β-Endorphin. Biological activity of synthetic analogs with analgesia inhibiting property in rat vas deferens and guinea pig ileum assays

Three synthetic analogs of human β-endorphin (β_h-EP) (I, [Gln⁸, Gly³¹]-β_h-EP-Gly-Gly-NH₂; II, [Arg^{9,12,24,28,29}]-β_h-EP and III, [Cys^11,26, Phe²⁷, Gly³¹]-β_h-EP), which have been shown to possess potent inhibiting activity to β_h-EP-induced analgesia, were assayed in rat vas deferens and guinea pig ileum bioassay systems. In the rat vas deferens assay, relative potencies of these analogs were β_h-EP, 100; I, 30; II, 40; III, 1, whereas in the guinea pig ileum assay: β_h-EP, 100; I, 184; II, 81; III, 163. From previous studies on their analgesia potency in mice and opiate receptor-binding activity in rat brain membranes, their activity in rat vas deferens correlates well with the analgesic potency and the activity from guinea pig ileum assay shows good correlations with that from the opiate receptor-binding assay.     

β-Endorphin

Three β_h-EP analogs which show different extents of alteration in analgesic potency by substitution of a single amino acid residue were assayed for their peripheral opioid activity and the binding to opioid μ-receptor to determine the relationships among the opioid activities obtained from different assays. In the guinea pig ileum assay, [Gln⁸]-β_h-EP showed a higher inhibitory activity than the parent peptide. [Tyr³¹]-analog had the same potency as β_h-EP, while [Trp²⁷]-analog retained only one fourth the potency of β_h-EP. Assayed on the vas deferens of the mouse and the rat, all three substituted β_h-EP analogs exhibited a lower potency than their parent peptide. Receptor binding assay using [³H]-dihydromorphine as the primary ligand showed that [Gln⁸]-analog had a binding potency 1.5-fold that of β_h-EP, while the potencies of [Tyr³¹]- and [Trp²⁷]-analogs were not significantly different from that of the parent peptide. No correlation in relative potency was found between vas deferens assays and their μ-receptor binding or analgesic activity. However, the relative potencies of binding to μ-receptor in [Gln⁸]- and [Tyr³¹]-analogs were found to be consistent with those of analgesic and guinea pig ileum assays, whereas the binding to β-EP receptor of all analogs appeared to be related to the charge properties of β-EP molecule.     

α1‐ and α2‐Adrenoceptor hyporesponsiveness in isolated bisected vas deferens of bile duct‐ligated rats

1 It has been suggested that cholestasis accompanied with changes in autonomic balance and hyporesponsiveness in muscarinic and adrenergic receptors of some organs, e.g. cardiovascular system. Increased plasma levels of epinephrine and norepinephrine has been shown during cholestasis suggesting augmented activity of sympathetic nervous system. In this study we evaluate both α₁ and α₂ responsiveness in isolated rat vas deferens, as a tissue with rich adrenergic innervations. 2 Epididymal and prostatic halves of vas deferens responsiveness have been studied to phenylephrine and clonidine respectively in three groups of un‐operated, sham‐operated (sham), and bile duct‐ligated (BDL) rats. 3 Our results indicate that in vas deferens of BDL animals, the concentration‐response curve of both phenylephrine and clonidine shifted to rightward compared to control group, while the position of concentration‐response curve of sham group did not change significantly (P > 0.05). EC₅₀ of phenylephrine and IC₅₀ of clonidine were increased showing a decreased responsiveness of tissue to phenylephrine (P < 0.05) and clonidine (P < 0.001) in BDL rats. 4 In this study, both subtype of α‐adrenoceptors (α₁ and α₂) has been studied in cholestatic rat vas deference. Our results showed that cholestasis induce hyporesponsiveness to phenylephrine and clonidine. These results are consistent with previous reports, suggesting the hyporesponsiveness of α₁‐adrenoceptors in pulmonary artery and papillary muscle and mesenteric beds. Our conclusion is that the cholestasis induces hyporesponsiveness to phenylephrine and clonidine in epididymal (α₁‐adrenoceptors) and prostatic (α₂‐adrenoceptors) halves of rat vas deferens respectively. Although the logical explanation to this hyporesponsiveness is the down regulation but it has been suggested that it is not because of down regulation.     

