RB101-mediated protection of endogenous opioids: potential therapeutic utility?

The endogenous opioids met- and leu-enkephalin are inactivated by peptidases preventing the activation of opioid receptors. Inhibition of enkephalin-degrading enzymes increases endogenous enkephalin levels and stimulates robust behavioral effects. RB101, an inhibitor of enkephalin-degrading enzymes, produces antinociceptive, antidepressant, and anxiolytic effects in rodents, without typical opioid-related negative side effects. Although enkephalins are not selective endogenous ligands, RB101 induces these behaviors through receptor-selective activity. The antinociceptive effects of RB101 are produced through either the mu-opioid receptor alone or through activation of both mu- and delta-opioid receptors; the antidepressant-like and anxiolytic effects of RB101 are mediated only through the delta-opioid receptor. Although little is known about the effects of RB101 on other physiologically and behaviorally relevant peptides, these findings suggest that RB101 and other inhibitors of enkephalin-degrading enzymes may have potential as novel therapeutic compounds for the treatment of pain, depression, and anxiety.

Influence of pain treatment by epidural fentanyl and bupivacaine on homing of opioid-containing leukocytes to surgical wounds.

Endogenous opioids released from leukocytes extravasating into injured tissue can interact with peripheral opioid receptors to inhibit nociception. Animal studies have shown that the homing of opioid-producing leukocytes to the injured site is modulated by spinal blockade of noxious input. This study investigated whether epidural analgesia (EDA) influences the migration of beta-endorphin (END) and/or met-enkephalin (ENK)-containing leukocytes into the subcutaneous wound tissue of patients undergoing abdominal surgery. In part I patients received general anesthesia combined either with intra- and postoperative EDA (with bupivacaine and fentanyl) or with postoperative patient controlled intravenous analgesia (PCIA; with the opioid piritramide). In part II patients received general anesthesia combined with either epidural fentanyl or bupivacaine which was continued postoperatively. Samples of cutanous and subcutanous tissue were taken from the wound site at the beginning, at the end and at various times after surgery, and were examined by immunohistochemistry for the presence of END and ENK. We found that (i) epidural bupivacaine, fentanyl and PCIA provided similar and clinically acceptable postoperative pain relief; (ii) compared to PCIA, epidural bupivacaine or fentanyl did not change the gross inflammatory reaction within the surgical wound; (iii) opioid-containing leukocytes were almost absent in normal subcutaneous tissue but migrated to the inflamed wound tissue in ascending numbers within a few hours, reaching a peak at about 24 h after surgery; (iv) compared to PCIA, EDA resulted in significantly decreased homing of END-containing leukocytes to the injured site at 24 h after surgery; and (v) the magnitude of this decrease was similar regardless of the epidural medication. These findings suggest that nociceptive but not sympathetic neurons are primarily involved in the attraction of opioid-containing leukocytes during early stages of inflammation.

Amphetamine triggers an increase in met-enkephalin simultaneously in brain areas and immune cells.

We analyzed effects of amphetamine on proenkephalin-derived peptides in brain areas and immune cells in rats. Acute, as well as a repeated amphetamine treatment, decreased the concanavalin-A-induced lymphocyte proliferation, concomitantly with an increase of free met-enkephalin in nucleus accumbens, prefrontal cortex, spleen, thymus and splenic macrophages. Proenkephalin protein increased in prefrontal cortex, thymus (32 kDa isoform), nucleus accumbens and spleen (44 kDa isoform), while proenkephalin mRNA levels decreased in brain stem. The influence of met-ENK in key brain areas for sensitization and in immune organs is consistent with the idea that changes on met-ENK could underlie amphetamine's effects on brain and IS.

Withdrawal following sufentanil/propofol and sufentanil/midazolam. Sedation in surgical ICU patients: correlation with central nervous parameters and endogenous opioids.

PURPOSE: Patients in the ICU after long-term administration of an opioid/hypnotic often develop delirium. To assess the nature of this phenomenon, patients in a surgical ICU following ventilatory support and sedation with an opioid/hypnotic/sedative were studied. METHODOLOGY: Following sufentanil/midazolam (group 1; n =14) or sufentanil/propofol (group 2; n =15) sedation, patients were evaluated for changes in mean arterial blood pressure and heart rate, the activity of the central nervous system (sensory evoked potentials, spectral edge frequency of EEG), and the endogenous opioids plasma concentrations (beta-endorphin, met-enkephalin). Data obtained were correlated with the individual intensities of withdrawal symptoms 6-, 12-, and 24 h following sedation. RESULTS: Following a mean duration of ventilation of 7.7 days (+/-3.6 SD) in groups 1 and 3.5 (+/-1.7 SD) in group 2, withdrawal intensities peaked within the 6th hour after cessation. Plasma beta-endorphin and met-enkephalin levels were low during sedation, and only the sufentanil/midazolam group demonstrated a postinhibitory overshoot. Withdrawal symptom intensities demonstrated an inverse correlation with beta-endorphin and met-enkephalin levels, a direct linear correlation with amplitude height of the evoked potential, and blood pressure and heart rate changes. Withdrawal intensities did not correlate with EEG power spectral edge frequency. CONCLUSION: The endorphinergic system is suppressed when a potent exogenous opioid like sufentanil is given over a long period of time. Following sedation, abstinence symptoms seem to be related to postinhibitory increased endorphin synthesis. This is mostly seen in the combination of sufentanil/midazolam. In addition, an increase in the amplitude of the sensory-evoked potential suggests a postinhibitory excitatory state within the nociceptive system.

