Development of super-sensitive assay for hormone secretion from single cells

三磷酸腺苷与神经递质共同储存在神经细胞的突触小泡中，与激素共同储存在分泌小泡中。众所周知，ATP与多种激素共同释放，包括来自胰岛β细胞的胰岛素和来自嗜铬细胞的儿茶酚胺。我们以胰岛β细胞为模型系统，建立了灵敏的激素释放监测方法。为了监测单个β细胞的胰岛素分泌，在培养中将单个β细胞包裹在Fura-2负载的小牛肺动脉内皮细胞(CPAE)中，CPAE细胞可以检测到ATP。CPAE细胞对甲苯丁胺或海人藻酸不反应胞浆钙浓度的升高，但对ATP的[Ca^&lt；2+&gt；]i升高而不脱敏，且呈剂量依赖关系。短期应用10 mM的甲苯磺丁胺可使共培养的β细胞和邻近的CPAE细胞内[Ca~(2+)]i升高。三磷酸腺苷受体阻断剂苏拉明抑制甲苯丁胺诱导的…CPAE细胞[Ca^&lt；2+&gt；]i升高更多，但对β细胞[Ca^&lt；2+&gt；]i升高无明显抑制作用，证实胰岛素促分泌剂诱导的CPAE细胞共培养钙反应是由β细胞释放的ATP介导的。因此，以CPAE细胞为报告细胞的共培养体系可用于监测单个完整的胰岛β细胞的胰岛素分泌。该系统还表明，当胰岛β细胞受到刺激时，它们对真实的三磷酸腺苷作出反应，提示三磷酸腺苷可能通过P_2嘌呤受体(P_2受体)作用于胰岛β细胞来刺激胰岛素的分泌。我们通过筛选大鼠胰岛cDNA文库，获得了编码P&lt；2X-4&gt；家族第四个成员(P&lt；2X-4&gt；)的基因。大鼠P&lt；2X-4&gt；是一个由388个氨基酸组成的蛋白质，有两个可能的跨膜片段。P_&lt；2X-4&gt；m RNA广泛表达于脑和外周组织，包括多种内分泌组织，在多种激素分泌细胞系中也有表达。我们在非洲爪哇卵母细胞中异源表达了P_&lt；2X-4&gt；，并对其药理作用进行了研究，发现其类似物可激活阳离子选择性离子通道。激动剂效价顺序为：ATP&GT；ADP&GT；2-甲硫基-三磷酸腺苷(2MeSATP)；&GT；α-β-亚甲-三磷酸腺苷(α-β-亚甲-三磷酸腺苷)。这些结果表明，P&lt；2X-4&gt；在包括内分泌细胞在内的非神经细胞以及神经细胞中介导了细胞外ATP诱导的生物效应。较少

ATP is costored with neurotransmitters in the synaptic vesicles of neuronal cells and with hormones in the secretory vesicles. ATP is known to be coreleased with various hormones, including insulin from pancreatic beta-cells and catecholamine from chromaffin cells. We have developed the sensitive assay which can moniter the released hormone, using pancreatic beta-cells as a model system. To monitor insulin secretion from single beta-cells, a single beta-cell was surrounded in culture by fura-2 loaded calf pulmonary artery endothelium (CPAE) cells, which can detect the ATP.CPAE cells did not respond with a rise in cytoplasmic calcium concentration ([Ca^<2+>]i) to either tolbutamide or kainate, but did respond with a rise in [Ca^<2+>]i to ATP without desensitization and in a dose-dependent manner. A brief application of tolbutamide (10mM) increased [Ca^<2+>]i in both the beta-cell and the adjacent CPAE cells in coculture. Suramin, an ATP receptor blocker, inhibited the tolbutamide-induce … More d elevation in [Ca^<2+>]i in the CPAE cells, but did not inhibit the elevation in [Ca^<2+>]i in the beta-cell, confirming that the insulin secretagogue-induced Ca^<2+> response in CPAE cells in coculture is mediated by ATP released from the beta-cell. The coculture system using CPAE cells as reporter cells is, therefore, useful for monitoring insulin secretion from single intact beta-cells.We have also shown by this system that upon stimulation of pancreatic beta-cells, they respond to the realesd ATP,suggesting that ATP may act on pancreatic beta-cells to stimulate insulin secretion through P_2 purinoreceptors (P_<2X> receptor). We isolated cDNA encoding a fourth member (P_<2X-4>) of the P_<2X> family by screening a rat pancreatic islet cDNA library. Rat P_<2X-4> is a protein of 388 amino acids and has two putative transmembrane segments. P_<2X-4> mRNA is widely expressed in brain and peripheral tissues, including various endocrine tissues and it is also expressed in various hormone-secreting cell lines. We have heterologously expressed the cloned P_<2X-4> in Xenopus laevis oocytes and have characterized its pharmacological properties ATP,its analogs and ADP activate cation-selective ion channels. The order of agonist potency is ATP>ADP>2-methyl-thioATP (2MeSATP) >>alphabeta-methelene-ATP (alphabetameATP). These results suggests that P_<2X-4> mediates extracellular ATP-induced biological effects in non-neuronal cells including, endocrine cells, as well as in neuronal cells. Less