Synthesis, conformation and biological activity of dermorphin and deltorphin I analogues containing N-alkylglycine in place of residues in position 1, 3, 5 and 6.

Syntheses are described of new dermorphin and [D-Ala2]deltorphin I analogues in which the phenylalanine, the tyrosine or the valine residues have been substituted by the corresponding N-alkylglycine residues. Structural investigations by CD measurements in different solvents and preliminary pharmacological experiments were carried out on the resulting peptide-peptoid hybrids. The contribution from aromatic side chain residues is prominent in the CD spectra of dermorphin analogues and the assignment of a prevailing secondary structure could be questionable. In the CD spectra of deltorphin analogues the aromatic contribution is lower and the dichroic curves indicate the predominance of random conformer populations. The disappearance of the aromatic contribution in the [Ntyr1,D-Ala2]-deltorphin spectrum could be explained in terms of high conformational freedom of the N-terminal residue. The kinetics of degradation of the synthetic peptoids digestion by rat and human plasma enzymes were compared with that of [Leu5]-enkephalin. The binding to opioid receptors was tested on crude membrane preparations from CHO cells stably transfected with the mu- and delta-opioid receptors. The biological potency of peptoids was compared with that of dermorphin in GPI preparations and with that of deltorphin I in MVD preparations. All the substitutions produced a dramatic decrease in the affinity of the peptide-peptoid hybrids for both the mu- and delta-opioid receptors. Nval5 and/or Nval6 containing hybrids behaved as mu-opioid receptor agonists and elicit a dose-dependent analgesia (tail-flick test) when injected i.c.v. in rats.     

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.     

Topographical requirements for delta opioid ligands: presence of a carboxyl group in position 4 is not critical for deltorphin high delta receptor affinity and analgesic activity.

To investigate the role of the carboxyl group in deltorphin molecules, we have synthesized three new analogues in which the acidic amino acid residues in position 4 of the deltorphins were replaced by non-acidic but hydrophilic amino acids residues. The three analogues, [Ser4]-, [Gln4]-, and [Cys4]-deltorphin, all are as potent or more potent than either deltorphin I or II at delta opioid receptors and possess good delta selectivities. The excellent correlation between their in vitro delta receptor potencies and their intrathecal antinociception activity forms a strong argument for involvement of those receptors in spinal nociceptive modulation in the rats.     

Post-translational amino acid racemization in the frog skin peptide deltorphin I in the secretion granules of cutaneous serous glands

The dermal glands of the South American hylid frog Phyllomedusa bicolor synthesize and expel huge amounts of cationic, alpha-helical, 24- to 33-residue antimicrobial peptides, the dermaseptins B. These glands also produce a wide array of peptides that are similar to mammalian hormones and neuropeptides, including a heptapeptide opioid containing a D-amino acid, deltorphin I (Tyr-DAla-Phe-Asp-Val-Val-Gly NH2). Its biological activity is due to the racemization of L-Ala2 to D-Ala. The dermaseptins B and deltorphins are all derived from a single family of precursor polypeptides that have an N-terminal preprosequence that is remarkably well conserved, although the progenitor sequences giving rise to mature opioid or antimicrobial peptides are markedly different. Monoclonal and polyclonal antibodies were used to examine the cellular and ultrastructural distributions of deltorphin I and dermaseptin B in the serous glands by immunofluoresence confocal microscopy and immunogold-electron microscopy. Preprodeltorphin I and preprodermaseptins B are sorted into the regulated pathway of secretion, where they are processed to give the mature products. Deltorphin I, [l-Ala2]-deltorphin I and dermaseptin B are all stored together in secretion granules which accumulate in the cytoplasm of all serous glands. We conclude that the L- to D-amino acid isomerization of the deltorphin I occurs in the secretory granules as a post-translational event. Thus the specificity of isomerization depends on the presence of structural and/or conformational determinants in the peptide N-terminus surrounding the isomerization site.     

