The influence of experimental inflammation and axotomy on leucine enkephalin (leuENK) distribution in intramural nervous structures of the porcine descending colon.

BACKGROUND: The enteric nervous system (ENS), located in the intestinal wall and characterized by considerable independence from the central nervous system, consists of millions of cells. Enteric neurons control the majority of functions of the gastrointestinal tract using a wide range of substances, which are neuromediators and/or neuromodulators. One of them is leucine-enkephalin (leuENK), which belongs to the endogenous opioid family. It is known that opioids in the gastrointestinal tract have various functions, including visceral pain conduction, intestinal motility and secretion and immune processes, but many aspects of distribution and function of leuENK in the ENS, especially during pathological states, remain unknown. RESULTS: During this experiment, the distribution of leuENK - like immunoreactive (leuENK-LI) nervous structures using the immunofluorescence technique were studied in the porcine colon in physiological conditions, during chemically-induced inflammation and after axotomy. The study included the circular muscle layer, myenteric (MP), outer submucous (OSP) and inner submucous plexus (ISP) and the mucosal layer. In control animals, the number of leuENK-LI neurons amounted to 4.86 ± 0.17%, 2.86 ± 0.28% and 1.07 ± 0.08% in the MP, OSP and ISP, respectively. Generally, both pathological stimuli caused an increase in the number of detected leuENK-LI cells, but the intensity of the observed changes depended on the factor studied and part of the ENS. The percentage of leuENK-LI perikarya amounted to 11.48 ± 0.96%, 8.71 ± 0.13% and 9.40 ± 0.76% during colitis, and 6.90 ± 0.52% 8.46 ± 12% and 4.48 ± 0.44% after axotomy in MP, OSP and ISP, respectively. Both processes also resulted in an increase in the number of leuENK-LI nerves in the circular muscle layer, whereas changes were less visible in the mucosa during inflammation and axotomy did not change the number of leuENK-LI mucosal fibers. CONCLUSIONS: LeuENK in the ENS takes part in intestinal regulatory processes not only in physiological conditions, but also under pathological factors. The observed changes are probably connected with the participation of leuENK in sensory and motor innervation and the neuroprotective effects of this substance. Differences in the number of leuENK-LI neurons during inflammation and after axotomy may suggest that the exact functions of leuENK probably depend on the type of pathological factor acting on the intestine.

Leucine-enkephalin promotes wound repair through the regulation of hemidesmosome dynamics and matrix metalloprotease.

The skin responds to environmental stressors by coordinated actions of neuropeptides and their receptors. An endogenous peptide for δ-opioid receptor (DOPr), Leu-enkephalin (L-ENK), is expressed in the skin and its expression is altered in pathological conditions. Although the importance of DOPr is rapidly gaining recognition, the molecular mechanisms underlying its effects on wound healing are largely undefined. We show here that L-ENK induced activation of Erk, P90(RSK), and Elk-1 and promoted the disruption of hemidesmosomes and the expression of matrix metalloprotease (MMP)-2 and MMP-9, important processes for wound healing. Treatment with Erk inhibitor blocked activation of P90(RSK) and Elk-1 and significantly blunted wound repair. Therefore, our results suggest that activation of Erk and its downstream effectors, P90(RSK) and Elk-1, are critical for DOPr-mediated skin homeostasis.

Oxidative and nitrative modifications of enkephalins by human neutrophils: effect of nitroenkephalin on leukocyte functional responses.

