Biology of Reproduction

The role of ion channel activity in the response of rat pituitary lactotrophs and gonadotrophs to dopamine (DA) and GnRH, respectively, was investigated. Single lactotrophs and gonadotrophs were unambiguously identified with the reverse hemolytic plaque assay, and recordings of membrane potential and current were obtained using whole-cell and single-channel patch-clamp techniques. In lactotrophs, DA inhibited spontaneous electrical activity by activating a K conductance that hyperpolarized the cells. A 50 pS K channel underlies this response and was activated following agonist binding to a D, type receptor via a "direct" interaction with a pertussis toxin-sensitive G-protein. In gonadotrophs, GnRH triggers rhythmic hyperpolarizations due also to a K conductance increase. The K channel underlying the GnRH response is an apamin-sensitive, Ca+-activated channel. Although both agonists produce hyperpolarizations in their respective target cells via K channel activation, differences in intracellular calcium responses probably discriminate the stimulatory (GnRH) and inhibitory (DA) actions on hormone secretion. Each K channel type plays a different role in modulating the intracellular Ca levels to yield these actions. INTRODUCTION A wide variety of neurotransmitters and peptides that reach the anterior pituitary gland via the hypothalamic-hypophysial portal system are known to influence the secretion of hormones. Dopamine (DA) appears to be the primary regulator of prolactin secretion exerting a tonic inhibitory action upon lactotrophs [1]. In contrast, LH and FSH secretion from gonadotrophs is stimulated by the action of GnRH [2]. While much is known about the coupling between receptors for these agents and second messenger systems, less is known about the ion channel effectors and electrical events associated with signal transduction in individual cell types. This is, in part, due to the difficulty of identifying specific cell types in a mixed population for individual study by single-cell experimental methods. Recently, our laboratories have overcome this problem by combining the reverse hemolytic plaque assay (RHPA) to identify secretory subtypes [3] with measurements of membrane potentials and ion currents from whole cells and isolated membrane patches [4]. Using these methods, we have explored the actions of DA and GnRH on electrical activity in their respective target cells. Our observations indicate that changes in the activity of ion channels in identified lactotrophs and gonadotrophs induced by these secretogogues can both be fundamental to and occur in parallel with the process of excitation-secretion coupling. The present article reviews and expands upon previously published observations [5-7]. 'This work was supported by NIH grants NS18788, AR17803, and HD12629, a grant from the W.M. Keck Foundation, and awards from the Mellon Foundation and the McKnight Foundation. 2Correspondence: Dr. Gerry S. Oxford, Department of Physiology, University of North Carolina, Box 7545, 04 Medical Science Research Bldg., Chapel Hill, NC 27599. FAX: (919) 966-6927. MATERIALS AND METHODS Cells were dissociated from anterior pituitary glands of either male (gonadotroph) or cycling female (lactotroph) Sprague-Dawley rats using either collagenase (lactotrophs) or collagenase and trypsin (gonadotrophs) according to previously published protocols [5, 7, 8]. Lactotrophs and gonadotrophs were identified by the RHPA [3] using antibodies to rat prolactin [6] or bovine LH [7], respectively, and maintained in culture for 1-4 days prior to recording. Gigaseal whole-cell or single-channel techniques [4] were employed to record from RHPA-positive cells. For whole-cell experiments, the external bath contained 150 mM NaCI, 25 mM CaC12, 2.5-5 mM KCl, 1 mM MgC1,, 8-10 mM glucose, 10 mM HEPES buffer (pH 7.4). The intracellular pipette solution contained 120-130 mM potassium aspartate, 20 mM KCI, 0.1 mM guanosine triphosphate (GTP), 2 mM ATP, and 10-20 mM HEPES buffer (pH 7.4). For single-channel measurements in cell-attached patches, the pipette contained 150 mM KCI, 3.1 mM MgC12, and 15 mM HEPES buffer. Secretogogues and other agents were applied directly to the solution bathing the cells or by using a U-tube [9]. All experiments were performed at room temperature (20250C).