Semicarbazide Substitution Enhances Enkephalins Resistance to Ace Induced Hydrolysis

In our previous study on [Met5]-enkephalin analogues, [Met5]-enkephalin semicarbazide was found as a new enkephalin amide that produces antinociception even in ACE (Angiotensin Converting Enzyme) exposure in vivo. In the present work we examined the corresponding [Leu5]-enkephalin derivatives to confirm the influence of semicarbazide substitution. To prevent the enkephalins biodegradation animals were pretreated with a mixture of peptidase inhibitors. As assessed by tail-flick test no significant difference was detected between the produced antinociception by the [Leu5]-enkephalin derivatives. Based on our results both semicarbazide and ethylamide groups could preserve the provided analgesia after captopril (ACE inhibitor) omission from the peptidase inhibitors mixture. This work confirms that semicarbazide substitution on enkephalins yields ACE resistance antinociceptive peptides, nevertheless it may necessarily not enhance the peptides analgesic potencies.     

Molecular-dynamics simulations of [Met5]- and [D-Ala2,Met5]-enkephalins. Biological implication of monomeric folded and dimeric unfolded conformations.

To investigate the biologically active conformation of enkephalin, molecular-dynamics simulations were applied to [Met5]- and [D-Ala2,Met5]-enkephalins. The dynamic trajectory of monomeric extended [Met5]-enkephalin was analysed in terms of relative mobility between respective torsions of backbone chain. After 10 ps of the dynamics simulation, the conformational transition was converged into a stationary state among the beta-bend folded forms, where they are stabilized by several intramolecular hydrogen-bond formations. Similar conformational transition was also observed in the dynamics simulation of [D-Ala2,Met5]enkephalin, which is a more mu-receptor-specific peptide than [Met5]enkephalin. The geometrical correspondence between the monomeric enkephalin conformation in the stationary state and morphine molecule (a mu-specific rigid opiate) was surveyed by virtue of the triangular substructures generated by choosing three functional atoms in each molecule, and good resemblances were observed. On the other hand, the dynamics simulation of the antiparallel extended [Met5]enkephalin dimer showed a trajectory different from that of the monomeric one. Two intermolecular hydrogen bonds at Tyr1 (NH3+)...Met5(CO2-) end residues were held throughout the 100 ps simulation, the dimeric structure being consequently kept. The conformational transition of the backbone chains from the antiparallel extended form to the twisted one took place via an intermediate state. Many conformations revealed during the dynamics simulation showed that the relative orientations of each two Tyr1, Gly3, Phe4 and Met5 residues in the dimer are nearly related by a pseudo-C2-symmetry respectively, and both halves of the dimer structure could be further fitted to the monomeric folded enkephalin conformation. The monomeric and dimeric conformations of enkephalin at their stationary states are discussed in relation to the substrate-specificity for mu- and delta-opioid receptors.     

Radioiodinated [D-Ala2, Met5] enkephalin. Preparation, purification by high performance liquid chromatography and binding characteristics

125I[D-Ala2, Met5] enkephalin with high specific activity (122-185 Ci/mmol) was prepared and purified by Sep-Pak C18 reverse phase cartridge followed by high performance liquid chromatography (HPLC). HPLC at pH 3.0 resolved 125I[D-Ala2, Met5] enkephalin into two fractions, which ran as a single spot in thin-layer chromatography with the same Rf values. Alkaline hydrolysates of the HPLC-purified fractions showed a single spot corresponding to monoiodotyrosine standard when analysed by thin-layer chromatography. Binding kinetics of the tracer was found to approach equilibrium after 30 min at 24 degrees. Scatchard analysis of the saturation equilibrium binding studies gave an equilibrium dissociation constant of 3.58 nM and the number of binding site of 30 fmol/mg protein. Enkephalin analogs were capable of displacing 125I[D-Ala2, Met5] enkephalin binding from the rat brain plasma membrane. The effective concentration of [D-Ala2, Met5] enkephalin and [D-Ala2, Leu5] enkephalin for 50% inhibition of 125I[D-Ala2, Met5] enkephalin binding was estimated to be 79 nM and 23 nM, respectively. Both substance P and gastrin tetrapeptide failed to displace the 125I[D-Ala2, Met5] enkephalin binding to any significant extent. The 125I[D-Ala2, Met5] enkephalin prepared by the present procedure is therefore a useful tracer. This method of preparing radioiodinated peptide may be applicable to other enkephalin analogs or neuropeptides in general.     

