Received for publication July 23, 2008.
Revised October 28, 2008.
Accepted for publication October 28, 2008.

In vitro and in vivo pharmacological characterization of the neuropeptide S receptor antagonist [D-Cys(tBu)⁵]NPS

Abstract

Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as 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 HEK293 cells stably expressing the mouse NPSR (HEK293_mNPSR), however, the peptide inhibited in a concentration dependent manner 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 µM) produced a concentration dependent and parallel rightward shift of the concentration response curve to NPS showing a pA₂ value of 6.44. 10 µM [D-Cys(tBu)⁵]NPS did not affect signalling at seven NPSR unrelated G-protein coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmole intracerebroventricularly reduced the percent of animals losing the RR in response to diazepam 15 mg/kg and their sleeping time. [D-Cys(tBu)⁵]NPS (1-10 nmoles) 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 nmole 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 due to the selective activation of the NPSR protein.

Key words: NPSR, NPSR knock out mice, Neuropeptide S, [D-Cys(tBu)5]NPS, calcium mobilization, righting reflex recovery