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.     

Effect of pertussis toxin treatment on the down-regulation of opiate receptors in neuroblastoma X glioma NG108-15 hybrid cells

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with 10 nM [D-Ala2,D-Leu5] enkephalin (DADLE) results in a reduction of cell-surface opiate delta receptors. Whether opiate receptor internalization requires the activation of the guanine nucleotide-binding protein (Ni) is unclear. Hence, activation of Ni was attenuated by treating hybrid cells with 100 ng/ml pertussis toxin (PT) for 3 h, which resulted in a decrease in DADLE's ability to inhibit adenylate cyclase activity. Despite this prior treatment with PT, chronic exposure of these cells to 10 nM DADLE resulted in a time-dependent decrease in both [3H]diprenorphine and [3H]DADLE binding. This reduction in 3H-ligand binding in cells previously treated with PT represented internalization of the receptors because translocation was observed of bound [3H]DADLE from plasma membrane fractions to the lysosomal fractions in the Percoll gradients. Thus, opiate receptors internalize without activation of Ni. The internalization of opiate receptors was not accompanied by Ni. By measuring the amount of the 41-kDa alpha subunit being labeled by PT with [32P]NAD+, it was determined that plasma membrane preparations, of both the control cells and cells treated with 10 nM of DADLE for 4 h, contained equal concentrations of Ni, 2 pmol of Ni/mg of protein. Additionally, there was no measurable alteration in the amount of PT substrate in the lysosomal fractions of the DADLE-treated cells as compared to that of control cells. Chronic DADLE treatment resulted in a decrease in Km value of NAD+ in the ADP-ribosylation of 41-kDa subunit of Ni. In summary, opiate receptors internalize as agonist-receptor complexes without the guanine nucleotide-binding component.     

A spectrin-dependent ATPase of the human erythrocyte membrane

Removal of spectrin from erythrocyte membranes results in the simultaneous loss of a calcium-stimulated, magnesium-dependent ATPase with an apparent KD for Ca2+ of 1 microM. This ATPase activity with high Ca2+ affinity is specifically reconstituted by addition of purified spectrin to spectrin-depleted membranes, and the reconstituted activity is directly proportional to the amount of spectrin that is reassociated with the membranes. Spectrin binding and activation of the high Ca2+ affinity Mg2+-ATPase are proportionally inhibited by thermal denaturation, trypsin digestion, or treatment of the membranes with thiol-reactive reagents. Binding of calmodulin to the Ca2+ pump ATPase requires that calmodulin contains bound ca2+. By contrast, spectrin binding to the erythrocyte membrane is Ca2+-independent. Direct assay of calmodulin is purified spectrin and absence of chlorpromazine inhibition of reconstitution demonstrate that activation of the high Ca2+ affinity ATPase resulting from spectrin binding is not a result of contamination of spectrin by calmodulin. Additional evidence that the spectrin-activated ATPase is an entity separate and distinct from the Ca2+ pump is provided by other characteristics of the activation phenomenon. It is suggested that spectrin constitutes part of an ATPase which may function as a component of the "cytoskeleton" controlling erythrocyte shape and membrane flexibility.     

Photolabile opioid derivatives of D-Ala2-Leu5-enkephalin and their interactions with the opiate receptor

Photolabile derivatives of D-Ala2-Leu5-enkephalin were prepared by synthetic procedures in which a 2-nitro-4-azidophenyl group is linked to the terminal carboxyl group of the enkephalin by means of an ethylenediamine or ethylenediamine beta-alanine spacer. These peptides bind to opiate receptors with nanomolar affinities and inhibit electrically stimulated contractions of the mouse vas deferens and adenylate cyclase activity of NG108-15 neuroblastoma x glioma hybrid cell membranes. Both inhibitions are reversed by the opiate antagonist naloxone. Photolysis of the ligands bound to rat brain membranes results in the loss of approximately 50% of the receptor sites. This decrease in receptor number is blocked by naloxone and requires light. A photolabile [3H]enkephalin derivative labels an equivalent number of sites under similar irradiation conditions.     