Structure of two adrenal polypeptides containing multiple enkephalin sequences

Two enkephalin-containing polypeptides of 4000 and 5000 daltons have been isolated from extracts of bovine adrenal medulla. Each polypeptide was purified to homogeneity and subjected to sequence analysis. The entire primary structure of the 4000-dalton polypeptide was established by a combination of automated Edman degradation and chemical analysis of its tryptic peptides. The polypeptide contains two copies of the [Met]-enkephalin sequence, one at the amino terminus and the other at the carboxyl terminus. Chemical analysis of the tryptic peptides and automated Edman degradation of the 5000-dalton polypeptide indicated the presence of a [Leu]enkephalin sequence at the carboxyl terminus and an internal [Met]enkephalin sequence. Both of the above enkephalin-containing polypeptides appear to be intermediates in the biosynthesis of the enkephalins.     

Comparative Studies of the Binding of Dimeric and Monomeric Enkephalins to Neuroblastoma-Glioma NG108–15 Cells

Binding activity of the enkephalin dimer [D-Ala2, Leu5-NH-CH2-]2 (DPE2) to NG108-15 hybrid cells was compared to that of the monomer [D-Ala2, Leu5]enkephalin amide (DALEA). At 25 degrees C, the values of the apparent affinity constant for DPE2, measured to intact and lysed cells and membranes, was 5.0 (+/- 0.09) X 10(9) M-1 for n = 28 experiments, as compared to 0.9 (+/- 0.08) X 10(9) M-1 (n = 16) for DALEA. At 4 degrees C, the binding affinity of DPE2 decreased by 43% and that of DALEA by 33%. An important difference between the binding of DPE2 and DALEA was that, after necessary corrections for difference in maximal "bindability" of the respective tritiated enkephalins, the molar binding capacity for DALEA was twofold higher than for DPE2, although mutual cross-displacement studies indicated that binding occurred to one class of noninteracting homogeneous receptors. The binding capacity for intact and lysed cells and membranes was 20 (+/- 2) X 10(-11) M for DPE2 and 43 (+/- 2) X 10(-11) M for DALEA. The enkephalin monomers [D-Ala2, D-Leu5]enkephalin (DADLE) and [D-Ala2, Met5]enkephalin amide (DAMEA) showed binding characteristics similar to those of DALEA.     

Tetraethylammonium facilitates the stimulation-evoked loss of the enkephalins from the myenteric plexus of guinea-pig ileum

The effects of electrical field stimulation on the contents of [Met]enkephalin and [Leu]enkephalin were determined in myenteric plexus-longitudinal muscle preparations of the guinea-pig small intestine. Cycloheximide (0.1 mM) was present in all experiments to prevent de nouveau biosynthesis. The two enkephalins were separated by high performance liquid chromatography and assayed on the mouse vas deferens. Stimulation with submaximal pulses (50 mA, 0.5 ms) at a frequency of 10 Hz caused maximal losses of about 35% of [Met]enkephalin and [Leu]enkephalin after 3 h (108000 pulses). The plot of log (enkephalin content) against number of pulses was steeper during the first 30 min than during the later periods. Tetraethylammonium bromide (TEA, 10 mM) increased the [Met]enkephalin and [Leu]enkephalin contents of the non-stimulated preparations by about 50%. When the preparations were stimulated in the presence of TEA at 50 mA and 1 Hz, the plots of loss of enkephalins against number of pulses were linear until the maximum of about 50% was reached. Compared with stimulation in the absence of TEA, the rate constant was 8 times greater for [Leu]enkephalin and 20 times greater for [Met]enkephalin. The absolute losses per pulse were about 13 times greater for [Leu]enkephalin and 27 times greater for [Met]enkephalin than in the absence of TEA. In the presence of bacitracin and a mixture of dipeptides, the enzymatic degradation of the enkephalins was sufficiently suppressed to cause an overflow of 30-60% of the enkephalins lost from their stores into the perifusing Krebs solution. Until it is possible to determine the preformed precursors, which are present in large quantities, the kinetics relationship between these precursors and the enkephalins cannot be investigated. A similar dilemma exists for the relationship between "released' enkephalins and the losses from their stores.     

