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

催乳素(PRL)是所有脊椎动物脑下垂体的一种高度多功能的激素。它在调节鱼类的盐分和水分平衡(渗透压调节)方面具有重要作用，包括罗非鱼，这是一种可以在淡水和海水之间自由移动的物种(它是泛盐类)。在稀释的淡水环境中，血液离子水平往往会下降，PRL通过增加血液渗透压和降低皮肤和其他渗透调节表面的水和离子渗透性来应对这种情况。与这些行为一致的是，在淡水中保存的罗非鱼中，催乳素细胞比在海水中保存的罗非鱼显示出更大的活性。当罗非鱼从海水转移到淡水中时，血液PRL也会上升，并随着血液渗透压的下降而保持升高。罗非鱼PRL细胞似乎对细胞外渗透压的变化有直接反应，因为当罗非鱼脑垂体体外孵育时，随着介质渗透压的降低，PRL的释放增加。渗透压感受器是指细胞对渗透压信号作出适当的生理反应，这种反应是通过称为信号转导通路的机制引起的。钙在这些途径中被广泛使用，作为第二信使，介导细胞对特定外部信号的反应。罗非鱼PRL细胞的渗透调节特性为研究渗透压信号转化为渗透调节反应(即PRL分泌)的途径提供了一个极好的、可能是唯一的模型系统。硬骨鱼与其他脊椎动物不同，PRL细胞被分离到脑垂体的前部，形成几乎均匀的团块，很容易分离出来用于器官和分散细胞的培养。这种解剖结构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，可以增强我们对其他较难获得的渗透敏感(和渗透压调节)内分泌和神经内分泌系统(例如，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)的调控。本研究的长期目标是通过改变罗非鱼PRL细胞的渗透环境来确定参与调节PRL产生和释放的机制。目前的研究建立在本实验室以前的工作基础上，旨在构建更详细的渗透感受细胞信号转导图景。其具体目的是确定细胞外渗透压降低引起的PRL释放增加是否由细胞大小增加导致细胞内游离钙([Ca~(2+)]i)增加所触发。假说是，细胞外渗透压的下降导致水被动流入PRL细胞，从而增加细胞体积；这启动了细胞外钙的内流和[Ca~(2+)]i的升高，从而触发了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起[Ca~(2+)]i的升高和/或PRL释放的增加；3)细胞大小和PRL释放的增加是否依赖于细胞外钙的进入。为了达到这些目的，将使用体外灌流孵育技术，通过视频捕获图像、同源放射免疫分析和显微荧光分光光度法分别测量单个细胞的大小、PRL释放和细胞内钙离子浓度([Ca2]i)。检验这些假说的一般方法包括利用内源性PRL释放调节剂和药物来操纵渗透信号转导的各个步骤。这项拟议的研究将提供对渗透感受所涉及的信号转导过程的更好理解，渗透感受是一种感觉通道，由于缺乏合适的模型系统，其充分理解一直受到阻碍。使用罗非鱼PRL细胞作为模型提供了独特的研究优势，这是研究人员在解决其他脊椎动物渗透压调节系统中类似问题时所没有的。

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.