β-Endorphin: peripheral opioid activity of homologues from six species

The peripheral opioid activity of six homologous β-endorphins (β-EPs) were assayed on the guinea pig ileum and the vas deferens of the mouse, the rat and the rabbit. In the guinea pig ileum assay, human β-EP (βh-EP) was less potent than camel, turkey, and ostrich β-EPs, of the same potency as equine β-EP and more active than des-acetyl salmon β-EP. In the rat vas deferens, mammalian β-EPs showed higher activity than those from the bird and the fish, whereas in the mouse vas deferens assay, βh-EP is more active than those from other species. In the rabbit vas deferens, however, all homologous β-EPs show very weak activity. The relative potency of β-EP homologues obtained from rat vas deferens assay is in good correlation with the analgesic potency, while the receptor binding activity does not correlate with any of the four bioassays, but appears to be related to the charge properties of the peptides.     

Pharmacological profiles of β-funaltrexamine (β-FNA) and β-chlornaltrexamine (β-CNA) on the mouse vas deferens preparation

The profiles of action of β-funaltrexamine (β-FNA) and β-chlornaltrexamine (β-CNA) have been assessed in the mouse vas deferens preparation. β-FNA, but not β-CNA, demonstrated a reversible agonist action that appeared to be mediated via κ-receptor interaction. β-CNA produced an irreversible antagonism of μ-, κ- and δ-mediated agonist actions, whereas β-FNA irreversibly antagonized μ-mediated agonist effects only. This selective action of β-FNA could also be seen following administration in vivo. β-CNA and particularly β-FNA should prove valuable in the elucidation of multiple opioid receptors.     

β-Endorphin

A double-headed analog of human β-endorphin (β_h-EP), N, N'-bis (β-endorphinyl)-cystine (II), has been synthesized by the solid-phase method, along with β_h-EP-Cys(CH₂CONH₂)-OH (I) and (Tyr³¹]-β_h-EP (III). Their relative potencies in a radioreceptor-binding assay were: B_h-EP, 100; II, 235; I, 170; and III, 204. In the tail-flick test for analgesic activity their relative potencies were: β_hEP, 100; II, 86; I, 93; and III, 116.     

Prazosin has low potency at α1A‐adrenoceptors and high potency at α1D‐adrenoceptors in rat vas deferens

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β-Endorphin

Inhibition of binding of β_h-endorphin or Leu-enkephalin by β_h-endorphin analogs of various chain lengths in membrane preparations of the neuroblastoma x glioma NG108–15 cells has been investigated. The removal of even a single residue from the C-terminus results in the inability of the resulting peptide to completely displace β_h-endorphin. In addition, the proportion of nondisplaceable binding increases with decreasing chain length.     

β-Endorphin

Ostrich β-endorphin has been synthesized by the solid-phase method. Opiate activity in a radioreceptor binding assay is about seven times that of human β-endorphin. Structural differences within positions 6–15 account for the increased binding potency.     

Functional and radioligand binding characterization of the α1L‐adrenoceptor subtype of the human vas deferens

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Selective ligands for opioid receptors: β-Cyclopropylalanyl containing analogs of enkephalin

