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.     

3H]guanidinoethylmercaptosuccinic acid binding to tissue homogenates. Selective labeling of enkephalin convertase

[3H]Guanidinoethylmercaptosuccinic acid (GEMSA), a potent inhibitor of enkephalin convertase, binds to membrane and soluble fractions of tissue homogenates saturably and reversibly with a KD of 6 nM. Specific binding accounts for greater than 95% of total binding. The highest levels of [3H]GEMSA binding occur in the pituitary gland and the brain, with much lower levels in peripheral tissues. GEMSA, guanidinopropylsuccinic acid, 2-mercaptomethyl-3-guanidinothiopropionic acid, aminopropylmercaptosuccinic acid, [Leu] enkephalin-Arg, and [Met]enkephalin-Arg inhibit [3H] GEMSA binding to crude rat brain homogenates, to crude bovine pituitary homogenates, and to pure enkephalin convertase with equal potencies. Their Ki values against [3H]GEMSA binding are similar to their Ki values against enkephalin convertase activity. EDTA and 1,10-phenanthroline markedly inhibit both binding and enzymatic activity. The ratio of the Vmax for 5-dimethylaminonaphthalene-1-sulfonyl-Phe-Leu-Arg to the Bmax (maximal number of binding sites) for [3H]GEMSA is about 2,000 min-1 in both pure enzyme preparations and crude tissue homogenates. [3H] GEMSA binding activity is found only in fractions containing enkephalin convertase during enzyme purification from bovine pituitary by L-arginine affinity chromatography. These data confirm that [3H]GEMSA binds only to enkephalin convertase in crude homogenates under our assay conditions. CoCl2 activates enzyme activity without altering the Ki of GEMSA against enzymatic hydrolysis and weakly inhibits [3H] GEMSA binding by increasing the KD.     

Enkephalin hydrolysis by mouse plasma in vitro.

Hydrolysis of [Leu]- and [Met]enkephalin was determined in samples of pooled whole mouse plasma in vitro by using HPLC-ECD to measure accumulation of Tyr-containing metabolites. More Tyr-Gly-Gly accumulated from [Met]enkephalin than from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in mouse plasma was approximately half that of [Leu]enkephalin. Comparisons of metabolite formation in the presence versus the absence of inhibitors with high selectivity for various peptidases demonstrated that a bestatin-sensitive aminopeptidase, presumably aminopeptidase M, as well as enkephalinase and angiotensin converting enzyme, participate in the hydrolysis of enkephalin in mouse plasma.     

Interaction of enkephalins and des-tyrosyl-enkephalins with synaptosomal plasma membrane vesicles: enkephalin binding and inhibition of proline transport

Leucine- and methionine-enkephalins inhibit the Na+-dependent transport of proline into plasma membrane vesicles derived from synaptosomes. Glycine transport is weakly inhibited by enkephalins whereas there is no inhibition of transport of glutamic acid, aspartic acid, or gamma-aminobutyric acid. The inhibition of proline uptake is observed with des-tyrosyl-enkephalins but not with morphine, dynorphin(1-13), or beta-endorphins. Furthermore, enkephalin-induced inhibition of proline transport is not antagonized by naloxone. [Leu]enkephalinamide and modified [Leu]enkephalins with greater selectivity for the delta-subclass of enkephalin binding sites are less effective than [Leu]enkephalin in the inhibition of proline transport. Specific binding of [3H]Leu-enkephalin to the plasma membrane vesicles is demonstrated, and des-Tyr-[Leu]enkephalin competes with Leu-enkephalin for [Leu]enkephalin binding sites. The similarity in the concentrations of des-Tyr-[Leu]enkephalin required to compete for specific [Leu]enkephalin binding and to inhibit proline transport suggests that a specific subclass of enkephalin binding sites, distinguished by their recognition of both the enkephalins and their des-tyrosyl derivatives, may be associated with the synaptic proline transport system.     

