Amperometric detection of quantal secretion from patch-clamped rat pancreatic beta-cells

Serotonin (5-HT) is taken up in insulin granules and co-released with insulin on stimulation of pancreatic islet beta-cells. Based on these observations, we have used microcarbon fiber amperometry to examine secretogogue-induced 5-HT release from rat beta-cells preloaded for 4-16 h with 5-HT and then exposed to a bath solution containing 10 microM forskolin. In response to local application of KCl (60 mM) or tolbutamide (50-200 microM), we recorded barrages of amperometric events. Each amperometric event consisted of a short pulse of current measurable at electrode voltages that catalyze 5-HT oxidation. With either secretogogue, release was calcium-dependent. On combining amperometry with perforated patch whole-cell recording, we found that barrages of such events were well coupled in time and graded in intensity with depolarization-induced Ca2+ currents and well correlated with increases in membrane capacitance. In cell-attached patch recording, amperometric events evoked by application of tolbutamide followed the closure of ATP-sensitive K+ channels and coincided with the onset of electrical activity. These experiments suggest that amperometry is a useful technique for studying, in real time, the dynamic aspects of stimulus-secretion coupling in beta-cells. Their performance was facilitated by the design of a new carbon fiber electrode (ProCFE) described within.