 nmol/rat. The protective effect was abolished in both ulcer models by bilateral cervical vagotomy. N G-nitro-L-arginine, an inhibitor of NO synthase, reduced the protective action in ethanol-induced, but not in indomethacin-induced gastric damage. The results suggest that activation of supraspinal δ and μ-opioid receptors resulted in inhibition of gastric mucosal lesions elicited by ethanol or indomethacin. The gastroprotective action is independent from the effect of opioids on acid secretion. Vagal nerve is involved in conveying the central action to the periphery. The mechanism of the gastroprotective effect of opioids is different in ethanol- and indomethacin-ulcer models: prostaglandins and nitric oxide are likely to be involved in the protective action of opioid peptides in ethanol-, but not in the indomethacin-ulcer model.

The Mu 3 opiate receptor subtype

The mu 3 opiate receptor subtype has been characterized by various binding assays as opiate alkaloid selective (eg, morphine) and opioid peptide (eg, methionine enkephalin) insensitive. The binding is monophasic, saturable, and stereospecific, as well as naloxone reversible. This opiate receptor subtype has been found on human and invertebrate tissues, demonstrating that it has been conserved during evolution. Furthermore, in numerous reports, this receptor is coupled to constitutive nitric oxide release. In this regard, for example, morphine immune downregulating activities parallels those actions formerly attributed to nitric oxide. Thus, this opiate receptor represents an addition to mu receptor heterogeneity that offers an explanation for the difference in actions of opioid peptides and opiate alkaloids in physiological systems transcending analgesia.

Streptococcus parasanguis pepO encodes an endopeptidase with structure and activity similar to those of enzymes that modulate peptide receptor signaling in eukaryotic cells.

Studies in our laboratory have identified two fimbria-associated adhesins, FimA and Fap1, of Streptococcus parasanguis FW213. In this study, we isolated and sequenced DNA fragments linked to fimA to determine if they contained additional factors associated with adherence, virulence, or survival in the host. An open reading frame just upstream and divergently transcribed from the fimA operon was identified and named pepO. Northern hybridization indicated that pepO is transcribed as a monocistronic message. pepO encodes a predicted 631-amino-acid protein with a molecular mass of approximately 70.6 kDa. PepO contains the essential motif HEXXH, typical of many zinc-dependent metalloproteases and metallopeptidases. PepO has significant sequence identity to mammalian metallopeptidases, including endothelin-converting enzyme, which converts a potent vasoconstrictor into its active form, and neutral endopeptidase (NEP), which is involved in terminating the activity of opioid peptides. The opioid peptide metenkephalin is a natural substrate of NEP. Cell extracts of FW213 cleaved metenkephalin at the same site as does NEP, while an extract from an insertionally inactivated pepO mutant did not. These results indicate that FW213 pepO encodes an enzyme with activity similar to that of known mammalian endopeptidases. Phylogenetic analysis of PepO and its homologues suggests lateral genetic exchange between bacteria and eukaryotes.

Opioid peptides as possible neuromodulators of the afferent synaptic transmission in the frog semicircular canal

Vestibular receptors of the frog, Rana temporaria, were examined for the effect of bath-applied opioid peptide leu-enkephalin, its synthetic analogue dalargin and the specific opiate antagonist naloxone. Multiunit afferent activity of the whole vestibular nerve was recorded in an in vitro preparation. Leu-enkephalin (0.005–100 nM) and dalargin (0.1–100 nM) depress the resting discharge frequency. Naloxone (10 nM–1 μM) antagonizes responses induced by leu-enkephalin and dalargin that suggests a specific action of opioid peptides. Leu-enkephalin and delargin inhibit the excitatory action of l-glutamate. The effects of opioid peptides on l-glutamate-induced responses are unaffected by Co 2+ block of transmitter release from hair cells that could speak in favour of the postsynaptic nature of these responses. At the same time, the other possible site of action of opioid peptides, such as efferent system, can not be excluded. The results indicate that opiate receptors are present in hair cells and that the neurotransmitter l-glutamate is involved in opiate action at the peripheral vestibular system of the frog. We suggest that opioid peptides may act as a neuromodulator in this system.