Photoaffinity inactivation of the enkephalin receptor.

An arylazide enkephalin derivative, [D-Ala2,Met5]enkephalin-Tyr-N-(2-nitro-4-azidophenyl) ethylenediamine (ETN), has been synthesized. In the dark, it inhibited the binding of [3H]enkephalinamide to enkephalin receptor-rich NG-108 cell membranes with an I50 = 2.2 X 10(-8) M or KI = 7 X 10(-9) M, assuming competitive inhibition. Photolysis of membranes in the presence of ETN caused irreversible inactivation of the enkephalin receptor, but inactivation was prevented by the addition of enkephalin, the half-effective concentration being 3 x 10(-9) M. ETN appears to be an effective photoaffinity label for the enkephalin receptor.     

Degradation of low-molecular-weight opioid peptides by vascular plasma membrane aminopeptidase M

Since both aminopeptidases and angiotensin I-converting enzyme are reported to degrade circulating enkephalins, we have examined the degradation of low-molecular-weight opioid peptides by a vascular plasma membrane-enriched fraction previously shown to contain both angiotensin I-converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). Except for an enkephalin analog resistant to amino-terminal hydrolysis, [D-Ala2]enkephalin, the purified vascular plasma membrane preferentially degraded low-molecular-weight opioids by hydrolysis of the N-terminal Tyr-1--Gly-2 bond. Enkephalin degradation was optimal at pH 7.0 and was inhibited by the aminopeptidase inhibitors amastatin (I50 = 0.08 microM), bestatin (9.0 microM) and puromycin (80 microM). Maximal rates of hydrolysis, calculated per mg plasma membrane protein, were highest for the shorter peptides (18.3, 15.6 and 16.6 nmol/min per mg for Met5-enkephalin, Leu5-enkephalin and Leu5-enkephalin-Arg6, respectively) and decreased with increasing peptide length (0.7 nmol/min per mg for dynorphin (1-13)). No significant hydrolysis of beta- and gamma-endorphin was detected. Km values decreased significantly with increasing peptide length (Km = 72.9 +/- 2.7, 43.6 +/- 4.7 and 21.4 +/- 0.9 microM for Met5-enkephalin, Leu5-enkephalin-Arg6 and Met5-enkephalin-Arg6-Phe7, respectively). However, no further decreases were seen with even larger sequences, i.e., dynorphin(1-13). Other peptides hydrolyzed by the plasma membrane aminopeptidase (angiotensin III, kallidin and hepta(5-11)-substance P) inhibited enkephalin degradation in a competitive manner. Thus, localization, specificity and kinetic data are consistent with identification of aminopeptidase M as a vascular enzyme with the capacity to differentially metabolize low-molecular-weight opioid peptides within the microenvironment of vascular cell surface receptors. Such differential metabolism may play a role in modulating the vascular effects of peripheral opioids.     

X-ray diffraction studies of enkephalins. Crystal structure of [(4''-bromo) Phe4,Leu5]enkephalin.

In order to investigate the structure-activity relationship of [Leu5]- and [Met5]enkephalins, [(4'-bromo)Phe4, Leu5]-, [(4'-bromo)Phe4, Met5]- and [Met5] enkephalins were synthesized and crystallized. The crystal structure of [(4'-bromo) Phe4, Leu5]- enkephalin was determined by X-ray diffraction method using the heavy atom method and refined to R = 0.092 by the least-squares method. The molecule in this crystal took essentially the same type I' beta-turn conformation found in [Leu5]enkephalin [Smith & Griffin (1978) Science 199, 1214-1216). On the other hand, the preliminary three-dimensional Patterson analyses showed that the most probable conformations of [(4'-bromo)Phe4,Met5]- and [Met5]enkephalins are both the dimeric extended forms. Based on these insights, the biologically active conformation of enkephalin was discussed in relation to the mu- and delta-receptors.     

