Stimulus-Secretion Coupling Mechanisms for Rapid, Nongenomic Corticosteroid Actions in the Teleost Prolactin Cell Model System

North Carolina State University, Raleigh NC

Investigators

Abstract

Prolactin has well over 300 known functions in vertebrates. It regulates virtually every aspect of physiology including osmoregulation, behavior, growth and metabolism, reproduction and immune function. The diversity and number of actions of prolactin is paralleled by the complexity with which the hormone is regulated. The investigators will address the novel mechanisms by which the steroid, cortisol, rapidly inhibits prolactin release from the pituitary gland of an important euryhaline food fish, the tilapia (Oreochromis mossambicus). Previously, they showed cortisol acts within minutes to inhibit prolactin release in tilapia. This discovery as well as others have altered the prevailing consensus that the effects of steroid hormones are mediated solely through their ability to alter the expression of genes, a process that typically requires hours or days to occur. Over the past decade it has become increasingly apparent that all classes of steroids rapidly regulate various organ systems. Cortisol and other glucocorticoids modulate hormone secretion, neuronal excitability, behavior, cell morphology, and carbohydrate metabolism in various vertebrates within seconds or minutes. Unlike other classes of steroids, however, most rapid glucocorticoid actions produce inhibitory responses. Despite abundant evidence for rapid glucocorticoid effects, the cell-signaling mechanisms mediating their actions are poorly understood. This is due, in part, to the inherent difficulty of studying inhibitory rather than stimulatory responses and the lack of suitable, native model systems to address the cell biology underlying rapid glucocorticoid actions. Over mammals and other vertebrates, fishes present an important advantage for the study of prolactin cell function--prolactin cells are segregated as a nearly homogenous mass that is easily separated for study. Prolactin baseline secretory activity can be easily manipulated to study potentially important stimulators and inhibitors of prolactin cell function and the cell-signaling pathways that mediate their action. The investigators show cortisol acts at the membrane, independent of gene expression to rapidly inhibit prolactin release by reducing two cellular messengers, cAMP and calcium. These actions may occur through a specific high-affinity pituitary membrane receptor, and involve reductions in voltage-gated calcium channel activity and influx of extracellular calcium. Studies also demonstrate the steroid may directly act at the membrane to suppress phospholipase C, an enzyme critical to regulating cellular calcium in vertebrates. In the present proposal, four specific objectives will address in further detail the mechanisms mediating rapid, nongenomic effects of cortisol, including several components never previously explored in vertebrates. The first objective will address the type of receptor that cortisol may bind to rapidly modulate prolactin secretion. The second will examine whether the steroid acts to rapidly alter the membrane electrical properties of prolactin cells to reduce voltage-sensitive calcium channels either directly or through increasing potassium ion conductances across the cell membrane. The third aim will test whether cortisol inhibits phospholipase C activity, inositol triphosphate production, calcium release from intracellular-sensitive pools, and activity of protein kinases in events that lead to rapid reductions in prolactin release. The fourth objective will explore whether the steroid might rapidly modify growth factor signaling to regulate prolactin release. These studies will employ a combination of methods to study cell-signaling, including both cell and tissue culture, pharmacological manipulations, bioimaging, hormone receptor-binding, immunoassays and electrophysiology. Completion of the proposed studies will advance the knowledge of rapid, nongenomic actions of steroids. This is a new and growing discipline in the field of endocrinology, regulatory biology and medicine. Specifically, the research should lead to development of a comprehensive model describing the signaling pathways mediating rapid actions of a "stress hormone" known to influence, and possibly impair, several physiological processes including memory, behavior, reproduction, and immune function. As cortisol and prolactin exert opposing actions on hydromineral balance in fish, the detailed workings underlying osmoregulation, an ancient and universal process critical to physiological adaptation, will also be advanced.