Synthesis of [D-Ala2,4'-125I-Phe3,Glu4]deltorphin and characterization of its delta opioid receptor binding properties.

Both [D-Ala2,Glu4]Deltorphin and [D-Ala2,4'-I-Phe3,Glu4]Deltorphin are highly selective ligands for delta, relative to mu, opioid receptors. Radiolabeled [D-Ala2, 4'-125I-Phe3,Glu4]Deltorphin ([125I]Deltorphin) was prepared with a specific activity of 2200 Ci/mmol from [D-Ala2, 4'-NH2-Phe3, Glu4]Deltorphin through a diazonium salt intermediate. The inhibition of [125I]Deltorphin binding to rat brain membranes by ligands selective for mu, delta, and kappa opioid receptors is consistent with binding by the radioligand to a single site having the properties of a delta opioid receptor. The results of these studies are in good agreement with those obtained by structurally different delta opioid receptor ligands. The similarity between the delta receptor site labeled by [125I]Deltorphin and those labeled by other delta receptor agonists, in contrast to differences seen by in vivo studies of their analgesic effects, is discussed.     

A high affinity, highly selective ligand for the delta opioid receptor: [3H]-[D-Pen2, pCl-Phe4, d-Pen5]enkephalin

Binding characteristics of a new, conformationally constrained, halogenated enkephalin analogue, [3H]-[D-penicillamine2, pCl-Phe4, D-penicillamine5]enkephalin ([3H]pCl-DPDPE), were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C determined a dissociation constant (Kd) of 328 +/- 27.pM and a receptor density (Bmax) of 87.2 +/- 4.2 fmol/mg protein. Kinetic studies demonstrated biphasic association for [3H]pCl-DPDPE, with association rate constants of 5.05 x 10(8) +/- 2.5 x 10(8) and 0.147 +/- 10(8) +/- 0.014 x 10(8) M-1 min-1. Dissociation was monophasic with a dissociation rate constant of 2.96 x 10(-3) +/- 0.25 x 10(-3) min-1. The average Kd values determined by these kinetic studies were 8.4 +/- 2.7 pM and 201 +/- 4 pM. Competitive inhibition studies demonstrated that [3H]pCl-DPDPE has excellent selectively for the delta opioid receptor. [3H]pCl-DPDPE binding was inhibited by low concentrations of ligands selective for delta opioid receptor relative to the concentrations required by ligands selective for mu and kappa sites. These data show that [3H]pCl-DPDPE is a highly selective, high affinity ligand which should be useful in characterizing the delta opioid receptor.     

A novel opioid structure which accepts protonated as well as non-protonated nitrogen: a family of pure, delta receptor selective antagonists.

Conventional opioids including opioid peptides require an "opioid" nitrogen which exists in protonated state while interacting with the receptor. In the present paper we demonstrate that the Tyr-Pro-Gly-Phe-Leu-Thr hexapeptide sequence accepts N-terminal substituents such as N-t-Boc, N-phenylacetyl and N-diphenylacetyl where the N cannot become protonated, as well as "traditional" substitutions such as N,N-diallyl, where protonation is likely under physiological conditions. The opioid peptides bearing these substituents are pure antagonists of medium affinity (Ke values in the mouse vas deferens bioassay against [Met5]-enkephalin are in the 3 x 10(-7)-4 x 10(-6) M range) with a high delta receptor preference (50-350-fold delta over mu selectivity ratios).     

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.     

N,N-dialkyl-4-[(8-azabicyclo[3.2.1]-oct-3-ylidene)phenylmethyl]benzamides, potent, selective delta opioid agonists

A series of N,N-dialkyl-4-(9-aryltropanylidenemethyl)benzamides was prepared. The lead compounds, 15a and 15c, exhibited extremely high affinity for the delta opioid receptor with excellent selectivity versus the micro opioid receptor. They were full agonists at the delta opioid receptor, as assessed by stimulation of GTPgammaS binding, and displayed antinociceptive activity.     

Monoclonal antiidiotypic antibodies against delta opioid receptors as an electron microscopy probe.

Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface 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.     

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.     

