Double-enkephalins—Synthesis,activity on guinea-pig ileum,and analgesic effect

We have synthesized enkephalin analogues in which C-terminal methionine or leucine residues are replaced by a second active fragment of the enkephalin analogue. Synthesis of two compounds is described: in one, two fragments of a D-Ala²-enkephalin analogue are connected by a -NH-NH-bridge, and in the other, three methylene groups are incorporated between the amino groups. The first compound is a very potent inhibitor of electrically induced contractions of guinea-pig ileum and produces a strong analgesia when administered intraperitoneally in mice. The second compound is less active on the ileum and fails to produce analgesia after systemic injection. The double-enkephalins may interact with μ-receptors.     

Alterations within the endogenous opioid system in mice with targeted deletion of the neutral endopeptidase ('enkephalinase') gene

The biological inactivation of enkephalins by neutral endopeptidase (enkephalinase, NEP, EC3.4.24.11) represents a major mechanism for the termination of enkephalinergic signalling in brain. A pharmacological blockade of NEP-activity enhances extracellular enkephalin concentrations and induces opioid-dependent analgesia. Recently, knockout mice lacking the enzyme NEP have been developed [Lu et al., J. Exp. Med. 1995;181:2271-2275]. The present study investigates the functional consequences and biochemical compensatory strategies of a systemic elimination of NEP activity in these knockout mice. Using biochemical and behavioural tests we found that the lack of NEP activity in brain is not compensated by enhanced activities of alternative enkephalin-degrading enzymes. Also no change in enkephalin biosynthesis was detectable by in situ methods quantifying striatal proenkephalin-mRNA levels in NEP-deficient mice compared with wildtype. Only a 21% reduction of mu receptor density in crude brain homogenates of NEP knockout mice was observed, while delta- and kappa-opioid receptor densities were unchanged. This receptor downregulation was also confirmed functionally in the hot-plate paradigm. NEP knockouts developed normally, but showed enhanced aggressive behaviour in the resident-intruder paradigm, and altered locomotor activity as assessed in the photobeam system. Thus, although NEP plays a substantial role in enkephalinergic neurotransmission, the biochemical adaptations within the opioid system of NEP-deficient mice are of only modest nature.     

Differential effects on active avoidance performance and locomotor activity of two major enkephalin metabolites, tyr-gly-gly and des-tyr-[leu]enkephalin

We examined the effects of two enkephalin metabolites, des-tyr-[leu]enkephalin and tyr-gly-gly, on one-way active avoidance conditioning in mice. These metabolites are products of the two major enkephalin hydrolyzing enzymes in plasma, aminopeptidase and angiotensin converting enzyme. Like [leu]enkephalin from which it may be formed, tyr-gly-gly impaired avoidance acquisition, and its dose-response function for this effect was U-shaped. Also like [leu]enkephalin, tyr-gly-gly did not alter locomotor activity. On the other hand, des-tyr-[leu]enkephalin, at the doses tested, was without effect on avoidance conditioning but produced decreased locomotion. These data suggest that the tyrosine end of the enkephalin molecule may be important for its effects on conditioning. Because of their low opioid potencies, it is unlikely that the behavioral actions of tyr-gly-gly and des-tyr-[leu]enkephalin are mediated through opioid receptors.     

Endogenous methionine enkephalin may play an anticonvulsant role in the seizure-susceptible El mouse

After the intracisternal injection of three protease inhibitors which prevent the degradation of methionine enkephalin (amastatin, Des-Pro²-bradykinin, and phosphoramidon) and a mixture of these protease inhibitors, we investigated the effect on convulsive seizures in the seizure-susceptible El mouse. We also measured the cerebral methionine enkephalin content by high-performance liquid chromatography coupled with radioimmunoassay. Protease inhibitors significantly decreased both the incidence of seizures and the seizure score in El mice in a dose-dependent manner. This anticonvulsant effect was reversed by naloxone (2 mg/kg, sc). The cerebral methionine enkephalin content increased significantly after the administration of protease inhibitors in comparison with saline injection. These findings suggest that it was not protease inhibitors but instead increase of endogenous methionine enkephalin that reduced the incidence of seizures and the seizure score in El mice. Together with our previous data, the present findings support our hypothesis that a deficit in anticonvulsant endogenous methionine enkephalin is involved in the pathogenesis of seizures in the El mouse.     