The synthesis and resolution of the amino acid β-cyclopropylalanine (Cpr) and its incorporation into four enkephalin analogs is reported. The analogs prepared were: Tyr - l - Cpr - Gly - Phe - Pen (des - COOH - Nle = n - pentylamide = Pen) (l -Cpr²-Pen⁵-ENK), Tyr-d -Cpr-Gly-Phe-Pen (d -Cpr²-Pen⁵-ENK), l -Cpr-Tyr-d -Ala-Gly-Phe-Pen (l -Cpr⁰-d -Ala²-Pen⁵-ENK) and d -Cpr-Tyr-d -Ala-Gly-Phe-Pen (d -Cpr⁰-d -Ala²-Pen⁵-ENK). Each was tested for its ability to inhibit the field stimulated guinea pig ileum (GPI) and rat vas deferens (RVD) and the results compared to the effect d -Ala²-d -Leu⁵-enkephalin (DADLE) has on the same preparations. The results show that at concentrations up to 10^-5 m all four analogs, as well as DADLE, are full agonists on the GPI preparation. The concentrations necessary to produce a 50% inhibition of the twitch response were, DADLE, 3.5 °× 10^-8 m ; l - Cpr⁰-d -Ala²-Pen⁵-ENK, 6.0 × 10^-8 m ; d -Cpr²-Pen⁵-ENK, 1.1 × 10^-7 m ; l -Cpr²-Pen⁵-ENK, 1.2 × 10^-6 m and d -Cpr⁰-d -Ala²-Pen⁵-ENK, > 10^-5 m . On RVD a different result was observed with only DADLE (1.3 × 10^-6 m ) and l -Cpr⁰-Pen⁵-enkephalin (1.8 × 10^-6 m ) showing full agonist activity. d -Cpr²-Pen⁵-ENK was a partial agonist (29 · 5% inhibition of the twitch at 10^-5 m ) while d -Cpr⁰-d -Ala²-Pen⁵-ENK and l -Cpr²-Pen⁵-ENK did not inhibit the twitch at concentrations up to 10^-5 m . These compounds which were inactive or of low potency on each preparation were also tested as antagonists. Only d -Cpr²-Pen⁵-ENK was an antagonist (pA₂ = 6.09) versus DADLE on RVD while d -Cpr⁰-d -Ala²-Pen⁵-ENK was inactive as an antagonist on both GPI and RVD. d -Cpr²-Pen⁵-ENK, therefore, represents the first enkephalin analog to be categorized as a mixed agonist-antagonist.     

A New β2-Adrenoceptor Agonist with α1-Adrenoceptor Blocking Properties

Abstract: The phenylethanolamine D2343 exhibits a dualistic adrenoceptormediated effect, i.e. a β-agonistic effect combined with an α-antagonistic one. Tracheal smooth muscles and heart preparations were used to gauge the agonistic effect on adrenergic β-receptors. Rabbit aorta and rat vas deferens were used to determine the α-adrenoceptor blocking activity. The β-adrenoceptor activity of D2343 was classified as β₂-type with about the same efficacy as the β₂-selective terbutaline on tracheal muscle. The effect on isolated heart preparations was greater than that produced by terbutaline. The α-receptor blocking capacity was directed against the α₁-receptor type and was of nearly the same potency as for phentolamine.     

Conformational investigation of α, β-dehydropeptides

The crystal structure of Ac-Pro-ΔVal-NHCH₃ was examined to determine the influence of the α,β-dehydrovaline residue on the nature of peptide conformation. The peptide crystallizes from methanol-diethyl ether solution at 4° in needle-shaped form in orthorhombic space group P2₁2₁2₁ with a= 11.384(2) Å, b = 13.277(2) Å, c = 9.942(1) Å. V = 1502.7(4) ų Z = 4, D_m= 1.17 g cm^?3 and D_c=1.18 g cm^?3 The structure was solved by direct methods using SHELXS-86 and refined to an R value of 0.057 for 1922 observed reflections. The peptide is found to adopt a β-bend between the type I and the type III conformation with φ₁=?68.3(4)°, ψ₁=? 20.1(4)°, φ₂=?73.5(4)°= and Ψ₂=?14.1(4)°=. An intramolecular hydrogen bond between the carbonyl oxygen of ith residue and the NH of (i+ 3)th residue stabilizes the β-bend. An additional intermolecular N.,.O hydrogen bond joins molecules into infinite chains. In the literature described crystal structures of peptides having a single α,β-dehydroamino acid residue in the (i+ 2) position and forming a β-bend reveal a type II conformation.     

β-Endorphin: analgesic and receptor binding activity of non-mammalian homologs

Analgesic potencies of turkey, ostrich and des-acetyl salmon β-endorphins have been measured in the tail-flick test and binding affinities determined by radioreceptor assay. The duration of analgesia and the slope of the dose-response curves generated by these peptides are similar to those elicited by mammalian β-endorphins. This suggests that they act in vivo and in vitro on the same population of opiate receptors. The ratio of binding to analgesic potencies observed for these peptides varies nearly sixfold. Structure-activity analysis suggests that a basic side-chain at position 9 is required in order to produce a high opiate activity both in vivo and in vitro. A reexamination of the biological activities of camel β-endorphin shows that the analgesic potency and binding affinity of this peptide are respectively 1.7 and 2.7 times higher than human β-endorphin. His-27 and/or Gln-31 may contribute to this increased potency. The dissociation of radioreceptor binding affinity from analgesic potency in these naturally occurring β-endorphin homologs suggests that either the conditions under which the binding assay is performed mask the true binding potency in the brain or that, once bound to the appropriate receptor, these homologs do not possess equal ability to produce biological effects.     

