Characterization of [125I]AR-M100613, a high-affinity radioligand for delta opioid receptors

AR-M100613 ([I]-Dmt-c[-D-Orn-2-Nal-D-Pro-D-Ala-]) is the iodinated analog of a cyclic casomorphin previously shown to be a potent antagonist at the delta opioid receptor. Specific [125I]AR-M100613 binding to rat whole brain membranes was saturable, reversible, and best fit to a one-site model (Kd = 0.080 +/- 0.008 nM, Bmax = 45.2 +/- 4.4 fmol/mg protein). [125I]AR-M100613 binding was displaced with high affinity by the delta opioid receptor ligands SNC-80, Deltorphin II and DPDPE but not the mu or kappa-selective receptor ligands DAMGO and U69593. Residual non-selective binding of [125I]AR-M 100613 to mu opioid receptors is blocked by the addition of CTOP to the assay buffer. [35S]GTPgammaS binding assays indicate that AR-M100613 is a potent, selective, and reversible antagonist for delta opioid receptors in rat brain membranes. The high-affinity, high specific activity, low nonspecific binding and antagonist profile of [125I]AR-M100613 favor its use as a radiochemical probe for delta opioid receptors.     

Inhibition of mu and delta but not kappa opioid binding to membranes by Fab fragments from a monoclonal antibody directed against the opioid receptor

Fab fragments from a monoclonal antibody, OR-689.2.4, directed against the opioid receptor, selectively inhibited opioid binding to rat and guinea pig neural membranes. In a titratable manner, the Fab fragments noncompetitively inhibited the binding of the mu selective peptide [D-Ala2,(Me)Phe4,Gly(OH)5][3H] enkephalin and the delta selective peptide [D-Pen2,D-Pen5] [3H]enkephalin (where Pen represents penicillamine) to neural membranes. In contrast, kappa opioid binding, as measured by the binding of [3H]bremazocine to rat neural membranes and guinea pig cerebellum in the presence of mu and delta blockers, was not significantly altered by the Fab fragments. In addition to blocking the binding of mu and delta ligands, the Fab fragments displaced bound opioids from the membranes. When mu sites were blocked with [D-Ala2,(Me)Phe4,Gly(OH)5]enkephalin, the Fab fragments suppressed the binding of [D-Pen2,D-Pen5][3H]enkephalin to the same degree as when the mu binding site was not blocked. The Fab fragments also inhibited binding to the mu site regardless of whether or not the delta site was blocked with [D-Pen2,D-Pen5]enkephalin. This monoclonal antibody is directed against a 35,000-dalton protein. Since the antibody is able to inhibit mu and delta binding but not kappa opioid binding, it appears that this 35,000-dalton protein is an integral component of mu and delta opioid receptors but not kappa receptors.     

125I][D-Ala2]deltorphin-I: a high affinity, delta-selective opioid receptor ligand.

The selective delta opioid agonist [D-Ala2]deltorphin-I was radioiodinated and the product purified using reverse phase HPLC. The binding characteristics and distribution profile of [125I][D-Ala2]deltorphin-I were assessed in mouse brain using homogenate binding techniques and quantitative autoradiography. [125I][D-Ala2]deltorphin-I bound with high affinity to a single class of sites (KD = 0.5 nM) in brain membrane preparations and striatal sections. Competition studies indicated that [125I][D-Ala2]deltorphin-I was selectively labeling delta opioid receptors as shown by the ratio of apparent affinities for mu and delta receptors (KI mu/KI delta = 1388). The autoradiographical distribution profile of [125I][D-Ala2]deltorphin-I binding sites was also consistent with that of other delta-selective radioligands. The data indicate that [125I][D-Ala2]deltorphin-I binds to delta opioid receptors with high affinity and selectivity. Because of its very high specific activity, it can be detected rapidly with high sensitivity by autoradiographic emulsion.     

Biphasic competition between opiates and enkephalins: does it indicate the existence of a common high affinity ("mu-1") binding site?

