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

催乳素在脊椎动物中有300多种已知功能。它几乎调节生理的方方面面，包括渗透调节、行为、生长和新陈代谢、生殖和免疫功能。催乳素作用的多样性和数量与荷尔蒙调节的复杂性是平行的。研究人员将探讨类固醇皮质醇快速抑制一种重要的泛盐类食用鱼罗非鱼(Oreochromis Mossambi)脑下垂体催乳素释放的新机制。此前，他们显示皮质醇在几分钟内就能抑制罗非鱼催乳素的释放。这一发现以及其他发现改变了普遍的共识，即类固醇激素的影响完全是通过它们改变基因表达的能力来调节的，这一过程通常需要几个小时或几天的时间才能发生。在过去的十年里，越来越明显的是，所有类别的类固醇都能迅速调节各种器官系统。皮质醇和其他糖皮质激素在几秒钟或几分钟内调节各种脊椎动物的激素分泌、神经元兴奋性、行为、细胞形态和碳水化合物代谢。然而，与其他类型的类固醇不同，大多数快速的糖皮质激素作用会产生抑制反应。尽管有大量证据表明糖皮质激素的快速作用，但调节其作用的细胞信号机制却知之甚少。这在一定程度上是由于研究抑制性反应而不是刺激性反应的内在困难，以及缺乏合适的、天然的模型系统来研究糖皮质激素快速作用的细胞生物学。与哺乳动物和其他脊椎动物相比，鱼类为研究催乳素细胞功能提供了一个重要的优势--催乳素细胞被分离成几乎均匀的团块，很容易分离出来进行研究。催乳素的基线分泌活性可以很容易地被操纵，以研究催乳素细胞功能的潜在重要刺激和抑制因子，以及介导其作用的细胞信号通路。研究人员发现，皮质醇作用于细胞膜，与基因表达无关，通过减少两个细胞信使cAMP和钙来迅速抑制催乳素的释放。这些作用可能通过特定的高亲和力脑垂体膜受体发生，涉及电压门控钙通道活性的降低和细胞外钙的内流。研究还表明，类固醇可能直接作用于细胞膜，抑制磷脂酶C，磷脂酶C是调节脊椎动物细胞钙的关键酶。在本提案中，四个具体目标将进一步详细阐述皮质醇快速、非基因组效应的调节机制，包括几种以前从未在脊椎动物中探索过的成分。第一个目标将解决皮质醇可能结合的受体类型，以快速调节催乳素的分泌。第二项研究将研究类固醇是否会直接或通过增加细胞膜上的钾离子电导来迅速改变催乳素细胞的膜电性，从而减少电压敏感的钙通道。第三个目标将测试皮质醇是否在导致催乳素释放迅速减少的事件中抑制磷脂酶C活性、三磷酸肌醇的产生、细胞内敏感池中钙的释放以及蛋白激酶的活性。第四个目标将探索类固醇是否可以迅速改变生长因子信号来调节催乳素的释放。这些研究将采用多种方法研究细胞信号，包括细胞和组织培养、药理操作、生物成像、激素受体结合、免疫分析和电生理学。这些研究的完成将促进对类固醇快速、非基因组作用的了解。这是内分泌学、调节生物学和医学领域的一门新兴学科。具体地说，这项研究应该开发一个全面的模型，描述一种“应激激素”的快速作用的信号通路，这种激素可以影响甚至可能损害包括记忆、行为、生殖和免疫功能在内的几个生理过程。由于皮质醇和催乳素对鱼类体内的水矿物质平衡起着相反的作用，渗透调节的详细工作原理也将被推进，渗透调节是一种古老而普遍的过程，对生理适应至关重要。

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.