Ryan R R, Weber H C, Mantey S A, Hou W, Hilburger M E, Pradhan T K, Coy D H, Jensen R T
Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
J Pharmacol Exp Ther. 1998 Oct;287(1):366-80.
Neither the native ligand nor the cell biology of the bombesin (Bn)-related orphan receptor subtype 3 (BRS-3) is known. In this study, we used RT-PCR to identify two human lung cancer lines that contain sufficient numbers of native hBRS-3 to allow study: NCI-N417 and NCI-H720. In both cell lines, [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) stimulates [3H]inositol phosphate. In NCI-N417 cells, binding of 125I-[DTyr6,betaAla11,Phe13,Nle14]Bn(6-14) was saturable and high-affinity. [DPhe6,betaAla11,Phe13,Nle14]Bn(6-14) stimulated phospholipase D activity and a concentration-dependent release of [3H]inositol phosphate (EC50 = 25 nM) and intracellular calcium (EC50 = 14 nM); the increases in intracellular calcium were primarily from intracellular stores. hBRS-3 activation was not coupled to changes in adenylate cyclase activity, [3H]-thymidine incorporation or cell proliferation. No naturally occurring Bn-related peptides bound or activated the hBRS-3 with high affinity. Four different bombesin receptor antagonists inhibited increases in [3H]inositol phosphate. Using cytosensor microphysiometry, we found that [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) caused concentration-dependent acidification. The results show that native hBRS-3 receptors couple to phospholipases C and D but not to adenylate cyclase and that they stimulate mobilization of intracellular calcium and increase metabolism but not growth. The discovery of human cell lines with native, functional BRS-3 receptors, of new leads for a more hBRS-3-specific antagonist and of the validity of microphysiometry as an assay has yielded important tools that can be used for the identification of a native ligand for hBRS-3 and for the characterization of BRS-3-mediated biological responses.
蛙皮素（Bn）相关孤儿受体亚型3（BRS-3）的天然配体和细胞生物学特性均不明确。在本研究中，我们利用逆转录聚合酶链反应（RT-PCR）鉴定出两个人肺癌细胞系，即NCI-N417和NCI-H720，它们含有足够数量的天然人BRS-3以供研究。在这两个细胞系中，[DPhe6，βAla11，Phe13，Nle14]Bn（6-14）均可刺激[3H]肌醇磷酸的生成。在NCI-N417细胞中，125I-[DTyr6，βAla11，Phe13，Nle14]Bn（6-14）的结合具有饱和性且亲和力高。[DPhe6，βAla11，Phe13，Nle14]Bn（6-14）可刺激磷脂酶D的活性以及[3H]肌醇磷酸（EC50 = 25 nM）和细胞内钙（EC50 = 14 nM）的浓度依赖性释放；细胞内钙的增加主要源于细胞内储存库。人BRS-3的激活与腺苷酸环化酶活性、[3H]-胸苷掺入或细胞增殖的变化无关。没有天然存在的Bn相关肽能以高亲和力结合或激活人BRS-3。四种不同的蛙皮素受体拮抗剂可抑制[3H]肌醇磷酸的增加。利用细胞传感器微生理学方法，我们发现[DPhe6，βAla11，Phe13，Nle14]Bn（6-14）可引起浓度依赖性酸化。结果表明，天然人BRS-3受体与磷脂酶C和D偶联，但不与腺苷酸环化酶偶联，并且它们可刺激细胞内钙的动员并增加代谢，但不促进生长。具有天然功能性BRS-3受体的人细胞系的发现、更具人BRS-3特异性拮抗剂的新线索的发现以及微生理学方法作为一种检测方法的有效性，产生了重要的工具，可用于鉴定人BRS-3的天然配体以及表征BRS-3介导的生物学反应。
