Secretion of peptide hormones is a poorly understood, yet vitally important process. For example, type II diabetes is associated with insufficient insulin secretion. However, many aspects of insulin secretion remain a mystery. A significant problem in studying hormone secretion is the lack of methods for monitoring peptides. The overall goal of the work is to develop electrochemical and electrophoresis-based methods for monitoring peptides secreted from single b-cells and single islets of Langerhans with high time resolution. Microelectrodes will be used to detect secretion of insulin and 5-hydroxytryptamine (5-HT) from single cells. Insulin detection will be based on oxidation at a carbon fiber electrode modified with a recently developed ruthenium oxide-type catalyst. The electrochemistry of insulin at the new electrode will be characterized including determination of oxidation sites on insulin. 5-HT, which can be detected at an unmodified carbon fiber electrode, will be investigated as a possible marker for insulin secretion. As the electrochemical methods are developed, they will be applied to fundamental studies of peptide secretion including: 1) correlation of secretion with cytoplasmic Ca2+ concentration, 2) spatial distribution of exocytosis sites, 3) characterization of compound exocytosis events, 4) effect of extracellular conditions on extrusion of vesicular contents, and 5) effect of metabolic state on exocytosis. In addition, rapid competitive immunoassays, performed using capillary electrophoresis with laser-induced fluorescence detection will be developed for insulin, glucagon, and somatostatin. The assays will be incorporated into an on-line sampling system to allow monitoring of these hormones released from single islets of Langerhans. Assays with analysis times less than 2 seconds are possible which should allow for high time resolution using this method.
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