Enkephalin knockout male mice are resistant to chronic mild stress

Enhanced stress reactivity or sensitivity to chronic stress increases the susceptibility to mood pathologies such as major depression. The opioid peptide enkephalin is an important modulator of the stress response. Previous studies using preproenkephalin knockout (PENK KO) mice showed that these animals exhibit abnormal stress reactivity and show increased anxiety behavior in acute stress situations. However, the consequence of enkephalin deficiency in the reactivity to chronic stress conditions is not known. In this study, we therefore submitted wild‐type (WT) and PENK KO male mice to chronic stress conditions, using the chronic mild stress (CMS) protocol. Subsequently, we studied the CMS effects on the behavioral and hormonal level and also performed gene expression analyses. In WT animals, CMS increased the expression of the enkephalin gene in the paraventricular nucleus (PVN) of the hypothalamus and elevated the corticosterone levels. In addition, WT mice exhibited enhanced anxiety in the zero‐maze test and depression‐related behaviors in the sucrose preference and forced swim tests. Surprisingly, in PENK KO mice, we did not detect anxiety and depression‐related behavioral changes after the CMS procedure, and even measured a decreased hormonal stress response. These results indicate that PENK KO mice are resistant to the CMS effects, suggesting that enkephalin enhances the reactivity to chronic stress.     

Structural-functional relations of short analogs of enkephalin

The similarity of action of narcotic analgesics and opioid peptides is due to activation of a common opiate receptor as the primary step in initiating biochemical chains responsible for diverse morphine-like effects. The most widely used assays for opioid and analgesic activities are presented and evaluated. Approximately 180 short enkephalin analogues (di-, tri- and tetrapeptides), described in the literature, are systematized and their opioid and systemic analgesic activities compared with methionine-enkephalin and morphine as the reference compounds, respectively. The analysis of structure-opioid activity relationships among these enkephalin analogues substantiates the hypothesis that only a limited N-terminal region of the peptide molecule is essential for the binding of opioid peptides to the subclass of opiate receptors interacting with narcotic alkaloids (mu-receptors). An attempt has been made to identify minimal structural elements responsible for the mu-receptor activation. Shortening of the molecule and modification of its elements are examined with regard to the mu- and delta-receptor selectivity. It is emphasized that the aromatic structure of the C-terminal region of the peptide is not obligatory for the mu-receptor binding. Modifications of short enkephalin analogues which might confer them antagonistic properties are reviewed. The correlation between the ability of short enkephalin analogues to interact with mu-receptors and their antinociceptive properties is discussed along with some structural features pertinent to the analgesic effect after systemic administration of peptides. On the basis of this analysis, peptides containing no more than four amino acids are considered as the most probable morphine-like analgesics.     

Metabolism and absorption enhancement of methionine enkephalin in human nasal epithelium

The objective of this study was to investigate absorption enhancing approaches for systemic delivery of methionine enkephalin via the nose. Absorption promotion of methionine enkephalin in the presence of protease inhibitors (bestatin, puromycin) and absorption enhancers (glycocholate, dimethyl-beta-cyclodextrin) were investigated in human nasal epithelium. Co-administration of the peptide with protease inhibitors and absorption enhancers resulted in a remarkable increase in Met-Enk permeation (4- to 94-fold). The increase was proportional to transepithelial resistance reduction and permeation of paracellular marker dye. Perturbation of the epithelial tight junctions seen in vitro may not occur in vivo due to mucus protection and mucociliary clearance.     

