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

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 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.

Fig. 1.

Calcium mobilization assay performed on HEK293_mNPSR cells. Concentration-response curves to NPS and [d-Cys(tBu)⁵]NPS (top). Inhibition-response curve to [d-Cys(tBu)⁵]NPS (0.1 nM–100 μM) against the stimulatory effect of 10 and 100 nM NPS (bottom). Data are mean ± S.E.M. of four experiments.

Fig. 2.

Calcium mobilization assay performed on HEK293_mNPSR cells. Concentration-response curve to NPS obtained in the absence (control) and in the presence of increasing concentrations of [d-Cys(tBu)⁵]NPS (0.1–100 μM) (top); the corresponding Schild plot is shown at bottom. Data are mean ± S.E.M. of four experiments.

Fig. 3.

Typical PCR analysis of tail biopsies DNA from offspring obtained by mating heterozygous NPSR(+/-) mice. PCR product of 418 bp was from the homozygous wild-type mice [NPSR(+/+)]; a 599-bp DNA fragment was amplified from homozygous knockout mice [NPSR(-/-)]. The heterozygous NPSR(+/-) mice showed both the PCR products.

Fig. 4.

Recovery of righting reflex in Swiss mice. Effects elicited by intracerebroventricularly injected NPS (0.1 nmol) and [d-Cys(tBu)⁵]NPS (1–10 nmol) alone or coinjected on the percentage of animals losing the righting reflex in response to 15 mg/kg i.p. diazepam (left) and on their sleeping time (right). Sleeping time is defined as the amount of time between the loss and regaining of the righting reflex. Data are mean ± S.E.M. of 16 mice per group. *, p < 0.05 versus saline, according to Kruskal-Wallis and Dunn's test (left) or one-way analysis of variance followed by Dunnett's test for multiple comparisons (right).

Fig. 5.

Recovery of righting reflex in NPSR(+/+) and NPSR(-/-) mice. Effects elicited by 1 nmol i.c.v. injected NPS on the sleeping time of animals losing of the righting reflex in response to 15 mg/kg i.p. diazepam. Sleeping time is defined as the amount of time between the loss and regaining of the righting reflex. Data are mean ± S.E.M. of 10 to 12 mice per group. *, p < 0.05 versus saline, according to the Student's t test.