Neuroendocrine-modulated epithelial HCO3- transport

描述(由申请人提供)： 该项目的长期目标是确定说明上皮性HCO3转运的机制和相关的调节级联反应，特别关注男性生殖道。精子在输精管中的成熟、储存和激活需要不同的PHS。上皮性阴离子转运疾病，如囊性纤维化，会导致男性不育。我们建立了研究完整的猪输精管、新鲜分离的人和猪组织、原代细胞培养和永生化上皮细胞中离子转运的方案。这些细胞系统中的每一个都显示出在完整组织中观察到的特性。这套非凡的实验系统将用于实现以下具体目标。目的1：测试不同的HCO3转运模型。最简单的模型有一个Na？共转运体和一个Na？？共转运蛋白在基底膜，CFTR在顶膜。输精管细胞中还存在其他的HCO3转运蛋白。我们将确定这些成分的功能贡献，并具体确定输精管分泌功能是否存在节段性差异。目的2：确定通过输精管上皮调节HCO3分泌的途径。我们假设生理递质不同地调节Cl-和HCO3-的分泌，以获得具有不同pH的管腔液体容量。去甲肾上腺素和腺苷刺激PKA，而催产素刺激PKC实现阴离子分泌。缓激肽通过一种未知的机制刺激阴离子分泌，这需要环氧合酶的活性，这种反应可以通过睾酮预处理来增强。我们将首先关注缓激肽，以阐明引起这些观察的受体(S)和胞浆途径(S)，并确定其对HCO3-和/或Cl-分泌的相对影响。此外，我们将确定激动剂刺激的信号通路，这些通路被整合在一起，积极地改变精子暴露在其中的流体环境。目的3：建立永生化人输精管细胞系。我们建立的猪输精管上皮细胞系是一种有价值的研究工具。然而，类似的人类细胞系将具有更大的价值，因为有许多针对人类组织使用的工具可用。因此，我们将开发另一种用于研究的细胞系，可以外推到人体导管。目的4：在体内评价上皮细胞HCO3转运的动态变化。输精管管腔pH值尚未在任何物种中得到系统评估，尽管这一参数对精子的储存和激活至关重要。这一结果将为体外试验确定的转运机制提供生殖生理学基础。这些研究的结果将确定药物干预的靶点，以调节管腔pH值，最直接地应用于男性生育。我们将建立一个机制模型(S)来解释上皮性HCO3运输的急性调节，该模型可以扩展到其他身体系统并与之进行比较。 男性生殖道内的细胞主动调节内液的pH值，这对精子成熟和激活非常重要。我们开发并将使用一些来自人类和猪生殖管的实验系统来确定这些细胞如何调节精子接触的液体的pH值和体积。这些研究的结果将确定治疗男性不育症或实施男性避孕的方法。

DESCRIPTION (provided by applicant): The long-term goal of this project is to determine the mechanisms and associated regulatory cascades that account for epithelial HCO3- transport with particular focus on the male reproductive tract. Distinct pHs are required for sperm maturation, storage and activation in the deferent duct. Diseases of epithelial anion transport such as cystic fibrosis result in male infertility. We established protocols to study ion transport in the intact porcine vas deferens, in freshly isolated human and porcine tissues, in primary cell cultures, and in immortalized epithelial cells. Each of these cell systems exhibits properties observed in the intact tissue. This remarkable set of experimental systems will be used to achieve the following specific aims. Aim 1: To test alternative models of HCO3- transport. The simplest model has a Na? cotransporter and a Na?? cotransporter in the basolateral membrane, and CFTR in the apical membrane. Additional HCO3- transporters are present in vas deferens cells. We will determine the functional contribution of these components and specifically determine whether there is segmental variation in vas deferens secretory function. Aim 2: To determine pathways that modulate HCO3- secretion across vas deferens epithelium. We hypothesize that physiological transmitters differentially regulate Cl- and HCO3- secretion to achieve luminal fluid volumes with distinct pH. Norepinephrine and adenosine stimulate PKA whereas oxytocin stimulates PKC to achieve anion secretion. Bradykinin stimulates anion secretion by an undetermined mechanism that requires cyclooxygenase activity and the response is enhanced by testosterone pretreatment. We will focus first on bradykinin to elucidate the receptor(s) and cytosolic pathway(s) that accounts for these observations and determine the relative effects on HCO3- and/or Cl- secretion. Further, we will determine the agonist-stimulated signaling pathways that are integrated to actively modify the fluid environment to which sperm are exposed. Aim 3: To develop an immortalized human vas deferens cell line. The porcine vas deferens epithelial cell line that we developed is a valuable research tool. However, greater value will be associated with an analogous human cell line because numerous tools that are targeted for use with human tissues are available. Thus, we will develop another cell line for studies that can be extrapolated for inferences to the human duct. Aim 4: To assess, in vivo, dynamic changes in epithelial HCO3- transport. Vas deferens lumen pH has not been systematically assessed in any species although this parameter is critical for sperm storage and activation. Results will provide a foundation in reproductive physiology for the transport mechanisms that are identified by in vitro assays. Results from these studies will identify targets for pharmacological interventions to modulate luminal pH with the most direct application to male fertility. We will establish a mechanistic model(s) to account for acute modulation of epithelial HCO3- transport that can be extended to, and compared with, other bodily systems. Cells lining the male reproductive tract actively regulate the pH of the internal solution, which is important for sperm maturation and activation. We developed and will use a number of experimental systems from human and pig reproductive duct to determine how these cells regulate the pH and volume of the fluid to which sperm are exposed. Results from these studies will identify methods to treat male infertility or to implement male contraception.