Amperometry at a carbon fiber microelectrode was used to monitor secretion of peptide hormone from single melanotrophs of the intermediate lobe of the rat pituitary. The method is based on electrochemical oxidation of tryptophan and tyrosine residues of small proopiocortin-derived peptides secreted from these cells. For single-cell measurements, the electrode, which had a sensing diameter of approximately 9 microns and a total tip diameter of 30 microns, was positioned approximately 1 micron away from single melanotrophs. When cells were stimulated by application of 64 mM K+, a series of randomly occurring current spikes with an average area of 34 +/- 6 fC was observed. The current spikes were strongly dependent on the presence of Ca2+. Current spikes of nearly identical area and shape were also elicited by mechanical stimulation. Cyclic voltammograms obtained from cell releasates confirmed that the substance detected was a tryptophan- or tyrosine-containing peptide. On the basis of amperometric tests of the most abundant peptides in melanotrophs, it is concluded that the current spikes are due to detection of primarily alpha-melanocyte stimulating hormone. The spike area corresponds to 0.32 amol of alpha-melanocyte stimulating hormone. It is concluded that the current spikes represent detection of concentration pulses that are expected following exocytosis events.

Spatially resolved measurements of exocytosis in pancreatic beta-cells were made using amperometry with 1-microm radius electrodes. These measurements revealed that certain portions of a cell actively undergo exocytosis following stimulation with depolarizing agents, but other regions are inactive. The amperometric measurements were compared to measurements made with the membrane indicator dye, FM1-43, which showed uneven increases in fluorescence around the surface of the cell, with amperometric secretion being detected only at the brightest regions. In some instances, a large number of exocytotic events were detected from one electrode position. The number of events was larger than what would be expected based on the number of vesicles that could fit under an electrode of the dimensions used. These results suggest a mechanism of vesicle traffic that allows multiple fusions at a small membrane area.

The amperometric and cyclic voltammetric detection of a-melanocyte stimulating hormone (MSH), 0-endorphin, and corticotropin-like intermediate lobe peptide (CLIP), all proopiocortin (POC) derived peptides secreted from melanocytes of the pituitary intermediate lobe, at carbon fiber microelectrodes was investigated. For amperometry, it was found that all of these peptides could be detected; however, fouling of the electrodes reduced the response of the electrode after successive application of the peptide in flow injection experiments. The fouling wa5 apparently due to oxidation of tyrosine in the peptides as similar results were found for tyrosine but not tryptophan. The effect of fouling could be reversed if the electrode was electrochemically treated by scanning from -1 .OV to +1.OV at 300 V/s for 2 min between application of the peptides. Using cyclic voltammtery at 800 V/s, it was possible to distinguish MSH, which had a peak shaped voltammogram, from the other POC peptides, which had relatively flat voltammetric waves at this scan rate. The scan rate dependence of the peak current for MSH revealed that the voltammetry was adsorption controlled. As a result, in a monitoring application, where voltammograms are continuously obtained with a fixed interval between them, decreasing the interval increases the temporal resolution but decreases the sensitivity for MSH. It was found that when monitoring the current in the potential range of 0.90 to 1 .OOV, the temporal response to MSH was dependent upon the potential window used for scanning. Using high scan rates and a potential window of 0 to 1.2 V, it was possible to monitor exocytosis from single melanocytes and use the voltammogram to demonstrate detection of MSH from the cells.