A MONITOR OF SECRETION FROM SINGLE PANCREATIC BETA-CELLS

ATP is known to be coreleased with insulin from pancreatic beta-cells. 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 an elevation in cytoplasmic calcium concentration ([Ca2+](i)) to either tolbutamide (100 mu mol/l) or kainate (1 mmol/l) but did respond with an elevation in [Ca2+](i) to ATP (0.1-10 mu mol/l) without desensitization and in a dose-dependent manner. A brief application of tolbutamide (10 mu mol/l) increased [Ca2+](i) in both the beta-cell and the adjacent CPAE cells in co-culture, Suramin (100 mu mol/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 beta-cell, confirming that the insulin secretagogue-induced Ca2+ response in CPAE cells in co-culture is mediated by ATP released from the beta-cell. When co-culture of the beta-cell and CPAE cells was stimulated by kainate (1 mmol/l) and then tolbutamide (10 mu mol/l), the CPAE cells showed elevations in [Ca2+](i) in response to kainate and tolbutamide during elevation in [Ca2+](i) in the beta-cell. This Ca strongly suggests that insulin secretion as well as an increase in [Ca2+], in response to different agents, i.e., kainate and tolbutamide, occurs in a single beta-cell. A long exposure of tolbutamide (100 mu mol/l, 4 min) resulted in a long-lasting elevation in [Ca2+](i) in the beta-cell. During the elevation in [Ca2+](i) induced by tolbutamide in the beta-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 beta-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 beta-cells.