Isolation of subunits, α, β and γ of the complex taipoxin from the venom of Australian taipan snake (Oxyuranus s. scutellatus): characterization of β taipoxin as a potent mitogen

The venom of Australian taipan snake (Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of α, β and γ subunits. This report describes the high pressure liquid chromatography (HPLC) separation of α, β (β-1 and β-2) and γ subunits from taipan crude venom. The fractions containing the taipoxin subunits were further purified to obtain homogeneous proteins. The toxicity in mice showed the α subunit as most toxic, the γ subunit as moderately toxic and the β-1 and β-2 subunits were nontoxic. The proteins β-1 and β-2 were found to be mitogenic having neurotrophic activity on PC12 cells in culture similar to nerve growth factor. Immunologically, α, β-1, β-2 and γ subunits were found to be different, showing cross reactivity, and β-1 and β-2 were found to be identical for biological properties and molecular weight. Further characterization of unexpected mitogenic activity of β subunits is underway.     

Conformational investigation of αβ-dehydropeptides

Solution conformations of three series of model peptides, homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHcH₃ (Xaa = Val, Phe, Leu, Abu. Ah) as well as αβ-unsaturated Ac-Pro-ΔXaa-NHCH₃ [Δ Xaa =ΔVal, (Z)-ΔPhe, (Z)-ΔLeu, (Z)-ΔAbu] were investigated in CDCl₃ and CH₂Cl₂ by ¹H-, ¹³C-NMR, and FTIR spectroscopy. NH stretching absorption spectra, solvent shifts Δδ for NH (Xaa) and NHCH₃ on going from CDCl₃ to (CD₃)₂SO, diagnostic interresidue proton NOEs, and trans-cis isomer ratios were examined. These studies performed showed the essential difference in conformational propensities between homochiral peptides (L-Xaa) on the one hand and heterochiral (D-Xaa) and αβ-dehydropeptides (ΔXaa) on the other. Former compounds are conformationally flexible with an inverse γ-bend, a β-turn, and open forms in an equilibrium depending on the nature of the Xaa side chain. Conformational preferences of heterochiral and αβ-dehydropeptides are very similar, with the type-II β-turn as the dominating structure. There is no apparent correlation between conformational properties and the nature of the Xaa side chain within the two groups. The β-turn formation propensity seems to be somewhat greater in αβ-unsaturated than in heterochiral peptides, but an estimation of β-folded conformers is risky.     

Association of β-agonists with corresponding β2- and β1-adrenergic pentapeptide sequences

Synthesized β₁- and β₂-pentapeptide sequences corresponding to published adrenoceptor transmembrane activation site subtypes were investigated in vitro for selectivity in association for drug ligands of known selectivity. Both nuclear magnetic resonance spectroscopy and molecular mechanics demonstrated that structural differences among the corresponding pentapeptide activation-site sequences can explain agonist selectivity. Results suggest the agonists bind across the activation site loop on the second transmembrane α-helix by dipole/dipole interactions between a ligand and the peptide. Since electrostatic interactions within the membrane may determine the rate of intercellular ion flux, agonist association across the activation site sequence could thereby decrease electrostatic resistance to positive ion flux into the cell. Interactions between the peptides and the ligands may provide insight into the structures and mechanisms involved in association of ligands for the identical sequences on the β-adrenoreceptors.     

Structure-activity studies of analogs of β, γ-methylene-ATP at P2X-purinoceptors in the rabbit ear central artery

