Neuroprotective Potential of Biphalin,Multireceptor Opioid Peptide,Against Excitotoxic Injury in Hippocampal Organotypic Culture

Biphalin is a dimeric opioid peptide that exhibits affinity for three types of opioid receptors (MOP, DOP and KOP). Biphalin is undergoing intensive preclinical study. It was recognized that activation of δ-opioid receptor elicits neuroprotection against brain hypoxia and ischemia. We compare the effect of biphalin and morphine and the inhibition of opioid receptors by naltrexone on survival of neurons in rat organotypic hippocampal cultures challenged with NMDA. Findings: (1) 0.025–0.1 μM biphalin reduces NMDA-induced neuronal damage; (2) biphalin neuroprotection is abolished by naltrexone; (3) reduced number of dead cells is shown even if biphalin is applied with delay after NMDA challenge.     

Structure-activity relationships of arodyn, a novel acetylated kappa opioid receptor antagonist.

We previously reported that the novel dynorphin A (Dyn A, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln) analog arodyn (Ac[Phe(1,2,3),Arg(4),d-Ala(8)]Dyn A-(1-11)NH(2), Bennett, M.A., Murray, T.F. & Aldrich, J.V. (2002) J. Med. Chem. vol. 45, pp. 5617-5619) is a kappa opioid receptor-selective peptide [K(i)(kappa) = 10 nm, K(i) ratio (kappa/mu/delta) = 1/174/583] which exhibits antagonist activity at kappa opioid receptors. In this study, a series of arodyn analogs was prepared and evaluated to explore the structure-activity relationships (SAR) of this peptide; this included an alanine scan of the entire arodyn sequence, sequential isomeric d-amino acid substitution in the N-terminal 'message' sequence, NMePhe substitution individually in positions 1-3, and modifications in position 1. The results for the Ala-substituted derivatives indicated that Arg(6) and Arg(7) are the most important residues for arodyn's nanomolar binding affinity for kappa opioid receptors. Ala substitution of the other basic residues (Arg(4), Arg(9) and Lys(11)) resulted in lower decreases in affinity for kappa opioid receptors (three- to fivefold compared with arodyn). Of particular interest, while [Ala(10)]arodyn exhibits similar kappa opioid receptor binding as arodyn, it displays higher kappa vs. mu opioid receptor selectivity [K(i) ratio (kappa/mu) = 1/350] than arodyn because of a twofold loss in affinity at mu opioid receptors. Surprisingly, the Tyr(1) analog exhibits a sevenfold decrease in kappa opioid receptor affinity, indicating that arodyn displays significantly different SAR than Dyn A; [Tyr(1)]arodyn also unexpectedly exhibits inverse agonist activity in the adenylyl cyclase assay using Chinese hamster ovary cells stably expressing kappa opioid receptors. Substitution of NMePhe in position 1 gave [NMePhe(1)]arodyn which exhibits high affinity [K(i)(kappa) = 4.56 nm] and exceptional selectivity for kappa opioid receptors [K(i) ratio (kappa/mu/delta) = 1/1100/>2170]. This peptide exhibits antagonistic activity in the adenylyl cyclase assay, reversing the agonism of 10 nm Dyn A-(1-13)NH(2). Thus [NMePhe(1)]arodyn is a highly kappa opioid receptor-selective antagonist that could be a useful pharmacological tool to study kappa opioid receptor-mediated activities.     

Synthesis and biological evaluation of new biphalin analogues with non-hydrazine linkers

Biphalin is a potent opioid peptide agonist, with a palandromic structure, composed of two enkephalin-like active fragments connected tail to tail by a hydrazine linker (Tyr-D-Ala-Gly-Phe-NH-NH<-Phe<-Gly<-D-Ala<-Tyr). This study presents the synthesis and in vitro bioassays of six new biphalin analogues with three different non-hydrazine linkers, some of which have higher binding affinity and bioactivity than biphalin.     

Use of hydrophilic diamines for bridging of two opioid peptide pharmacophores. Synthesis and receptor binding of two new analogues.

The bivalent ligand approach, which assumes that two pharmacophores are connected by a spacer, was used to design receptor type-selective ligands for opioid receptors. The first two opioid peptide bivalent ligands with different spacer lengths containing different numbers of hydroxyl groups, (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-)2 (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-CHOH-)2, were synthesized and their binding to mu, delta, and kappa opioid receptors was characterized. Both analogues were found to possess high opioid in vitro activities. The length of the hydrophilic spacer does not affect the affinity for delta receptors, whereas shorter spacer length increases affinity for mu and even more so for kappa receptors. Thus receptor type-selective peptides for opioid receptors can be designed using the bivalent approach.     