Displacement from brain membranes of labeled opiates by low concentrations of enkephalins and of labeled enkephalins by low concentrations of opiates has been previously explained by the existence of a common high affinity site termed mu-1. An alternative interpretation of the same results is that the trough seen in the low concentration zone of the displacement curves represents cross binding of mu and delta opioid ligands to delta and mu receptors, respectively. In three sets of experiments with brain membranes, the size of the trough is shown to be dependent on the labeled ligand used: The ratio between the size of troughs seen with [3H]D-Ala, D-Leu enkephalin and with [3H]morphine varies with experimental conditions (storage of membranes at 4 degrees C for 72 h), with ratio of mu:delta receptors (e.g. in thalamus and cortex which are enriched in mu and delta sites, respectively) and with pretreatment of membranes with naloxonazine. These results can not be explained by a common high affinity site, but rather by binding of [3H]D-Ala, D-Leu enkephalin to mu and of [3H]morphine to delta opioid receptors.     

14 beta-(Bromoacetamido)morphine irreversibly labels mu opioid receptors in rat brain membranes

The binding properties of 14 beta-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM [3H] [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the mu binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The mu receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the delta-selective peptide [3H] [D-penicillamine2,D-penicillamine5]enkephalin (DPDPE) and (-)-[3H]bremazocine in the presence of mu and delta blockers, selective for kappa binding sites. Under conditions where 90% of the 0.25 nM [3H]DAGO binding sites were blocked, 80% of the 0.8 nM [3H]naloxone binding and 50% of the 0.25 nM 125I-labeled beta h-endorphin binding were inhibited by BAM alkylation. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the mu site did not afford protection.2+hese studies have demonstrated that when a disulfide bond     

Affinity Labeling Mu Opioid Receptors With Novel Radioligands

1. A series of novel opiate ligands based upon 6α-naloxamine have been examined in opioid receptor binding assays.2. Coupling an ethylamine spacer alone to 6-α-naloxamine gave a compound with relatively poor affinity for mu opioid receptors compared to naloxone, although it retained high affinity for kappa₁ opioid receptors. Coupling a benzoyl group significantly increased the affinity. The presence at the 4-position of the benzoyl moiety of an amino-(NalAmiBen) or an azido-substituent (NalAziBen) did not significantly effect the affinity at mu receptors. However, iodinating the benzoyl moiety at the 3-position increased the affinity of the derivatives.3. Two compounds were radiolabeled and evaluated in receptor binding assays. Both radioligands labeled sites in CHO cells stably transfected with the mouse MOR-1 clone. The amino coupound [¹²⁵I]NalAmiBen and the azido derivative [¹²⁵I]NalAziBen reversibly bound to membranes from CHO cells transfected with MOR-1 with high affinity in the dark. Exposure of [¹²⁵I]NalAmiBen to UV did not alter the reversibility of binding, but exposure of [¹²⁵I]NalAziBen to UV light led to the covalent coupling of the radioligand to the receptor. When run on SDS-PAGE, [¹²⁵I]NalAziBen binding showed a band at approximately 70–80 kDa. A control corresponding to nonspecific binding failed to reveal any labeling. No bands were observed from membranes labeled with [¹²⁵I]NalAmiBen.     

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.     

Opioid receptors in rat cardiac sarcolemma: effect of phenylephrine and isoproterenol

The present study demonstrates the presence of opioid receptors in the rat cardiac sarcolemma isolated by the hypotonic LiBr-shock procedure. Opioid binding was measured by using [3H]U69 593, [3H](2-D-penicillamine,5-D-penicillamine)enkephalin ([3H]DPDPE) or [3H][D-Ala2,MePhe4,Gly-(ol)5]enkephalin ([3H]DAGO) as selective radioligands for K, delta and mu opioid receptors, respectively. Both the K- and delta-selective ligands exhibited highly specific (75-86%) binding, saturable at a concentration of about 20 nM. No specific binding for the selective agonist DAGO was observed. A marked increase in both [3H]U69 593 and [3H]DPDPE binding was observed after incubation of the sarcolemma with the alpha-adrenoceptor agonist phenylephrine or with the beta-adrenoceptor agonist isoproterenol. These stimulatory effects were associated with an increase in the Bmax values, a decrease in the Kd values, and were completely antagonized by the respective antagonists phentolamine and propranolol.     

Probing the opioid receptor complex with (+)-trans-superfit. I. Evidence that [D-Pen2,D-Pen5]enkephalin interacts with high affinity at the delta cx binding site.

