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Endomorphins interact with the substance P (SP) aminoterminal SP (1-7) binding in the ventral tegmental area of the rat brain
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Biological Research on Drug Dependence.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Biological Research on Drug Dependence.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Biological Research on Drug Dependence.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
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2008 (English)In: Peptides, ISSN 0196-9781, Vol. 29, no 10, 1820-1824 p.Article in journal (Refereed) Published
Abstract [en]

We have recently identified a specific binding site for the tachykinin peptide substance P (SP) fragment SP1-7 in the rat spinal cord. This site appeared very specific for SP1-7 as the binding affinity of this compound highly exceeded those of other SP fragments. We also observed that endomorphin-2 (EM-2) exhibited high potency in displacing SP1-7 from this site. In the present work using a [H-3]-labeled derivative of the heptapeptide we have identified and characterized [H-3]-SP1-7 binding in the rat ventral tegmental area (VTA). Similarly to the [H-3]-SP1-7 binding in the spinal cord the affinity of unlabeled SP1-7 to the specific site in VTA was significantly higher than those of other SP fragments. Further, the tachykinin receptor NK-1, NK-2 and NK-3 ligands showed no or negligible binding to the identified site. However, the mu-opioid peptide (MOP) receptor agonists DAMGO, EM-1 and EM-2 did, and significant difference was observed in the binding affinity between the two endomorphins. As recorded from displacement curves the affinity of EM-2 for the SP1-7 site was 4-5 times weaker than that for SP1-7 but about 5 times higher than that of EM-1. The opioid receptor antagonists naloxone and naloxonazine showed weak or negligible binding. it was concluded that the specific site identified for SP1-7 binding in the rat VTA is distinct from the MOP receptor although it exhibits high affinity for EM-2.

Place, publisher, year, edition, pages
2008. Vol. 29, no 10, 1820-1824 p.
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97822DOI: 10.1016/j.peptides.2008.05.014ISI: 000260284100024OAI: oai:DiVA.org:uu-97822DiVA: diva2:172905
Available from: 2008-11-21 Created: 2008-11-21 Last updated: 2014-03-31Bibliographically approved
In thesis
1. Characterization of Substance P (SP) Aminoterminal SP (1-7) Binding in Brain Regions and Spinal Cord of the Male Rat: Studies on the Interaction with Opioid Related Pathways
Open this publication in new window or tab >>Characterization of Substance P (SP) Aminoterminal SP (1-7) Binding in Brain Regions and Spinal Cord of the Male Rat: Studies on the Interaction with Opioid Related Pathways
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Binding sites for substance P(1-7), SP(1-7) have been identified and characterized for the first time in crude membrane fraction from rat CNS using tritiated ([3H]) SP(1-7) as tracer. These putative receptors were investigated in relation to their affinity for tachykinins, opioid peptides and sigma receptor ligands. [3H]-SP(1-7) specifically binds to high affinity binding sites identified as receptor targets for the heptapeptide SP (1-7). Two distinct binding sites were observed in the spinal cord. One site is recognized by high affinity for SP(1-7) with a Kd of 0.5 nM, whereas the other site showed low affinity for the heptapeptide (Kd=12 nM). In the brain, the binding of SP(1-7) fitted a single site binding model with a Kd of 4.4 nM and a Ki of 4.2 nM. Further, using the spinal cord membranes the binding of [3H]-SP (1-7) was weakly displaced by SP and other N-terminal fragments thereof and no or negligible affinity was observed for ligands of the NK-1, NK-2 and NK-3 tachykinin receptors, C-terminal SP(5-11), Tyr-w-MIF-1 or the mu-opioid receptor antagonists naloxone and naloxonazine. On the other hand it was significantly displaced by endomorphin-2, DAMGO, and Try-MIF-1 and exhibit some affinity for MIF-1, ß-casomorphin and endomorphin-1. However, only endomorphin-2, DAMGO and Tyr-MIF-1 showed affinity in the close range of the native peptide SP(1-7). The affinity of endomorphin-2 for the spinal cord site was 10 times lower than that of SP(1-7) but more than 100 times higher than the affinity recorded for endomorphin-1. Tyr-MIF-1 but not Tyr-w-MIF-1 showed similar affinity as endomorphin-2 for SP(1-7) site. All peptides exhibiting high affinity at the SP(1-7) site, have a phenylalanine or a leucine residue in their C-terminal structure.

Further, synthetic analogues of SP(1-7) were tested for their affinity for the SP(1-7) receptor in the rat spinal cord. An important finding here was that the receptor-ligand-interaction was favoured by the C-terminal region of SP(1-7). Residues at positions 5-7 appeared crucial for binding to the specific SP(1-7) site. The presence of the amidated Phe7 residue was extremely critical for binding to the SP(1-7) site.The analogue Gln5-Gln6-Phe7-NH2 was almost equipotent with the parent peptide in the SP (1-7) receptor binding assay.

Furthermore, the SP(1-7)-amide potently and dose dependently reduced several signs of the reaction to morphine withdrawal and was significantly attenuated by the addition of the sigma agonist SK-10047.

In conclusion, the work presented in this thesis has contributed the characterization of the properties of highly selective binding sites for SP(1-7) in the rat spinal cord and VTA. These sites appear to be distinct from the µ-opioid receptor or any of the known neurokinin receptors. The study further indicates that the SP(1-7)-amide mimics the effect of the nativ heptapeptide and that the mechanisms for its action involve a sigma receptor site.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 81 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 85
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-9401 (URN)978-91-554-7349-5 (ISBN)
Public defence
2008-12-12, B41, BMC, Husargatan, Uppsala, 13:15
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Supervisors
Available from: 2008-11-21 Created: 2008-11-21Bibliographically approved

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