Antinociceptive effects of [D-Ala2]deltorphin II, a highly selective delta agonist in vivo

The present study has characterized the antinociceptive actions of [D-Ala2]deltorphin II following intracerebroventricular (i.c.v.) administration in the mouse tail-flick test. [D-Ala2]deltorphin II produced dose- and time-related antinociception, with maximal effects at +10 min and significant antinociception which lasted for 40-60 min. [D-Ala2]deltorphin II was 13-fold more potent than i.c.v. [D-Pen2, D-Pen5]enkephalin (DPDPE), a second highly selective delta agonist, and approximately equipotent with i.c.v. morphine in producing antinociception. The antinociceptive effects of i.c.v. [D-Ala2]deltorphin II and DPDPE, but not those of morphine, were antagonized by the selective delta antagonist, ICI 174,864. In contrast, pretreatment with the non-equilibrium mu antagonist, beta-funaltrexamine blocked morphine antinociception, but failed to antagonize [D-Ala2]deltorphin II and DPDPE antinociception. These data indicate that [D-Ala2]deltorphin II produced its antinociceptive effects at a supraspinal delta receptor. [D-Ala2]deltorphin II appears to be the most appropriate delta opioid agonist currently available for studies in vivo and support the involvement of delta receptors in supraspinal antinociception.     

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.     

Transformation of the amyloidogenic peptide amylin(20-29) into its corresponding peptoid and retropeptoid: access to both an amyloid inhibitor and template for self-assembled supramolecular tapes

The highly amyloidogenic peptide sequence of amylin(20-29) was transformed into its corresponding peptoid and retropeptoid sequences to design a novel class of beta-sheet breaker peptides as amyloid inhibitors. This report describes the synthesis of the chiral peptoid building block of L-isoleucine, the solid phase synthesis of the peptoid and retropeptoid sequences of amylin(20-29), and the structural analysis of these amylin derivatives in solution by infrared spectroscopy, circular dichroism, and transmission electron microscopy. It was found that the peptoid sequence did not form amyloid fibrils or any other secondary structures and was able to inhibit amyloid formation of native amylin(20-29). Although the retropeptoid did not form amyloid fibrils it had only modest amyloid inhibitor properties since supramolecular tapes were formed.     

D-Ala2]deltorphin II analogs with high affinity and selectivity for delta-opioid receptor.

Nine [D-Ala2]deltorphin II (DL-II:Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2) analogs having various aliphatic amino acids at positions 5 and 6 were synthesized to gain more information about the role of hydrophobic Val5,6 residues for the delta-opioid receptor selectivity. Binding assays of analogs replaced by Ala demonstrated the importance of hydrophobic Val5,6 residues in DL-II for delta-affinity and selectivity, and especially critical importance of Val5 residue for higher delta-selectivity. By enhancing the hydrophobicity of residues at positions 5 and 6, we have developed analogs with very high delta-affinity and selectivity over those of DL-II, e.g., [Ile5,6], [norleucine5,6] and [gamma-methyl-leucine5,6]DL-II, which will be useful as delta-selective ligands for investigation of the physiological role of opioid receptors.     

Radioiodinated [D-Ala2, Met5] enkephalin. Preparation, purification by high performance liquid chromatography and binding characteristics

125I[D-Ala2, Met5] enkephalin with high specific activity (122-185 Ci/mmol) was prepared and purified by Sep-Pak C18 reverse phase cartridge followed by high performance liquid chromatography (HPLC). HPLC at pH 3.0 resolved 125I[D-Ala2, Met5] enkephalin into two fractions, which ran as a single spot in thin-layer chromatography with the same Rf values. Alkaline hydrolysates of the HPLC-purified fractions showed a single spot corresponding to monoiodotyrosine standard when analysed by thin-layer chromatography. Binding kinetics of the tracer was found to approach equilibrium after 30 min at 24 degrees. Scatchard analysis of the saturation equilibrium binding studies gave an equilibrium dissociation constant of 3.58 nM and the number of binding site of 30 fmol/mg protein. Enkephalin analogs were capable of displacing 125I[D-Ala2, Met5] enkephalin binding from the rat brain plasma membrane. The effective concentration of [D-Ala2, Met5] enkephalin and [D-Ala2, Leu5] enkephalin for 50% inhibition of 125I[D-Ala2, Met5] enkephalin binding was estimated to be 79 nM and 23 nM, respectively. Both substance P and gastrin tetrapeptide failed to displace the 125I[D-Ala2, Met5] enkephalin binding to any significant extent. The 125I[D-Ala2, Met5] enkephalin prepared by the present procedure is therefore a useful tracer. This method of preparing radioiodinated peptide may be applicable to other enkephalin analogs or neuropeptides in general.     

