The primary objectives of this study are to gain a firm understanding of the mechanism of excitability in endocrine cells and to determine the mechanism by which stimulators or inhibitors of hormone secretion alter the electrical activity of the cells they act upon. To accomplish these goals, voltage clamp experiments will be performed initially on a transformed cell line from the anterior pituitary, GH3 cells, and later on preparations of dispersed cells and primary cultures of the following endocrine cells: pancreatic Beta -cells, cells of the anterior pituitary, the pars intermedia of the pituitary and the adrenal cortex. Macroscopic and single channel current measurements made with the patch clamp technique will be used to identify and characterize the membrane conductances responsible for excitability in these cells. Channel identification will be determined primarily by studying ion selectivity, pharmacology, single channel conductance and kinetics. Following these preliminary experiments, we will study the mechanisms by which the following secretagogues alter the membrane potential of the cells they act upon: glucose on Beta-cells, thyrotropin releasing factor on anterior pituitary cells, dopamine on cells of the pars intermedia and angiotensin II on adrenal cortical cells. We will determine if these agents activate ionic channels in a manner similar to acetylcholine, or if they alter, either directly or indirectly, the properties of existing channels. This information will hopefully lead to a better understanding of the mechanism of stimulus-secretion coupling in endocrine cells. A final objective of this proposal is to utilize GH3 cells as an alternative to the squid axon for biophysical studies of channel function. In particular, we will measure macroscopic conductance, single channel behavior and gating current of the Na channels in these cells. The objective of these experiments will be to determine a kinetic model of the Na channel which is consistent with the data obtained from all three measurements.