Mu1: a very high affinity subtype of enkephalin binding sites in rat brain

Displacement studies of [3H]-[D-Ala2-MePhe4-Gly-ol5]-enkephalin ([3H]-DAGO) and [3H]-[D-Ala2-D-Leu5]-enkephalin ([3H]-DADL) by the corresponding unlabeled ligands show that there are at least three classes of sites which bind these enkephalin analogs with high affinity. Using computer modeling, the introduction of the third site significantly improved the goodness of fit in ten consecutive experiments. These sites appear to correspond to the mu, delta and mu 1 sites, with mean dissociation constants of 11, 1.3 and 0.9 nM for DADL and 2.5, 300 and 0.3 nM for DAGO, respectively.     

Further characterization of the in vitro hydrolysis of [Leu]- and [Met]enkephalin in rat plasma: HPLC-ECD measurement of substrate and metabolite concentrations.

Hydrolysis of [Leu]- and [Met]enkephalin was determined in whole rat plasma in vitro by using HPLC-ECD to measure Tyr, Tyr-Gly and Tyr-Gly-Gly formation. Although [Leu]- and [Met]enkephalin did not differ in Tyr or Tyr-Gly accumulation, the amount of Tyr-Gly-Gly resulting from [Met]enkephalin hydrolysis was greater than that resulting from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in plasma was slightly shorter than that of [Leu]enkephalin. By comparing metabolite formation in the presence and absence of peptidase inhibitors with high selectivity for their respective enzymes, these studies demonstrated that aminopeptidase M and angiotensin converting enzyme are the major peptidases that hydrolyze enkephalins in rat plasma.     

Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.     

Mu and delta receptors: their role in analgesia in the differential effects of opioid peptides on analgesia

Utilizing the mouse tail-flick assay, the rank order of analgesic potency for various opioids (i.c.v.) is beta h-endorphin greater than D-Ala2-D-Leu5-enkephalin greater than morphine greater than D-Ala2-met-enkephalinamide much greater than met-enkephalin much greater than leu-enkephalin. Assuming mu receptor mediation of analgesia, there is an affinity and analgesic potency (ie: D-Ala2-Leu5-enkephalin has 1/7 the affinity of morphine for the mu receptor but is 18X more potent as an analgesic). Additionally, sub-analgesic doses of various opioid peptides have opposite effects on analgesic responses. Leu-enkephalin, D-Ala2-D-Leu5-enkephalin or beta h-endorphin potentiate morphine or D-Ala2-met-enkephalinamide analgesia whereas met-enkephalin or D-Ala2-met-enkephalinamide antagonize opioid-induced analgesia. Using the enkephalins as the prototypic delta ligands (100 fold selective) and based on their effects on analgesia, we suggest that Leu-enkephalin-like peptides interact with the delta receptor as an "agonist" to facilitate and met-enkephalin-like peptides as an "antagonist" to attenuate analgesia. Given the biochemical evidence of a coupling between mu and delta receptors, we suggest that the mechanism of facilitation or attenuation of analgesia by the enkephalins is a direct in vivo consequence of this coupling. Further, the analgesic potencies of various opioid ligands can be better correlated to the combination of their simultaneous occupancy of mu and delta receptors.     

Antinociceptive effect of [Met5]enkephalin semicarbazide is not affected by dipeptidyl carboxypeptidase‐I