Monoclonal antibodies to leucine enkephalin

Monoclonal antibodies to leucine enkephalin have been produced after fusion of mouse myeloma cells with spleen cells from hyper-immune mice. Hybrid clones 2D1 and SL1 were characterised using radioimmunoassay and an enzyme-linked immunosorbent assay. The antibody 2D1 was of low affinity and showed a maximum sensitivity of 0.1ng. The antibody binds equally well to the sulphated leucine enkephalin and to methionine enkephalin. It does not cross-react with dynorphin, methionine enkephalin-arg-phe or oxidised methionine enkephalin. The hybrid clone SL1 appears to be specific for leucine enkephalin. Preliminary immunocytochemical studies have shown that both antibodies bind specifically to leucine enkephalin in defined areas of the central nervous system.     

DPen2-[DPen5]enkephalin, a delta opioid receptor-selective analog of [Leu]enkephalin, impairs avoidance learning in an automated shelf-jump task in rats

Both [Leu]enkephalin and DPen2-[DPen5]enkephalin, a delta opioid receptor selective analog of [Leu]enkephalin, impaired acquisition of an automated shelf-jump response in rats. A similar level of impairment was produced by equimolar doses of the two enkephalins. As is seen for [Leu]enkephalin when tested in a one-way active avoidance task, the dose-response function for the impairment produced by DPen2-[DPen5]enkephalin in the automated shelf-jump task is U-shaped. These results, together with our previous findings that DPen2-[DPen5]enkephalin and [Leu]enkephalin both impair acquisition of a one-way active avoidance response in mice, and that [Leu]enkephalin impairs acquisition of that same response in rats, support our suggestion that delta opioid receptors are implicated in the effects of [Leu]enkephalin on conditioning. In addition, these results indicate that the involvement of delta opioid receptors in acquisition impairment extends to two species of rodents and to two different avoidance conditioning tasks.     

Attenuation of Enkephalin Activity in Neuroblastoma × Glioma NG108—15 Hybrid Cells by Phospholipases

The role of membrane phospholipids in enkephalin receptor-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma X glioma NG108-15 hybrids was studied by selective hydrolysis of lipids with phospholipases. When NG108-15 cells were treated with phospholipase C from Clostridium welchii at 37 degrees C, an enzyme concentration--dependent decrease in adenylate cyclase activity was observed. The basal and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF-5'-guanylylimidodiphosphate (Gpp(NH)p)-sensitive adenylate cyclase activities. Further, Leu5-enkephalin inhibition of basal or PGE1-stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level. [3H]D-Ala2-Met5-enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment. Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p. Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N-component. Further, treatment of NG108-15 hybrid cell membrane with phospholipase C at 24 degrees C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding. The reduction in enkephalin potency could be reversed by treating NG108-15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate. The enkephalin activity in NG108-15 cells was not altered by treating the cells with phospholipase A2 o phospholipase C from Bacillus cereus. Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity.     

Differential effects of carboxyfullerene on MPP+/MPTP-induced neurotoxicity

The effects of carboxyfullerene on a well-known neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenyl-pyridinium (MPP+) were investigated. In chloral hydrate-anesthetized rats, cytosolic cytochrome c was elevated in the infused substantia nigra 4 h after an intranigral infusion of MPP+. Five days after local application of MPP+, lipid peroxidation (LP) was elevated in the infused substantia nigra. Furthermore, dopamine content and tyrosine hydroxylase (TH)-positive axons were reduced in the ipsilateral striatum. Concomitant intranigral infusion of carboxyfullerene abolished the elevation in cytochrome c and oxidative injuries induced by MPP+. In contrast, systemic application of carboxyfullerene did not prevent neurotoxicity induced by intraperitoneal injection of MPTP. In mice, systemic administration of MPTP induced a dose-dependent depletion in striatal dopamine content. Simultaneous injection of carboxyfullerene (10 mg/kg) actually potentiated MPTP-induced reduction in striatal dopamine content. Furthermore, systemic administration of carboxyfullerene (30 mg/kg) caused death in the MPTP-treated mice. An increase in the striatal MPP+ level and reduction in hepatic P450 level were observed in the carboxyfullerene co-treated mice. These data showed that systemic application of carboxyfullerene appears to potentiate MPTP-induced neurotoxicity while local carboxyfullerene has been suggested as a neuroprotective agent. Furthermore, an increase in striatal MPP+ level may contribute to the potentiation by carboxyfullerene of MPTP-induced neurotoxicity.     

