ATP is known to be coreleased with insulin from pancreatic β-cells. To monitor insulin secretion from single β-cells, a single β-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 an elevation in cytoplasmic calcium concentration ([Ca2+]i) to either tolbutamide (100 mumol/l) or kainate (1 mmol/l) but did respond with an elevation in [Ca2+]i to ATP (0.1–10 μmol/l) without desensitization and in a dose-dependent manner. A brief application of tolbutamide (10 μmol/l) increased [Ca2+]i in both the β-cell and the adjacent CPAE cells in co-culture. Suramin (100 μmol/l), an ATP-receptor blocker, inhibited the tolbutamide-induced elevation in [Ca2+]i in the CPAE cells but did not inhibit the elevation in [Ca2+]i in the β-cell, confirming that the insulin secretagogue-induced Ca2+ response in CPAE cells in co-culture is mediated by ATP released from the β-cell. When co-culture of the β-cell and CPAE cells was stimulated by kainate (1 μmol/l) and then tolbutamide (10 μmol/l), the CPAE cells showed elevations in [Ca2+]i in response to kainate and tolbutamide during elevation in [Ca2+]i in the β-cell. This strongly suggests that insulin secretion as well as an increase in [Ca2+]i in response to different agents, i.e., kainate and tolbutamide, occurs in a single β-cell. A long exposure of tolbutamide (100 μmol/l, 4 min) resulted in a long-lasting elevation in [Ca2+]i in the β-cell. During the elevation in [Ca2+]i, induced by tolbutamide in the β-cell, secretion was observed immediately after its application and again after some resting time. This temporal analysis shows that the secretory response of a single intact β-cell rapidly desensitizes despite the [Ca2+]i remaining elevated during the secretagogue stimulation and that it can return, after some desensitized period, in an intact cell. The present co-culture system using CPAE cells as reporter cells is, therefore, useful for monitoring insulin secretion from single intact β-cells.

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