Chromaffin cells will be used as a model for studying stimulus- secretion coupling. Release of catecolamines from these cells, which is dependent on an increase in intracellular calcium levels, can result from either depolarization of the membrane (voltage-mediated) or an activation of cholinergic receptors (receptor- or agonist- mediated). It is not known, however, at what point other than an increase in intracellular calcium the signaling pathways underlying the two modes of stimulation leading to secretion converge. The objective of this study is to answer this question by using a high temporal resolution technique to examine the two pathways, and to determine whether the two pathways are similarly modulated by common second messenger systems. The whole-cell "tight seal" voltage-clamp method will be employed to measure ionic currents and to assay secretion. Secretion is associated with the fusion of cytoplasmic vesicles with the plasma membrane. These fusion events increase total membrane area which is reflected as an increase in membrane capacitance. Thus, by simultaneously recording membrane capacitance and conductance, it is possible to determine the activity of ion channels and secretion of catecholamines simultaneously. Furthermore, recent advances in calcium measurement with fluorescent dyes make it possible to assay intracellular calcium levels over the same brief period of time. The ability to correlate transmembrane ionic currents and changes in intracellular calcium levels with the process of secretion by exocytosis provides a uniquely powerful combination of approaches to the cellular mechanisms controlling exocytosis at the single cell level. The results of this research will provide more detailed knowledge of the sequence of events which couple stimulation to cellular secretion.