A variety of data support the existence of an opioid receptor complex composed of distinct but interacting mu cx and delta cx binding sites, where "cx" indicates "in the complex." The ability of subantinociceptive doses of [Leu5]enkephalin and [Met5]enkephalin to potentiate and attenuate morphine-induced antinociception, respectively, is thought to be mediated via their binding to the delta cx binding site. [D-Pen2,D-Pen5]Enkephalin also modulates morphine-induced antinociception, but has very low affinity for the delta cx binding site in vitro. In the present study, membranes were depleted of their delta ncx binding sites by pretreatment with the site-directed acylating agent, (3S,4S)-(+)-trans-N-[1-[2-(4-isothiocyanato)phenyl)-ethyl]-3-methy l-4- piperidyl]-N-phenylpropaneamide hydrochloride, which permits selective labeling of the delta cx binding site with [3H][D-Ala2,D-Leu5]enkephalin. The major findings of this study are that with this preparation of rat brain membranes: a) there are striking differences between the delta cx and mu binding sites; and b) both [D-Pen2,D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin exhibit high affinity for the delta cx binding site.     

Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (mu) and enkephalin (delta) binding sites

Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of mu and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of [3H]-D-Ala2-D-Leu5-enkephalin (DADLE) in the presence of 10(-5) M D-Pro4-morphiceptin (to block the mu receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of [3H]-dihydromorphine, together with the higher potency of morphine analogues to displace [3H]-naloxone binding established the presence of mu sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of [3H]-DADLE binding. The observed heterogeneity of opioid receptors in cultured SH-SY5Y cells would serve as an excellent model for the biochemical and pharmacological characterization of brain opiate receptors.     

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.     

Differential knockdown of delta-opioid receptor subtypes in the rat brain by antisense oligodeoxynucleotides targeting mRNA.

Two antisense oligodeoxynucleotides (A-ODN), targeting delta-opioid receptor mRNA (DOR) and two mismatch ODN sequences (mODN) were continuously infused for 24 days into the lateral brain ventricles of Wistar rats. The density of delta-opioid receptors in rat brain homogenates was measured by saturation binding experiments using four selective ligands, two agonists ([D-Ala2, Glu4]-deltorphin and DPDPE) and two antagonists (Dmt-Tic-OH and naltrindole), and by immunoblotting SDS solubilized receptor protein. In brain membranes of mODN or saline-infused rats, the rank order of delta-opioid receptor density, calculated by Bmax values of the four delta-opioid receptor ligands, was: [D-Ala2, Glu4]deltorphin approximately Dmt-Tic-OH approximately naltrindole (86-118 fmo/mg protein) > DPDPE (73.6+/-6.3 fmol/mg protein). At the end of the 24 day infusion of A-ODN targeting DOR nucleotide sequence 280299 (A-ODN280-299), the Bmax of DPDPE (62.4+/-3.2 fmol/mg protein) was significantly higher than that of Dmt-Tic-OH (31.5+/-3.9 fmol/mg protein). Moreover, both the Kd value for DPDPE saturation binding and the Ki value for Dmt-Tic-OH displacement by DPDPE were halved. In contrast, an A-ODN treatment targeting exon 3 (A-ODN741-760) decreased the specific binding of [D-Ala2, Glu4]deltorphin and Dmt-Tic-OH significantly less (67%-81%) than the binding of DPDPE (53%), without changes in DPDPE Ki and KD values. No A-ODN treatment modified the specific binding of the micro-opioid agonist DAMGO and of the k-selective opioid receptor ligand U69593. On the Western blot of solubilized striatum proteins, A-ODN(280-299) and A-ODN(741-760) downregulated the levels of the DOR protein, whereas the corresponding mODN were inactive. The 24-day infusion of A-ODN(280-299) inhibited the rat locomotor response to [D-Ala2, Glu4]deltorphin but not to DPDPE. Intracerebroventricular (i.c.v.) infusion of A-ODN(741-760) reduced the locomotor responses to both delta-opioid receptor agonists, whereas mODN infusion never affected agonist potencies. In conclusion, these results demonstrate that 24-day continuous i.c.v. infusion of A-ODN targeting the nucleotide sequence 280-299 of DOR can differentially knockdown delta1 and delta2 binding sites in the rat brain.     

