Abstract
Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as the NPS receptor (NPSR). In the frame of a structure-activity study performed on NPS Gly5, the NPSR ligand [d-Cys(tBu)5]NPS was identified. [d-Cys(tBu)5]NPS up to 100 μM did not stimulate calcium mobilization in human embryonic kidney (HEK) 293 cells stably expressing the mouse NPSR; however, in a concentration-dependent manner, the peptide inhibited the stimulatory effects elicited by 10 and 100 nM NPS (pKB, 6.62). In Schild analysis experiments [d-Cys(tBu)5]NPS (0.1–100 μM) produced a concentration-dependent and parallel rightward shift of the concentration-response curve to NPS, showing a pA2 value of 6.44. Ten micromolar [d-Cys(tBu)5]NPS did not affect signaling at seven NPSR unrelated G-protein-coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmol i.c.v. reduced the percentage of animals losing the RR in response to 15 mg/kg diazepam and their sleeping time. [d-Cys(tBu)5]NPS (1–10 nmol) was inactive per se but dose-dependently antagonized the arousal-promoting action of NPS. Finally, NPSR-deficient mice were similarly sensitive to the hypnotic effects of diazepam as their wild-type littermates. However, the arousal-promoting action of 1 nmol NPS could be detected in wild-type but not in mutant mice. In conclusion, [d-Cys(tBu)5]NPS behaves both in vitro and in vivo as a pure and selective NPSR antagonist but with moderate potency. Moreover, using this tool together with receptor knockout mice studies, we demonstrated that the arousal-promoting action of NPS is because of the selective activation of the NPSR protein.
Footnotes
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This work was supported by the University of Ferrara [FAR Grant]; the Italian Ministry of University [Grant PRIN 2006]; and the National Institutes of Health National Institute of Mental Health [Grant MH71313].
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V.C. and A.R. contributed equally to this work.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.143867.
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ABBREVIATIONS: NPS, neuropeptide S; NPSR, NPS receptor; SHA 66, 3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid benzylamide; SHA 68, 3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide; HEK, human embryonic kidney; HEK293mNPSR, HEK293 cells expressing the mouse NPSR; RR, righting reflex recovery; NPSR(-/-), NPSR knockout mice; PCR, polymerase chain reaction; CHO, Chinese hamster ovary.
- Received July 23, 2008.
- Accepted October 28, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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