Neutrophils play a major role in acute inflammation by generating reactive oxygen/nitrogen species. Opioid peptides, including enkephalins, are present at inflammation sites. Neutrophils contribute to protect against inflammatory pain by releasing opioid peptides. In this investigation, the ability of human polymorphonuclear cells to induce oxidative and nitrative modifications of Leu-enkephalin has been investigated in vitro. Activated human neutrophils mediate the oxidation of Leu-enkephalin resulting in the production of dienkephalin. In the presence of nitrite at concentrations observed during inflammatory and infectious process (10-50 µM), nitroenkephalin, a nitrated derivative of Leu-enkephalin, is additionally formed. The yield of nitroenkephalin increases with nitrite concentration and is significantly inhibited by the addition of catalase or 4-aminobenzoic acid hydrazide (ABAH), a specific inhibitor of peroxidases. These results suggest that neutrophils induce nitration of Leu-enkephalin by a mechanism that is dependent on myeloperoxidase activity and hydrogen peroxide. Oxidative/nitrative modifications of Leu-enkephalin have been also evidenced when cells were treated with the NO-donor molecule, DEANO. The nitrated enkephalin has been examined for its effect on leukocyte functional responses. The data reveal that nitroenkephalin at micromolar concentrations inhibits superoxide anion generation and degranulation of azurophilic granules of human polymorphonuclear cells. Moreover, nitroenkephalin inhibits spontaneous apoptosis of neutrophils, as evaluated by measuring caspase-3 activity. Collectively, our data indicate that the nitrated enkephalin attenuates neutrophil activation and promotes the short-term survival of these cells, suggesting a possible role of the nitrocompound in the efficiency and resolution of inflammatory processes.

Plasma endogenous enkephalin levels in early systemic sclerosis: clinical and laboratory associations.

OBJECTIVE: Met- and leu-enkephalins are endogenous opioid neuropeptides with potent analgesic, vasoactive, immunomodulatory and anti-apoptotic properties. We hypothesised that clinical or immunological variables of early systemic sclerosis (SSc) might be correlated to plasma enkephalin levels. METHODS: Plasma samples were collected at study entry of the Genetics versus Environment in Scleroderma Outcomes Study (GENISOS) cohort (early SSc, n=116). Plasma met-enkephalin and leu-enkephalin levels (microg/ml) were measured by high performance liquid chromatography (HPLC) and correlated to clinical and laboratory parameters in the GENISOS database. Statistical analyses were performed by nonparametric Wilcoxon rank sum tests and Pearson correlation coefficients. RESULTS: Significantly lower plasma met-enkephalin levels were associated with anti-topoisomerase-I seropositivity (6+8.3 vs. 14.9+22.8 microg/ml, p=0.02). Plasma leu-enkephalin levels were significantly higher in SSc patients with digital pulp loss (95.6+130 vs. 64.9+101 microg/ml, p=0.02). Lower mean plasma met-enkephalin levels and inversely higher leu-enkephalin levels were noted in SSc patients with Raynaud's phenomena (p=NS). CONCLUSION: The associations of plasma enkephalin levels to immunologic or clinical pathologies may underscore their vasogenic or fibrogenic significance and potential as therapeutic targets in early SSc.

Endogenous signaling complexity in neuropeptides- leucine- and methionine-enkephalin.

1. In addition to his many fine contributions in furthering our understanding of the neurochemical action of ecosanoids, catchelomines, steroids, anandamines, cannabinoids, endorphins, and the many modifications made to these neural factors, twenty years ago Julius Axelrod published a noteworthy paper concerning the nature of neuropeptides and their potential for multiple neurophysiological effects (Redgate et al., 1986). 2. In that report, Axelrod and coworkers described the neurological actions of the then recently discovered leucine- and methionine-enkephalins, and their biological functions which were novel, atypical, and in possession of neurological effects that were significantly "much more than additive." 3. In this short communication I would like to expand on this observation concerning the "additive effects" contained within the amino acid sequence of the atypical neurotransmitter peptides leucine- and methonine-enkephalin.

Topography and associations of leu-enkephalin and luteinizing hormone-releasing hormone neuronal systems in the human diencephalon.