A study of the effect of the irreversible delta receptor antagonist [D-Ala2,Leu5,Cys6]-enkephalin on delta cx and delta ncx opioid binding sites in vitro and in vivo.

Several lines of data support the existence of two classes of delta receptors: the delta cx binding site, which is the delta binding site of the mu-delta opioid receptor complex, and the delta ncx, which is the noncomplexed delta receptor. [D-Ala2,Leu5,Cys6]Enkephalin (DALCE) is an extended analog of [Leu5]enkephalin, which has been shown to bind irreversibly to delta receptors via the terminal cysteine by formation of a disulfide bond with the receptor. In vivo studies have shown that DALCE produces short-lived antinociceptive actions, followed by long-term antagonism of delta receptor-mediated antinociception. The major goal of the present study was to examine the effect of DALCE on the delta cx and delta ncx binding sites in vitro and in vivo. Intracerebroventricular administration of 40 micrograms DALCE failed to decrease [3H][D-Ala2,D-Leu5]enkephalin binding to the delta cx and delta ncx binding sites. Pretreatment of membranes with DALCE in vitro greatly reduced the Bmax of the delta ncx binding site, without significantly altering the Bmax of the delta cx binding site. These findings suggest that when administered in vivo, DALCE fails to distribute uniformly throughout the brain, and that it therefore binds covalently to opioid receptors mostly in the periventricular regions. Viewed collectively, these data support the hypothesis that DALCE acts as a selective delta ncx antagonist, and that the delta ncx binding site, which is sensitive to DALCE, is most likely synonymous with the recently described delta 1 receptor.     

Synthesis and receptor binding characteristics of [D-Ala2, cysteamine 5] enkephalin, a thiol-containing probe for structural elements of opiate receptors

For the elucidation of structural elements in the opiate receptors, a thiol-containing enkephalin analog [D-Ala2, cysteamine 5]enkephalin, and its dimeric analog were synthesized and evaluated in the radio-ligand receptor binding assays using rat brain membranes. The dimeric analog was very potent in both delta and mu assays. Comparison of receptor affinities of the thiol-containing enkephalin with those of standard mu or delta receptor specific ligands suggested that the mu receptor contains an essential thiol group which may interact with the thiol group at the C-terminus of the enkephalin analog. It also appears that no metal-ion site, postulated for the delta receptors, is present in the delta binding site.     

Interaction of [3H](–)-SKF-10,047 with Brain σ Receptors: Characterization and Autoradiographic Visualization

The sigma opiates differ from other opiates in their stimulatory and psychotomimetic actions. The sigma opiate [3H](-)-SKF-10,047 has been used to characterize sigma receptors in rat nervous tissue. Binding of [3H](-)-SKF-10,047 to rat brain membranes was of high affinity, saturable, and reversible. Scatchard analysis revealed the apparent interaction of this drug with two distinct binding sites characterized by affinities of 0.03 and 75 nM (5 mM Tris-HCl buffer, pH 7.4, at 4 degrees C). Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the high-affinity binding site is the mu opiate receptor. The lower-affinity site (revealed after suppression of mu and delta receptor binding) has been identified as the sigma opiate/phencyclidine receptor. In vitro autoradiography has been used to visualize neuroanatomical patterns of receptors labeled using [3H](-)-SKF-10,047 in the presence of normorphine and [D-Ala2,D-Leu5]enkephalin to block mu and delta interactions, respectively. Labeling patterns differ markedly from those for mu, delta, or kappa receptors. The highest densities (determined by quantitative autoradiography) are found in the medial portion of the nucleus accumbens, amygdaloid nucleus, hippocampal formation, central gray, locus coeruleus, and the parabrachial nuclei. Receptors in these structures could account for the stimulatory, mood-altering, and analgesic properties of the sigma opiates. Although not the most selective sigma opiate ligand, [3H](-)-SKF-10,047 binds to sigma opiate receptors in brain, and this interaction can be readily distinguished from its interactions with other classes of brain opiate receptors.     