Cutting edge: fasting-induced hypoleptinemia expands functional regulatory T cells in systemic lupus erythematosus

Fasting is beneficial in the prevention and amelioration of the clinical manifestations of autoimmune diseases including systemic lupus erythematosus. The mechanisms responsible for these effects are not well understood. During fasting, there is a dramatic reduction of the levels of circulating leptin, an adipokine with proinflammatory effects. Leptin also inhibits CD4(+)CD25(+)Foxp3(+) regulatory T cells, which are known to contribute significantly to the mechanisms of peripheral immune tolerance. In this study, we show that fasting-induced hypoleptinemia in (NZB × NZW)F(1) lupus-prone mice induced an expansion of functional regulatory T cells that was reversed by leptin replacement. The specificity of the findings was indicated by the lack of these effects in leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice. These observations help to explain the beneficial effects of fasting in autoimmunity and could be exploited for leptin-based immune intervention in systemic lupus erythematosus.     

Comparison of the behavioral effects of β-endorphin and enkephalin analogs

The behavioral effects of β-endorphin, enkephalin analogs, morphine and etorphine were briefly compared. In the tail-flick test in mice and in the wet shake test in rats, β-endorphin and D-Ala²-D-Leu⁵-enkephalin had equal antinociceptive activity; D-Ala² -Met-enkephalinamide and D-Leu⁵-enkephalin were less active. The order of activity of the enkephalin analogs and opiate alkaloids for stimulating locomotor activity in mice paralleled their analgesic activities; β-endorphin, however, had only minimal stimulatory actions. Morphine sulfate, 50 μg injected into the periaqueductal gray, produced hyperactivity but this effect was not observed with etorphine or opioid peptides. By contrast, “wet dog” shakes was observed with the opioid peptides but not with either opiate alkaloid. These heterogenous behavioral responses, which were all antagonized by naloxone, indicate that multiple types of receptors mediate the effects of opiates in the central nervous system.     

Naloxone-precipitated jumping in mice pretreated with acute injections of opioids.

Naloxone induced jumping was examined in mice pretreated with single dose of narcotic agonist (morphine, heroin, LAAM, methadone), mixed agonist-antagonist (pentazocine, cyclazocine, buprenorphine), or an enkephalin analog (D-met², pro⁵)-enkephalinamide. Acute sensitization to naloxone, as demonstrated by jumping, was observed after pretreatment with the narcotics, the enkephalin analog, and to a lesser degree after cyclazocine and pentazocine. Mice pretreated with buprenorphine did not jump in response to naloxone. This procedure may be of value in the rapid identification of drugs with a propensity to produce morphine-like physical dependence.     

Tyr-MIF-1 and Met-enkephalin share a saturable blood-brain barrier transport system

Previous studies have shown that methionine enkephalin and Tyr-MIF-1 are transported from the brain to the blood by a saturable, stereospecific, carrier-mediated process. It was not established by these studies whether Tyr-MIF-1 and methionine enkephalin were transported by the same system or by separate, but overlapping systems. This issue was investigated in anesthetized mice receiving injections containing both 131I-methionine enkephalin and 125I-Tyr-MIF-1 into the lateral ventricle of the brain. Mice were decapitated and the brain to blood transport rate was derived from the residual counts in the brain. It was found that in individual mice, the transport rate for Tyr-MIF-1 correlated highly with the transport rate for methionine enkephalin but not with the transport of iodide. This shows that the transport of Tyr-MIF-1 is closely coupled to the transport of methionine enkephalin but dissociable from the brain to blood transport of iodide. Furthermore, the inability of varying doses of Tyr-MIF-1 or of methionine enkephalin to preferentially self-inhibit is radiolabeled form in comparison with the other peptide shows that, functionally, only a single system exists. Aluminum, a noncompetitive inhibitor of Tyr-MIF-1 transport, was also without preferential inhibition. Thus, under the conditions of these studies, only a single system could be functionally demonstrated for the transport of both Tyr-MIF-1 and methionine enkephalin.     

