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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
Uppsala universitet, Medicinska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap, Avdelningen för biologisk beroendeforskning.
2008 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis , 2008. , s. 81
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 85
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-9401ISBN: 978-91-554-7349-5 (tryckt)OAI: oai:DiVA.org:uu-9401DiVA, id: diva2:172908
Disputas
2008-12-12, B41, BMC, Husargatan, Uppsala, 13:15
Opponent
Veileder
Tilgjengelig fra: 2008-11-21 Laget: 2008-11-21 Sist oppdatert: 2018-01-13bibliografisk kontrollert
Delarbeid
1. Endomorphin-1 and endomorphin-2 differently interact with specific binding sites for substance P (SP) aminoterminal SP (1-7) in the rat spinal cord
Åpne denne publikasjonen i ny fane eller vindu >>Endomorphin-1 and endomorphin-2 differently interact with specific binding sites for substance P (SP) aminoterminal SP (1-7) in the rat spinal cord
Vise andre…
2006 Inngår i: Peptides, Vol. 27, nr 4, s. 753-759Artikkel i tidsskrift (Fagfellevurdert) Published
Identifikatorer
urn:nbn:se:uu:diva-97821 (URN)
Tilgjengelig fra: 2008-11-21 Laget: 2008-11-21bibliografisk kontrollert
2. Endomorphins interact with the substance P (SP) aminoterminal SP (1-7) binding in the ventral tegmental area of the rat brain
Åpne denne publikasjonen i ny fane eller vindu >>Endomorphins interact with the substance P (SP) aminoterminal SP (1-7) binding in the ventral tegmental area of the rat brain
Vise andre…
2008 (engelsk)Inngår i: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 29, nr 10, s. 1820-1824Artikkel i tidsskrift (Fagfellevurdert) 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.

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Identifikatorer
urn:nbn:se:uu:diva-97822 (URN)10.1016/j.peptides.2008.05.014 (DOI)000260284100024 ()
Tilgjengelig fra: 2008-11-21 Laget: 2008-11-21 Sist oppdatert: 2018-01-13bibliografisk kontrollert
3. Small peptides mimicking substance P (1-7) and encompassing a C-terminal amide functionality
Åpne denne publikasjonen i ny fane eller vindu >>Small peptides mimicking substance P (1-7) and encompassing a C-terminal amide functionality
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2008 (engelsk)Inngår i: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 42, nr 1, s. 31-37Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Some of the biological effects demonstrated after administration of substance P (SP) in vivo can indirectly be attributed to the fragmentation of the undecapeptide to its N-terminal bioactive fragment SP1–7. This heptapeptide (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) is a major bioactive metabolite from SP that frequently exerts similar biological effects as the parent peptide but also, in several cases, completely opposite actions. Specific binding sites for the heptapeptide SP1–7 that are separate from the SP preferred NK receptors have been identified. In this study we demonstrate that (a) the C-terminal part of the SP metabolite SP1–7 is most important for binding as deduced from an Ala scan and that a replacement of Phe7 for Ala is deleterious, (b) truncation of the N-terminal amino acid residues of SP1–7 delivers peptides with retained binding activity, although with somewhat lower binding affinities than SP1–7 and (c) a C-terminal amide group as a replacement for the terminal carboxy group of SP1–7 and for all of the truncated ligands synthesized affords approximately 5–10-fold improvements of the binding affinities.

Emneord
Substance P (1–7), SP (1–7), SP1–7, Substance P, Structure–activity relationships (SAR), Ala scan, Peptidomimetics
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-97823 (URN)10.1016/j.npep.2007.11.002 (DOI)000254231100003 ()18093649 (PubMedID)
Tilgjengelig fra: 2008-11-21 Laget: 2008-11-21 Sist oppdatert: 2018-01-13bibliografisk kontrollert
4. The C-terminal amidated analogue of the Substance P (SP) fragment SP (1-7) attenuates the expression of naloxone- precipitated withdrawal in morphine dependent rats
Åpne denne publikasjonen i ny fane eller vindu >>The C-terminal amidated analogue of the Substance P (SP) fragment SP (1-7) attenuates the expression of naloxone- precipitated withdrawal in morphine dependent rats
Vise andre…
2009 (engelsk)Inngår i: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 30, nr 12, s. 2418-2422Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

We previously demonstrated that intracerebroventricular (i.c.v.) administration of the substance P (SP) aminoterminal fragment SP(1-7) attenuates the expression of morphine withdrawal in the male rat. In this study we have used a synthetic analogue of this peptide, i.e. the SP(1-7) amide showing higher binding potency than the native heptapeptide, in a similar experimental set-up. Thus, Wistar male rats were made tolerant to morphine by daily injections of the opiate during 8 days. Following peptide administration (i.c.v.) and a subsequent naloxone challenge a variety of physical syndromes of withdrawal were recorded. We observed that the SP(1-7) amide potently and dose-dependently reduced several signs of reaction to morphine withdrawal. Interestingly, the effect of the peptide amide was significantly attenuated by the addition of the sigma agonist (+)-SKF-10047. We conclude that the SP(1-7) amide mimics the effect of the native SP fragment and that the mechanisms for its action involve a sigma receptor site.

Emneord
Morphine, Opiate, Substance P (SP), SP1-7 amide, Rat, Withdrawal, Sigma receptor
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-97824 (URN)10.1016/j.peptides.2009.08.009 (DOI)000272903900044 ()19686790 (PubMedID)
Tilgjengelig fra: 2008-11-21 Laget: 2008-11-21 Sist oppdatert: 2018-01-13bibliografisk kontrollert

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