Opiate receptor binding in the brain of rat during stress

Two types of opioid receptors were studied in the brain of rats: Delta (for endogenous opiate) and mu (for exogenous opiates). 3H derivates: D-Ala2-enkephalin and Naloxone were used as labeled ligands. The results obtained were calculated by computer program for automatic estimation of the data using approximation equations. An increase of binding delta receptors is observed in both types of stress (2-8 times), while to the mu receptors the binding is less effective mainly after irradiation. These data suggest that a close interaction exists between sympathoadrenal system and opioid mechanisms during stress.     

Endogenous ligands for sigma opioid receptors in the brain ("sigmaphin"): evidence from binding assays

Two endogenous ligands which interact preferentially with the sigma opioid receptors were identified from the guinea-pig brain extract in a Sephadex G-50 fractionation. These two ligands inhibited more potently the binding of [3H]SKF-10047 to sigma opioid receptors than [3H]naloxone to mu opioid receptors, [3H]ethylketocyclazocine to kappa opioid receptors and [3H]DADLE to delta opioid receptors. In the phencyclidine receptor assay, these two ligands were almost inactive. Incubation of these ligands with trypsin destroyed at least 50% of the activities in the sigma opioid receptor assay. Both ligands inhibited the sigma binding in a dose-dependent manner. The inhibition could be eliminated when the two ligands were removed from incubation media by extensive washings. It is therefore concluded that sigma opioid receptors are not phencyclidine receptors and that endogenous ligands for sigma opioid receptors may exist in the brain.     

An affinity label for delta-opioid receptors derived from [D-Ala2]deltorphin I.

A series of potential affinity label derivatives of the amphibian opioid peptide [D-Ala2]deltorphin I were prepared by incorporation at the para position of Phe3 (in the 'message' sequence) or Phe5 (in the 'address' sequence) of an electrophilic group (i.e. isothiocyanate or bromoacetamide). The introduction of the electrophile was accomplished by incorporating Fmoc-Phe(p-NHAlloc) into the peptide, followed later in the synthesis by selective deprotection of the Alloc group and modification of the resulting amine. While para substitution decreased the delta-opioid receptor affinity, selected analogs retained nanomolar affinity for delta receptors. [D-Ala2,Phe(p-NCS)3]deltorphin I exhibited moderate affinity (IC50=83 nM) and high selectivity for delta receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in delta-receptor affinity (80- and >1200-fold, respectively, compared with [D-Ala2]deltorphin I). In the 'address' sequence, the Phe(p-NH2)5 derivative showed the highest delta-receptor affinity (IC50=32 nM), while the Phe(p-NHCOCH2Br)5 and Phe(p-NCS)5 peptides displayed four- and tenfold lower delta-receptor affinities, respectively. [D-Ala2,Phe(p-NCS)3]deltorphin I exhibited wash-resistant inhibition of [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) binding to delta receptors at a concentration of 80 nM. [D-Ala2, Phe(p-NCS)3]deltorphin I represents the first affinity label derivative of one of the potent and selective amphibian opioid peptides, and the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the 'message' sequence of the peptide.     

Receptor autoradiography of mu and delta opioid peptide receptors in spontaneously hypertensive rats.

The receptor autoradiographic distribution of opioid peptide receptors in spontaneously hypertensive rats (SHR) was compared to that of Sprague-Dawley (SD) rats, using the highly selective mu and delta opioid receptor ligands, [3H]DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol) and [3H]DPDPE ([D-Pen2,D-Pen5]enkephalin), respectively. Although the distribution of these binding sites was similar in both strains, SHR showed significantly higher binding densities of mu receptors in 16 of 27 areas examined. These included the patch and matrix components of the caudate-putamen (CPu), olfactory tubercle, endopiriform nucleus, anterior cingulate cortex, ventral tegmental area lateroposteral thalamic nucleus and the ventral part of the dentate gyrus. In contrast, SHR had lower [3H]DAGO binding sites in the CA1 of the hippocampus. Conversely, SHR showed higher binding densities of delta receptors in 7 of 20 areas examined, including the CPu, CA2 and CA3 areas of the hippocampus and the central grey. High-to-low lateromedial gradients of striatal delta receptors were observed in both strains. Because opioid peptides are known to participate in locomotive behavior in rodents and in the control of blood pressure, the present results support a role of opioid peptidergic systems in the manifestation of hyperactivity and hypertension observed in SHR.     

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.