Delta9-tetrahydrocannabinol releases and facilitates the effects of endogenous enkephalins: reduction in morphine withdrawal syndrome without change in rewarding effect.

Recent studies have suggested that cannabinoids might initiate the consumption of other highly addictive substances, such as opiates. In this work, we show that acute administration of Delta9-tetrahydrocannabinol in mice facilitates the antinociceptive and antidepressant-like responses elicited by the endogenous enkephalins protected from their degradation by RB 101, a complete inhibitor of enkephalin catabolism. This emphasizes the existence of a physiological interaction between endogenous opioid and cannabinoid systems. Accordingly, Delta9-tetrahydrocannabinol increased the release of Met-enkephalin-like material in the nucleus accumbens of awake and freely moving rats measured by microdialysis. In addition, this cannabinoid agonist displaced the in vivo [3H]diprenorphine binding to opioid receptors in total mouse brain. The repetitive pretreatment during 3 weeks of Delta9-tetrahydrocannabinol in mice treated chronically with morphine significantly reduces the naloxone-induced withdrawal syndrome. However, this repetitive administration of Delta9-tetrahydrocannabinol did not modify or even decrease the rewarding responses produced by morphine in the place preference paradigm. Taken together, these behavioural and biochemical results demonstrate the existence of a direct link between endogenous opioid and cannabinoid systems. However, chronic use of high doses of cannabinoids does not seem to potentiate the psychic dependence to opioids.

Thalidomide treatment in chronic constrictive neuropathy decreases endoneurial tumor necrosis factor-alpha, increases interleukin-10 and has long-term effects on spinal cord dorsal horn met-enkephalin.

Thalidomide reduces thermal hyperalgesia and mechanical allodynia in chronic constrictive sciatic nerve injury (CCI). Since thalidomide mainly inhibits tumor necrosis factor alpha (TNF-alpha) synthesis with less well defined effects on other cytokines, we investigated the effect of the drug on the expression of the proinflammatory cytokines TNF-alpha, interleukin-1beta (IL-1beta) and interleukin 6 (IL-6), and of the anti-inflammatory cytokine interleukin-10 (IL-10) in the lesioned rat sciatic nerve. The increase of endoneurial TNF-alpha during the first week after CCI was reduced after thalidomide treatment, as shown with immunohistochemistry and enzyme-linked-immunosorbent assay. In contrast, endoneurial IL-1beta-immunoreactivity (IR) and IL-6-IR were not altered by thalidomide treatment, nor was macrophage influx. Recruitment of epineurial IL-10 immunoreactive macrophages as well as the recovery of injury-induced depletion of endoneurial IL-10-IR was enhanced by thalidomide treatment. To control for central plasticity as another factor for the effects of thalidomide, the spinal cord was analyzed for changes in neurotransmitters. The decrease in CGRP-IR and SP-IR in the dorsal horn of operated animals was not influenced by treatment. In contrast, the increase in met-enkephalin observed in the dorsal horn of operated animals was further enhanced in the thalidomide-treated animals. The study elucidates some of the complex alterations in CCI and its modulation by thalidomide, and provides further evidence for a possible therapeutic benefit of cytokine-modulating substances in the treatment of neuropathic pain.

Intravenous injections of the ribosome inactivating protein trichosanthin did not affect methionine enkephalin and beta-endorphin levels in the mouse brain and pituitary.

The effect of trichosanthin on levels of the neuropeptides beta-endorphin (beta-EP) and methionine enkephalin (met-EK) in the mouse brain and pituitary was investigated. Mature male ICR mice were divided into two groups. One group received intravenous injections of physiological saline and served as the control. Another group received daily intravenous injections of trichosanthin (0.2 mg/25g/injection) for three consecutive days. The animals were sacrificed four hours after the last injection. Their brains were dissected into three regions: A (thalamus and hypothalamus), B (cerebral cortex) and C (cerebellum and brainstem) and their pituitaries were collected. The samples were then extracted and assayed for beta-EP and met-EK by specific radioimmunoassays. It was found that there were no statistically significant changes in the levels of the two neuropeptides in the pituitary and the brain regions except for the level of beta-EP in brain region A.

The effect of haloperidol on met-enkephalin, beta-endorphin, cholecystokinin and substance P in the pituitary, the hypothalamus and the striatum of rats during aging.