Although several studies indicated that leu-enkephalin controls gonadal function, the morphological substrate of this modulation is unknown. To reveal potential interaction sites between leu-enkephalin and LH-releasing hormone (LHRH) in the hypothalamus, the distribution and connections of leu-enkephalin-immunoreactive (IR) and LHRH-IR systems were examined in the human diencephalon using double-label immunohistochemistry. First the leu-enkephalin-IR and LHRH-IR neural elements were mapped, then the maps of the two different neurotransmitter systems were superimposed unveiling the overlapping areas. The putative juxtapositions between leu-enkephalin-IR and LHRH-IR structures were revealed with double label immunocytochemistry. Close contacts were detected in the medial preoptic area and in the infundibulum/median eminence. In these areas, diaminobenzidine-silver-intensified, black leu-enkephalin-IR fibers abutted fusiform, brown, diaminobenzidine-labeled LHRH neurons often forming multiple contacts. Examination of semithin sections of these close associations with the aid of oil immersion revealed no cleft between the contacting LHRH-IR and leu-enkephalin-IR elements. Our findings indicate that the juxtapositions between LHRH-IR and leu enkephalin-IR neurons may be functional synapses forming the morphological substrate of the leu-enkephalin-modulated LHRH secretion in the human diencephalon. Moreover, the wide distribution of leu-enkephalin-IR elements suggests leu-enkephalin control of other diencephalic functions as well.

A possible role for an enkephalinergic system in the internal defense mechanism of Biomphalaria alexandrina exposed to Schistosoma mansoni.

The present study documents that in Bioniphalaria alexandrina coordinated responses to Schistosoma mansoni infection are modulated by receptor-mediated opioid signals. Rather comprehensive tests in susceptible and resistant snails have demonstrated: I- the presence of an endogenaus opioids in the snail hemolymph (in particular, Leu-enkephalin-like material). II- in vitro treatment of snail hemocytes with synthetic Leu-enkephalin analogue (DADLE) resulted in the modulation of cellular adherence, and phagocytic activity. III- the addition of Naloxone, either alone or in combination whith DADLE, generally reduced hemocyte activity indicating opioid-receptor-mediated mechanism. V- the presence of DADLE or Naloxone modulated the level of IL-2-, TNF-gamma- and FNF-alpha-like molecules in S. mansoni resistant and susceptible snails. Specifically, DADLE and DADLE in combination with Naloxone generally were found to be capable of modulating resistant snail hemocytes at concentrations of 10(-6) and 10(-8) M. Similar actions after incubation with the same concentrations were not detected in the susceptible snails. These observations demonstrate the existence of a complete opioid system in B. alexandrina, associated with susceptibility and resistance to S. mansoni infection, the results suggest the role of such opioid system in molecular signaling within the host and in host-parasite interactions.

Involvement of dynorphin in immobilization stress-induced antinociception in the mouse.

The effect of antiserum against [Met(5)]-enkephalin, [Leu(5)]-enkephalin, beta-endorphin, or dynorphin A-(1-13) administered intracerebroventricularly (i.c.v.) or intrathecally (i. t.) on immobilization-induced antinociception was studied in ICR mice. Antinociception was assessed by the tail-flick assay. Immobilization of the mouse increased inhibition of the tail-flick response at least 1 h. The i.c.v. or i.t. injection with antiserum against dynorphin A-(1-13) at the dose of 200 microg significantly attenuated immobilization-induced inhibition of the tail-flick response. However, antiserum against [Met(5)]-enkephalin, [Leu(5)]-enkephalin, or beta-endorphin did not affect the immobilization stress-induced antinociception. Furthermore, i.c.v. or i.t. injection with nor-binaltorphimine (Nor-BNI; from 1 to 20 microg) effectively inhibited immobilization stress-induced inhibition of the tail-flick response in a dose-dependent manner. However, beta-FNA (from 0.5 to 2 microg) or naltrindole (from 1 to 20 microg) administered i.c.v. or i.t. did not affect immobilization stress-induced antinociception. Our results suggest that supraspinally and spinally located dynorphin appears to be involved in the production of immobilization stress-induced antinociception via stimulating kappa-opioid receptors.