Enkephalins and Opiate Antagonists Control Calmodulin Distribution in Neuroblastoma-Glioma Cells

The calcium binding protein calmodulin and the opiate receptor binding sites are unevenly distributed in various subcellular fractions of neuroblastoma-glioma NG108-15 cells. The crude mitochondrial-membrane fraction of these cells contains two membrane fractions that are separable by sucrose gradient centrifugation. These two differ in the content of both calmodulin and opiate receptors. Leucine enkephalin and D-Ala2-methionine enkephalinamide decrease the amount of membrane-bound calmodulin in the NC108-15 cells in a time- and dose-dependent manner, whereas the opiate antagonists naloxone and levallorphan have an opposite effect. Naloxone blocks the effect of leucine enkephalin and dextrallorphan has no significant effect. The opiate alkaloids entorphine and phenazocine induce changes similar to that of the enkephalins whereas morphine is inactive even at high concentrations. The alteration in the amount of membrane-bound calmodulin after a short incubation (15 min) with the enkephalins or with naloxone is reflected as an opposite change in the amount of calmodulin in the cell cytosol. Naloxone and levallorphan also increase the number of opiate receptors in NG108-15 cells but dextrallorphan has no such effect. Modulation of the intracellular distribution of calmodulin by opioid peptides and alkaloids may control the activity of various membrane-bound and cytosolic systems that are calmodulin- and/or calcium-dependent.     

Are the regulatory cation-binding sites of opiate receptors a part of potential-dependent calcium channels of neuromembranes?

Characteristics of 45Ca2+ transport into rat brain synaptosomes have been studied. The value of an equilibrium constant of the Ca2+ binding at a specific site of neuromembrane's potential-stimulated calcium channel was determined (KM = 0,27 +/- 0,04 mM). Stability constants of several metal-ion complexes with the cation-binding regulatory site at high- and low-affinity receptors for [D-Ala2, D-Leu5]enkephaline and morphine high-affinity receptors of rat brain membranes were estimated. Comparative analysis of the constants calculated along with the results of investigation of a calcium antagonist, Verapamil, and Zn2+ ions influence on various types of opiate receptors allow to conclude that regulatory cation-binding sites of enkephaline and morphine receptors are not identical to Ca2+-binding centres of potential-stimulated calcium channels of the neuromembrane.     

Enkephalin and alpha-adrenergic receptors: evidence for both common and differentiable regulatory pathways and down-regulation of the enkephalin receptor

Several clones of neuroblastoma-glioma NG108-15 hybrid cells were used to reveal whether the regulation of opiate receptor density interacts with the regulation of alpha-adrenergic or acetyl-choline receptors. Low density of alpha-adrenergic receptors in 3 selected clones was accompanied with similar reduction in the density of enkephalin receptors but not in muscarinic acetyl-choline receptors. Yet opiate antagonists that increased the number of opiate receptors in the parent NG108-15 cells in a stereospecific manner had no similar effect on the number of alpha-adrenergic receptors. Moreover, the stable enkephalin analogue D-ala-2-methionine enkephalinamide, but not the opiate alkaloid morphine, decreased the binding of 3H-DAMEA to the membranes and induced down-regulation of enkephalin receptors. Yet DAMEA had no effect on the binding of the alpha-adrenergic antagonist 3H-yohimbine. The study suggests that alpha-adrenergic and enkephalin receptors may share some common regulatory pathways but opiate peptides and antagonists selectively decrease or increase the density of enkephalin receptors, respectively, with no effect on alpha-adrenergic receptor density.     

pH Selectivity of N-Ethylmaleimide Reactions with Opiate Receptor Complexes in Rat Brain Membranes

N-Ethylmaleimide (NEM) decreases opiate agonist binding presumably by blocking crucial sulfhydryl (SH) groups at receptor binding sites. At physiological pH, NEM decreased GTP and manganese regulation but increased sodium effects on [3H]D-Ala2-Met5-enkephalinamide (D-Ala enk) binding to rat brain membranes. To determine the apparent pK values of putative SH groups in opiate receptors that react with NEM, rat brain membranes were incubated with 100-250 microM NEM in buffers ranging from pH 4.5 to 8.0. Results showed that lowering pH below 6.5 reduced the NEM effect on opiate receptor functions and that the apparent pK values of NEM-reacting SH groups in binding and regulatory sites ranged between 5.4 to 6.0. Most of the total SH groups in brain membranes continued to react with NEM at low pH, so that when nonspecific SH groups were blocked by incubating membranes at pH 4.5 with NEM, opiate receptors became sensitive to very low concentrations (1 microM) of NEM.     