Region-dependent G-protein activation by mu-, delta 1- and delta 2-opioid receptor agonists in the brain: comparison between the midbrain and forebrain

The ability of selective mu- ([D-Ala2, NHPhe4, Gly-ol]enkephalin: DAMGO), delta1- ([D-Pen2, Pen5]enkephalin: DPDPE) and delta2- ([D-Ala2]deltorphin II: DELT II) opioid receptor agonists to activate G-proteins in the midbrain and forebrain of mice and rats was examined by monitoring the binding of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). The levels of [35S]GTPgammaS binding stimulated by DAMGO in the mouse and rat midbrain were significantly greater than those by DPDPE or DELT II. However, relatively lower levels of stimulation of [35S]GTPgammaS binding by all of the agonists than would have been predicted from the receptor densities were observed in either the limbic forebrain or striatum of mice and rats. The effects of DAMGO, DPDPE and DELT II in all three regions were completely reversed by selective mu-, delta1- and delta2-antagonists, respectively. The results indicate that the levels of mu-, delta1- and delta2-opioid receptor agonist-induced G-protein activation in the midbrain are in good agreement with the previously determined distribution densities of each receptor type. Furthermore, the discrepancies observed in the forebrain might reflect differential catalytic efficiencies of receptor-G-protein coupling.     

8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one derivatives as orphanin FQ receptor agonists.

A series of 8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan+ ++-4-one derivatives 1 was studied with respect to the binding affinity for the orphanin FQ (OFQ) and opioid (mu, kappa, delta) receptors. The influence of stereochemistry as well as the substitution pattern of the phenyl-ring in position 1 on the affinity for the orphanin FQ receptor and selectivity to opioid (mu, kappa, delta) receptors is discussed. The most interesting compound 1c was tested for its anxiolytic-like properties in vivo.     

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.     

Optically pure (-)-4-[(N-allyl-3-methyl-4-piperidinyl)phenyl-amino]-N,N-diethylbenzami de displays selective binding and full agonist activity for the delta opioid receptor

The optical isomers of 4-[(N-allyl-3-methyl-4-piperidinyl)phenylamino]-N,N-diethylbenzamide+ ++ (3) have been prepared and tested in both binding and functional assays. The data show that (-)-3 is responsible for the delta opioid activity demonstrated by the racemic material. This compound displays a binding affinity of 5.5 nM for the delta opioid receptor as well as a 470-fold delta versus mu selectivity. Importantly, (-)-3 is a full agonist at the delta receptor in comparison with SNC-80 (2). Taken together, the data suggest that (-)-3 behaves more like the prototypical delta agonists, BW373U86 or SNC-80, and less like the peptidomimetic compound SL-3111 (5).     

Synthesis and binding properties of specific photoaffinity ligands for mu and delta opioid receptor subtypes

We report the synthesis and binding properties of specific photoaffinity ligands for mu and delta opioid receptor subtypes. These ligands are derived from DAGO: Tyr-D-Ala-Gly-NMePhe-Gly-ol, a mu selective probe and DTLET: Tyr-D-Thr-Gly-Phe-Leu-Thr, a delta selective probe by modifying the Phe 4 residue. These modifications are: i) a nitro group on the para position of Phe ring as Phe(4 NO2) or Nip, ii) an azido group as Phe(4 N3) or AZ. Pharmacological responses on mouse vas deferens (delta sites) and guinea pig ileum (mu sites), as well as competition experiments with [3H] DAGO and [3H] DTLET on crude rat brain membranes have been performed. The nitro group on the phenyl ring of the Phe residue preserves the affinity and selectivity of each probe: NipDAGO for the mu sites, NipDTLET for the delta ones. However the nitro probes do not appear to be photoactivable by u.v. irradiation. Likewise, azidation of the phenyl ring of the Phe residue does not change the receptor selectivity of each probe, but AZDAGO has less affinity than its parent molecule DAGO, while AZDTLET has more affinity than DTLET. These compounds are photoactivable and provide an efficient tool to characterize and isolate the different receptor subtypes, especially the delta site.     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.     

Activation of central opioid receptors may induce gastric mucosal defence in the rat.