Low-dose targeted complement inhibition protects against renal disease and other manifestations of autoimmune disease in MRL/lpr mice

Complement appears to play a dual role in the progression of systemic lupus erythematosus, serving a beneficial role in enhancing immune complex clearance, while serving a pathogenic role in inducing local inflammation. To investigate these different roles of complement in a therapeutic setting, MRL/lpr mice were treated with the targeted murine C3 complement inhibitor, CR2-Crry, from 16 to 24 wk of age (after the development of proteinuria). The targeting moiety, CR2, binds to C3 breakdown products deposited at sites of complement activation and has the potential to provide complement inhibition locally without causing systemic inhibition. Administration of CR2-Crry i.v., at a dose of 0.25 mg once a week, was associated with a significant survival benefit, improved kidney function, and a significant reduction in glomerulonephritis and renal vasculitis. The presence of skin lesions and lung bronchiolar and vascular inflammation was also dramatically reduced by CR2-Crry treatment. CR2-Crry treatment also resulted in a significant reduction in autoantibody production, as measured by anti-dsDNA Ab levels, and did not cause an increase in circulating immune complex levels. These effects on autoimmunity and circulating immune complexes represent significant potential advantages over the use of Crry-Ig in MRL/lpr mice, a systemic counterpart of CR2-Crry. CR2-Crry localized preferentially to the kidneys in 16-wk MRL/lpr mice with a kidney-localized half-life of approximately 24 h. Thus, targeted complement inhibition at the C3 level is an effective treatment in murine lupus, even beginning after onset of disease.     

DSLET (D-ser2-leu5-enkephalin-Thr6) produces analgesia on the hot plate by mechanisms largely different from DAGO and morphine-like opioids

Fifteen generations of selective breeding were used to produce lines (strains) of mice which differ markedly from one another in levorphanol-induced antinociception on the hot plate assay. These are the high antinociceptive response (HAR) and low antinociceptive response (LAR) selection lines, which now differ by over 5-fold in the i.p. dose of levorphanol doubling control (no drug or saline) latency scores. We sought to determine if these large genetically-mediated differences in antinociceptive sensitivity bred into these selection lines with i.p. levorphanol would generalize equally to a series of enkephalin analogues known to differ in their selectivity for mu and delta opioid receptors. DAGO (D-ala2, MePhe4, Gly-ol5 enkephalin), a highly mu selective agent, produced a 67-fold difference between HAR and LAR mice in the slopes of the dose-response curves on the hot plate assay, while DSLET (D-ser2, leu enkephalin Thr6), a delta selective agent, only produced a 5.4-fold difference via the i.c.v. route. DADLE (D-ala, D-ser enkephalin) a slightly delta preferring ligand, was found to be intermediate (17.4-fold difference). These findings demonstrate that selective breeding has been quite successful in altering those genes which control analgesia due to mu selective agents, while relatively little change has occurred in those genes which control analgesia due to delta agonists. Thus, analgesia mediated by the former has been genetically dissociated from analgesia mediated by the latter, implying that DAGO has mechanisms of action largely dependent of DSLET on the hot plate assay. These findings are consistent with the contention that the mu receptor mediates analgesia produced by DAGO, while a different receptor (presumably delta) mediates much of the analgesic effects of DSLET.     