ADP-ribosylation of adenylate cyclase by pertussis toxin. Effects on inhibitory agonist binding

Adenylate cyclase in NG108-15 (neuroblastoma X glioma hybrid) cells is responsive to both stimulatory and inhibitory ligands. Bordetella pertussis toxin (PT) catalyzes the ADP-ribosylation of a 41,000-Da peptide believed to be a subunit of the putative guanyl nucleotide-binding protein (Gi) involved in cyclase inhibition and abolishes inhibitory effects of opiate agonists. In studying the effects of PT on opiate receptors, we found that [3H]enkephalinamide binding was reduced by approximately 90% in membranes prepared from cells incubated with PT compared to control membranes. Agonist affinity, assessed by enkephalinamide competition for [3H]diprenorphine-binding sites, was markedly reduced in cells incubated with PT. Furthermore, inhibition by guanylylimidodiphosphate of ligand binding to opiate receptors was reduced following treatment with PT. The number of opiate receptors assessed by [3H]diprenorphine binding was unaltered by PT. These data are consistent with the hypothesis that PT-catalyzed ADP-ribosylation impairs the interaction of Gi with the inhibitory receptor-ligand complex, effectively uncoupling the inhibitory receptor from Gi and the cyclase catalytic unit.     

Assessment of the role of endogenous regulators in the activation of Ca-ATPase in erythrocyte membranes

The contribution of calmodulin and protein kinases A or C to the activation of membrane Ca-ATPase was studied on saponin-permeabilized rat erythrocytes. In the presence of all endogenous regulators, the dependence of the Ca-ATPase activity of Ca2+ concentration was described by a bell-shaped curve with a maximum at 2-5 microM Ca2+; K0.5 = 0.43 microM Ca2+. Washing of erythrocyte membranes with 5-10 microM Ca2+ maintained up to 75% of the ATPase activity, while washing with EGTA (2 mM) decreased the activity, on the average, 5-fold, and increased K0.5 up to 0.54-0.6 microM Ca2+. An addition of an EGTA extract to washed membranes restored up to 75% of the original ATPase activity, while calmodulin restored about 40% of the original Ca-ATPase activity and decreased K0.5 to 0.23-0.3 microM Ca2+. The calmodulin inhibitor R24571 failed to alter the Ca-ATPase activity in permeabilized erythrocytes but slightly diminished it in reconstituted membranes. The protein kinase C inhibitors H7 and polymyxin increased the Ca-ATPase activity in permeabilized red cells and suppressed it in reconstituted membranes. The data obtained suggest that in native red cell membranes Ca-ATPase is activated by regulator(s) dependent on Ca2+ and protein kinase which are other than calmodulin.     

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.     

Down regulation of enkephalin (delta) receptors. Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue D-Ala2-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to delta-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the alpha 2-adrenergic ligand, [3H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of delta-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.     

Control mechanisms governing the infectivity of Chlamydia trachomatis for HeLa cells: the role of calmodulin

Adhesion of the obligate intracellular bacterium Chlamydia trachomatis to host cells is associated with a flux of Ca2+ across the cell membrane, and infection is enhanced by treatment of host cells with Ca2+ ionophore. The possibility that Ca2+ might interact with host cell Ca2+ regulatory proteins to promote chlamydial infection was investigated. Treatment of HeLa 229 cells with the calmodulin inhibitors pimozide, trifluoperazine, chlorpromazine, promethazine or haloperidol reduced chlamydial infectivity as measured by inclusion counting or the specific incorporation of [3H]threonine. The inhibitory effect was reversible, dose-related and shown to be associated with impairment of chlamydial adhesion and uptake by the host cells. This effect was clearly distinguished from the delayed maturation of chlamydiae due to continuous exposure to calmodulin inhibitors which may result from a decrease in the availability of high energy compounds from the host cells necessary for chlamydial growth. The possible mechanisms for calmodulin-mediated chlamydial endocytosis are discussed.     

