[35S]GTPγS binding stimulated by endomorphin-2 and morphiceptin analogs

doi:10.1016/j.bbrc.2006.04.079

Biochemical and Biophysical Research Communications

Volume 345, Issue 1, 23 June 2006, Pages 162-168

https://doi.org/10.1016/j.bbrc.2006.04.079 Get rights and content

Abstract

The ability of several μ-selective opioid peptides to activate G-proteins was measured in rat thalamus membrane preparations. The μ-selective ligands used in this study were three structurally related peptides, endomorphin-1, endomorphin-2 and morphiceptin, and their analogs modified in position 3 or 4 by introducing 3-(1-naphthyl)-d-alanine (d-1-Nal) or 3-(2-naphthyl)-d-alanine (d-2-Nal). The results obtained for these peptides in [³⁵S]GTPγS binding assay were compared with those obtained for a standard μ-opioid agonist DAMGO. [d-1-Nal³]Morphiceptin was more potent in G-protein activation (EC₅₀ value of 82.5 ± 4.5 nM) than DAMGO (EC₅₀ = 105 ± 9 nM). [d-2-Nal³]Morphiceptin, as well as endomorphin-2 analogs substituted in position 4 by either d-1-Nal or d-2-Nal failed to stimulate [³⁵S]GTPγS binding and were shown to be potent antagonists against DAMGO. It seems that the topographical location of the aromatic ring of position 3 and 4 amino acid residues can result in a completely different mode of action, producing either agonists or antagonists.

Section snippets

Materials and methods

Peptide synthesis. Peptides were synthesized by a standard solid-phase procedure as described before [11], using techniques for butyloxycarbonyl (Boc)-protected amino acids on p-methylbenzhydrylamine (MBHA) resin (100–200 mesh, 0.8 mM/g, Novabiochem, La Jolla, USA). Fifty percent trifluoroacetic acid (TFA) in dichloromethane was used for deprotection of Boc-groups and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) was employed to facilitate coupling. Simultaneous

Results

The competitive radioligand binding experiments and μ-opioid receptor activation of the G-proteins in the functional [³⁵S]GTPγS assays were used to compare the binding characteristics of several μ-opioid selective peptides and their analogs. The μ-selective ligands used in this study were Tyr-d-Ala-Gly-MePhe-Gly-ol (DAMGO), structurally related peptides: endomorphin-1, endomorphin-2, and morphiceptin, and the analogs of endomorphin-2 and morphiceptin modified in position 3 or 4 by introducing

Discussion

Since their discovery in 1997, endomorphins are probably the most extensively studied opioid peptides. Many analogs of endomorphins have been synthesized in an attempt to develop new peptide analgesics, as well as to determine their pharmacological properties. The presence of an aromatic amino acid in positions 3 and 4 is crucial for an effective binding to the μ-opioid receptor [18]. A close structural similarity of endomorphin-2 and another atypical opioid peptide, morphiceptin, which both

Acknowledgments

This work was supported by the grant from the French government (Bourse du Gouvernement français) (to J.F.), the grant from the Conseil Régional de Haute Normandie (to J.F.), and the grant from the Centre National de la Recherche Scientifique (CNRS, France). The authors thank Jozef Cieslak for his excellent technical assistance.

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Citation Excerpt :
Mu-opioid receptors are activated by Vitex agnus-castus methanol extracts [1202]. Binding of [[35]S] GTPgammaS is stimulated by endomorphin-2 and morphiceptin analogs [343]. An aequorin luminescence-based calcium assay gives pharmacologically relevant data for mu and delta-opioid agonists without involving radioactivity or animal tissues [344].
This paper is the 29th consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning 30 years of research. It summarizes papers published during 2006 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular–biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurological disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17).
The spinal antinociceptive effects of endomorphins in rats: Behavioral and G protein functional studies
2008, Anesthesia and Analgesia
Endomorphin-1 and endomorphin-2 are endogenous peptides that are highly selective for μ-opioid receptors. However, studies of their functional efficacy and selectivity are controversial. In this study, we systematically compared the effects of intrathecal (i.t.) administration of endomorphin-1 and -2 on nociception assays and G protein activation with those of [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO), a highly effective peptidic μ-opioid receptor agonist.
Male Sprague-Dawley rats were used. Acute and inflammatory pain models were used to compare the duration and magnitude of antinociception. Agonist-stimulated [³⁵S]GTPγS binding was used to observe the functional activity at the level of the receptor-G protein in both spinal cord and thalamic membranes. In addition, antagonists selective for each receptor type were used to verify the functional selectivity of endomorphins in the rat spinal cord.
After i.t. administration, endomorphin-1 and -2 produced less antinociceptive effects than DAMGO in the model of acute pain. Concentration–response curves for DAMGO-, endomorphin-1-, and endomorphin-2-stimulated [³⁵S]GTPγS binding revealed that both endomorphin-1 and -2 produced less G protein activation (i.e., approximately 50%–60%) than DAMGO did in the membranes of spinal cord and thalamus. In addition, i.t. endomorphin-induced antinociception was blocked by μ-opioid receptor selective dose of naltrexone (P < 0.05), but not by δ- and κ-opioid receptor antagonists, naltrindole and nor-binaltorphimine (P > 0.05).
Endomorphins are partial agonists for G protein activation at spinal and thalamic μ-opioid receptors. Both in vivo and in vitro measurements together suggest that DAMGO is more effective than endomorphins. Spinal endomorphins' antinociceptive efficacy may range between 53% and 84% depending on the intensity and modality of the nociceptive stimulus.
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^☆: Abbreviations: Boc, butyloxycarbonyl; BSA, bovine serum albumin; GDP, guanosine diphosphate; GPCRs, G protein-coupled receptors; GTP, guanosine triphosphate; GTPγS, guanosine-5′-O-(3-thio)triphosphate; [³⁵S]GTPγS, guanylyl 5′-[γ-[³⁵S]thio]-triphosphate; MBHA, p-methylbenzhydrylamine; TBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TFA, trifluoroacetic acid.

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[35S]GTPγS binding stimulated by endomorphin-2 and morphiceptin analogs☆

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgments

Life Sci.

J. Biol. Chem.

Bioorg. Med. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Pharmacol.

G proteins: transducers of receptor-generated signals

Annu. Rev. Biochem.

Go mediates the coupling of the μ opioid receptor to adenylyl cyclase in cloned neural cells and brain

Proc. Natl. Acad. Sci. USA

μ and δ receptors belong to a family of receptors that are coupled to potassium channels

Proc. Natl. Acad. Sci. USA

The GTP-binding protein, Go, regulates neuronal calcium channels

Nature

Endogenous opioids: biology and function

Annu. Rev. Neurosci.

Identification of two related pentapeptides from the brain with potent opiate agonist activity

Nature

[³⁵S]GTPγS binding stimulated by endomorphin-2 and morphiceptin analogs☆