1. Haloperidol increased the Met-enk level in the striatum at all age groups. However, the Met-enk level was decreased in AL of young and middle-aged rats by the drug. 2. Haloperidol elevated the beta-end level in AL and CCK level in NIL in young rats only. 3. The SP content in NIL was decreased by haloperidol in all age groups. 4. With regard to the effect of aging, Met-enk level in AL of middle-aged rats was higher than that in young rats. The beta-end level in AL also increased in old rats. 5. Aging modified the haloperidol effect on beta-end level in AL and CCK level in NIL as the effect was only observed in young rats. 6. In addition, aging caused a blunted response of Met-enk level to haloperidol in the striatum but an increased response of SP content to haloperidol in the NIL.

Specific molecular mass detection of endogenously released neuropeptides using in vivo microdialysis/mass spectrometry.

The specific molecular detection of the endogenous neuropeptides methionine ([Met]5) enkephalin and neurotensin released in vivo in rat brain has been accomplished using microdialysis and mass spectrometry. Microdialysis probes were implanted in specific brain regions and were used to collect samples from brain extracellular fluids in unanesthetized, freely moving animals. Microelectrospray/tandem mass spectrometry was used to achieve molecular-specific identification of the neuropeptides with a sensitivity in the amol/microliters range. Measurements of the amounts of neuropeptides in the dialysates obtained from studies of KCl-stimulated release showed that [Met]5-enkephalin from the globus pallidus/ventral pallidum region was present at a level of approximately 4-6 fmol/10 microliters of dialysate and neurotensin from the hypothalamus of approximately 500 amol in 10 microliters of dialysate. In this manuscript, we present the first data of a mass- and molecular-specific detection and quantitation of individual neuropeptides released in response to either intracerebrally or systemically administered compounds.

[The effect of reaferon on the opioid systems of chronically alcoholized rats]. / Vliianie reaferon na opioidnye sistemy khronicheski alkogolizirovannykh krys.

Chronic alcoholic intoxication is followed by a fall in 3H-(D-ala-2,D-leu-5)-enkephalin affinity of mu-opioid receptors with their unchanged concentrations in the rat brain cortex, by reductions in the tissue and plasma levels of beta-endorphine and met-enkephalin. A daily administration of reaferon in a dose of 10,000 IU during two fortnights completely restored both the binding affinity of the receptors and the concentrations of the peptides tested: those of beta-endorphine in the adenohypophysis and plasma and those of metenkephalin in the adrenals and plasma.

Effects of acute xylene exposure on the enkephalinergic neuromodulatory system in rats.

Xylene is a neurotoxic organic solvent widely used in industry. However, the neurochemical mechanism of its action on the central nervous system is to date relatively unknown. In this work, the effect of subacute xylene exposure on met-enkephalin like immunostaining in different brain regions is described. Acute treatment with xylene generates a reduction in immunostaining for met-enkephalin in the globus pallidus, the olfactory tubercule and the hypothalamic medial preoptic area, without changes in the parietal cortex, caudatus-putamen and the central amygdaloid nuclei. It is suggested that enkephalins could play a role in xylene neurotoxic mechanism in the brain.

Effect of subacute toluene administration on the enkephalinergic neuromodulatory system in rats and protective action of ganglioside treatments.

Gangliosides perform protective functions in the central nervous system. This paper describes a study of the effect of ganglioside administration on toluene neurotoxicity. Rat brain met-enkephalin immunostaining in the central amygdaloid nuclei showed changes in rats treated simultaneously with gangliosides and toluene with respect to rats treated with toluene alone. It is suggested that gangliosides prevent toluene neurotoxicity at this level, leading to hypothetical neurobehavioral changes.

[The effect of the aminopeptidase inhibitor bestatin on the enkephalin level in the rat brain]. / Vliianie ingibitora aminopeptidaz bestatina na uroven' énkefalinov v mozge krys.

Met- and leu-enkephalin contents in midbrain (including hypothalamus) and striatum of rats were determined by radioimmunoassay after bestatin (racemate) injection (200 g, i.c.v.). It was found that bestatin administration influenced the midbrain met-enkephalin content, values and directions of the changes observed being dependent upon the time after the injection. The data obtained confirm the participation of aminopeptidase in enkephalin inactivation and present evidence for the possibility of regional variations of enkephalin catabolism pathways in the brain.

[Changes in the number of neuromediator receptors on murine lymphocytes in ontogeny]. / Izmenenie chisla retseptorov k neiromediatoram na limfotsitakh myshei v ontogeneze.

It has being shown by radiological methods of evaluation of the binding sites with adrenergic, cholinergic and neuropeptide agents that during mice ontogenesis the number of binding sites to these agents is progressively declined. The cAMP accumulation by lymphoid cells after contact with adrenaline is declined also. The regularities shown may reflect processes of the immune change during the age.