Falls H Douglas, Dayton Brian D, Fry Dennis G, Ogiela Christopher A, Schaefer Verlyn G, Brodjian Sevan, Reilly Regina M, Collins Christine A, Kaszubska Wiweka
Metabolic Disease Research, Global Pharmaceutical Products Division, Abbott Laboratories, R4MJ, Bldg. AP9A, 100 Abbott Park Road, Abbott Park, IL 60064-6099, USA.
J Mol Endocrinol. 2006 Aug;37(1):51-62. doi: 10.1677/jme.1.01943.
Ghrelin, a 28 amino acid, octanoylated peptide, is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to various endocrine functions, including stimulation of GH release, ghrelin has been characterized as an important regulator of energy homeostasis. Ghrelin administration has been shown to increase adiposity in rodents and stimulate food intake in humans. Studies suggest that these orexigenic effects are mediated primarily through GHS-R expression in hypothalamic and pituitary neuronal pathways. In this context, GHS-R has been recognized as a potential target for the treatment of GH deficiency and body weight disorders. Cell lines provide convenient in vitro systems to identify and characterize potential pharmacophores and to analyze GHS-R functional activity. While recombinant cell lines that overexpress GHS-R have served as effective research tools for these studies, such cell lines may differ in signaling response to ghrelin compared with hypothalamic or pituitary cells expressing GHS-R. We show here that a cell line derived from a rat anterior pituitary adenoma, RC-4B/C, expresses endogenous GHS-R as judged by reverse transcriptase-PCR. In a Ca(2+)mobilization assay, RC-4B/C cells demonstrate a dose-dependent increase in intracellular [Ca(2+)] on stimulation with rat ghrelin and a related peptide agonist, hexarelin (EC(50), 1.0 nM and 1.7 nM respectively), but are unresponsive to treatment with inactive des-octanoyl rat ghrelin. A subclone, RC-4B/C.40, with a more robust and stable ghrelin response, was isolated from the parental population of cells to allow further analysis of GHS-R signal transduction. Using pertussis toxin and the phospholipase C inhibitor U-73122, we show that ghrelin signals through the Gq pathway in the RC-4B/C.40 cells. We also demonstrate that the ghrelin-induced rise of intracellular [Ca(2+)] in RC-4B/C.40 cells involves initial Ca(2+)release from intracellular stores followed by a sustained elevation that occurs via influx of extracellular Ca(2+) through ion channels. In addition, unlike observations reported in recombinant cell systems, the RC-4B/C.40 cells do not exhibit a high level of GHS-R constitutive activity as determined in a phosphatidylinositol hydrolysis assay. Overall, the data presented here suggest that the RC-4B/C parental and RC-4B/C.40 cells provide novel in vitro systems for the characterization of GHS-R pharmacophores and ghrelin signaling.
胃饥饿素是一种由28个氨基酸组成的辛酰化肽，是生长激素促分泌素受体（GHS-R）的内源性配体。除了各种内分泌功能，包括刺激生长激素释放外，胃饥饿素还被认为是能量稳态的重要调节因子。研究表明，给予胃饥饿素可增加啮齿动物的肥胖程度，并刺激人类的食物摄入。研究表明，这些促食欲作用主要通过下丘脑和垂体神经元途径中的GHS-R表达介导。在这种情况下，GHS-R已被认为是治疗生长激素缺乏症和体重紊乱的潜在靶点。细胞系为鉴定和表征潜在药效基团以及分析GHS-R功能活性提供了方便的体外系统。虽然过表达GHS-R的重组细胞系已成为这些研究的有效工具，但与表达GHS-R的下丘脑或垂体细胞相比，此类细胞系对胃饥饿素的信号反应可能有所不同。我们在此表明，源自大鼠垂体前叶腺瘤的细胞系RC-4B/C通过逆转录聚合酶链反应（RT-PCR）检测显示表达内源性GHS-R。在钙动员试验中，RC-4B/C细胞在用大鼠胃饥饿素和相关肽激动剂六肽促生长素（分别为EC50，1.0 nM和1.7 nM）刺激后，细胞内[Ca2+]呈剂量依赖性增加，但对无活性的去辛酰化大鼠胃饥饿素处理无反应。从亲代细胞群体中分离出一个对胃饥饿素反应更强且更稳定的亚克隆RC-4B/C.40，以进一步分析GHS-R信号转导。使用百日咳毒素和磷脂酶C抑制剂U-73122，我们表明胃饥饿素在RC-4B/C.40细胞中通过Gq途径发出信号。我们还证明，胃饥饿素诱导的RC-4B/C.40细胞内[Ca2+]升高涉及细胞内储存的Ca2+的初始释放，随后是通过离子通道的细胞外Ca2+内流导致的持续升高。此外，与重组细胞系统中报道的观察结果不同，RC-4B/C.40细胞在磷脂酰肌醇水解试验中未表现出高水平的GHS-R组成型活性。总体而言，本文提供的数据表明，RC-4B/C亲代细胞和RC-4B/C.40细胞为表征GHS-R药效基团和胃饥饿素信号传导提供了新的体外系统。