Calmodulin regulation of the ATP-dependent calcium uptake by inverted vesicles prepared from rabbit synaptosomal plasma membranes

Calmodulin has been shown to activate the ATP-dependent Ca2+ uptake in inside-out vesicles which have been prepared from rabbit synaptosomal plasma membranes by the methodology of Gill et al. (Gill, D.L., Grollman, E.F. and Kohn, L.D. (1981) J. Biol. Chem. 256, 184-192). Following extensive washings of these membranes with EGTA/EDTA solutions, the Ca2+ uptake activity demonstrated an affinity for calmodulin of 30 nM and an affinity for Ca2+ of 2 microM. The activity was completely inhibited by the anticalmodulin compound R24571 (Ki congruent to 8 microM). The molecular weight of the ATPase molecule, revealed by a combination of the [125I]calmodulin overlay technique and [32P]phosphoenzyme electrophoresis, was 145 000. The overlay technique also revealed that the mechanism of activation is via a direct binding of calmodulin to the pump molecule.     

Exercise conditioning increases rat myocardial calcium uptake

To investigate potential mechanisms underlying the enhanced myocardial performance consequent to exercise training, the adrenergic receptors of myocardial tissue and Ca2+ uptake into sarcoplasmic reticulum-enriched fractions from exercise conditioned animals were compared with that of sedentary controls. Female Wistar rats were exercised by swimming 30 min (5 days/wk) for 12 wk. Exercise conditioning was effective in producing myocardial hypertrophy, as reflected by an increase in heart weight (1.179 +/- 0.022 vs. 1.031 +/- 0.020 g, P less than 0.001) and heart weight-to-body weight ratio (3.29 +/- 0.06 vs. 2.77 +/- 0.05 X 10(-3), P less than 0.001) but no difference in body weight. Despite the myocardial hypertrophy, neither the affinity nor the density of the alpha 1-adrenergic receptors or the beta-adrenergic receptors determined by Scatchard analysis of the ligands [3H]prazosin and [3H]dihydroalprenolol were significantly different between the two groups. The basal Ca2+ uptake into the sarcoplasmic reticulum was also similar (9.90 +/- 0.97 vs. 9.04 +/- 0.75 nmol/mg protein/min), but the addition of calmodulin produced a significantly greater increment in Ca2+ uptake into sarcoplasmic reticulum from the exercised-conditioned animals (1.90 +/- 0.23 vs. 1.21 +/- 0.19 nmol/mg protein/min, P less than 0.03). The adenosine triphosphatase (ATPase) activities of the sarcoplasmic reticulum-enriched fractions of the two groups were similar. We conclude that exercise conditioning produces an enhancement of calmodulin-mediated calcium uptake that is independent of any effect on Ca2+-ATPase.     

Studies on the effects of glucose in vitro and of the glycaemic state in vivo on the binding characteristics of mu, delta and kappa opiate receptors in mouse brain.

The effect of glucose on the binding characteristics of opiate receptor subtypes was investigated in brain membranes from normoglycaemic lean Aston (C57BL/6J) mice using [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO), [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) and [3H]U69,593 as selective ligands for mu, delta and kappa opiate receptors respectively. The equilibrium dissociation constants (Kd) and maximal binding capacities (Bmax) of [3H]DAMGO and [3H]DPDPE were unaltered by 20mM glucose in vitro. Similarly, [3H]U69,593 binding was not modified by increasing the concentration of glucose from 0 to 20mM (P between 0.10 and 0.05), or by the presence of 20mM fructose and of 20mM 3-O-me-glucose, a non-metabolisable sugar, in the incubation medium. The nonselective opiate ligand, [3H]diprenorphine, bound with similar affinity and binding capacity to brain membranes prepared from control and streptozotocin-diabetic Swiss (CD1) mice. The addition of 20mM glucose or of 20mM fructose in vitro induced no changes in their binding parameters. The affinity and binding capacity of [3H]U69,593 to STZ-diabetic Swiss mouse brain membranes was not significantly different to that of normoglycaemic controls; 20mM glucose in vitro had no effect on ligand binding to kappa sites in STZ-diabetic mouse brain membranes. We conclude that glucose does not interact directly with the opiate receptor to modfy it in such as way as could explain the altered sensitivity to different opioid agonists seen in obese and hyperglycaemic animal models in vivo.     

Opiate binding to membrane preparations of neuroblastoma x glioma hybrid cells NG108-15: effects of ions and nucleotides.

Interaction of a number of opiate agonists with the opiate receptors in NG108-15 cell membranes is influenced by ions, as well as certain nucleotides. Steady state binding of [³H]leu-enkephalin is increased by Mg⁺⁺ and decreased by Na⁺, GMP-P(NH)P, GTP, GDP, ITP and IMP-P(NH)P. Half-maximal inhibition produced by GMP-P(NH)P occurred at 4.6 μM. The dissociation of [³H]leu- and [³H]met-enkephalin, as well as [³H]etorphine, from these opiate receptors was also shown to be altered by both ions and nucleotides.     

