The functional coupling between membrane electrical events and exocytotic release of hormones from endocrine cells is a key element in the physiology of secretion in endocrine glands. In the case of pituitary hormone secretion the membrane ion channels involved and the molecular mechanisms coupling stimulatory and inhibitory secretogogue receptors to these ion channels are only now being revealed. This task is complicated due to the presence of cellular subtypes secreting more than one hormone and interactions between more than one regulator of secretion. Many of the key secretagogue receptors belong to the superfamily of seven membrane spanning domain receptors that transduce ligand binding into cellular action via guanine nucleotide binding proteins (G-proteins). Among such receptors are those for dopamine (D2 type), the primary regulator of prolactin secretion, and somatostatin which governs inhibition of growth hormone release. The principle objective of this study is to employ patch clamp electrophysiological methods to examine the changes in ion channel function which underlie the actions of these and other agonists on individual pituitary cells in culture, and to elucidate the coupling mechanisms, both common and unique, which regulate excitation-secretion coupling in secretory subtypes. Normal rat anterior pituitary cells will be identified using the reverse hemolytic plaque assay (RHPA) and a newly developed method, the sequential cell immunoblot assay (SCIBA) to unambiguously discriminate cell types and quantify changes in secretion at the single cell level. In addition we will examine cell lines in which normal and mutated dopamine receptors are recombined with other elements of the signal transduction pathway. Key issues to be addressed are (1) the identity of G-proteins coupling D2 receptors to ion channels, (2) the mechanisms involved in this coupling, (3) the structural determinants of coupling specificity of receptors and G-proteins, (4) the nature of the relative immunity of D2 receptors to desensitization, and (5) the precise role of ion channel and ion concentration changes in the regulation of hormone secretion. This research will lead to a clearer understanding of the events underlying regulated hormone secretion in pituitary cells. In addition, the common identity of dopamine receptor subtypes in both pituitary and brain suggest that these studies may provide mechanistic insight into the actions of these neurotransmitters in affective disorders and neural regulation of vegetative functions.
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