IONIC MECHANISMS RELATED TO SECRETION IN PITUITARY CELLS

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A description of the functional coupling between membrane electrical events and exocytotoic 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 and molecular 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 is common to both processes, and the involvement of regenerative electrical behavior has been now documented for pituitary and pancreatic cells. However, in the case of hormone secretion from normal pituitary cells the membrane ion channels likely responsible for the relevant electrical behavior, the pools for elevated calcium, and the molecular mechanisms by which secretogogue receptors are coupled to ion channel behavior and secretion have yet to be definitively elucidated. This task has been made more complicated by the recent identification of pituitary cellular subtypes which may secrete either a single hormone or co-release two hormones. The principle objective of this study is to examine the changes in ion channel function triggered by known secretory stimulants and/or inhibitors in individual anterior pituitary cells in tissue culture, and to determine the intracellular coupling mechanisms, both common and unique, which regulate secretion in secretory subtypes. Cells of the GH3 line and normal rat anterior pituitary cells will be used. The secretory "fingerprint" of the latter will be determined in living cultures by the reverse hemolytic plaque assay (RHPA), an immune hemolysis procedure which we have refined. Regulation of ion channels and secretion will be investigated with 4 technical approaches. (1) The patch voltage clamp technique will be applied to these cells in both single channel and whole cell recording modes to examine the response of particular classes of ion channels to directly applied secretogogues. Intracellular dialysis and excised patch methods will be employed to control the intracellular ionic and second-messenger environment to assess the role of PI metabolites and Gproteins in receptor channel coupling. (2) Changes in intracellular Ca++ will be measured in single cells during secretogogue action using spectroscopic determination of fura-2 fluorescence changes. (3) We will develop and employ a fluorescent method based on complement-induced liposome lysis to detect specific hormone secretion from individual cells. A microbilayer immunolytic probe for hormone secretion will also be explored. (4) Determination of the degree of intercellular coupling among pituitary cells in culture will be done with dual patch electrical measurements and dye transfer studies. The influence of secretogogue action on these processes will be examined in a pilot study. This research will lead to a clearer understanding of the events underlying hormone secretion in endocrine cells. It will also provide a framework in which to understand membrane properties and abnormalities associated with pathologies of hormone secretion.