(4-Carboxamido)phenylalanine is a surrogate for tyrosine in opioid receptor peptide ligands

(S)-4-(Carboxamido)phenylalanine (Cpa) is examined as a bioisosteric replacement for the terminal tyrosine (Tyr) residue in a variety of known peptide ligands for the mu, delta and kappa opioid receptors. The Cpa-containing peptides, assayed against cloned human opioid receptors, display comparable binding affinity (Ki), and agonist potency (EC50) to the parent ligands at the three receptors. Cpa analogs of delta selective peptides show an increase in delta selectivity relative to the mu receptor. Cpa is the first example of an amino acid that acts as a surrogate for Tyr in opioid peptide ligands, challenging the long-standing belief that a phenolic residue is required for high affinity binding.     

Dynorphin A analogs containing a conformationally constrained phenylalanine derivative in position 4: reversal of preferred stereochemistry for opioid receptor affinity and discrimination of kappa vs. delta receptors

Analogs of the opioid peptide [D-Ala8]dynorphin A-(1-11)NH2 containing optically pure (R)- and (S)-2-aminotetralin-2-carboxylic acid (Atc) in position 4 were synthesized and evaluated for opioid receptor affinity. These peptides are the first reported dynorphin A analogs containing a conformationally constrained amino acid in place of the important aromatic residue Phe4. By incorporating resolved Atc isomers, the opioid receptor affinity and the stereochemistry of the constrained residue could be unambiguously correlated. Both Dyn A analogs containing Atc in position 4 retained nanomolar affinity for kappa and mu opioid receptors. Unexpectedly the peptide containing (R)-Atc, corresponding to a conformationally constrained D-Phe analog, displaying higher affinity for both kappa and mu receptors than the peptide containing (S)-Atc. In contrast [D-Phe4,D-Ala8]Dyn A-(1-11)NH2 exhibited significantly lower affinity for kappa and mu receptors than the parent peptide, as expected. Conformational restriction of the Phe4 sidechain or incorporation of D-Phe in position 4 had the largest effect on delta receptor affinity, yielding compounds with negligible affinity for these receptors. Thus, there appear to be distinctly different structural requirements for this residue for kappa vs. delta receptors, and it is possible to completely distinguish between these two receptors by changing a single residue in Dyn A.     

Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin

We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions.     

Reciprocal opioid-opioid interactions between the ventral tegmental area and nucleus accumbens regions in mediating mu agonist-induced feeding in rats

Feeding elicited by the mu-selective agonist, [D-Ala2, M-Phe4, Gly-ol5]-encephalin administered into the nucleus accumbens is blocked by accumbal pre-treatment with mu, delta1, delta2 and kappa, but not mu1 opioid antagonists. Correspondingly, mu-agonist-induced feeding elicited from the ventral tegmental area is blocked by ventral tegmental area pre-treatment with mu and kappa, but not delta opioid antagonists. A bi-directional opioid-opioid feeding interaction has been firmly established such that mu-agonist-induced feeding elicited from the ventral tegmental area is blocked by accumbal naltrexone, and that accumbal mu-agonist-induced feeding is blocked by naltrexone pre-treatment in the ventral tegmental area. To determine which opioid receptor subtypes mediate the regional bi-directional opioid-opioid feeding interactions between these two sites, the present study examined the dose-dependent ability of either general (naltrexone), mu (beta-funaltrexamine), kappa (nor-binaltorphamine) or delta (naltrindole) opioid antagonists administered into one site to block mu-agonist-induced feeding elicited from the other site. General, mu and kappa, but not delta opioid receptor antagonist pre-treatment in the ventral tegmental area dose-dependently reduced mu-agonist-induced feeding elicited from the nucleus accumbens. General, mu and delta, and to a lesser degree kappa, opioid receptor antagonist pre-treatment in the nucleus accumbens dose-dependently reduced mu-agonist-induced feeding elicited from the ventral tegmental area. Thus, multiple, but different opioid receptor subtypes are involved in mediating opioid-opioid feeding interactions between the nucleus accumbens and ventral tegmental area regions.     