Influencia del ácido oleico sobre la actividad aminopeptidasa de astrocitos de rata

Changes in fatty acid composition of membrane lipids induce modifications on the activity of several enzymes and membrane transporters. Glial cells possess aminopeptidases which are located in the plasma membranes. Aminopeptidases are generally zinc-metalloenzymes which hydrolyze peptide bonds near the N-terminal end of peptides and polypeptides. The importance of these enzymes is based on their major role in protein metabolism and in the regulation of circulating hormones and biologically active peptides. We study the effects of oleic acid on several aminopeptidase activities in primary cultures of rat astroglia, using aminoacyl-beta-naphthylamides as substrates. Oleic acid inhibits Ala-, Cys-, Leu- and Tyr-aminopeptidase activities, but not modifies Arg- and pGlu-aminopeptidase activities. Oleic acid modulates aminopeptidase activities in astrocytes. This could be related with intercellular communication and molecular transport processes, in which the astrocyte function has been involved. Furthermore, oleic acid might modulate the action of opioid peptides and steroid hormones on astroglial cells.

The relationship between structure and activity among opioid peptides

Since the discovery and isolation of the endogenous opioid peptides Leu- and Met-enkephalin, structural studies have been focused on deducing the bioactive conformation of the peptide ligands. Theoretically, linear peptides can have many different backbone conformations, yet early, X-ray studies on enkephalin and its analogues showed only two different backbone conformations: extended and single β-bend. More recent reports include a third conformation for Leu-enkephalin and constrained opioid peptides from two ‘new’ classes (i.e. cyclic and ‘allaromatic’ peptides). In this report the relationship between solid-state X-ray structure and opioid peptide activity is examined. The N-terminal amine nitrogen and the two aromatic rings have previously been identified as structural features important to the biological activity of opioid peptides. From X-ray studies we find that the distances between the centroids of the aromatic rings, and between the N-terminal amine nitrogen and the centroid of the phenylalanine ring, vary over a large range. There is a discernible relationship, however, between the separation of the two rings and their orientation that correlates with activity.

Altered responses to female odors in parasitized male mice: neuromodulatory mechanisms and relations to female choice

There is accumulating evidence that females may preferentially select parasite-free or -resistant males. Minimal attention has, however, been paid to the mate preferences and responses of the parasitized male hosts themselves. Here, we considered the effects of parasitic infection on male host mate responses, the neuromodulatory correlates of these responses, and the relations of these responses to female mate choice. Using an odor “preference” test, we examined the effects of different stages of an acute, sub-clinical infection with the naturally occurring, enteric, single host, protozoan parasite, Eimeria vermiformis, on the responses of male mice, Mus musculus domesticus, to the odors of estrous females along with the responses of uninfected females to the parasitized males. At 4 days post-infection (non-infective, pre-patent stage) E. vermiformis-infected male mice showed a significantly decreased preference for the odors of estrous females, whereas at 10 days post-infection (infective, patent stage) infected males showed a significantly increased preference for the odors of estrous females. Parasitized males displayed no significant changes in their responses to the odors of non-estrous females, supporting effects on the reproductively related responses of the host. In parallel, estrous females displayed a reduced interest in the odors of infected males. Least interest was expressed in the odors of the patent, infective males, consistent with the avoidance of contagion. Using selective opioid peptide receptor agonists and antagonists we found evidence that enhanced kappa opioid peptide (e.g., dynorphin) activity was related to the decreased sexual interest of the pre-infective males, while augmented delta opioid peptide (e.g., enkephalin) activity was associated with the enhanced responses of the infective males to females. We further showed that acute kappa opiate administration reduced the responses of uninfected males to females and that uninfected females displayed modified responses to the odors of uninfected males subject to acute modifications of opioid activity. We suggest that these differential shifts in endogenous opioid activity in the parasitized males are associated with and, or related to alterations in neuro-immune and endocrine functions. These findings show that parasitic infection can have, depending on the stage of infection and associated neuromodulatory changes, either significant facilitatory or inhibitory effects on male host preferences for and responses to females.

