IONIC MECHANISMS RELATED TO SECRETION IN PITUITARY CELLS

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A description of the functional coupling between membrane electrical events and the exocytotic release of hormones from endocrine cells is of key importance to understanding the physiology of secretion in endocrine glands. Much less is known about the ionic mechanisms involved in excitation-secretion coupling in these systems than about the functionally analogous release of neurotransmitter molecules from nerve terminals. A general requirement for increased intracellular calcium and for regenerative electrical behavior is common to both processes, but in the case of hormone secretion the membrane ionic channels likely responsible for the relevant electrical behavior have yet to be determined. The principle objective of this study is to characterize the changes in ionic channel function triggered by known secretory stimulants and/or inhibitors in individual anterior pituitary cells and by neuromodulators in dorsal root ganglion neurons in tissue culture. The voltage clamp technique will be applied to these cells using "giga-seal" methods for patch and whole cell recording. The project will focus on four elements. (1) Examination of the details of the action of thyrotropin-releasing hormone (TRH) on Ca-activated and voltage-dependent K channels in GH3 cells using measurements of macroscopic and single channel currents coupled to manipulation of phosphotidylinositide metabolism. (2) Determination of the actions of a variety of secretogogues on ionic currents in rat pituitary cells segregated into secretory types by gradient separation methods. Cell types will be identified by immunocytochemistry. (3) Probe the mechanisms involved in "down modulation" of Ca channels in DRG cells by neurotransmitters using intracellular dialysis and single channel recording. (4) Simultaneously measure electrical activity and hormone release from single pituitary cells in culture using a reverse hemolytic plaque assay during application of secretogogues. This research will lead to a clearer understanding of the molecular events underlying hormone release and channel modulation. It will also provide a framework in which to examine membrane properties and abnormalities associated with pathologies of hormone secretion and neurotransmitter release.