[Role of neuropeptides and "pain substances" in the formation of humoral mechanisms of experimental and postoperative pain]. / Rol' neiropeptidov i "bolevykh substantsii" v formirovanii gumoral'nykh mekhanizmov éksperimental'noi i posleoperatsionnoi boli.

Humoral mechanisms of pain caused by different factors vary. The authors compare blood concentrations of "painful substances" in experimental dogs and in patients suffering from postoperative pain relieved by electroacupuncture for assessing the role of these substances: serotonin, histamine, prostaglandin F2 alpha (PGF), and neuropeptides beta-endorphine, methionine- and leucine-enkephalines. Serotonin, histamine, and PGF participated in the nociception process in an equal measure both in dogs and humans. Notable differences were observed for neuropeptides, which can be explained by species-specific differences and by probable contribution of other neuropeptides to mechanisms of experimental and postoperative pain.

[Effect of enkephalins on the liver secretory function]. / Vliianie énkefalinov na sekretornuiu funktsiiu pecheni.

A decrease in the bile flow and bile acids, proteins and lipids secretion in enkephalines infusion into the portal vein was revealed. The bile secretion changes under the effect of enkephaline seem to result from the latter effect upon metabolic processes in hepatocytes.

Mouse skin reactions following fractionated irradiation with carbon ions.

PURPOSE: Skin reactions in the mouse leg following various daily doses given with 290 MeV/u carbon ions were investigated. MATERIALS AND METHODS: Seven different LET (linear energy transfer) values ranging from 14 to 100keV/microm were selected. The fractionation schedules were 1-, 2-, 4- and 8-daily fractions. The isoeffect doses to produce moist desquamation on the dose-response curves were calculated with 95% confidence limits. RESULTS: The isoeffect doses for carbon ions of 14 and 20 keV/microm increased with an increase in the number of fractions up to 4 fractions, but became constant when the number of fractions further increased to 8 fractions. This leveling off in isoeffect dose was more prominent for 40 keV/microm. Recovered dose per fraction was largest for 2 fractions of the 14keV/microm carbon beam. The isoeffect doses for 50, 60, 80 and 100keV/microm consistently increased with an increase in the number of fractions and did not show saturation up to 8 fractions. RBE (relative biological effectiveness) increased linearly with LET for all fractionation schedules. CONCLUSIONS: These results suggest that daily fractionation with carbon ions could spare radiation damage in patients, and that changes the fractionation schedule affect clinical outcome.

Conformational sampling of bioactive conformers: a low-temperature NMR study of 15N-Leu-enkephalin.

Conformational studies of enkephalins are hampered by their high flexibility which leads to mixtures of quasi-isoenergetic conformers in solution and makes NOEs very difficult to detect in NMR spectra. In order to improve the quality of the NMR data, Leu-enkephalin was synthesized with 15N-labelled uniformly on all amide nitrogens and examined in a viscous solvent medium at low temperature. HMQC NOESY spectra of the labelled Leu-enkephalin in a DMSOd6/H2O) mixture at 275 K do show numerous NOEs, but these are not consistent with a single conformer and are only sufficient to describe the conformational state as a mixture of several conformers. Here a different approach to the structure-activity relationships of enkephalins is presented: it is possible to analyse the NMR data in terms of limiting canonical structures (i.e. beta- and gamma-turns) and finally to select only those consistent with the requirements of delta selective agonists and antagonists. This strategy results in the prediction of a family of conformers that may be useful in the design of new delta selective opioid peptides.

Enkephalinergic nerve terminals target inhibitory interneurons in the rat hippocampus.

