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

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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