Electrochemical detection of exocytosis at single rat melanotrophs.
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.
Paras
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CD
Department of Chemistry, University of Florida, Gainesville 32611-7200, USA.
Kennedy
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RT
D000818:Animals; D002244:Carbon; D002478:Cells, Cultured; D004563:Electrochemistry; D005089:Exocytosis / Q000502:physiology*; D008297:Male; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism*; D008839:Microelectrodes; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000521:alpha-MSH / Q000032:analysis / Q000378:metabolism*
D000521:alpha-MSH; D002244:Carbon