Using correlated light and electron microscopic preembedding enkephalin immunocytochemistry combined with post-embedding GABA immunogold staining, we found morphological evidence of a direct connection between the enkephalinergic and GABAergic systems in the rat hippocampus. Enkephalin-immunoreactive boutons were found to be presynaptic to GABA-immunoreactive postsynaptic profiles, establishing type 2 symmetrical synapses on GABA-positive cell bodies and dendritic shafts in strata radiatum and lacunosum moleculare of the CA1 region. Thirty-six percent of all studied postsynaptic targets (n = 40) were non-pyramidal, including all somatic (n = 7) and 47% of the dendritic (n = 13) postsynaptic targets. The remaining 64% consisted of pyramidal dendritic shafts and spines. These results support previous physiological experiments suggesting that the opioidergic system takes part in disinhibitory processes in the hippocampal formation.

Hypothalamo-septal enkephalinergic fibers terminate on AMPA receptor-containing neurons in the rat lateral septal area.

A large number of septal neurons express alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA)-type excitatory glutamate receptors. It has been demonstrated that in the mediolateral part of the rat lateral septum, calbindin-containing neurons are heavily innervated by hypothalamic, enkephalinergic fibers forming exclusively asymmetric synaptic contacts. This connection was suggested to be excitatory. In order to further elucidate this hypothesis, the aim of the present study was to determine whether these enkephalinoceptive neurons express GluR1 and GluR2/3 AMPA receptor subunits. Correlated light and electron microscopic analysis, using single immunostaining for GluR1 and GluR2/3, and double immunostaining for Leu-enkephalin and GluR1 or GluR2/3, was performed on vibratome sections of the rat lateral septal area. The studies revealed that while GluR1 is mainly associated with dendritic and somatic spines, GluR2/3 is mostly present in the perisomatic area. Leu-enkephalin boutons establish asymmetric synaptic contacts at the level of the soma and initial dendrites of both of these cells. A semiquantitative analysis showed that these enkephalin-targeted cells represent 50% of the total number of both GluR1 and GluR2/3-containing lateral septal neurons. These results suggest that: (1) AMPA receptor-expressing neurons appear to be the exclusive recipient of hypothalamic Leu-enkephalin boutons; (2) these enkephalinoceptive neurons contain both GluR1 and GluR2/3 AMPA receptor subunits; however, (3) only the GluR2/3 subtype, located in the perisomatic area, may be associated with Leu-enkephalin-containing inputs.

Evidence for localized and discrete roles for enkephalins during memory formation in the chick.

This study examined effects on memory formation produced by [Leu]enkephalin and [Met]enkephalin administration in 2 regions of the 2-day-old chick brain involved in memory formation: the intermediate medial hyperstriatum ventrale (IMHV) and the lobus parolfactorius (LPO). Basal concentrations of endogenous [Leu]enkephalin and [Met]enkephalin were determined for 5 brain regions, and effects of 1-trial peck-avoidance training on enkephalin concentrations were examined in the IMHV and LPO. [Leu]enkephalin was amnestic when administered in the IMHV but not in the LPO. In contrast, [Met]enkephalin may be amnestic when administered in the LPO but not in the IMHV. Training decreased [Met]enkephalin concentration in the LPO but not in the IMHV. Training had no effect on [Leu]enkephalin concentration in either the IMHV or the LPO. Thus, amnestic effects of [Leu]- or [Met]enkephalin administration are brain-region specific. Regional activity of endogenous [Met]enkephalin during memory formation is consistent with localized amnestic effects produced by [Met]enkephalin administration.

[Possible mechanism of the analgesic effect of interleukin-2 in central nervous system].

The monoclonal antibody against IL-2R (Tac) could not block the analgesic effect of IL-2, and IL-2 mutant that could not bind to beta subunit of IL-2 receptor still had capability of increasing the pain threshold of rats. All these facts suggest that the analgesic effect of IL-2 in CNS is not mediated through the IL-2 receptor, and that the immune and analgesic effects of IL-2 are mediated through different receptor mechanisms. It is suggested that there are common antigenic determinants and similar structure between IL-2 and endogenous opioid peptides (EOP). This implies that the analgesic effect of IL-2 might be mediated by interaction between IL-2 and opioid receptors in CNS. Using radioimmunoassay the contents of EOP of different nuclei were measured at different times after injecting IL-2 into the lateral ventricle of rats. The results suggested that the analgesic effect of IL-2 may be related to beta-EP and LEK in arcuate hypothalamic nucleus, paraventricular hypothalamic nucleus and locus ceruleus.