Comparative structure-function studies with analogs of dynorphin-(1-13) and [Leu5]enkephalin

Analogs of dynorphin-(1-13) with modifications in the enkephalin segment were compared with correspondingly modified analogs of [Leu5]enkephalin in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assay as well as in mu- and delta-receptor selective binding assays. The obtained results indicate that a) the enkephalin binding domain of the dynorphin (kappa) receptor has structural requirements which are distinct from those of the enkephalin binding site at the mu-receptor and b) the introduction of an identical conformational constraint in [Leu5]enkephalin and in the enkephalin segment of dynorphin-(1-13) produces a superpotent agonist in both cases. Fluorescence energy transfer measurements with the active [4-tryptophan]analogs of dynorphin-(1-13) and [Leu5]enkephalin and with dynorphin-(1-17) demonstrated a more extended conformation of the N-terminal tetrapeptide segment in [Trp4]dynorphin-(1-13) than in [Trp4, Leu5]enkephalin as well as the absence of an interaction between the N- and C-terminal segments of dynorphin-(1-17).     

Sigma and opioid receptors in human brain tumors

Human brain tumors (obtained as surgical specimens) and nude mouse-borne human neuroblastomas and gliomas were analyzed for sigma and opioid receptor content. Sigma binding was assessed using [3H]1,3-di-o-tolylguanidine (DTG), whereas opoid receptor subtypes were measured with tritiated forms of the following: mu, [D-ala2,mePhe4,gly-ol5]enkephalin (DAMGE); kappa, ethylketocyclazocine (EKC) or U69,593; delta, [D-pen2,D-pen5]enkephalin (DPDPE) or [D-ala2,D-leu5]enkephalin (DADLE) with mu suppressor present. Binding parameters were estimated by homologous displacement assays followed by analysis using the LIGAND program. Sigma binding was detected in 15 of 16 tumors examined with very high levels (pmol/mg protein) found in a brain metastasis from an adenocarcinoma of lung and a human neuroblastoma (SK-N-MC) passaged in nude mice. kappa opioid receptor binding was detected in 4 of 4 glioblastoma multiforme specimens and 2 of 2 human astrocytoma cell lines tested but not in the other brain tumors analyzed.     

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.     

Cystamine-enkephalin dimer. Syntheses and biological activities of enkephalin analogs containing cystamine and cysteamine

A cystamine-enkephalin dimer, containing two molecules of [D-Ala2, Leu5] enkephalin cross-linked at the COOH-terminal leucine residue with cystamine, (NH2-CH2-CH2-S-)2, has been synthesized in order to examine directly the dimerization effect of an enkephalin molecule on the opiate receptor interactions. In a comparison of potencies against [3H]-[D-Ala2,D-Leu5] enkephalin (3H-DADLE) and [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (3H-DAGO) as delta and mu tracers, respectively, enkephalin dimer showed a very high affinity, especially for the delta opiate receptors. Dimer was almost threefold more potent than DADLE, which is one of the most utilized delta ligand to date. When the binding affinity of cystamine-dimer was compared with that of its reduced thiol-monomer, namely [D-Ala2,Leu5,cysteamine6] enkephalin, the increment in affinity was four to fivefold for both delta and mu receptors. The results strongly indicate that the dimeric enkephalin is more potent presumably due to the simultaneous interaction with the two binding sites of the opiate receptors.     

Quantitative autoradiographic distribution of meptazinol-sensitive binding sites in rat brain

1. Meptazinol is an interesting opioid-producing naloxone-reversible analgesia with few cardiovascular and respiratory effects. Recent studies indicate that mu 1 opioid receptors mediate meptazinol analgesia. Using a computerized autoradiographic subtraction technique, we have examined the regional distribution of meptazinol-sensitive [3H][D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) binding and compared this with the distribution of mu 1 binding determined by competition with low [D-Ala2,D-Leu5]enkephalin (DADL) concentrations. 2. Meptazinol and DADL lowered [3H]DAGO to similar extents in most brain regions studied. The greatest levels of inhibition were observed in the periaqueductal gray, interpeduncular nucleus, thalamus, hypothalamus, and hippocampus. Low levels of inhibition were found in the temporal and frontal cortex. The correlation between the inhibition of [3H]DAGO binding by meptazinol and that by DADL was high (r = 0.83), consistent with the binding of meptazinol to mu 1 sites.     

