The objective of this study is to analyze, at the level of the individual cell and the cell membrane, the role of ions and ion transport in the regulation, by specific secretagogues and inhibitors, of luteinizing hormone (LH) and prolactin (PRL) secretion. Dispersed normal rat anterior pituitary cells will be maintained in culture. Individual gonadotrophs and lactotrophs will be identified by reverse hemolytic plaque techniques. Double-barreled (open-tip and ion-selective barrels) microelectrodes and patch clamp techniques will be used to study membrane potentials and resistances, intracellular Ca2+, K+, and Na+ activities, and ionic channels in the gonadotroph membrane. The effects of gonadotrophin releasing hormone (GnRH) on these parameters and the contributions of external and internal calcium to GnRH-induced increases in cytosolic Ca2+ will be explored. The role of cytosolic Ca2+ in ion-channel gating and the gating, conductance and kinetics of channels that respond directly to GnRH will be particularly examined. Additional factors that stimulate LH secretion (high external K+, the calcium ionophore A23187 and Na+-channel activators) will be similarly studied. In parallel experiments with isolated identified lactotrophs, Ca2+, K+ and Na+-channels in the cell membrane will be studied by patch-clamp. Ca2+ and K+-channel characteristics will be examined in detail under control conditions and in the presence of an inhibitor (dopamine, DA) and of stimulators (thyrotrophin releasing hormone, TRH, and vasoactive intestinal peptide, VIP) of PRL secretion. A method for determining cytosolic Ca2+ activity from the kinetics of K+-channels will be explored and results obtained will be validated with Ca2+-selective microelectrodes. Action potentials in isolated lactotrophs will be examined. Effects of estrogen pre-treatment (cultured cells) and of altered biological states on gonadotroph and lactotroph electrophysiology will be studied. Ultimately, the results obtained should contribute to the development of therapeutic strategies for handling problems related to human fertility and reproduction.