Ultrastructural evidence for convergence of enkephalin and adrenaline-containing axon terminals on common targets and their presynaptic associations in the rat nucleus locus coeruleus.

The endogenous opioid peptide, enkephalin, and epincphrine are distributed in varicose processes throughout the nucleus locus coeruleus (LC) in the dorsolateral tegmentum of the rat brain. In this brain region, micro-opioid and alpha-2-adrenergic receptors have been shown to share the same potassium channel suggesting that they may be co-localized in the same terminal or that they may be present in terminals that innervate the same target neuron. However, this has not been demonstrated at the ultrastructural level. Thus, the present study combined the immunocytochemical localization of the opioid peptide leucine5-enkephalin (ENK) and the epinephrine synthesizing enzyme, phenylethanolamine-N-methyltransferase (PNMT) in the same section of tissue within the LC at the electron microscopic level. At the light microscopic level, both ENK and PNMT varicose processes were dense and overlapped the region known to contain the noradrenergic cell bodies and dendrites of the LC. However, the morphological features of the two immunolabeled fiber types appeared different in 30-microns thick coronal sections. PNMT-labeled process were thin, beaded and ramified within the coronal plane. ENK-immunoreactive fibers, however, were more punctate in appearance and processes joining these puncta were not often evident in the frontal plane examined. Varicose fibers immunolabeled for either ENK or PNMT were confirmed to be axons and axon terminals by electron microscopy. Both types contained small clear as well as large dense core vesicles and formed heterogeneous types of synaptic specializations with postsynaptic targets. A common feature encountered in dually labeled tissue sections was convergence of the separately labeled axon terminals on common targets. Another common feature was the apposition of PNMT-labeled axon terminals with ENK-immunoreactive axon terminals that formed synaptic contacts in the plane of section examined. Although numerous ENK and PNMT-labeled axon terminals were identified in similar regions of the neuropil, few terminals were found to contain both labels. These findings indicate that the opioid peptide ENK and epinephrine may elicit concerted actions on common noradrenergic neurons in the LC via separate sets of afferents.

Stimulatory effect of intracerebroventricular met- and leu-enkephalin on corticosterone secretion in rats.

The significance of enkephalins for the function of the hypothalamic-pituitary-adrenal (HPA) axis, in spite of many efforts, is still elusive. We investigated the effect of leucine- and methionine-enkephalin on the HPA activity in conscious rats. These enkephalins, given intracerebroventricularly (i.c.v.) increased dose-dependently the activity of the HPA axis, measured indirectly through serum corticosterone levels. On a molar basis, leu-enkephalin exerted a stronger effect that met-enkephalin. Naloxone, an opioid receptor antagonist, given i.c.v. prior to enkephalins almost abolished the corticosterone response to met-enkephalin and significantly impaired the response to leu-enkephalin. Prazosin, an alpha 1-adrenergic antagonist, considerably reduced the increase in serum corticosterone levels induced by both enkephalins. Pretreatment of rats with yohimbine, an alpha 2-adrenergic antagonist, also considerably reduced the corticosterone response to met-enkephalin and significantly diminished the response induced by leu-enkephalin. Naloxone and yohimbine inhibited to the same extent the corticosterone response to met- and leu-enkephalin. This suggests an interaction between presynaptic opioid and alpha 2-receptors in regulation of the HPA function. Propranolol, a beta-adrenergic antagonist, given i.c.v. did not alter the corticosterone levels raised by met- and leu-enkephalin. These results indicate that both met- and leu-enkephalin increase the activity of the HPA axis in rats and both central opioid and adrenergic alpha-receptors are involved in this stimulation.