Opiate receptor knockout mice define mu receptor roles in endogenous nociceptive responses and morphine-induced analgesia.

Morphine produces analgesia at opiate receptors expressed in nociceptive circuits. mu, delta, and kappa opiate receptor subtypes are expressed in circuits that can modulate nociception and receive inputs from endogenous opioid neuropeptide ligands. The roles played by each receptor subtype in nociceptive processing in drug-free and morphine-treated states have not been clear, however. We produced homologous, recombinant mu, opiate receptor, heterozygous and homozygous knockout animals that displayed approximately 54% and 0% of wild-type levels of mu receptor expression, respectively. These mice expressed kappa receptors and delta receptors at near wild-type levels. Untreated knockout mice displayed shorter latencies on tail flick and hot plate tests for spinal and supraspinal nociceptive responses than wild-type mice. These findings support a significant role for endogenous opioid-peptide interactions with mu opiate receptors in normal nociceptive processing. Morphine failed to significantly reduce nociceptive responses in hot plate or tail flick tests of homozygous mu receptor knockout mice, and heterozygote mice displayed right and downward shifts in morphine analgesia dose-effect relationships. These results implicate endogenous opioid-peptide actions at mu opiate receptors in several tests of nociceptive responsiveness and support mu receptor mediation of morphine-induced analgesia in tests of spinal and supraspinal analgesia.

The distribution of methionine-enkephalin and leucine-enkephalin in the brain and peripheral tissues. 1977.

A method is described for the rapid extraction of opioid peptides from the brain and other tissues. The method is based on acid extraction of tissues followed by adsorption of the extract onto Amberlite XAD‐2 resin. Elution with methanol separates the enkephalins and α‐endorphin from β‐endorphin. Over 90% of the opioid peptide activity isolated from brain and gut of several species by our method was due to methionine‐ and leucine‐enkephalin. In contrast, the major opioid peptide activity recovered from the pituitary was due to peptides of much greater mol. wt. than the enkephalins. An opioid peptide with properties unlike those of the known endorphins or enkephalins was present in brain extracts. This peptide, termed ∈‐endorphin, has an apparent mol. wt. of 700 to 1200; it constituted between 5 to 10% of the total opioid activity in our extracts. A differential assay of methionine‐ and leucine‐enkephalin was made either by destroying methionine‐enkephalin activity with cyanogen bromide or by separating the peptides by thin layer chromatography. The ratio of methionine‐enkephalin to leucine‐enkephalin varied greatly in different brain regions. The highest proportions of leucine‐enkephalin were found in the cerebral cortex and hippocampus. Formaldehyde perfusion and fixation of the brain in vivo had no significant effect on the brain content of enkephalin, indicating that proteolytic breakdown is not a major problem in the extraction of these peptides. It is suggested that the enkephalins may have a neurotransmitter role in both brain and peripheral tissues and that methionine‐ and leucine‐enkephalin may subserve separate neuronal functions.

Cocaine and Inhibition of Nitric Oxide Synthesis Produce Opioid-Mediated Antinociception

Cocaine and nitric oxide are known to influence the perception of pain. The present study sought to determine if the endogenous opioid peptide system participates in cocaine-induced antinociception and antinociception produced by antagonism of nitric oxide. Pain perception was measured using the hot plate test. Administration of cocaine (25 mg/kg) to male rats resulted in a significant increase in reaction time in the hot plate test, which was reversed by treatment with 3, 10, and 30 mg/kg of the opiate antagonist, naloxone. In rats that were not treated with cocaine, doses of 30 and 60 mg/kg of naloxone significantly reduced hot plate reaction time. Treatment of animals with the nitric oxide synthase inhibitor, Nωnitro-L-arginine, produced a significant increase in response time to the hot plate, which was reversed by administration of naloxone. These data indicate that antinociception produced in the rat by cocaine appears to have a supraspinal component and to involve activation of endogenous opioid peptide activity in the brain. The results also suggest a tonic inhibition of endogenous opioid peptide activity by nitric oxide, which when antagonized, results in diminished response to pain.