Characterization of Opioid Receptor Subtypes in Solution

Stable opioid receptor binding activity that retains distinct subtype specificities (mu, delta, and kappa) has been obtained in high yields in digitonin extracts of rat brain membranes that had been preincubated with Mg2+ prior to solubilization. The dependence on Mg2+ ions for receptor activity is also expressed in the soluble state, where the presence of Mg2+ leads to high-affinity and high-capacity opioid peptide binding to the delta, mu, and kappa sites (the latter subtype measured by the binding of [3H]dynorphin1-8). Binding of opiate alkaloids to soluble receptor sites is less dependent on Mg2+ than is opioid peptide binding. Soluble opioid binding activity shows the same sensitivity to Na+ ions and guanine nucleotides as the membrane-bound receptor. The ligand-receptor interactions give evidence of strong positive cooperativity, which is interpreted in terms of association-dissociation of receptor subunits on ligand binding in solution. Binding of enkephalin peptides is associated with the large macromolecules present (apparent Stokes radii greater than 60 A), whereas both those and several small species present (less than 60 A) bind opiate alkaloids and dynorphin1-8.     

Selective activation of opioid receptors differentially affects lordosis behavior in female rats

The effects of opioid peptides that are highly selective ligands for mu receptors (morphiceptin). delta receptors (delta-receptor peptide), kappa receptors (dynorphin 1-9), and the mu/delta complex (beta-endorphin), were tested on lordosis behavior in ovariectomized rats primed with estrogen and progesterone. Intracerebroventricular infusions of beta-endorphin or morphiceptin both inhibited and facilitated lordosis in a dose-dependent fashion whereas all doses of delta-receptor peptide facilitated lordosis. Dynorphin 1-9 had no significant effect at any dose, although a trend toward increased lordosis quotients was observed 30 min after infusion. The effects of beta-endorphin, morphiceptin, and delta-receptor peptide were reversed with naloxone, although naloxone alone had no effect on lordosis behavior. These results indicate that the specific activation of opioid receptor subtypes differentially affects lordosis behavior. It appears that binding to high-affinity mu 1 receptors exerts an inhibitory influence on lordosis, whereas binding to low-affinity mu 2 receptors or delta receptors exerts a facilitatory influence. Binding to kappa receptors does not appear to affect lordosis behavior.     

Bivalent opioid peptide analogues with reduced distances between pharmacophores

To investigate the role of distance between two opioid peptide pharmacophores on in vitro and in vivo activities, three new bivalent opioid analogues have been synthesized in which the dipeptide Tyr-D-Phe was connected with diamine moieties ("bridges"). The analogue with a hydrazine bridge has high receptor affinity to mu, kappa, and delta receptor types, as well as potent and long acting antinociceptive activity after intraperitoneal administration.     

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.     

Brain and Spinal Cord Distribution of Biphalin: Correlation with Opioid Receptor Density and Mechanism of CNS Entry

Abstract: Biphalin [(Tyr-d -Ala-Gly-Phe-NH)₂] is a bivalent, opioid peptide containing two pharmacophores linked by a hydrazine bridge. When administered intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception. Biphalin has also been shown to cross both the blood-brain and blood-cerebrospinal fluid barriers. To understand the basis of biphalin's potency, regional brain and spinal cord distribution studies with [¹²⁵I-Tyr¹]biphalin were performed 5, 20, and 40 min after intravenous bolus injections. A statistically greater amount of [¹²⁵I-Tyr¹]-biphalin was detected in the nucleus accumbens compared with other brain regions (p < 0.05). This correlates with the high density of δ- and μ-opioid receptor mRNA and binding sites shown to be expressed in the nucleus accumbens. Also, a statistically greater amount of [¹²⁵I-Tyr¹]biphalin was detected in two other circumventricular organs, the choroid plexus and pituitary, when compared with other brain regions. These studies provide evidence that biphalin can reach not only brain sites, but also spinal sites to elicit antinociception. The overall CNS distribution of [¹²⁵I-Tyr¹]biphalin was decreased with naloxone, d -Phe-Cys-Tyr-d -Trp-Arg-Thr-Pen-Thr-NH₂, or naltrindole pretreatment, showing that biphalin detected in the brain and spinal cord is binding to δ- and μ-opioid receptors. Additional in situ brain perfusion experiments identified a saturable component contributing to CNS entry of [¹²⁵I-Tyr¹]biphalin, which could be described by Michaelis-Menten kinetics with a K_m of 2.6 ± 4.8 µM, V_max of 14.6 ± 2.89 pmol^?1·min^?1·g^?1, and K_d of 0.568 ± 0.157 µl·min^?1·g^?1. Brain entry of [¹²⁵I-Tyr¹]biphalin was sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and l -phenylalanine, suggesting use of the large neutral amino acid carrier. This work provides evidence that biphalin is a promising, potent analgesic that has a unique mechanism for reaching both spinal and supraspinal opioid receptor sites.     