Direct Demonstration of the Activation of UDP-N-Acetylgalactosamine: [GM3]N-Acetylgalactosaminyltransferase by Cyclic AMP

Treatment of NG108-15 cells in culture with the opiate peptide [D-Ala2,D-Leu5]enkephalin produces maximal inhibition of cyclic AMP synthesis in less than 15 min. The activity of [GM3]:N-acetylgalactosaminyltransferase is similarly inhibited, but maximal inhibition is not observed for at least 30 min following the addition of [D-Ala2,D-Leu5]enkephalin. Conversely, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine rapidly potentiates the intracellular accumulation of cyclic AMP and, in a more gradual fashion, increases [GM3]:N-acetylgalactosaminyltransferase activity. The reductions in the activity of [GM3]:N-acetylgalactosaminyltransferase that occur following treatment of NG108-15 cells with indomethacin argues for a direct role of cyclic AMP in the observed changed in [GM3]:N-acetylgalactosaminyltransferase activity. By adding low concentrations of cyclic AMP (but not cyclic GMP) to microsomes derived from neonatal rat brain, we were able to demonstrate a dose-dependent phosphorylation of membrane protein and subsequent doubling of [GM3]:N-acetylgalactosaminyltransferase activity.     

Distinction between Endoplasmic Reticulum-Type and Plasma Membrane-Type Ca2+ Pumps (Partial Purification of a 120-Kilodalton Ca2+-ATPase from Endomembranes)

Two biochemical types of Ca2+-pumping ATPases were distinguished in membranes that were isolated from carrot (Daucus carota) suspension-cultured cells. One type hydrolyzed GTP nearly as well as ATP, was stimulated by calmodulin, and was resistant to cyclopiazonic acid. This plasma membrane (PM)-type pump was associated with PMs and endomembranes, including vacuolar membranes and the endoplasmic reticulum (ER). Another pump ("ER-type") that was associated mainly with the ER hydrolyzed ATP preferentially, was insensitive to calmodulin, and was inhibited partially by cyclopiazonic acid, a blocker of the animal sarcoplasmic/ER Ca2+ pump. Oxalate stimulation of Ca2+ accumulation by ER-type, but not PM-type, pump(s) indicated a separation of the two types on distinct compartments. An endomembrane 120-kD Ca2+ pump was partially purified by calmodulin-affinity chromatography. The purified polypeptide bound calmodulin reacted with antibodies to a calmodulin-stimulated Ca2+ pump from cauliflower and displayed [32P]phosphoenzyme properties that are characteristic of PM-type Ca2+ pumps. The purified ATPase corresponded to a phosphoenzyme and a 120-kD calmodulin-binding protein on endomembranes. Another PM-type pump was suggested by a 127-kD PM-associated protein that bound calmodulin. Thus, both ER- and PM-type Ca2+ pumps coexist in most plant tissues, and each type can be distinguished from another by a set of traits, even in partially purified membranes.     

Interaction of D-Ala2-[Tyr-3,5-3H]enkephalin(5-D-Leu) with opiate receptors of rat brain

Some kinetic features of D-Ala2-[Tyr-3.5-3H]enkephalin (5-D-Leu) binding to opiate receptors of rat brain were studied. It was shown that the Leu-enkephalin D analog interacts with the high and low affinity binding sites of opiate receptors, the equilibrium constants being equal to 0.71 and 8.4 nM, respectively. The rate constant for the label association with the high affinity binding sites in 2 . 10(8) M-1 min-1; those for the label dissociation from the opiate receptor binding sites with high and low affinities are 7.2 . 10(-3) and 0.16 min-1, respectively. Hence, the half-life time of these complexes is 95.7 and 4.3 min, respectively. Na+, K+ and Li+ markedly decrease the specific finding of the label, while Mg2+, Mn2+ and Ca2+ at the concentrations studied markedly increase its specific binding. It is concluded that the Leu-enkephalin D-analog under study acts as a morphine agonist and reveals a much higher affinity for rat brain opiate receptors than does Leu- or Met-enkephalin. This makes it a useful tool for study of the enkephalin reception under normal and pathological conditions.