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.
Department of Medicine (Jewish Hospital) and Cell Biology/Physiology, Washington University Medical Center, St. Louis, Missouri 63110, USA.
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D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D008297:Male; D008564:Membrane Potentials; D015221:Potassium Channels / Q000187:drug effects; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012701:Serotonin / Q000378:metabolism*; D014044:Tolbutamide / Q000494:pharmacology
D007004:Hypoglycemic Agents; D015221:Potassium Channels; D005576:Colforsin; D012701:Serotonin; D011189:Potassium Chloride; D014044:Tolbutamide