Influence of peptidase inhibitors on the apparent agonist potency of delta selective opioid peptides in vitro.

Several peptides of diverse structure, reported to possess high affinity and selectivity for the delta opioid receptor, were studied using the mouse isolated vas deferens preparation to determine the effect of peptidase inhibition on their apparent potency. The peptides evaluated included [Leu5] enkephalin, the cyclic enkephalin analogs [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Pen2,p-F-Phe4,D-Pen5]enkephalin (F-DPDPE), the linear enkephalin analogs [D-Ala2,D-Leu5]enkephalin (DADLE) and [D-Ser2(O-tBu), Leu5,Thr6]enkephalin (DSTBULET), and the naturally occurring amphibian peptides Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (dermenkephalin), Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (deltorphin I) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (deltorphin II). Concentration-response curves were determined for each peptide in the absence and presence of a combination of the peptidase-inhibiting agents bacitracin, bestatin, and captopril. A wide range of potencies was observed, both in the control state and in the presence of peptidase inhibition. The synthetic enkephalin analogs demonstrated small increases in potency with peptidase inhibition (no increase in the case of DPDPE), whereas the naturally occurring peptides were markedly increased in potency, up to as much as 123-fold for dermenkephalin. In the presence of peptidase inhibition, deltorphin II was the most potent peptide tested (IC50 = 1.13 x 10(-10) molar), and as such is the most potent delta opioid agonist reported to date. Stability to metabolism must be considered in the design and evaluation of in vitro experiments using peptides of this type.     

Ligand binding profiles of delta-opioid receptor in human cerebral cortex membranes: evidence of delta-opioid receptor heterogeneity

In this study, receptor binding profiles of opioid ligands for subtypes of opioid delta-receptors were examined employing [3H]D-Pen2,D-Pen5-enkephalin ([3H]DPDPE) and [3H]Ile(5,6)-deltorphin II ([3H]Ile-Delt II) in human cerebral cortex membranes. [3H]DPDPE, a representative ligand for delta1 sites, labeled a single population of binding sites with apparent affinity constant (Kd) of 2.72 +/- 0.21 nM and maximal binding capacity (Bmax) value of 20.78 +/- 3.13 fmol/mg protein. Homologous competition curve of [3H]Ile-Delt II, a representative ligand for delta2 sites, was best fit by the one-site model (Kd = 0.82 +/- 0.07 nM). Bmax value (43.65 +/- 2.41 fmol/mg) for [3H]Ile-Delt II was significantly greater than that for [3H]DPDPE. DPDPE, [D-Ala2,D-Leu5]enkephalin (DADLE) and 7-benzylidenaltrexone (BNTX) were more potent in competing for the binding sites of [3H]DPDPE than for those of [3H]Ile-Delt II. On the other hand, deltorphin II (Delt II), [D-Ser2,Leu5,Thr6]enkephalin (DSLET), naltriben (NTB) and naltrindole (NTI) were found to be equipotent in competing for [3H]DPDPE and [3H]Ile-Delt II binding sites. These results indicate that both subtypes of opioid delta-receptors, delta1 and delta2, exist in human cerebral cortex with different ligand binding profiles.     

Synthesis, biology, NMR and conformation studies of the topographically constrained delta-opioid selective peptide analogs of [beta-iPrPhe(3)]deltorphin I.