Opioid receptor selectivity of peptide models of beta-endorphin

Two peptides, designed to contain structural models of the proposed hydrophilic linker domain (residues 6-12) and amphiphilic alpha-helical domain (residues 13-29) in beta-endorphin, have been tested for their abilities to mimic the opioid receptor selectivity profile of the natural hormone. In competitive binding assays employing guinea-pig brain membranes, both peptides displayed a much higher affinity for mu- and delta-opioid receptors than for kappa opioid receptors. Relative to beta-endorphin, the peptide models were 2-3 times more potent in the mu and kappa receptor binding assays, and about equipotent in the delta receptor binding assay. In guinea-pig ileum assays, one peptide was equipotent to beta-endorphin and the other was twice as potent. Like beta-endorphin, their actions on this tissue were highly sensitive to naloxone antagonism, indicating that they were mediated by mu receptors and not kappa receptors. In view of the design of the two peptide models, and their minimal homology to the natural hormone, these results provide additional evidence in support to our proposal for the functional conformation of beta-endorphin.     

RTI-4614-4: an analog of (+)-cis-3-methylfentanyl with a 27,000-fold binding selectivity for mu versus delta opioid binding sites.

The objective of this study was to determine the binding affinities of (+/-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]- N-phenylpropanamide-HCI (RTI-4614-4), which is an analog of (+)-cis-3-methylfentanyl for opioid receptor subtypes. The Ki values (nM) of this agent for opioid receptor subtypes were as follows: mu (0.0055), delta (148), kappa 1 (84.8), kappa 2a (2275), and kappa 2b (22.3). The selectivity of this agent for the mu binding site was 27,000 vs. the delta binding site, 15,400 vs. the kappa 1 binding site, 413,700 vs the kappa 2a and 4,054 vs the kappa 2b binding site. In contrast, two other fentanyl analogs, N-(2-(4-methylpyridinyl))-N-(1-phenethyl-4-piperidinyl) 2-furamide and N-(2-pyrazinyl)-N-(1-phenethyl-4-piperdinyl)2-furamide had considerably higher Ki values at, and were less selective for, the mu binding site. Since RTI-4614-4 is composed of a mixture of four stereoisomers, the resolution of these isomers should permit identification of an extremely potent and selective agent for the opioid mu receptor.     

Prejunctional effects of opioids in the perfused mesentery of the rat and rabbit: interactions with alpha 2-adrenoceptors.

Prejunctional effects of opioids were examined in the perfused mesentery of two species: the rat and rabbit. Use of agonists selective for subtypes of mu, delta, and kappa opioid receptors produced no effect on contractile responses to adrenergic nerve stimulation in the rat perfused mesentery, except for small effects of the kappa agonist EKC, which may be non specific. In contrast, mu, delta and kappa receptors appear to be present in the rabbit. The mu selective agonist, DAMGO, kappa agonist, ethylketocyclazocine, and delta agonists, DPDPE and [Leu5]-enkephalin, all produced significant inhibition of contractile responses to transmural nerve stimulation. The inhibitory effect was greatest for ethylketocyclazocine. To test the possibility that prejunctional activation of alpha 2 adrenoceptors with endogenous norepinephrine might decrease the activity of prejunctional opioid receptors in the rabbit, inhibitory effects of delta and kappa selective agonists were tested in the presence of 10(-7) M yohimbine. Inhibitory responses of the kappa selective agonist ethylketocyclazocine were enhanced, while that of delta selective agonists [Leu5]-enkephalin and DPDPE remained unchanged when yohimbine was present. Thus, the effects of opioids vary and depend on the tissue and receptor subtypes they act upon. Furthermore, the enhanced inhibitory effect of opioid receptor activation in the presence of yohimbine is not found for all opioid receptors.     