Dipeptidyl carboxypeptidase‐I is an enzyme involved in the biological degradation of enkephalins. It has been suggested that C‐terminal amidation of enkephalins enhances their resistance to dipeptidyl carboxypeptidase‐I‐mediated biodegradation. In this study, a novel [Met5]enkephalin amide (MEA) analogue [Met5]enkephalin (ME)‐semicarbazide synthesized by another laboratory in our group was assessed for its antinociceptive effects compared with ME‐ethylamide, MEA and ME, using tail flick test. To protect the administered drugs from biodegradation, rats were pretreated with peptidase inhibitors including amastatin, phosphoramidon and captopril. Then captopril (dipeptidyl carboxypeptidase‐I inhibitor) was deleted from the peptidase inhibitors' combination for evaluating in vivo resistance of the synthetic drugs to dipeptidyl carboxypeptidase‐I. According to the results, ME‐semicarbazide and MEA were resistant enough to dipeptidyl carboxypeptidase‐I to exert their strong antinociception following intrathecal administration even in the absence of captopril, whereas the antinociceptive effects produced by ME‐ethylamide (10 nmol) were abolished in rats not pretreated with captopril, indicating that significant amounts of the ME‐ethylamide were degraded by dipeptidyl carboxypeptidase‐I. Replacement of the amide moiety of MEA with semicarbazide provides a new ME derivative, with high analgesic effects as well as more resistance to dipeptidyl carboxypeptidase‐I‐mediated biodegradation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Binding characteristics of dermorphin, and [dermorphin1-7]-beta c-endorphin in rat brain membranes

Dermorphin and a camel beta-endorphin (beta c-EP) analog in which residues 1-7 correspond to the dermorphin sequence ([Dermorphin1-7]-beta c-EP) have been investigated with respect to their receptor binding characteristics using human and camel beta-EP as reference peptides. Tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin, ethylketocyclazocine and human beta-endorphin were used as primary ligands in the rat brain membrane preparation for radioreceptor assay. Camel beta-endorphin was the most potent peptide in all experiments. [Dermorphin1-7]-beta c-EP is significantly less potent towards 3H-ethylketocyclazocine and 3H-[D-Ala2, D-Leu5]-enkephalin but is as potent towards 3H-dihydromorphine and 3H-human beta-endorphin. Dermorphin itself weakly displaces tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin and ethylketocyclazocine (potency relative to camel beta-EP, 1-4%) but it is more potent (9%) in competition with tritiated human beta-endorphin. Dermorphin and the [Dermorphin-1-7]-beta c-EP appear to interact preferentially with mu opiate receptors.     

The occurrence and receptor specificity of endogenous opioid peptides within the pancreas and liver of the rat. Comparison with brain.

Our observations that opioid peptides have direct effects on islet insulin secretion and liver glucose production prompted a search for endogenous opiates and their receptors in these peripheral tissues. Mu-, delta- and kappa-receptor-active opiates were demonstrated in brain, pancreas and liver extracts by displacement studies using selective ligands for the three opiate receptor subtypes [( 3H][D-Ala2,MePhe4,Gly5-ol]enkephalin, [3H][D-Ala2,D-Leu5]enkephalin and [3H]dynorphin respectively). Receptor-active opiates in brain extracts exhibited a stronger preference for delta-opiate-receptor sites than for mu and kappa sites. Pancreatic extract opiates demonstrated a similar activity at mu and delta sites, but substantially less at kappa sites. Liver extracts displayed similar selectivity for all three sites. The affinities of the receptor-active opiates for mu-, delta- and kappa-receptor subtypes displayed a rank order of potency: brain much greater than pancreas greater than liver. Total immunoreactive beta-endorphin and [Met5]enkephalin levels in liver and hepatocytes were greater than those in brain. Immunoreactive [Met5]enkephalin levels in pancreas were similar to, but beta-endorphin levels were substantially higher than, those in brain. Delta and kappa opiate-binding sites of high affinity were identified in crude membrane preparations of islets of Langerhans, but no specific opiate-binding sites could be demonstrated in liver membrane preparations. Immunoreactive dynorphin and beta-endorphin were demonstrated by immunogold labelling in rat pancreatic islet cells. No positive staining of liver sections for opioids was observed. These results suggest that the tissue content of opiate-receptor-active compounds in the pancreas and the liver is very significant and could contribute to the regulation of normal blood glucose levels.     

