In vitro and in vivo pharmacological characterization of the neuropeptide s receptor antagonist [D-Cys(tBu)5]neuropeptide S.
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 microM 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 (pK(B), 6.62). In Schild analysis experiments [D-Cys(tBu)(5)]NPS (0.1-100 microM) produced a concentration-dependent and parallel rightward shift of the concentration-response curve to NPS, showing a pA(2) 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.
Camarda
Valeria
V
Department of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, Ferrara, Italy.
Rizzi
Anna
A
Ruzza
Chiara
C
Zucchini
Silvia
S
Marzola
Giuliano
G
Marzola
Erika
E
Guerrini
Remo
R
Salvadori
Severo
S
Reinscheid
Rainer K
RK
Regoli
Domenico
D
Calò
Girolamo
G
D000818:Animals; D002460:Cell Line; D006801:Humans; D008297:Male; D051379:Mice; D009479:Neuropeptides / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors*
C515062:NPSR1 protein, mouse; D009479:Neuropeptides; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; C535232:neuropeptide S, 5-Cys(tBu)-; C506531:neuropeptide S, human