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NEUROPHARMACOLOGY

In Vitro and in Vivo Pharmacological Characterization of the Neuropeptide S Receptor Antagonist [D-Cys(tBu)⁵]Neuropeptide S

Department of Experimental and Clinical Medicine, Section of Pharmacology, Neuroscience Center, National Institute of Neuroscience (V.C., A.R., C.R., S.Z., G.M., D.R., G.C.) and Department of Pharmaceutical Sciences and Biotechnology Center (E.M., R.G., S.S.), University of Ferrara, Ferrara, Italy; and Department of Pharmaceutical Sciences, University of California, Irvine, California (R.K.R.)

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 (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. 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.

Address correspondence to: Dr. Girolamo Calò, Department of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, via Fossato di Mortara, 19, 44100 Ferrara, Italy. E-mail: g.calo{at}unife.it