Structure-function analysis of the cloned opiate receptors: peptide and small molecule interactions.

Opiate receptors mediate the physiological actions of opioid peptides and the clinical effects of the synthetic opioid agonists and antagonists. Site-directed mutagenesis studies have revealed regions of opiate receptors that are essential for ligand recognition, and this could aid the design of more selective opioid ligands.

Purification and characterization of a secreted T cell beta-endorphin endopeptidase.

Primary murine CD4+ and CD8+ T cells and the EL-4 thymoma cell line secrete a metal-dependent, thiol endopeptidase that hydrolyzes β-endorphin at essentially equivalent rates at one of either of two peptide bonds (leu17-phe18 or phe18-lys19) yielding β-endorphin1–17 (γ-endorphin) or β-endorphin1–18 and their corresponding C-terminal fragments β-endorphin18–31 and β-endorphin19–31. This endopeptidase was purified to homogeneity from EL-4 cell conditioned medium (>1,000 fold, 26% yield, a single band of ≈110 kDa via SDSPAGE). The enzyme also cleaves dynorphin A1–7 and dynorphin B1–13 but not Leuenkephalin, dynorphin A1–9, γ-endorphin, or β-endorphin18–31. The endopeptidase is expressed constitutively in EL-4 cells and upon anti-CD3 activation of CD4+ and CD8+ T cells. Based on cleavage sites identified in a number of opioid peptide substrates, it can be concluded that the enzyme exhibits a complex specificity with a preference for cleaving on the amino side of hydrophobic or basic residues of larger peptide substrates. The presence of such an extracellularly acting peptidase clearly could be important in attenuating or modifying the biological activity of opioid peptides in their role as extracellular chemical signals between cells.

Action of opioid peptides on the rat adrenal cortex: stimulation of steroid secretion through a specific μ opioid receptor

While there have been several studies on the actions of opioid peptides on adrenocortical steroidogenesis, the results of these studies have failed to resolve the question as to whether these peptides exert a direct action on the adrenal cortex. The present studies were designed to address this question directly, using collagenase-dispersed rat zona glomerulosa and zonae fasciculata/reticularis cells incubated in vitro. The results obtained clearly show that the opioid peptides tested (beta-endorphin, Leu-enkephalin, Met-enkephalin, and its long-acting analogue, DALA) all exerted a significant stimulatory effect on aldosterone secretion by zona glomerulosa cells and all, except Leu-enkephalin, stimulated corticosterone secretion by inner zone cells. The response was shown to be inhibited by naloxone. There did not appear to be a significant interaction between the effects of ACTH and the opioid peptides on adrenocortical cells. Studies using specific agonists for opioid receptor subtypes (DAMGO, DPDPE and U-50488H, specific for mu, delta and kappa receptors respectively) showed that the effect of opioid peptides on the zona glomerulosa appeared to be mediated exclusively by mu receptors while the response of inner zone cells was mediated by both mu and, to a lesser extent, kappa receptors. Finally, studies on the second messenger systems activated by the opioid peptides and the receptor agonists showed that these peptides act to increase labelling of inositol trisphosphate, and strongly suggest that, in the rat adrenal cortex, both mu and kappa opioid receptors are linked to the activation of phospholipase C.