Replacement of Phe3 in the endogenous delta-opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid beta-isopropylphenylalanine (beta-iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand-binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [beta-iPrPhe3]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe3 residue in [beta-iPrPhe3]deltorphin I provided the most desirable biological properties with binding affinity (IC50 = 2 nM), bioassay potency (IC50 = 1.23 nM in MVD assay) and exceptional selectivity for the delta-opioid receptor over the mu-opioid receptor (30 000). Further conformational studies based on two-dimensional NMR and computer-assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr1 and (2S,3R)-beta-iPrPhe3 residues adopt trans side-chain conformations, and the linear peptide backbone favors a distorted beta-turn conformation.     

Spinal antinociceptive effects of [D-Ala2]deltorphin II, a novel and highly selective delta-opioid receptor agonist.

Pharmacological assays in isolated tissues and binding tests have recently shown that two peptides, with the sequence Tyr-D-Ala-Phe-Asp-(or Glu)- Val-Val-Gly-NH2, isolated from skin extracts of Phyllomedusa bicolor and named [D-Ala2]deltorphin I and II, respectively, possess a higher affinity and selectivity for delta-opioid receptors than any other known natural compound. Since much evidence supports the role of spinal delta-opioid sites in producing antinociceptive effects, we investigated whether analgesia might be detected by direct spinal cord administration of [D-Ala2]deltorphin II (DADELT II) in the rat. The thermal antinociceptive effects of intrathecal DADELT II and dermorphin, a potent mu-selective agonist, were compared at different postinjection times by means of the tail-flick test. The DADELT II produced a dose-related inhibition of the tail-flick response, which lasted 10-60 min depending on the dose and appeared to be of shorter duration than the analgesia produced in rats after intrathecal injection of dermorphin (20-120 min). The analgesic effect of infused or injected DADELT II was completely abolished by naltrindole, the highly selective delta antagonist. These results confirm the involvement of delta receptors in spinal analgesic activity in the rat.     

The study of degradation of peptides in human plasma and serum by the ERIAD high pressure liquid chromatography-mass spectrometry method

The proteolysis of bradykinin, [Leu5]enkephalin and some of its synthetic analogs. [D-Ala2,Leu5]enkephalin and [D-Ala2,Leu5]enkephalinyl-Arg, by human blood plasma and serum enzymes was investigated. The degradation products were identified. Based on the kinetic data, the principal pathways of degradation and its limiting steps were established.     

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.     

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.     

Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.     

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.     

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.     

Circular-dichroic spectra of vasopressin analogues and their cyclic fragments.

The circular dichroic spectra of [Arg8]vasopressin, [Mpr1, Arg8]vasopressin, [Mpr1, D-Arg8]-vasopressin, pressinamide, deaminopressinamide, tocinamide, deaminotocinamide, [Leu4, D-Arg8]-vasotocin, [Mpr1, Leu4, D-Arg8]vasotocin and [Phe2, Lys8]vasopressin have been studied. All these substances showed a characteristic positive dichroic band at about 225 nm due to the presence of tyrosine in sequence position 2. The intensity of this band was affected by interactions between the tyrosine side-chain and other structural elements in the molecule, such as the Na-amino group, the side-chain of phenylalanine in position 3 and the linear C-terminal peptide. Analysis of the response of this band to structural modifications of the molecule and change in the solvent (particularly comparing neutral aqueous solutions with hexafluoroacetone solutions) allowed some conformational conclusions. The linear C-terminal tripeptide is probably situated over the cyclic portion of the molecule both in vasopressin and oxytocin substances. Its steric interaction with the tyrosine side-chain seems to be particularly efficient in molecules containing D-arginine in position 8. In the vasopressin series the stacking interaction of neighbouring aromatic amino acid residues furthermore limits the conformational freedom of the tyrosine side-chain and also probably distorts the dihedral angles of residues 1-3 in comparison with oxytocin. The interactions of phenylalanine and arginine with tyrosine relatively decrease the conformational effects of the primary amino group. Consequently the local conformation of vasopressin in the region of the tyrosine residue is more rigid and less sensitive to changes in medium than that of oxytocin. The circular dichroic spectra did not show any basic conformational differences in the backbone peptide chain of oxytocin and vasopressin substances. A weak negative disulphide band at about 290 nm could be observed in the spectra of both series of substances.