The effect of different opioid peptides on acidified ethanol- and indomethacin-induced gastric mucosal lesions was studied following intracerebroventricular (i.c.v.) administration. It was found that both the selective delta opioid receptor agonists--deltorphin II, [D-Ala(2), D-Leu(5)]-enkephalin (DADLE), [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE)-, mu-opioid receptor agonist--[D-Ala(2), Phe(4), GlyT-ol]-enkephalin (DAGO)--as well as beta-endorphin inhibited the mucosal damage induced by both ethanol and indomethacin in pmolar dose range. In contrast, the gastric acid secretion was not influenced by DADLE in the dose of 16 nmol/rat and only a slight reduction (40%) was induced by DAGO in the dose of 1.9 nmol/rat. The protective effect was abolished in both ulcer models by bilateral cervical vagotomy. N(G)-nitro-L-arginine, an inhibitor of NO synthase, reduced the protective action in ethanol-induced, but not in indomethacin-induced gastric damage. The results suggest that activation of supraspinal delta and mu-opioid receptors resulted in inhibition of gastric mucosal lesions elicited by ethanol or indomethacin. The gastroprotective action is independent from the effect of opioids on acid secretion. Vagal nerve is involved in conveying the central action to the periphery. The mechanism of the gastroprotective effect of opioids is different in ethanol- and indomethacin-ulcer models: prostaglandins and nitric oxide are likely to be involved in the protective action of opioid peptides in ethanol-, but not in the indomethacin-ulcer model.     

Single residue modifications of the delta opioid receptor selective peptide, [D-Pen2,D-Pen5]-enkephalin (DPDPE). Correlation of pharmacological effects with structural and conformational features

Six analogs of the highly delta opioid receptor selective, conformationally restricted, cyclic peptide [D-Pen2,D-Pen5]enkephalin, Tyr-D-Pen-Gly-Phe-D-PenOH (DPDPE), were synthesized and evaluated for opioid activity in rat brain receptor binding and mouse vas deferens (MVD) smooth muscle assays. All analogs were single amino acid modifications of DPDPE and employed amino acid substitutions of known effects in linear enkephalin analogs. The effect on binding affinity and MVD potency of each modification within the DPDPE structural framework was consistent with the previous reports on similarly substituted linear analogs. Conformational features of four of the modified DPDPE analogs were examined by 1H NMR spectroscopy and compared with DPDPE. From these studies it was concluded that the observed pharmacological differences with DPDPE displayed by diallyltyrosine1-DPDPE ([DAT1]DPDPE) and phenylglycine4-DPDPE ([Pgl4]DPDPE) are due to structural and/or conformational differences localized near the substituted amino acid. The observed enhanced mu receptor binding affinity of the carboxamide terminal DPDPE-NH2 appears to be founded solely upon electronic differences, the NMR data suggesting indistinguishable conformations. The observation that the alpha-aminoisobutyric acid substituted analog [Aib3]DPDPE displays similar in vitro opioid behavior as DPDPE while apparently assuming a significantly different solution conformation suggests that further detailed conformational analysis of this analog will aid the elucidation of the key structural and conformational features required for action at the delta opioid receptor.     

Comparison of the endogenous heptapeptide Met-enkephalin-Arg6-Phe7 binding in amphibian and mammalian brain

In previous communications [4, 38] we published that [3H]Met-enkephalin-Arg6-Phe7 (MERF) binds to opioid (kappa2 and delta) and sigma2 sites in frog and rat brain membrane preparations, however no binding to kappa1 sites could be established. In the present paper we compare the frog, rat and guinea pig brain membrane fractions with respect to their MERF binding data. No qualitative differences were found between the three species but specific binding of labelled MERF was maximal in frog brain and lowest in guinea pig brain, which corresponds to their kappa2 opioid receptor distribution. The naloxone resistant binding was also present in all investigated species and varied from 25% in frog and guinea pig cerebrum, to 50% in rat cerebrum and cerebellum, but no naloxone inhibition was found in guinea pig cerebellum where no kappa2 opioid receptors have been found. The presence of sigma2-like receptor was demonstrated in each investigated membrane fraction with displacement experiments using (-)N-allyl-normetazocine as competitor of tritiated MERF. It was shown that this site was responsible for 60-80% of [3H]MERF binding. The remaining part of the naloxone resistant labelled MERF binding could be displaced only with endogenous opioid peptides as met-enkephalin, dynorphin and beta-endorphin. The eventual physiological role of multiple MERF receptors is discussed.     

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.     

Synthesis and binding affinities of 4-diarylaminotropanes, a new class of delta opioid agonists

A series of 4-diarylaminotropanes has been prepared. Both endo and exo diastereomeric forms bound to the delta opioid receptor but the endo isomers were more potent and selective versus the mu opioid receptor than the exo isomers. The most potent delta opioid agonist (14) exhibited a delta opioid Ki of 0.2 nM and was 860-fold selective over mu.     

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.