Enzymatic cleavage prior to antibody incubation as a method for neuropeptide immunocytochemistry

When deplasticized Epon sections were treated with endo- and/or exopeptidases prior to incubation with antibodies, the neuropeptide immuno-reactivity of secretory nerves was often altered in a predictable way. Cleavage of neurosecretory material in octopus nerves by trypsin and carboxypeptidase-B enhanced enkephalin-like immunoreactivity, while Molluscan neuropeptide-like immunoreactivity was prevented by tryptic cleavage. The enzyme effects indicated the occurrence of a heptapeptide (Tyr-Gly-Gly-Phe-Met/Leu-Arg-Phe) that contains both the enkephalin and the Molluscan neuropeptide sequence. Vasopressin terminals of the rat neurohypophysis, which presumably contain enkephalin precursor sequences, exhibited enkephalin-like immunostaining after tryptic cleavage. ACTH/beta-endorphin cells of the rat intermediate pituitary, which synthesize the enkephalin sequence at the N-terminus of Beta-endorphin, exhibited enkephalin=like immunoreactivity when sections were treated with alpha-chymotrypsin or trypsin, but not after incubation with leucine-aminopeptidase or carboxypeptidase-B. Enkephalin-like immunostaining could not be induced in any way in ACTH/beta-endorphin cells of the anterior pituitary. Enzymatic cleavage may give additional information in immunocytochemical localization studies on neuropeptide sequences in secretory nerves and hormonal granules.     

Interleukin-12 exhibits potent antiviral activity in experimental herpesvirus infections.

The prophylactic and therapeutic efficacy of interleukin-12 was studied by using murine models of herpes simplex virus infection. Prophylactic administration consisted of two intraperitoneal doses of interleukin-12 given 48 and 24 h prior to infection. Therapeutic intraperitoneal administration of interleukin-12 commenced 6 h after the mice were infected with herpes simplex virus and was continued daily for a total of 5 days. Interleukin-12 therapy improved the survival rates of mice with systemic herpes simplex virus infection compared with those of placebo-treated infected mice. Subcutaneous administration of interleukin-12 also improved the rate of survival of mice after systemic herpes simplex virus infection, although higher doses were required to give comparable effects. Combined prophylactic and therapeutic administration of interleukin-12 produced the greatest effect on survival after an otherwise lethal systemic infection. Intraperitoneal administration of interleukin-12 for 2 days before and 3 days after systemic infection with herpes simplex virus resulted in survival of 80% of the mice. These surviving mice were resistant to subsequent reinfection with herpes simplex virus. Such resistance was apparently specific for herpes simplex virus infection, since a second group of survivors succumbed to a lethal infection with murine cytomegalovirus. Infectious virus was recovered from lumbar ganglia explants dissected from survivors of prophylactic interleukin-12 therapy and cultured for 5 days in vitro, suggesting that interleukin-12 treatment did not prevent the establishment of latent herpes simplex virus infection. One action of interleukin-12 may be to enhance natural killer cell-mediated clearance of the virus. However, interleukin-12 therapy was also effective in mice carrying the beige mutation, which reduces natural killer cell lytic activity, suggesting that interleukin-12 has additional activities in vivo.     

Effects of Toxoplasma gondii (Gleadle strain) on the host-parasite relationship in trichinosis.

The effects of concurrent infection with Toxoplasma gondii on the host-parasite relationship in trichinosis were studied. Infected mice showed a delay in expulsion of Trichinella spiralis adults from the gut. Persisting adult female worms were fecund but the numbers of larvae recovered from the muscles were not increased. Increased resistance to the systemic phase of trichinosis was shown by reduced numbers of muscle larvae after intravenous injection of newborn larvae in animals with toxoplasmosis as compared with control mice. There were no differences in small bowel pathology of trichinous mice with and without toxoplasmosis but inflammation around muscle cysts of T. spiralis was reduced in mice with toxoplasmosis. The eosinophilia which normally develops in mice with trichinosis was suppressed by concurrent toxoplasmosis. Trichinella infection did not alter the numbers of T. gondii cysts recovered from the brain 4 weeks after infection. It is suggested that the delay in expulsion of adult worms, decrease in muscle inflammation around T. spiralis cysts, and inhibition of eosinophilia result from immune suppression, while the reduction in numbers of muscle larvae after intravenous injection of newborn larvae reflects enhanced nonspecific resistance to infection in toxoplasmosis.