Stimulus-Synthesis/Secretion Coupling: The Tilapia Prolactin Cell as a Model Osmoreceptor

催乳素（PRL）是所有脊椎动物脑垂体的一种高度通用的激素。 它在调节鱼类的盐和水平衡（盐调节）方面具有重要作用，包括罗非鱼，Oreochromis mossambicus，一种可以在淡水和海水之间自由移动的物种（它是广盐性的）。 在稀释的淡水环境中，血液离子水平趋于下降，PRL通过增加血液渗透压和降低皮肤和其他皮肤调节表面的水和离子渗透性来对抗这种情况。 与这些行动相一致，PRL细胞表现出更大的活动，在罗非鱼举行的淡水比在海水中举行。 当罗非鱼从海水移到淡水时，血液PRL也会升高，并随着血液渗透压的下降而保持升高。 罗非鱼PRL细胞似乎是直接响应于细胞外渗透压的变化，因为PRL释放增加，介质渗透压降低时，罗非鱼垂体腺在体外孵育。 术语“渗透压受体”是指以适当的生理反应响应渗透信号的细胞，所述生理反应通过称为信号转导途径的机制诱发。 钙（Ca+2）作为第二信使广泛用于这些途径，介导对特定外部信号的细胞反应的诱导。 罗非鱼PRL细胞的渗透感受性特性提供了一个极好的和可能独特的模型系统，用于研究渗透信号被转导成渗透调节反应的途径（即，PRL分泌）。与其他脊椎动物不同的是，在硬骨鱼中，PRL细胞被分离到垂体的前部，几乎是均匀的团块，很容易分离用于器官和分散细胞培养。这种解剖学排列极大地促进了分泌机制的表征，并提供了一个非常有用的模型，可以增加我们对其他较难获得的敏感的监管的一般理解。（和神经调节）内分泌和神经内分泌系统（例如，在哺乳动物中参与加压素和催产素释放的下丘脑-神经垂体大细胞系统）。本研究的目的是探讨罗非鱼PRL细胞渗透环境的变化对PRL产生和释放的调控机制。目前的研究建立在本实验室以前的工作基础上，旨在构建一个更详细的图片中的信号转导受体细胞。 具体的目的是确定PRL释放的上升是否响应于细胞外渗透压摩尔浓度的降低是由细胞大小的增加引起的，这导致细胞内游离Ca+2（[Ca 2 +]i）的增加。 假设是细胞外渗透压的下降导致水被动流入PRL细胞，增加细胞体积;这引发细胞外Ca 2+的流入和[Ca 2 +]i的上升，从而触发PRL释放的增加。 为了解决这个问题，将进行实验以确定：1）细胞大小的增加是否导致[Ca 2 +]i的增加; 2）细胞大小的增加是否引发[Ca 2 +]i的升高和/或PRL释放的增加;以及3）暴露于低渗培养基后细胞大小和PRL释放的增加是否依赖于细胞外Ca 2+的进入为了实现这些目标，将使用体外灌注-孵育技术，其允许分别通过视频捕获的图像、同源放射免疫测定和显微荧光光谱测定来测量单个细胞大小、PRL释放和细胞内钙浓度（[Ca 2 +]i）。 一般的方法，用于测试所提出的假设包括利用内源性调节PRL释放和药理学试剂来操纵的各个步骤pueretically参与的渗透信号的转导。拟议中的研究将提供一个更好的理解的信号转导过程中涉及的接收，一种感觉的方式，其充分理解已受到阻碍，缺乏一个合适的模型系统。使用罗非鱼PRL细胞作为模型提供了独特的研究优势，研究人员无法解决其他脊椎动物的哺乳动物神经调节系统中的类似问题。

Prolactin (PRL) is a highly versatile hormone of the pituitary gland of all vertebrates. It has essential actions in the regulation of salt and water balance (osmoregulation) in fish, including the tilapia, Oreochromis mossambicus, a species which can move freely between fresh water and seawater (it is euryhaline). In the diluting freshwater environment, blood ion levels tend to fall and PRL acts to counter this by increasing blood osmolality and reducing the water and ion permeability of skin and other osmoregulatory surfaces. Consistent with these actions, PRL cells show greater activity in tilapia held in fresh water than in those held in seawater. Blood PRL also rises and remains elevated as blood osmolality declines when tilapia move from seawater to freshwater. The tilapia PRL cell appears to be directly responsive to changes in extracellular osmolality inasmuch as PRL release increases as medium osmolality is reduced when tilapia pituitary glands are incubated in vitro. The term "osmoreceptor" refers to cells that respond to osmotic signals with an appropriate physiological response that is evoked through mechanisms called signal transduction pathways. Calcium (Ca+2) is widely used in these pathways as a second messenger that mediates the induction of cellular responses to specific external signals. The osmoreceptive properties of the tilapia PRL cells offer an excellent and possibly unique model system for investigating the pathways through which an osmotic signal is transduced into an osmoregulatory response (i.e., PRL secretion). In teleosts, unlike in other vertebrates, PRL cells are segregated into the anterior portion of the pituitary as a nearly homogeneous mass, which is easily separated for organ and dispersed-cell culture. This anatomical arrangement greatly facilitates the characterization of secretory mechanisms, and provides a highly useful model that can augment our general understanding of the regulation of other less-accessible osmosensitive (and osmoregulatory) endocrine and neuroendocrine systems (e.g. the hypothalamo-neurohypophysial magnocellular systems involved in vasopressin and oxytocin release in mammals).The long-term objective of this research is to identify the mechanisms involved in the regulation of the production and release of PRL by changes in the osmotic environment of the tilapia PRL cell. The present studies build on previous work in this laboratory, and are designed to construct a more detailed picture of signal transduction in osmoreceptive cells. The specific aim is to determine whether the rise in PRL release in response to reduced extracellular osmolality is triggered by an increase in cell size that leads to an increase in intracellular free Ca+2 ([Ca2+]i). The hypothesis is that a fall in extracellular osmolality leads to the passive influx of water into the PRL cell that increases cell volume; this initiates an influx of extracellular Ca2+ and a rise in [Ca2+]i, which triggers the increase in PRL release. In order to address this question, experiments will be conducted to determine: 1) whether an increase in cell size leads to an increase in [Ca2+]i; 2) whether an increase in cell size initiates a rise in [Ca2+]i and/or an increase in PRL release; and 3) whether an increase in cell size and PRL release following exposure to hyposmotic medium is dependent on the entry of extracellular Ca2+.To achieve these aims, in vitro perifusion-incubation techniques will be used which allow measurements of individual cell size, PRL release, and intracellular calcium concentration ([Ca2+]i) through video-captured images, homologous radioimmunoassays, and microspectrofluorometry, respectively. The general approach for testing the proposed hypotheses consists of utilizing endogenous regulators of PRL release and pharmacological agents to manipulate the various steps putatively involved in the transduction of the osmotic signal. The proposed research will provide a better understanding of the signal transduction processes involved in osmoreception, a sensory modality whose full understanding has been hampered by the lack of a suitable model system. The use of the tilapia PRL cell as a model offers distinct investigative advantages unavailable to researchers addressing similar questions in other vertebrate osmoregulatory systems.