Camarda Valeria, Rizzi Anna, Ruzza Chiara, Zucchini Silvia, Marzola Giuliano, Marzola Erika, Guerrini Remo, Salvadori Severo, Reinscheid Rainer K, Regoli Domenico, Calò Girolamo
Department of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, Ferrara, Italy.
J Pharmacol Exp Ther. 2009 Feb;328(2):549-55. doi: 10.1124/jpet.108.143867. Epub 2008 Oct 29.
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.
神经肽S（NPS）被确定为一种孤儿受体的内源性配体，该受体现被称为NPS受体（NPSR）。在对NPS Gly5进行的构效关系研究中，确定了NPSR配体[D-Cys(tBu)(5)]NPS。高达100微摩尔的[D-Cys(tBu)(5)]NPS不会刺激稳定表达小鼠NPSR的人胚肾（HEK）293细胞中的钙动员；然而，该肽以浓度依赖性方式抑制了10和100纳摩尔NPS引起的刺激作用（pK(B)，6.62）。在希尔德分析实验中，[D-Cys(tBu)(5)]NPS（0.1 - 100微摩尔）使NPS的浓度-反应曲线产生浓度依赖性和平行的右移，显示pA(2)值为6.44。10微摩尔的[D-Cys(tBu)(5)]NPS对七种与NPSR无关的G蛋白偶联受体的信号传导没有影响。在小鼠翻正反射（RR）恢复试验中，脑室内注射0.1纳摩尔的NPS可降低因15毫克/千克地西泮而失去RR的动物百分比及其睡眠时间。[d-Cys(tBu)(5)]NPS（1 - 10纳摩尔）本身无活性，但能剂量依赖性地拮抗NPS的促觉醒作用。最后，NPSR缺陷小鼠对地西泮催眠作用的敏感性与它们的野生型同窝小鼠相似。然而，在野生型小鼠而非突变小鼠中可检测到1纳摩尔NPS的促觉醒作用。总之，[D-Cys(tBu)(5)]NPS在体外和体内均表现为一种纯的、选择性的NPSR拮抗剂，但效力中等。此外，使用该工具并结合受体敲除小鼠研究，我们证明了NPS的促觉醒作用是由于NPSR蛋白的选择性激活。