Synthesis and biological activity of opioid peptides containing nucleoamino acid residues

Synthesis and biological activity of opioid peptides containing nucleoamino acid residues

Role of Opioid Peptides in the Regulation of Cytokine Production by Murine CD4 + T Cells

The presence of the opioid peptides α- and β-endorphin (-End) but not methionine enkephalin (Met-enk) in in vitro cultures of purified CD4 + T cells, stimulated with concanavalin A in the presence of irradiated spleen cells, resulted in a threefold stimulation of IL-2, IL-4, and IFN-γ production. The stimulating effect was dependent on the concentration of the peptides and reached optimal values in the dose range from 10 -12 to 10 -10 M. Similar results were obtained when purified CD4 + T cells were stimulated with immobilized anti-CD3, indicating a direct effect of opioid peptides on CD4 + T cells. Moreover, in this system a twofold enhancement of IL-6, but not IL-1, secretion was observed. These stimulatory effects were not mediated through opioid receptors since the peptide fragment β-End 6-31 that lacks the N-terminal opioid receptor binding part was still stimulatory. This is in agreement with our finding that β-End did not affect cAMP, as described for the triggering of classical opioid receptors. Experiments undertaken to reveal the mechanism of action of opioid peptides suggest an overall enhancement of lymphokine production: (1) enhancement of IL-4 production occurred also in the presence of excess IL-2; and (2) neither IL-1 receptor-antagonizing protein nor anti-IL-6 were capable to abrogate the stimulatory effect on IL-2 and IL-4 production. Finally, the presence and activity of opioid receptors in cultures of CD4 + T cells were substantiated by the fact that the opioid receptor antagonist naloxone by itself enhanced cytokine synthesis, which points to the endogenous production by lymphocytes of down-regulating opioid peptides.

Opioid peptide release evoked by noxious stimulation of the hind instep of rats

Previous studies showed that opioids and opioid peptides modulate vascular reactions such as plasma extra-vasation and vasodilatation through inhibition of substance P release from peripheral nerve endings of primary afferent fibers, and suggested the existence of endogenous opioid peptide activity related to regulation of inflammatory responses. Here we examined the effect of heat stimulation and antidromic stimulation of primary afferent fibers on the release of immunoreactive opioid peptides into the perfusate of the subcutaneous space in the rat instep. Antidromic stimulation of sectioned sciatic and saphenous nerves did not have any significant effect on the release of Met-enkephalin (Met-EK), while immersion of the hind paw in hot water (47°C) for 30 min caused an increase in Met-EK release into the perfusate. High-pressure liquid chromatography of the perfusate revealed that noxious heat stimulation induced increase in release of Leu-enkephalin (Leu-EK) as well as Met-EK, although the maximal concentration of Leu-EK was less than 16% of that of Met-EK. No β-endorphin was detected in the perfusate before, during or after heating. We conclude that noxious heat stimulation mainly leads to increase in Met-EK, and that this peptide originates mainly, from peripheral cells containing opioid peptides such as immune cells and/or Merkel cells, not from primary afferent fibers.

Evidence for Involvement of Endogenous Opioid Peptides in Altered Nociceptive Responses of Mice Infected with Eimeria vermiformis

Parasite modification of host behavior is a well established phenomenon; however, little is known about the modulatory mechanisms regulating such effects. This study examined the relationship between Eimeria vermiformis infection, nociceptive responses, and endogenous opioid peptide activity in male RML mice. Infected mice displayed increases in centrally mediated antinociceptive responses (i.e., analgesia, measured as the latency of a foot-lifting response to a 50 C surface) throughout the prepatent period. The level of analgesia declined following onset of patency on day 8 postinfection (PI). Opioids were implicated in the increased antinociceptive response of the infected mice as the response was blocked by administration of a prototypic opiate antagonist, naloxone (1.0 mg/kg), on day 7 PI when maximum levels of analgesia were noted. This indicates that the analgesia evident in the parasitized mice was associated with increased opioid activity. Analgesia is one of a variety of behaviors influenced by changes in opioid activity, thus these observations provide further support for the contention that other parasite-induced alterations in host behavior may, in part, be the result of alterations in the activity of opioid modulatory systems.