NPY-induced feeding: pharmacological characterization using selective opioid antagonists and antisense probes in rats

The ability of neuropeptide Y to potently stimulate food intake is dependent in part upon the functioning of mu and kappa opioid receptors. The combined use of selective opioid antagonists directed against mu, delta or kappa receptors and antisense probes directed against specific exons of the MOR-1, DOR-1, KOR-1 and KOR-3/ORL-1 opioid receptor genes has been successful in characterizing the precise receptor subpopulations mediating feeding elicited by opioid peptides and agonists as well as homeostatic challenges. The present study examined the dose-dependent (5-80 nmol) cerebroventricular actions of general and selective mu, delta, and kappa1 opioid receptor antagonists together with antisense probes directed against each of the four exons of the MOR-1 opioid receptor gene and each of the three exons of the DOR-1, KOR-1, and KOR-3/ORL-1 opioid receptor genes upon feeding elicited by cerebroventricular NPY (0.47 nmol, 2 ug). NPY-induced feeding was dose-dependently decreased and sometimes eliminated following pretreatment with general, mu, delta, and kappa1 opioid receptor antagonists. Moreover, NPY-induced feeding was significantly and markedly reduced by antisense probes directed against exons 1, 2, and 3 of the MOR-1 gene, exons 1 and 2 of the DOR-1 gene, exons 1, 2, and 3 of the KOR-1 gene, and exon 3 of the KOR-3/ORL-1 gene. Thus, whereas the opioid peptides, beta-endorphin and dynorphin A(1-17) elicit feeding responses that are respectively more dependent upon mu and kappa opioid receptors and their genes, the opioid mediation of NPY-induced feeding appears to involve all three major opioid receptor subtypes in a manner similar to that observed for feeding responses following glucoprivation or lipoprivation.     

Synthesis and biological activity of the first cyclic biphalin analogues

Biphalin is a linear octapeptide with strong opioid activity. Its structure is based on two identical sequences derived from enkephalins joined C-terminal to C-terminal by an hydrazide bridge (Tyr-D-Ala-Gly-Phe-NH-NH<--Phe<--Gly<--D-Ala<--Tyr). In this study we present the design, synthesis, and biological evaluation of the first cyclic biphalin analogues. d-Alanine residues in positions 2, 2' of the parent peptide were replaced by d- and l-cysteine and an intramolecular disulfide bond between the cysteine thiol groups was introduced. We obtained two cyclic analogues with quite different biological profiles.     

Conformation-activity relationships of opioid peptides with selective activities at opioid receptors

The discovery of endogenous opioid peptides 25 years ago opened up a new chapter in efforts to understand the origins and control of pain, its relationships to other biological functions, including inflammatory and other immune responses, and the relationships of opioid peptides and their receptors to a variety of undesirable or toxic side effects often associated with the nonpeptide opiates such as morphine including addiction, constipation, a variety of neural toxicities, tolerance, and respiratory depression. For these investigations the need for potent and highly receptor selective agonists and antagonists has been crucial since they in principle allow one to distinguish unequivocally the roles of the different opioid receptors (mu, delta, and kappa) in the various biological and pathological roles of the opioid peptides and their receptors. Conformational and topographical constraint of the linear natural endogenous opioid peptides has played a major role in developing peptide ligands with high selectivity for mu, delta, and kappa receptors, and in understanding the conformational, topographical, and stereoelectronic structural requirements of the opioid peptides for their interactions with opioid receptors. In turn, this had led to insights into the three-dimensional pharmacophore for opioid receptors. In this article we review and discuss some of the developments that have led to potent, selective, and stable peptide and peptidomimetic ligands that are highly potent and selective, and that have delta agonist, mu antagonist, and kappa agonist biological activities (other authors in this issue will discuss the development of other types of activities and selectivities). These have led to ligands that provide unique insight into opioid pharmacophores and the critical roles opioid ligands and receptor scan play in pain, addiction, and other human maladies.     

Exploration of universal cysteines in the binding sites of three opioid receptor subtypes by disulfide-bonding affinity labeling with chemically activated thiol-containing dynorphin A analogs.

A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of mu and delta opioid receptors with SNpys-containing enkephalins [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys(Npys)-containing analogs of dynorphin A, namely, [D-Ala2, Cys(Npys)8]dynorphin A-(1-9) amide (1) and [D-Ala2, Cys(Npys)12]dynorphin A-(1-13) amide (2). When rat (mu and delta) or guinea pig (kappa) brain membranes were incubated with these Cys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (mu), deltorphin II (delta), and U-69593 (kappa), the number of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label mu receptors (EC50 = 27-33 nM), but also label delta receptors fairly well (160-180 nM). However, for kappa receptors they showed drastically different potencies as to affinity labeling; i.e., EC50 = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled kappa receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in mu, delta, and kappa receptors, whereas analog 2 only labels the Cys residues conserved in mu and delta receptors.