The significance of mu- and delta-receptors in rat pancreatic islets for the opioid-mediated insulin release

The binding and the insulinotropic effects of enkephalin analogs and of morphine were investigated in rat pancreatic islets. Binding of [3H]Met-enkephalin was saturable, specific and reversible; the rank order for inhibition competition of [3H]Met-enkephalin binding by various compounds was Met-enkephalin = D-Ala2-MePhe4, Met(0)ol enkephalin) greater than Leu-enkephalin greater than morphine with half-maximal inhibitory constants (IC50) of approx. 0.3, 0.3, 100 and greater than 100 nM, respectively. Both the natural enkephalins exerted their insulinotropic effect only at stimulatory glucose concentrations. They had a dual action; whereas insulin secretion was increased at low enkephalin concentration, this effect was reversed at higher concentrations. However, the various enkephalins exerted this effect at different concentrations; only the EC50 values (half-maximal effective concentrations) of their insulinotropic effect were in the same range as the IC50 values of inhibition of [3H]met-enkephalin binding. Cysteamine pretreatment of rats (depletion of somatostatin containing D-cells and decrease in somatostatin secretion) did not change the Met-enkephalin effect on insulin secretion. In contrast to Met-enkephalin, binding of [3H]morphine to islets was not saturable, and morphine had no effect on insulin secretion unless at unphysiologically high concentrations. The data, therefore, indicate that: mu-receptors (affinity for morphine) do not play a role in rat pancreatic islets; delta-receptors (binding site for Met-enkephalin when mu-receptors are not present) mediate the insulinotropic effect of low Met-enkephalin concentrations; and the insulinotropic action of Met-enkephalin is not mediated indirectly via the paracrine effect of an inhibition of somatostatin secretion.     

Endogenous opioids and the growth regulation of a neural tumor

Endogenous opioid systems (endogenous opioids and their receptors) are known to participate in the regulation of tumor growth. The present study was conducted to examine whether [Met5]-enkephalin influences the growth of transplanted neuroblastoma, and to explore the role of other opioid peptides in carcinogenesis. A/Jax mice were inoculated with 10(6) S20Y cells and received daily injections of [Met5]-enkephalin. Dosages of 0.5 to 30 mg/kg delayed tumor appearance and prolonged survival of these mice; antitumor effects were blocked by concomitant injections of naloxone. Daily administration (10 mg/kg) of [Leu5]-enkephalin had no effect on neurotumor growth. [D-Ala2, D-Leu5]-enkephalin and ethylketocyclazocine, ligands selective for delta and kappa receptors, respectively, also did not influence neuro-oncogenesis. These results demonstrated the potent growth inhibiting effects of the naturally occurring opioid pentapeptide, [Met5]-enkephalin, and substantiate reports identifying and characterizing an opioid receptor (i.e., zeta) for which [Met5]-enkephalin is the most potent ligand.     

Patterns of product inhibition for bacterial nitrite reductase

PO +Dehydrophenylalanine (delta Phe) having the E-configuration (delta EPhe ; phenyl and C = O cis) was incorporated into [Leu5]-enkephalin in order to restrict its conformation. Compared with the Z-isomer, in the radio-ligand receptor binding assays, [D-Ala2, delta EPhe4 , Leu5] enkephalin showed drastically decreased potency for the delta and mu opiate receptors, i.e., 260- and 150-fold loss of affinity, respectively. The results strongly indicate that the opiate receptors require the Z-configuration (phenyl and C = O, trans) of the delta Phe4 residue and may require a specific interrelationship between the aromatic rings of the Tyr1 and Phe4 residues in the molecule for binding. The conformation of [Leu5]-enkephalin specific for the delta receptors was analyzed and a comparison made with its crystal structure recently elucidated.     

Enkephalins interfere with early phases of voluntary ethanol drinking

The relationship between the opiate peptides Leu-enkephalin and [D-Ala2-Met5]enkephalinamide (DAME) and the initial expression and maintenance of ethanol preference was studied in male Wistar rats. Subcutaneous administration of both peptides prior to the first choice test between water and ethanol induced reductions on ethanol intake and subsequently on total fluid intake. Leu-enkephalin treatment also diminished ethanol preference in the day of treatment and in consecutive days. Neither Leu-enkephalin nor DAME treatments modified rats sucrose preference or intake. The results suggest that the enkephalins studied, when administered in the early phases of ethanol preference, interfere with the mechanisms involved in the propensity to drink ethanol.