Antinociceptive interactions of opioid delta receptor agonists with morphine in mice: supra- and sub-additivity.

In this study, the antinociceptive interactions of fixed ratio combinations of intracerebroventricularly (i.c.v.) given morphine and subantinociceptive doses of the delta agonists, [D-Pen2, D-Pen5]enkephalin (DPDPE), [D-Ala2, Glu4]deltorphin (DELT) or [Met5]enkephalin (MET) were examined using the mouse warm water tail flick test. When morphine was coadministered with DPDPE or DELT in a 4:1 and 9:1 mixture, respectively, a synergistic antinociceptive effect was observed. In contrast, when morphine was coadministered with MET in a 1:2 fixed ratio mixture, a subadditive interaction occurred. These results demonstrate both positive and negative modulatory interactions of delta agonists with morphine in an antinociceptive endpoint and that these interactions can be either supra- or subadditive. The data support the concept of a functional interaction between opioid mu and delta receptors and a potential regulatory role for the endogenous ligands of the opioid delta receptor.     

Facile incorporation of urea pseudopeptides into protease substrate analogue inhibitors

A new procedure that employs a one-pot, oxidative Hofmann rearrangement to incorporate a urea linkage into peptide backbones is detailed herein. This methodology was used to replace the scissile peptide bonds of [Leu5]enkephalin and a hexapeptide HIV-1 protease substrate. The [Leu5]enkephalin analogue was found to inhibit cleavage of hippurylhistidylleucine (HHL) by porcine kidney angiotensin-converting enzyme (PK-ACE) with a 0.88 mM IC50 value, comparable to the Michaelis constant of [Leu5]enkephalin with the same enzyme. The HIV-1 protease substrate analogue was shown to inhibit HIV-1 protease with an IC50=34 microM.     

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.     

Delta and mu opiate receptor probes: fluorescent enkephalins with high receptor affinity and specificity

The fluorescent amino acid, L-1-pyrenylalanine (Pya) was incorporated into [D-Ala2,Leu5]enkephalin and its methyl ester at position 4 or 5. Pya-enkephalins showed strong fluorescent intensity and displayed high binding affinity for opiate receptors. Pya4-enkephalins showed high specificity for the mu receptors, while Pya5-enkephalins showed high specificity and selectivity for the delta receptors. Particularly, [D-Ala2,Pya5]enkephalin was as potent as the most utilized delta-specific ligand of [D-Ala2,D-Leu5]enkephalin (DADLE), and yet its delta-selectivity was about 5-times greater than that of DADLE. Thus, Pya-enkephalins per se can be utilized as a fluorescent probe or tracer for the opiate receptor-binding assays.     

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.     

Synthesis and biological activity of [Leu5]enkephalin derivatives containing D-glucose

The synthesis of some [Leu5]enkephalin derivatives is described in which D-glucose has been linked to the opioid pentapeptide through the ester bond involving the carboxyl function at the C-terminal with C-1 or C-6 of the D-glucopyranose moiety. Enkephalin derivatives were assayed for opioid activity and found to be full agonists in bioassays based on inhibition of electrically evoked contractions of the guinea pig ileum (GPI) and of the mouse vas deferens (MVD). The obtained results suggest that the opioid activity of the tested glucoconjugates depend upon the ester bond position in the molecule. Whereas 1-O conjugate 5 was somewhat more potent than [Leu5]enkephalin in the GPI assay, the 6-O conjugates, with the exception of 1-O-benzyl derivative 11, were considerably less potent. All enkephalin derivatives were delta-receptor selective; in particular, the acetylated analog 8 was three times more delta-receptor selective than [Leu5]enkephalin.     