Structure-activity studies using naphthylmethyl analogs of β, γ-methylene-ATP were conducted at the P_2X-purinoceptor that mediates contraction of the rabbit ear central artery by ATP, α, β-m-ATP. On the adenine base, substitution at the C²-position (WRC-0440) increased the agonist potency 2-fold and substitution at the C⁸-position (WRC-0431) did not change agonist potency, and both compounds had the same maximal response as β, γ-m-ATP, whereas substitution at the N⁶-position (WRC-0416) abolished activity. On the D-ribose sugar, substitution on the 2′-hydroxyl generated a partial agonist (WRC-0479), which had a maximal effect of only 39% of that of β-γ-m-ATP. Attempts to substitute the 3′-hydroxyls by naphthylmethyl failed, but substitution by p-methoxybenzyl (WRC-0617) did not change potency or the maximal response. Cyclic substitution of both the 2′- and 3′-hydroxyls by naphthylmethylidine (WRC-0498) had no effect on the agonist potency or the maximal response relative to β-γ-m-ATP. On the β, γ-methylenetriphosphonate chain, substitution on the methylene linkage by naphthylmethyl (WRC-0433) had no effect on agonist potency, but the maximal response increased to 122% that of β-γ-ATP. However, the contractile response to WRC-0433 was not desensitized by α, β-γ-ATP (contractile responses to all other agonists were abolished by α-β-γ-ATP pretreatment), but was blocked by the α₁ antagonist prazosin (10^?6 M). WRC-0433 appears to act at a prejunctional site that mediates ATP-induced release of norepinephrine. Purine nucleotides with substituents at the 2′-position of the ribose sugar could provide a lead to the generation of P_2X-purinoceptor antagonists. © 1995 Wiley-Liss, Inc.     

Differences in postsynaptic α-Adrenoceptor Populations Between Isolated Cat Urethra and Various Other Isolated Tissues.

Abstract This study was undertaken with the aim of determining whether the postsynaptic α-adrenergic receptor population of the cat urethra differed from that of other isolated tissues (rabbit aorta and rat vas deferens) and if so the possibility of selectively affect these receptors. For this purpose the substances 2-methylammonio-1-(spiro[cyclopentane-1, 1′-indene]-3′-yl)ethanol(KABI2023), noradrenaline (NA) and dopamine (DA) were used. KABI 2023 and NA acted as full agonists on all three tissues investigated. DA was a full agonist on the vas deferens but was almost inactive on urethra. The contractile response of urethra to KABI 2023 was of an α-adrenergic nature, as it could be blocked with phentolamine. Compared with NA, KABI 2023 showed a 10 times higher selectivity for the receptors of urethra than for those of aorta. Affinity constants (log KB) for phentolamine and haloperidol with use of the various agonists were estimated. The affinity of phentolamine was found to be significantly different when using NA and KABI 2023 as agonists on the urethra but not on the aorta. Corresponding findings were obtained with haloperidol. On the vas deferens a greater difference in log KB values than that on the urethra was found. On the basis of the results, it is suggested that the population of postsynaptic α-adrenoceptors in the urethra (cat) differs from that in the aorta (rabbit). On the vas deferens a heterogenicity of postsynaptic receptors seem to exist which makes the interpretation of the results more difficult on this organ. In the presence of phentolamine and haloperidol the maximum responses to NA were potentiated on the aorta and vas deferens, but not on the urethra. Investigations to evaluate this difference are now in progress.     

Conformational investigation of α,β-dehydropeptides

Conformations of three series of model peptides: homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHCH₃ (Xaa=Phe, Val, Leu. Abu. Ala) as ivell as α,β-dehydro Ac-Pro-ΔXaa-NHCH_s [ΔXaa = (Z)-ΔPhe, ΔVal. (Z)-ΔLeu, (Z)-ΔAbu] were investigated by CD spectroscopy in 2 % dichloromethanecyclohexane, trifluoroethanol. water. and occasionally in other solvents. The spectra of homochiral peptides show a significant solvent dependence. Folded structures are present in 2% dichloromethane-cyclohexane and unordered ones occur in water. The folded conformers are of the inverse γ-turn type for all the peptides but Ac-Pro-L-Phe-NHCH₃ for which the type-I β-turn is preferred. The changes in the spectra of the heterochiral peptides are limited. The compounds adopt the typc-II β–turn in 2% dichloromethanecyclohexane, represented by class B spectra, and retain this conformation in water as well as in fluorinated alcohols but not always to a full extent. The CD spectra of the unsaturated peptides in 2%, dichloromethanecyclohexane, although they cannot be assigned to any common spectral class, must be attributed to the βII-turn conformation as determined for these coinpounds by NMR and IR spectroscopy. The CD spectra of dehydropeptides exhibit a considerable solvent dependence and suggest unordered structures in water.