Penetration of phospholipid monolayers by opioid peptides and opiate drugs (agonists and antagonists)

The monomolecular film technique was used to compare the specific interaction of opioid peptides (Leu-enkephalin, Met-enkephalin, dynorphin A and β-endorphin) and opiate agonists (alkaloids such as levorphanol and ethylketocyclazocine) and opiate antagonists (naloxone and diprenorphine) with various phospholipid films (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and dioleoylphosphatidylglycerol). We were able to demonstrate the interaction of opiate drugs and opioid peptides (0.1 μM) with both zwitterionic and negatively charged phospholipids up to very high surface pressures. In the presence of a phospholipid monolayer, the surface activity of opioid peptides became much greater than that observed at the air-water interface. In the case of opiate drugs, the phenomenon was different and these drugs penetrate the monolayer films at a low pressure in comparison with opioid peptides, even at very high concentration. We have also evaluated the penetration of the antagonists naloxone and diprenorphine; the interaction of these was very low.

Opioids and testicular activity in the frog, Rana esculenta.

The presence and activity of brain, pituitary and testicular beta-endorphin (beta-EP)-like material have been studied in the frog, Rana esculenta, using reverse-phase high-pressure liquid chromatography, coupled with radioimmunoassay and immunocytochemistry. In-vivo and in-vitro treatments with naltrexone were carried out to assess the putative physiological activity of opioid peptides. beta-EP(1-31) and (1-27), together with their acetylated forms, have been identified in brain, pituitary and testis. In particular, beta-EP(1-31) concentrations peaked during July in the brain and pituitary, whilst in testes maximum concentrations were found in April and November. beta-EP immunoreactivity was present in the brain within the nucleus preopticus and nucleus infundibularis ventralis while positive fibres in the retrochiasmatic regions projected to the median eminence. In the testis, interstitial cells, canaliculi of the efferent system, spermatogonia and spermatocytes showed positive immunostaining for beta-EP. In intact animals, naltrexone treatment increased plasma and testicular androgen levels and this effect was confirmed in in-vitro incubations of minced testes. Naltrexone also induced a significant increase in germ cell degeneration. Our results indicated that an opioid system modulates the hypothalamus-pituitary-gonadal axis in the frog, Rana esculenta and, for the first time, we have shown that the testicular activity of a non-mammalian species may be regulated by opiates locally.

Cardiovascular and Other Physical Effects of Acute Administration of Naltrexone in Autistic Children

ABSTRACT The physical side effects of acutely administered naltrexone (0.5, 1.0, 1.5, and 2.0 mg/kg, orally) were compared with placebo in 13 children (3-12 years old) with autism. Heart rate, systolic blood pressure, mean arterial blood pressure, and axillary body temperature were obtained before and 1 h after placebo or drug administration. Serum concentrations of the liver enzymes glutamic-oxaloacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT) were obtained 1 h and 24 h after drug. In comparison with placebo, no significant effects of any of the four doses of naltrexone were found on any of these measures. Three hours after administration, there were no significant effects of acute naltrexone on EKG parameters (heart rate, axis, PR, QRS, QT, or QTc) compared to predrug values. These data support the safety of acute administration of naltrexone on vital signs, EKG, and liver function in preadolescent children with autism and are consistent with studies in healthy normotensive adults. In view of prior findings that naltrexone can alter cardiovascular function in certain pathologic states (including some that involve increased opioid peptide activity) and findings of increased opioid activity in some autistic individuals, the absence of cardiovascular effects of naltrexone in autistic children is tentatively suggestive of the safety of acutely administered naltrexone. Since chronic high doses of naltrexone can increase liver transaminase levels in adults, it remains advisable for clinicians to monitor liver function in children receiving chronic naltrexone treatment. Other adverse effects of acute naltrexone in these children appeared to be minimal.