Identification of an opioid receptor subunit carrying the mu binding site

The enkephalin affinity reagent [3H]Tyr-D-Ala-Gly-Phe-Leu-CH2Cl [( 3H]DALECK) was synthesized. It exhibited high-affinity reversible binding, at pH 7.4, to both mu and delta opioid receptor sites in rat brain membranes. At pH 8.1, nanomolar levels of [3H]DALECK produced an irreversible labeling in synaptic membranes, essentially only in one subunit of 58 000 daltons. The irreversible phase of the reaction reduced the subsequent binding of a mu-selective enkephalin derivative but not that of a delta-selective one. It is concluded that a mu subunit of the opioid receptor exists, can be alkylated specifically, and is of Mr 58 000.     

Dimeric pentapeptide enkephalin: a novel probe of delta opiate receptors

A dimeric pentapeptide enkephalin (DPE2) consisting of two molecules of [D-Ala 2, Leu 5] enkephalin linked at C-terminal leucine with ethylenediamine, (H-Tyr-D-Ala-Gly-Phe-Leu-NH-Ch2)2 is a bivalent ligand for the delta enkephalin receptors of rat brain and neuroblastoma-glioma hybrid (NG108-15) cells. This new enkephalin analog shows dramatically increased affinity in radioligand assays using whole brain membranes when delta but not mu specific radioligands are employed. When membranes from NG108-15 cells are used, the dimer shows greatly increased activity irrespective of the mu or delta specificity of the tracer. The dimer DPE2 shows a four-fold, "sodium shift" in its IC50 for competition with [3H]naloxone, suggestive of agonist behavior. Agonist activity was confirmed by demonstrating that DPE2 inhibits cyclic AMP production in prostaglandin E1 stimulated NG108-15 cells, and by demonstrating very high potency in the mouse vas deferens bioassay. DPE2 binds to the same delta sites as the delta-selective monomer [D-Ala2, D-Leu5] enkephalin, since the two ligands show complete crossdisplacement. Radiolabeled 3H-DPE2 shows a five-fold higher affinity constant, a 2.5-fold higher association rate constant, and a two-fold lower dissociation rate than the monomer. These results are consistent with the hypothesis that the dimeric pentapeptide enkephalin can bridge two delta receptors. This enkephalin dimer provides a valuable new probe of opiate receptors and their organization in cell membranes.     

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.     

Enkephalin is liberated from metorphamide and dynorphin A1-8 by endo-oligopeptidase A, but not by metalloendopeptidase EC 3.4.24.15.

It has been previously reported that both the cysteinyl-endo-oligopeptidase A and the metalloendopeptidase EC 3.4.24.15 are able to generate enkephalin from a number of enkephalin-containing peptides, including dynorphin A1-8. The present study shows that only endo-oligopeptidase A is able to generate [Leu5]enkephalin and [Met5]enkephalin from dynorphin A1-8 and from metorphamide respectively. It is also shown that endo-oligopeptidase A neither hydrolyses the specific EC 3.4.24.15 substrate alpha-N-benzoyl-Gly-Ala-Ala-Phe p-aminobenzoate, nor is inhibited by the specific EC 3.4.24.15 inhibitor N-[1(RS)-carboxy-2-phenylethyl]-alpha-Ala-Ala-Phe p-aminobenzoate.     

Photoreactive enkephalin analogue: [D-Ala2, p-N3-Phe4-Met5]-enkephalin. Synthesis, purification by high performance liquid chromatography and characterization

A photoreactive [D-Ala2, p-N3-Phe4-Met5]enkephalin was synthesized by classical solution peptide synthetic methods. The hydroxysuccinimide ester was used in all the coupling steps in the presence of a weak base, triethylamine. The deprotected enkephalin analogue was purified on high performance liquid chromatography using a Waters, C18 muBondapak reverse phase column and its purity was assessed by thin-layer chromatography. The composition of the analogue was determined and confirmed by elemental analysis and amino acid analysis. Its photoreactivity was demonstrated by the time dependent ultraviolet spectral changes on exposure to light. Competition receptor binding showed that [D-Ala2, p-N3-Phe4-Met5]enkephalin was 4-fold more potent than [D-Ala2, Met5]-enkephalin in competing for binding to the enkephalin binding site. The data presented suggest that this photoreactive enkephalin analogue may be suitable for use in the in situ photoaffinity labeling of the enkephalin receptor.