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Capacitance measurements of exocytosis were combined with carbon fibre amperometry for time-resolved measurements of the properties of secretion in single, insulin-secreting, mouse pancreatic beta-cells pre-loaded with the amine serotonin (5-HT). Glucose-induced electrical activity was associated with the appearance of brief and transient amperometric currents reflecting the serotonin co-released with insulin. The integrated amperometric responses resulting from voltage-clamp depolarisations were proportional to the corresponding increase in cell capacitance. Both parameters exhibited U-shaped relationships to the membrane potential with maximums around 0 mV. There was a variable latency (40-730 ms, average 230 ms) between the onset of the depolarisation and the amperometric current. During high-frequency repetitive stimulation, a progressive decrease in the exocytotic capacity ("depression") was observed. This was paralleled by a corresponding reduction of the amperometric responses. Using the carbon fibre to map the beta-cell for release sites indicated that exocytosis was confined to the part of the cell containing the highest density of secretory granules. Two types of amperometric responses were observed. In about 50% of the cells, a smooth increase was observed with no discernible discrete events. In the remaining cells, the amperometric records contained large spikes. These were ten or more times larger than that expected for the fusion of individual secretory granules. We propose that these large spikes reflect the exocytosis of multigranular complexes formed inside the beta-cell prior to exocytosis.

Type

Journal article

Journal

Pflugers Arch

Publication Date

03/2000

Volume

439

Pages

634 - 645

Keywords

Action Potentials, Animals, Calcium, Carbon, Electric Conductivity, Exocytosis, Glucose, Insulin, Islets of Langerhans, Mice, Microelectrodes, Patch-Clamp Techniques, Reaction Time, Serotonin, Temperature